International Review of Research in Mental Retardation: Neurotoxicity and Developmental Disabilities

Neurodevelopmental Effects of Alcohol THOMAS M. BURBACHER AND KIMBERLY S. GRANT DEPARTMENT OF ENVIRONMENTAL AND OCCUPATIONAL HEALTH SCIENCES SCHOOL OF PUBLIC HEALTH AND COMMUNITY MEDICINE WASHINGTON NATIONAL PRIMATE RESEARCH CENTER AND CENTER ON HUMAN DEVELOPMENT AND DISABILITY UNIVERSITY OF WASHINGTON SEATTLE, WASHINGTON

I.

INTRODUCTION

The goal of this chapter is to provide an overview of the developmental eVects of prenatal exposure to alcohol in the forms of ethanol and methanol. All alcohols have a similar chemical structure. The three most commonly known alcohols are ethyl alcohol (ethanol), methyl alcohol (methanol), and isopropyl alcohol (isopropanol). Isopropanol is better known as rubbing alcohol, a common item in most American homes. Human exposures to toxic levels of isopropanol are uncommon and have not been reported in pregnant women. Health eVects data from animal studies suggest that isopropanol has low acute and chronic toxicity and is not a teratogen or developmental neurotoxicant (Kapp et al., 1996). Most research on the fetal eVects of maternal alcohol exposure has focused on the compounds ethanol and methanol. The physical chemical properties of these two agents are displayed in Table I. Methanol (H3C–OH), a component of many products, including alternative motor fuels, antifreeze, glass cleaner, paints, and varnishes, is the simplest alcohol with a chain consisting of a carbon atom with three hydrogen atoms attached. Ethanol (H3C–CH2–OH), the psychoactive ingredient in alcoholic beverages that results in intoxication, has a chain that is two times as long. While similar exposure scenarios exist for these two compounds (occupational exposure, intentional ingestion), the primary routes of exposure that are related to developmental eVects are quite diVerent. Ethanol is an ancient drug that is widely accepted throughout the world, consumed in the form of alcoholic beverages to achieve a pleasant state of euphoria or relaxation. Although women working in professions such as nurses, assemblers, janitors, INTERNATIONAL REVIEW OF RESEARCH IN MENTAL RETARDATION, Vol. 30 0074-7750/06 $35.00

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Copyright 2006, Elsevier Inc. All rights reserved. DOI: 10.1016/S0074-7750(05)30001-2

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Thomas M. Burbacher and Kimberly S. Grant TABLE I PHYSICAL CHEMICAL PROPERTIES a AND METHANOL

OF

ETHANOL

Name

Methanol

Ethanol

Synonyms Structure Formula CAS RN Molecular weight Melting point Boiling point Flash point Physical appearance

Methyl alcohol, wood alcohol H3C–OH CH4O 67‐56‐1 32.04
98 C 64.6 C 12 C Clear, colorless, very mobile, flammable liquid with pungent, slightly alcoholic odor Miscible with water and most other organic solvents Industrial and laboratory solvent, to denature ethanol, chemical reagent, antifreeze octane booster in gasoline, requisite for hydrogen fuel cell technology

Ethyl alcohol, alcohol H3CCH2OH C2H6O 64‐17‐5 46.07
114 C 78.5 C 13 C Clear, colorless, very mobile, flammable liquid with pleasant alcoholic odor and burning taste Miscible with water and most other organic solvents Alcoholic beverages Industrial and laboratory solvent In pharmaceutical preparations and perfumes Antiseptic Octane booster in gasoline

Miscibility Principal uses

a From ‘‘The Merck Index, An Encyclopedia of Chemicals, Drugs, and Biologicals’’ (2001). Merck & Co., Inc, Whitehouse Station, New Jersey.

clinical lab technicians, and housekeepers can also be exposed to ethanol (Seta et al., 1988), a recent review found that low blood alcohol levels resulting from occupational exposure do not represent a risk to pregnant women (Irvine, 2003). In contrast, exposure to methanol occurs almost exclusively in industrial and laboratory settings. Women working as assemblers, janitors, clinical laboratory technicians, machine operators, and mechanics can be exposed to methanol via dermal absorption and inhalation (Seta et al., 1988). Since 1988, methanol has received attention as a low‐emission, high‐performance motor fuel and the primary fuel source for vehicles powered by hydrogen‐based fuel cell technology (Gold & Moulis, 1988; Fuller et al., 1997). If methanol‐based fuels were used on a widespread basis in the future, there would be public exposure on streets, refueling stations, and garages. This chapter draws from both the human and animal literature to explore the consequences of prenatal exposure to ethanol and methanol on the behavioral development of exposed

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oVspring. For ethanol, the weight of evidence to determine the risk of adverse eVects on development from prenatal exposure comes largely from prospective, longitudinal studies of human maternal–infant pairs. While a large volume of supportive studies using animal models does exist for ethanol, a comprehensive review of these studies was considered beyond the scope of this chapter. Excellent reviews of the teratogenic eVects of alcohol using animal models are available (see Guerri & Hannigan, 1996). For methanol, few reports are available describing exposure eVects in human infants. The weight of evidence to determine the risk of adverse eVects on development from prenatal exposure to methanol, therefore, comes largely from studies using animal models.

II.

ETHANOL

Ethanol (EtOH) is a small molecule compound that is soluble in water and lipids, easily passing through cell membranes in the body (Ramchandani et al., 2001). Ethanol selectively concentrates in highly vascularized organs such as the lungs and the brain and these organs have higher EtOH concentrations after exposure. Metabolism of EtOH is dependent on enzymes in the liver that initiate its metabolic breakdown. Ethanol is metabolized by the enzyme alcohol dehydrogenase (ADH) to acetaldehyde, which, in turn, is metabolized by the enzyme, aldehyde dehydrogenase, to acetic acid, and ultimately to water and carbon dioxide. Women metabolize EtOH diVerently from men and have higher blood levels of EtOH due to higher fat content, smaller body size, and less gastric‐ADH activity (Frezza et al., 1990; Seitz et al., 1993). In general, women are at increased risk for EtOH‐induced brain injury (see review by Prendergast, 2004). Female alcoholics show measurable brain shrinkage after shorter periods of EtOH exposure than male subjects (Mann et al., 1992). The enhanced sensitivity of women to the adverse eVects of alcoholism is not limited to the brain but includes higher rates of advanced liver disease and other EtOH‐related disorders (Morgan & Sherlock, 1977). Prenatal exposure to EtOH is the leading cause of preventable birth defects and mental retardation in the United States, if not the world. The consumption of beer, wine, or spirits during pregnancy can have a profound impact on the processes of normal child development. The eVects of EtOH are dose‐dependent and children born to alcoholic or EtOH‐abusing mothers are at the highest risk for poor developmental outcome (Stratton et al., 1996). Developmental eVects are widespread and range from structural malformations (skeleton, heart, kidney) to delays in physical growth and deficits in neurobehavioral development (learning, memory, language,

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and social behavior) (American Academy of Pediatrics, 2000; Mattson & Riley, 1998; Mattson et al., 2001). The United States has made great strides in educating women of reproductive age about the dangers of drinking EtOH during pregnancy. The rate of drinking among pregnant women in the United States declined from 16.3% in 1995 to 12.8% in 1999 (MMWR, 2002). The rate of binge‐drinking and frequent EtOH abuse rates, however, remained stable during the same period (2.7 and 3.3%, respectively). A.

Historical Perspective

Throughout history, maternal drinking during pregnancy has been suspected of causing adverse eVects in exposed oVspring. Mentioned by the Hebrews in the Book of Judges 13:4, the harmful eVects of drinking have been known since biblical times when couples were forbidden to drink wine on their wedding night so that aVected infants would not be conceived (Haggard & Jellinek, 1942). A report on drunkenness to the British House of Commons in the 1800s indicated that oVspring of alcoholic women were frequently ‘‘born weak and silly . . . shriveled and old, as though they had numbered many years’’ (Goodacre, 1965). In 1900, a published report noted that alcoholic women had increased rates of spontaneous miscarriage and delivery of stillborn infants and exposed oVspring were at high risk for epilepsy (Sullivan, 1900). Although historical writings warned of the dangers of drinking during pregnancy, physicians during the post‐prohibition era in the United States dismissed these concerns as moralism and interest in the subject sharply declined (Warner & Rosett, 1975). In the late 1960s, a team of French investigators published a report indicating that children born to alcoholic mothers shared a number of distinguishing physical characteristics (Lemoine et al., 1968). International attention was not, however, directed at this constellation of birth defects until the clinical observations of Jones and Smith were published in 1973. These investigators coined the term ‘‘fetal alcohol syndrome’’ (FAS) to describe the prenatal and postnatal growth deficiencies and physical malformations observed in infants born to alcoholic mothers. The death of one of the study subjects allowed for the first necropsy of an FAS infant and neuropathology results confirmed that there was severe damage to both neuronal and glial cells as well as absence of the corpus callosum. The pioneering work of these Seattle dysmorphologists triggered new clinical and scientific interest in EtOH as a serious teratogen and prospective research programs were initiated in the cities of Seattle, Detroit, Cleveland, Atlanta, and Pittsburgh (Streissguth et al., 1981; Jacobson et al., 1993a; Greene et al., 1991a; Coles et al., 1991; Day et al., 1989).

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Clinical Features of Fetal Alcohol Syndrome and Related Disorders

The most serious outcome for the children of drinking mothers is fetal alcohol syndrome (FAS). As detailed in the 1996 report from the Institute of Medicine (IOM) (Stratton et al., 1996), clinical diagnosis of this syndrome is based on three required criteria: (1) a deficiency in prenatal and postnatal physical growth, (2) impairment of the central nervous system (CNS), and (3) specific craniofacial malformations. The diagnostic criteria for FAS and alcohol‐related eVects from the 1996 IOM report are displayed in Table II. The diagnosis of FAS requires that the height, weight, and/or length of the child be less than the 10th percentile. Physical growth in FAS infants is characterized by growth retardation and may include reductions in birth weight, loss of weight over time not associated with nutrition, and body weight that is disproportional to height, length, and head circumference. Damage to the CNS is required for the FAS diagnosis and eVects in oVspring include mental retardation, neurological abnormalities, or developmental delays. The diagnosis of FAS also requires the presence of a distinct facial dysmorphology, often involving abnormalities of the eyes and the nose (see Figs. 1 and 2). In addition to microcephaly, facial features of the syndrome include short palpebral fissures, flattened midface, indistinct philtrum, thin upper lip, and upturned nose (Abel, 1984). Common features of the FAS face that are not required for a formal diagnosis include epicanthal folds, low nasal bridge, minor ear anomalies, and micrognathia. In addition to the diagnostic criteria set forth in the 1996 IOM report, a second set of clinical diagnostic criteria have been published by Astley and Clarren (2000). This system is known as the Washington criteria and is based on a four‐digit code that corresponds to the requisite diagnostic features of FAS. While vastly helpful in accurate diagnoses of aVected infants, a number of weaknesses with both coding schemes have been pointed out (Hoyme et al., 2005). Limitations include failing to adequately integrate the family/genetic history of the child into the criteria, confusing terminology, and inadequate definitions of clinical diagnoses (encephalopathy, neurobehavioral disorder). Hoyme and colleagues (2005) have proposed a clarification of the 1996 IOM criteria to enhance the identification and treatment of children with fetal alcohol spectrum disorders. These changes allow for more accurate diagnoses in routine clinical settings. Many children born to mothers who drink heavily during pregnancy do not meet the criteria for a formal diagnosis of FAS. It is now well recognized that there can be significant behavioral changes in children born to drinking

6 DIAGNOSTIC CRITERIA

Thomas M. Burbacher and Kimberly S. Grant TABLE II* FETAL ALCOHOL SYNDROME (FAS) RELATED EFFECTS (IOM, 1996)

FOR

AND

ALCOHOL‐

Fetal Alcohol Syndrome 1. FAS with confirmed maternal alcohol exposurea A. Confirmed maternal alcohol exposurea B. Evidence of a characteristic pattern of facial anomalies that includes features such as short palpebral fissures and abnormalities in the premaxillary zone (flat upper lip, flattened philtrum, and flat midface) C. Evidence of growth retardation, as in at least one of the following: ‐ low birth weight for gestational age ‐ decelerating weight over time not due to nutrition ‐ disproportional low weight to height D. Evidence of CNS neurodevelopmental abnormalities, as in at least one of the following: ‐ decreased cranial size at birth ‐ structural brain abnormalities (microcephaly, partial or complete agenesis of the corpus callosum, cerebellar hypoplasia) ‐ neurological hard or soft signs (as age‐appropriate), such as impaired fine motor skills, neurosensory hearing loss, poor tandem gait, poor eye–hand coordination 2. FAS without confirmed maternal alcohol exposure B, C, and D as previously stated 3. Partial FAS with confirmed maternal alcohol exposure A. Confirmed maternal alcohol exposurea B. Evidence of some components of the pattern of characteristic facial anomalies Either C or D or E C. Evidence of growth retardation, as in at least one of the following: ‐ low birth weight for gestational age ‐ decelerating weight over time not due to nutrition ‐ disproportional low weight to height D. Evidence of CNS neurodevelopmental abnormalities, as in: ‐ decreased cranial size at birth ‐ structural brain abnormalities (microcephaly, partial or complete agenesis of the corpus callosum, cerebellar hypoplasia) ‐ neurological hard or soft signs (as age‐appropriate), such as impaired fine motor skills, neurosensory hearing loss, poor tandem gait, poor eye–hand coordination E. Evidence of a complex pattern of behavior or cognitive abnormalities that are inconsistent with developmental level and cannot be explained by familial background or environment alone, such as learning diYculties; deficits in school performance; poor impulse control; problems in social perception; deficits in higher‐level receptive and expressive language; poor capacity for abstraction or metacognition; specific deficits in mathematical skills; or problems in memory, attention, or judgment. Alcohol‐Related EVects Clinical conditions in which there is a history of maternal alcohol exposure,a,b and where clinical or animal research has linked maternal alcohol ingestion to an observed outcome. There are two categories, which may co‐occur. If both diagnoses are present, then both diagnoses should be rendered:

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TABLE II (continued ) 4. Alcohol‐related birth defects (ARBD) List of congenital anomalies, including malformations and dysplasias Cardiac Atrial septal defects Aberrant great vessels Ventricular septal defects Tetralogy of Fallot Skeletal Hypoplastic nails Clinodactyly Shortened fifth digits Pectus excavatum and carinatum Radioulnar synostosis Klippel–Feil syndrome Flexion contractures Hemivertebrae Camptodactyly Scoliosis Renal Aplastic, dysplastic, hypoplastic kidneys Ureteral duplications Horseshoe kidneys Hydronephrosis Ocular Strabismus Refractive problems secondary to small globes Retinal vascular anomalies Auditory Conductive hearing loss Neurosensory hearing loss Other Virtually every malformation has been described in some patient with FAS. The etiologic specificity of most of these anomalies to alcohol teratogenesis remains uncertain. 5. Alcohol‐related neurodevelopmental disorder (ARND) Presence of: A. Evidence of CNS neurodevelopmental abnormalities, as in any one of the following: ‐ decreased cranial size at birth ‐ structural brain abnormalities (microcephaly, partial or complete agenesis of the corpus callosum, cerebellar hypoplasia) ‐ neurological hard or soft signs (as age‐appropriate), such as impaired fine motor skills, neurosensory hearing loss, poor tandem gait, poor eye–hand coordination and/or: B. Evidence of a complex pattern of behavior or cognitive abnormalities that are inconsistent with developmental level and cannot be explained by familial background or environment alone, such as learning diYculties; deficits in school performance; poor impulse control; problems in social perception; deficits in higher‐level receptive and expressive language; poor capacity for abstraction or metacognition; specific deficits in mathematical skills; or problems in memory, attention, or judgment. a

A pattern of excessive intake characterized by substantial, regular intake, or heavy episodic drinking. Evidence of this pattern may include frequent episodes of intoxication, development of tolerance or withdrawal, social problems related to drinking, legal problems related to drinking, engaging in physically hazardous behavior while drinking, or alcohol‐related medical problems, such as hepatic disease. b As further research is completed and as, or if, lower quantities or variable patterns of alcohol use are associated with ARBD or ARND, these patterns of alcohol use should be incorporated into the diagnostic criteria. *Reprinted with permission from ‘‘Fetal Alcohol Syndrome: Diagnosis, Epidemiology, Prevention, and Treatment’’ (1996) by the National Academy of Sciences, courtesy of the National Academies Press, Washington, DC.

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FIG. 1. Facial features associated with fetal alcohol syndrome in the young child. Reprinted with permission from Streissguth, A. P., & Little, R. E. (1994). ‘‘Unit 5 Alcohol, Pregnancy, and Fetal Alcohol Syndrome: Second Edition’’ of the Project Cork Institute Medical School Curriculum (slide lecture series) on Biomedical Education: Alcohol Use and Its Medical Consequences, produced by Dartmouth Medical School.

FIG. 2. Infants and young children with fetal alcohol syndrome. Reprinted with permission from Streissguth, A. P., Landesman‐Dwyer, S., Martin, D. C., & Smith, D. W. (1980). Teratogenic eVects of alcohol in humans and laboratory animals. Science, 209(18), 353–361.

mothers without the full expression of FAS. These children may have mild to severe impairments in attention, memory, and adaptive behavior but lack the dysmorphic facial features required for a formal diagnosis of FAS. This condition has been historically referred to as fetal alcohol eVects (FAE). In 1996, the IOM proposed the terms alcohol‐related neurodevelopmental disorder (ARND) and alcohol‐related birth defects (ARBD) to describe non‐FAS children with a history of maternal EtOH exposure and poor developmental outcomes that have been clinically or scientifically linked to in utero EtOH exposure (Stratton et al., 1996). In an attempt

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to unify the diverse terms being used in the EtOH literature, the term fetal alcohol spectrum disorder (FASD) was proposed by Barr and Streissguth (2001) to denote all clinical and pathological manifestations of EtOH teratogenicity. Sampson and colleagues evaluated the incidence of FAS and ARND using two longitudinal U.S. studies (in Seattle and Cleveland) and a large, prospective study of infants born in Roubaix, France (Sampson et al., 1997). Results from the Seattle cohort of primarily white, married, middle‐class women yielded an FAS incidence of 2.8 per 1000 live births. The incidence of FAS in the Cleveland study with a low‐SES, inner‐city sample was 4.6 per 1000 while the incidence of FAS in Roubaix was 2.3 per 1000 live births. The authors noted that mothers of children diagnosed with FAS in Seattle, Cleveland, and Roibaix were likely to be alcoholic and poor. The prevalence of ARND was also calculated for the Seattle cohort, yielding a combined rate of FAS and ARND of 9.1/1000 live births. This finding implies that nearly one child in every 100 births is aVected by maternal EtOH consumption, a disquieting figure that underscores the magnitude of this public health problem. C.

Neurodevelopmental Effects of Ethanol Consumption During Pregnancy

It is now well established that prenatal exposure to EtOH can lead to a continuum of neurodevelopmental eVects in infants, children, and adolescents (Mattson & Riley, 1998). In keeping with the landmark principles of teratology laid out by Wilson (1977), the eVects of prenatal EtOH exposure are dependent on dose, timing, and conditions (e.g., age of mother) of exposure. Fetal alcohol syndrome is typically associated with heavy drinking or alcoholism during pregnancy. A drink is defined as 0.5 oz absolute alcohol (AA), whether it is in the form of beer, wine, or spirits. Although definitions vary, light drinking is usually defined as approximately one drink per day, moderate drinking as two drinks per day, and heavy drinking as 3.5 or more drinks per day (Abel et al., 1998). 1. PATTERN OF CONSUMPTION

Children born to alcoholic women may vary in their developmental outcome based on the pattern of maternal EtOH consumption. Peak blood EtOH levels of alcoholics diVer, depending on whether drinks are steadily consumed throughout the day or consumed in a single binge episode (Gladstone et al., 1996). The binge pattern of drinking refers to the consumption of 5 to 6 drinks on some occasions and is typically seen in women who may drink heavily on the weekend but abstain during the week. The

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increased threat that binge drinking poses to normal fetal development was demonstrated in a study of moderate to heavy drinking in a large, inner‐city sample of pregnant women in Detroit (Jacobson et al., 1998a). This study found that the most serious eVects of EtOH were observed in children whose mothers consumed more than five drinks per occasion at least once a week, indicating that a pattern of heavy, intermittent consumption placed the fetus at greatest risk for poor developmental outcome. Other cohort studies have also demonstrated that the sudden and significant increases in blood EtOH associated with binge drinking pose a greater danger to the developing fetus than do lower‐dose, chronic exposure scenarios (Streissguth et al., 1994b). A recent study of binge drinking during pregnancy found that exposed children were 1.7 times more likely to have an IQ in the range of mental retardation and 2.5 times more likely to have delinquent behavior in the classroom (Bailey et al., 2004). Data from comparative studies of EtOH exposure and pregnancy outcome have also demonstrated that in mice, rats, and monkeys, a single binge exposure or a series of binge exposures can result in significant physical and neurobehavioral abnormalities in exposed oVspring (Clarren et al., 1992; Goodlett & Eilers, 1997; Goodlett et al., 1990; Sulik et al., 1986). These results indicate that maternal peak blood EtOH values and not total volume consumed is most predictive of the teratogenic risk associated with prenatal drinking. Based on the available data, the intermittent binge pattern of heavy drinking with its attendant peaks in blood EtOH is associated with the greatest expression of neurotoxicity in exposed oVspring. 2. TIMING OF EXPOSURE

Timing of exposure is another important variable in defining the risk that maternal drinking poses to oVspring development. The groundbreaking work by Sulik (1984, 2005) demonstrated that the critical period for the induction of EtOH‐induced facial malformations in the mouse occurs very early in gestation (day 7) and lasts for a brief period of time (a few hours). As illustrated in Fig. 3, when EtOH exposure occurs on day 7 in gestation, the mouse fetus exhibits facial characteristics that are similar to children with FAS (small head, short palpebral fissures, and long upper lip with deficient philtrum). The limited time period during which the craniofacial area is sensitive to the eVects of EtOH may be one of the factors that explain why most women who are chronic alcoholics do not give birth to infants with facial dysmorphia. Research using macaque monkeys as an animal model of FAS found evidence that a critical period for craniofacial defects may exist in nonhuman primates as well (Astley et al., 1999). The greatest number of craniofacial alterations in young pigtailed macaques exposed to EtOH occurred when exposure took place on gestation day 19 or 20 (average gestation length is

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FIG. 3. Facial characteristics of fetal mouse exposed to ethanol. Reprinted with permission from Dr. Kathy Sulik, Department of Cell and Developmental Biology and Bowles Center for Alcohol Studies, The University of North Carolina, Chapel Hill.

175 days). Ethanol‐induced skeletal changes were diYcult to detect at birth, increased substantially at 6 months, and then gradually lessened as the animals reached 12 and 24 months of age. The authors note that studies in two animal species (monkey and mouse) support the notion of a critical period for induction of facial dysmorphia resulting from prenatal EtOH exposure. 3. NEUROBEHAVIORAL PROFILES ASSOCIATED WITH PRENATAL ETHANOL EXPOSURE

Not every child exposed to EtOH during prenatal development will display developmental deficits. Those that are affected can express deficits across a broad range of neurobehavioral domains (Jacobson & Jacobson, 2002; Mattson & Riley, 1998; Mattson et al., 2001). In the first published report on FAS, the investigators noted tremors and a weak sucking reflex in one infant while a second infant displayed marked developmental retardation (Jones & Smith, 1973). A third infant became cyanotic within 5 hours of birth, dying within 5 days. This report provided the first clear demonstration that infants with a history of high‐level gestational exposure can be accurately classified at birth. Data from early studies on maternal drinking and oVspring development indicated that a number of parameters were adversely aVected by exposure. Little and colleagues found that the birth weight of infants born to alcoholic women who drank during pregnancy was, on average, 493 grams less than that of control infants (Little et al., 1980).

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Published in the same year, data from a small study using the Bayley Scales of Infant Development indicated that EtOH‐exposed infants had significantly lower scores on scales of mental and motor development (Golden et al., 1982). A number of prospective, longitudinal research studies have provided the lion’s share of the data available on prenatal alcohol exposure and neurodevelopmental disability. To provide a perspective that emphasizes the long‐ term nature of alcohol neurotoxicity in exposed children, over years and even over decades, the general findings of larger cohort studies are examined in the following section. a. Seattle. In 1974, the Seattle Longitudinal Prospective Study on Alcohol and Pregnancy was initiated to study the eVects of socially acceptable levels of maternal drinking on pregnancy outcome and infant development in Washington state (Streissguth et al., 1981). A group of 1529 women, primarily white, married, and middle‐class, were identified from local hospitals and screened for participation in the study. Two cohorts of infants were examined. One cohort consisted of 163 infants with mothers who drank, on average, 2 or more drinks per day or who had a history of binge drinking (5 or more drinks per occasion). Study infants were examined by dysmorphologists and two newborns in this sample were diagnosed with full FAS (Hanson et al., 1978). There was a significant relationship between level of prenatal exposure to EtOH and the presence of physical features compatible with FAS. Infants with features compatible with FAS did not receive a formal FAS diagnosis but did express some of the growth anomalies associated with this syndrome (fetal growth retardation, microcephaly, and facial dysmorphia). The diagnosis of ‘‘features compatible with FAS’’ was found in 19% of infants with mothers who drank 4 or more drinks per day and 11% of children whose mothers reported drinking 2 to 4 drinks per day. The second Seattle cohort consisted of approximately 500 infants who were selected based on maternal traits, eVectively dividing the group between infants born to heavier drinkers (two or more drinks/day) and infants born to women who drank infrequently or abstained (Streissguth et al., 1981). Close to 80% of the women in this cohort reported drinking at some point during pregnancy and infants born to mothers consuming more than two drinks per day were overrepresented in the sample (36%). A binge style of drinking (five or more drinks on any one occasion) was reported by 39% of women in early pregnancy and 25% continued this pattern during midpregnancy. Only eight women in this cohort reported a significant problem with alcohol, emphasizing that most subjects in this group were not drinking at levels associated with chronic alcohol abuse and alcoholism. Increased EtOH consumption during pregnancy was related to decreased neonatal growth patterns, particularly for birth weight, length, and head circumference

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(Martin et al., 1980). While eVects on early postnatal growth were related to prenatal alcohol exposure in a dose‐dependent fashion, these diVerences were not present after 8 months of age (Streissguth et al., 1980). Subsequent data analyses revealed a lack of association between physical growth parameters and prenatal alcohol exposure from 8 months through 14 years of age, suggesting that the early size eVects documented in this cohort were transient and physical growth was not permanently disrupted (Sampson et al., 1994). The Brazelton Neonatal Assessment Scale (BNAS), commonly used in clinical and research settings, measures a range of abilities, including reflex strength, behavioral state, and general reactivity. On day 1 of life, Seattle investigators found alcohol use during midpregnancy was related to poorer habituation and increased levels of low arousal on the BNAS (Streissguth et al., 1983). The authors note that the levels of drinking associated with diminished performance on the Brazelton were clearly within the socially acceptable range (2–4 drinks per day). It is interesting to note that in this cohort, 474 behavioral outcomes were studied across the first 7 years of life and habituation to light in early infancy showed the strongest relationship with maternal drinking (Streissguth et al., 1993). On day 2 of life, head turning and sucking behaviors were evaluated using operant test procedures to study newborn conditioning (a form of simple learning) (Martin et al., 1977). Although maternal EtOH exposure alone was not related to performance, the combination of maternal drinking and smoking did significantly impact early learning abilities in exposed infants. Mental and psychomotor processing was evaluated in this cohort at 8 months of age with the Bayley Scales of Infant Development (Streissguth et al., 1980). Prenatal EtOH exposure had significant eVects on behavioral development when mothers drank, on average, 4 or more drinks per day while infants born to women drinking 2 to 4 drinks per day were not adversely aVected. These data suggest that the threshold of drinking associated with adverse behavioral eVects in infancy is somewhere between 2 and 4 drinks per day. At 4 years of age, decrements in IQ were documented in the EtOH‐ exposed children from the Seattle cohort (Streissguth et al., 1989). Results support a threshold relationship where 3 drinks per day during pregnancy was associated with a 5‐point drop in IQ. Assessment of fine and gross motor development revealed that more errors, longer performance time, longer latency to correct errors, and poorer balance were associated with maternal EtOH consumption (Barr et al., 1990). Children exposed to approximately one drink per day during early pregnancy made more errors on a test of manual dexterity while time to completion was increased in children exposed to approximately three drinks per day. Reaction time and attention

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were measured with a vigilance test where children were asked to press a button when a small cat appeared on a screen (Streissguth et al., 1984). Alcohol‐exposed children made more errors of omission and commission on this task and frequently took longer to respond than did unexposed controls, suggesting a fundamental slowing of mental processing speed. At 7.5 years, further vigilance testing revealed attentional decrements and slower reaction times in children with a history of prenatal EtOH exposure (Streissguth et al., 1986). Errors of commission (responding when no stimulus was present) was the performance variable most highly correlated with prenatal EtOH exposure. Drinking during pregnancy was also related to longer reaction times, further emphasizing decrements in speed of cognitive operations. In general, the magnitude of the EtOH eVect on childhood attention increased with higher levels of exposure and a pattern of maternal binge drinking. Further testing at 7.5 years focused on the assessment of IQ, learning, and classroom behavior (Streissguth et al., 1990). Although most children were performing in the range of normal intelligence, consumption of two drinks or more per day during midpregnancy was associated with a 7‐point decrement in IQ. The IQ subtests most aVected by EtOH exposure were Arithmetic, Digit Span, and Block Design. On tests of academic achievement, arithmetic emerged as the area most aVected by a history of prenatal EtOH exposure but reading was also negatively impacted. A binge pattern of maternal drinking (greater than five drinks on any one occasion) was associated with a 1‐ to 3‐month delay in learning math and reading by the end of the first grade. Ratings of classroom behavior revealed that prenatal EtOH exposure was related to distractibility, diYculty with retention and recall, and poor organizational skills. Deficits in problem‐solving, the ability most strongly aVected by gestational EtOH exposure, and speed of information processing were primarily associated with a binge‐style drinking pattern. Findings from this study suggest that heavy gestational exposure to alcohol aVects memory, attention, and abstract problem solving and that by school age; these children are having diYculties with learning and adaptive classroom behavior. Eighty‐two percent of the original Seattle cohort was evaluated on measures of school achievement, adaptive behavior, and social competence in early adolescence (Olson et al., 1997). Study results demonstrated a subtle but significant relationship between maternal drinking and antisocial behavior, school problems, and negative self‐perception. Behavioral dysfunction was most strongly related to a pattern of maternal binge drinking and the level of drinking before recognition of pregnancy, not the level maintained during midpregnancy. At a 14‐year follow‐up, young adults were also

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evaluated using a number of psychometric tests measuring attention, memory, reading, and arithmetic (Streissguth et al., 1994a,b). Maternal binge or massed drinking was the pattern of EtOH exposure most highly associated with deficits in reading proficiency and numerical problem solving during adolescence. Although not all exposed subjects were aVected, maternal drinking during pregnancy was linked to problems with response inhibition, focused and sustained attention, and spatial learning in a dose‐dependent manner. The pattern of drinking associated with the highest risk to exposed oVspring was one in which drinks were clustered or massed. Scores on tests of arithmetic and phonological processing were also related to prenatal EtOH exposure in a dose‐dependent fashion. The attentional/memory deficits observed in these adolescent subjects were correlated (0.67) with neurobehavioral eVects at 7 years of age. For children born to heavy drinkers, 91% of those who scored poorly in arithmetic at 7 years continued to score poorly on arithmetic at 14 years. Results from the latest published study of this cohort indicate that at 21 years of age, a history of prenatal EtOH exposure has placed these young adults at risk for experiencing EtOH‐related problems (Baer et al., 2003). Subjects were given the Alcohol Dependency Scale (ADS), and items such as passing out, blackouts, and physical illness were related to prenatal EtOH exposure. In contrast, actual alcoholic beverage consumption rates were not related to maternal drinking. These findings suggest that prenatal EtOH exposure is associated with negative consequences of heavy drinking in adulthood but is not related to patterns of behavior that reflect compulsive use or addiction. b. Detroit. A prospective study of prenatal EtOH exposure and cognitive development was conducted in Detroit, Michigan, with a sample of primarily poor, inner‐city women who received obstetrical care from a local maternity hospital (Jacobson et al., 1991). Recruitment of pregnant women took place from 1986 to 1989 and enrollment into the study was based on alcohol consumption during pregnancy. The cohort consisted of approximately 416 infants who were born to women enrolled in the study. Fifty‐one percent of women enrolled in the study were drinking around the time of conception and 10% continued during pregnancy. Sixteen percent of the mothers enrolled (n ¼ 67) abstained from alcohol while 74% (n ¼ 308) were light drinkers (less than one drink per day). Five percent of the mothers (n ¼ 22) were moderate drinkers (one to two drinks per day) while 3% (n ¼ 12) were heavy (two to four drinks per day) and 2% (n ¼ 7) were very heavy (over 4 drinks per day) drinkers. Birth weight and crown–rump length data were obtained from this cohort to evaluate the eVects of gestational EtOH exposure on fetal growth (Jacobson et al., 1994a). Heavily exposed infants weighed an

16

Thomas M. Burbacher and Kimberly S. Grant

average of 509 grams less at birth and were 4 cm shorter (crown–rump length) than unexposed controls. The eVects of ETOH exposure on fetal growth were primarily limited to those women who drank in excess of 4 drinks per day, supporting a threshold for prenatal EtOH eVects on early growth. Infants from this cohort were tested on visual recognition memory at 6.5 and 12 months and cross‐modal transfer at 12 months (a measure of memory that involves the transfer of sensory information between two modalities) (Jacobson et al., 1993b). The results indicated that basic recognition memory, both within and across sensory modalities, was normal in EtOH‐exposed infants. Although maternal drinking did not adversely impact early memory, a closer examination of the data revealed that the duration of visual fixations was longer in exposed subjects on both tasks. These findings suggest that ethanol‐exposed infants may be processing information more slowly than unexposed controls, requiring longer periods of time to encode test stimuli in memory. Infants born to mothers who drank at least two drinks per day were twice as likely to show deficits in speed of processing information. The threshold for an alcohol eVect on early information processing speed was approximately one or more drinks/day. Additional testing at 6.5 months utilized a visual expectancy test paradigm that relies on the shifting of visual gazes from one physical location to another (Jacobson et al., 1994b). Prenatal ethanol exposure was associated with slower response times and a reduction in the number of fast responses, corroborating initial results that suggested a delay or deficit in mental processing speed within this cohort of EtOH‐exposed infants. Slowed response times and fewer fast responses (measures of early cognitive dysfunction) were evident in infants born to mothers who averaged at least one drink per day. At 1 year of age, infants from this cohort were tested using the Bayley Scales of Infant Development to evaluate early mental and psychomotor development (Jacobson et al., 1993a). Higher levels of maternal drinking were associated with poorer scores on the Bayley and no threshold for safe drinking was ascertained. Substandard scores on the Bayley (1 SD below the sample mean) were primarily related to maternal consumption of 4 or more drinks per day around conception or one drink or more per day during pregnancy. The threshold for an alcohol eVect on early mental performance was approximately one drink per day. Functional impairment during infancy, when examined over all test measures, was not present in the oVspring of women who consumed less than one drink per day during pregnancy (Jacobson et al., 1998c). Deficits were greatest in exposed infants born to heavy drinkers who were older than 30 years of age and those born

NEURODEVELOPMENTAL EFFECTS OF ALCOHOL

17

to mothers with a binge pattern of EtOH consumption (five drinks per occasion at least once a week). Additional neurobehavioral evaluations were undertaken with this cohort when the children reached 7 years of age. Subjects were tested on measures of learning and memory and preliminary data suggest that greater levels of prenatal EtOH exposure were related to deficits in executive functioning, focused attention, and flexible problem solving (Jacobson et al., 1998a). From a psychosocial perspective, EtOH‐exposed children frequently lacked interpersonal social skills and acted aggressively in the classroom, emphasizing the toll of maternal drinking on social behavior in exposed oVspring (Jacobson et al., 1998b). A 2004 report published on this cohort focused on the relationship between prenatal EtOH exposure and IQ at 7.5 years, with a special emphasis on maternal age, alcohol abuse history, and home environment as moderating variables (Jacobson et al., 2004). Overall results indicated that while there was no EtOH eVect on full‐scale IQ, specific deficits in attention, arithmetic, and working memory were evident. Certain subgroups of children emerged from the analysis as more vulnerable to the adverse eVects of EtOH exposure because of advanced maternal age, greater maternal alcohol abuse, and/or being raised in a home lacking intellectual stimulation. c. Atlanta. In a prospective study in Atlanta, 103 subjects were recruited from a prenatal care clinic that primarily served low‐SES, inner‐city women from 1980 to 1986 (Coles et al., 1985). Data were collected on maternal drinking behavior around conception and during pregnancy so that the eVects of drinking cessation on developmental outcome could be examined. Women were recruited into one of three experimental groups: those who reported consuming an average of 3.5 drinks/day during pregnancy (n ¼ 26), those who drank, on average, four drinks/day but stopped by midpregnancy (n ¼ 22), and those who never drank at all (n ¼ 55). Infants were weighed, measured, examined for dysmorphia, and evaluated for neurobehavioral eVects during the neonatal period. Three of the infants in this study received dysmorphia scores that classified them as FAE but there were no eVects of prenatal EtOH exposure on measures of physical growth. The results from the Brazelton Neonatal Behavioral Assessment Scale indicated that infants exposed to EtOH at any point during gestation exhibited abnormal reflexes, immature motor skills, and greater activity levels when compared to unexposed controls. Infants born to women who stopped alcohol consumption during midpregnancy had better scores on measures of state control, need for stimulation, motor tonicity, tremulousness, and asymmetries in reflexive behavior than did infants born to mothers who continued to drink. The data support the concept that abstinence in later pregnancy is associated with behavioral gains in exposed newborns and that infants with a history of chronic fetal exposure are at the greatest risk for behavioral eVects in the

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neonatal period. A subset of the group tested in early infancy was reexamined at 6 months of age using the Bayley Scales of Infant Development (Coles et al., 1987). Poor scores from early infancy measures on the Brazelton (days 1–3) were associated with reduced mental and psychomotor scores on the Bayley. The findings indicate that abnormal behavior is present from the first days of postnatal life in EtOH‐exposed newborns and that changes in neonatal behavior may be predictive of later deficits in cognitive and motor behaviors. A subset of the original cohort from Atlanta was retested at approximately 6 years of age to evaluate facial dysmorphia, physical growth parameters, measures of cognition, academic progress, and adaptive behavior (Coles et al., 1991). There was a high correlation between neonatal dysmorphia scores, including microcephaly, and results from a physical exam at 6 years of age. Results from cognitive testing indicated that academic achievement scores were lower in children with a history of prenatal EtOH exposure but the greatest performance deficits were associated with a pattern of maternal drinking that persisted throughout pregnancy. Math and reading skills were significantly lower in both experimental groups (continued‐to‐drink and stopped‐drinking) but children from the continued‐to‐drink group also showed deficits in sequential processing and general intellectual functioning. Ratings of adaptive behavior were similar between exposed and unexposed children. The overall results suggest that EtOH‐induced dysmorphias are persistent over early childhood and mothers who continue to drink throughout pregnancy place their oVspring at the highest risk for neurodevelopmental eVects. At 7 years of age, impulsivity, attention, and internalizing/externalizing behaviors were evaluated in these children (Brown et al., 1991). Hyperactivity and impulsive behavior were not observed in the general cohort but children in the continued‐to‐drink group had diYculty with mental concentration (a measure of sustained attention) and were more often described by teachers as having attentional and behavioral problems in the classroom. Externalizing behaviors (aggression, destructiveness, inattention) were significantly linked to prenatal EtOH exposure but internalizing behaviors such as anxiety and depression were only weakly associated with maternal drinking history. The authors suggest that current levels of maternal EtOH use and the quality of the caretaking environment are more influential on internalizing behaviors than prenatal EtOH exposure. Study results once again support the idea that mothers who quit drinking during midpregnancy confer significant developmental advantages to their oVspring when compared to mothers who continue to drink throughout pregnancy. The latest published reports from the Atlanta cohort describe studies performed on a subset of children at approximately 15 years of age (Coles

NEURODEVELOPMENTAL EFFECTS OF ALCOHOL

19

et al., 2002; Riley et al., 2003). Alcohol‐exposed children with dysmorphia were compared to alcohol‐exposed children without dysmorphia and children not exposed to alcohol prenatally. Subjects were tested to examine the eVects of prenatal EtOH exposure on growth, cognition/sustained attention, and behavioral problems during adolescence. Facial dysmorphia persisted in ETOH‐exposed teenagers but there was no evidence of continuing deficits in aspects of physical growth such as weight and height. Subjects with a history of prenatal exposure but no dysmorphia performed as well as did unexposed subjects on tests of sustained attention and IQ. In contrast, dysmorphic individuals had lower IQ scores and more diYculty solving arithmetic problems. A similar pattern was found on tests of sustained attention where errors of omission (failing to detect a stimulus when presented) were particularly high in the dysmorphic group, suggesting a specific deficit in visual perception. Neonatal scores on the dysmorphia exam and the Brazelton were predictive of IQ, achievement scores (including math), and sustained attention during adolescence in this cohort. Study results support the diagnostic sensitivity of neonatal dysmorphia exams and infant behavior scales in predicting long‐term intellectual and cognitive outcomes in EtOH‐exposed individuals. d. Cleveland. The Cleveland Prospective Alcohol‐in‐Pregnancy Study was launched in 1980 to evaluate the rate of alcoholism in a general population of pregnant women and to study the reproductive eVects of EtOH on pregnancy outcome (Sokol et al., 1981). Data from 2913 low‐SES women seeking prenatal care from a city hospital were collected during the first year of the study. This sample was eVectively divided into two experimental groups based on scores from the Michigan Alcoholism Screening Test (MAST) given at the initial visit. This test is a composite of 25 questions that are primarily related to the psychosocial aspects of problem drinking and scores of 5 or more are considered indicative of EtOH abuse. Women with MAST scores of 5 or more were selected to represent heavy drinkers (MAST positive) and women with MAST scores lower than 5 were enrolled to represent light drinkers (MAST negative). Drawn from the larger prospective study, a cohort of infants from MAST‐ positive pregnancies (n ¼ 176) and MAST‐negative pregnancies (n ¼ 183) were examined for the presence of physical anomalies during the neonatal period (Ernhart et al., 1987). Twenty‐six percent of the mothers enrolled in the infancy study (n ¼ 95) abstained from alcohol while 45% (n ¼ 161) were light/moderate drinkers (fewer than two drinks per day). Seventeen percent of the mothers (n ¼ 60) drank 4 or fewer drinks per day, 6% (n ¼ 23) drank six or fewer drinks per day, and 5% of the women (n ¼ 20) drank more than six drinks per day. Teratogenic eVects were dose‐dependent and the highest counts of congenital anomalies and facial dysmorphia were seen in children

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exposed to EtOH during the embryonic period. The most frequently noted items were ptosis, cleft palate, and cleft lip. The greatest risk for facial dysmorphia was related to maternal drinking of 6 or more drinks per day around the time of conception. No clear threshold could be identified for craniofacial abnormalities since these eVects were observed in infants born to women who consumed fewer than 6 drinks per day. The Cleveland cohort was evaluated several times during infancy and early childhood (6 mo, 1 yr, 2 yr, 3 yr, 4 yr 10 mo) by trained observers who visited each child in their home (Boyd et al., 1991; Greene et al., 1991a). Tallies of dysmorphia obtained during the neonatal period were strongly related to indices of maternal drinking during pregnancy. Birth weight was also aVected by maternal EtOH consumption during pregnancy, although this relationship was less robust. Tests of mental development, IQ, and sustained attention were used to evaluate neurobehavioral outcome in these children. Excluding the 1 confirmed case of FAS, multiple analyses relating maternal EtOH use and childhood outcome did not find evidence of neurobehavioral deficits on any test measure. Heavily exposed children performed as well as children born to light drinkers on test measures of cognition and intelligence. These results suggest that prenatal EtOH exposure, at levels not resulting in FAS, does not impair the development of normal childhood intelligence. A longitudinal analysis of physical growth in this cohort found evidence of an alcohol eVect on size at birth but this eVect was attenuated in the preschool years, providing little evidence of persistent growth deficits in EtOH‐exposed children (Greene et al., 1991b). No reports have been published for this cohort since 1991. e. Pittsburgh. The Pittsburgh study was designed to examine the eVects of prenatal ETOH and marijuana use on infant growth and development. The sample consisted of approximately 650 low‐SES women participating in the Maternal Health Practices and Child Development Project from 1983 to 1985 (Day et al., 1989). During the first trimester, 31% of the mothers enrolled (n ¼ 199) abstained from alcohol while 37% (n ¼ 240) were light drinkers ( >2.9 drinks per week). Eight percent of the mothers (n ¼ 49) were moderate drinkers (three to six drinks per week) while 24% (n ¼ 154) were heavy drinkers (one or more drinks per day). Rates of heavy drinking decreased from 24% during the first trimester to 5% during the last trimester. Within 2 days of delivery, oVspring from live‐born singleton births (n ¼ 595) were measured, weighed, and examined by trained nurses for physical anomalies. Prenatal EtOH exposure was related to an increased risk of low birth weight, length, and head circumference under the 10th percentile, and the presence of minor physical malformations. The average birth weight of infants exposed to EtOH throughout pregnancy was 815 grams lower than the birth weight of unexposed control infants. These adverse eVects were

NEURODEVELOPMENTAL EFFECTS OF ALCOHOL

21

primarily associated with drinking during the first trimester, particularly during months 1 and 2, and illustrate the increased vulnerability of the fetus to EtOH exposure during early pregnancy. At 8 months of age, infants from this cohort were weighed and measured again to examine eVects of exposure on physical development (Day et al., 1990). There was a significant relationship between EtOH use throughout pregnancy and weight and length of infants at this age. Growth and morphology deficits remained persistent in exposed infants, particularly in subjects whose mothers drank continuously or during the last 2 trimesters of pregnancy. A month later (9 months of age), infants were tested on the Bayley Scales of Infant Development. Results from the Bayley did not indicate an association between rates of maternal drinking and delays in mental or psychomotor development (Richardson et al., 1995). At 3 years of age, children prenatally exposed to EtOH remained smaller on measures of weight, height, and head circumference and these eVects were dose‐dependent (Day et al., 1991). Growth eVects were primarily associated with 1 or more drinks/day during the second and third trimester. The number of minor physical malformations was also elevated in children who were exposed to 1 or more drink/day during the first trimester of pregnancy. The authors note that while the growth eVects associated with gestational alcohol exposure are modest and lack clinical significance for the individual child, they suggest the long‐term disruption of physical growth in this population. Children from the Pittsburgh cohort were tested at ages 6 and 10 years on measures of IQ and academic achievement (Goldschmidt et al., 1996, 2004). At age 6 years, consumption of one or more drinks per day during the second trimester was associated with reduced scores in arithmetic, reading, and spelling. Follow‐up at 10 years found that teachers rated children with first‐ and second‐trimester EtOH exposure more poorly on measures of classroom performance than unexposed controls. Binge drinking (four or more drinks per occasion) during midpregnancy was associated with lower reading recognition and comprehension and poorer teacher ratings of academic achievement. Additional testing at this age was undertaken to provide information on learning and memory, abstract reasoning, mental flexibility, eye–hand coordination, and information processing (Richardson et al., 2002). Prenatal EtOH use was associated with significantly poorer performance on tests of design and story memory as well as verbal learning. These eVects were associated primarily with levels of maternal drinking at four or more drinks per day during the first and second trimesters of pregnancy. Exposed oVspring at age 10 were physically smaller than their unexposed counterparts, expressing a pattern of growth deficits that has been consistent since birth (Day et al., 1999). Prenatal exposure to EtOH during the first trimester was related to reduced height and head circumference and exposure

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during the second trimester was associated with lighter body weight. The data suggest that the period of greatest vulnerability for EtOH eVects on oVspring size is during early gestation when fetal cells are rapidly multiplying. Reports from the Pittsburgh cohort have described results from examinations of children at 14 years of age. Weight, height, and head circumference continued to be aVected by prenatal EtOH exposure (Day et al., 2002). On average, the oVspring of heavy drinkers were 16 lbs lighter than the offspring of abstainers. EVects were dose‐dependent and found at levels of exposure less than 1 drink/day. Results of cognitive tests show that prenatal EtOH exposure was associated with deficits in learning and memory that were specific to the verbal domain (Willford et al., 2004). These results are consistent with the cognitive findings at the 10‐year follow‐up and suggest long‐term losses in the intellectual potential of children exposed to light (< 0.4 drinks/day) to moderate (<.89 drinks/day) levels of EtOH during the first trimester of pregnancy. f. Global EVorts at Defining the Consequences of Fetal Ethanol Exposure. There are a number of noteworthy eVorts at the international level designed to elucidate the neurobehavioral consequences of fetal EtOH exposure. Using an interdisciplinary approach to clinical assessment, the long‐term outcome of children with FAS has been studied in Berlin, Germany (Steinhausen & Spohr, 1998; Spohr et al., 1993). Results indicate that facial dysmorphia associated with FAS diminished over time but other growth eVects, such as microcephaly, remained clinically detectable. The authors note that mental retardation was the greatest long‐term neurobehavioral deficit in FAS children and that these intellectual losses were not significantly improved with environmental enrichment measures. A number of neuropsychological problems also persisted across childhood into adolescence. Hyperkinetic disorders, emotional disturbances, stereotypic behavior, and sleep abnormalities were frequently reported in young adults with FAS and both parents and teachers consistently rated attention deficits and impaired social relationships as the greatest problems for EtOH‐exposed subjects. Remarkably, over 63% of the subjects in this study suVered from at least one psychiatric disorder. This rate of psychopathology, in conjunction with alcohol‐related cognitive and social deficits, makes adaptive, independent living an unrealistic goal for many young people with FAS. Research projects on prenatal EtOH exposure and oVspring eVects in South Africa and Russia have been described in published symposium proceedings (Riley et al., 2003). Dr. Sandra Jacobson outlined new findings from a study in Western Cape Province, South Africa, designed to evaluate the neurobehavioral consequences of maternal drinking in this highly exposed population of infants. There is a very high rate of FAS in this

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23

mixed‐ancestry population, partially owing to the now outlawed dop system which paid a portion of a farm worker’s wages in wine (Croxford & Viljoen, 1999; May et al., 2000). At 6.5, 12, and 13 months after birth, study subjects were examined on a battery of tests shown historically to be sensitive to alcohol exposure. Visual acuity, visual recognition memory, complexity of play, the A not B paradigm, and a numerosity test were used to measure important attributes of early sensory and cognitive development. Preliminary data indicate that prenatal EtOH exposure was related to fetal growth retardation (smaller size at birth) and deficits in visual acuity (Carter et al., 2001). At 6.5 months, a history of maternal drinking was associated with slower information processing and at 12 months, exposure was related to deficits in visual recognition memory. Prenatal EtOH exposure was also related to performance deficits on the A not B paradigm and numerosity tests. These results suggest that high‐level intrauterine EtOH exposure aVects basic intellectual processes early in postnatal life. In the same symposium (Riley et al., 2003), Dr. Sarah Mattson presented results from a collaborative project in Russia designed to study the rates of FAS in Moscow orphanages and boarding schools and compare the neurobehavioral profile of FAS children in Moscow with FAS children in the United States. Results indicate that 7.9% of children who were evaluated were positive for FAS, a very high rate of this disorder. Neurobehavioral evaluations included a Russian version of the WISC to test IQ and the scores of FAS children were significantly lower than controls. The subtest scores for Comprehension, Arithmetic, Vocabulary, and Coding were lower in FAS subjects. Teachers and governesses rated children with FAS as exhibiting more attention problems in the classroom but rates of hyperactivity were equivalent to controls. Additional cognitive testing in a subgroup of subjects with FAS revealed that they had problems with spatial memory (location of objects) but not in concentration abilities or visual memory. g. A Collective Look at AVected OVspring. The five longitudinal, prospective studies already detailed have generated a rich body of knowledge on the neurodevelopmental eVects of prenatal EtOH exposure. A summary of these findings as well as highlights of each research program are provided in Table III. In the last 30 years, extensive clinical and scientific information has been gathered on the neurobehavioral consequences of prenatal exposure to EtOH. Jacobson and Jacobson (2002) have provided a summary of these data and deficits in three basic dimensions of behavior emerged: arithmetic, attention, and socioemotional competence. According to the authors, children with a history of intrauterine EtOH exposure tend to lack the ability to stay focused and attentive over time and have diYculty analyzing problems and forming eVective response strategies. As EtOH‐aVected children

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TABLE III PRENATAL ETHANOL EXPOSURE AND NEUROBEHAVIORAL DEVELOPMENT Effects found in:

Infancy

Childhood

Adolescence

Seattle Physical Growth Yes No No Behavioral Development Yes Yes No □ EVects are dose‐dependent and occur at socially acceptable/moderate levels of drinking. □ EVects are long‐term and represent permanent neurobehavioral deficits. □ Binge style drinking poses greatest risk to the fetus. Detroit Physical Growth Yes No data No data Behavioral Development Yes Yes No data □ Binge style drinking poses greatest risk to the fetus. □ Older mothers (>30 yrs) are more likely to have an aVected infant. □ EVects during infancy associated with 1 or more drinks/day during gestation. Atlanta Physical Growth Yes Yes No Behavioral Development Yes Yes Yes □ Abstinence from drinking during later pregnancy reduces risk of aVected infant. □ Only subjects with facial dysmorphia have persistent neurobehavioral deficits. Cleveland Physical Growth Yes Yes No data Behavioral Development Yes Yes No data □ 6 or more drinks/day around conception associated with greatest risk for facial dysmorphia. □ Levels of exposure that do not produce FAS do not result in neurobehavioral deficits. Pittsburgh Physical Growth Yes Yes Yes Behavioral Development No Yes Yes □ Drinking during the first trimester poses greatest overall risk to the fetus. □ Light to moderate drinking during early pregnancy associated with long‐term eVects on physical and mental development.

mature, deficits in social behavior become more pronounced and are often expressed in the form of aggression in the classroom, impaired social judgments, and antisocial/delinquent behavior. A history of prenatal exposure may place young adults at risk for abusing EtOH themselves and experiencing EtOH‐related dependency problems (Baer et al., 2003), underscoring the intergenerational nature of EtOH abuse and the long‐term consequences of prenatal exposure.

NEURODEVELOPMENTAL EFFECTS OF ALCOHOL

D.

25

Ethanol‐Induced Brain Injury

Although prenatal ethanol exposure can have adverse consequences on multiple organ systems, the most dramatic eVects are clearly on the central nervous system. Autopsy reports of aVected children have demonstrated that serious, widespread brain damage is frequently attendant to gestational EtOH exposure. Since the identification of the FAS 30 years ago, there have been many eVorts to identify the areas of the brain most sensitive to alcohol during gestation and the mechanisms that may be responsible for the disruption of neural development. Although no specific pattern of malformations can be identified, adverse eVects on brain size, corpus callosum, basal ganglia, cerebellum, and neural glial cells have been documented (Clarren, 1986; Roebuck et al., 1998). Since 1995, the persistent eVects of prenatal alcohol exposure on the brain have been studied with magnetic resonance imaging (MRI), a quantitative tool that allows structural examination of the size and volume of brain structures in living subjects (reviewed by Mattson et al., 2001; Riley et al., 2004). The most consistent finding in these studies has been a reduction in the size of the cranial vault (microcephaly) of children with a history of heavy prenatal EtOH exposure. Consistent with behavioral observations that indicate EtOH‐exposed children have balance and motor impairments, reductions in cerebellar volume have been documented. The corpus callosum, the fiber tract that connects the two hemispheres of the brain, may be the neural structure most aVected by EtOH exposure. The absence or thinning of the corpus callosum is a common neuroanatomical defect associated with intrauterine EtOH exposure and may significantly contribute to problems with cognition, motor skills, and bimanual coordination. In a study of adults with FAS or FAE, a thinning of the corpus callosum was associated with functional motor losses while a thickened callosum was linked to deficits in executive functioning (the ability to plan and execute problem‐solving strategies) (Bookstein et al., 2002). The basal ganglia, important for limb and eye movements and cognition, are also vulnerable to gestational EtOH exposure and show marked size reductions in EtOH‐exposed children. New whole‐brain analytic techniques have been applied to imaging data from children with a history of heavy prenatal EtOH exposure. Results indicate changes in the shape and size of the corpus callosum, a disproportionate reduction in temporal lobe size, changes in the symmetry of the gray matter in parts of the temporal lobe, and increased gray matter/decreased white matter in the perisylvian cortices of the temporal and parietal lobes. Significant abnormalities in whole brain shape, such as narrowing of the temporoparietal regions and reduced growth of the frontal lobes, have also been documented. These research findings indicate that EtOH‐induced brain

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malformations may be regionally specific and suggest that neurobehavioral deficits characteristic of FAS/FAE children may be tied to distinct areas of neural injury. Groundbreaking research has demonstrated that EtOH may interfere with the glutamate and gamma‐aminobutyric (GABA) neurotransmitter systems by triggering widespread cell death in the developing rat brain (Ikonomidou et al., 1999, 2000). The greatest period of vulnerability for neuronal loss coincides with the period of synaptogenesis, also known as the brain growth spurt. This research suggests that EtOH exposure during the brain growth spurt in humans, from approximately the sixth month of gestation to several years after birth, can cause the loss of millions of developing nerve cells. The authors postulate that these losses may explain the diverse range of neurobehavioral deficits and reduced brain size commonly observed in children aVected by prenatal alcohol exposure. Work published by Olney and colleagues (2001) has demonstrated that a single intoxicating dose of EtOH, lasting for several hours, is suYcient to trigger a massive loss of brain cells in developing rats and mice. Ethanol has the potential to trigger widespread apoptotic neurodegeneration through blockade of NMDA glutamate receptors and excessive activation of GABA (A) receptors. This massive cell death, induced by EtOH, may be an important mechanism in the etiology of FAS and may play a defining role in the expression of neurobehavioral deficits in FAE and related disorders (Olney et al., 2002).

III.

METHANOL

Methanol is one of the most commonly used chemicals in American industry (National Library of Medicine, Toxicology Information Program). Also referred to as methyl alcohol and wood alcohol, methanol (MeOH) is an important industrial solvent that is necessary in the production of consumer goods such as solid fuels (Sterno), antifreeze, and photocopying fluids. It is also used in the pharmaceutical and agricultural industries and in the manufacture of ethylene glycol, methyl halides, methacrylates, and methylamines (Von‐Burg, 1994). As described in the federal Hazardous Substances Data Bank, MeOH is required to produce chemical intermediates such as formaldehyde and acetic acid and is used in products as diverse as paints, plastic bottles, and contemporary fabrics (http://toxnet.nlm.nih.gov). Within the last two decades, the utilization of MeOH as an alternative motor fuel has been explored in both public and private sectors. Recent attention has focused on the use of MeOH as a primary fuel source for vehicles powered by hydrogen‐based fuel cell technology (Fuller et al., 1997).

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27

One of the most important characteristics of MeOH is that it is a low‐ emission, high‐performance combustible motor fuel. Increased use of MeOH as a fuel source could lead to improved air quality by reducing the hydrocarbon emissions responsible for increased atmospheric ozone concentrations and, ultimately, global warming (Gold & Moulis, 1988). If methanol is used in the production of new fuels, there could be widespread exposure to the public, primarily from inhalation of MeOH vapors (Carson et al., 1987). While the eVects of acute high‐dose MeOH poisoning in adults have been well chronicled, little is known about eVects of chronic, low‐dose exposure, particularly in sensitive subgroups such as pregnant women (International Programme on Chemical Safety, 1997). The eVects of acute high‐dose MeOH exposure have been characterized from clinical cases of human poisoning and comparative toxicology work with rodents and monkeys. The time course and progression of MeOH toxicity in humans have been documented in detail (Bennett et al., 1953; Tephly & McMartin, 1984). In brief, the individual typically experiences a short period of intoxication, followed by a period in which no symptoms of intoxication or toxicity are noted. This asymptomatic period is followed by symptoms of poisoning, such as headache, nausea, vomiting, loss of equilibrium, severe abdominal pain, and diYculty in breathing. These symptoms can be followed by coma and death. Neurological abnormalities, including focal cranial nerve deficits, optic atrophy, and a Parkinsonian‐like syndrome, usually involving symptoms such as rigidity, tremor, and impaired balance, have been reported (Guggenheim et al., 1971; Ley & Gali, 1983; Riegel & Wolf, 1966). Findings from occupational health research indicate that workplace exposure to MeOH may have links to the immediate or delayed onset of this Parkinsonian‐like disorder (Tanner, 1992). In 2002, this syndrome was reported in a physicist after chronic exposure to MeOH in the laboratory (Finkelstein & Vardi, 2002). There are numerous reports on the metabolism and disposition of MeOH in rodents and nonhuman primates (see reviews by Tephly, 1991; Tephly & McMartin, 1984). Data on human subjects are limited to clinical observations in cases of MeOH poisoning and experimental exposure studies in healthy volunteers. The absorption of MeOH is rapid following oral ingestion, inhalation of MeOH vapor, or dermal contact. Once absorbed, MeOH distributes readily to all organs and tissues roughly in proportion to their water content (Yant & Schrenk, 1937). Metabolism is the predominant route of elimination at low or moderately high doses of MeOH. Methanol is first converted to formaldehyde which rapidly undergoes oxidation to formate (formic acid). Formate then enters the folate biochemical pathway and is eventually oxidized to carbon dioxide (CO2). Formate is considered the toxic metabolite of MeOH, responsible for disturbances of the visual system and

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metabolic acidosis, the cause of death in highly exposed subjects (Jacobsen & McMartin, 1986).

A.

Exposure Scenarios for Pregnant Women

In the context of MeOH as an alternative motor fuel, most exposures of pregnant women would take place on highways, urban streets, during refueling, and in private garages. For each scenario involving MeOH‐based fuel, calculations have been made as to the extent of predicted MeOH exposure for the average adult (Gold & Moulis, 1988). Even in situations likely to result in the highest exposures, such as refueling (23–38 ppm) and hot‐soak emissions in private garages (192–383 ppm), the length of exposure is relatively brief and anticipated levels of MeOH exposure fall below those associated with clinical neurotoxicity. The current threshold limit value for MeOH for an 8‐hour work day, 40 hours per week, is a time‐weighted average (TWA) of 230 ug/m3 or 200 ppm (American Conference of Governmental Industrial Hygienists, 1990). In addition to maternal inhalation exposure, prenatal exposure to MeOH can also occur through the maternal consumption of adulterated alcoholic beverages, fruit, vegetables, and food/ drinks that have been artificially sweetened with aspartame. In the case of tainted alcohol, it is not possible to disentangle the eVects of MeOH from ethanol.

B.

Neurodevelopmental Effects of Exposure

1. HUMAN INFANTS

Until recently, there were no cases in the published medical literature of developmental neurotoxicity in human infants associated with prenatal MeOH exposure. In 2004, the first case report was described in which an infant was exposed to MeOH during gestation through maternal exposure (Belson & Morgan, 2004). The level of blood MeOH was tested in the newborn (61.6 mg/dL) and death due to severe intraventricular bleeding occurred on postnatal day 4. The mother remained in a state of metabolic acidosis despite treatment and died 10 days after delivery. Postnatal exposure was reported in an infant who was fed a mixture of formula and windshield cleaner that contained MeOH. The infant was hospitalized and appeared to recover without long‐term neurological damage (Brent et al., 1991). In Egypt, a cluster of infant deaths following immunization were due to metabolic acidosis from MeOH poisoning (Darwish et al., 2002). These deaths and possibly other infant deaths in this farming community were due to the excessive topical application of MeOH following immunization.

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Methanol is an eVective anti‐inflammatory and antipyretic agent and concern over adverse reactions to vaccines led to the misuse of MeOH by well‐ meaning medical personnel. 2. STUDIES WITH THE RODENT ANIMAL MODEL

Laboratory studies of prenatal MeOH exposure using rodent animal models have reported numerous signs of teratogenicity in MeOH‐exposed pups. Pregnant rats exposed by inhalation to either MeOH or ethanol, in concentrations ranging from 5000 to 20,000 ppm, delivered oVspring with an increased number of malformations (primarily cervical ribs and urinary or cardiovascular defects) (Nelson et al., 1985). At equivalent doses, the treatment‐related eVects were more pronounced in litters exposed to MeOH than those exposed to EtOH. Bolon and colleagues exposed pregnant mice to 5000, 10,000, or 15,000 ppm MeOH vapor and at the two higher doses, near‐term fetuses showed an increased number of resorptions, fetal malformations such as neural/ocular defects, cleft palate, hydronephrosis, and dysmorphic limbs as well as reduced fetal weights (Bolon et al., 1993). EVects were dependent on when exposure occurred during embryonic development. Exposure during gestation days 7 through 9, coinciding with neural tube development and closure, resulted in neural tube defects and ocular lesions. In contrast, exposure during gestation days 9 through 11, the period of likely neural tube reopening, was associated with malformations of the paw and digits. No MeOH‐related eVects were observed in the group exposed to 5000 ppm. Rogers et al. (1993) exposed pregnant mice to concentrations of MeOH vapor ranging from 1000 to 15,000 ppm on days 6 through 15 of gestation. In pups exposed to 5000 ppm MeOH or higher, an increase in the number of exencephalies and cleft palate was observed while pups exposed to 7500 ppm MeOH or higher exhibited increases in embryo/fetal death. At 10,000 ppm and above, reduced fetal weight was documented. A significant increase in the proportion of fetuses per litter with cervical ribs was noted at 2000 ppm, providing evidence that the 2000 ppm dose was the Lowest Observed Adverse EVect level (LOAEL). Based on these data, the No Observed Adverse EVect Level (NOAEL) would be 1000 ppm. Subsequent work from this investigative team has demonstrated that in the mouse, gastrulation and early organogenesis are periods in which the fetus is particularly sensitive to the teratogenic eVects of inhaled MeOH (Rogers & Mole, 1997). A study of oral MeOH exposure in rats (1.6, 0.9, 0.6% v/v) indicated significant eVects on litter size and neonatal and postnatal mortality (Abel & Bilitzke, 1992). The rate of postnatal deaths in exposed pups was very high and reached 100% in the highest dose group (1.6%). In a separate study of oral MeOH exposure, a single dose resulted in fetal growth deficits and a

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dose‐dependent increase in congenital malformation such as undescended testes, exophthalmia, and anophthalmia (Youssef et al., 1997). Neurobehavioral evaluations of rodents developmentally exposed to MeOH are few in number. In a study by Infurna and Weiss (1986), pregnant rats were exposed to MeOH in the drinking water at 20,000 ppm on either gestational day (GD) 15–17 or GD 17–19. Exposed pups displayed diYculties in suckling behavior and in finding nesting material from the home‐cage. Stanton et al. (1995) exposed pregnant rats to MeOH at a concentration of 15,000 ppm, via inhalation, on GD 7–19. A broad‐based battery of behavioral tests was given to the oVspring including motor activity, T‐maze learning, olfactory learning, acoustic startle, and passive avoidance learning. The exposed pups did not show MeOH‐related impairments on any of the neurobehavioral measures. The only adverse eVect observed in this study was reduced birth weight. Weiss et al. (1996) exposed pregnant rats to either 4500 ppm MeOH or 0 ppm via inhalation for 6 hours daily. Maternal exposure began on GD 6 and both pups and dams were exposed until postnatal day 21. Suckling behavior, odor discrimination, and motor activity were measured prior to weaning. Adult functioning was measured with two operant procedures, the fixed‐ratio wheel‐running procedure and a stochastic spatial discrimination task. While the eVects in exposed oVspring were subtle, MeOH exposure influenced a number of neurobehavioral endpoints. In the preweaning phase of testing, a treatment‐related eVect was observed on the motor activity test. On the operant measures, both tests showed evidence of performance decrements due to MeOH exposure but the diVerences were not robust. 3. METHANOL EXPOSURE FROM ASPARTAME (ARTIFICIAL SWEETENER)

As previously noted, the consumption of aspartame‐sweetened food products and beverages results in MeOH exposure (Butchko et al., 2002). The release of MeOH occurs when aspartame is absorbed and metabolized during digestion (Stegink et al., 1983). In a review chapter of aspartame ingestion during pregnancy, the authors were unable to definitively comment on the extent of prenatal MeOH loading from maternal ingestion but it is generally regarded to be insignificant (Pitkin, 1984). In an attempt to investigate the eVects of postnatal aspartame exposure, infant stumptail macaque monkeys were fed high levels of aspartame in their formula for 9 months (Reynolds et al., 1984). Aspartame exposure was unrelated to developmental parameters such as physical growth, serum chemistry, urinalysis, hematology, and brain wave patterns. These monkeys were also evaluated on tests of hearing and cognition and no deficits in performance were reported (Suomi, 1984).

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4. METHANOL RESEARCH IN A NONHUMAN PRIMATE MODEL

A series of studies conducted in our laboratory were designed to characterize MeOH metabolism, blood clearance, and distribution kinetics prior to and during pregnancy, and to evaluate subsequent reproductive and oVspring developmental outcome, after long‐term, low‐dose maternal exposure to MeOH vapor in nonhuman primates (Burbacher et al., 1999, 2004a,b). The two‐cohort study design used adult female Macaca fascicularis monkeys exposed to 0, 200, 600, or 1800 ppm MeOH vapor for 2 hours/day, 7 days/ week prior to and during pregnancy. Female monkeys were bred to nonexposed male breeders and 34 liveborn oVspring were delivered at the Infant Primate Research Laboratory at the University of Washington. The 34 oVspring were evaluated during the first 9 months of life using a test battery that included procedures largely adapted from studies with human infants. As outlined in Table IV, test procedures to evaluate MeOH eVects on fetal mortality and malformations, oVspring size at birth, newborn health, neonatal behavioral responses, visually coordinated reaching, visual acuity, gross motor skills, spatial and visual memory, development of social behavior, learning, and postnatal physical growth were utilized in this study. Maternal exposure to MeOH resulted in peak methanol blood levels of 2 to 10 times above background. Blood methanol levels returned to baseline before 8 hr post‐exposure. MeOH exposure was not associated with overt TABLE IV OFFSPRING TEST BATTERY USED TO EVALUATE DEVELOPMENTAL EFFECTS OF PRENATAL METHANOL EXPOSURE OVspring test

Postnatal age at test

Newborn Size Medical Treatments Newborn Health Exam Neonatal Behavioral Scale Object Retrieval Visual Acuity Motor Milestones Object Permanence Visual Recognition Memory Social Behavior Observations Physical Growth Spatial Discrimination & Reversal Nonmatch‐to‐sample

Birth Birth to 9 months Birth Day 1 to 13 Weeks 2 to 6 Weeks 1 to 12 Weeks 2 to 7 months Weeks 2 to 3½ months Days 190 to 220 (postconception age) Week 2 to 7 months Birth to 9 months Months 5 to 7 Month 8 to 9

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maternal toxicity, reproductive loss, oVspring congenital malformations, or a reduction in the size of the oVspring at birth. Previous studies have reported increases in fetal mortality or malformations following prolonged daily exposures at high concentrations of MeOH vapor: over 10,000 ppm for rats (Infurna & Weiss, 1986; Nelson et al., 1985; Stanton et al., 1995) and over 2000 ppm for mice (Rogers et al., 1993). The 2‐hour exposure period used in the present study was most likely too brief to cause increases in maternal/fetal mortality or malformations, even at the 1800 ppm exposure concentration. Methanol exposures were, however, associated with a reduction in the length of pregnancy. In this study, the duration of pregnancy for all of the MeOH exposure groups (average: 160 day) was significantly shorter than the control group (average: 168 days). These results suggest that MeOH exposure during pregnancy may influence the hormonal control of the onset of labor at exposure concentrations that do not aVect overall fetal growth. The reduced gestation lengths of the MeOH‐exposed infants may reflect the premature activation of the fetal HPA axis that controls timing of birth. The basis for such an eVect is unknown but it could represent the direct action of MeOH on the fetal neuroendocrine system or an indirect action of MeOH on the maternal uterine environment. Independent of the specific biological mechanism, the reduced pregnancy durations of MeOH‐exposed dams suggest a subtle but systematic disturbance in the timing of labor and delivery. On most developmental assessments, the MeOH‐exposed infants performed as well as controls. However, treatment effects were found on two test measures: visually guided reaching and recognition memory. Results indicated that prenatal MeOH exposure is associated with a delay in early sensorimotor development as measured by the infant’s ability to reach for, grasp, and retrieve a small object during the first month of life. As illustrated in Fig. 4, this eVect was only observed in male subjects, suggesting a sex‐ specific eVect on the development of visually directed reaching. The delay for males was dose related and ranged from approximately 9 days for the 200 ppm MeOH subjects to more than 2 weeks for the 600 ppm and 1800 ppm exposure groups. The Fagan Test of Infant Intelligence is used with human infants to study visual recognition memory and provides an early measure of information processing, attention, and memory (Fagan & Detterman, 1992). When familiar (previously seen) stimuli are paired with novel (new) stimuli, normal human and macaque monkeys will typically prefer to view the novel stimuli. Novelty scores are interpreted as evidence of visual recognition memory as some attributes of the familiar stimuli must be encoded in memory for the novelty response to occur. Deficits in visual recognition memory have been reported for groups of infant monkeys at high risk for poor developmental

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FIG. 4. Results of Object Retrieval Test. Infant monkey reaching toward and retrieving a small toy to receive the applesauce reward. The ability to accurately reach for, grasp, and pick up the test objects is scored by trained observers.

outcome (methylmercury exposure; Gunderson et al., 1986, 1988), low birth weight (Gunderson et al., 1989), and failure‐to‐thrive (Gunderson et al., 1987). The results of the Fagan Test were examined by problem type. Methanol‐exposed infants performed as well as controls on problems using abstract geometric patterns, but all MeOH‐exposed groups were unable to solve more diYcult test problems using complex social stimuli (monkey faces). A schematic of the apparatus and the data from the social memory problems are displayed in Fig. 5. Prenatal MeOH exposure did not retard physical growth rates during the first 9 months of life. Our later studies, however, indicated that growth retardation in females may be a delayed eVect of high‐dose MeOH exposure. This eVect was not observed as a general decrease in the growth of females as a group, but as a ‘‘wasting syndrome’’ in two of the seven female oVspring in the 1800 ppm exposure group after 1 year of age. The syndrome was severe and resulted in the euthanasia of both of the females. Results of assays for simian retroviruses, blood chemistry, CBC, liver, kidney, thyroid, and pancreatic function were unremarkable. The results of clinical blood tests and autopsy examinations did not provide evidence as to the etiology of this puzzling syndrome. In summary, the results of our study suggest that maternal inhalation of MeOH may be related to a subtle but systematic disturbance in the timing of labor and delivery in pregnant nonhuman primates. While no clear pattern of adverse eVects was found on neurobehavioral development, visually coordinated reaching and early memory was disrupted in exposed

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FIG. 5. Results of Visual Recognition Memory Assessment using social (faces) stimuli. Infants are presented with visually engaging stimuli and then tested for memory of these images. Visual fixations to the test stimuli (on the right and left) are recorded by trained research staV via foot switches. Visual preferences for novel stimuli are interpreted as evidence of memory for previously seen (i.e., familiar) visual targets.

oVspring. The results from this study demonstrate that chronic in utero MeOH exposure, at subclinical levels, is not associated with frank teratogenic eVects but does alter the course of behavioral development in young monkeys.

IV.

EVALUATING THE RISK FROM PRENATAL EXPOSURE TO ETHANOL AND METHANOL

For ethanol, the weight of evidence to determine the risk of adverse eVects on development from prenatal exposure comes largely from prospective, longitudinal studies of human maternal–infant pairs. The results from these studies support several general conclusions. First, the developmental eVects of EtOH are dose‐dependent. The more a pregnant woman drinks, the greater the likelihood that she will give birth to an aVected infant. In addition, at high levels of exposure, the damage to the fetal nervous system from gestational EtOH exposure can be severe and may result in frank mental retardation and long‐term neurodevelopmental disability. The most serious eVects are generally seen in infants born to women who report consuming an average of four drinks or more per day during pregnancy. At lower levels of exposure, EtOH eVects are subtler and may take the form of small reductions in physical size, behavioral problems in school, and diYculty with mental concentration. These eVects are generally associated with an average of one to two drinks per day during pregnancy, which would result in

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maternal blood alcohol levels above background for only a brief period of time each day. Second, the developmental eVects of EtOH are related to the pattern of maternal drinking. Results indicate that women who exhibit a binge or massed pattern of drinking, particularly in early pregnancy, place their oVspring at the greatest risk for neurobehavioral deficits. Third, there are clear biological advantages to limiting fetal exposure and abstaining from EtOH consumption during late pregnancy, even if drinking occurred during early and midpregnancy. Abstinence during late pregnancy is associated with improved developmental outcomes in exposed oVspring. Not all children who are exposed to EtOH during gestation are equally aVected and many children with histories of heavy maternal exposure appear to be clinically normal. All of the factors that are associated with this range of responses even at high exposure levels are not known but some may have to do with maternal characteristics such as age (there is greater fetal vulnerability in older mothers), nutrition, and other drug use. New data indicate that a positive and stimulating home environment is associated with improved outcome in alcohol‐exposed children (Jacobson et al., 2004). In addition, experimental research with animals suggests that the use of Vitamin C and E may help reduce the adverse eVects of ethanol on the developing fetus (Cohen‐Kerem & Koren, 2003). In terms of understanding the dose–response relationship, the use of average number of drinks per day may poorly characterize the actual risks of maternal drinking. Abel (1998, 1999) has strongly cautioned that the use of such a metric may vastly underestimate the actual exposure of the fetus. Abel argued that most drinking in pregnant women occurs on 1 or 2 days of the week so that the actual consumption per drinking day is significantly greater than what the average would suggest. Given this pattern of drinking, the adverse developmental eVects of EtOH are not the result of one or two drinks per day but, instead, the result of seven to 14 drinks on a drinking day during the week. In 2000, the American Academy of Pediatrics Committee on Substance Abuse and Committee on Children with Disabilities recommended that women who are pregnant or who are planning a pregnancy should abstain from alcohol because ‘‘there is no known safe amount of alcohol consumption during pregnancy.’’ Noted in the Committee’s recommendations were the findings from a large prospective study of the relationship between EtOH intake and birth weight (Mills et al., 1984). Alcohol intake and birth weight data collected on over 30,000 pregnancies indicated a reduction in mean birth weight ranging from 14 grams for women who drank less than 1 drink per day to 165 grams for those who consumed three to five drinks daily. The Committee recommended that significant eVorts should be made to educate pediatricians, health care professionals, and their patients as well as elementary, junior, and high school students concerning the harmful eVects of

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alcohol consumption during pregnancy. The Committee also encouraged pediatricians to assume a leadership role in public education campaigns aimed at ‘‘decreasing the incidence of FAS through reduction in alcohol use by pregnant women’’ (American Academy of Pediatrics, 2000). For methanol, the weight of evidence to determine the risk of adverse eVects on development from prenatal exposure comes largely from studies using animal models. The current Environmental Protection Agency (EPA) risk assessment for methanol is restricted to oral intake (Integrated Risk Information System or IRIS). A no‐observed‐eVect level (NOAEL) of 500 mg/kg/day was calculated based on a 90‐day subchronic study of rats gavaged daily with 0, 100, 500, or 2500 mg/kg/day methanol. Animals were exposed to methanol for 42 to 90 days before sacrifice. Results indicated a reduction in the brain weights of animals in the highest dose group, resulting in a NOAEL at the 500 mg/kg/day dose. Research published in 2004 indicates that a dose of at least 3400 mg/kg/day oral MeOH on GD 7 results in craniofacial abnormalities and skeletal defects in fetal mice (Rogers et al., 2004). As displayed in Fig. 6, malformations in exposed subjects included

FIG. 6. Facial dysmorphia in fetal mice exposed to methanol. Controls are shown in images A and E. Facial malformations induced from maternal oral methanol exposure include holoprosencephaly with single naris and micrognathia (B), cleft lip (C), maxillary and mandibular hypoplasia (B–D, F, H), lateral facial cleft (G), low‐set ears (B–D, F–H), and gross facial dysgenesis (D, F, H). Reprinted with permission from Dr. John Rogers, Reproductive Toxicology Division, National Health and Environmental EVects Laboratory, OYce of Research and Development, United States Environmental Protection Agency, Research Triangle Park, North Carolina.

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holoprosencephaly, cleft lip, maxillary and mandibular hypoplasia, facial cleft, low‐set ears, and gross facial dysgenesis. These results are particularly noteworthy as they demonstrate that the craniofacial defects associated with MeOH exposure are strikingly similar to those associated with EtOH exposure. This suggests that the frank teratogenic eVects associated with oral intake may be similar between the two compounds. While no EPA risk assessment is available for inhalation exposure to methanol, the data from our laboratory would indicate that the reference concentration would be quite low. Similar to the results for ethanol, the results from our studies show that exposure to methanol that briefly increases blood methanol levels above background each day is associated with subtle developmental eVects in exposed oVspring. These results indicate that protection of the fetus from chronic exposure to methanol in the environment would require much more stringent exposure standards than those currently in use for occupational exposure. Future studies should focus eVorts on further elucidating the neuroendocrine eVects of methanol that may interfere with full‐term delivery as well as early perceptual–motor and visual memory eVects on oVspring. Studies should use various animal models and focus on the dose–response characteristics of chronic low‐level exposure during pregnancy. ACKNOWLEDGMENTS The authors acknowledge the dedicated assistance of Amy Voltin, Noelle Liberato, and the staV of the Infant Primate Research Laboratory at the University of Washington. We also thank Drs. Ann Streissguth, John Rogers, Kathy Sulik, and Christian EckhoV for providing first‐rate photographic images and assistance with figures and tables. This project was supported by funds from the National Institutes of Health, Grants RO1 ES03745, RO1 ES06673, P51HD02274, P51RR00166, and P30ES07033.

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Gunderson, V. M., Grant‐Webster, K. S., Burbacher, T. M., & Mottet, N. K. (1988). Visual recognition memory deficits in methylmercury exposed Macaca fascicularis infants. Neurotoxicology and Teratology, 10, 373–379. Gunderson, V. M., Grant‐Webster, K. S., & Fagan, J. F. (1987). Visual recognition memory in high‐ and low‐risk infant pigtailed macaques (Macaca nemestrina). Developmental Psychology, 23, 671–675. Gunderson, V. M., Grant, K. S., Burbacher, T. M., Fagan, J. F., 3rd, & Mottet, N. K. (1986). The eVect of low‐level prenatal methylmercury exposure on visual recognition memory in infant crab‐eating macaques. Child Development, 57, 1076–1083. Gunderson, V. M., Grant‐Webster, K. S., & Sackett, G. P. (1989). Deficits in visual recognition memory in low‐birthweight infant pigtailed monkeys (Macaca nemestrina). Child Development, 60, 119–127. Haggard, H. W., & Jellinck, E. M. (1942). ‘‘Alcohol Explored.’’ Garden City, New York. Hannigan, J. H. (1996). What research with animals is telling us about alcohol‐ related neurodevelopmental disorder. Pharmacology, Biochemistry, and Behavior, 55(4), 489–499. Hanson, J. W., Streissguth, A. P., & Smith, D. W. (1978). The eVects of moderate alcohol consumption during pregnancy on fetal growth and morphogenesis. The Journal of Pediatrics, 92, 457–460. Hoyme, H. E., May, P. A., Kalberg, W. O., Kodituwakku, P., Gossage, J. P., Trujillo, P. M., Buckley, D. G., Miller, J. H., Aragon, A. S., Khaole, N., Viljoen, D. L., Jones, K. L., & Robinson, L. K. (2005). A practical clinical approach to diagnosis of fetal alcohol spectrum disorders: Clarification of the 1996 Institute of Medicine criteria. Pediatrics, 115, 39–47. Ikonomidou, C., Bittigau, P., Ishimaru, M. J., Wozniak, D. F., Koch, C., Genz, K., Price, M. T., Stefovska, V., Horster, F., Tenkova, T., Dikranian, K., & Olney, J. W. (2000). Ethanol‐induced apoptotic neurodegeneration and fetal alcohol syndrome. Science, 287, 1056–1060. Ikonomidou, C., Bosch, F., Miksa, M., Bittigau, P., Vockler, J., Dikranian, K., Tenkova, T. I., Stefovska, V., Turski, L., & Olney, J. W. (1999). Blockade of NMDA receptors and apoptotic neurodegeneration in the developing brain. Science, 283, 70–74. Infurna, R., & Weiss, B. (1986). Neonatal behavioral toxicity in rats following prenatal exposure to methanol. Teratology, 33, 259–265. International Programme on Chemical Safety, Environmental Health Criteria 196, Methanol, World Health Organization, 1997. Irvine, L. F. (2003). Relevance of the developmental toxicity of ethanol in the occupational setting: A review. Journal of Applied Toxicology, 23, 289–299. Jacobsen, D., & McMartin, K. E. (1986). Methanol and ethylene glycol poisonings. Mechanism of toxicity, clinical course, diagnosis, and treatment. Medical Toxicology, 1, 309–334. Jacobson, J. L., & Jacobson, S. W. (2002). EVects of prenatal alcohol exposure on child development. Alcohol Research & Health: The Journal of the National Institute on Alcohol Abuse and Alcoholism, 26, 282–286. Jacobson, J. L., Jacobson, S. W., Sokol, R. J., & Ager, J. W., Jr. (1998a). Relation of maternal age and pattern of pregnancy drinking to functionally significant cognitive deficit in infancy. Alcoholism: Clinical and Experimental Research, 22, 345–351. Jacobson, S. W., Jacobson, J. L., Sokol, R. J., Martier, S. S., Ager, J. W., & Kaplan, M. G. (1991). Maternal recall of alcohol, cocaine, and marijuana use during pregnancy. Neurotoxicology and Teratology, 13, 535–540. Jacobson, J. L., Jacobson, S. W., Sokol, R. J., Martier, S. S., Ager, J. W., & Kaplan‐Estrin, M. G. (1993a). Teratogenic eVects of alcohol on infant development. Alcoholism: Clinical and Experimental Research, 17, 174–183.

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PCBs and Dioxins HESTIEN J. I. VREUGDENHIL AND NYNKE WEISGLAS‐KUPERUS DEPARTMENT OF PEDIATRICS DIVISION OF NEONATOLOGY, ERASMUS MC–SOPHIA CHILDREN’S HOSPITAL UNIVERSITY MEDICAL CENTER, ROTTERDAM, THE NETHERLANDS

I. A.

NEUROTOXICOLOGY OF PCBs AND DIOXINS

Chemical Properties and Deposition

PCBs and polychlorinated dibenzo‐para‐dioxins (PCDDs) and polychlorinated dibenzo‐furans (PCDFs) (the latter two are summarized as dioxins) are polyhalogenated aromatic hydrocarbons with comparable molecular structures. They consist of a biphenyl ring and, depending on the number and position of chlorine atoms on the two rings, there are 209 theoretically possible PCB discrete chemical compounds, called congeners, and 210 diVerent dioxin congeners (75 PCDDs and 135 PCDFs). Their basic structure is presented in Fig. 1. PCBs were commercially produced as complex mixtures (under trade names such as Aroclor, Clophen, Phenoclor) for a variety of applications, such as dielectric fluids for capacitors and transformers, heat transfer fluids, hydraulic fluids, lubricating and cutting oils, and as additives in pesticides, paints, adhesives, sealants, carbonless copy paper, flame retardants, organic dilutents, and plastics. Their commercial utility was based largely on their chemical and physical stability, including low flammability and their miscibility with organic compounds. The total amount of PCBs produced worldwide from 1929 to the 1980s, when most countries reduced or stopped the production, has been estimated at approximately 1.5 million metric tons (de Voogt & Brinkman, 1989; WHO, 1989). In 1982, it was estimated that 31% had been released to the environment and 65% was still in use or in storage, or deposited in landfills (Tanabe, 1988). Moreover, PCBs can be formed unintentionally as by-products in a variety of chemical processes that contain chlorine and hydrocarbon sources. Dioxins are generally formed as unwanted and often unavoidable byproducts during the synthesis of a wide array of commercial chemical INTERNATIONAL REVIEW OF RESEARCH IN MENTAL RETARDATION, Vol. 30 0074-7750/06 $35.00

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Copyright 2006, Elsevier Inc. All rights reserved. DOI: 10.1016/S0074-7750(05)30002-4

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Hestien J. I. Vreugdenhil and Nynke Weisglas-Kuperus

FIG. 1. Molecular structures of PCBs, PCDDs, and PCDFs.

products, especially those based on chlorinated aromatics, precursors, and intermediates. Moreover, they are formed during various combustion processes, such as burning of solid waste from municipal incinerators. B.

Toxic Mechanisms

The first mechanism that was described for toxic eVects of PCBs and dioxins was, after entering cells, their interaction with a cytoplasmic receptor protein, the aryl‐hydrocarbon (Ah) receptor (Safe & Goldstein, 1989). Depending on the positions of the chlorine atoms on the biphenyl ring

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structure (ortho, meta, or para position), and consequently the planar shape, the diVerent compounds bind, to a certain extent, to this receptor. Dioxins as well as dioxin-like PCBs (coplanar PCBs; having no chlorine atom on the ortho position) are recognized as potent compounds to interact with the Ah receptor (Safe, 1994). Furthermore, two other groups of PCBs can be distinguished based on the ability to interact with the Ah receptor, mono‐ ortho‐substituted PCBs (weak dioxin-like) and ortho‐substituted PCBs (nondioxin-like PCBs). Mono‐ortho‐substituted congeners have one chlorine atom on the ortho position and are intermediate in their ability to interact with Ah receptors. Ortho‐substituted have more than one ortho‐substitution on the biphenyl ring, which reduces the planarity of the molecule and reduces the ability to interact with the Ah receptor (Kafafi et al., 1993). Both non‐ortho‐substituted (coplanar compounds) and ortho‐substituted PCBs are toxic. Their mechanism of toxicity, however, is likely to be diVerent. As described previously, toxicity of coplanar compounds appears to be mediated by the Ah receptor (Safe, 1994). The toxic potency of a coplanar PCB congener is reflected in a toxic equivalent factor (TEF), based on its ability to bind the Ah receptor relative to the binding ability of the most potent dioxin, TCDD (Safe, 1990; Van den Berg et al., 1998a). For noncoplanar PCBs, the ability of the TEF to predict their neurotoxic potency is low (Giesy & Kannan, 1998; Shain et al., 1991). Since 1995, there is growing evidence that especially non-dioxin-like PCBs and weak dioxin-like PCBs and their metabolites, such as hydroxylated PCBs, may produce a wide spectrum of neurotoxic eVects, while dioxin-like PCBs may have less activity in the central nervous system (CNS) (Fischer et al., 1998; Korach et al., 1988; Shain et al., 1991). C.

CNS Effects

Neurochemical studies have shown that many elements of the CNS, and especially of the developing CNS, are susceptible to exposure to PCBs and dioxins, including cellular and synaptic processes and endocrine systems (Brouwer et al., 1995, 1999; Mariussen & Fonnum, 2001; Tilson & Kodavanti, 1998). These aspects will be further discussed in the following text. At the cellular level, PCBs‐induced alteration in markers for neuronal and glial cell development have been reported in several brain areas in rats that were perinatally exposed to a PCB mixture (Morse et al., 1996a). The levels of these markers for structural and functional brain development were altered in a complex manner, depending on age, sex, or brain region of the animal. The changes were suggestive of neuronal damage or death and were reported in several areas of the brain, including the lateral olfactory tract, striatum, prefrontal cortex, and in the cerebellum and brain stem (Morse et al., 1996a). Perturbations were also reported on intracellular calcium

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Hestien J. I. Vreugdenhil and Nynke Weisglas-Kuperus

homeostatic mechanisms and second messenger systems that play a role in neuronal growth and normal physiology of cells (Kodavanti & Tilson, 1997; Kodavanti et al., 1993, 1994; Shafer et al., 1996). EVects of exposure to PCBs on synaptic processes included an inhibition of the synaptic transmission assessed in the dendate gyrus of the cerebral cortex in adult rats (Gilbert & Crowley, 1997) as well as in the hippocampus (Niemi et al., 1998) and in the visual cortex in prenatally exposed rats (Altmann et al., 1995). Synaptic transmission can be measured by means of long‐term potentiation, which is a model of synaptic plasticity that is suggested to be related to learning and memory at the synaptic level (McNaughton, 1993). Several brain neurotransmitter systems have been shown to be aVected by exposure to PCBs and dioxin, including dopamine, serotonine, glutamate, GABA, and cholinergic systems (Eriksson et al., 1991; Mariussen & Fonnum, 2001; Mariussen et al., 1999; Morse et al., 1996b; Seegal et al., 1997). EVects of perinatal exposure on dopaminergic systems have been documented most thoroughly. It appeared that in rats, developmental exposure to PCBs can result in opposite alterations in brain dopamine concentrations, depending on the type of the congener. For example, perinatal exposure to ortho‐substituted PCBs led to decreases in brain dopamine, whereas perinatal exposure to a coplanar PCB congener resulted in elevated concentrations of dopamine (Brouwer et al., 1995; Seegal et al., 1997). D.

Other Toxic Effects

PCBs and dioxins, and especially one type of PCB metabolites, the hydroxylated PCB metabolites, are presently known as endocrine disrupters. Multiple PCB congeners may impact upon multiple endocrine systems that may communicate with each other and are involved in fetal CNS development. These complex mechanisms of actions have not been much studied, and their role in developmental neurotoxic PCB and dioxin eVects remains largely unknown. Most information is available on thyroid hormone changes, generally including decreases in plasma thyroid hormone levels in fetal and neonatal rats as well as in plasma of the women of the Dutch cohort and their children, 2 weeks after birth (Brouwer et al., 1995, 1998; Koopman‐Esseboom et al., 1994b). Moreover, interactions with the steroid hormone system are suggested, due to PCB‐ and dioxin‐induced changes in steroid hormone homeostasis or to endocrine-like actions of these contaminants, particularly during development (Golden et al., 1998). Estrogenic (Bitman & Cecil, 1970; Kester et al., 2000; Korach et al., 1988), anti‐estrogenic (Amin et al., 2000; Jansen et al., 1993; Kramer et al., 1997; Moore et al., 1997), and anti‐androgenic (Hany et al., 1999) eVects have been

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51

described in in vivo and in vitro studies, possibly depending on congener type and/or metabolite.

II.

HUMAN EXPOSURE TO PCBs AND DIOXINS

Ninety percent of human exposure to PCBs and dioxins occurs through the diet, with food of animal origin being the predominant source (i.e., background exposure) (Furst et al., 1992). Contamination of food is primarily caused by deposition of emissions of various sources on farmland and water (waste incineration, production of chemicals) followed by bioaccumulation in the food chains, in which they are particularly aYliated with fat. Other sources may include contaminated feed for cattle, chicken, and farmed fish, improper application of sewage sludge, flooding of pastures, and waste eZuents (Furst et al., 1992). Since PCBs and dioxins are lipid‐soluble and are only slowly degraded, with half‐lifetimes in humans ranging from 1.8 to 9.9 years (Steele et al., 1986; Taylor & Lawrence, 1992), these compounds accumulate in adipose tissue. During pregnancy, PCBs and dioxins are transferred through the placenta and are able to cross the blood–brain barrier, exposing the fetus during a vulnerable time of CNS development (Masuda et al., 1978). PCBs have been detected in brain tissue of stillborn babies, exposed to environmental levels of PCBs, from 17 weeks of gestational age onward (Lanting et al., 1998a). A breast‐fed infant is additionally exposed to relatively large amounts of PCBs and dioxins, since these compounds are excreted in breast milk. For example, PCB levels were still approximately four times higher in 42‐month‐old children that were breast‐fed during infancy than in their formula‐fed counterparts that were predominantly prenatally exposed to PCBs and dioxins (Patandin et al., 1997). Since these neurotoxic compounds are able to interact with many processes of the CNS, including neurotransmitters and hormones that mediate brain development, the developing CNS is considered to be especially vulnerable to exposure to these neurotoxic compounds. Hence, prenatally, the CNS may be most vulnerable to harmful eVects of exposure to these compounds. Prenatal exposure can be regarded as chronic exposure of the developing brain. Postnatally, the CNS continues to develop rapidly, doubling in weight in the first year of life, reaching 90% of its adult size by 5 years of age. Much of this increase is due to an increase in neuronal maturation, production of glial cells, outgrowth of dendrites and axons, formation of synapses, and myelination of axons (Dodgson, 1962). Moreover, extensive cell death and synapse elimination takes place postnatally. These postnatal maturation processes

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Hestien J. I. Vreugdenhil and Nynke Weisglas-Kuperus

may be especially vulnerable to adverse eVects of lactational exposure to PCBs and dioxins. The maturation rates vary for diVerent brain structures. Therefore, lactational exposure to PCBs and dioxins can be hypothesized to cause structure‐related functional diVerences, depending on the time window of exposure. For example, during the first 2 years of life in humans, functional cortical activity increases earliest in the sensorimotor and occipital cortices, before 3 to 6 months, the auditory and visual association cortices from 4 to 7 months, and latest in the frontal cortex, after 6 to 12 months (Chiron et al., 1992; Chugani et al., 1987). Moreover, timing of maximum brain growth, maximum synaptic density, dendritic arborizations, and myelination, all occur first in primary motor and sensory areas and later in the frontal cortex (Barkovich & Kjos, 1988; Barkovich et al., 1988; Becker et al., 1984; Huttenlocher & Dabholkar, 1997; Mrzljak et al., 1990).

A.

Perinatal Exposure to PCBs and Dioxins and Neurodevelopmental Outcomes

1. ACCIDENTAL EXPOSURE

Two accidents (‘‘Yusho,’’ Japan, 1968, and ‘‘Yu Cheng,’’ Taiwan, 1979) clearly showed the neurotoxic potential of prenatal exposure to these compounds. Large populations were accidentally exposed for relatively short periods to rice oil that was contaminated during the manufacturing process with heat transfer fluids containing PCBs, PCDFs, and polychlorinated quarterphenyls (PCQs). Children born to exposed Yusho mothers were described as dull and inactive at 6 years of age and had IQs averaging 70 (Harada, 1976). Cognitive functions were more thoroughly addressed in the Yu Cheng cohort (n ¼ 118), showing consistent cognitive delays of 5 points from 4 to 7 years of age compared to a matched control group (Chen et al., 1992; Lai et al., 1994). In children born up to 6 years after the incident, cognitive abilities were comparably aVected (Chen et al., 1992; Lai et al., 1994). Moreover, in 7‐ to 12‐year‐old Yu Cheng children, latencies and amplitudes of the P300 peak of an auditory event related potential, reflecting CNS mechanisms that evaluate and process relevant stimuli, were respectively longer and decreased in the exposed oVspring compared to their matched controls (Chen & Hsu, 1994). The measured P300 latencies in that study were inversely correlated with IQs. In the Yu Cheng cohort at 6, 7, 8, and 9 years of age, more spatially related cognitive abilities were diVerently aVected in boys and girls. Only the exposed boys scored lower than their nonexposed matched controls (Guo et al., 1995). These results, therefore, may have provided the

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53

first evidence of sex steroid hormone–modulating eVects of PCBs and dioxins on cognitive development in humans. 2. ENVIRONMENTAL EXPOSURE

The neurodevelopmental eVects described in the Yusho and Yu Cheng cohorts leave little doubt that high levels of prenatal exposure to mixtures of PCBs and dioxins result in neurotoxic eVects of these compounds in humans. Subtle neurodevelopmental eVects of perinatal exposure to PCBs and dioxins have also been described in several cohorts of children that were perinatally exposed to environmental levels of PCBs and dioxins (Darvill et al., 2000; Jacobson & Jacobson, 1996; Koopman‐Esseboom et al., 1996; Patandin et al., 1999b; Rogan & Gladen, 1991; Walkowiak et al., 2001). In these cohort studies, neurological, cognitive, and psychomotor aspects have been studied prospectively. The largest PCB cohorts include two cohorts that were selected based on maternal consumption of PCB‐contaminated fish from the North American Great Lakes: the Lake Michigan cohort (n ¼ 313) that was recruited between 1980 and 1981 (Jacobson et al., 1984a,b) and the more recently (1991–1994) recruited Oswego cohort (n ¼ 293) (Lonky et al., 1996). Another large cohort study has been executed in North Carolina, consisting of 912 mother–infant pairs that were recruited from a general population between 1978 and 1982 (Rogan et al., 1986a). In Europe, the main cohort studies include cohorts in Denmark, The Netherlands, and Germany. The two Danish cohorts were recruited in the Faeroe Islands: the first cohort consists of 435 children born between 1986 and 1987 (Grandjean et al., 1992), the second cohort was recruited from 1994 to 1995 (n ¼ 192), as part of a multicenter cohort study in which the Dutch PCB/dioxin study and a German study participated as well. The Danish cohorts are diVerent from other Northern European cohorts, due mainly to local dietary habits that include consumption of pilot whale blubber and whale meat. In these children, PCB levels were higher compared to levels in Northern Europe, whereas dioxin levels were comparable (Grandjean et al., 1995). The Dutch cohort (n ¼ 418) (Koopman‐Esseboom et al., 1994a) and German cohort (n ¼ 171) (Winneke et al., 1998), respectively, recruited between 1990–1992 and 1994–1995, both consist of mother–infant pairs that were drawn from the general population. The cohorts had similar inclusion criteria and used similar neurodevelopmental tests. In the Dutch cohort, however, restrictions were applied on the number of included breast‐fed children to study lactational exposure to PCBs and dioxins more thoroughly. Half of the recruited population had been breast‐fed for at least 6 weeks during infancy and the other half was fed with formula milk in which PCBs and dioxins were not

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Hestien J. I. Vreugdenhil and Nynke Weisglas-Kuperus

detectable. The formula‐fed children represent children that were exposed mainly prenatally to PCBs and dioxins. The study design, inclusion and exclusion criteria, and PCB and dioxin measurements applied in the Dutch PCB/dioxin study will be presented in more detail. 3. NEURODEVELOPMENTAL EFFECTS

Neonatal neurological eVects of prenatal exposure to PCBs include deficits such as poorer autonomic regulation and more abnormal reflexes (Jacobson et al., 1984b; Lonky et al., 1996), hypotonia (Huisman et al., 1995a; Rogan et al., 1986b), and hyporeflexia (Rogan et al., 1986b). At 18 months of age, prenatal exposure to PCBs was negatively associated with the neurological condition in the Dutch PCB/dioxin cohort (Huisman et al., 1995b); however, this adverse eVect was not seen on the neurological condition in these children at 42 months of age (Lanting et al., 1998b). Assessment of standardized developmental tests, measuring general cognitive and psychomotor abilities, showed negative eVects of prenatal exposure to PCBs on psychomotor abilities until 2 years of age in the North Carolina (Gladen et al., 1988; Rogan & Gladen, 1991) and in the Dutch cohort at 3 months of age (Koopman‐Esseboom et al., 1996). Cognitive eVects of prenatal exposure to PCBs were seen at 7 months of age (Winneke et al., 1998) and more pronounced negative eVects were seen on more matured general cognitive abilities measured at 42 months (Patandin et al., 1999b; Walkowiak et al., 2001) and at 11 years of age (Jacobson & Jacobson, 1996). In the North Carolina study, however, prenatal exposure to PCBs was not related to cognitive and psychomotor abilities at 3, 4, and 5 years of age (Gladen & Rogan, 1991). Adverse eVects of prenatal exposure to PCBs have also been described on more specific cognitive domains, such as processing time, attention, and memory skills (both verbal and numerical auditory memory) in children at 4 years of age (Jacobson et al., 1990, 1992). Moreover, negative relations between prenatal PCB exposure and verbal comprehension skills at 42 months of age (Patandin et al., 1999b) and 11 years of age (Jacobson & Jacobson, 1996) have been described, in addition to verbal IQs and concentration skills (Jacobson & Jacobson, 1996). EVects of lactational or postnatal exposure to PCBs and dioxins have been detected in a few studies. In the Dutch cohort, psychomotor abilities at 7 months of age were decreased in children that were breast‐fed with relatively high concentrations of PCBs and dioxins (Koopman‐Esseboom et al., 1996). At 42 months of age, in the German cohort, negative eVects of postnatal exposure have been described on general cognitive abilities (Walkowiak et al., 2001).

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55

The results of the neurodevelopmental studies from birth to 42 months of age in the Dutch PCB/dioxin cohort are summarized in Table I.

III.

BEHAVIORAL ANIMAL STUDIES

The potential of subtle neurodevelopmental eVects of perinatal exposure to environmental levels of PCBs and dioxins seen in human studies is supported by the results of behavioral animal studies. Perinatal exposure to PCBs and dioxins has been related to several motor deficits, including impaired development of the righting reflex in rats and in mice with impaired ability to remain on a rotating rod (Overmann et al., 1987; Thiel et al., 1994). Moreover, in mice, perinatal exposure to a dioxin-like PCB congener was related with ‘‘spinning’’ behavior, diminished grip strength, and ability to traverse a wire rod (Tilson et al., 1979). Perinatal exposure to a PCB mixture resulted in impairment on several tasks that involve acquisition or recollection of spatial information, including impaired performance on spatial (based on the location of an object) discrimination reversal tasks (Bowman et al., 1978; Schantz et al., 1989, 1991) and decreased accuracy on a spatial delayed alteration task in monkeys (Levin et al., 1988; Schantz et al., 1991). In both tasks, memory and attentional processes are involved. Since the accuracy deficit did not worsen with increasing delay, the eVect was interpreted not as a memory impairment but rather as failure of attentional processes (Schantz et al., 1991). Monkeys that were perinatally exposed to a mixture of PCBs also performed diVerently on a fixed interval scale (Mele et al., 1986). In this task, a range of functions is assessed including inhibitory processes, maximal response rates, and temporal organization of behavior (Rice, 1988). The exposed monkeys showed disruptions in the temporal pattern of responding and slight elevations in their response rate (Mele et al., 1986). It has been suggested that in some of these behavioral deficits, processes related to the prefrontal cortex are involved in the mechanism of neurotoxic action of PCBs, potentially including mesocortical dopaminergic projections that terminate in the prefrontal cortex (Schantz et al., 1989, 1991). The deficit patterns on the discrimination reversal learning task (Schantz et al., 1989, 1991) and on the delayed spatial alteration showed similarities with deficits of monkeys with lesions to the dorsolateral area of the prefrontal cortex (Goldman et al., 1971). However, the current knowledge on brain structure‐related eVects of perinatal exposure to PCBs is too limited to support the hypothesis of prefrontal cortex involvement in the mechanism of eVect.

TABLE I SIGNIFICANT ASSOCIATIONS BETWEEN PERINATAL EXPOSURE TO PCBS AND DIOXINS AND NEURODEVELOPMENTAL OUTCOMES FROM 2 WEEKS TO 42 MONTHS OF AGE IN THE DUTCH COHORT Age

Cohorta

Neurological condition

2 wks

RþG

SPCBbreast milk, Total TEQ

Psychomotor development PDI, Bayley Scales of Infant Abilities Psychomotor development PDI, Bayley Scales of Infant Abilities Neurological condition

3m

R

SPCBmaternal

7m

R

Dioxin TEQ

18 m

RþG

SPCBmaternal/cord

42 m

RþG

SPCBmaternal/cord

42 m

R

SPCBmaternal/cord

Outcome variable

Exposure variable

56 General cognitive abilities K‐ABCc: Cognitive, Sequential & Simultaneous processing scale Verbal comprehension Reynell Developmental Language Scales

EVect description Higher breast milk levels of PCBs and dioxins were related with lower NOSb and higher incidence of hypotonia (Huisman et al., 1995a). Higher prenatal exposure was related with lower PDI scores (Koopman‐Esseboom et al., 1996). The highest exposed BF children (33%) scored lower than the less exposed BF children and comparable to FF children (Koopman‐Esseboom et al., 1996) Higher prenatal exposure was related to lower NOSb (Huisman et al., 1995a). Higher prenatal exposure was related with lower scores on the three scales. EVects were more pronounced in the FF group, lacking significance in the BF group (Patandin et al., 1999b). Higher prenatal exposure was significantly related with lower scores in the FF group, and not in the BF group (Patandin et al., 1999b).

Attentional processes Free play observation

42 m

R

SPCBmaternal/cord

Reaction time and sustained attention Computerized vigilance task

42 m

R

SPCBcord/ SPCB42 months

Problem Behavior Teacher CBCLd

42 m

R

Behavior Groninger Behavioral Scale (GBO)

42 m

R

SPCBmaternal/cord, SPCBbreast milk, Total TEQ SPCB42 months

Higher prenatal exposure was related with less episodes of high-level play, suggestive of less attentional abilities (Patandin et al., 1999c). Prenatal PCB exposure was related with more errors in the beginning of the task, suggestive of less focused attention (Patandin et al., 1999c). SPCB levels at 42 months were related with longer reaction times and a higher slope, suggestive of less sustained attention (Patandin et al., 1999c). Prenatal PCB and dioxin exposure was related to a higher prevalence of Withdrawn/Depressed behavior (Patandin et al., 1999c). SPCB levels at 42 months were related with a higher score on the GBO questionnaire, indicating more hyperactive behavior (Patandin et al., 1999c).

57

SPCB ¼ Sum of PCBs, IUPAC nos. 118, 138, 153, and 180 in maternal plasma measured during pregnancy, in cord plasma, or in breast milk. SPCB42 months ¼ Sum of PCBs, IUPAC nos. 118, 138, 153, and 180, measured in plasma of 42-month old children, representing mainly lactational transfer of maternal PCBs (the breast‐fed group) and in part during gestation (the formula‐fed group) [Patandin, 1997 #59]. TEQ ¼ Toxic Equivalent; Total TEQ ¼ sum of TEQs of 8 dioxin like PCBs (IUPAC nos. 77, 105, 118, 126, 156, and 169) and 17 dioxin congeners measured in breast milk. Shaded cells are results that give evidence of negative eVects of lactational exposure. a R ¼ Rotterdam and G ¼ Groningen cohort. b NOS ¼ Neurological optimality score. c K‐ABC ¼ Dutch Kaufman Assessment Battery for Children. d CBCL ¼ Child Behavior Checklist.

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Hestien J. I. Vreugdenhil and Nynke Weisglas-Kuperus IV.

A.

HUMAN NEURODEVELOPMENTAL PCB AND DIOXIN RISK ASSESSMENT

Prenatal Versus Postnatal Exposure to PCBs and Dioxins

Although much larger quantities of PCBs and dioxins are transferred to the child postnatally through lactation than prenatally, human epidemiological studies suggest more pronounced neurodevelopmental eVects of prenatal exposure to PCBs and dioxins compared to postnatal exposure to these compounds. However, several animal studies have shown profound behavioral impairments induced by postnatal exposure to low levels of a mixture of ortho‐substituted PCB mixtures that is representative of the PCB mixture found in human milk. In these monkeys, impaired performance was seen on spatial learning tasks, including impairment in learning a delayed spatial alteration task (Rice, 1998a, 1999) and more perseverative responding (Rice, 1999; Rice & Hayward, 1997). Moreover, slower acquisition of a fixed interval task and inability to inhibit inappropriate responding have been associated with postnatal exposure to PCB mixtures (Rice, 1997, 1999). These impairments suggest a discrimination learning deficit and diYculty in adaptively changing response patterns—deficits that are suggestive of involvement of prefrontal cortex processes in the neurotoxic mechanism of PCBs and dioxins (Rice, 1999). EVects of lactational exposure on these functions need to be addressed more thoroughly in the human studies. In human studies, addressing neurodevelopmental eVects of lactational exposure to PCBs and dioxins is complex. Breast milk contains several substances, such as several long‐chain polyunsaturated fatty acids, that are not available in formula milk. These acids are important constituents of the structural lipids of nonmyelinated cell membranes in the developing nervous system and are essential for growth, function, and integrity (Innis, 1994), and may therefore be important for optimal brain development. A meta‐ analysis of studies that addressed neurodevelopmental benefits of breast‐ feeding provided evidence for enhanced early cognitive development that sustained through childhood and adolescence (Anderson et al., 1999), taking into account a number of studies which suggested that diVerences in cognitive development were attributable to the generally associated diVerences in social economic conditions. The latter aspect forms another complicating feature of assessing neurodevelopmental eVects of lactational exposure: in Western societies, parents who choose to breast‐feed their child are likely to be diVerent in several parental and home environmental conditions. These aspects may influence the susceptibility to harmful eVects of perinatal exposure to PCBs and dioxins. The results of prenatal exposure to PCBs and dioxins on general cognitive abilities at 42 months of age in the Dutch cohort

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may illustrate the complexity of exploring eVects of exposure to PCBs and dioxins in breast‐fed children. At 42 months of age, adverse eVects of prenatal exposure to these compounds were more pronounced in the formula‐fed group compared to the breast‐fed group of children (Patandin et al., 1999b). B.

Sex Steroid–Related Behavioral PCB and Dioxins Effects

Neurotoxic eVects of perinatal exposure to PCBs and dioxins that cause developmental deficits may be mediated by endocrine‐disrupting properties of PCBs and dioxins. For example, steroid hormones play a mediating role in CNS development and influence not only reproductive but also nonreproductive behaviors that show sex diVerences (Fitch & Denenberg, 1998; Matsumoto, 1991). In animals, some eVects of perinatal exposure to PCBs and dioxins on nonreproductive behaviors have been reported. For example, a feminizing eVect on sweet preference was found in male rats that were perinatally exposed to a PCB mixture representative to PCBs found in human milk. In their female counterparts, sweet preference was not aVected (Hany et al., 1999). In contrast, prenatal exposure to a dioxin (TCDD) and coplanar (dioxin-like) PCBs decreased sweet preference in female rats, which can be interpreted as a masculinizing eVect in females. In the exposed males, no change in sweet preference was seen (Amin et al., 2000). The animal studies suggest both feminizing and masculinizing eVects of perinatal exposure to PCBs and dioxins on sex‐specific behavior, which may suggest steroid hormone–mediated eVects of PCB and dioxin exposure. In human studies, eVects of perinatal exposure to PCBs and dioxins on nonreproductive sex‐specific behavior have hardly been addressed. The only study that provided some evidence for steroid hormone–mediated behavioral eVects of prenatal exposure to PCBs and dioxins is the study in the highly exposed children of the Yu Cheng cohort. In this cohort, more spatially related cognitive abilities, which generally show some sex diVerences, were diVerently aVected in boys and girls. Only the exposed boys scored lower than their nonexposed matched controls (Guo et al., 1995). C.

Neurodevelopmental Interstudy Differences

The results of epidemiological studies that address neurodevelopmental eVects of perinatal exposure to environmental levels of PCBs show inconsistencies both between cohorts and within cohorts at diVerent ages. These diVerences in outcome do not necessarily undermine conclusions that prenatal exposure to environmental levels of PCBs is related to subtle harmful eVects on child neurodevelopment. The diVerences could be related to a

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Hestien J. I. Vreugdenhil and Nynke Weisglas-Kuperus

number of factors, including diVerences in exposure assessment techniques, diVerences in composure of environmental PCB mixtures, and diVerences in exposure levels. Moreover, diVerences in parental and home environmental conditions or the occurrence of other neurotoxic agents, which may confound relationships between exposure and neurodevelopmental outcome, may have led to diVerences in results. Additionally, diVerent neurodevelopmental outcome variables have been used in the cohort studies. Furthermore, addressing neurodevelopmental eVects of perinatal exposure to PCBs and dioxins as well as comparison of eVects assessed by diVerent cohorts at diVerent ages is complicated by the fact that the outcome variables are developmental qualities. EVects of perinatal exposure to PCBs and dioxins may not become evident until further maturation of the child. Some of these issues will be discussed in the following text. 1. EXPOSURE LEVELS

Exposure levels in the cohorts are diYcult to compare since, in the earlier American studies, diVerent assessment techniques have been used compared to the later initiated studies. In an eVort to compare exposure levels of several cohorts, median levels of PCB153 in maternal blood were used for comparison (Longnecker et al., 2003). This congener is always among the PCB congeners present at the highest concentration and constitutes a large proportion of the PCBs mixtures in all studies. That study showed that the median Dutch PCB153 level (0.10 mg/g lipid) was comparable to the median level in the Lake Michigan cohort (0.12 mg/g lipid), the North Carolina cohort (0.08 mg/g lipid), and the German median levels (0.14 mg/g lipid). The median exposure level in the Faeroe Islands cohort (recruited between 1994 and 1995) was 3 to 4 times higher than in these studies (0.45 mg/g lipid). 2. CONFOUNDING VARIABLES AND POTENTIAL DIFFERENCES IN SUSCEPTIBILITY TO EFFECTS OF PCB AND DIOXIN EXPOSURE

In the epidemiological studies, subjects could not be randomly assigned to predetermined levels of exposure or type of feeding during infancy. Samples were based on volunteer mother–infant pairs and parents were free in choosing the type of infant feeding they preferred because of acceptable ethical concerns. Therefore, all cohort studies have made eVorts to assess potential confounding variables, to adjust for these variables when studying the relation between perinatal exposure to PCBs and dioxins and neurodevelopment. In Western societies, the relation between perinatal exposure to PCBs and dioxins and neurodevelopment is often confounded by parental and home environmental conditions. Due to the physical stability and accumulation of PCBs and dioxins in human tissues, PCB and dioxin body burdens are

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strongly related to maternal age at birth. Mothers at older age who give birth to a child are often higher educated and have higher IQs than women at younger age who give birth to a child. Maternal age may also reflect other aspects of social economic conditions as well as psychosocial age‐related attributes (Stein, 1985). Child development is a process in which structural changes and environmental experiences influence each other mutually. For example, many cognitive skills, including IQs, verbal and spatial abilities, and perceptual speed (Alarcon et al., 1998; Plomin & Loehlin, 1989; Posthuma et al., 2001), have been shown to be under genetic influences. Environmental or psychosocial aspects, such as intellectual stimulation, organization of the home environment, verbal responsivity of the parents, variability of daily experience, and parental involvement also influence cognitive development (Bradley et al., 2001). In animal studies, environmental aspects influenced cortical diVerentiation and dendritic formation, thereby changing the functional connectivity of the nervous system. Several lines of evidence point toward the relationship between dendritic and synaptic changes and experiences and, more specifically, learning (Devoogd et al., 1985; Moser et al., 1994; Rosenzweig & Bennett, 1996; WolV et al., 1995). Moreover, numerous animal studies showed that environmental enrichment can compensate for and possibly even reverse some of the adverse eVects of developmental insults (Brenner et al., 1985; Diamond et al., 1975; Kolb, 1989). These studies suggest potential for structural and functional recovery throughout the cortical maturation period in animals. In humans, evidence of neural plasticity throughout the maturation period may be supported by results of studies in low birth weight children. These studies reported that in children at high biological risk, favorable early parental and home characteristics could compensate for or mask developmental delays (Landry et al., 1997; PfeiVer & Aylward, 1990; Weisglas‐ Kuperus et al., 1993). Hence, these genetic and environmental conditions are important predictors of cognitive development and can additionally be important in determining the vulnerability of an individual child or a given population to the eVects of neurotoxicants. Favorable parental and home environmental conditions may protect some groups against negative neurodevelopmental eVects of perinatal PCB and dioxin exposure. Evidence for this hypothesis is seen in the Dutch study showing less pronounced eVects of prenatal exposure to PCBs in breast‐fed children compared to formula‐fed children at 42 months of age (Patandin et al., 1999b). A reanalysis in the Lake Michigan cohort similarly showed that prenatal exposure to PCBs was related with lower IQs at 11 years of age in predominantly the group of formula‐fed children (n ¼ 56, 31%) (Jacobson & Jacobson, 2001). In the North Carolina cohort, prenatal exposure to PCBs

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Hestien J. I. Vreugdenhil and Nynke Weisglas-Kuperus

was not related to later cognitive and motor development from 3 to 5 years of age, in contrast to the Lake Michigan and Dutch study. In the North Carolina cohort, a relatively high proportion of the population was breast‐ fed during infancy (88%). Moreover, the average years of college education in the North Carolina cohort was 3 years and in the Lake Michigan cohort 1 year. In the Dutch cohort, 40% of the mothers have finished high school and 30% of them finished professional and university training. Some of the diVerences in neurodevelopmental eVects of PCB exposure between study centers can therefore be hypothesized to be related to cohort diVerences in the levels of these conditions that are important to child development. 3. CONFOUNDING BY OTHER NEUROTOXIC COMPOUNDS

Due to the correlational feature of the epidemiological studies addressing eVects of perinatal exposure to PCBs and dioxins, relations between these compounds and outcome are potentially related to exposure to other neurotoxic compounds, such as methyl mercury and lead. For example, in the Faeroe study, in which the local diet consists predominantly of fish and fish products, PCB and dioxin levels were seen to be relatively high compared to other European studies, as were the levels of methyl mercury. Significant relations between prenatal exposure to PCBs and reaction time and (semantic) memory skills appeared to be mainly attributable to prenatal exposure to methyl mercury compounds (Grandjean et al., 2001). However, in children exposed to high levels of methyl mercury, eVects of prenatal exposure to PCBs on these outcome variables were more pronounced than in children exposed to lower levels of methyl mercury, suggesting a potential interaction between these neurotoxic compounds in their neurodevelopmental eVects. In the Lake Michigan cohort, mothers were selected based on their diet history on Lake Michigan PCB‐contaminated fish. Fish and other aquatic species often form the source of exposure to PCBs as well as other neurotoxic compounds, such as methyl mercury. The relations between neurodevelopment and prenatal PCB exposure as described in the Lake Michigan studies, therefore, may have been confounded by exposure to this compound. However, based on the congruence between the results of animal studies and several human cohort studies, it has been suggested that the deficits observed in the Lake Michigan studies result, at least in part, from PCB exposure (Rice, 1995). In contrast to the Lake Michigan study, the North Carolina, Dutch, and German cohorts were recruited from the general public which may reduce the risk of confounding by methyl mercury. In the Netherlands, PCB and dioxin exposure occurs mainly through dietary intake of predominantly dairy products, as well as processed food and meat and fish products (Patandin et al., 1999a). In the Dutch PCB/dioxin cohort, lead and

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cadmium levels in blood samples drawn from 18-month old children (n ¼ 151) were relatively low (Weisglas‐Kuperus et al., 2000) and not related to cognitive outcome at 42 months of age (Patandin et al., 1999b). 4. NEURODEVELOPMENTAL TESTS AND THE DEVELOPMENT OF COGNITIVE ABILITIES

The cohort studies also show diVerences in the neurodevelopmental testing protocols that were used to explore neurotoxic eVects of perinatal exposure to PCBs and dioxins. Neurodevelopmental assessment has occurred at diVerent ages using diVerent test materials, which complicates comparison of the diVerent cohorts, especially due to the developmental nature of cognitive and motor abilities and since some aVected functions may not become apparent until a more mature age. Most prospective longitudinal studies have assessed general cognitive and motor abilities by means of developmental tests that were reassessed repeatedly through childhood. Performance on the developmental tests reflects, especially at a more mature age, a broad range of domains of function, including memory, visuospatial abilities, verbal and quantitative reasoning, and attentional aspects. Although general cognitive development seems to be the most relevant outcome variable in risk assessment studies, because of its predictive feature for later outcome, general cognitive ability indices may be too general to assess subtle eVects of exposure to neurotoxic compounds. The general cognitive score can obscure important individual diVerences in specific cognitive profiles, since children with diVerent cognitive profiles can have comparable scores on this outcome variable. Moreover, it can be reasoned that general cognitive scores reflect the product of learning, which is strongly related to social economic aspects, rather than processes of learning. The development of general cognitive abilities may progress at diVerent rates. Reported negative eVects of prenatal exposure to PCBs at diVerent assessment times within one cohort do not resolve the question of whether the same children are aVected in their abilities at the diVerent assessment periods. Consequently, risk assessment studies into eVects of perinatal exposure to PCBs and dioxins on cognitive and motor abilities in children may benefit from addressing the level and course of the development of these abilities. Early PCB and dioxin exposure may induce changes in brain structures that continue to influence neurodevelopment during maturation, resulting in delayed eVects of functions that develop later in childhood. Especially when eVects of lactational exposure to PCBs and dioxins are addressed, structure‐ related functional diVerences potentially, depending on the time window of exposure, can be hypothesized due to diVerences in maturation rates of diVerent brain structures. The exploration of neurotoxic eVects of perinatal exposure to neurotoxic agents therefore should address more specific

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domains of cognitive functioning that can be assessed at a more mature age. These domains are not suYciently measured by developmental or IQ tests, since most domains are indicated by too few items to provide reliable measurement of domain‐specific performance.

V.

THE DUTCH PCB AND DIOXIN STUDY AT SCHOOL AGE

Children enrolled in the Dutch PCB/dioxin cohort (Rotterdam and Groningen cohort) were invited to participate in follow‐up assessment at 6 to 7 years of age and half of the Rotterdam cohort was invited at 9 years of age as well. General cognitive and motor abilities, gender role‐play behavior, and neuropsychological functions were assessed. A neurophysiological assessment was also included. A.

Aims of the Study at School Age

The general aim of this study was to describe neurodevelopmental eVects of perinatal exposure to environmental levels of PCBs and dioxins in normal Dutch children at school age, as well as on the development of general cognitive and motor abilities from 3 to 84 months of age. In addition, the goal was to gain more insight into potential compensating eVects of parental and home environmental conditions and breast‐feeding, as well as into neurotoxic mechanisms of eVects of perinatal exposure to these compounds. B.

Subjects and Inclusion and Exclusion Criteria

A total of 418 healthy mother–infant pairs were recruited from June 1990 to June 1991. Half of the study population was recruited in Rotterdam (n ¼ 207), a highly industrialized and densely populated area, and the other half in Groningen (n ¼ 211), a semi‐urban area in The Netherlands. Healthy pregnant women were asked by their obstetrician or midwife to participate in a prospective neurodevelopmental study. The cohort consists of Caucasian mother–infant pairs. Pregnancy and delivery had been without complications; instrumental deliveries or caesarian sections were excluded. Only first or second at term‐born infants (37–42 weeks of gestation) who had no congenital anomalies or diseases were included. Because of these criteria, the cohort of children can be presumed to be at relatively low risk for neurodevelopmental deficits. To study the eVects of prenatal as well as postnatal PCB and dioxin exposure, it was aimed to include an equal number of women who intended to breast‐feed their child for at least 6 weeks (BF) and women who intended to

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use formula‐feeding (FF). All infants in the FF group received formula from a single batch (Almiron M2, Nutricia NV, Zoetermeer, The Netherlands) from birth until 7 months of age. In this formula, concentrations of both PCBs and dioxins were below the detection limit. The medical ethics committee of the University Hospital Rotterdam/ Sophia Children’s Hospital, and the Academical Hospital Groningen approved the study design and the parents gave informed consent. C.

Exposure Measurements

The exposure variables that were used in these studies included PCB levels in maternal and cord plasma. Maternal plasma samples were collected from the mothers during the last month of pregnancy and cord plasma samples were collected directly after birth. These samples were analyzed by means of gas chromatography with electron capture detection (GC‐ECD) for four PCB congeners, International Union for Pure and Applied Chemistry (IUPAC) numbers 118, 138, 153, and 180 (Burse et al., 1989; Koopman‐ Esseboom et al., 1994a). Two weeks after delivery a 24‐hour representative breast milk sample was collected from the mothers who were breast‐feeding their children. These samples were analyzed for 17 most abundant dioxins (PCDDs and PCDFs) and three dioxin-like PCBs (IUPAC nos. 77, 126, 169) by means of gas chromatography–high‐resolution mass spectometry (GC‐HRMS). In these samples, 23 non-dioxin-like PCBs (IUPAC nos. 28, 52, 66, 70, 99, 101, 105, 118, 128, 137, 138, 141, 151, 153, 156, 170, 177, 180, 183, 187, 194, 195, and 202) were measured by GC‐ECD (Koopman‐Esseboom et al., 1994a). Toxic potency of the mixture of dioxins and dioxin-like PCBs was expressed by using the toxic equivalent factor approach (Van den Berg et al., 1998b). Prenatal exposure to PCBs is defined as the sum of the concentrations of the four PCB congeners measured in maternal plasma and in cord plasma. PCB and dioxin concentrations in breast milk were assessed shortly after birth and form an indirect measure of prenatal exposure (Rogan et al., 1986a). Postnatal exposure to PCBs and dioxins through lactation was estimated in the BF group by multiplying breast milk levels of PCBs, dioxin-like PCB TEQs (dioxin toxic equivalents), and dioxin TEQs with the numbers of weeks of breast‐feeding. D.

Cognitive and Motor Abilities at School Age

At school age, the Dutch version of the McCarthy Scales of Children’s Abilities (Van der Meulen & Smirkovsky, 1985) was used to assess the general cognitive abilities (General Cognitive Index) and memory and motor

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skills in children of the Rotterdam and Groningen cohort (n ¼ 418). From the original cohort, 90% (n ¼ 376) were willing to participate in this follow‐ up (mean age 6.7 years þ 0.3). The data of four children were excluded from the data analysis because of potential confounding pathology. Prenatal PCB and dioxin levels were comparable for the nonparticipating and participating children. Multiple linear regression analysis showed that, adjusted for confounding variables, prenatal exposure to PCBs and dioxins was not significantly related to cognitive and motor development at school age. Moreover, eVects of prenatal exposure on cognitive and motor abilities were not statistically diVerent for the two feeding groups. However, it appeared that eVects of prenatal exposure to PCBs and dioxins on cognitive and motor abilities were modified by parental and home environmental conditions (maternal age, parental education level and verbal IQ, and HOME score). The parental and home environmental conditions were strongly related to each other. Older maternal age was related to a higher parental education level and verbal IQ and higher scores on the HOME questionnaire, conditions that are considered to be relatively more favorable to child development. The impact of adverse eVects of prenatal exposure to PCBs and dioxins on cognitive and motor abilities was suggested to increase as parental and home environmental conditions were lower. In children raised in relatively more favorable parental and home environmental conditions, subtle eVects of prenatal exposure to PCBs and dioxins were not detectable. EVect modifications of PCB and dioxin exposure by maternal age, parental education level and verbal IQ, and HOME scores could not be explored simultaneously in one regression analysis due to the problem of multicollinearity. The results did not show evidence of negative eVects of lactational exposure to PCBs and dioxins on cognitive and motor outcome, nor of eVect modification of lactational exposure by parental and home environmental characteristics. We concluded that neurotoxic eVects of prenatal exposure to environmental levels of PCBs and dioxins may persist into school age and may result in subtle cognitive and motor delays. The results of this study suggest that parental and home environmental conditions influence the consequences of the neurotoxic eVects on cognitive and motor development (Vreugdenhil et al., 2002a). E.

The Development of General Cognitive and Motor Abilities from 3 to 84 Months of Age

A disadvantage of studying relations between perinatal exposure to PCBs and dioxins and cognitive and motor abilities at a certain age is that the developmental course of these abilities is not captured. Therefore, eVects of perinatal exposure to PCBs, measured in maternal plasma, on

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the development of cognitive and motor abilities, as assessed in the Rotterdam cohort at 3, 7, 18, 42, and 84 months of age, were described using the method of random regression modeling (RRM). Moreover, important predictors of general cognitive and motor development from 3 to 84 months of age were identified in this study. Data on cognitive and motor abilities were available for all analyzable children (excluding children with potential confounding pathology, n ¼ 3). In the initial RRM models, all selected variables of potential relevance to cognitive and motor development were included. In these models, higher levels of prenatal PCB exposure were significantly related to a lower level of cognitive and motor development from 3 to 84 months of age. In this study, the problem of multicollinearity when including the four previously described interaction variables of prenatal PCB exposure and parental and home environmental variables simultaneously in the regression model was solved by centering these variables as well as their main terms. Simultaneous inclusion of these variables showed that eVects of prenatal exposure to PCBs on the level of cognitive development were significantly modified by maternal age, overruling eVect modification by the other parental and home environmental conditions. In children born to younger mothers, eVects of prenatal exposure to PCBs on cognitive development were suggested to be more pronounced than in children born to older mothers, a condition that is likely to reflect more favorable parental and home environmental conditions for child development. Prenatal PCB levels, and modification by maternal age, along with parental education level and verbal IQ and HOME scores, were important determinants of the level of cognitive development. Motor development was eYciently estimated by prenatal PCB levels, including its modification by HOME scores along with parental education levels. EVects of prenatal PCB exposure on motor development were more pronounced when the HOME scores were lower. The results provided no evidence of negative eVects of lactational exposure to PCBs on cognitive or motor development and neither were maternal (prenatal) thyroid hormone levels related to these outcomes. These results provided evidence of adverse eVects of prenatal exposure to PCBs on the level of cognitive and motor development, eVects that may be modified by conditions that are favorable to child development. Compared to the large positive eVects of more optimal parental and home environmental conditions, the negative eVects of prenatal PCB exposure on cognitive development from 3 to 84 months of age were relatively small. EVects of prenatal exposure were more pronounced for motor than for cognitive development. Motor development may therefore be a more sensitive outcome to detect prenatal exposure to PCBs and related compounds than is cognitive development (Vreugdenhil et al., 2002c).

68 F.

Hestien J. I. Vreugdenhil and Nynke Weisglas-Kuperus Sex‐Specific Play Behavior

As part of the first follow‐up study at school age, play behavior was assessed by means of the Pre‐School Activity Inventory (PSAI) in the Rotterdam cohort (Golombok & Rust, 1993). The PSAI assesses masculine and feminine play behavior scored on three subscales: Masculine, Feminine, and Composite. One hundred sixty PSAI questionnaires were returned (mean age þ SD: 7.5 years þ 0.4). Higher prenatal PCB levels were related with less masculinized play behavior in boys and with more masculinized play behavior in girls. Higher prenatal dioxin levels, available for BF children, were associated with more feminized play in boys as well as in girls, assessed by the Feminine scale. There was no evidence that lactational exposure to PCBs and dioxins was related to play behavior in the total BF group nor in boys and girls separately. The results are suggestive of steroid hormone involvement in the neurotoxic mechanism of action of prenatal exposure to environmental levels of PCBs and dioxins (Vreugdenhil et al., 2002b).

G.

Neuropsychological Functions

Half of the Rotterdam cohort, the lowest prenatally exposed (p25; n ¼ 26) and the highest prenatally exposed children (p75; n ¼ 26) of both feeding groups (total n ¼ 104) were invited to participate in neuropsychological and neurophysiological assessments at 9 years of age. From the invited children, 80% (n ¼ 83) were willing to participate in this follow‐up study (mean age þ SD: 9.2 þ 0.2). Exposure levels of the participating and nonparticipating children were comparable. The assessment of neuropsychological functions included the Rey Complex Figure task, the Auditory–Verbal Test, the Simple Reaction Time task, and the Tower of London (TOL). Prenatally high‐exposed children had, adjusted for confounding variables, longer reaction times and more variation in their reaction times, and lower scores on the TOL than did prenatally low‐exposed children. On the latter task, assessing predominantly executive or planning functions, in contrast to the other tasks, children that were BF for a long period (>16 weeks) scored significantly lower than did FF children. The results of this study are suggestive of multifocal or diVuse neurotoxic eVects of prenatal exposure to PCBs and related compounds. For lactational exposure, the negative eVect on the TOL scores may suggest that processes related to the prefrontal cortex are involved in the neurotoxic mechanism of exposure to PCBs and related compounds. This can be hypothesized since the frontal cortex shows a delayed maturation rate compared to other brain regions and developing brain structures are more

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vulnerable to exposure to PCBs and dioxins. A complex task such as the TOL may also serve as a sensitive outcome parameter to assess neurotoxic eVects of early exposure to PCBs and related compounds (Vreugdenhil et al., 2004a). H.

Neurophysiological Endpoints

The P‐300 is considered to be a cognitive component of event‐related brain potentials and occurs with a latency of about 300 milliseconds when a person is actively processing (‘‘attending to’’) incoming stimuli. Prenatally high‐exposed children had significantly longer P‐300 latencies than prenatally low‐exposed children, adjusted for confounding variables. The results gave no evidence of diVerences in P‐300 latencies related to lactational exposure to PCBs and dioxins. Instead, a longer duration of breast‐feeding (>16 weeks) was associated with shorter P‐300 latencies compared to children that were BF for 6 to 16 weeks and to FF children. No diVerences in P‐300 amplitudes were seen relative to prenatal or postnatal exposure to PCBs and dioxins or to the duration of breast‐feeding. These results suggest that prenatal exposure to PCBs and dioxins delays CNS mechanisms that evaluate and process relevant stimuli at school age, whereas breast‐feeding accelerates these mechanisms (Vreugdenhil et al., 2004b).

VI. A.

DISCUSSION

Neurotoxic Mechanisms

Prenatal exposure to PCBs and dioxins can be regarded as chronic exposure of the developing CNS and many processes of the CNS are likely to be sensitive to exposure to PCBs and dioxins, including neuronal and glial cells, neurotransmitters, and endocrine systems (Brouwer et al., 1995, 1999; Mariussen & Fonnum, 2001; Tilson & Kodavanti, 1998). Consequently, eVects of prenatal exposure to PCBs and dioxins are likely to be of multifocal or diVuse nature. The results of the Dutch PCB/dioxin study and other prospective human PCB studies suggest eVects of prenatal exposure to PCBs on several neurodevelopmental outcome variables, including general cognitive and motor development (Gladen et al., 1988; Jacobson & Jacobson, 1996; Koopman‐Esseboom et al., 1996; Patandin et al., 1999b; Rogan & Gladen, 1991; Vreugdenhil et al., 2002a; Walkowiak et al., 2001; Winneke et al., 1998), verbal comprehension skills (Jacobson & Jacobson, 1996; Patandin et al., 1999b), processing speed (Grandjean et al., 2001; Vreugdenhil et al., 2004a), attention and concentration (Jacobson & Jacobson, 1996;

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Patandin et al., 1999c; Vreugdenhil et al., 2004a), memory skills (Jacobson & Jacobson, 1996; Vreugdenhil et al., 2002a), planning or executive functions (Vreugdenhil et al., 2004a), and on a neurophysiological endpoint that assesses processing and evaluation of auditory stimuli (Vreugdenhil et al., 2004b). In the neuropsychological study, scores on some tests were not related to perinatal exposure to PCBs and dioxins. This may reflect diVerences in sensitivity of neuropsychological tests to measure subtle neurotoxic eVects of perinatal exposure to neurotoxic compounds. The diVerence in sensitivity in a relatively small cohort may aVect the power to detect eVects and may, therefore, result in missing eVects of perinatal exposure to PCBs and dioxins (increasing Type II errors). Postnatally, maturation of diVerent areas in the brain occurs at diVerent rates. The frontal cortex shows the slowest maturation rate. Since developing CNS structures are known to be especially vulnerable to adverse eVects of exposure to PCBs and dioxins, structure‐related eVects of lactational exposure can be hypothesized. Some evidence in support of this hypothesis can be found in the finding that performance on the TOL was the only outcome that was suggested to be related to lactational exposure to PCBs. In planning or executive functions, processes of the prefrontal cortex are especially involved, in which higher cortical functions from several areas of the brains are integrated. In monkeys that were only exposed to PCBs through lactation, PCB‐induced behavioral deficits were also suggestive of prefrontal cortex involvement (Rice, 1999). Moreover, brain dopaminergic systems have been shown to be aVected (Mariussen et al., 2001; Seegal et al., 1997) by exposure to PCBs and some major dopaminergic pathways are known to serve the prefrontal cortex (Saper et al., 2000). Another important aspect of neurotoxic eVects of prenatal exposure to PCBs and dioxins is that the neurodevelopmental consequences of neurotoxic actions of prenatal exposure to environmental levels of PCBs and dioxins may be influenced by parental and home environmental conditions. Global measurements of cognitive and motor abilities are relatively strongly related to parental and home environmental conditions. The results of the Dutch PCB/dioxin study are suggestive of compensation of negative eVects of perinatal exposure on cognitive and motor development in children raised in more favorable parental and home environmental conditions or of cumulative deficits in children raised in less favorable conditions. These results may be in line with animal studies which show a positive impact of an enriched environment on the eVects of brain lesions (Brenner et al., 1985; Diamond et al., 1975; Kolb, 1989) and with some human studies addressing eVects of perinatal exposure to lead and methyl mercury (Bellinger et al., 1988; Winneke & Kraemer, 1984) and the outcome in very low birth

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weight children (Landry et al., 1997; PfeiVer & Aylward, 1990; Weisglas‐ Kuperus et al., 1993). Neurotoxic eVects of perinatal exposure to PCBs and dioxins may be mediated by hormone‐disrupting properties of PCBs and dioxins, for example, in regard to steroid and thyroid hormone systems. The (sex‐specific) eVects of perinatal exposure to PCBs and dioxins on childhood play behavior suggest mediation of behavioral eVects of prenatal PCB and dioxin exposure by the sex‐steroid hormone system. However, evaluation of the relation between prenatal steroid hormone status and PCB and dioxin exposure is needed to further confirm these findings. In this cohort, maternal and infant thyroid hormone levels were related to maternal levels of PCBs and dioxins. Prenatal alterations in prenatal thyroid hormone levels may cause long‐lasting neurodevelopmental deficits (Porterfield, 1994; Rovet & Ehrlich, 1995). However, in the Dutch PCB dioxin study, maternal thyroid hormone status was not statistically related to the level of cognitive and motor development from 3 to 84 months of age. The presently used analyses, therefore, do not provide evidence that prenatal thyroid hormone status is one of the key mechanisms in the neurotoxic eVects of prenatal exposure to PCBs and dioxins on general cognitive and motor development. Animal studies show diVerences in the neurotoxic eVects of nonplanar PCBs and dioxins and dioxin-like PCBs (Fischer et al., 1998; Tilson & Kodavanti, 1997). Humans are exposed to complex mixtures of PCBs and dioxins and their related compounds, such as hydroxylated PCBs. Not finding associations between outcome variables and TEQs or total TEQs may suggest that neurotoxic eVects of PCBs and dioxins were not mediated by the Ah receptor, as is in line with animal studies that report more pronounced neurotoxic actions of nonortho‐substituted PCB congeners than of dioxins and dioxin-like PCBs. The studies presented here show more pronounced eVects on general cognitive and motor abilities of the four nonplanar PCBs (IUPAC nos. 118, 138, 153, and 180) as assessed in plasma compared to the dioxin TEQs and total TEQs assessed in breast milk. However, based on these results, we believe that we cannot diVerentiate eVects of diVerent types of congeners, since the levels of diVerent types of congeners were strongly related (Koopman‐Esseboom et al., 1994a). Moreover, dioxins as well as a more elaborate number of PCB congeners were assessed in breast milk that was available for only half of the cohort. Analyses in this subpopulation may have increased the risk of Type II errors, which may consequently increase the risk of missing associations. However, in regard to play behavior, some evidence of diVerent neurotoxic eVects of PCBs and dioxins can be hypothesized. Prenatal exposure to the sum of the four nonplanar PCBs was suggested to be related with opposite eVects in boys and girls on masculine play behavior, whereas higher levels of prenatal

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exposure to dioxins, expressed in TEQs, were related to more feminized play behavior in both boys and girls in a similar direction. In conclusion, the mechanisms of neurotoxic eVects of prenatal exposure to PCBs and dioxins may include multifocal, or diVuse, neurodevelopmental impairments. Due to diVerences in the maturation of the CNS, lactational exposure may be related to more focal eVects, in which processes related to the prefrontal cortex are suggested to be involved. Neurotoxic eVects on neurodevelopmental outcome that is more strongly related to parental and home environmental conditions may be modified by these conditions. Moreover, steroid hormones are suggested to be involved in the neurotoxic mechanism of eVects of prenatal exposure to PCBs on a sex‐specific nonreproductive behavior. B.

Is Breast‐Feeding Safe?

Although BF children are exposed to relatively large amounts of PCBs and dioxins, negative eVects of lactational exposure to PCB and dioxins are only suggested on the scores on the TOL. The results of this study, therefore, may indicate that eVects of prenatal exposure to PCBs and dioxins are more pronounced than eVects of exposure to PCBs and dioxins through lactation. This is in agreement with most of the human studies that address perinatal exposure to environmental levels of PCBs and dioxins (Gladen et al., 1988; Jacobson & Jacobson, 1996; Jacobson et al., 1990; Rogan & Gladen, 1991). Only two studies have described adverse eVects of lactational exposure on scores on these developmental tests. Lactational exposure to PCBs and dioxins was related to lower psychomotor abilities at 7 months of age (Koopman‐ Esseboom et al., 1996) in the Dutch study and to lower general cognitive abilities at 42 months of age in the German cohort (Walkowiak et al., 2001). There are some methodological aspects that should be considered in risk assessment studies that address neurodevelopmental eVects of lactational exposure to PCBs and dioxins, especially in Western societies. Based on the studies described in the previous paragraph, it can be hypothesized that negative eVects of lactational exposure may, similarly to eVects of prenatal exposure, be counteracted or masked by optimal parental and home environmental conditions. In The Netherlands, comparable to most Western societies, the parents’ choice for breast‐feeding their child generally also reflects diVerences in, for example, levels of parental and home environmental conditions. Studies that explore subtle adverse eVects of lactational exposure to PCBs and dioxins may therefore benefit from more advanced modeling techniques in which the interrelationships of these neurodevelopmental determinants and exposure can be more properly modeled. Moreover, more insight is needed into potential beneficial eVects of breast‐feeding, such as

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the eVects of brain‐stimulating substances that are provided by breast milk and not by formula milk. The design and aims of the studies described in this paragraph are not adequate to address this aspect of breast‐feeding. However, the results of the neurophysiological study presented in this paragraph may suggest positive eVects of a longer duration of breast‐feeding in which potentially brain‐stimulating eVects of substances in breast milk are involved. Animal studies show evidence of profound neurodevelopmental eVects in monkeys that were exposed only to low levels of PCBs and dioxins through lactation (Rice, 1997, 1999; Rice & Hayward, 1997, 1999). These results indicate the potential for neurodevelopmental eVects of lactational exposure to PCBs and dioxins in humans. Moreover, these behavioral deficits in animals were suggestive of prefrontal cortex involvement. Since structure‐ related eVects of lactational exposure can be hypothesized considering maturation diVerences of brain structures, risk assessment studies that address lactational exposure should include a more elaborate neuropsychological test battery and larger study populations in which children were breast‐fed for longer durations than in the Dutch cohort (median of breast‐feeding duration 16 weeks). This may increase the knowledge of neurotoxic eVects of lactational exposure as well as help to diVerentiate eVects of prenatal and lactational exposure to PCBs and dioxins. Although infants are exposed to relatively large amounts of PCBs and dioxins through lactation, neurodevelopmental eVects of prenatal exposure to environmental levels of PCBs and dioxins were generally more pronounced. Subtle eVects of postnatal exposure to PCBs and dioxins were detected on one of the neurodevelopmental outcome variables that were explored in the Dutch PCB/dioxin cohort. On the other hand, the neurophysiological data may suggest beneficial eVects of a longer duration of breast‐ feeding in which potentially brain‐stimulating eVects of substances in breast milk are involved. These results do not warrant restrictions on breast‐feeding or reductions of the period of breast‐feeding in the Western societies. Neurodevelopmental eVects of lactational exposure to PCBs and dioxins and eVects of breast milk brain‐stimulating substances should be studied more thoroughly, using advanced modeling techniques in addition to addressing specific cognitive domains in larger cohorts as well as animal research. C.

Magnitude of Estimated Neurodevelopmental Effects

The magnitude of neurodevelopmental eVects that were associated with PCBs and dioxin exposure is relatively small in the Dutch cohort, and is not likely to be clinically relevant to the individual child. The level of cognitive development from 3 to 84 months of age, for example, in children born to

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younger mothers, was approximately 3 points lower in high prenatally exposed children (75% equivalent) compared to their low-exposed counterparts (25% equivalent). Under less favorable parental and home environmental conditions, however, the magnitude of cognitive and motor decrements may be larger. The Rotterdam cohort consists of families willing to participate for at least 7 years in this study. Parental and home environmental characteristics of this group are therefore likely to be more advantaged than in the average Dutch population or in populations in which educational possibilities or potential for cognitive stimulation are limited. When considering these subtle eVects in a large population, a lower average IQ shifts the distribution and increases the number of individuals who can be classified as retarded (IQ < 85). Additionally, it decreases the number of gifted and exceptionally gifted individuals (IQ > 130). For example, if the average IQ is shifted by 5 points (in a normal distribution with a mean of 100 and a standard deviation of 15), the number of children that score below 70 increases by a factor of 2 (Rice, 1998b). Neurodevelopmental eVects of perinatal exposure to PCBs and dioxins are detectable in a cohort of normal children. The magnitude of the eVects is relatively small and not likely to be clinically relevant to the individual child. The magnitude of neurodevelopmental eVects may be somewhat larger in populations in which conditions for child development are less favorable. For the whole society, however, these subtle decrements may have long‐term consequences.

VII.

FUTURE PERSPECTIVES

The epidemiological PCB studies described in this chapter draw attention to a number of important aspects that should be considered in this type of prospective follow‐up risk assessment studies that address eVects of perinatal exposure to PCBs and dioxins on neurodevelopmental outcome. First, the results of these studies may illustrate the importance of nonrandom attrition of the subjects of the original cohort. Ten percent of the original cohort was lost to follow‐up at school age. Although exposure levels were not statistically diVerent between participating and nonparticipating children, the latter group was significantly more formula‐fed, breast‐fed for shorter periods, and maternal age, parental education level, and verbal IQ were significantly lower in this lost to follow‐up group. Since developmental eVects of prenatal exposure to PCBs and dioxins may be influenced by parental and home environmental conditions, this is an important change in the study population. At preschool age, adverse eVects of prenatal PCB

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exposure on cognitive development were seen in the total cohort, whereas at school age, significant adverse eVects were seen only when parental and home characteristics were less optimal. The higher mean levels of these background variables in the population at school age might explain that no eVect of prenatal PCB exposure was seen in the total cohort, adjusting for the mean population levels of the confounders. Therefore, changes in the distribution of these variables in a cohort are a point of great attention in prospective follow‐up studies that address neurodevelopmental risks of perinatal exposure to PCBs and dioxins, since it may cause missing neurodevelopmental eVects in older children. Second, the choice of neurodevelopmental outcomes to detect adverse eVects of prenatal and lactational exposure to PCBs and dioxins should be addressed with great care in risk assessment studies. For example, general cognitive abilities, as measured with developmental tests, may not be the most sensitive outcome to detect neurotoxic eVects of lactational exposure to PCBs and dioxins since this outcome is particularly sensitive to parental and home environmental conditions. The process of learning or more specific neuropsychological functions as well as motor development may be more sensitive outcomes in risk assessment studies addressing subtle eVects of lactational exposure to neurotoxicants. Third, due to the complex interrelationships of various neurodevelopmental determinants and maternal PCB and dioxin levels, risk assessment studies may benefit from using sophisticated statistical modeling techniques. Moreover, these analyses make it possible to address the developmental course of functions in addition to addressing the level of functions at a certain point during the development of those functions. Fourth, due to diVerences in eating habits or area diVerences in environmental PCB and dioxin mixtures, populations worldwide are exposed to dissimilar mixtures of PCBs and dioxins. For example, results from the Oswego Study indicated that the cord blood of women who consumed Lake Ontario fish contained a significantly higher proportion of the most heavily chlorinated PCBs relative to non‐fish eaters. Levels of PCBs of lighter chlorination as well as the total PCB levels were similar in these groups (Stewart et al., 1999). Moreover, the cord blood levels of the highly chlorinated PCBs correlated more strongly with breast milk PCB levels than lower chlorinated PCBs. The results of the neurodevelopmental analyses in this cohort (children from birth to 12 months of age) showed some evidence of more pronounced neurodevelopmental eVects of exposure to the higher chlorinated PCBs (Darvill et al., 2000). The initial findings of this study as well as the results of laboratory studies therefore suggest that risk assessment studies can benefit from addressing more thoroughly the eVects of diVerent types of congeners to which children are exposed.

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Fifth, environmental levels of PCBs and dioxins are generally declining, due to worldwide control of sources, regulations of disposal practices, elimination of production, and natural attenuation. In The Netherlands, eVorts to minimize dioxin emissions, as were performed from the late 1980s, clearly show decreasing levels of PCBs and dioxins in food in the past 10 years (Liem et al., 2000). In breast milk, dioxin levels even decreased up to 50% during the past decade (Liem et al., 2000; Van Leeuwen & Malisch, 2002) (see Fig. 2). Children, however, are perinatally exposed to a large number of other potentially neurotoxic-persistent environmental pollutants such as heavy metals, pesticides and insecticides, flame retardants, and cleansers. For example, although breast milk levels of PCBs and dioxins have decreased over the past years, an increase is seen in the levels of another group of persistent organic pollutants, polybrominated diphenyl ethers (PBDEs) (Meironyte et al., 1999). PBDEs are used as flame retardants and are presently applied throughout the world. The chemical structure of the PBDEs resembles the structure of PCBs and dioxins and their neurotoxic properties have been recognized (Darnerud et al., 2001). Furthermore, of the over 80,000 chemicals that are used in commerce and industry, only a small proportion has undergone testing for neurodevelopmental toxicity. PCBs and dioxins are among the few contaminants subjected to extensive research exploring their neurotoxic properties and neurodevelopmental consequences for humans exposed to environmental levels of these compounds. The subtle neurodevelopmental decrements detected in the prospective follow‐up studies in healthy‐born children who were perinatally exposed to relatively low levels, environmental levels of PCBs and dioxins may be illustrative for the

FIG. 2. Temporal trends of levels of PCDD/PCDF in human milk (Van Leeuwen & Malisch, 2002).

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Saper, C. B., Iversen, S., & Frackowiak, R. S. (2000). The association areas of the cerebral cortex and the cognitive capacities of the brain. In E. R. Kandell, J. H. Schwartz, & T. M. Jessel (Eds.), Principles of Neural Science (p. 361). New York: McGraw-Hill. Schantz, S. L., Levin, E. D., & Bowman, R. E. (1991). Long‐term neurobehavioral eVects of perinatal polychlorinated biphenyl (PCB) exposure in monkeys. Environmental Toxicology and Chemistry, 10, 747–756. Schantz, S. L., Levin, E. D., Bowman, R. E., Heironimus, M. P., & Laughlin, N. K. (1989). EVects of perinatal PCB exposure on discrimination‐reversal learning in monkeys. Neurotoxicology and Teratology, 11, 243–250. Seegal, R. F., Brosch, K. O., & Okoniewski, R. J. (1997). EVects of in utero and lactational exposure of the laboratory rat to 2,4,20 ,40 ‐ and 3,4,30 ,40 ‐tetrachlorobiphenyl on dopamine function. Toxicology and Applied Pharmacology, 146, 95–103. Shafer, T. J., Mundy, W. R., Tilson, H. A., & Kodavanti, P. R. (1996). Disruption of inositol phosphate accumulation in cerebellar granule cells by polychlorinated biphenyls: A consequence of altered Ca2þ homeostasis. Toxicology and Applied Pharmacology, 141, 448–455. Shain, W., Bush, B., & Seegal, R. (1991). Neurotoxicity of polychlorinated biphenyls: Structure‐ activity relationship of individual congeners. Toxicology and Applied Pharmacology, 111, 33–42. Steele, G., Stehr‐Green, P., & Welty, E. (1986). Estimates of the biologic half‐life of polychlorinated biphenyls in human serum. New England Journal of Medicine, 314, 926–927. Stein, Z. A. (1985). A woman’s age: Childbearing and child rearing. American Journal of Epidemiology, 121, 327–342. Stewart, P., Darvill, T., Lonky, E., Reihman, J., Pagano, J., & Bush, B. (1999). Assessment of prenatal exposure to PCBs from maternal consumption of Great Lakes fish: An analysis of PCB pattern and concentration. Environmental Research, 80, S87–S96. Tanabe, S. (1988). PCB problems in the future: Foresight from current knowledge. Environmental Pollution, 50, 5–28. Taylor, P. R., & Lawrence, C. E. (1992). Polychlorinated biphenyls: Estimated serum half lives. British Journal of Industrial Medicine, 49, 527–528. Thiel, R., Koch, E., Ulbrich, B., & Chahoud, I. (1994). Peri‐ and postnatal exposure to 2,3,7,8‐ tetrachlorodibenzo‐p‐dioxin: EVects on physiological development, reflexes, locomotor activity, and learning behavior in Wistar rats. Archives of Toxicology, 69, 79–86. Tilson, H. A., Davis, G. J., McLachlan, J. A., & Lucier, G. W. (1979). The eVects of polychlorinated biphenyls given prenatally on the neurobehavioral development of mice. Environmental Research, 18, 466–474. Tilson, H. A., & Kodavanti, P. R. (1997). Neurochemical eVects of polychlorinated biphenyls: An overview and identification of research needs. Neurotoxicology, 18, 727–743. Tilson, H. A., & Kodavanti, P. R. (1998). The neurotoxicity of polychlorinated biphenyls. Neurotoxicology, 19, 517–525. Van den Berg, M., Birnbaum, L., Bosveld, A. T., Brunstrom, B., Cook, P., Feeley, M., Giesy, J. P., Hanberg, A., Hasegawa, R., Kennedy, S. W., Kubiak, T., Larsen, J. C., van Leeuwen, F. X., Liem, A. K., Nolt, C., Peterson, R. E., Poellinger, L., Safe, S., Schrenk, D., Tillitt, D., Tysklind, M., Younes, M., Waern, F., & Zacharewski, T. (1998a). Toxic equivalency factors (TEFs) for PCBs, PCDDs, PCDFs for humans and wildlife. Environmental Health Perspectives, 106, 775–792. Van den Berg, M., Birnbaum, L., Bosveld, A. T. C., Brunstrom, B., Cook, P., Feeley, M., Giesy, J. P., Hanberg, A., Hasegawa, R., Kennedy, S. W., Kubiak, T., Larsen, J. C., van

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Leeuwen, F. X., Liem, A. K., Nolt, C., Peterson, R. E., Poellinger, L., Safe, S., Schrenk, D., Tillitt, D., Tysklind, M., Younes, M., Waern, F., & Zacharewski, T. (1998b). Toxic equivalency factors (TEFs) for PCBs, PCDDs, PCDFs for humans and wildlife [see comments]. Environmemtal Health Perspectives, 106, 775–792. Van der Meulen, B. F., & Smirkovsky, M. (1985). Mos. 2 1/2 –8 1/2 McCarthy Ontwikkelingsschalen. The Netherlands: Swets & Zeitlinger B.V. Van Leeuwen, F. X. R., & Malisch, R. (2002). Results of the third round of the WHO‐ coordinated exposure study on the levels of PCBs, PCDDs, and PCDFs in human milk. Organohalogen Compounds, 56, 311–316. Vreugdenhil, H. J., Lanting, C. I., Mulder, P. G., Boersma, E. R., & Weisglas‐Kuperus, N. (2002a). EVects of prenatal PCB and dioxin background exposure on cognitive and motor abilities in Dutch children at school age. Journal of Pediatrics, 140, 48–56. Vreugdenhil, H. J., Slijper, F. M., Mulder, P. G., & Weisglas‐Kuperus, N. (2002b). EVects of perinatal exposure to PCBs and dioxins on play behavior in Dutch children at school age. Environmental Health Perspectives, 110, A593–A598. Vreugdenhil, H. J. I., Duivenvoorden, H. J., & Weisglas‐Kuperus, N. (2002c). Neurodevelopmental EVects of Perinatal Exposure to Environmental Levels of PCBs and Dioxins in Children at School Age. Rotterdam, the Netherlands: Erasmus University Thesis. Vreugdenhil, H. J. I., Mulder, P. G., Emmen, H. H., & Weisglas‐Kuperus, N. (2004a). EVects of perinatal exposure to PCBs on neuropsychological functions in the Rotterdam cohort at 9 years of age. Neuropsychology, 18, 185–193. Vreugdenhil, H. J. I., Van Zanten, G. A., Brocaar, M. P., Mulder, P. G. H., & Weisglas‐ Kuperus, N. (2004b). Prenatal PCB exposure and breast‐feeding aVect auditory P300 latencies in 9‐year‐old Dutch children. Developmental Medicine and Child Neurology, 46, 398–405. Walkowiak, J., Wiener, J. A., Fastabend, A., Heinzow, B., Kramer, U., Schmidt, E., Steingruber, H. J., Wundram, S., & Winneke, G. (2001). Environmental exposure to polychlorinated biphenyls and quality of the home environment: EVects on psychodevelopment in early childhood. Lancet, 358, 1602–1607. Weisglas‐Kuperus, N., Baerts, W., Smrkovsky, M., & Sauer, P. J. (1993). EVects of biological and social factors on the cognitive development of very low birth weight children. Pediatrics, 92, 658–665. Weisglas‐Kuperus, N., Patandin, S., Berbers, G. A., Sas, T. C., Mulder, P. G., Sauer, P. J., & Hooijkaas, H. (2000). Immunologic eVects of background exposure to polychlorinated biphenyls and dioxins in Dutch preschool children. Environmental Health Perspectives, 108, 1203–1207. WHO (1989). Levels of PCBs, PCDDs, and PCDFs in breast milk: Results of the WHO‐ coordinated interlaboratory quality control studies and analytical field studies. Environmental Health Series, 34. Winneke, G., Bucholski, A., Heinzow, B., Kramer, U., Schmidt, E., Walkowiak, J., Wiener, J. A., & Steingruber, H. J. (1998). Developmental neurotoxicity of polychlorinated biphenyls (PCBS): Cognitive and psychomotor functions in 7‐month‐old children. Toxicology Letters, 102–103, 423–428. Winneke, G., & Kraemer, U. (1984). Neuropsychological eVects of lead in children: Interactions with social background variables. Neuropsychobiology, 11, 195–202. WolV, J. R., Laskawi, R., Spatz, W. B., & Missler, M. (1995). Structural dynamics of synapses and synaptic components. Behavioural Brain Research, 66, 13–20.

Interactions of Lead Exposure and Stress: Implications for Cognitive Dysfunction DEBORAH A. CORY‐SLECHTA ENVIRONMENTAL AND OCCUPATIONAL HEALTH SCIENCES INSTITUTE, A JOINT INSTITUTE OF ROBERT WOOD JOHNSON MEDICAL SCHOOL, UNIVERSITY OF MEDICINE AND DENTISTRY OF NEW JERSEY, AND OF RUTGERS, THE STATE UNIVERSITY OF NEW JERSEY, PISCATAWAY, NEW JERSEY

I.

HISTORY AND CURRENT UNDERSTANDING OF LEAD EFFECTS

Although the toxicity of lead (Pb) was recognized as far back as the time of the ancient Romans (Hernberg, 2000), the versatility of this metal virtually guaranteed its continued application. Throughout the years, Pb was utilized in cosmetics such as rouge and mascara, as a spermicide, broadly as a sugar and preservative for cooking and wines, and for pewter, coins, and ceramic glazes. Lead pipes served as the basis of the plumbing system that supplied the Roman Empire with water. The high levels of exposure and Pb body burdens at that time as a consequence of these uses has repeatedly been documented in analyses of bone lead levels of the ancient Romans. New uses of Pb emerged during the Industrial Revolution, with the manufacture of ammunition and glassware and the introduction of printing processes, among others. But by far the greatest increase in worldwide distribution of Pb, and consequently of human exposure, arose as a result of two new applications in the 1920s. The first was the incorporation of Pb into gasoline in the United States as a highly eYcacious anti‐knock agent. This occurred despite the presence of warnings of potential health hazards, such as those issued in the Surgeon General’s report of 1926 (Rosner & Markowitz, 1985). Leaded gasoline was critical to the development and success of the automotive industry. Lead was also incorporated at about the same time into paint as a pigment, again resulting in broad distribution in the environment through the application of such paints in homes, buildings, and other structures. INTERNATIONAL REVIEW OF RESEARCH IN MENTAL RETARDATION, Vol. 30 0074-7750/06 $35.00

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It was almost 50 years later that recognition of the adverse health eVects resulting from Pb exposure reached a level of concern suYcient to provoke a phase‐out of Pb from gasoline (1973) and paint (1978) in the United States and the implementation of public screening programs for elevated Pb exposure. These actions were prompted largely by deaths resulting from acute lead poisoning in children. Over the period 1951 to 1953, for example, 94 pediatric deaths due to lead poisoning were documented in New York City, Cincinnati, St. Louis, and Baltimore. Similar cases were reported in other countries as well, with such fatalities typically occurring at blood lead (PbB) levels of 80 mg/dL and above. Moreover, contrary to existing beliefs at the time, reports began to emerge indicating that children who survived acute lead encephalopathy, rather than fully recovering, were left with residual and permanent sequelae such as mental retardation, recurrent seizures, cerebral palsy, and optic atrophy (Byers & Lord, 1943; Perlstein & Attala, 1966). These events made two facets of Pb toxicity evident. First, they demonstrated that the central nervous system was a critical target organ for Pb. Second, they made clear that children were particularly vulnerable to these eVects. Based on these findings, consequent eVorts aimed at understanding the health hazards associated with Pb in children focused predominantly on the central nervous system and associated behavioral manifestations, with two specific hazards serving as the focus. The first was whether chronic low‐level Pb exposure was reliably associated with diminutions of cognitive function, and, a second was the blood Pb (PbB) levels associated with such eVects, i.e., the nature of the corresponding dose–eVect relationship. The initial studies in children designed to address these questions were largely cross‐sectional in design and focused on the validity of an association between elevated PbB and decrements in IQ, with IQ generally measured using standardized psychometric tests. Even when considered in light of their limitations, these studies suggested that PbBs as low as 30 mg/ dL could be associated with detrimental eVects, a significant departure from the 80 mg/dL level. Subsequently, prospective cohort studies were initiated in the United States as well as in various other countries, which enrolled pregnant women and longitudinally tracked Pb exposure levels and outcomes in oVspring at various intervals thereafter (D. Bellinger et al., 1987, 1989; D. C. Bellinger et al., 1992; Dietrich et al., 1991, 1993b, 2001; Fulton et al., 1987; McMichael et al., 1988; Wasserman et al., 1997). As these studies proceeded, their collective findings provided accumulating and compelling evidence for an inverse relationship between elevated PbBs and IQ score, indeed, with similar magnitudes of eVects detected across divergent populations. Based on the outcome of these longitudinal studies, with their greater

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power to detect Pb eVects and to control significant potential confounding variables, the PbB level of concern for IQ reductions in children was lowered to 10 mg/dL by the CDC in 1991, where it has since remained. The World Health Organization set a similar level of concern. More recent studies have been directed to questions of whether a threshold PbB level exists for an association between Pb exposure and cognitive deficits, and to determining what specific behavioral processes aVected by Pb contribute to the decrements in cognition (D. Bellinger et al., 1987, 1989; D. C. Bellinger et al., 1992; Dietrich et al., 1991, 1993a, 2001; Fulton et al., 1987; McMichael et al., 1988; Wasserman et al., 1997). These newer eVorts provide evidence for cognitive deficits at PbB levels lower than 10 mg/dL in children and also demonstrate deficits in working memory and behavioral flexibility as additional components of the behavioral toxicity (Canfield et al., 2003, 2004). Paralleling these prospective cohort studies have been an extensive number of experimental studies aimed at further evaluating the behavioral and neurotoxicity arising from Pb exposure, and determining the mechanisms by which they occur. Importantly, both the nature of the observed eVects of Pb and the PbB levels at which eVects occur in experimental studies are remarkably similar to those reported for human populations (Cory‐Slechta, 1984, 1988, 1995a, 2003; Rice, 1996). In addition, the toxicokinetics of Pb shows significant similarities across species. This further enhances the credibility of findings from the experimental models.

II.

Pb EXPOSURE IN THE CONTEXT OF ENVIRONMENTALLY REALISTIC CONDITIONS

Both the human and experimental studies of Pb were critical for establishing an association between elevated Pb exposure and reduced cognitive function. They have also been informative with respect to neurobiological and behavioral mechanisms of action. However, the environmental realities of Pb exposure can be quite diVerent from the conditions under which it has been examined to date. First, environmental chemical exposures do not occur in isolation: human populations are exposed to mixtures of chemicals, the nature and levels of which may change dynamically across time. The resultant mixtures could modify the eVects of Pb in ways that are currently unknown, by adding or potentially even reversing its eVects. Second, Pb exposures occur in a context of many other potential host or extrinsic risk‐modifying factors, including genetic background, age, gender, developmental period of exposure, nutritional status, lifestyle, stress levels,

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and underlying disease state, among others. As with mixtures, these other variables may also modify the eVects of Pb exposure, by increasing or decreasing eVects on Pb toxicity. At the present time, almost nothing is known about the ability of such factors to modify Pb risk. In fact, our current understanding of the eVects of Pb on cognitive function, and virtually all other outcomes, is based almost exclusively on studies of its eVects in isolation. While many of the prospective cohort studies have had the potential to examine risk modification, such variables are generally controlled statistically, and thus potential risk modification is not generally evaluated (Burns et al., 1999; Dietrich et al., 1993a,b; Gomaa et al., 2002; Tong & Lu, 2001; Wasserman et al., 2000). This is often attributed to sample sizes deemed insuYcient to produce reliable interaction evaluation. In experimental studies, Pb is almost invariably studied in isolation from other potential risk modifiers, and most often in healthy young rodents that are almost exclusively males. Thus, the extent to which our current understanding of Pb realistically reflects its toxicity or adverse eVects on cognitive functions may not be accurate, particularly if additive, synergistic, or potentiated interactions occur with other chemical exposures or risk factors. This chapter describes our observations on one such new interaction, that of environmental stress with Pb, as determined in experimental models. It presents the rationale for hypothesizing such an interaction, followed by highlights of some current studies based on this hypothesis. Notably, this interaction of Pb with environmental stress resulted in a profile of eVects that diverges notably from those produced by Pb exposure alone. These interactions are relevant to understanding the role of Pb as a contributor to human disease and dysfunction, to our understanding of mechanisms of neurotoxicity and other target organ toxicities associated with Pb, and, moreover, may be particularly pertinent to criteria for screening programs for the prevention of adverse eVects of Pb.

III. RISK MODIFIERS FOR Pb NEUROTOXICITY: ENVIRONMENTAL STRESS AS A CASE STUDY A.

Elevated Lead Exposure Preferentially Impacts Low Socioeconomic Status Populations

After the removal of Pb from gasoline in the 1970s, PbB levels of the U.S. population declined markedly, as has been documented across successive years in the National Health and Nutrition Examination Surveys, a national representative survey of the civilian U.S. population (Brody et al., 1994; MahaVey et al., 1982; Pirkle et al., 1998). Indeed, the prevalence of elevated PbBs (
10 mg/dL) in children between 1 and 5 years of age has

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continued to decline from an estimated 88% as reported in the 1976–1980 NHANES II survey to values of 2.2% based on 1999–2000 estimates (Meyer et al., 2003). When considered in relation to various demographic factors, however, a diVerent story emerges, since levels of elevated PbB in children actually diVer markedly in relation to race/ethnicity, income level, and residence type. Based on NHANES III reports from 1994, the percentage of elevated PbB values in African‐American children, for example, at 22%, was five times higher than that of the general population, while the percentage of children with elevated PbB levels from low‐income families living in pre‐1946 homes, residences endemic to our inner cities, was 16%, almost four‐fold higher than in the overall population (Pirkle et al., 1998). Although the diVerences in percentages have since narrowed, the disparitites across racial/ethnic groups remain, with 2001 figures of 2% for white non‐Hispanic children, 9% for black non‐Hispanic (four‐fold higher), and 6% (almost three‐fold higher) for Hispanic children (Meyer et al., 2003). Figure 1 shows these diVerential levels of elevated PbBs in relation to race/ethnicity across various PbB clusterings based on 2001 United States data. Total numbers of black non‐ Hispanic and Hispanic children exceed those of white non‐Hispanics in every PbB category shown. As Fig. 1 shows, elevated Pb exposure has now become a demographically circumscribed health problem, one of particular concern for socioeconomically disadvantaged, medically underserved inner-city minority children who reside in old houses with Pb‐based paints. Indeed, PbB histories that include levels in excess of 40 mg/dL still occur in many low socioeconomic (SES)

FIG. 1. Numbers of children with elevated PbB levels (>10 mg/dL) in relation to racial and ethnic groups across various blood lead level (BLL) groupings based on 2001 U.S. data. Unknown category represents individuals who did not identify race/ethnicity. From Meyer et al. (2003).

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inner-city children. These are populations that lack the economic aZuence and social capital to successfully lobby for Pb abatement programs. Because of the vicious cycle of poverty, the parents and grandparents of these children often lived in these same neighborhoods; thus they too experienced the highest Pb exposures in the country, making the cycle of poverty coincide with the cycle of Pb exposure and maintaining Pb exposure as an intergenerational public health problem in these populations. Even as this problem endures, at least one study suggests that not only do Pb eVects in children occur even below 10 mg/dL and that there may be no threshold for deficits in IQ, but that these problems can be of even greater magnitude than eVects occurring at higher PbBs (Canfield et al., 2003). B.

Low SES Is Already a Known Risk Factor for Various Diseases and Behavioral Dysfunctions

What is particularly notable about the current demographics of elevated Pb exposure is that low SES is already a well‐documented risk factor for adverse health impacts and behavioral and neurological dysfunctions, even after other pertinent covariates have been statistically controlled. In adults, for example, the risk of mortality, the prevalence of numerous diseases, increased blood pressure, and the prevalence of schizophrenia (Dohrenwend, 1990) and depression (Hirschfeld & Cross, 1982) have all been shown to be inversely related to employment grade, occupational status, income, and years of education (Adelstein, 1980; Dyer et al., 1976; Marmot et al., 1984; Pappas et al., 1993; Pincus et al., 1987). In addition to Pb poisoning, links between low SES and greater health risk in children have been reported for vision problems, otitis media, hearing loss, cytomegalic inclusion disease, and iron deficiency anemia (Starfield, 1982). Lower SES children also have higher levels of mental retardation, learning disabilities, emotional and behavioral problems, and deficits in language, memory, and attentional capacities (Anderson & Armstead, 1995; Ardila & Rosselli, 1995). Low SES has been described as the underlying basis of academic risk in minority children (Arnold & DoctoroV, 2003). C.

Links Between Low SES, Stress, and Cortisol

1. ELEVATED STRESS IS POSTULATED TO UNDERLIE THE INCREASED INCIDENCE OF DISEASE AND DYSFUNCTION ASSOCIATED WITH LOW SES

One hypothesis that has been proposed to account for the association between lower SES and the increased prevalence of adverse health outcomes and behavioral dysfunctions is a greater exposure of these populations to

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environmental and psychosocial stressors (Lupien et al., 2001). Stress has been defined as life events that require adaptation (Selye, 1950) or a state in which the individual perceives that the demands of the environment exceed the ability to cope (Adler et al., 1994). Lower SES individuals report greater exposures to stressful life events as well as greater impacts of these stresses on their lives (Baum et al., 1999; Dohrenwend, 1973; Marmot & Wilkinson, 1999, 2001; Taylor & Seeman, 1999). The physical and social environments associated with low SES, characterized by deteriorating housing, high crime and violence rates, increased drug use, fewer two‐parent families, and chronic unemployment, no doubt contribute to this. Low SES populations are subjected to a greater frequency of threatening and uncontrollable events in life, higher levels of environmental hazards and violence, and lower levels of family stability (Bradley & Corwyn, 2002). The corresponding lower income levels may mean inability to consistently aVord required expenses, such as home mortgage or rent payment, food, day care, transportation, or health insurance, contributing to the ongoing cycle of poverty. There is a chronic strain associated with unstable employment situations and persistent economic hardships. The hypothesis that stress contributes to the greater prevalence of disease and dysfunction in low SES populations is based on numerous studies showing that, like low SES, higher levels of stress are associated with poorer health outcomes, including hypertension, cardiovascular disease and death, the extent of chronic illness, and altered immune function, among others (Brosschot et al., 1992; Calabrese et al., 1987; Kennedy et al., 1988; Rahe & Lind, 1971; Wyler et al., 1971). Increased stress adversely aVects mood and cognitive function (Lupien & McEwen, 1997) and correlates with measures of anxiety and depression (Vinokur & Selzer, 1975). Increased stress is also associated with poorer academic achievement (Harris, 1972) and impairments in cognitive functions, including attention and memory functions (Mueller, 1976). Stress may actually exert its most detrimental eVects in children and have eVects that are particularly long‐lived (Cohen et al., 1973; Tennes & Kreye, 1985). 2. STRESS STIMULI ACTIVATE THE HYPOTHALAMIC– PITUITARY–ADRENAL AXIS

Stressful stimuli are known to activate adrenal cortical glucocorticoids, an eVect considered an adaptive response to stress, as shown schematically in Fig. 2 (left side) (Vazquez, 1998). Physiological or psychological stressors cause release of corticotropin‐releasing hormone (CRH) and arginine vasopression (AVP) from the periventricular nucleus (PVN) of the hypothalamus. This stimulates release by the adrenal pituitary of adrenocorticotropin (ACTH), which then acts on adrenal cortex receptors to elevate plasma glucocorticoids that subsequently activate glucocorticoid receptors,

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FIG. 2. Schematic depicting the influence of low SES producing stress that then acts on the hypothalamic–pituitary–adrenal (HPA) axis (left side). Physiological or psychological stressors cause release of corticotropin‐releasing hormone (CRH) and arginine vasopression (AVP) from the periventricular nucleus (PVN) of the hypothalamus. This stimulates release by the adrenal pituitary of adrenocorticotropin (ACTH), which then acts on adrenal cortex receptors to elevate plasma glucocorticoids that subsequently activate glucocorticoid receptors, including those in brain. Feedback loops to pituitary, hypothalamus, and hippocampus regulate the secretion of corticosteroids. Hippocampal regulation of the HPA axis is particularly significant in relation to cognitive outcome. The HPA axis interacts extensively with the mesocorticolimbic dopamine system (right outset) of the brain. In this system, the nucleus accumbens receives dopaminergic input from neurons in the ventral tegmental area as well as glutamatergic projections, both NMDA and AMPA/kainite‐mediated, from the septo‐hippocampal system and from the prefrontal cortex. The prefrontal cortex also receives dopaminergic input from the ventral tegmental area and glutamatergically mediated information from the septo‐hippocampal system. Notably, both the hippocampus and prefrontal cortex have been reported to play an inhibitory role over HPA axis function (Diorio et al., 1993; Figueiredo et al., 2003; Jacobson & Sapolsky, 1991; Sullivan & Gratton, 1999). Increased stress resulting in elevated cortisol levels can impact cognitive function through interactions of the HPA axis with the mesocorticolimbic system. As indicated, Pb exposure also impacts the mesocorticolimbic systems of the brain as well as HPA axis function, as illustrated here.

including those in the brain. (Cortisol is the main glucocorticoid of human and nonhuman primates, whereas corticosterone is the main glucocorticoid of the rat.) Feedback loops to pituitary, hypothalamus, and hippocampus regulate the secretion of glucocorticoids. Hippocampal

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regulation of the HPA axis is particularly significant, given its critical role in numerous cognitive functions. In fact, hippocampectomy or transaction of the fornex is known to elevate basal HPA activity (Jacobson & Sapolsky, 1991). Glucocorticoids act via two types of receptors: Type I, or mineralocorticoid (MR) receptors, and Type II, or glucocorticoid (GR) receptors. In brain, MR receptors are located primarily in limbic regions, including the septo‐hippocampal system and amygdala. In contrast, GR receptors are distributed throughout brain. GR receptors in particular are activated by higher levels of corticosterone such as those associated with stress (Joels & de Kloet, 1994), while low basal corticosterone levels activate MR receptors. 3. LOW SES IS ASSOCIATED WITH INCREASED CORTISOL

The higher levels of stress in low SES populations are thought to produce chronic elevation of associated stress hormones such as glucocorticoid, a presumption supported by an increasing number of studies. Low SES children from 6 to 10 years of age living in Montreal had higher morning salivary cortisol levels than did children from more aZuent families (Lupien et al., 2001). In another study, elevated salivary cortisol levels in children were associated with lower SES as well as the mother’s extent of depressive symptomatology, eVects that emerged as early as 6 years of age (Lupien et al., 2000). In adults, job strain and the expression of anger were associated with elevation of free cortisol early in the working day (Steptoe et al., 2000). A 2002 study reported that family SES was inversely related to initial cortisol levels in a population of young adult African‐American males (Kapuku et al., 2002). 4. ELEVATED GLUCOCORTICOIDS ARE ASSOCIATED WITH INCREASED DISEASE AND DYSFUNCTION

Again, in concert with stress as the potential mediator of increased disease and dysfunction in low SES populations, increasing evidence also documents a relationship between chronic elevation of glucocorticoids and disease and dysfunction, with sustained elevations of cortisol reported to increase resistance to insulin, and to cause hypertension, hypercholesterolemia, arteriosclerosis, and immunosuppression (Munck et al., 1984). In a review, Kristenson and colleagues (Kristenson et al., 2004) describe the fact that in several studies, an SES gradient can be demonstrated for all causes of mortality and for diseases that include coronary heart disease, diabetes, gastrointestinal disease, respiratory diseases, arthritis, and adverse birth outcomes.

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A.

BOTH Pb AND STRESS CAN PRODUCE COGNITIVE DEFICITS

Stress During Development, Sustained Cognitive Deficits, and Alterations in Structure and Function of the Brain and the HPA Axis

Particularly important in the current context is that circulating glucocorticoids can cross the blood–brain barrier to act on GR and MR receptor sites in the central nervous system. In studies in humans, various measures of stress/anxiety in mid‐pregnancy were inversely related to developmental indices in oVspring at 8 months of age as measured on the Bayley Scales. Moreover, morning cortisol levels of mothers were also inversely related to developmental outcomes at both 3 and 8 months of age (Buitelaar et al., 2003). O’Connor and colleagues (O’Connor et al., 2003) report an association between higher levels of self‐assessed maternal anxiety and depression and higher rates of behavioral and emotional problems (conduct problems, emotional problems, hyperactivity/inattention) in their children at 81 months of age after controlling for several potentially confounding variables. EVect levels were generally comparable in males and females, and similar eVects had been described in this cohort at 47 months of age, indicating their persistence. Using a naturally stressed cohort of mothers in Quebec who had been exposed to an ice storm, it was reported that the level of prenatal anxiety could account for 11 to 12% of the variance in Bayley Mental Development Index (MDI) scores and productive language abilities, and for 17% of the variance in receptive language abilities in 2‐year‐olds (Laplante et al., 2004). EVects from both nonhuman primate and rodent studies provide evidence that the nature of stress eVects on cognition depends upon the parameters of stress exposure, oVspring gender, and the behavioral paradigm used (i.e., stress eVects reflect risk modification), and that significant individual diVerences in responsiveness occur as well. Prenatal stress consisting of exposure of mothers to unpredictable noise during mid‐ to late gestation, for example, altered social behaviors of juvenile monkeys (18‐month‐old oVspring), elevated basal cortisol and ACTH hormone levels, and produced higher levels of stress‐induced ACTH (Clarke & Schneider, 1993; Clarke et al., 1994; Coe et al., 2003). OVspring of monkeys that were subjected to noise stressors during diVerent periods of gestation were reported to exhibit reduced attention span and neuromotor capabilities during the first month of life (Schneider et al., 2002). Aspects of both learning and memory were evaluated in these oVspring at 32 to 34 months of age using a nonmatching to sample memory task. In this case, monkeys of prenatally

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stressed mothers required the same number of trials to criterion as their nonstressed counterparts, and exhibited no alterations in accuracy across diVerent delay values of up to 120 s, suggesting no residual deficits in either acquisition or memory (Schneider et al., 2001). However, even at 120 s delay values, these monkeys maintained accuracy levels ranging from approximately 83 to 86%, i.e., they exhibited minimal reductions in memory. It would be important to determine whether more challenging memory conditions, where delays suYcient to reduce accuracy levels to chance, for example, would reveal memory deficits. Nevertheless, this cohort of primates did sustain excess levels of metabolites of norepinephrine and dopamine in cerebrospinal fluid consistent with residual changes in brain catecholamine function. In addition, several studies have shown decreases in hippocampal volume of 10 to 12% and reductions in neurogenesis in male and female oVspring of monkeys that had been exposed to acoustic startle either in mid‐ or late gestation for 25% of the gestational period. Basal and stress‐induced cortisol levels were also elevated in stressed oVspring (Coe et al., 2003). Magnetic resonance imaging scans of corpus callosum revealed decreases in volume in male stressed oVspring and increases in females, relative to controls (Coe et al., 2002). Studies in rodents provide evidence for cognitive dysfunction, but also of improved performance, as well as individual diVerences in stress responsivity, again dependent upon experimental parameters. For example, female oVspring whose dams were exposed to corticosterone in drinking water throughout gestation and lactation exhibited improvements in learning in a water maze paradigm at 21, 30, and 90 days of age, as well as in active avoidance measured at 15 months of age (Catalani et al., 2002). In another study, gestational restraint stress had no eVect on object recognition memory and improved performance of female oVspring in a radial arm maze, eliminating the gender diVerences seen in controls (Bowman et al., 2004). In contrast to those findings, however, another study reported that prenatal stress retarded the acquisition of reversal learning (Weller et al., 1988) and of spatial learning in a water maze (Lemaire et al., 2000; Nishio et al., 2001; Szuran et al., 2000). One of the more complete studies (Vallee et al., 1999) demonstrated increased numbers of errors in a radial arm maze at 22 months of age in male oVspring from dams subjected to restraint stress during the final week of gestation. In addition, stressed oVspring showed an accelerated age‐related decline in the HPA axis response to stress and, at least at one time point of measurement, elevated basal corticosterone levels. Encompassing both improvements and adverse eVects, de Kloet and Oitzl (de Kloet & Oitzl, 2003) observed that maternal separation stress in rats significantly increased, relative to controls, the percentage of rats

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exhibiting impaired performance on a water maze learning task, but also the percentage exhibiting no impairment, thus moving performers toward both extremes of the distribution. Both outcomes resulted from a decrease in the number of partially impaired performers. How this related specifically to gender was not described. Such findings underscore the widely reported individual diVerences in stress responsivity and recovery that, no doubt, also contribute to the inconsistencies across studies (Dellu et al., 1996; Garcia & Armario, 2001; Irwin et al., 1989; Kabbaj et al., 2000; Liu et al., 1997; Tomie et al., 1987). B.

Pb Exposure and Deficits in Cognitive Function

Like low SES, and in some cases stress, elevated PbB levels in children have been associated with adverse cognitive eVects in several prospective epidemiological studies based on psychometric measures of intelligence (Needleman, 1993; Schwartz, 1994; S. Tong et al., 1996), with reductions in IQ scores in response to increasing PbBs. Similar eVects have been reported in cohorts from several diVerent countries and thus diVerent environmental conditions, including Boston and Cleveland in the United States, Australia, Scotland, and Yugoslavia (D. Bellinger et al., 1987, 1989; D. C. Bellinger et al., 1992; Dietrich et al., 1991, 1993a, 2001; Fulton et al., 1987; McMichael et al., 1988; Wasserman et al., 1997), indicating the generality of the eVects, findings that ultimately served as the basis for the designation of a level of 10 mg/dL of Pb in blood as a level of concern. Later studies suggest not only that Pb‐induced reductions in IQ may occur at PbBs even below 10 mg/dL (Canfield et al., 2003), but that the nature of the dose–eVect curve is not linear, since the magnitude of the deficit at PbBs <10 mg/dL actually exceeded that occurring above 10 mg/dL. Moreover, in examining children whose lifetime average PbB was 7.2 mg/dL (range: 0–20 mg/dL), deficits in tests of spatial working, spatial memory span, shifting behavior, and behavioral flexibility were observed using the Cambridge Neuropsychological Testing Automated Battery, even after controlling for IQ test scores (Canfield et al., 2004). Such findings have correspondences in learning impairments in experimental animal models (D. A. Cory‐Slechta, 1995b, 1997) that also demonstrate the selectivity of Pb eVects on learning (Cohn et al., 1993). One such example utilized a multiple schedule of repeated learning (RL) and performance (P), a behavioral paradigm in which RL and performance tasks (components) alternated across the course of an experimental session, with diVerent stimulus conditions signaling which component is currently in eVect. The RL component requires completion of a sequence of responses for reinforcement, with the correct sequence changing in each successive test session, allowing learning to be measured repeatedly across sessions. The P

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component also requires completion of a sequence of responses for reward, but the specific sequence remains constant across sessions. Both the RL and the P components require intact motor and sensory capabilities, as well as appropriately motivated subjects, but learning per se is only required during the RL component. Figure 3 contrasts behavior of a control (top) with a chronic Pb‐exposed (from weaning) rat (bottom) (Cohn et al., 1993). In each set, correct responses cumulate vertically in the top tracing, time is represented horizontally, and pips indicate reinforcement delivery, while the bottom tracing shows the concurrent errors that occurred. Control performance was typically characterized by a relatively high level of accuracy during the first P component (P1), a steady rate of food rewards and relatively few errors. The transition to the RL component (RL1) is accompanied by increased errors and a decline in the number of food rewards earned, as the correct sequence

FIG. 3. Cumulative records of performance on a multiple schedule of repeated learning and performance over the course of a behavioral session from left to right in a rat exposed chronically to distilled water (top record) vs 250 ppm lead acetate drinking solutions (bottom). The multiple schedule involved a repeated learning (RL) component which required learning a new 3‐response sequence during each behavioral session. These alternated with a performance (P) component in which the 3‐response sequence remained constant across sessions. The top tracing of each record shows correct responses which cumulate vertically. Each pip depicts the delivery of a reinforcer for correctly completing the sequence of 3 responses required for reward. The pen reset to the baseline with each transition between the P and RL components. The bottom tracing shows errors that occurred during the components. The lead‐treated rat earned virtually no food deliveries, i.e., completed no correct sequences, during the RL components of the session despite normal accuracy levels in the P component under conditions where no learning was required (P component). From Weiss & Cory‐Slechta (1994).

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of responses for this specific session is learned. Behavior during the second P component (P2) is again composed of a steady rate of food rewards and relatively few errors. The second presentation of the RL component (RL2) is marked by both a gradual increase in the rate at which food rewards are earned and a decrease in the number of errors relative to levels in RL1, consistent with a gradual learning of the correct sequence for this session. A selective eVect of Pb on learning processes per se, as distinct from nonspecific behavioral changes, is clear in the bottom tracings, with behavior during both presentations of the P component unimpaired, whereas no evidence of learning during either presentation of the RL component was observed, in that virtually no food deliveries were obtained. This was not due to a reduction in rates of responding, since a very high and sustained rate of errors occurred throughout RL1 and RL2.

V.

DO Pb EXPOSURE AND STRESS INTERACT?

As has been noted, the greatest elevations in PbB levels now occur in low SES populations, the same populations hypothesized to experience the highest levels of environmental stress and presumed chronic elevations of glucocorticoids. As has been described, both Pb and low SES stress are also associated with similar behavioral deficits in terms of cognitive dysfunction. This raises the obvious question as to whether Pb and stress interact. Specifically, does Pb alter the stress response, and thereby have ramifications for all systems that interact with the HPA axis? Or, conversely, does environmental stress alter the impact of Pb exposure, including its cognitive eVects, those of greatest concern in children? Either outcome could change the profile of alterations observed for each risk factor, and, moreover, such interactions could also have significant implications for human health risk assessment and for remediation and screening programs. A.

The Mesocorticolimbic Dopamine System as a Common Substrate for Pb and Stress

Biological plausibility for an interaction between Pb and stress derives from the fact that both act on the mesocorticolimbic dopamine system of the brain. In the mesocorticolimbic system (Fig. 2, right outset), the nucleus accumbens (NAC) receives dopaminergic input from neurons in the ventral tegmental area as well as glutamatergic projections, both NMDA and AMPA/kainite‐mediated, from the septo‐hippocampal system and from the prefrontal cortex (PFC). The PFC also receives dopaminergic input

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from the ventral tegmental area and glutamatergically mediated information from the septo‐hippocampal system. Notably, both hippocampus and PFC have been reported to play an inhibitory role over HPA axis function (Diorio et al., 1993; Figueiredo et al., 2003; Jacobson & Sapolsky, 1991; Sullivan & Gratton, 1999). The mesocorticolimbic dopamine/glutamate system plays a key role in the mediation of complex cognitive/executive functions, including learning, memory, and attention (Goethals et al., 2004; Kane & Engle, 2002; Tanji & Hoshi, 2001). Deficits in mesocorticolimbic system function are also implicated in the pathophysiology of schizophrenia (Coyle, 1996; Pennartz et al., 1994; Robbins et al., 1989), a neurological dysfunction also more prevalent with low SES and with stress and linked to Pb exposure (M. G. A. Opler et al., 2004). That cognitive function depends critically upon interactions of the stress hormone corticosterone with mesocorticolimbic dopamine systems was demonstrated in a study in rats showing that adrenalectomy disrupted a mesocorticolimbic‐associated working memory task in rats, i.e., a delayed nonmatching‐to‐sample, with this deficit being corrected by systemic replacement of corticosterone (Mizoguchi et al., 2004). Moreover, interactions occur between the HPA axis and mesocorticolimbic dopamine systems at multiple levels. Interactions related to NAC dopaminergic function (Barrot et al., 2000; Piazza et al., 1996) have been shown that have significant behavioral impacts for the mediation of rewarding properties of drugs of abuse and other stimuli (Marinelli & Piazza, 2002). Corticosteroids/stress interactions with glutamate systems in PFC (Bagley & Moghaddam, 1997; Diorio et al., 1993; Jackson & Moghaddam, 2001; Lowy et al., 1993; Moghaddam, 1993, 2002; Meaney & Aitken, 1985) suggest that medial PFC is a target site for negative feedback eVects of glucocorticoids on stress‐induced HPA axis activity. The interactions of the HPA axis with the hippocampus may be particularly significant, given the interactions between corticosteroids and excitatory amino acids in this region and their consequences for learning and memory (Barnes et al., 1977; de Kloet et al., 1998; Dellu et al., 1994; Eichenbaum, 1997; Kerr et al., 1994; Lupien et al., 2002; McEwen, 2001; McEwen & Sapolsky, 1995; McEwen et al., 1968; Monk & Nelson, 2002; Palvides et al., 1996; Sapolsky, 1992; Sapolsky et al., 1985, 1986; Watanabe et al., 1992, 1995). Pb exposure also impacts mesocorticolimbic systems. Indeed, this system appears to be a preferential target of Pb exposures initiated postweaning, in contrast to the nigrostriatal system, the other major long‐length dopamine system of the brain. In rats, PbB levels averaging 16 to 28 mg/dL were associated with decreased levels of D2 dopamine receptor and dopamine transporter binding in NAC after only 2 weeks of Pb exposure, a time

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when detection of increases in brain Pb levels would be diYcult, and these reductions persisted through the 12‐month Pb exposure protocol (Pokora et al., 1996). In addition, postweaning Pb exposures increase dopamine release in NAC (Zuch et al., 1998). Moreover, at least for postweaning Pb exposure, alterations in mesocorticolimbic dopamine/glutamate function appear to mediate aspects of the associated behavioral toxicity that occurs with this Pb exposure regimen (Bauter et al., 2002; D. Cory‐Slechta et al., 2000, 2002; D. A. Cory‐Slechta et al., 1997c, 1998, 2000) as embodied by changes in Fixed Interval (FI) schedule‐controlled behavior, and in learning, measured using a multiple schedule of repeated learning and performance. Both FI performance and learning have been shown to be sensitive to Pb exposure in response to a range of diVerent exposure regimens, and thus are not restricted to one developmental period of exposure (D. A. Cory‐Slechta, 1984; Rice, 1992, 1993). Microinjection of the irreversible dopamine antagonist EEDQ into NAC, but not into dorsal striatum (terminal projection region for neurons of the nigrostriatal dopamine system), of normal rats markedly reduced rates of responding on the FI schedule, which then recovered over the next 2 to 3 days, consistent with the turnover of DA receptor proteins. These findings confirmed the importance of the nucleus accumbens to mediation of FI performance under normal, non‐Pb conditions. Further, injection of intermediate doses of dopamine itself into NAC increased FI response rates, as does Pb. Similarly, injection of the N‐methyl‐D‐aspartate glutamatergic antagonist MK‐801 into NAC also mimicked the eVect of postweaning Pb exposure on FI performance (D. Cory‐Slechta et al., 2000). Thus, increased dopamine and blockade of NMDA‐mediated glutamate function in NAC appear to be mechanisms of Pb‐induced increases in FI response rates, consistent with the maintenance of dopamine/glutamate balance for normal nucleus accumbens function. With respect to Pb eVects on the multiple RL and P baseline, we have shown that infusion of MK‐801 into NAC, i.e., blockade of NMDA‐mediated glutamatergic input into NAC, mimics the pattern of Pb eVects on this paradigm (Bauter et al., 2003), i.e., MK‐801 infusions selectively decrease accuracy in the RL component, and do so by decreasing perseverative errors, with no eVects on accuracy in the P component, a pattern of eVects identical to that seen with Pb on this baseline (see Fig. 3) (Cohn et al., 1993; D. A. Cory‐Slechta et al., 1997a, 1999). Other Pb exposure regimens also impact the mesocorticolimbic dopamine system of the brain. Exposures beginning at parturition caused an impairment of receptor‐mediated regulation of dopamine synthesis in NAC but not in dorsal striatum, and decreased concentrations of dopamine metabolites in NAC and ventral tegmental area (Lasley & Lane, 1988).

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Pb exposures beginning at 2 to 6 days of age augmented levels of NAC dopamine release in response to KCl perfusion in microdialysis studies (Devoto et al., 2001). Pb exposure has also been repeatedly shown to adversely impact glutamatergic systems, including those of hippocampal structures. Exposures beginning at gestational day 15 to 16 decreased hippocampal glutamate and GABA release, resulting in a U‐shaped dose–eVect function (Lasley & Gilbert, 2002). Pb exposures beginning at conception, from weaning on, or beginning at conception and ending at weaning, all altered hippocampal glutamate release (Lasley et al., 1999). Furthermore, glutamatergic NMDA receptors have repeatedly been described to change in response to Pb exposures across multiple brain regions (D. A. Cory‐Slechta et al., 1997b; Lasley et al., 2001; Ma et al., 1998; Nihei et al., 2000; Zhang et al., 2002).

VI.

A.

EXPERIMENTS ADDRESSING Pb–STRESS INTERACTIONS

Experimental Design and Methods

Given the demographic overlap of elevated Pb burden and low SES/high stress, combined with correspondences in their sites of actions in the central nervous system and associated behavioral profiles, the hypothesis that these risk factors would interact served as the basis for experiments in our laboratory. Two other prior brief reports were supportive. In one, oVspring of both genders exposed to Pb (0.3% Pb acetate) through lactating dams exhibited increased corticosterone levels at postnatal day 30 (Yu et al., 1996). In another, exposure to 220 ppm Pb during gestation and lactation increased basal corticosterone levels in 35‐day‐old males and exacerbated the eVects of a mild stressor (intraperitoneal injection) with respect to corticosterone and behavioral responses (Virgolini et al., 1999, 2004). One other consideration that contributed to the rationale was the potential consequences when multiple risk factors target a common system/network of the brain. The brain may readily be able to compensate for the eVects of an individual chemical or risk factor itself acting on a particular target system of the brain. However, when multiple target or functional sites within that particular system are attacked by diVerent mechanisms (Pb in conjunction with other neurotoxicants and/or extrinsic or intrinsic host risk factors), the system may no longer be able to homeostatically re‐regulate itself, thereby leading to sustained or cumulative damage. Figure 4 provides a hypothetical example featuring a dopamine terminal. Four concurrent insults are portrayed. While all four target the dopamine terminal, they do

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FIG. 4. Schematic depicting the multiple‐hit hypothesis as applied to a dopamine terminal within the central nervous system. Four concurrent insults are depicted that occur at diVerent target sites of the dopamine terminal: Insult A aVecting the vesicular transporter, Insult B aVecting the metabolism of tyrosine to DOPA, Insult C breakdown of DOPAC, and Insult D aVecting the DA transporter. The multiple‐hit hypothesis here presumes that the brain may readily be able to compensate for the eVects of an individual chemical itself acting on a particular target system of the brain. However, when multiple target or functional sites within that particular system are attacked by diVerent mechanisms (multiple chemical exposures or chemical exposures combined with other risk factors), the system may no longer be able to homeostatically re‐regulate itself, thereby leading to sustained or cumulative damage. From Cory‐Slechta (2005).

so by diVerent mechanisms, i.e., at diVerent sites of the system. Here, for example, insult A targets the vesicular monoamine transporter, insult B attacks the enzyme converting tyrosine to DOPA, insult C the metabolism of DOPAC to HVA, and insult D the dopamine transporter that takes dopamine back up from the synaptic cleft post‐release. This multiplicity of insults occurring concurrently at multiple sites within the system may constrict the range and flexibility of compensatory mechanisms, thereby compromising the integrity of the system. As a consequence, multiple risk factors acting simultaneously could have eVects that are more robust, more rapid in onset, or even diVer in character from eVects produced by a single risk factor.

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One issue given consideration in the experimental design was the relevant Pb and stress exposure models to examine. Aspects of the current demographics of human Pb exposure are important to consider. Pb exposure, like poverty, now constitutes a cycle, with low SES mothers experiencing both high levels of stress and also having the highest Pb exposure levels. The Pb body burden accumulated over life, as well as the impacts of stress on the mother, including that experienced during gestation, are passed on to her children. Thereafter, these children, who are highly likely to remain in the cycle of poverty, will continue to be exposed to Pb over their lifetimes as well, and will also begin to experience similar environmental stresses associated with low SES conditions. While a complete understanding of critical exposure periods for behavioral and neurochemical alterations has not yet been fully defined, it is clear that the neurotoxic eVects of Pb are by no means limited to exposures beginning early in development. Our own studies demonstrate enduring behavioral and neurochemical eVects of Pb, even when exposure of rats is initiated only after weaning (Cohn et al., 1993; D. A. Cory‐Slechta, 1990; D. A. Cory‐Slechta & Weiss, 1985). Similarly, susceptibility to the impacts of stress appears to have few restrictions, but like Pb, the nature of the outcome varies markedly depending upon gender, the timing of stress, its predictability, and its duration. Consequently, there are numerous experimental Pb exposure and stress models that can be examined with respect to potential interactions of these risk factors. Our initial study focused primarily on maternal contributions. It administered Pb through gestation and lactation combined with (i) maternal stress alone, and with (ii) maternal stress followed by adult oVspring stress. Moreover, Pb exposure was initiated 2 months prior to breeding to ensure that an elevated Pb body burden, as in the human environment, was sustained prior to pregnancy (D. A. Cory‐Slechta et al., 1987). This design allowed a determination of the contribution of maternal Pb alone and of maternal stress as distinct from continuous stress that occurs across the lifetime, and also provided the ability to assess potentially dormant eVects associated with the maternal contributions. In this study, rat dams were exposed to 0 or 150 ppm Pb acetate in drinking water for 2 months prior to breeding and throughout lactation. A subset of dams were subjected to 45‐min of restraint stress 3 times daily on gestational days 16 and 17. This resulted in oVspring derived from four diVerent treatment conditions: (1) 0/NS, no stress, no Pb exposure; (2) 0/S, maternal stress, no Pb exposure; (3) 150/NS, maternal Pb exposure, no stress; and (4) 150/S, maternal Pb exposure and maternal stress. OVspring were weaned at 21 days of age, and, using 1 male or female per litter to preclude litter‐specific eVects, behavioral and neurochemical

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endpoints known to be altered by Pb exposure were evaluated at diVerent time points thereafter. Behavioral assessment included performance on a Fixed Interval (FI) 1-min schedule of reinforcement that began at 50 to 60 days of age. This baseline has consistently been demonstrated to be sensitive to Pb across various exposure periods and species (D. A. Cory‐Slechta, 1984, 1992; Rice, 1988, 1992). FI performance was thus deemed likely to be sensitive to modulation in this study, since we had never previously evaluated maternal‐only Pb exposures for behavioral toxicity. FI performance in children has been reported to be a surrogate for impulsivity (Darcheville et al., 1992, 1993), one of the clinical symptom domains of attention deficit disorder, with high FI response rates correlating with impulsive choices and low rates with self‐controlled choices in behavioral paradigms that oVer small rewards after short delays (impulsive) vs larger rewards after longer delays (self‐controlled choice). On the FI 1‐min schedule of food reinforcement, a 45 mg food pellet delivery followed the first lever press response occurring at least 1 min after the preceding food delivery, with responses occurring during the 1‐min interval itself having no programmed consequences. Reinforcement delivery also initiated the next 1‐min FI. Sessions ended following the completion of the 1‐min interval in progress 30 min after the session began, or after a total of 32 min, whichever occurred first. Behavioral sessions were carried out 5 to 6 days per week. Standard performance measures were computed from each session for every animal: (1) overall response rate, or total number of responses divided by total session time; (2) mean post‐reinforcement pause time (PRP), i.e., the mean time to the occurrence of the first response in an interval; and (3) mean running rate, i.e., the rate of responding calculated with the PRP subtracted out. Once stable FI performance was established, tail blood was collected immediately after a behavioral session for determination of basal corticosterone levels. During the course of FI testing, the eVects of varied stress exposures imposed prior to behavioral test sessions were evaluated, as will be described. Following that FI session, stress‐induced changes in corticosterone levels were also evaluated in oVspring. One week after the termination of behavioral testing, regional levels of brain catecholamines were determined. Highlights of findings from these experiments are described in the following text to illustrate how Pb exposure can modify stress‐related responses, how stress can modulate the eVects of Pb exposure, as well as examples of potentiated eVects of combined Pb þ stress, i.e., eVects occurring in the absence of an eVect of either variable alone. Also evident is the complexity of risk modification, since the nature of the eVects of Pb alone and stress alone and of Pb þ stress exhibited very diVerent profiles in relation to gender,

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brain region, time of measurement, specific neurotransmitter, etc. Pb eVects modified by stress in these studies are indicated by the absence of a comparable eVect in the Pb þ stress group. Similarly, stress eVects modified by Pb are indicated by the absence of a corresponding eVect of stress in the Pb þ stress group. Potentiated eVects of Pb þ stress occur when no eVects of Pb alone or stress alone occur, but an eVect is seen with the combination.

B.

Outcomes

1. GENERAL EFFECTS IN OFFSPRING

Body weights of pups at weaning, at 2 months of age, and over the course of behavioral testing revealed highly significant gender diVerences, but no eVects of Pb or stress or any interaction between them. No diVerences in litter size, weights, or gestational length occurred. HPLC determinations of brain catecholamines were determined from a subset of oVspring at postnatal day 21 and described elsewhere and also in later adulthood in additional subsets of rats (D. A. Cory‐Slechta et al., 2004). 2. HIGHLIGHTS OF EFFECTS IN MALE OFFSPRING

a. Basal Corticosterone Maternal Pb exposure alone significantly increased plasma basal corticosterone levels in male oVspring, as shown in Fig. 5 (main eVect of Pb, p ¼ 0.01). Since these eVects were measured prior to any adult stresses, and well into adulthood, with Pb exposure having ended at 21 days of age, they must be viewed as permanent alterations. The Pb‐induced increases were of comparable magnitude in stressed vs nonstressed groups, with an approximate doubling of values in each Pb group relative to corresponding control. These findings have significant implications with respect to mechanisms by which Pb exposure could act, on both the nervous system and other target organs, given the adverse health eVects associated with elevated corticosterone and its impact on catecholamine systems that not only mediate complex cognitive function, but that also play a key role in drug abuse and dysfunctions which are presumed to underlie schizophrenia. b. Behavior Maternal Pb exposure alone (150/NS) initially (i.e., weeks 1–8, prior to the first adult stress challenge) markedly decreased FI overall response rates, as shown in Fig. 6, relative to all other conditions, whereas stress alone (0/S) had no eVects. Initially, Pb þ stress (150/S) produced rates comparable to those of controls (0/NS), but by week 5, response rates in this group appeared to plateau, while those of control and stress alone groups continued to increase (Pb by stress, p < 0.0001). (Decreases seen at week 9 reflect the eVect of pre‐FI session restraint stress; its impact was blunted by

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FIG. 5. Group mean ± SE basal corticosterone levels of male oVspring measured at 6 months of age in groups subjected to maternal exposure to 0 or 150 ppm as indicated, with or without combined maternal stress as indicated (NS ¼ no stress; S ¼ stress). Outcomes of post hoc tests as indicated following the repeated measures analysis of variance. From D. A. Cory‐Slechta et al. (2004).

Pb.) By week 11, rates of the maternal Pb group (150/NS) reached levels comparable to those of the Pb þ stress group (150/S), and by week 14, no significant group diVerences were any longer detectable. These findings demonstrate a Pb by stress interaction that is dynamic across time. c. Neurotransmitter Changes Permanent changes in brain catecholamine systems were evident in male oVspring, as highlighted here for NAC in Fig. 7 (n ¼ 9–13/gp). They reflected regional diVerences in response to Pb alone, to stress alone, and to Pb þ stress. DiVerential changes in the Pb þ stress group relative to Pb or stress alone were found for dopamine and its metabolite DOPAC and for serotonin (5‐HT). In the case of dopamine, for example, stress alone (0/S) decreased levels relative to control (0/NS), whereas no corresponding eVect was seen with Pb þ stress (150/S), indicating that Pb modified this eVect of stress (Pb by stress interaction). Similarly, Pb alone (150/NS) had no eVect and stress alone (0/S) significantly reduced levels of DOPAC, whereas Pb þ stress (150/S) increased levels relative to control (0/NS) and stress alone (0/S), again consistent with a modification of the stress eVect by Pb. Changes in HVA, another dopamine metabolite, did not correspond to those seen with DOPAC, since levels were increased by Pb (150/NS), but not by Pb þ stress (150/S). Findings with HVA demonstrate a

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FIG. 6. Group mean ± SE overall response rates on the FI schedule of male oVspring of dams exposed to 0 or 150 ppm Pb exposure with or without maternal stress (NS ¼ no stress; S ¼ stress) across the course of 20 weeks of testing, with behavioral test sessions carried out 5 days per week. Interaction eVect from the repeated measures analysis of variance as indicated. From D. A. Cory‐Slechta et al. (2004).

Pb eVect modified by maternal stress. The diVerential DOPAC and HVA results also suggest that intra‐ and extracellular metabolism of dopamine may be diVerentially aVected by these treatments (cf. DOPAC vs HVA). In the case of 5‐HT, only Pb alone (150/NS) increased levels, thus its eVects were modified by stress (cf. 150/S). d. Relationships Between NAC Dopamine and Basal Corticosterone Levels To begin to evaluate potential mechanisms of eVect from these findings, simple linear regression analyses were initially employed. Interestingly, when basal corticosterone levels were regressed against neurotransmitter changes, a dichotomy in the nature of the emergent relationships was observed in control (0/NS; Fig. 8, left) vs stressed (0/S; Fig. 8 right) adult male oVspring. Levels of basal corticosterone increased in controls (left) with increasing NAC dopamine levels (p ¼ 0.03; n ¼ 12), whereas an inverse function, although not statistically significant, was seen in response to stress (right; p ¼ 0.158; n ¼ 12). These findings, although not conclusive, at least suggest the possibility of a permanent stress‐induced modification of the nature of the glucocorticoid–dopamine interactions in NAC. Thus, corticosterone

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FIG. 7. Group mean ± SE levels of dopamine (DA, top left), its metabolites DOPAC (top right) and HVA (bottom left), and of 5‐HT (bottom right) in ng/ml protein in nucleus accumbens of adult male oVspring of dams exposed 0 or 150 ppm Pb exposure with or without maternal stress (NS ¼ no stress; S ¼ stress). In these plots, 0/NS ¼ no maternal Pb, no maternal stress; 0/S ¼ no maternal Pb, maternal stress; 150/NS ¼ maternal Pb exposure, no maternal stress; 150/S ¼ maternal Pb exposure and maternal stress. Main eVects and interactions from repeated measures analyses of variance as indicated. From D. A. Cory‐Slechta et al. (2004).

increases may be able to modify neurochemical mechanisms that mediate FI performance (D. Cory‐Slechta et al., 2000, 2002; D. A. Cory‐Slechta et al., 1996, 1998), underscoring the importance of experiments aimed at verifying and further defining altered corticosterone as a mechanism of Pb eVects.

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FIG. 8. Dopamine levels in nucleus accumbens in relation to basal corticosterone levels of adult male oVspring of control dams (left) or dams exposed to maternal stress (right panel). Each data point represents an individual animal within the designated group. p values indicate outcome of simple linear regression analyses, with corresponding regression summary and r2 values as indicated. D. A. Cory‐Slechta, unpublished data.

3. HIGHLIGHTS OF EFFECTS IN FEMALE OFFSPRING

Gender diVerences in response to both Pb and stress as well as to their interaction were prevalent and notable in several outcome measures. While such findings have been reported by others for stress (Faraday, 2002; Shalev & Weiner, 2001), the potential for gender‐associated diVerences in response to Pb exposure remains largely unknown, although a human study now points to an elevated susceptibility of males to the cognitive deficits associated with Pb (Ris et al., 2004). a. Basal Corticosterone As in dams (D. A. Cory‐Slechta et al., 2004) and male oVspring (Fig. 5), maternal Pb exposure alone increased basal corticosterone levels in female oVspring (Fig. 9) by values of 26 and 61%, respectively, eVects that were of comparable magnitude statistically in the 150/NS and 150/S groups. Again, such findings underscore the potential importance of altered corticosterone as a mechanism of Pb eVects on the central nervous system. b. Behavior Females generally maintained far lower FI overall response rates than did males, and also exhibited a pattern of Pb/stress‐related changes that diVered substantially from that of males. In females, stress alone (0/S) increased FI overall response rates relative to all other treatments (Fig. 10), eVects that were evident within the first week of testing and were sustained over the 16 weeks of behavioral testing (Pb by stress interaction, p ¼ 0.03). Whereas Pb alone (150/NS) produced notable decrements in FI

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FIG. 9. Group mean ± SE basal corticosterone levels of female oVspring measured at approximately 8 months of age in groups subjected to maternal exposure to 0 or 150 ppm Pb exposure as indicated, with or without combined maternal stress as indicated (NS ¼ no stress; S ¼ stress). Main eVect from the repeated measures analysis of variance as indicated. From D. A. Cory‐Slechta et al. (2004).

FIG. 10. Group mean ± SE overall response rates on the FI schedule of female oVspring of dams exposed to 0 or 150 ppm Pb exposure with or without maternal stress (NS ¼ no stress; S ¼ stress) across the course of 20 weeks of testing with behavioral test sessions carried out 5 days per week. Interaction eVect from the repeated measures analysis of variance as indicated. From D. A. Cory‐Slechta et al. (2004).

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FIG. 11. Overall response rates on the FI schedule in relation to basal corticosterone levels of adult female oVspring of control dams (left) or dams exposed to maternal stress (right panel). Each data point represents an individual animal within the designated group. p values indicate outcome of simple linear regression analyses, with corresponding regression summary and r2 values as indicated. D. A. Cory‐Slechta, unpublished data.

response rates in males, it had no eVects in females. Pb þ stress (150/S) in females significantly decreased response rates, such that the largest group diVerences were seen between stress alone (S/0) and Pb þ stress groups (150/S), suggesting a potent modification of the stress response by Pb in females in the context of behavior. c. Relationships Between Basal Corticosterone Levels and Behavior Linear regression analyses were used as a first attempt at determining potential relationships between changes in basal corticosterone levels and behavior. Figure 11 shows an interesting dichotomy in the resulting relationships in females in that FI response rates (week 8 data) in control females (0/NS; left panel) declined (although not significantly) with increasing basal corticosterone levels, whereas a significant increase was seen in stressed (S/0) female oVspring (right panel), with no adult stress having occurred at this time point. Such findings show that altered corticosterone levels can be related, at least statistically, to FI performance, and suggest a rationale for examining the ability of glucocorticoid antagonists to reverse stress‐induced increases in FI response rates. d. Neurotransmitter Changes Permanent changes in neurotransmitter levels were also observed in adult female oVspring with the important determinant (Pb, stress, or Pb  stress) diVering by region and with a profile of eVects diVering from that observed in males. Interactions were especially notable in the PFC for dopamine (Fig. 12, left panel; Pb by

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FIG. 12. Group mean ± SE levels of dopamine in ng/ml protein measured in prefrontal cortex (left), nucleus accumbens (middle), and striatum (right) of adult female oVspring of dams exposed to 0 or 150 ppm Pb exposure with or without maternal stress (NS ¼ no stress; S ¼ stress). In these plots, 0/NS ¼ no maternal Pb, no maternal stress; 0/S ¼ no maternal Pb, maternal stress; 150/NS ¼ maternal Pb exposure, no maternal stress; 150/S ¼ maternal Pb exposure and maternal stress. Main eVects and interaction eVects from the repeated measures analysis of variance as indicated. From D. A. Cory‐Slechta et al. (2004).

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stress, p ¼ 0.006), with dopamine levels increased in post hoc tests only by Pb þ stress, a presumably permanent and potentiated (i.e., occurring in the absence of an eVect of either Pb or stress alone) consequence of these treatments, and a notable eVect given the reported significance of PFC for inhibitory control of stress responses (Brake et al., 2000; Deutch et al., 1990; Figueiredo et al., 2003). In the striatum (right panel; Pb  stress interaction), while both Pb (significantly) and stress (marginally) increased dopamine levels, no such eVect occurred in the Pb þ stress group. NAC changes (middle panel) reflected the eVects of Pb per se, with significant and comparable reductions in both the 150/NS and 150/S groups. 4. STRESS RESPONSIVITY IN ADULT OFFSPRING

a. Male Adult OVspring Stress Procedures At week 9 of FI testing of males, restraint stress was imposed using a procedure similar to that employed with dams except that it involved only a single 45‐min session and was followed by FI performance evaluation. In addition, tail blood was collected following the FI session for corticosterone determinations. During week 12, a saline injection was administered i.p. immediately before the FI session. Finally, an intruder was introduced into the home cage for 15 min during week 19 immediately prior to the FI session. b. EVects on Male FI Performance and Corticosterone Levels i. FI PERFORMANCE. DiVerential alterations in stress responsivity as manifested in subsequent FI performance were most evident in males in response to an intruder stress (Fig. 13), where statistical analysis confirmed a main eVect of Pb as well as a Pb by stress interaction. Post hoc assessments indicated that the most pronounced suppression of FI response rate occurred in the group exposed only to maternal Pb exposure, since this group was the only one diVering from control. In contrast, the group exposed to combined maternal Pb þ stress did not diVer from control. Thus, when combined with stress, the Pb eVects were modified. ii. STRESS‐INDUCED CORTICOSTERONE. For males, stress‐induced corticosterone levels were examined only after the first stress challenge (restraint stress), but these also yielded notable group diVerences, in this case, related primarily to maternal Pb alone, which significantly attenuated the stress‐ induced elevation in corticosterone levels seen in all three other groups, as shown in Fig. 14, reflecting a Pb by stress interaction. c. Female Adult OVspring Stress Procedures The first stress challenge of females, occurring during week 17 of FI testing, consisted of a single 45‐min session of restraint stress using the same procedure described previously for dams. The second stressor, imposed during week 18 of FI testing, was a 15‐min locomotor activity session with placement into the apparatus serving as a novel environment. The third stressor, imposed during week 22 of FI

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FIG. 13. Group mean ± SE overall response rates on the FI schedule of male oVspring of dams exposed to 0 or 150 ppm Pb exposure with or without maternal stress (NS ¼ no stress; S ¼ stress) following an intruder stress (introduction of a strange male into the home cage for 15 min). Data are plotted as percent of control based on the mean rates from the two FI sessions preceding the stress. Main eVects and interactions in the corresponding repeated measures analysis of variance as indicated. D. A. Cory‐Slechta, unpublished data.

testing, consisted of a cold challenge that involved placement of animals in their home cages (without the bedding) in a 4  C temperature‐controlled room for 20 min. Immediately after completion of each stress challenge, FI performance was evaluated as usual. Stress challenges were carried out during the diestrus I phase of the estrous cycle to ensure constant and low estrogen levels, since fluctuations in the estrogen cycle can alter both endogenous and released corticosterone levels, and, moreover, stress responsivity can vary with the stage of the estrous cycle (S. M. Anderson et al., 1996; Viau & Meaney, 1991). The FI session was followed in each case by collection of tail blood for corticosterone determinations. d. EVects on Female FI Performance and Corticosterone Levels i. FI PERFORMANCE. The eVects of restraint stress imposed prior to the test session during week 17 on FI performance measures and corticosterone levels as percent of control are shown in Fig. 15. Restraint stress resulted in a marked reduction in FI overall response rates of greater than 80% across groups (top left), but the magnitude of the decrement diVered by Pb/stress conditions, as indicated by a Pb‐stress interaction in the corresponding repeated measures analysis of variance (F(1,28) ¼ 10.47, p ¼ 0.003). Post hoc assessments revealed that the reductions in FI response rate were

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FIG. 14. Group mean ± SE levels of restraint‐induced corticosterone levels plotted as a percent of basal corticosterone levels in adult male oVspring of dams exposed 0 or 150 ppm Pb exposure with or without maternal stress (NS ¼ no stress; S ¼ stress). In these plots, 0/NS ¼ no maternal Pb, no maternal stress; 0/S ¼ no maternal Pb, maternal stress; 150/NS ¼ maternal Pb exposure, no maternal stress; 150/S ¼ maternal Pb exposure and maternal stress. Outcomes of post hoc tests as indicated following the repeated measures analysis of variance. D. A. Cory‐ Slechta, unpublished data.

attenuated by approximately 10 to 15% in the 150/S group relative to both maternal stress alone (0/S) and to maternal Pb alone (150/NS). Markedly similar changes were noted in run rate values on the FI schedule (F(1,28) ¼ 5.74, p ¼ 0.024), where rates were reduced by greater than 75% across groups (bottom left), but attenuated in the 150/S group relative to both maternal stress alone (0/S) and to maternal Pb alone (150/NS). These findings are consistent with a combined Pb þ stress eVect that diVers from eVect seen in response to each variable alone. PRP times on the FI schedule (top right) were significantly elevated by restraint stress imposed prior to the session, with increases of approximately 700% of control. Again, however, a Pb by stress interaction was confirmed (F(1,28) ¼ 14.03, p ¼ 0.0008) as a result of

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increases in the 0/S group that were approximately three‐fold higher than those observed in control (0/NS) and Pb þ stress (150/S) groups. In addition, levels in the Pb alone group (150/NS) were marginally increased in comparison to both control (p ¼ 0.06) and to the 150/S group (p ¼ 0.08). Here, Pb significantly modified a stress‐induced response. ii. STRESS‐INDUCED CORTICOSTERONE. Basal corticosterone values of female oVspring were significantly elevated in response to maternal Pb exposure per se, with absolute values of 162.6 ± 23.8 for the 0/NS group, 169.4 ± 18.8 for the 0/S group, 238.4 ± 46.7 for the 150/NS group, and 278.8 ± 41.6 for the 150/S group, as previously reported (D. A. Cory‐Slechta et al., 2004). As expected, restraint stress resulted in dramatic elevations in corticosterone levels to values of 200 to 300% of basal determinations (Fig. 15, bottom right). Group mean ± S.E. absolute values in ng/ml were: 470.61 ± 68.02 for the 0/NS group, 463.38 ± 59.55 for the 0/S group, 481.27 ± 67.60 for the 150/NS group, and 498.49 ± 63.56 for the 150/S group. The increases in this case, perhaps because of the intensity of the stressor, did not result in any statistically significant diVerences in relation to Pb or stress or to their interaction.

VII. CONSIDERATIONS ARISING FROM THE DIFFERENTIAL EFFECTS OF MATERNAL Pb, MATERNAL STRESS, AND MATERNAL Pb þ STRESS Several aspects of these findings are relevant to our understanding of the human health implications of Pb, the mechanisms by which Pb induces cognitive dysfunction, and to the design of prevention screening programs for elevated Pb exposure.

FIG. 15. Group mean ± SE levels of FI performance and corticosterone levels following restraint stress in adult female oVspring of dams exposed to 0 or 150 ppm Pb exposure with or without maternal stress (NS ¼ no stress; S ¼ stress). In these plots, 0/NS ¼ no maternal Pb, no maternal stress; 0/S ¼ no maternal Pb, maternal stress; 150/NS ¼ maternal Pb exposure, no maternal stress; 150/S ¼ maternal Pb exposure and maternal stress. Depicted are overall response rate (top left), run rate (bottom left), postreinforcement pause time (top right), and stress‐induced corticosterone levels (bottom right). Data for FI performance are plotted as percent of control, with the mean value of the two sessions preceding the restraint stress serving as control. Main eVects and interactions in the corresponding repeated measures analysis of variance as indicated. From Virgolini et al., 2005.

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Deborah A. Cory-Slechta Relevance to Human Health, Mechanisms of Pb Effects, and Prevention Screening Programs for Pb

1. Pb EXPOSURE CAN PERMANENTLY ALTER CORTICOSTERONE LEVELS

As shown in Figs. 4 and 8, maternal Pb exposure per se resulted in permanent elevations in basal corticosterone in oVspring. It is not yet clear whether these eVects represent a direct eVect of Pb on some component of the limbic–HPA axis, or whether an indirect eVect on the HPA axis occurs through Pb‐induced changes in mesocorticolimbic DA or glutamate functions. Regardless of direction, however, such findings are notable since they could suggest that elevated Pb burden increases vulnerability of low SES populations to disease and dysfunction through chronic elevations of cortisol. As has been noted, prolonged elevation of cortisol has been proposed as a mechanism contributing to the increased incidence of disease and dysfunction associated with low SES, populations also experiencing the highest levels of Pb exposure. We have since determined that Pb exposure initiated only at weaning in the rat (21 days of age) decreases basal corticosterone levels. It also increases stress‐induced corticosterone levels and behavioral responsivity (measured as the impact of various stress challenges administered prior to the measurement of FI performance), confirming that Pb‐induced changes in corticosterone are not limited to early periods of development. Collectively, these findings demonstrate that Pb exposure alters HPA axis function and therefore can also interfere with the ability to respond to stress, one of the most important adaptation systems of the body.

2. STRESS AND Pb CAN MODIFY EACH OTHER’S EFFECTS, AND POTENTIATED EFFECTS OF Pb þ STRESS CAN OCCUR

These findings are critical given the significance of the stress response as an adaptive compensatory mechanism. For example, dopamine levels in the PFC of female oVspring were increased only in response to combined Pb þ stress group (Fig. 11). A Pb/stress interaction in females was also noted in FI performance following restraint stress (Fig. 15), where a Pb‐induced eVect, increases in PRP time, was modified by stress, and thus were not observed in the Pb þ stress females. Collectively, these results raise questions about our current understanding of the human health eVects of Pb, since experimental studies typically examine Pb in isolation from other risk factors, and cohort studies generally control for potential risk modifiers in order to examine the nature and magnitude of the eVect of Pb alone.

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3. Pb‐INDUCED CHANGES IN CORTICOSTERONE AS A MECHANISM OF ITS BEHAVIORAL AND NEUROCHEMICAL TOXICITY

Because of the broad impact of corticosterone on brain neurotransmitter systems, and specifically its eVects on mesocorticolimbic circuits, the current findings also suggest that Pb‐induced changes in corticosterone could play a mechanistic role in the adverse behavioral and neurochemical eVects of Pb exposure. Our previous studies show a key role for NAC dopamine systems and glutamatergic inputs from hippocampus to NAC in mediating normal and Pb‐induced changes in FI performance and in learning (D. A. Cory‐Slechta, 1993; D. A. Cory‐Slechta et al., 1998, 1999). Figure 12 shows, in stressed female oVspring, a linear relationship between basal corticosterone and FI response rates. Together, these findings raise the question as to whether Pb‐induced alterations in HPA axis function lead to mesocorticolimbic system alterations that then produce the neurochemical and behavioral changes associated with Pb exposure. If so, blockade of Pb‐induced corticosterone changes would reverse Pb‐induced behavioral and neurochemical eVects and chronic elevation of corticosterone should mimic these eVects. 4. THE NATURE OF Pb/STRESS INTERACTION IS NOT EASILY PREDICTABLE

Importantly, the nature of the Pb/stress interaction is not easily predictable. Numerous factors influence the extent to which these interactions occur, including gender and developmental period of Pb exposure (maternal vs postweaning), brain region, and time of measurement. Since the nature of eVects can change with time (e.g., in male oVspring, prefrontal cortical dopamine levels were decreased by Pb at 21 days of age, but not in adulthood [D. A. Cory‐Slechta et al., 2004]; group diVerences in behavior also changed across time), interpretations based on changes at a single time point in such studies cannot necessarily be generalized. Thus, while it would certainly be desirable, it may be unrealistic to expect a simple, uniform generalizable relationship encompassing the interaction between Pb exposure and stress. 5. INTERPRETATIONS OF Pb þ STRESS EFFECTS AS A ‘‘REVERSAL’’ OR ‘‘ANTAGONISM’’

Interpretations of Pb þ stress eVects as a ‘‘reversal’’ or ‘‘antagonism’’ of the eVects of either variable alone could be inaccurate or misleading. Data from a single time point in a dynamic process may be misleading, as has been noted, since diVerent patterns of eVects could be seen subsequently. Second, it is likely to be the case that even while absolute levels of measurement from

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a combined Pb þ stress appear to restore outcomes to levels comparable to control, the eVects in the Pb þ stress group may well reflect quite diVerent operative neurochemical, corticosteroid, and/or behavioral mechanisms compared to normal and thus not constitute true reversals. Equivalent eVects in the control and Pb þ stress groups could also mask dormant toxicity. 6. IMPLICATIONS FOR RISK ASSESSMENT

The findings from this study already have implications for risk assessment, since elevated corticosterone levels associated with Pb per se could signal that Pb exposure produces an enhanced state of stress and thus increased susceptibility to disease and dysfunction. Pb also significantly modified the nature of the stress response. Moreover, eVects of Pb þ stress are, for some measures, greater than those of either factor alone. Understanding the nature and mechanisms of these interactions has significance not only for a full evaluation of human health risks, but also for the determination of any therapeutic and screening strategies that might attenuate the impacts of these factors.

VIII. A.

FUTURE RESEARCH NEEDS

What Parameters of Pb Exposure and of Stress Are Important to Interactions?

These beginning evaluations of the modification of the neurotoxicity of Pb by stress raise numerous questions that need to be pursued. One of the first is what parameters of Pb exposure and environmental stress might yield interactive eVects of an additive or greater nature. It is already clear that combined maternal Pb þ stress can produce permanent changes in important functions in our experimental models. For example, only combined Pb þ stress led to what appeared to be permanent increases in prefrontal cortical dopamine levels in female oVspring. PFC dopamine is critical to the mediation of cognitive functions, including learning and memory (Goethals et al., 2004; Kane & Engle, 2002; Tanji & Hoshi, 2001), and plays a feedback function over HPA axis function (Figueiredo et al., 2003; Jacobson & Sapolsky, 1991). In addition, PFC dopamine is considered to be involved in the pathology of schizophrenia (Costa et al., 2004; Lewis et al., 2004; Volk & Lewis, 2002; Weinberger et al., 2001). A report linking Pb exposure to schizophrenia is interesting in this regard (M. G. Opler et al., 2004), particularly given the critical role that maternal/neonatal stress has been postulated to serve in the etiology of this disabling neurological dysfunction (Walker & Diforio, 1997). Indeed, such studies raise the intriguing

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question, which could be addressed in cross‐sectional and prospective cohort study follow‐ups, of the role that Pb exposure may play in schizophrenia or in other behavioral processes in which this system is thought to play a role, including attention deficit and drug abuse. Mesocorticolimbic circuits are also critical to the mediation of the rewarding property of drugs of abuse (Bonci et al., 2003; Everitt & Wolf, 2002), and, interestingly, drug abuse incidence has been reported to be higher in low SES populations (Furr‐ Holden & Anthony, 2003). Moreover, corticosterone appears to be crucial to the acquisition of drug use, since in one report, self‐administration only occurred when corticosterone levels exceeded a threshold level. Increased corticosterone also enhances sensitivity to low doses of cocaine, suggesting such exposures can increase individual vulnerability to cocaine use (Goeders, 2002).

B.

How Does Combined Pb þ Stress Impact Cognitive Function?

An additional and critical question with respect to human health risk assessment is whether combined exposure to Pb and stress act synergistically or in some other fashion to modify complex cognitive function, particularly learning, since, as has been noted, both Pb and stress individually have been demonstrated to be a risk factor for adverse eVects on cognition. The findings described here and in Cory‐Slechta et al. (D. A. Cory‐Slechta et al., 2004) provide experimental support for reports from the Cincinnati (Ris et al., 2004) and Port Pirie (McMichael et al., 1992; S. Tong et al., 2000) prospective cohort studies in children of more pronounced eVects of Pb in subjects with low SES backgrounds.

C.

Is Altered HPA Axis Function a Mechanism of Lead‐Induced Neurotoxicity and Cognitive Dysfunction?

The apparently permanent impact of Pb exposure, both maternal only (here and in D. A. Cory‐Slechta et al., 2004) and that occurring later in development (postweaning, data in preparation), raises the question as to whether a mechanism by which Pb impacts cognitive function is indirectly via alterations in HPA axis structure and/or function. As has been noted, Pb‐associated changes in dopamine and glutamatergic components of the mesocorticolimbic system are well documented, but the associated mechanisms of these alterations remain unclear. The current findings raise the possibility that they may be indirect manifestations of altered HPA axis function produced by Pb that consequently change the nature of its

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interactions with mesocorticolimbic systems and thereby alter aspects of cognitive function that this system controls. Studies using adrenalectomy or HPA antagonists to preclude such eVects, or administration of chronic corticosterone to mimic such eVects, will be useful to address this issue. Such studies will need to examine the diVerential impacts of diVerent Pb exposure regimens, diVerent stressors, and diVerent measures of cognitive function. D.

Is Attention Deficit a Behavioral Mechanism of the Cognitive Deficits Associated with Pb Exposure or with Stress?

Both Pb and stress are associated with cognitive function, but in neither case are the associated behavioral mechanisms yet known. For Pb, one repeatedly asserted hypothesis is that the cognitive dysfunction occurs via deficits in attentional processes (D. Bellinger et al., 1994), based on reports of associations between increasing Pb levels and negative ratings for inattentiveness, distractibility, impulsivity, and lack of persistence by teachers and parents (Fergusson et al., 1988; Needleman et al., 1979; Yule et al., 1981) in children. In one study (D. Bellinger et al., 1994), dentin Pb correlated with two of four factors constituting the theoretical framework for attention described by Mirsky et al. (Mirsky, 1987; Mirsky et al., 1991): focus–execute and shift of attention. Pb‐associated increases in exposed children in errors in a serial choice reaction time paradigm were described as resembling clinical observations in attention deficit disorder (Winneke et al., 1989). Experimental studies have attempted to further delineate a role for attention deficit in response to Pb and to determine which constituents of this behavioral construct may be aVected. Sustained attention, one of the three symptom domains embodied in the clinical diagnosis of attention deficit, does not appear to be particularly vulnerable to Pb, whereas impulsivity/aversion to delay is impacted by Pb and clearly merits further evaluation (Brockel & Cory‐Slechta, 1998, 1999a,b; D. A. Cory‐Slechta, 2003). As with Pb, the role of attention deficits in stress‐related changes in cognition are not yet well understood and have been examined only to a limited extent, with most of the studies including confounds. In a case‐ control study (McIntosh et al., 1995), mothers of children with attention deficit hyperactivity disorder (ADHD) reported greater psychological stress during pregnancy. In a cohort of 348 8‐year‐old children, psychosocial risk was associated with higher scores on the Child Behavior Check List for behavioral problems related to attention, delinquency, and aggression. In this study, stressful family circumstances included a combination of genetic and psychosocial factors. Children of mothers exposed to wartime stress

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compared to those born 2 years later similarly exhibited higher ratings of child behavior problems, although stress during pregnancy in this study was not evaluated with a validated instrument (Meijer, 1985). A community‐ based study found that mothers with enhanced anxiety at 32 weeks of gestation were more than twice as likely to have children with elevated behavioral problems at 4 years of age, including inattention/hyperactivity after controlling for other covariates, an eVect that was of equal strength in both genders (O’Connor et al., 2002). Limitations of this study included the fact that maternal anxiety was based on self‐report questionnaires and eVect sizes were modest. Also in relation to stress, another study examined cortisol levels post‐assessment of behavior (normalized Weschler Intelligence Scale for Children [WISC] and Tests of Variables of Attention, a test of sustained attention) in 43 Korean boys with diagnosed ADHD (Hong et al., 2003). In that study, patients exhibiting decreased post‐test cortisol levels also had higher commission and omission errors during certain portions of the test than did those exhibiting increased cortisol levels. A review concluded that prenatal psychosocial stress might be a risk factor for ADHD, but that further studies are warranted to examine this relationship (Linnet et al., 2003). Similar conclusions are appropriate for Pb exposure. Thus, the extent to which attention deficit contributes as a behavioral mechanism of eVect to the cognitive deficits associated with either Pb or stress cannot yet be discerned. Not only are additional studies examining potential relationships warranted, but, ultimately, such studies must diVerentiate contributions of diVerent response classes of attention deficit in any such eVects, i.e., whether they involve alterations in impulsivity, and/or in sustained attention, and/or in activity levels. E.

Should Screening Efforts for Elevated Pb Burden Include Maternal Assessments?

The outcomes of even the currently limited studies of Pb þ stress described here raise important issues with respect to screening for elevated PbBs. Currently, such screening is carried out in children. However, two observations here suggest that maternal PbB screening may be just as significant and warranted as later measures in children. The first observation was that maternal Pb exposure alone permanently increased basal corticosterone in oVspring of both genders. Moreover, maternal Pb combined with maternal stress resulted in some eVects that were not seen in response to maternal Pb alone or maternal stress alone, such as increased frontal cortical dopamine levels in female oVspring. Such findings point to a need to prevent exposures as early as possible during development.

126 F.

Deborah A. Cory-Slechta How Do Other Risk Modifiers Interact with Pb?

Also to be considered is that these studies have examined the potential interaction of Pb exposure of only one other risk modifier, environmental stress. But there are multiple other potential risk modifiers, such as other neurotoxic chemicals (PCBs, pesticides, or heavy metals) and both extrinsic and intrinsic host factors (genetic background, gender, intercurrent disease state, nutritional status, obesity, and smoking). Amazingly, almost no information on how Pb exposure may interact with these other factors is currently available. It is particularly surprising, for example, to consider the fact that so little is known about the potential diVerential eVects of Pb by gender. While gender diVerences in the outcome of stress are extensively described, our results are among the few experimental studies that report diVerences in relation to gender in response to maternal Pb exposure. Indeed, few prospective studies report whether Pb‐associated changes in IQ are diVerential with respect to gender, since, in most cases, the eVects of gender are statistically controlled. The two exceptions provide conflicting results of greater sensitivity (Ris et al., 2004; S. Tong et al., 2000).

G.

A Multi‐Hit Hypothesis: A Biological Basis for Enhanced Effects of Combined Risk Factors

Given the number of risk modifiers, the question that arises is what risk factors might be most important to study in conjunction with Pb. Of particular concern might be those that act on the same regions/systems of the brain as does Pb, a concept embodied in the multi‐hit hypothesis described previously (Fig. 4) that was part of the rationale for beginning these eVorts. It hypothesizes that the brain may readily be able to compensate for the eVects of an individual chemical or risk factor itself acting on a particular target system of the brain. However, when multiple target or functional sites within that particular system are attacked by diVerent mechanisms (Pb in conjunction with other neurotoxicants and/or extrinsic or intrinsic host risk factors), the system may no longer be able to homeostatically re‐regulate itself, thereby leading to sustained or cumulative damage. Thus, risk factors that act on regions of the brain in common with the neurotoxicant of interest would be candidates. Equally important to remember is that behavioral function is critically dependent upon the interactions of the networks and systems of the brain. The mesocorticolimbic system of the brain interacts with various other regions and neurotransmitter systems, including those with receptors for excitatory amino acids, histamine, GABA, and with both nicotinic and

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muscarinic cholinergic receptors. Thus, direct eVects on dopamine system function are very likely to secondarily target other systems and regions of brain with which dopamine systems interact, thereby oVering the potential to amplify eVects of chemical exposures. In fact, it is interesting to consider the possibility that the network/systems operation of the brain may underlie its sensitivity to many neurotoxicants through amplification and distribution of damage.

H.

Other Neurotoxicants and Risk Modifiers

Pb, of course, is but one of many environmental and occupational chemicals that adversely impact nervous system structure and/or function, and for which risk modification is likely to occur. Indeed, our findings add to a growing literature, not only in neurotoxicology (Ankarberg et al., 2004; Hojo et al., 2002; Noraberg & Arlien‐Soborg, 2000; Zareba et al., 2002), but in other areas of toxicology and environmental health that demonstrate similar risk modifications. In the context of neurotoxicants with potential roles in developmental disabilities, eVorts will first be required to understand and consequently prioritize the most plausible risk modifiers for these neurotoxicants and thereafter determine the extent to which they may modify eVects on complex cognitive behaviors.

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Developmental Disabilities Following Prenatal Exposure to Methyl Mercury from Maternal Fish Consumption: A Review of the Evidence GARY J. MYERS DEPARTMENTS OF NEUROLOGY AND PEDIATRICS, UNIVERSITY OF ROCHESTER SCHOOL OF MEDICINE AND DENTISTRY, ROCHESTER, NEW YORK

PHILIP W. DAVIDSON STRONG CENTER FOR DEVELOPMENTAL DISABILITIES, UNIVERSITY OF ROCHESTER SCHOOL OF MEDICINE AND DENTISTRY, ROCHESTER, NEW YORK

CONRAD F. SHAMLAYE MINISTRY OF HEALTH, VICTORIA, MAHE, REPUBLIC OF SEYCHELLES

I. A.

INTRODUCTION

Mercury

Mercury (Hg) is a natural element in the earth’s crust. It enters the human environment from both natural and anthropogenic sources. Natural sources provide the largest environmental contribution and include volcanic eruptions, degassing of the earth’s surface, and evasion from bodies of water (WHO, 1990; United States Environmental Protection Agency, 1997). Anthropogenic sources contribute a smaller amount, but they are the component over which mankind has some control. These human sources are diverse and include the burning of fossil fuels and refuse, cremation, smelting, and waste or loss from industrial use. In addition, mercury is widely used in manufacturing and medicine. Large quantities of elemental mercury are used to produce caustic soda and chlorine gas and by paper mills. Both INTERNATIONAL REVIEW OF RESEARCH IN MENTAL RETARDATION, Vol. 30 0074-7750/06 $35.00

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industries lose significant amounts into the environment. Elemental mercury forms an amalgam with gold and is used extensively in small gold mining operations. Mercury has been mined for thousands of years in many places around the world. The mine at Almaden in Spain operated continuously for over 2000 years and, when active, brought over 10,000 tons of mercury to the surface each year (WHO, 1990). For economic reasons, it and most other mines are now closed. The amount of mercury in our atmosphere and immediate environment has been slowly increasing over the past century (Agency for Toxic Substances and Disease Registry, 1999; Benoit et al., 2003). Mercury has three oxidation states. The zero oxidation state (Hg0) is usually referred to as ‘‘elemental mercury’’ and is either a liquid or vapor. Mercury can lose one electron, in which case it binds to another mercury atom and is referred to as mercurous (Hgþ). The mercurous oxidation state is unstable and either binds to other compounds or dissociates into an atom of Hg0 or the mercuric oxidation state (Hg2þ), where two electrons are lost. Both mercurous and mercuric can form numerous chemical compounds. The oxidation states of Hg0, Hgþ, and Hg2þ are generally referred to as ‘‘inorganic’’ mercury. They can be readily interconverted in the body and in the environment. Most analytical methods do not distinguish among these forms. The mercuric oxidation state can form a variety of stable organic mercury compounds. B.

Mercury Toxicity

In the human body, there is no known physiologic use for any form of Hg. All forms of mercury are toxic in suYcient quantity and their health eVects vary with the form, dose, manner of exposure, and a variety of other variables (Clarkson et al., 2003). Some forms aVect primarily the nervous system while others damage the kidneys or lungs. Elemental and inorganic forms of mercury can cause acrodynia, a complex of symptoms and signs that includes autonomic instability with redness and pain in the distal extremities, hypertension, photophobia, and general apathy (Warkany, 1966). Acrodynia was common during the first half of the 20th century when mercurials were commonly used to treat intestinal parasites and as teething powders. After the association between Hg exposure and acrodynia was recognized and the use of mercury compounds to treat children reduced, the condition rapidly disappeared and is rarely seen now. The toxicity of the organic form MeHg was recognized in the 1940s (Buckell et al., 1993). Its toxicity in children was recognized a few years later, as will be described. The three common forms of human exposure to Hg include dental amalgams, fish consumption, and vaccines (Clarkson, 2002). Each of these

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exposures is to a diVerent chemical form of mercury. Dental amalgams, at the time of placement and during mastication, release small amounts of elemental Hg vapor into the oral cavity and some enters the bloodstream through either the lungs or the gastrointestinal tract. All fish contain MeHg in concentrations that vary with age and size, and when they are consumed, it is readily absorbed in the gastrointestinal tract. Many vaccines contain thimerosal, a preservative that is composed of about 50% ethylmercury. When vaccines are administered, the mercury remains in the body. There has been public concern about Hg exposure for many years, and the concern appears to be growing. This may reflect the general increased interest in environmental pollution that has taken place in recent years, a better understanding of the consequences of exposure to Hg, an overall increased interest in pollution, or any number of other factors. Poisoning by various forms of Hg is well known (Buckell et al., 1993; Warkany, 1966). However, several studies have reported that exposure to levels previously thought to be harmless may have adverse eVects on child development (Cordier et al., 2002; Cox et al., 1989; Grandjean et al., 1997; Kjellstrom et al., 1986, 1989; Marsh et al., 1987; McKeown‐Eyssen et al., 1983; Ramirez et al., 2000, 2003). These reports of adverse associations are derived from epidemiological studies in which nearly all of the children studied were clinically normal on testing. However, the studies compared children with diVerent levels of exposure and reported subtle diVerences between the groups on certain outcome tests. These diVerences are not apparent in individual children. These findings raise a legitimate concern that we may be approaching or have already reached a level of mercury exposure where subtle adverse developmental eVects may begin to appear. These reports deserve our careful attention because of their public health implications. They also deserve close scrutiny since epidemiological studies of long‐term low‐dose exposures are known to be complex to carry out and their findings are often open to varied interpretations (Mink et al., 2004). C.

Methyl Mercury Toxicity

Among toxic exposures to mercury, the organic form of MeHg is of particular concern because it is highly toxic and small amounts occur naturally in the environment. Mercury released into the atmosphere from natural or anthropogenic sources circulates widely and can be deposited at very distant sites. Thus, even places that are very distant from direct sources of natural or industrial mercury discharges will have some level of mercury. In aquatic systems, various microorganisms such as bacteria methylate a small portion of the inorganic mercury to the organic form. Once MeHg is present in bacteria, it is then bioaccumulated and bioconcentrated up the food chain.

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Consequently, all aquatic organisms, including fresh‐ and saltwater fish, contain some level of MeHg and the millions of people worldwide who consume fish or seafood are exposed. Most fish contain relatively small amounts of MeHg. For example, open‐ ocean fish generally have mercury levels that are below 0.5 parts per million (ppm) in their flesh (ATSDR, 1999). These levels are usually considered background since they can occur in fish when there is no evidence of overt local pollution. This background level of MeHg in fish does not appear to have changed over the past century despite increasing industrialization (Kraepiel et al., 2003; Miller et al., 1972). The excretion of MeHg from most living organisms, including fish, is relatively slow. As a result, predators near the top of the food chain, such as shark, marlin, and sailfish, can accumulate MeHg levels of 1 ppm or more in their flesh even in the absence of any overt mercury pollution. At the very top of the food chain are large ocean mammals such as whales that can accumulate up to 3 ppm of mercury in their flesh, of which half is MeHg and the remainder inorganic (Julshamn et al., 1987). Human exposure to MeHg can occur in ways apart from fish consumption. Organic Hg compounds including MeHg have been used since about 1914 as fungicides to preserve seed grain for planting (ATSDR, 1999; Buckell et al., 1993; WHO, 1990). During the time that MeHg was used to treat seed grain, there were numerous episodes of human poisoning (ATSDR, 1999). Such use is now banned. The first report of prenatal MeHg poisoning was from Sweden and involved the consumption of treated grain (Engleson & Herner, 1952). However, it was the massive industrial pollution in Japan during the 1950s and 1960s that brought MeHg to worldwide attention (Harada, 1968; Smith & Smith, 1975). In the early 1970s in Iraq, there was an outbreak of MeHg poisoning from the consumption of fungicide‐treated wheat intended for planting. Research following this episode led to the hypothesis that lower levels of prenatal MeHg exposure, such as those achieved by consuming fish containing background levels of MeHg, might present a risk to the developing brain (Cox et al., 1989). Following publication of this hypothesis, a number of epidemiological studies were carried out to determine if MeHg exposure from the consumption of fish with only background levels of MeHg actually does harm the fetal brain. This research focused primarily on prenatal exposure since both clinical experience and experimental studies indicate the developing brain is especially sensitive to the toxic eVects of MeHg (ATSDR, 1999). The interpretation of these studies continues to be controversial. Some scientists feel the hypothesis is confirmed while others are more cautious in their interpretation of the data. Since 2000, those who believe the hypothesis is proven have claimed that large numbers of U.S. children are at significant developmental

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risk from fish consumption. The first assertion appeared in a report on human exposure to MeHg by the National Research Council (NRC) (2000). The committee stated: ‘‘Available consumption data and current population and fertility rates indicate that over 60,000 newborns annually [in the U. S.] might be at risk for adverse neurodevelopmental eVects from in utero exposure to MeHg.’’ (p. 325). The statement appeared in the report summary, but there was no explanation in the text or documentation as to how this number was derived. Subsequently, the estimated number of U.S. children at risk was claimed to be 300,000 (MahaVey et al., 2004a; Rice, 2003) and subsequently over 600,000 (MahaVey, 2004b; Trasande et al., 2005). If these statements are true, MeHg should account for a substantial portion of all children with developmental disabilities and public health eVorts to reduce fish consumption would be expected to substantially improve children’s health. The rest of this chapter focuses on a review of research on MeHg exposure from fish consumption and examines the evidence from poisoning and epidemiological studies that such exposure might be a major cause of developmental disabilities in U.S. children.

II.

REPORTED CASES OF PRENATAL METHYL MERCURY POISONING

Fewer than 100 cases of prenatal MeHg poisoning have been documented in the world literature. Only a small number of these reports include reliable exposure data. These poisonings occurred either with consumption of MeHg‐treated seed grain or seafood contaminated with industrial waste. Poisoning with consumption of MeHg‐treated seed grain is clearly due to MeHg, but industrial contamination of seafood is usually a mixture of toxins, as it was at Minamata (Nomura, 1968). Table I summarizes the reported cases and they are discussed in more detail in the following text. Although there are several reports that claim there were a larger number of prenatal cases in Japan, only the ones reported by Harada in 1968 appear to be documented (Harada, 1972, 1995; Murakami, 1972; Shephard, 1976) A.

Prenatal Poisoning from Consumption of Methyl Mercury‐Treated Seed Grain

In 1952, Engleson and Herner reported a Swedish family that consumed MeHg‐treated grain. Urine levels of Hg were elevated in the father, mother, and a 1‐year‐old child. The father and child had clinical evidence of

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Gary J. Myers et al. TABLE I DOCUMENTED CASES OF PRENATAL (FETAL OR CONGENITAL) METHYL MERCURY POISONING

Exposure source Contaminated grain

Contaminated fish

Location

Na

Maternal symptoms

Exposure level

Reference

Sweden

1

None

Unknown

Iraq

23 11

Ataxia, vision change, death Paresthesias

1

None

All >564 ppb blood 16–598 ppm hair Unknown

Davis et al. (1996)

22

Paresthesias 4 mothers Paresthesias

Unknown

Harada (1968)

293 ppm

Saito (2004a)

United States Minamata, Japan Niigata, Japan

1

Engleson & Herner (1952) Amin Zaki et al. (1974) Marsh et al. (1978)

Number is based on children with a neurological score >4, but it is not clear if all had functional neurological impairment. a

poisoning, but the mother did not. The mother was pregnant when the grain was consumed. The infant appeared normal at birth and did not have elevated Hg in the urine. However, she had severe mental retardation. No follow‐up data were reported. In the winter of 1971–1972, an epidemic of poisoning from MeHg‐treated seed grain occurred in Iraq (Bakir et al., 1973). Similar Hg poisonings had already occurred in Iraq and physicians recognized the clinical picture early. Soon after the first cases were identified, studies of the exposure began. Prenatal exposure was determined by measuring the level of total Hg (THg) in maternal hair. Total Hg in hair is composed of over 80% organic Hg, mainly MeHg. The amount of THg deposited in the hair appears to be stable over time and has been shown to correspond well to the blood level. Hair grows at a known rate of about 1.1 cm a month and by cutting the hair sample into short segments for analysis, the timing and degree of exposure can be determined with reasonable accuracy (Phelps et al., 1980). Some investigators believe that mercury enters hair and brain by similar mechanisms and thus hair mercury measurements accurately reflect brain exposure. Hair is the only biological marker that has been directly correlated with Hg concentrations in the brain (Cernichiari et al., 1995a). Following the Iraq poisoning, Amin‐Zaki and colleagues (Amin‐Zaki et al., 1974, 1976) reported on a number of children exposed to MeHg in utero. Six of them manifested evidence of poisoning and ‘‘the lowest

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measured blood level in infants associated with signs of poisoning was 564 ppb.’’ Subsequently, Amin‐Zaki and colleagues reported 5‐year follow‐up of 32 infants with prenatal MeHg exposure (Amin‐Zaki et al., 1979). All of the infants with symptoms or signs had maternal peak hair Hg concentrations above 100 ppm. At age 5 years, 5 of the children had cerebral palsy and 18 had speech delay. Marsh and colleagues reported 81 children from Iraq with prenatal exposure, of whom some had neurological impairment (Marsh et al., 1987). Four children having prenatal exposure of 405 ppm in maternal hair or greater were clearly neurologically impaired while an additional seven had neurological scores above 4 and might have been impaired. Snyder (Snyder, 1971) reported one case of prenatal MeHg exposure from the United States. A family in New Mexico fed MeHg‐ contaminated grain to their hog and subsequently consumed the pork. The mother was pregnant when the pork was consumed. The level of exposure was measured only in her urine, but other family members had hair mercury levels of 186, 329, 1398, and 2436 ppm (Davis et al., 1994). The infant was born with severe neurological problems including seizures and died at age 21 years. B.

Prenatal Poisoning from Consumption of Contaminated Seafood

In the 1950s, the consequences of a massive industrial pollution at Minamata, Japan, were first recognized. A local chemical factory had been dumping untreated waste directly into Minamata Bay for many years, but in 1954, a mysterious neurological disease was first recognized in the area. The disorder was initially named Minamata disease (MD) because it was presumed to be an infectious disorder. Following several years of investigation, it was determined that MD was secondary to pollution and resembled MeHg poisoning. The fish and shellfish from Minamata Bay were reported to have MeHg levels in their flesh as high as 47 ppm (Tsubaki & Irukayama, 1977). It was not clear why human illness did not occur earlier since the factory had been dumping waste since 1935. However, two possible reasons were finally determined. In the 1950s, the company changed the chemical catalyst for acetaldehyde production to one that resulted in a greater production of MeHg as a by‐product. In addition, the factory increased the total production of acetaldehyde steadily from the late 1940s to 1960 (Nomura, 1968). The neurological findings of MD were ascribed primarily to MeHg, but the actual exposure was to multiple toxins present in the factory discharge. In addition to mercury, the factory waste was known to contain large

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amounts of silicon, iron, aluminum, calcium, magnesium, potassium, sodium, ammonia, copper, arsenic, manganese, chlorine, phosphorus, sulfur, and lead (Harada, 1972). Several of these toxins were originally considered to be the cause of MD. However, the patients’ symptoms were very similar to those of patients with known poisoning by MeHg and it was eventually ascribed to that cause alone. The contribution, if any, of the concomitant toxic exposures was never determined. In 1968, Harada reported 22 cases of fetal MD (Harada, 1968). The children were from six of the seven small fishing villages where cases of postnatal poisoning were found. All of the children reported had severe neurological deficits, including microcephaly, intellectual disability, cerebral palsy, and seizures. The children’s mothers appeared to be healthy, but five mothers reported having mild sensory symptoms. Among the 22 children described, 17 were from families whose occupation was fishing and seafood was a major component of their diet. In most cases, there were other family members with clinical MeHg poisoning. The children’s prenatal exposure levels were not determined because all of them were over 1 year of age when Harada first evaluated them. However, mercury levels were measured in the hair of the children and their mothers when they were evaluated. The children’s hair mercury levels ranged from 5 to 100 ppm and the mothers’ from 1 to 191 ppm. The hair mercury content of healthy infants under 1 year of age living in the Minamata district at that time ranged from 0 to 158 ppm. Following that report, there were other children who were said to have congenital MeHg poisoning, but the details of those cases have not been reported. In the mid‐1960s, a similar industrial pollution occurred near Niigata, Japan. Local health care workers recognized the cause of the poisoning early and MeHg levels were measured during pregnancy or shortly after delivery (Tsubaki & Irukayama, 1977). Health authorities oVered mothers with a hair Hg level of over 50 ppm the opportunity to terminate their pregnancies (Watanabe & Satoh, 1996). Only one infant at Niigata was formally diagnosed with congenital MeHg poisoning. Her hair THg level, measured shortly after birth, was 77 ppm. Her mother’s hair THg level measured at the same time was 293 ppm (Saito, 2004a). One other child was suspected of having prenatal poisoning, but this was not oYcially confirmed (Saito, 2004a). There were 12 mothers with hair Hg levels measured between 51 and 115 ppm who were pregnant and chose to deliver their infants. At delivery, all of those infants and their mothers appeared healthy. The children were examined by a pediatrician at age 5 years and reported to be normal (Moriyama & Takizawa, 2001). The nature of the evaluation was not specified. Adult follow‐up of those subjects was reported in 2004 (Saito et al., 2004b). Three of them completed middle school, six completed

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high school, and one each completed nursing school, business school, and university. Although MD appears to be poisoning with MeHg, those aVected were actually exposed to a mixture of toxic chemicals that included MeHg. The last subject with clinical symptoms from consumption of fish or seafood reported was born in Japan in 1965 (Saito, 2004a). Although there have been media reports that similar industrial pollution has occurred elsewhere, such as China, no cases of prenatal MeHg poisoning from consuming fish or seafood apart from those in Japan have been documented. Whether MeHg exposure from fish consumption in the absence of other toxins might cause similar findings is diYcult to determine from the industrial poisonings in Japan.

III.

DEVELOPING A HYPOTHESIS ABOUT PRENATAL LOW‐LEVEL METHYL MERCURY EXPOSURE

The episodes of poisoning from contaminated seed grain and seafood previously outlined led to the hypothesis that levels of exposure which have little or no detectable eVect on the mother can seriously damage the developing brain. However, of interest is that among the reported cases from Japan and elsewhere, there were no children with mild or moderate disability. There was one epidemiological study carried out at Minamata that tried to determine if there was such a spectrum. The study compared schoolchildren from the Minamata area with those from schools where minimal exposure was believed to have occurred (Fujino et al., 1976). The authors looked at the prevalence of abnormal mental and motor functions among school students. Their findings raised the intriguing possibility that varying levels of disability might have followed prenatal MeHg exposure in Japan. However, the study was not able to link the findings exclusively to MeHg because the exposure was to a mixture of toxins over several years and was not directly measured. In addition, there were some limitations in the survey methodology which included not measuring exposure in the children studied and pollution later found in the control area. Even so, the study raised the possibility of a spectrum of disability related to MeHg exposure. The poisoning in Iraq provided an opportunity to determine whether this hypothesis was correct. The outbreak was an acute exposure that took place over 3 to 4 months. In the winter of 1971–1972, the Iraq government imported over 70,000 metric tons of seed grain treated with MeHg (Bakir et al., 1973). The grain arrived after the planting season and some of it was ground into flour and consumed. The exposure was only to MeHg

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and the victims varied in the amount they consumed. An estimated 50,000 people were exposed, with over 6000 hospitalizations and 450 deaths documented (Bakir et al., 1973; Greenwood, 1985). The amount consumed varied within and among families and, consequently, it presented a unique opportunity to examine the eVects of prenatal exposure of varying degrees. Following the outbreak, a group of investigators from the University of Rochester undertook a study of children who were in utero during the exposure (Marsh et al., 1987). Mothers who were known to be pregnant during the exposure were interviewed in their homes. The children’s developmental milestones, such as age at which they first walked, were ascertained and the children examined neurologically. The children’s prenatal Hg exposure was determined by measuring the THg in the mother’s hair growing during pregnancy. The women in Iraq allow their hair to grow very long and, when the Hg in their hair was measured by segmental analysis, it provided an accurate picture of their exposure over the prior one or more years. When the association between the children’s prenatal exposure and their developmental milestones and neurological findings was examined, it showed a dose–response relationship (Cox et al., 1989). That relationship indicated that maternal hair levels as low as 10 ppm might be associated with adverse eVects on a child’s neurodevelopment. It was known that people who consume fish with no overt Hg contamination could have THg levels of 10 ppm or above in their hair (Airey, 1983; Matthews, 1983). Consequently, the studies in Iraq raised the question of whether these levels of exposure actually pose a risk to the child’s development. The Iraq studies were not adequate to answer this question because fish consumption was minimal and there were other study limitations (Marsh, 1994). Studies of people who actually consume fish were needed to confirm the hypothesis. Since millions of people around the world consume fish daily, it is possible to study this issue directly (Vannuccini, 2003).

IV. EPIDEMIOLOGICAL STUDIES OF FISH‐CONSUMING POPULATIONS A number of epidemiological studies were designed to test the hypothesis that the levels of exposure achieved by consuming fish that contain only background levels of contamination actually present a risk to the developing fetal brain. Investigators postulated that if low‐level exposure to MeHg did have adverse eVects on the developing brain that the manifestations

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would be subtle. Consequently, the study would require detailed evaluations of a large cohort in which individual fish consumption varied (and, hence, MeHg exposure) and careful control for the many covariates already known to have a significant influence on child development. Although many people consume fish, finding a population with a suitable exposure that could be studied in such detail was a diYcult task. So far, epidemiological studies have been reported from Brazil, Canada, French Guiana, New Zealand, Peru, the Philippines, and the Faroe and Seychelles Islands (Cordier et al., 2002; Davidson et al., 1998; Grandjean et al., 1997; Kjellstrom et al., 1986, 1989; Marsh et al., 1995; McKeown‐Eyssen et al., 1983; Myers et al., 2003; Ramirez et al., 2000, 2003). These epidemiological studies present a number of challenges to investigators and all of the studies have limitations. After finding a population with a suitable exposure, the investigators must measure it accurately. Studies that use surrogate measures of prenatal exposure, such as maternal hair measured when the children are several years old, are diYcult to interpret (Murata et al., 1999). Next, the cohort must agree to the study and reside in a setting where a study can be carried out. Ideally, the population should be studied repeatedly over time. Other limitations can include logistical issues, limited numbers of subjects, exposure to multiple coexisting potential toxins, unmeasured covariates, and other design limitations (Davidson et al., 2004). Ensuring reliable data collection presents diYculties that can, to a great degree, be overcome by rigorous study design, but there remain the issues of how to categorize and analyze the data and interpret and communicate the findings. Table II summarizes the epidemiological studies that have been reported to date. The table includes the authors’ opinion about whether the study shows an adverse association between low‐level prenatal MeHg exposure and various endpoints or not. Some investigators report associations and others do not. Among those reporting an association, the endpoints studied and reported to show an adverse association with prenatal MeHg exposure have varied. Among these studies, there are three that looked at relatively large populations over time and had suYcient power to detect subtle adverse changes in endpoints if they were present. These three studies have received the most attention and they will be discussed in more detail. The other studies are of interest, but a variety of factors, such as smaller cohorts, limited control of covariates, or single evaluations, makes them more diYcult to interpret. These limitations also make it diYcult to be confident that any changes detected might be causally related to prenatal MeHg exposure.

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TABLE II SUMMARY OF EPIDEMIOLOGY STUDIES EVALUATING PRENATAL (FETAL) METHYL MERCURY EXPOSURE Country/Cohort

Exposure source

N

Authors’ conclusion

Exposure

Reference Grandjean et al. (1999b) McKeown‐Eyssen et al. (1983) Cordier et al. (2002) Grandjean et al. (1997) Steuerwald et al. (2000) Kjellstrom et al. (1986) Kjellstrom et al. (1989) Marsh et al. (1995) Ramirez et al. (2000, 2003) Myers et al. (1995a) Davidson et al. (1998) Myers et al. (2003)

381

þ

Mean 11 ppm**

Canada

Gold mining Fish

234

þ

Mean 6 ppm**

French Guiana

Fish

378

þ

Faroes Main

Whale meat

1022

þ

Means 1.4 to 10.2 ppm** 1‐350 ppb*

Faroes PCB

Whale meat

182

þ

1‐102 ppb*

New Zealand 4y

Fish

79

þ

1‐89 ppm**

New Zealand 6y

Fish

350

þ

1‐89 ppm**

Peru

Fish

131

–

1‐30 ppm**

Philippines

78

þ

0‐130 ppb*

Seychelles Pilot

Gold mining Fish

781

–

1‐37 ppm**

Seychelles Main

Fish

789

–

1‐27 ppm**

Brazil

*, cord blood; **, maternal hair. Authors’ conclusion: þ indicates that the authors interpreted the study to show an adverse association between prenatal MeHg exposure and the endpoints studied.

A.

New Zealand

The first large study of prenatal low‐level MeHg exposure was reported from New Zealand (Kjellstrom et al., 1986, 1989). The cohort children were exposed to prenatal MeHg when their mothers consumed fish and chips. Fish and chips is widely consumed in New Zealand and is often made using shark meat. Some of the shark contained up to 4 ppm of Hg, a level not generally considered as background (Mitchell et al., 1982). The investigators reported adverse associations between the level of prenatal Hg exposure and several of their endpoints. The original study was published in a Swedish technical bulletin and it is not clear that it was peer reviewed (Marsh, 1994; U.S. EPA, 1997).

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However, the data were later reanalyzed and published in the peer‐reviewed literature (Crump et al., 1998). The study was complicated by several design issues that have been discussed elsewhere in detail (Marsh, 1994). These included a small sample size, the inclusion of children from three diVerent ethnic groups, each with a distinct and diVerent culture (Maori, European, and Pacific Islanders), and testing of controls and subjects at diVerent ages. When the reanalysis was done, an adverse association was again found. However, it was present only when one child with a very high level of exposure (89 ppm) was excluded. The National Academy of Sciences (NRC) chose to include this study in its review (2000), but some investigators continue to be cautious about how to interpret it. There are two large ongoing studies that are generally recognized as having adequate study designs and subject numbers that they might be able to determine whether prenatal MeHg exposure from consumption of fish containing background levels of MeHg might adversely aVect child development. One study is in the Republic of Seychelles and the other in the Faeroe Islands. Both studies have longitudinal designs and have been published extensively in the peer‐reviewed literature. B.

Seychelles Islands

In 1989–1990, the University of Rochester team that carried out the Iraq study initiated a longitudinal study of a population in the Seychelles Islands, the Seychelles Child Development Study (SCDS). The SCDS enrolled a main cohort that consisted of 779 children with prenatal exposure to MeHg from maternal fish consumption. In Seychelles, the ocean fish contains background Hg levels that average 0.3 ppm and the population does not consume sea mammals (Cernichiari et al., 1995b). At enrollment, the mothers reported consuming fish on average with 12 meals each week. Prenatal exposure was estimated by determining total and inorganic mercury in maternal hair growing during pregnancy. The prenatal exposure averaged 6.9 ppm with a range of 1 to 27 ppm. There was no evidence of exposure to other toxins and measured levels of lead and polychlorinated biphenyls (PCBs) were within normal limits (Davidson et al., 1998). A variety of covariates were measured, including maternal intelligence and the family’s socioeconomic status. Each family was visited at home to assess the child’s home environment using the Caldwell‐Bradley Home Observation of Measurement of the Environment (HOME). Each family participated in six test batteries through the child’s first 10.5 years of life. The children were evaluated when they were 6, 19, 29, 66, 107, and 128 months of age. Testing started with global measures and became increasingly focused on specific cognitive processes as the children matured (Davidson et al., 1995,

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1998; Myers et al., 1995b, 2003; Shamlaye et al., 1995). Through 2005, over 60 primary outcomes have been measured and the association of each with prenatal MeHg exposure examined using both linear and nonlinear models (Axtell et al., 1998, 2000; Huang et al., 2005). Evaluations over the first 9 years of the children’s lives found four statistically significant associations between prenatal MeHg exposure and specific outcomes. One association was adverse (the grooved pegboard using the nonpreferred hand at 9 years of age), two were beneficial (the Preschool Language Scale Total Language Score at 66 months and the Connors Teacher Rating Scale at 9 years of age), and one was diYcult to categorize (males had lower activity scores on the Bayley Scales of Infant Development–Infant Behavior Rating Scale at 29 months of age). The authors concluded that these associations were consistent with chance and that the study provided no support for an adverse association between child neurodevelopment and prenatal exposure to MeHg from maternal consumption of ocean fish at the levels being studied (Myers et al., 2003). The study was not able to rule out adverse eVects above 10 ppm in maternal hair because the cohort had a limited number of children with exposures in that range. The study also could not exclude the possibility that subtle adverse eVects might occur and be detected as the children mature. The study is ongoing and continues to be double blinded. Evaluations are planned for the cohort as they reach their teen years.

C.

Faeroe Islands

In 1986–1987, the Faeroe Islands research team enrolled a main cohort of 1022 children. The children’s prenatal exposure was from the maternal consumption of seafood during pregnancy. Fish in the Faeroe Islands are relatively low in Hg content, but the population also consumes whale meat and this was the primary source of Hg (Grandjean et al., 1997). Whale meat can contains up to 3 ppm of Hg, with equal parts MeHg and inorganic (Andersen et al., 1987). They measured prenatal exposure to Hg in both maternal hair and in cord blood taken at delivery. The geometric mean for the cord blood concentration of Hg was 22.9 ppb (interquartile range was 13.4–41.3 ppb with a maximum of 350 ppb) and for the maternal hair Hg 4.27 ppm (interquartile range 2.6–7.7 ppm with 15% over 10 ppm) (Grandjean et al., 1997). Whale blubber is also consumed in the Faeroes and it contains PCBs and other toxins. The investigators measured exposure to PCBs in umbilical cord tissue and reported elevated levels. The relationship of PCBs that are lipophilic measured in cord tissue that has virtually no fat to values present in other tissues is not clear.

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A number of covariates, such as maternal intelligence and socioeconomic status, were measured. The children were examined at 7 and 14 years of age, using an extensive battery of neuropsychological and neurophysiological tests. Following the 7‐year evaluations, the investigators reported the association between prenatal MeHg exposure measured in cord blood and 20 neuropsychological endpoints (Grandjean et al., 1997). Eleven of these endpoints had a significant association on one of three analytical models using ‘‘. . .a one‐tailed statistical significance level of 0.05. . .’’ (p. 425). These included adverse associations ‘‘. . .in the domains of language, attention, memory, and to a lesser extent in visuospatial and motor functions’’ (p. 417). Associations with maternal hair mercury levels were said to be present, but not as strong, and were not reported in detail. The contribution of PCBs and other toxins to these findings was addressed statistically in that and subsequent papers (Grandjean, 2004; Grandjean & Budtz‐Jorgensen, 1999; Grandjean et al., 1997, 2001). The investigators concluded that the adverse findings on multiple endpoints were primarily related to the MeHg exposure and that these eVects could be detected at a few weeks of life (Grandjean et al., 1997, 1998; Steuerwald et al., 2000). After the results of the 7‐year evaluations were published, oYcials in the Faeroes directed the investigators to tell the subjects their Hg values. Subsequently, the study was no longer double blind. The subjects were evaluated a second time at age 14 years. Only neurophysiological endpoints have been reported so far after that evaluation (Grandjean et al., 2004; Murata et al., 2004). These reports describe finding an association between prenatal MeHg exposure and several parameters of brainstem auditory evoked responses (BAERs). The authors reported ‘‘latencies of peak III and V increased by about 0.012 ms when the cord blood mercury doubled.’’ They also reported an association between prenatal MeHg exposure and heart rate variability as measured by the R–R interval on the EKG. However, they did not find an association between prenatal MeHg exposure and blood pressure (BP) that was reported at age 7 years (Sorensen et al., 1999). D.

Differences Between the Seychelles and Faeroe Islands Studies

These two studies were both well designed and executed and reached diVerent conclusions about the risk to a child’s neurodevelopment following prenatal MeHg exposure. Both studies examined large populations with significant MeHg exposure, used extensive test batteries, and included

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a large number of covariates. However, there are important diVerences between them. Perhaps most striking are the diets and exposure. In the Seychelles, the diet is fish, fruits, and vegetables while the Faeroes diet includes whale meat and blubber. Consuming whale blubber leads to exposure to other toxins such as PCBs, cadmium, pesticides, and other persistent organic pollutants. In addition, the Faeroe Islands are in the North Atlantic while the Seychelles are near the equator and essential micronutrients in fish, such as omega 3 fatty acids, are reported to diVer, depending upon the temperature of the sea. Fatty acids and micronutrients have been shown to have beneficial eVects on child development (Grantham‐McGregor & Ani, 1999; Koletzko et al., 2001). There are also genetic diVerences between the cohorts. The heritage of the Faeroes is Scandinavian while that of Seychelles is primarily African. In addition, there are a number of diVerences in the study designs, covariates measured, statistical analysis plan, and other aspects of the studies that may account for the diVerent findings.

V. A.

INTERPRETING THE AVAILABLE DATA

Governmental Interpretations

Using the data from the studies described, health and environmental authorities have tried to establish what they consider safe levels of MeHg exposure. In 1990, an expert panel organized by the World Health Organization concluded that ‘‘. . .a prudent interpretation of the Iraqi data implies that a 5% risk may be associated with a peak mercury level of 10–20 m{ts}/g [ppm] in the maternal hair’’ (WHO, 1990, p. 103). In 1997, the U.S. Environmental Protection Agency (EPA) reviewed the data (U.S.E.P.A., 1997) and determined that the reference dose (RfD) should be set at 0.1 mg/kg/day. This translates to a maternal hair level of approximately 1.2 ppm, or a blood level of 5.8 ppb. The EPA defines an RfD as ‘‘a safe dose to consume daily over a lifetime. . ..’’ To establish the RfD, the EPA determined what they felt was the lowest level of exposure associated with any adverse eVect (58 ppb measured in blood). They then took one‐tenth of that number so there would be a safety or uncertainty factor. They based their determination on the Iraq study initially, but when the Faeroe Islands study was published in 1997, they recalculated the RfD. Although the studies in Iraq and the Faeroe Islands diVered markedly, the RfD determination was identical. The FDA in 1979 established an ADI (acceptable daily intake) for Hg in adults of 0.4 ug/kg/day (Tollefson & Cordel, 1986). In 1999, the Agency for Toxic Substances and Disease Registry (ATSDR) set the permissible

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exposure level at 0.3 mg/kg/day (ATSDR, 1999), based on data from the Seychelles studies. At the international level, the Joint FAO/WHO Committee on Food Additives met in 2003 and determined that 1.5 ug of MeHg/kg bw/week or 0.21 ug/kg/day could be consumed daily over a lifetime. In Japan, the Ministry of Health, Labour, and Welfare established a PTWI (permissible tolerable weekly intake) in 1973 that translates to about 0.4 ppm of THg, or 0.3 ppm of MeHg. These determinations of a safe dose to consume by varying health authorities are all based on individual interpretations of the same data. Some authorities based their determination on one study. The EPA based their RfD on the Faroes study while ATSDR used the Seychelles study. Others, such as the Joint FAO/WHO Committee on Food Additives, considered both studies. Despite these diVerent approaches and the use of diVerent uncertainty factors, the determinations are fairly similar. It is not clear presently which determination is most appropriate to protect human health. B.

Benchmarks

When one determines the benchmark dose from the various studies, they too seem to fall within a narrow range. Nearly all of the outcomes from the various studies on which a benchmark dose has been determined indicate that an exposure level around 10–20 ppm in maternal hair may carry a risk to the infant. This is shown in Fig. 1 (Clarkson & Strain, 2004). Benchmark dose calculations from the Faeroe Islands fall in the lower part of this range, while those from the Seychelles fall in the upper part of the range (Crump et al., 2000). C.

Factors Contributing to the Differing Interpretations

Numerous factors probably contribute to these diVerences in interpretation. However, the following may be especially important. 1. EXPOSURE BIOMARKER

Toxins can be measured in many biological media and mercury is no exception. Selecting the biomarker that most accurately defines the exposure is critical. With MeHg, the critical organ is the brain, but there is no direct way to measure brain levels in living subjects. Hair measurements have been the standard for many years and in both the Seychelles and Faeroe Islands, exposure was measured in maternal hair growing during pregnancy. However, in the Faeroe Islands, exposure was also measured in cord blood. Cord blood recapitulates exposure that occurs during the last few weeks of pregnancy. A bolus exposure occurring earlier in pregnancy, such as from

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FIG. 1. Benchmark evaluations of epidemiological studies. NOAEL, no observed adverse eVect level. There are two calculations from New Zealand, depending on whether the single child with the highest exposure is included (1) or excluded (2). Reprinted with permission from Clarkson, T. W., & Strain, J. J. (2004). Methyl mercury: Loaves versus fishes. Seychelles Medical Dental Journal, 7(1), 64.

consumption of a whale meal, would not be detected by measuring cord blood. If hair is long enough and analyzed in segments, it can recapitulate exposure during the entire 9 months of pregnancy. Consequently, a larger exposure at any time during the pregnancy would appear in hair. Autopsy studies have confirmed that maternal hair Hg levels correlate highly with brain levels (Cernichiari et al., 1995a). The association of cord blood mercury and brain levels has not been reported. Presently, the choice of the optimal biomarker is controversial. 2. CONCOMITANT EXPOSURES

Most human exposures are to mixtures of toxins, such as occurred with the pollution from factories in Japan. Although the toxicity of one toxin may predominate, there may be contributions from the other toxins present. For example, there are reports that concomitant exposure to MeHg and PCBs may have synergistic toxicity (Risher et al., 2003; Stewart et al., 2003). Investigators with the Faeroes study report their findings are mainly associated with MeHg exposure and that they can statistically factor out the contribution of other toxins. Some authorities feel that statistical methods can accomplish this while others do not.

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3. OUTCOME MEASURES

The selection of the most sensitive outcome to measure presents a special problem. Studies in the Iraq poisoning showed a dose–response relationship between exposure levels and the attainment of developmental milestones and neurological findings on examination (Cox et al., 1989). However, with low‐ level exposure from fish consumption, investigators are looking for more subtle developmental diVerences. The Seychelles and Faeroe Island studies approached the outcome measures diVerently. In the Seychelles, subjects were evaluated first for mild diVuse cognitive changes, such as general intelligence, neurological findings, and milestones. As the children matured, testing focused on increasingly specific motor and cognitive functions and processes. In the Faeroe Islands, testing focused on domain‐specific functions and neurophysiological tests for vision, hearing, and central processing from the beginning (White et al., 1993). The problem selecting an outcome is highlighted by the National Research Council (NRC) review (2000). The committee was charged with reviewing the available data and asked to determine ‘‘. . . the appropriateness of the critical study, end points of toxicity, and uncertainty factors used by EPA in the derivation of the reference dose for MeHg’’ (p. xii). The endpoint that had the lowest Benchmark Determination (BMDL) turned out to be the McCarthy Scales of Children’s Abilities (MSCA) from the New Zealand study. However, the NRC committee rejected it, based on the limitations of that study (p. 285). The next lowest BMDL was on the Continuous Performance Test (CPT) from the Faeroe Islands study. The committee also rejected it because of technical diYculties in test administration (p. 286). The third lowest BMDL was on the Boston Naming Test (BNT) from the Faeroe Islands. The panel chose the BNT stating that it was ‘‘. . . the most sensitive, reliable end point’’ (p. 299). Using the BMDL from the BNT, the panel concluded that the EPA’s RfD was appropriate. However, selection of the BNT raises additional concerns. The BNT was developed to detect aphasia and brain damage in adults and is not a standard part of child neuropsychological testing. No biological reason has been proposed as to why the BNT should be particularly sensitive to prenatal MeHg exposure. The Faeroe Islands investigators themselves have reported concerns about the BNT. In 1997, they wrote, ‘‘. . . especially for the Boston Naming Test, the PCB concentration appeared to be an important predictor’’ (Grandjean et al., 1997, p. 425). They later stated ‘‘. . . the cord PCB concentration was associated with deficits on the Boston Naming Test. . .’’ and ‘‘. . . the association between cord PCB and cord‐blood mercury (r ¼ 0.42) suggested possible confounding’’ (Grandjean et al., 2001, p. 305). Selecting an appropriate outcome to focus upon presents a substantial

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challenge to investigators and those interpreting their studies to use in making public policy. 4. STATISTICAL ANALYSIS

There are many approaches that can be taken to analyze the data and investigators must choose. The Seychelles study established a priori a linear analysis plan that they considered the primary analysis. Subsequent nonlinear and other analyses were considered confirmatory or secondary. The Faeroe Islands study performed three diVerent analyses on the data from their 7‐year‐old evaluations and reported all three models (Grandjean et al., 1997) Among the models, there were 11 endpoints that showed statistically significant adverse associations with prenatal MeHg exposure. Interpreting these findings was challenging because the significant endpoints were not consistent among the diVerent models. In addition to the factors already highlighted, there are numerous others such as the selection and measurement of covariates that make epidemiological studies challenging to carry out and to interpret (Davidson et al., 2004).

VI.

WHAT CONSTITUTES A DEVELOPMENTAL DISABILITY?

The term ‘‘developmental disability’’ is a political one. The definition presented by the Center for Communicable Diseases (CDC) of the U. S. Department of Health and Human Services is ‘‘Developmental disabilities are a diverse group of severe chronic conditions that are due to mental and/ or physical impairments. People with developmental disabilities have problems with major life activities such as language, mobility, learning, self‐help, and independent living. Developmental disabilities begin anytime during development up to 22 years of age and usually last throughout a person’s lifetime.’’ (CDC, 2005). The subtle diVerences in cognitive and motor performance that have been reported to have an association with prenatal MeHg exposure are all far below the level of clinical significance (see this volume’s Introduction). As an example, the diVerence in finger‐tapping reported to be present between high‐ and low‐exposure groups in the Faeroe Islands consisted of four finger taps using both hands in 15 seconds. Tapping with either hand individually was nearly identical and there was substantial overlap in scores between the two groups (Grandjean et al., 1998). Finger‐tapping and the other reported findings from epidemiological studies are statistical associations that have no clinical relevance for an individual. Even so, it is important to know whether prenatal MeHg exposure can consistently be shown to be associated with a change in these endpoints. If so, it would strengthen the conclusion that

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there is a causal relationship between low‐level prenatal MeHg exposure and adverse eVects on the developing nervous system. However, the diVerences reported to date by epidemiological studies could not be considered developmental disabilities since they would have no impact on the child’s ability to function. VII.

DOES EXCEEDING THE EPA’S RFD PLACE A CHILD AT DEVELOPMENTAL RISK?

If we accept that the reported associations between prenatal MeHg exposure and child development are indeed true, although this is by no means a certainty, would they constitute a developmental disability? Several papers have equated exceeding the RfD as placing the child at developmental risk (MahaVey et al., 2004a; Trasande et al., 2005). To understand whether this is likely to be true, it is helpful to examine the definition of the RfD. As noted earlier, the RfD is the dose that can be consumed daily over a lifetime without a risk of adverse eVects. The RfD determined by the EPA for MeHg includes a safety or uncertainty factor of 10, that is, the EPA determined what they believed was the lowest level of exposure thought to be associated with adverse eVects and divided that number by 10. The lowest level thought to be associated with harm to the fetus is, of course, open to substantial controversy. It seems unlikely that exceeding the RfD, even on a regular basis, would place the child at significant risk of any clinical health problems. To illustrate the clinical impact of the reported changes, one of the strongest pieces of evidence for a detectable change among subjects of varying prenatal MeHg exposure from the Faeroe Islands study is on the BAERs at 14 years of age (Murata et al., 2004). Murata and colleagues reported that the latency of wave III and V increased by 0.012 ms for each doubling of the cord Hg exposure. There are no data to suggest that a change of 1/100,000th of a second would constitute a significant impairment in auditory processing and it would not be considered a developmental disability. Thus, there is no evidence that exceeding the RfD, even on a regular basis, would place the child at risk of having a developmental disability. VIII.

HOW DID THE NRC AND THE EPA DETERMINE THE RISK TO UNITED STATES CHILDREN?

The process by which the NRC determined that 60,000 U.S. children were at risk from MeHg exposure was explained in a letter from the committee chair to the U.S. Food and Drug Administration dated December 1, 2000, and later published (MahaVey et al., 2004a). The determination was made as follows:

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Start with the RfD determined by the EPA

0.1 ug/kg/d

Take the population of U.S. women of childbearing age (15–44 years). [Based on the U.S. Census Bureau data from 1989]

60,208,000

Take the percentage of women who reported consuming fish [based on the Continuing Surveys of Food Intake by Individuals conducted in 1989–1990, this is approximately 30.5%]

18,363,440

Take the number of those women who reported consuming over 100 grams of fish per day (approximately 5%)

918,172

Take the birth rate for U.S. women

65.6/1000

Multiply the number of women consuming over 100 grams of fish per day by the birth rate for U.S. women

60,232

This formula determines the number of U.S. children who might exceed the RfD established by the EPA. It does not determine the number of children who might have a developmental disability from MeHg exposure secondary to maternal consumption of fish. Indeed, the number was obtained without requiring that there be even one child with a developmental disability secondary to MeHg exposure from fish consumption. Subsequently, the number of U.S. children at risk was increased to 300,000, based on a recalculation of this sequence of assumptions using more recent data from the 2000 NHANES survey (MahaVey et al., 2004a; Rice, 2003). Not surprisingly, fish consumption was substantially higher in the NHANES survey since health authorities had encouraged it for its reported cardiac benefits. The number was later increased to over 600,000 U.S. children at risk based on the recognition that fetal hemoglobin binds more tightly to MeHg (Trasande et al., 2005). This had been known for many years (Amin‐Zaki et al., 1976), but was apparently not considered when the EPA originally determined its RfD.

IX.

CONCLUSIONS

Mercury is ubiquitous in our environment and the amount that we come in contact with is increasing. The organic form of MeHg is very toxic to the central nervous system. All fish contain small amounts of MeHg and everyone who consumes fish or seafood is exposed to it. Once inside the body, MeHg readily crosses the placenta and the blood–brain barrier and enters the central nervous system. Methyl mercury poisoning episodes have clearly demonstrated that the developing fetal brain is especially sensitive and can be seriously impaired, even when the mother has no symptoms.

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The events in Japan confirm that consuming fish that is heavily contaminated with MeHg can damage the developing central nervous system. However, interpreting the Japanese experience is complicated by the very high exposures (at Minamata, some fish contained up to 50 ppm of MeHg or 100 times the background level usually found) and the presence of other toxins in the factory waste. Even so, studies of poisoning in Iraq suggested that a level of prenatal exposure of around 10 ppm measured in maternal hair might adversely aVect the developing fetus. Individuals who consume fish with only background levels of MeHg can achieve a hair level of 10 ppm or greater. However, there are no reported cases of mild disability after prenatal MeHg exposure and no reports of poisoning from fish consumption outside of Japan. The evidence that low levels of MeHg exposure from fish consumption might adversely aVect a child’s neurodevelopment presently comes only from epidemiology studies in Iraq and elsewhere. As has been noted, these studies are diYcult to carry out and equally hard to interpret. To date, these studies have not been conclusive and the evidence that MeHg exposure at the levels achieved by consuming fish that is not overtly polluted adversely aVects the fetal brain is very controversial. The level of MeHg exposure at which adverse eVects to the developing brain first occur is not presently known. DiVerent health and environmental authorities interpret the epidemiological studies of MeHg exposure from maternal fish and seafood consumption diVerently and the risk of fetal neurological harm from this source requires further study. There is general agreement among health authorities that fish consumption has significant benefits. Fish is an important source of protein, long chain fatty acids, and micronutrients. The American Heart Association encourages the consumption of two fish meals a week to maintain a healthy heart (AHA, 2005). Consequently, it is important to maintain a balanced view of fish consumption. Although fish consumption carries theoretical risks from MeHg exposure, it also carries proven benefits. Many scientists are not prepared to make the multiple assumptions necessary to arrive at the conclusion that large numbers of U.S. children are presently at significant risk for a developmental disability from exposure to low‐dose MeHg. There is agreement, however, that the high‐quality study done in the Faeroe Islands shows conclusively that the consumption of whale meat and blubber can have adverse health eVects and health authorities there have appropriately discouraged its consumption by women of childbearing age (Weihe et al., 2005). However, the Faeroes study does not clarify which toxin present in whales causes the harm. Since millions of people around the world consume fish daily, it should be possible to study this issue directly without extrapolating from populations having unusual

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diets, exposures to multiple toxins, or overt poisoning. Although current evidence does not confirm health risks at these low levels of exposure, it does seem prudent to reduce the anthropogenic sources contributing to Hg in our environment since, at some level of exposure, there will be quantifiable health risks. Most health authorities agree that fish consumption has clear health benefits and should be encouraged. We do not believe it is scientifically sound to use the EPA’s RfD as a level that, if exceeded, leads to a developmental disability. Nor do we believe that current evidence supports the presence of subtle adverse eVects on child development from the consumption of ocean fish at prenatal exposures below 10 ppm measured in maternal hair. Ongoing research should help to clarify the level of exposure that is associated with subtle diVerences in children’s neurodevelopment. REFERENCES Airey, D. (1983). Total mercury concentrations in human hair from 13 countries in relation to fish consumption and location. Science of the Total Environment, 31, 157–180. AHA (American Heart Association) (2005). Fish and omega‐3 fatty acids. http://www. americanheart.org/presenter.jhtml?identifier¼4632 accessed 5/22/05. Amin‐Zaki, L., Elhassani, S., Majeed, M. A., Clarkson, T. W., Doherty, R. A., & Greenwood, M. (1974). Intra‐uterine methylmercury poisoning in Iraq. Pediatrics, 54, 587–595. Amin‐Zaki, L., Elhassani, S., Majeed, M. A., Clarkson, T. W., Doherty, R. A., Greenwood, M. R., & Giovanoli‐Jakubczak, T. (1976). Perinatal methylmercury poisoning in Iraq. American Journal of Diseases of Children, 130, 1070–1076. Amin‐Zaki, L., Majeed, M. A., Elhassani, S. B., Clarkson, T. W., Greenwood, M. R., & Doherty, R. A. (1979). Prenatal methylmercury poisoning. Clinical observations over five years. American Journal of Diseases of Children, 133, 172–177. Andersen, A., Julshamn, K., Ringdal, O., & Morkore, J. (1987). Trace elements intake in the Faroe Islands. II. Intake of mercury and other elements by consumption of pilot whales (Globicephalus meleanus). Science of the Total Environment, 65, 63–68. ATSDR (Agency for Toxic Substances and Disease Registry) (1999). Toxicological profile for Mercury. U. S. Department of Health and Human Resources. Division of Toxicology/ Toxicology Information Branch, Atlanta, Georgia. Axtell, C. D., Cox, C., Myers, G. J., Davidson, P. W., Choi, A. L., Cernichiari, E., Sloane‐ Reeves, J., Shamlaye, C. F., & Clarkson, T. W. (2000). Association between methylmercury exposure from fish consumption and child development at five and a half years of age in the Seychelles Child Development Study: An evaluation of nonlinear relationships. Environmental Research, 84, 71–80. Axtell, C. D., Myers, G. J., Davidson, P. W., Choi, A. L., Cernichiari, E., Sloane‐Reeves, J., Shamlaye, C., Cox, C., & Clarkson, T. W. (1998). Semiparametric modeling of age at achieving developmental milestones after prenatal exposure to methylmercury in the Seychelles child development study. Environmental Health Perspectives, 106, 559–563. Bakir, F., Damluji, S. F., Amin‐Zaki, L., Murtadha, M., Khalidi, A., Al‐Rawi, N. Y., Tikriti, S., Dhahir, H. I., Clarkson, T. W., Smith, J. C., & Doherty, R. A. (1973). Methylmercury poisoning in Iraq. Science, 181, 230–241.

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mercury from fish. Stage 2. Interviews and psychological tests at age 6. Solna, National Swedish Environmental Board, 112 pp (Report no. 3642). Koletzko, B., Agostoni, C., Carlson, S. E., Clandinin, T., Hornstra, G., Neuringer, M., Uauy, R., Yamashiro, Y., & Willatts, P. (2001). Long chain polyunsaturated fatty acids (LC‐ PUFA) and perinatal development. Acta Paediatrica, 90, 460–464. Kraepiel, A. M., Keller, K., Chin, H. B., Malcolm, E. G., & Morel, F. M. (2003). Sources and variations of mercury in tuna. Environmental Science & Technology, 37, 5551–5558. MahaVey, K. R., Clickner, R. P., & Bodurow, C. C. (2004a). Blood organic mercury and dietary mercury intake: National health and nutrition examination survey, 1999 and 2000. Environmental Health Perspectives, 112, 562–570. MahaVey, K. (2004b). Session VI‐ Methyl Mercury: Exposure EVects in Susceptible Groups. Impact on Children. The Toxicology Forum 29th Annual Winter Meeting. February 2–4, 2004, Washington, DC. http://www.toxforum.org/Winter_2004.pdf accessed 5/17/05. Marsh, D. O., Clarkson, T. W., Cox, C., Myers, G. J., Amin‐Zaki, L., & Al‐Tikriti, S. (1987). Fetal methylmercury poisoning. Relationship between concentration in single strands of maternal hair and child eVects. Archives of Neurology, 44, 1017–1022. Marsh, D. O. (1994). Organic Mercury: Clinical and neurotoxicological aspects. Handbook of Clinical Neurology, 20, 413–429. Marsh, D. O., Turner, M. D., Smith, J. C., Allen, P., & Richdale, N. (1995). Fetal methylmercury study in a Peruvian fish‐eating population. Neurotoxicology, 16, 717–726. Matthews, A. D. (1983). Mercury content of commercially important fish of the Seychelles and hair mercury levels of a selected part of the population. Environmental Research, 30, 305–312. McKeown‐Eyssen, G. E., Ruedy, J., & Neims, A. (1983). Methyl mercury exposure in northern Quebec. II. Neurologic findings in children. American Journal of Epidemiology, 118, 470–479. Miller, G. E., Grant, P. M., Kishore, R., Steinkruger, F. J., Rowland, F. S., & Guinn, V. P. (1972). Mercury concentrations in museum specimens of tuna and swordfish. Science, 175, 1121–1122. Mink, P. J., Goodman, M., Barraj, L. M., Imrey, H., Kelsh, M. A., & Yager, J. (2004). Evaluation of uncontrolled confounding in studies of environmental exposures and neurobehavioral testing in children. Epidemiology, 15(4), 385–393. Mitchell, J. W., Kjellstrom, T. U., & Reeves, R. L. (1982). Mercury in takeaway fish in New Zealand. New Zealand Medical Journal, 95, 112–114. Moriyama, H., & Takizawa, Y. (2001). Assessing low‐dose eVects on children exposed in utero to methylmercury—Protocol for on‐the‐spot re‐inspection for Minamata and Niigata data at early stage. In Y. Takizawa & M. Osame (Eds.), Methylmercury poisoning in Minamata and Niigata, Japan (pp. 54–60). Tokyo: Japan Health Association. Murakami, U. (1972). The eVect of organic mercury on intrauterine life. Advances in Experimental Medicine & Biology, 27, 301–336. Murata, K., Weihe, P., Renzoni, A., Debes, F., Vasconcelos, R., Zino, F., Araki, S., Jorgensen, P. J., White, R. F., & Grandjean, P. (1999). Delayed evoked potentials in children exposed to methylmercury from seafood. Neurotoxicology & Teratology, 21, 343–348. Murata, K., Weihe, P., Budtz‐Jorgensen, E., Jorgensen, P. J., & Grandjean, P. (2004). Delayed brainstem auditory evoked potential latencies in 14‐year‐old children exposed to methylmercury. Journal of Pediatrics, 144, 177–183. Myers, G. J., Davidson, P. W., Cox, C., Shamlaye, C. F., Palumbo, D., Cernichiari, E., Sloane‐ Reeves, J., Wilding, G. E., Kost, J., Huang, L., & Clarkson, T. W. (2003). Prenatal methylmercury exposure from ocean fish consumption in the Seychelles child development study. Lancet, 361, 1686–1692.

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Environmental Agents and Autism: Once and Future Associations SUSAN L. HYMAN DEPARTMENT OF PEDIATRICS (GOLISANO CHILDREN’S HOSPITAL AT STRONG), UNIVERSITY OF ROCHESTER SCHOOL OF MEDICINE AND DENTISTRY, ROCHESTER, NEW YORK

TARA L. ARNDT DEPARTMENT OF ENVIRONMENTAL MEDICINE, UNIVERSITY OF ROCHESTER SCHOOL OF MEDICINE AND DENTISTRY, ROCHESTER, NEW YORK

PATRICIA M. RODIER DEPARTMENT OF OBSTETRICS AND GYNECOLOGY, UNIVERSITY OF ROCHESTER SCHOOL OF MEDICINE AND DENTISTRY, ROCHESTER, NEW YORK

I.

INTRODUCTION

Autism is a neurodevelopmental disorder whose symptoms are manifest along a continuum, hence, the common use of the term, Autism Spectrum Disorders (ASD). The Diagnostic and Statistical Manual—IV (American Psychiatric Association, 1994) defines a general set of symptoms related to deficits in social reciprocity, communication, and repetitive behaviors that can be clustered into four specific diagnoses within the spectrum. However, the heterogeneity of clinical presentations even within those subgroups suggests that there is no single etiology or neurobiologic cause. Evidence for the genetic contribution to the etiology of autism was reported as early as 1977 (Folstein & Rutter, 1977). The genetics of autism has been an active area of epidemiologic and molecular genetic pursuit (Bailey et al., 1995; Stodgell et al. [rev.], 2001b). The support for a genetic component to the etiology of autism is based on the observation of recurrence in siblings of almost 4% (Chakrabarti and Fombonne, 2001), which is much greater than the risk for the disorder in the general population. The concordance for INTERNATIONAL REVIEW OF RESEARCH IN MENTAL RETARDATION, Vol. 30 0074-7750/06 $35.00

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Copyright 2006, Elsevier Inc. All rights reserved. DOI: 10.1016/S0074-7750(05)30005-X

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autism is 60% in identical twins, with some symptoms of autism present in over 92% of the identical twin of an aVected individual (Bailey et al., 1995). Larger twin studies will be an important avenue to investigate the role of genetic and environmental factors in the etiology of autism. The role of environmental agents and the environment itself in the development of the symptoms of autism has been the source of speculation and hypothesis generation since Leo Kanner first described autism in 1943 (Kanner, 1943). Indeed, some of the hypotheses generated based on clinical observations in his original cases remain areas of study to this day, the genetic contribution being a prime example. Some of the early etiologic hypotheses have succumbed to advances in behavioral and medical science, however. This chapter summarizes the hypotheses related to role of the environment in autism. It will not only examine how environmental factors might be etiologically related to autism but also how the thinking around environmental influences shaped treatment for and beliefs about the disorder.

II.

THE FAMILY ENVIRONMENT

In 1943, Kanner first reported a case series of children who had social isolation, atypical language development, and intense and unusually ritualized behaviors (Kanner, 1943). He noticed that, among his first 11 patients with this symptom complex, there seemed to be a pattern of cold and obsessive parents, professional families, and atypical attachment of parent and child. He did note that he thought the disorder was of organic origin. The idea of a broader phenotype, where family members could share some lesser characteristics with the aVected family member and still be subthreshold for the diagnosis, was not yet conceptualized (Dawson et al., 2002). The prevailing explanations for behavioral disorders in the years after World War II lay in psychological theories related to the relationship of an individual with his/her parents. This was specifically applied to autism by Bruno Bettelheim, a self‐proclaimed expert in child psychology and development whose Ph.D. was in philosophy. He advanced the hypothesis that autism was the result of maternal rejection (Bettelheim, 1967). He advocated that aVected individuals could only be helped by removal from their families and application of treatments that involved both language‐based therapies and physical management to repair a dramatically abnormal bonding experience with a ‘‘refrigerator’’ mother. The Orthogenic School he ran in Chicago was the site of much of this treatment. Later review of the case histories of his patients suggests many would not have been diagnosed with

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autism as defined by Kanner. The belief that autism was based on poor parenting, however, was the prevailing theory explaining autism through the 1950s and into the 1960s. A generation of families suVered shame and guilt because they thought that they provided a socially toxic environment that caused their child’s autism. It is possible that the disorder was underdiagnosed because of the stigma of blame for the parents in addition to the lack of acceptable or eVective treatments. Insight‐oriented psychotherapy and other language‐based psychodynamic interventions, which were the treatments used by psychiatrists in the 1950s and 1960s, did not address the core symptoms of the disorder and were not successful in teaching people with autism how to interact socially.

III. A.

CONSIDERATION OF BIOLOGIC ETIOLOGIES

The Prenatal Environment

1. OBSTETRIC COMPLICATIONS

By the end of the 1950s, the biologic bases for many neurologic and psychiatric disorders were being investigated. For example, the role of asphyxia, metabolic instability, and placental compromise during gestation and delivery were increasingly linked with developmental disabilities. The National Institute for Child Health funded the largest study to date to identify the factors associated with birth injury and later neurologic handicap in the Collaborative Perinatal Project. Between 1959 and 1961, exhaustive data were collected on over 50,000 pregnancies. For seven years, targeted neurologic disorders manifesting in childhood were prospectively monitored in this sample. Autism was one of them (Torrey et al., 1975). At that time, the diagnostic criteria for autism were relatively vague and the distinction from childhood psychosis and mental retardation was not always clear. Only 14 children were reported to have had autism in this birth cohort. (The prevalence would be less than 3:10,000 if all children with autism were identified.) When the obstetrical data were analyzed for this subset of children, the presence of bleeding in pregnancy compared to controls was the only obstetric finding that almost reached statistical significance. It was noted that more fathers of children with autism were employed as chemists than would have been expected by chance. This finding was rediscovered in a modern sample examining the potential for cognitive profiles of technical professions such as engineers and computer scientists as opposed to social workers that might be associated with a genetic predisposition to autism (Wheelwright & Baron‐Cohen, 2001). This modern interpretation of the observation suggested that a genetic predisposition for features of autism

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in family members related to enhanced visual problem‐solving skills rather than an occupational exposure might be the association leading to autism in the oVspring. Subsequent to the Collaborative Perinatal Project, investigators probed the potential association of obstetric factors with autism using retrospective examination of birth records and birth histories of children later diagnosed with autism compared to cohort controls and unaVected siblings. These more recent studies had the benefit of more discrete diagnostic criteria for autism, so cases defined as having autism probably represented a more homogeneous group. The families who participated in an epidemiological study in Nova Scotia (Bryson et al., 1988) reported a higher incidence of neonatal respiratory distress in children with autism. However, no consistent findings related to gestational age, birth weight, asphyxia, or obstetric intervention has been identified across studies. It has been noted that increased obstetric risk characterizes the families of children with autism (cases and unaVected siblings compared to controls), suggesting that the obstetric findings are not causal for the autism but are somehow related to a genetic and/or environmental risk in the family (Table I). Although no consistent associations of individual events with autism have emerged when the obstetric and neonatal histories are examined, the neuroanatomical findings reported in brains of people with autism indicate that the abnormalities most often seen are of prenatal origin. Bauman and Kemper (1985) reported increased hippocampal cellular density and decreased cellularity of the cerebellum. Rodier et al. (1996) reported hypoplasia of brain stem nuclei. Bailey et al. (1998) identified heterotopias and atypical development of the inferior olive in some cases of autism. Both autopsy and imaging studies have identified the cerebellum as abnormal in some cases of autism. The most consistent pathologic finding is decreased number of Purkinje cells in the cerebellum (Bailey et al., 1998; Bauman & Kemper, 1985). Courchesne has reported hypoplasia of the cerebellar vermis on MRI, although this finding is variable (Courchesne et al., 1988). The neuroanatomic studies have not identified a specific brain region involved in all cases or pinpointed a time in prenatal development that the neurologic insult occurred. However, the reports all identify abnormalities that occurred between the first weeks and last trimester of gestation. The periventricular leukomalacia, cysts, and ventricular dilatation associated with asphyxic injury do not appear in the histology of idiopathic autism (Bailey et al., 1998). Prenatal events related to atypical brain development and subsequent atypical function are likely to play a role in the etiology of autism. How genetic factors, environmental embryologic events, and obstetric optimality may interact is an area deserving of further study.

TABLE I SEVERAL STUDIES IDENTIFY AN INCREASED RATE OF OBSTETRIC AND PERINATAL COMPLICATIONS IN BOTH CHILDREN WITH ASD AND SIBLINGS, SUGGESTING A FAMILIAL RISK Authors

Cases

Controls

Diagnosis

Glasson, Bower, Petterson et al. (2004)

465 ASD

481 Sibs 1313 population controls

Record review, DSM–IV criteria

Hultman, Sparen, Cnattangius et al. (2002)

408 ASD

Matched control 2040

Swedish Medical Birth Registry

Zwaigenbaum, Szatmari, Jones et al. (2002) Juul‐Dam, Townsend, & Courchesne (2001)

78 ASD

88 Sibs

ADI/ADOS diagnosis

61 Autism 13 PDD, NOS

Report of Final Natality Statistics, 1995

ADI/ADOS

110 Autism

50 Down syndrome

ADI, ADOS

Bolton, Murphy, MacDonald et al. (1997)

Findings Cases had older parents, were firstborns, fetal distress, Apgar <6 at 1 minute, fetal distress, induction, threatened abortion, labor <1 hour, c‐section, maternal anesthesia. Sibs were more similar to cases than controls Cases had increased maternal smoking in early pregnancy, c‐section, small for gestational age, lower Apgar scores, maternal birth outside of Europe or North America, and more congenital malformations Decreased obstetric optimality in both groups Autism group had increased maternal uterine bleeding, lower maternal vaginal infection, or use of contraceptives during conception. PDD, NOS group had increased hyperbilirubinemia Strong relationship of optimality and rate of disorder in first‐degree relatives

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2. PRENATAL INFECTIONS

One of the techniques used to identify causative factors for a disorder involves epidemiologic surveillance. This often occurs after an initial clinical observation leads to a hypothesis associating an event with an outcome. This is particularly valuable in the case of infectious agents that occur in circumscribed epidemics. Clinical correlation of disorders led to the identification of congenital rubella as a risk factor for autism. In the late 1960s, an epidemic of rubella swept the United States. Over 20,000 pregnant women delivered fetuses exposed to maternal rubella in utero. Chess (1971) made the association of intrauterine infection with a syndrome that included visual impairment, motor impairment, deafness, heart disease, mental retardation, and autism. In this sample of rubella‐exposed cases, autism appeared to occur only in children with multiple handicaps (Chess & Fernandez, 1980). In a study specifically designed to identify the critical periods for eye defects, deafness, mental retardation, and heart malformations after rubella exposure, Ueda et al. (1979) collected data on the time of onset of rash in mothers who contracted rubella. The result was that oVspring with multiple symptoms came mainly from those exposed within the first 8 weeks post‐conception. The same study showed that mothers whose oVspring had severe mental retardation had onset of rash in the second to fifth week post‐conception. The severity of the epidemic was such that the research necessary to develop and test a vaccine was accelerated. By 1972, a rubella vaccine was in widespread use in the United States and soon became mandated for public school attendance. Rubella all but disappeared. Thirty years later, the causative association of brain damage from maternal rubella infection during critical periods of intrauterine life was extrapolated to implicate immunization in the second year of life with attenuated virus as a cause of autism with regression (Wakefield et al., 1998). This paper was retracted by most of the authors in 2004 (Murch et al., 2004). It remains an important citation given the influence it exerted on public health practice and further research. Other fetal infections associated with diVuse brain damage, cerebral palsy, visual impairment, and deafness are also reported to result in symptoms of autism. Cytomegalovirus (Stubbs et al., 1984) and herpes simplex encephalitis (DeLong et al., 1981) have both been reported in cases of autism. It is not well understood if the diVuse nature of the underlying neurologic insult results in symptoms of autism or whether the infection specifically interrupts development in brain regions producing autistic behaviors in these case reports. The potential role of antibodies to neural proteins secondary to viral infection as a cause of idiopathic autism has been hypothesized by Singh and colleagues (1998). They reported increased levels of peripheral

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antibodies to myelin basic protein, measles, and HSV 6 (herpes simplex virus 6). This may represent a nonspecific immune response to infection or may indicate a pathological response to common viral infections in children at risk for autism. a. Novel Infectious Agents. The overwhelming majority of people who have autism do not have visual or hearing impairment like the children exposed to rubella infection in utero described by Chess in the 1970s. The brains of people with autism that have been examined do not contain pathological evidence of inflammation, tissue necrosis, or destruction that would be expected to follow conventional infections during the developmental period (Bailey et al., 1998). More recently identified viral agents, such as Borna disease virus, have been documented to infect rats at varying stages of gestation and neonatal life without evidence of inflammation. Behavioral sequelae reminiscent of autism, such as altered taste preferences, disordered circadian rhythm, decreased imitation in play, increased distress vocalization, and disordered emotional reactivity, have been attributed to neonatal infection with this virus (Hornig & Lipkin, 2001). Neonatal exposure to Borna disease virus causes rodents to have altered cerebellar histology, although in the rodent model, the morphological finding was disorganization and thinning of the granule cell layer (Hornig & Lipkin, 2001). The rats also had loss of granule cells in the dentate gyrus with neonatal and adult infection (Hornig & Lipkin, 2001). Viral exposure in utero in the rodent does not result in serious maternal illness. No evidence of exposure to Borna disease virus has been identified in humans with autism. The possibility that an infectious agent leads to atypical brain development either through primary infection, genetic predisposition in combination with infection, or maternal response to the infection’s altering brain development remains an avenue for future research. b. Response to Maternal Infection. The concept of an infectious agent that acts in a novel fashion, perhaps through maternal immune response without pathological signs of inflammation, and results in behavioral symptoms is being pursued in the study of other neuropsychiatric disorders such as schizophrenia (Brown & Susser, 2002). It has been proposed that the maternal immune response, possibly mediated through cytokines, results in altered social behavior in the oVspring in a mouse model in which the mother is infected with influenza during gestation (Shi et al., 2005). It is not fetal infection with the influenza virus, but the maternal response to viral infection that is being investigated. This type of explanation might relate to the observation of clustering of births of children with autism in certain seasons as well as the diYculty in replicating the finding in other locations at other times. c. Bacterial Overgrowth in the Colon. Since 1990, much attention in the lay community has turned to potential causes of autism in the internal environment. A hypothesis has been generated that overgrowth of specific bacteria

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in the intestine results in exacerbation of symptoms of autism secondary to exposure to clostridial toxins (Song et al., 2004). Studies that include sampling in the context of gastroenterologic evaluation have not identified pathological bacteria (Horvath et al., 1999). One group reported an alteration in the subspecies of clostridia found in the stool of a community sample of children with autism (Song et al., 2004). The model proposed does not include a mechanism for how the diVerence in bacterial species might result in the symptoms of autism but suggests that treatment with antibiotics might reduce symptoms. This is an unproven therapy that has serious implications for the potential emergence of antibiotic‐resistant bacteria.

B.

Seasonality of Births

Although few people with autism have a history of catastrophic infection during prenatal or neonatal life; it was clinically observed in regions around the world that there seemed to be a seasonality to the birth dates of children with autism. It was suggested that women pregnant in the winter would be at greater risk for influenza or other viral infections in their second trimester and that some sequelae of mild infection might predispose the infant to have autism. Retrospective reports of mild illness during pregnancy are not a particularly accurate way to collect data. Families of children with special needs may have diVerent recall than do families of typically developing children. A seasonal diVerence in the births of children with autism is not consistently identified (Bolton et al., 1992, Landau et al., 1999). Prospective population‐based studies that are currently examining the epidemiology of autism may be able to answer this question.

C.

Internal Environment

1. METABOLIC IMBALANCE

One of the most important public health advances of the 20th century was the identification that some disorders that result in mental retardation can be prevented by neonatal screening and dietary correction of inborn errors of metabolism. Phenylketonuria (PKU) was identified as a clinical syndrome in the 1930s when a Finnish physician found a reducing substance in the urine of a set of siblings with mental retardation (Centerwall & Centerwall, 2000). In this autosomal recessive disorder, aVected individuals cannot metabolize the amino acid phenylalanine. Toxic amounts result in brain damage that leads to mental retardation, seizures, and autism. This brain damage is entirely preventable if dietary restriction of phenylalanine is enforced from early infancy through childhood. Although PKU is a genetic

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disorder that does not manifest neurologic harm until there is toxic exposure to a dietary metabolite, it is a model of how disruption of the internal metabolic environment can result in symptoms of autism. EVective screening and dietary treatment have eliminated this as a cause of autism in children in the United States. This model, however, has been invoked in the pursuit of several hypotheses related to the presence of metabolic abnormalities that might result in the toxic accumulation of metabolites. These include the hypothesis that there is a purine‐responsive subtype of autism (Page & Coleman, 2000), the hypothesis that children with autism cannot excrete phenolic amines properly (Alberti, 1999), the hypothesis that children with autism cannot properly excrete dietary opioid compounds that act like false neurotransmitters when absorbed across a ‘‘leaky gut’’ (Reichelt et al., 1990), and the hypothesis that children with autism have a defect in their biochemical response to oxidative stress (James et al., 2004). No specific metabolic abnormality has yet been confirmed in children with idiopathic autism. Hypotheses regarding metabolic causes of autism are the basis for many complementary and alternative treatments that are popular at this time. 2. YEAST OVERGROWTH IN THE COLON

A popular intervention for a number of chronic disorders is the elimination of refined sugar from the diet, with the intention of decreasing the hospitality of the colon for Candida albicans (Crook, 1986). This theory was specifically applied to autism after the report of a pair of brothers who had atypical onset of symptoms of autism and motor symptoms that were associated with metabolites identified as related to yeast in their urine organic acids (Shaw, 1995). Although no conventional evidence of yeast overgrowth has been identified on endoscopy of children with autism (Horvath et al., 1999), this remains a popular lay hypothesis as a cause or exacerbation of the symptoms of autism. The observation of increased metabolites associated with the presence of yeast might have been incidental findings, might have been real but secondary to other characteristics of the underlying disorder, or might have been related to etiology (Shaw, 1995).

D.

External Environment

1. EXAMINING ETIOLOGIES

a. When an Epidemic Is Considered. In the 1990s, a controversy erupted over whether there was an increasing rate of diagnosis of autism and related disorders. The prevailing scientific thought regarding etiology supported, and continues to support, a multifactorial genetic disorder. If, indeed, there

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was a rapid rise in prevalence from the 4 to 6 per 10,000 that had been reported prior to 1993 to 40 to 60/10,000 as reported in 2001 (Chakrabarti and Fombonne, 2001), other theories of causation would need to be invoked or incorporated into a scientific explanation that focused on a primarily genetic etiology. 2. INCREASING PREVALENCE VS EPIDEMIC

It has not been established by epidemiologic studies whether, indeed, there is a genuine increase in the prevalence of ASD or whether the reported increase is an artifact. Increased awareness by pediatricians and psychologists resulting in earlier diagnosis and diagnosis of milder cases may be adding to the apparent increase in number of cases. The appearance of increasing numbers of diagnoses may result from improved interventions, which lead to more patients being presented to service delivery systems. A more inclusive diagnosis adopted with publication of DSM–IV in 1994 has surely contributed to the present prevalence data (Yeargin‐Allsopp et al., 2003). An administrative data set derived from schools and the state network of disability‐related support in California reported an 800‐fold increase in the diagnosis of autism and a 1000‐fold increase in the diagnosis of PDD between 1987 and 1998 (California Department of Developmental Services, 1999). This was not an epidemiologic study that utilized case finding and corroboration of the diagnoses, but rather, a report on the number of individuals with autism or PDD diagnoses receiving services from a regional cohort of providers. At the beginning of this time period, educational services for children with autism were minimal. With the passage of federal legislation identifying autism as a separate handicapping condition for educational purposes and research and publications noting eVective treatment for symptoms of autism, discrete educational programs for autism were established for children from the time of birth to the age of 21 in many areas. The communication to parents of entitlement for services was enhanced in this decade by advocacy groups and by the advent of the Internet. Croen et al. (2003) questioned whether some of the increased numbers reflected reclassification of people previously diagnosed as having mental retardation as now having autism. Dramatically increased numbers of children referred for services for treatment of symptoms of autism may be due to increased awareness on the part of parents and practitioners, so it is important not to mistake educational or service referrals for epidemiologic documentation of an increasing prevalence. Publication of the incidence of autism in one geographic area, Olmsted County, Minnesota, between 1976 and 1997 pinpointed an increase in the incidence to the time period that the

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definition of autism was modified with the adoption of the DSM–IV (Barbaresi et al., 2005). 3. EPIDEMIOLOGIC PRINCIPLES IN EXAMINING INCREASING PREVALENCE

To identify the actual number of aVected individuals in a population for accurate population statistics, studies need to utilize case‐finding strategies to identify all aVected individuals in the population. This requires population screening for the disorder with in‐depth assessment of all individuals who are screen positive. Groups in Sweden and in England have used this approach (Arvidsson et al., 1997; Baird et al., 2000; Chakrabarti and Fombonne, 2001). A diVerent approach to case finding was taken by Yeargin‐Allsopp and others (2003) in Atlanta. They abstracted all school records on all 3‐ to 10‐year‐old children known to the school districts in Atlanta and five surrounding counties, all specialty medical clinics in that region, and all providers of services to people with developmental disabilities. They then applied DSM–IV criteria and rediagnosed all participants by record review. Current initiatives funded by the Center for Disease Control and Prevention will be examining prevalence of autism and related disorders in sites across the United States using case‐finding methodology. Although a dramatic increase in prevalence of autism over the past 20 years cannot be objectively documented because of the confounds in design and definition in the earlier studies, prospective data collection will allow for tracking future trends. At least one study has identified a plateau in reports of new cases, suggesting that the increase may, in part, have been due to ‘‘catch‐up’’ diagnosis of older children (Lingam et al., 2003). Whether the reported increased prevalence of autism is based on a true increase in the number of children with autism or increased identification of cases, it has resulted in increased interest in the role of environmental factors in the etiology of autism. This is something of a change in conceptualization of basic research needs after the hope inspired by the Human Genome Project in the 1990s had focused etiologic research in autism on genetics. 4. ENVIRONMENTAL INVESTIGATION: BRICK TOWNSHIP, NEW JERSEY

Reports of clusters of cases of autism have been the focus of targeted studies looking for specific teratogens associated with the regions in question. The recognition of clusters of aVected individuals often results in hypothesis generation for environmental scientists. They can then test these hypotheses in the laboratory or by looking for replication in other populations. In 2001, the Centers for Disease Control and Prevention (Bertrand

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et al., 2001) reported their investigation of a suspected cluster of cases of autism in Brick Township, New Jersey. This area was of interest because the water supply was obtained from a region in proximity to a Super Fund site, where there had been an attempt to remove toxic chemicals. Using case‐ finding methodology in this area and a control site elsewhere in New Jersey, the investigators were not able to find diVerences in prevalence rates between sites. They did identify a higher rate of both of autism (40 per 10,000) and PDD (67 per 10,000) in 3‐ to 10‐year‐old children than had been reported previously in North America. Although it is certainly plausible that an early environmental exposure might result in ASD, the communities investigated in this study were not documented as true clusters and have not been informative as to environmental agents that might be associated with autism.

E.

Iatrogenic/Medical Interventions

1. MMR VACCINE

As investigators examined the data related to the reported increased rates of ASD, many investigators suspected that events that occurred around the time when symptoms of autism were noticed by parents might play a role. Regression in language and other milestones is reported in 30% of children with autism (Tuchman & Rapin, 1997). Families recalling regressions have reported that they occur before 2 years of age in about 23 of situations and by age 3 in 95% (Tuchman & Rapin, 1997). An event routinely introduced into the second year of life in the late 1970s through early 1980s was the measles, mumps, rubella (MMR) vaccination. A temporal association of the MMR with the increased rates of autism was proposed (Wakefield et al., 1998). Wakefield et al. (1998) reported that the families and physicians in the cases they reported with gastrointestinal symptoms recalled that the autistic regression began after MMR immunization. These families and/or their practitioners recalled onset of diarrhea or other intestinal symptoms after immunization and sought gastrointestinal evaluation (Wakefield et al., 1998). The first author suggested that the attenuated virus was responsible for intestinal pathology that might be associated with or responsible for the onset of the behavioral symptoms. Epidemiologic data do not support the hypotheses associating MMR immunization with autism (Demicheli et al., 2005). Dales et al. (2001) reviewed the prevalence data before and after introduction of MMR vaccination in California. MMR was licensed in 1971 and was in wide use by 1976. Immunization for MMR has been mandatory in most states since the 1970s. Dales et al. (2001) determined that the increase in reported cases of

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ASD began before the general use of MMR and continued to rise after vaccination rates had become stable. Similarly, studies in the United Kingdom (Taylor et al., 1999) did not identify a relationship between the introduction of MMR immunization and the increased prevalence of autism or a temporal association with the time of vaccination. In Denmark, immunization policy allows for parental choice. Review of their national immunization data and autism registry identified a prevalence of ASD that was identical in vaccinated and unvaccinated children (Madsen et al., 2002). Barbaresi et al. identified the increased prevalence of autism to occur over 14 years after introduction and standard use of the MMR vaccine in Olmsted County (Barbaresi et al., 2005). This increased incidence was noted at the time diagnostic criteria were modified. These studies collectively indicate that in children with ASD, in general, as well as the subset with reported regression, the MMR vaccination does not play a causative role on a population level. The recommendation for childhood vaccination with MMR has not been altered (Demicheli et al., 2005). There is no evidence that the immunologic burden is greater with trivalent vaccination (OYt et al., 2002). No evidence has been published supporting separation of trivalent MMR vaccine into individual components to diminish proposed developmental sequelae. 2. MERCURY

Because of a concern that vaccines in general might be a route of post‐ natal exposure to toxic agents, and knowing that some vaccines contain thimerosal (which includes the organic mercurial ethylmercury chloride), a new hypothesis was proposed. It suggested that mercury poisoning was the cause of regression leading to autism (Bernard et al., 2001). The MMR vaccine does not and has never contained thimerosal as a preservative because it is an attenuated live virus vaccine that would be damaged by exposure to a preservative like thimerosal. Attenuated live virus vaccines provoke the production of antibodies to an engineered portion of a virus, rather than the naturally occurring virus itself. The attenuated virus does not lead to infection of the host, but the resulting antibodies protect the host from the wild‐type virus. Immunizations for diphtheria, tetanus, pertussis, Haemophilus influenzae, and hepatitis do not require live attenuated viruses to produce immunity, so a preservative could be used. The ethylmercury‐based preservative, thimerosal, has been in use since the 1930s (Stratton et al., 2001). Mercury occurs in several forms: organic mercury compounds (including methyl mercury and ethylmercury), inorganic mercury compounds, and elemental (inorganic) mercury. Each form is metabolized diVerently by the body and results in diVerent symptoms with exposure. The form of exposure

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(inhaled as a vapor versus ingested in food) also aVects the symptoms produced (Table II). Because the vaccines that contained mercury were given starting at birth to 2 months of age and continuing until the second year, epidemiologic studies cannot concentrate on associated regression. The health records data set in Denmark allowed analysis of recorded ASD status before and after thimerosal was removed from vaccines in 1992. The rate of recorded diagnoses of autism began to increase in 1990 and continued to increase over the next 10 years of data collection (Madsen et al., 2003). These epidemiologic data strongly contradict the proposal that any association exists between thimerosal exposure and autism. New childhood vaccinations for hepatitis B and H. influenzae became increasingly common in the early 1990s, but Madsen et al. (2003) found no association of ASD and estimates of cumulative exposure. The blood level of ethylmercury in infants after immunization has been documented to be lower than the acceptable safe level of methyl mercury (Pichichero, 2002), a more neurotoxic compound (Magos et al., 1985). The Institute of Medicine expert panel agreed that although methyl mercury is a known cause of brain damage at toxic levels in the developing fetus and infant, the scientific literature does not contain evidence that supports the allegation that thimerosal administered in childhood vaccines has an association with autism (Stratton et al., 2001). Assessment of the metabolic pathways important to the detoxification of heavy metals continues to be studied to determine whether children with autism have a predisposition to abnormal elimination of mercury. Compared to controls, children with autism have been reported to have an enhancement of glutathione synthesis and oxidative reduction in response to supplemental vitamin B12 (James et al., 2004). The relationship of these data to the theory of mercury intoxication as a cause of autism remains hypothetical.

F.

Known Teratogens Associated with Autism

1. THALIDOMIDE

An association between early thalidomide exposure and symptoms of autism was discovered by Miller and Stro¨ mland and colleagues (Miller, 1991; Miller and Stro¨ mland, 1993) in the early 1990s. They were examining individuals exposed to thalidomide in utero for brain stem abnormalities related to ocular control. They observed that there was an abnormally high rate of autism in this group. Indeed, a rate of 30% was identified for people whose injury took place in the third week of gestation. Rodier et al. (1996)

TABLE II MERCURY EXPOSURE: SOURCE AND SYMPTOMS (DAVIDSON ET AL., 2004; ZHANG, 1984) Type of mercury Vapor, inorganic (elemental Hg)

Organic (monomethyl Hg)

Ethyl Hg

Name of disorder

Source

Mad hatter’s disease (occupational exposure) Acrodynia (children) Minamata disease

Industrial exposure, religious sects, cosmetics, dental amalgam, historically, exposure for children in teething powders

Bizarre behavior, gingivitis, tremor in adults Children: Irritability, failure to thrive, photophobia, erythema of hands and feet

Fish consumption as either primary or gestational exposure or breast milk

Parasthesias, speech impairment, ataxia, visual field constriction, hearing impairment Prenatal exposure: cerebral palsy, mental retardation, seizures Lower‐level exposure: one study suggests learning and attention problems at school age with prenatal exposure, another did not confirm these findings Most common symptoms are headache, loss of appetite, and muscle weakness There are no studies of symptoms after prenatal or neonatal exposure.

?

Thimerosal preservative in vaccines

Symptoms

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noted that the only neural structures forming at that time were the brain stem motor nuclei. Abnormal development of the oculomotor and abducens nuclei could explain the increased rate of eye movement abnormalities seen in the adults exposed to thalidomide in utero and abnormal development of the facial nucleus could explain the facial hypotonia seen in the same cases. Because limb bud development is also interrupted in fetal exposure to thalidomide, it was quickly terminated as a treatment of nausea in pregnancy in the 1960s. Exposure to thalidomide during gestation was not a major cause of autism, but as a known teratogen that is associated with an increased incidence of autism, it is an important clue in identifying potential environmental causes. 2. VALPROIC ACID

Thalidomide is not teratogenic in rodents (Schumacher et al., 1968), so a compound that causes similar birth defects, valproic acid, was administered to rats to investigate the potential relationship of early teratogen exposure and neuropathologic findings in brains of people with autism. Rodier et al. (1996) reported that the brains of rats exposed to valproic acid in utero in early gestation had similar hypoplasia of brain stem nuclei to that found in a case of idiopathic autism. The brains of animals exposed at the time of neural tube closure not only had decreased cellularity of the brain stem nuclei, but had a subsequent decreased number of Purkinje cells in the cerebellum as a downstream event (Ingram et al., 2000). This is another similarity to human idiopathic autism. Stanton et al. (2001) have demonstrated that animals exposed to valproate during neural tube closure have a characteristic enhancement of acquisition of eye blink conditioning. Autism is the only disorder studied thus far in which eye blink conditioning is changed in this fashion (Sears et al., 1994). This is additional evidence that an embryologic insult at this early stage in development, when the only neurons that have developed are destined for the brain stem, might be associated with findings of autism. Similar histologic findings in the brain stem in a strain of mice with a knockout mutation of the HOXA1 gene that regulates early brain stem development (Carpenter et al., 1993) suggested that the early gestational exposure to valproic acid might be altering brain development at the time this gene is exerting its action. Stodgell et al. (2001a) have determined that administration of valproic acid can drive expression of HOXA1 to abnormally high levels both at the time it is expressed normally and at slightly earlier or later times, when the gene is transcriptionally silent under normal circumstances (Stodgell et al., 2001b). Valproic acid is the second known pharmaceutical agent associated with autism with prenatal exposure. It has been reported that 11% of children

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whose mothers were prescribed valproic acid during pregnancy have autism in one series of 57 children (Moore et al., 2000). Physical features that result from prenatal exposure (most likely, in the first 8 weeks of pregnancy) include epicanthal folds, a broad nasal bridge, a short nose with anteverted nares, a long upper lip, and low‐set, posteriorly rotated ears (Williams et al., 2001). Prenatal valproate exposure is not a major cause of autism, because few mothers of children with autism are treated with this agent for seizures, migraine prophylaxis, or mood disorders. However, neurologists, internists, psychiatrists, obstetricians, and other practitioners treating adults must be aware of the teratogenicity of this agent when they prescribe it to women of childbearing age. Thalidomide and valproate exposure during pregnancy account for only a small number of cases of autism. They tell us, however, that exposure of the embryo to a toxic substance can increase the risk of autism. We can use these cases to learn more about the mechanisms by which development can be disrupted, whether it is due to interaction of a teratogen with a genetic predisposition or by action of the teratogen on neural elements themselves. 3. MISOPROSTOL

The prostaglandin misoprostol causes contractions and can induce uterine evacuation in the first trimester. For that reason, it has been administered to induce abortion, typically in the sixth week of pregnancy (Pastuszak et al., 1998). Thalidomide was administered for symptoms of nausea as early as the third to fourth week of pregnancy. It is known that both thalidomide and misopristol exposure increase the risk of dysfunction of the muscles of facial expression, as well as of the lateral rectus muscle of the eye. This facial diplegia is called Moebius syndrome, and people with idiopathic Moebius syndrome exhibit a rate of autism of about 25% (Johansson et al., 2001). The syndrome can be produced by mutation in genes leading to abnormal brain stem development or by embryologic events that interrupt brain stem development. The embryonic events need not act on early developmental genes. In the case of misoprostol, the event is probably ischemia. Children who come to term after early exposure to misoprostol use are at markedly increased risk for Moebius syndrome and comorbid autism (Bandim et al., 2003; Gonzalez et al., 1993, 1998). The association of misoprostol‐induced Moebius syndrome with autism has already been replicated (Marquez‐Dias et al., 2004). It provides evidence that an early injury to the region of the brain stem where the facial nucleus and the abducens nucleus are located can be significant in the etiology of autism, whether the maldevelopment is genetic or environmental in origin.

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4. ALCOHOL

Fetal alcohol syndrome may be the most common environmental cause of mild mental retardation in the United States (Nanson, 1992). A history of maternal alcohol use is not commonly found among children with idiopathic autism. There are cases reported of children with comorbid fetal alcohol syndrome and autism, however (Kielinen et al., 2004). Since both autism and fetal alcohol syndrome are not rare, it is plausible that the comorbidity reported is a chance occurrence, as suggested by Fombonne (2002). Larger epidemiologic samples would be necessary to confirm alcohol as an environmental risk factor for autism. Primary prevention of developmental disabilities would include counseling women of childbearing age not to drink alcohol if there is any chance of pregnancy.

IV.

INTERFACE OF INTERNAL AND EXTERNAL ENVIRONMENTS

Genetic studies evaluating candidate genes have not uniformly obtained the same associations. Linkage studies have identified many chromosomal regions that appear to be slightly linked to ASD, but none that is strongly linked. Risch and others (1999) have interpreted these results to mean that many genes, not just a few, are involved in autism. Theoretically, another factor that could lower the significance level of linkage loci would be the variable presence of environmental factors that interact with genotype to produce the autism phenotype. Le Couteur and colleagues (1996), investigating twin pairs, came to the same conclusion by finding that the variance in expression of symptoms within monozygotic pairs was as great as the variance between pairs. There is no question that there is a genetic component in the etiology of autism. That has been the major thrust of research through the past decade and continues to be a very important area of pursuit. However, studies combining the eVects of gene and environment have become more important since 2000. Environmental factors may either protect persons at genetic risk for autism or promote the occurrence of symptoms of autism. These factors might be protective, as in the case of supplemental folic acid during gestation preventing spina bifida (Wald et al., 1991). Environmental factors may also contribute to the development of symptoms, as has been demonstrated for thalidomide, valproic acid, and misoprostol. It is not yet known whether factors such as these are ever suYcient to induce autism by themselves or whether they exert their action through their eVects on the action of specific susceptibility genes.

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Investigation of the mechanism of gene action of the HOXA1 gene has identified that the teratologic eVects of maternal valproic acid exposure in a rodent model occur through alteration of gene function (Stodgell et al., 2001a). The disruption of brain development both in utero and in the first years of life on the basis of environmental disregulation of gene function is an area of active pursuit. The possible disruption of neurotransmitter formation and the resultant directions for brain development by pesticides, the possibility of heavy metal exposures altering cell adhesion and repulsion, and the investigation of how exposures might alter regional brain development are all areas currently under investigation through Environmental Protection Agency–funded research (National Institute of Environmental Health and Safety, 2004).

V.

SUMMARY AND CONCLUSIONS

As we broaden our understanding of the potential interplay of social environment, prenatal environment, postnatal environment, immune response to the environment, teratogenic exposures, and genetic predisposition, the scientific community is examining an increasing number of possible influences on the etiology of autism. Some environmental factors (such as the maternal lack of involvement) have been clearly disproven, while others (such as infections) are being reevaluated in the light of scientific progress and new knowledge. What is clear is that for treatment and prevention of symptoms to be appropriately targeted, scientific understanding of the etiology and neurobiology of autism is critically needed. Research combining epidemiology, clinical studies, and basic science is necessary to address that common goal. ACKNOWLEDGMENTS Drs. Hyman and Rodier and Ms. Arndt are supported in part by U19HD35466, a Collaborative Program of Excellence in Autism. Drs. Hyman and Rodier are also supported by U54MH066397, a STAART (Studies to Advance Autism Research and Treatment) Center.

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Endocrine Disruptors as a Factor in Mental Retardation BERNARD WEISS DEPARTMENT OF ENVIRONMENTAL MEDICINE UNIVERSITY OF ROCHESTER MEDICAL CENTER ROCHESTER, NEW YORK

I. A.

INTRODUCTION

What Are Endocrine Disruptors?

The term ‘‘endocrine disruption’’ soared to prominence with publication of a book, Our Stolen Future (Colborn et al., 1996). It presented in detail the argument that many chemical contaminants in the environment interfere with the biological function of hormones by blocking, mimicking, displacing, or acting through a variety of other mechanisms to subvert their natural roles. Although the argument rested primarily on observations in wildlife and on laboratory experiments, its cogency for human health was undeniable: the same chemicals and hormones are found in humans. Endocrine‐disrupting chemicals (EDCs) include many types of substances encompassing many diVerent chemical structures acting through diverse biological mechanisms to exert their eVects. The latter include estrogenic, androgenic, and anti‐estrogenic anti‐androgenic eVects, and altered thyroid function. Although disorders of reproductive function first brought these agents to our attention, their potential to interfere with brain development is now seen as at least equally significant for public health. All twelve of the chemicals listed as persistent organic pollutants, the ‘‘dirty dozen,’’ which were the subject of a global treaty to abolish their use, earned their labels, in part, because of their potency as endocrine disruptors. This list contained the following organochlorine compounds: dioxins, furans, PCBs, DDT, hexachlorobenzene, chlordane, toxaphene, dieldrin, aldrin, endrin, heptachlor, and mirex. The Centers for Disease Control and Prevention (CDC, 2003) has published exposure data for 116 chemicals from NHANES 1999–2000. The data INTERNATIONAL REVIEW OF RESEARCH IN MENTAL RETARDATION, Vol. 30 0074-7750/06 $35.00

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include statistics on the distribution of blood or urine levels for each environmental chemical. All have been shown, directly or indirectly, to modify endocrine function. They include: *Polycyclic aromatic hydrocarbons (PAHs) *Dioxins, furans, and coplanar PCBs *Non‐coplanar PCBs *Phytoestrogens (e.g., soy) *Selected organophosphate pesticides *Organochlorine pesticides *Carbamate pesticides *Herbicides *Pest repellents and disinfectants *Metals (lead, mercury, cadmium) *Cotinine (nicotine metabolite) *Phthalates This list illustrates a crucial principle: we now carry in our bodies a broth of chemicals to which our forebears were never exposed. Although we seek, by a variety of maneuvers, to isolate the contributions of individual agents in our research, we cannot totally extract them from this baseline confounding. We do not know which combinations are additive, which antagonistic, or which are synergistic. It challenges logic to accept the proposition that the sum of their biological eVects is zero, the assumption that underlies the setting of current exposure standards.

B.

Hormones and Toxicology

Endocrine systems serve an essential function: they enable signals from a specific organ to evoke a dynamic, coordinated response in a distant target tissue. Their role, generally, is to maintain homeostasis. In some instances, however, the signal originates outside the body. This possibility is the core issue generating concerns about endocrine‐disrupting chemicals. Three properties of EDCs claimed particular force. One arose from the roles played by hormones in fetal development. As we already knew from our experience with lead, mercury, alcohol, and other chemicals, exposure levels that exert minimal or transient eVects in adults can produce lasting damage during development. Many toxic chemicals pass easily from mother to fetus and from breast milk to the neonate. The second concern arose from the tendency of many endocrine disruptors to persist in the environment because they resist degradation, bioaccumulate in the food

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chain as they ascend predator levels, and are stored in the body’s fat depots, where they are slowly metabolized and excreted; the half‐life in humans of the prototypical dioxin, tetrachlorodibenzo‐para‐dioxin or TCDD, is 7 to 10 years. When fat is mobilized during pregnancy or lactation, the fetus or neonate may be exposed to substantially elevated levels. Other EDCs, such as phthalates, are so ubiquitous in the environment that exposure is constant although their half‐lives may range from weeks to months. The third property generating concern was the geographical mobility of such agents. Arctic species, such as polar bears, are heavily contaminated with chemicals generated far from the original sites of discharge into the environment (Skaare et al., 2002). The contaminants are carried to the arctic by global weather patterns and by the toxic loads in the bears’ customary prey. Hormones occupy a crucial role during development. Hormonal mechanisms act as arbiters of brain structure and neurochemistry. Gonadal hormones direct and modulate the processes underlying sexual diVerentiation of the brain. Thyroid hormones regulate neuronal and glial proliferation as well as cell migration and diVerentiation. Glucocorticoids play essential roles in several aspects of normal brain development, especially through their enduring eVects on the hypothalamic–pituitary–adrenal (HPA) axis and its role in behavior. Basic research in neuroendocrinology provides the basis both for these linkages and for the health concerns evoked by EDCs. Because endocrine disruption is such a new aspect of environmental toxicology, however, our current knowledge of its scope and manifestations in human populations remains scattered. And, because of our incomplete grasp of these properties, this chapter provides a basic window on the EDC issue rather than a comprehensive survey of its role in human health. Much of it, necessarily, will come from animal experiments and observations in wildlife. We will cover only a few chemical classes, some of the implications of which for humans have at present to be hypothesized rather than confirmed. Furthermore, the mechanistic relevance of some EDCs to neurobehavioral toxicity has not been established; dioxin, for example, probably exerts such toxic eVects by mechanisms apart from its hormonal eVects. Perhaps the main reason EDCs became the subjects of such profound unease is their implications for human reproductive function, especially in males. Falling sperm counts (see Swan et al., 2000), along with rising rates of testicular cancer, cryptorchidism, and hypospadias, have generated questions about the possible contribution of environmental chemicals to these trends. Sharpe and Skakkebaek (1993) first proposed such a link. In 2003, Skakkebaek (2003) advanced the hypothesis more directly:

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EXAMPLES

OF

TABLE I ENDOCRINE‐DISRUPTING CHEMICALS

Phthalates (plasticizers): testicular poisons Alkylphenols (detergents): estrogenic properties Bisphenol A (coatings): estrogenic properties Organochlorine pesticides (e.g., DDT): estrogenic properties PCBs: thyroid hormone dysfunction Brominated flame retardants (PBDEs): thyroid dysfunction Dioxins: anti‐androgenic properties Vinclozolin (fungicide): anti‐androgen Phytoestrogens (plants): estrogenic properties Other persistent organic pollutants (e.g., PAHs)

There is evidence that poor semen quality, testicular cancer, undescended testes and hypospadias are symptoms of one underlying entity, testicular dysgenesis syndrome (TDS), which may be increasingly common due to adverse environmental influences. Experimental and epidemiological studies suggest that TDS is the result of disruption of embryonal programming and gonadal development during fetal life.

Sources of endocrine disruptors abound. They can be found in the form of pesticides, such as insecticides, herbicides, and fungicides used in agriculture, from where, as now recognized, they drift into homes or pollute drinking water supplies. They can leach out of plastic containers holding intravenous fluids or even food and water (the alkylphenols). They are present in can linings and in dental sealants (Bisphenol A). They have been found in toys that young children place into their mouths (phthalates). Dispersed into aquatic environments, they can contaminate fish. Table I is a list of the more prominent disruptors already identified and their primary hormonal targets. C.

Neurobehavioral Toxicity as an Issue

Unambiguous connections between EDCs and human neurobehavioral function exist primarily for the PCBs, as described in the chapter by Vreugdenhil and Weisglas‐Kuperus in this volume. The paucity of extensive human data on EDCs compels us to rely on animal models to gauge the relevance for human function. Yet, although hormones govern much of brain development and behavior, the literature devoted to neurobehavioral endpoints and EDCs, even for animal models, is dismayingly sparse. And, even for those, few have explored eVects based on complex behavioral repertoires and brain morphometry. Fewer still have featured exposure levels consistent with those measured in human populations. Schantz and Widholm (2001), in a discussion of these issues, noted the wide gaps in our knowledge of how EDCs influence cognitive function.

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FIG. 1. How gonadal hormones control sexual diVerentiation of the brain during development.

Links between impaired endocrine function and behavioral disorders are hardly a novel phenomenon. Sexual diVerentiation of the brain, for example, is known to be guided primarily by the actions of gonadal hormones (Fig. 1). Disturbing the appropriate balance of these hormones during development by exogenous agents is documented to produce morphological, neurochemical, and behavioral abnormalities. This delicate balance is persuasively illustrated by the behavioral correlates of the genetic virilizing disease, congenital adrenal hyperplasia. Females with this disorder, which exposes them to high levels of androgens during gestation as well as postnatally, exhibit behavioral patterns indicating both masculinization and defeminization (Berenbaum, 1999). For example, as children, they tend to show male preferences for toys; they are more aggressive than normal girls; they show greater spatial abilities than normal girls; and they are less interested in feminine appearance. Over a broad range of behaviors, development becomes more prototypically male because of a shift in the balance of gonadal hormones. Vreugdenhil and Weisglas‐ Kuperus, in their chapter in this book, note a similar but less extreme tendency correlated, in girls, with PCB/dioxin exposure.

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In writing about the hazards posed by environmental contaminants, McEwen (1987) observed, ‘‘Exogenous mimics can play havoc with brain development and diVerentiation.’’ McEwen’s statement supports a compelling argument for assessing the developmental neurotoxicity of endocrine disruptors. Sexually dimorphic behaviors, for at least some EDCs, may prove especially sensitive indicators because the magnitude of sex diVerences themselves, independent of eVects on the individual sexes, oVer endpoints that are too frequently analyzed superficially. Experiments with PCBs and dioxins (exemplified by 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin [TCDD]) oVer a variety of examples indicating that, during early brain development, males and females are diVerentially susceptible to their eVects, a diVerentiation that provides important clues about their toxic mechanisms. Despite the limited epidemiological and experimental data bearing directly on EDCs, however, everything else that is known about developmental neurotoxicity and endocrine influences on brain development should evoke disquiet about the extent of human exposure to these contaminants. But the relevance of EDCs for mental retardation (MR), as it is generally understood, requires a subtlety of argument that professionals will need to translate for the public. Endocrine disruption is unlikely to emerge in arresting examples, such as the methyl mercury catastrophe in Minamata, Japan, or in other mass chemical disasters. Exposures to EDCs typically result not in blatant clinical signs but in eVects distributed over populations. As in other reviews in this volume, the key argument for relevance to public health and welfare is a shift in the population distribution of some functional property, such as IQ score or incidence of delinquency. Figure 2 illustrates the argument (cf. Weiss, 2000). What are the defining characteristics of MR? Among them is an IQ below 70, although a classification of MR may also include deficient social skills and limited sensory and motor function. Substances classified as EDCs, and other environmental contaminants as well, may promote an elevated risk of MR in the population by inducing relatively small changes in the IQ distribution. Figure 2 shows that a shift in population IQ of only 3% substantially elevates the number of individuals in that population scoring below 70. Such a small shift, whether the criterion is IQ, propensity for delinquency, age of puberty, or other socially important measures, confronts society with profound questions. The risks of exposure in this case are assumed by society rather than by the individual, because such a small shift for the individual incurs relatively minor costs. For the society as a whole, the costs are substantial. Herrnstein and Murray (1994), for example, calculated that a three‐point shift in mean IQ would be translated into a 20% change in the number of families on welfare and exhibiting other social pathologies.

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FIG. 2. Population consequences of a shift in mean IQ score of 3%.

II.

THE CURRENT SCENE

The chemicals considered in this chapter were chosen not only because they have been identified as widespread environmental contaminants but also to illustrate how, for some, their potential as health threats surfaced unexpectedly. The absence of a predictive algorithm, in fact, led in 1996 to the formation of an Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC), under the auspices of the Environmental Protection Agency, to make recommendations on how to develop a screening and testing program for EDCs requested by Congress. The discussion that follows highlights those EDCs that have yielded data pertinent to or that carry implications for developmental neurotoxicity. It will mainly treat eVects attributable to gonadal hormone function; thyroid function is discussed in the chapter by Vreugdenhil and Kuperus‐Weisglas. A.

Dioxins and Polychlorinated Biphenyls (PCBs)

Members of this chemical class pervade the environment and human tissues. They include PCBs, dioxins, and dibenzofurans. The halogenated dioxins and dibenzofurans are not commercial chemicals but by‐products emitted in the combustion of wastes such as plastics and as inadvertent contaminants in the manufacture of products such as paper and herbicides. The PCBs were used in many industrial processes, for example, as dielectric fluids for insulation, as plasticizers, as components of carbonless copy paper,

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FIG. 3. Basic structures of chlorinated compounds acting as neurotoxicants.

and in many other ways. This class comprises a large family. It includes 75 polychlorinated dibenzodioxin (PCDD) isomers, 135 polychlorinated dibenzofurans, and 209 PCB isomers (Fig. 3). They are notable for their long persistence in the environment and resistance to degradation. They become increasingly concentrated in tissues, particularly in fat, as they ascend the food chain. Dioxins represent the subclass of endocrine disruptors that has received the closest scrutiny in toxicology. One particular congener, TCDD, has become the reference compound for this chemical class, and the focus of several thousand studies, ranging from its molecular mechanisms to its eVects in exposed human populations. Because of its status as a model compound, its developmental neurotoxicity will be examined in detail. 1. BACKGROUND

Ever since its identification as a component of Agent Orange and its notorious label as the ‘‘most potent man‐made chemical,’’ dioxin (or TCDD) has evoked fierce controversy over its threats to health. Most, if not all, of its actions are held to be mediated through the aryl hydrocarbon receptor (AhR), a cytoplasmic protein that translocates chemicals of this basic structure to the nucleus. Besides its toxic potency, one of the reasons it evokes health concerns is its accumulation in the food chain and quite long half‐life, about 10 years, in humans. Its extended residence in the body is attributable to its lipophilic properties and slow metabolism and excretion. Because of these properties and its pervasive distribution, all humans carry some dioxin in their bodies. DeVito et al. (1995) attempted to estimate human background body burdens of dioxin and similar chemicals (dibenzo‐p‐dioxins, dibenzofurans, and coplanar PCBs, all of which bind

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to the AhR) by summing their TCDD equivalents (TEFs) on the basis of relative toxic potency compared to TCDD. They arrived at a value of 13 ng/kg, a value that has fallen steadily since 1995 due to attempts to control release of these compounds into the environment. AhR is expressed in most organs and cells in the body. Petersen et al. (2000) showed that mRNAs encoding AhR and structural analogs are widely distributed throughout the brain and brain stem. Their pervasiveness in brain signifies an important role for these receptors in brain function that remains to be explained. TCDD has aroused an extensive research eVort because it is seen as emblematic of human exposure to a wide array of agents belonging to similar chemical classes, such as the polycyclic aromatic hydrocarbons formed during the combustion of fossil fuels and garbage and which also bind to AhR. Most of what we know specifically about TCDD in humans is the result of accidents such as one that occurred in Seveso, Italy, in 1975 (Mocarelli et al., 2000) or high‐level occupational exposures (Neuberger et al., 1999). The Seveso accident, occurring in a plant manufacturing pesticides, exposed some of the surrounding population to high doses of TCDD. The most intriguing outcome was a reduction in male births from those fathers exposed during adolescence. The Yusho and Yu‐cheng episodes in Japan and Taiwan (Aoki, 2001; Rogan & Gladen, 1992; see also Vreugdenhil & Weisglas‐Kuperus, this volume) exposed the victims to a complex mixture of PCBs, PCDDs, and PCDFs. Many experimental studies stemmed from recognition that TCDD acts an endocrine disruptor. These eVects seem most pronounced when exposure occurs in utero, although the data from Mocarelli et al. (2000) indicate that adolescence in humans may be a sensitive period as well. Laboratory experiments show the developing male rat reproductive system to be sensitive even to relatively modest doses of TCDD. The sensitivity is reflected by reproductive tract abnormalities such as abnormal prostate growth, by reduced sperm counts, and by impaired copulatory performance, even at doses down to 64 ng/kg (Mably et al., 1992), not too far from the estimated 1995 ‘‘background’’ body burden of 13 ng/kg. Lactational exposure in rats to TCDD also partially demasculinizes and feminizes sexual behavior in adult male oVspring (Bjerke et al., 1994). Kakeyama et al. (2003) found impaired copulatory performance in male rat oVspring whose dams received 800 ng/kg TCDD on gestational day (GD) 15. They attributed the eVects to altered frontal cortex function. TCDD alters reproductive tract development in female rats as well (Gray et al., 1997). For example, single doses on GD 15 in these studies delayed puberty, induced clefting of the clitoris, and led to formation of a ‘‘thread’’ of tissue across the opening of the vagina.

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It is still the case that one of the least studied consequences of TCDD exposure, and, in fact, exposure to endocrine disruptors in general, is its developmental neurotoxicity. One reason, certainly, is that other health endpoints, such as cancer, have received more attention. Investigators such as MacLusky et al. (1998) and Schantz and Widholm (2001), however, have tried to arouse recognition that impaired neuroendocrine development can also induce changes in brain structure and cognitive function. Even now, only a handful of studies, except for the PCBs, have targeted complex behavior. Schantz and Bowman (1989) conducted a pioneering study in monkeys exposed prenatally to TCDD. Although the exposed oVspring displayed retarded learning of shape reversals, they performed equivalently to controls on spatial and color reversals. The dose administered to the pregnant monkeys (0.126 ng/kg per day) can be calculated to have resulted in a body burden of 19 ng/kg, equivalent to the lowest dose used in the studies that will be described and close to estimated human background levels a decade ago. 2. ROCHESTER STUDIES

a. Behavioral EVects. Given the universal dissemination of dioxins in human tissues and their potency as developmental toxicants, the relative handful of studies devoted to eVects on neurobehavioral development struck us as a marked inconsistency with their potential health consequences. We were impressed in particular by their eVects on copulatory behavior, which implied alterations in sexual diVerentiation of the brain and provided the impetus for the studies described in this chapter. Here, these findings serve three purposes. One, they indicate the scope of possible adverse eVects of current EDC exposures in human populations. Two, they demonstrate the necessity of directing analyses at sexually dimorphic postnatal consequences arising from prenatal exposures to EDCs. Three, they illustrate the nonlinear dose–response patterns seen frequently in toxicology but especially with endocrine disruptors. All of the studies treated pregnant rats with TCDD administered orally on a designated GD. For our behavioral measures, we chose schedule‐controlled operant behavior (SCOB) because it provides a powerful tool for examining neurobehavioral function. In this class of behavioral procedures, a relationship is defined between the behavior of a subject and its consequences in a defined environment. Human behavior, in most environments, is also governed by its consequences, and operant behavior oVers a means for emulating such conditions, especially the lack of predictability. SCOB provides numerous procedures for the analysis of learning, performance, and memory (Weiss & O’Donoghue, 1994), as well as providing the ability to tailor tasks to model complex cognitive activities in humans.

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Under both transitional and steady‐state conditions, SCOB studies have been used extensively to detect and interpret outcomes of exposures to pharmacologic and toxicologic agents, including developmental exposures. Two behavioral studies are described in this chapter. Both administered doses low enough to allow comparisons with estimates of human exposure to PCDDs. One experiment examined the reinforcing potency of access to running wheels (Markowski et al., 2001). In sexually mature female rats, running wheel activity follows a 4‐ to 5‐day cycle, corresponding to the stage of the estrous cycle. We hypothesized that measuring motivation to run, rather than the usual routine of simply recording spontaneous running, would provide a more sensitive index of estrous cycle aberrations. We based our hypothesis on data from Gray et al. (1997) indicating reproductive system abnormalities in females exposed prenatally to TCDD, although at higher doses. Because our interest lay in more subtle eVects at low doses, we designed an experiment to assess motivation to run in specially designed wheels, based on the proposition that it would serve as a responsive measure of perinatal endocrine disruptor exposure. For this cohort, TCDD was administered on GD 18, which corresponds to the onset of neuronal synaptogenesis and myelination, development of the catecholamine systems that mediate many reward behaviors, and the neonatal testosterone surge. The running wheels were designed to provide a wheel of great enough diameter (60 cm) to permit running on a virtually flat surface constructed of parallel rods spaced at 15‐degree intervals. To rotate the wheel, the rat had to thrust against one of the rods with a hind limb, while positioning the forelegs on other rods to supplement or support the more powerful hind leg thrust. It maintained rotation by coordinating a sequence of similar movements. An electric clutch brake mounted on the axle of the apparatus regulated free rotation of the wheel. An operant response lever and cue light were located inside the wheel, near the running position. A magnetic reed switch tabulated wheel revolutions and allowed calculation of the revolution rate and distribution in time. Adult female oVspring were trained to earn brief opportunities to run by pressing a lever on a fixed‐ratio (FR) schedule of reinforcement. The FR requirement was increased at 5‐session intervals in the sequence: FR1, 2, 5, 10, 20, and 30. This is a procedure useful for probing motivational limits. We saw a significant main eVect of TCDD on the number of wheel revolutions per session (see Fig. 4). Exposed females also pressed the lever at a slower rate and, consequently, earned significantly fewer run opportunities per session. However, when exposed females did earn an opportunity to run, their rotation speeds equaled those of controls, suggesting that these results represent a motivational rather than a motor deficit. Despite the significant behavioral eVects, vaginal cytology indicated that females from all of the

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FIG. 4. Wheel revolutions by female rats exposed to TCDD prenatally. To unlock the wheel brake for a 20‐sec period, the rat had to press a lever a specified number of times (the FR or fixed ratio) (Markowski et al., 2001).

exposure groups were following normal 4‐ to 5‐day cycles and that estrous cycle phase did not alter their FR behavior. The consistency of the dose–response relationship, which for some FRs took on a quadratic form, prompted further analysis with Benchmark Dose software (BMDS, USEPA). Benchmark doses refer to values derived from modeling the dose–response function, then calculating from it a value that corresponds to a specified eVective dose. It is replacing the traditional measures of toxicity such as the No‐Observed‐Adverse‐EVect‐Level (NOAEL) because it incorporates the entire dose–response function instead of relying on a single number. The Benchmark calculations presented here are for the first session during the FR5 condition (Fig. 5). The benchmark dose (BMD 10 ¼ ED 10) is 7.66 ng/kg TCDD and represents a dip of 10% from the modeled zero dose. The 95% lower bound (BMDL) is about 5.70 ng/kg. In practice, EPA would apply a safety or uncertainty factor to this value to calculate a Reference Dose (RfD), the daily dose that over a lifetime would be presumed free of adverse health eVects. If we follow current EPA procedures, and apply an Uncertainty Factor of 100 to the lower bound to calculate a RfD, current human body burdens would be seen as substantially higher. In a subsequent experiment, Hojo et al. (2002) administered TCDD on GD 8. This day was chosen to immediately precede neural tube closure,

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FIG. 5. Benchmark Dose plot for FR5 performance for access to running wheels. BMD designates the dose at which revolutions declined by 10% from controls. BMDL is the 95% lower bound (Markowski et al., 2001).

the subsequent expansion and diVerentiation of brain regions, and the appearance of AhR in brain on GD 10 (Abbott et al., 1995). Sex diVerences in SCOB prompted our experimental design. Van Haaren and his coworkers (van Hest et al., 1989) have documented consistent male– female diVerences in operant performance. For example, under ratio schedules, the performance of castrated males resembles the lower response rates more typical of control females, suggesting the influence of testosterone. Females, on the other hand, tend to respond more eYciently than do males under a DRL reinforcement schedule. For such reasons, we tested the animals from this experiment with a multiple schedule of food reinforcement. Two diVerent components, an FR 11 and a DRL 10‐sec (diVerential reinforcement of low response rate), schedule alternated within each session. The FR schedule specifies that every nth response is reinforced. It typically engenders high rates of responding. In the incremental FR condition, the FR value was increased every 4 days in an ascending series of values ranging between 1 and 71. Such a progression allowed us to study the transition‐state performances that occur in response to changes in experimental conditions. Transition‐state performances are of particular interest because they reflect the ability of the subject to learn, adapt, or adjust to changing environmental circumstances. The rate and form of such behavioral adjustments may indicate an adverse eVect not seen under final steady‐state conditions, during which compensatory factors have had an opportunity to emerge. A transition‐state procedure can also be viewed as a dynamic challenge that requires the subject to adjust to a new set of circumstances and may

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thereby reveal deficits or vulnerabilities not seen under steady‐state conditions. Unmasking silent toxicity can be achieved using behavioral or other forms of challenges, such as pharmacologic agents or conditions that impose stress on the subject. Such challenges have been used to reveal delayed neurotoxicity after developmental exposures to neurotoxic agents, as well as to evaluate its mechanisms. In a multiple schedule, two or more simple schedules of reinforcement are presented in successively alternating components, with unique stimulus conditions, such as visual or auditory stimuli, signaling which component is in eVect. Typically, the performance of a well‐trained rat in which good discriminative control has been established switches between the components so that responding in each component resembles that seen in a rat trained only under that specific schedule. Discriminative control is a sensitive measure of cognitive function; it requires the subject to focus attention to the stimulus conditions and to respond appropriately. A DRL component was combined with an FR component in the present study. Under a DRL schedule, a clock begins at the onset of the component and after each lever press. Only a press emitted after the specified interval (10 sec in this experiment) has elapsed is reinforced with a food pellet. If the rat responds too early, the clock is reset, and the 10‐sec waiting period begins again. Under this contingency, then, lower rates of responding yield higher rates of pellet delivery. In contrast, high FR rates yield high rates of food delivery. In this experiment, we expected to see high rates of responding in the FR component and low rates in the DRL component. A multiple schedule oVers several advantages. In a sense, it may be thought of as a way to assay multitasking ability, an ability essential to teenagers in the 21st century. By combining schedules of potentially diVerent sensitivities to the exposure agent, we increase the likelihood of measuring exposure eVects. Interpreting the nature of the toxic response may be facilitated by comparing the results across the component schedules because one component schedule acts as a baseline control for the other. Performance on the two components may suggest sensory deficits, may implicate cognitive processes involved in complex learning and memory; or may suggest a role for a specific neurochemical involvement or other mechanisms of action. Because gonadal hormones can influence diVerences in responding between males and females in operant behaviors such as lever‐pressing, neurotoxicants that disturb the organizational eVects of these hormones on brain development could potentially produce enduring performance changes. Should developmental TCDD exposure interfere with sexual diVerentiation of the brain, we would expect to observe an altered pattern of sex diVerences in behavior.

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Normal male rats, for example, tend to emit higher overall response rates than do females under ratio schedules or under schedules that diVerentially reinforce high rates of responding. Under ratio schedules, the performance of castrated males resembles the lower response rates more typical of control females, suggesting the influence of testosterone (van Hest et al., 1989). Females, on the other hand, tend to respond more eYciently than do males under a DRL reinforcement schedule. To maximize food reinforcement, subjects must lever‐press at high rates under the FR component and low rates (at least 10‐sec between individual responses) under the DRL schedule. A unique visual cue indicated which component was active. We observed sexually dimorphic eVects for each schedule component. Under FR 11, we found a significant exposure‐by‐sex interaction. Figure 6 shows that TCDD reduced the response rates of exposed males but increased the response rates of exposed females compared to same‐sex controls. The same exposure‐by‐sex interaction was observed for the DRL component, with exposed males responding more like control females and exposed females responding more like control males. Figure 7 shows Benchmark Dose plots, based on male–female diVerence scores, for both components. The BMD 10 values were 2.77 and 2.97 ng/kg for the FR

FIG. 6. Sex diVerences in fixed‐ratio (FR) responding. The abscissa specifies the number of responses required for delivery of a 45‐mg food pellet. Male oVspring showed a lowering of response rate as a function of TCDD dose. Female oVspring showed an increase in rate (Hojo et al., 2002).

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FIG. 7. Benchmark Dose plot showing sex diVerences in fixed‐ratio (FR) performance. The ordinate represents male–female diVerences in response rates (responses per minute) (BMD and BMDL as in Fig. 5. Hojo et al., 2002).

and DRL components, respectively. These fall below current estimates of human body burdens. 3. MORPHOLOGICAL EFFECTS

We also undertook measures of cortical thickness because sexually dimorphic patterns of cortical lateralization have been documented in both human and animal brains (Zareba et al., 2002). Rat males tend to exhibit right hemisphere dominance compared to females, while females exhibit more diVuse lateralization patterns and greater left hemisphere bias compared to males. Similar asymmetries exist in humans. Because prenatal TCDD exposure causes demasculinization of male rat reproductive structure and function, we decided to examine the eVect of prenatal TCDD on dimorphic cortical lateralization. For morphometry, animals from the control and 180 ng/kg groups were sampled at postnatal day 90. The analysis of gross brain size during fixation with Bouin’s solution showed no statistically significant diVerences between the control and exposed group, nor between sexes, so the lateralization measures are not confounded with size. Patterns of cortical lateralization were changed by GD 8 exposure. Figure 8 plots the results of measurements in the brains of female oVspring in the 180 ng/kg group. A mean width was calculated for each region shown. The diVerence between right and left hemisphere was calculated using the formula R–L (%) ¼ (R–L/R þ L/2)  100. The figure,

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FIG. 8. Changes in brain lateralization indices after gestational exposure to 180 ng/kg TCDD. The numbers adjacent to each bar refer to specific brain areas. The length of each bar is calculated as right – left/100 (Zareba et al., 2002).

based on the brains of female oVspring, shows a pronounced tendency to reversal of dominance in a number of areas. The same tendency was also apparent in male oVspring, but not to the same extent. 4. IMPLICATIONS OF DIOXIN’S NEUROBEHAVIORAL EFFECTS

We can draw the following conclusions from these data:  Sexually dimorphic behaviors are sensitive to doses in the range of

recent human levels.  Exposures in utero may produce global, permanent alterations in be-

havior and brain anatomy.  Behavioral changes reflect altered patterns of brain development.

In their review of dioxin neurotoxicity, Kakeyama and Tohyama (2003) suggested that it exerts such eVects by disrupting gonadal and thyroid hormone function, as well by ‘‘neural‐disrupting action’’ on neural transmission and neural network formation. Such a role would logically be attributed to the AhR. Although the role of the AhR in brain development remains obscure, it may hold the key. For example, gamma‐aminobutyric acid (GABA) cells in at least one sexually dimorphic brain area, the preoptic area, expressed the AhR gene (Hays et al., 2002). What such findings teach us is that direct, simple associations between exposures to endocrine disruptors and their consequences will prove more challenging than we think.

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EMERGING HAZARDS

This section discusses two chemical agents that are currently arousing intense debate because they are so ubiquitous in human tissue and the environment. Unlike pesticides, say, they are not limited to one sphere of use, but are present in a wide range of consumer products and are produced in staggering quantities. Unlike pesticides, too, they have escaped close scrutiny and regulation. The two agents are Bisphenol A and phthalates. They deserve even closer scrutiny, perhaps, than pesticides. A.

Bisphenol A

1.

EXPOSURES AND SOURCES

Bisphenol A (BPA) is a chemical monomer found in epoxy resins, polycarbonate plastics (including many food and beverage containers), in dental prostheses and sealants, in the linings of metal cans used to preserve foods, in baby bottles, and even in the clear plastic cages used to house laboratory animals. U.S. Food and Drug Administration research has found that BPA leaches from infant formula cans into infant formulas (Biles et al., 1997). It also leaches from can linings and microwave ovenware into food products. It has been detected in 95% of human urine samples (Kuklenyik et al., 2003). And, to the dismay of experimenters, it also is released from the polycarbonate cages used to house laboratory animals (Howdeshell et al., 2003). Enough BPA was released as a result of being housed in used polycarbonate cages to produce a 16% increase in uterine weight in prepubertal female mice relative to females housed in used polypropylene cages. U.S. production alone comes to about 800 million pounds annually. Bisphenol A possesses both estrogen agonist and antagonist properties, but can also act as an antiandrogen, blocking the action of dihydrotestosterone. In the mouse, it has led to enlarged prostate glands, reduced sperm production, and testicular (Sertoli cell) damage. In female mice, it advances puberty, alters mammary gland morphology, and, stemming from in utero exposure, produces anomalies in female reproductive organ development. Especially intriguing because of the current obesity epidemic, low doses of BPA also produce elevations in body weight (Howdeshell et al., 1999). Such eVects occur at administered doses (2.4 mg/kg) estimated to correspond to human exposure levels. To discuss how and whether environmental levels of BPA are relevant to neurobehavioral endpoints, it is useful to again review current exposure standards. In EPA practice, a measure of toxic potency, such as the Lowest Observed Adverse EVect Level (LOAEL) is derived from a dose–response function. The LOAEL corresponds to the lowest dose at which an adverse

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eVect was detected. Typically, it is divided by 1000 to provide a margin of safety adequate to protect public health. This value is designated as the RfD. The LOAEL for BPA was determined as 50 mg/kg per day, and the RfD as 50 mg/kg per day. For BPA, the predominant measures of toxicity have been linked to reproductive function. Neurobehavioral studies are more recent additions to the literature. Such data indicate that adverse eVects can occur at exposure levels far lower than those interfering with reproductive function, and even at levels close to the RfD. 2. NEUROBEHAVIORAL OUTCOMES

Bisphenol A has attracted and been the topic of a considerable neurobehavioral literature. Two features of this literature claim our attention. One is dose. Neurobehavioral measures reveal eVects at exposure levels far below those that have been considered free of eVects based on measures such as the rat uterotrophic assay (Ashby, 2001). Based on such endpoints, the current EPA RfD is 50 mg/kg per day, which is 0.1% of the LOAEL, 50 mg/kg. Neurobehavioral approaches have detected eVects at doses below the RfD. The second, striking feature of this literature is the scope of neurobehavioral eVects observed. They extend far beyond those that would be expected to result from an agent whose primary actions were believed to derive from its estrogenic properties. One measure investigated much too infrequently in neurotoxicology is maternal behavior. Unlike sexual performance, which has been studied extensively with EDCs, its expression is more subtle and its measurement more demanding. Its implications are more subtle as well, because aberrant maternal behavior, resulting from fetal exposure, adversely aVects neonatal development, leading to aberrant behavior in the subsequent generation of oVspring, a phenomenon termed epigenetic transmission. Palanza et al. (2002) administered 10 mg/kg BPA to pregnant mice during GD 14–18. One group of female oVspring was then mated, and their maternal behavior scored on PND 2–15. Other females were treated with BPA during adulthood. Treated females showed several changes in maternal behavior. For example, they spent less time nursing their pups; they spent more time grooming; they spent more time outside the nest. Unexpectedly, females exposed to BPA both prenatally and as adults showed many fewer eVects. The important feature here is dose; 10 mg/kg BPA is 20% of the RfD, which is supposed to be without eVects in humans. Social behaviors have also been the object of study. Dessi‐Fulgheri et al. (2002) administered BPA to pregnant Sprague‐Dawley rats, a strain with low sensitivity to estrogenic chemicals. One group of rats received 40 mg/kg BPA from GD 10 to PND 21. Another group received 400 mg/kg BPA from

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GD 14 to PND 6. An extensive inventory of play behaviors in male oVspring was surveyed on PND 35, 45, and 55. Both exposure regimens resulted in altered patterns of behavior. For some measures, the low‐dose regimen produced greater eVects than the high‐dose regimen, i.e., a U‐shaped dose– response function. Such functions appear frequently in toxicology, especially in the EDC literature. The same group also undertook a study of social and sexual behaviors (Farabollini et al., 2002). They administered 40 mg/kg per day of BPA to pregnant and lactating dams using a cross‐fostering design. Rats exposed prenatally (from mating to birth) were raised by control mothers. Rats exposed postnatally (from birth to weaning) came from mothers treated with BPA only during lactation. Four series of observations were conducted once the rats reached 100 days of age: (1) response to the introduction into the home cage of an intruder rat; (2) sexual orientation, defined as visits to an area containing an animal of the opposite sex; (3) male sexual performance; (4) female sexual performance. In females, perinatal BPA tended to increase proceptive behaviors. In males, prenatal exposure increased the frequency of defensive behaviors directed at the intruder male. The influence of BPA on sexual behavior varied with the exposure protocol. BPA treatment generally impaired sexual performance. Prenatal exposure increased intromission latency. Postnatal exposure increased the number of intromissions before ejaculation. The eVects observed in this study were subtle rather than blatant, which would be expected given a dose that lies below the RfD. The same dose, 40 mg/kg per day was also administered by Adriani et al. (2003) to pregnant rats from mating to weaning. One of the endpoints they used is designed to assess ‘‘impulsive’’ behavior. It was measured in an operant chamber with two nose‐poke response devices when the oVspring reached adulthood. One, e.g., the left nose cone, was programmed to deliver a standard 45‐mg food pellet for each snout insertion. The other, the right nose cone, was programmed to deliver five pellets, but only after a specified delay between 10 and 100 sec. Impulsive responding is defined as a shift in preference for the single, immediate reward as the delay increases in duration. In this experiment, BPA attenuated the shift, a finding suggested by the authors to implicate serotonergic mechanisms because selective serotonin reuptake inhibitors, such as fluoxetine, induce a similar eVect. Kawai et al. (2002) also observed a U‐shaped function. They administered BPA to pregnant mice during GD 11–17 in doses of 2 and 20 mg/kg daily, doses consistent with current human exposure (Schonfelder et al., 2002). As endpoints, they chose the response in male oVspring, held in a neutral enclosure, to intruders introduced into the enclosure. They recorded responses at 8, 12, and 16 weeks of age.

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At both BPA doses, testis weights were significantly lower at 12 weeks of age. At 8 weeks of age, contact time, a measure of aggression, was elevated in the treated animals. Testosterone concentrations, although lower in the treated groups, did not fall significantly, and could not account for the behavioral eVects. Two studies by Kubo et al. (2001, 2003) examined sex diVerences in behavior and brain structure following perinatal exposure to BPA. Kubo et al. (2001) administered 1.5 mg/kg to rat dams from GD 1 to PND 21. Open‐field behavior was observed at 6 weeks of age and passive avoidance performance at 7 weeks of age. At 20 weeks of age, the brains were removed and two structures examined. One, the sexually dimorphic nucleus of the preoptic area (SDN‐POA) is much larger in male than in female rats. The second, the locus coeruleus (LC) is larger in females. Female oVspring from control mothers were more active than male oVspring, a typical finding. BPA essentially erased the diVerence. Male controls exhibited longer latencies than females to enter the dark chamber in the passive avoidance procedure, but those exposed to BPA did not. BPA exposure produced no changes in relative SDN‐POA volume, but eliminated sex diVerences in LC volume; it was reduced in females and increased in males. The second study by Kubo et al. (2003) turned to lower doses, administering 30 or 300 mg/kg from GD 1 to PND 21 to the dams to bracket the RfD of 50 mg/kg per day. Anogenital distance was not aVected, nor did the experimenters detect any diVerences in reproductive organ development. The low dose but not high dose of BPA significantly feminized male open‐ field behavior and reduced the number of intromissions in a test of sexual performance (cf., the low‐dose eVect in Dessi‐Fulgheri et al., 2002). As with the first study, SDN‐POA sex diVerences remained intact, but LC volume and cell number, greater in female controls, were reversed by both doses of BPA. The validity of these observations was supported by data from groups of rats treated with diethylstilbestrol and trans‐resveratrol, both of which are estrogenic. Funabashi et al. (2004) also examined brain morphology following prenatal BPA exposure. They dissolved BPA in drinking water to attain a daily dose from mating to weaning of approximately 2.5 mg/kg. Although high compared to most of the other BPA behavioral studies, it is still 1/20 of the NOAEL based on conventional reproductive endpoints. Funabashi et al. chose to examine sex diVerences in two structures, the SDN‐POA and the bed nucleus of the stria terminalis (BST). Both structures are larger in males, but the SDN‐POA shows sex diVerences in the number of corticotrophin‐releasing hormone (CRH), with females exhibiting higher values. The authors found a similar result in BST. BPA did not alter sex diVerences in SDN‐POA, but eliminated them in BST; the number of CRH

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neurons increased in male oVspring brains and decreased in female brains. The implications of such a finding may not be clear, but eliminating sex diVerences in brain anatomy has significant consequences. MacLusky et al. (2005), in a paper that challenges conventional views, examined the formation of dendritic spine synapses in the hippocampus. They noted that, because standard tests of estrogenicity, such as the rat uterotrophic assay, show weak bioactivity, it has been assumed that human BPA exposure is too low to elicit significant estrogenic responses. The studies cited previously, and others as well, demonstrate neurobehavioral eVects at exposure levels as much as 1000 below those required to stimulate uterine growth. These eVects are generally sexually dimorphic; their expression diVers in males and females, and, in some instances, results in elimination of sex diVerences. Such findings imply that BPA, like the dioxin data cited earlier, interferes with sexual diVerentiation of the brain during early development. MacLusky et al. reasoned that if BPA inhibits sexual diVerentiation of the rodent brain, its influence might also be seen in other indices of estradiol on neural structures. They chose to examine, in adult female rats, hippocampal pyramidal cells in an area known as CA1 by measuring dendritic spine synapse density. They based their experiment on the fact that estradiol induces a rapid increase in this density measure. In this study, adult female rats were treated with estradion‐17ß or the oil vehicle one week after undergoing ovariectomy. Selected animals were treated simultaneously with BPA at doses of 40, 120, and 400 mg/kg. A second experiment administered estradiol alone, BPA alone at a dose of 300 mg/kg, or the combination. In both experiments, the animals were sacrificed 30 min after injection. The brains were then processed for stereology. Estradiol almost doubled spine synaptic density. BPA inhibited hippocampal synapse formation in a dose‐dependent fashion. The dose of 400 mg/kg almost completely eliminated the estradiol eVect and regression analysis determined an ED50 for BPA of 117 mg/kg. Even the lowest dose, 40 mg/kg, produced a significant lowering of spine density. This dose is below the EPA RfD of 50 mg/kg per day. The European Commission TDI (Tolerable Daily Intake) of 10 mg/kg per day is based on a 500‐fold safety margin over the LOAEL, a figure derived from three‐generation rat reproductive toxicity assessments. It is only lower by a factor of four than the lowest dose used by MacLusky et al. These authors conclude: ‘‘The ability of BPA to block the eVects of estrogen on [CA1 spine density] raises the possibility that chronic environmental exposure to BPA might interfere with estrogen eVects on the development and function of the brain, inhibiting normal sex diVerences in non‐reproductive behavior . . . as well as exacerbating the negative impact on the aging brain of declining gonadal hormone levels. . ..’’

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The conclusion that emerges from an examination of the BPA literature is that current exposure standards are wholly inadequate for guarding human health. Traditional assay methods (such as Ashby, 2001) yield values that are magnitudes greater than those derived from neurobehavioral assessments. If a dose of 10 mg/kg, as used by Palanza et al. (2002) in their study of maternal behavior, were chosen as the LOAEL, and, by convention, divided by 1000 to obtain a RfD of 10 ng/kg, the latter value would fall significantly below current human exposure levels. B.

Phthalate Esters

Phthalates comprise a class of chemicals used as softeners, or plasticizers, in polyvinyl chloride (PVC, vinyl) products, including children’s toys, decorating and building products, blood bags, and in solvents and other additives in a wide range of consumer products, including cosmetics, personal care products, wood finishes, and insecticides. The ink used to print on plastic, foil‐packed products frequently contains phthalates, as do the adhesives used in packaging. In these applications, phthalates migrate or dissolve into the products they enclose. They are also found in products such as baby formula, cheese, margarine, and potato chips because phthalates are fat soluble. In addition, they gravitate to fat cells and breast milk. They are perhaps the most abundant synthetic chemical contaminant in the environment. Production of the most prevalent phthalate ester, di(2‐ethylhexyl) phthalate, or DEHP, is about 4 million tons annually. The European Commission has prohibited the sale of toys and childcare articles often placed in the mouth by children less than 3 years of age that are made of soft PVC containing more than 0.1% by weight of six phthalates. The current literature oVers only fragments of information about the developmental neurotoxicity of phthalates. They are included in this chapter because they are universally distributed in human tissues, because they are now linked to a collection of hormonally linked disorders falling within the definition of testicular dysgenesis syndrome described earlier, and because their anti‐androgenic eVects, disclosed by a variety of measures, provide a compelling reason for believing that they can exercise significant eVects on neurobehavioral development. In the last few years, transgenerational studies on the reproductive toxicity of the phthalate esters have demonstrated that several of them produce reproductive organ malformations in male rat oVspring after in utero and neonatal treatment. Fisher (2004) noted that exposing rats in utero to dibutyl phthalate (DBP) produces a syndrome resembling the testicular dysgenesis syndrome described earlier. It includes reduced spermatogenesis, cryptorchidism, hypospadias, and hyperplastic cells. Gray et al. (2000) administered DEHP to pregnant rats

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during the presumed period of sexual diVerentiation (GD 14 to PND 3). The male oVspring displayed a variety of eVects that are defined as demasculinization and feminization. These include a shortened anogenital distance at birth, retained nipples, cleft phallus with hypospadias, undescended testes, and small to absent sex accessory glands. These toxic outcomes presumably arise from the anti‐androgenic properties of certain of the phthalates. The androgens testosterone and its metabolite, dihydrotestosterone, are produced by fetal males and determine the male phenotype. Sexual diVerentiation of the brain and behavior is a product of both estradiol, produced by conversion of testosterone via aromatase (Fig. 1), and androgens themselves; the latter apparently are responsible for sexually dimorphic play behaviors. Another EDC with anti‐androgenic properties, the widely used fungicide vinclozolin, transformed male rat pup play behavior to female patterns after administration during the early postnatal period (Hotchkiss et al., 2002). One study, of a preliminary nature, suggested that developmental exposure to DEHP may produce behavioral consequences. Arcadi et al. (1998) administered low doses to pregnant rats from GD 1 to PND 21. At one month of age, they measured performance of female oVspring on a beam‐ walking test. To avoid the combination of a loud tone and bright light, the rats had to walk along a narrow beam to reach a dark box, at which point the stimuli ceased. Their data showed a dose‐related increase in the time needed to traverse the beam. The doses used by these investigators are far lower than those used by others, so these eVects need to be replicated. They did not explain why they studied only females, given that DEHP is viewed as toxic to male reproductive structure and function. A comprehensive investigation by Moore et al. (2001) points to the directions in which queries about phthalate developmental neurotoxicity should be pursued. They dosed pregnant rats with varying doses of DEHP from GD 3 through PND 21. Their survey of outcomes, like those of Gray et al. (2000), demonstrated reduced anogenital distance, areola and nipple retention, undescended testes, and incomplete preputial separation (an external sign of puberty in the rat). In addition, they found prostate deformities, reduced sperm counts, penile malformations, and other markers of TDS. But the most relevant finding for questions about phthalate neurotoxicity was impaired sexual behavior. DEHP‐exposed males, at about 2.5 months of age, exhibited deficient copulatory performance; they failed to mount, intromitt, or ejaculate. The authors termed these eVects ‘‘demasculinized sexual behaviors.’’ They note that these eVects cannot be attributed to lowered circulating testosterone levels, which maintain adult copulatory performance in rats at levels even 1/3 of normal, or to their other findings. Instead, they write, ‘‘the most likely explanation is that in utero

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and lactational DEHP exposure causes incomplete sexual diVerentiation of the CNS.’’ From a neurobehavioral perspective, our current understanding of their actions indicates that phthalates possess the capacity to alter brain development and its functional expression. Whether they do so in humans at prevailing environmental exposure levels is not settled; the question has not been investigated. New data, however, show correlations between maternal levels of phthalate metabolites in urine and anogenital distance in infant boys (Swan et al., 2005); the higher the maternal levels, the shorter the distance, a finding that is a sign of demasculinization or feminization. These findings suggest the possibility that prenatal exposure to phthalates may alter brain development, a process in which androgens play a crucial role. Behavioral endpoints are rarely linked to a single variable. However, some behaviors, such as rough‐and‐tumble play, are specifically organized by androgens. The critical time in the rat for organization of play is the early neonatal period, when interactions of androgens with the AR set up the sex diVerence observed in juveniles (Meaney, 1989; Meaney & McEwen, 1986). Increased male rough‐and‐tumble play is specifically organized by the stimulation of ARs in the amygdala region of the brain (Meaney, 1989) and is not dependent on activational eVects of steroids (Meaney & McEwen, 1986). Further, androgen receptor dysfunctions, such as testicular feminized mutant males, neonatal castration, and neonatal exposure to the androgen‐receptor antagonist flutamide, decrease the expression of play by juvenile males, whereas administration of estradiol agonists does not increase play in either male or female rats. Therefore, rat rough‐and‐tumble play is unique in that it is clearly organized by androgens themselves during the neonatal period and is not influenced by activational eVects of other steroid hormones. Anti‐androgens may also influence maternal responses. Tomaszycki et al. (2001) studied maternal responses to separation–rejection vocalizations emitted by infant rhesus monkeys following prenatal administration of flutamide. Flutamide is a drug used to treat prostate tumors because it interferes with the action of testosterone. Flutamide treatment early in gestation (before GD 70) reduced the likelihood that mothers would respond to their male oVspring, perhaps because these infants also displayed more female‐typical vocalizations; rhesus mothers are more likely to respond to male infant cries. These findings provide an example of how an organism’s behavior guides the response of its social environment to the organism. This is an important result because it shows how alterations in sex‐specific behaviors can also modify or even determine the response of conspecifics, especially the mother, to the individual. Exploration of the neurobehavioral consequences of developmental exposure to phthalates deserves a high priority. We have learned enough about its

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biological, especially endocrine‐disrupting eVects, to oVer a compelling argument that these consequences will emerge in exposed humans.

IV.

ENDOCRINE DISRUPTION AND NEUROBEHAVIORAL DEVELOPMENT

When endocrine disruptors came to be perceived, in the 1990s, as a serious environmental threat to human health, their ability to interfere with early development was viewed as their primary source of concern. That view is bolstered by a constantly expanding literature and even by studies such as those by MacLusky et al. (1998, 2005) showing low‐dose acute eVects in adults. The adult studies merely expand our view of possible outcomes. The sheer breadth of neurotoxic possibilities uncovered so far, however, means that our comprehension of endocrine disruptor health risks is severely limited. This chapter, given the narrow scope of current knowledge, is hardly more than a beginner’s guide to the topic. What we do understand about this class of chemical agents is the subtle, often unexpected ways in which it leaves its message. This chapter is designed to foster an appreciation of such unexpected consequences. REFERENCES Abbott, B. D., Birnbaum, L. S., & Perdew, G. H. (1995). Developmental expression of two members of a new class of transcription factors: I. Expression of aryl hydrocarbon receptor in the C57BL/6N mouse embryo. Developmental Dynamics, 204, 133–143. Adriani, W., Seta, D. D., Dessi‐Fulgheri, F., Farabollini, F., & Laviola, G. (2003). Altered profiles of spontaneous novelty seeking, impulsive behavior, and response to D‐ amphetamine in rats perinatally exposed to bisphenol A. Environmental Health Perspectives, 111, 395–401. Aoki, Y. (2001). Polychlorinated biphenyls, polychlorinated dibenzo‐p‐dioxins, and polychlorinated dibenzofurans as endocrine disrupters—What we have learned from Yusho disease. Environmental Research, 86, 2–11. Arcadi, F. A., Costa, C., Imperatore, C., Marchese, A., Rapisarda, A., Salemi, M., Trimarchi, G. R., & Costa, G. (1998). Oral toxicity of bis(2‐ethylhexyl) phthalate during pregnancy and suckling in the Long‐Evans rat. Food and Chemical Toxicology, 36, 963–970. Ashby, J. (2001). Increasing the sensitivity of the rodent uterotrophic assay to estrogens, with particular reference to bisphenol A. Environmental Health Perspectives, 109, 1091–1094. Berenbaum, S. A. (1999). EVects of early androgens on sex‐typed activities and interests in adolescents with congenital adrenal hyperplasia. Hormones and Behavior, 35, 102–110. Biles, J. E., White, K. D., McNeal, T. P., & Begley, T. H. (1997). Determination of the diglycidyl ether of bisphenol A and its derivatives in canned foods. Journal of Agricultural and Food Chemistry, 47, 1965–1969. Bjerke, D. L., Brown, T. J., MacLusky, N. J., Hochberg, R. B., & Peterson, R. E. (1994). Partial demasculinization and feminization of sex behavior in male rats by in utero and lactational

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exposure to 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin is not associated with alterations in estrogen receptor binding or volumes of sexually diVerentiated brain nuclei. Toxicology and Applied Pharmacology, 127, 258–267. CDC (2003). Second National Report on Human Exposure to Environmental Chemicals. Atlanta, Georgia: Department of Health and Human Services. Colborn, T., Dumanoski, D., & Myers, J. P. (1996). Our stolen future. New York: Dutton. Dessi‐Fulgheri, F., Porrini, S., & Farabollini, F. (2002). EVects of perinatal exposure to bisphenol A on play behavior of female and male juvenile rats. Environmental Health Perspectives, 110(Suppl. 3), 403–407. DeVito, M. J., Birnbaum, L. S., Farland, W. H., & Gasiewicz, T. A. (1995). Comparisons of estimated human body burdens of dioxinlike chemicals and TCDD body burdens in experimentally exposed animals. Environmental Health Perspectives, 103, 820–831. Farabollini, F., Porrini, S., Della Seta, D., Bianchi, F., & Dessi‐Fulgheri, F. (2002). EVects of perinatal exposure to bisphenol A on sociosexual behavior of female and male rats. Environmental Health Perspectives, 110(Suppl. 3), 409–414. Fisher, J. S. (2004). Environmental anti‐androgens and male reproductive health: Focus on phthalates and testicular dysgenesis syndrome. Reproduction, 127, 305–315. Funabashi, T., Kawaguchi, M., Furuta, M., Fukushima, A., & Kimura, F. (2004). Exposure to bisphenol A during gestation and lactation causes loss of sex diVerence in corticotropin‐ releasing hormone‐immunoreactive neurons in the bed nucleus of the stria terminalis of rats. Psychoneuroendocrinology, 29, 475–485. Gray, L. E., Jr., Ostby, J., Furr, J., Price, M., Veeramachaneni, D. N., & Parks, L. (2000). Perinatal exposure to the phthalates DEHP, BBP, and DINP, but not DEP, DMP, or DOTP, alters sexual diVerentiation of the male rat. Toxicological Sciences, 58, 350–365. Gray, L. E., Wolf, C., Mann, P, & Ostby, J. S. (1997). In utero exposure to low doses of 2,3,7,8‐ tetrachlorodibenzo‐p‐dioxin alters reproductive development of female Long Evans hooded rat oVspring. Toxicology and Applied Pharmacology, 146, 237–244. Hays, L. E., Carpenter, C. D., & Petersen, S. L. (2002). Evidence that GABAergic neurons in the preoptic area of the rat brain are targets of 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin during development. Environmental Health Perspectives, 110(Suppl. 3), 369–376. Herrnstein, R. J.., & Murray, C. (1994). The bell curve. New York: Free Press. Hojo, R., Stern, S., Zareba, G., Markowski, V. P., Cox, C., Kost, J. T., & Weiss, B. (2002). Sexually dimorphic behavioral responses to prenatal dioxin exposure. Environmental Health Perspectives, 110, 247–254. Hotchkiss, A. K., Ostby, J. S., Vandenbergh, J. G., & Gray, L. E., Jr. (2002). An environmental antiandrogen, vinclozolin, alters the organization of play behavior. Physiology and Behavior, 79, 151–156. Howdeshell, K. L., Hotchkiss, A. K., Thayer, K. A., Vandenbergh, J. G., & vom Saal, F. S. (1999). Exposure to bisphenol A advances puberty. Nature, 401, 763–764. Howdeshell, K. L., Peterman, P. H., Judy, B. M., Taylor, J. A., Orazio, C. E., Ruhlen, R. L., Vom Saal, F. S., & Welshons, W. V. (2003). Bisphenol A is released from used polycarbonate animal cages into water at room temperature. Environmental Health Perspectives, 111, 1180–1187. Kakeyama, M., Sone, H., Miyabara, Y., & Tohyama, C. (2003). Perinatal exposure to 2,3,7,8‐ tetrachlorodibenzo‐p‐dioxin alters activity‐dependent expression of BDNF mRNA in the neocortex and male rat sexual behavior in adulthood. Neurotoxicology, 24, 207–217. Kakeyama, M., & Tohyama, C. (2003). Developmental neurotoxicity of dioxin and its related compounds. Industrial Health, 41, 215–230. Kawai, K., Nozaki, T., Nishikata, H., Aou, S., Takii, M., & Kubo, C. (2002). Aggressive behavior and serum testosterone concentration during the maturation process of male

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mice: The eVects of fetal exposure to bisphenol A. Environmental Health Perspectives, 111, 175–178. Kubo, K., Arai, O., Omura, M., Watanabe, R., Ogata, R., & Aou, S. (2003). Low dose eVects of bisphenol A on sexual diVerentiation of the brain and behavior in rats. Neuroscience Research, 45, 345–356. Kubo, K., Arai, O., Ogata, R., Omura, M., Hori, T., & Aou, S. (2001). Exposure to bisphenol A during the fetal and suckling periods disrupts sexual diVerentiation of the locus coeruleus and of behavior in the rat. Neuroscience Letters, 304, 73–76. Kuklenyik, Z., Ekong, J., Cutchins, C. D., Needham, L. L., & Calafat, A. M. (2003). Simultaneous measurement of urinary bisphenol A and alkylphenols by automated solid‐phase extractive derivatization gas chromatography/mass spectrometry. Analytical Chemistry, 75, 6820–6825. Mably, T. A., Moore, R. W., Goy, R. W., & Peterson, R. E. (1992). In utero and lactational exposure of male rats to 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin. 2. EVects on sexual behavior and the regulation of luteinizing hormone secretion in adulthood. Toxicology and Applied Pharmacology, 114, 108–117. MacLusky, N. J., Brown, T. J., Schantz, S., Seo, B. W., & Peterson, R. E. (1998). Hormonal interactions in the eVects of halogenated aromatic hydrocarbons on the developing brain. Toxicology and Industrial Health, 14, 185–208. MacLusky, N. J., Hajszan, T., & Leranth, C. (2005). The environmental estrogen bisphenol‐A inhibits estrogen‐induced hippocampal synaptogenesis. Environmental Health Perspectives, 113, 675–679. Markowski, V. P., Zareba, G., Stern, S., Cox, C., & Weiss, B. (2001). Altered operant responding for motor reinforcement and the determination of benchmark doses following perinatal exposure to low‐level 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin. Environmental Health Perspectives, 109, 621–627. McEwen, B. S. (1987). Steroid hormones and brain development: Some guidelines for understanding actions of pseudohormones and other toxic agents. Environmental Health Perspectives, 74, 177–184. Meaney, M. J., & McEwen, B. S. (1986). Testosterone implants into the amygdala during the neonatal period masculinize the social play of juvenile female rats. Brain Research, 398, 324–328. Meaney, M. J. (1989). The sexual diVerentiation of social play. Psychiatric Development, 7, 247–261. Mocarelli, P., Gerthoux, P. M., Ferrari, E., Patterson, D. G., Jr., Kieszak, S. M., Brambilla, P., Vincoli, N., Signorini, S., Tramacere, P., Carreri, V., Sampson, E. J., Turner, W. E., & Needham, L. L. (2000). Paternal concentrations of dioxin and sex ratio of oVspring. Lancet, 355, 1858–1863. Moore, R. W., Rudy, T. A., Lin, T. M., Ko, K., & Peterson, R. E. (2001). Abnormalities of sexual development in male rats with in utero and lactational exposure to the antiandrogenic plasticizer Di(2‐ethylhexyl) phthalate. Environmental Health Perspectives, 109, 229–237. Neuberger, M., Rappe, C., Bergek, S., Cai, H., Hansson, M., Jager, R., Kundi, M., Lim, C. K., Wingfors, H., & Smith, A. G. (1999). Persistent health eVects of dioxin contamination in herbicide production. Environmental Research, 81, 206–214. Palanza, P. L., Howdeshell, K. L., Parmigiani, S., & vom Saal, F. S. (2002). Exposure to a low dose of bisphenol A during fetal life or in adulthood alters maternal behavior in mice. Environmental Health Perspectives, 110(Suppl. 3), 415–422. Petersen, S. L., Curran, M. A., Marconi, S. A., Carpenter, C. D., Lubbers, L. S., & McAbee, M. D. (2000). Distribution of mRNAs encoding the arylhydrocarbon receptor,

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arylhydrocarbon receptor nuclear translocator, and arylhydrocarbon receptor nuclear translocator‐2 in the rat brain and brainstem. Journal of Comparative Neurology, 427, 428–439. Rogan, W. J., & Gladen, B. C. (1992). Neurotoxicology of PCBs and related compounds. Neurotoxicology, 13, 27–35. Schantz, S. L., & Bowman, R. E. (1989). Learning in monkeys exposed perinatally to 2,3,7,8‐ tetrachlorodibenzo‐p‐dioxin (TCDD). Neurotoxicology and Teratology, 11, 13–19. Schantz, S. L., & Widholm, J. J. (2001). Cognitive eVects of endocrine‐disrupting chemicals in animals. Environmental Health Perspectives, 109, 1197–1206. Schonfelder, G., Wittfoht, W., Hopp, H., Talsness, C. E., Paul, M., & Chahoud, I. (2002). Parent bisphenol A accumulation in the human maternal–fetal–placental unit. Environmental Health Perspectives, 110, A703–A707. Sharpe, R. M., & Skakkebaek, N. E. (1993). Are estrogens involved in falling sperm counts and disorders of the male reproductive tract? Lancet, 341, 1392–1395. Skaare, J. U., Larsen, H. J., Lie, E., Bernhoft, A., Derocher, A. E., Norstrom, R., Ropstad, E., Lunn, N. F., & Wiig, O. (2002). Ecological risk assessment of persistent organic pollutants in the arctic. Toxicology, 181–182, 193–197. Skakkebaek, N. E. (2003). Testicular dysgenesis syndrome. Hormone Research, 60(Suppl. 3), 49. Swan, S. H., Elkin, E. P., & Fenster, L.. (2000). The question of declining sperm density revisited: An analysis of 101 studies published 1934–1996. Environmental Health Perspectives, 108, 961–966. Swan, S. H., Main, K. M., Fan Liu, Stewart, S. L., Kruse, R. L., Calafat, A. M., Mao, C. S., Redmon, J. B., Ternand, C. L., Sullivan, S., & Teague, J. L. (2005). Anogenital distance— A marker of fetal androgen action—is decreased in male infants following phthalate exposure during pregnancy. Environmental Health Perspectives, 113, 1056–1061. Tomaszycki, M. L., Davis, J. E., Gouzoules, H., & Wallen, K. (2001). Sex diVerences in infant rhesus macaque separation–rejection vocalizations and eVects of prenatal androgens. Hormones and Behavior, 39, 267–276. van Hest, A., van Haaren, F., & van de Poll, N. E. (1989). Perseverative responding in male and female Wistar rats: EVects of gonadal hormones. Hormones and Behavior, 23, 57–67. Weiss, B., & O’Donoghue, J. (Eds.) (1994). Neurobehavioral toxicity: Analysis and interpretation. New York: Raven Press. Weiss, B. (2000). Vulnerability of children and the developing brain to neurotoxic hazards. Environmental Health Perspectives, 108(Suppl. 3), 375–381. Zareba, G., Hojo, R., Zareba, K. M., Watanabe, C., Markowski, V. P., Baggs, R. B., & Weiss, B. (2002). Sexually dimorphic alterations of brain cortical dominance in rats prenatally exposed to TCDD. Journal of Applied Toxicology, 22, 129–137.

The Neurotoxic Properties of Pesticides HERBERT L. NEEDLEMAN SCHOOL OF MEDICINE, UNIVERSITY OF PITTSBURGH PITTSBURGH, PENNSYLVANIA

I.

INTRODUCTION

Every year a billion pounds or more of pesticides (herbicides, insecticides, fungicides, and other agents) are added to homes, water, food, and soil. Many of these compounds work by damaging the nervous systems of insects. They are neurotoxins. Because their large numbers of actively dividing and diVerentiating neural cells enhance susceptibility to toxicant‐induced damage, embryos, fetuses, and young children are at particular risk of toxicity. This concern extends well beyond the theoretical for two reasons: pesticides cross the placental barrier and neurogenesis continues through childhood. Despite the staggering volume of chemicals in widespread use as pesticides, the study of their neurotoxicity is a relatively young discipline. In the late nineteenth century, the major pesticides in use were lead, sulfur compounds, and arsenicals, all highly toxic agents. With the expansion of the synthetic chemical industry after World War II, DDT, Phenoxyacetic acid (2‐4‐D), and carbamates were produced and used in large amounts. DDT, introduced in 1930, saved thousands of lives from malaria and typhus while increasing crop yields around the world. Because of these factors, it was used widely and, until the 1960s, it was considered harmless. By 1955, 90% of all pesticides in use were commercially synthesized. Pyrethrum, a natural pesticide derived from chrysanthemums, has been in use since 1850, but has been rapidly replaced in commercial use by synthetic pyrethroids. These pyrethroids also have demonstrated neurotoxic properties. Organochlorine pesticides, because of their persistence in the environment, began to be replaced by organophosphates in the 1970s. Pesticides in common use are shown in Table I. The neurotoxicity of lead, a metal long in use as a pesticide, was recognized at least as far back as the second century BC, with the warning by the INTERNATIONAL REVIEW OF RESEARCH IN MENTAL RETARDATION, Vol. 30 0074-7750/06 $35.00

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Copyright 2006, Elsevier Inc. All rights reserved. DOI: 10.1016/S0074-7750(05)30007-3

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Pesticide

Type

Aldicarb Carbaryl Acephate Azinphos‐methyl Chlorpyrifos Cypermethrin Permethrin

Carbamate Carbamate OP OP OP Pyrethrin Pyrethrin

Est. Pounds applied US 1992

Est. Pounds applied US 1997

4,022,468 4,570,414 3,389,865 2,548,867 14,764,535 228,082 1,068,598

4,227,552 4,857,542 2,462,354 2,091,014 13,463,879 187,991 1,066,056

Est. Acres treated US 1992

Est. Acres treated US 1997

14,033,279 46,573,318 13,106,811 12,911,243 110,605,402 11,177,200 99,367,875

15,250,029 43,533,709 14,825,546 12,859,058 109,548,948 Not available 94,909,695

Greek physician Nikander that ‘‘Lead makes the mind give way.’’ It has now been abandoned as a pesticide. Organophosphates (OPs) are the commonest pesticides in current use. Their neurotoxicity first came to notice in the 1930s. In southern states, triorthocresyl phosphate (TOCP) was added to Ginger Jake, a popular soft drink. Because prohibition was in eVect, and because the drink contained some alcohol, consumption was widespread. Between 20,000 and 50,000 men who imbibed were aZicted with tremor and paralysis. This condition, often permanent, became known as the ‘‘Ginger Jakes.’’

II.

EXPOSURE

Over 34,000 pesticides are registered by the Environmental Protection Agency (EPA) and over 1 billion pounds of conventional pesticides are used annually in the United States. Their use and dispersion has grown exponentially. In 1955, 3% of all corn and soy crops were treated with herbicides. By 1985, the number had grown to 95%. In addition to agricultural applications, pesticides are used inside housing and schools, on lawns, and on pets. Approximately 60 million pounds of OP pesticides are applied to U.S. agricultural crops annually; nonagricultural uses account for about 17 million pounds per year. Exposures may occur by ingestion, inhalation, or dermal contact. Farm workers, applicators, and manufacturers of these pesticides may have higher exposures, but ingestion of food contaminated with OP pesticides and contact during residential application are the primary sources of exposure for the general population.

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Today’s high levels of pesticide use have not changed substantially since the mid‐1990s, although there has been some shift in the mix of agents produced. Little attention was given to the toxicity of pesticides until relatively recently. In 1963, Rachel Carson published Silent Spring, and awakened the public to the dire eVects of organochlorine pesticides, such as DDT, on avian reproduction. After a period of struggle, DDT was banned altogether in the United States because of its persistence in the environment, with resultant bioaccumulation and mammalian toxicity. Later, dibromochloropropane (DBCP) was shown to cause irreversible damage to human spermatogenesis, with resultant sterility among exposed workers. Carbamates (used extensively since the 1950s) and OPs are now among the most commonly used classes of insecticides in the United States. Pesticide use in homes, schools, food, and water provide ample opportunity for human exposure. Approximately 90% of households use pesticides. In Florida, detectable levels of pesticides were found in 88 to 100% of houses. The average home used 7.5 pesticides. In one survey, 93% of California schools reported pesticide use. Of those pesticides used, 54% were classified as neurotoxicants (Lewis, Fortmann, & Camman, 1994). Children’s exposure to pesticides, because of their behavior and diet, is greater than that of adults. In the large Centers for Disease Control and Prevention (CDC) survey of exposures, the 50th percentile for diethylphosphate, a metabolite of chlorpyrifos, was 1.4 ng/ml for children, 1.0 for adults. Contamination of foods continues to be an important source for human exposure. Surveys by the EPA and the Consumers Union showed detectable pesticide residues on over half of vegetables tested; anywhere from 9 to over 20 diVerent pesticides were detected. Water supplies frequently contain residues. The U.S. Geological Survey (USGS) tested 5000 water samples from 1991–1995. At least one pesticide was detected in every stream sampled, and half of the ground water samples were positive. (U. S. Geological Survey, 1997) A survey in 1995 by the Environmental Working Group tested the drinking water of 29 Midwestern cities and found levels in excess of federal standards of 35% for cyanazine and 17% for atrazine. They estimated that in the 6‐week testing period, 10,000 infants in the test cities drank formula constituted with drinking water over the standard (Cohen, Wiles, & Bondoc, 1995). Atrazine degrades into three metabolites: desisopropyl atrazine, desethylatrazine, and dialkylatrazine; all display similar toxicity as the parent molecule. A 1990 USGS found desethylatrazine in 98% of all surface water samples and desisopropyl atrazine in 90%. In addition to the neurotoxicity of organochlorines, they have been shown to have eVects on the endocrine system. Semen quality has been shown to be impaired by exposure to alachlor and atrazine (Swan, Kruse, Liu, Barr, Drobnis, Redmon, Wang, Brazil, & Overstreet, 2003), and feminization

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of male frogs has been detected (Hayes, Haston, Tsui, Hoang, HaeVele, & Vonk, 2002). In farm areas, exposure is not limited to pesticide applicators. A study of migrant farm workers in California found elevated levels of diazinon, chlorpyrifos, and malathion in household dust and on the hands of resident children. (Bradman, Harnly, Draper, Seidel, Teran, Wakeham, & Neutra, 1997). Reports to U.S. poison control centers provide an estimate of high‐dose exposures. Over 123,000 clinical cases of home‐based pesticide exposure were recorded; the major toxicant was OPs. Despite the ban on a single OP, chlorpyrifos (CPF), for household use, its use has been replaced by other OPs. Chlorpyrifos continues to be used in farming, on golf courses, and in new home foundations. An unknown but sizable number of homes have the product on the shelf and continue to use it.

III.

SETTING PESTICIDE STANDARDS

Regulatory standards for pesticides are constructed from two databases: experimental studies, primarily of rodents, and observational data from adult humans. The former director of the Neurotoxicolgy Division of the EPA clearly stated the limitations of adult data and makes a case for the particular vulnerability of children and the need for longitudinal studies: Risk assessors should be aware that chemical‐induced neurotoxicity in adults may not always be a good predictor of developmental neurotoxicity. Adverse eVects on the developing nervous system can occur prior to conception up to the time of sexual maturity, depend on the time of exposure relative to a critical state of nervous system development, can be seen at any time during the lifespan of the organism, may lead to delayed onset or latent eVects, and may elicit compensatory mechanisms that obscure underlying neurotoxicity. Hugh Tilson (2000).

In 1993, after five years of discussion, the National Academy of Sciences issued a groundbreaking report, ‘‘Pesticides in the Diets of Infants and Children.’’ This report, in addition to delineating the biological and behavioral diVerences that result in increased childhood exposure and sensitivity to pesticides, recommended that the EPA modify its decision‐making process for setting tolerances to acknowledge the health eVects of pesticide exposure as opposed to agricultural practices. Despite this clear instruction, no changes were made. As a consequence, Congress unanimously passed the Food Quality Protection Act. This law called for an additional 10‐fold safety factor to be used to define reference doses of pesticides to protect children. There are now three safety factors, each requiring a 10‐fold reduction: one

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to accommodate the extrapolation from animals to humans, one to accommodate the variances of sensitivity among individuals, and one for children. No observed eVect levels (NOEL) for OP pesticides, data required for regulation, have been set on the basis of rodent studies that measure peripheral blood activity of acetylcholinesterase (AChE), a target of OP. Studies that have examined neurotoxicity employed coarse and insensitive measures of brain weight, gross malformations, and neurological function as estimates of eVect. Other studies, to be presented later, show alterations in rodent neurochemistry, structure, and behavior in the absence of AChE alterations. Exposure to neurotoxic chemicals does not begin at birth. Lead (Bellinger, Leviton, Waternaux, Needleman, & Rabinowitz, 1987), PCBs (Jacobson & Jacobson, 1997), and mercury (Grandjean, Weihe, White, & Debes, 1999) are among a group of toxicants in which intrauterine exposure has been found to impair later infant development. OPs, carbamates, and pyrethroids, agents that are designed to damage insect nervous systems, find similar toxic targets in humans. Since 2000, studies have found high detection rates of OPs in newborns.

IV.

THE NEUROTOXICITY OF PESTICIDES

About 80 OPs are in use as pesticides. Of these, about 50 account for 70% of the production. Because of their lethal properties, four OPs have been used as military weapons. Three were employed by the German army in World War II (tabun, sarin, soman); VX was developed by Britain and the United States.

A.

Organophosphates

OPs have a number of toxic properties. Perhaps the most prominent is that they interfere with the breakdown of acetylcholine (ACh), a vital and widely distributed neurotransmitter. ACh carries stimuli across the synapse. After it conveys its message, it is metabolized and inactivated by enzymes known as AChEs. If they persist in the synapse, hyperstimulation results. AChE‐inhibiting pesticides are absorbed by three routes: the skin, the lungs, and the gut. Because of their lipophilicity, they are absorbed to a considerable degree into the brain. Their accumulation in fat stores is responsible for relapsing episodes of toxicity that may occur after external exposure has ceased (Baldi, Lebailly, Mohammed‐Brahim, Letenneur, Dartigues, & Brochard, 2003).

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OPs interact with two diVerent groups of compounds that break them down; both are esterase. A‐esterases hydrolyze and thereby detoxify the compound. Paroxonase is a member of this group. AChE is a B‐esterase. The toxicity of OPs is not due solely to their interaction with AChE. Noncholinergic toxicity is discussed later in the chapter. By interfering with AChE, persistence of the neurotransmitter acetylcholine ensues, resulting in hyperstimulation of synapses. (Slotkin, 1999; Whitney, Seidler, & Slotkin, 1995). OPs, in general, inhibit AChE irreversibly, whereas carbamates’ eVects are reversible. Acute toxicity with OPs results in overstimulation of muscarinic and nicotinic receptors which are present in the peripheral nervous system and, to some degree, within the central nervous system. In addition to down‐ regulation of muscarinic receptors, CPF lead to altered brain RNA concentrations and inhibition of DNA synthesis within 4 hours of treatment in all brain regions. Symptoms have commonly included excessive salivation, vomiting, tachycardia, lethargy, muscle weakness, hypertonia, and respiratory distress. Some OPs lead to a delayed long‐term polyneuropathy in adults involving tingling, weakness, and ataxia of the lower limbs, with resultant paralysis (Abdiou‐Donia & Lapadula, 1990; Lotti, 2002). The degree to which symptoms among individuals with low‐level chronic exposure correspond with levels of AChE or cholinesterase inhibition is not clear. Nevertheless, chronic pesticide exposure has been linked to anxiety and irritability (Metcalf & Holmes, 1969), as well as diYculty in concentration, word finding, alertness, and hand–eye coordination. (Bowers & Sim, 1964) Decreased AChE activity is not the sole mechanism of OP neurotoxicity; the correlation between cholinesterase and pseudocholinesterase activity and neuronal damage is not strong (Klassen, Amdur, & Doull, 1986). In addition to their action on AChE, OPs have been found to aVect adenyl cyclase (Song, Seidler, Saleh, Zhang, Padilla, & Slotkin, 1997). This is a molecule essential in energy metabolism and brain signaling. OPs thereby interfere with cell development. These eVects may be expressed in altered behavior such as increased maze‐running time and errors (dichlorvos) (Schulz, Nagygymatenyi, & Desi, 1995) or impaired open field activity (diazinon) (Spyker & Avery, 1977). OPs are alkylating agents, and have been reported to have mutagenic properties. They have also been reported to interfere with immune function (Moriya, Ohta, Watanabe, Miyazawa, Kato, & Shirasu, 1983; Newcombe, 1992). Important contributions to understanding the noncholinergic toxic mechanisms of OPs have been made by the toxicology group at Duke University. They measured markers for oligodendrocytes (neuronal support cells),

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neuronal cell bodies, and developing axons after giving CPF to rodents. Prenatal administration elicited an immediate increase in myelin basic protein and neurofilament markers. At postnatal day 30, there were deficits in the biomarkers. Early postnatal administration of CPF evoked no changes, but treatment on postnatal day 11–14 produced reductions in all three biomarkers by postnatal day 30 (Garcia, Seidler, & Slotkin, 2003). The same group found persistent changes in gender‐specific performance in maze‐running tasks after early and late administration of CPF at low doses (Levin, Nakajima, Christopher, Seidler, & Slotkin, 2001). When they gave CPF to neonatal rats, they found no immediate eVect on T‐lymphocyte function. When the rats were followed into adulthood, T‐cell replication rates were significantly impaired. The authors suggest that the delayed changes were due to lasting alterations of neural function (Navarro, Basta, Seidler, & Slotkin, 2001). Alterations in synaptic development and neuronal activity in cholinergic and catacholaminergic synapses were reported after administration of subtoxic doses of CPF (Dam, Garcia, Seidler, & Slotkin, 1999). DNA synthesis was also aVected (Dam, Seidler, & Slotkin, 1998). EVects on serotonergic signals were also reported by this group (Aldridge, Seidler, Meyer, Thillai, & Slotkin, 2003). After administration of CPF to pregnant dams on gestational days 17–20, the time of peak neurogenesis, offspring females showed impaired accuracy and slower habituation in maze running. The authors conclude that ‘‘[P]renatal exposure induces long‐term changes in cognitive performance that are distinctively gender specific’’ (Levin, Addy, Baruah, Elias et al., 2002). A growing body of data indicates that OPs may have widespread eVects at lesser doses. This has been reviewed by Eskenazi, Bradman, and Castorina (1999). A single OP exposure of adequate dose may result in axonal damage after 8 to 12 days. Acute intoxication is not the only consequence. Lesser doses administered over time produce delayed but similar damage (Sharp, Eskenazi, Harrison, Callas, & Smith, 1986). A single dose of sarin, a powerful OP, given to nonhuman primates, yielded abnormal EEGs one year later in 2 of 3 monkeys. Workers who had a single symptomatic exposure to sarin, but who presented no clinical sequelae, displayed abnormal quantitative EEGs years after the exposure (Burchfiel & DuVy, 1982).

B.

Carbamates

Carbamates are widely used as insecticides, fungicides, and herbicides. They act by inhibiting AChE but, unlike OPs, their action is reversible. Because they are water soluble, their use in crops containing a high water content is not licensed. Despite this, outbreaks of poisoning have occurred

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when the agent was used in watermelons and cucumbers. A severe outbreak of aldicarb poisoning occurred on July 4, 1985, when over 1300 individuals were poisoned by eating contaminated watermelon. Victims displayed motor twitching, vomiting, bradycardia, hyperventilation, and lacrimation within 30 minutes of eating the fruit. In Jamaica, a carbamate, methomyl, was mistaken for salt and introduced into bread. Five people were stricken; three died. Because the carbamates do not permanently attach to AChE, once exposure has ended, symptoms usually remit within 3 to 6 hours. C.

Pyrethroids

Pyrethroids are synthetic molecules that chemically resemble the natural pesticide secreted by chrysanthemums. Although generally regarded as safe, pyrethroids are not innocuous; they both activate and inactivate gates of sodium channels, resulting in prolonged opening, with membrane depolarization, repetitive discharges leading to excess nervous system stimulation and hyperexcitatory symptoms. (Narahashi, 1996). Some widely used pyrethroid compounds, particularly isomers of Type II, have toxicity comparable to many OPs (Soderlund, Smith, & Lee, 2000). Both Type I and Type II pyrethroids produce potent sympathetic stimulation. They frequently are marketed in mixtures with OPs, and may have synergistic toxic eVects.

V. A.

HUMAN EFFECTS OF PESTICIDE EXPOSURE

Effects on Children

Epidemiologic study of the impact of pesticide exposure during pregnancy and early childhood has been limited. Past studies have uniformly classified human pesticide exposures on the basis of occupation or self‐reported use of relevant products. Such approaches to exposure classification are rife with error. Research on children has generally focused on the oVspring of agricultural workers, but urban exposures, primarily from household use of pesticides, can be quite high. Despite the formidable body of experimental evidence on the neurotoxic eVects and mechanisms of pesticide action, the amount of empirical information about pesticides and childhood neurobehavioral development is slender. This is true for four reasons: the absence of rigorous epidemiological studies in appropriate samples of infants and children, the lack of animal studies that use behavior as an endpoint, the lack of sensitive behavioral measures in those studies attempted, and the absence of long‐term studies of behavioral outcome (Weiss, 1997).

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B.

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Effects on Adults

What data exist suggest that neurobehavioral eVects are a likely outcome. Studies of workers exposed to OPs emphasize the need for long‐term follow‐ up. Stephens et al. compared a group of 146 farmers exposed to OPs by dipping sheep to controls and found deficits in attention, impaired information processing, and a greater need for psychiatric services (Stephens, Spurgeon, & Calvert, 1995). Savage and coworkers compared a group of 100 pesticide applicators who had experienced an episode of intoxication. All were considered recovered, but significant deficits in IQ, fine motor function, card sorting, and a general impairment score were found (Savage, Keefe, Mounce, Heaton, Lewis, & Burcar, 1988). Burchfiel and DuVy (1982) measured quantitative EEGs in workers after a single exposure to sarin and found long‐term alterations. Perera and colleagues reported on a cohort study of 263 inner city mothers, measuring plasma CPF and exposure to polycyclic aromatic hydrocarbons (PAH) by personal air monitors. Intrauterine exposure to CPF was significantly (inversely) related to birth weight and body length in this multiethnic urban population, after adjustment for covariates. Polycyclic aromatic hydrocarbons had similar eVects (Perera, Rauh, Tsai et al., 2003). Berkowitz et al. examined 404 births and found that OP exposure, when coupled with low paroxynase activity, was associated with smaller head size (Berkowitz, Wetmur, Birman‐Deych, Obel, Lapinski, Godbold, Holzman, & WolV, 2004). Eskenazi et al. found no diVerence in growth, but did find a reduced gestational duration in a large sample of oVspring of mothers in an agricultural community (Eskenazi, Harley, Bradman, Weitzen, Jewell, Barr, Furlong, & Holland, 2004). No long‐term follow‐up studies of neurobehavioral performance in exposed children has been published to date. The late eVects of early exposure to toxins is an area that is beginning to receive attention. Baldi and colleagues conducted a prospective study of 1507 workers and examined the association between occupational exposure to pesticides and neurobehavioral function. Exposed subjects had lower cognitive function than referents. The risk for Parkinson’s disease and dementia of the Alzheimer’s type were 5.6 (1.47–21.6), and 2.39 (1.02–5.63), respectively (Baldi et al., 2003). VI.

SUMMARY OF WHAT IS KNOWN AND WHAT REMAINS TO BE DISCOVERED

Each of three recognized neurotoxic pesticides, OPs, carbamates, and pyrethroids, is widely distributed in nature, and each has been found in human tissue. Organophosphates, the most toxic and most widely

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distributed, have been shown in animal studies to have impacts beyond AChE inhibition and to alter rodent behavior at low tissue doses. Human data on low‐dose eVects are sparse, but prenatal exposure to OPs has been reported to aVect gestational duration, newborn length, and head size. No neurobehavioral data are presently available to address the question of the impact of these neurotoxins on infants and children. Reference doses are currently constructed from examination of inappropriate and coarse outcomes: peripheral rodent blood AChE levels and gross measures of brain anatomy. For informed regulation, adequate and accurate understanding of the toxic properties of these agents is essential. This will require studies of the fine structure of immature rodent brains, dendritogenesis, and the development and migration of neurons after exposure to low doses of these toxicants, dosed separately and in combination. Behavioral data are also needed. The sensitivity of the immature brain cannot be ignored. The pesticide industry, in an attempt to undermine the 10‐fold safety factor, has contracted for studies of healthy young adults given oral doses of CPF. The number of subjects in these studies ranged between 7 and 50. It is not surprising that no eVect was reported: the power to find an eVect was in the range of 0.2. These studies are worthless. A few cohort studies of children at community exposures, examining neurobehavioral outcome measures, are under way, and should provide data useful in the intelligent regulation of these agents. REFERENCES Abdiou‐Donia, M., & Lapadula, D. (1990). Mechanisms of organophosphorus ester‐induced delayed neurotoxicity Type I and II. Annual Review of Pharmacology and Toxicology, 30, 405–440. Aldridge, J., Seidler, F., Meyer, A., Thillai, I., & Slotkin, T. (2003). Serotonergic systems targeted by developmental exposure to chlorpyrifos: EVects during diVerent critical periods. Environmental Health Perspectives, 111, 1736–1743. Baldi, I., Lebailly, P., Mohammed‐Brahim, B., Letenneur, L., Dartigues, J., & Brochard, P. (2003). Neurodegenerative diseases and exposure to pesticides in the elderly. American Journal of Epidemiology, 157, 409–414. Bellinger, D., Leviton, A., Waternaux, C., Needleman, H., & Rabinowitz, M. (1987). Longitudinal analyses of prenatal and postnatal lead exposure and early cognitive development. New England Journal of Medicine, 316, 1037–1043. Berkowitz, G., Wetmur, J., Birman‐Deych, E., Obel, J., Lapinski, R., Godbold, J., Holzman, I., & WolV, M. (2004). In utero pesticide exposure, maternal paraxonase activity, and head circumference. Environmental Health Perspectives, 112, 388–391. Bowers, M., & Sim, V. (1964). Some behavioral changes in man following anticholinesterase administration. Journal of Nervous and Mental Diseases, 138, 383–389. Bradman, M., Harnly, M., Draper, W., Seidel, S., Teran, S., Wakeham, O., & Neutra, R. (1997). Pesticide exposures to children from California’s Central Valley: Results of a pilot study. Journal of Exposure and Analytical Environmental Epidemiology, 7, 217–234.

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Burchfiel, J., & DuVy, F. (1982). Organophosphate neurotoxicity: Chronic eVects of sarin on the electroencephalogram of monkey and man. Neurobehavioral Toxicology and Teratology, 4, 767–768. Cohen, B., Wiles, R., & Bondoc, E. (1995). Weed killers by the glass: A citizens’ tap water monitoring project in 29 cities. Washington, DC: Environmental Working Group. Dam, K., Garcia, S., Seidler, F., & Slotkin, T. (1999). Neonatal chlopfyrifos exposure alters synaptic development and neuronal activity in cholinergic and catacholaminergic pathways. Brain Research and Developmental Brain Research, 116, 9–20. Dam, K., Seidler, F., & Slotkin, T. (1998). Developmental toxicity of chlorprifos: Delayed targeting of DNA synthesis after repeated administration. Brain Research and Developmental Brain Research, 108, 39–45. Eskenazi, B., Bradman, A., & Castorina, R. (1999). Exposure of children to organophosphate pesticides and their potential adverse health eVects. Environmental Health Perspectives, 107(Suppl. 3), 409–419. Eskenazi, B., Harley, K., Bradman, A., Weitzen, E., Jewell, N., Barr, O., Furlong, C., & Holland, N. (2004). Association of in utero organophosphate pesticide exposure and fetal growth and length of gestation in an agricultural population. Environmental Health Perspectives, 112, 1116–1124. Garcia, S., Seidler, F., & Slotkin, T. (2003). Developmental neurotoxicity elicited by prenatal or postnatal chlorpyrifos exposure: EVects on neurospecific proteins indicate changing vulnerabilities. Environmental Health Perspectives, 111, 297–303. Grandjean, P., Weihe, P., White, R., & Debes, F. (1998). Cognitive performance of children prenatally exposed to ‘‘safe’’ levels of methylmercury. Environmental Research, 77, 165–172. Hayes, T., Haston, K., Tsui, M., Hoang, A., HaeVele, C., & Vonk, A. (2002). Herbicides: Feminization of male frogs in the wild. Nature, 419, 895–896. Jacobson, J., & Jacobson, S. (1997). Evidence for PCBs as neurodevelopmental toxicants in humans. Neurotoxicology, 18, 415–424. Klassen, C. D., Amdur, M. O., & Doull, J. (1986). Toxicology: The basic science of poisons. New York: Macmillan. Levin, E., Addy, N., Baruah, A., Elias, A., Christopher, N. C., Seidler, F. J., & Slotkin, T. A. (2002). Prenatal chlopyrifos exposure in rats causes persistent behavioral alterations. Neurotoxicology and Teratology, 24, 733–741. Levin, E., Nakajima, A., Christopher, N., Seidler, F., & Slotkin, T. (2001). Persistent behavioral consequences of neonatal chlorpyrifos exposure in rats. Brain Research and Developmental Brain Research, 130, 83–89. Lewis, D., Fortmann, R., & Camman, D. (1994). Evaluation of methods for monitoring the potential exposure of small children to pesticides in the residential environment. Archives of Environmental Contamination and Toxicology, 26, 37–46. Lotti, M. (2002). Promotion of organophosphate induced delayed polyneuropathy by certain esterase inhibitors. Toxicology, 182, 245–248. Metcalf, D., & Holmes, J. (1969). EEG, psychological, and neurological alternations in humans with organophosphorus exposure. Annals New York Academy of Sciences, 160, 357–365. Moriya, M., Ohta, T., Watanabe, K., Miyazawa, T., Kato, K., & Shirasu, Y. (1983). Further mutagenicity studies on pesticides in bacterial reversion assay systems. Mutation Research, 116, 185–216. Narahashi, T. (1996). Neuronal ion channels as the target site of insecticides. Pharmacology and Toxicology, 79, 1–14. Navarro, H., Basta, P., Seidler, F., & Slotkin, T. (2001). Neonatal chloropyrifos administration elicits deficits in immune function in adulthood: A neural eVect? Brain Research Developmental Brain Research, 130, 249–252.

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Newcombe, D. (1992). Immune surveillance, organophosphorus exposure, and lymphomagenesis. Lancet, 339, 539–541. Perera, F., Rauh, V., Tsai, W‐Y., Kinney, P., Camann, D., Barr, D., Bernert, T., Garfinkel, R., Tu, Y. H., Diaz, D., Dietrich, J., & Whyatt, R. M. (2003). EVects of transplacental exposure to environmental pollutants on birth outcomes in a multiethnic population. Environmental Health Perspectives, 111, 201–205. Savage, T., Keefe, T., Mounce, L., Heaton, R., Lewi, J., & Burcar, P. (1988). Chronic neurological sequelae of acute organophosphate pesticide poisoning. Archives of Environmental Health, 43, 38–45. Schulz, H., Nagygymatenyi, L., & Desi, I. (1995). Lifetime exposure to dichlorvos aVects behavior of mature rats. Human and Experimental Toxicology, 14, 721–726. Sharp, D., Eskenazi, B., Harrison, R., Callas, P., & Smith, A. (1986). Delayed health hazards of pesticide exposure. Annual Review of Public Health, 7, 441–471. Slotkin, T. (1999). Developmental cholinotoxicants: Nicotine and chlopyrifos. Env. Health Persp., 107(Suppl. 1), 71–80. Soderlund, D., Smith, T., & Lee, H. (2000). DiVerential sensitivity of sodium channel isoforms and sequence variants to pyrethroid insecticides. Neurotoxicology, 21, 127–137. Song, X., Saleh, F., Zhang, J., Padilla, S., & Slotkin, T. (1997). Cellular mechanisms for developmental toxicity of chlorpyrifos: Targeting the adenyl cyclase signaling cascade. Toxicology and Applied Pharmacology, 145, 182–191. Spyker, J., & Avery, D. (1977). Neurobehavioral eVect of prenatal exposure to the organophosphate Diazinon in mice. Journal of Toxicology and Environmental Health, 3, 989–1002. Stephens, R., Spurgeon, A., Calvert, I., Beach, J., Levy, L. S., Berry, H., & Harrington, J. M. (1995). Neuropsychological eVects of long‐term exposure to organophosphates in sheep dip. Lancet, 345, 1135–1139. Swan, S., Kruse, R., Liu, F., Barr, D., Drobnis, E., Redmon, J., Wang, C., Brazil, C., & Overstreet, J. (2003). Semen quality in relation to biomarkers of pesticide exposure. Environmental Health Perspectives, 111, 1478–1484. Tilson, H. (2000). Neurotoxicology risk assessment guidelines: Developmental neurotoxicology. Neurotoxicology, 21, 189–194. U. S. Geological Survey (1997). Pesticides in surface and ground water of the United States. Pesticides National Synthesis Project. (http://www.water.wr.usgs.gov/pnsp/gwsw1.html). Weiss, B. (1997). Pesticides as a source of developmental disabilities. Mental Retardation and Developmental Disabilities Research Review, 3, 246–256. Whitney, K., Seidler, F., & Slotkin, T. (1995). Developmental neurotoxicity of chlorpyrifos: Cellular mechanisms. Toxicology and Applied Pharmacology, 134, 53–62.

Parental Smoking and Children’s Behavioral and Cognitive Functioning MICHAEL WEITZMAN, MEGAN KAVANAUGH, AND TODD A. FLORIN AMERICAN ACADEMY OF PEDIATRICS CENTER FOR CHILD HEALTH RESEARCH AND DEPARTMENT OF PEDIATRICS, UNIVERSITY OF ROCHESTER SCHOOL OF MEDICINE AND DENTISTRY, ROCHESTER, NEW YORK

I.

INTRODUCTION

Tobacco smoke exposure of fetuses and children remains common in the United States, despite a reported 33% reduction in smoking during pregnancy over the past decade (Mathews, 2001). It has been convincingly established that parental smoking contributes to many child health problems, such as low birth weight, asthma, respiratory infections, otitis media, and sudden infant death syndrome (SIDS) (Etzel, Balk, Bearer, Miller, Shea, Simon, Falk, Miller, Rogan, & Hendrick, 1997; Jacobs, JoVe, Knight, Kulig, & Rogers, 2001). Both animal model and human epidemiologic studies also strongly suggest that prenatal and early passive exposure to tobacco smoke leads to negative behavioral and neurocognitive eVects in children, and there are plausible biologic mechanisms through which this may occur. To date, these negative neurodevelopmental and behavioral associations are far less well recognized by the pediatric, child development, and public health communities than are the respiratory and SIDS associations. An expanding body of literature indicates that maternal smoking during pregnancy and early childhood is associated with neurotoxic eVects on children. This literature suggests that such exposure results in increased rates of children’s behavior problems and psychiatric disorders, and may also lead to subtle intellectual decrements and neurocognitive impairments. This exposure is common, rarely confined to the prenatal period, and is often associated with other factors adversely aVecting behavioral and cognitive outcomes in children. Lower maternal educational achievement and socioeconomic status, increased rates of maternal depression and anxiety disorder, and alcohol and psychoactive drug use are all more common among women who smoke INTERNATIONAL REVIEW OF RESEARCH IN MENTAL RETARDATON, Vol. 30 0074-7750/06 $35.00

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during pregnancy or during the childbearing and childrearing years. Each of these factors, and possibly others as yet uncovered, may compound the negative eVects of children’s tobacco exposure on child development. Other childhood environmental exposures, such as to lead, have well‐ documented neurodevelopmental eVects. Still others, such as to methylmercury, have suspected but as yet unconfirmed adverse neurodevelopmental eVects. In both cases, substantial clinical, public health, and environmental policy development have been implemented to reduce children’s exposure. In contrast, children’s prenatal and early passive exposure to tobacco smoke is extremely prevalent and may result in serious neurocognitive and behavioral problems, but there has been a lack of corresponding preventative actions. This chapter reviews the literature on the eVects of tobacco exposure, both prenatal and postnatal, on children’s behavior and cognition.

II.

A.

CHARACTERISTICS OF WOMEN ASSOCIATED WITH MATERNAL SMOKING

Prevalence

The estimated prevalence of smoking during pregnancy varies, depending on the source of the data and maternal characteristics of the study population, and has been declining in the recent past (Chandra, 1995; Department of Health and Human Services, 2001; Division of Vital Statistics, 1992; Fried, Prager, MacKay, & Xia, 2003; Martin, Brady, Ventura, Menacker, & Park, 2002; Ventura, Martin, Mathews, & Clarke, 1996). In 2001, approximately 23% of women of childbearing age smoked (V. Fried et al., 2003). The most recent national data based on birth records indicates that 12.2% of pregnant women smoke, a 37% decline from 1987, the first year such information was collected (Martin et al., 2002). Smoking during pregnancy varies by ethnicity: American Indians, including Aleuts and Eskimos (20.0%), Hawaiian Asians (14.4%), White (13.2%), Black (9.1%), Hispanic (3.5%), and Asian (2.8%) (Martin et al., 2002). Prenatal smoking is greatest during late teen years (19.2%) (Martin et al., 2002). The percentage of pregnant women who smoke varies greatly by education: 9 to 11 years of education, 25.5%; 12 years, 16.4%; 13 to 15 years, 9.1%; and 16 or more years, 2.0% (Martin et al., 2002). In addition, more than one‐ third of all U.S. children are regularly exposed to environmental tobacco smoke as assessed by serum cotinine levels (Pirkle, Flegal, Bernett, Brody, Etzel, & Maurer, 1996; Schuster, Franke, & Pham, 2002). Many epidemiologic studies rely on parent‐reported smoking behavior rather than assessment of biomarkers to characterize fetal and child exposure.

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This is likely to result in underreporting of exposure, introducing an important bias for studies: some children who are exposed are misclassified or their exposure is underestimated, thereby likely underestimating the eVect of tobacco exposure (Olds, 1997).

B.

Associated Secondary Risk Exposures

Infants who are exposed to maternal smoking during pregnancy are at increased risk for other toxic exposures. Smoking mothers are more likely to drink and use illicit drugs (HoVman, Welte, & Barnes, 2001; Jacobson, J., Jacobson, S., Sokol, Martier, Ager, & Shankaran, 1994; Miller, Boudreaux, & Regan, 1995; National Institute on Drug Abuse [NIDA], 1993). Women smokers also diVer from nonsmoking women in a number of important psychosocial characteristics (Koren, 1999). Higher rates of unwanted pregnancies (Hellerstedt, Pirie, Lando, Curry, McBride, Grothaus, & Nelson, 1998), diYculties coping with stress, and lower self‐esteem (Siqueira, Diab, Bodian, & Rolnitzky, 2000) are found more commonly among smoking women compared to nonsmoking women. Women who smoked during pregnancy have been found to be more likely to report marital diYculties and more likely to physically discipline their infants (Morales, Marks, & Kumar, 1997). Smoking mothers are less likely to breastfeed their infants (Lanting, Fidler, Huisman, Touwen, & Boersma, 1994; Niemela & Jarvenpaa, 1996; Olds, 1997). Women who smoke also are more anxious, change jobs more frequently, and divorce more often than women who do not smoke (Yerushalmy, 1971). Associations between cigarette smoking and mental illness are not confined to women who smoke during pregnancy. Cigarette smoking is associated with psychiatric disorders among adolescents and adults in the general population (Brook, Cohen, & Brook, 1998; Hill, Lowers, Locke‐Wellman, & Shen, 2000; Kandel et al., 1997; Yerushalmy, 1971). Persons with mental illness are about twice as likely to smoke as other persons (Lasser, Boyd, Woolhandler, Himmelstein, McCormick, & Bor, 2000).

C.

Genetics

Data suggest that dopamine‐related genes are associated with smoking (Muneoka, Ogawa, Kamei, Mimura, Kato, & Takigawa, 2001; Richardson & Tizabi, 1994), and abnormalities in the dopaminergic‐reward pathways have been implicated in substance abuse and addictive behaviors (Muneoka et al., 2001). Dopamine D2 receptor gene variants have been found to be associated with alcoholism, drug dependency, obesity, smoking, pathological gambling, attention‐deficit hyperactivity disorder (ADHD), Tourette

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syndrome, as well as other compulsive behaviors (Blum, Wood, Braverman, Chen, & Sheridan, 1995). Linkage studies indicate that there are several possible smoking‐associated genes, which include cytochrome P450 subfamily polypeptide 6 (CYP2A6); dopamine D1, D2, and D4 receptors; dopamine transporter; and serotonin transporter genes (Arinami, Ishiguro, & Onaivi, 2000; Blum, Sheridan et al., 1995; Comings & Blum, 2000; Comings, Ferry et al., 1996; Comings, Gade et al., 1997). Genetic aspects of behavioral problems that are autosomally linked to smoking are likely to be transmitted equally from smoking mothers and smoking fathers. Studies to date, however, have found that maternal smoking is more strongly associated with adverse developmental outcomes than is paternal smoking (Eskenazi & Castorina, 1999; Trasti, Vik, Jacobsen, & Bakketeig, 1999). If parental smoking were simply a marker for genetically driven behavioral problems in children, then studies should find that maternal and paternal smoking contribute equally to adverse outcomes in children, which has not been the case. Epidemiological studies of child development have demonstrated that the number of risk factors present in a child’s life increases the likelihood of adverse outcomes for that child (Escalona, 1982; SameroV & Chandler, 1975; SameroV & Seifer, 1983). Both nature and nurture are responsible for developmental outcomes. Socioeconomic and familial factors sometimes overshadow the role of biology in producing emotional diYculties and intellectual disability (Ramey, Bryant, Sparling, & Wasik, 1984). The ‘‘transactional model’’ (SameroV, 1987) of child development supposes that a child’s initial biological makeup, including genotype, is not fully expressed at birth, but only develops during an interactive process with the environment. Particularly relevant to the topic at hand are findings that demonstrate that the relationship between smoking during pregnancy and low birth weight babies is modified by polymorphisms in two maternal metabolic genes, CYP1A1 and GSTT1 (Wang et al., 2002) and that children with two copies of a dopamine transporter (DAT) polymorphism are the ones likely to develop ADHD in the presence of maternal smoking (Kahn, Khoury, Nichols, & Lanphear, 2003).

III. A.

POTENTIAL PATHWAY FOR ADVERSE EFFECTS

Low Birth Weight

In 1957, Simpson reported on the adverse eVect of maternal smoking on birth weight, and multiple studies have confirmed this finding (Kleinman & Madans, 1985; Kramer, 1987a; Macarthur & Knox, 1988; MacMahon,

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Alpert, & Salber, 1965). These studies show a direct dose–response relationship (Kleinman & Madans, 1985; Kline, Stein, & Hutzler, 1987). The eVect of prenatal exposure on birth weight is more attributable to intrauterine growth retardation than to preterm delivery. Kramer and associates (1990) estimated the eVect of prenatal maternal smoking as a 5% reduction in relative weight per pack of cigarettes smoked per day, and Meyer and Comstock (1972) reported that the eVect of maternal cigarette smoking on infant birth weight was an average reduction of 150 to over 300 g. Both maternal smoking and paternal smoking are associated with lower birth weight, but maternal smoking is associated with more of an eVect on birth weight than paternal smoking (Eskenazi & Castorina, 1999; Matsubara, Kida, Tamakoshi, Wakai, Kawamura, & Ohno, 2000). Cigarette smoking is the single most important factor aVecting birth weight in developed countries (Kramer, 1987b). Only one study we are aware of has found no eVect of passive exposure to cigarette smoking among nonsmoking mothers (Haug et al., 2000). Reduction of smoking during pregnancy has been shown to improve infant birth weight (Sexton & Hebel, 1984). Prenatal maternal smoking aVects the fetus in a number of ways that may result in chronic hypoxia. Placental vascular resistance increases when women smoke during pregnancy (Howard, Hosokawa, & Maguire, 1987; Lehtovirta & Forss, 1978). Nicotine is a vasoconstrictor. In addition, exposure to nitric oxide in cigarette smoke can cause prostacyclin deficiency, which, in turn, may aVect the uteroplacental blood flow and contribute to the impaired fetal nutrition of babies born to women who smoke (Ulm, Plockinger, Pirich, Gryglewski, & Sinzinger, 1995). Maternal smoking during pregnancy also is associated with alterations of protein metabolism and enzyme activity in fetal cord blood (Jauniaux, Biernaux, Gerlo, & Gulbis, 2001). These may be secondary to irreversible changes in cellular function of the trophoblast and may contribute to fetal growth restriction. Cigarette smoking during pregnancy also transiently lowers maternal uterine blood flow and reduces the flow of oxygen from the uterus to the placenta (Morrow, Ritchie, & Bull, 1988). Increased levels of carboxyhemoglobin are found in both maternal and fetal blood when the mother smokes during pregnancy, and this can lead to fetal hypoxia because carboxyhemoglobin replaces oxyhemoglobin that normally releases oxygen to the fetal tissues (Soothill, Morafa, Ayida, & Rodeck, 1996). The fetus suVers chronic hypoxic stress as a consequence of maternal smoking, as evidenced by elevated neonatal hematocrit levels (Bush et al., 2000). Poor intrauterine growth has a lasting eVect on subsequent development of children (Dunn, Mcburney, Ingram, & Hunter, 1976). Low birth weight infants are at increased risk of emotional and behavioral problems (Barros,

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Huttly, Victora, Kirkwood, & Vaughan, 1992; McCarton, 1998; Pharoah, Stevenson, Cooke, & Stevenson, 1994). The sequelae of low birth weight also include intellectual disability and hyperactivity (Breslau & Chilcoat, 2000). Breslau and colleagues (Breslau, Chilcoat, Johnson, Andreski, & Lucia, 2000) found an increase in neurologic abnormalities or soft signs, defined as deviations in motor, sensory, and integrative functions that do not signify localized brain dysfunction, such as cranial nerve abnormalities, lateralized dysfunction, or the presence of pathologic reflexes, among low birth weight children. These findings were, in turn, associated with increased risk for lower IQ and learning disorders among children. Low birth weight is also associated with an increased risk of reading and math disabilities (Johnson & Breslau, 2000). It remains unclear whether the modest decrements in birth weight associated with maternal smoking have neurobehavioral consequences among those who are not born prematurely or of substantially low birth weight, but data exist showing that even among all children born with normal birth weight, on average, those with greater birth weights have higher IQs than those with lower birth weights (Matte, Bresnahan, Begg, & Susser, 2001). B.

In Utero Brain Growth

Maternal smoking has been shown to increase the likelihood that a child will be born with a small head circumference (Kallen, 2000). Children born to smoking mothers may experience catch‐up growth in weight and partial catch‐up growth in length, but diVerences in head circumference persist to at least 5 years of age (Vik, Jacobsen, Vatten, & Bakketeig, 1996). No diVerence in head circumference measurements has been found when women who were pregnant stopped smoking prior to 32 weeks’ gestation, as compared to the head circumferences of children of nonsmoking mothers (Lindley, Becker, Gray, & Herman, 2000).

IV.

NEUROCOGNITIVE AND BEHAVIORAL OUTCOMES ASSOCIATED WITH MATERNAL SMOKING

At least four comprehensive reviews of the animal model and epidemiologic studies of maternal prenatal tobacco smoke’s eVects on brain development, behavior, and neurocognitive functioning of children have been published in the past decade (Ernst, Moolchan, & Robinson, 2001; National Cancer Institute, 1999; Olds, 1997; Weitzman, Byrd, Aligne, & Moss, 2002). Cigarette smoke is comprised of more than 2000 chemical compounds (J. Johnson et al., 2000), and only a relative few of these have been studied

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for their biologic activity (Dube & Green, 1982; Naeye, 1992). Both animal model and human epidemiologic studies have focused primarily on nicotine. A.

Animal Models

Animal studies provide experimental models where a toxic exposure can be isolated to the prenatal period and isolated to tobacco exposure. The majority of the animal research has focused on the toxic eVects of nicotine, all of the studies have focused on exposures in the prenatal period, and the findings have been quite consistent despite the wide variability of study designs employed (Ernst et al., 2001). Animal studies have confirmed that nicotine at doses not high enough to cause intrauterine growth retardation still acts as a neuro‐teratogen, altering rodent brain development and behavior (Hellstrom‐Lindahl & Nordberg, 2002). Nicotine exposure to the prenatal brain may prematurely stimulate the shift from neuronal proliferation to diVerentiation, a shift normally occurring later in development. Thus, nicotine exposure during brain development may act as a cholinergic signal mimicking the trophic eVects of acetylcholine, which mediates such cellular eVects as cell–cell communication, neuronal pathfinding, cell cycle and mitosis, locomotion, immune functions, amino acid absorption, and trophic functions (Wessler, Kirkpatrick, & Racke, 1998). As the relative density and distribution of nicotine receptors change during the course of the prenatal development, nicotine may elicit diVerent eVects at diVerent developmental stages of the human brain. For example, in a more mature nervous system, nicotinic acetylcholine receptors (nAChRs) modulate neurotransmitter release eliciting a short‐term response, which becomes desensitized with continued stimulation. During development, however, stimulation of nAChRs leads to communication with genes that control cell replication, diVerentiation, growth, and death (Hellstrom‐Lindahl & Norberg, 2002). Prenatal nicotine exposure significantly increases adrenergic receptor binding in the cerebral cortex of adult animals (Navarro, Slotkin, Tayyeb, Lappi, & Seidler, 1990; Navarro et al., 1990; Peters, 1984; Tizabi & Perry, 2000). In mouse experiments where the dose and timing of nicotine were varied, Nasrat and colleagues (Nasrat, Alhachim, & Mahmood, 1986) demonstrated that doses of nicotine equivalent to 20 cigarettes per day in humans resulted in shortening of the gestational period, particularly when that exposure occurred during the second and third trimesters. Similar to the findings of human studies, prenatal exposure to nicotine in animal studies consistently is associated with lower birth weight in oVspring (Bassi, Rosso, Moessinger, Blanc, & James, 1984; Leichter, 1989, 1995; Miller, Boudreaux, & Regan, 1995). Animal studies also demonstrate that in rats, mice, and

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guinea pigs, there is increased postnatal motor activity associated with in utero nicotine exposure (Ajarem & Ahmad, 1998; Johns, Louis, Becker, & Means, 1982). Such studies also have found attention and memory deficits in maze task performance (Levin, Briggs, Christopher, & Rose, 1993; Peters & Ngan, 1982; Yanai, Pick, Rogelfuchs, & Zahalka, 1992) and mild deficits in learning (Johns, Louis, Becker & Means, 1982; Genedani, Bernardi, & Bertolini, 1983; Levin et al., 1993). In many cases, these studies have found alterations in attention, memory, and learning that are consistent with ADHD, but not all studies have produced such findings (Bertolini, Bernardi, & Genedani, 1982; Paulson et al., 1993). Such contradictory findings may reflect the small magnitude of the cognitive eVects in animals or diVerences in experimental methodology (Ernst et al., 2001). Nicotine suppresses DNA synthesis in newborn rat brains, especially in the cerebellum (Slotkin, Greer, Faust, Cho, & Seidler, 1986). It also reduces dopaminergic activity in the oVspring of nicotine‐exposed pregnant females in the ventral tegmental area, nucleus accumbens, and striatum. A reduction in dopamine in animal models has produced cognitive deficits and significant alteration in attentional processes. Cognitive diseases in children, such as ADHD and autism, have been treated with dopamine modulators with success (Nieoullon, 2002). The reduction in dopaminergic activity by nicotine during gestation thus may have lasting cognitive eVects on oVspring. Nicotine also reduces the uptake of serotonin (Thomas, Garrison, Slawecki, Ehlers, & Riley, 2000) and is typically associated with rat hyperactive behavior (Muneoka et al., 2001; Richardson & Tizabi, 1994). Thus, nicotine in experimental animals has been shown to alter in utero growth and oVsprings’ cognitive and motor performance, DNA synthesis, and neurotransmitter function associated with mood. B.

Epidemiologic Studies

Observational studies involving humans using both cross‐sectional and longitudinal data also suggest negative developmental consequences of children’s prenatal and early passive exposure to tobacco smoke. These studies have used samples that are ethnically, culturally, and socially diverse. These studies support the view that children’s behavior and cognition are adversely aVected by prenatal and early childhood tobacco exposure. Many employ multivariate statistical analyses to control for numerous potential confounders of the association between prenatal tobacco exposure and children’s behavior and cognition. Fried and colleagues (Fried, Oconnell, & Watkinson, 1992) have made a substantial contribution to the literature regarding prenatal substance use and subsequent childhood outcomes using the Ottawa Prenatal Prospective Study (OPPS), a longitudinal study of 698

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women giving birth to children between 1980 and 1983. Women were interviewed regarding substance use prior to and during pregnancy and regarding a large number of pregnancy, neonatal, and early childhood outcome variables. The earliest follow‐up studies occurred at 12 and 24 months of age. Prenatal maternal cigarette smoking was significantly associated with lower mental scores at 12 months of age and altered auditory response at 12 and 24 months. After adjusting for confounders, the same research team found poorer language development and lower cognitive scores at both 36 and 48 months in 133 children from the OPPS exposed to tobacco prenatally (Fried & Watkinson, 1990). Fried and associates (1992) were able to continue to follow this cohort, and found similar results in children 60 and 72 months of age. The latest studies of the same cohort of children demonstrated dose‐dependent associations between prenatal tobacco exposure and lower language and reading scores, poorer performance of visuoperceptual tasks, and lower global intelligence scores using the Wechsler Intelligence Scale for Children (WISC) in 9‐ to 12‐ year‐old children (Fried & Watkinson, 2000; Fried, Watkinson, & Gray, 1998; Fried, Watkinson, & Siegel, 1997). Fried and colleagues thus have used the OPPS cohort to successfully track the neurocognitive eVects of prenatal tobacco exposure from the neonatal period into adolescence. C.

Adverse Behavior Outcomes in Children of Smoking Mothers

Studies of children whose mothers smoked during pregnancy have consistently demonstrated that such children have higher rates of behavior problems than those not exposed. Olds (1997), for example, noted that 10 of 11 human studies reviewed found increased rates of child behavior problems and ADHD‐like behaviors, even after controlling for many potential confounders (Day, Richardson, Goldschmidt, & Cornelius, 2000; Dunn, Mcburney, Ingram, & Hunter, 1977; Fergusson, Horwood, & Lynskey, 1993; Fried & Watkinson, 1990; Hardy & Mellits, 1972; Kristjansson, Fried, & Watkinson, 1989; Milberger, Biederman, Faraone, Chen, & Jones, 1996; Naeye & Peters, 1984; Rantakallio, Laara, Isohanni, & Moilanen, 1992; Streissguth, Barr et al., 1986; Streissguth, Martin et al., 1984; Wakschlag et al., 1997; Walker, Rosenberg, & Balaban‐Gil, 1999; Weitzman, Gortmaker, & Sobol, 1992). These studies vary from the newborn period up through adolescence. Day and associates performed a longitudinal study of 672 children, examining the relationship between prenatal tobacco exposure and development of behavior problems at age 3 using various measures of behavior, activity level, attention, impulsivity, and self‐report of smoking. Increases in scores on the oppositional behavior, immaturity, emotional instability, physical aggression, and activity scales were significantly associated with prenatal tobacco exposure

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(Day et al., 2000). Fergusson and colleagues (1993) explored eVects of maternal smoking before and after pregnancy and behavioral outcomes at a later stage of life in 8‐, 10‐, and 12‐year‐old children. They followed a birth cohort of 1265 New Zealand children longitudinally for 15 years. After adjustment for confounding variables, maternal smoking during pregnancy was associated with increases in rates of behavioral problems in this sample, as measured by conduct disorder, attention deficit, and total disruptive behavior scores. Wakschlag and associates (1997) looked specifically at the occurrence of conduct disorder in boys 13 to 18 years of age whose mothers smoked during pregnancy. Data from 177 boys, ages 7 to 12 years at first assessment, who were followed longitudinally for 6 years, demonstrated that mothers who smoked more than a half a pack of cigarettes daily during pregnancy were more than 4 times more likely to have a child with conduct disorder than mothers who did not smoke during pregnancy. These studies, and the many others in the literature, strongly suggest that maternal smoking during pregnancy is independently associated with adverse behavior outcomes in children of all ages. D.

Newborns and Preschoolers

Fried et al. (1987) reported increases in hypertonicity and heightened tremors and startles among neonates who were prenatally exposed to tobacco compared to neonates born to nonsmokers. Longo (1977) found evidence for neonatal hyperactivity among oVspring of smoking mothers. In a sample of 27 nicotine‐exposed and 29 unexposed full‐term newborn infants, Law and associates (2003) determined nicotine exposure by maternal self‐report and maternal salivary cotinine. The NICU Network Neurobehavioral Scale (NNNS), a validated scale developed by the National Institutes of Health to study prenatal drug exposure, was administered to measure neurobehavioral function. After adjustment for covariates, the authors found dose–response relationships between higher maternal salivary cotinine and newborn hyperexcitability, hypertonicity, and increased stress/abstinence signs. Reijneveld and associates (2002), in a study of 3179 children aged 1 to 6 months from Holland, interviewed parents of a national community‐based sample regarding excessive infant crying using 10 published definitions and risk factors including parental employment, living area, lifestyle, and obstetric history. Their study found maternal smoking to be the strongest and most consistent predictor of excessive crying, reaching statistically significant positive odds ratios for 6 of 10 definitions, compared to parental unemployment (3 of 10), urban environment (2 of 10), and infant hospitalization after delivery (2 of 10). They concluded that if maternal smoking indeed causes excessive crying, it may

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explain between 2.1 and 17.2% of excessive crying during this age period, depending upon the definition of excessive crying employed. Kelmanson and associates (Kelmanson, Erman, & Litvina, 2002) conducted a study involving 250 randomly selected 2‐ to 4‐month‐old infants whose mothers completed the Early Infancy Temperament Questionnaires. Infants born to smoking mothers had more frequent fussy periods, more protesting behavior at face washing and bath, indiVerent attitudes to the mother when held by a new person, extreme reactions during diapering and bowel movements, less attention to the parent during parent/infant play activity, and more sensitivity to wet diapers. They also were characterized by more intensive reactions compared to the babies of nonsmokers. Brook and associates (Brook, Brook, & Whiteman, 2000) reported that maternal smoking during pregnancy was associated with negativity in 2‐year‐old children. Williams and associates (Williams et al., 1998) reported on a prospective longitudinal study of 5342 5‐year‐old children in which maternal smoking during pregnancy was associated with externalizing behavior problems in a dose‐dependent fashion. Behavior problems were assessed using the Child Behavior Check List (CBCL), which defines externalizing behavior problems such as argues a lot, demands a lot of attention, destroys own things or things belonging to others, disobedient at home, gets into many fights, lying, screams a lot, sudden changes in mood, stubborn or irritable, or temper tantrums. Smoking more than 20 cigarettes per day at the first clinic visit was associated with a 2.6 times greater chance of externalizing behavioral problems in the child at 5 years of age compared to no cigarette use. These studies, like the studies noted in the following text of older children, suggest that prenatal tobacco exposure may increase the risk for ADHD, Oppositional Defiant Disorder, and Conduct Disorder. E.

School‐Age Children and Adolescents

Weitzman and colleagues (1992) in a longitudinal study involving 2256 U.S. children, ages 4 to 11 years, found that children who were exposed postnatally and both prenatally and postnatally were more likely to have behavior problems, even after controlling for numerous potential confounders. In this study, there was evidence of a dose–eVect response and the tobacco exposure eVect was not limited to any particular area of children’s behavior, such as antisocial behaviors, anxiety, depression, hyperactivity, or easy distractibility. For example, among children whose mothers smoked both during and after pregnancy, there were 1.17 additional problems independently associated with smoking less than a pack per day and 2.04 additional problems of smoking a pack or more per day. Extreme behavior scores on the Behavior Problem Index were 1.41 times more likely if the

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mother smoked less than a pack per day and 1.54 times more likely if she smoked a pack or more per day. Fergusson and colleagues (1998) also found maternal smoking during pregnancy to be associated with increased rates of behavior problems in a longitudinal study of a birth cohort of 1265 children up to age 18 years in New Zealand. This study used both teacher and mother reports, thereby eliminating the potential problem that smoking mothers may be less tolerant of children’s behaviors and more likely to report them as abnormal. A clear dose–response relationship between cigarettes smoked during pregnancy and disruptive behaviors, conduct disorder, and attention deficit was found after adjusting for confounding variables. Rantakallio and colleagues (1992) found an association between prenatal cigarette smoking and later delinquency in the Finnish birth cohort study, a population of 12,068 pregnant mothers who gave birth to 12,058 liveborn children in two provinces of Finland in 1966, and Wakschlag and colleagues (1997), in a later prospective study in the United States, found that boys ages 7 to 12 were more likely to be referred for psychiatric care for conduct disorder if their mothers smoked during pregnancy. In contrast, Silberg and colleagues (2003) found no increased rates of conduct disorder among adolescents with mothers who smoked. Maughan and colleagues (2001) used data from the 1970 British Birth Cohort Study, a prospective study of all children born in England, Scotland, and Wales in the first week of April 1970, to examine the links between maternal smoking in pregnancy and antisocial behavior in oVspring. This study found a strong dose–response relationship between the extent of pregnancy smoking and childhood‐onset conduct disorders, but no links with adolescent‐onset antisocial behaviors. Controls for mothers’ subsequent smoking history modified this picture, suggesting that the prime risks for early‐onset conduct problems may be associated with persistent maternal smoking or correlates of persistent smoking rather than with pregnancy smoking per se. Wakschlag and colleagues (2002) reviewed the literature on in utero exposure to maternal smoking as a risk factor for conduct disorder and delinquency and concluded that the association is independent of confounders, present across diverse contexts, and consistent with basic science, but that methodologic limitations preclude causal conclusions. They say that the research to date provides consistent support for, but not proof of, an etiologic role for prenatal smoking in the development of antisocial behavior. F.

Cognitive Impairments

Prenatal exposure to maternal smoking has been shown to adversely aVect children’s performance on intelligence and achievement tests, as well as performance in school, although the findings in this area are not as consistent

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as those for increased rates of behavior problems. Butler and Goldstein (1973) demonstrated that children whose mothers smoked 10 or more cigarettes per day were on average between 3 and 5 months delayed in reading, mathematics, and general ability when compared to oVspring of nonsmokers. A number of studies (Dunn et al., 1977; Fergusson et al., 1993; Fogelman & Manor, 1988; Fried et al., 1992; Naeye & Peters, 1984; Rantakallio, 1983; Rantakallio & Koiranen, 1987) demonstrate similar eVects, while some found eVects to virtually disappear after controlling for confounders (Baghurst, Tong, Woodward, & McMichael, 1992; Fergusson & Lloyd, 1991; Mcgee & Stanton, 1994). In families in which mothers smoked during some but not all pregnancies, exposed children performed worse on intelligence tests than did their unexposed siblings (Fergusson & Lloyd, 1991). For example, at 8 years of age, children whose mothers did not smoke during the first trimester of pregnancy scored a mean IQ of 104 on the WISC‐R compared to a mean IQ of 97 in children whose mothers smoked more than 20 cigarettes. Similarly, children of women who quit smoking during pregnancy have been found to score higher on tests of cognitive ability than children whose mothers smoke throughout pregnancy (Fried & Watkinson, 1990). As has been noted, Fried’s OPPS (1988) is the most comprehensive examination of the association between intrauterine exposure to smoke and later developmental outcomes. This study has provided longitudinal data regarding auditory processing, reading, and language development. Fried and Watkinson (1988) found that infants born to maternal smokers have decreased rates of auditory habituation and increased sound thresholds. The children in this study at 12 and 24 months showed decreased responsiveness on auditory‐related items on the Bayley Scales of Infant Development associated with prenatal cigarette exposure. By ages 3 and 4 years, language development as assessed by standardized tests was found to be adversely aVected by maternal cigarette smoking (Fried et al., 1992). These findings were dose‐related, and persisted in follow‐up studies through age 12 years (Fried & Watkinson, 2000; Fried et al., 1992, 1997, 1998). A study by Olds and colleagues (1994a) estimated the eVect of prenatal smoking on cognitive function after controlling for many potential confounders: smoking 10 or more cigarettes per day during pregnancy was independently associated with an average 4.35‐point decrease in Stanford–Binet IQ scores. The same investigators (1994b) also demonstrated that intervention with quality, long‐term prenatal nurse home visitation can oVset the impairment in intellectual functioning associated with substantial maternal smoking during pregnancy. While this study did not estimate how much of the prevention in intellectual disability was due to smoking cessation itself, the data suggested that the increase in IQ was, in part, due to a reduction in maternal smoking during pregnancy.

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A number of epidemiological studies have focused on the association between exposure to smoke and subsequent symptoms of ADHD. Linnet and colleagues (2003) reviewed 24 studies published between 1973 and 2002 assessing the relationship between prenatal exposure to nicotine and the risk of developing behavior problems related to ADHD in childhood. They concluded that this literature demonstrated that nicotine exposure during pregnancy is a risk factor for ADHD‐related disorders among children. We will review several key studies among them. Denson and colleagues (1975), in a case‐control study, showed hyperactivity to be associated in a dose–response manner with maternal smoking. Milberger and colleagues (1996, 1998) also employed a case‐control study and found that prenatal tobacco exposure contributes to children’s ADHD. Data from 140 6‐ to 17‐year‐old boys with DSM–III‐R ADHD were compared with 120 normal comparison subjects. Twenty‐two percent of the ADHD children had maternal history of prenatal smoking compared with 8% of control subjects. Furthermore, the positive association persisted after adjustment for socioeconomic status, parental IQ, and parental ADHD status. Mick and colleagues (2002) investigated the association between ADHD and prenatal exposure to maternal cigarette smoking using a retrospective, hospital‐based, case‐control study of 280 ADHD cases and 242 non‐ADHD cases. ADHD cases were 2.1 times more likely to have been exposed to cigarettes in utero than were the non‐ADHD controls. Wasserman and colleagues (2001) examined the association between children’s behavior problems using the CBCL and maternal smoking in Yugoslavia, a country where there is no smoking–social class gradient. Mothers were enrolled during pregnancy in a prospective study of lead exposure and the children were assessed using the CBCL at ages 4, 4, and 5 years. Blood lead level, measured twice a year, was associated slightly with the delinquent scale. Smoking was associated with worse scores on almost all of the CBCL subscales for boys and with somatic complaints for girls, controlling for lead, HOME scores, maternal education, age, gender, lead exposure, and birth weight. This study adds to the literature because it controls for lead and because it includes noneconomically or socially disadvantaged families.

H.

School Performance

Rantakallio and Koiranen (1987) reported data from 1819 Finnish children in the 1966 birth cohort previously discussed demonstrating that parental smoking is associated with lower mean scores on ‘‘theoretical subjects

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based on school reports.’’ Byrd and Weitzman (1994) demonstrated that children of smoking parents are more likely to repeat kindergarten or first grade, using the U.S. National Health Interview Survey. Survey data from 9996 children, ages 7 to 17 years, were analyzed to determine factors independently associated with increased risk of grade retention. Exposure to household smoking increased the risk of grade retention by 1.4 times. This study, however, was cross‐sectional, and did not distinguish prenatal from childhood passive exposure to tobacco.

V.

RESEARCH IMPLICATIONS

Listed here are a series of 10 questions that emanate from the findings to date in this profoundly important field: 1. What additional information can be learned from animal model studies, what are the limitations of extrapolations from such data to eVects on children, and what additional animal model studies are indicated? What hypotheses suggested by human studies can better be tested in animal studies? 2. What additional information can be derived from further human epidemiologic investigation, what samples and study designs are indicated, and how would we develop such studies? What hypotheses suggested by animal studies can be tested in epidemiologic studies? 3. Are the adverse neurocognitive and behavioral eVects associated with tobacco exposure due to prenatal or early childhood passive exposure, or to both prenatal and postnatal exposure? This is a very diYcult question to answer using human observational epidemiologic data, as a very small minority of women smoke only during pregnancy. Animal model studies to date have focused on prenatal smoke exposure’s eVects on subsequent neurocognitive functioning of oVspring. Studies in animals that are not exposed in utero, but are exposed after birth would help answer this very important question. Moreover, there are diVerent and more chemicals that youngsters are exposed to from environmental tobacco smoke than from direct smoking. Therefore, it is possible that some of these diVerential exposures may have diVering eVects. 4. Is there a unique neurobehavioral signature associated with prenatal and early passive exposure to tobacco smoke, or is it variable and does it vary by stage of development at which the fetus or child is exposed? 5. Do tobacco eVects vary by whether the exposure is acute or chronic, and is there a critical period of exposure? Similarly, is the eVect modified by other environmental exposures, such as to lead and mercury?

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6. Do neurobehavioral problems change over the course of childhood or are they static, and whether changing or static are their eVects likely to be more deleterious at diVerent stages of children’s lives? 7. There is evidence of a dose–eVect response between children’s tobacco exposure and various domains of behavior and neurocognitive functioning. Do the slopes of these relationships change over the range of exposure? 8. Are there children who are especially vulnerable to tobacco exposure, such as those growing up in poverty, or with mothers who are poorly educated or depressed, or children with intellectual impairments such as already low IQ, ADHD, or specific learning disabilities? Several studies, as noted earlier, suggest that there are genetic as well as social characteristics that make certain children more vulnerable than others, but a more robust literature clearly is needed. 9. Are the adverse neurocognitive and behavioral eVects reversible if mothers reduce or stop smoking, and if yes, are there times when, or by when it is especially important to reduce or curtail exposure? For example, does reduction or curtailment of tobacco exposure lead to decreased rates of behavior problems or decrements in IQ? Similarly, are there environmental control mechanisms that eVectively could reduce children’s exposure to tobacco smoke? 10. Are there parenting strategies or social support interventions that can overcome the biologic eVects of tobacco exposure? What are the eVects of early intervention on children who have been exposed to maternal smoking?

VI.

SUMMARY

A causal argument rests on the accumulation of evidence along five major domains: biologic plausibility, consistency, temporality, dose–response gradient, and strength of association (Last, 2001). Our review of the literature on maternal smoking and subsequent child neurocognitive functioning indicates evidence in each of these domains. As described, there are multiple biologically plausible mechanisms by which prenatal and passive tobacco smoke exposure could result in neurocognitive and behavioral alterations. At this time, the large number of epidemiologic studies and the consistency of their findings concerning increased rates of behavioral problems provide a broad base of consistency across populations and across various study designs and endpoint measures. The temporal sequence of exposure preceding outcome, while somewhat cloudy in some human studies, is clearly evident in the animal models. Numerous studies also support a dose–response relationship, i.e., the greater the exposure, the higher the likelihood that children will demonstrate adverse behaviors. Moreover, the animal

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data provide a strong body of evidence of diminished neurocognitive performance associated with prenatal exposure while eliminating the possibility of misattributing these alterations to confounding with social or psychological characteristics that frequently accompany parental smoking. Some remain skeptical (Ernst et al., 2001; National Cancer Institute, 1999; Olds, 1997; Ramsay & Reynolds, 2000) of research demonstrating adverse eVects of smoking during pregnancy. As noted elsewhere, diVerences exist between smoking and nonsmoking mothers that might explain adverse outcomes among the oVspring of smoking mothers, i.e., heavy and moderate smokers receive less prenatal care, recognize their pregnancies later, and report more symptoms of depression than do nonsmokers and light smokers (SameroV & Seifer, 1983). These diVerences suggest that smokers are more likely to be depressed and less likely to practice health‐promoting behaviors for themselves or their children. However, more than a dozen observational studies have controlled for numerous potential confounders, including depression and substance abuse, and the association between smoking and adverse child neurocognitive outcomes remained. Intervention studies and animal models also potently contradict the contention that maternal smoking simply is a proxy for other factors responsible for adverse neurocognitive and behavioral outcomes. The magnitude of the adverse behavioral and neurocognitive eVects of tobacco exposure for individual children remains unclear, and some available measurements seem modest, i.e., a decrement of 4 to 5 IQ points (Olds, Henderson, and Tatelbaum, 1994b) and an odds ratio of approximately 1.5 for adverse developmental or behavioral outcomes. Yet, considerable public resources have been directed to other problems having a similar magnitude of eVect and, given the vast numbers of children aVected, the net eVect on a societal level would be expected to be quite large. It also is essential that we recognize that an insult of this type and magnitude, when coupled with other risks that tend to cluster among a significant percentage of exposed children, may have substantial eVects on functioning and quality of life across the life span. While many questions remain, both animal model and human epidemiologic data clearly suggest a causal relationship between prenatal tobacco exposure and adverse behavioral and neurocognitive eVects on children.

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Neurobehavioral Assessment in Studies of Exposures to Neurotoxicants DAVID C. BELLINGER CHILDREN’S HOSPITAL BOSTON HARVARD MEDICAL SCHOOL AND HARVARD SCHOOL OF PUBLIC HEALTH CAMBRIDGE, MASSACHUSETTS

I.

INTRODUCTION

In studies of children’s chronic, low‐level exposures to environmental neurotoxicants, the greatest concern, on a population basis, tends to be increased morbidity rather than increased mortality. While fatal poisonings do, unfortunately, still occur occasionally (Kaufman, Staes, & Matte, 2003), exposure standards are generally not set at levels that fall just short of fatal doses but at levels that are presumed to pose, over a lifetime of exposure, little appreciable risk to an individual’s health and well‐being. To an increasing extent, such standards are based on neurobehavioral toxicities as the critical health endpoints, a trend illustrated most clearly for lead (CDC, 1991) and methylmercury (Rice, 2004). Whereas the LD50 (dose at which the case fatality rate is 50%) is unambiguous as an index of adverse eVect, however, interpreting subtle decrements in performance on a neurobehavioral test as adverse eVects from which children must be protected is controversial. In most instances, the decrements do not correspond to diagnostic criteria, and aVected children do not ‘‘have’’ a disease in the usual sense. The eVects could be characterized as being low in severity but high in prevalence (Aylward, 2002). In the biomarker continuum model of the National Research Council (1989), they are indications of ‘‘altered structure or function’’ rather than clinical disease. Another aspect that complicates interpretation is that exposure‐associated changes in performance tend to represent quantitative deviations from expected behavior rather than qualitative deviations and performance generally remains ‘‘within normal limits.’’ In other words, in contrast to a tumor, which is qualitatively diVerent from normal tissue morphology, the changes represent quantitative variations in functions or INTERNATIONAL REVIEW OF RESEARCH IN MENTAL RETARDATION, Vol. 30 0074-7750/06 $35.00

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behaviors that are normally present (intelligence, problem‐solving, social interactions, motor coordination, etc.). Some observers argue that any deficit in children’s performance is worrisome because it could represent the tip of the iceberg, while others argue that a deficit that does not represent a clinically defined impairment is of little import for a child’s well‐being. From the latter perspective, an exposure should be viewed as warranting concern only if it results in an increased prevalence of children who meet clinically based criteria for a diagnosis such as mental retardation or attention‐deficit hyperactivity disorder. This chapter discusses issues germane to data collection, analysis, and interpretation in studies of the neurotoxicity of environmental chemical exposures.

II. A.

DATA COLLECTION

Differences Between a Clinical Assessment and a Research Assessment

In a clinical assessment, the referral question that brings a child to attention is the major determinant of the tests administered. Typically, the concern is the child’s dysfunction in a particular setting or in performing certain complex tasks, such as reading or mathematics. The goal of the assessment is therefore to identify the primary and secondary deficits so that an appropriate remedial plan can be formulated. Although the evaluation might include brief assessments of the major domains of function (e.g., language, memory, executive functions, visual–spatial function, attention), a ‘‘flexible’’ approach will be adopted so that the tests administered are tailored to the child’s specific presenting complaint. Additional tests, perhaps requiring multiple testing sessions, might be included as the evaluation unfolds and the examiner formulates and tests new hypotheses. This process is thus directly analogous to the ‘‘rule in, rule out’’ approach to the diVerential diagnosis applied in other areas of medical decision making. The constraints that structure a research assessment are diVerent. The data collection experience must be standardized so that all children are administered the same set of tests in the same order, under similar conditions. The need to implement such a ‘‘one size fits all’’ approach usually results in the collection of information of limited clinical utility for a particular child. Typically, a single test session of 3 hours, often less, and sometimes under suboptimal field conditions, is feasible. Some have pushed this standardization even further, taking the procrustean step of constructing a generic battery that can be applied in the study of any neurotoxicant. Examples include the Pediatric Environmental Neurobehavioral Test

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Battery (Amler & Gibertini, 1996), the Adult Environmental Neurobehavioral Test Battery (Amler, Anger, & Sizemore, 1995), the Behavioral Evaluation for Epidemiology Studies Test Battery (Echeverria, Heyer, Bittner, Rohlman, & Woods, 2002), the Cambridge Neuropsychological Testing Automated Battery (Luciana, 2003), and the WHO Neurobehavioral Core Test Battery (Anger, Cassitto, Liang, Amador, Hooisma, Chrislip, Mergler, Keifer, Hortnagl, & Fournier, 1993). These batteries are human analogues of batteries assembled for testing neurotoxicity in animals, such as the functional observation battery (Moser, 2000). To employ such a generic assessment battery, an investigator must accept important trade‐oVs because the same battery might not be the best choice under all circumstances. In assembling a battery, an investigator must weigh many considerations in deciding how best to allocate the limited evaluation time available for an individual child. The elements of an optimal battery for a particular study will vary, depending on the state of knowledge about the neurotoxicant of interest (limited knowledge would indicate the use of a broad‐based, exploratory battery to generate hypotheses, while extensive knowledge would indicate the use of a highly focused battery for confirmatory testing of specific hypotheses in restricted functional domains); characteristics of the target population (e.g., age distribution, sociodemographic and cultural context); the presumed mechanism of neurotoxicity (e.g., diVuse versus focal brain injury, including the localization of focal injury); the pattern of exposure (chronic versus acute; episodic versus stable); and the purpose of the investigation (e.g., to define the scope and severity of a public health problem versus to generate new neuropsychological knowledge about brain–behavior relationships). The Faeroe Islands (Grandjean, Weihe, White, Debes, Araki, Yokoyama, Murata, Sorensen, Dahl, & Jorgensen, 1997) and Seychelles Islands (Myers, Davidson, Cox, Shamlaye, Palumbo, Cernichiari, Sloane‐Reeves, Wilding, Kost, Huang, & Clarkson, 2003) methylmercury studies illustrate some diVerences in the ways that investigators assemble a test battery. For the 7‐year assessment conducted in the Faeroe Islands study, White and colleagues (White, Debes, Dahl, & Grandjean, 1994) used what was known about the neuropathological lesions of Minamata disease patients to identify the functional domains likely to be aVected if the neuropathological eVects of low‐dose exposure are milder forms of the injuries that result from high‐dose exposure. To be selected, a test also had to have been shown to be sensitive to known focal brain damage. As a result, the battery consisted largely of tests designed to assess specific functions, such as confrontational naming, word list learning, and visual memory. In contrast, in designing the battery for the 5.5‐year assessment for the Seychelles Islands study, Davidson and colleagues (Davidson, Myers, Cox, Shamlaye, Sloane‐Reeves, Cernichiari, Marsh,

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Clarkson, & Tanner, 1994) applied a less theory‐based approach, reviewing the human and animal literatures on low‐dose methylmercury exposure and identifying eight domains in which toxicities have been reported: global– cognitive, visual–perceptual, speech–language, visual memory, visual attention, neuromotor/neurologic, social–emotional, and learning–achievement. This strategy resulted in a battery that included more apical or omnibus tests, such as the Wechsler Intelligence Scale for Children—Third Edition, which yields IQ scores. In addition, both groups of investigators applied many of the same practical criteria in choosing tests (age and cultural appropriateness, psychometric properties such as reliability and validity, scoring flexibility, examiner training needs). In selecting the tests to administer to the Seychellois children at 9 years of age, Myers et al. (2003) applied a blended approach. While retaining many apical tests in the battery, they also included several of the domain‐focused tests that had been administered to the Faeroese children at 7 years of age. This ‘‘best of both worlds’’ approach increased the opportunities for interstudy comparison of results. B.

The Range of Domains That Can Be Assessed

One of the major challenges both in implementing and in interpreting measures of neurobehavioral function associated with exposure to chemical pollutants is the sheer number of endpoints that can potentially be measured in a study. Factor analyses of scores on comprehensive test batteries generally identify at least seven broad classes of function: language and communicative functions, attention or concentration, memory, problem solving (including the executive functions of organization and planning), spatial/ perceptual skills, sensory functions, motor skills, and emotional/adaptive functions (Fiedler, 1996; Larrabbe, 2000; White, Gerr, Cohen, Green, Lezak, Lybarger, Mack, Silbergeld, Valciukas, Chappell, & Hutchinson, 1994). Other endpoint domains that are frequently of interest, particularly in pediatric studies, include general intelligence and achievement (e.g., academic skills such as reading and mathematics). Furthermore, each of these broad classes can be subdivided. In the language domain, expressive skills can be distinguished from receptive skills. Expressive skills can themselves be subdivided: speech production (articulation and oromotor coordination), morpho‐phonemics, morpho‐syntactics, confrontational naming, fluency, sentence construction, narrative (discourse production), and expository writing. With respect to attention, at least five independent aspects can be distinguished: the abilities to screen out irrelevant stimuli, shift attention from one stimulus to another, sustain attention, retain information while manipulating or transforming it, as well as the consistency of attention over time (Mirsky & Duncan, 2001). The subdivision process, which results in a

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branching tree structure, has no logical terminus. At some point, however, it passes from the practical realm of clinical assessment to the realm of basic psychological theory and research. The universe of neurobehavioral endpoints that can be assessed is thus eVectively almost unlimited, constrained only by the interests, imagination, and ingenuity of the investigator. Because each domain can be deconstructed into increasingly specific component skills, the practical decision an investigator must make is the level within this tree structure on which to focus in order to generate data that best address the research question. If the goal is to estimate the societal costs of an exposure, good assessment choices would be tests of known validity and reliability that assess domains near the top of the tree structure (i.e., apical tests), meaning tests of endpoints whose importance is widely recognized and for which economists have worked out algorithms for monetizing deficits (i.e., reading level or IQ). If the goal is to understand the neuropsychological bases for exposure‐related diVerences on apical endpoints, the focus should be on reliable and valid tasks that assess more specific, componential functions, in other words, functions that are lower in the tree structure. The farther down one goes in the tree, the less certain will be the interpretation of the functional significance of any exposure‐related diVerences observed. Moreover, it is generally the case (although not invariably) that the lower the level one targets, the more experimental will be the instruments, with less secure knowledge about norms and psychometric characteristics. Psychologists are essentially in a ‘‘chicken and egg’’ position, trying to assess, in studies with major public health implications, systems about which new knowledge is continuing to accrue as part of basic psychological research. As new theoretical models are developed, new tests based on them, in turn, are constructed. For example, a recent trend in intelligence test construction is a shift to tests for which items are selected on the basis of current models of information processing and away from tests for which items are selected empirically, based solely on their ability to generate desirable distributions of performance within a population (the psychometric tradition) (Lezak, 1995). While this process advances the field of neuropsychology, it must seem rather perverse to those who rely on neurobehavioral data to shape rational and defensible public policy. The solution to this problem is not obvious. Waiting until complete, veridical models of neurobehavioral function and brain–behavior relationships are worked out before relying on such endpoints as bases for risk assessment is a prescription for inaction. This dilemma is faced in all areas of clinical medicine, however. The best available model of a disease process, however inaccurate and incomplete, provides the strongest basis for developing preventive and therapeutic strategies.

268 C.

David C. Bellinger Intelligence Tests versus Domain‐Focused Tests

The psychometric characteristics of intelligence tests are generally stronger and more fully characterized than are those of neurobehavioral tests more narrowly focused on specific domains (e.g., visual–spatial skills, executive functions, memory). Moreover, tests considered to assess the same putative domain can vary substantially in sensitivity (Chouinard & Braun, 1993). In addition, many neuropsychological tests were developed for use in a clinical evaluation, in which a primary goal is to discriminate individuals who are functioning within normal limits from individuals who have clinically significant impairments. As a result, such tests might be relatively insensitive in a setting in which most individuals perform within the normal range, which is the case in the typical population‐based study of a neurotoxicant exposure. It is increasingly common to find arguments that the concept of intelligence, in general, and tests of psychometric intelligence, in particular, should be abandoned in favor of tests of specific functions that are chosen for inclusion in an assessment battery on the basis of current findings in cognitive neuroscience (Ardila, 1999). Lezak (1984) drew an analogy between averaging performance across disparate neuropsychological domains to derive a full‐scale IQ (or any summary index) and calculating an average motor strength of the four limbs of an individual with a broken leg. The useful information, she argued, lies in the measurements of the components rather than in the average value. Lezak (1995) went so far as to state that IQ, as a score, is ‘‘inherently meaningless’’ and should be discarded, and that ‘‘composite scores of any kind have no place in neuropsychological assessment’’ (p. 24). While no one can define exactly what intelligence is, apart from the tautology ‘‘it is what intelligence tests measure,’’ a large literature attests to the robust associations between IQ and important measures of life success, such as school grades, years of education, job success, social status, and income (Neisser, Boodoo, Bouchard, Boykin, Brody, Ceci, Halpern, Loehlin, PerloV, Sternberg, & Urbina, 1996; Sternberg, Grigorenko, & Bundy, 2001). Moreover, IQ is central to many definitions of mental retardation. Based on the associations evident on a population basis, Weiss (2000) estimated that a 3% rise in the population IQ would be associated with reductions of 10 to 25% in a variety of outcomes that entail enormous societal costs, such as out‐of‐wedlock birth, low birth weight, welfare recipiency, and failure to complete high school. In addition, unlike changes on specific neuropsychological tests, IQ has the advantage that accepted practices exist for calculating the costs of loss in function, and thus the economic benefits to society of policies that reduce a neurotoxicant exposure. For

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example, Grosse and colleagues (Grosse, Matte, Schwartz, & Jackson, 2002) estimated that the change in the mean blood lead level of U.S. preschool children between 1976 and 1999 (based on NHANES) was 15.1 mg/dL. Based on published meta‐analyses, they further estimated that children’s IQ scores were increased between 0.185 and 0.323 points per 1 mg/ dL decline, resulting in an increase of 2.2 to 4.7 IQ points over this period. Converting IQ points to worker productivity and then to discounted lifetime earnings, they estimated that, for each cohort of 2‐year‐olds, the savings corresponds to $110 to $319 billion. Historically, cognitive functioning has been the primary focus of assessment batteries applied in studies of environmental neurotoxicants. The reasons probably include the ready availability of assessment tools for cognitive assessment, the value assigned in the culture to optimizing cognitive health, and the availability of econometric algorithms for calculating costs and benefits of cognitive deficits. With growing interest in the possibility that environmental chemicals function as endocrine disruptors, the scope of assessment is broadening to include nonreproductive sexually dimorphic behaviors (Rogan & Ragan, 2003; Sandberg, Vena, Weiner, Beehler, Swanson, & Meyer‐Bahlburg, 2003; Weiss, 2002). For example, sex diVerences in play behavior among Dutch school‐aged children were observed as a function of perinatal exposure to polychlorinated biphenyls (PCBs) and dioxins (Vreugdenhil, Slijper, Mulder, & Weiglas‐Kuperus, 2002). Specifically, higher PCB levels in boys were associated with less masculine and more feminine play, whereas, in girls, higher exposures were associated with more masculine play. New research vistas continue to open up with regard to endpoint domains, even for neurotoxicants as well studied as lead. For instance, several investigators have begun to explore lead as a risk factor for antisocial behavior (Dietrich, Ris, Succop, Berger, & Bornschein, 2001; Needleman, Reiss, Tobin, Biesecker, & Greenhouse, 1996). In 2002, Needleman and colleagues (Needleman, MacFarland, Ness, Feinberg, & Tobin, 2002) reported that adjudicated delinquents have significantly higher bone lead levels than do controls. Stretesky and Lynch (2001) found that homicide rates at the county level are significantly associated with air lead levels. In a series of ecologic analyses, Nevin (2000) found that rates of several forms of socially undesirable behaviors, including homicide, were time‐linked to lead production. Although care must be exercised in drawing causal inferences from such data, experimental studies in the mouse (Donald, Cuther, & Moore, 1987), rat (Holloway & Thor, 1987), monkey (Laughlin, Bushnell, & Bowman, 1991), and cat (Li, Han, Gregg, Kemp, Davidow, Louria, Siegel, & Bogden, 2003) also suggest a link between lead exposure and aggression.

270 D.

David C. Bellinger Infant Assessments

Scores measured in infancy and, to a lesser extent, in the preschool years, are important exceptions to the generalization that neurobehavioral test scores are fairly stable over time and possess strong predictive validity. Because early neurodevelopment is highly dynamic, saltatory, and nonlinear, both within and across domains (Darrah, Hodge, Magill‐Evans, & Kembhavi, 2003), it presents the assessor with a moving target. This presents two challenges. The first is interpreting the meaning of apparent performance deficits in the absence of the context provided by a measure of baseline performance. This can be a problem regardless of an examinee’s age, but it is especially problematic when the assessment is conducted during a period of rapid developmental change. The second challenge is distinguishing change over time that is expected as part of normal development from that which represents a deviation from expectation, reflecting, for instance, the impact of a neurotoxicant exposure. One reason why scores on tests of infant development tend to be, at best, only modestly associated with later IQ is the restricted range of ways, primarily limited to sensory–motor behaviors, in which an infant’s knowledge can be probed, and the lack of continuity between these response modalities and the ones that can be exploited as children get older. For instance, an infant’s knowledge must be inferred from changes in sucking or activity patterns in response to stimuli, or from looking preferences as assessed using habituation or visual expectancy paradigms (Rose, Feldman, & Jankowski, 2003). As a result, certain late‐maturing higher‐order skills that might be particularly sensitive to neurotoxicant exposures cannot be assessed in infants (e.g., reading, complex problem solving including hypothesis formation and testing, executive functions such as planning, organizing, and strategizing skills). The apparent emergence of a deficit only long after an insult has been referred to as a child ‘‘growing into a lesion’’ (Segalowitz & Hiscock, 2002), meaning that the impairment lies silent until the brain structures or networks that suVered damage have matured and the impact on function can be assessed (Anderson, Antoine, Damasio, Tranel, & Damasio, 1999). What appears to represent plasticity might therefore be an artifact of a mismatch between function assessed and the age at assessment. On the other hand, neurobehavioral test scores in infancy do possess strong concurrent validity and can be interpreted in much the same way as birth weight. Although not predictive of later weight, it is highly informative as an index of a newborn’s general health status. In addition, assessing outcomes in infancy has some potential advantages over delaying assessment until children are older and certain functional domains can be assessed more easily. Because neurobehavioral outcomes are sensitive to so many other biological and social

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environmental factors (see Section II.B), reducing the amount of time between neurotoxicant exposure and outcome assessment will reduce the influence of intervening, possibly confounding, factors, thus both increasing the strength of and reducing bias in the estimate of the neurotoxicant’s contribution. Investigators are increasingly turning to the use of tests that are homologous to those used with animal models, particularly nonhuman primates (Sharbaugh, Viet, Fraser, & McMaster, 2003; see also Rice’s chapter in this book). The use of tests that can be applied across species provides obvious advantages when the goal is to draw inferences about human risk from the results of studies on animal models. Among the major tasks being explored in this regard are delayed response (A‐not‐B) (Diamond, 2000), which is purported to assess aspects of executive functions, and operant conditioning tasks (Paule, Chelonis, BuValo, Blake, & Casey, 1999), which assess aspects of learning and memory. It seems improbable that exposure to a neurotoxicant will produce an invariant set of neurobehavioral findings that serve, in eVect, as a behavioral phenotype or ‘‘signature’’ injury. First, neurobehavior represents such a highly organized, final common pathway, influenced by a myriad of factors, that the specific expressions of a neurotoxicant’s impact will likely vary somewhat, depending on the context within which a particular child is exposed (see section II.C). Second, for many contaminants, the biology underlying neurotoxicity is extraordinarily complex, particularly with respect to developmental exposures, with multiple processes aVected at multiple levels. For example, lead neurotoxicity includes alterations in apoptosis, excitotoxicity, neurotransmitter storage and release, second messenger system, oxidative phosphorylation, cerebrovascular endothelial cell function, and glial cell function (both astroglia and oligodendroglia) (Lidsky & Schneider, 2003). Under such circumstances, one would expect the injury to be multifocal and diVuse, with any resulting neurobehavioral deficits being pervasive rather than specific, that is, involving many functional systems. The likelihood that the pattern of deficits would be pathognomonic, identifying the child as having been exposed to lead rather than to another neurotoxicant, seems slight (although theoretically possible). A final, methodological impediment to identifying a behavioral signature is the lack of equivalence, across tests of diVerent domains, in sensitivity (Chouinard & Braun, 1993). Inferences about diVerential deficits in ability should not be drawn unless the tests used to assess the diVerent domains in question are matched in terms of characteristics such as true score variance (Chapman & Chapman, 1978). If children exposed to a particular neurotoxicant show deficits in, for instance, visual–motor integration but not in confrontational naming, this might be because the test used to assess visual–motor

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integration has stronger psychometric characteristics than does the test used to assess confrontational naming.

III.

DATA ANALYSIS

Although many pitfalls complicate the process of drawing inferences from studies of neurobehavioral toxicities, three threats to validity will be discussed: an inflation of the studywise Type I error rate that results when a test battery includes many tests that yield many scores, the potential for confounding bias, and a form of model misspecification that results when possible eVect modification by covariates is ignored. A.

Inflation of Type I Error Rate

A child’s neurobehavioral test scores are correlated with one another, both within and across tests. In the case of nested scores, such as Verbal IQ and Full‐Scale IQ or Verbal IQ and the subtest scores that contribute to it, the correlations might approach 0.9. A 2‐hour battery that includes several tests might generate 100 or more separate scores. Applying a Type I error rate (a) of 0.05, one would expect 5 or more (depending on the degree of intercorrelation) tests of association with the exposure of interest to be statistically significant, even if the null hypothesis is true. How, then, should this possibility be taken into account in the interpretation of the results of the analyses? Some solutions involve imposing a stricter criterion on hypothesis testing based on the number of tests conducted, such as the Bonferroni family of methods (Rosenthal & Rubin, 1984). Methods such as the Bonferroni correction strike many as a straightforward solution, but one that is excessively conservative and rather mindless. It does not discriminate between the p values generated for neurobehavioral tests that diVer in their reliability and validity, in their importance with respect to the primary study hypotheses, or in terms of the extent to which a given test was included in the battery for exploratory or confirmatory purposes. Other approaches that attempt to examine the big picture generated by all of the analyses are more appealing, although all have limitations. Some investigators prioritize analyses hierarchically, so that subscale scores are not examined unless more global scores indicate a significant association with exposure. In a study that employs the WISC‐III, for example, Verbal IQ and Performance IQ would be analyzed in relation to exposure only if Full‐Scale IQ were significantly associated with exposure. The subtests that contribute to Performance IQ would only be analyzed if Performance IQ were found to be associated with exposure, and so on. This approach would be conservative, however, if the

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eVects of a neurotoxicant are not diVuse but highly specific. The eVect might not be suYciently large to aVect aggregated scores such as Full‐Scale IQ, and its dilution by being combined with scores in unaVected domains would result in an investigator never examining subtest scores. Myers et al. (2003) examined the distribution of p values for the hypothesis tests conducted, demonstrating that while some p values approached the specified a, overall the distribution did not depart significantly from what would be expected under the null hypothesis. This approach has appeal, although this, of course, cannot identify true and spurious associations and, furthermore, might be misleading if an investigator has included assessments of a large number of domains that are unaVected by the neurotoxicant of interest. The few tests for which a is approached or exceeded might be ignored as chance findings. Neuropsychologists face a similar problem in trying to identify abnormal findings in a clinical evaluation that includes multiple tests. Statistically based methods have been developed that take into account reliability of the tests, heterogeneity of findings within and across domains and levels (global, domain, test), and empirical Bayes techniques (Ingraham & Aiken, 1996; Miller & Rowling, 2001; Thomas, Siemiatycki, Dewar, Robins, Goldberg, & Armstrong, 1985). Such methods might be adapted for use in studies of neurotoxicant exposures. Some epidemiologists contend that adjustments would be appropriate if the data set consisted of randomly generated numbers but that they are not needed, and are likely to result in errors of inference, when applied to actual data (Rothman, 1990; Savitz & Olshan, 1995). In this view, it makes little sense to require that an investigator who chooses to measure more variables be, in eVect, penalized by having to apply one value for a to evaluate a particular score, while another investigator, who measured only that one score, applies a less conservative value for a. It is possible that the estimated coeYcient for the association of interest is exactly the same in the two studies but is interpreted to be significant only in the latter study. While the best method for controlling Type I error at the level of the individual study remains elusive, the best method at a broader level is the same as it has always been—replication of a finding in diVerent studies. B.

Confounding Bias

Because neurobehavior represents a high level of coordinated neurological function, it is exquisitely sensitive to disruption by many biologic and environmental factors. Distinguishing the eVects, if any, of the neurotoxicant of interest from the eVects of these other factors can pose a formidable challenge, particularly when the various eVects are entangled with one another. This entanglement, or confounding, is present when exposure to

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the neurotoxicant co‐occurs with exposure to a factor that, itself, is causally associated with the outcome of interest (but does not lie in the putative causal pathway linking the neurotoxicant to the outcome). Under these circumstances, failure to control for this factor will result in a biased (invalid) estimate of the association between the neurotoxicant and the outcome. Confounding is thus study‐specific rather than a property, per se, of the association. Therefore, it can, depending on the circumstances within a study cohort, lead to either an overestimate or an underestimate of the association between the neurotoxicant and the outcome. For instance, in most cohorts of children, higher exposure to lead is correlated with lower social class and other risk factors for less optimal neurodevelopment. Therefore, failing to control for social class, or measuring it poorly, will suggest that the decline in neurodevelopment with increasing lead exposure is greater than it is likely to be. In such cohorts, the crude, or unadjusted coeYcient for lead might be two or more times greater than the adjusted coeYcient. In at least two lead study cohorts, however (Port Pirie, Cincinnati), higher lead exposures were associated with higher social class standing. In the Kosovo cohort, families of smelter managers were able to live in luxury apartments in proximity to the smelter while families of smelter laborers resided some distance away (Wasserman, Liu, Lolacono et al., 1997). At the time that the Boston cohort was recruited, well‐to‐do families were purchasing older housing in urban neighborhoods and conducting extensive renovations of leaded surfaces, with the result that prenatal exposures to lead were greater among infants from families of higher social class (Bellinger, Leviton, Waternaux, Needleman, & Rabinowitz, 1987). In both the Kosovo and Boston cohorts, therefore, the coeYcient for lead adjusted for social class was greater than the unadjusted coeYcient. For a given neurotoxicant, the pattern of confounding can vary from cohort to cohort, depending on the route of exposure. In the most common scenario for lead, in which higher exposures are due to lead‐painted surfaces in poor repair, children with higher lead levels will be those whose families are lower in social class. In Edinburgh, Scotland, where the primary route of exposure was to lead leached from lead plumbing, children’s blood lead levels were not related to social class (Fulton, Thomson, Hunter, Raab, Laxen, & Hepburn, 1987). A similar absence of confounding by social class would be expected in settings in which the primary route of exposure is airborne lead. The typical approaches to controlling for confounding bias are to restrict the sample to a single stratum of a known confounder, stratify the analysis by strata of the confounder, or to adjust for the confounder by including a term for it in a regression analysis. Sample restriction is eVective, but will limit the generalizability of the findings, as well as exclude the possibility of

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testing for eVect modification by the factor. Stratified analysis can be useful but is not feasible under many circumstances. For instance, the more confounded the exposure and stratification factor are, the more frequent will be empty cells. In most settings, it is necessary to control for more than one potential confounder, and the sample sizes needed to stratify simultaneously on multiple confounders become prohibitive. Therefore, the most common approach to controlling confounding bias is multiple regression (or analysis of covariance), often involving adjustment of the outcome for the potential confounder and then regressing the residuals on the neurotoxicant exposure of interest. It is important to recognize that, in certain scenarios, this can be very conservative. In the case of lead and social class, for instance, when terms for both factors are included in a model, all of the outcome variance shared by lead and social class is apportioned to social class. As mentioned earlier, children of lower social class tend to live in environments richer in lead. Therefore, social class is a proxy for lead exposure opportunities. Controlling for this portion of social class would therefore be inappropriate, representing over‐control (Bellinger, Leviton, & Waternaux, 1989). What one wants to control for is the component of social class that is not a proxy for lead exposure (although it might be correlated with lead exposure) and which contributes in a causal fashion to neurodevelopment. A similar argument can be applied to the problem of how parental IQ should be handled in analyses of lead studies. While parental IQ is clearly strongly predictive of child IQ (Bouchard & McGue, 1981), it is assumed that the parent’s measured IQ is an accurate estimate of the genetic potential passed on to a child. If the child is being raised in a lead‐contaminated environment, however, it is likely that the parent was as well, so that the parent’s IQ was likely reduced by his or her own exposure to lead in childhood. As a result, measured parental IQ is not a pure measure of the child’s genetic endowment but reflects a generational eVect of lead. Adjusting for it when estimating the association between the child’s lead burden and IQ is necessary, but the analyst must remain aware that this might be conservative, attenuating the estimate. Methylmercury poses a diVerent challenge. The primary route of exposure to this neurotoxicant is fish consumption, so the children of women who consume greater amounts of fish during pregnancy tend to be exposed to more methylmercury than are children of women who do not eat fish (Schober, Sinks, Jones, Bolger, McDowell, Osterloh, Garrett, Canady, Dillon, Sun, Joseph, & MahaVey, 2003). Greater fish consumption presumably also entails greater exposure to nutrients in fish that promote neurodevelopment, such as the polyunsaturated fatty acids docosahexenoic acid and arachidonic acid (Carlson & Neuringer, 1999; Daniels, Longnecker, Rowland, & Golding, 2004). This confounding cannot be eliminated by

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sample restriction, such as by studying children of women who accumulate methylmercury by another route than fish consumption. Nor can it be addressed by adding a term to a regression equation. What one would want to do is to control for the characteristics that lead some women to consume more fish than others. Because, in most circumstances, it is precisely the same behavior that increases methylmercury exposure that also increases exposure to the beneficial constituents of fish, adjusting for fish consumption would not be helpful. Kjellstrom and colleagues (Kjellstrom, Kennedy, Wallis, Stewart, Friberg, Lind, Wutherspoon, & Mantell, 1989) developed a clever solution to this problem, matching women who consumed more than 3 fish meals per week and who had hair mercury levels >6 mg/g to women who consumed equivalent amounts of fish but who had hair mercury levels <6 mg/g. In this way, fish consumption and methylmercury intake were dissociated and, as a result, confounding reduced. It might not have been completely eliminated, however, because these groups still diVered in that something led the group with hair mercury levels <6 mg/g to eat fish with lower levels of methylmercury. Because methylmercury is found largely in fish muscle and long‐chain polyunsaturated fatty acids (PUFAs) in lipids, it is possible that diVerences in the choices of fish species to consume resulted in the fetuses in the two groups being exposed to diVering levels of PUFAs and other beneficial nutritional constituents of fish. C.

Effect Modification

In contrast to confounding, which is a bias and thus something to be eliminated, eVect modification is an inherent characteristic of an association. It is present when the magnitude of the association between an exposure and an outcome varies across strata of a third factor. This could result when the third factor potentiates or mitigates the impact of the exposure of interest. For instance, the association between asbestos exposure and bronchogenic carcinoma is much greater among smokers (relative risk of 92 compared to nonsmokers without asbestos exposure) than it is among nonsmokers (relative risk of 10) (SelikoV, Hammond, & Churg, 1968). Similarly, the neurodevelopment of preschool children from families of higher social class standing appears to recover from higher prenatal lead exposures to a greater extent than does the neurodevelopment of children from families of lower standing (Bellinger, Leviton, & Sloman, 1990). Failure to identify and characterize eVect modification will thus result in an incomplete and potentially misleading understanding of the nuances and complexities of the association. By including only main eVect terms for a neurotoxicant and a covariate in a regression model, the analyst assumes, in eVect, that the coeYcient for the neurotoxicant (i.e., the change in the health

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endpoint for each unit change in exposure) is constant across all strata of the covariate. If the covariate is an eVect modifier, however, this coeYcient for the neurotoxicant will underestimate the magnitude of the association in the stratum of the covariate in which the association between the exposure and endpoint is greatest and overestimate the magnitude of the association in strata in which the association is weakest. An important corollary of the default assumption that a covariate is not an eVect modifier is that the coeYcient for the neurotoxicant is assumed to be the same in study samples in which the distributions of covariates diVer, i.e., assumed to have the same value. If the coeYcient does vary depending on the values of certain covariates, however, and study samples diVer in the values of these covariates, it follows that the coeYcient for the neurotoxicant estimated in the diVerent study samples, assuming no measurement error, should not be the same. In other words, if social class is an eVect modifier of low‐level lead neurotoxicity, as it appears to be (Bellinger, 1995), we should not expect that all studies will estimate the same coeYcient for lead unless the study samples are equivalent in the social class distribution (and all other eVect modifiers). EVect modification should thus be considered as a potential explanation for inconsistency across studies in the study findings (Bellinger, 2000). The possibility of eVect modification is acknowledged in current approaches to risk assessment. The biomarker model of the National Research Council includes the possibility that the rate or likelihood of disease progression following exposure is not the same in all people, and exposure standards are set so that the most susceptible subgroups of the population are protected. Often this is accomplished by the applying to the critical dose a modifying or uncertainty factor (e.g., 3 or 10) because variation in subgroup susceptibility has rarely been characterized quantitatively. Making consideration of eVect modification an integral element of data analysis holds the promise of improving the scientific grounding of this step of a risk assessment, enabling the establishment of exposure standards that are more precisely linked to actual levels of risk. Unfortunately, most studies are not designed with explorations of eVect modification in mind. Rather, the motivating question is more likely to be, ‘‘Is neurotoxicant X at dose Y associated with neurobehavioral deficits in children’’ than to be, ‘‘Which subgroups of children show neurobehavioral deficits following exposure to neurotoxicant X at dose Y?’’ Analyses of eVect modification require much greater sample sizes than do analyses of main eVects (Pearce, 1989), so unless the study has been designed to address eVect modification, it is unlikely that the relevant analyses will have adequate statistical power. Among the classes of possible eVect modifiers that have been evaluated in pediatric neurobehavioral toxicities studies are social environment, nutrition, genetics, and co‐exposures. In terms of social environment, the factors

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considered have included social class, parent IQ, HOME score, and sex (Bellinger et al., 1990; Davidson, Myers, Shamlaye, Cox, Gao, Axtell, Morris, Sloane‐Reeves, Cernichiari, Choi, Palumbo, & Clarkson, 1999; Davidson, Myers, Shamlaye, Cox, & Wilding, 2004; McMichael, Baghurst, Vimpani, Robertson, Wigg, & Tong, 1992; Tong, McMichael, & Baghurst, 2000; Winneke & Kramer, 1984). In an interesting application of eVect modification, Wallace, Reitzenstein, and Withers (2003) found that exposure of rats to methylazoxymethanol reduced the magnitude of the enhancement of cortical thickness typically seen in response to being raised in an enriched environment. In fact, the dose at which this reduction in experience‐dependent plasticity was observed was well below the dose at which altered cortical morphogenesis was observed, suggesting that it was a more sensitive index of subtle injury. In terms of nutrition, RuV and colleagues (RuV, Markowitz, Bijur, & Rosen, 1996) reported that as blood lead level was reduced in 18‐ to 30‐ month‐old children, neurodevelopmental scores increased, but only among children who were iron‐suYcient. In the Seychelles Child Development Study, for some neurodevelopmental outcomes, better scores were associated with higher prenatal methylmercury levels, attributable to maternal fish consumption (Davidson et al., 1999; Myers et al., 2003). Clarkson and Stain (2003) speculated that these seemingly paradoxical findings would result if any adverse eVects of methylmercury at the low levels experienced in this cohort were outweighed by the influence of micronutrients in fish that promote optimal neurodevelopment, such as long‐chain PUFAs, iodine, iron, and choline. Little work has been done to investigate the role of genetic polymorphisms in modulating the impact of neurotoxicants in children, with most such work focusing on lead. Among adult lead workers carrying the apolipoprotein E4 allele, tibia lead level was more strongly inversely related to neurobehavioral function than it was in adult lead workers without an E4 allele (Stewart, Schwartz, Simon, Kelsey, & Todd, 2002), although in a study of children, those with an E4 allele had better early neurodevelopment than did children without an E4 allele, and genotype did not modify the association between lead and neurodevelopment (Wright, Hu, Silverman, Tsaih, Schwartz, Bellinger, Ettinger, Weiss, & Hernandez‐Avila, 2003). Another polymorphism of interest is amino levulinic acid dehydratase (ALA‐D). Adult male workers with the wild‐type allele (ALA‐D‐1‐1 genotype) had higher urinary ALA levels and poorer scores on some neurobehavioral tests, suggesting that the variant ALA‐D‐2 allele might be protective (Chia, Yap, & Chia, 2004). Another study suggested a link between ALA‐D allele and risk of amyotrophic lateral sclerosis in adults (Kamel, Umbach, Lehman, Park, Munsat, Shefner, Sandler, Hu, & Taylor, 2003). Another polymorphism

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under investigation with regard to lead neurotoxicity is the vitamin D receptor (Haynes, Kalkwarf, Hornung, Wenstrup, Dietrich, & Lanphear, 2003).

IV. A.

DATA INTERPRETATION

The Meaning of Small Decrements in Performance on Neurobehavioral Tests

An observer applying a strictly clinical perspective might argue that the goal of a regulatory standard for an environmental chemical is to prevent impairments that bring children to medical attention because they meet diagnostic criteria and thus are cases of a ‘‘disease.’’ A small exposure‐ associated decrement on a neurobehavioral test score would therefore be considered unimportant because, even among the children with higher environmental exposures, function generally remains within normal limits defined by the clinical criteria, and minor variations within the range of normal function are thought to carry relatively little import, at least in terms of a child’s utilization of health care resources. From this perspective, when neurobehavioral toxicity is considered to be the critical health endpoint, risk assessments should use as the point of departure categorical diagnoses or clinical entities, such as autism spectrum disorders, learning disabilities, attention deficit hyperactivity disorder (ADHD), or mental retardation. In response to this clinical perspective, some argue that the instruments used to assess neurobehavioral function are so crude and indirect as measures of brain function that a chemical’s impact must be fairly robust to aVect a child’s performance on them. As a result, such tests are therefore likely to underestimate the scope and severity of a chemical’s true neurobehavioral toxicity, reflecting, as mentioned earlier, only the tip of the iceberg. From this standpoint, any exposure‐related decrement in a test score that is statistically significant should be considered worrisome. A limitation of this approach, however, is that it does not accord any weight to the magnitude of the performance change associated with exposure, making sample size the critical determinant of whether or not a particular study is interpreted as demonstrating neurotoxicity. Even an infinitesimal diVerence in group means or deviation of a slope from 0, whether due to a very small true eVect or entirely to a chance imbalance due to random measurement errors, will be statistically significant if the sample size is suYciently large. Therefore, it is necessary to place the observed exposure‐related performance diVerence into a context that aids interpretation of its importance. Are only eVects that correspond to diagnostic criteria ‘‘important’’? This is a particularly vexing

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problem when a diagnosis is made on the basis of a continuously distributed characteristic, such as IQ. If the magnitude of the apparent eVect of exposure to a contaminant is dose‐dependent, then at low doses the change in IQ will be small, insuYcient in magnitude to result in a child of normal premorbid IQ being classified as mentally retarded post‐exposure. It might only be at much higher doses that the resulting impairment of IQ is dramatic enough to result in such classification. Where should the line be drawn between trivial and important eVect? (Much of the following discussion is based on Bellinger, 2004.) B.

Reduced Performance on a Neurobehavioral Test as a Proxy for a Clinically Significant Impairment

A performance deficit on a neuropsychological test typically takes the form of reduced function in a specific isolated skill, such as remembering strings of digits, providing the names of objects depicted in line drawings, or reproducing abstract designs using colored blocks. How well a child performs these tasks might seem to indicate little about the child’s ability to function in the ‘‘real world.’’ In contrast, diagnostic criteria tend to be stated in terms of an inability to fulfill the expectations for behavior in specific settings or relationships, focusing on deficits that are large enough to result in ‘‘harmful dysfunctions’’ (Wakefield, 1997). For instance, the criteria for ADHD include the requirement that, ‘‘Some impairment from the symptoms is present in two or more settings (e.g., at school and at home),’’ and that, ‘‘There must be clear evidence of clinically significant impairment in social, academic, or occupational functioning’’ (APA, 1994, p. 84). To have a reading disorder, a child must present with impairment that ‘‘significantly interferes with academic achievement or activities of daily living that require reading skills’’ (APA, 1994, p. 50). Although a deficit on a neurobehavioral test is unlikely to represent a harmful dysfunction in the sense that it brings a child to clinical attention, it can, nevertheless, be a surrogate for such a deficit or a prodrome, a sign that a child is in the early stages of developing a harmful dysfunction. Consider the relationship between handwriting and the ability to insert pegs into a board, a task commonly included in neurobehavioral assessment batteries. Many school assignments require children to compose narratives. To do this well requires the integration of diverse skills, including conceptualization of the theme, development of a sequence of sentences that convey it, and production of the letters that compose the words. A child for whom the last skill, the physical act of writing, is diYcult and eVortful will be able to devote relatively fewer cognitive resources to theme development and composition, placing him or her at a disadvantage in carrying out this type of assignment. Despite

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the increasing use of word processing even among children of primary school‐ age, handwriting thus continues to be a skill of considerable importance for a child’s success in the school setting. Assessing it is diYcult, however. Collecting a writing sample requires at least 15 minutes (and often considerably more), and coding it in ways that capture, in quantitative terms, the nature and severity of any deficiencies in penmanship might require an hour or more. Coders must learn the criteria, become demonstrably proficient in their application, and maintain proficiency over time. In contrast to a writing sample, a pegboard is simple to administer and to score. The Grooved Pegboard, for example, takes approximately 90 seconds to administer, requiring little training on the part of the examiner. Moreover, a child’s score (time to complete) is objective and available immediately. In a follow‐up study of 8‐year‐old children with a congenital heart defect (Bellinger, Wypij, duPlessis, Rappaport, Jonas, Wernovsky, & Newberger, 2003b), a writing sample was obtained and the Grooved Pegboard administered. The time needed to complete the pegboard was highly correlated with all three dimensions of handwriting skill, letter formation, size, and alignment and spacing, for which scores were derived using a labor‐intensive coding method (Johnson & Carlisle, 1996). A child’s performance on the Grooved Pegboard provides, in an extremely eYcient manner, a considerable amount of information about his or her handwriting skill. Children’s visual–motor integration skills are frequently assessed using design–copying tasks, such as the Rey–Osterrieth Complex Figure (ROCF). The ROCF was administered to the same sample of children from whom handwriting samples were collected, and copies were blindly classified into 5 categories based on overall goodness‐of‐organization and style (Bellinger, Bernstein, Kirkwood, Rappaport, & Newburger, 2003a). The ability to produce a good copy varied greatly among children, but one might ask, ‘‘Who cares?’’ Copying figures is not a skill that is explicitly called upon in a child’s natural settings so a deficit in the ability to do so would not seem to be harmful dysfunction. ROCF category, however, was associated, in a monotonic manner, with the percentage of children receiving remedial services in school. Fewer than 10% of children with the best ROCF performance required such academic supports versus more than 50% of children with the worst performance. A child’s performance on the ROCF thus appears to provide information germane to the criteria applied in making the resource allocation and treatment decisions attendant to learning disability, which represents a clearly harmful dysfunction for the child and a substantial cost to society. One might ask, ‘‘Why not simply use need for remedial academic services as the study endpoint?’’ rather than something that is as tedious to score as the ROCF? Typically, an investigator is interested not only in establishing the

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final outcome, such as failure to make adequate progress in school, but in discerning the reasons for the child’s failure. What is it that impedes progress? It is by examining the patterns in a child’s scores on tests such as the ROCF that such questions can be answered. By administering a large battery of neuropsychological tests, Taylor, Burant, and Holding (2002) were able to identify underlying neuropsychological constructs that mediate the association between very low birth weight and academic achievement (for reading, verbal working memory; for mathematics, perceptual planning). Similarly, Bellinger and colleagues (Bellinger, Bernstein, Kirkwood, Rappaport, & Newburger, 2003a) determined that the poor performance of children with congenital heart defects on the ROCF was likely attributable more to impaired visual–perceptual skills than to impaired executive functions or motor control skills. C.

Dynamic Nosology and the Risk of False‐Negative Errors of Inference

Rather than being etiologically based, many diagnoses in pediatric neurology, psychiatry, and neuropsychology are syndromal. They are made on the basis of observable signs and symptoms and lack confirmatory laboratory findings that identify a causative factor (e.g., a chromosomal anomaly, cytomegalovirus). Such diagnoses are essentially a system of verbal shorthand for referring to the most commonly observed symptom complexes, obviating the need to describe in toto a child’s presenting signs and symptoms. Providing something with a name is a powerful psychological act, however (Whorf, 1956), and we must be careful not to imbue such linguistic conveniences with too much significance, allowing them to constrain our observations and formulations (Kushner, Bastian, Turner, & Burns, 2003). Frequently, syndromal diagnoses are assigned when an individual’s score on some continuously distributed characteristic exceeds (or falls short of) some value. It is not quite correct to say that one person ‘‘has’’ the disease, while another person with a score just below (or above) the cutoV does not (and thus, is ‘‘within normal limits’’). Rather than asking, ‘‘Does he have it?’’ it is more appropriate to ask ‘‘How much of it does he have?’’ (Rose, 1993). The distributions of the characteristics underlying childhood disorders such as depression, delinquency, and dyslexia are normal and not bimodal, as would be expected if the population consists of two distinct subgroups, the normal and the abnormal (Shaywitz, Escobar, Shaywitz, Fletcher, & Makuch, 1992; Van den Oord, Pickles, & Waldman, 2003). Patients who carry a diagnosis of unipolar major depressive disorder but have only ‘‘subthreshold depressive’’ symptoms are, nevertheless, not in remission. Such symptoms are associated with increased psychosocial

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disability, more rapid relapse, and a poorer long‐term prognosis (Judd, 2002). In order to allocate treatment resources, clinicians classify individuals categorically (as diseased or not diseased) based on discrete cutoV values; they rely on continuous measurements to track a patient’s improvement after initiation of treatment (e.g., the degree of glycemic control in a patient with diabetes). An important characteristic of syndromal diagnoses is that they are dynamic. Diagnostic criteria for syndromal conditions change over time as experience accrues with the symptom complex. For some disorders, this takes the form of ‘‘splitting,’’ so that what was previously thought to represent a single condition is resolved into a family of related but distinct conditions. Such partitioning is commonplace throughout medicine, illustrated most strikingly by the practice of attaching the prefix ‘‘non‐’’ to create labels for newly recognized clinical entities (e.g., non‐insulin‐dependent diabetes mellitus, non‐A non‐B hepatitis, non‐Hodgkin’s lymphoma) (Ellman & Feinstein, 1993). Two neurobehavioral disorders that have been partitioned in this way are autism spectrum disorder (ASD) and ADHD, each of which now includes multiple subtypes or variants. As evidence that the partitioning process continues, current debate focuses on whether both inattentive subtype (Barkley, 2003) and ADHD that is comorbid with oppositional defiant/conduct disorder (Banaschewski, Brandeis, Heinrich, Albrecht, Brunner, & Rothenberger, 2003) should be considered distinct disorders rather than ADHD variants. Absent the emergence of new disease processes or advances in the understanding of disease pathogenesis, this process reflects a change in the way clinicians sort and classify patients’ signs and symptoms rather than a change in the signs and symptoms themselves. The evolution in nosology can also involve ‘‘lumping,’’ particularly in the case of syndromal diseases with considerable phenotypic variability. Diseases that, in the past, were considered unrelated are recognized as variants on a single theme when a pathogenic process that links them all is discerned. An example is the 22q11 microdeletion syndrome, first identified in the mid‐1990s (Glover, 1995). Children with this syndrome were formerly identified as cases of Shprintzen syndrome, DiGeorge syndrome, velocardiofacial syndrome, or conotruncal face anomaly syndrome. Which diagnosis was assigned largely depended on whether a child was seen by an endocrinologist, an immunologist, a cardiologist, or a dysmorphologist. Similarly, Duchenne’s muscular dystrophy, Becker’s muscular dystrophy, and X‐linked dilated cardiomyopathy, were, until recently, considered to be clinically distinct diseases until genomic research revealed that they constitute a family of dystrophinopathies. All are caused by mutations of the gene coding for dystrophin, a skeletal muscle protein (Burke, 2003).

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The fluidity of nosology is relevant to neurobehavioral toxicity because a neurotoxicant might cause a pattern of signs and symptoms that clinicians have not reified as a formal ‘‘diagnosis’’ by assigning it a consensus label. As noted, the neurobehavioral eVects of chemical exposures generally diVer quantitatively rather than qualitatively from normal function. One implication of this is that exposure to a neurotoxicant is unlikely to produce a unique ‘‘signature injury’’ in the way that vinyl chloride produces a deviation from normal cell morphology, angiosarcoma of the liver, that is rarely seen in the absence of this exposure. Even if an exposure were to produce a unique pattern of neurobehavioral deficits, however, the more infrequent the exposure is, the less likely it is that pattern will be assigned a name and enter the clinical lexicon as a new diagnosis. Nevertheless, a symptom complex that lacks a name can be just as burdensome to a child and family as one for which a label is available. Goldberg (2000) cautioned against interpreting syndromal classifications too literally, saying that, ‘‘rather than carving nature as the joints, we appear to be drawing lines in the fog.’’ D.

Implications of the Fact That the Population Distribution of a Characteristic Moves as a Whole

A small change in the mean signals predictable accompanying changes in the proportions of individuals in the source population who fall into the tails of the distribution, where individuals who meet diagnostic criteria are found. Thus, the importance of a shift in group mean lies not in what it indicates about the average change among members of the study sample, but what it implies about the changes in the tails of the distribution in the population from which the study sample was drawn. This principle has been amply demonstrated in other areas of epidemiologic research. Using data for 52 populations from around the world, Rose and Day (1990) calculated, for several diseases, the correlation between the central tendency of the distribution of the underlying health indicator and the proportion of individuals with values of the indicator that exceed the clinical cutoV used to sort people into patients and nonpatients. The correlation between the median systolic blood pressure in a population and the prevalence of hypertension in the same population was 0.85; for mean body mass index in a population and the prevalence of obesity in the same population, the correlation was 0.94; for mean alcohol intake in a population and the prevalence of alcoholism in the same population, the correlation was 0.97. Moreover, for all of these characteristics, the slopes of the best‐fit regression lines were steep, indicating that, in comparing two populations, a small diVerence between the population means for a health indicator is associated with a surprisingly large diVerence in the prevalence of clinical

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cases in the two populations. The mean value of a population distribution is thus highly informative about the proportion of individuals in the population who have deviant values, about whom clinicians are most concerned. An important corollary of this principle is that a change in the mean value will produce a predictable change in the prevalence of clinically defined cases in the source population. For example, based on the scatterplot of mean systolic blood pressure and the prevalence of hypertension in the 52 populations, one could predict that a reduction of 1 mm Hg in mean systolic blood pressure would be associated with a reduction of 1% in the prevalence of hypertension. If the prevalence were 15%, a 5‐mm Hg reduction in mean systolic blood pressure, a reduction that is insignificant for an individual, would result in 33% decline in the prevalence of hypertension, from 15 to 10%. On this basis, Rose (1981) estimated that an intervention that reduced the mean blood pressure in a population by as little as 2 to 3 mm Hg (a population‐based strategy) would be as eVective in reducing the prevalence of hypertension in that population as providing drug therapy for all individuals with clinically defined hypertension (a high‐risk strategy). A preventive measure that brings much benefit to the population might thus oVer little benefit to each individual in the population, a principle that Rose labeled the ‘‘prevention paradox’’ (Rose, 1985). Paradoxical or not, this logic underlies many public health interventions, such as immunizations against infectious childhood diseases and the use of seatbelts, that prevent diseases or events that are relatively rare but which, for an aVected individual, can cause considerable morbidity. Although Rose’s predictions were based solely on cross‐sample comparisons, they have been powerfully validated by longitudinal measurement of secular trends in the relationship between group mean and the prevalence of deviant cases within the same population (Laaser, Breckenkamp, Ullrich, & HoVmann, 2001; Whittington & Huppert, 1996). In the realm of neurotoxicity studies, this principle has been demonstrated most clearly for lead. Meta‐analyses suggest that a doubling of childhood blood lead, from 10 to 20 mg/dL, is associated with a 1‐ to 3‐point decline in IQ (IPCS, 1995; Pocock, Smith, & Baghurst, 1994; Schwartz, 1994), a change that is well within the standard error of measurement (SEM) of IQ tests. Reducing the mean blood lead level in the population would be unlikely to provide discernible benefit to any randomly selected child in the population, but the prevention paradox predicts that the benefit to the population would be substantial in terms of reducing the prevalence of IQ scores associated with need for remedial academic services, reduced lifetime earning potential, etc. What is sometimes overlooked, moreover, is that if a distribution retains its shape and variance, an increase in the area of the lower tail will be accompanied by a mirror image decrease in the area of the

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upper tail, in this case, a reduction in the prevalence of high IQ scores (Weiss, 1988). This argument need not be made solely on statistical grounds but can be illustrated using the data from two studies. In the study of Needleman and colleagues (Needleman, Gunnoe, Leviton, Reed, Peresie, Maher, & Barrett, 1979), the median verbal IQ scores of the high and low dentine lead groups diVered by fewer than 10 points, but the proportion of children in the high‐lead group with scores below 80 was threefold higher than the proportion in the low‐lead group. Similarly, no child in the high‐lead group had a score above 125, while 5% of children in the low‐lead group did (Needleman, Leviton, & Bellinger, 1982). In the study of Bellinger et al. (1987), comparison of the cumulative frequency distributions for residualized Mental Development Index scores (Bayley Scales) of 2‐year‐old children, stratified by the concentration of lead in umbilical cord blood (low: <3 mg/dL; medium: 6–7 mg/dL; high: >10 mg/dL), showed that the distribution rose much more rapidly for the high‐lead group, with the percentage of children scoring 10 to 20 points below expected two to three times greater than the corresponding percentages in the mid‐ and low‐lead groups. In considering the interpretation of the eVect sizes reported in neurobehavioral toxicity studies, some observers conclude that they are ‘‘in the noise’’ because they are generally smaller than the SEMs of the tests used to detect them. This claim warrants consideration. The SEM is an estimate of the measurement error in a child’s observed score on a test. Inversely related to the reliability coeYcient of the test, it is used to define the range within which a child’s true score is likely to fall (e.g., the 95% confidence interval is observed score  1.96 SEM). Does an appeal to the SEM provide a sound basis for concluding that, for example, a 3‐point deficit in the mean IQ of an exposed group, compared to an unexposed group, is meaningless? One would have to assume that the true mean scores are equivalent in the exposed and unexposed groups but that, by chance, the measured IQs of children in the exposed group are systematically underestimated (the children’s true scores fall in the upper region of the confidence intervals around the children’s observed scores) and that the measured IQs of children in the unexposed group are systematically overestimated (the true scores fall in the lower region of the confidence intervals around the children’s observed scores). That the direction of measurement error would be suYciently correlated with exposure status to produce this result seems highly unlikely (more specifically, the probability would be <0.05). The SEM is an important statistical concept for interpreting an individual’s scores, but it is misleading to invoke it to draw inferences about the clinical significance of a diVerence between group means. This latter task demands a diVerent level of analysis, one that focuses on population risk rather than on individual risk.

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The crucial distinction between individual and population risk is relevant in another way to studies of environmental neurotoxicants. Clinicians focus on the relative risk of adverse outcome in a patient who presents with a particular risk factor profile. For instance, the relative risk of producing a child with Down syndrome is 50‐fold greater for a woman over 44 years of age than it is for a woman under 30 years of age (incidence rates of 34.6/1000 births vs 0.7/1000 births) (Alberman & Berry, 1979). Epidemiologists, however, focus on the population attributable risk. Although the risk of Down syndrome is much lower among younger women, as a result of the much greater birth rate of younger women, 51% of infants with Down syndrome are born to women under 30 years of age. This is because attributable risk is the product, across exposure strata, of the products of relative risk and prevalence. Thus, ‘‘a large number of people at a small risk may give rise to more cases of disease than the small number who are at high risk’’ (Rose, 1985, p. 431). Applying this principle to neurotoxicants, more cases of adverse neurobehavioral outcome are likely to arise among children with neurotoxicant body burdens that are in the middle portion of the distribution than in the upper tail of the distribution. This provides another reason why public health eVorts should be focused not only on decreasing the numbers of children with body burdens that place them in the upper tail but on shifting the entire distribution toward lower levels. E.

Brain–Behavior Relationships

Those who rely on neurobehavioral data in setting exposure standards frequently ask questions such as, What does a low score on test X mean in terms of the site of damage in the brain? Apparently, many would feel more comfortable basing a standard on test X if poor performance mapped, in an isomorphic manner, onto the underlying neural substrate (poor performance on test X means damage to site Y, and only site Y, in the brain; poor performance on test A means damage to site B, and only site B, etc.). Unfortunately, the state of the science provides relatively little satisfaction in this regard. Most people would agree that having a complete brain is better than having only part of a brain, but the pediatric neurology literature is replete with case studies of children with dramatic cortical malformations, such as hydrocephalus, porencephalic cysts, and cerebellar agenesis, who have normal or near‐normal cognitive function (Chugani, Muller, & Chugani, 1996). These cases led one investigator to entitle a paper, ‘‘How much brain does a mind need?’’ (Lebeer, 1998). In adults, the site and size of a lesion, such as those produced by stroke, often correlate well with the nature and severity of any residual functional impairments, but such correlations are typically not observed when brain injury occurs in children.

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Chapman and colleagues (Chapman, Max, Gamino, McGlothlin, & CliV, 2003) reported that although children who suVered a stroke had discourse skills that were significantly worse than those of matched controls, neither the site nor the size of the lesion was associated with the severity of language impairment. The literature on very low birth weight indicates that the presence of an abnormality on a standard MRI study correlates poorly with the presence of minor functional disturbances such as slight reductions in IQ or motor incoordination (although morphological correlates of major impairments, such as cerebral palsy and severe visual impairments, can be identified) (Cooke & Abernathy, 1999; Krageloh‐Mann, Toft, Lundig, Andresen, Pyrds, & Lou, 1999; Rushe, Rifkin, Stewart, Townsend, Roth, Wyatt, & Murray, 2001; Skranes, Vik, Nilsen, Smevik, Andersson, & Brubakk, 1997). Only 30 to 60% of unselected children with frank mental retardation or significant developmental delay have positive findings on high‐resolution CT or MRI studies, generally consisting of disorders of ventral induction, abnormalities of migration, and white matter disorders (Schaefer & Bodensteiner, 1998). Children with clinically identified learning disabilities, ASDs, and ADHD generally do not show focal structural abnormalities (Filipek, 1999; Frank & Pavlakis, 2001). Given that these and similar disorders are currently thought to reflect abnormalities in the brain networks that underlie cognition and behavior, functional imaging modalities, such as f MRI, positron emission, and magnetic resonance spectroscopy (MRS), are likely to be more informative than structural imaging. For example, Trope and colleagues (Trope, Lopez‐Villegas, Cecil, & Lenkinski, 2001) found that in a small sample of children with elevated blood lead levels, MRI examinations were uninformative, but in vivo MRS spectroscopy studies showed that markers of metabolism in frontal gray matter (the N‐acetylaspartate/creatine and phosphocreatine ratios) were significantly lower than control levels. These labor‐intensive and costly methods are of limited applicability in field epidemiological studies of low‐level neurotoxicant exposures, given the typical sample sizes required to detect subtle eVects. One property of neurobehavioral tests that complicates their interpretation is that no test assesses a single functional domain. Although tests are marketed as measures of memory, problem solving, visual–spatial skills, or language, good performance on any test requires the competent coordination of a variety of skills. Consider the Developmental Test of Visual–Motor Integration (the VMI) (Beery, 1989). The specificity of the test name notwithstanding, poor performance on the VMI can result not only from a deficit in visual–motor integration, but from deficits anywhere in the long chain of skills called upon in the process of copying line drawings: visual– spatial analysis (ability to see part–whole relationships), grapho–motor

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control (the manual dexterity needed to control the pencil tip), cross‐modal integration of visual and motor information, planning and sequencing (the use of space), behavioral modulation (impulsivity), or motivation (the extent of fatigue or lack of interest in cooperating). Knowing only that a child achieved a poor score provides little insight into the underlying vulnerability (or vulnerabilities) responsible. Only if the VMI score is examined in the context of the results of many other tests that assess diVerent mixes of the skills called upon can one test alternative hypotheses, the approach typically applied in a clinical evaluation. Is it necessary, however, in order to formulate sound public policy, to dissociate the component skills and identify the most vulnerable elements? It depends on the goal of the study, and it might not be necessary in order to achieve a result that dramatically improves public health. Understanding only the key exposure pathway, John Snow halted the spread of cholera in nineteenth‐century London simply by dismantling the Broad Street pump. He knew nothing of the underlying disease pathogenesis, however, and another 30 years passed before Koch identified the Vibrio cholerae. Another factor complicating the process of drawing inferences about brain–behavior relationships in children exposed to neurotoxicants is that the brain continues to undergo substantial changes well into the second decade of postnatal life. The changes include the establishment of hemispheric dominance, the completion of myelination (particularly in the frontal lobes), synaptic pruning, and synaptic reorganization. As a result, the neurobehavioral impact of a lesion will likely diVer if it is acquired in adulthood, when it perturbs a fully developed system, than if it is acquired prenatally or in the early postnatal period, when it perturbs a system in which developmental processes are still ongoing (Bernstein, 1994; Tager‐ Flusberg, 2000). In other words, a brain that has been exposed to, for instance, methylmercury, from the outset of its development is not a brain in which some parts are normal and others abnormal (Oliver, Johnson, KarmiloV‐Smith, & Pennington, 2000). The brain as a whole is likely to have developed diVerently than it would have in the absence of methylmercury exposure, as suggested by the fact that the distribution of brain lesions indicates highly diVuse damage in fetal Minamata disease patients but highly localized lesions (calcarine fissure of the occipital cortex, cerebellum) in adult Minamata disease patients (Burbacher, Rodier, & Weiss, 1990). In some cases, the increased plasticity of developing systems might reduce the impact of an insult (Aram & Eisele, 1992). For example, children with agenesis of the corpus callosum do not express the same disconnection syndrome (‘‘split brain’’) seen in adults who undergo callosal resection (Chugani et al., 1996). In other cases, the impact of the insult might be greater on the developing organism than the adult because of its myriad,

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cumulating downstream eVects on both structure and function. Lesions that spare language in proficient speakers can impair language acquisition, suggesting that the neural substrate for language processing is not as highly localized in children as in adults and that, to some extent, the specific regions of the brain enlisted to solve a particular problem change with age (Stiles, 2000). Thus, while adults with acquired, focal lesions in the right parietal cortex often perform poorly on design‐copying tasks such as the VMI or the ROCF (Weintraub, 2000), it does not necessarily follow that poor performance on design‐copying tasks following developmental exposure to a neurotoxicant necessarily implies that the neurotoxicant caused a right parietal lesion in children. In any event, neurobehavioral test performance does not indicate what function was served by the part of the brain that was damaged by insult, but rather what the remaining brain can do in the face of this damage (Stein, 1987). The distinction is perhaps subtle but reflects an important diVerence in perspective. Even in the absence of an insult, the brain–behavior relationships underlying complex cognitive processes appear to diVer substantially in adults and children. An f MRI study of performance on a verbal fluency task identified the expected regions of activation in both children and adults (left inferior frontal cortex, left middle frontal gyrus), but cortical activation was much more widespread in children than in adults, particularly in the right hemisphere (right inferior frontal gyrus) (Gaillard, Hertz‐Pannier, Mott, Barnett, LeBihan, & Theodore, 2000). Similarly, prefrontal cortical activity during memory and attention tasks, assessed using f MRI, is more diVuse in children than in adults (Casey, Giedd, & Thomas, 2000). These diVerences between children and adults appear to be due not simply to age‐related diVerences in competence, but to age‐related diVerences in functional neuroanatomy. In an f MRI study, comparing visual lexical processing in adults and 7‐ to 10‐year‐olds, diVerent patterns of activation were found in children and adults, even when the two age groups were matched in terms of accuracy on the task (Schlagger, Brown, Lugar, Visscher, Miezin, & Petersen, 2002). The results of a study by Herbert and colleagues (Herbert, Ziegler, Makris, Bakardjiev, Hodgson, Adrien, Kennedy, Filipek, & Caviness, 2003) should give one pause when interpreting the results of studies using morphometric structural MR imaging. Children with developmental language disorder were found, upon segmentation analysis, to have a larger mean total brain volume than control children, reflecting largely an increased volume of cerebral white matter. This suggests that, in some cases at least, performance might be a function more of the eYciency and organization of neural networks than of simply the sizes of individual structures involved in the networks. Much remains to be learned about brain–behavior relationships in children, but ‘‘bigger is better,’’ a high‐technology version of

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phrenology, is clearly not a useful basis on which to draw inferences. The prevalence of detectable structural abnormalities on neuroimaging is likely to be very low among children with the subtler neurobehavioral deficits typically associated with environmental chemical exposures. Highly regular brain not behavior correlations no doubt exist. What is at issue is the vast gulf between our models of these relationships and the relationships themselves. The complexity of the brain systems that underlie performance on complex neurobehavioral tasks can be illustrated by the results of a volumetric MRI study comparing dyslexic and adult control males (Brown, Eliez, Menon, Rumsey, White, & Reiss, 2001). Rather than being restricted to those brain regions traditionally considered to be involved in language processes, group diVerences were surprisingly extensive, implicating the involvement of widely distributed neural networks rather than a strict localization of function. DiVerences were found in both cortical and subcortical regions, including the left posterior superior temporal gyrus (STG) and the temporo–parieto–occipital region; the left inferior, middle, superior, and medial temporal regions; the right parieto–occipital junction and STG; the left orbital gyrus and frontal pole; inferior and superior frontal gyrus (bilateral); head of caudate and thalamus (bilateral); and the semilunar modules of the cerebellum. Our knowledge of neural correlates of behavior is evolving and deepening rapidly. Clearly, the assumption of a simple isomorphism between a deficit as complex as performance on a neurobehavioral test and dysfunction in a specific brain structure is much too simple. For example, in Mirsky and Duncan’s (2001) neuropsychological model, attention includes 5 separate components. In the assessment battery these investigators assembled to assess these components, a reaction time task is used to assess the ‘‘sustain’’ and the ‘‘stabilize’’ components. Accuracy pertains to the sustain component of attention, while the variance of reaction time pertains to the stabilize component. One often reads in papers that a neurotoxicant‐related impairment on a reaction time task suggests a deficit in attention and, specifically, injury to the frontal lobes of the brain. While this is probably not incorrect, it is certainly only a small part of the story. Even a simple reaction time task involves the coordination of multiple processes, and each process might depend on diVerent brain regions. Stuss, Binns, Murphy, and Alexander (2002) demonstrated that, functionally and anatomically, the anterior frontal area contains at least three distinct attentional processes. Compared to controls and to patients with nonfrontal lesions, patients with lesions in the superior medial frontal area had slower reaction times; patients with left dorsolateral lesions tended to make more false‐positive errors (commission); and patients with right dorsolateral lesions appeared to have

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diYculty distinguishing targets from nontargets, producing high rates of both false‐positive and false‐negative errors; the performance of patients with inferior medial frontal lesions was similar to that of controls. In addition to a complex function such as attention calling upon a variety of brain regions, it is also the case that the same brain region can subserve multiple complex functions. For example, functional imaging studies suggest that a prefrontal network involving the mid‐dorsolateral, mid‐ventrolateral, and dorsal anterior cingulated cortex is activated in a wide variety of tasks, including perception, response selection, executive control, working memory, episodic memory, and problem solving (Duncan & Owen, 2000). A final reason to be cautious about requiring that the neural substrate underlying performance on a neurobehavioral test be clearly worked out before the test can be used in the risk assessment process is the apparent high prevalence of incidental abnormal findings on MRI. In a study conducted to provide normative data on brain structures, nearly one in five (18%) asymptomatic adult volunteers had such findings (Katzman, Dagher, & Petronas, 1999). The great majority of findings (83%) were not thought to require referral for follow‐up evaluations, but the prevalence of abnormalities is startling. A risk assessor must thus decide whether a neurobehavioral impact of a toxicant should be discounted as unimportant if a structural brain correlate of the impact cannot be identified, and, conversely, whether a structural impact should be considered important even if a neurobehavioral correlate (i.e., impaired function) cannot be identified. Based on our present understanding, it would seem prudent to accord greater importance to the former eVects than to the latter eVects.

V.

CONCLUSION

Work at the interface between neuropsychology and neurotoxicology has progressed far since the days in the not‐so‐distant past in which investigators used simple t tests to compare the test scores of children classified as ‘‘exposed’’ to a neurotoxicant to those classified as ‘‘unexposed.’’ The advances have occurred in all phases of study design, including sample specification and sampling strategy; the breadth, depth, and sophistication of neurodevelopmental assessments; the precision and accuracy of analytical laboratory measurements of exposure biomarkers; and the elegance of the biostatistical analyses applied to the data. Some of the responsibility for the increased rigor is, no doubt, due to the intense scrutiny such studies often receive due to the potential regulatory and economic implications of the findings. When the stakes are so high, the

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standards of evidence must also be high, and the bar has been raised accordingly. The necessarily observational nature of this research has consistently posed the most formidable challenges to the interpretation of study findings. Strictly speaking, to answer the question of whether exposure to a particular contaminant aVects children’s development requires that all other determinants of the outcome of interest have been measured accurately and appropriately incorporated into the statistical modeling. This is a tall order. As one consequence, such studies are, by necessity, multidisciplinary. The idea of a ‘‘lone wolf’’ investigator in this field is not feasible (and never really was). The aggregation into research teams of individuals who bring diverse skills to apply, including neuropsychology, toxicology, analytical chemistry, epidemiology, and biostatistics, is another factor contributing to the increase in study rigor over past decades. It can be expected that the scope of the expertise represented on teams will continue to expand as the questions asked increase in subtlety and complexity. Nutritionists, geneticists, sociologists and, no doubt, scientists from yet other disciplines will be included on future teams as other perspectives are recognized as important in the search for valid characterizations of the associations between environmental contaminant exposures and child development. REFERENCES Alberman, E., & Berry, C. (1979). Prenatal diagnosis and the specialist in community medicine. Community Medicine, 1, 89–96. American Psychiatric Association (APA). (1994). Diagnostic and statistical manual of mental disorders. (4th ed.). Washington, DC: American Psychiatric Press. Amler, R. W., & Gibertini, M. (1996). Pediatric environmental neurobehavioral test battery. Atlanta, GA: U.S. Department of Health and Human Services. Amler, R. W., Anger, W. K., & Sizemore, O. J. (1995). Adult environmental neurobehavioral test battery. Atlanta, GA: U.S. Department of Health and Human Services. Anderson, S. W., Antoine, B., Damasio, H., Tranel, D., & Damasio, A. R. (1999). Impairment of social and moral behavior related to early damage in human prefrontal cortex. Nature Neuroscience, 2, 1032–1037. Anger, W. K., Cassitto, M. G., Liang, Y.‐X., Amador, R., Hooisma, J., Chrislip, D. W., Mergler, D., Keifer, M., Hortnagl, J., & Fournier, L. (1993). Comparison of performance from three continents on the WHO‐recommended neurobehavioral core test battery. Environmental Research, 62, 125–147. Aram, D. M., & Eisele, J. A. (1992). Plasticity and recovery of higher cognitive functions following early brain injury. In I. Rapin & S. J. Segalowitz (Eds.), Handbook of Neuropsychology (Vol. 6, pp. 73–92). New York: Elsevier. Ardila, A. (1999). A neuropsychological approach to intelligence. Neuropsychology Review, 9, 117–236. Aylward, G. P. (2002). Cognitive and neuropsychological outcomes: More than IQ scores. Mental Retardation and Developmental Disabilities, 8, 234–240. Banaschewski, T., Brandeis, D., Heinrich, H., Albrecht, B., Brunner, E., & Rothenberger, A. (2003). Association of ADHD and conduct disorder—Brain electrical evidence for

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the existence of a distinct subtype. Journal of Child Psychology and Psychiatry, 44, 356–376. Barkley, R. A. (2003). Issues in the diagnosis of attention‐deficit/hyperactivity disorder in children. Brain & Development, 25, 77–83. Bellinger, D. C. (1995). Interpreting the literature on lead and child development: The neglected role of the ‘‘experimental system.’’ Neurotoxicology and Teratology, 17, 201–212. Bellinger, D. C. (2000). EVect modification in epidemiologic studies of low‐level neurotoxicant exposures and health outcomes. Neurotoxicology and Teratology, 22, 133–140. Bellinger, D. C. (2004). What is an adverse eVect? A possible resolution of clinical and epidemiological perspectives on neurobehavioral toxicity. Environmental Research, 95, 394–405. Bellinger, D., Leviton, A., & Sloman, J. (1990). Antecedents and correlates of improved cognitive performance in children exposed in utero to low levels of lead. Environmental Health Perspectives, 89, 5–11. Bellinger, D., Leviton, A., & Waternaux, C. (1989). Lead, IQ, and social class. International Journal of Epidemiology, 18, 180–185. Bellinger, D. C., Bernstein, J. H., Kirkwood, M. W., Rappaport, L. A., & Newburger, J. W. (2003a). Visual–spatial skills in children following open‐heart surgery. Journal of Developmental and Behavioral Pediatrics, 24, 169–179. Bellinger, D., Leviton, A., Waternaux, C., Needleman, H. L., & Rabinowitz, M. (1987). Longitudinal analyses of pre‐ and postnatal lead exposure and early cognitive development. New England Journal of Medicine, 316, 1037–1043. Bellinger, D. C., Wypij, D., duPlessis, A. J., Rappaport, L. A., Jonas, R. A., Wernovsky, G., & Newberger, J. W. (2003b). Neurodevelopmental status at eight years in children with dextro‐transposition of the great arteries: The Boston Circulatory Arrest Trial. Journal of Thoracic and Cardiovascular Surgery, 126, 1385–1396. Bernstein, J. H. (1994). Assessment of developmental toxicity: Neuropsychological batteries. Environmental Health Perspectives, 102(Suppl. 2), 141–144. Berry, K. (1989). Developmental Test of Visual–Motor Integration.. Bouchard, T. J., & McGue, M. (1981). Familial studies of intelligence: A review. Science, 212, 1055–1059. Brown, W. E., Eliez, S., Menon, V., Rumsey, J. M., White, C. D., & Reiss, A. L. (2001). Preliminary evidence of widespread morphological variations of the brain in dyslexia. Neurology, 56, 781–783. Burbacher, T. M., Rodier, P. M., & Weiss, B. (1990). Methylmercury developmental neurotoxicity: A comparison of eVects in humans and animals. Neurotoxicology and Teratology, 12, 191–202. Burke, W. (2003). Genomics as a probe for disease biology. New England Journal of Medicine, 349, 969–974. Carlson, S. E., & Neuringer, M. (1999). Polyunsaturated fatty acid status and neurodevelopment: A summary and critical analysis of the literature. Lipids, 34, 171–178. Casey, B. J., Giedd, J. N., & Thomas, K. M. (2000). Structural and functional brain development and its relation to cognitive development. Biological Psychology, 54, 241–257. Centers for Disease Control (CDC) (1991). Preventing lead poisoning in young children. Atlanta: U.S. Public Health Service. Chapman, L., & Chapman, J. (1978). The measurement of diVerential deficit. Journal of Psychiatric Research, 14, 301–311. Chapman, S. B., Max, J. E., Gamino, J. F., McGlothlin, J. H., & CliV, S. N. (2003). Discourse plasticity in children after stroke: Age at injury and lesion eVects. Pediatric Neurology, 29, 34–41.

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From Animals to Humans: Models and Constructs DEBORAH C. RICE ENVIRONMENTAL AND OCCUPATIONAL HEALTH PROGRAM DEPARTMENT OF HEALTH AND HUMAN SERVICES, MAINE CENTER FOR DISEASE CONTROL AND PREVENTION, AUGUSTA, MAINE

I.

INTRODUCTION

This chapter focuses on tests in animal models that assess the domains aVected in developmental disabilities. There are many choices of test procedures to assess a multiplicity of functional domains, from so‐called ‘‘screening tests’’ that are capable of detecting relatively severe impairment to very sophisticated tests that provide detailed information on sensory, motor, and cognitive capabilities. Screening batteries have been developed by national and international agencies to detect and characterize developmental neurotoxicity, such as the U.S. Environmental Protection Agency Developmental Neurotoxicity Test (EPA, 1998) and those developed by the Organization for Economic Cooperation and Development (OECD, 1999). These batteries include tests of locomotor activity and crude assessment of learning, sensory, and motor integration. Considerable eVort was devoted to validation and standardization of such tests, including comparison among various batteries (Buelke‐ Sam et al., 1985; Catalano, McDaniel, & Moser, 1997; Elsner et al., 1986; Tilson et al., 1997; Vorhees, 1985). These screening batteries are not designed to, nor are they capable of, characterizing in detail types of impairment produced in specific domains, or indeed, in many instances, of identifying the domains aVected. Thus, extrapolation from results of screening tests to specific deficits in children is problematic. Nonetheless, identification of neurotoxicity with screening procedures indicates that the chemical under study is probably also neurotoxic to the developing human. Various functional domains may be assessed in detail, using a variety of tests suitable for rodents and other animals. Fine and gross motor function INTERNATIONAL REVIEW OF RESEARCH IN MENTAL RETARDATION, Vol. 30 0074-7750/06 $35.00

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may be assessed in various ways, as well as numerous aspects of sensory function. Cognitive domains that are aVected in developmental disabilities may also be assessed using animal models. There are numerous tests available for assessing learning and memory. In addition, however, the components of executive function can be examined in animal models: attention, distractibility, impulsivity, adaptability, and temporal and spatial organization of behavior. Although children are capable of more complex behavior than are animals, the basic functional domains constituting these behaviors can nonetheless be assessed in animal models. However, many of these testing procedures require extensive training of the animals and considerable expertise on the part of the experimental researcher. Interpretation of the results from animal experiments requires an understanding of the diVerences between humans and the species under investigation, as well as the appropriateness of the dosing regimen to exposure in humans. It is, of course, crucial to understand the diVerences in nervous system anatomy and physiology and their potential contribution to species diVerences. In addition, metabolic diVerences between humans and other species may be important determinants of eVects. The parent compound may be metabolized to a less toxic or nontoxic chemical, or to the toxic entity, and these conversions may be species‐specific. The rate of metabolism is also important. Typically, laboratory species metabolize and/or excrete chemicals more quickly than humans, often by many times. Therefore, the external dose administered to the animal often produces a lower body burden following repeated exposure than the same dose would in humans. If at all possible, the body burden (blood or tissue levels of the active agent) should be determined in the animal model, so that the appropriate comparison can be made to human body burden. EVects of developmental neurotoxicants may be observed at approximately the same body burden in humans and experimental animal models, whereas the external dose necessary to produce eVects in animals can overestimate the dose (exposure) required to produce eVects in humans (Rice, de DuVard, DuVard, Iregren, Satoh, & Watanabe, 1996a). The body burden of a toxicant can be a determinant of the type of toxicity produced. High doses may not be good predictors of eVects at lower levels of exposure. For example, high levels of lead produce encephalopathy in both humans and animals. It was impossible to predict, based on this massive response to toxic insult, the fact that, at lower body burdens, lead produces deficits in attention and impulse control. The developmental period during which exposure occurs may also be an important determinant of the type and severity of damage produced. The nervous system develops by a series of processes that are exquisitely choreographed temporally and spatially. The timing of these processes during fetal and postnatal developments is known for humans and experimental

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animals (Rice & Barone, 2000) and diVers among species. For example, it is important to keep in mind, when designing experiments or interpreting results, that some events that are prenatal in humans occur postnatally in rats and mice. Experiments should be designed to mimic the timing of human exposure as much as possible. This chapter discusses a sampling of tests that may be used to characterize the neuropsychological eVects of developmental neurotoxicants in animal models. The environmental chemicals that have been the most studied with regard to developmental neurotoxic eVects are lead, methylmercury, and polychlorinated biphenols (PCBs). A number of longitudinal prospective studies have characterized the consequences of exposure to these agents, including decreased IQ, attention problems, impulsivity, distractibility, perseverative behavior, and sensory and motor impairment. Therefore, eVects of these chemicals on tasks assessing these functional domains are used as examples of the use of animal models to elucidate specific deficits. For these chemicals, eVects in humans and animals are remarkably congruent. This chapter is not a review of the neurotoxic eVects of chemicals in animals. It is also not a compendium of available techniques. A number of books and workshops have addressed methodological issues in behavioral toxicology (cf. Weiss & Elsner, 1996; Weiss & O’Donoghue, 1994). The intention of this chapter is simply to present examples of the techniques available to identify and characterize neurotoxic eVects produced by developmental exposure in animal models.

II.

TESTS OF COGNITION

Perhaps the most typically measured endpoint in studies of the neuropsychological eVects of exposure to environmental chemicals in children is standardized tests of intelligence, or IQ. Lead (Canfield, Henderson, Cory‐Slechta, Cox, Jusko, & Lanphear, 2003; Rice, 1996b), methylmercury (Kjellstro¨ m et al., 1989), and PCBs (Schantz, Widholm, & Rice, 2003) produce IQ deficits. There are no standardized intelligence tests per se for animals. However, aspects of intelligence, including learning ability and memory, can readily be assessed in almost any species. Other aspects of cognition, including so‐called executive functions such as impulse control, attention, and ability to respond appropriately to the consequences of one’s actions, may also be assessed in animal models, often using tasks the same as or similar to those used in humans (Paule, 1990; Paule, Chelonis, BuValo, Blake, & Casey, 1999). These behavioral domains are often impaired by exposure to neurotoxic agents, perhaps more frequently than IQ. Impairment in these specific domains has important consequences in terms of ability to learn and function in society.

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A wide variety of tasks may be used to assess learning ability in animal models. Avoidance tests have a long history of use with rodents, usually rats. In an active avoidance task, the subject has to learn to move from one compartment to another to avoid a shock. In passive avoidance, the subject is required to stay in a nonpreferred lit compartment rather than moving to a preferred unlit one in order to avoid being shocked. (Rats are nocturnal and will move to the dark compartment preferentially.) These tests do not tax the learning ability of the rat, and therefore assess only gross cognitive impairment. In addition, general arousal level will influence performance on this task, since increased arousal will make it more likely that the animal will move (advantageous for active avoidance and disadvantageous for passive avoidance). Somatosensory impairment will also particularly confound the results of these tasks. Therefore, results must be interpreted with caution in the absence of information on other factors that may influence performance. Perhaps a more sensitive but still simple test of learning is the discrimination task. Discrimination tasks are usually assessed in the visual domain, even in rodents, despite the fact that the spatial vision of laboratory rodents is relatively poor compared to ours (see section on sensory function). In such tasks, the subject learns to choose one stimulus rather than another (or others). For example, the subject may have to respond to a specific shape (nonspatial discrimination) to be reinforced. Discrimination performance may be assessed using automated computer‐controlled equipment or non‐ automated procedures such as mazes. Discrimination tasks have proved sensitive to developmental exposure to lead (Rice, 1996b), methylmercury (Buelke‐Sam et al., 1985; Elsner et al., 1988), and PCBs (Rice, 1999b). However, the sensitivity of discrimination tasks in detecting toxicant‐ induced impairment is dependent upon task diYculty: there may be no eVect on tasks that are simple for the subject to learn. For example, rats exposed to lead during development were impaired on a diYcult but not easy nonspatial discrimination task (Winneke, Brockhaus, & Baltissen, 1977) (Fig. 1). A series of discrimination problems may also be presented. Normal animals will typically learn successive discrimination problems more quickly. This ‘‘learning set formation,’’ or learning to learn, represents the ability of the organism to take advantage of past exposure to a particular set of rules. Monkeys exposed developmentally to lead displayed impaired ability to learn successive problems more quickly as the experiment progressed, in addition to impaired acquisition of the individual discrimination tasks (Lilienthal, Winneke, Brockhaus, & Malik, 1986). Requiring the subject to perform a reversal of the original discrimination problem is often more sensitive to toxicant‐induced impairment than the

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FIG. 1. Number of errors on a simple and diYcult visual discrimination task in rats. Lead‐ treated rats were impaired on a task that was diYcult for controls but not on one that was easy. From Winneke et al., 1977.

initial acquisition of the task. In a discrimination reversal task, the formerly correct stimulus becomes the incorrect one and vice versa. In the nonspatial version of the task, the relevant stimulus dimension is form or color, for example, rather than the position of stimuli. Typically, the subject is required to perform a series of such reversals, which is indicative of how quickly the subject learns that the rules of the game change in a predictable manner. Nonspatial discrimination reversal performance was impaired by developmental exposure to lead (Bushnell & Bowman, 1979; Rice, 1985; Rice & Gilbert, 1990a; Rice & Willes, 1979). At lower or moderate body burdens of lead, monkeys were impaired on discrimination reversal problems even though they were not impaired on the initial acquisition of the task (Rice, 1985; Rice & Willes, 1979) (Fig. 2). Acquisition of performance (learning) can be assessed in any number of other tasks, including tasks designed to measure other domains, such as memory. A task that has been used frequently in behavioral toxicology is spatial alternation. In this task, the subject is required to alternate responses between locations (levers in a computer‐controlled task or alleys in a maze in a non‐automated task), with no cues provided to indicate correct location. This task requires the subject to abandon the position that has just produced a reward on the preceding trial, and thus requires the ability to adapt to an

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FIG. 2. Performance of lead‐treated (filled circles) and control (open circles) monkeys on the initial acquisition (0) and series of 20 reversals of a visual discrimination task. Treated monkeys were not impaired on acquisition of the task, but failed to learn the reversal task over successive reversals as quickly as did controls. Introduction of 150 extra trials on the correct stimulus for reversal 5 disrupted both groups to an equal extent, with treated monkeys making more errors per reversal for reversals 6 to 10. Five hundred extra trials following reversal 10 disrupted the performance of both groups.

imposed and arguably unnatural rule. Delays may be implemented between opportunities to respond to assess spatial memory. The ability to learn this task was impaired in monkeys exposed developmentally to PCBs (Rice & Hayward, 1997) or lead (Rice & Gilbert, 1990b; Rice & Karpinski, 1988), and rats exposed to PCBs (Schantz, Seo, Moshtaghian, Peterson, & Moore, 1996; Schantz, Seo, Wong, & Pessah, 1997). The repeated acquisition task is a task designed to assess the ability to learn new information repeatedly. This task requires the subject to learn a new sequence of responses multiple times, for example, in every daily session. The sequence may be spatial (learning to respond on a series of levers in a specific sequence) or nonspatial (learning to respond in sequence to a set of colors or shapes). Such a task assesses the ability of the subject to learn and remember new information and ignore previously learned response patterns. Learning, memory, attention, and adaptability are all components of this task. This task proved sensitive to lead exposure in rats (Cohn, Cox, & Cory‐Slechta, 1993). In particular, rats were not impaired on the performance

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component of the schedule, which did not change across sessions, but were impaired on the acquisition component, which required learning a new sequence every session. Lead exposure in children is associated with an inability to follow complex sequences of directions (Needleman et al., 1979). Although repeated acquisition performance is not directly comparable, both deficits indicate an inability to hold information online and to organize behavior in time or space. Deficits in such abilities are indicative of impairment in executive function. Another type of task that requires repeated learning and adaptation to changing environmental contingencies is concurrent schedules of reinforcement. In a concurrent schedule, diVerent ‘‘rules’’ are in eVect on diVerent levers (usually two) at the same time. The subject should learn to apportion responding according to the schedule contingencies. For example, if one lever pays oV with a reinforcer twice as often following a response as a second lever, the most adaptive strategy is to respond on the first lever twice as often. In addition, the relative payoV of the levers can be changed by the experimenter within a session or across a number of sessions, and the ability of the subject to adapt to the new contingencies can be determined. Rats exposed developmentally to methylmercury exhibited impairment in their ability to respond to changing contingencies during old age (Newland, Reile, & Langston, 2004). Impairment in transitions was also observed in monkeys exposed to either lead or methylmercury (Newland, Yezhou, Lodgberg, & Berlin, 1994). When relative reinforcement densities were changed, treated monkeys changed their behavior slowly, not at all, or in the wrong direction. B.

Memory

There are many tasks that may be used to assess memory. Retention of passive or active avoidance performance (described previously) in rodents may be assessed following a delay, typically 24 hours. This is a crude test of memory, however, and negative results (no memory impairment) should be interpreted with caution. A test of spatial learning and memory commonly used with rodents is the Morris water maze task. The subject is put into a circular pool, and must locate a submerged platform to escape the water. The time to locate the platform on subsequent trials is considered an indication of memory. The location of the platform may also be changed to assess ability to learn a new location (and cease searching for the old). This task has proved sensitive to toxicant exposure (Viberg, Fredriksson, & Eriksson, 2003). This test is sensitive to hippocampal damage in rodents (D’Hooge & De Deyn, 2001; Redish & Tourtezky, 1998). It should not be assumed that the results of this task measure all aspects of ‘‘memory,’’ but rather only very specific abilities.

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A task that is often used to assess spatial memory is delayed spatial alternation. As has been discussed, this task not only measures memory but also requires the subject to choose a location diVerent from the one that last produced a reinforcement, which may be considered a measure of adaptability. This task may be tested in a maze or using automated equipment (response buttons or levers). This task has proved sensitive to developmental exposure to PCBs in monkeys (Levin, Schantz, & Bowman, 1998) and rats (Schantz, Moshtaghian, & Ness, 1995), as well as lead in monkeys (Rice & Gilbert, 1990b; Rice & Karpinski, 1988) and rats (Cory‐Slechta, Pokora, & Widzowski, 1991) (Fig. 3). Another test of spatial memory used with rats is radial arm maze performance. The apparatus consists of a central compartment with alleys (typically eight) radiating from it like spokes of a wheel. Food reinforcers may be placed in some or all of the compartments. In some studies, reinforcements are placed in the same compartments on every test session, to test the subjects’ ability to remember across longer periods of time (so‐called reference memory). Other compartments may be baited on some trials or sessions

FIG. 3. Average latency to respond on a T‐maze delayed alternation task in male and female rats exposed to a high or low dose one of three ortho‐substituted PCBs. Exposed females had latencies similar to those of males, suggesting a masculinizing eVect. PCB‐treated female but not male rats also had more total errors than did controls. These results highlight the importance of testing both sexes, as well as the importance of assessing a number of performance measures from a study, which may provide diVerent types of information. From Schantz et al., 1995.

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and not others (working memory). The most eYcient response pattern is to enter each alley only once. The time taken to collect all reinforcements and the number of unbaited arms entered or re‐entered are determined. This task has proved sensitive to developmental dioxin (Seo, Powers, Widholm, & Schantz, 2000) and methylmercury (Dore´ et al., 2001; Goulet, Dore´ , & Mirault, 2003) exposure. Individual PCB congeners were tested in the same laboratory on both radial arm maze and delayed alternation (Schantz et al., 1995, 1996, 1997). Interestingly, the congeners impaired performance on one test and not the other; which test was aVected was congener‐specific. This suggests that these tasks measure diVerent behavioral domains, and highlights the importance of assessing behavior on a number of tasks, even tasks that purportedly measure the same aspect of behavior (such as spatial memory). A task that is used more often with monkeys than with rodents, and is also suitable for use in children, is the delayed matching to sample task. In the nonspatial version of the task, the subject is presented with a particular stimulus (such as color or form) to remember. Following a delay period, a number of unique stimuli are presented, including the sample stimulus. The requirement is to choose the (previously) sample stimulus. The spatial version of this task requires a match to a previously presented position. Monkeys exposed developmentally to lead were not impaired in the acquisition of either a nonspatial or spatial matching to sample task (Rice, 1984), but lead‐treated monkeys performed more poorly than did controls under delay conditions. Control monkeys reached chance performance at longer delay values than treated monkeys. C.

Attention

Attentional deficits are a common consequence of developmental exposure to environmental toxicants in humans, including lead (Fergusson, Fergusson, Horwood, & Kinzett, 1988), methylmercury (Grandjean et al., 1997), and PCBs and dioxins (Patandin, Veenstra, Mulder, Sewnaik, Sauer, & Weisglas‐Kuperus, 1999). Attentional deficits may also result from developmental exposure to drugs such as maternal tobacco smoking, cocaine, or marijuana (Fried & Smith, 2001; Fried & Watkinson, 2001; Leech, Richardson, Goldschmidt, & Day, 1999; Richardson, Ryan, Willford, Day, & Goldschmidt, 2002). Assessment of attentional processes in animals has received considerable attention over many years. This section will describe only a few of the available procedures. For an excellent review of the literature, see Bushnell, 1998. A task that has proved sensitive to developmental toxicant exposure in a number of studies is a vigilance task, which is typically assessed using a

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computer for stimulus presentation and recording of responses (Grandjean et al., 1997; Stewart et al., 2003a; Winneke, Brockhaus, Collet, & Kraemer, 1989). The child is asked to respond to one stimulus and not to respond to others. Typical stimuli are animal pictures for younger children or letters for older ones. Stimuli are presented one at a time. The main dependent variable is reaction time, or the time between presentation of the stimulus and the response. The child is told to respond as quickly as possible. Errors of commission (false positives) and omission (false negatives) should also be recorded. This task is one form of a signal detection task. Another form of signal detection task used to measure attention in animals is also called the go–no go procedure. In this task, the subject is asked to respond when one stimulus type is presented and to withhold responding when a diVerent one (or none) is presented. This task is often used to study the ability of the subject to detect or discriminate between stimuli. This test may be used in both humans and animals (including rodents) to test sensory thresholds. This task has been used in both humans and rodents to test attention using suprathreshold stimuli (Bushnell, Benignus, & Case, 2003; Oshiro, Krantz, & Bushnell, 2001). A task similar to the signal detection task is the complex reaction time task, which requires the subject to respond to a particular stimulus rather than to others presented simultaneously. A similar task that is probably less sensitive to attentional deficits is a simple reaction time task, in which the subject is required to respond as quickly as possible to presentation of an invariant stimulus. Monkeys exposed to methylmercury from birth to adulthood were not impaired on a simple reaction time task or a series of complex reaction time tasks in either reaction time or accuracy (Rice, 1998a). Similarly, developmental lead exposure did not aVect performance on a simple reaction time task (Rice, 1988). However, treated monkeys did display an increased incidence of failure to respond to the stimulus within a specified time period. In addition, several groups of lead‐ or methylmercury‐exposed monkeys were tested on a number of very diYcult sensory assessments, which required response to stimuli at or near threshold. The reaction times of treated monkeys were not diVerent from those of controls, in terms of average response time or distribution of responses. Nor did they change across the course of the often lengthy session, indicating that attention was sustained for long periods of time. These negative results are in contrast to the eVects of lead or methylmercury on performance on vigilance tasks in children (Grandjean et al., 1997; Winneke et al., 1989). It may be that eVects would have been observed on a vigilance task in lead‐exposed animals, since the eVects in children were on errors of commission (i.e., responding to nontarget stimuli) rather than reaction time, which may reflect impairment in

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impulse control rather than attention. On the other hand, increased reaction times were observed on a simple reaction time task in lead‐exposed children (Needleman, 1987). Parsing the behavioral mechanisms responsible for deficits on tasks meant to assess attention is diYcult, in either animal models or humans. Increased errors of commission may be the result of failure to inhibit inappropriate responding rather than to attentional deficits (see following text). Sensory deficits, particularly in higher‐order sensory processing, may masquerade as attentional problems. Obviously, motor deficits would interfere with performance on reaction time tasks. Impaired learning ability may be interpreted as attentional deficits. It is important to test subjects, human or animal, until stable performance is attained, and cognition should be assessed independently on other tasks. D.

Distractibility

A behavioral domain that is linked to attention is distractibility: the lack of ability to stay on task in the presence of irrelevant stimuli. Lead‐exposed children were rated by their teachers as being more distractible (Needleman et al., 1979; Yule, Urbanowicz, Lansdown, & Millar, 1984). On a rating scale used in a study in New Zealand, the ‘‘attention’’ subscale included ‘‘short attention span’’ as well as ‘‘inattention, easily distracted’’; both were impaired as a function of increased lead exposure (Fergusson et al., 1988). Distractibility may be tested directly by the systematic introduction of irrelevant cues, and/or by identifying systematic response patterns associated with irrelevant stimulus dimensions. For example, irrelevant cues were introduced into a nonspatial discrimination task in two studies of lead‐exposed monkeys (Rice, 1985; Rice & Gilbert, 1990a). Even though lead‐treated monkeys had learned the task as well as controls, they performed more poorly after introduction of irrelevant cues, and attended to (responded on) the irrelevant cues in systematic ways. E.

Impulsivity

One of the hallmarks of developmental lead exposure is inability to inhibit inappropriate behavior (Needleman et al., 1979; Rice, 1996b). Increased impulsivity as a result of developmental PCB (Patandin et al., 1999; Stewart et al., 2003a) or lead (Raab, Thomson, Boyd, Fulton, & Laxen, 1990) exposure is evidenced by increased errors of commission on a vigilance task. Developmental methylmercury exposure was associated with increased reaction time and omission errors; eVect on commission errors was not reported (Grandjean et al., 1997).

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There are a number of ways to measure the ability of animals to inhibit inappropriate responding. In many cognitive tasks assessed in experimental animals, opportunities to make a response choice are separated by short periods (inter‐trial intervals) during which responding is neither punished nor rewarded. Similarly, incorrect responses are often punished with a short ‘‘time out’’ period in addition to withholding a reinforcement (reward). Increased responding during these periods indicates failure of impulse inhibition. For example, developmental lead exposure produced increased responding in schedule components in which responding was never reinforced in monkeys (Rice, 1992a; Rice, Gilbert, & Willes, 1979) and rats (Angell and Weiss, 1982). A simple task that assesses the ability to inhibit responding is the DRL (diVerential reinforcement of low rate) schedule of reinforcement. This schedule has been used in animals for decades, initially in the pharmaceutical industry and, more recently, in the study of the eVects of environmental chemicals. Under this schedule, the subject is required to wait a specified amount of time between responses (or between reinforcement and the next response) to be reinforced; responding before the elapsed time results in the time requirement’s being reset. This schedule is suitable for many species, including rodents, monkeys, and humans. In a study of monkeys exposed postnatally to PCBs, control animals learned over the course of a number of sessions to space the majority of responses at least 30 sec apart, the time required to be rewarded (Rice, 1998b) (Fig. 4). In contrast, the average interval between responses for the PCB‐treated monkeys was still less than 30 sec even after 50 days of testing. In consequence, PCB‐treated monkeys received fewer reinforcements and made more nonreinforced responses. In other words, they failed to inhibit responding even when punished for not doing so. Lead‐exposed rats also performed more poorly than did controls on a DRL task (Alfano and Petit, 1981; Dietz, McMillan, Grant, & Kimmel, 1978). This schedule has also been used in children exposed to PCBs. Failure to inhibit responding was associated independently with in utero exposure to PCBs or methylmercury, or postnatal exposure to lead in school‐age children (Paul Stewart, personal communication). Thus, there was congruence between animal and human data on the same schedule for the eVects of developmental PCB exposure. EVects on IQ identified in early childhood were not present as the children got older (Stewart et al., 2003b). The eVects observed on this schedule indicate that the DRL schedule is measuring something diVerent from IQ. The use of the DRL in this study was based directly on results from the monkey study, and exemplifies the potential for a two‐way fertilization between epidemiological and experimental research.

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FIG. 4. The mean time between responses (inter‐response time or IRT) and the ratio of reinforced/nonreinforced responses for controls and monkeys exposed postnatally to PCBs. An IRT of at least 30 sec was required for reinforcement. The mean IRT of the treated group was still less than 30 sec after 50 sessions (days) of testing. Their response pattern was less eYcient that the control group; they made many more responses for each reinforcement. From Rice, 1998b.

Another schedule that has been used extensively to assess the eVects of drugs and chemicals on behavior is the fixed interval (FI) schedule of reinforcement. Under this schedule, the subject is required to make one response after a specified elapsed time to be reinforced. Unlike the DRL schedule, premature responding under the FI schedule has no scheduled consequences. After the subject learns the schedule, responding is typically characterized by a pause at the beginning of the interval, followed by a gradually accelerating rate of response terminating in reinforcement. Developmental lead exposure resulted in increased rates of response in both

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monkeys (Rice, 1992a; Rice et al., 1979) and rats (Angell and Weiss, 1982; Cory‐Slechta, Weiss, & Cox, 1983, 1985) (Fig. 5). Similarly, monkeys exposed developmentally to PCBs exhibited increased rates of response (Rice, 1997a). The FI schedule does not specifically punish high rates of response. Nonetheless, high response rates represent at least ineYcient behavior, and may be viewed as representing failure of response inhibition.

F.

Perseveration

Another nonadaptive response pattern is perseveration—persisting in response patterns that do not pay oV or have ceased to pay oV. Perseveration represents failure to adapt appropriately to environmental contingencies or changes in those contingencies. A test used in humans that assesses perseveration and adaptability is the Wisconsin Card Sort Test. In this test, the experimenter presents a sample card, and then several possible test cards that match the sample card in one or more domain. Responding correctly depends on the ability to generalize whether the relevant stimulus domain is color, number, or shape, which must be inferred from the consequences of responses (verbal feedback on whether the choice was correct or not). The investigator may change the relevant stimulus class at any time, and the subject must infer the new rule by whether responses are identified as correct or incorrect. Concurrent blood lead levels in children were associated with an increase in perseverative errors on this task (Chiodo, Jacobson, & Jacobson, 2004; Stiles & Bellinger, 1993). That is, more highly lead‐exposed children continued to respond as if the experimenter had not changed the ‘‘rule’’ of which stimulus class was relevant. Perseveration may be assessed in animals in a number of ways. In the delayed nonspatial discrimination task, already described, the relevant stimulus class was also changed, similar to the Wisconsin Card Sort Test. Lead‐ exposed monkeys attended to the newly irrelevant stimulus class longer than did controls (Rice, 1985; Rice & Gilbert, 1990a). Marked perseverative behavior was observed in a spatial delayed alternation task in monkeys exposed developmentally to lead (Rice & Gilbert, 1990b; Rice & Karpinski, 1988). In that task, the monkey was required to alternate responses between two buttons. If the monkey responded incorrectly, a response had to be made on the opposite correct button before a response on the alternate button was considered correct. Delays were interspersed to assess short‐term memory. At even very short delays, lead‐treated monkeys perseverated on the same button, responding incorrectly for sometimes hours at a time without switching to the other button (Fig. 6). Increased perseverative behavior on a delayed alternation task has also been observed in rats

FIG. 5. The number of responses per second emitted on an FI schedule by lead‐exposed (triangles) and control (asterisks) monkeys. Monkeys were exposed to lead from birth onward (Group 2), during infancy only (Group 3), or beginning after infancy (Group 4). Lead‐treated monkeys in all groups responded at a higher rate. Lead exposure during infancy only was suYcient to produce this eVect, as was exposure beginning after infancy. This suggests that eVects are irreversible, and that exposure during infancy is not necessary for lead‐induced impairment. From Rice, 1992a.

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FIG. 6. The session length and total number of incorrect responses on a delayed spatial alternation task in controls (triangles) and monkeys exposed to a low dose (Xs) or higher dose (inverted triangles) of lead from birth. The session ended after 100 correct responses. Each incorrect response extended the session by one response. Treated monkeys made many perseverative errors, in some cases, hundreds in a row, which resulted in much longer session lengths as well as an increased number of errors.

exposed to lead (Cory‐Slechta et al., 1991) and in monkeys exposed developmentally to PCBs (Rice & Hayward, 1997). Lead‐exposed monkeys also perseverated for position on the concurrent discrimination task (Rice, 1992b). Treated monkeys responded incorrectly

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on the previously correct position more often than did controls, even though position was irrelevant in the task. Lead‐exposed monkeys also perseverated on a previously correct position on the nonspatial delayed matching to sample task already discussed (Rice, 1984). Detection of the types of deficits identified on the tasks discussed often required detailed analysis of response sequences and error patterns. Simply tallying errors would have provided little or no insight into the specific functional domains aVected by toxicant exposure.

III.

SENSORY FUNCTION

There are many chemicals that produce sensory deficits in humans and animals, including metals, solvents, and pesticides. Animal models, including rodents, can provide useful information concerning sensory system impairment. Extrapolation of results from evaluation of sensory system function in animals is relatively straightforward for a couple of reasons. First, if the stimulus presentation is well controlled, the variance of the control group for the function being measured should be relatively small. This allows smaller changes in sensory system function to be detected in treated groups compared to other endpoints such as learning or memory. Second, the results are readily interpretable, and usually can be extrapolated to humans in a relatively straightforward manner. Although sensory function varies among species in sensitivity and the range in which stimuli can be detected, sensory loss in one species predicts a similar loss in another species. A.

Vision

Humans and other Old World primates have better spatial vision than most other animals, as well as trichromatic color vision. There are important diVerences with respect to visual function between nocturnal rodents (rats and mice) and humans. Half of the primate (including human) brain is devoted to visual processing. In contrast, rodents rely largely on olfaction and audition, as well as somatosensory information via the vibrissae (whiskers) to receive information about the environment. Humans also have binocular vision, and therefore good depth perception. We thus are able to detect and interpret the visual world in considerable detail. The spatial frequencies that delineate objects are analyzed by our visual system by sorting them into their sine wave components. Therefore, determining the ability of the visual system to detect sine waves over a number of frequencies assesses the fundamental function of the system, at least in the orientation of testing (usually, vertical sine waves). The ability to detect high

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frequencies may be considered roughly equivalent to tests of visual acuity, which measure only one point on the frequency threshold curve. The spatial visual function of macaque monkeys is almost identical to that of humans (Fig. 7). In contrast, the ability of nocturnal rodents to detect spatial frequencies is poor. Albino animals, including albino rats, have abnormal visual anatomy and physiology, and therefore poor spatial vision compared to normally pigmented members of their species. Rats and mice also have retinas composed almost exclusively of rods, and therefore have little or no color vision. Rodents also do not have binocular vision. Therefore, rodents have limited utility for the study of some aspects of human visual function, although basic mechanisms of toxicity may be studied, as has been done for lead, for example (Fox et al., 1994, 1997, 1998). Spatial vision was assessed in our laboratory in several groups of monkeys exposed to methylmercury or lead during various developmental periods. To perform the task, the monkey sat in a restraining chair in a light‐tight chamber facing two oscilloscopes. On one was a sine wave grating, and the other displayed a blank field of equal average luminance. The monkey was required to press a button corresponding to the scope displaying the grating. For each of a number of spatial frequencies (a few wide bars to many narrow bars on the screen), the contrast between the lightest and darkest part of the grating was varied systematically across trials, and the contrast at which the monkey could not distinguish the scope displaying the grating from the blank scope was determined. That point was considered the threshold for that frequency. Monkeys exposed to moderate levels of methylmercury exhibit deficits in high‐frequency spatial vision under high luminance conditions, whether exposure was prenatal only (Burbacher et al., 2005), postnatal only (Rice & Gilbert, 1982), or pre‐ plus postnatal (Rice & Gilbert, 1990c). When tested under very low luminance conditions (comparable to dim starlight) that

FIG. 7. Left panel: Contrast sensitivity (CS) functions for a number of species. The CS is the inverse of the minimum contrast between the light and dark gradations across a sine wave grating that can be distinguished by the subject; higher contrast sensitivity represents better spatial vision. Note the three y‐axes. Right panel top: CS of humans and macaque monkeys under five luminance conditions. CS is best in bright light and worst under very low luminance conditions. Right panel bottom: CS of control monkeys (solid lines) under high and low luminance conditions, and two monkeys exposed to methylmercury from birth. One methylmercury‐treated monkey (left) had a high spatial frequency deficit under high luminance conditions, and a small deficit across all frequencies under low luminance conditions. The other monkey (right) had very impaired CS at high luminance at all but very low frequencies as well as impairment in CS across frequencies at low luminance. This monkey navigated her environment in an apparently normal manner, including in the large exercise cages she shared with others. From Rice, 1990.

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required a significant period of dark adaptation (rod vision), spatial vision was impaired across most frequencies (Rice & Gilbert, 1982, 1990c). Deficits in spatial visual function were also reported in children exposed to methylmercury in utero (Altmann et al., 1998) and adults exhibiting methylmercury poisoning (Mukuno et al., 1981). Motion detection is another important aspect of visual function. Humans sacrifice motion sensitivity in favor of spatial and color vision. Predatory species, such as domestic cats, have superior motion vision compared to humans, but cats have poorer spatial vision than do humans. Although cats have been used extensively to study the physiology of vision, they have been little used in toxicology. The eVect of developmental methylmercury or lead exposure on motion vision was also studied in monkeys using forced‐choice discrimination, with one screen displaying a sine wave flicker and the other an unflickering blank field. The behavioral paradigm was the same as that used for testing of spatial vision. Methylmercury exposure resulted in superior temporal vision under low luminance conditions (Rice & Gilbert, 1990c). This finding was interpreted as possibly resulting from selective remodeling of the visual system, with damage to the parvocellular system (important for high‐frequency spatial vision) allowing for expansion of the magnocellular system (important for detection of flicker). In contrast, developmental lead exposure produced no eVect on spatial vision but deficits in motion vision (Rice, 1998c). It was important to test several aspects of visual function to characterize or, in some cases, even to detect, deficits in visual function. One of the hallmarks of methylmercury poisoning in adults is constriction of visual fields (Takeuchi & Eto, 1999). This finding was replicated in adult macaque monkeys exposed chronically to methylmercury during adulthood (Merigan, 1980). Deficits in visual fields were not observed in monkeys in our laboratory as young adults, based on an assessment using a simple non‐ automated system. Monkeys fixated on a treat in the center of a cardboard circle, and treats were moved in from the sides until the monkey moved its gaze to the treats. This procedure tested only the binocular visual field, not the field for each eye, and was undoubtedly much less sensitive than automated systems designed to test visual fields. Nonetheless, some individuals exhibited mild constriction of visual fields during old age compared to age‐matched controls (Rice, 1996c). This suggests a delayed eVect of methylmercury many years after cessation of methylmercury exposure, and/or an interaction of previous methylmercury exposure and aging (Rice, 1996c). Only Old World monkeys, apes, and humans have a color discrimination system based on three receptor types, which are sensitive to red, green, or blue light. Our discrimination of color is based on ‘‘averaging’’ the input of these receptor types, which is why combinations of many diVerent

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frequencies can look like the same shade to us. Other monkeys may have only two receptor types, and other animals may have one or none. The most common defect of color vision in humans is red–green color blindness, which is typically caused by a genetic deficit that results in abnormal red or green receptors. In contrast, solvent exposure in adults produces a characteristic loss of blue–yellow color vision (Campagna et al., 2001), which is associated with damage to external retinal layers, or more extensive color vision loss, which may include damage to internal retinal layers and/or optical nerve (Geller & Hudnell, 1997). Color vision loss has been demonstrated in monkeys exposed to acrylamide (Merigan, 1989). Testing for color vision deficits predictive of those in humans would preferably be performed in animals with comparable color vision systems, particularly if the type of deficit is unknown. If human data suggest a particular type of color vision deficit that may be explored in other species, for example, New World monkeys with two receptor types, then those species may be used. Birds have very good color vision, and have long been used in the study of behavior and behavioral pharmacology. They have a tetrachromatic visual system, which uses oil droplet filters in the detection of various wavelengths. The diVerential eVect of a toxicant on mammals versus birds is diYcult to predict, making birds a less desirable model than primates for color vision research. However, birds have been used to study the eVects of drugs on color vision and other aspects of visual function (Bradley & Blough, 1993). B.

Audition

Unlike the visual system, the anatomy and physiology of the auditory system of mammals is similar across species. (As is true for the visual system, however, albino animals may have impaired auditory function.) Frequencies are detected individually by excitation of receptors at a specific location on the cochlear membrane. Rats and Old World monkeys can hear frequencies about twice as high as humans, with low‐ and mid‐frequency detection being similar. A simple test of audition is often included in screening tests of the neurotoxicity of chemicals using rats. The startle response to a loud noise is a test of the overall integrity of the nervous system, and may reveal deafness or serious hearing impairment as well as motor impairment. The response may be dependent upon the stimulus parameters (Marable & Maurissen, 2004). Often, the frequency or frequencies delivered and amplitude (loudness) are not well characterized. However, this basis approach can be used to determine hearing thresholds over a range of frequencies using well‐controlled stimulus presentation. A loud stimulus that would elicit a startle is preceded by a stimulus of the same frequency but lower amplitude.

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If the first stimulus is detected (heard), the animal does not startle, or the startle is attenuated. The threshold can be determined by varying the amplitude of the first stimulus. This ‘‘pre‐pulse inhibition’’ paradigm has been used to assess the ototoxicity of a variety of agents. For example, low‐ frequency hearing deficits were identified in rats developmentally exposed to a PCB congener (Crofton & Rice, 1999). A standard clinical test suitable for use in adults or children is pure‐tone audiology, the determination of the threshold for a number of frequencies. Pure tones are delivered through an earphone to each ear separately. Typically, a number of amplitudes are presented at each frequency, and the person indicates when a tone is detected. The lowest amplitude (loudness) detected is considered the threshold. Both developmental lead (Schwartz & Otto, 1987) and methylmercury (Murata, Araki, Yokoyama, Uchida, & Fujimura, 1993) exposure produce deficits in detection of pure tones in children. Monkeys have been used to examine the eVects of developmental exposure to lead and methylmercury on pure‐tone thresholds. The monkey sat in a primate chair with earphones, so that stimuli could be presented to each ear independently in a controlled manner. The monkey indicated detection of a tone by breaking contact with a metal bar (a signal detection task). This method of response allows determination of the latency to respond, which provides important information concerning behavioral control and threshold. (Latencies should increase close to threshold, and should be reasonably consistent across presentations of the same amplitude and frequency combinations.) Thresholds were determined in each ear over a number of frequencies. Monkeys exposed to methylmercury from birth to adulthood exhibited increased thresholds at high frequencies (Rice & Gilbert, 1992) (Fig. 8), as did monkeys exposed continuously to lead from birth (Rice, 1997b), whereas monkeys exposed pre‐ plus postnatally exhibited impaired hearing over a wide range of frequencies. Pure‐tone thresholds provide only basic, first‐level information concerning auditory function. An individual may have normal pure‐tone detection and still have diYculty distinguishing speech, for example. Speech is comprised of generally small but rapid changes in frequency and amplitude. It is technically straightforward to test frequency or amplitude ‘‘diVerence thresholds’’ (ability to detect changes in frequency or amplitude) in animals exposed to toxicants, although this has not typically been done. DiVerence thresholds for frequency were a more sensitive indicator of auditory impairment following aminoglycoside antibiotic exposure in monkeys (Stebbins, Clark, Pearson, & Weiland, 1973). On the other hand, amplitude diVerence thresholds were unimpaired in guinea pigs in the presence of deficits in pure tone thresholds (Prosen, Moody, Stebbins, & Hawkins, 1981).

FIG. 8. Thresholds for pure tones in each ear for control and five monkeys dosed with methylmercury from birth. Only monkey 34 had normal detection of pure tones. Other monkeys had varying degrees of middle‐ to high‐frequency hearing loss, and could not be tested at the higher frequencies tested in controls. Monkey 34 had the most impaired visual function of any in her methylmercury‐exposed cohort (Fig. 7). The results of these studies highlight individual sensitivity: in this case, sensitivity within a subject for impairment in two sensory systems. Individual sensitivity among subjects is routinely observed in toxicology experiments. From Rice & Gilbert, 1992.

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An alternative approach is to study the eVects of toxicants on language discrimination directly. A test that is used clinically to evaluate auditory (language) processing is the determination of the infant’s ability to distinguish ba and da and, at a later age, bi and di. Monkeys can also discriminate human speech sounds. In a study with rhesus monkeys, it was reported that developmental lead exposure impaired the ability of young monkeys to discriminate speech sounds (da and pa) using an electrophysiological procedure (Molfese et al., 1986). Lead‐exposed children are impaired on the Seashore Rhythm Test (Needleman et al., 1979), which requires the subject to discriminate whether pairs of tone sequences are the same or diVerent. Lead‐exposed children also have a decreased ability to identify words when frequencies were filtered out (i.e., when information was missing [Dietrich, Succop, Berger, & Keith, 1992]). Interestingly, rats are apparently also able to discriminate between human speech sounds using the same mechanisms used by humans (Reed, Howell, Sackin, Pizzimenti, & Rosen, 2003; Toro, Trobalon, & Sebastian‐Galles, 2005). This observation presents new possibilities for studying the eVects of toxicants on auditory processing using rodents. This would allow information of significant relevance to humans to be collected in an inexpensive and well‐studied toxicological model.

C.

Somatosensory Function

Somatosensory information is composed of several modalities—light touch, pressure, pain, temperature, vibration, and joint proprioception. These modalities are subserved by diVerent receptor types, and associated nerve fibers may be either myelinated or unmyelinated. DiVerent receptor or nerve types may be diVerentially sensitive to impairment by toxic agents. Many neurotoxicants produce impairment of somatosensory function in humans, which has been most completely documented in adults. Lead produces a peripheral neuropathy that is characterized by both motor and sensory impairment (Seppalainen, Hernberg, Vesanto, & Koch, 1983; Zimmermann‐Tansella, Campara, D’Andrea, Savonitto, & Tansella, 1983 ). A characteristic symptom of methylmercury poisoning is paresthesia in the distal extremities (‘‘stocking and glove’’ paresthesia), as well as perioral paresthesia (Tsubaki & Irukayama, 1977; WHO, 1990). In fact, a number of metals, in addition to solvents and pesticides, produce impairment of somatosensory function as a consequence of peripheral neuropathy (Rice, 1999b). Somatosensory function in humans is typically assessed clinically using crude procedures and uncontrolled stimuli (Rice, 1997c). This may include asking the individual whether she or he detects light touch, pinprick, or a

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tuning fork placed against the skin. These procedures may identify large deficits, but are not adequate to detect less severe changes in function. Screening tests in rodents may include relatively crude assessment of somatosensory function, including infliction of pain/pressure (pinching the feet with the fingers or forceps), pain (electric shock), or pain/heat (hot water). Reacting to the stimulus or drawing away from it is considered to indicate detection. As with other sensory system testing, assessment of subclinical impairment requires careful control of stimulus presentation and a well‐defined response. Probably the easiest stimulus to control is vibration. Depending on the frequency, vibration is detected by several types of end organs and nerve fibers, and so may be diVerentially aVected by neurotoxic exposure. Vibration sensitivity was assessed in monkeys exposed to lead beginning at birth, or exposed to methylmercury either postnatally or pre‐ plus postnatally (Rice & Gilbert, 1995). The monkey’s hand was positioned so that the tip of the middle finger made slight contact with a dull needle. The needle was vibrated, with the frequency and amplitude controlled precisely by computer. The monkey signaled detection of the stimulus by breaking contact with a bar with the other hand. Developmental lead exposure resulted in moderately elevated thresholds at higher frequencies, whereas methylmercury exposure produced severe impairment in some individuals. A similar technique was used to determine somatosensory impairment produced by acrylamide and misonidazole following adult exposure in monkeys (Maurissen, Weiss, & Davis, 1983; Maurissen et al., 1981). This procedure can be used in humans with little or no modification. It could also be modified for use with other species. In addition, other modalities could be tested using similar procedures.

D.

Olfaction and Taste

These senses receive little attention in developmental behavioral toxicity testing, so the degree to which they may be aVected is largely unknown. These senses may be impaired or have other abnormalities in syndromes such as autism, and may contribute to strange food preferences or aversions (P. Rodier, personal communication). The perception of the taste of food is largely dependent on olfaction; there are only five types of taste receptors (sweet, sour, bitter, salt, and umami). Olfaction is an important sense in rats and mice, as evidenced by the large olfactory bulb. An olfactory discrimination task may be used with rodent pups as a simple test of learning (Buelke‐Sam et al., 1985); the pup is required to find a location containing its home‐cage bedding. Olfactory ability could be assessed using test procedures

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similar to those used for other modalities. Testing requires adequate control of stimulus presentation, but that is true for all sensory system testing.

IV.

MOTOR FUNCTION

Motor function may somewhat arbitrarily be divided into gross movement, including postural control and stability, and fine motor function. Both may be aVected by exposure to a variety of chemicals as a result of occupational exposure, including metals, solvents, and pesticides. Motor development was assessed in infants in a number of epidemiological studies of the eVects of developmental exposure to neurotoxicants using an infant assessment battery, including methylmercury (Grandjean et al., 2001), PCBs (Huisman et al., 1995; Stewart, Reihman, Lonky, Darvill, & Pagano, 2000), and drugs of abuse (Morrow et al., 2001). Many studies included assessment of motor function in young children using the Bayley Scales of Infant Development, which includes tests of postural stability, and gross and fine motor function (e.g., Bellinger, Leviton, Needleman, Waternaux, & Rabinowitz, 1986). There are a number of procedures available in rodents for assessment of gross movement (Ossenkopp, Kavaliers, & Sanberg, 1996). Probably the one most often used in toxicology is the ability to stay on a ‘‘rotorod,’’ which is a cylinder that can be rotated at various speeds. The latency to fall from the rod is recorded. Additional tasks include climbing a rope, an inclined plane, or a screen. Swimming ability may also be assessed. Such tests would seem to be directly comparable to motor tests in children, and can be interpreted in a straightforward manner. For example, developmental PCB exposure in rats results in impairment on rotorod performance (Roegge et al., 2004) and methylmercury exposure produced deficits in swimming ability and posture (Spyker et al., 1972). Another common test used in rodents is grip strength, the measurement of the ability of the rodent to hold onto a rod being pulled away by an experimenter. The force required for the rodent to break its hold with the front or hind feet is determined. This is a semiquantitative measure of strength (and somatosensory function) that will probably detect moderate neuropathy, but is not a well‐controlled procedure and therefore would not be expected to detect small deficits. Another test of gross motor integrity is hindlimb foot splay. The rodent is dropped from a specific height, and the distance between the inked hind paws is measured. Grip strength, rotorod performance, and hind limb foot splay may be diVerentially sensitive to toxicant exposure (Gilbert & Maurissen, 1982; Youssef & Santi, 1997). Locomotion is also typically measured as part of a screening battery for developmental neurotoxicity in rodents. Locomotor activity may be

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considered an apical test, integrating the output of the entire nervous system (sensory, motor, arousal, cognitive, etc.). It may also be influenced by toxicity in other organ systems; if the animal feels ill, for example, motor activity will likely be aVected. Thus, locomotor activity is not a specific test, in that it may be aVected by many factors not directly related to nervous system toxicity. In addition, it provides little or no information concerning what behavioral domains may be aVected by a particular exposure. Locomotion may be recorded electronically using photocells, either in a rectangular area (‘‘open field’’) or in a ‘‘figure‐eight maze,’’ which is an intersecting alley shaped like a figure‐eight (Vorhees, 1985). An alternative method is to place the subject in an open field sectioned into grids and manually observe the number of grids entered. In addition to horizontal movement, rearing is often quantified separately. Total activity may also be measured using some kind of transducer under the cage containing the subject; this method will also record grooming, scratching, etc. Rodents typically engage in higher levels of both horizontal and rearing activity when first introduced into a novel environment, which is interpreted as exploratory behavior. Activity decreases over time; usually, there is a marked decrease in activity over an hour or so (Fig. 9). This is referred to as habituation, and

FIG. 9. Habituation of motor activity in normal rats across time from six diVerent testing laboratories using various measuring devices. Data are normalized to percentage of initial activity. Habituation was observed in all laboratories, despite marked diVerences in experimental conditions and total activity ‘‘counts.’’ From Crofton & MacPhail, 1996.

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is often considered to be indicative of cognition. Decreased habituation has been observed as a result of developmental exposure in mice to PCBs (Eriksson & Fredriksson, 1996) or polybrominated diphenyl ether (PBDE) flame retardants (Viberg, Fredriksson, & Eriksson, 2004). Normal rodents also spend the majority of their time near the sides of the enclosure rather than in the middle. All of these variables may be influenced by exposure to a toxicant. Any toxicant will aVect locomotor performance at some dose, because the animal is ill. This is also true for any neurotoxicant, which at some dose will produce central nervous system depression. Stimulants such as amphetamine produce increased locomotion at lower doses, and decreased locomotor behavior at high, toxic doses (Buelke‐Sam et al., 1985). This same pattern results from developmental exposure to lead in rodents (Rice et al., 1979). However, increased locomotion in rodents is not comparable to ‘‘hyperactivity’’ in children (attention deficit hyperactivity disorder or ADHD). This syndrome is characterized by increased impulsivity and attention problems, although increased activity may also be present. A DRH (diVerential reinforcement of high rate) schedule of reinforcement was used to assess performance in aging rats exposed developmentally to methylmercury (Newland & Rasmussen, 2000). The rats were required to respond on a lever a specified number of times within a short time period. As the treated rats aged, they were increasingly less able to perform the task. This may well represent motor impairment that was not apparent upon observation of the rats, although cognitive deficits may also be at least partially responsible. The eVects of prenatal lead exposure were studied on gross motor movement and strength during adulthood in monkeys (Newland, Yezhou, Logdberg, & Berlin, 1996). Monkeys were required to pull back a bar under load on either an FI schedule or a fixed ratio (FR) schedule, the latter engendering a high rate of response. Treated monkeys made fewer completed responses under the FR schedule but not the FI schedule, which generates a lower response rate. This suggests that eVects were due to motor impairment rather than some other factor, such as motivation. Postural stability may also be sensitive to exposure to developmental toxicants in children. Commercially available instruments measure several elements of postural sway under various experimental conditions. Lead exposure aVects a number of aspects of postural stability in children (Bhattacharya, Shukla, Bornschein, Dietrich, & Keith, 1990; Despre´ s et al., 2005). It would be diYcult to measure postural stability directly in four‐ footed animals, so that indirect evidence, such as impaired motor dexterity, is relied upon. A number of motor behaviors may be tested in preweaning

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rodents, for example, for which the ontogenetic development has been determined (see review by Rice & Barone, 2000). Fine motor control may also be sensitive to impairment by exposure to developmental neurotoxicants. Lead (Chiodo et al., 2004; Stiles & Bellinger, 1993) and methylmercury (Grandjean et al., 1997) aVect fine motor performance in children, for example. Fine motor control was assessed in monkeys exposed developmentally to methylmercury (Rice, 1996c). Monkeys retrieved fruit pieces from a series of compartments with varying depths. Methylmercury‐treated monkeys took longer to retrieve the fruit, and some had diYculty retrieving from the deeper compartments. In contrast, these monkeys did not exhibit deficits in gross arm movement, as measured by an automated reaction time experiment (Rice, 1996c). These monkeys also exhibited an intension tremor in their hands during old age (unpublished), which is characteristic of methylmercury poisoning. Since these monkeys had somatosensory impairment in their fingers, it is quite likely that the fine motor impairment was due, at least in part, to somatosensory deficits.

V.

CONCLUSIONS

There is a wealth of testing procedures for assessing cognitive, sensory, and motor function in animal models. These range from screening tests or batteries to very sophisticated and detailed assessment of functional abilities of the subject. Many of the same cognitive domains can be studied in animals as those that have been found sensitive to toxicant exposure in children: learning, memory, adaptability, attention, impulsivity, distractibility, and perseverative behavior. Motor function can be assessed in animal models by numerous techniques, and results are directly applicable to humans. The visual system of typical animal models (nocturnal rodents) lacks good color and spatial vision; therefore, important aspects of human vision are diYcult to assess. However, visual loss in rodents may nonetheless be predictive of loss in the same function in humans, and mechanisms of toxicity can be productively studied in rodents. For senses other than vision, the physiological processes of animals and humans are the same or at least very similar, such that deficits identified in animals are readily extrapolated to humans. It is important to be cognizant of certain caveats, however. Simple (screening) tests may be nonspecific or insensitive (or both). If the cognitive capability of the animal is not suYciently taxed, the test will be insensitive to all but gross impairment. Failure to assess a number of behavioral domains risks not detecting even relatively severe impairment. Lack of good stimulus

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control for sensory system assessment results in increased variability and therefore decreased sensitivity. Similarly, assessment of motor performance requires adequate control of both response requirement and motor response. Finally, contribution of dosing and dosing regimen, developmental period of exposure, and diVerences in metabolic capabilities between humans and animal models to outcome must be kept in mind, and understood as well as possible, in the interpretation of results from studies in animal models of developmental neurotoxicity of environmental chemicals.

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Maurissen, J. P. J., Conroy, P. J., Passalacqua, W., Von Burg, R., Weiss, B., & Sutherland, R. M. (1981). Somatosensory deficits in monkeys treated with misonidazole. Toxicology and Applied Pharmacology, 57, 119. Maurissen, J. P. J., Weiss, B., & Davis, H. T. (1983). Somatosensory thresholds in monkeys exposed to acrylamide. Toxicology and Applied Pharmacology, 71, 266. Merigan, W. H. (1980). Temporal modulation sensitivity of macaque monkeys. Vision Research, 20, 953–959. Merigan, W. H. (1989). Chromatic and achromatic vision of macaques: Role of the P pathway. Journal of Neuroscience, 9, 776–783. Molfese, D. L., Laughlin, N.K, Morse, P. A., Linnville, S. E., Wetzel, W. F., & Erwin, R. J. (1986). Neuroelectrical correlates of categorical perception for place of articulation in normal and lead‐treated rhesus monkeys. Journal of Clinical and Experimental Neuropsychology, 8, 680–696. Morrow, C. E., Bandstra, E. S., Anthony, J. C., Ofir, A. Y., Xue, L., & Reyes, M. L. (2001). Influence of prenatal cocaine exposure on full‐term infant neurobehavioral functioning. Neurotoxicology and Teratology, 23, 533–544. Mukuno, K., Ishakawa, S., & Okamura, R. (1981). Grating test of contrast sensitivity in patients with Minamata disease. British Journal of Ophthalmology, 65, 284–290. Murata, K., Araki, S., Yokoyama, K., Uchida, E., & Fujimura, Y. (1993). Assessment of central, peripheral, and autonomic nervous system functions in lead workers: Neuroelectrophysiological studies. Environmental Research, 61, 323–336. Needleman, H. L. (1987). Introduction: Biomarkers in neurodevelopmental toxicology. Environmental Health Perspectives, 74, 149–152. Needleman, H. L., Gunnoe, C., Leviton, A., Reed, R., Peresie, H., Maher, C., & Barrett, P. (1979). Deficits in psychologic and classroom performance of children with elevated dentine lead levels. New England Journal of Medicine, 300, 689–695. Newland, M. C., & Rasmussen, E. B. (2000). Aging unmasks adverse eVects of gestational exposure to methylmercury in rats. Neurotoxicology and Teratology, 22, 819–828. Newland, M. C., Reile, P. A., & Langston, J. L. (2004). Gestational exposure to methylmercury retards choice in transition in aging rats. Neurotoxicology and Teratology, 26, 179–194. Newland, M. C., Yezhou, S., Logdberg, B., & Berlin, M. (1994). Prolonged behavioral eVects of in utero exposure to lead or methyl mercury: Reduced sensitivity to changes in reinforcement contingencies during behavioral transitions and in steady state. Toxicology and Applied Pharmacology, 126, 6–15. Newland, M. C., Yezhou, S., Logdberg, B., & Berlin, M. (1996). In utero lead exposure in squirrel monkeys: Motor eVects seen with schedule‐controlled behavior. Neurotoxicology and Teratology, 18, 33–40. Organization for Economic Co‐Operation and Development (OECD) (1999). Organization for Economic Co‐Operation and Development TG 426—Developmental Neurotoxicity Study. OECD Environment Health and Safety Publications. Environment Directorate, Organization for Economic Co‐Operation and Development.. Oshiro, W. M., Krantz, Q. T., & Bushnell, P. J. (2001). Characterizing tolerance to trichloroethylene (TCE): EVects of repeated inhalation of TCE on performance of a signal detection task in rats. Neurotoxicology and Teratology, 23, 617–628. Ossenkopp, K.‐P., Kavaliers, M., & Sanberg, P. R. (Eds.) (1996). Measuring movement and locomotion: From invertebrates to humans. Austin, TX: R. G. Landes. Patandin, S., Veenstra, J., Mulder, P. G. H., Sewnaik, A., Sauer, P. J. J., & Weisglas‐Kuperus, N. (1999). Attention and activity in 42‐month‐old Dutch children with environmental exposure to polychlorinated biphenyls and dioxins. In S. Patandin (Ed.), EVects of

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International Review of

RESEARCH IN MENTAL RETARDATION EDITED BY

LARAINE MASTERS GLIDDEN

DEPARTMENT OF PSYCHOLOGY ST. MARY’S COLLEGE OF MARYLAND ST. MARY’S CITY, MARYLAND

Board of Associate Editors Philip Davidson UNIVERSITY OF ROCHESTER SCHOOL OF MEDICINE AND DENTISTRY

Elisabeth Dykens VANDERBILT UNIVERSITY

Michael Guralnick UNIVERSITY OF WASHINGTON

Richard Hastings UNIVERSITY OF WALES, BANGOR

Linda Hickson COLUMBIA UNIVERSITY

Connie Kasari UNIVERSITY OF CALIFORNIA, LOS ANGELES

William McIlvane E.K. SHRIVER CENTER

Glynis Murphy LANCASTER UNIVERSITY

Ted Nettelbeck UNIVERSITY OF ADELAIDE

Marsha M. Seltzer UNIVERSITY OF WISCONSIN-MADISON

Jan Wallander SOCIOMETRICS CORPORATION

Contributors

Numbers in parentheses indicate the pages on which the authors’ contributions begin.

Tara L. Arndt (171), Developmental of Environmental Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642 David C. Bellinger (263), Children’s Hospital Boston, Harvard Medical School and Harvard School of Public Health, Cambridge, Massachusetts 02115 Thomas M. Burbacher (1), Department of Environmental and Occupational Health Sciences, School of Public Health and Community Medicine, Washington National Primate Research Center and Center on Human Development and Disability, University of Washington, Seattle, Washington 98195 Deborah A. Cory-Slechta (87), Environmental and Occupational Health Sciences Institute, A Joint Institute of Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, and of Rutgers, the State University of New Jersey, Piscataway, New Jersey 08854 Philip W. Davidson (141), Strong Center for Developmental Disabilities, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642 Todd A. Florin (237), American Academy of Pediatrics, Center for Child Health Research and Department of Pediatrics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642 Kimberly S. Grant (1), Department of Environmental and Occupational Health Sciences, School of Public Health and ix

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Community Medicine, Washington National Primate Research Center and Center on Human Development and Disability, University of Washington, Seattle, Washington 98195 Susan L. Hyman (171), Department of Pediatrics (Golisano Children’s Hospital at Strong), University of Rochester School of Medicine and Dentistry, Rochester, New York 14642 Gary J. Myers (141), Departments of Neurology and Pediatrics, University of Rochester, School of Medicine and Dentistry, Rochester, New York 14642 Megan Kavanaugh (237), American Academy of Pediatrics, Center for Child Health Research and Department of Pediatrics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642 Herbert L. Needleman (225), School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15213 Deborah C. Rice (301), Environmental and Occupational Health Program, Department of Health and Human Services, Maine Center for Disease Control and Prevention, Augusta, Maine 04333 Patricia M. Rodier (171), Department of Obstetrics and Gynecology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642 Conrad F. Shamlaye (141), Ministry of Health, Victoria, Mahe, Republic of Seychelles Hestien J. I. Vreugdenhil (47), Department of Pediatrics, Division of Neonatology, Erasmus MC–Sophia Children’s Hospital, University Medical Center, 3015 GJ Rotterdam, The Netherlands Nynke Weisglas-Kuperus (47), Department of Pediatrics, Division of Neonatology, Erasmus MC–Sophia Children’s Hospital, University Medical Center, 3015 GJ Rotterdam, The Netherlands Bernard Weiss (195), Department of Environmental Medicine, University of Rochester Medical Center, Rochester, New York 14642 Michael Weitzman (237), American Academy of Pediatrics, Center for Child Health Research and Department of Pediatrics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642

Foreword

In April 2005, the American Psychologist published a review article entitled Environmental Toxicants and Developmental Disabilities coauthored by Susan Koger, Ted Schettler, and Bernard Weiss. It is no coincidence that Bernard Weiss is one of the three guest editors of the current special theme volume on Neurotoxicity and Developmental Disabilities, and that the other two guest editors, Phil Davidson and Gary Myers, are cited extensively in the article as are many of the other contributors to this volume of the International Review. This volume is timely in its publication and it represents up‐to‐date findings summarized by the leading researchers in the field. Phil Davidson and his co‐ editors have done a masterful job in assembling them and putting together a comprehensive overview of neurotoxicity and developmental disabilities. Steve Schroeder, one of the pioneers in this field, calls this volume seminal in his Foreword. I second that judgment. Not only is the research seminal, it is also of extraordinary practical importance to the lives of children and adults around the world. Koger, Schettler, and Weiss (2005) describe a cumulative risk model in which exposure to neurotoxicants combines with poverty, inadequate schooling, poor prenatal care, and other risk factors to add to the percentage of decline in intellectual performance of exposed individuals. In the United States, neighborhood violence is likely to be part of this model, and in some other countries violence from war, exposure to infectious diseases, and malnutrition can be added to the list. To the extent that we can understand and reduce or eliminate any of these risks, we improve quality of life for the individual and for society. Phil Davidson and his co-editors have done a materful job in getting together to provide a comprehensive overview of neurotoxicity and developmental disorders. LARAINE MASTERS GLIDDEN SERIES EDITOR xi

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REFERENCE Koger, S. M., Schettler, T., & Weiss, B. (2005). Environmental toxicants and developmental disabilities. American Psychologist, 60, 243–255.

Foreword

This volume includes the work of some of the pioneers and best thinkers of the field of neurobehavioral toxicology related to developmental disabilities. It is a seminal work that should be on the shelf of every serious researcher and student of developmental disabilities. In 1975, Bernard Weiss (Weiss and Laties, 1975) edited one of the first volumes on behavioral toxicology, which gave birth to a new sub‐field in toxicology. This was a significant change because it pointed to behavior, in addition to organ or cellular or molecular functions, as an end‐point in the study of toxicity. In 1979, Needleman et al. (1979) published a paper in the New England Journal of Medicine on the eVects of low‐level lead exposure on the IQ and attention deficits of school children in Chelsea and Sommerville, both boroughs of Boston. Although this was not the first paper on the topic, it contained sophisticated methodology that set the gold standard for neurotoxicity studies for decades to come. The bar had been raised, and neurobehavioral toxicology became a respectable scientific discipline. In 1987, I edited a monograph for the AAMR Monograph Series on Toxic Substances and Mental Retardation (Schroeder, 1987). The field of research in mental retardation and developmental disabilities had hardly been aware of the burgeoning field of neurotoxicology. In 1990, two papers, one by me and one by Gary Myers, were published in this International Review series on methodological issues in the study of the eVects of heavy metals on mental retardation (Myers and Marsh, 1990; Schroeder, 1990). In 1997, a special issue of MRDD Research Reviews, edited by Phil Davidson et al. (1997) was published, entitled, ‘‘Neurotoxins and Developmental Disabilities.’’ In 2000, Environmental Health Perspectives (Schroeder, 2000) published a supplement on neurotoxicity and developmental disabilities in children. All of the above papers and volumes attempted to merge the field of MRDD research

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with research in neurotoxicity. Were they successful? In my opinion, the answer is, ‘‘not very.’’ Why? In my opinion, one of the main reasons that we have not had more truly interdisciplinary research on neurotoxicity is that these fields have divided the responsibility between several competing government agencies, advocacy groups from industry, environmental advocacy groups, and other fragments of the Social Policy Establishment, but have not suYciently consulted the people most aVected, i.e., parents and families with MRDD. While all of these groups are important and necessary, none by itself is suYcient to get the job done. Government agencies like NIEHS and EPA have focused on high quality basic science research on neurotoxicity, looking for the basic mechanism for some neurotoxin, developing ever more sophisticated methods for assessing its eVects in animal models and in humans, controlling for confounding factors, and alternative explanations. Often the studies have over‐controlled for confounds, to the point that their results are of marginal relevance to the target population. Why? Because these variables are rarely controlled well in the real world. The important variables may be multiple, having both proximal and distal eVects, immediate and delayed, as has been well discussed by Weiss (Weiss and Reuhl, 1994). Bellinger (Chapter 9) discusses neurobehavioral measures as indices of risk. The development of neurobehavioral test batteries to measure neurotoxic eVects in humans and animals has become a growth industry. But how eVective are such shotgun approaches in matching their measures to the specific behavioral phenotype of a particular toxin? Few have aimed at studying the underlying neural mechanisms of such specific toxic eVects. The work of Cory‐Slechta and Rice in the current volume (Chapters 3, 10) are notable exceptions. More work of this sort is needed to translate the risk of impairment of basic internal indices of neural functions into external indices of environmental risk. The current risk models used by environmental regulatory agencies tend to focus on threshold eVects and dose response functions of certain neurotoxins and on setting and enforcing environmental regulatory standards. Yet we know that many environmental neurotoxins have no threshold or hockey stick function. The environmental regulatory approach is important but not suYcient. Why? For the simple reason that over half of the populations of the earth in Asia, Africa, and the Far East have almost totally ignored environmental pollution control in their economic development plans. Having just spent two years in the Middle East and having attended several such conferences, I can personally attest that this is the case. Yet environmental pollution is a global problem. There will always be a rapacious industrialist lobby trying to

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influence politicians to roll back standards, just as has occurred in the U.S. in recent Bush Administration. The emphasis of government agencies needs to be not only on detection and regulation of infractions, but also on prevention, and health promotion. All of them are necessary. Fortunately, it appears that the Centers for Disease Control and Prevention is refocusing its mandate on detection, prevention, and health promotion. Health promotion and disease prevention has been a public health mantra in the U.S. for many years. Unfortunately, it has had a limited eVect because the focus has been only on health education rather than upon inducing environmental change. Fortunately, new technologies have been developed to prioritize and measure environmental changes in communities (CDC, 2002; Rootman et al. 2001). This is a tall order, but a necessary one. Where did the development of the technology for measurement of environmental concerns and of environmental change come from? You guessed it, from research on the disability movement over the past 30 years. This should be a lesson to us. The link between developmental disabilities and environmental neurotoxicity research can come from the partnership between researchers, the consumers and families aVected, and the Social Policy Establishment. In 1975 there was no national Education of All the Handicapped Act or Birth‐to‐Three Program for infants and children at risk for disability. There is now, thanks to the partnership between parents, professionals, and families joining together to pass these laws. The same movement can join to focus the attention of our country on the most serious threat to our children ever. Sound basic science, relevant translational research, and a strong social policy commitment in partnership with consumers and families can do it. We must. We have no alternative. STEPHEN R. SCHROEDER UNIVERSITY OF KANSAS REFERENCES CDC (U. S. Centers for Disease Control and Prevention) (2002). Syndemics overview: What procedures are available for planning and evaluating initiatives to prevent syndemics? The National Center for Chronic Disease Prevention and Health Promotion Syndemics Prevention Network. Available online at www.cdc.goc/syndemics. Davidson, P. W., Myers, G. J., & Schroeder, S. R. (Eds.) (1997). Environmental Toxins and Developmental Disabilities. Mental Retardation and Developmental Disabilities Research Reviews. (Vol. 3, pp. 221–278). Myers, G. J., & Marsh, D. O. (1990). The role of methyl mercury toxicity in mental retardation. In N. W. Bray (Ed.), International Review of Research in Mental Retardation. (Vol. 16, pp. 33–50). New York: Academic Press.

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Needleman, H., Gunnoe, C., Leviton, A., et al. (1979). Deficits in psychologic and classroom performance of children with elevated dentine levels. New England Journal of Medicine, 300, 689–695. Rootman, I., Goodstadt, M., Hyndman, B., McQueen, D., Potvin, L., Springett, J., & Ziglio, E. (Eds.) (2001). Evaluation in health promotion: Principles and perspectives (pp. 241–270). Copenhagen, Denmark: World Health Organization – Europe. Schroeder, S. R. (Ed.) (1987). Toxic substances and mental retardation: Neurobehavioral toxicology and teratology. Washington, DC: AAMD Monograph Series No. 7. Schroeder, S. R. (1990). Methodological issues in specifying neurotoxic risk factors for developmental delay: Lead and cadmium as prototypes. In N. Bray (Ed.), International review of research in mental retardation. (Vol. 18, pp. 1–31). New York: Academic. Schroeder, S. R. (2000). Mental retardation and developmental disabilities influenced by neurotoxic insults. Environmental Health Perspectives, 108(Suppl. 3), 395–399. Weiss, B., & Laties, V. G. (1975). Behavioral toxicology. New York: Plenum Press. Weiss, B., & Reuhl, K. (1994). Delayed neurotoxicity: A silent toxicity. In I. W. Chang (Ed.), Principles of Neurotoxicology. (pp. 765–781). New York: Dekker.

Preface

It has been some time since any review has addressed the coupling between neurotoxicity and mental retardation. Schroeder’s Foreword summarizes previous reviews and argues for an updated, comprehensive survey. We underook this volume to achieve such a goal. Our introduction will emphasize several important points. First, the topic is very complex. Many variables influence developmental outcomes following exposures to neurotoxicants. Developmental eVects depend not only on the particular neurotoxicants, but also on the timing of the exposure; e.g., during gestation (and when), during early childhood, later in childhood, during adolescence, or even during adulthood. The pattern of exposure is critical; was it chronic, intermittent, or acute? Dose is fundamental to toxicity, of course. But dose is not necessarily directly related to response magnitude or character; nonlinear dose-response relationships are common, and may reflect compensatory mechanisms at higher exposure levels that are not provoked at lower doses. Additional complications arise because exposures rarely occur in isolation. For example, fish consumption has been linked by some research to adverse developmental outcomes attributed to their methylmercury content (see Myers for a review). But many aquatic environments are contaminated with chemicals or prey species that introduce other neurotoxicants, making it diYcult to scientifically untangle the specific contributions of particular chemicals to developmental outcomes. Moreover, environmental conditions other than chemicals may modify developmental risks. Such outcomes have been called eVect modification by Bellinger and are reviewed in Chapter 9. A significant issue is the nature of the relationship between dose and response and how it is expressed. High doses of neurotoxicants may inflict devastating damage to the CNS. High dose exposures have typically resulted from poisoning episodes and have led to severe mental retardation and other developmental disabilities in both individual cases and in populations.

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Lower exposure levels, such as those resulting from longterm exposure to neurotoxicants widely distributed in the environment, more typically cause more subtle, mild deficits of far less severity than mental retardation. These deficits may have minimal adverse clinical consequences for any exposed individual, but may shift the distribution of aVected traits, such as IQ, in a population (Weiss, Bellinger). Such reasoning and data have driven regulatory decisions in both toxicology and clinical medicine. Third, most of the literature addressing the consequences of neurotoxic exposures is focused on adverse developmental eVects. There is no research to document the consequences of exposures to neurotoxicants among persons who already suVer from mental retardation. People with mental retardation secondary to congenital chromosomal or genetic abnormalities may have a higher risk of adverse exposure eVects. Too little is known about such risks. Fourth, we have almost no information regarding adverse developmental consequences of exposures to numerous chemical substances common to our environment. We know a great deal about the neurotoxicity of lead (Cory-Slechta), Mercury (Myers, Davidson, and Weiss), PCBs (Vreugdenhil and Weisglas-Kuperus), and ethanol (Burbacher). But data are rare or absent concerning many substances that are ubiquitous in the environment, such as pthalates (Weiss), methanol (Burbacher and Grant), pesticides (Needleman) and tobacco (Weitzman, Kavanaugh, and Florin), especially in humans. Much more work is needed to determine the degree to which these substances pose developmental risks. Moreover, as Cory-Schlecta points out for lead, even for those neurotoxicants that have been investigated extensively, new data often emerge to change our estimates of the risks posed by exposure. Human epidemiological studies are very expensive, time-consuming, and subject to many limitations. Animal studies oVer an appeal to circumvent some of these problems. But, as Rice points out, few parallels can be drawn directly between human and animal data, in large part due to a lack of correspondence between the doses chosen for experimental studies and those prevailing in the environment. The lack of concordant outcome measures amplifies the discrepancies. Too many experimental studies sacrifice the possibility of extrapolation to simply demonstrate toxic potential. Among the most intriguing of the reviews in this volume is the review of environmental exposures and autism spectrum disorders by Hyman and Rodier. Their work highlights the principle that the consequences of neurotoxic exposures during early development are seen in diVerent guises at diVerent development stages and, in fact, that such consequences of an exposure could remain silent until later in life. Developmental neurotoxicology is an emerging discipline. The heightened public awareness of exposure to toxic substances in the environment should induce our funding agencies to accelerate support for research to identify

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and quantify risks. Many years and large sums of money are needed to clarify those risks associated with just the limited collection of substances now under study, but thousands of chemicals thought to possess neurotoxic eVects remain unexplored (Weiss and Landrigan, 2000). This book was conceived to advance that agenda. PHILIP W. DAVIDSON GARY J. MYERS BERNARD WEISS UNIVERSITY OF ROCHESTER SCHOOL OF MEDICINE AND DENTISTRY REFERENCES Weiss, B., and Landrigan, P. J. (2000). The developing brain and the environment: An introduction. Environ Health Perspect, 108(Suppl 3), 373–374.

Index

A Abstinence in alcohol/neurodevelopment, 35 during pregnancy, 17–18 Acetylcholine (ACh), pesticide neurotoxicity and, 229–230 Acetylcholinesterase (AChE), pesticide neurotoxicity and, 229–232, 234 ADHD. See Attention-deficit hyperactive disorder Adrenocorticotropic hormone (ACTH), in lead exposure/stress, 93–94, 96 ADS. See Alcohol Dependency Scale Adult stress responsivity, in lead exposure/ stress, 115–119 Adverse behavior, maternal smoking outcomes and, 245–246 Adverse eVects pathway, in parental smoking, 240–242 Ah. See Aryl-hydrocarbon Alcohol, in autism, 188 Alcohol Dependency Scale (ADS), in alcohol/ neurodevelopmental eVects, 15 Alcohol, neurodevelopmental eVects Alcohol Dependency Scale in, 15 brain injury in, 25–26 apoptosis and, 26 FAE and, 25–26 FAS and, 25–26 GABA and, 26 MRI studies and, 25 EtOH and, 1, 2, 3–26 historical perspective of, 3, 4 metabolism of, 3 FAS and, 5, 6–7 ARBD in, 7, 8 ARND in, 7, 8–9 339

clinical features of, 5–9 CNS impairment in, 5–6 diagnostic criteria in, 6–7 FASD in, 8–9 growth deficiencies in, 5–6 MeOH in, 1, 2, 3, 26–34 from aspartame, 30 environment and, 26–27 Fagan test for, 32 infants and, 28–29 LOAEL and, 29 metabolism of, 26–28 pregnancy and, 32–34 primate models and, 31–34 rodent models and, 29–30 test batteries for, 31, 33 toxicity of, 26–27 in pregnancy/EtOH, FAE and, 8, 17 pregnancy/EtOH and, 9–24 abstinence during, 17–18 ADS in, 15 Bayley Scales of Infant Development in, 11–12, 13, 15, 16, 18, 21 binge drinking in, 10, 14, 21 birthweight in, 11–12 BNAS in, 13, 17, 19 consumption patterns in, 9–10, 12, 19–20 dysmorphias in, 18–19, 20, 36–37 exposure timing in, 10–11, 20–22 FAE and, 8, 17 FAS and, 10, 12, 20, 22–23 IQ tests in, 13–14, 19, 21, 23 MAST scores in, 19 maternal impairment in, 16–17 neurobehavioral profiles in, 10–11, 11–24 postnatal testing in, 13–15, 15–16, 21–23 prenatal exposure in, 20, 23–24 smoking in, 13, 20

340 Alcohol, neurodevelopmental eVects (cont.) vigilance tests and, 14 pregnancy/MeOH in behavioral tests and, 30 MeOH exposure in, 28, 30 rodent models, 29–30 properties of, 2 risk evaluation in, 34–37 abstinence in, 35 dose dependency in, 34–35 drinking patterns in, 35 recommendations for, 35–37 risks of, 1–3 gender diVerences in, 3–4 prenatal exposure in, 3–4, 9–24 Alcohol-related birth defects (ARBD), in alcohol/neurodevelopment, 7, 8 Alcohol-related neurodevelopmental disorder (ARND), in alcohol/ neurodevelopment, 7, 8–9 Altered HPA axis, in lead exposure/stress, 123–124 American Heart Association (AHA), in methyl mercury risk assessment, 144, 161–162, 163 Animal behavior studies, PCBs/dioxins in, 55, 58, 61 Animal models in models/constructs, 302–303 in neurobehavioral assessment, 271 parental smoking and, 243–244, 251 Antisocial behavior, maternal smoking and, 247–248 Apoptosis, in brain injury, 26 ARBD. See Alcohol-related birth defects ARND. See Alcohol-related neurodevelopmental disorder Aryl-hydrocarbon (Ah) receptors endocrine disruptors and, 202–203, 211 PCBs/dioxins and, 48–49 ASD. See Autism Spectrum of Disorders Aspartame, in alcohol/neurodevelopment, 30 Attention, in models/constructs, 309–311 Attention deficit, in lead exposure/stress, 124–125 Attention-deficit hyperactive disorder (ADHD) maternal smoking and, 239, 240, 244, 245, 250, 252

index in neurobehavioral assessment, 279, 280, 283, 288 Audition, models/constructs and, 321–324 Autism Spectrum of Disorders (ASD), 188 autism/environmental agents and, 171, 182, 183, 184, 188 in neurobehavioral assessment, 283 Autism/environmental agents, 171–172 ASD and, 171, 182, 183, 184 etiologies of, 173–188 alcohol and, 188 antibodies to neural proteins and, 176–177 bacterial colonic overgrowth and, 177–178 birth seasonality and, 177–178 borna virus and, 177 brain anomalies and, 174 Collaborative Perinatal Project and, 173–174, 175 DSM-IV and, 180, 181 endoscopy and, 179 environmental investigation and, 180–181 examination of, 179–180 herpes virus and, 177 HOXA1 gene and, 186 iatrogenic medical interventions and, 182–184 maternal infections and, 177 mercury-containing vaccines and, 183–184, 185 metabolic imbalance and, 178–179 misoprostol and, 186–187 MMR vaccine and, 182–183 Moebius syndrome, 187 obstetric complications and, 173–174, 175 PDD in, 180, 182 PKU and, 178–179 prenatal infections and, 176–178 prevalence v. epidemic in, 180–181 rubella and, 176 teratogens and, 184–188 thalidomide and, 184, 186 valproic acid and, 186–187 yeast colonic overgrowth and, 179 family environment in, 172–173 genetics v., 171

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341

internal/external environment interfaces in, 188–189 ASDs and, 188 folic acid and, 188 HOXA1 gene and, 189 linkage studies and, 188 spina bifida and, 188

B Bacterial colonic overgrowth, in autism/ environmental agents, 177–178 BAER. See Brainstem auditory evoked responses Basal corticosterone, in lead exposure/stress, 107, 108, 111, 112, 113 Bayley Scales/infant development in alcohol/neurodevelopment, 11–12, 13, 15, 16, 18, 21 cognitive deficit studies and, 96 in methyl mercury exposure, 154 Behavioral outcomes, PCBs/dioxins and, 204–209, 211 Behavioral tests, pregnancy/MeOH and, 30 Benchmark Determination/lowest (BDML), in methyl mercury exposure, 159 Benchmark Dose (BMD), in PCBs/dioxins, 206, 207, 209–210 Benchmarks, in methyl mercury exposure, 157, 158 Binge drinking, in alcohol/neurodevelopment, 10, 14, 21 Biomarkers in methyl mercury exposure, 157–158 in neurobehavioral assessment, 277 Birth seasonality, in autism/environmental agents, 177–178 Birthweight. See also Low birth weight pregnancy/EtOH and, 11–12 Bisphenol A (BPA) in endocrine disruptors, 212–217 sources of, 212 Blood lead (PbB) levels. See also Lead exposure/stress interactions in lead exposure/stress, 88–89, 91, 98, 100, 101–102 BMD. See Benchmark Dose BNAS. See Brazelton Neonatal Assessment Scale

BNT. See Boston Naming Test Borna virus, in autism/environmental agents, 177 Boston Naming Test (BNT), in methyl mercury exposure, 159 BPA. See Bisphenol A Brain anomalies, in autism/environmental agents, 174 Brain growth, in utero, parental smoking and, 242, 244 Brain injury, in alcohol/neurodevelopmental eVects, 25–26 Brain-behavior relationships, in neurobehavioral assessment, 287–292 Brainstem auditory evoked responses (BAER), in methyl mercury exposure, 155 Brazelton Neonatal Assessment Scale (BNAS), pregnancy/EtOH and, 13, 17, 19 Breast-feeding PCBs/dioxins and, 51, 53–54, 64–65, 74–76 risk assessment and, 58–59, 62 safety considerations and, 72–73

C Carbamates, pesticide neurotoxicity and, 231–232, 233–234 CBCL. See Child Behavior Check List CCD (Center for Communicable Diseases) definition, of developmental disability, 160 Central nervous system in lead exposure/stress interactions, 88, 94, 96 of PCBs/dioxins, 49–50 Central nervous system impairment, in FAS, 5–6 Characteristics distribution change, in neurobehavioral assessment, 284–287 Child behavior, parental smoking and, 245–246 Child Behavior Check List (CBCL), parental smoking and, 247, 250 Chlorpyrifos (CPF), pesticide neurotoxicity and, 230–231, 233 Clinical features, of FAS, 5–9

index

342 Clinical v. research data collection, in neurobehavioral assessment, 264–266 CNS. See Central nervous system Cognition tests, models/constructs and, 303–317 Cognitive deficit studies, in lead exposure/ stress, 89, 90, 96–100 Cognitive function, in lead exposure/ stress, 123 Cognitive impairments, parental smoking and, 248–249 Cognitive/motor abilities, PCBs/dioxins and, 65–66, 66–67, 74 Cognitive/motor development, in PCBs/ dioxins studies, 61, 63–64 Collaborative Perinatal Project, in autism/ environmental agents, 173–174, 175 Color detection, models/constructs and, 320–321 Concurrent schedules, in cognition tests, 307 Confounding bias, in neurobehavioral assessment, 273–276 Congeners, in PCBs/dioxins, 47 Consumption patterns, pregnancy/EtOH and, 9–10, 12, 19–20 Continuous Performance Test (CPT), in methyl mercury exposure, 159 Corticosterone levels in lead exposure/stress, 120 stress-induced, 115–117, 119 Cortisol, in lead exposure/stress, 92–95 CPF. See Chlorpyrifos CPT. See Continuous Performance Test

DiVerential reinforcement of high rate (DRH) tasks, in models/constructs, 328 DiVerential reinforcement of low rate (DRL) tasks in models/constructs, 312–313 in PCBs/dioxins, 207–209 Direct dose response, in parental smoking, 241 Discrimination learning, in cognition tests, 304–305 Discrimination reversal, in cognition tests, 305–306 Distractibility, in models/constructs, 311 Domain ranges, in neurobehavioral assessment, 266–267, 271–272 Dopamine, PCBs/dioxins and, 50, 55 Dopamine terminals, in lead exposure/stress, 103–104 Dose dependency, in risk evaluation, 34–35 Dosing regimen, in models/constructs, 330 DRH. See DiVerential reinforcement of high rate tasks Drinking patterns, in risk evaluation, 35 DRL. See DiVerential reinforcement of low rate tasks DSM-IV, in autism/environmental agents, 180, 181 Dynamic nosology, in neurobehavioral assessment, 282–284 Dysmorphias, in alcohol/neurodevelopment, 18–19, 20, 36–37

E D Data analysis, in neurobehavioral assessment, 272–279 Data collection, in neurobehavioral assessment, 264–272 Data interpretation, in neurobehavioral assessment, 279–293 Decrement meaning, in neurobehavioral assessment, 279–280 Default assumptions, in neurobehavioral assessment, 277 Developmental disability, in methyl mercury exposure, 160–161 Diagnostic criteria, FAS and, 6–7

EDC. See Endocrine disrupting chemicals EDSTAC. See Endocrine Disruptor Screening and Testing Advisory Committee EVect modification, in neurobehavioral assessment, 276–279 Endocrine disrupting chemicals (EDC), in endocrine disruptors, 196, 198, 204, 213–214 Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC), neurobehavioral toxicity and, 201 Endocrine disruptors, in mental retardation BPA in, 212–217 LOAELs and, 212–213, 217

index neurobehavioral outcomes and, 213–217 RfDs and, 213, 214, 215 sexual eVects of, 212, 213–214, 215, 216 social behaviors and, 214 sources of, 212 EDCs and, 196, 198, 204, 213–214 glucocortoids and, 197 HPA axis and, 197 neurobehavioral toxicity and, 198–201 EDSTAC and, 201 IQ distributions in, 200, 201 PCBs in, 198 sexual diVerentiation of brain in, 199–200, 208 NHANES and, 195–196 PCBs/dioxins in, 197, 201–211 AhR and, 202–203, 211 behavioral eVects of, 204–209, 211 BMDs and, 206, 207, 209–210 DRLs and, 207–209 environmental exposures, 201, 203 FRs and, 205–206, 207–209 morphological eVects of, 210–211 PCDDs and, 202, 203 reproductive abnormalities and, 203, 205 RfDs and, 206 SCOB studies and, 204–205, 207 TCDD and, 202–206, 208, 209, 210, 211 treadmill studies and, 205, 206 persistent organic pollutants and, 195, 196–197 phthalate esters in, 217–220 sexual eVects of, 217–218 social behaviors and, 219–220 phthalates, 197, 198 properties of, 196–197 reproductive system and, 197 sources of, 197, 198, 212 Endoscopy, autism/environmental agents and, 179 Environment, in alcohol/neurodevelopment, 26–27 Environment interfaces, in autism/ environmental agents, 188–189 Environmental exposures, in PCBs/dioxins, 76, 201, 203 Environmental neurotoxicants, in neurobehavioral assessment, 263–264, 271

343 Epidemiological studies, on parental smoking, 244–245 Ethanol (EtOH) alcohol/neurodevelopment and, 1, 2, 3–26 historical perspective of, 3 metabolism of, 3 Etiologies, in autism/environmental agents, 173–188 EtOH. See Ethanol Executive functions, in cognition tests, 303 Experimental methods, in lead exposure/ stress, 103–107 Exposure levels in PCBs/dioxins studies, 60 pesticide neurotoxicity and, 226–228 Exposure measurements, PCBs/dioxins and, 65 Exposure timing, in pregnancy/EtOH, 10–11, 20–22

F FAE. See Fetal alcohol eVects Faeroe Islands study, in methyl mercury exposure, 154–155 Fagan test, in alcohol/neurodevelopment, 32 Family environment, in autism/environmental agents, 172–173 FAS. See Fetal alcohol syndrome FASD. See Fetal alcohol spectrum disorder Fetal alcohol eVects (FAE) in alcohol/neurodevelopment, 8, 17, 25–26 pregnancy/EtOH and, 8, 17 Fetal alcohol spectrum disorder (FASD), in alcohol/neurodevelopment, 8–9 Fetal alcohol syndrome (FAS) alcohol/neurodevelopment and, 5, 6–7, 25–26 pregnancy/EtOH and, 10, 12, 20, 22–23 Fetus, parental smoking and, 241 FI. See Fixed interval Fish consumption, in methyl mercury exposure, 150–156 Fixed interval (FI) schedules, in models/ constructs, 313–314, 315 Fixed interval (FI) testing, in lead exposure/ stress, 106, 109, 111, 112, 113, 115–117, 118–119

index

344 Fixed-ratio (FR) schedules, in endocrine disruptors, 205–206, 207–209 Folic acid, in autism/environmental agents, 188 Food chains, in methyl mercury exposure, 143–145 Food Quality Protection Act, pesticide neurotoxicity and, 228–229 FR. See Fixed-ratio schedules Functional domains, models/constructs and, 301–302

G Gamma-aminobutyric acid (GABA), alcohol/ neurodevelopment and, 26 Generic testing batteries, in neurobehavioral assessment, 264, 265–266, 269 Genetic polymorphisms, in neurobehavioral assessment, 278–279 Genetics in autism/environmental agents, 171 maternal smoking and, 239–240 Gestation period, maternal smoking and, 243 Glucocorticoids endocrine disruptors and, 197 in lead exposure/stress, 93–95 Go-no go tests, models/constructs and, 310 Growth deficiencies, in FAS, 5–6

H Habituation, models/constructs and, 327–328 Health screening, in lead exposure/stress, 120–122 Herpes virus, in autism/environmental agents, 177 Hierarchical data analysis, in neurobehavioral assessment, 272 HOME (Home Observation of Measurement of the Environment) scores maternal smoking and, 250 in methyl mercury exposure, 153–154 PCBs/dioxins and, 66–67 HOXA1 gene, in autism/environmental agents, 186, 189 HPA. See Hypothalamic pituitary adrenal axis

Human exposure accidental, 52–53 children and, 51–52, 54 environmental, 53–54 food contamination and, 51–52 neurodevelopment and, 54–55, 56–57 in PCBs/dioxins, 51–55 perinatal, 52–55 pesticide neurotoxicity and, 232–233 postnatal testing and, 51–52, 54 pregnancy and, 51–52 Hypothalamic pituitary adrenal (HPA) axis in endocrine disruptors, 197 in lead exposure/stress, 93–95, 96, 101 Hypothesis development, in methyl mercury exposure, 149–150

I Iatrogenic medical interventions, in autism/ environmental agents, 182–184 Impulsivity, in models/constructs, 311–314 Inclusion/exclusion criteria, in PCBs/dioxins studies, 64–65 Infants alcohol/neurodevelopment and, 28–29 in neurobehavioral assessment, 270–272 Intelligence v. domain-focused tests, in neurobehavioral assessment, 268–269 Interpretation, in models/constructs, 302 IQ distributions, endocrine disruptors and, 200, 201 IQ scores in lead exposure/stress, 98 in maternal smoking, 252, 253 in neurobehavioral assessment, 272–273, 275, 279–281, 285–286 in PCBs/dioxins studies, 61, 66, 73–74 IQ studies, in lead exposure/stress, 88 IQ tests, in alcohol/neurodevelopment, 13–14, 19, 21, 23

L Lead exposure/stress interactions, 87–127 CNS and, 88, 94, 96 cognitive deficit studies and, 89, 90, 96–100 ACTH in, 96

index Bayley scales, 96 HPA axis and, 96 IQ scores in, 98 MRI studies in, 97 PbB levels in, 98, 100 RL component in, 98–100 cognitive/executive functions in, 101 experimental methods in, 103–107 adult stress responsivity and, 115–119 basal corticosterone and, 107, 108, 111, 112, 113 corticosterone/stress-induced and, 115–117, 119 dopamine terminals and, 103–104 female oVspring results and, 111–115 FI testing and, 106, 109, 111, 112, 113, 115–117, 118–119 male oVspring results and, 107–111 multiple-hit hypothesis and, 104 NAC dopamine and, 109–110, 111 neurotransmitters and, 114–115 future research in, 122–127 altered HPA axis in, 123–124 attention deficit in, 124–125 cognitive function in, 123 maternal Pb screening in, 125 multi-hit hypothesis in, 126–127 parameters in, 122–123 risk modifiers in, 126, 127 health screening in, 120–122 corticosterone levels and, 120 Pb/stress combinations and, 120 risk assessment and, 122 history of, 87–89, 94, 96 IQ studies and, 88 low SES populations and, 100, 105 mesocorticolimbic dopamine system and, 94, 100–103 HPA axis in, 101 NAC in, 101, 102–103 NMDA in, 100, 102, 103 PbB levels in, 101–102 risk factors in, 89–90 risk modifiers in, 90–95 ACTH and, 93–94 cortisol and, 92–95 environmental hazards and, 93 glucocorticoids and, 93–95 HPA axis and, 93–95 low SES populations and, 90–92

345 race/ethnicity and, 91 stress symptoms and, 92, 94 Linkage studies, in autism/environmental agents, 188 LOAEL. See Lowest observed adverse eVect level Locomotion, models/constructs and, 327–328 Low birth weight parental smoking and, 240–242 in PCBs/dioxins studies, 61 Low SES populations, in lead exposure/stress, 90–92, 100, 105 Lowest observed adverse eVect level (LOAEL) in alcohol/neurodevelopment, 29 endocrine disruptors and, 212–213, 217

M Magnetic resonance imaging (MRI) studies in alcohol/neurodevelopment, 25 in lead exposure/stress, 97 in neurobehavioral assessment, 288, 290–292 MAST. See Michigan Alcoholism Screening Test Maternal impairment, in pregnancy/ EtOH, 16–17 Maternal infections, in autism/environmental agents, 177 Maternal lead screening, in lead exposure/ stress, 125 Maternal smoking characteristics, parental smoking and, 238–240 McCarthy Scales of Children’s Abilities (MSCA) in methyl mercury exposure, 159 PCBs/dioxins and, 65–66 Measles, mumps, rubella (MMR) vaccine, in autism/environmental agents, 182–183 Memory, in models/constructs, 307–309 Mercury. See also Methyl mercury/prenatal exposure; Total mercury in hair (THg) environmental impact of, 141–142 oxidation states of, 142 sources of, 142–143 toxicity of, 142–143 Mercury-containing vaccines, in autism/ environmental agents, 183–184, 185

346 Mesocorticolimbic dopamine system, lead exposure/stress and, 94, 100–103 Metabolic imbalance, in autism/ environmental agents, 178–179 Metabolism, of EtOH, 3 Methanol (MeOH) in alcohol/neurodevelopment, 1, 2, 3, 26–34 metabolism of, 26–28 Methanol (MeOH) exposure, in pregnancy/ MeOH, 28, 30 Methyl mercury/prenatal exposure, 141–164 data interpretation in, 156–160 BDML and, 159 benchmarks and, 157, 158 biomarkers and, 157–158 BNT and, 159 concomitant exposures and, 158 CPT and, 159 governmental, 156–157 MSCA and, 159 statistical analysis and, 160 developmental disability in, 160–161 CCD definition in, 160 epidemiological studies in BAERs and, 155 Bayley Scales and, 154 experimental considerations in, 150–151, 152 Faeroe Islands and, 154–155 fish-consuming populations and, 150–156 New Zealand and, 152–153 results variations in, 154–156 Seychelles Islands and, 153–154 food chains and, 143–145 HOME scores and, 153–154 human risk assessment of, 144, 161–162 AHA and, 163 hypothesis development in, 149–150 mercury and environmental impact of, 141–142 oxidation states of, 142 sources of, 142–143 toxicity of, 142–143 PCBs and, 154–155 reported cases of, 145–149 Minamata disease in, 147–149, 150, 163 seafood in, 145–147, 150–156 seed grain in, 145–147, 149–150 RfD and, 156–157

index EPA definition of, 161, 162, 164 THg in hair and, 146, 148, 150, 158 toxicity of, 143–145 Michigan Alcoholism Screening Test (MAST) scores, in alcohol/ neurodevelopment, 19 Minamata disease in methyl mercury exposure, 147–149, 150, 163 in neurobehavioral assessment, 289 Misoprostol, in autism/environmental agents, 186–187 MMR. See Measles, mumps, rubella Models/constructs animal models in, 302–303 attention in, 309–311 go-no go tests and, 310 signal detection tests and, 310 vigilance tests and, 309–310 cognition tests in, 303–317 concurrent schedules in, 307 discrimination learning in, 304–305 discrimination reversal in, 305–306 executive functions in, 303 repeated acquisition in, 306–307 distractibility in, 311 dosing regimen in, 330 functional domains and, 301–302 impulsivity in, 311–314 DRL task and, 312–313 FI schedules and, 313–314, 315 response times and, 312–313 interpretation of, 302 memory in, 307–309 Morris water maze task and, 307–308 radial arm maze task and, 308–309 motor function in, 326–329 DRH schedules and, 328 gross/fine movement and, 328–329 habituation and, 327–328 locomotion and, 327–328 rotorod and, 326 perseveration in, 314–317 Wisconsin Card Sort Test and, 314 screening batteries and, 301 sensory function in, 317–326 audition and, 321–324 color detection and, 320–321 motion detection and, 320

index

347

olefaction/taste and, 325–326 somatosensory function and, 324–325 vision and, 317–321 Moebius syndrome, in autism/environmental agents, 187 Morphological eVects, of PCBs/ dioxins, 210–211 Morris water maze task, in models/ constructs, 307–308 Motion detection, models/constructs and, 320 Motor function gross/fine movement and, 328–329 in models/constructs, 326–329 MRI. See Magnetic resonance imaging MSCA. See McCarthy Scales of Children’s Abilities Multi-hit hypothesis, in lead exposure/stress, 104, 126–127

N NAC. See Nucleus accumbens NAC dopamine, in lead exposure/stress, 109–110, 111 Neural protein antibodies, in autism/ environmental agents, 176–177 Neurobehavioral assessment, of neurotoxicant exposure altered structure/function in, 263–264 data analysis in, 272–279 biomarker models in, 277 confounding bias in, 273–276 default assumptions in, 277 eVect modification in, 276–279 genetic polymorphisms and, 278–279 hierarchical, 272 IQ scores and, 272–273, 275 type 1 error rate and, 272–273 WISC-III in, 272–273 data collection in, 264–272 adjustments to, 273 animal models and, 271 clinical v. research, 264–266 domain ranges and, 266–267, 271–272 generic testing batteries and, 264, 265–266, 269 infants and, 270–272 intelligence v. domain-focused tests and, 268–269

IQ scores and, 266, 268–269, 270 multiple v. single testing sessions and, 264 theoretical models and, 267 WISC-III and, 266, 272 data interpretation in, 279–293 ADHD and, 279, 280, 283, 288 ASD and, 283 brain-behavior relationships and, 287–292 characteristics distribution change and, 284–287 decrement meaning in, 279–280 dynamic nosology and, 282–284 individual v. population risk and, 287 IQ scores and, 279–281, 285–286 Minamata disease and, 289 MRI performance studies and, 288, 290–292 ROCF and, 281–282 significant impairment and, 280–282 study design and, 292–293 systematic errors and, 286–287 VMI and, 288–290 environmental neurotoxicants and, 263–264, 271 Neurobehavioral outcomes, in endocrine disruptors, 213–217 Neurobehavioral profiles, pregnancy/EtOH and, 10–11, 11–24 Neurobehavioral toxicity, in endocrine disruptors, 198–201 Neuronal/glial cell markers, PCBs/dioxins and, 49 Neuropsychological function tests, in PCBs/ dioxins studies, 68–69 Neurotoxic contamination, in PCBs/dioxins studies, 62–63 Neurotoxic eVects, in children, parental smoking and, 237–238 Neurotoxic mechanisms, of PCBs/dioxins, 48–49, 69–72 Neurotoxic properties, of pesticides ACh and, 229–230 AChE and, 229–232, 234 carbamates and, 231–232, 233–234 common pesticides and, 225–226 CPF and, 230–231, 233 exposure and, 226–228 Food Quality Protection Act for, 228–229

index

348 Neurotoxic properties, of pesticides (cont.) human exposure and, 232–233 NOEL and, 229 OPs and, 229–232, 233–234 pyrethroids and, 232, 233–234 setting standards for, 228–229 Neurotransmitters, in lead exposure/ stress, 114–115 New Zealand study, in methyl mercury exposure, 152–153 NHANES, endocrine disruptors and, 195–196 NMDA, in lead exposure/stress, 100, 102, 103 No observed eVect levels (NOEL), pesticide neurotoxicity and, 229 Nucleus accumbens (NAC), mesocorticolimbic dopamine system and, 101, 102–103

O Obstetric complications, in autism/ environmental agents, 173–174, 175 Olefaction/taste, models/constructs and, 325–326 Organophosphates (OP), pesticide neurotoxicity and, 229–232, 233–234

P Parental smoking, behavioral/cognitive functioning. See also Smoking adverse eVects pathway in, 240–242 ADHD and, 245 child behavior and, 245–246 direct dose response in, 241 fetus and, 241 low birth weight, 240–242 in utero brain growth and, 242, 244 maternal smoking characteristics in, 238–240 ADHD in, 239, 240 genetics, 239–240 prevalence in, 238–239 secondary risk exposures in, 239 maternal smoking outcomes in ADHD and, 244, 245, 250, 252 adverse behavior and, 245–246

animal models and, 243–244, 251 antisocial behavior and, 247–248 CBCL and, 247, 250 cognitive impairments and, 248–249 epidemiological studies and, 244–245 gestation period and, 243 HOME scores and, 250 IQ scores and, 252, 253 newborns/preschoolers and, 246–247 prenatal exposure and, 243–244 school performance and, 250–251 school-age children and, 247–248 Pb. See Lead PbB. See Blood lead levels PCBs in endocrine disruptors, 198 in methyl mercury exposure, 154–155 PCBs/dioxins, 47–77 animal behavior studies in, 55, 58, 61 dopamine in, 55 spinning in, 55 chemical properties of, 47–48 congeners in, 47 CNS eVects of, 49–50 dopamine and, 50 neuronal/glial cell markers and, 49 Dutch school age study in, 64–69 breast-feeding and, 64–65, 74–76 cognitive/motor abilities and, 65–66, 66–67, 74 environmental exposure in, 76 exposure measurements and, 65 HOME scores and, 66–67 inclusion/exclusion criteria and, 64–65 IQs and, 66 neuropsychological function tests and, 68–69 risk assessment in, 74 RRM models and, 67 sex-specific behavior and, 68 endocrine disrupters in dopamine and, 50 steroid hormones and, 50–51 as endocrine disruptors, 197, 201–211 human exposure to, 51–55 accidental, 52–53 breast-feeding in, 51, 53–54 children and, 51–52, 54 environmental, 53–54 food contamination and, 51–52

index neurodevelopmental eVects of, 54–55, 56–57 perinatal, 52–55 postnatal testing in, 51–52, 54 pregnancy and, 51–52 human risk assessment in, 58–64 breast-feeding and, 58–59, 62 prenatal v. postnatal exposure and, 58–59 interstudy diVerences in, 59–64 cognitive/motor development and, 61, 63–64 exposure levels and, 60 IQs and, 61 low birth weight and, 61 neurotoxic contamination and, 62–63 susceptibility and, 60–62 neurotoxic mechanisms of, 48–49, 69–72 Ah receptors in, 48–49 breast-feeding safety in, 72–73 IQs in, 73–74 perinatal exposure and, 71 postnatal exposure and, 73–74 prenatal exposure and, 69–72 TEF in, 49 sex-steroid eVects in, 59 PCDD. See Polychlorinated dibenzodioxin PDD, in autism/environmental agents, 180, 182 Perinatal exposure, PCBs/dioxins and, 71 Perseveration, in models/constructs, 314–317 Persistent organic pollutants, in endocrine disruptors, 195, 196–197 Pesticides/commonly used, pesticide neurotoxicity and, 225–226 Phenylketonuria (PKU), autism/ environmental agents and, 178–179 Phthalate esters, in endocrine disruptors, 217–220 Phthalates, in endocrine disruptors, 197, 198 PKU. See Phenylketonuria Polychlorinated dibenzodioxin (PCDD), in endocrine disruptors, 202, 203 Postnatal exposure, in PCBs/dioxins, 73–74 Postnatal testing, in pregnancy/EtOH, 13–15, 15–16, 21–23 Pregnancy in EtOH/neurodevelopment, 9–24 MeOH and, 32–34 Prenatal exposure

349 in maternal smoking, 243–244 in PCBs/dioxins, 69–72 in pregnancy/EtOH, 20, 23–24 Prenatal infections, in autism/environmental agents, 176–178 Prenatal v. postnatal exposure, in PCBs/ dioxins, 58–59 Prevalence, of maternal smoking, 238–239 Primate models. See also Models/constructs MeOH and, 31–34 Properties of alcohols, 2 of endocrine disruptors, 196–197 Pyrethroids, pesticide neurotoxicity and, 232, 233–234

R Race/ethnicity, in lead exposure/stress, 91 Radial arm maze task, in models/ constructs, 308–309 Recommendations, for risk evaluation, 35–37 Reference Dose (RfD) definition of, 161, 162, 164 in endocrine disruptors, 213, 214, 215 in methyl mercury exposure, 156–157 PCBs/dioxins in, 206 Repeated acquisition, in cognition tests, 306–307 Repeated learning (RL) component, in cognitive deficit studies, 98–100 Reported cases, of methyl mercury prenatal exposure, 145–149 Reproductive abnormalities, PCBs/dioxins and, 203, 205 Reproductive systems, endocrine disruptors and, 197 Response times, in models/ constructs, 312–313 Results variations, in methyl mercury exposure, 154–156 Rey-Osterrieth Complex Figure (ROCF), in neurobehavioral assessment, 281–282 Risk assessment in alcohol/neurodevelopment, 34–37 in lead exposure/stress, 122 in methyl mercury exposure, 144, 161–162 in PCB/dioxin studies, 74 in PCBs/dioxins, 58–64

index

350 Risk factors in alcohol/neurodevelopment, 1–3 gender diVerences and, 3–4 in lead exposure/stress, 89–90 in prenatal exposure, 3–4, 9–24 Risk modifiers, in lead exposure/stress, 90–95, 126, 127 Risk/individual v. population, in neurobehavioral assessment, 287 RL. See Repeated learning ROCF. See Rey-Osterrieth Complex Figure Rodent models MeOH and, 29–30 in pregnancy/MeOH, 29–30 Rotorod, models/constructs and, 326 RRM models, in PCBs/dioxins studies, 67 Rubella, autism/environmental agents and, 176

S Schedule-controlled operant behaviour (SCOB), in PCBs/dioxins, 204–205, 207 School performance, in maternal smoking, 250–251 Screening batteries, models/constructs and, 301 Seafood, in methyl mercury exposure, 145–147, 150–156 Secondary risk exposures, in maternal smoking, 239 Seed grain, in methyl mercury exposure, 145–147, 149–150 Sensory function, in models/constructs, 317–326 Sex-specific behavior, in PCBs/dioxins studies, 68 Sex-steroid eVects, in PCBs/dioxins exposure, 59 Sexual diVerentiation, of brain, in endocrine disruptors, 199–200, 208 Sexual eVects in endocrine disruptors, 212, 213–214, 215, 216 of phthalate esters, 217–218 Seychelles Islands study, in methyl mercury exposure, 153–154

Signal detection tests, models/constructs and, 310 Significant impairment, in neurobehavioral assessment, 280–282 Smoking. See also Parental smoking, behavioral/cognitive functioning in pregnancy/EtOH, 13, 20 Social behaviors in endocrine disruptors, 214 phthalate esters and, 219–220 Somatosensory function, models/constructs and, 324–325 Sources, of endocrine disruptors, 197, 198, 212 Spina bifida, in autism/environmental agents, 188 Spinning, PCBs/dioxins and, 55 Standards, for pesticides, pesticide neurotoxicity and, 228–229 Statistical data analysis, in methyl mercury exposure, 160 Steroid hormones, in PCBs/dioxins studies, 50–51 Stress symptoms, in lead exposure/ stress, 92, 94 Study design, in neurobehavioral assessment, 292–293 Susceptibility, in PCBs/dioxins studies, 60–62 Systematic errors, in neurobehavioral assessment, 286–287

T TCDD. See 2,3,7, 8-Tetrachlorodibenzo-p-dioxin TEF. See Toxic equivalent factor Teratogens, autism/environmental agents and, 184–188 Test batteries, for MeOH, 31, 33 Testing sessions/multiple v. single, in neurobehavioral assessment, 264 2,3,7,8-Tetra-chlorodibenzo-p-dioxin (TCDD), PCBs/dioxins in, 202–206, 208, 209, 210, 211 Thalidomide, in autism/environmental agents, 184, 186 Theoretical models. See also Models/ constructs

index

351

in neurobehavioral assessment, 267 THg. See Total mercury in hair Total mercury in hair (THg), methyl mercury exposure and, 146, 148, 150, 158 Toxic equivalent factor (TEF), in PCBs/ dioxins exposure, 49 Toxicity of MeOH, 26–27 of methyl mercury, 143–145 Treadmill studies, endocrine disruptors and, 205, 206 Type 1 error rates, data analysis and, 272–273

Vision, models/constructs and, 317–321 Visual-Motor Integration Test (VMI), in neurobehavioral assessment, 288–290 VMI. See Visual-Motor Integration Test

W Wechsler Intelligence Scale for Children Third Edition (WISC-III), in neurobehavioral assessment, 266, 272–273 WISC-III. See Wechsler Intelligence Scale for Children - Third Edition Wisconsin Card Sort Test, models/constructs and, 314

V Valproic acid, in autism/environmental agents, 186–187 Vigilance tests, in alcohol/ neurodevelopment, 14

Y Yeast colonic overgrowth, autism/ environmental agents and, 179

Contents of Previous Volumes

Volume 1

Volume 2

A Functional Analysis of Retarded Development SIDNEY W. BIJOU

A Theoretical Analysis and Its Application to Training the Mentally Retarded M. RAY DENNY

Classical Conditioning and Discrimination Learning Research with the Mentally Retarded LEONARD E. ROSS

The Role of Input Organization in the Learning and Memory of Mental Retardates HERMAN H. SPITZ Autonomic Nervous System Functions and Behavior: A Review of Experimental Studies with Mental Defectives RATHE KARPER

The Structure of Intellect in the Mental Retardate HARVEY F. DINGMAN AND C. EDWARD MEYERS Research on Personality Structure in the Retardate EDWARD ZIGLER

Learning and Transfer of Mediating Responses in Discriminating Learning BRYAN E. SHEPP AND FRANK D. TURRISI

Experience and the Development of Adaptive Behavior H. CARL HAYWOOD AND JACK T. TAPP

A Review of Research on Learning Sets and Transfer or Training in Mental Defectives MELVIN E. KAUFMAN AND HERBERT J. PREHM

A Research Program on the Psychological Effects of Brain Lesions in Human Beings RALPH M. REITAN

Programming Perception and Learning for Retarded Children MURRAY SIDMAN AND LAWRENCE T. STODDARD

Long-Term Memory in Mental Retardation JOHN M. BELMONT

Programming Instruction Techniques for the Mentally Retarded FRANCES M. GREENE

The Behavior of Moderately and Severely Retarded Persons JOSEPH E. SPRADLIN AND FREDERIC L. GIRARDEAU

Some Aspects of the Research on Mental Retardation in Norway IVAR ARNIJOT BJORGEN

Author Index-Subject Index

353

354

contents of previous volumes

Research on Mental Deficiency During the Last Decade in France R. LAFON AND J. CHABANIER

A Theory of Primary and Secondary Familial Mental Retardation ARTHUR R. JENSEN

Psychotherapeutic Procedures with the Retarded MANNY STERNLIGHT

Inhibition Deficits in Retardate Learning and Attention LAIRD W. HEAL AND JOHN T. JOHNSON, JR.

Author Index-Subject Index

Volume 3 Incentive Motivation in the Mental Retardate PAUL S. SIEGEL Development of Lateral and Choice-Sequence Preferences IRMA R. GERJUOY AND JOHN J. WINTERS, JR. Studies in the Experimental Development of Left-Right Concepts in Retarded Children Using Fading Techniques SIDNEY W. BIJOU Verbal Learning and Memory Research with Retardates: An Attempt to Assess Developmental Trends L. R. GOULET Research and Theory in Short-Term Memory KEITH G. SCOTT AND MARCIA STRONG SCOTT

Growth and Decline of Retardate Intelligence MARY ANN FISHER AND DAVID ZEAMAN The Measurements of Intelligence A. B. SILVERSTEIN Social Psychology and Mental Retardation WARNER WILSON Mental Retardation in Animals GILBERT W. MEIER Audiologic Aspects of Mental Retardation LYLE L. LLOYD Author Index-Subject Index

Volume 5 Medical-Behavioral Research in Retardation JOHN M. BELMONT Recognition Memory: A Research Strategy and a Summary of Initial Findings KEITH G. SCOTT

Reaction Time and Mental Retardation ALFRED A. BAUMEISTER AND GEORGE KELLAS

Operant Procedures with the Retardate: An Overview of Laboratory Research PAUL WEISBERG

Mental Retardation in India: A Review of Care, Training, Research, and Rehabilitation Programs J. P. DAS

Methodology of Psychopharmacological Studies with the Retarded ROBERT L. SPRAGUE AND JOHN S. WERRY

Educational Research in Mental Retardation SAMUEL L. GUSKIN AND HOWARD H. SPICKER

Process Variables in the Paired-Associate Learning of Retardates ALFRED A. BAUMEISTER AND GEORGE KELLAS

Author Index-Subject Index

Volume 4

Sequential Dot Presentation Measures of Stimulus Trace in Retardates and Normals EDWARD A. HOLDEN, JR.

Memory Processes in Retardates and Normals NORMAN R. ELLIS

Cultural-Familial Retardation FREDERIC L. GIRARDEAU

contents of previous volumes

355

German Theory and Research on Mental Retardation: Emphasis on Structure LOTHAR R. SCHMIDT AND PAUL B. BALTES

Placement of the Retarded in the Community: Prognosis and Outcome RONALD B. MCCARVER AND ELLIS M. CRAIG

Author Index-Subject Index

Physical and Motor Development of Retarded Persons ROBERT H. BRUININKS

Volume 6 Cultural Deprivation and Cognitive Competence J. P. DAS Stereotyped Acts ALFRED A. BAUMEISTER AND REX FOREHAND Research on the Vocational Habilitation of the Retarded: The Present, the Future MARC W. GOLD Consolidating Facts into the Schematized Learning and Memory System of Educable Retardates HERMAN H. SPITZ An Attentional-Retention Theory of Retardate Discrimination Learning MARY ANN FISHER AND DAVID ZEAMAN Studying the Relationship of Task Performance to the Variables of Chronological Age, Mental Age, and IQ WILLIAM E. KAPPAUF Author Index-Subject Index Volume 7 Mediational Processes in the Retarded JOHN G. BORKOWSKI AND PATRICIA B. WANSCHURA The Role of Strategic Behavior in Retardate Memory ANN L. BROWN Conservation Research with the Mentally Retarded KERI M. WILTON AND FREDERIC J. BOERSMA

Subject Index

Volume 8 Self-Injurious Behavior ALFRED A. BAUMEISTER AND JOHN PAUL ROLLINGS Toward a Relative Psychology of Mental Retardation with Special Emphasis on Evolution HERMAN H. SPITZ The Role of the Social Agent in Language Acquisition: Implications for Language Intervention GERALD J. MAHONEY AND PAMELA B. SEELY Cognitive Theory and Mental Development EARL C. BUTTERFIELD AND DONALD J. DICKERSON A Decade of Experimental Research in Mental Retardation in India ARUN K. SEN The Conditioning of Skeletal and Autonomic Responses: Normal-Retardate Stimulus Trace Differences SUSAN M. ROSS AND LEONARD E. ROSS Malnutrition and Cognitive Functioning J. P. DAS AND EMMA PIVATO Research on Efficacy of Special Education for the Mentally Retarded MELVINE E. KAUFMAN AND PAUL A. ALBERTO Subject Index

356 Volume 9 The Processing of Information from Short-Term Visual Store: Developmental and Intellectual Differences LEONARD E. ROSS AND THOMAS B. WARD Information Processing in Mentally Retarded Individuals KEITH E. STANOVICH Mediational Process in the Retarded: Implications for Teaching Reading CLESSEN J. MARTIN Psychophysiology in Mental Retardation J. CLAUSEN Theoretical and Empirical Strategies for the Study of the Labeling of Mentally Retarded Persons SAMUEL L. GUSKIN The Biological Basis of an Ethic in Mental Retardation ROBERT L. ISAACSON AND CAROL VAN HARTESVELDT Public Residential Services for the Mentally Retarded R. C. SCHEERENBERGER Research on Community Residential Alternatives for the Mentally Retarded LAIRD W. HEAL, CAROL K. SIGELMAN, AND HARVEY N. SWITZKY Mainstreaming Mentally Retarded Children: Review of Research LOUIS CORMAN AND JAY GOTTLIEB Savants: Mentally Retarded Individuals with Special Skills A. LEWIS HILL

contents of previous volumes Visual Pattern Detection and Recognition Memory in Children with Profound Mental Retardation PATRICIA ANN SHEPHERD AND JOSEPH F. FAGAN III Studies of Mild Mental Retardation and Timed Performance T. NETTELBECK AND N. BREWER Motor Function in Down’s Syndrome FERIHA ANWAR Rumination NIRBHAY N. SINGH Subject Index

Volume 11 Cognitive Development of the Learning-Disabled Child JOHN W. HAGEN, CRAIG R. BARCLAY, AND BETTINA SCHWETHELM Individual Differences in Short-Term Memory RONALD L. COHEN Inhibition and Individual Differences in Inhibitory Processes in Retarded Children PETER L. C. EVANS Stereotyped Mannerisms in Mentally Retarded Persons: Animal Models and Theoretical Analyses MARK H. LEWIS AND ALFRED A. BAUMEISTER An Investigation of Automated Methods for Teaching Severely Retarded Individuals LAWRENCE T. STODDARD

Volume 10

Social Reinforcement of the Work Behavior of Retarded and Nonretarded Persons LEONIA K. WATERS

The Visual Scanning and Fixation Behavior of the Retarded LEONARD E. ROSS AND SUSAM M. ROSS

Social Competence and Interpersonal Relations between Retarded and Nonretarded Children ANGELA R. TAYLOR

Subject Index

contents of previous volumes The Functional Analysis of Imitation WILLIAM R. MCCULLER AND CHARLES L. SALZBERG Index

357 Autonomy and Adaptability in Work Behavior of Retarded Clients JOHN L. GIFFORD, FRANK R. RUSCH, JAMES E. MARTIN, AND DAVID J. WHITE Index

Volume 12 An Overview of the Social Policy of Deinstitutionalization BARRY WILLER AND JAMES INTAGLIATA Community Attitudes toward Community Placement of Mentally Retarded Persons CYNTHIA OKOLO AND SAMUEL GUSKIN Family Attitudes toward Deinstitutionalization AYSHA LATIB, JAMES CONROY, AND CARLA M. HESS Community Placement and Adjustment of Deinstitutionalized Clients: Issues and Findings ELLIS M. CRAIG AND RONALD B. MCCARVER

Volume 13 Sustained Attention in the Mentally Retarded: The Vigilance Paradigm JOEL B. WARM AND DANIEL B. BERCH Communication and Cues in the Functional Cognition of the Mentally Retarded JAMES E. TURNURE Metamemory: An Aspect of Metacognition in the Mentally Retarded ELAINE M. JUSTICE Inspection Time and Mild Mental Retardation T. NETTELBECK

Issues in Adjustment of Mentally Retarded Individuals to Residential Relocation TAMAR HELLER

Mild Mental Retardation and Memory Scanning C. J. PHILLIPS AND T. NETTELBECK

Salient Dimensions of Home Environment Relevant to Child Development KAZUO NIHIRA, IRIS TAN MINK, AND C. EDWARD MEYERS

Cognitive Determinants of Reading in Mentally Retarded Individuals KEITH E. STANOVICH

Current Trends and Changes in Institutions for the Mentally Retarded R. K. EYMAN, S. A. BORTHWICK, AND G. TARJAN Methodological Considerations in Research on Residential Alternatives for Developmentally Disabled Persons LAIRD W. HEAL AND GLENN T. FUJIURRA A Systems Theory Approach to Deinstitutionalization Policies and Research ANGELA A. NOVAK AND TERRY R. BERKELEY

Comprehension and Mental Retardation LINDA HICKSON BILSKY Semantic Processing, Semantic Memory, and Recall LARAINE MASTERS GLIDDEN Proactive Inhibition in Retarded Persons: Some Clues to Short-Term Memory Processing JOHN J. WINTERS, JR. A Triarchic Theory of Mental Retardation ROBERT J. STERNBERG AND LOUIS C. SPEAR Index

358

contents of previous volumes

Volume 14

Volume 15

Intrinsic Motivation and Behavior Effectiveness in Retarded Persons H. CARL HAYWOOD AND HARVEY N. SWITZKY

Mental Retardation as Thinking Disorder: The Rationalist Alternative to Empiricism HERMAN H. SPITZ

The Rehearsal Deficit Hypothesis NORMAN W. BRAY AND LISA A. TURNER Molar Variability and the Mentally Retarded STUART A. SMITH AND PAUL S. SIEGEL Computer-Assisted Instruction for the Mentally Retarded FRANCES A CONNERS, DAVID R. CARUSO, AND DOUGLAS K. DETTERMAN

Developmental Impact of Nutrition on Pregnancy, Infancy, and Childhood: Public Health Issues in the United States ERNESTO POLLITT The Cognitive Approach to Motivation in Retarded Individuals SHYLAMITH KREITLER AND HANS KREITLER Mental Retardation, Analogical Reasoning, and the Componential Method J. MCCONAGHY

Procedures and Parameters of Errorless Discrimination Training with Developmentally Impaired Individuals GIULO E. LANCIONI AND PAUL M. SMEETS

Application of Self-Control Strategies to Facilitate Independence in Vocational and Instructional Settings JAMES E. MARTIN, DONALD L. BURGER, SUSAN ELIAS-BURGER, AND DENNIS E. MITHAUG

Reading Acquisition and Remediation in the Mentally Retarded NIRBHAY N. SINGH AND JUDY SINGH

Family Stress Associated with a Developmentally Handicapped Child PATRICIA M. MINNES

Families with a Mentally Retarded Child BERNARD FARBER AND LOUIS ROWITZ

Physical Fitness of Mentally Retarded Individuals E. KATHRYN MCCONAUGHY AND CHARLES L. SALZBERG

Social Competence and Employment of Retarded Persons CHARLES L. SALZBERG, MARILYN LIKINS, E. KATHRYN MCCONAUGHY, AND BENJAMIN LINGUGARIS/KRAFT Toward a Taxonomy of Home Environments SHARON LANDESMAN Behavioral Treatment of the Sexually Deviant Behavior of Mentally Retarded Individuals R. M. FOXX, R. G. BITTLE, D. R. BECHTEL, AND J. R. LIVESAY Behavior Approaches to Toilet Training for Retarded Persons S. BETTISON Index

Index

Volume 16 Methodological Issues in Specifying Neurotoxic Risk Factors for Developmental Delay: Lead and Cadmium as Prototypes STEPHEN R. SCHROEDER The Role of Methylmercury Toxicity in Mental Retardation GARY J. MYERS AND DAVID O. MARSH Attentional Resource Allocation and Mental Retardation EDWARD C. MERRILL

contents of previous volumes Individual Differences in Cognitive and Social Problem-Solving Skills as a Function of Intelligence ELIZABETH J. SHORT AND STEVEN W. EVANS Social Intelligence, Social Competence, and Interpersonal Competence JANE L. MATHIAS Conceptual Relationships between Family Research and Mental Retardation ZOLINDA STONEMAN Index Volume 17 The Structure and Development of Adaptive Behaviors KEITH F. WIDAMAN, SHARON A. BORTHWICK-DUFFY, AND TODD D. LITTLE Perspectives on Early Language from Typical Development and Down Syndrome MICHAEL P. LYNCH AND REBECCA E. EILERS The Development of Verbal Communication in Persons with Moderate to Mild Mental Retardation LEONARD ABBEDUTO Assessment and Evaluation of Exceptional Children in the Soviet Union MICHAEL M. GERBER, VALERY PERELMAN, AND NORMA LOPEZ-REYNA Constraints on the Problem Solving of Persons with Mental Retardation RALPH P. FERRETTI AND AL R. CAVALIER Long-Term Memory and Mental Retardation JAMES E. TURNURE Index Volume 18 Perceptual Deficits in Mildly Mentally Retarded Adults ROBERT FOX AND STEPHEN OROSSIII

359 Stimulus Organization and Relational Learning SAL A. SORACI, JR. AND MICHAEL T. CARLIN Stimulus Control Analysis and Nonverbal Instructional Methods for People with Intellectual Disabilities WILLIAM J. MCILVANE Sustained Attention in Mentally Retarded Individuals PHILLIP D. TOMPOROWSKI AND LISA D. HAGER How Modifiable Is the Human Life Path? ANN M. CLARKE AND ALAN D. B. CLARKE Unraveling the ‘‘New Morbidity’’: Adolescent Parenting and Developmental Delays JOHN G. BORKOWSKI, THOMAS L. WHITMAN, ANNE WURTZ PASSINO, ELIZABETH A. RELLINGER, KRISTEN SOMMER, DEBORAH KEOUGH, AND KERI WEED Longitudinal Research in Down Syndrome JANET CARR Staff Training and Management for Intellectual Disability Services CHRIS CULLEN Quality of Life of People with Developmental Disabilities TREAVOR R. PARMENTER Index

Volume 19 Mental Retardation in African Countries: Conceptualization, Services, and Research ROBERT SERPELL, LILIAN MARIGA, AND KARYN HARVEY Aging and Alzheimer Disease in People with Mental Retardation WARREN B. ZIGMAN, NICOLE SCHUPF, APRIL ZIGMAN, AND WAYNE SILERMAN

360 Characteristics of Older People with Intellectual Disabilities in England JAMES HOGG AND STEVE MOSS Epidemiological Thinking in Mental Retardation: Issues in Taxonomy and Population Frequency TOM FRYERS Use of Data Base Linkage Methodology in Epidemiological Studies of Mental Retardation CAROL A. BOUSSY AND KEITH G. SCOTT Ways of Analyzing the Spontaneous Speech of Children with Mental Retardation: The Value of Cross-Domain Analyses CATHERINE E. SNOW AND BARBARA ALEXANDER PAN Behavioral Experimentation in Field Settings: Threats to Validity and Interpretation Problems WILLY-TORE MRCH Index

Volume 20 Parenting Children with Mental Retardation BRUCE L. BAKER, JAN BLACHER, CLAIRE B. KOPP, AND BONNIE KRAEMER Family Interactions and Family Adaptation FRANK J. FLOYD AND CATHERINE L. COSTIGAN Studying Culturally Diverse Families of Children with Mental Retardation IRIS TAN MINK Older Adults with Mental Retardation and Their Families TAMAR HELLER A Review of Psychiatric and Family Research in Mental Retardation ANN GATH

contents of previous volumes A Cognitive Portrait of Grade School Students with Mild Mental Retardation MARCIA STRONG SCOTT, RUTH PEROU, ANGELIKA HARTL CLAUSSEN, AND LOIS-LYNN STOYKO DEUEL Employment and Mental Retardation NEIL KIRBY Index

Volume 21 An Outsider Looks at Mental Retardation: A Moral, a Model, and a Metaprincipal RICHARD P. HONECK Understanding Aggression in People with Intellectual Disabilities: Lessons from Other Populations GLYNIS MURPHY A Review of Self-Injurious Behavior and Pain in Persons with Developmental Disabilities FRANK J. SYMONS AND TRAVIS THOMPSON Recent Studies in Psychopharmacology in Mental Retardation MICHAEL G. AMAN Methodological Issues in the Study of Drug Effects on Cognitive Skills in Mental Retardation DEAN C. WILLIAMS AND KATHRYN J. SAUNDERS The Behavior and Neurochemistry of the Methylazoxymethanol-Induced Microencephalic Rat PIPPA S. LOUPE, STEPHEN R. SCHROEDER, AND RICHARD E.TESSEL Longitudinal Assessment of Cognitive-Behavioral Deficits Produced by the Fragile-X Syndrome GENE S. FISCH Index

contents of previous volumes Volume 22 Direct Effects of Genetic Mental Retardation Syndromes: Maladaptive Behavior and Psychopathology ELISABETH M. DYKENS Indirect Effects of Genetic Mental Retardation Disorders: Theoretical and Methodological Issues ROBERT M. HODAPP The Development of Basic Counting, Number, and Arithmetic Knowledge among Children Classified as Mentally Handicapped ARTHUR J. BAROODY The Nature and Long-Term Implications of Early Developmental Delays: A Summary of Evidence from Two Longitudinal Studies RONALD GALLIMORE, BARBARA K. KEOGH, AND LUCINDA P. BERNHEIMER Savant Syndrome TED NETTELBECK AND ROBYN YOUNG The Cost-Efficiency of Supported Employment Programs: A Review of the Literature ROBERT E. CIMERA AND FRANK R. RUSCH Decision Making and Mental Retardation LINDA HICKSON AND ISHITA KHEMKA ‘‘The Child That Was Meant?’’ or ‘‘Punishment for Sin?’’: Religion, Ethnicity, and Families with Children with Disabilities LARAINE MASTERS GLIDDEN, JEANNETTE ROGERS-DULAN, AND AMY E. HILL Index Volume 23 Diagnosis of Autism before the Age of 3 SALLY J. ROGERS The Role of Secretin in Autistic Spectrum Disorders AROLY HORVATH AND J. TYSON TILDON

361 The Role of Candidate Genes in Unraveling the Genetics of Autism CHRISTOPHER J. STODGELL, JENNIFER L. INGRAM, AND SUSAN L. HYMAN Asperger’s Disorder and Higher Functioning Autism: Same or Different? FRED R. VOLKMAR AND AMI KLIN The Cognitive and Neural Basis of Autism: A Disorder of Complex Information Processing and Dysfunction of Neocortical Systems NANCY J. MINSHEW, CYNTHIA JOHNSON, AND BEATRIZ LUNA Neural Plasticity, Joint Attention. and a Transactional Social-Orienting Model of Autism PETER MUNDY AND A. REBECCA NEAL Theory of Mind and Autism: A Review SIMON BARON-COHEN Understanding the Language and Communicative Impairments in Autism HELEN TAGER-FLUSBERG Early Intervention in Autism: Joint Attention and Symbolic Play CONNIE KASARI, STEPHANNY F. N. FREEMAN, AND TANYA PAPARELLA Attachment and Emotional Responsiveness in Children with Autism CHERYL DISSANAYAKE AND MARIAN SIGMAN Families of Adolescents and Adults with Autism: Uncharted Territory MARSHA MAILICK SELTZER, MARTY WYNGAARDEN KRAUSS, GAEL I. ORSMOND, AND CARRIE VESTAL Index

Volume 24 Self-Determination and Mental Retardation MICHAEL L. WEHMEYER

362 International Quality of Life: Current Conceptual, Measurement, and Implementation Issues KENNETH D. KEITH Measuring Quality of Life and Quality of Services through Personal Outcome Measures: Implications for Public Policy JAMES GARDNER, DEBORAH T. CARRAN, AND SYLVIA NUDLER Credulity and Gullibility in People with Developmental Disorders: A Framework for Future Research STEPHEN GREENSPAN, GAIL LOUGHLIN, AND RHONDA S. BLACK Criminal Victimization of Persons with Mental Retardation: The Influence of Interpersonal Competence on Risk T. NETTELBECK AND C. WILSON The Parent with Mental Retardation STEVE HOLBURN, TIFFANY PERKINS, AND PETER VIETZE Psychiatric Disorders in Adults with Mental Retardation STEVE MOSS Development and Evaluation of Innovative Residential Services for People with Severe Intellectual Disability and Serious Challenging Behavior JIM MANSELL, PETER MCGILL, AND ERIC EMERSON The Mysterious Myth of Attention Deficits and Other Defect Stories: Contemporary Issues in the Developmental Approach to Mental Retardation JACOB A. BURACK, DAVID W. EVANS, CHERYL KLAIMAN, AND GRACE IAROCCI Guiding Visual Attention in Individuals with Mental Retardation RICHARD W. SERNA AND MICHAEL T. CARLIN Index

contents of previous volumes Volume 25 Characterizations of the Competence of Parents of Young Children with Disabilities CARL J. DUNST, TRACY HUMPHRIES, AND CAROL M. TRIVETTE Parent–Child Interactions When Young Children Have Disabilities DONNA SPIKER, GLENNA C. BOYCE, AND LISA K. BOYCE The Early Child Care Study of Children with Special Needs JEAN F. KELLY AND CATHRYN L. BOOTH Diagnosis of Autistic Disorder: Problems and New Directions ROBYN YOUNG AND NEIL BREWER Social Cognition: A Key to Understanding Adaptive Behavior in Individuals with Mild Mental Retardation JAMES S. LEFFERT AND GARY N. SIPERSTEIN Proxy Responding for Subjective Well-Being: A Review ROBERT A. CUMMINS People with Intellectual Disabilities from Ethnic Minority Communities in the United States and the United Kingdom CHRIS HATTON Perception and Action in Mental Retardation W. A. SPARROW AND ROSS H. DAY Volume 26 A History of Psychological Theory and Research in Mental Retardation since World War II DONALD K. ROUTH AND STEPHEN R. SCHROEDER Psychopathology and Intellectual Disability: The Australian Child to Adult Longitudinal Study BRUCE J. TONGE AND STEWART L. EINFELD

contents of previous volumes Psychopathology in Children and Adolescents with Intellectual Disability: Measurement, Prevalence, Course, and Risk JAN L. WALLANDER, MARIELLE C. DEKKER, AND HANS KOOT Resilience, Family Care, and People with Intellectual Disabilities GORDONGRANT, PAULRAMCHARAN, AND PETER GOWARD Prevalence and Correlates of Psychotropic Medication Use among Adults with Developmental Disabilities: 1970–2000 MARIA G. VALDOVINOS, STEPHEN R. SCHROEDER, AND GEUNYOUNG KIM Integration as Acculturation: Developmental Disability, Deinstitutionalization, and Service Delivery Implications M. KATHERINE BUELL Cognitive Aging and Down Syndrome: An Interpretation J. P. DAS Index

363 CARMICHAEL OLSON, AND GERALYN R. TIMLER Memory, Language Comprehension, and Mental Retardation EDWARD C. MERRILL, REGAN LOOKADOO, AND STACY RILEA Reading Skills and Cognitive Abilities of Individuals with Mental Retardation FRANCES A. CONNERS Language Interventions for Children with Mental Retardation NANCY C. BRADY AND STEVEN F. WARREN Augmentative and Alternative Communication for Persons with Mental Retardation MARYANN ROMSKI, ROSE A. SEVCIK, AND AMY HYATT FONSECA Atypical Language Development in Individuals with Mental Retardation: Theoretical Implications JEAN A. RONDAL Index

Volume 27

Volume 28

Language and Communication in Individuals with Down Syndrome ROBIN S. CHAPMAN

Promoting Intrinsic Motivation and Self-Determination in People with Mental Retardation EDWARD L. DECI

Language Abilities of Individuals with Williams Syndrome CAROLYN B. MERVIS, BYRON F. ROBINSON, MELISSA L. ROWE, ANGELA M. BECERRA, AND BONITA P. KLEIN-TASMAN Language and Communication in Fragile X Syndrome MELISSA M. MURPHY AND LEONARD ABBEDUTO On Becoming Socially Competent Communicators: The Challenge for Children with Fetal Alcohol Exposure TRUMAN E. COGGINS, LESLEY B. OLSWANG, HEATHER

Applications of a Model of Goal Orientation and Self-Regulated Learning to Individuals with Learning Problems PAUL R. PINTRICH AND JULIANE L. BLAZEVSKI Learner-Centered Principles and Practices: Enhancing Motivation and Achievement for Children with Learning Challenges and Disabilities BARBARA L. MCCOMBS Why Pinocchio Was Victimized: Factors Contributing to Social Failure in People with Mental Retardation STEPHEN GREENSPAN

364 Understanding the Development of Subnormal Performance in Children from a Motivational-Interactionist Perspective JANNE LEPOLA, PEKKA SALONEN, MARJA VAURAS, AND ELISA POSKIPARTA Toward Inclusion Across Disciplines: Understanding Motivation of Exceptional Students HELEN PATRICK, ALLISON M. RYAN, ERIC M. ANDERMAN, AND JOHN KOVACH Loneliness and Developmental Disabilities: Cognitive and Affective Processing Perspectives MALKA MARGALIT The Motivation to Maintain Subjective Well-Being: A Homeostatic Model ROBERT A. CUMMINS AND ANNA L. D. LAU Quality of Life from a Motivational Perspective ROBERT L. SCHALOCK Index Volume 29 Behavioral Phenotypes: Going Beyond the Two-Group Approach ROBERT M. HODAPP Prenatal Drug Exposure and Mental Retardation ROBERT E. ARENDT, JULIA S. NOLAND, ELIZABETH J. SHORT, AND LYNN T. SINGER Spina Bifida: Genes, Brain, and Development JACK M. FLETCHER, MAUREEN DENNIS, HOPE NORTHRUP, MARCIA A. BARNES, H. JULIA HANNAY, SUSAN H. LANDRY, KIM COPELAND, SUSAN E. BLASER,

contents of previous volumes LARRY A. KRAMER, MICHAEL E. BRANDT, DAVID J. FRANCIS The Role of the Basal Ganglia in the Expression of Stereotyped, Self-Injurious Behaviors in Developmental Disorders HOWARD C. CROMWELL AND BRYAN H. KING Risk Factors for Alzheimer’s Disease in Down Syndrome LYNN WARD Precursors of Mild Mental Retardation in Children with Adolescent Mothers JOHN G. BORKOWSKI, JULIE J. LOUNDS, CHRISTINE WILLARD NORIA, JENNIFER BURKE LEFEVER, KERI WEED, DEBORAH A. KEOGH, AND THOMAS L. WHITMAN The Ecological Context of Challenging Behavior in Young Children with Developmental Disabilities ANITA A. SCARBOROUGH AND KENNETH K. POON Employment and Intellectual Disability: Achieving Successful Employment Outcomes KAYE SMITH, LYNNE WEBBER, JOSEPH GRAFFAM, AND CARLENE WILSON Technology Use and People with Mental Retardation MICHAEL L. WEHMEYER, SEAN J. SMITH, SUSAN B. PALMER, DANIEL K. DAVIES, AND STEVEN E. STOCK Index

Contents

Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Foreword by Laraine Masters Glidden . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Foreword by Stephen R. Schroeder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ix xi xiii xvii

Neurodevelopmental Effects of Alcohol Thomas M. Burbacher and Kimberly S. Grant I. II. III. IV.

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ethanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evaluating the Risk from Prenatal Exposure to Ethanol and Methanol . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 3 26 34 37

PCBs and Dioxins Hestien J. I. Vreugdenhil and Nynke Weisglas-Kuperus I. II. III. IV. V. VI. VII.

Neurotoxicology of PCBs and Dioxins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Human Exposure to PCBs and Dioxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Behavioral Animal Studies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Human Neurodevelopmental PCB and Dioxin Risk Assessment . . . . . . . . . . . . . . . . The Dutch PCB and Dioxin Study at School Age . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

v

47 51 55 58 64 69 74 77

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Interactions of Lead Exposure and Stress: Implications for Cognitive Dysfunction Deborah A. Cory-Slechta I. History and Current Understanding of Lead EVects. . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Pb Exposure in the Context of Environmentally Realistic Conditions . . . . . . . . . . . III. Risk Modifiers for Pb Neurotoxicity: Environmental Stress as a Case Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Both Pb and Stress Can Produce Cognitive Deficits . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Do Pb Exposure and Stress Interact? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Experiments Addressing Pb–Stress Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Considerations Arising from the DiVerential EVects of Maternal Pb, Maternal Stress, and Maternal Pbþ Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. Implications and Future Research Needs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

87 89 90 96 100 103 119 122 127

Developmental Disabilities Following Prenatal Exposure to Methyl Mercury from Maternal Fish Consumption: A Review of the Evidence Gary J. Myers, Philip W. Davidson, and Conrad F. Shamlaye I. II. III. IV. V. VI. VII. VIII. IX.

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reported Cases of Prenatal Methyl Mercury Poisoning . . . . . . . . . . . . . . . . . . . . . . . . Developing a Hypothesis About Prenatal Low-Level Methyl Mercury Exposure Epidemiological Studies of Fish-Consuming Populations . . . . . . . . . . . . . . . . . . . . . . . Interpreting the Available Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . What Constitutes a Developmental Disability? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Does Exceeding the EPA’s RfD Place a Child at Developmental Risk? . . . . . . . . . How Did the NRC and the EPA Determine the Risk to United States Children? Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

141 145 149 150 156 160 161 161 162 164

Environmental Agents and Autism: Once and Future Associations Susan L. Hyman, Tara L. Arndt, and Patricia M. Rodier I. II. III. IV. V.

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Family Environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Consideration of Biologic Etiologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Interface of Internal and External Environments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary and Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

171 172 173 188 189 189

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Endocrine Disruptors as a Factor in Mental Retardation Bernard Weiss I. II. III. IV.

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Current Scene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Emerging Hazards. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Endocrine Disruption and Neurobehavioral Development . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

195 201 212 220 220

The Neurotoxic Properties of Pesticides Herbert L. Needleman I. II. III. IV. V. VI.

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Setting Pesticide Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Neurotoxicity of Pesticides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Human EVects of Pesticide Exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary of What Is Known and What Remains to Be Discovered . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

225 226 228 229 232 233 234

Parental Smoking and Children’s Behavioral and Cognitive Functioning Michael Weitzman, Megan Kavanaugh, and Todd A. Florin I. II. III. IV.

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Characteristics of Women Associated with Maternal Smoking . . . . . . . . . . . . . . . . . . Potential Pathway for Adverse EVects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neurocognitive and Behavioral Outcomes Associated with Maternal Smoking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Research Implications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

237 238 240 242 251 252 253

Neurobehavioral Assessment in Studies of Exposures to Neurotoxicants David C. Bellinger I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Data Collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

263 264 272

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IV. Data Interpretation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

279 292 293

From Animals to Humans: Models and Constructs Deborah C. Rice I. II. III. IV. V.

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tests of Cognition. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sensory Function. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Motor Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

301 303 317 326 329 330

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contents of Previous Volumes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

339 353