Biological Monitoring: Prospects in Occupational and Environmental Medicine. Deutsche Forschungsgemeinschaft (DFG) Copyright © 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-27795-7
Deutsche Forschungsgemeinschaft Kennedyallee 40, D-53175 Bonn, Federal Republic of Germany Postal address: D-53175 Bonn Telefon: ++49/228/885-1 Telefax: ++49/228/885-2777 E-Mail:
[email protected] Internet: http://www.dfg.de
Translator: Julia A. Handwerker-Sharman
This book was carefully produced. Nevertheless, editors, authors and publisher do not warrant the information contained therein to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate.
Library of Contress Card No.: applied for A catalogue record for this book is available from the British Library. Die Deutsche Bibliothek – CIP-Cataloguing-in-Publication-Data A catalogue record for this publication is available from Die Deutsche Bibliothek ISBN 3-527-27795-1 © 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim (Federal Republic of Germany), 2002 Printed on acid-free paper. All rights reserved (including those of translation in other languages). No part of this book may be reproduced in any form – by photoprinting, microfilm, or any other means – nor transmitted or translated into machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law. Cover Design and Typography: Dieter Hüsken Composition: K + V Fotosatz, 64743 Beerfelden Printing: betz-druck gmbh, Darmstadt Bookbinding: J. Schäffer GmbH & Co. KG, Grünstadt Printed in the Federal Republic of Germany.
Contents
1 1.1
Foreword . . . . . . . . . . . . . . . . . . . . . .
IX
The Importance of Biological Monitoring The Development and Importance of Biological Monitoring in the DFG and MAK Commission . . . .
1
Dietrich Henschler
2 2.1
Internal Exposure and Haemoglobin Adducts Biological Monitoring in Occupational and Environmental Medicine – The Present State of the Art and Future Prospects
. .
5
Metabolic Profiling – A Way of Better Understanding External and Internal Exposure to Organic Stubstances . . . . . . . . . . . . . . .
16
Jürgen Angerer
2.2
Albert W. Rettenmeier
2.3
Biological Monitoring of Arylamines and Nitroarenes . . . . . . . . . . . . . . . . . . .
24
Gabriele Sabbioni
V
Contents
3 3.1
DNA Adducts Genetic Cancer Susceptibility and DNA Adducts: Studies in Smokers and Coke Oven Workers . . . .
35
Magarita Rojas, Kroum Alexandrov, Helmut Bartsch and Berthold Spiegelhalder
3.2
The Detection of DNA Adducts in Biological Monitoring . . . . . . . . . . . . . .
46
Werner K. Lutz and Martin G. Maisch
3.3
32
P-Postlabelling HPLC Analysis of DNA Adducts in Breast Tissue . . . . . . . . . . . . . . . . . .
57
Wolfgang Pfau
3.4
Studies of 8-Hydroxy-2'-Deoxyguanosine: A Biomarker for Oxidative DNA Damage in vivo? . .
68
Boleslaw Marczynski, Jürgen Hölzer and Michael Wilhelm
4 4.1
Susceptibility Improved Methods of Phenotyping and Effect Monitoring for Evaluating the Risk to the Individual, using GSTT1 as an Example
. . .
78
Genetic Polymorphisms of Sulfotransferases as Susceptibility Parameters . . . . . . . . . . . .
84
Ernst Hallier
4.2
Hansruedi Glatt
4.3
Genotyping and Phenotyping, Using NAT2 as an Example . . . . . . . . . . . . . . . . . . .
96
Klaus Golka and Meinolf Blaszkewicz
4.4
New High-thoughput Technology in the Diagnostic Screening of Susceptibility Factors . Ricarda Thier, Thomas Brüning and Yon Ko
VI
103
Contents
5 5.1
Cytogenetic Parameters Biological Monitoring with Cytogenetic Methods . . . 110 Günter Obe, Helga Fender and Gisela Wolf
5.2
Examples of the Use of Three-colour Chromosome Painting in Cytogenetic Biomonitoring . . . . . . . . 121 Erich Gebhart, Irmgard Verdorfer and Susann Neubauer
5.3
The Comet Assay as a Biological Monitoring Test . . . 130 Günter Speit, Oliver Merk and Andreas Rothfuß
6 6.1
Immunology Immunoglobulins as Markers of Long-term Exposure to Allergenic Substances . . . . . . . . . . . . . . . 140 Hans Drexler
6.2
Immunological Effects of Polymorphic Key Enzymes
. 146
Jürgen Lewalter
7 7.1
Epidemiology Evaluation of Exposure in Epidemiological Studies . . 169 Kurt Ulm
7.2
Possibilities and Limitations of the Molecular Epidemiology of Workplace Exposures . . . . . . . . 175 Kurt Straif
8
Summary . . . . . . . . . . . . . . . . . . . . . . 191 Jürgen Angerer and Helmut Greim
9
Authors . . . . . . . . . . . . . . . . . . . . . . . 199
VII
Foreword
On 9th and 10th March 2000, at the invitation of the DFG (Deutsche Forschungsgemeinschaft) in Bonn, a symposium was held on the possibilities offered by biological monitoring in occupational and environmental medicine. Also discussed was the question of collaboration at a national level between the various disciplines in this field. These talks were prompted among other things by the following considerations. Not least thanks to the activities of the DFG, over the last 30 years biological monitoring has attained a high niveau in Germany. This has been assisted by the fact that the German legislation on the protection of health and safety at work immediately implemented the results of the work carried out by the Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area. As a result, in Germany the effectiveness of these preventive measures in the individual could be shown in practice, which in turn stimulated further research in this field. Germany can therefore claim to take a leading role in the field of biological monitoring, based on research and practice. New possibilities of biological monitoring in the form of biochemical and biological effect markers, such as e.g. protein and DNA adducts or cytogenetic parameters, may enable us to further improve the prevention of diseases caused by hazardous substances. Little is known to date, however, about the diagnostic meaningfulness of these parameters. For this reason, the experts from various fields, above all toxicology, occupational medicine, immunology, human genetics, analysis and epidemiology, should be brought together to clarify the importance of these parameters in future for the prevention of disease. In this context, what needs to be done to maintain or re-attain Germany‘s leading role in this important and dynamically developing field of research should also be evaluated. According to the wishes of the participants, this first exchange of ideas, made possible by the symposium, should form the nucleus for further research. The DFG, worthily represented in our field by Dr. Beate Konze-Thomas and her successor Dr. Armin Krawisch, is offered my thanks for makIX
Foreword ing this symposium possible. I would also particularly like to thank the DFG for all it has done over the past 45 years for research in the field of occupational-medical toxicology and thus for the working man. We also gratefully acknowledge the financial support of the translation of this work by the DFG Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area (Chairman: Helmut Greim). J. Angerer
X
1
The Importance of Biological Monitoring
1.1
The Development and Importance of Biological Monitoring in the DFG and MAK Commission Dietrich Henschler *
1.1.1 Forerunners The method of biological monitoring is much older than its name. Biological monitoring was first used 130 years ago, with the determination of salicyluric acid in urine to monitor the therapy of rheumatism with huge doses of salicylic acid. The name Biological Monitoring comes from the Englishspeaking countries, where it has been used for about 50 years; there is no equivalent German term. Biological monitoring relies on analytical methods for the determination of chemicals. Progress in biological monitoring can therefore only be achieved if progress is made in the field of analytical chemistry. Another driving force is the need in practice to know the concentrations of substances in the organism for the purpose of risk prevention. Occupational hygiene has taken on a pioneering role in this field. As early as 1890, the levels of lead in the blood and urine of workers from factories with exposure to lead were determined to protect workers at risk against episodes of acute lead poisoning; this led to the setting of tolerance thresholds for lead in blood and urine. A milestone in the progress made in this field were the investigations of Robert Kehoe (1933) into the absorption, distribution, storage and excretion of small doses of lead, which were designed to ensure the safety of tetraethyllead used as an additive in high performance motor fuels. Another exceptional early example of successful biological monitoring is the monitoring of employees exposed to trichloroethylene carried out since 1953 in Sweden. The determination of the main metabolite, trichloroacetic acid, in urine was provided by the manufacturer of the solvent and degreasing plants, and systematically carried out at regular intervals in all exposed workers. The Fujiwara method for trichloroacetic acid, which was robust and validated early on, confirmed after long years of experience the MAK value of 30 ml/m3 introduced very early in that country. *
Institut für Toxikologie der Universität Würzburg, Versbacher Str. 9, 97078 Würzburg
Biological Monitoring: Prospects in Occupational and Environmental Medicine. Deutsche Forschungsgemeinschaft (DFG) Copyright © 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-27795-7
1
1 The Importance of Biological Monitoring A third example is making use of effect parameters. The application of organophosphate compounds as insecticides in agriculture and to combat malaria led in the 1940s and 1950s to numerous cases of acute intoxication, some of which were lethal. In 1951 Wilson published his theories about the molecular mechanism of the intoxication: the irreversible binding of the phosphate ester residue to the serine in the catalytic centre of acetylcholinesterase. Practicable methods for the determination of the acetylcholinesterase activity in blood were rapidly developed which reliably show when hazardous amounts of organophosphate compounds have been incorporated. Since it was founded in 1955, the DFG Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area (MAK Commission) has recognized the value of biological monitoring and followed the examples given above in its endeavours. In the very first List of MAK Values (issue I, 1958), the possibilities, importance and value of the procedure are mentioned in the foreword.
1.1.2 Organization of the Commission From the beginning there has been a working group within the MAK Commission called “Analyses of Hazardous Substances in Air of Work Areas”. In 1975 a new working group was set up, “Analyses of Hazardous Substances in Biological Materials”. The head of this group was Jürgen Angerer, in Erlangen. Both groups published the methods they developed in continuous loose-leaf collections. The collection for the analyses in biological materials grew much more rapidly, which illustrates the particular interest of the analysts in the methods with a biological focus. After intensive preparatory consultation, in 1979 a new working group, “Setting of Threshold Limit Values in Biological Material”, was set up under the headship of Gerhard Lehnert, in Erlangen. This group carried out several pilot studies with suitable substances with the aim of defining threshold limit values in biological materials. At the same time comparable efforts were being carried out by the European Community and the TLV Committee in the USA. It was stipulated from the beginning – in analogy to the MAK values – that comprehensive scientific documentation was to be drawn up for the setting of threshold limit values. The first example of a biological threshold limit value was drawn up by H. M. Bolt. The name given to the new category was “Biological Tolerance Value for Occupational Exposures” (BAT value). The Commission introduced the first BAT values into the List of MAK Values in 1982, with an extensive explanatory introduction; it was then called “Maximum Concentration Values at the Workplace and Biological Tolerance Values for Occupational Exposures”, from 1992 shortened to the “List of MAK and BAT Values”. Since 1983, exten2
1.1 The Development and Importance of Biological Monitoring sive scientific documentation has been published at irregular intervals corresponding with the introduction of new BAT values in the list (Verlag Chemie VCH/Wiley, Weinheim). A few years after their introduction by the MAK Commission, the BAT values were incorporated in the official regulations of the AGS (Committee for Hazardous Substances) of the BMA (Federal Ministry for Labour) in the form of the technical regulation TRGS 903; this stipulates the carrying out of analyses in biological materials under certain conditions.
1.1.3 Research Activities of the MAK Commission Several members of the Commission have made significant experimental contributions to the further development of the system. From 1980–1989 a series of research projects focusing on carcinogenic substances at the workplace received financial support from the DFG as part of a special promotion programme. As a result, Germany played a pioneering role in the research into biological monitoring.
1.1.4 Criteria BAT values differ from MAK values as follows: • BAT values protect the individual; MAK values, however, as samples are taken from the ambient air, apply to collectives. • BAT values are absolute peak values, while MAK values can be taken as average values over time. • Observance of BAT values should guarantee that no (biological) changes occur that are adverse to the health. • Observance of a BAT value for one substance should not lead to amplification of the effects of other substances. • BAT values, determined according to the rules of toxicology, should correlate with the corresponding MAK values. • The effects observed within the range of BAT values should be fully reversible. For this reason, BAT values are not given for carcinogenic substances. Instead, “Exposure equivalents for carcinogenic substances” (EKAs) are given for defined concentrations/doses of this class of substance. 3
1 The Importance of Biological Monitoring
1.1.5 Peripheral Conditions BAT values relate to “internal exposure”. Depending on the effects studied and the database, either the substance itself, its metabolites, the products of the reaction of the substance and/or metabolites with endogenous structures (e.g. adducts with DNA or haemoglobin), or functional changes are determined. The biological matrices investigated to date are: blood, urine, faeces, skin, hair, tissues and exhaled air. While MAK values are valid without having to take the physical condition of the worker into consideration, BAT values take into account a series of particular influences: physical stress, which is manifest in changing respiratory volumes and thus in different rates of absorption of the substances; the personal care taken by each worker, personal hygiene at work and outside; previous illnesses; age and sex; type and extent of differences in metabolism (polymorphism); and changes in toxicokinetic behaviour, above all in the excretion of substances and their metabolites.
1.1.6 Conclusions Since their introduction into the German List of MAK Values, 44 BAT values and 13 EKA values for carcinogenic substances have been included. Their number will probably remain far below the number of MAK values (about 750 substances). The main reasons for this: only for a limited number of substances have the necessary toxicokinetic data been evaluated and suitable analytical methods drawn up. Nevertheless, biological monitoring and the BAT values are an important instrument of occupational health care. The underlying principle of protection of the individual puts into practice a maxim of the national constitution (article 2), which puts protection of the individual on the same level as protection of the collective. In this important sector of occupational health care, the Commission has carried out decisive and internationally recognized pioneering work.
4
2
Internal Exposure and Haemoglobin Adducts
2.1
Biological Monitoring in Occupational and Environmental Medicine – The Present State of the Art and Future Prospects Jürgen Angerer *
Biological monitoring is a measure used in occupational and environmental medicine in the protection of the individual against the harmful effects of toxic substances. It is used to estimate the extent to which a person has been exposed to a substance and the resulting effects on the person‘s health (Zielhuis 1980, Angerer & Gündel 1996, Kommission Human-Biomonitoring des Umweltbundesamtes 1996, Schaller & Angerer 1998). Biological monitoring today differentiates between dose monitoring, biochemical effect monitoring and biological effect monitoring. Dose monitoring is the determination of the hazardous substance or its metabolites in body fluids. Biochemical effect monitoring is the quantification of the products of the reaction of mutagenic substances with germ plasm. Proteins and their adducts with mutagenic substances are regarded as a surrogate for DNA. We speak of biological effect monitoring when the first reactions of the body to the exposure are detectable, e. g. changes in enzyme activity or genetic parameters. The predictive importance for the effects on the health increase in the order dose monitoring, biochemical effect monitoring, biological effect monitoring (Figure 1). Biological monitoring supplements the determination of hazardous substances in workplace air and in the various environmental media, and in addition has numerous advantages. Simplified, it can be said that the main advantage of biological monitoring is that it provides information about whether and to what extent a person absorbs hazardous substances from his environment. This is of great importance in particular in the area of environmental medicine, as all over the country chemicals are determined in all kinds of materials, such as wood, building materials, the dust collected by vacuum cleaners etc., without previously a relationship being evaluated between the level of hazardous substances in these media and the dose taken up by the person. *
Institut für Arbeits-, Sozial- und Umweltmedizin, Universität Erlangen-Nürnberg, Schillerstr. 25/29, 91054 Erlangen
Biological Monitoring: Prospects in Occupational and Environmental Medicine. Deutsche Forschungsgemeinschaft (DFG) Copyright © 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-27795-7
5
2 Internal Exposure and Haemoglobin Adducts
Figure 1: Monitoring hazardous substances.
The advantages offered by biological monitoring were recognized early on in Germany and since the end of the 1960s continuously developed. Responsible for this to a large extent is certainly the DFG Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area, which made great advances in the analytical field (Analyses of Hazardous Substances in Biological Materials 1976–1999). In 1980 the Commission was the first body world-wide to evaluate threshold limit values for internal exposure to hazardous substances, the so-called Biological Tolerance Values for Occupational Exposures (BAT) (see BAT value documentation). Today this work is supplemented by the ‘Humanbiomonitoring’ Commission of the Federal Environmental Agency, who evaluate threshold limit values and reference values for the exposure to hazardous substances of the general population (Kommission Human-Biomonitoring des Umweltbundesamtes 1996). This Commission specifically uses the analytical basis created by the DFG. The German Society of Occupational Medicine and Environmental Medicine must also be mentioned, which, in 1982, began to carry out external quality control in the form of an intercomparison programme for occupational-medical and environmental-medical, toxicological analyses (Schaller et al. 1984). In retrospect it seems a good thing that at the beginning of the 1980s the European Community withdrew its involvement in the evaluation and implementation of biological monitoring at the workplace. Before this, all guidelines and regulations of the European Community pertaining to pro6
2.1 Biological Monitoring in Occupational and Environmental Medicine tection against the effects of hazardous substances at the workplace included biological monitoring (Angerer & Schaller 1990). Afterwards, the guidelines of the European Community no longer included notes on the implementation of biological monitoring for the protection of the individual against disease. It is only now that this kind of preventive health protection has been resumed (guideline 98/24/EG). This means that while our European partners have remained more or less at the level of the guidelines for lead (guideline 82/605/EG), which stipulate biological monitoring for cases of lead poisoning, in Germany biological monitoring has been developed further, so that today we take a leading position in this field, or at least we could take a leading position. This led to the question posed in this symposium: how can we in Germany maintain and further our leading position in the development of biological monitoring and make this clear internationally? The prerequisite for biological monitoring is the availability of sensitive, specific analytical procedures with which the various parameters in body fluids can be determined. It is therefore no coincidence that the introduction of new methods of instrumental analysis have always also led to innovations in biological monitoring. In the 1960s it was atomic absorption spectrometry which first allowed the analysis of metals in the concentration range in question. From the mid 1970s the ever cheaper GC/MS combinations allowed organic substances to be determined down to the pg/l range. These analytical developments and the progress made are documented in the collection of methods Analyses of Hazardous Substances in Biological Materials of the DFG Commission (Analysen in biologischem Material 1976 – 1999). This collection of methods represents the analytical state of the art in the field of occupational and environmental medicine. With these methods it is possible to carry out dose monitoring for many substances. Increasingly, also methods for biochemical effect monitoring are being included in the collection. Naturally this collection of methods also contains the classic methods of biological effect monitoring, such as the determination of d-aminolaevulinic acid in urine in the case of exposure to lead. Not included at present are methods for the determination of DNA adducts. Not yet included are susceptibility markers. The latter will in future, however, as decreed by the DFG Commission, be included in the collection of methods. Cytogenetic parameters and immunological parameters are also not included in this collection of methods. In the case of dose monitoring, today we can determine practically all the relevant metals in body fluids (Tab. 1). In addition to merely detecting the metals, today we are working towards analysing them in their different bonding states. With so-called species analysis it is possible, for example, to separate carcinogenic inorganic arsenic compounds from less toxic organic arsenic compounds and to detect them in urine. Here, we are still right at the beginning of exciting new possibilities. Among the organic substances, solvents and also important groups of substances such as aromatic amines, aromatic nitro compounds and PAHs can be determined (Tab. 2). 7
2 Internal Exposure and Haemoglobin Adducts Table 1: Biological monitoring of metals. Metals
Blood
Urine
Aluminium Antimony Arsenic Barium Beryllium Cadmium Chromium Cobalt Lead Mercury Molybdenum Nickel Palladium Platinum Selenium Silver Thallium Vanadium
+ – – – + + + + + + – + – + + – – –
+ + + + + + + + + + + + + + + + + +
Others CO-Hb Fluoride ALA (lead)
+ – –
– + +
Table 2: Biological monitoring of organic substance. Solvent
Blood
Urine
Aliphatic hydrocarbons Aromatic hydrocarbons Halogenated hydrocarbons Alcohols, ketones PAHs Aromatic amines Aromatic nitro compounds
+ + + + – (+) (+)
– – – + + + +
Table 3 shows the spectrum of solvent metabolites that can be routinely determined in urine today. Persistent organic chlorines, which can be determined in blood and urine in very low concentrations, are more of environmental-medical relevance (Tab. 4). 8
2.1 Biological Monitoring in Occupational and Environmental Medicine Table 3: Biological monitoring of solvents. Substance
Parameters
Aromatic substances Benzene
S-Phenylmercapturic acid Muconic acid o-Cresol Methylhippuric acid Mandelic acid, phenylglyoxylic acid
Toluene Xylenes Ethylbenzene, styrene Chlorobenzenes
Chlorophenols
Aliphatic substances n-Hexane
Hexanedione
Halogenated hydrocarbons Trichloroethylene
Trichloroacetic acid
Glycol ethers Ethylene glycol monomethyl ether Ethylene glycol monoethyl ether Ethylene glycol monobutyl ether
2-Methoxyacetic acid 2-Ethoxyacetic acid 2-Butoxya cetic acid
Carbon disulphide
TTCA
Vinyl chloride
Thiodiglycolic acid
Table 4: Biological monitoring of persistent organic chlorines. Organic chlorines
Blood
Urine
DDT, DDE Hexachlorocyclohexanes Polychlorinated biphenyls (PCBs) Hexachlorobenzene Pentachlorophenol Chlorophenols (CPs) Chlorobenzenes (CBs)
+ + + + + – –
– + – – + + +
For the non-persistent pesticides, we are beginning to develop methods for biological monitoring. Nevertheless, I would like to show you the capability of dose monitoring with an example from the environmental-medical range. Food analysts and the respective authorities previously believed that pesticides are barely taken up from foodstuffs. In a joint project supported by the BMBF (Ministry of Education and Science), we evaluated, among other things, a very sensitive method for the determination of organophosphate metabolites in urine. In the meantime, with this method we have investigated over 1000 persons from the general population, including about 500 adults 9
2 Internal Exposure and Haemoglobin Adducts
Figure 2: Exposure of the adult general population to the organic phosphates dimethylphosphate (DMP), dimethylthiophosphate (DMTP), dimethyldithiophosphate (DMDTP), diethylphosphate (DEP), diethylthiophosphate (DETP) and diethyldithiophosphate (DEDTP).
(Figure 2). In about 80 % of the general population we detected dimethylphosphate and dimethylthiophosphate in the urine. If we consider the 95th percentiles, which are around 100 lg/g creatinine, the concentrations are impressive (Hardt & Angerer 2000). The reference value for the excretion of PCP in urine in the general population is 8 lg/l. The reference value for the concentration of lead in blood is 80 lg/l. We have therefore come to the conclusion that the exposure to organic phosphates in the general population is relatively homogenous. The same is also true for pyrethroids, but the detectable concentrations are lower by about two orders of magnitude (Hardt et al. 1999). Our assumption at present is that both organic phosphates and pyrethroids are taken up mainly with food. This insight is currently causing the authorities responsible a few headaches. It must also be noted here that for most of the non-persistent pesticides there are at present no suitable methods available for biological monitoring. In most cases we do not even know anything about the metabolism of the pesticides in man. To conclude, it can be said that in the field of dose monitoring we are able today to detect exposure to hazardous substances down to the environmental-medical range. The spectrum of parameters is already impressive, but must be continually extended. For certain groups of substances, such as e. g. pesticides, there are still great gaps in our knowledge. Progress has been made also in biochemical effect monitoring. Over the last few years much work has been done in the field of protein adducts, in particular haemoglobin adducts. Taken together, the numerous studies carried out in man provide the following simplified picture. By means of Edman degradation, the adducts bound to N-terminal valine are separated off. With this method, above all the alkylating substances 10
2.1 Biological Monitoring in Occupational and Environmental Medicine Table 5: Biochemical effect monitoring – protein adducts in man. Alkylating substances e.g. Ethylene oxide Butadiene Acrylonitrile Acrylamide Propylene oxide Styrene Aromatic amines Aromatic nitro compounds PAHs Others e.g. Dimethylformamide Benzoquinone
such as ethylene oxide, styrene, butadiene and acrylonitrile can be detected. The adducts of aromatic amines and the adducts of PAHs are cleaved hydrolytically from their bond with cysteine. Of course also the adducts of aromatic nitro compounds can be determined in the form of their reduction products (Tab. 5) (see e. g. Angerer et al. 1998, Bolt 1996, Ehrenberg et al. 1996, Farmer et al. 1996, Farmer H. 1995, Müller et al. 1998, Neumann et al. 1995, Pastorelli et al 1999, Sabbioni et al. 1996, Schütze et al. 1995, Sepai et al. 1995, Severi et al. 1994, Skipper et al. 1990, Tannenbaum 1991, Thier et al. 1999, Van Welie et al. 1992, Waidyanatha et al. 1998). Today Hb adducts can be determined in the leading laboratories routinely and very sensitively. In the blood of smokers, for example, hydroxyethylvaline and cyanoethylvaline can be determined. This is not the case in non-smokers. In addition, smokers and non-smokers both have not inconsiderable amounts of methylvaline in the blood, which is formed as a result of endogenous methylation (Bader et al. 1995). With the detection of haemoglobin adducts we are also learning more about the metabolism of substances. Dimethylformamide is an important organic solvent which can cause severe damage to the liver. Whether it is also mutagenic or carcinogenic is still unclear. We have discovered that with occupational exposure to DMF, the amounts of mercapturic acids of this solvent excreted in the urine of man are much greater than those found in experiments with animals. This means, among other things, that in man a methylisocyanate can be formed as an intermediate from DMF which can bind not only with glutathione, but also with haemoglobin or DNA (Figure 3). To test this hypothesis we did not add isothiocyanate to the globin, the usual Edman degradation procedure, in order to induce a ring formation reaction. Instead, we merely heated the potentially formed haemo11
2 Internal Exposure and Haemoglobin Adducts
Figure 3: Hypothetical, simplified metabolism of DMF.
globin adduct in an acidic milieu, on the assumption that the isocyanate, if it had actually been formed and had formed a ring adduct, would then be cleaved from haemoglobin. The results are shown in Figure 4. In ten persons exposed to DMF, we detected much higher adduct concentrations than the background exposure (Angerer et al. 1998). In the meantime we have confirmed these results with a large collective of persons occupationally exposed to DMF. This result shows on the one hand that DMF may be mutagenic, and on the other hand that the assumed formation of methylisocyanate is really possible. Our next aim is therefore to try to detect also the DNA adducts resulting from exposure to DMF. Progress has therefore been made in the determination of haemoglobin adducts, and further progress is possible in the future. This applies to both the monitoring function and the knowledge gained from the detection of adducts. For biochemical effect monitoring, our aim is also to be able to determine DNA adducts by means of instrumental analysis. This applies to the DNA adducts in blood and their products of degradation which are excreted in urine. 8-Hydroxy-2-deoxyguanosine is such a nucleoside, and is determined today by many researchers. Today the detection of chemically modified nucleosides looks very promising for various reasons. As we know, the four DNA bases react with mutagenic chemicals to form defined compounds, which are removed from the DNA strand by a repair mechanism and excreted in the urine. Determination of these compounds in the urine must make it possible to quantify substance-specific biochemical effects. 12
2.1 Biological Monitoring in Occupational and Environmental Medicine
Figure 4: Adducts formed in persons exposed to DMF and in controls.
New preparation methods and specific enrichment techniques make this target seem within reach. In particular when combined with LC/MS/ MS. The combination of HPLC and mass spectrometry, aimed at for decades, today seems technically possible and should allow great progress to be made, particularly in the field of biological monitoring. The future prospects are therefore as follows: 1. New analytical methods, such as ICP/MS and LC/MS/MS, will open up new possibilities for biological monitoring. 2. Progress will be made in particular in the preparation of the biological material and the enrichment of the analytes, for example on tailor-made columns and by using on-line techniques. 3. In the field of dose monitoring we will extend the substance boundaries. Methods for the determination of pesticides in biological material are feasible. We will also gain further insights into the metabolism of the various hazardous substances in man. 4. It is of central importance for occupational and environmental medicine to further extend biochemical effect monitoring. This applies for Hb adducts and in particular of course for DNA adducts of mutagenic substances. The biochemical effect markers are closer to the ultimate toxic substance than mere dose monitoring. 13
2 Internal Exposure and Haemoglobin Adducts References Angerer, J.; Bader, M.; Krämer, A.: Ambient and biochemical effect monitoring of workers exposed to ethylene oxide, Int Arch Occup Environ Health 71, 14–18 (1998). Angerer, J.; Göen, T.; Krämer, A.; Käfferlein, H. U.: N-Methylcarbamoyl adducts at the N-terminal valin of globin in workers exposed to N,N-dimethylformamide. Arch. Toxicol. 72: 309–313 (1998). Angerer, J.; Gündel, J.: Biomonitoring and occupational medicine. Possibilities and limitations, Ann. Ist. Super Sanità 32 (2): 199–206 (1996). Angerer, J.; Schaller, K. H. (eds): Analysen in biologischem Material. Deutsche Forschungsgemeinschaft, Ringbuchsammlung Lieferung 1–13, Wiley-VCH Verlag, Weinheim (1976–1999). Angerer, J.; Schaller, K. H.: Auswirkungen der Harmonisierung des sozialen Arbeitsschutzes in der Europäischen Gemeinschaft auf die Prävention toxisch bedingter Gesundheitsschäden in der Bundesrepublik Deutschland, vorgetragen auf der: Jahrestagung der Deutschen Gesellschaft für Arbeitsmedizin e.V.; Frankfurt-Hoechst, 28.–31. Mai (1990). Bader, M.; Lewalter, J.; Angerer, J.: Analysis of N-akylated amino acids in human hemoglobin: evidence for relevated N-methylvaline levels in smokers, Int. Arch. Occup. Environ. Health 67: 237–242 (1995). Bolt, H. M.: Butadiene and isoprene: future studies and implications, Toxicology 113, 356–360 (1996). EG-Richtlinie/Council Directive of 12 June 1989 on the introduction of measures to encourage improvements in the safety and health of workers at work (89/291/EEC), Official Journal of European Communities No. L 183/1 (1989). EG-Richtlinie/Council Directive of 27 November 1980 on the protection of workers from the risks related to exposure to chemical, physical and biological agents at work (80/ 1107/EEC), Official Journal of the European Communities No. L 327/8 (1980). EG-Richtlinie/Council Directive of 28 July 1982 on the protection of workers from the risks related to exposure to metallic lead and its ionic compounds of work (first individual Directive within the meaning of Article 8 of Directive 80/1107/EEC) (82/605/ EEC), Official Journal of European Communities No. L 247/12 (1982). Ehrenberg, L.; Granath, F.; Törnqvist, M.: Macromolecule Adducts as Biomarkers of Exposure to Environmental Mutagens in Human Populations, Environ. Health Perspect, 104 (3), 423–428 (1996). Farmer, P. B.: Monitoring of human exposure to carcinogens through DNA and protein adduct determination, Toxicol. Lett.; 82/8, 757–762 (1995). Farmer, P. B.; Sepai, O.; Lawrence, R.; Autrup, H.; Sabro Nielsen, P.; Vestergard, A. B.; Waters, R.; Leuratti, C.; Jones, N. J.; Stone, J.; Baan, R. A.; van Delft, J. H. M.; Steenwinkel, M. J. S. T.; Kyrtopoulos, S. A.; Souliotis, V. L.; Theodorakopoulus, N.; Bacalis, N. C.; Natarajan, A. T.; Tates, A. D.; Haugen, A.; Andreassen, A.; Øvrebø, S.; Shuker, D. E. G.; Amaning, K. S.; Schouft, A.; Ellul, A.; Garner, R. C.; Dingley, K. H.; Abbondandolo, A.; Merlo, F.; Cole, J.; Aldrich, K.; Beare, D.; Capulas, E.; Rowley, G.; Waugh, A. P. W.; Povey, A. C.; Haque, K.; Kirsch-Volders, M.; Van Hummelen, P.; Castelain, P.: Biomonitoring human exposure to environmental carcinogenic chemicals, Mutagenesis, 11 (4), 363–381 (1996). Hardt, J.; Angerer, J.: Determination of dialkyl phosphates in human urine using gas chromatography-mass spectrometry, J. Anal. Toxicol.; 678–684 (2000). Hardt, J.; Heudorf, U.; Angerer, J.: Zur Frage der Belastung der Allgemeinbevölkerung durch Pyrethroide – Kurzmitteilung, Umweltmed. Forsch. Prax.; 4 (1), 54–55 (1999). Kommission „Human-Biomonitoring“ des Umweltbundesamtes: Human-Biomonitoring: Definitionen, Möglichkeiten und Voraussetzungen sowie Qualitätssicherung und
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2.1 Biological Monitoring in Occupational and Environmental Medicine Konzept der Referenz- und Human-Biomonitoring-Werte in der Umweltmedizin, Bundesgesundheitsbl.; 6, 205–244 (1996). Müller, M.; Krämer, A.; Angerer, J.; Hallier, E.: Ethylene oxide protein adduct formation in humans: Influence of glutathione-S-transferase polymorphisms, Int. Arch. Occup. Environ. Health, 71, 499–502 (1998). Neumann, H. G.; Albrecht, O.; van Dorp, C.; Zwirner-Baier, I.: Macromolecular adducts caused by environmental chemicals, Clin. Chem. 41/12, 1835–1840 (1995). Pastorelli, R.; Guanci, M.; Restano, J.; Berri, A.; Micoli, G.; Minoia, C.; Alcini, D.; Carrer, P.; Negri, E.; La Vecchia, C.; Fanelli, R.; Airoldi, L.; Seasonal effect on airborne pyrene, urinary 1-hydroxypyrene, and benz(a)pyrene diol epoxide-hemoglobin adducts in the general population, Cancer Epidemiol. Biomarkers Prev.; 8, 561–565 (1999). Sabbioni, G.; Wei, J.; Liu, Y. Y.: Determination of hemoglobin adducts in workers exposed to 2,4,6-trinitrotoluene, J. Chromatogr. B. Biomed. Appl.; 682, 243–248 (1996). Schaller, K. H.; Angerer, J.: Biomonitoring in der Umweltmedizin, Umweltmed. Forsch. Prax.; 3 (3): 168–175 (1998). Schaller, K. H.; Angerer, J.; Lehnert, G.; Valentin, H.; Weltle, D.: Externe Qualitätssicherung arbeitsmedizinisch-toxikologischer Untersuchungen in der Bundesrepublik Deutschland, Arbeitsmed. Sozialmed. Präventivmed.; 19, 79–84 (1984). Schütze, D.; Sepai, O.; Lewalter, J.; Miksche, L.; Henschler, D.; Sabbioni, G.: Biomonitoring of workers exposed to 4,4’-methylenedianiline or 4,4’-methylenediphenyl diisocyanate, Carcinogenesis, 16, 573–582 (1995). Sepai, O.; Henschler, D.; Sabbioni, G.: Albumin adducts, hemoglobin adducts and urinary metabolites in workers exposed to 4,4’-methlylenediphenyl diisocyanate, Carcinogenesis 16, 2583–2587 (1995). Severi, M.; Pauwels, W.; Van Hummelen, P.; Roosels, D.; Kirsch-Volders, M.; Veulemans, H.: Urinary mandelic acid and hemoglobin adducts in fiberglass-reinforced plastics workers exposed to styrene, Scand. J. Work Environ. Health, 20, 451–458 (1994). Skipper, P. L.; Tannenbaum, S. R.: Protein adducts in the molecular dosimetry of chemical carcinogens, Carcinogenesis, 11 (4), 507–518 (1990). Tannenbaum, S. R.: Hemoglobin-carcinogen adducts as molecular biomarkers in epidemiology, in: Ernster, L. (eds): Xenobiotics and Cancer, Japan SGL Soc. Press, Tokyo, Taylor & Francis Ltd. London, pp. 351–360 (1991). Thier, R.; Lewalter, J.; Kempkes, M.; Selinski, S.; Bruning, T.; Bolt, H. M.: Haemoglobin adducts of acrylonitrile and ethylene oxide in acrylonitrile workers, dependent on polymorphisms of the glutathione transferases GSTT1 and GSTM1, Arch. Toxicol.; 73, 197–202 (1999). Van Welie, R. T. H.; van Dijck, R. G. J. M.; Vermeulen, N. P. E.: Mercapturic acids, protein adducts, and DNA adducts as biomarkers of electrophilic chemicals, Crit. Rev. Toxicol.; 22 (5/6), 271–306 (1992). Waidyanatha, S.; Yeowell-O‘Connell, K.; Rappaport, S. M.: A new assay for albumin and hemoglobin adducts of 1,2- and 1,4-benzoquinones, Chem. Biol. Interact. 115: 117–139 (1998). Zielhuis, R. L.: Recent and potential advances applicable to the protection of workers’ health: Biological Monitoring. Presented at the international seminar Assessment of toxic agents at the workplace – Roles of ambient and biological monitoring. Workshop, Luxemburg, 8–12 December (1980).
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2.2
Metabolic Profiling – A Way of Better Understanding External and Internal Exposure to Organic Substances Albert W. Rettenmeier *
For the substances given in the List of MAK and BAT Values which have biological threshold limit values or exposure equivalents, the internal dose or internal exposure is at present mainly determined on the basis of a single parameter. For the organic substances the concentration of a metabolite in urine is usually used. In some cases the internal exposure is also deduced from the concentration of the substance itself in blood or urine. For carcinogenic substances haemoglobin adducts can also be used as exposure parameters. In those cases in which it is possible to determine a metabolite directly responsible for the critical toxic effects, this procedure is satisfactory. In addition to those substances which form haemoglobin adducts, an example of such a substance is the solvent n-hexane. The metabolism of n-hexane is shown in Figure 1. n-Hexane is metabolized in several steps to 2,5-hexanedione. This is a metabolite that we are able to analyse, and is not only from a quantitative point of view of particular importance. It is also the metabolite which is directly responsible for the critical toxic effect – in this case peripheral polyneuropathy. Its concentration in urine is a measure of the health risk resulting from the exposure to n-hexane. For many substances, however, the critical toxic effects are not caused by a metabolite in the dominant metabolic pathway, but by metabolites that are quantitatively of much less importance. Often several toxic effects must be taken into account when monitoring the external and internal exposure; they may be caused by metabolites from different metabolic pathways. It must be borne in mind that the various metabolic pathways • • • *
16
compete with one another during metabolism, may become saturated if the substance is present in sufficient quantities, are subject to intra-individual and inter-individual fluctuations,
Institut für Hygiene und Arbeitsmedizin, Universitätsklinikum Essen, Hufelandstr. 55, 45147 Essen
2.2 Metabolic Profiling – A Way of Better Understanding
Figure 1: Metabolism of n-hexane.
•
and can be promoted or inhibited by previous or accompanying exposures.
A much more accurate picture of the internal exposure and the risks associated with it is therefore obtained if not only one metabolite, but metabolites from various metabolic pathways can be used for biological monitoring, in other words, if the metabolic profile can be drawn up. Metabolic profiling is the name given to the quantitative determination of metabolic products from the various metabolic pathways of endogenous or exogenous substances. The aim of metabolic profiling is to •
comprehensively determine the quantitative importance of the various metabolic pathways and thus the exposure and, if possible, the effects, 17
2 Internal Exposure and Haemoglobin Adducts • •
highlight deviations from the “normal” patterns of excretion, and thus reveal the individual risks resulting from exposure at an early stage.
The metabolic profile is determined mainly by the functional state of the cells involved in metabolism. The metabolic profile thus reflects the current functional state of these cells. Metabolic profiling was used first of all for detecting congenital metabolic disturbances. Abnormal profiles for organic acids excreted in urine can indicate the presence of metabolic defects (Rashed et al. 1997). In addition, metabolic profiling is used mainly to explain the mechanisms of action of drugs and to estimate the risk of side-effects associated with them. The latter is shown in the example below. Figure 2 shows the complex metabolic degradation of the antiepileptic drug valproic acid (VPA) (Rettenmeier et al. 1989). In the 1970s and 1980s a small proportion of the patients treated with valproic acid suffered severe liver intoxication, which led to death in more than 100 cases. Epidemiological studies showed that in particular infants and small children were affected who were treated at the same time with other anti-epileptic drugs, in particular those which induced cytochrome P 450. The cause of the liver damage was thought to be on the one hand the increased formation of hepatotoxic valproic acid metabolites, such as the terminally unsaturated metabolite 4-ene-VPA (metabolite 1) and 2,4-diene-VPA (metabolite 7). On the other hand, also defective or decompensating metabolic pathways seemed to play a causal role. This applies in particular to b-oxidation, which is usually the quantitatively most important phase I metabolic pathway of valproic acid, leading to the formation of 3-oxo-VPA (metabolite 12). The discovery that the terminal desaturation is a cytochrome P 450-dependent reaction, which can be induced by anticonvulsive co-medication with phenobarbital or phenytoin, was decisive for understanding the hepatotoxic effects of valproic acid (Rettie et al. 1987). After an adequate analytical procedure had been developed for metabolic profiling after administration of valproic acid (Rettenmeier 1989), it was investigated whether patients potentially at risk from a therapy involving valproic acid could be identified on the basis of the profile of the metabolites in urine after a single oral dose of valproic acid (Rettenmeier et al. 1990). Most of the 22 paediatric patients included in the study were undergoing anticonvulsive therapy with phenobarbital when valproic acid was administered. The diagrams in Figure 3 show the relative amounts of the hepatotoxic metabolite 4-ene-VPA and the oxidation product 3-oxo-VPA excreted by the individual patients. The concentration of the hepatotoxic metabolite was on average 1–2 % of the concentration of the oxidative metabolite. Excretion of the hepatotoxic 4-ene-VPA metabolite was found to be higher in most patients undergoing therapy with phenobarbital (dark col18
2.2 Metabolic Profiling – A Way of Better Understanding
Figure 2: Metabolism of valproic acid.
19
2 Internal Exposure and Haemoglobin Adducts
a)
b)
c) Figure 3: Relative recovery of 4-ene-VPA and 3-oxo-VPA in urine after single oral doses of valproic acid (in % of the renally eliminated products of valproic acid). The final diagram shows the relationship between the terminal/subterminal oxidation products of valproic acid (cytochrome P 450-dependent products) and the products of mitochondrial b-oxidation.
20
2.2 Metabolic Profiling – A Way of Better Understanding umn) than in other patients. In 2 of the 22 patients comparatively little 3oxo-VPA was formed. In these two patients the ratio of cytochrome P 450dependent terminal and subterminal oxidation products to b-oxidation products was weighted clearly in favour of the former. This indicated impairment of mitochondrial b-oxidation. An explanation for this finding is the low levels of carnitine in these patients. Carnitine is necessary for transporting fatty acids, one of which is also valproic acid, into the mitochondria. By drawing up the metabolic profile it was therefore possible for this relatively small collective of 22 patients to detect both the inducing effect of medication and also a metabolic disturbance. Both factors increase the risk of hepatotoxicity. The interesting thing about this was that the metabolic disturbance was recognized not in the increased formation of a metabolite, but in its decreased formation. Metabolic profiling therefore proved a suitable procedure for identifying early on patients at risk from therapy involving valproic acid. Although in analogy metabolic profiling would seem to be suitable for detecting the risks to the individual from exposure to chemicals at the workplace, to date there are only few studies available which investigate this possibility. For example, studies of the metabolism of trichloroethylene (Bernauer et al. 1996). In the case of trichloroethylene oxidative and reductive metabolic pathways compete with one another; the capacity of the oxidative metabolic pathway is 1000 to 10000 times greater than that of the reductive
Figure 4: Metabolism of trichloroethylene (simplified) [acc. Dekant W. et al. 1984].
21
2 Internal Exposure and Haemoglobin Adducts pathway. The two metabolic pathways lead to different toxic effects. While the products of oxidative metabolism cause central nervous and hepatotoxic effects, the products of the reductive, glutathione-dependent pathway are responsible for the nephrotoxic and nephro-carcinogenic effects. If with high levels of exposure the oxidative metabolic pathway becomes saturated, the reductive metabolic pathway gains in importance and the risk of kidney damage increases. The ultimate carcinogenic agents formed via the b-lyase reaction cannot be determined. Other metabolites of the reductive metabolic pathway, such as 1,2-dichlorovinyl-cysteine, 2,2-dichlorovinyl-cysteine and also 1,2-dichlorovinyl mercapturic acid, have been detected, however, in experimental animals and also in the urine of exposed persons, and are thus open to analysis. It is recommended at present that the oxidative metabolites trichloroethanol and trichloroacetic acid are used to determine the exposure. By analysing the products from both oxidative and reductive metabolism, it would be possible, over and above evaluating the risk from trichloroethylene itself and the oxidative metabolites, to better estimate the nephrotoxic and nephro-carcinogenic risk. Determination of the metabolites of oxidative and reductive metabolism, however, requires separate analytical procedures. Another example of where metabolic profiling can be put to good use is the monitoring of the health of workers exposed to ethylene glycol monobutyl ether. Ethylene glycol monobutyl ether is oxidized in 2 main steps via the corresponding aldehyde to butoxyacetic acid, which causes the haemolytic effects of this glycol ether. Ethylene glycol monobutyl ether has a much shorter half-life than its short-chain homologues ethylene glycol monomethyl ether and ethylene glycol monoethyl ether, probably as a result of the formation of an unusual glutamine conjugate of butoxyacetic acid, which, as a readily water-soluble product, is rapidly eliminated (Rettenmeier et al. 1993). There is evidence that this glutamine conjugate, which is usually involved in the excretion of butoxyacetic acid in amounts of up to 70%, is not formed in some persons or is formed in only very small amounts. At the same level of exposure this may result in an increased risk of developing haemolysis. Monitoring the exposure by determining butoxyacetic acid, even after previous hydrolysis of the conjugate, is unsuitable for identifying these persons at risk. In the meantime a method has been developed which allows the free butoxyacetic acid and the glutamine conjugate to be simultaneously determined together with the other metabolites and thus a complete metabolic profile to be drawn up in one analytical process (Müller & Rettenmeier 1999). These few examples clearly illustrate the advantages of metabolic profiling for the monitoring of exposure and its effects. Metabolic profiling, however, involves complicated analytical procedures and requires detailed knowledge of the metabolism of the substance.
22
2.2 Metabolic Profiling – A Way of Better Understanding There is a need for further research in particular: 1. In the area of metabolic degradation, which for many substances used at the workplace is only incompletely understood. As most metabolites are not commercially available, they must be synthesized, which is often very difficult. 2. In the development of analytical methods for drawing up metabolic profiles. Generally only GC/MS and LC/MS methods can be used, as many of the metabolites, which in some cases are present only in very small concentrations, can only be determined with special mass spectrometric methods. 3. In the development of computer programs which can process the profile data and identify abnormal profiles. Reference ranges and cut-off values for the critical metabolites must be determined in field studies and the laboratory and by using data from experiments with animals. References Bernauer, U.; Birner, G.; Dekant, W.; Henschler, D.: Biotransformation of trichloroethene: dose-dependent excretion of 2,2,2-trichloro-metabolites and mercapturic acids in rats and humans after inhalation, Arch. Toxicol.; 70, 338–346 (1996). Dekant, W.; Metzler, M.; Henschler, D: Novel metabolites of trichloroethylene through dechlorination reactions in rats, mice and humans, Biochem. Pharmacol.; 33, 2021– 2027 (1984). Müller, G.; Rettenmeier, A. W.: Simultaneous GC/MS determination of butoxyacetic acid and its glutamine conjugate in urine of workers exposed to 2-butoxyethanol, 6th European Meeting on Mass Spectrometry in Occupational and Environmental Health, Stockholm, September 1–3 (1999). Rashed, M. S.; Bucknall, M. P.; Little, D.; Awad, A.; Jacob, M.; Alamoudi, M.; Alwattar, M.; Ozand, P. T.: Screening blood spots for inborn errors of metabolism by electrospray tandem mass spectrometry with a microplate batch process and a computer algorithm for automated flagging of abnormal profiles, Clin. Chem.; 43, 1129–1141 (1997). Rettenmeier, A. W.; Hennigs, R.; Wodarz, R.: Determination of butoxyacetic acid and Nbutoxyacetylglutamine in urine of lacquerers exposed to 2-butoxyethanol, Int. Arch. Occup. Environ. Health, 65, 151–153 (1993). Rettenmeier, A. W.; Howald, W. N.; Levy, R. H.; Witek, D. J.; Gordon, W. P.; Porubek, D. J.; Baillie, T. A.: Quantitative metabolic profiling of valproic acid in humans using automated GC/MS techniques, Biomed. Environ. Mass Spectrom.; 18, 192–199 (1989). Rettenmeier, A. W.; Lebherz, J.; Wodarz, R. et al.: Bestimmung der Valproinsäuremetaboliten nach einmaliger oraler Belastung: Ein Weg zur Früherkennung potentieller Risikopatienten? in: Epilepsie 89, Wolf, P. (eds), S. 421–425, Einhorn-Presse Verlag, Reinbek (1990). Rettie, A. E.; Rettenmeier, A. W.; Howald, W. N.; Baillie, T. A.: Cytochrome P-450-catalyzed formation of D4-VPA, a toxic metabolite of valproic acid, Science, 235, 890–893 (1987).
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2.3
Biological Monitoring of Arylamines and Nitroarenes Gabriele Sabbioni *
2.3.1 Introduction Arylamines and nitroarenes are important intermediates in the production of plastics and polyurethanes. Employees in many factories are subjected to chronic low-level exposure to a wide variety, often a mixture, of arylamines, nitroarenes and arylisocyanates (Fig. 1). We have developed methods to measure the haemoglobin (Hb) adducts of over 50 arylamines and nitroarenes (Sabbioni 1992, Sabbioni 1994a, Sabbioni 1994 b, Sabbioni & Sepai 1999, Sabbioni & Beyerbach 1995, Schütze et al. 1995, Sepai et al. 1995a, Sepai et al. 1995 b, Sabbioni et al. 1996). With these methods we can assess exposure to low levels of these chemicals. Since in most cases the air monitoring values were below the detection limit, we used the protein adduct levels to estimate the daily dose, which was then compared to the TD50 (i.e. daily dose which yields tumours in 50% of rodents). Biological samples were collected from groups of workers. With each study we endeavoured to collect control samples from unexposed clerical or medical staff. Blood samples were prepared using current methods (Sabbioni & Beyerbach 1995, Schütze et al. 1995, Sepai et al. 1995a). The Hb, plasma and urine samples were stored at –20 8C. Samples hydrolysed with an acid or base were extracted at basic pH into organic solvents, derivatised with perfluorinated acid anhydride and analysed by gas chromatography-mass spectrometry (GC-MS), in the negative chemical ionization (NCI) mode. For each compound a calibration line was established at five concentrations covering the expected levels of adducts or metabolites in the samples.
*
24
Walther-Straub-Institut für Pharmakologie und Toxikologie, Ludwig-MaximiliansUniversität München, Nussbaumstraße 26, 80336 München
2.3 Biological Monitoring of Arylamines and Nitroarenes
Figure 1: DNA adducts and protein adducts of arylamines, nitroarenes, and aryisocyanates.
2.3.2 Biomonitoring of Protein Adducts 2.3.2.1 Workers Exposed to 2-Methylaniline and Aniline A high incidence of bladder cancer was reported in employees from a factory producing rubber. Blood samples were available from 73 workers of this factory. The Hb adducts of 2-methylaniline (2MA), aniline (A) and 4aminobiphenyl (4ABP) were investigated. Extremely high amounts of 2MA (0.1–200 ng/g Hb) and of A (0.1–35 ng/g Hb) were found (Ward et al. 1996). The levels of 4ABP were equivalent to those in the controls. The use of environmental air monitoring as a means of dose estimation has a number of drawbacks: (i) it is often not sensitive enough, (ii) does not give an indication of the effective dose but an indication of the inhaled dose, and (iii) gives no indication of the metabolism of the compound in question. Hb adduct levels are dosimeters for the bioavailability of reactive xenobiotics or their metabolites and possibly also a dosimeter for the DNA adducts at the site of tumour formation. Furthermore, with the knowledge from animal data it is possible to estimate the daily dose from the measured Hb adduct levels. However, it is necessary to make the following assumptions: 25
2 Internal Exposure and Haemoglobin Adducts • The adduct levels result from steady-state exposures (Ac), Ac = 0.5 × A×Ter (Where A is the average daily increment per total Hb, and Ter is the lifetime of an erythrocyte.) Thus, to calculate the single dose the adduct level has to be divided by 60 (Tannenbaum et al.1986). • Modified Hb has the same life-span as unmodified Hb and the adducts are stable to repair mechanisms. • The pharmacokinetics of the xenobiotic compound is comparable in rats and humans. Taking 2MA as an example, the amount of adduct associated with Hb can be divided by 0.00059, which is the proportion of an administered dose found associated with the Hb in rats (Sabbioni 1992, Birner & Neumann 1988). Thus for a 70 kg individual with 200 ng 2 MA/g Hb, the dose is 68 lg 2 MA/kg/day. In the absence of epidemiological data, animal experiments must be used to characterize the health risk to man. The daily dose given to rodents that causes a fifty percent greater likelihood of the development of a tumour is termed the TD50. The human exposure dose expressed as a percentage of the rodent potency dose (TD50) has been termed the HERP index (Ames et al. 1987, Goodman & Wilson 1991, Crabtree et al. 1991, Gold et al. 1992, Talaska et al. 1994). The HERP index is very useful, as long as the many assumptions that are required are not ignored. Using the HERP index, it was possible to rank the risk or hazard potential for the workers exposed to the levels of amines for which we have established an internal exposure dose from the adduct levels. The daily dose of 2MA for the workers with the highest adduct levels in this study was 1/300 of the TD50 value determined for rats (Gold et al. 1993). 2.3.2.2 Workers Exposed to 4,4'-Methylenedianiline (MDA) or 4,4'-Methylenediphenyl Diisocyanate (MDI) Other groups of workers investigated were exposed to MDA or MDI (Schütze et al. 1995). The levels of MDA and MDI in air were below the detection limits. However, adducts and metabolites were detected in a high percentage of the samples. Hb adducts of MDA were found in 97 % and Nacetyl-MDA (AcMDA) in 65 % of the MDA workers. Hb adducts of MDA were found in 38 % of the MDI workers. In workers exposed to MDI, the N-acetylated compound (AcMDA) was found only in one worker (Sepai et al. 1995a). Only the adducts of the primary amine from the hydrolysis of MDI were analysed in this study. The presence of MDI adducts or other isocyanate metabolites were not investi26
2.3 Biological Monitoring of Arylamines and Nitroarenes gated. Urine, collected at the same time as the blood samples from these MDA and MDI workers, was extracted at alkaline pH with and without previous acid treatment. MDA and AcMDA were found in the urine of 84 % MDA workers and 78 % MDI workers. In order to release MDA and AcMDA from possible conjugates, urine was treated under strong acidic conditions. Following this procedure, higher levels of MDA were found than the sum of MDA and AcMDA after base extraction alone. Urinary metabolites are indicators of recent exposure: it is not advisable to estimate an average daily dose from these values as there is a likelihood of over or under-estimation (Cocker et al. 1994), that is a HERP of 0.3 %. In rats 0.044 % of the MDA dose was found as Hb adducts (Sabbioni & Schütze 1998). Assuming that the pharmacokinetics of MDA are similar in rats and humans, the daily MDA dose of these workers is at least 10 000 times below the TD50 (Gold et al. 1993) found for rats (i.e. a HERP of 0.01 %). 2.3.2.3 Workers Exposed to 2,4,6-Trinitrotoluene (TNT) Another study involved the biomonitoring of workers employed in a Chinese 2,4,6-trinitrotoluene (TNT) factory (Sabbioni et al. 1996, Liu et al. 1995). The factory controls were fire fighters, white collar workers, security guards and the director. The blood was collected by the Medical Department of the factory. Hb from the workers was hydrolysed with sodium hydroxide, extracted with methylene chloride and analysed by GC-MS with negative chemical ionization. The 4-amino-2,6-dinitrotoluene (4ADNT) levels of the workers were up to 522 ng/g Hb. The highest levels were found in the screening and loading group. For 2-amino-4,6-dinitrotoluene (2ADNT) the highest level was 14.7 ng/g Hb. Hb adducts of TNT were found in all the factory controls. This indicates that there is general contamination of the factory, since the Hb of our German laboratory workers was free of 2ADNT and 4ADNT. The Hb adducts determined in the present study were compared with the air levels and skin levels. The air and skin concentrations were measured in the same workplace, but at a different time than the blood collection. The adduct levels in the exposed workers related more closely to the skin contamination than to air concentrations, indicating that skin contamination is the main source of the internal dose. Cataracts are sometimes the first and only sign of adverse health effects in workers exposed to TNT. Health records show that 29 of 126 exposed workers from this factory have developed cataracts (Liu et al. 1995). The prevalence of cataracts correlates with the increase in TNT-Hb adducts. No cataracts were found when the TNT-Hb level was below 30 ng/g Hb (as determined by ELISA), even for individuals who had been employed for up to 20 years in this factory. The three subjects with the highest 27
28
Scheme 2.
Scheme 1.
2 Internal Exposure and Haemoglobin Adducts
2.3 Biological Monitoring of Arylamines and Nitroarenes level of adducts > 300 ng/g Hb were all diagnosed as having cataracts. It is possible that adducts occur with lens proteins of exposed workers which are similar to the protein adducts of TNT. This is the subject of a future study.
2.3.2.4 Women with Polyurethane-Coated Breast Implants Exposed to 2,4-Toluenediamine (24TDA) Another topic of concern is the non-occupational exposure to aromatic amines released from medical devices made of polyurethane (PU). We detected the presence of degradation products of PU – namely monomeric toluenediamines: 2,4-toluenediamine (24TDA), a suspected human carcinogen, and 2,6-toluenediamine (26TDA) – in the blood and urine of patients with PU-covered breast implants up to two years after the operation (Sepai et al. 1995b). From our results we can estimate a potential risk from these implants. Following a lag of approximately 20 days, where no TDAs above background levels were detected in the plasma, the levels of both 24TDA and 26TDA rose, reaching a maximum of 4.4 (2.1) ng/ml plasma for 24TDA (26TDA), and then remained at those levels for over 180 days. Most TDA was covalently bound to the plasma proteins, especially albumin. An adduct level of 4.4 ng TDA/ml plasma corresponds to a daily dose of 70 400 ng for a 60 kg person. This was calculated by comparison with the adduct level found in the plasma of rats dosed with radioactive TDA (Grantham et al 1978) and a steady state to single dose conversion factor for albumin adducts of about 29 (Sabbioni et al. 1987). This value is about 300 times larger than the daily dose estimated for two implants by an Expert Panel of the Canadian Medical Association (Expert panel of the Canadian Medical Association 1991). The Expert Panel expects five additional cases of breast cancer in 10 million patients with two implants. Our risk estimation is, therefore, around 1500 additional breast cancer cases in 10 million women. This additional risk is, of course, minute when 1 in 10 woman are likely to develop breast cancer in the normal western population. However, the risk from 24TDA was only related to breast cancer. We should keep in mind that the primary sites of action of this suspected carcinogen are the liver and the kidneys. This indicates that the risk of liver cancer may be of more concern. Our calculated dose level is about 1200 times lower than the TD50 in rats (Gold et al. 1993), that is a HERP of 0.08 %.
2.3.2.5 Conclusions Ambient monitoring is often a poor measure of exposure in man. The internal dose takes into account different modes of exposure, metabolism and 29
2 Internal Exposure and Haemoglobin Adducts individual susceptibilities. The presence of Hb adducts demonstrates the bioavailability of N-hydroxy-arylamines, the key intermediates for the subsequent biochemical effects. We found haemoglobin adducts in different groups of workers, although sometimes the levels in air were below the detection limit. From the measured Hb adduct levels we estimated the daily dose, which was then compared to rodent carcinogenic potency data. The HERP indexes for the arylamines we studied were 0.3 % for 2MA, 0.01 % for MDA and 0.08 % for 24TDA. The genotoxic risk resulting from these chemicals, without taking into account synergistic effects, is possibly comparable to the hazard from the amount of formaldehyde in conventional home air (HERP index of 0.6 %) (Ames et al. 1987).
2.3.3 Biomonitoring of DNA Adducts using HPLC/MS/MS To relate the exposure with the DNA damage it is essential to measure the DNA adducts derived from environmental and endogenous carcinogens in tissues from animals and man (Poirier & Beland 1997). The DNA adduct levels are typically in the range of 1 in 106 to 1 in 109 normal nucleotides. Therefore, highly sensitive techniques are required that can analyse the only small amounts of DNA (1–300 lg) which are available in studies with human material. The 32P-postlabelling assay is the method most widely used for the analysis of DNA adducts (Talaska et al. 1992, Beach & Gupta 1992, Izzotti 1998, Phillips & Castagnaro 1999). This assay uses c-32P-labelled adenosine triphosphate to incorporate a highly radioactive reporter group into the nucleotides. After enzymatic hydrolysis of DNA and the postlabelling procedure, the nucleotides are separated from normal nucleotides by TLC or HPLC and visualized using radioautography or in-line scintillation counting. These procedures can detect adducted nucleotides very sensitively. The major drawbacks are (Phillips & Castagnaro 1999): • the inability to characterize unknown adducts, • susceptibility to false positives or false negatives, • poor reproducibility, • difficult interpretation of the spots on the 2-dimensional and 4-dimensional TLC plates, • extensive method validation is required for reliable quantitative performance. In recent years, HPLC/MS/MS has assumed an important role in bioanalytical chemistry in terms of structure characterization, trace level detection, and quantification. The major advantages of HPLC/MS/MS are: 30
2.3 Biological Monitoring of Arylamines and Nitroarenes • analyses using isotopically labelled internal standards, • exceptional selectivity and specificity, • additional evidence of characteristic retention times, • the potential for quantitative analyses without chemical derivatization reactions, • there is no longer any need to work with radioisotopes. In view of these features, a number of laboratories have been interested in replacing the 32P-postlabelling methods with the new HPLC/MS/MS technique (Andrews et al. 1999, Beland et al 1999, Doerge et al. 1999, Gangl et al. 1999). In general the following principles have been adopted to develop a method for the characterization and quantification of DNA adducts: • the synthesis of standards, • the use of the synthesized standards to optimize chromatographic and detection conditions, • the evaluation of detection limits in vitro, • the application of the methodology to an in vivo system. Recently, HPLC/MS analyses have successfully been conducted with the dG-C8 adduct of 4-aminobiphenyl in rodents. Doerge et al. (Doerge et al. 1999) developed a quantitative isotope dilution method for analysis of N(deoxyguanosine-8-yl)-4-aminobiphenyl (dG-C8-4-ABP), which is the principal nucleoside adduct of 4-aminobiphenyl (4-ABP)-modified DNA. Column switching valves were used to perform on-line sample concentration and clean up. This permitted direct analysis of enzymatic DNA hydrolysates using narrow-bore liquid chromatography. Electrospray ionization (ESI)-MS detection was performed by monitoring [M+H]+ (m/z = 435) and two fragment ions (m/z = 319, 195) characteristic for dG-C8-4-ABP, along with [M+H]+ (m/z = 444) and a fragment ion (m/z = 328) for the deuterated internal standard. The detection limit for dG-C8-4-ABP in DNA hydrolysates was ~10 pg on-column. For a sample containing 100 lg DNA, this corresponds to 0.7 dG-C8-4-ABP adducts in 107 normal nucleotides. Doerge et al. 1999 analysed calf thymus DNA modified in vitro and hepatic DNA isolated from mice treated in vivo with two dose levels (0.1 or 1.0 mg/kg body weight) of 4-ABP. The adduct level for these mice was 5 and 30 dG-C8–4-ABP adducts in 107 normal nucleotides. The intra-assay precision varied from 9.7 to 17.7 % relative standard deviation. In another recent study Gangl et al. (1999) applied capillary liquid chromatography/microelectrospray mass spectrometry to the detection of deoxyribonucleoside adducts of the food-derived mutagen 2-amino-3methylimidazo[4,5-f]quinoline (IQ) from in vitro and in vivo sources. Selective reaction monitoring techniques with a triple-quadrupole mass spectrometer enabled the sensitive and specific detection of IQ adducts in vitro and in animals. The detection limit was 1 adduct in 107 unmodified bases using 300 lg DNA. The DNA adducts N-(deoxyguanosin-8-yl)-2-amino3-methylimidazo[4,5-f]quinoline and 5-(deoxyguanosin-N 2-yl)-2-amino-331
2 Internal Exposure and Haemoglobin Adducts methyl-imidazo[4,5-f]quinoline were found in kidney tissues of chronically treated cynomolgus monkeys.
2.3.4 Conclusions Mass spectrometric ionization and detection methods are continuously being developed further. It is reasonable to expect further improvements in HPLC/MS instrumentation technology. Possibly, the HPLC/MS techniques will achieve the sensitivity of current radio-chemical detection methods. Sensitive HPLC/MS assays will enable the parallel use of the different adduct detection techniques. Mass spectrometry will be the most important tool for confirming adduct structures and recognizing new adducts in animal experiments, and possibly also in man. The levels of detection in HPLC/MS/MS studies could be further improved by optimizing the HPLC side of the analytical system, i.e. the coupling of micro HPLC with microspray. In addition, there is still room for improvements in the handling (enzymatic digestion, extraction) of the DNA to increase the yields and the reproducibility of DNA adduct recovery. Thus, in future, it may be possible to detect adducts in the range of 1 adduct in 108–109 normal bases. This detection limit is necessary to match the sensitivity of the postlabelling assay which has been used in biomonitoring studies of populations exposed to carcinogens. References Ames, B. N.; Magaw, R.; Gold, L. S.: Ranking possible carcinogenic hazards, Science, 236, 271–280 (1987). Andrews, C. L.; Vouros, P.; Harsch, A.: Analysis of DNA adducts using high-performance separation techniques coupled to electrospray ionization mass spectrometry, J. Chromatogr. A, 856, 515–526 (1999). Beach, A. C.; Gupta, R. C.: Human biomonitoring and the 32P-postlabeling assay, Carcinogenesis, 13, 1053–1074 (1992). Beland, F. A.; Doerge, D. R.; Churchwell, M. I.; Poirier, M. C.; Schoket, B.; Marques, M.: Synthesis, characterization, and quantitation of a 4-aminobiphenyl DNA adduct standard, Chem. Res. Toxicol.; 12, 68–77 (1999). Birner, G.; Neumann, H.-G.: Biomonitoring of aromatic amines. II: Hemoglobin binding of some monocyclic aromatic amines, Arch. Toxicol.; 62, 110–115 (1988). Cocker, J.; Nutley, B. P.; Wilson, H. K.: A biological monitoring assessment of exposure to methylene dianiline in manufacturers and users, Occup. Environ. Med.; 51, 519– 522 (1994). Crabtree, H. C.; Hart, D.; Thomas, M. C.; Witham, B. H.; McKensie, I. G.; Smith, C. P.: Carcinogenic ranking of aromatic amines and nitro compounds, Mutat. Res.; 264, 155–162 (1991). Doerge, D. R.; Churchwell, M. I.; Marques, M. M.; Beland, F. A.: Quantitative analysis of 4-aminobiphenyl-C8-deoxyguanosyl DNA adducts produced in vitro and in vivo using HPLC-ES-MS, Carcinogenesis, 20, 1055–1061 (1999).
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2.3 Biological Monitoring of Arylamines and Nitroarenes Expert panel of the Canadian Medical Association: Safety of polyurethane-covered, breast implants, Can. Med. Assoc. J.; 145, 1125–1128 (1991). Gangl, E. T.; Turesky, R. J.; Vouros, P.: Determination of in vitro and in vivo-formed DNA adducts of 2-amino-3-methylimidazo[4,5-f]quinoline by capillary liquid chromatography/microelectrospray mass spectrometry, Chem. Res. Toxicol.; 12, 1019–1027 (1999). Gold, L. S.; Manley, N. B.; Slone, T. H.; Garfinkel, G. B.; Rohrbach, L.; Ames, B. N.: The fifth plot of the carcinogenic potency database: results of animal bioassays published in the general literature through 1988 and by the National Toxicology Program through 1989, Environ. Health Perspect.; 100, 65–135 (1993). Gold, L. S.; Slone, T. H.; Stern, B. R.; Manley, N. B.; Ames, B. N.: Rodent carcinogens: setting priorities, Science, 258, 261–265 (1992). Goodman, G.; Wilson, R.: Quantitative prediction of human cancer risk from rodent carcinogenic potencies: a closer look at the epidemiological evidence for some chemicals not definitively carcinogenic in humans, Regul. Toxicol. Pharmacol.; 14, 118–146 (1991). Grantham, P. H.; Mohan, L.; Benjamin, T.; Roller, P. P.; Miller, J. R.; Weisburger, E. K.: Comparison of the metabolism of 2,4-toluenediamine in rats and mice, J. Environ. Pathol. Toxicol.; 3, 149–166 (1978). Izzotti, A.: Detection of modified DNA nucleotides by postlabeling procedures, Toxicol. Methods, 8, 175–205 (1998). Liu, Y.-Y.; Yao, M.; Fang, J.-L.; Wang, Y.-W.: Monitoring human risk and exposure to trinitrotoluene (TNT) using haemoglobin adducts as biomarkers, Toxicol. Lett.; 77, 281–287 (1995). Phillips, D. H.; Castagnaro, M.: on behalf of the trial participants. Carcinogenesis, 14, 301–315 (1999). Poirier, M. C.; Beland, F. A.: Aromatic amine DNA adduct formation in chronically-exposed mice: considerations for human comparison, Mutat. Res.; 376, 177–184 (1997). Sabbioni, G.; Beyerbach, A.: Biomonitoring of arylamines: haemoglobin adducts of aniline derivatives, J. Chromatog. B.; 667, 75–83 (1995). Sabbioni, G.; Schütze, D.: Hemoglobin binding of bicyclic aromatic amines, Chem. Res. Toxicol. 11, 471–483 (1998). Sabbioni, G.; Sepai, O.: Comparison of hemoglobin binding, mutagenicity and carcinogenicity of arylamines and nitroarenes, Chimia, 49, 374-380 (1995), and 53, 456 (1999). Sabbioni, G.; Hemoglobin binding of arylamines and nitroarenes: molecular dosimetry and quantitative structure activity relationships, Environ. Health Perspect.; 102 (Suppl 6), 61–67 (1994B). Sabbioni, G.; Hemoglobin binding of nitroarenes and quantitative structure-activity relationships. Chem. Res. Toxicol.; 7, 267–274 (1994A). Sabbioni, G. Quantitative structure activity relationship of the N-oxidation of aromatic amines Chem.-Biol. Interact.; 81, 91–117 (1992). Sabbioni, G.; Skipper, P. L.; Büchi, G.; Tannenbaum, S. R.: Isolation and characterization of the major serum albumin adduct formed by aflatoxin B1 in vivo in rats, Carcinogenesis, 8, 819–824 (1987). Sabbioni, G.; Wei, J.; Liu, Y.-Y.: Determination of hemoglobin adducts in workers exposed to 2,4,6-trinitrotoluene, J. Chromatogr. B.; 682, 243–248 (1996). Schütze, D.; Sepai, O.; Lewalter, J.; Miksche, L.; Henschler, D.; Sabbioni, G.: Biomonitoring of workers exposed to 4,4'-methylenedianiline or 4,4'-methylenediphenyl diisocyanate, Carcinogenesis, 16, 573–582 (1995). Sepai, O.; Czech, S.; Eckert, P.; Henschler, D.; Sabbioni, G.: Exposure to toluene diamines from polyurethane-coated breast implants, Toxicology Lett.; 77, 371–378 (1995A).
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2 Internal Exposure and Haemoglobin Adducts Sepai, O.; Henschler, D.; Sabbioni, G.: Albumin adducts, hemoglobin adducts and urinary metabolites in workers exposed to 4,4‘-methylenediphenyl diisocyanate, Carcinogenesis, 16, 2583–2587 (1995B). Talaska, G.; Roh, J. H.; Getek, T. J.: 32P-Postlabelling and mass spectrometric methods for analysis of bulky, polyaromatic carcinogen-DNA adducts in humans, Chromatogr.; 580, 293–323 (1992). Talaska, G.; Schamer, M.; Casetta, G.; Tizzani, A.; Vineis, P.: Carcinogen-DNA adducts in bladder biopsies and urothelial cells: a risk assessment exercise, Cancer Lett.; 84, 93–97 (1994). Tannenbaum, S. R. Bryant, M. S.; Skipper, P. L.; Maclure, M.: Hemoglobin adducts of tobacco-related aromatic amines: application to molecular epidemiology, Banbury Rep.; 26, 63–75 (1986). Ward, E. M.; Sabbioni, G.; DeBord, D. G.; Teass, A. W.; Brown, K.; Talaska, G.; Roberts, D.; Ruder, A.; Streicher, R. P.: Biological monitoring of aromatic amine exposures at a chemical plant with a known bladder excess, J. Nat. Cancer Inst.; 88, 1040–1052 (1996).
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3
DNA Adducts
3.1
Genetic Cancer Susceptibility and DNA Adducts: Studies in Smokers and Coke Oven Workers Magarita Rojas, Kroum Alexandrov, Helmut Bartsch and Bertold Spiegelhalder *
Preventive strategies must identify individuals susceptible to cancer as a result of the combination of exposure to carcinogens, cancer-predisposing genes and a lack of protective factors. To this aim, in case-control studies we determined PAH-DNA adducts as a measure of exposure to tobacco smoke and as susceptibility markers, together with genetic polymorphism in drug-metabolizing enzymes related to CYP1A1, GSTM1 and GSTT1 genes. (+)-anti-Benzo(a)pyrene diol-epoxide (BPDE)-DNA adduct levels were quantified in white blood cell (WBC) and lung tissue DNA. CYP1A1 polymorphism and GSTM1 or GSTT1 gene deletion was analysed in genomic DNA from lung parenchyma and white blood cells. Results from lung cancer patients and coke oven workers exposed to PAHs allowed CYP1A1GSTM1 genotype combinations to be correlated with BPDE-DNA adduct levels. Smokers with the homozygous CYP1A1 variant and GSTM1 null had the highest adduct levels and were, as shown in Japanese smokers, most susceptible to lung cancer. On the basis of this short review, we conclude that BPDE-DNA adduct levels resulting from genotype combinations that represent a risk may serve as markers for identifying most susceptible individuals.
3.1.1 Introduction The characterization of genetic determinants for cancer susceptibility is important for understanding the pathogenesis of the disease and for preventive measures. There is growing evidence that there is a group of predisposing polymorphic genes, like those involved in carcinogen metabolism and repair, which may increase cancer in certain subjects, even if they are *
Division of Toxicology and Cancer Risk Factors, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg
Biological Monitoring: Prospects in Occupational and Environmental Medicine. Deutsche Forschungsgemeinschaft (DFG) Copyright © 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-27795-7
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3 DNA Adducts exposed to only low levels of carcinogens in the environment (Caporaso & Goldstein 1995, Vineis et al. 1994, Vineis 1997). Preventive strategies must therefore identify these vulnerable members in our society, in particular those with an unfavourable combination of high carcinogen exposure, cancer-predisposing genes and a lack of (dietary) protective factors. Thus, molecular epidemiology faces the difficult task of analysing individuals exposed to carcinogens for a combination of ‘risk’ genotypes associated with higher cancer susceptibility. Rather than taking cancer as an endpoint, combinations of cancer-predisposing genes can then be explored using DNA adducts as intermediate risk markers. With this approach it should be possible to better define gene-environment interaction and facilitate the identification of high-risk subjects within populations exposed to carcinogens. Some current case-control studies of environmentally induced lung cancer related to cigarette smoking or PAH exposure in coke oven workers are briefly summarized below. The literature cited is not exhaustive, and the reader is referred to articles published earlier (Bartsch & Hietanen 1996, Bartsch 1996, Bartsch et al. 1995, Kriek et al. 1998).
3.1.2 Material and Methods DNA was isolated from normal lung tissue obtained from untreated lung cancer patients undergoing surgery. Blood samples from male coke oven workers were obtained from a plant in France in 1995. Occupational exposure in the year of blood sampling was in the range from <0.15 to >4 lg/m3 benzo(a)pyrene (BP). All samples were coded, and white blood cells were prepared on Ficoll and frozen before DNA extraction. DNA was extracted from non-tumourous lung tissue using proteinase K/RNase digestion and a modified phenol extraction procedure (Alexandrov et al. 1992). The DNA from white blood cells (either lymphocytes or lymphocyte + monocyte fraction (LMF)) was isolated as described (Rojas et al. 1995). 0.2–1 mg DNA was used for analysis of BP-tetrols by high-performance liquid chromatography combined with fluorimetric detection (HPLC-FD) (Alexandrov et al. 1992, Rojas et al. 1994), allowing quantification of (+)-anti-BP diol-epoxide (BPDE)-DNA adducts (detection limit 0.2 BPDE-DNA adducts per 108 nt). In the lung cancer studies, PCR/RFLP-based analysis of CYP1A1 gene polymorphisms and GSTM1 gene deletion was carried out as described (Cascorbi et al. 1996, Brockmöller et al. 1993, Volkenandt et al. 1993, Arand et al. 1996). An allele carrying only a T to C transition 1194 bp downstream of exon 7 in the 3'-flanking region, leading to a Mspl-restriction site (m1), was termed *2A. An allele with m1 plus a mutation in exon 7 leading to an lle/Val-exchange at codon 462 (m2) due to a A to G transition at nt 4889 was termed *2B. 36
3.1 Genetic Cancer Susceptibility and DNA Adducts The nomenclature for the polymorphisms in the GSTM1 and GSTT1 genes used are as follows (Garte & Crosti 1998): the GSTM1*1 active genotype comprises the following functional allele configurations: GSTM1*1A/ *1A, GSTM*1B/*1B, GSTM1*1A/*2, and GSTM1*1B/*2. The non-functional null or deleted allele is GSTM1*2, and the corresponding deficient phenotype was termed GSTM1 null phenotype. Similarly, GSTT1*1 represents the active genotype, while the non-functional genotype null or deleted allele was denoted as GSTT1*2.
3.1.3 Results and Discussion 3.1.3.1 Bulky DNA Adducts in Human Lung Carcinogenesis and Disease Susceptibility Cigarette smoking is the greatest risk factor for lung cancer, but drug-metabolizing enzymes, which often display genetic polymorphism and convert lung carcinogens from the occupational environment or tobacco into DNAbinding metabolites in target cells, can modulate intermediate effect markers, e.g. DNA adducts, and ultimately the cancer risk. There is substantial evidence that bulky, mostly PAH-derived DNA adducts are of significance for the onset of lung carcinogenesis in smokers and workers exposed to PAHs (Kriek et al. 1998). Having developed a sensitive and specific HPLCFD procedure for BPDE-DNA adduct detection in human lung tissue and LMF (Rojas et al. 1994), the aims of our ongoing studies are (i) to identify specific genotype combinations that lead to high BPDE-DNA adduct levels in smokers and workers exposed to PAHs and (ii) to use the characterized markers for early detection of individuals susceptible to lung cancer.
Figure 1: Principal metabolic pathways of BP in human lung leading to the formation of the ultimate carcinogenic metabolite BPDE, which reacts with DNA if not detoxified by glutathione S-transferases (GSTs); the resulting DNA adducts lead to the initiation of lung carcinogenesis.
37
3 DNA Adducts Among the many DNA adducts found in smokers’ lungs, we concentrated on the polycyclic aromatic hydrocarbon BP, because it is an important carcinogenic constituent in tobacco smoke, polluted air and in the occupational environment. The mechanism by which BP interacts with DNA, activates oncogenes and initiates carcinogenic processes, involves the formation of one of the enantiomeric BP-diol-epoxides (BPDE). The biologically most active enantiomer is the (+)-anti-BPDE, a major ultimate carcinogen which can now be quantified using our HPLC-FD technique. In human lung, cytochrome P4501A1 is one of the enzymes that converts polycyclic PAHs into DNA-binding metabolites, as shown for BP (Fig. 1). BP-3-hydroxylase (AHH) is a marker for CYP1A1-related enzyme activity in human lung. Glutathione S-transferases (GSTs) including GSTM1 in the liver and GSTM3 in the lung detoxify reactive diol-epoxide intermediates (Fig. 1). Our previous studies revealed that tobacco smoke has great effects on carcinogen-metabolizing enzymes in the human lung (Bartsch 1996).
3.1.3.2 Smoking Enhances BPDE-DNA Adduct Levels in White Blood Cells of Coke Oven Workers Exposed to PAHs As a result of the sensitivity of the HPLC-FD method, the level of BPDEDNA adducts could be determined in white blood cells from coke oven workers exposed to PAHs. The aim was to see whether (i) smoking enhances the binding of PAHs to DNA and (ii) whether CYP1A1-GSTM1 genotype combinations can act as modifiers of DNA adduct levels. Groups of coke oven workers exposed to PAHs and controls who were not exposed, each made up of smokers and non-smokers, were investigated (Rojas et al. 1995). The BPDE-DNA levels in the white blood cells of workers was 15 times higher than in those from the controls. However, the most important finding was that smoking increased the adduct levels in workers exposed to PAHs. There was a 200-fold interindividual variation in smoking workers, which was only 6-fold in non-smoking workers. The enhancing effect of smoking on DNA adduct levels in white blood cells from workers exposed to PAHs was confirmed recently (van Schooten et al. 1995). These increased levels and the high variability of BPDE-DNA adducts in smoking workers indicate genetic variations in PAH metabolism and DNA-adduct formation. This is supported by our observation of BPDEDNA adduct dependence on specific CYP1A1-GSTM1 genotype combinations (see below). As the same synergistic effects may occur in the lung, this would provide an explanation for the enhancing effect of smoking in PAH-associated occupational lung cancer. Recent studies showed that DNA-adduct levels in the white blood cells of smokers correlate with adduct levels in lung tissue of lung cancer patients (Tang et al. 1995, Wiencke et al. 1995). The regulation of CYP1A1 expression is complex and also involves transcriptional control elements which regulate enzyme induction. These have not 38
3.1 Genetic Cancer Susceptibility and DNA Adducts fully been characterized at the molecular level. Therefore, a genotype/phenotype approach was applied to examine the BPDE-DNA adduct levels in lung cancer tissue from patients with high CYP1A1 inducibility (Bartsch 1996). This phenotype was measured by immunohistochemical staining with a monoclonal antibody, while the GSTM1 inactive was determined by PCR. Compared to the respective wild type, smokers with similar cigarette consumption had 100-fold higher BPDE-DNA adduct levels in bronchial tissue (Bartsch 1996) when they were both GSTM1 null and highly inducible for CYP1A1. This large difference was not seen in lung parenchymal tissue. Although GSTM1 is not expressed in human lung, GSTM3-related activity is found in this tissue which seems to be co-regulated with the GSTM1 form (Nakajima et al. 1995). Thus, individuals with nulled GSTM1 genotype suffer from impaired detoxification of tobacco carcinogens, both qualitatively because of the absence of GSTM1 in the body and low expression of GSTM3 in the lung, and quantitatively because of the overall lower GST activity. This effect of GSTM1 null on lung PAH adduct levels was also seen in a Finnish cohort of lung cancer patients (Bartsch & Hietanen 1996). In current smokers, the GSTM1 gene deletion resulted in a 10 % increase in total bulky DNA adduct levels in the lung, whereas in exsmokers it was as much as 2.5-fold. This increase in DNA adduct levels is compatible with results from a meta-analysis of lung cancer patients with GSTM1 deficiency. In smokers the relative risk was found to increase to 1.4 for lung cancer of all major histological subtypes. This increased risk would account for 17 % of all new cases of lung cancer in smokers annually, as a result of the high relevance of the GSTM1 null genotype that occurs in about 50 % of Caucasians (McWilliams et al. 1995). A study in human cell lines revealed that GSTM1 deletion is associated with high inducibility by TCDD of the CYP1A1 gene transcription (Vaury et al. 1995). Although the underlying mechanism is not fully understood, this observation in vitro and our data for the genotype dependence of PAH adduct levels in man underline the importance of GYP1A1/GSTM1 as risk modifiers for tobacco-associated DNA damage and lung cancer. The latter is supported by case-control studies in Japan and by our recent genotyping results (see below). Results from our earlier work suggested that PAHs present in tobacco smoke induce pulmonary CYP1A1 gene expression only in certain individuals. As a consequence, the generation of DNA-reactive metabolites of tobacco carcinogens in lung target cells should be affected by polymorphic genes whose products are involved in the activation and detoxifying reactions of PAHs. Therefore we subsequently examined whether there is a correlation between the CYP1A1-related catalytic activity in the lungs of smokers and the level of PAH-DNA adducts that is thought to be critical for the onset of lung carcinogenesis. Initially, we used the sensitive method of 32P-postlabelling for detecting tobacco smoke-associated DNA adducts. Then, because of the low specificity of this method, an improved analytical 39
3 DNA Adducts procedure using HPLC-FD was developed to quantify BPDE after its binding to cellular DNA in man (Alexandrov et al. 1992). Both methods were then applied to the lung parenchyma of smokers to determine the level of DNA adducts; in the same lung samples microsomal CYP1A1-related enzyme (AHH) activity was measured. A positive, highly significant correlation (0.91 P <0.001) was found between the pulmonary enzyme activity and the level of BPDE-DNA adducts. The level of total bulky PAH-DNA adducts (determined by 32P-postlabelling) also correlated with AHH, but less closely. Surprisingly, in this study with Italian smokers, the BPDE-DNA adducts alone accounted for about 20% of the total bulky adducts in smokers’ lungs. These positive correlations explain why a variation in inducibility or the genetic polymorphism of CYP1A1-related enzyme activity act as modifiers for the lung cancer risk in smokers, and also implicate CYP1A1 in the formation and binding of BPDE to lung DNA in smokers.
3.1.3.3 CYP1A1/GSTM1 Genotype Dependence of BPDE-DNA Adduct Levels and Risk for Smoking-Related Lung Cancer In Japanese populations, a significant correlation between susceptibility to lung cancer and homozygosity for the CYP1A1 Mspl allele (*2A/*2A) was reported (Kawajiri et al. 1990, Nakachi et al. 1991, Nakachi et al. 1993). Hayashi et al. (Hayashi et al. 1992) found exon-7 mutation (*2B) to be associated with squamous cell and small cell lung cancer. Case-control studies revealed that Japanese individuals with the susceptible CYP1A1 (*2A/*2A or *2B/*2B) genotype combined with GSTM1 null were at remarkably higher risk, particularly for Kreyberg I type and squamous-cell carcinoma of the lung at a low-dose level of cigarette smoke (Kihara & Noda 1995). Individuals with homozygous CYP1A1 (*2A/*2A) were found to be relatively resistant to tobacco-related lung cancers when combined with GSTM1 active, but were highly susceptible when combined with GSTM1 null, with a 7- to 8-fold increased risk (Kihara & Noda 1995). In addition, survival of lung cancer patients with the ‘risk’ gene CYP1A1 (*2A/*2A or *2B/*2B) combined with a GSTM1 null genotype was remarkably shortened, compared with that of patients with the wild type allele combination (Goto et al. 1996), confirming earlier results based on phenotyping (Bartsch et al. 1990). These studies suggested a genetic basis for the association of high CYP1A1 metabolic activity and GSTM1 null genotypes with increased lung cancer incidence, but the underlying molecular mechanisms regulating this effect are still not well understood (discussed in Rojas et al. 1998). Therefore, the levels of BPDE-DNA adducts were analysed by HPLC-FD in nontumourous lung tissue from 20 lung cancer patients and in white blood cells from 20 coke oven workers exposed to PAHs, all current tobacco smokers. CYP1A1 mutations and GSTM1 deletion polymorphisms in each subject were analysed in genomic DNA by PCR/RFLP (Cascorbi 1996). 40
3.1 Genetic Cancer Susceptibility and DNA Adducts Table 1: Classification of CYP1A1-GSTM1 genotype combinations in groups with low, medium and high levels of BPDE-DNA adducts; the data were compiled for lung parenchymal DNA from 20 smoking lung cancer patients and for WBC DNA from 20 PAH-exposed (asymptomatic) coke oven workers (extracted from Rojas et al. 1998). Smokers with the combination of mutated homozygous CYP1A1 and GSTM1 null had the highest PAH-DNA adduct levels and were shown earlier to be at high risk for squamous cell carcinoma (SCC) of the lung in Japanese populations, even at a low dose of cigarette smoke (Nakachi and Imai 1996; for details see text). Genotype combination
CYP1A1 CYP1A1 CYP1A1 CYP1A1 *0/*0 CYP1A1 CYP1A1 CYP1A1 a) b)
*1/*1-GSTM1 active *1/*2A-GSTM1 active *1/*1-GSTM1 *0/*0 *1/*2A or *2B-GSTM1
BPDE adducts per 108 nt (median)
Odds ratios for genetic susceptibility of Japanese smokers for SCC
Lung DNA from lung cancer patients (n = 20)
WBC-DNA from coke oven workers (n = 20)
Low cigarette dose
High cigarette dose
<0.2 <0.2 0.7 n.d. b)
<0.2 <0.2 0.7 0.6
1.0 (*0.5) a) (*3.0) a) (*3.0) a)
9.0 13.0 11.8 16.3
44.0 n.d. n.d.
16.0 n.d. 41.0
20.0 n.d. n.d.
*2A/*2A-GSTM1 *0/*0 6.4 *2A/*2B-GSTM1 *0/*0 1.9 *2B/*2B-GSTM1 *0/*0 n.d.
Approximate values extracted from Nakachi and Imai 1996. Not determined.
Our results showed, for the first time, a clear effect of the combination of CYP1A1 and GSTM1 genotypes on the formation of BPDE-DNA adducts in the human lung and white blood cells (Table 1): (i) Subjects with the GSTM1 active genotype were not found to have any detectable BPDEDNA adducts (<0.2 per 108 nt), (ii) those with CYP1A1*1/*1 or who were heterozygous for GYP1A1*2A or *2B and with a GSTM1 null combination had low but significantly increased (P <0.00005) levels of BPDE-DNA adducts, and (iii) those with the CYP1A1*2A/*2A mutant allele from the GSTM1 null group had the highest BPDE-DNA adduct levels. A graphical representation of these data is shown in Figure 2. The consistent results in the two pilot study groups demonstrated that the combination of homozygous mutated CYP1A1 and GSTM1 null led, at a similar or even lower smoke dose, to a stronger increase in BPDE-DNA adduct levels than found in subjects with CYP1A1 and GSTM1 wild type. In support of our observation, Soni et al. (1998) reported higher bulky DNA 41
3 DNA Adducts
Figure 2: Genotype dependence of BPDE-DNA adduct levels in the lung (smokers) and white blood cells (WBC) (coke oven workers); M = mutated; 0 = deleted.
adduct levels in the lymphocytes of smokers who had deficient GSTM1 enzyme activity. Our data also provide, for the first time, an understanding of the mechanisms shown in previous epidemiological studies that correlated these ‘risk’ genotypes in Japanese workers with increased smoking-related lung cancers particularly at lower levels of cigarette consumption (Nakachi & Imai 1996) and shortened survival of lung cancer patients (Goto et al. 1996). Although homozygous carriers of the Mspl-mutation of CYP1A1 are very rare among Caucasians (<1%) (Cascorbi et al. 1996), a Scandinavian study found the combined CYP1A1 (Mspl) and GSTM1 null genotype significantly over-represented in patients with squamous-cell carcinoma of the lung (Alexandrie et al. 1994). Our findings are also consistent with the prevalence of G:C ? T:A transversion mutations found in the p53 tumour suppressor gene of tobacco-associated lung tumours. This suggests PAH-related mutational damage occurs more frequently in persons who are GSTM1 null (Ryberg et al. 1994). Furthermore, the coincidence of mutational hotspots in p53 and BPDE-related adduct hotspots suggests that BPDE and structurally related PAH diol-epoxides are involved in the transformation of human lung tissue in smokers (Denissenko et al. 1996). In conclusion, the examination of BPDE-DNA adducts resulting from the GSTM1 and the CYP1A1 polymorphisms and their interaction with other susceptibility markers, such as mutagen sensitivity and impaired DNA-repair capacity (Hsu 1987), will help to identify high-risk subjects among smokers and subjects occupationally and/or environmentally exposed to PAHs. There is growing evidence that predisposing polymorphic genes, involved in carcinogen metabolism and repair, increase the cancer risk in certain subjects (Caporaso & Goldstein 1995), even when they are 42
3.1 Genetic Cancer Susceptibility and DNA Adducts exposed to only low levels of carcinogens (Vineis et al. 1994, Vineis 1997). It is therefore necessary within preventive strategies to identify these vulnerable members in our society, in particular those with unfavourable combinations of carcinogen exposure, predisposing genes and a lack of protective factors.
3.1.3.4 Acknowledgements We acknowledge the collaboration and contributions during various phases of our studies on lung cancer patients and coke oven workers of G. Auburtin and L. Mayer (Institut National de L‘Environment Industriel et des Risques, Verneuil en Hallate, France), I. Cascorbi, J. Brockmöller and I. Roots (University Clinic Charité, Humboldt University, Berlin, Germany), and H. Vainio, S. Anttila and K. Husgafvel-Pursiainen (Institute of Occupational Health, Helsinki, Finland).
References Alexandrie, A. K.; Sundberg, M. I.; Seidegard, J. et al.: Genetic susceptibility to lung cancer with special emphasis on CYP1A and GSTM1: a study on host factors in relation to age at onset, gender and histological cancer types, Carcinogenesis, 15, 1785– 1790 (1994). Alexandrov, K.; Rojas, M.; Geneste, O. et al.: An improved fluorometric assay for dosimetry of benzo(a)pyrene diol-epoxide-DNA adducts in smoker‘s lung: comparison with total bulky adducts and aryl hydrocarbon hydroxylase activity, Cancer Res., 52, 6248–6253 (1992). Arand, M.; Muhlbauer, R.; Hengstler, J. et al.: Multiplex polymerase chain reaction protocol for the simultaneous analysis of the glutathione S-transferase GSTM1 and GSTT1 polymorphisms, Anal. Biochem. 236, 184–186 (1996). Bartsch, H.; Hietanen, E.; Petruzzelli, S. et al.: Possible prognostic value of pulmonary Ah-locus-linked enzymes in patients with tobacco-related lung cancer, Int. J. Cancer, 46, 185–188 (1990). Bartsch, H.; Hietanen, E.: The role of individual susceptibility in cancer burden related to environmental exposure, Environ. Hlth. Perspect., 104 (S3), 569–577 (1996). Bartsch, H.; Rojas, M.; Alexandrov, K. et al.: Metabolic polymorphism effecting DNA binding and excretion of carcinogens in humans, Pharmacogenetics, 5, 84–90 (1995). Bartsch, H.: DNA adducts in human carcinogenesis: etiological relevance and structure-activity relationship, Mutat. Res., 340, 67–79 (1996). Brockmöller, J.; Kerb, R.; Drakoulis, N. et al.: Genotype and phenotype of glutathione S-transferase class l isoenzymes in lung cancer patients and controls, Cancer Res., 53, 1004–1011 (1993). Caporaso, N.; Goldstein, A.: Cancer genes – single and susceptibility – exposing the difference, Pharmacogenetics, 5, 59–63 (1995). Cascorbi, I.; Brockmöller, J.; Roots, I.: A C4887A polymorphism in exon 7 of human CYP1A1: population frequency, mutation linkages, and impact on lung cancer susceptibility, Cancer Res., 56, 4965–4969 (1996). Denissenko, M. F.; Pao, A.; Tang, M. S. et al.: Preferential formation of benzo(a)pyrene adducts at lung cancer mutational hotspots in p53, Science, 274, 430–432 (1996).
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3 DNA Adducts Garte, S. A.; Crosti, F. A.: Nomenclature system for metabolic gene polymorphisms, in: Vineis, P.; Lang, M.; Caporaso, N. et al. (eds.): Metabolic polymorphism and cancer, IARC Sci. Publ., Lyon, IARC (1998). Goto, M.; Yoneda, S.; Yamamoto, M. et al.: Prognostic significance of germ line polymorphisms of the GYP1A1 and glutathione S-transferase genes in patients with nonsmall cell lung cancer, Cancer Res., 56, 3725–3730 (1996). Hayashi, S.-I.; Watanabe, J.; Kawajiri, K.: High susceptibility to lung cancer analyzed in terms of combined genotypes of P4501A1 and Mu-class glutathione S-Transferase genes, Jpn. J. Cancer Res., 83, 866–870 (1992). Hsu, T. C.: Genetic predisposition to cancer with special reference to mutagen sensitivity, in vitro cellular and developmental biology, Tissue Culture Ass. Inc., Vol. 23(9), 591–603 (1987). Kawajiri, K.; Nakachi, K.; Imai, K. et al.: Identification of genetically high risk individuals to lung cancer by DNA polymorphism of the cytochrome P4501A1 gene, FEBS Lett., 263, 131–133 (1990). Kihara, M.; Noda, K.: Risk of smoking for squamous and small cell carcinomas of the lung modulated by combinations of CYP1A1 and GSTM1 gene polymorphisms in a Japanese population, Carcinogenesis, 16, 2331–2336 (1995). Kriek, E.; Rojas, M.; Alexandrov, K. et al.: Polycyclic aromatic hydrocarbon-DNA adducts in humans: relevance as biomarkers for exposure and cancer risk, Mutat. Res., 400, 215–231 (1998). McWilliams, J. E.; Sanderson, B. J. S.; Harris, E. L. et al.: Glutathione S-transferase M1 (GSTM1) deficiency and lung cancer risk, Cancer Epidemiol. Biomarkers Prev., 4, 589–594 (1995). Nakachi, K.; Imai, K.; Hayashi, S.-I. et al.: Genetic susceptibility to squamous cell carcinoma of the lung in relation to cigarette smoking dose, Cancer Res., 51, 5177–5180 (1991). Nakachi, K.; Imai, K.; Hayashi, S.-I. et al.: Polymorphisms of the CYP1A1 and glutathione S-transferase genes associated with susceptibility to lung cancer in relation to cigarette dose in a Japanese population, Cancer Res., 53, 2994–2999 (1993). Nakachi, K.; Imai, K.: Epidemiology of genetic susceptibility and cigarette smoking in lung carcinogenesis by CYP1A1 polymorphisms, Gann Monograph on Cancer Res., 44, 195–205 (1996). Nakajima, T.; Elovaara, E.; Anttila, S. et al.: Expression and polymorphism of glutathione S-transferase in human lungs: risk factors in smoking-related lung cancer, Carcinogenesis, 16, 707–711 (1995). Rojas, M.; Alexandrov, K.; Auburtin, G. et al.: Anti-benzo(a)pyrene diol-epoxide-DNA adduct levels in peripheral mononuclear cells from coke oven workers and the enhancing effect of smoking, Carcinogenesis, 16, 1373–1376 (1995). Rojas, M.; Alexandrov, K.; Cascorbi, I. et al.: High benzo(a)pyrene diol-epoxide DNA adduct levels in lung and blood cells from subjects with combined CYP1A1 MspI/ MspI-GSTM1*0/*0 genotypes, Pharmacogenetics, 8, 109–118 (1998). Rojas, M.; Alexandrov, K.; van Schooten, F.-J. et al.: Validation of a new fluorometric assay for benzo(a)pyrene diol-epoxide-DNA adducts in human white blood cells: comparison with 32P-postlabeling and ELISA, Carcinogenesis, 15, 557–560 (1994). Ryberg, D.; Kure, E.; Lystad, S. et al.: p53 mutations in lung tumors: relationship to putative susceptibility markers for cancer, Cancer Res., 54, 1551–1555 (1994). Soni, M.; Madurantakan, M.; Krishnaswamy, K.: Glutathione S-transferase Mu (GST Mu) deficiency and DNA adducts in lymphocytes of smokers, Toxicology, 126, 155– 162 (1998). Tang, D.; Santella, R. M.; Blackwood, A. M. et al.: A molecular epidemiological casecontrol study of lung cancer, Cancer Epidemiol. Biomarkers Prev., 4, 341–346 (1995).
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3.1 Genetic Cancer Susceptibility and DNA Adducts Van Schooten, F. J.; Jongeneelen, F. J.; Hillebrand, M. J. X. et al.: Polycyclic aromatic hydrocarbon-DNA adducts in white blood cell DNA and 1-hydroxypyrene in the urine from aluminum workers: Relation with job category and synergistic effect of smoking, Cancer Epidemiol. Biomarkers Prev., 4, 69–77 (1995). Vaury, C.; Laine, R.; Noguiez, P. et al.: Human glutathione S-transferase M1 null genotype is associated with a high inducibility of cytochrome P450 1A1 gene transcription, Cancer Res., 55, 5520–5523 (1995). Vineis, P.; Bartsch, H.; Caporaso, N. et al.: Genetically based N-acetyltransferase metabolic polymorphism and low-level environmental exposure to carcinogens, Nature, 369, 154–156 (1994). Vineis, P.: Molecular epidemiology: low-dose carcinogens and genetic susceptibility, Int. J. Cancer, 71, 1–3 (1997). Volkenandt, M.; Dicker, A. P.; Banerjee, D. et al.: Methods in molecular biology, PCR protocols: current methods and applications, Totowa NJ, Humana Press (1993). Wiencke, J. K.; Kelsey, K. T.; Varkonyi, A. et al.: Correlation of DNA adducts in blood mononuclear cells with tobacco carcinogen-induced damage in human lung, Cancer Res., 55, 4910–4914 (1995).
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3.2
The Detection of DNA Adducts in Biological Monitoring Werner K. Lutz and Martin G. Maisch *
3.2.1 Introduction DNA adducts are formed as a result of the covalent binding with DNA of chemically reactive substances or their reactive metabolic intermediates. DNA adducts can lead to mutations, and therefore the formation of DNA adducts can be seen as an early stage in the process of chemical mutagenesis and carcinogenesis. For biological monitoring, DNA adducts are therefore being considered not only as exposure markers, but also as biochemical effect markers. For the biological monitoring of exposure, the most suitable source of DNA are probably white blood cells. Other sources, such as exfoliated cells of the epithelium of the urinary tract taken from urine or smears of the oral mucosa, are usually not sensitive enough as a result of the limited amount of DNA obtained. Material from biopsies and surgical excision can only be obtained in exceptional cases. The most important methods for the detection of DNA adducts are listed in Table 1. The first analysis of a human DNA sample to be published was based on an antiserum against benzo[a]pyrene–DNA adducts (Perera et al. 1982). Other immunological analyses are available for the DNA adducts of 2-acetylaminofluorene, aflatoxin, cisplatin, 8-methoxypsoralen, methylating agents, and polycyclic aromatic hydrocarbons (PAHs). As a result of cross-reactions and the problems this brings with it for the quantification of the adducts, over the past few years antibodies have been used less often for directly determining adduct concentrations, but more often for the enrichment of adducts using affinity chromatography. Parallel to the immunological methods of detection, the 32P-postlabelling method was developed (Randerath et al. 1981). Later on, electrochemical (Floyd et al. 1986), fluorescence spectroscopic (Weston et al. 1989, Alexandrov et al. 1992) and mass spectrometric methods were developed. Sev*
46
Lehrstuhl für Toxikologie der Universität Würzburg, Versbacher Str. 9, 97078 Würzburg
3.2 The Detection of DNA Adducts in Biological Monitoring Table 1: Methods for determining DNA adducts and their detection limits (acc. R.C. Garner, Mutat Res 402 [1998] 67–75, modified). Methods
Relative detection for the following limit (adducts/ DNA sample nucleotides) weights (lg)
Immunoassay (no longer used for direct quantification) Slot-blot assay 3/105 USERIA 1/107 ELISA competitive 3/107 ELISA non-competitive 1/107
1 25 50 1
32
P-postlabelling (certain restrictions) Standard method 1/107 After enrichment of the adducts (nuclease P1, 1/109 extraction, chromatography) Fluorescence spectroscopy (great restrictions) With low temperatures Synchronous fluorescence Line narrowing analysis
10 10
3/107 3/107 1/108
1000 100 1000
1/107
50
3/109
100
3/108 1/107
100 100
Electrochemical detection (great restrictions)
Mass spectrometry (slight restrictions) GC-MS of the carcinogen (after cleavage of the DNA) GC-MS of alkylated bases LC-MS of nucleoside adducts
eral volumes containing congress papers (Bartsch et al. 1988, Gledhill & Mauro 1991, Bartsch et al. 1993, Hemminki et al. 1994) and reviews (Perera 1988, Schut & Shiverick 1992, Hemminki 1995, Schell et al. 1995, Talaska et al. 1996, Poirier 1997, Wild & Pisani 1997, Garner 1998) describe the possibilities and limitations of DNA adduct analysis. The detection limits for the adduct concentrations in DNA that can be reached with the various methods and the amounts of DNA required are also shown in Table 1. To evaluate how this can be used in biological monitoring it is necessary to know that the background exposure to polycyclic aromatic hydrocarbons taken up via the food and air leads to adduct concentrations in the range of 1–10 in 108 nucleotides (Schoket 1999) and that in general about 10 lg DNA can be isolated from the leukocytes in 1 millilitre blood. For these reasons the 32P-postlabelling method seems particularly well-suited for the biological monitoring of environmental exposure in man. 47
3 DNA Adducts When evaluating the various methods, not only quantitative aspects, however, but also qualitative aspects are of importance, in particular the question of the amount of structural information yielded by the analysis. The postlabelling method is very sensitive, but does not provide any information about the chemical structure. Mass spectrometry (MS) would be the most suitable method in this respect. Both methods are described below.
3.2.2 Methods 3.2.2.1 The
32
P-Postlabelling Method
a) Detection principle DNA is enzymatically hydrolyzed to deoxynucleoside-3'-phosphates (from now on referred to as 3'-nucleotides). Compared to the normal 3'-nucleotides, the 3'-nucleotide adducts are enriched (see below) and phosphorylated enzymatically at the 5' position with 32P-phosphate from [c-32P]ATP. During thin-layer chromatography the nucleotide carcinogen adducts are chromatographically different from the normal nucleotides and after twodimensional chromatography they can be detected on the thin-layer chromatographic plate as radioactive spots and quantified by measuring the radioactivity (most easily by means of electronic autoradiography). The method is described in detail in Gupta 1996. b) Enrichment of the adducts In the “enrichment” procedure usually used, the 3'-nucleotides (normal nucleotides plus the corresponding adducts) obtained after DNA hydrolysis are treated with nuclease P1 (Reddy & Randerath 1986). This enzyme removes the phosphate group at the 3' position. The nucleosides formed are no longer substrates for enzymatic phosphorylation at the 5' position and are therefore no longer radioactively labelled. Many adducts are poor substrates for nuclease P1; they retain the 3' phosphate group and can be 32Pphosphorylated at the 5' position and thus determined. Unfortunately, not all adducts are nuclease P1-resistent. Well-suited are the adducts of polycyclic aromatic hydrocarbons. Most adducts determined in human samples have therefore belonged to this class of substances. Also various adducts from a, b-unsaturated aldehydes resulting from oxidative stress can be processed in this way. If this is not possible, enrichment must be carried out by means of extraction (Gupta 1985) or by using chromatographic procedures. c) Applications The first adducts detected in the DNA from the oral mucosa of a smoker and a non-smoker were presented at a congress at the end of 1983 (Ran48
3.2 The Detection of DNA Adducts in Biological Monitoring derath et al. 1984). In 1986 DNA adducts were detected in placental tissue (Everson et al. 1986). At the same time, the detection of DNA adducts in the peripheral lymphocytes of coke-oven workers was reported for the first time (Haugen et al. 1986). Most publications deal with exposure to polycyclic aromatic hydrocarbons as a result of smoking at the workplace, and due to general air pollution. This focusing on PAHs results from the fact that the adducts formed can be easily enriched with nuclease P1 and the phosphorylation reaction has a high yield. With other adducts the situation is usually not as favourable. In such cases it is often too much effort to optimize the methods available, so that many published data can only be interpreted qualitatively (aromatic amines, styrene, certain drugs; overview in Lutz 2000).
3.2.2.2 Mass Spectrometry Mass spectrometric analysis, unlike 32P-postlabelling, also provides structural information. The disadvantages are its lower sensitivity and the lack of volatility. Particularly for the analysis of nucleoside adducts the latter presents a great problem. The combination of gas chromatography and mass spectrometry (GCMS), implemented successfully for decades, is therefore used only to analyse (small) base adducts or after cleaving the adduct-forming substance. Examples are the aromatic amine 4-aminobiphenyl (Lin et al. 1994, Culp et al. 1997) and the heterocyclic amine PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) (Friesen et al. 1994). Base adducts were detected in human samples for example by identifying methylated (Shuker & Bartsch 1994) and 2-hydroxyethylated guanine (Ranasinghe et al. 1998, Wu et al. 1999), and malonaldehyde-modified guanine (Rouzer et al. 1997). Over the past few years the combination of liquid chromatography with mass spectrometry (LC-MS) has been improved and the analysis of nucleoside adducts after electrospray ionization (ESI) and tandem mass spectrometry (MS/MS) developed further. Examples of this are the deoxyguanosine modifications caused by the heterocyclic amines PhIP and IQ (3methyl-3H-imidazo[4,5-f]quinolin-2-amine) (Rindgen et al. 1995, Gangl et al. 1999), 4-aminobiphenyl (Doerge et al. 1999), malonaldehyde (Chaudhary et al. 1996), and the analysis of 1,N6-etheno-deoxyadenosine (edAdo) (Chen et al. 1999). The last study above describes the only analysis known to us of nucleoside adducts in DNA from a human sample (placenta), and yields an adduct concentration in the range of 2.5 edAdo per million dAdo.
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3 DNA Adducts
3.2.3 Research Required for Improving Methods 3.2.3.1 Situation in Germany The postlabelling method is used in about a dozen laboratories in Germany, and experiences with it are constantly exchanged in regular meetings. DNA samples from in vitro incubation or experiments with animals predominate, in connection with the question of the genotoxic potential of known or potential mutagens and carcinogens. Publications of the original data from the biological monitoring of DNA adducts in man are rare and come almost exclusively from the research group under the leadership of H. Bartsch at the Deutsches Krebsforschungszentrum in Heidelberg. The mass spectrometric analysis of DNA adducts from human samples is, world-wide, still in its infancy. With regard to methodological developments, Germany is about 5 years behind the USA. This is due, in part, also to the cost of high-performance equipment and the problem of financing without preliminary data. The initiative of the DFG in the summer of 1999 to provide high-performance equipment concentrated (unfortunately) on the acquisition of Fourier transform and time-of-flight mass spectrometry, which is less suitable for the analysis of nucleoside adducts. The use of mass spectrometry to analyse DNA adducts was previously limited to the structural verification of base adducts (e.g. Pfau et al. 1997) or was carried out in a laboratory that specialized in spectrochemical analysis (Schrader & Linscheid 1997, Siethoff et al. 1999). Mass spectrometry should be used more often in molecular-epidemiological studies. In our opinion both postlabelling (PL) and mass spectrometry (MS) should be further developed for the biological monitoring of DNA adducts in man, as the two methods complement each other well with regard to sensitivity and structural information. Increased collaboration of the various working groups would be desirable in the exchange of samples and synthesized adduct standards. The development of further methods of enrichment and analysis should also be promoted.
3.2.3.2 The
32
P-Postlabelling Method
Thanks to the high specific activity of 32P (7000 Ci/mmol), this method is extremely sensitive: a few lg DNA may be sufficient to detect an adduct concentration in the range of 1 in 109 nucleotides. Another advantage is that no expensive equipment is needed and that with the use of disposable materials the danger of contamination is relatively low. The accuracy and reproducibility of the determination of adduct concentrations must, however, be improved. Intercomparison programmes in 15 laboratories in which defined samples with relatively high adduct concentrations were analysed, yielded coefficients of variation, after “outliers” 50
3.2 The Detection of DNA Adducts in Biological Monitoring had been excluded, of up to 100% (Phillips & Castegnaro 1993, Phillips et al. 1999). Even within one laboratory the adduct concentration determined can differ greatly from batch to batch, in particular in the range of the detection limit. For a detailed discussion of the various sources of error, reference is made to the review of Lutz (Lutz 2000). For correct quantification, various positive and negative controls (including internal and additive standards) must be carried out. Few of the data published to date meet the requirements of good analytical practice with regard to quantification. If these quality criteria can be fulfilled, thanks to its high sensitivity and the low amount of sample material needed, postlabelling will be able to secure a place for itself, and the synthesis of adduct standards to optimize reproducibility and detection limit will be worthwhile aims.
3.2.3.3 Mass Spectrometry The methodological development of the mass spectrometric analysis of DNA adducts for biological monitoring will probably focus on deoxynucleoside adducts, as with base adducts there is the problem of contamination with RNA bases and the question of recovery after base splitting. Problematic areas during the analysis of nucleoside adducts are the enrichment of the adducts in small volumes and the detection limits that can then be reached. The areas of application of mass spectrometry in biological monitoring will remain limited for the time being to high exposures/ high adduct concentrations (for example in the case of treatment with cytostatic drugs) and/or cases where large amounts of DNA are available (for example after surgery). The insights gained from this, together with the expected developments in the equipment, will help make it possible in future to be able to carry out the biological monitoring of DNA adducts also after environmental exposure by analysing leukocyte DNA from just a few millilitres of blood.
3.2.4
Research Required in Various Subject Areas
3.2.4.1 Background DNA Adducts Endogenous agents and processes (Lutz 1990) can lead to DNA modifications, which can be detected with postlabelling. Some of these DNA modifications are known as “I-compounds” (indigenous = endogenous). These are large, structurally not identified nucleotide modifications, whose occurrence – even in untreated animals – depends on many factors, such as age, sex, organ, nutrition and oxidative processes (Randerath et al. 1992, Randerath et al. 1999). In addition to endogenous DNA modifications, exposure 51
3 DNA Adducts to unavoidable exogenous carcinogens also leads to detectable DNA adducts, e.g., exposure to polycyclic aromatic hydrocarbons in food and general air pollution. The total background damage is in the range of about 1 damaged nucleotide in 105 nucleotides (Gupta & Lutz 1999). Detection of background exposure with postlabelling is by no means merely a disturbance or disadvantage. It provides a possible reference value on the basis of which additional exposure, for example at the workplace, can be evaluated as an incremental (cancer) risk. This question is of importance also in connection with the evaluation of MAK values for chemical carcinogens of Group 5, as it must be decided at which level of exposure an increment above background becomes significant. An increase in background adducts after the treatment of animals with test substances could be (mis)interpreted as the production of adducts by the substance. A structural elucidation of such DNA modifications would therefore be of great interest also for this reason.
3.2.4.2 Interindividual Differences As mentioned earlier, most adduct-forming substances must be metabolically activated to produce chemically reactive intermediates. The concentration of these so-called “ultimate” carcinogens is therefore dependent upon the activity of the activating and detoxifying enzymes involved. The repair of the adducts formed is also catalysed by enzymes to a large extent. The activity of all these enzymes differs from individual to individual, depending on genetically determined factors and exogenous influences, such as enzyme induction as a result of smoking or medicines. The same level of exposure can therefore cause completely different adduct concentrations in different people. Lung tissue from smokers and non-smokers examined for DNA adducts caused by polycyclic aromatic hydrocarbons revealed, for example, that the adduct concentrations in the group of smokers were on average 3 times higher than in the non-smokers, but the range of values overlapped to such an extent that some smokers had lower values than many non-smokers. At present, the enzymes suspected of being involved in this variability are under investigation and many molecular-epidemiological studies examine the relationship between enzyme polymorphism and the incidence of cancer. As markers of biochemical effects, DNA adducts are highly suitable for this purpose. In addition, also investigations into the chemo-prevention of carcinogenesis or the optimization of medication in the individual, in particular in the chemotherapy of tumours, are worthwhile aims.
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3.2 The Detection of DNA Adducts in Biological Monitoring 3.2.4.3 The Course of DNA Adduct Concentrations after Exposure The adduct concentration determined in a DNA sample is the result of adduct formation, adduct repair and “adduct dilution” due to DNA replication (the newly synthesized strand does not contain any adducts on formation). An adduct concentration is therefore the result of an equilibrium which is also governed to a high degree by the rate of cell division and thus the lifetime of the cells. The longer the cell lives, the longer the period in which the exposure leaves its mark in the form of DNA adducts. In a sample of peripheral leukocytes the lifetime depends on the cell type. Sub-populations of lymphocytes can have a lifetime of several years, while peripheral granulocytes survive for a few days at the most. Differences between lymphocytes and total leukocytes are therefore understandable. It is also understandable that a change in the cell population sampled can simulate changes in the genotoxic effects. To investigate such questions, also analyses of the corresponding haemoglobin adducts may be of use. Hb adducts are not “repaired”, and the lifetime of erythrocytes is clearly defined. The kinetics of Hb adducts is therefore easier to interpret than that of DNA adducts.
3.2.4.4 DNA Adducts as Exposure Markers or Effect Markers? During DNA replication DNA adducts can lead to the insertion of a wrong base in the daughter strand. After further replication the false pairing can become “fixed” via base pair substitution. For a proto-oncogen or tumour suppressor gene this mutation could be one step in the process of carcinogenesis. A correlation between adduct concentration and cancer risk could therefore be postulated. It is a long way from a DNA adduct to a tumour and the probability that step by step a cell will become malignant depends on many other, individually determined factors. A correlation between the primary DNA damage and the probability of cancer developing can therefore be assumed for a large collective, but not for an individual. Our studies of PAH-DNA adducts in the normal mucous membranes and peripheral lymphocytes of patients with tumours of the pharynx and larynx surprisingly yielded a correlation between cigarette consumption and the adduct concentration in the mucous membranes, but not in the peripheral lymphocytes (Maisch et al. 2000). This example shows that more work is needed before DNA adduct data can be reliably interpreted as a biomarker.
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3 DNA Adducts References Alexandrov, K.; Rojas, M.; Geneste, O.; Castegnaro, M.; Camus, A. M.; Petruzzelli, S.; Giuntini, C.; Bartsch, H.: An improved fluorometric assay for dosimetry of benzo(a)pyrene diol-epoxide-DNA adducts in smokers’ lung – comparisons with total bulky adducts and aryl hydrocarbon hydroxylase activity, Cancer Res., 52, 6248–6253 (1992). Bartsch, H.; Hemminki, K.; O’Neill, I. K. (eds): Methods for detecting DNA damaging agents in humans: applications in cancer epidemiology and prevention, IARC Sci. Publ.; vol. 89, pp. 219 (1988). Bartsch, H.; Kadlubar, F.; O‘Neill, I. (eds): Biomarkers in Human Cancer – Part II. Exposure Monitoring and Molecular Dosimetry, Environ. Health Perspect., 99, pp. 312 (1993). Chaudhary, A. K.; Reddy, G. R.; Blair, I. A.; Marnett, L. J.: Characterization of an N6-oxopropenyl-2’-deoxyadenosine adduct in malondialdehyde-modified DNA using liquid chromatography/electrospray ionization tandem mass spectrometry, Carcinogenesis, 17, 1167–1170 (1996). Chen, H.-J. C.; Chiang, L.-C.; Tseng, M.-C.; Zhang, L. L.; Ni, J.; Chung, F.-L.: Detection and quantification of 1,N6-ethenoadenine in human placental DNA by mass spectrometry, Chem. Res. Toxicol.; 12, 1119–1126 (1999). Culp, S. J.; Roberts, D. W.; Talaska, G.; Lang, N. P.; Fu, P. P.; Lay J. O. Jr.; Teitel, C. H.; Snawder, J. E.; Von Tungeln, L. S.; Kadlubar, F. F.: Immunochemical, 32P-postlabeling and GC/MS detection of 4-aminobiphenyl-DNA adducts in human peripheral lung in relation to metabolic activation pathways involving pulmonary N-oxidation, conjugation, and peroxidation, Mutat. Res., 378, 97–112 (1997). Doerge, D. R.; Churchwell, M. I.; Marques, M. M.; Beland, F. A.: Quantitative analysis of 4-aminobiphenyl-C8-deoxyguanosyl DNA adducts produced in vitro and in vivo using HPLC-ES-MS, Carcinogenesis, 20, 1055–1061 (1999). Everson, R. B.; Randerath, E.; Santella, R. M.; Cefalo, R. C.; Avitts, T. A.; Randerath, K.: Detection of smoking-related covalent DNA adducts in human placenta, Science, 231, 54–57 (1986). Floyd, R. A.; Watson, J. J.; Harris, J.; West, M.; Wong, P. K.: Formation of 8-hydroxydeoxyguanosine, hydroxyl free radical adduct of DNA in granulocytes exposed to the tumor promoter, tetradecanoylphorbolacetate, Biochem. Biophys. Res. Commun., 137, 841–846 (1986). Friesen, M. D.; Kaderlik, K.; Lin, D.; Garren, L.; Bartsch, H.; Lang, N. P.; Kadlubar, F. F.: Analysis of DNA adducts of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in rat and human tissues by alkaline hydrolysis and gas chromatography/electron capture mass spectrometry: validation by comparison with 32P-postlabeling, Chem. Res. Toxicol.; 7, 733–739 (1994). Gangl, E. T.; Turesky, R. J.; Vouros, P.: Determination of in vitro- and in vivo-formed DNA adducts of 2-amino-3-methylimidazo[4,5-f]quinoline by capillary liquid chromatography/microelectrospray mass spectrometry, Chem. Res. Toxicol., 12, 1019–1027 (1999). Garner, R. C.: The role of DNA adducts in chemical carcinogenesis, Mutat. Res., 402, 67–75 (1998). Gledhill, B. L.; Mauro, F.: New Horizons in Biological Dosimetry. New York, Chichester, Brisbane, Toronto, Singapore: Wiley-Liss (1991). Gupta, R. C.: 32P-Postlabeling for detection of DNA adducts, in: Pfeifer, G. P. (ed): Technologies for Detection of DNA Damage and Mutations, New York: Plenum Press, pp. 45–61 (1996).
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3.2 The Detection of DNA Adducts in Biological Monitoring Gupta, R. C.; Lutz, W. K.: Background DNA damage from endogenous and unavoidable exogenous carcinogens: a basis for spontaneous cancer incidence? Mutat. Res., 424, 1–8 (1999). Gupta, R. C.: Enhanced sensitivity of phosphorus-32 postlabeling analysis of aromatic carcinogen:DNA adducts, Cancer Res., 45, 5656–5662 (1985). Haugen, A.; Becher, G.; Benestad, C.; Vahakangas, K.; Trivers, G. E.; Newman, M. J.; Harris, C. C.: Determination of polycyclic aromatic hydrocarbons in the urine, benzo(a)pyrene diol epoxide-DNA adducts in lymphocyte DNA, and antibodies to the adducts in sera from coke oven workers exposed to measured amounts of polycyclic aromatic hydrocarbons in the work atmosphere, Cancer Res., 46, 4178–4183 (1986). Hemminki, K.; Dipple, A.; Shuker, D. E. G.; Kadlubar, F. F.; Segerbaeck, D.; Bartsch, H.: DNA Adducts: Identification and biological significance, IARC Sci. Publ., 125, pp. 478 (1994). Hemminki, K.: DNA adducts in biomonitoring, J. Occup. Environ. Med., 37, 44–49 (1995). Lin, D.; Lay, J. O. Jr. Bryant, M. S.; Malaveille, C.; Friesen, M.; Bartsch, H.; Lang, N. P.; Kadlubar, F. F.: Analysis of 4-aminobiphenyl-DNA adducts in human urinary bladder and lung by alkaline hydrolysis and negative ion gas chromatography-mass spectrometry, Environ. Health Perspect., 102 Suppl. 6, 11–16 (1994). Lutz, W. K.: DNA-Addukte als Genotoxizitätsmarker für ein biologisches Monitoring, Arbeitsmed. Sozialmed. Umweltmed., 35, 303–311 (2000). Lutz, W. K.: Endogenous genotoxic agents and processes as a basis of spontaneous carcinogenesis, Mutat. Res., 238, 287–295 (1990). Maisch, M.; Vock, E. H.; Preisler, V.; Hoppe, F.; Vamvakas, S.; Helms, J.; Lutz, W. K.: Biomonitoring of DNA adducts in lymphocytes and normal mucosa of pharynx and larynx in cancer patients as potential indicators of smoking dose, duration, or age at tumor manifestation, Naunyn-Schmied. Arch. Pharmacol., 361 Suppl, R170 (2000). Perera, F. P.; Poirier, M. C.; Yuspa, S. H.; Nakayama, J.; Jaretzki, A.; Curnen, M. M.; Knowles, D. M.; Weinstein, I. B.: A pilot project in molecular cancer epidemiology: determination of benzo(a)pyrene-DNA adducts in animal and human tissues by immunoassays, Carcinogenesis, 3, 1405–1410 (1982). Perera, F. P.: The significance of DNA and protein adducts in human biomonitoring studies, Mutat. Res., 205, 255–269 (1988). Pfau, W.; Schulze, C.; Shirai, T.; Hasegawa, R.; Brockstedt, U.: Identification of the major hepatic DNA adduct formed from the food mutagen 2-amino-9H-pyrido[2,3-b]indole (A alpha C), Chem. Res. Toxicol., 10, 1192–1197 (1997). Phillips, D. H.; Castegnaro, M.; participants on behalf of the trial participants: Standardization and validation of DNA adduct postlabelling methods: report of interlaboratory trials and production of recommended protocols, Mutagenesis, 14, 301–315 (1999). Phillips, D. H.; Castegnaro, M.: Results of an interlaboratory trial of 32P-postlabelling, IARC Sci. Publ., 124, 35–49 (1993). Poirier, M. C.: DNA adducts as exposure biomarkers and indicators of cancer risk, Environ. Health Perspect., 105 Suppl. 4, 907–912 (1997). Ranasinghe, A.; Scheller, N.; Wu, K. Y.; Upton, P. B.; Swenberg, J. A.: Application of gas chromatography/electron capture negative chemical ionization high-resolution mass spectrometry for analysis of DNA and protein adducts, Chem. Res. Toxicol., 11, 520– 526 (1998). Randerath, K.; Li, D. H.; Nath, R.; Randerath, E.: Exogenous and endogenous DNA modifications as monitored by 32P-postlabeling – relationships to cancer and aging, Exp. Gerontol., 27, 533–549 (1992).
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3 DNA Adducts Randerath, K.; Randerath, E.; Agrawal, H. P.; Reddy, M. V.: Biochemical (postlabelling) methods for analysis of carcinogen-DNA adducts, IARC Sci. Publ., 59, 217–231 (1984). Randerath, K.; Randerath, E.; Zhou, G.-D.; Li, D.: Bulky endogenous DNA modifications (I-compounds) – possible structural origins and functional implications, Mutat. Res., 424, 183–194 (1999). Randerath, K.; Reddy, M. V.; Gupta, R. C.: 32P-Labeling test for DNA damage, Proc. Natl. Acad. Sci. USA, 78, 6126–6129 (1981). Reddy, M. V.; Randerath, K.: Nuclease P1-mediated enhancement of sensitivity of 32Ppostlabeling test for structurally diverse DNA adducts, Carcinogenesis, 7, 1543–1551 (1986). Rindgen, D.; Turesky, R. J.; Vouros, P.: Determination of in vitro formed DNA adducts of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine using capillary liquid chromatography/electrospray ionization/tandem mass spectrometry, Chem. Res. Toxicol., 8, 1005–1013 (1995). Rouzer, C. A.; Chaudhary, A. K.; Nokubo, M.; Ferguson, D. M.; Reddy, G. R.; Blair, I. A.; Marnett, L. J.: Analysis of the malondialdehyde-2'-deoxyguanosine adduct pyrimidopurinone in human leukocyte DNA by gas chromatography/electron capture/negative chemical ionization/mass spectrometry, Chem. Res. Toxicol., 10, 181–188 (1997). Schell, C.; Popp, W.; Kraus, R.; Vahrenholz, C.; Norpoth, K.: 32P-postlabeling analysis of DNA adducts in different populations, Toxicol. Lett., 77, 299–307 (1995). Schoket, B.: DNA damage in humans exposed to environmental and dietary polycyclic aromatic hydrocarbons, Mutat. Res., 424, 143–154 (1999). Schrader, W.; Linscheid, M.: Styrene oxide DNA adducts: in vitro reaction and sensitive detection of modified oligonucleotides using capillary zone electrophoresis interfaced to electrospray mass spectrometry, Arch. Toxicol., 71, 588–595 (1997). Schut, H. A. J.; Shiverick, K. T.: DNA adducts in humans as dosimeters of exposure to environmental, occupational, or dietary genotoxins, FASEB J., 6, 2942–2951 (1992). Shuker, D. E. G.; Bartsch, H.: Detection of human exposure to carcinogens by measurement of alkyl-DNA adducts using immunoaffinity clean-up in combination with gas chromatography-mass spectrometry and other methods of quantitation, Mutat. Res., 313, 263–268 (1994). Siethoff, C.; Feldmann, I.; Jakubowski, N.; Linscheid, M.: Quantitative determination of DNA adducts using liquid chromatography/electrospray ionization mass spectrometry and liquid chromatography/high-resolution inductively coupled plasma mass spectrometry, J. Mass Spectrometry, 34, 421–426 (1999). Talaska, G.; Cudnik, J.; Jaeger, M.; Rothman, N.; Hayes, R.; Bhatnagar, V. J.; Kayshup, S. J.: Development and application of non-invasive biomarkers for carcinogen-DNA adduct analysis in occupationally exposed populations, Toxicology, 111, 207–212 (1996). Weston, A.; Manchester, D. K.; Poirier, M. C.; Choi, J. S.; Trivers, G. E.; Mann, D. L.; Harris, C. C.: Derivative fluorescence spectral analysis of polycyclic aromatic hydrocarbon-DNA adducts in human placenta, Chemical Research In Toxicology, 2, 104–108 (1989). Wild, C. P.; Pisani, P.: Carcinogen-DNA and carcinogen-protein adducts in molecular epidemiology, IARC Sci. Publ., 142, 143–158 (1997). Wu, K.-Y.; Scheller, N.; Ranasinghe, A.; Yen, T.-Y.; Sangaiah, R.; Giese, R.; Swenberg, J. A.: A gas chromatography/electron capture/negative chemical ionization high-resolution mass spectrometry method for analysis of endogenous and exogenous N7-(2hydroxyethyl)guanine in rodents and its potential for human biological monitoring, Chem. Res. Toxicol., 12, 722–729 (1999).
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P-Postlabelling HPLC Analysis of DNA Adducts in Breast Tissue
Wolfgang Pfau *
Most carcinogenic substances lead to their biological effects only after enzymatic metabolism in the body, so-called metabolic activation. The reactive metabolites formed are, for example, in the case of polycyclic aromatic hydrocarbons (PAHs), dihydrodiol epoxides. In the case of aromatic amino compounds, hydroxylamine intermediates are formed. These metabolites represent the early stages of electrophiles; they react among other things with the nucleophilic centres of DNA (Figure 1). As a result of covalent binding DNA adducts are formed. These DNA adducts can then lead to mutations or strand breaks, if they are not repaired. If this damage occurs in the right genes – oncogenes or tumour suppressor genes – a tumour can be initiated or tumor growth stimulated (Hemminki et al. 1994). DNA adducts are therefore of decisive importance in chemical carcinogenesis and the qualitative and quantitative analysis of DNA adducts is a relevant problem. As it is assumed that the formation of DNA adducts in leukocytes takes place via the same electrophilic metabolites as the binding of the carcinogenic substances to the DNA of the target organ of the carcinogenic effects, the value determined is also called the target dose (Neumann 1994, Hemminki et al. 1995). In addition to the amount actually taken up, the internal dose, also other individual parameters, such as enzyme activity and toxicokinetics are taken into account. The individual metabolic capacity, with regard to both enzymatic activation and detoxification, plays an important role (Vineis et al. 1999). Determination of the phenotype or genotype of polymorphic enzymes is therefore an important supplement to biological monitoring. Described below is a pilot study of the relationship between the DNA adduct level in the mammary gland and the N-acetyltransferase genotype. Suitable sources of DNA for biological monitoring are in particular non-invasively accessible cells (leukocytes, exfoliated epithelial cells (bladder, mouth, intestine)). There are numerous descriptions of biological monitoring studies which use DNA adduct analysis to determine exposure at *
Arbeitsgruppe Toxikologie und Umweltmedizin, Umweltmedizin Hamburg e.V., Vogt-Kölln-Straße 30, 22527 Hamburg, Germany
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Figure 1: Nucleophilic sites in the DNA bases and preferred binding sites of PAHs, aromatic amines (AA) and alkylating carcinogens (A).
the workplace, via the environment or food (Schoket et al. 1999, Lutz 2000). Highly accurate results are achieved by determining the DNA adduct levels in biopsy samples from the (suspected) target organ of the carcinogenic effects; for example PAH adducts in the bronchial epithelium of smokers (Phillips et al. 1988), or aromatic amine adducts in the bladder epithelium (Vineis et al. 1994, Rothman et al. 1996). DNA adduct levels are generally very low, so that highly sensitive methods are necessary for such analyses.
3.3.1 DNA Adduct Analysis In vitro or in animal experiments the DNA adduct levels can reach about 0.01% (one adduct in 104 nucleotides). In human tissue samples, however, adduct levels of 0.1 to 100 adducts per cell (one adduct in 107–1010 nucleotides) are detected (Phillips 1997). These are usually DNA adducts of unknown chemical structure, and, in addition, man is always exposed to complex mixtures of carcinogenic substances, so that numerous different DNA adducts are observed. Only very few analytical methods have the necessary sensitivity and specificity (Table 1). Many carcinogens, and therefore often also the DNA adducts (or their hydrolysates), have stronger or weaker fluorescent properties which allow these substances to be detected very sensitively. The use of all fluorescent techniques is, however, limited to fluorescent molecules, i.e. to polycyclic aromatic DNA adduct-inducers (Jankowiak & Small 1989, Alexandrov et al. 1992). 58
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P-Postlabelling HPLC Analysis of DNA Adducts in Breast Tissue
Table 1: Methods for DNA adduct analysis. Method
Sensitivity 1)
Comments
References
3
10 100
labelled substances must be synthesized; cannot be used in biological monitoring only fluorescent compounds, complicated apparatus only fluorescent compounds
Phillips 1990, Lin et al. 1992
H/HPLC C/HPLC
14
FLNS 2) HPLC/fluorescence GC/MS
100 10
Immunoassay
1
32
0.1
P-Postlabelling
1) 2)
derivatization necessary, cleavage from adduct adduct structure must be known, antibodies must be prepared most suitable for large, aromatic molecules
Jankowiak & Small 1989, Vineis et al. 1994 Giese 1997, Friesen et al. 1996, Poirier 1993 Randerath et al. 1981, Phillips 1997
approximate detection limit in adducts per 108 nucleotides Fluorescence Line Narrowing Spectroscopy
Immunochemical methods also reach a very high level of sensitivity and have already been used in numerous molecular-epidemiological studies to determine DNA adducts (Santella 1999). One advantage here is that by selecting the antibody, also statements can be made about the chemical structure of DNA adducts. Although it was first assumed that with monoclonal antibodies against benzo[a]pyrene-modified DNA only the DNA adducts of BaP would be detected, it was soon discovered that these antibodies also bind the very similar DNA adducts of other PAHs (Poirier 1993). The DNA adduct levels determined with these antibodies are regarded today as parameters for the total DNA damage induced by PAHs (Perera 1996). One disadvantage is that first of all such antibodies must be prepared, i.e. the structure of the DNA adduct being sought must be known, and must also be available in sufficient amounts. Technical developments over the past few years have greatly improved the sensitivity of mass spectrometric analysis. GC/MS and FAB/MS are used regularly in the chemical analysis and elucidation of the structures of synthetic DNA adduct standards (Giese 1997). The methods are sufficiently sensitive to allow hydrolysis products of DNA adducts to be detected; after alkaline hydrolysis and derivatization, 2-amino-1-methyl-6phenylimidazo[4,5-b]pyridine-DNA (PhIP-DNA) adducts could be detected in human tissue samples by means of GC/MS (Friesen et al. 1994). Electrospray LC/MS was used successfully to detect malondialdehyde adducts. The advantage of these methods is the structural information which is obtained from the molecular weight of the adduct or even the fragmentation pattern. 59
3 DNA Adducts The 32P-postlabelling method (Randerath et al. 1981) is at present the most sensitive analytical method for detecting DNA adducts. This method comprises the following steps: 1) 2) 3) 4) 5) 6) 7)
Isolation of the DNA, Enzymatic hydrolysis of the DNA to 3'-phospho nucleotides, Enrichment of the modified nucleotides (nuclease P1 digestion or butanol extraction SPE), 5'-Phosphorylation with T4-polynucleotide kinase and [c-32P]-ATP, Chromatographic separation of the labelled nucleotide adduct bisphosphates, Detection by means of autoradiography and Quantification by determining the Cerenkov radiation.
The advantages of this method are: • • • • •
its extremely high sensitivity (one adduct/10 9 normal nucleotides), with only a small amount of DNA necessary (1–10 lg), its specificity for DNA adducts as a result of two to three discriminating enzymatic steps, the wide range of adducts for which it can be used, including DNA adducts of unknown structure, and that no complicated apparatus is needed.
However, this method (like every analytical method) leads to only relative quantitative results if calibration with standard adducts is not carried out. A disadvantage of this method was the low resolution of chromatographic separation. We have been able to eliminate this deficit by introducing an HPLC method of analysis (Pfau & Phillips 1991).
3.3.2 HPLC Analysis of
32
P-Labelled DNA Adducts
With exposure to exogenous substances, man is almost always exposed to complex mixtures of genotoxic compounds. These are, for example, complex mixtures of polycyclic aromatic hydrocarbons (exhaust fumes, emissions from industrial plants such as coking plants or blast furnaces, coal fires, coal tar), polycyclic aromatic amines or nitro compounds (diesel fumes) or heterocyclic aromatic amines (fried or grilled food), or highly complex mixtures of various carcinogenic substances (tobacco smoke). To analyse the adducts in the body formed after exposure to these mixtures, not only highly sensitive methods are necessary, but also the chromatographic methods used must have a high resolution to be able to separate 60
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P-Postlabelling HPLC Analysis of DNA Adducts in Breast Tissue
Figure 2: Separation of 32P-labelled DNA adducts by reversed-phase HPLC: taking as an example ten adducts of very similar structure, which were formed as a result of the binding of ten anti-dihydrodiol epoxides of PAHs (A) (Pfau & Phillips 1991) and of DNA adducts which were formed by six heterocyclic aromatic amines (B) (Brockstedt & Pfau 1999).
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3 DNA Adducts the DNA adducts from one another and thus identify the biologically relevant components of these mixtures (Autrup 1991, Dale & Garner 1996). An HPLC method was therefore developed to supplement the conventional DC technique, which is of inadequate resolution. With a high resolution (base line separation) it was possible to analyse 32P-labelled DNA adducts of the diol epoxides of ten structurally very similar PAHs (Figure 2A, Pfau & Phillips 1992). Chromatographic separation of this mixture of 32P-labelled adducts by means of ion exchange DC produced a diagonal zone of radioactivity, as is similarly detected with 32P-postlabelling analysis of DNA from tissue samples, e.g. from the lungs of heavy smokers. This is in agreement with the assumption that these diagonal zones contain complex mixtures of very similar DNA adducts which are not separated. This new HPLC method has been used in various studies to characterize DNA adducts: for example to analyse PAH-induced DNA adducts (dibenzo[a,h]anthracene (Lecoq et al 1992), benzo[b]fluoranthene (Pfau et al. 1992a, Weyand et al. 1993), chrysene (Pfau et al. 1996)) benzo[g]chrysene (Giles et al. 1996), dibenzo[a,j]anthracene (Baer-Dubowska et al. 1995) or benzo[c]phenanthrene (Giles et al. 1995). In the meantime this method has been used also for the separation of adduct mixtures formed by various complex mixtures containing PAHs: artificial PAH mixtures (Hughes et al. 1993, Zhan et al. 1995), coal tar (Hughes et al. 1993, Culp & Beland 1994), diesel fumes (King et al. 1994, Savela et al. 1995), used motor oil (Carmichael et al. 1991) or coking residues (Weyand & Wu 1995). By combining thin-layer and HPLC analysis, individual PAH adducts in these mixtures were identified, in other words the biologically active components of these mixtures were characterized. By means of 32P-postlabelling/HPLC analysis Savela et al. (1995), for example, were able to show that chrysene is one of the biologically relevant components of diesel fumes. It was also shown that 32P-postlabelling/HPLC analysis can be used for DNA adducts which are formed by other classes of carcinogenic substances: polycyclic aromatic nitro compounds (King et al. 1994, Zhan et al. 1995), heterocyclic amines (Pfau & Brockstedt 1999, Figure 2B), bile (Scates et al. 1993), DNA adducts resulting from pharmaceuticals (aristolochic acid, tamoxifen), chlorinated aliphatic hydrocarbons or reactive oxygen species (Carmichael et al. 1992). Parallel to this and also as a consequence of it, other HPLC separation methods have been described which have also proved suitable for separating 32P-labelled DNA adducts with a high resolution and sensitivity (Lau & Baird 1991, Möller et al. 1993, Otteneder et al. 1999). These methods have to date, however, not been employed so widely. It was found that the HPLC methods are always less sensitive than the DC methods. This is a result of the detection technique: while autoradiographic methods allow long exposure times, the detection time in the online HPLC radioactivity counter is limited to a few seconds.
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P-Postlabelling HPLC Analysis of DNA Adducts in Breast Tissue
3.3.3 DNA Adducts in Breast Tissue Investigations by Li et al. (1996) showed that higher DNA adduct levels can be detected in the healthy breast tissue surrounding the tumour in patients with breast cancer than in the breast tissue of reduction mammoplasty patients (control group). We also analysed the DNA adduct level in the mammary gland and found a relationship between this biomarker and the N-acetyltransferase status (Pfau et al. 1998). Enzymatic N-acetylation is a detoxification reaction of aromatic amines. Persons with high N-acetyltransferase (NAT) activity, so-called fast acetylators, can therefore better protect themselves e.g. against the toxic effects of aromatic amines. The activity of NAT2 is genetically determined; about 60 % of Caucasians have a low level of enzyme activity (slow acetylators). This can be shown by molecular biological analysis of a DNA sample: fast acetylators have at least one wild-type allele, persons with two mutated alleles of the NAT2 gene are regarded as slow acetylators. RFLP analysis can be used to investigate this. As a result of mutation, a restriction site is missing in the NAT2 gene. Mutated and wild-type alleles can be detected using PCR amplification, restriction digestion and gel electrophoresis and thus homozygote WT, heterozygote and homozygote mutants, i.e. slow acetylators, can be identified. While this polymorphism has been known for a long time for the NAT2 enzyme, genetic polymorphism was described only recently for
Figure 3: Frequency of the wild-type allele of NAT2 (NAT2*4) and of the NAT1 alleles NAT1*4, NAT1*10 and NAT1*14 depending on the DNA adduct level (1st–4th quartiles) in breast tissue from the population investigated.
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3 DNA Adducts NAT1 (Badawi et al. 1995). Accordingly, also for this enzyme we investigated the most frequent mutations in this gene in the patients. Breast tissue samples from breast cancer patients and reduction mammoplasty patients were investigated in co-operation with the Institute of Cancer Research in England (Table 2, Pfau et al. 1998): Higher DNA adduct levels are found in slow acetylators (NAT2), for NAT1 there was no significant correlation. Figure 3 shows the relationship found in the population investigated (n=42) between the DNA adduct level and the frequency of (NAT) alleles. For NAT2 the wild-type allele (NAT2*4) was detected most often in the quartile with the lowest adduct levels and, with decreasing tendency, this allele no longer occurred in the patients with the highest adduct levels in the breast tissue DNA. For NAT1, however, the wild-type allele was most frequent in patients with high adduct levels; mutant alleles showed a tendency to be more frequent in tissue with lower adduct levels. A similar relationship is described between DNA adducts in the bladder epithelium and NAT polymorphism (Rothman et al. 1996, Badawi et al. 1995). Also with cancer of the bladder, an increased risk was found for slow N-acetylators (Perera 1997, Vineis et al. 1999). Several studies have been published which describe a possible relationship between the risk of breast cancer and NAT activity. It was found that the acetylation status alone did not influence the risk of breast cancer (Vineis et al. 1999). According to Ambrosone et al. (1996) there is an increased risk of developing breast cancer for smokers after the menopause in particular if they are the slow N-acetylator type; this result could, however, not be confirmed in a recently published study. In the patients we investigated, smoking does not seem to play a role in the formation of DNA adducts. The medical records showed that over 80 % of the patients were non-smokers. The relationship between the DNA adduct level and the NAT status is still significant even after stratification for the smoking habits (Table 2). A few studies investigated the relationship between the risk of breast cancer and the consumption of fried and grilled meat, taking into account
Table 2: Relationship between DNA adduct level and N-acetylator polymorphism. NAT 2
n
NAT2 slow 1) adduct level 2)
NAT2 fast 1) adduct level 2)
p 3)
All patients Reduction mammoplasty patients Non-smokers
42 31
83.0 (9–414.6) 121.0 (9–414.6)
39.7 (0–91) 51.0 (0–91)
0.0053 0.0052
19
120.0 (36–414.6)
36.5 (0–91)
0.015
1)
N-acetylator type (NAT2) deduced from genotype. Average adduct level per 109 nucleotides, range in brackets. 3) p values were determined using two-sided Mann-Whitney analysis of the adduct level. 2)
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P-Postlabelling HPLC Analysis of DNA Adducts in Breast Tissue
the N-acetylation status of the patients. While in two studies an increased relative risk was observed for women who often consumed well-fried meat and were of the slow acetylator type (NAT2), this could not be confirmed in other studies. Numerous results indicate that also genotoxic components play a role in the carcinogenesis of the mammary gland. It is also probable that these are external factors, such as environmental and/or nutritional influences. Polymorphism of the substance-metabolizing enzymes certainly also influences the risk of developing breast cancer (Agundez et al. 1995). References Agundez, J. A. G.; Ladero, J. M.; Olivera, M.; Abildua, R.; Roman, J. M.; Benitez, J.: Genetic analysis of the arylamine N-acetyltransferase polymorphism in breast cancer patients, Oncology, 52, 7–11 (1995). Alexandrov, K.; Rojas, M.; Geneste, O.; Castegnaro, M.; Camus, A. M.; Petruzzelli, S.; Giuntini, C.; Bartsch, H.: An improved fluorometric assay for dosimetry of benzo(a)pyrene diol-epoxide-DNA adducts in smokers’ lung – comparisons with total bulky adducts and aryl hydrocarbon hydroxylase activity, Cancer Res.; 52, 6248–6253 (1992). Ambrosone, C. B.; Freudenheim, J. L.; Graham, S.; Marshall, J. R.; Vena, J. E.; Brasure, J. R.; Michalek, A. M.; Laughin, R.; Nemoto, T.; Gillenwater, K. A.; Harrington, A. M.; Shields, P. G.: Cigarette smoking, N-acetyltransferase 2 genetic polymorphisms, and breast cancer, JAMA, 276, 1494–1501 (1996). Autrup, H.: Human exposure to genotoxic carcinogens: methods and their limitations, J. Cancer Res. Clin. Oncol., 117, 6–12 (1991). Badawi, A. F.; Hirvonen, A.; Bell, D. A.; Lang, N. P.; Kadlubar, F. F.: Role of aromatic amine acetyltransferases, NAT1 and NAT2, in carcinogen-DNA adduct formation in the human urinary bladder, Cancer Res., 55, 5230–5237 (1995). Baer-Dubowska, W.; Nair, R. V.; Cortez, C.; Harvey, R. G.; DiGiovanni, J.: Covalent DNA adducts formed in mouse epidermis from dibenz[a,j]anthracene: evidence for the formation of polar adducts, Chem. Res. Toxicol., 8, 292–301 (1995). Brockstedt, U.; Pfau, W.: Separation of 32P-labeled nucleotide adducts formed by food derived carcinogenic aminoimidazoazarenes using reversed-phase high performance liquid chromatography, Chromatographia, 50, 547–552 (1999). Carmichael, P. L.; Ni Shé, M.; Phillips, D. H.: DNA adducts in human and mouse skin maintained in short-term culture and treated with petrol and diesel engine lubricating oils, Cancer Lett., 57, 229–235 (1991). Carmichael, P. L.; Ni Shé, M.; Phillips, D. H.: Detection and characterization by 32P-postlabelling of DNA damage induced by an oxygen free-radical-generating system, Carcinogenesis, 13, 1127–1135 (1992). Culp, S. J.; Beland, F. A.: Comparison of DNA adduct formation in mice fed coal tar or benzo[a]pyrene, Carcinogenesis, 15, 247–252 (1994). Dale, C. M.; Garner, R. C.: Measurement of DNA adducts in humans after complex mixture exposure, Food Chem. Toxicol., 34, 905–919 (1996). Friesen, M. D.; Kaderlik, K.; Lin, D.; Garren, L.; Bartsch, H.; Lang, N. P.; Kadludar, F. F.: Analysis of DNA adducts of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in rat and human tissues by alkaline hydrolysis and gas chromatography/electron capture mass spectrometry: Validation by comparison with 32P-postlabeling, Chem. Res. Toxicol., 7, 733–738 (1994). Giese, R. W.: Detection of DNA Adducts by electron capture mass spectrometry, Chem. Res. Toxicol., 10, 255–270 (1997).
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3 DNA Adducts Giles, A. S.; Seidel, A.; Phillips, D. H.: In vitro reaction with DNA of the Fjord-region diol epoxides of benzo[g]chrysene and benzo[c]phenanthrene as studied by 32P-postlabelling. Chem. Res. Tox., 8, 591–599 (1995). Giles, A. S.; Seidel, A.; Phillips, D. H.: Covalent DNA adducts formed in mouse epidermis by benzo[g]chrysene, Carcinogenesis, 17, 1331–1336 (1996). Hemminki, K.; Autrup, H.; Haugen, A.: DNA and protein adducts, Toxicology, 101, 41– 53 (1995). Hemminki, K.; Dipple, A.; Shuker, D. E. G.; Kadlubar, F. F.; Segerbäck, D.; Bartsch, H.: IARC, DNA adducts – identification and biological significance, In: IARC Scientific Publications No 125, Lyon, 217–228 (1994). Hughes, N. C.; Pfau, W.; Hewer, A.; Jacob, J.; Grimmer, G.; Phillips, D. H.: Covalent binding of polycyclic aromatic hydrocarbon components of coal tar to DNA in mouse skin, Carcinogenesis, 14, 135–144 (1993). Jankowiak, R.; Small, G. J.: Fluorescence line narrowing spectroscopy in the study of chemical carcinogenesis, Anal. Chem., 61, 1023–1032 (1989). King, L. C.; George, M.; Gallagher, J.; Lewtas, J.: Separation of 32P-postlabelled DNA adducts of PAH and nitrated PAH, Chem. Res. Toxicol., 7, 503 (1994). Lau, H. H. S.; Baird, W. M.: Detection and identification of benzo[a]pyrene DNA adducts by [35S]phosphorothioate labeling and HPLC, Carcinogenesis, 12, 885–893 (1991). Lecoq, S.; Pfau, W.; Grover, P. L.; Phillips, D. H.: HPLC separation of 32P-postlabelled DNA adducts formed from dibenzo[a,h]anthracene in skin, Chem. Biol. Inter., 85, 173–185 (1992). Li, D.; Wang, M.; Dhingra, K.; Hittelman, W. N.: Aromatic DNA adducts in adjacent tissues of breast cacer patients: clues to breast cancer etiology, Cancer Res., 56, 287– 293 (1996). Lutz, W. K.: DNA-Addukte als Genotoxizitätsmarker für ein biologisches Monitoring, Arbeitsmed. Sozialmed. Umweltmed., 35, 303–311 (2000). Möller, L.; Zeisig, M.; Vodicka, P.: Optimization of an HPLC method for analyses of 32 P-postlabeled DNA adducts, Carcinogenesis, 14, 1343–1348 (1993). Neumann, H.-G.: Biomonitoring, in: Marquardt, H.; Schäfer, S. (Hrsg.): Lehrbuch der Toxikologie, BI-Wissenschaftsverlag, Mannheim, 737–749 (1994). Otteneder, M.; Eder, E.; Lutz, W. K.: 32P-postlabelling analysis of DNA adducts of styrene 7,8-oxide at the O6-position of guanine, Chem. Res. Tox., 12, 93–99 (1999). Perera, F. P.: Molecular epidemiology: Insights into cancer susceptibility, risk assessment, and prevention, J. of the Natl. Cancer Inst., 88, 496–509 (1996). Pfau, W.; Seidel, A.; Phillips, D. H.; Oesch, F.; Grover, P. L.: 32P-Postlabelling/HPLC analysis of DNA adducts formed from chrysene and its metabolites, Journal of Polycyclic Compounds, 10, 219–226 (1996). Pfau, W.; Phillips, D. H.: Improved reversed-phase high-performance liquid chromatographic separation of 32P-labelled nucleoside 3',5'-bisphosphate adducts of polycyclic aromatic hydrocarbons, J. Chromat., 570, 65–76 (1991). Pfau, W.; Hughes, N. C.; Grover, P. L.; Phillips, D. H.: HPLC analysis of DNA adducts formed by benzo[b]fluoranthene, Cancer Letters, 65, 159–167 (1992). Pfau, W.; Stone, E. M.; Brockstedt, U.; Carmichael, P. L.; Marquardt, H.; Phillips, D. H.: DNA adducts in human breast tissue: Influence of NAT 2 genotype, Cancer Epidem. Biom. Prev., 7, 1019–1025 (1998) Phillips, D. H.: Detection of DNA modifications by the 32P-postlabelling assay, Mutat. Res., 378, 1–12 (1997). Phillips, D. H.; Hewer, A.; Martin, C. N.; Garner, R. C.; King, M. M.: Correlation of DNA adduct levels in human lung with cigarette smoking, Nature, 336, 790–792 (1988).
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P-Postlabelling HPLC Analysis of DNA Adducts in Breast Tissue
Poirier, M. C.: Antisera specific for carcinogen-DNA adducts and carcinogen modified DN: applications for detection of xenobiotics in biological samples, Mutation Research, 288, 31–38 (1993). Randerath, K.; Reddy, M. V.; Gupta, R. C.: 32P-Postlabeling test for DNA damage, Proc. Natl. Acad. Sci. USA, 78, 6126–6129 (1981). Rothman, N.; Bhatnagar, V. K.; Hayes, R. B.; Zenser, T. V.; Kashyap, S. K.; Butler, M. A.; Bell, D. A.; Lakshmi, V.; Jaeger, M.; Kashyap, R.; Hirvonen, A.; Schulte, P. A.; Dosemeci, M.; Hsu, F.; Parikh, D. J.; Davies, B. B.; Talaska, G.: The impact of interindividual variation in NAT2 activity on benzidine urinary metabolites and urothelial DNA adducts in exposed workers, Proc. Natl. Acad. Sci. USA, 93, 5084–5089 (1996). Santella, R. M.: Immunological methods for the detection of carcinogen-DNA damage in humans, Cancer Epidem. Biom. Prev., 8, 733–739 (1999). Savela, K.; King, L.; Gallagher, J.; Lewtas, J.: 32P-Postlabelling and HPLC separation of DNA adducts formed by diesel extracts in vitro and in mouse skin and lung after topical treatment, Carcinogenesis, 16, 2083–2089 (1995). Scates, D. K.; Spigelman, A. D.; Nugent, K. P.; Phillips, R. K. S.; Venitt, S.: DNA adducts, detected by 32P-postlabelling in DNA treated in vitro with bile from patients with familial adenomatous polyposis and from unaffected controls, Carcinogenesis, 14, 1107–1110 (1993). Schoket, B.: DNA damage in humans exposed to environmental and dietary polycyclic aromatic hydrocarbons, Mutat. Res., 424, 143–153 (1999). Vineis, P.; Bartsch, H.; Caporaso, N.; Harrington, A. M.; Kadlubar, F. F.; Landi, M. T.; Malaveille, C.; Shields, P. G.; Skipper, P.; Talaska, G.; Tannenbaum, S. R.: Genetically based N-acetyltransferase metabolic polymorphism and low-level environmental exposure to carcinogens, Nature, 369, 154–156 (1994). Vineis, P.; Malats, N.; Lang, M.; d‘Errico, A.; Caporaso, N.; Cuzick, J.; Boffeetta (eds): Metabolic polymorphism and susceptibility to cancer, IARC Scientific Publications, No 148, Lyon (1999). Weyand, E. H.; Cai, Z.; Rice, J. E.; He, Z.; LaVoie, E. J.: Detection of the major DNA adducts of benzo[b]fluoranthene in mouse skin: role of phenolic dihydrodiols, Chem. Res. Toxicol., 6, 568–577 (1993). Weyand, E. H.; Wu, Y.: Covalent binding of PAH components of manufactured gas plant residue to mouse lung and forestomach DNA, Chem. Res. Toxicol., 8, 955–962 (1995). Zhan, D.; Herreno Saenz, D.; Chiu, L.; van Tungelen, L. S.; Wu, Y.; Lewtas, J.; Fu, P. P.: Separation of 32P-labelled 3',5'-bisphosphate nucleotides of polycyclic aromatic hydrocarbon anti-diol epoxides and derivatives, J. Chromatography, A 710, 149–157 (1995).
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3.4
Studies of 8-Hydroxy-2'-Deoxyguanosine: A Biomarker for Oxidative DNA Damage in vivo? Boleslaw Marczynski *, Jürgen Hölzer ** and Michael Wilhelm *, **
3.4.1 Background Most genotoxic substances cause genomic damage as a result of the formation of covalent adducts and the induction of strand breaks in the DNA. If repair does not take place before replication, such lesions can lead to mutations. Earlier investigations in cell-free systems, cell cultures and animal experiments indicate that the development of cancer is influenced mainly by the formation of reactive oxygen species in the presence of various noxae (Loft & Paulsen 1996, Jaurand 1997, Beckman & Ames 1998). Reactive oxygen species are the superoxide anion, hydrogen peroxide and the hydroxyl radical. Also singlet oxygen, ozone and nitrogen monoxide (NO) are under consideration. Hydroxyl radicals are considered to be the most reactive oxygen radicals; they can cause direct oxidative DNA damage (Floyd 1990, Collins 1999). There are numerous physiological (e.g. respiration) and pathophysiological processes (e.g. inflammation) in which reactive oxygen species occur.
** Berufsgenossenschaftliches Forschungsinstitut für Arbeitsmedizin (BGFA), RuhrUniversität Bochum, Bürkle-de-la-Camp-Platz 1, 44789 Bochum, Germany ** Abteilung für Hygiene, Sozial- und Umweltmedizin, Ruhr-Universität Bochum, Universitätsstr. 150, 44801 Bochum, Germany
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3.4.2 Oxidative DNA Damage 3.4.2.1 Endogenous Causes The reactive oxygen species can be physiologically compensated for to a large extent by corresponding oxidation-reduction processes. A certain amount of endogenously caused oxidative DNA damage is, however, always present (Ames & Gold 1991). This amount increases when the reactive oxygen species increase, as a result of pathophysiological processes such as inflammation (Loft & Poulsen 1996, Jaurand 1997) and the lack of reducing enzymes and antioxidants (Halliwell 1998).
3.4.2.2 Exogenous Causes Ionizing radiation, and many inorganic and organic chemicals or their metabolites induce oxidation-reduction processes, which then as a result of an imbalance between oxidants and antioxidants lead to oxidative stress (Halliwell 1998). In the DNA, single and/or double strand breaks and oxidative adducts can thus be produced (Anderson et al. 1998, Halliwell 1998).
Figure 1: Oxidative adducts of pyrimidine bases.
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Figure 2: Oxidative adducts of purine bases.
Of the DNA nucleosides, the pyrimidine derivatives (cytidine and thymidine, Figure 1) and the purine derivatives (adenosine and guanosine, Figure 2), guanine is the most reactive and thus predestined to be the main site of action for hydroxyl radicals.
3.4.3 8-Hydroxy-2'-Deoxyguanosine, a Promutagenic Base In the presence of hydroxyl radicals, usually the carbon atom at position 8 of the guanine is hydroxylated. The product formed is 8-hydroxy-2'-deoxyguanosine (8-OH-dG). As a result of the chemical reactiveness of guanine and the permanent production of reactive oxygen species in physiological cell metabolism, 8-OH-dG is continuously present in the DNA. It is therefore continuously produced after DNA repair as an endogenous “waste product”. This steady state is, however, disturbed in the presence of exogenous oxygen radical inducers (chemical carcinogens, ionizing radiation and others) (Collins 1999). In such cases too much 8-OH-dG is generated, which leads to the quantitative overloading of the repair mechanisms of the cell (Figure 3). One of the possible consequences of this overloading is malignant transformation at the cellular level. Spontaneous endogenous DNA damage that is not repaired is probably the main factor responsible for the aging process in higher organisms. 70
3.4 Studies of 8-Hydroxy-2'-Deoxyguanosine
Figure 3: Oxidative DNA damage in the “steady state”.
The 8-OH-dG adduct is a promutagenic base, which allows malignant transformation to take place as a result of mutagenic G ? T transversion (Moriya 1993). As a result of the great increase in the formation of the promutagenic 8-hydroxyguanine base, the probability increases that mutagenic G ? T transversion will take place during cell division. 8-OH-dG adducts and other oxidative DNA adducts can be removed from the DNA by DNA repair enzymes (DNA glycosylases) (Tchou & Grollman 1993, Pouget et al. 1999). An increase in the formation of free radicals leads not only to an increase in the formation of 8-OH-dG adducts and DNA strand breaks, but also influences the repair capacity (Epe et al. 1996). The level of 8-OHdG adducts can be reduced by the formamidopyrimidine derivative (after reduction of the product formed from guanine and the hydroxyl radical) (Halliwell 1998). In this case, 8-OH-dG adducts cannot be detected by HPLC with an electrochemical detector.
3.4.4 Determination of 8-OH-dG (Method) 8-OH-dG was first determined in the 1980s using HPLC and an electrochemical detector (Floyd et al. 1986, Kasai et al. 1986). Several different techniques (HPLC, GC-MS and 32P-postlabelling) have since been developed for quantifying 8-OH-dG adducts. As Ravanat et al. (1995) and Schu71
3 DNA Adducts Table 1: 8-OH-dG adducts in the lymphocytes of control persons from various European countries: 1–5: from Collins (1999) (n ≤ 9 per country). 6: our own data (n = 61), in white blood cells (n = 61). Country
1 2 3 4 5 6
France Ireland Spain The Netherlands Great Britain Germany
8-OH-dG/105dG (Mean) Total
Men
Women
0.45 0.76 0.33 0.59 0.62 0.55
0.43 1.14 0.33 0.72 0.81 0.58 (0.61)*
0.47 0.37 0.33 0.46 0.43 0.53 (0.51)*
* Median.
ler et al. (1997) reported, the determination of 8-OH-dG using GC-MS and 32 P-postlabelling produces values that are too high. As a result of great inaccuracies in the determination of oxidative DNA adducts at first, the leading scientists in this field met in Aberdeen, Scotland, in 1997 to discuss new strategies for improving the methods (Collins et al. 1997). This led to the development of new methods (Collins 1999, Pouget et al. 1999). The newly developed and established methods (HPLC/UV/ECD) for the determination of 8-OH-dG in DNA from white blood cells allow the results from different laboratories to be compared. The levels determined are in the range from >0.2 8-OH-dG/105 dG, as seen in the lymphocytes of control persons from various European countries (Collins 1999, Table 1). The average number of adducts that we determined in a control collective (0.55 8-OH-dG/105 dG) (Marczynski et al. 2000) is similar to the values found in persons from The Netherlands. Conspicuous are the high values for male persons from Great Britain and Ireland, and the low number of DNA adducts in persons from the Mediterranean region (Spain). To date the HPLC/UV method with electrochemical detector has proved the most reliable (Collins et al. 1997). Intercomparison programmes to compare the results from various laboratories have yet to be carried out.
3.4.5 Suitability of 8-OH-dG as a Biomarker The steady-state levels for the 8-OH-dG adducts in human DNA are often greater than one 8-OH-dG adduct per 106 dG bases. Compared with other DNA changes (such as e.g. specific DNA adducts, which can be formed from PAH metabolites) oxidative DNA adducts are, therefore, much more 72
3.4 Studies of 8-Hydroxy-2'-Deoxyguanosine frequent. Many authors therefore assume that continuous damage to DNA by “reactive species” is a main cause of the development of cancer with age (Beckman & Ames 1998). As part of biological monitoring, adduct levels can be determined in white blood cells, urine and other tissues. As the target tissues are generally not accessible, white blood cells are often investigated as surrogate cells. As new white blood cells are continuously being formed in the blood, the adduct levels determined represent only a steady-state level. Determination of the adduct level in white blood cells does not show which period of exposure is covered. Taking into account the different lifetimes of the various types of blood cells, it must be assumed that both short-term and long-term effects are included. As 8-OH-dG is hardly metabolized in the organism and is renally eliminated, determination of the substance in urine is also an ideal diagnostic instrument for the biological monitoring of persons exposed to certain carcinogenic substances. Under physiological conditions 8-OH-dG is eliminated daily with the urine (Shigenaga & Ames 1991, Loft & Paulsen 1996). The meaningfulness of 8-OH-dG adduct levels in human tissues and body fluids is limited by several factors. • The correlation between the 8-OH-dG levels in blood/urine and target organs is unclear. • Artificial DNA oxidation takes place during isolation/analysis (Helbock et al. 1998). • The relationship between 8-OH-dG and the incidence of cancer shows that for some types of cancer (e.g. kidney cell carcinomas) the level of adducts is increased (Okamoto 1994), for other types (breast cancer) the levels are unchanged (Nagashima et al. 1995; detailed information on 8-OHdG with other diseases: Loft & Paulsen 1996). • The suitability of lymphocytes as surrogate cells has to date not been investigated with sufficiently large collectives. •
Increased 8-OH-dG adduct levels are not substance specific.
• The influence of nutritional habits, the consumption of alcohol and cigarettes, and sport on 8-OH-dG adduct levels, compared with specific exposures to hazardous substances, is unclear (Kiyosawa et al. 1990, Hanaoka et al. 1993, Inoue et al. 1993, Lagorio et al. 1994, Schins et al. 1995, Marczynski et al. 1997, Takahashi et al. 1997).
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3.4.6 Suitability of 8-OH-dG as a Biomarker for Oxidative DNA Damage after Exposure to PAHs – First Results Despite the enormous amount of progress made in detecting DNA adducts, there are relatively few publications about the DNA adduct levels of carcinogenic substances after exposure at the workplace. Most publications are about the adducts of polycyclic aromatic hydrocarbons (Eder 1999). As a result of the non-standardized analytical procedures, which are not specially for this type of determination, there are considerable deficits in the early recognition and prevention of PAH-induced diseases. As man is exposed to PAHs, sometimes in large amounts, via tobacco smoke, food and the environment, the results can often not be clearly interpreted. Recent discussion has focused in particular on oxidative processes as an effect of exposure to PAHs. There is evidence that carcinogenic substances such as benzo[a]pyrene undergo oxidation-reduction reactions and produce free radicals as a result of a redox-cycling process (Martinez et al. 1995, Canova et al. 1998). These carcinogenic substances can therefore cause the oxidative modification of macromolecules such as DNA by the formation of free radicals, and thus contribute towards cancer development. In the investigations of 8-OH-dG adducts, the formation of DNA strand breaks (single strand, double strand, alkali-labile sites) is of particular interest. For this reason, we investigated for the first time the relationship between 8-OH-dG adduct levels and DNA strand breaks (by means of the comet assay; method according to Pouget et al. 1999 and Speit et al. 1999). The results revealed in the workers investigated a significant increase in oxidative DNA damage after high-level exposure to PAHs as a result of the formation of DNA strand breaks and 8-OH-dG DNA adducts (Marczynski et al. 2002). The increased DNA adduct level were observed together with the increased tail moment. The earlier results lent support to the hypothesis that exposure to PAHs leads to the increased formation of reactive oxygen species in the white blood cells of exposed persons. These results depend greatly, however, on the collectives investigated. The results for this biomarker can only be evaluated exactly together with the results of ambient and biological monitoring and data for genetic polymorphism. In the future the relationship between oxidative DNA damage after exposure to PAHs and the formation of anti-BPDE-DNA adducts should be compared. This adduct plays an important role in the carcinogenesis of benzo[a]pyrene (Rojas et al. 1994).
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3.4.7 Summary It can be assumed from the database available that the increased formation of reactive oxygen species plays a role in the formation of tumours in exposed persons. The determination of 8-OH-dG is a highly sensitive genotoxicological method. It is hoped that it will improve the early recognition and evaluation of health impairments, and prevent certain diseases, e.g. malignant tumours, and that it can be used routinely in biological monitoring. Oxidative DNA adducts could therefore be used as indirect markers, in addition to biological monitoring, for mutagenic and carcinogenic substances. It must, however, be borne in mind that oxidative DNA adducts are unspecific and the adduct levels can be affected by numerous factors (e.g. life-style). Conclusion: The determination of 8-OH-dG adduct levels is a promising attempt at evaluating the effects of carcinogenic substances in man. The meaningfulness of this parameter must still, however, be validated. References Ames, B. N.; Gold, L. S.: Endogenous mutagens and the causes of aging and cancer, Mutat. Res., 250, 3–16 (1991). Anderson, D.; Yu, T.aW.; McGregor, D.aB.: Comet assay responses as indicators of carcinogen exposure, Mutagenesis, 13, 539–555 (1998). Beckman, K. B.; Ames, B. N.: Oxidative DNA damage: assessing its role in cancer and aging, in: Aruoma, O. J.; Halliwell, B. (eds): DNA and free radicals: Techniques, mechanisms & applications. OICA International (1998). Canova, S.; Degan, P.; Peters, L. D.; Livingstone, D. R.; Voltan, R.; Venier, P.: Tissue dose, DNA adducts, oxidative DNA damage and CYP1A-immunopositive proteins in mussels exposed to waterborne benzo[a]pyrene, Mutat. Res., 399, 17–30 (1998). Collins, A. R.; Cadet, J.; Epe, B.; Gedik, C.: Problems in the measurement of 8-oxoguanine in human DNA, report of a workshop, DNA oxidation, held in Aberdeen, UK, 19–21 January, 1997. Carcinogenesis, 18, 1833–1836 (1997). Collins, A. R.: Oxidative DNA damage, antioxidants, and cancer, BioEssays, 21, 238– 246 (1999). Eder, E.: DNA-Addukte krebserregender Stoffe in der Umwelt und am Arbeitsplatz: Bildung, Nachweis und Bedeutung, Umwelt Forsch. Prax., 4, 323–334 (1999). Epe, B.; Ballmaier, D.; Adam, W.; Grimm, G. N.; Saha-Möller, C. R.: Photolysis of N-hydroxypyridinethiones: a new source of hydroxyl radicals for the direct damage of cell-free and cellular DNA, Nucleic Acids Res., 24, 1625–1631 (1996). Floyd, R. A.; Watson, J. J.; Wong, P. K.; Altmiller, D. H.; Rickard, R. C.: Hydroxy-free radical adduct of deoxyguanosine: sensitive detection and mechanisms of formation, Free Rad. Res. Comm., 1, 163–172 (1986). Floyd, R. A.: The role of 8-hydroxyguanine in carcinogenesis, Carcinogenesis, 11, 1447– 1450 (1990). Halliwell, B.: Can oxidative DNA damage be used as a biomarker of cancer risk in humans? Problems, resolutions and preliminary results from nutritional supplementation studies, Free Rad. Res., 29, 469–486 (1998). Hanaoka, T.; Tsugane, S.; Yamano, Y.; Takahashi, T.; Kasai, H.; Natori, Y.; Watanabe, S.: Quantitative analysis of 8-hydroxyguanine in peripheral blood cells: An application for asbestosis patients, Int. Arch. Occup. Environ. Health, 65, 215–217 (1993).
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3 DNA Adducts Helbock, H. J.; Beckman, K. B.; Shigenaga, M. K.; Walter, P. B.; Woodall, A. A.; Yeo, H. C.; Ames, B. N.: DNA oxidation matters: The HPLC-electrochemical detection assay of 8-oxo-deoxyguanosine and 8-oxo-guanine, Proc. Natl. Acad. Sci. USA, 95, 288–293 (1998). Inoue, T.; Mu, Z.; Sumikawa, K.; Adachi, K.; Okochi, T.: Effect of physical exercise on the content of 8-hydroxydeoxyguanosine in nuclear DNA prepared from human lymphocytes, Jpn. J. Cancer Res., 84, 720–725 (1993). Jaurand, M.-C.: Mechanisms of fiber-induced genotoxicity, Environ. Health Perspect., 105 (Suppl. 5), 1073–1084 (1997). Kasai, H.; Crain, P. F.; Kuchino, Y.; Nishimura, S.; Ootsuyama, A.; Tanooka, H.: Formation of 8-hydroxyguanine moiety in cellular DNA by agents producing oxygen radicals and evidence for its repair, Carcinogenesis, 7, 1849–1851 (1986). Kiyosawa, H.; Suko, M.; Okudaira, H.; Murata, K.; Chung, M. H.; Kasai, H.; Nashimura, S.: Cigarette smoking induces formation of 8-hydroxydeoxyguanosine, one of the oxidative DNA damages in human peripheral leukocytes, Free Rad. Res. Commun., 11, 23–27 (1990). Lagorio, S.; Tagesson, C.; Forastiere, F.; Iavarone, I.; Axelson, O.; Carere, A.: Exposure to benzene and urinary concentrations of 8-hydroxydeoxyguanosine, a biological marker of oxidative damage to DNA, Occup. Environ. Med., 51, 739–743 (1994). Loft, S.; Paulsen, H. E.: Cancer risk and oxidative DNA damage in man, J. Mol. Med., 74, 297–312 (1996). Marczynski, B.; Rozynek, P.; Elliehausen, H.-J.; Korn, M.; Baur, X.: Detection of 8-hydroxydeoxyguanosine, a marker of oxidative DNA damage, in white blood cells of workers occupationally exposed to styrene, Arch. Toxicol., 71, 496–500 (1997). Marczynski, B.; Rihs, H.-P.; Rossbach, B.; Hölzer, J.; Angerer, J.; Scherenberg, M.; Hoffmann, G.; Brüning, T.; Wilhelm, M.: Formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine and DNA strand breaks in white blood cells of workers exposed to polycyclic aromatic hydrocarbons in coke and in graphite-electrodes production: comparison with ambient monitoring, urinary metabolites and enzyme polymorphisms, Carcinogenesis, 23, 273–281 (2002). Marczynski, B.; Scherenberg, M.; Hölzer, J.; Schlösser, S. T.; Hoffmann, G.; Wilhelm, M.: 8-Hydroxydeoxyguanosine and strand breaks formation in white blood cell DNA of workers highly exposed to polycyclic aromatic hydrocarbons, Naunyn-Schmiedeberg‘s Arch. Pharmacol., 361 (Suppl.) 4, 612 (2000). Martinez, P. G.; Livingstone, D. R.: Benzo[a]pyrene-dione-stimulated oxyradical production by microsomes of digestive gland of the common mussel, Mytilus edulis L.; Mar. Environ. Res., 39, 185–189 (1995). Moriya, M.: Single-stranded shuttle phagemid for mutagenesis studies in mammalian cells: 8-Oxoguanine in DNA induces targeted G·C?T·A transversions in simian kidney cells, Proc. Natl. Acad. Sci. USA, 90, 1122–1126 (1993). Nagashima, M.; Tsuda, H.; Takenoshita, S.; Nagamachi, Y.; Hirohashi, S.; Yokota, J.; Kasai, H.: 8- ydroxydeoxyguanosine levels in DNA of human breast cancer are not significantly different from those of non-cancerous breast tissues by the HPLC-ECD method, Cancer Lett., 90, 157–162 (1995). Okamoto, K.; Toyokuni, S.; Uchida, K.; Ogawa, O.; Takenewa, J.; Kahehi, Y.; Kinoshita, H.; Hattori-Nakakuki, Y.; Hiai, H.; Yoshida, O.: Formation of 8-hydroxy-2'-deoxyguanosine and 4-hydroxy-2-nonenal-modified proteins in human renal-cell carcinoma, Int. J. Cancer, 58, 825–829 (1994). Pouget, J.-P.; Ravant, J.-L.; Douki, T.; Richard, M.-J.; Cadet, J.: Measurement of DNA base damage in cells exposed to low doses of c-radiation: comparison between the HPLC-EC and comet assays, Int. J. Radiat. Biol., 75, 51–58 (1999).
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3.4 Studies of 8-Hydroxy-2'-Deoxyguanosine Ravanat, J.-L.; Turesky, R. J.; Gremaud, E.; Trudle, L. J.; Stadler, R. H.: Determination of 8-oxoguanine in DNA by gas-chromatography mass-spectrometry and HPLC-electrochemical detection – overestimation of the background level of the oxidized base by the gas-chromatography mass-spectrometry assay, Chem. Res. Toxicol., 8, 1039–1045 (1995). Rojas, M.; Alexandrov, K.; von Schooten, F.-J.; Hillebrand, M.; Kriek, E.; Bartsch, H.: Validation of a new fluorometric assay for benzo[a]pyrene diolepoxide-DNA adducts in human white blood cells: comparison with 32P-postlabeling and ELISA, Carcinogenesis, 15, 557–560 (1994). Schins, R. P. F.; Schilderman, P. A. E. L.; Borm, P. J. A.: Oxidative DNA damage in peripheral blood lymphocytes of coal workers, Int. Arch. Occup. Environ. Health, 67, 153– 157 (1995). Schuler, D.; Ottender, M.; Sagelsdorff, P.; Eder, E.; Gupta, R. C.; Lutz, W. K.: Comparative analysis of 8-oxo-2'-deoxyguanosine in DNA by 32P- and 33P-postlabelling and electrochemical detection, Carcinogenesis, 18, 2367–2371 (1997). Shigenaga, M. K.; Ames, B. N.: Assays for 8-hydroxy-2’-deoxyguanosine: A biomarker of in vivo oxidative DNA damage, Free Radical. Biol. Med., 10, 211–216 (1991). Speit, G.; Trenz, K.; Schütz, P.; Rothfuß, A.; Merk, O.: The influence of temperature during alkaline treatment and electrophoresis on results obtained with the comet assay, Toxicol. Letters, 110, 73–78 (1999). Takahashi, K.; Pan, H.; Kasai, H.; Hanaoka, T.; Feng, Y.; Liu, N.; Zhang, S.; Xu, Z.; Tsuda, T.; Yamato, H.; Higashi, T.; Okubo, T.: Relationship between asbestos exposure and 8-hydroxydeoxyguanosine levels in leucocytic DNA of workers at a Chinese asbestos-material plant, Int. J. Occup. Environ. Health, 3, 111–119 (1997). Tchou, J.; Grollman, A. P.: Repair of DNA containing the oxidatively-damaged base, 8oxoguanine. Mutat. Res., 299, 277–287 (1993).
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4.1
Improved Methods of Phenotyping and Effect Monitoring for Evaluating the Risk to the Individual, using GSTT1 as an Example Ernst Hallier *
Chemical substances are metabolized in the human organism in several successive or parallel, enzyme-dependent steps. In most cases this leads to “detoxification”, i.e. the metabolic product is less toxic than the initial substance. There are, however, also examples of “toxification”, i.e. the transformation of a chemical into a toxic intermediate. Recently, genetic factors governing the different disposition of the individual, in particular enzyme polymorphism, have been the subject of much discussion in occupational and environmental medicine. An hereditary genotype anchored in the genetic information leads to the different expression of an enzyme. The high or low level of activity of this enzyme, and in some cases even the lack of it this causes, is called the phenotype. The individual genotype can be determined today using molecular biological methods (PCR), while the phenotype can be determined from the rate of conversion of certain test substrates. Enzyme polymorphism has been known to pharmacology for a long time; research into the subject has developed into its own field of science, pharmacogenetics. The best known example of enzyme polymorphism is the lack of glucose-6-phosphate-dehydrogenase, which can lead to haemolytic anaemia after the administration of anti-malaria medicines and sulfonamides. Other polymorphisms lead to a reduction in the therapeutic effectiveness or to an increased risk of undesired side-effects of medicines (Nebert 1997). Also in occupational medicine and toxicology there have been a few examples in the meantime of the different risk to the individual from chemicals as a result of enzyme polymorphism, in particular, polymorphism of N-acetyltransferase 2. Persons with low enzyme activity (slow acetylators) have an increased risk when exposed to aromatic amines of developing methaemoglobinaemia, and after years of exposure to these substances have a higher risk of developing bladder cancer than fast acetylators (Cartwright et al. 1982, Lewalter & Miksche 1991). *
Georg-August-Universität Göttingen, Institut für Arbeits- und Sozialmedizin, Waldweg 37, 37073 Göttingen Biological Monitoring: Prospects in Occupational and Environmental Medicine. Deutsche Forschungsgemeinschaft (DFG) Copyright © 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-27795-7
4.1 Improved Methods of Phenotyping and Effect Monitoring Important enzymes of phase II metabolism are the glutathione S-transferases (GSTs) (Hayes & Pulford 1995). The GSTs conjugate electrophilic compounds or metabolites with glutathione, a metabolic step that in almost all cases has a detoxifying effect; there are, however, important exceptions. In view of this, polymorphisms of these enzymes which can reduce the detoxifying capacity of the individual affected are being investigated in more depth. To date, polymorphism of three GSTs are known. These are GSTM1 (Seidegard et al. 1988), GSTT1 (Hallier et al. 1990, Pemble et al. 1994) and GSTP1 (Henderson et al. 1998). The polymorphism of GSTM1 is based on a gene deletion, which leads to a loss in enzymatic activity (Seidegard et al. 1988). GSTM1 conjugates e.g. epoxides of polycyclic aromatics with glutathione; it is therefore very important as a detoxifying enzyme (Hayes & Pulford 1995). In the European population about 50 % of people have a GSTM1 deficiency (Daly et al. 1993). There are, however, clear ethnic differences; more than 90 % of the Polynesian population are GSTM1-deficient, while in Germany only 50 % are (Hayes & Pulford 1995, Schulz et al. 1999 a). It has been shown in numerous studies that there is a relationship between GSTM1 deficiency and the occurrence of cancer of the lung, bladder and colon (Seidegard et al. 1990, Bell et al. 1993, Daly et al. 1993, Zhong et al. 1993). GSTT1 produces, like the other GSTs, mainly inactive metabolites. The enzyme can, however, also metabolize substances such as dichloromethane to produce genotoxic intermediates (Thier et al. 1996). Investigations of GSTT1 began with the detection of the polymorphism of glutathione-dependent conjugation reactions in erythrocytes (Peter et al. 1989, Hallier et al. 1990). As substrates, in addition to methylchloride and methylbromide, also ethylene oxide and dichloromethane were investigated (Föst et al. 1991, Hallier et al. 1994). GSTT1 is expressed both in the liver and in the erythrocytes (Meyer et al. 1991, Schröder et al. 1992). In vitro experiments with lymphocytes revealed a protective effect of GSTT1, when lymphocytes were incubated with methylbromide, ethylene oxide or dichloromethane and investigated for sister chromatid exchange (SCE) (Hallier et al. 1993). After sequencing the cDNA of GSTT1, a PCR method was established and validated by phenotyping the erythrocytes (Pemble et al. 1994). Molecular epidemiological studies have to date revealed only few relationships between malignant diseases and GSTT1 polymorphism. A significant increase in the odds ratio for the development of a myelodysplastic syndrome (MDS) was described for GSTT1-negative patients (Chen et al. 1996). Subsequent studies by other research groups could not find this relationship (Atoyebi et al. 1997, Basu et al. 1997, Schulz et al. 1999a). In more recent studies it was found that several polymorphisms of different enzymes must be regarded together. In a study of therapy-induced leukaemia, patients with mammary carcinomas were conspicuous. In 50 % of the cases investigated, both GSTT1 and GSTM1 were found to be deleted (Schulz et al. 1999b). Also with regard to sensitization against thimer79
4 Susceptibility osal, an important preservative, this double deletion of GSTT1 and GSTM1 seems to be a risk factor (Westphal et al. 2000). What importance do these polymorphisms – which are of great scientific interest – have for practical occupational medicine? There is no average worker; a truism also confirmed by the metabolism of chemical substances in all its facets. A few basic facts must first be discussed, however, before we go into more depth. It must be clearly differentiated between consideration of the individual risk and the collective risk. This can be demonstrated e.g. using the GSTs; there are many studies available of the relationships between GSTM1 polymorphism and malignant diseases. Numerous studies found that individuals of GSTM1-negative genotype were more frequent among patients with certain malignant diseases. The individual risk for GSTM1-negative genotypes was usually increased by a factor of two, in a few cases also once by a factor of three (Rebbeck 1997). Unlike the changes in certain tumour suppressor genes, which cause great increases in the individual cancer risk, this is a small increase. When considering the effects on large collectives, it must be taken into account that the GSTM1 deficiency in Europe affects about 50 % of the population and the GSTT1 deficiency between 10 % and 30 %, while changes in tumour suppressor genes are rare. GST polymorphisms are therefore of importance for the evaluation of the risk for the whole population. At present, determination of the various polymorphisms is not a suitable instrument for assessing the risk to the individual in industry. The exposure situation is too varied and our knowledge about the concrete effects of polymorphisms is too incomplete. Nevertheless, it is possible to show the occupational-medical toxicological relevance of GSTT1 polymorphism, also for individual risk assessment, with a case study. Two workers, who were accidentally poisoned with methylbromide, produced very different symptoms, despite the same level of exposure: the patient with a negative GSTT1 genotype merely suffered transient symptoms such as nausea, while the patient with a positive GSTT1 genotype produced severe and lasting neurotoxic symptoms, and since the accident has been confined to a wheelchair. Responsible for this is thought to be the GSTT1-dependent metabolism of methylbromide to form a neurotoxic metabolite (Garnier et al. 1996). To give a prognosis of the individual‘s susceptibility to the acute neurotoxic effects of methylbromide, it would be necessary to know to what extent the individual transforms methylbromide into the toxic metabolite. Determination of the genotype by means of PCR provides only a rough division of the population into two groups; those persons who have the gene, and those in which the gene is deleted. The actual enzyme activity, in other words the phenotype, differs greatly, however, within a population both for GSTT1 (Schröder et al. 1996) and for GSTM1 (Bell et al. 1992). To date it has not been possible to group people even roughly into homozygote and heterozygote carriers of the marker by determining the genotype with PCR. 80
4.1 Improved Methods of Phenotyping and Effect Monitoring For assessing the risk to the individual, practicable methods for determining the phenotype must therefore be developed; these can be used as biological monitoring procedures in occupational and environmental medicine. Development of these methods is, however, time-consuming and expensive, as they include chemical-analytical and biochemical procedures. Nevertheless, there has already been some success in this field. The currently used rough classification into GSTT1 phenotypes on the basis of the measured substrate conversion in blood samples (Hallier et al. 1993) can be replaced by a new and more exact procedure for assaying the product (Müller et al. 1999). As a result of its methodological simplicity, determination of the genotype is suitable as a screening method for large collectives, e.g. in molecular epidemiology. For assessing the risk to the individual, however, determination of the phenotype is imperative, in order to evaluate the actual extent of enzyme activity. Research into the following areas is therefore still required: Considerable research is still needed for: • factors which modulate the genetically determined enzyme status (induction, inhibition, intraindividual variability) • the development of practicable and standardized methods of biological monitoring for evaluating the individual enzyme activity and kinetics.
References Atoyebi, W.; Kusec, R.; Fidler, C.; Peto, T. E.; Boultwood, J.; Wainscoat, J. S.: Glutathione S-transferase gene deletions in myelodysplasia, Lancet, 349, 1450–1451 (1997). Basu, T.; Gale, R. E.; Langabeer, S.; Linch, D. C.: Glutathione S-transferase theta 1 (GSTT1) gene defect in myelodysplasia and acute myeloid leukaemia, Lancet, 349, 1450 (1997). Bell, D. A.; Taylor, J. A.; Paulson, D. F.; Robertson, C. N.; Mohler, J. L.; Lucier, G. W.: Genetic risk and carcinogen exposure: a common inherited defect of the carcinogen-metabolism gene glutathione S-transferase M1 (GSTM1) that increases susceptibility to bladder cancer, J. Natl. Cancer Inst., 85, 1159–1164 (1993). Bell, D. A.; Thompson, C. L.; Taylor, J.; Miller, C. R.; Perera, F.; Hsieh, L. L.; Lucier, G. W.: Genetic monitoring of human polymorphic cancer susceptibility genes by polymerase chain reaction: application to glutathione transferase mu, Environ. Health Perspect., 98, 113–117 (1992). Cartwright, R. A.; Glashan, R. W.; Rogers, H. J.; Ahmad, R. A.; Barham-Hall, D.; Higgins, E.; Kahn, M. A.: Role of N-acetyltransferase phenotypes in bladder carcinogenesis: A pharmacogenetic epidemiological approach to bladder cancer, Lancet, II, 842–846 (1982). Chen, H.; Sandler, D. P.; Taylor, J. A.; Shore, D. L.; Liu, E.; Bloomfield, C. D.; Bell, D. A.: Increased risk for myelodysplastic syndromes in individuals with glutathione transferase theta 1 (GSTT1) gene defect, Lancet, 347, 295–297 (1996). Daly, A. K.; Thomas, D. J.; Cooper, J.; Pearson, W. R.; Neal, D. E.; Idle, J. R.: Homozygous deletion of the gene for glutathione S-transferase M1 in bladder cancer, Br. Med. J., 307, 481–482 (1993).
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4 Susceptibility Föst, U.; Hallier, E.; Ottenwälder, H.; Bolt, H. M.; Peter, H.: Distribution of ethylene oxide in human blood and its implications for biomonitoring, Human Exp. Toxicol., 10, 25–31 (1991). Garnier, R.; Rambourg-Schepens, M. O.; Müller, A.; Hallier, E.: Glutathione transferase activity and formation of macromolecular adducts in two cases of acute methyl bromide poisoning, Occ. Environm. Med., 53, 211–215 (1996). Hallier, E.; Deutschmann, S.; Reichel, C.; Bolt, H. M.; Peter, H.: A comparative investigation of the metabolism of methyl bromide and methyl iodide in human erythrocytes, Int. Arch. Occup. Environm. Health, 62, 221–225 (1990). Hallier, E.; Langhof, T.; Dannappel, D.; Leutbecher, M.; Schröder, K.; Goergens, H. W.; Müller, A.; Bolt, H. M.: Polymorphism of glutathione conjugation of methyl bromide, ethylene oxide and dichloromethane in human blood: influence on the induction of sister chromatid exchanges (SCE) in lymphocytes, Arch. Toxicol., 67, 173–178 (1993). Hallier, E.; Schröder, K. R.; Asmuth, K.; Dommermuth, A.; Aust, B.; Goergens, H. W.: Metabolism of dichloromethane (methylene chloride) to formaldehyde in human erythrocytes: influence of polymorphism of glutathione transferase Theta (GST T1-1), Arch. Toxicol., 68, 423–427 (1994). Hayes, J. D.; Pulford, D. J.: The glutathione S-transferase supergene family: Regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance, Crit. Rev. Biochem. Mol. Biol., 30, 445–600 (1995). Henderson, C. J.; McLaren, A. W.; Moffat, G. J.; Bacon, E. J.; Wolf, C. R.: Pi-class glutathione S-transferase: regulation and function, Chem.-Biol. Interact., 111/112, 69–82 (1998). Lewalter, J.; Miksche, L. W.: Empfehlungen zur arbeitsmedizinischen Prävention expositions- und dispositionsbedingter Arbeitsstoff-Beanspruchungen, Verhandlungen der Deutschen Gesellschaft für Arbeitsmedizin, 31, 135–139 (1991). Nebert, D. W.: Polymorphisms in drug-metabolizing enzymes: What is their clinical relevance and why do they exist?, Am. J. Hum. Genet., 60, 265–271 (1997). Pemble, S. E.; Schröder, K. R.; Spencer, S. R.; Meyer, D. J.; Hallier, E.; Bolt, H. M.; Ketterer, B.; Taylor, J. B.: Human glutathione S-transferase Theta (GSTT1): cDNA cloning and the characterization of a genetic polymorphism, Biochem. J., 300, 271–275 (1994). Peter, H.; Deutschmann, S.; Reichel, C.; Hallier, E.: Metabolism of methyl chloride by human erythrocytes, Arch. Toxicol., 63, 351–355 (1989). Rebbeck, T. R.: Molecular epidemiology of the human glutathione S-transferase genotypes GSTM1 and GSTT1 in cancer susceptibility, Canc. Epidem. Biomark. Prev., 6, 733–743 (1997). Schröder, K. R.; Hallier, E.; Meyer, D. J.; Wiebel, F. A.; Müller, A. M. F.; Bolt, H. M.: Purification and characterization of a new glutathione S-transferase, class q, from human erythrocytes, Arch. Toxicol., 70, 559–566 (1996). Schröder, K. R.; Hallier, E.; Peter, H.; Bolt, H. M.: Dissociation of a new glutathione Stransferase activity in human erythrocytes, Biochem. Pharmacol., 43, 1671–1674 (1992). Schulz, T. G.; Haase, D.; Wörmann, B.; Schoch, C.; Schnittger, S.; Hiddemann, W.; Bünger, J.; Hallier, E.: Increased occurrence of GSTM1-deficiency in patients with AML or MDS and complex caryotype abnormalities, Naunyn Schmiedeberg‘s Arch. Pharmacol., 359 Suppl., R 150 (1999 a). Schulz, T. G.; Haase, D.; Schnittger, S.; Bünger, J.; Schoch, C.; Fonatsch, C.; Westphal, G. A.; Griesinger, F.; Wörmann, B.; Hallier, E.: Glutathione S-transferase (GST)-deficiency in de novo and therapy-induced hematologic malignancies, Arch. Pharm. Pharm. Med. Chem., 331 Suppl. 2, 43 (1999 b).
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4.1 Improved Methods of Phenotyping and Effect Monitoring Seidegard, J.; Pero, R. W.; Markowitz, M. M.; Roush, G.; Miller, D. G.; Beattie, E. J.: Isoenzyme(s) of glutathione transferase (class l) as a marker for the susceptibility to lung cancer, a follow up study, Carcinogen., 11, 33–36 (1990). Seidegard, J.; Vorachek, W. R.; Pero, R. W.; Pearson, W. R.: Hereditary differences in the expression of the human glutathione transferase active on trans-stilbene oxide are due to a gene deletion, Proc. Natl. Acad. Sci. USA, 85, 7293–7297 (1988). Thier, R.; Pemble, S. E.; Kramer, H.; Taylor, J. B.; Guengerich, F. P.; Ketterer, B.: Human glutathione S-transferase T1-1 enhances mutagenicity of 1,2-dibromoethane, dibromoethane and 1,2,3,4-diepoxybutane in Salmonella typhimurium, Carcinogen., 17, 163–166 (1996). Westphal, G. A.; Schnuch, A.; Schulz, T. G.; Reich, K.; Aberer, W.; Brasch, J.; Koch, P.; Wessbecher, R.; Sliska, C. H.; Bauer, A.; Hallier, E.: Homozygous gene deletions of the glutathione S-transferases M1 and T1 are associated with thiomersal sensitization, Int. Arch. Occup. Health, 73(6), 384–388 (2000). Zhong, S.; Wyllie, A. H.; Barnes, D.; Wolf, C. R.; Spurr, N. K.: Relationship between the GSTM1 genetic polymorphism and susceptibility to bladder, breast and colon cancer, Carcinogen., 14, 1821–1824 (1993).
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4.2
Genetic Polymorphisms of Sulfotransferases as Susceptibility Parameters Hansruedi Glatt *
4.2.1 Summary Sulfotransferases are involved in the metabolism of many chemical substances. They usually produce readily water-soluble metabolites that are easily excreted, and thus contribute to the detoxification of the substance. Investigations with test systems using recombinant organisms have shown, however, that sulfotransferases also form reactive, potentially mutagenic and carcinogenic metabolites relatively often. In man, eleven sulfotransferases are known to metabolize small xeno- and endobiotic molecules. For two forms, genetic polymorphisms affecting enzyme function have been detected. Most of the other forms have not been investigated for this. In view of their dual properties – they convert some substrates to more toxic compounds and others to less toxic ones – the existence of further polymorphisms for sulfotransferases is, however, likely. Research is needed to determine (a) which substances are converted by which sulfotransferases to less toxic or more toxic compounds, (b) to test the forms not yet investigated for genetic polymorphisms and (c) on the basis of the results to evaluate relationships between the sulfotransferase genotype and health risks in exposed individuals.
4.2.2 The Sulfotransferase Forms in Man Sulfotransferases transfer the sulfonyl group from the co-substrate 5'-phosphoadenosyl-3'-phosphosulfate to the nucleophilic groups (–OH, –NH2, –SH, –N ? O) of their substrates (Falany 1997). The reaction is called sulfonation, as a result of the group transferred; it is also known as sulfation, because during O-sulfonation (the most frequent reaction) a sulfate ester is *
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Deutsches Institut für Ernährungsforschung, Abteilung Ernährungstoxikologie, Arthur-Scheunert-Allee 114–116, 14558 Bergholz-Rehbrücke
4.2 Genetic Polymorphisms of Sulfotransferases formed. The sulfotransferases can be classified in two groups. The first group sulfonates macromolecules (tyrosine residues of proteins, lipids, carbohydrate moieties); most of these sulfotransferases are located in the membranes of Golgi’s apparatus. These enzymes are not known to metabolize small molecules. The second group sulfonates xenobiotic and endogenous substances of low molecular weight and includes only soluble enzymes (Falany 1997); eleven different forms are known in man. Their amino acid sequences are homologous and they can therefore be regarded as an enzyme superfamily (or products of a gene superfamily) (Weinshilboum et al. 1997, Yamazoe et al. 1994). SULT was the name chosen for the superfamily at the 2nd Sulfation Workshop (Ardmore 1993). Systematic classification is under way; although this has not yet been formally approved, it has already found widespread use for the human forms. The nomenclature for SULT follows the criteria used for classifying cytochromes P450. The names of genes are usually written in italics, gene products (mRNA, protein, cDNA) in normal script. Based on the degree of identity of the amino acid sequence derived from the cDNA, the SULT have been classified in families (e.g. SULT1, SULT2), subfamilies (e.g. SULT1A, SULT1B) and single gene products (e.g. SULT1A1, SULT1A2). If several different proteins are formed from one gene as a result of different mRNA splicing, they are represented by a small letter after the name (e.g. SULT2B1a, SULT2B1b). The various SULT forms in man (1A1, 1A2, 1A3, 1B1, 1C1, 1C2, 1E1, 2A1, 2B1a, 2B1b, 4A1) differ greatly in particular in their substrate specificity and tissue distribution. The trivial names, which are based on substrates, are often misleading. The classic “phenol sulfotransferase” (1A1), for ex-
Figure 1: Mutagenicity of 2-nitropropane in V79-derived cells in which heterologous SULT have been stably expressed. The frequency of mutations at the hprt locus was investigated. The mean values and standard deviations of 2 cultures are given.
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4 Susceptibility ample, metabolizes not only phenols, but also aromatic amines, aromatic hydroxylamines, alcohols, N-oxides and nitroalkanes (as nitronates); similarly 17-oestradiol is metabolized not only by “oestrogen sulfotransferase” (1E1), but also by e.g. 1A1 and 2A1 (Falany 1997).
4.2.3 Genetic Polymorphisms of SULT SULT1A1 (former names: thermostable or phenol-sulfating phenol sulfotransferase, P-PST) and 1A3 (thermolabile or catecholamine-sulfating phenol sulfotransferase, M-PST) are expressed e.g. in thrombocytes; their activity can be determined specifically with the substrates p-nitrophenol and dopamine, respectively (Raftogianis et al. 1997). Thus, simple procedures for phenotyping are available. They were used long before the genes were identified. The interindividual variability of 1A3 was found to be slight, that of 1A1, however, great (by a factor of >50). In the meantime a genetic explanation has been found to be involved in this variation. All of the 1A3-cDNAs isolated to date were found to have the same sequence; different 1A1 sequences have, however, been discovered (Coughtrie et al. 1999, Engelke et al. 2000, Jones et al. 1995, Ozawa et al. 1998, Raftogianis et al. 1997). Of particular importance is a G ? A exchange in codon 213, which leads to an amino acid exchange (Arg ? His) (Raftogianis et al. 1997). The enzyme variant SULT1A1*His has less favourable kinetic properties and is less stable than the enzyme variant 1A1*Arg. In the population of Potsdam we found allele frequencies of 63 % for 1A1*Arg and 37 % for 1A1*His (Engelke et al. 2000). Similar allele frequencies were observed in the USA (Ozawa et al. 1998, Raftogianis et al. 1997) and in Great Britain (Coughtrie et al. 1999). Functionally relevant polymorphism is also known for SULT1A2 (Brix et al. 1998, Engelke et al. 2000, Raftogianis et al. 2000). Base exchanges in codons 7 and 235 lead to amino acid exchanges (Ile ? Thr and Asn ? Thr). Usually both exchanges occur together. Primarily the exchange in codon 235 is of importance for the functioning of the enzyme. 1A2*Asn has much more favourable kinetic properties than 1A2*Thr (Brix et al. 1998, Raftogianis et al. 2000). In Potsdam we found allele frequencies of 62 % for 1A2*Asn and 38 % for 1A2*Thr (Engelke et al. 2000). The genes SULT1A1 and 1A2 are located next to each other on the same chromosome (8.5 kb between last exon of 1A1 and first exon of 1A2); there is a close relationship between their polymorphisms (Engelke et al. 2000). It is notable that the alleles of the highly active variants (1A1*Arg and 1A2*Asn) or those of the less active variants (1A1*His and 1A2*Thr) are almost always found together on one chromosome. Little is known about the tissues in which 1A2 is expressed. The cDNA was isolated from the liver; the corresponding mRNA must therefore be present. However, the expression of 1A2 mRNA in the liver seems to be about 100 times lower than that of 1A1. 86
4.2 Genetic Polymorphisms of Sulfotransferases Of the other SULT forms only 2A1 and to a small extent 1E1 were investigated for interindividual differences. For 2A1, a bimodal activity distribution was observed in the liver; this cannot be attributed to differences in the coding section (Wood et al. 1996). The highest expression of SULT1B1 was found at the protein level in the colon. We observed considerable differences in tissue samples from different test persons; the reasons for this variation are still unknown. The other SULT forms (1C1, 1C2, 2B1a, 2B1b, 4A1) were described for the first time between 1998 and 2000. They are known mostly only at the nucleic acid level; there are no data available for their expression at the protein and enzyme activity level in the human organism. Also nothing is known about their physiological function, interindividual variability and possible genetic polymorphisms.
4.2.4 Toxification and Detoxification of Chemicals by SULT: Differences between Allelic Variants Numerous xenobiotics are excreted as glucuronides and sulfo conjugates (as sulfates or – less often – as sulfamates or thiosulfates). For this reason, the conjugation reactions are usually associated closely with metabolic detoxification. This way of looking at things is increasingly proving to be too simple, however. In the meantime it has become clear that at least sulfation (O-sulfonation) has considerable potential for producing a more toxic compound. There are chemical reasons for this. The sulfate group is highly electrophilic and, therefore, in suitable positions (where the resulting cation is stabilized as a result of mesomerism or inductive effects), forms a good leaving group. During this heterolysis a strongly electrophilic cation is generated which can form a covalent bond with cellular macromolecules. If it binds with DNA, mutations can be induced and tumours initiated. As early as the 1960s it was demonstrated that the activation of the hepatocarcinogen 2-acetylaminofluorene in the rat takes place via sulfation (Nhydroxylation followed by O-sulfonation) (DeBaun et al. 1968). In the following decades, however, this activation mechanism was largely ignored. The main reason for this was probably the fact that by this time in vitro tests with external activation systems (e.g. Ames test) had gained a central role in the detection of mutagenic substances and in the characterization of their activation modes. Activation via sulfation cannot, however, be reliably determined with external metabolic systems, as reactive sulfate esters, as a result of their charge, do not pass easily through membranes, in particular if they have short lifespans, and therefore do not reach the target cells (Glatt 1997). The detectability of SULT-induced activation has improved in recent years, as it is now possible by genetic engineering to express SULT 87
4 Susceptibility directly in the target cells of established mutagenicity test systems (e.g. Salmonella strains of Ames, V79 cells from the Chinese hamster) (Glatt 1997, Glatt et al. 1998, Glatt et al. 2000, Jones et al. 1995, Kreis et al. 2000). In our working group alone – in which most of the studies were carried out – over 100 substances were shown to be mutagenic after SULT-dependent activation. It is characteristic that often only one or a few SULT forms lead to activation of a substance, while many other forms are inactive with the substance (but activate other substances). This applies when various SULT forms from one species are compared (Glatt et al. 1998, Glatt et al. 2000), or orthologous forms from different species (Glatt et al. 1998, Glatt et al. 2000) and even allelic variants (alloenzymes) of the same form from one species. Described below, and illustrated with examples, is only the last of these. 2-Nitropropane (Figure 1) and 2,4-dinitrobenzyl alcohol (Figure 2) are activated by SULT 1A1*Arg more efficiently by several orders of magnitude than by its allelic variant, 1A1*His. Likewise, N-hydroxy-2-acetylaminofluorene (Figure 3) and N-hydroxy-2-acetylamino-5-phenylpyridine (Figure 4) are activated more strongly by 1A2*Asn than by its allelic variant 1A2*Thr. N-Hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, a metabolite of the heterocyclic amine 2-amino-1methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), which is formed from amino acids and creatine when foods are heated, is activated very efficiently by the two SULT forms, 1A1 and 1A2; the alloenzymes 1A1*Arg and 1A2*Asn are much more active, however, than 1A1*His and 1A2*Thr.
Figure 2: Mutagenicity of 2,4-dinitrobenzyl alcohol in Salmonella typhimurium TA1538-derived strains engineered for expression of allelic variants of human SULT1A1. Investigated was the reversion to histidine prototrophy. The mean values and standard deviations of 3 plates are given.
88
4.2 Genetic Polymorphisms of Sulfotransferases
Figure 3: Mutagenicity of N-hydroxy-2-acetylaminofluorene in Salmonella typhimurium TA1538-derived engineered for expression of allelic variants of human SULT1A2. The mean values and standard deviations of 3 plates are given.
Figure 4: Mutagenicity of N-hydroxy-2-acetylamino-5-phenylpyridine in Salmonella typhimurium TA1538-derived strains expressing allelic variants of human SULT1A2. The mean values and standard deviations of 3 plates are given.
89
4 Susceptibility
4.2.5 Relationship between SULT Genotype and Health Risks The observation that an alloenzyme activates a certain substance substantially more efficiently than another alloenzyme may suggest that individuals with homozygous coding for the highly active alloenzyme are at greater risk from the substance than those who do not code for it, or code for it only in a single copy. This supposition must, however, be tested in man in vivo, as the same enzyme may catalyze multiple, toxifying as well as detoxifying, biotransformation steps of the substance, and also as it competes with other enzymes, which can lead to a greater or lesser risk, depending on the product formed and the location of the enzyme. It must also be stressed that a certain genotype for a xenobiotic-metabolizing enzyme is not in itself a risk factor, but only in combination with exposure to specific substances. Depending on the type of exposure, the same genotype could have either favourable or adverse effects. The data described above suggest that the presence of the alloenzyme SULT1A1*Arg increases the risk represented by certain aromatic amines and 2-nitropropane. With e.g. exposure to benzene, however, the risk could be expected to be reduced, as phenol, catechol and hydroquinone (which are regarded as proximal mutagens/carcinogens of benzene) are transformed by SULT1A1 into easily excretable conjugates. Our particular interest focuses on nutritional toxicology. A few (but not all) epidemiological studies have revealed positive associations between the incidence of mammary and colon carcinomas and high meat consumption, in particular if the meat is well-fried. This finding suggests that heterocyclic amines could be of aetiological importance. As SULT1A1 is involved in the activation of heterocyclic amines (see above), is expressed in the colon mucosa and the mamma, and is genetically polymorphic, epidemiologic studies should be carried out to investigate relationships between SULT1A1 genotype, dietary habits and occurrence of neoplasias at these sites. We intend to carry out such investigations as part of the EPIC project (European Prospective Investigation into Cancer and Nutrition). In the EPIC study, data will be collected from several hundred-thousand persons for dietary habits, life-style and health; various physiological parameters will be determined and blood samples taken (Riboli 1992). As this prospective study was started only recently, there are not yet sufficient data available on the incidence of cancer. When establishing the genotyping of SULT1A1 and 1A2, comparisons were therefore made between groups which differed in those health-relevant physiological parameters which could already be determined, such as blood pressure and body weight. Such an investigation would be of interest as SULT metabolizes not only xenobiotics, but also plays an important role in the regulation of numerous hormones (iodothyronines; catecholamines; some peptides; sexual, mineral and cortico steroids; melatonin; eicosanoids); also cholesterol and cholic acids are among the SULT substrates 90
4.2 Genetic Polymorphisms of Sulfotransferases Table 1: SULT1A1 and 1A2 genotype in men of normal weight and obese men (n = 692) from the Potsdam area. Genotype
SULT1A1 *Arg/*Arg *Arg/*His *His/*His SULT1A2 *Asn/*Asn *Asn/*Thr *Thr/*Thr
Number of individuals normal weight
obese
152 149 45 145 156 45
121 178 47 116 176 54
Odds ratio b
1.000 1.501 1.244 1.000 1.253 1.343
(reference value) (1.086–2.075) (0.817–2.110) (reference value) (1.019–1.955) (0.943–2.395)
a
A Body Mass Index (BMI) of 20–25 and > 30, respectively, were the criteria for normal weight and obesity. b Obese persons compared to persons of normal weight. The confidence interval is given in brackets.
(Falany 1997, Kester et al. 1999). No relationship was observed between high blood pressure and the SULT1A1 and 1A2 genotypes. In obese persons, however, the alleles 1A1*His and 1A2*Thr were more frequent (Table 1). It is not yet known whether a SULT function is the reason for this association or whether it is the result of a coupled polymorphism in a neighbouring gene. Parallel to our working group, Coughtrie et al. (1999) established the genotyping of SULT1A1. They observed an increase in the frequency of the 1A1*Arg allele with increasing age (Table 2 a); when we investigated our test persons using the same criteria we found the same trend (Table 2 b). As these are exploratory investigations, the results should be regarded as preliminary. Nevertheless, the relationship between SULT1A genotype and obesity on the one hand (see above) and the known relationship between obesity and reduced life expectancy on the other hand could speak for this possibility. Should the relationship between SULT1A genotype and age be confirmed, this would mean that polymorphism of SULT1A1, SULT 1A2 or of a neighbouring gene influences life expectancy. It would then be interesting to find out whether there are differences in the frequency of specific causes of death between the SULT1A genotypes and whether SULT 1A1 substrates are of aetiological importance. Apart from this first study, there are no other studies available of the frequency of SULT1A genotypes in relation to incidences of disease or other health-related parameters. Now that the methods for determining the genotype have been developed, many such data can be expected to become available shortly. Such data will be particularly interesting from a toxicological point of view if at the same time a relationship is established with the exposure to substances that are either toxified or detoxified by SULT1A1 or 1A2. 91
4 Susceptibility Table 2: Dependency on age of the frequency of various SULT1A1 genotypes. Age group (years)
Genotype frequency
Allele frequency
*Arg/*Arg
*Arg/*His
*His/*His
*Arg
*His
n
n
n
%
%
%
%
%
a) Coughtrie et al. (1999) trend test p = 0.017. 12–39 32 40–49 21 50–59 19 60–69 25 70–99 40
British test persons (men and women, n = 293). Armitrage
b) Men from the Potsdam 12–39 0 40–49 70 50–59 120 83 60–69 1 70–99 0
area (n = 692). Age classified as in Coughtrie et al. – 0 – 0 – – 36.6 96 50.3 25 13.1 61.8 38.6 146 46.9 45 14.5 62.1 43.7 85 44.7 22 11.6 66.1 – 0 – 0 – –
1
39.0 38.9 47.5 51.0 58.8
41 25 16 20 22
50.0 46.3 40.0 40.8 32.4
9 8 5 4 6
11.0 14.8 12.5 8.2 8.8
64.0 62.0 67.5 71.4 75.0
36.0 38.0 32.5 28.6 25.0 (1999). – 38.2 37.9 33.9 –
Oldest person 64 years old.
4.2.6 Conclusions and Prospects By means of heterologous expression, exactly defined xenobiotic-metabolizing enzymes in man can be made available for metabolic and toxicological investigations. It can then be investigated whether they metabolize a certain substance. If the enzymes are expressed directly in target cells of toxicological test systems, it can also immediately be tested whether this metabolism leads to the formation of cytotoxic or mutagenic products. This information is of importance because many toxicologically active metabolites are so reactive and short-lived that they are difficult to determine with chemico-analytical methods. For example, various reactive sulfate conjugates react with water to regenerate the initial compound (Glatt 1997, Landsiedel et al. 1998). If an enzyme is found which metabolizes the substance in question (and at the same time possibly converts it into a more toxic or less toxic compound), and if genetic polymorphisms are known for this enzyme, it can be further investigated whether the allelic variants differ in this metabolic activity. If this is the case and test persons are available who have been exposed to this substance (e.g. as a result of medicinal therapy or at the workplace), it is advantageous to investigate and compare the kinetics, metabolite profiles, and pharmacological and toxicological effects in the 92
4.2 Genetic Polymorphisms of Sulfotransferases various genotypes separately. Not only the actual hazardous effects, but also biological and biochemical biomarkers (such as DNA adducts) may be used as toxicological endpoints. If the enzyme in question forms a reactive metabolite, possible target organs can be deduced from its tissue distribution, which deserve particular consideration in toxicological and epidemiological investigations in man. Also looking at metabolic variation from the other end (variable effect/ kinetics ? polymorphisms) can lead to new insights. If certain enzyme forms are found to greatly contribute to the metabolism of a substance and if peculiarities are observed in the kinetics or effects in certain exposed individuals, this can be seen as a reason to genetically investigate the corresponding loci in these individuals to discover new, metabolically and toxicologically relevant polymorphisms. In the strategies presented, with the aid of in vitro test systems using recombinant organisms, genes are identified which represent candidate susceptibility factors for certain substances. Whether polymorphisms in the corresponding genes actually influence the risks to a notable degree, must then, however, be investigated in man in vivo. In the medium-term, humanized animal models in which the endogenous enzyme in question has been replaced by the various allelic variants of the orthologous human enzyme should become available as an additional test system. The advantage of these models will be that extensive experimental-toxicological investigations will become possible in a system in which the polymorphic human gene is integrated into the complexity of a whole organism. Once it is known how the individual genetic polymorphisms determine the susceptibility with various substances and substance classes, this knowledge can be used to better evaluate those substances which are responsible for the hazardous effects after complex exposures (for example via the environment and food). The outlined strategies can be used for various components of the substance-processing system (e.g. biotransformation, transport, repair). SULT forms should be of considerable interest here, as they affect the biotransformation of numerous substances and can convert them into both more toxic and less toxic compounds. Many aromatic amines, nitroarenes, secondary nitroalkanes and metabolic precursors of benzylic or allylic alcohols can – directly or after other metabolic reactions – be toxified by SULT; many substances used at the workplace and environmental contaminants belong to these substance classes. As all known human SULT forms, including major allelic variants, are already available in test systems using recombinant organisms, it can be investigated relatively easily which forms are canditates for susceptibility factors in pharmaco-toxicological studies with certain substances. It is to be expected that in the coming years further human SULT forms and further functionally relevant SULT polymorphisms will be discovered. Corresponding test systems using recombinant organisms can then be constructed. Parallel to this, high-performance procedures for genotyping for function93
4 Susceptibility ally relevant polymorphisms must be established. The use of this method in occupational medicine and environmental and nutritional toxico-epidemiology would seem to be very promising. References Brix, L. A.; Nicoll, R.; Zhu, X.; McManus, M. E.: Structural and functional characterisation of human sulfotransferases, Chem.-Biol. Interact., 109, 123–127 (1998). Coughtrie, M. W. H.; Gilissen, R. A. H. J.; Shek, B.; Strange, R. C.; Fryer, A. A.; Jones, P. W.; Bamber, D. E.: Phenol sulfotransferase SULT1A1 polymorphism: molecular diagnosis and allele frequencies in Caucasian and African populations, Biochem. J., 337, 45–49 (1999). DeBaun, J. R.; Rowley, J. Y.; Miller, E. C.; Miller, J.: Sulfotransferase activation of N-hydroxy-2-acetylaminofluorene in rodent livers susceptible and resistant to this carcinogen, Proc. Soc. Exp. Biol. Med., 129, 268–273 (1968). Engelke, C. E. H.; Meinl, W.; Boeing, H.; Glatt, H. R.: Association between functional genetic polymorphisms of human sulfotransferases 1A1 and 1A2, Pharmacogenetics, 10, 163–169 (2000). Falany, C. N.: Sulfation and sulfotransferases. 3. Enzymology of human cytosolic sulfotransferases, FASEB J., 11, 206–216 (1997). Glatt, H. R.; Davis, W.; Meinl, W.; Hermersdörfer, H.; Venitt, S.; Phillips, D. H.: Rat, but not human, sulfotransferase activates a tamoxifen metabolite to produce DNA adducts and gene mutations in bacteria and mammalian cells in culture, Carcinogenesis, 19, 1709–1713 (1998). Glatt, H. R.; Engelke, C. E. H.; Pabel, U.; Teubner, W.; Jones, A. L.; Coughtrie, M. W. H.; Andrae, U.; Falany, C. N.; Meinl, W.: Sulfotransferases: genetics and role in toxicology, Toxicol. Lett., 112/113, 341–348 (2000). Glatt, H. R.: Sulfation and sulfotransferases: 4. Bioactivation of mutagens via sulfation, FASEB J., 11, 314–321 (1997). Jones, A. L.; Hagen, M.; Coughtrie, M. W. H.; Roberts, R. C.; Glatt, H. R.: Human platelet phenolsulfotransferases: cDNA cloning, stable heterologous expression in V79 cells and identification of a novel allelic variant of the phenol-sulfating form, Biochem. Biophys. Res. Commun., 208, 855–862 (1995). Kester, M. H. A.; Kaptein, E.; Roest, T. J.; van Dijk, C. H.; Tibboel, D.; Meinl, W.; Glatt, H. R.; Coughtrie, M. W. H.; Visser, T. J.: Characterization of human iodothyronine sulfotransferases, J. Clin. Endocrinol. Metab., 84, 1357–1364 (1999). Kreis, P.; Brandner, S.; Coughtrie, M. W. H.; Pabel, U.; Meinl, W.; Glatt, H. R.; Andrae, U.: Human phenol sulfotransferases hP-PST and hM-PST activate propane 2-nitronate to a genotoxicant, Carcinogenesis, 21, 295–299 (2000). Landsiedel, R.; Pabel, U.; Engst, W.; Ploschke, J.; Seidel, A.; Glatt, H. R.: Chiral inversion of 1-hydroxyethylpyrene enantiomers mediated by enantioselective sulfotransferases, Biochem. Biophys. Res. Commun., 247, 181–185 (1998). Ozawa, S.; Tang, Y. M.; Yamazoe, Y.; Kato, R.; Lang, N. P.; Kadlubar, F. F.: Genetic polymorphisms in human liver phenol sulfotransferases involved in the bioactivation of N-hydroxy derivatives of carcinogenic arylamines and heterocyclic amines, Chem. Biol. Interact.; 109, 237–248 (1998). Raftogianis, R. B.; Wood, T. C.; Otterness, D. M.; Van Loon, J. A.; Weinshilboum, R. M.: Phenol sulfotransferase pharmacogenetics in humans: association of common SULT 1A1 alleles with TS PST phenotype, Biochem. Biophys. Res. Commun., 239, 298–304 (1997). Raftogianis, R. B.; Wood, T. C.; Weinshilboum, R. M.: Human phenol sulfotransferases SULT1A2 and SULT1A1: genetic polymorphisms, allozyme properties, and human liver genotype-phenotype correlations, Biochem. Pharmacol., 58, 605–616 (2000).
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4.2 Genetic Polymorphisms of Sulfotransferases Riboli, E.: Nutrition and cancer: background and rationale of the European Prospective Investigation into Cancer and Nutrition (EPIC), Ann. Oncol., 3, 783–791 (1992). Weinshilboum, R. M.; Otterness, D. M.; Aksoy, I. A.; Wood, T. C.; Her, C.; Raftogianis, R.: Sulfation and sulfotransferases: 1. cDNAs and genes, FASEB J., 11, 3–14 (1997). Wood, T. C.; Her, C.; Aksoy, I.; Otterness, D. M.; Weinshilboum, R. M.: Human dehydroepiandrosterone sulfotransferase pharmacogenetics: quantitative western analysis and gene sequence polymorphisms, J. Steroid. Biochem. Mol. Biol., 59, 467–478 (1996). Yamazoe, Y.; Nagata, K.; Ozawa, S.; Kato, R.: Structural similarity and diversity of sulfotransferases, Chem.-Biol. Interact., 92, 107–117 (1994).
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4.3
Genotyping and Phenotyping, Using NAT2 as an Example Klaus Golka * and Meinolf Blaszkewicz *
N-Acetyltransferases are xenobiotic-metabolizing enzymes of phase 2. To date, two N-acetyltransferases (NAT) have been detected, which differ both in their substrate specificity and in their expression in the various tissues. NAT1 is found in the cytosol and is detectable e.g. in the liver, intestine, lungs, kidneys and leukocytes. Until recently it was regarded as monomorphic. A typical substrate is p-aminobenzoic acid (PABA). Other substrates are p-aminosalicylic acid (PAS), anisidine and sulfanilamide. Of greater toxicological and clinical relevance and better researched is NAT2 (reviews in Evans 1989, Thier et al. 1999, Autrup 2000). Typical substrates are e.g. the anti-tuberculosis drug, isoniazid (INH); various sulfonamides; the aromatase inhibitor, aminoglutethimide; the drug used against leprosy, dapsone; salazosulfapyridine, used with inflammatory diseases of the intestine; the medicine against high blood pressure, hydralazine; the anti-arrhythmia drug, procaine amide; caffeine and aromatic amines. NAT2 is also found in the cytosol and is present e.g. in liver cells, but not in leukocytes. The half-life of the anti-tuberculosis drug, isoniazid, in serum is 1.5 hours in “rapid” acetylators, and 3 hours in “slow” acetylators. In “slow” acetylators treated with isoniazid, increased peripheral polyneuropathy was described. In “slow” acetylators given hydralazine, increased antinuclear antibodies were found. The acetylator status can be expected to affect therapy above all when the level of the drug in serum is responsible for the effect, such as in the case of e.g. hydralazine. After it became known that sulfonamides are also acetylated, above all sulfamethazine (not available in Germany as a medicine) and dapsone, a drug against leprosy, were used for determining the acetylator status. The use of drugs in phenotyping studies is, however, for ethical reasons, not unproblematic. The substrate most suitable for determining the acetylator phenotype in studies is caffeine. This method was first established by Grant et al. *
96
Institut für Arbeitsphysiologie der Universität Dortmund, Ardeystr. 67, 44139 Dortmund
4.3 Genotyping and Phenotyping, Using NAT2 as an Example (1983). The decisive advantage of this method for field studies is that for phenotyping the person only drinks two cups of ground coffee and gives a urine sample after about 2 hours. This is adjusted to pH 3.5 and can then be stored at –18 8C. Nevertheless, this elegant method did not at first become established in practice as only the caffeine metabolite not acetylated, 1-methylxanthine (1X), was commercially available, but not, however, the acetylated caffeine metabolite, 5-acetylamino-6-formylamino-3-methyluracil (AFMU), also needed as a reference standard. Its synthesis is difficult (Röhrkasten et al. 1997), even when precursors are available in laboratories with the appropriate equipment. Furthermore, the cut-off which separates slow and rapid acetylators, which is defined by the ratio AFMU/1X, cannot be transferred from one laboratory to another, as it varies in a range of about 0.5–1.0, possibly as a result of chromatographic conditions. There are still differences from laboratory to laboratory when reference substances of the same origin are used. Chromatography of the two metabolites extracted from the urine using HPLC, with an analysis time of about 60 minutes per sample, is time-consuming and complicated. After the introduction of the polymerase chain reaction it became possible to determine the genotype for N-acetyltransferase 2. In 1999, 14 alleles have been found which code for a “slow” acetylator (Wormhoudt et al. 1999). The “rapid” allele is dominant over a “slow” allele. The correlation between acetylator phenotype and acetylator genotype is greater than 90 % (Figure 1). The molar ratio of the caffeine metabolites AFMU and 1X excreted in urine determined for a certain allele constellation, has a consid-
Figure 1: Determination of the acetylator phenotype and acetylator genotype in 54 patients with bladder tumours (in 18 patients the acetylator phenotype was determined in urine samples 2 and 4 hours after caffeine intake).
97
4 Susceptibility erable range of distribution for the individual alleles. This indicates that also other factors can influence the metabolism of the two metabolites. This is in agreement with recent results from various research groups which showed that e.g. in the metabolism of benzidine also other enzymes are involved, such as e.g. prostaglandine synthase H (Degen et al. 1998) and, contrary to earlier belief, also NAT1 (Zenser et al. 1996). The distribution of the two acetylator types shows clear ethnic differences. 5 % of Eskimos, 15 to 22 % of the Chinese, 83 % of Egyptians and 90 % of Moroccans are “slow” acetylators. In Central Europe about 50 to 65 % of the general population are “slow” acetylators. In 1979 Lower et al. observed for the first time that collectives of patients with bladder tumours contained a higher proportion of “slow” acetylators than found in the general population. Later Cartwright (1982) was able to show that the proportion of “slow” acetylators was particularly high among those patients with bladder tumours who were occupationally exposed to aromatic amines. Cartwright found that 22 of the 23 patients with bladder tumours who were formerly occupationally exposed to aromatic amines (in particular benzidine) in a factory which produced dyes, were “slow” acetylators. In 1991 Lewalter & Miksche reported that 92 of the 331 workers from a benzidine plant that was closed in 1969 had developed bladder tumours. The proportion of “slow” acetylators among those with bladder tumours was 81.5 %. The proportion of “slow” acetylators among the 331 workers ever employed in the benzidine plant was 48 %. The basic metabolism of aromatic amines is shown in Figure 2. “Slow” acetylators acetylate less of the xenobiotics (e.g. aromatic amines) per unit of time than “rapid” acetylators. For this reason, in “slow” acetylators alternative oxidative metabolic pathways are increasingly followed. The end product of the oxidative metabolism of aromatic amines are arylnitrenium ions, which are very reactive and react with the DNA of bladder urothelium cells. In earlier studies, a higher proportion of patients with bladder tumours were found to be “slow” acetylators than were the controls. In the mid 1990s it was shown for the first time in two large collectives in Germany that the proportion of “slow” acetylators among patients with bladder tumours is no longer significantly higher than in the general population (Golka et al. 1996). A suggested reason for this is the discontinuation of the production of carcinogenic aromatic amines such as e.g. benzidine (1971) in Germany resulting in a reduced background exposure in the general population due to products dyed with such substances. This supposition is based on the knowledge from occupational medicine that urothelial bladder tumours induced by aromatic amines can have very long latency periods (in some cases up to 40 years and more). The “slow” acetylator status is, with occupational exposure to aromatic amines, meanwhile recognized as being a predisposition factor for bladder tumours. However, it is still unclear whether the different aromatic 98
4.3 Genotyping and Phenotyping, Using NAT2 as an Example
Figure 2: Aromatic amines: metabolic pathways and the assumed mechanism for the carcinogenesis of urothelial tumours (modified according to Lang & Kadlubar 1991).
amines (e.g. mononuclear, multinuclear) cause differences in the predisposition for bladder tumours depending on the acetylator status. The most important risk factor for bladder tumours is smoking. As cigarette smoke contains various aromatic amines, smokers with bladder tumours could be expected to be mainly “slow” acetylators. Yet over-representation of the slow acetylator status was not observed in all studies. It can be investigated in groups of persons known to have an increased risk of bladder tumours, whether there is a shift towards “slow” acetylators. Such a shift supports the assumption of the induction of bladder tumours by aromatic amines. In some painters with bladder tumours there was a marked increase in “slow” acetylators. 14 of 16 painters with bladder tumours were “slow” 99
4 Susceptibility
Figure 3: The percentage and absolute frequency of the “slow” acetylator phenotype in patients with urothelial tumours and proven occupational exposure to aromatic amines (n) and patients with urothelial tumours without known occupational exposure (n) and healthy persons (`) (from Schöps et al. 1997). In the study of Lewalter & Miksche, the control collective is the total collective of workers in a benzidine synthesis plant employed between 1951 and 1967 (160 “slow” and 171 “rapid” acetylators).
acetylators (Golka et al. 1997A). This lends support to the theory that bladder tumours in painters, who in earlier years (up to about 1960) were exposed to azo dyes, were induced by aromatic amines. It has been shown that the benzidine originally used as a coupling component in production can be released in the human organism from bioavailable (soluble) azo dyes on a benzidine basis. The carcinogenic aromatic amine released is regarded as the reason for the increased risk of bladder tumours in painters and varnishers. In a study of miners, another occupational group with an increased risk of bladder tumours, no shift towards “slow” acetylators was found (Golka et al. 1997B). This contradicts the assumption that aromatic amines are the cause of the increased risk of bladder tumours in miners. 100
4.3 Genotyping and Phenotyping, Using NAT2 as an Example The acetylator status has been linked with a whole series of other diseases, e.g. intestinal tumours. In most studies, in patients with intestinal tumours the “fast” acetylator status predominated. This was not true, however, for a collective of patients from in and around Dortmund, an industrial region in Germany where there is increased mortality from carcinomas of the colon (Roemer et al. 1999). A relationship has also been suggested between the acetylator status and various auto-immune diseases. The results to date have been contradictory, however, not least as a result of the small number of cases. In a more recent study with a larger number of patients with lupus erythematosus, an increase in the “slow” acetylator phenotype was found (v. Schmiedeberg 1999). In addition, the increased occurrence of toxic epidermal necrolysis after the administration of sulfonamides was described in “slow” acetylators (Wolkenstein et al. 1995). The distribution of “slow” acetylators in the general population with regard to age is inconspicuous. An important question is whether the prognosis for persons with bladder tumours is different depending on whether they are “slow” acetylators or “rapid” acetylators. In a study of 196 patients with bladder tumours it was found, however, that at least with bladder tumours classified as T1 and T2 there was no clinically relevant relationship between the acetylator status and the tumour classification (TNM), the grading (G) and the prognosis (Schöps et al. 1997). References Autrup, H.: Genetic polymorphisms in human xenobiotica metabolizing enzymes as susceptibility factors in toxic response, Mutat. Res., 464, 65–76 (2000). Cartwright, R. A.; Rogers, H. J.; Barkham-Hall, D.; Glasham, R. W.; Ahmad, R. A.; Higgins, E.; Kahn, M. A.: Role of acetyltransferase in bladder carcinogenesis: a pharmacogenetic epidemiological approach to bladder cancer, Lancet, 2, 842–846 (1982). Degen, G. H.; Schlattjan, J. H.; Mähler, S.; Föllmann, W.; Bolt, H. M.: Metabolismus und Genotoxizität von Benzidin in Urothel- und Samenblasenzellkulturen, in: Hallier, E.; Bünger, J. (Hrsg.): Gesundheitsgefahren durch biologische Arbeitsstoffe. Neuro-, Psycho- und Verhaltenstoxizität. Dokumentationsband über die 38. Jahrestagung der Deutschen Gesellschaft für Arbeitsmedizin und Umweltmedizin in Wiesbaden vom 11.–14. 05. 1998, S. 603–604, DGAUM, Lübeck (1998). Evans, D. A.: N-Acetyltransferase, Pharmacol. Ther., 42, 157 (1989). Evans, D. A., Eze, L. C., Whibley, E. J.: J. Med. Genetics, 22, 479–483 (1985). Golka, K.; Prior, V.; Blaszkewicz, M.; Cascorbi, I.; Schöps, W.; Kierfeld, G.; Roots, I.; Bolt, H. M.: Occupational history and genetic N-acetyltransferase polymorphism in urothelial cancer patients of Leverkusen, Germany, Scand. J. Work Environ. Health, 22, 332–338 (1996). Golka, K.; Kempkes, M.; Flieger, A.; Blaszkewicz, M.; Bolt, H. M.: Overrepresentation of the slow acetylator phenotype in painters suffering from urinary bladder cancer, Med. Lav., 88, 425–426 (1997 a). Golka, K.; Reckwitz, T.; Kempkes, M.; Cascorbi, I.; Blaszkewicz, M.; Reich, S. E.; Roots, I.; Sökeland, J.; Schulze, H.; Bolt, H. M.: N-Acetyltransferase 2 (NAT2) and glutathione S-transferase l (GSTM1) in bladder cancer patients in a highly industrialized area, Int. J. Occup. Environ. Health, 3, 105–110 (1997 b). Golka, K.; Seidel, T.; Rötzel, C.; Thier, R.; Geller, F.; Staude, G.; Bolt, H. M.: Untersuchungen zu beruflichen und außerberuflichen Risikofaktoren an Harnblasenkarzi-
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4 Susceptibility nompatienten in einem Industriegebiet der neuen Bundesländer, in: Hallier, E.; Bünger, J. (Hrsg.): Gesundheitsgefahren durch biologische Arbeitsstoffe. Neuro-, Psychound Verhaltenstoxizität. Dokumentationsband über die 38. Jahrestagung der Deutschen Gesellschaft für Arbeitsmedizin und Umweltmedizin in Wiesbaden vom 11.–14. 05. 1998, S. 407–410. DGAUM, Lübeck (1998). Grant, D. M.; Tang, B. K.; Kalow, W.: Polymorphic N-acetylation of caffeine metabolite, Clin. Pharmacol. Ther., 33, 355–359 (1983). Hanke, J.; Krajewska, B.: Acetylation phenotypes and bladder cancer, J. Occup. Med., 32, 917–918 (1990). Hanssen, H. P.; Agrawal, D. P.; Goedde, H. W.; Bucher, H.; Huland, H.; Brachmann, W.; Ovenbeck, R.: Association of N-acetyltransferase polymorphism and environmental factors with bladder carcinogenesis. Study in a north German population, Eur. Urol., 11, 263–266 (1985) Lang, N. P.; Kadlubar, F. F.: Aromatic and heterocyclic amine metabolism and phenotyping in humans, New Horizons in Biological Dosimetry, 33–47 (1991). Lewalter, J.; Miksche, L. W.: Empfehlungen zur arbeitsmedizinischen Prävention expositions- und dispositionsbedingter Arbeitsstoff-Beanspruchungen, Verh. Dt. Ges. Arbeitsmed., 31, 135–139 (1991). Lower, G. M.; Nilsson, T.; Nelson, C. E.; Wolf, H.; Gamsky, T. E.; Bryan, G. T.: N-Acetyltransferase phenotype and risk in urinary bladder cancer. Approaches in molecular epidemiology, Environ. Health Perspect., 29, 71–79 (1979). Roemer, H. C.; Roetzel, C.; Thier, R.; Zorn, U.; Reckwitz, T.; Golka, K.; Loehlein, D.: The distribution of two polymorphic enzymes in colon cancer cases in a highly industrialized area. Proceedings of the 2nd International Congress on Gastrointestinal Carcinogenesis in Ulm/Germany. Röhrkasten, R.; Raatz, P.; Kreher, R. P.; Blaszkewicz, M.: Synthesis of the caffeine metabolites 5-acetylamino-6-formylamino-3-methyluracil (AFMU) and 5-acetylamino-6amino-3-methyluracil (AAMU) on a preparative scale, Z. Naturforsch., 52 b, 1526– 1532 (1997). Schöps, W.; Prior, V.; Golka, K.; Blaszkewicz, M.; Cascorbi, I.; Roots, I.; Bolt, H. M.; Kierfeld, G.: Untersuchung zur klinischen Relevanz der Acetyliererphänotypisierung bei 196 Urotheltumorpatienten, Urologe [A], 35, 64–67 (1997). Thier, R.; Golka, K.; Brüning, T.; Bolt, H. M.: Genetische Suszeptibilität im Hinblick auf toxische Arbeitsplatz- und Umweltbelastungen. Bundesgesundheitsblatt – Gesundheitsforschung – Gesundheitsschutz, 42, 834–840 (1999). von Schmiedeberg, S.; Fritsche, E.; Rönnau, A. C.; Specker, C.; Golka, K.; Richter-Hintz, D.; Schuppe, H.-C.; Lehmann, P.; Ruzicka ,T.; Esser, C.; Abel, J.; Gleichmann, E.: Polymorphisms of the xenobiotic-metabolizing enzymes CYP1A1 and NAT-2 in systemic sclerosis and lupus erythematosus, Adv. Exp. Med. Biol., 455, 147–152 (1999). Weber, W. W.; Hein, D. W.; Litwin, A.; Lower, G. M. Jr.: Fed. Proc. (United States), 42, 3086–3097 (1983). Wolkenstein, P.; Carrière, V.; Charue, D.; Bastuji-Garin, S.; Revuz, J.; Roujeau, J.-C.; Beaune, P.; Bagot, M.: A slow acetylator genotype is a risk factor for sulphonamideinduced toxic epidermal necrolysis and Stevens-Johnson syndrome. Pharmacogenetics 5, 255–258 (1995). Wormhoudt L. W.; Commandeur J. N. M.; Vermeulen N. P. E.: Genetic polymorphisms of human N-acetyltransferase, cytochrome P450, glutathione-S-transferase, and epoxide hydrolase enzymes: relevance to xenobiotic metabolism and toxicity. Crit. Rev. Toxicol., 29, 59–124 (1999). Zenser, T. V.; Lakshmi, V. M.; Rustan, T. D.; Doll, M. A.; Deitz, A. C.; Davis, B. B.; Hein, D. W.: Human N-acetylation of benzidine: role of NAT1 and NAT2, Cancer Res., 56, 3941–3947 (1996).
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4.4
New High-throughput Technology in the Diagnostic Screening of Susceptibility Factors Ricarda Thier *, Thomas Brüning * and Yon Ko **
4.4.1 Summary An important reason for the different sensibility of each individual to chemical substances is the variability of the genes which code for xenobioticmetabolizing enzymes. Polymorphisms of various isoforms of phase I and also the phase II enzymes have become important for occupational and environmental medicine. Evaluation of these differences can be carried out at a genetic level. Light cycler assisted technology allows the rapid and simultaneous analysis of several polymorphisms of xenobiotic-metabolizing enzymes and other susceptibility factors by means of on-line detection and the quantification of specific gene amplification products using hybridization probes labelled with fluorescent substances. In view of the progress to be expected in this field such high-throughput methods will gain considerably in importance.
4.4.2 Introduction The susceptibility of the individual to chemical substances, in particular hazardous substances at the workplace and in the environment, is increasingly becoming the main focus of toxicological interest when investigating the development of chronic diseases (Zbinden 1992). Absorbed substances undergo step-by-step biotransformation, in the course of which metabolites can be formed which have a greater or smaller toxic or carcinogenic potential than the initial substance. The biologically active dose of these metabo** Institut für Arbeitsphysiologie der Universität Dortmund, Ardeystr. 67, 44139 Dortmund ** Medizinische Universitäts-Poliklinik Bonn, Wilhelmstr. 35–37, 53111 Bonn
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4 Susceptibility lites depends on the dose of exogenously absorbed substances and the performance of xenobiotic-metabolizing enzymes. A reason for the different metabolic performance of each individual is the variability of the genes which code for these xenobiotic-metabolizing enzymes. Mutations which occur in the population in a gene for a xenobiotic-metabolizing enzyme with a frequency of at least 1 %, are called polymorphisms (Bolt 1994). In occupational and environmental medicine, of the polymorphisms for xenobiotic-metabolizing enzymes of phase 1, above all various isoforms of cytochrome P450 (CYP) are important in this context, and of the enzymes of phase 2, in particular N-acetyltransferases (NAT) and glutathione transferases (GST) (Bolt 1994). From an occupational-medical point of view, the isoenzymes of GSTT1 and GSTM1 seem important in the metabolism of organic solvents, plastics monomers and intermediate polycyclic aromatic hydrocarbons. The influence of N-acetyltransferase 2 (NAT2) in the induction of urothelial carcinomas with exposure to aromatic amines is regarded as proven; the N-oxidation of aromatic amines, which causes them to become more toxic, takes place to a greater extent in “slow” acetylators. It is evident that polymorphisms of xenobiotic-metabolizing enzymes decisively modulate the effects of toxic substances. The relative risk associated with the interaction of exposure to hazardous substances and the presence of genetic polymorphism, and the incidence of this combination in the population are decisive for the contribution of polymorphisms of xenobiotic-metabolizing enzymes to the development of chronic diseases in that population. With complex diseases caused in part by exogenous risk factors, it is possible to limit the risk by reducing the exposure to hazardous substances of susceptible groups of persons. It is therefore clear that casuistic and epidemiological investigations can throw considerably more light on the genesis of chronic diseases resulting from exposure to chemical substances when they include data for genetically determined features of enzyme polymorphisms of xenobiotic-metabolizing enzymes. To date, however, only few studies have investigated more closely the interaction of genotype and exposure (”gene-environment interaction”). Polymorphisms of xenobiotic-metabolizing enzymes can be determined on the basis of the genotype; these investigations are carried out at DNA level. There are several procedures which allow the high throughput of samples. The applications of some of these have, however, not yet been completely tried and tested, and they are not yet available for the determination of the polymorphism of xenobiotic-metabolizing enzymes. For this reason, only the light cycler assisted real time polymerase chain reaction is mentioned here; the authors have gained experience with this procedure in the establishment of methods for the determination of polymorphisms of xenobiotic-metabolizing enzymes (Brüning et al. 1999, Harth et al. 2000, Ko et al. 2000). The main advantage of the method is the rapid procedure. Usually mutation analysis is carried out by means of polymerase chain reaction (PCR) and then restriction digestion (RFLP). At least one working day is 104
4.4 New High-throughput Technology in the Diagnostic Screening
Figure 1: Comparison of the time needed for mutation analysis using PCR-RFLP and light cycler assisted technology (GE: gel electrophoresis).
needed to carry out the procedure. Up to 96 samples (including the necessary controls) can be processed per batch (Figure 1). With real time PCR it is no longer necessary to carry out restriction digestion and gel electrophoresis. With the light cycler, PCR can be successfully completed in less than 2 hours. However, at present the device can take only 32 samples. In addition to its speed, another advantage of this method is the possibility of specific on-line quantification of the amplified gene fragment.
4.4.3 Principles of the Technology In addition to the gene fragment-specific primers, two sequence-specific hybridization probes are used, the first of which is labelled at its 3' end with fluorescein (detection probe) and the second (anchor probe) at its 5' end with a different fluorescent dye (Light-Cycler-Red 640 or 705, LC-640 or LC-705) (Figure 2). These two probes are selected so that within the amplified gene fragment they hybridize a maximum of 1 to 5 base pairs apart. The emission diodes of the light cycler (470 nm) cause the fluorescein in the detection probe to fluoresce. The green light emitted causes the fluorophore LC-640 or LC-705 in the anchor probe to emit red light (640 or 705 nm). This procedure is known as fluorescence resonance energy transfer (FRET). FRET can only take place when the two probes are situated next to each other. The reading is therefore taken at the end of the annealing phase of each PCR cycle. The emission of red light is proportional to the amount of the amplified DNA fragment and is determined at the appropriate wavelength for the light cycler. After the reading has been taken, the two hybridization probes are displaced by the Taq polymerase (extension phase of the PCR cycle). 105
4 Susceptibility
Figure 2: Principles of light cycler assisted mutation analysis.
Figure 3: Investigations of GSTM1 gene deletion polymorphism.
4.4.4 Use with Gene Deletion Polymorphisms (GSTM1 and GSTT1) This technology is used to investigate the polymorphisms of the glutathione transferases GSTT1 and GSTM1. Both GSTs are subject to gene deletion polymorphism, which is shown in a partial (heterozygote) or complete (homozygote carriers of the corresponding null allele) loss of the functional gene product (Wiebel et al. 1999). If at least one allele is present, during real time PCR there is an increase in fluorescence emission (LC640). If the DNA sample is from a homozygote null genotype (GSTM1*0/0 or GSTT1*0/0) the probes cannot hybridize and therefore no fluorescence is emitted. Heterozygote (GSTM1*1/0 and GSTT1*1/0) carriers cannot be 106
4.4 New High-throughput Technology in the Diagnostic Screening distinguished with this method from homozygote carriers of the functional gene (GSTT1*1/1 and GSTM1*1/1). To exclude false negative results, a housekeeping gene (here glycerin aldehyde phosphate dehydrogenase, GAPDH) is amplified at the same time in every sample using a different fluorophore (LC-705), which is detected at another wavelength of the device (Figure 3; Ko et al. 2000).
4.4.5 Use with Point Mutations (GSTP1) The polymorphism of GSTP1 is based on two point mutations. In codon 104 a base-pair substitution (A1404G) leads to the exchange of the amino acid isoleucine by valine. Several publications report that the mutant allele produces less gene product (Zimniak et al. 1994, Ali-Osman et al. 1997). As in the first example, in addition to the two primers, with which some of the GSTP1 gene is amplified, two probes are selected. The detection probe hybridizes directly in the area of the GSTP1 point mutation to be investigated. The anchor probe is situated next to it. After completion of PCR a melting curve is recorded for the amplification products. The melting temperature (temperature at which the hybridization between the probes and the amplification product breaks down) is determined by the length and sequence of the probes and the homology with the amplification product. If the sequence of a probe is not absolutely complementary to that of the amplification product, the hybridization breaks down at lower temperatures. This is the case with point mutation. The melting temperature for allele (a), which is completely complementary with the probe, is 65.5 8C, that for the mutant allele (b) is 62 8C. This results in a single maximum in the first derivative of the melting curve for homozygote DNA samples, but two maxima in the first derivative of the curve for heterozygote DNA (Figure 4). Other uses of this kind for point mutations have already been estab-
Figure 4: Determination of GSTP1 polymorphism (A1404G) using the melting curve.
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Figure 4 (continued)
lished for the enzymes cytochrome P450 1B1 and NAD(P)H-quinone oxidoreductase (NQO1) (Harth et al. 2000, Brüning et al. 1999).
4.4.6 Prospects In many cases several mutation analyses must be carried out when investigating a gene. It is possible to investigate up to two mutations in a sample at the same time, as the light cycler has several wavelengths for determining different emissions. Theoretically, two very closely situated mutations can be detected with one detection probe. This results in further melting points for differentiating between the alleles, but these must be far enough apart for clear differentiation. With light cycler assisted real time PCR it is theoretically possible to investigate in parallel over 30 different mutation sites in several genes. This is particularly interesting for clinical use when as many investigations as possible must be carried out very quickly. For parallel investigations it 108
4.4 New High-throughput Technology in the Diagnostic Screening is, however, necessary that both the annealing temperatures of all primers and those of all probes are more or less the same, otherwise neither specific amplification nor specific detection and quantification of the amplification products can be carried out. This technology is not limited to the investigation of polymorphisms of xenobiotic-metabolizing enzymes, but can be used in the same way also for polymorphisms of other genes. It is therefore possible to carry out parallel determinations of polymorphisms of xenobiotic-metabolizing enzymes and the polymorphisms of other genetically determined susceptibility factors for certain diseases. Among these are the polymorphisms which lead to defects in DNA repair (e. g. with xeroderma pigmentosum) and other gene defects, as have been recognized, for example, for breast cancer or other diseases. In view of the rapid progress in development to be expected in this field, high-throughput methods will become more and more important for genotyping polymorphisms. References Ali-Osman, F.; Akande, O.; Antoun, G.; Jia-Xi, M.; Buolamwini, J.: Molecular cloning, characterization, and expression in Escherichia coli of full-length cDNAs of three human glutathione S-transferase Pi gene variants, JBC, Vol. 272, No 15 (1997). Bolt, H. M.: Genetic and individual differences in the process of biotransformation and their relevance for occupational medicine, Med. Lav., 85, 37–48 (1994). Brüning, T.; Abel, J.; Koch, B.; Lorenzen, K.; Harth, V.; Donat, S.; Sachinides, A.; Vetter, H.; Bolt, H. M.; Ko, Y.: Real time-PCR analysis of the cytochrome P450 1B1 codon 432-polymorphism, Arch. Toxicol., 73, 427–430 (1999). Harth, V.; Donat, S.; Ko, Y.; Abel, J.; Vetter, H.; Brüning, T.: NAD(P)H quinone oxidoreductase 1 codon 609 polymorphism and its association to colorectal cancer, Arch. Toxicol., 73, 528–531 (2000). Ko, Y.; Koch, B.; Harth, V.; Sachinidis, A.; Thier, R.; Vetter, H.; Bolt, H. M.; Brüning, T.: Rapid analysis of GSTM1, GSTT1 and GSTP1 polymorphisms using real time PCR, Pharmacogenetics, 10, 271–274 (2000). Wiebel, F. A.; Dommermuth, A.; Thier, R.: The hereditary transmission of the glutathione transferase hGSTT1-1 conjugator phenotype in a large family, Pharmacogenetics, 9, 252–256 (1999). Zbinden, G.: The three eras of research in experimental toxicology, TIPS, 13, 221–223 (1992). Zimniak, P.; Nanduri, B.; Pikula, S.; Bandorowicz-Pikula, J.; Singhal, S. S.; Srivastava, S. K.; Awasthi, S.; Awasthi, Y. C.: Naturally occurring human glutathione S-transferase GSTP1-1 isoforms with isoleucine and valine in position 104 differ in enzymic properties, Eur. J. Biochem. Sep. 15, 224, 893–899 (1994).
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5
Cytogenetic Parameters
5.1
Biological Monitoring with Cytogenetic Methods Günter Obe *, Helga Fender ** and Gisela Wolf ***
5.1.1 Introduction Chromosomal aberrations are structural changes in chromosomes, which can be analysed using the light microscope. The prerequisite for the formation of chromosomal aberrations is damage to the chromosomal DNA. A significant increase in the frequency of chromosomal aberrations therefore indicates that a causative agent is mutagenic. This is particularly evident with exposure to ionizing radiation (IAEA 1986). Also after exposure to diverse chemical substances an increase in chromosomal aberrations was detected in exposed groups of persons (Ashby & Richardson 1985, Natarajan & Obe 1980, Obe & Beek 1982, Obe & Natarajan 1993, 1996, Obe 1993, Obe & Müller 1999). Chromosomal aberrations are the effects of exposure; the term effect monitoring is therefore used (Wolf et al. 1997). A specific exposure can be deduced from the type of chromosomal aberrations only to a limited extent. All the types of chromosomal aberrations found in exposed persons also occur spontaneously. Increased frequencies of chromosomal aberrations in human lymphocytes correlate with an increased cancer risk (Bonassi et al. 1995, Hagmar et al. 1998 a, 1998 b). Human neoplasms are often characterized by typical chromosomal aberrations, which evidently led to the formation of the neoplasms (Mitelman et al. 1997). This close correlation between chromosomal aberrations and cancer makes the analysis of chromosomal aberrations in man particularly important. Other chromosomal changes used in effect monitoring are micronuclei and sister chromatid exchanges (SCE). Micronuclei occur when fragments of chromosomes or more rarely whole chromosomes do not enter the daughter nuclei during mitosis but are surrounded by their own nuclear membrane. An increase in the frequency of micronuclei therefore indicates *** Universität Essen, FB 9 Genetik, Universitätsstraße 5, 45117 Essen *** Robert Koch Institut, Nordufer 20, 13353 Berlin *** Berufsgenossenschaftliche Klinik für Berufskrankheiten Falkenstein/Vogtland, 08219 Falkenstein, Postfach 11 53 Biological Monitoring: Prospects in Occupational and Environmental Medicine. Deutsche Forschungsgemeinschaft (DFG) Copyright © 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-27795-7
5.1 Biological Monitoring with Cytogenetic Methods that exposure leads to chromosomal aberrations and/or spindle disorders (Fenech 1998, Müller & Streffer 1994). SCE are homologous exchanges between sister chromatids whose frequency is increased by mutagenic agents (Morris 1991, Sandberg 1982, Schubert 1990, Tice & Hollaender 1984, Tucker et al. 1993, Wolff 1982, Bruckmann et al. 1999 a, 1999 b).
5.1.2 Methodological Aspects The analysis of chromosomal aberrations in peripheral lymphocytes is a cytogenetic method used world-wide for human population monitoring. Over 90 % of lymphocytes are in a pre-synthetic phase of the cell cycle (G0). The cells are stimulated in vitro with a mitogen (usually phytohaemagglutinin), enter the cell cycle (G1, S, G2 phase) and divide mitotically (Natarajan & Obe 1982, Obe & Beek 1982). The mitotic process is blocked in a metaphase-like condition (C-metaphases, called metaphases below) with the spindle poison colchicine (usually the derivative colcemid) which after appropriate preparation allows all chromosomes to be easily recognized with the light microscope. Various types of chromosomal aberrations and micronuclei can be analysed after staining of the preparations, for example with Giemsa stain. Chromosomal aberrations which affect both chromatids of a metaphase chromosome at homologous sites are called aberrations of chromosome type; these include dicentric chromosomes, ring chromosomes and reciprocal translocations. If only one chromatid is affected by the aberration, this is an aberration of chromatid type, such as chromatid interchange or exchange and chromatid breaks. Acentric fragments can be both chromosome breaks (aberrations of chromosomal type) and isochromatid breaks (aberrations of chromatid type). Sister chromatid exchanges can be seen only if the DNA in the chromatids has been differentially substituted, for example with 5-bromodeoxyuridine (Bruckmann et al. 1999 a, 1999 b, Morris 1991). Translocations are made visible after fluorescence in situ hybridization (FISH); during this process DNA probes for individual chromosomes are labelled with fluorochromes and then hybridized together with these. Special FISH methods allow different fluorescent labelling of all chromosome pairs (mFISH) or banding of individual chromosomes (mBAND) (Johannes et al. 1999, Marshall & Obe 1998). FISH methods are complicated and expensive, and are only slowly becoming established in cytogenetic population monitoring (Littlefield et al. 1998, Straume & Bender 1997). The frequency of chromosomal aberrations gives only a realistic picture of the in vivo state when determined exclusively in the first in vitro mitosis. Cell divisions in vitro lead to a selection against aberrant mitoses 111
5 Cytogenetic Parameters and thus to a reduction in the frequency of chromosomal aberrations. First in vitro mitoses can be detected when the cells are cultured in the presence of the base analogue bromodeoxyuridine (B), which, if present in excess, is included in the replicating DNA instead of thymine (T). After the first S-phase in vitro in the presence of B, both chromatids of a chromosome are substituted with B in one DNA strand (TB-TB) and are stained uniformly dark with Giemsa stain. After two S-phases in the presence of B one chromatid has the constitution TB and appears dark after staining with Giemsa stain, the other chromatid has the constitution BB and appears light in colour. Metaphases with differentially stained chromosomes are not evaluated for chromosomal aberrations as they have already passed two Sphases in vitro, but SCE can be analysed in these cells. Chromosomes in differentially stained preparations can often not be evaluated as well as preparations block stained with Giemsa stain. To be able to combine the advantage of easier evaluation with an indication of the progression of the cell cycle, an additional culture can be incubated with B in order to how many second in vitro mitoses are present. After cultivation for 48 hours, there
Figure 1: Influence of the number of analysed metaphases per person (sample size) on the variance of the results. 250, 250 + 250 (500), 500 + 500 (1000) metaphases were evaluated consecutively. Given is the percentage of persons (Percentage of persons) with 0.0 to 8.0 percent aberrant metaphases (Percentage of aberrant cells). The data are based on cytogenetic analysis of the lymphocytes of 85 men (average age 68.3 ± 4.5 years). The frequency of aberrations found (columns) was compared with the theoretically expected normal distribution (lines). Only first in vitro metaphases were evaluated.
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x¯ ±SD Individual values
Figure 2: Intraindividual variability in the number of dicentric chromosomes and centric ring chromosomes in 4 to 6 samples of 1000 metaphases from lymphocytes from 4 control persons. The highest value for person 1 consists of 1 ring chromosome, the highest value for person 2 consists of a rogue cell with 2 tricentric chromosomes, which were counted as 4 dicentric chromosomes. Only first in vitro metaphases were evaluated.
are usually less than 5 % second metaphases, which barely affect the results of aberration analysis. The quality of the data collected depends in particular on the number of mitoses evaluated. The influence of the size of the sample is shown in Figure 1. The variance of the data increases with decreasing sample size, that is, for samples containing 1000, 500 and 250 evaluated metaphases it decreases in that order. In addition, the intraindividual variability of the frequency of chromosomal aberrations is a factor not to be neglected. Figure 2 shows the fluctuation ranges of the frequencies of dicentric chromosomes and centric rings after analysis of 4 to 6 samples each of 1000 metaphases from lymphocyte cultures from 4 persons. Table 1 shows that not only the frequencies of dicentric chromosomes and centric rings fluctuate with the number of evaluated metaphases, but also the other types of chromosomal aberrations. Even significant differences can occur, as shown in Table 1 for the percentage of aberrant cells. Table 2 shows the frequency of chromosomal aberrations in lymphocytes from women exposed to perchloroethylene who worked in dry-cleaning premises (Fender 1993) and from workers from waste disposal sites (Fender & Wolf 1998) with the corresponding groups of control persons. In both of the exposed groups the frequency of chromosomal aberrations was found to be significantly increased. In the waste disposal site workers and the corresponding controls multi-aberrant cells were found, which contained more than 2 dicentric chro113
5 Cytogenetic Parameters Table 1: Frequencies of chromosomal aberrations in two groups of control persons after analysis of 1000 or 500 metaphases per person (Sample size). The values are given with their standard errors (± SE). Only first in vitro metaphases were evaluated. The values in the shaded field are significantly different (p ≤ 0.05 two-sided, U test according to Mann and Whitney).
Number of persons investigated Age Total number of metaphases analysed
Sample size 1000 cells
Sample size 500 cells
99 39.6 ± 9.8 (17–58) 99000
83 36.6 ± 8.6 (20–56) 41500
Number of aberrations/ Number of aberrations/ person person Mean value ± SE Mean value ± SE (mean value per 1000 cells) Chromatid breaks
642 6.5 ± 0.5
244 3.0 ± 0.4 (5.9)
Chromatid exchanges
58 0.6 ± 0.1
5 0.06 ± 0.02 (0.1)
Dicentric chromosomes and centric rings
119 1.2 ± 0.1 (5 rings)
23 0.3 ± 0.1 (0.6) (1 ring)
Additional acentric fragments
207 2.1 ± 0.3
98 1.2 ± 0.2 (2.4)
Multi-aberrant metaphases (cells with >2 dicentric chromosomes)
5 0.05 ± 0.03
3 0.04 ± 0.02 (0.07)
Metaphases with aberrations of chromatid type
665 6.7 ± 0.5
233 2.8 ± 0.4 (5.6)
Metaphases with aberrations of chromosome type
391 4.0 ± 0.3
126 1.5 ± 0.2 (3.0)
% of aberrant metaphases
1.1 ± 0.1
0.9 ± 0.1
114
5.1 Biological Monitoring with Cytogenetic Methods mosomes or several aberrations of chromatid type. Such multi-aberrant cells (rogue cells) are regularly found in very low frequencies; how they are formed is unclear (Ahuja & Obe 1994, Wolf et al. 1999). Rogue cells should be listed separately, otherwise the values for aberrations can be too high (see also person 2 in Figure 2), and may not result from the exposure under investigation. Table 3 shows an example of SCE frequencies in waste disposal site workers and controls. Although significant increases in the frequency of chromosomal aberrations were observed in the waste disposal site workers (Table 2), the SCE frequency was not increased. In the controls, however, smoking was found to have a significant influence on the SCE frequency. Smoking and the consumption of alcohol have been shown to lead to chromosomal damage and are regarded as influencing factors for cytogenetic population monitoring (Obe et al. 1982, Obe & Anderson 1987). Another parameter used in cytogenetic population monitoring are micronuclei, which occur as a result of chromosomal aberrations or spindle disorders after exposure to mutagenic substances. Micronuclei develop when during mitotic anaphase fragments or whole chromosomes are not moved to the cell poles but become surrounded by their own nuclear membrane. Micronuclei are analysed usually in binucleate cells, which in the presence of cytochalasin B are formed by suppression of cells, but not of nuclear division (Fenech 1998, Müller & Streffer 1994).
5.1.3 Standardization and Quality Control The standardization of human population monitoring has not yet been sufficiently achieved despite the efforts of national and international bodies over many years. It is, however, the prerequisite for comparable results. Standardization must not, however, be limited to sample preparation and analysis, but must include the whole study design (Hook 1982, IAEA 1986, Carrano & Natarajan 1988, Speit et al. 1994). Cytogenetic human population monitoring studies should be planned according to epidemiological criteria and carried out taking certain quality criteria into consideration. Important influencing factors for the mitotic activity of lymphocytes in the blood culture are the composition of the culture medium and the mitogen used (usually phytohaemagglutinin). An important prerequisite for reliable data is the analysis of first in vitro mitosis (M1) for chromosomal aberrations, of second in vitro mitosis (M2) for SCE and of binucleate cells for micronuclei. The statistical reference should be the person investigated, not the cells analyzed. The number of analysed cells should be 1000 M1 cells for chromosomal aberrations, 50 M2 cells for SCE and 1000 binucleate cells for micronuclei. Imperative is the simultaneous analysis of control persons, who must match the exposed group of persons as closely as possible, 115
5 Cytogenetic Parameters Table 2: Cytogenetic population monitoring after exposure to chemicals. The values are given with their standard errors (± SE). Only first in vitro metaphases (M) were evaluated. The values in the shaded fields are significantly increased relative to those for the corresponding control groups (p ≤ 0.05 two-sided, U test according to Mann and Whitney). Data from Fender (1993) and Fender and Wolf (1998). Group
Sex/number
Age in years
Exposure Duration in years
Current con- Mean total concentracentration a tion b [mg/m3] [mg/m3]
Employees in f/9 dry-cleaning premises (perchloro-ethylene)
47.9 ± 5.5 (40–56)
16.4 ± 3.9 (8.6–20.5)
257 ± 62.8 (144–348)
4842 ± 2282 (1909–8277)
Controls
f/9
40.0 ± 16.5 (21–56)
–
–
–
Waste disposal site workers
m/75 f/7
36.6 ± 10.1 18–59
5.1 ± 3.5 0.5–13
Controls
m/65 f/6
39.6 ± 9.1 21–59
–
a
b c d e f g h
Geometric mean from about 10 average personal shift concentrations at the workplace. Estimation of the total occupational exposure. With one exception, 1500 metaphases per person were evaluated. With one exception, 1000 metaphases per person were evaluated. With two exceptions, 1000 metaphases per person were evaluated. Including rogue cells. Excluding rogue cells. Aberrations of chromatid type.
116
5.1 Biological Monitoring with Cytogenetic Methods
Chromosome analysis Number of analysed metaphases
Dicentric Additional Multi-aberMetaphases with chromoacentric rant metaaberrations of somes and fragments phases centric rings (rogue chromochromatid cells) some type type
13 100 c
34 3.8 ± 0.7 per person
60 0 6.7 ± 1.2 per person
97 10.8 ± 1.2
101 11.2 ± 1.7
2.6 per 1000 M
4.6 per 1000 M
7.4 per 1000 M
7.7 per 1000 M
13 500 c
3 0.3 ± 0.1 0.2 per 1000 M
18 0 2.0 ± 0.8 1.4 per 1000 M
66 7.3 ± 0.9 4.9 per 1000 M
22 2.4 ± 1.0 0.7 ± 0.1 1.6 per 1000 M
81 830 d
338 f (3 rings) 4.1 ± 0.2
573 f 6.9 ± 1.0
603 7.3 ± 0.5
517 6.2 ± 0.5
1.4 ± 0.7
174 g 2.1 ± 0.2
302 g 3.6 ± 0.4
31 (4 rings) 0.4 ± 0.1 of these 7 M only with ctb h
106 f 1.5 ± 0.2 103 g 1.5 ± 0.2
235 f 3.3 ± 0.6 210 g 2.9 ± 0.5
11 0.2 ± 0.1 of these 8 M only with ctb h
355 5.0 ± 0.4
308 4.3 ± 0.4
0.9 ± 0.6
70 420 e
% of aberrant metaphases
1.5 ± 0.2
117
5 Cytogenetic Parameters Table 3: SCE per metaphase in lymphocytes of waste disposal site workers and control persons and the influence of cigarette smoking. The values for smokers and non-smokers are significantly different in the controls, but not in the waste disposal site workers (p < 0.01, t-test). 50 metaphases were evaluated (in 2 control persons 40 metaphases). The values are given with their standard deviations (± SD) (Fender and Wolf 1998). Waste disposal site workers
Controls
All persons
Smokers
Nonsmokers
All persons
Smokers
Nonsmokers
Number of persons investigated
54
31
23
50
26
24
Number of metaphases analysed
2700
1550
1150
2480
1290
1190
SCE/cell (mean value ± SD)
6.1 ± 1.4
6.2 ± 1.5
6.0 ± 1.2
6.2 ± 1.4
6.7 ± 1.5
5.7 ± 0.9
apart from the exposure. Historical controls are not suitable (Wolf 1993, Wolf et al. 1996, 1997, 1999). Wolf et al. (1997) discuss in detail standardization and quality management in cytogenetic population monitoring. References Ahuja, Y. R.; Obe, G.: Are rogue cells an indicator of cancer risk due to the action of bacterial restriction endonucleases? Mutation Res., 310, 103–112 (1994). Ashby, J.; Richardson, C. R.: Tabulation and assessment of 113 human surveillance cytogenetic studies conducted between 1965 and 1984, Mutation Res., 154, 111–133 (1985). Bonassi, S.; Abbondandolo, A.; Camurri, L.; Dal, L.; Pra, A.; De Ferrari, M.; Degrassi, F.; Forni, A.; Lamberti, L.; Lando, C.; Padovani, P.; Sbrana, I.; Veccio, D.; Putoni, R.: Are chromosome aberrations in circulating lymphocytes predictive for a future cancer onset in humans? Preliminary results of an Italian cohort study, Cancer Genet. Cytogenet., 79, 133–135 (l995). Bruckmann, E, Wojcik, A, Obe, G.: Sister chromatid differentiation with biotin-dUTP, Chromosome Res., 7, 185–189 (1999 a). Bruckmann, E.; Wojcik, A.; Obe, G.: X-irradiation of G1 CHO cells induces SCE which are both true and false in BrdU-substituted cells but only false in biotin-dUTP-substituted cells, Chrom. Res., 7, 277–288 (1999 b). Carrano, A. V.; Natarajan, A. T.: Considerations for population monitoring using cytogenetic techniques (ICPEMC Publication No. 14), Mutation Res., 204, 379–406 (1988). Fender, H.: Chromosomenanalystiche Untersuchungen bei Textilreinigern: Vergleich von Literaturdaten und eigenen Untersuchungen unter besonderer Berücksichtigung methodischer Aspekte, bga-Schriften, 3/1993, 71–76 (1993). Fender, H.; Wolf, G.: Cytogenetic investigations in employees from waste disposal sites, Toxicology Letters, 96/97, 149–154 (1998).
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5.1 Biological Monitoring with Cytogenetic Methods Fenech, M.: Important variables that influence base-line micronucleus frequency in cytokinesis-blocked lymphocytes – a biomarker for DNA damage in human populations, Mutation Res., 404, 155–165 (1998). Hagmar, L.; Bonassi, S.; Strömberg, U.; Brogger, A.; Knudsen, L. E.; Norppa, H.; Reuterwall, C.: Chromosomal aberrations in lymphocytes predict human cancer: a report from the European Study Group on Cytogenetic Biomarkers and Health (ESCH), Cancer Res., 58, 4117–4121 (1998 a). Hagmar, L.; Bonassi, S.; Strömberg, U.; Mikoczy, Z.; Lando, C.; Hansteen, I.-L.; Huici Montagud, A.; Knudsen, L.; Norppa, H.; Reuterwall, C.; Tinnerberg, H.; Brøgger, A.; Forni, A.; Högstedt, B.; Lambert, B.; Mitelman, F.; Nordenson, I.; Salmaa, S.; Skerfving, S.: Cancer predictive value of cytogenetic markers used in occupational health surveillance programs: A report from an ongoing study by the European Study Group on Cytogenetic Biomarkers and Health (ESCH), Mutation Res.; 405, 171–178 (1998b). Hook, E. B.: Perspectives in mutation epidemiology 2. Epidemiologic and design aspects of studies of chromosome breakage and sister-chromatid exchange (ICPEMC Working Paper 5/2), Mutation Res., 99, 373–382 (1982). IAEA (International Atomic Energy Agency) Technical Reports Series No. 260 Biological Dosimetry: Chromosomal Aberration Analysis for Dose Assessment. International Atomic Energy Agency, Vienna (1986). Johannes, C.; Chudoba, I.; Obe, G.: Analysis of X-ray induced aberrations in human chromosome 5 using high-resolution multicolour banding FISH (mBAND), Chromosome Res., 7, 625–633 (1999). Littlefield, L. G.; McFee, A. F.; Salomaa, S. I.; Tucker, J. D.; Inskip, P. D.; Sayer, A. M.; Lindholm, C.; Makinen, S.; Mustonen, R.; Sorensen, K.; Tekkel, M.; Veidebaum, T.; Auvinen, A.; Boice, J. D. Jr.: Do recorded doses overestimate true doses received by Chernobyl cleanup workers? Results of cytogenetic analyses of Estonian workers by fluorescence in situ hybridization, Radiation Res., 150, 237–249 (1998). Marshall, R.; Obe, G.: Application of chromosome painting to clastogenicity testing in vitro, Env. Molec. Mutagen., 32, 212–222 (1998). Mitelman, F.; Mertens, F.; Johannson, B.: A break point map of recurrent chromosomal rearrangements in human neoplasia, Nature Genet., 15, 417–474 (1997). Morris, S. M.: The genetic toxicology of 5-bromodeoxyuridine in mammalian cells, Mutation Res., 258, 161–188 (1991). Müller, W.-U.; Streffer, C.: Micronucleus Assays, in: Obe, G. (ed.): Advances in Mutagenesis Research, Vol. 5, Springer-Verlag, Berlin, pp. 4–134 (1994). Natarajan, A. T.; Obe, G.: Screening of human populations for mutations induced by environmental pollutants: Use of the human lymphocyte system, Ecotox.; Env. Safety, 4, 468–481 (1980). Natarajan, A. T.; Obe, G.: Mutagenicity testing with cultured mammalian cells: Cytogenetic assays. In: Heddle, J. A. (ed.) Mutagenicity: New Horizons in Genetic Toxicology, Academic Press, New York, pp. 171–213 (1982). Obe, G.: Chromosomenaberrationen in menschlichen peripheren Lymphozyten und ihre Bedeutung für das zytogenetische Populationsmonitoring, in: Arndt, D.; Obe, G. (eds.): Zytogenetische Methoden. BGA Schriften, 3/39, MMV Medizin Verlag, München (1993). Obe, G.; Anderson, D.: Genetic effects of ethanol, Mutation Res., 186, 177–200 (1987). Obe, G.; Beek, B.: The human leukocyte test system, in: de Serres, F. J.; Hollaender, A. (eds.): Chemical mutagens, principles and methods for their detection, Vol. 7, Plenum Press, New York, pp. 337–400 (1982). Obe, G.; Müller, W.-U.: Zytogenetik in der genetischen Toxikologie und Strahlenbiologie, Medizinische Gen., 3, 373–377 (1999).
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5 Cytogenetic Parameters Obe, G.; Natarajan, A. T.: Mutagenicity tests with cultured mammalian cells: Cytogenetic assays, in: Corn, M. (ed.): Handbook of Hazardous Materials, Academic Press, New York, pp. 453–461 (1993). Obe, G.; Natarajan, A. T.: Zytogenetische Methoden, in: Wichmann, H. E.; Schlipköter, H.-W.; Fülgraff, G. (Hrsg.): Handbuch der Umweltmedizin 4/96, Ecomed, pp. 1–15 (1996). Obe, G.; Vogt, H.-J.; Madle, S.; Fahning, A.; Heller, W.-D.: Double-blind study on the effect of cigarette smoking on the chromosomes of human peripheral blood lymphocytes in vivo, Mutation Res., 92, 309–319 (1982). Sandberg, A. A.: Sister Chromatid Exchange, Alan R. Liss, Inc., New York (1982). Schubert, I.: Sister chromatid exchanges and chromatid aberrations: a comparison, Biol. Zentbl., 109, 7–18 (1990). Speit, G.; Bauchinger, M.; Schmidt, E.; Gebhard, E.; Hüttner, E.; Obe, G.: Zytogenetische Analysen an menschlichen Populationen (Human Population Monitoring: HPM), Bundesgesundheitsblatt, 3, 118–119 (1994). Straume, T.; Bender, M. A.: Issues in cytogenetic biological dosimetry: emphasis on radiation environments in space, Radiation Res., 148, 60–70 (1997). Tice, R. R.; Hollaender, A. (eds.): Sister Chromatid Exchanges. 25 Years of Experimental Research. Part A, The Nature of SCEs. Part B, Genetic Toxicology and Human Studies, Plenum Press, New York (1984). Tucker, J. D.; Aulette, A.; Cimino, M. C.; Dearfield, K. L.; Jacobson-Kram, D.; Tice, R. R.; Carrano, A. V.: Sister-chromatid exchange: second report of the gene-tox program. Mutation Res., 297, 101–180 (1993). Wolf, G.: Chromosomenanalytische Untersuchungen nach chemischer Exposition: Methodische Aspekte: Eine kritische Bewertung von Literaturdaten, in: Arndt, D.; Obe, G. (eds.): Zytogenetische Methoden. BGA-Schriften, 3/93, MMV Medizin Verlag, München, 58–64 (1993). Wolf, G.; Fender, H.; Obe, G.: Standardisierung und Validierung zytogenetischer HPMStudien, in: Arndt, D.; Obe, G. (eds.): Methodische Fragen beim Human Population Monitoring in der Zytogenetik, RKI-Schriften, 1/96, MMV Medizin Verlag, München, 8–11 (1996). Wolf, G.; Obe, G.; Fender, H.: Standardisierung und Qualitätsmanagement im zytogenetischen Populationsmonitoring, in: Arndt, D.; Obe, G. (eds.): Qualitätssicherung in der Zyto- und Molekulargenetik, RKI-Schriften, MMV Medizin Verlag, München, 19–24 (1997). Wolf, G.; Pieper, R.; Obe, G.: Chromosomal alterations in peripheral lymphocytes of female cabin attendants, Int. J. Radiat. Biol., 75, 829–836 (1999). Wolff, S. (ed.): Sister chromatid exchange, John Wiley and Sons, New York (1982).
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5.2
Examples of the Use of Three-colour Chromosome Painting in Cytogenetic Biomonitoring Erich Gebhart *, Irmgard Verdorfer * and Susann Neubauer **
5.2.1 Introduction Up to a few years ago, only classical cytogenetic technique was available for evaluating chromosomal aberrations in population groups exposed to mutagenic substances. Basic rules for evaluating the damage and interpreting it were proposed and applied (Speit et al. 1994). A decisive disadvantage of these classic techniques was, however, the unreliable detection of those types of aberrations which, as a result of their longevity and practical-clinical importance in man, are of particular interest, namely reciprocal translocations. In the cytogenetic detection of radiation it was therefore decided to concentrate e. g. on the selective evaluation of the easily detectable dicentric chromosomes and ring chromosomes. With the introduction of chromosome in situ suppression (CISS) hybridization (Lichter et al. 1988, Cremer et al. 1990) – nowadays also called chromosome painting in the international literature – important progress was made. With this technique it is not only possible to reliably detect chromosomal rearrangements (Lucas et al. 1992, Tucker et al. 1993) and document their lifespan (Gebhart et al. 1996, Matsumoto et al. 1998), but also the complex rearrangements (Neubauer et al. 1997), which were previously hardly detectable and occur in unexpectedly large amounts, can be detected. As a result of the simple and rapid evaluation, the selective hybridization of selected chromosomes has proved to be particularly practicable. Today, however, it is possible to carry out complete analysis of the whole genome using multicolour FISH (Speicher et al 1996) or spectral karyotyping (Schröck et al. 1996). Both techniques, however, are considerably more time-consuming and require more materials than the three-colour painting technique described here.
** Institut für Humangenetik der Universität Erlangen-Nürnberg, Schwabachanlage 10, 91054 Erlangen ** Klinik für Strahlentherapie der Universität Erlangen-Nürnberg
121
5 Cytogenetic Parameters
5.2.2 Method Metaphases from 48-hour cultures of transformed lymphocytes from the peripheral blood of the test persons are prepared in accordance with the standard techniques. The metaphase chromosomes are then hybridized using the CISS technique (Lichter et al. 1988) with chromosome-specific DNA libraries labelled with fluorescent substances. The use of libraries of the chromosomes 1, 2 and 4 and a three-colour technique has proved very reliable (Gebhart et al. 1996). Each of the three “labelled” chromosome pairs then appears in the fluorescence microscope (given suitable filter combinations) in its own colour (red, green, yellow), and this allows the rapid and reliable microscopic detection of exchanged pieces of chromosomes. Additional counterstaining of all chromosomes with DAPI allows – after changing the filter – reliable recognition of centromere regions (e. g. in complex rearrangements) and also confirmation of dicentric chromosomes. Depending on the target group and aim of the investigation, per person 1000 to 3000 mitoses with clear chromosome labelling are analysed with the microscope and the observed aberrations are individually documented. All recognizable types of aberration are recorded, e. g. even fragments which have no recognizable relationship with rearrangements. The number of breaks per 1000 mitoses is, in our experience, suitable for group comparison. It is determined as the minimum number of breaks necessary for the formation of the observed aberrations. We do not use the correction factors occasionally suggested in the literature to increase the determined aberration rate to the level for the whole genome. It can be assumed with caution that the technique used in our studies detects a little over a third of the total aberrations in the corresponding genome. Sufficiently extensive data from multi-colour painting analysis for exact comparison are not yet available. Only such data will provide a reliable basis for comparison. Painting analyses with various groups of persons are described below; the four groups of persons so far investigated by us are represented in Table 1. In addition, data from studies with 15 patients with chromosome instability syndromes (12 ataxia teleangiectasia and 3 Nijmegen breakage syndrome) and 17 heterozygote carriers of these mutations were used for comparison as positive controls. These data are heterogenic in that some of them were obtained from lymphoblastoid cell lines.
122
5.2 Examples of the Use of Three-colour Chromosome Painting Table 1: Composition of the four groups of persons investigated (for more details see Verdorfer et al. 2001). Group
Number of persons
Number of analysed metaphases
Distribution of the sexes
Age (years) range/mean
Comments
Controls
32
71 000
7m/25 f
24–57 38
Students, office and laboratory personnel
Tumour patients
51
75 000
11m/40 f
33–74 58
37 mamma carcinomas 10 tumours of the head and neck 4 other carcinomas
Personnel from 42 medical departments which use radiation
91 000
19m/23 f
25–61 38
30 radiology 8 nuclear medicine 4 radiation physics
TNT workers
19 000
11m/8 f
32–57 52
–
19
5.2.3 The Results of Our Own Studies Comparison of the individual results shown in Figure 1 documents a high interindividual variability in the observed incidence of breaks in each of the investigated groups of persons. This observation is in accordance with earlier results both with conventional cytogenetic analysis (Gebhart et al. 1980) and also painting analyses in tumour patients who were exposed to radiation (Gebhart et al. 1996). In the control group, the total number of breaks per 1000 mitoses exceeded the upper limit of the standard deviation in some cases (Figure 1, group 1). Intensive questioning revealed that two of these persons were exposed to increased levels of radiation (four X-rays taken in the period before the investigation), and in another female person increased chromosome instability was found to be the probable cause. Around two thirds of the breaks detected in this group were the result of stable translocations (Figure 2). In a group of tumour patients much higher average incidences of breaks were found; the spontaneous incidence of aberrations in the lymphocytes did not depend on the type of tumour (Figure 1, group 2). The relatively high number of breaks that caused stable rearrangements (over 123
5 Cytogenetic Parameters
Figure 1: Total number of breaks per 1000 metaphases in the groups of persons investigated (each circle represents the incidence of breaks in a person). 1: control group; 2: tumour patients (dark symbols = mamma carcinoma; light symbols = tumours of the head and neck and other carcinomas); 3: personnel from medical departments that use radiation; 4: persons exposed to nitroaromatics; 5*: 12 ataxia teleangiectasia (AT) and 3 Nijmegen breakage syndrome (NBS) patients for comparison; (3 indicates data from lymphoblastoid cell lines); 6*: 11 AT heterozygote and 6 NBS heterozygote persons for comparison. * As a result of their heterogenic composition (various syndromes, different target cells) groups 5 and 6 are not to be regarded as “groups” in the sense of groups 1–4. In addition to the individual values of the various groups, the mean values (^) and standard deviations (I) of each groups are given.
124
5.2 Examples of the Use of Three-colour Chromosome Painting
Figure 2: The number of breaks per 1000 metaphases involved in translocations in the groups of persons investigated (explanations as in Figure 1).
80 %, Figure 2) indicates that the causal factors were active mainly some time ago. The high variability of the results in this group is noteworthy. This evidently reflects the variety of mechanisms in these patients which cause aberrations. The total incidence of breaks in ten of these persons exceeded the upper limit of the standard deviation for this group. The results for the group of personnel from medical departments which use radiation (Figure 1, group 3) were almost identical to those of the control group. Only one individual value was markedly increased: this was from a 61-year-old radiation physicist with a high lifetime dose. Three other values were only slightly above the upper limit of the standard devia125
5 Cytogenetic Parameters tion. 75 % of breaks were causally involved in the formation of stable rearrangements (Figure 2, group 3). The data for group 4 are taken from an occupational-medical study of persons exposed to increased levels of nitroaromatics while destroying military waste. In addition to the increased average value for the number of breaks per 1000 mitoses relative to that for the control group, in particular the high number of instable aberrations – indicating short-term exposure – was conspicuous. As comparison of the values in Figure 1 and Figure 2 reveals, in this group only about 57 % of the breaks were involved in stable rearrangements. This is the lowest percentage in all four groups. The supplementary data for patients with hereditary chromosome instability (group 5* in both Figures) and for heterozygote carriers of the corresponding mutations (group 6*) are, in view of the relatively small number of individuals and the heterogeneity of the material investigated (large amount of data from lymphoblastoid cell lines), merely listed for comparison. On the one hand, the very high incidence of spontaneous aberrations – in particular in the peripheral lymphocytes – in the patients is clear, on the other hand, the relatively low percentage of breaks involved in stable rearrangements (58 %) shows that in these patients their innate chromosome instability leads to the continuous formation of new, also short-lived, instable aberrations. The frequency of breaks in heterozygote carriers of the mutation is, on average, above that in controls (Neubauer et al. 2002). We also focused in particular on the complex rearrangements; these are aberrations which are formed when at least three or more breaks occur in at least two or more chromosomes. Such complex aberrations are found also in lymphocytes of control persons (only the persons of this group exceeded the upper limit value we set of x+2s, for which exposure to mutagenic substances could be determined). In the group of persons who used radiation for medical purposes 12 % were found to have incidences of breaks from complex aberrations above the threshold limit value, in the group of tumour patients this was 19 % and in the group of persons exposed to nitroaromatics 31.5 %. Also this type of aberration could therefore not contribute towards a clear differentiation of the groups investigated. Finally we would like to note that our first comparative studies using the multi-colour (= 24-colour) technique show that it is possible to use this method also for population monitoring. The time and materials needed for this technique, however, do not make it a clear winner over the described three-colour painting method as a screening method for biological monitoring, as is sometimes propagated. For the mitoses that can actually be detected it does, however, produce more reliable results.
126
5.2 Examples of the Use of Three-colour Chromosome Painting
5.2.4 Conclusions The examples of applications of chromosome painting for population monitoring described here illustrate the possibilities and limitations of this technique. The ability to detect reciprocal translocations is one of the main strengths of such investigations, not only because this type of aberration is of high practical importance in particular in tumour genetics, and also in clinical genetics, but because these stable aberrations also have a very long lifespan and are therefore informative indicators of exposure to mutagenic substances that took place some time ago (Lucas et al. 1992, Gebhart et al. 1996). As was shown with various groups of persons, this technique is also very suitable for discovering particularly marked individual exposures to mutagenic substances. For its systematic use in biological monitoring, however, more experience is needed, as truely comparable biomonitoring studies with three differently labelled chromosomes are lacking. Nevertheless, important insights can be gained from the data already available. If the persons in the control group with known exposure to mutagenic substances are excluded, the average frequency of breaks is 0.011 + 0.005. If the upper limit is set as the average value plus twice the standard deviation, that is 0.021 for control persons not exposed, the following can be said about the other groups of persons investigated. The frequency of breaks exceeded this threshold limit value in 23 of the 51 tumour patients (= 45 %), in 4 of the 42 persons from medical departments which use radiation (= 9.5 %) and in 13 of the 19 persons exposed to nitroaromatics (= 68 %). The incidence of breaks in all patients with hereditary chromosome instability from whom blood was available for investigation was highly above this threshold limit value. In the heterozygote carriers of these mutations it was five of 17 values. What practical consequences this has for the estimation of workplace risks and for the health of the affected individual must be discussed on the one hand from the point of view of population monitoring, as mentioned above (Speit et al. 1994), and on the other must await a fundamental increase in our experience of these new techniques. In the literature truely comparable studies are lacking. A Czech research group were able to differentiate between a group of medical personnel and control persons using a two-colour FISH technique with samples for chromosomes 2 and 4 (Rubes et al. 1998). Most of the available studies of other research groups which used chromosome painting as a technique of biological monitoring or biodosimetry investigated persons exposed to radiation (Lucas et al. 1992, Lucas 1997) and/or used only one label for the selected chromosomes (Tucker et al. 1993, Finnon et al 1995, Salassidis et al. 1995, 1998, Scarpato et al. 1997). Even if most of these studies are based on the same selection of chromosomes as in our study, namely 1, 2 and 4, there are numerous investigations with other chromosome combinations (e. g. Natarajan et al. 1992, Johnson et al. 1998, Pressl & Stephan 1998). This means that quite a large number of comparable systematic population monitoring studies are to date not yet available. 127
5 Cytogenetic Parameters Chromosomal aberrations have for a long time been regarded as valuable indicators of exposure to mutagenic substances in man in the sense of population monitoring; they are a way of detecting potentially harmful exposures and their biological effects in man. As their variability reflects the individuality of biological reactions in man – known to every human geneticist – in a very realistic way, they are without a doubt a suitable warning system. This is, of course, all the more valid when as relevant an aberration spectrum as possible can be covered, as the chromosome painting techniques allow. Its inclusion, however, in the comprehensive screening of exposed groups of persons still requires considerably more experience, which can only be achieved by including it in co-operative research programmes. In this context, in particular also the validity and practicability of the 24-colour painting technique should be examined more closely. What all cytogenetic methods cannot achieve in the same way as biochemical techniques, as a result of the variability inherent in the system mentioned above, is the biological monitoring of relevant occupational-medical exposures in the sense of an exact quantification of the effective dose. References Cremer, T.; Popp, S.; Emmerich, P.; Lichter, P.; Cremer, C.: Rapid metaphase and interphase detection of radiation-induced chromosome aberrations in human lymphocytes by chromosomal suppression in situ hybridization, Cytometry, 11, 110–118 (1990). Finnon, P.; Lloyd, D. C.; Edwards, A. A.: Fluorescence in situ hybridization detection of chromosomal aberrations in human lymphocytes: applicability to biological dosimetry, Int. J. Radiat. Biol., 68, 429–435 (1995). Gebhart, E.; Lösing, J.; Wopfner, F.: Chromosome studies on lymphocytes of patients under cytostatic therapy. I. Conventional chromosome studies in cytostatic interval therapy, Hum. Genet., 55, 53–63 (1980). Gebhart, E.; Neubauer, S.; Schmitt, G.; Birkenhake, S.; Dunst, J.: Use of a three-colour chromosome-in-situ-suppression (CISS) technique for the cytogenetic detection of past radiation exposure, Radiat. Res., 145, 47–52 (1996). Johnson, K. L.; Tucker, J. D.; Nath, J.: Frequency, distribution and clonality of chromosome damage in human lymphocytes by multi-color FISH, Mutagenesis, 13, 217–227 (1998). Lichter, P.; Cremer, T.; Borden, J.; Manuelidis, L.; Ward, D. C.: Delineation of individual chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries, Hum. Genet., 80, 224–234 (1988). Lucas, J. N.: Dose reconstruction for individuals exposed to ionizing radiation using chromosome painting, Radiat. Res., 148, S33–S38 (1997). Lucas, J. N.; Awa, A.; Straume, T.; Poggensee, M.; Kodama, Y.; Nakano, M.; Ohtaki, K.; Weier, H. U.; Pinkel, D.; Gray, J.; Littlefield, G.: Rapid translocation frequency analysis in humans decades after exposure to ionizing radiation, Int. J. Radiat. Biol., 62, 53–63 (1992). Matsumoto, K.; Ramsey, M. J.; Nelson, D. O.; Tucker, J. D.: Persistence of radiation-induced translocations in human peripheral blood determined by chromosome painting, Radiat. Res., 149, 602–613 (1998). Natarajan, A. T.; Vyas, R. C.; Darroudi, F.; Vermeulen, S.: Frequencies of X-ray-induced chromosome translocations in human peripheral lymphocytes as detected by in situ hybridization using chromosome-specific DNA libraries, Int. J. Radiat. Res., 61, 199– 203 (1992).
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5.2 Examples of the Use of Three-colour Chromosome Painting Neubauer, S.; Dunst, J.; Gebhart, E.: The impact of complex chromosomal rearrangements on the detection of radiosensitivity in cancer patients, Radiother. Oncol., 43, 189–195 (1997). Neubauer, S.; Arutyunyan, R.; Stumm, M.; Dörk, T.; Bendix, R.; Bremer, M.; Varon, R.; Sauer, R.; Gebhart, E.: Radiosensitivity of Ataxia Telangiectasia and Nijmegen Breakage syndrome homozygotes and heterozygotes as determined by three-color-FISH chromosome painting. Radiat. Res., 157, 312–321 (2002) Pressl, S.; Stephan, G.: Chromosome translocations detected by fluorescence in situ hybridization (FISH) – a useful tool in population monitoring? Toxicol. Lett., 96/97, 189– 194 (1998). Rubes, J.; Kucharova, S.; Vozdova, M.; Musilova, P.; Zudova, Z.: Cytogenetic analysis of peripheral lymphocytes in medical personnel by means of FISH, Mutat. Res., 412, 293–298 (1998). Salassidis, K.; Braselmann, H.; Okladnikova, N. D.; Pressl, S.; Stephan, G.; Snigiryova, G.; Bauchinger, M.: Analysis of symmetrical translocations for retrospective biodosimetry in radiation workers of the Mayak nuclear-industrial complex (Southern Urals) using FISH-chromosome painting, Int. J. Radiat. Biol., 74, 431–439 (1998). Salassidis, K.; Georgiadou-Schumacher, V.; Braselmann, H.; Müller, P.; Peter, R. U.; Bauchinger, M.: Chromosome painting in highly irradiated Chernobyl victims: a follow-up study to evaluate the stability of symmetrical translocations and the influence of clonal aberrations for retrospective dose estimation, Int. J. Radiat. Biol., 68, 257– 262 (1995). Scarpato, R.; Lori, A.; Panasiul, G.; Barale, R.: FISH analysis of translocations in lymphocytes of children exposed to Chernobyl fallout: preferential involvement of chromosome 10, Cytogenet. Cell Genet., 79, 153–156 (1997). Schröck, E.; du Manoir, S.; Veldman, T.; Schoell, B.; Wienberg, J.; Ferguson-Smith, M. A.; Ning, Y.; Ledbetter, D. H.; Bar-Am, I.; Soenksen, D.; Garini, Y.; Ried, T.: Multicolor spectral karyotyping of human chromosomes, Science, 273, 494–497 (1996). Speicher, M. R.; Ballard, S. G.; Ward, D. E.: Karyotyping human chromosomes by combinatorial multi-fluor FISH, Nature Genet., 12, 368–375 (1996). Speit, G.; Bauchinger, M.; Schmid, E.; Gebhart, E.; Hüttner, E.; Obe, G.: Zytogenetische Analysen an menschlichen Populationen (Human Population Monitoring: HPM), Bundesgesundheitsblatt, 3/94, 118–119 (1994). Tucker, J. D.; Ramsay, M. J.; Lee, D. A.; Minkler, J. L.: Validation of chromosome painting as a biodosimeter in human peripheral lymphocytes following acute exposure to ionizing radiation in vitro, Int. J. Radiat. Biol., 64, 27–37 (1993). Verdorfer, I.; Neubauer, S.; Letzel, S.; Angerer, J.; Arutyunyan, R.; Martus, P.; Wucherer, M.; Gebhart, E.: Chromosome painting for cytogenetic monitoring of occupationally exposed and non-exposed groups of human individuals. Mutat. Res., 491, 97–109 (2001).
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5.3
The Comet Assay as a Biological Monitoring Test Günter Speit *, Oliver Merk * and Andreas Rothfuß *
The determination of exposure to genotoxic/mutagenic agents at the workplace or in epidemiological studies is of central importance for biological monitoring. There are numerous genotoxicity tests available for such investigations; most experience has been gained with cytogenetic tests (Speit et al. 1994). None of the methods established so far can reliably cover the whole spectrum of possible exposures to mutagenic substances, as the sensitivity of the methods differs for the various types of primary DNA damage. For this reason in practice different methods are generally used in combination. In addition, new methods are continuously being developed and tested for routine use in biological monitoring (Perera & Whyatt 1994). A few years ago a new genotoxicity test was developed, the comet assay (single-cell gel electrophoresis) (for a review, see Tice 1995, Speit & Hartmann 1999), which has some decisive advantages over the tests used until then: • • • • • •
it is a very simple, rapid and sensitive test for the quantification of DNA damage, it covers a wide range of DNA damage, it detects DNA damage at the single-cell level, it can be carried out with practically any cell population, only very small cell quantities are needed, it can be carried out with proliferating and non-proliferating cells.
For biological monitoring studies it has the great practical advantage that not only peripheral blood can be investigated, but also e. g. cells from the mucous membranes of the cheeks and nose. The latter can, as the site of primary exposure (e. g. with air pollution), be of particular importance (Valverde et al. 1997). The comet assay is a microgel electrophoretic technique, for which a single-cell suspension (e. g. heparinized peripheral blood) is needed. The *
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Universitätsklinikum Ulm, Abteilung Humangenetik, Albert-Einstein-Allee 11, 89070 Ulm
5.3 The Comet Assay as a Biological Monitoring Test
Figure 1: The comet assay procedure.
procedure is shown in diagram form in Figure 1. Exact details on the procedure can be found in the reviews of Tice (1995) and Speit & Hartmann (1999). The cells are mixed with agarose which is spread on a slide previously coated with agarose. The cells fixed on the slide are lysed under alkaline conditions to destroy their membrane structures. After lysis, the slides are placed in a horizontal electrophoresis apparatus and exposed to the alkaline electrophoresis buffer (unwinding). Then brief electrophoresis is followed by neutralization and staining with ethidium bromide or another DNA-binding dye. The preparations are then evaluated with a fluorescence microscope. DNA damage leads to the migration of the chromosomal DNA to the anode, which is seen under the microscope as a “comet” (Figure 2). The length and/or amount of DNA migration serves as a measure of the degree of DNA damage. Evaluation is carried out either directly with the microscope or with the aid of image analysis equipment. Some parameters often used in practice are listed below. These are in the case of evaluation without image analysis: • • •
total length (image length), % of cells in different damage classes, % of cells with tail (“damaged cells”), 131
5 Cytogenetic Parameters
Figure 2: Microscopic view of cells after the comet assay has been carried out (A) Controls. (B) Cell with induced DNA damage for which increased DNA migration (“comet”) is visible.
and • • • •
in case of evaluation with image analysis: total length (image length), tail length, % of DNA in tail, tail moment.
Image analysis equipment is expensive, but is worth the investment if the test is to be used routinely. There are various systems available, some of which have proved extremely useful in practice. The parameter “tail moment” which is often used in the image analysis evaluation has the advantage that it takes into consideration both the length and the amount of DNA migration. However, there is still no agreement on a standard formula for the calculation of this parameter, which is very abstract – especially for persons without much experience with this test – and provides no direct measure of the level of DNA damage. The use of image analysis equipment is in no way essential. Direct evaluation can be regarded as sensitive and reliable. Measuring the length of the tail under the microscope by means of an ocular scale is, however, very time-consuming. Assignment of a result to one of several classes of damage and the calculation of the amount of DNA damage from the figures (Collins et al. 1995, 1997) is straightforward and can also be represented well graphically. It should be borne in mind that the expressions “damaged cell” and “undamaged cell” 132
5.3 The Comet Assay as a Biological Monitoring Test are relative terms based on the microscopic picture of the controls. Of course, even cells without a tail have a large amount of (spontaneous) DNA damage. There are many modified versions of the comet assay, which have been used by different groups. An important difference is the pH value of the electrophoretic buffer, which means that a different range of DNA damage is detected. In biological monitoring, however, only the alkaline version with a pH value of over 13 should be used, as this has proved to be the most sensitive. Under these conditions DNA strand breaks, alkali-labile sites and repair incisions can be detected with the comet assay. The effect in the comet assay is determined above all by the amount and type of DNA damage in a cell, but many methodological parameters can also affect the results: • • • • •
concentration of the low melting-point agarose, composition of the lysis solution, conditions of alkaline treatment (pH, duration, temperature), conditions of electrophoresis (voltage, current, duration, temperature), staining and evaluation (dye, excitation of fluorescence, magnification, parameters).
Figure 3 shows, for example, the influence of the duration of alkaline treatment and electrophoresis on the tail moment in peripheral human leukocytes. It can be seen that prolonging the duration leads to clear effects on the DNA (Figure 3 A) and that these effects are the result of the fact that increased DNA migration takes place in most of the cells (Figure 3 B). This simple experiment also shows that times that are too short can make the test insensitive. Particularly with biological monitoring investigations in which often only weak effects are to be expected, it is important to check the sensitivity of the test before the study, to avoid false negative results. The second important conclusion that can be drawn from this is that extremely well-defined and completely reproducible test conditions are necessary. Otherwise a variability in the measurements resulting from variable test conditions could greatly limit the reliability of the results. This is a particular problem when the comet assay is used for biological monitoring, as here – unlike with experimental in vitro and in vivo tests – in most cases neither the exposure conditions can be varied nor is it possible to carry out independent repeat tests. It is only possible in exceptional cases to determine a dose-effect relationship; this can, however, provide strong evidence of the biological relevance of the test results. To check the quality and stability of the protocol used, it can be helpful to include an internal standard. It is possible with no great effort, for example, to expose a larger cell sample to radiation, to immediately deep-freeze it in portions and then include individual samples in the tests. The results for this sample can provide valuable information about the quality of the test conditions and/or the quality of the evaluation. 133
5 Cytogenetic Parameters A
B
Figure 3: The influence of the duration of alkaline treatment (first time) and electrophoresis (second time) on DNA migration (tail moment) in peripheral human leukocytes.
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5.3 The Comet Assay as a Biological Monitoring Test The reliability of the results obtained with the comet assay depends to a great extent on how well the test is carried out. In addition to adequate methods, quality control should take other aspects into consideration. The first step is to carefully record the sampling, transport and storage of samples. To avoid dying or dead cells affecting the results of the comet assay, parallel determinations of the viability (vital staining) of the cells should be carried out. The comet assay should be carried out only with coded preparations and repeated determinations with two parallel preparations are recommended. External quality control, e. g. the exchanging of preparations between two experienced laboratories, can be very useful for the evaluation. To evaluate the results, the data must be analysed using suitable statistical methods. To date there is no generally recommended statistical test. Naturally the same requirements apply as for other genotoxicity tests. As normal distribution of the effects cannot be expected, non-parametric tests based on the median should be used. It must be borne in mind that the unit of exposure is not the cell but the individual and that the pooling of data for several individuals can lead to the loss of important information on variability. To allow the data to be interpreted, criteria for a positive test result should be laid down before the study is carried out. The biological importance of effects in the comet assay is not universally clear and probably very complex. Effects in the comet assay reflect early events (primary DNA alterations) which do not necessarily lead to mutations. The comet assay is therefore to be regarded as an indicator test and the effects found should generally be considered as biomarkers for exposure (of cellular DNA); the potential danger can be deduced (hazard identification), but not the risk. It cannot be seen from the comet itself which kind of DNA changes led to DNA migration and whether this reflects a mutagenic/carcinogenic potential of the exposure. There is no longer a sharp division between the detection of exposure and the detection of a biological effect when modifications of the test (see above) can detect specific DNA damage which is directly connected with mutagenicity and carcinogenicity. If the comet assay is to be used meaningfully in biological monitoring, the type of DNA damage to be detected in the test must be taken into consideration, and also how long after the end of exposure such damage can persist in the cells investigated and which confounders can influence the test result. It has been demonstrated in numerous in vitro and in vivo tests that the comet assay detects a wide range of DNA damage (review in Tice 1995). In addition to direct DNA strand breaks practically all known DNA alterations, such as e. g. oxidative DNA damage, alkylation and large adducts, can cause the induction of DNA migration. An important exception are crosslinks – both DNA-DNA and DNA-protein crosslinks (Merk & Speit 1999). Crosslinks are the only known kind of DNA damage that leads to the inhibition of DNA migration. With the standard protocol of the comet assay exposure to crosslinkers – which may be relevant mutagenic substances and carcinogens – are not detected. Various modifications of the 135
5 Cytogenetic Parameters comet assay for specifically detecting crosslinkers have been established, but they are unsuitable as an (additional) routine test in biological monitoring. If the exposure includes crosslinkers as well as other genotoxic agents, it is possible that a potential genotoxic effect is weakened by crosslinks. With regard to the persistence of the effects in the comet assay, several experimental studies have demonstrated that most of the DNA damage that contributes to DNA migration is repaired or eliminated within a few hours. Investigations in man have been able to show the persistence of effects over several days only in individual cases (Hartmann et al. 1995). As long as there is no specific information available about the long-term persistence of the relevant damage, it should be assumed it is rapidly removed. In practice this means that the comet assay should be carried out during a period of exposure or immediately after the end of a short-term or longterm exposure period. Investigations at a later point in time must be well justified, as there is a greater probability of false negative results. Possible confounders that can influence the test results are, first of all, all the known factors which are important also for other genotoxicity tests and are usually ascertained by means of a comprehensive questionnaire (Speit et al. 1994). One confounder that to date has only been described for the comet assay is heavy physical activity/sport (Hartmann et al. 1994). It should be checked that the participants in a study have not carried out any physical activity to the point of exhaustion during the week previous to blood sampling. Some classes of DNA damage can be detected highly specifically in the comet assay by means of damage-specific endonucleases. For this procedure the preparations are incubated with the enzyme after lysis at 37 8C. The enzymes cut the DNA at the modifications for which they are specific and the resulting increase in single-strand breaks can be quantified in the comet assay. With this modification of the comet assay, in combination with endonuclease III or formamidopyrimidine-DNA-glycosylase (FPG-protein) oxidatively damaged bases could be detected with great sensitivity (Collins et al. 1993, 1997, Dennog et al. 1996). The additional use of this modified comet assay in biological monitoring seems sensible and very promising when there is information available about possible induced DNA damage and this type of damage can be detected using specific endonucleases. With this procedure greater sensitivity and also greater specificity is achieved. The latter can be of great importance for the evaluation of the biological relevance of comet assay effects. Another modification that can be useful for biological monitoring is the use of DNA repair inhibitors. Provided induced damage is repaired by excision repair, the presence of repair inhibitors such as aphidicolin or cytosine arabinoside (alone or in combination with hydroxyurea) leads to the inhibition of repair synthesis and accumulation of incisions. These incisions are single strand breaks, which in the comet assay cause (additional) DNA migration. In experiments with cell cultures it was shown that repair inhibitors lead to an increase in DNA migration after treatment of the cells with 136
5.3 The Comet Assay as a Biological Monitoring Test UV radiation or various mutagenic/carcinogenic chemicals (Speit & Hartmann 1995, Martin et al. 1999). We were able to show in man that oxidative DNA damage in the peripheral blood is induced after hyperbaric oxygen therapy and can be detected in the comet assay (Dennog et al. 1996). This damage is repaired very rapidly in leukocytes after the end of exposure. If a repair inhibitor (aphidicolin) is immediately added to the blood samples, however, after incubation for two hours at 37 8C a marked increase in the primary effects is observed (Speit et al. 1998). Whether such a procedure – which should theoretically lead in many cases to an increase in sensitivity – will prove suitable for biological monitoring, has still to be investigated. In various investigations in man, the comet assay was used not only to detect (induced) DNA damage, but also to determine the repair capacity (Plappert et al. 1997, Buschfort et al. 1997, Alapetite et al. 1999). The blood samples of the test persons were treated with ionizing radiation or a mutagenic chemical and the effect determined in the comet assay. The blood samples were incubated under physiological conditions and analysed in the comet assay at different times after the end of exposure to determine the decrease in the effect with time as a measure of the repair capacity. Persons exposed long-term to radiation from the region around Chernobyl were found to have a reduced repair capacity (Plappert et al. 1997). The criticism must, however, be made that to date the reproducibility of such differences has not been sufficiently investigated and the biological importance of such differences is not known. More recent investigations carried out by our working group with blood from breast cancer patients with a BRCA1 mutation indicate that the comet assay determines mainly the speed with which the damage is eliminated, but not the exactness of repair. No differences in the repair capacity were found in the comet assay for the blood we investigated from patients with BRCA1 mutation after radiation, but there was a marked increase in chromosomal aberrations relative to in the control persons (Rothfuß et al. 2000). Further investigations into the repair capacity are needed before the use of this modification can be recommended in biological monitoring. To date, the comet assay has been compared with other genetic parameters in only a few biological monitoring studies (Vodicka et al. 1995, Hartmann et al. 1998, Sram et al. 1998, Somorovska et al. 2000). Further systematic investigations are needed before any statement can be made about the future importance of the comet assay in biological monitoring. Such studies should take into consideration as far as is possible the use of damage-specific enzymes and repair inhibitors. Particular value should be laid on the standardization of the experimental conditions and strict quality control. In previous investigations the great variability of the results in the comet assay was found to be problematical. The results for cell samples from the same person investigated at different times differed considerably (Collins et al. 1997). It must be investigated to what extent this variability is the result of the method and avoidable. All in all, as a result of its many 137
5 Cytogenetic Parameters advantages, the comet assay could play an important role in biological monitoring in future. Assuming careful planning and implementation of the study, it should be possible to reliably measure the differences between groups of persons. The comet assay will only find general acceptance if solid data are published and the results are not over-interpreted. Acknowledgements This study was supported by the environment and health programme (’Programm Umwelt und Gesundheit‘ – PUG) of the Karlsruhe research centre. References Alapetite, C.; Thrion, P.; Rochefordiere, A.; Cosset, J.; Moustacchi, E.: Analysis by alkaline comet assay of cancer patients with severe reactions to radiotherapy: defective rejoining of radioinduced DNA strand breaks in lymphocytes of breast cancer patients, Int. J. Cancer, 83, 83–90 (1999). Buschfort, C.; Müller, M. R.; Seeber, S.; Rajewski, M. F.; Thomale, J.: DNA excision repair profiles of normal and leukemic human lymphocytes: functional analysis at the single-cell level, Cancer Res., 57, 651–658 (1997). Collins, A. R.; Ai-guo, A.; Duthie, S. J.: The kinetics of repair of oxidative DNA damage (strand breaks and oxidised pyrimidines) in human cells, Mutation Res., 336, 69–77 (1995). Collins, A. R.; Dusinska, M.; Franklin, M.; Somorovska, M.; Petrovska, H.; Duthie, S.; Filion, L.; Panayiotidis, M.; Roslova, K.; Vaughan, N.: Comet assay in human biomonitoring studies: reliability, validation and applications, Environ. Mol. Mutagen., 30, 139–146 (1997). Collins, A. R.; Duthie, S. J.; Dobson, V. L.: Direct enzymic detection of endogenous oxidative base damage in human lymphocyte DNA, Carcinogenesis, 14., 1733–1735 (1993). Dennog, C.; Hartmann, A.; Frey, G.; Speit, G.: Detection of DNA damage after hyperbaric oxygen (HBO) therapy, Mutagenesis, 11, 605–609 (1996). Hartmann, A.; Fender, H.; Speit, G.: Comparative biomonitoring study of workers at a waste disposal site using cytogenetic tests and the comet (single cell gel) assay, Environ. Mol. Mutagen., 32, 17–24 (1998). Hartmann, A.; Herkommer, K.; Glück, M.; Speit, G.: DNA-damaging effect of cyclophosphamide on human blood cells in vivo and in vitro studied with the single-cell gel test (comet assay), Environ. Mol. Mutagen., 25(3), 180–187 (1995). Hartmann, A.; Plappert, U.; Raddatz, K.; Grünert-Fuchs, M.; Speit, G.: Does physical activity induce DNA damage? Mutagenesis, 9(3), 269–272 (1994). Martin, F. L.; Cole, K. J.; Orme, M. H.; Grover, P. L.; Phillips, D. H.; Venitt, S.: The DNA repair inhibitors hydroxyurea and cytosine arabinoside enhance the sensitivity of the alkaline single-cell gel electrophoresis (comet) assay in metabolically-competent MCL-5 cells, Mutation Res., 445, 21–43 (1999). Merk, O.; Speit, G.: Detection of crosslinks with the comet assay in relationship to genotoxicity and cytotoxicity, Environ. Mol. Mutagen.; 33, 167–172 (1999). Perera, F. P.; Whyatt, R. M.: Biomarkers and molecular epidemiology in mutation/cancer research. Mutation Res., 313, 117–129 (1994). Plappert, U.; Stocker, B.; Fender, H.; Fliedner, T. M.: Changes in the repair capacity of blood cells as a biomarker for chronic low-dose exposure to ionizing radiation, Environ. Mol. Mutagen., 30(2), 153–160 (1997). Rothfuß, A.; Schütz, P.; Bochum, S.; Volm, T.; Eberhardt, E.; Kreienberg, R.; Vogel, W.; Speit, G.: Induced micronucleus frequencies in peripheral lymphocytes as a screen-
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5.3 The Comet Assay as a Biological Monitoring Test ing test for carriers of a BRCA1 mutation in breast cancer families, Cancer Res., 60, 390–394 (2000). Somorovska, M.; Szabova, E.; Vodicka, P.; Tulinska, J.; Barancokova, M.; Fabry, R.; Kubova, J.; Riegerova, Y.; Petrovska, M.; Liskova, A.; Rausova, K.; Dusinska, M.; Collins, A.: Biomonitoring of genotoxic risk in workers in a rubber factory: comparison of the comet assay with cytogenetic methods and immunology, Mutation Res., 445(2), 181–192 (2000). Speit, G.; Hartmann, A.: The contribution of excision repair to the DNA effects seen in the alkaline single cell gel test (comet assay), Mutagenesis, 10(6), 555–559 (1995). Speit, G.; Hartmann, A.: The comet assay (single-cell gel test) in: Methods in Molecular Biology, Vol. 113: DNA Repair protocols: Eukaryotic Systems, Humana Press, Totowa (1999). Speit, G.; Bauchinger, M.; Schmid, E.; Gebhart, E.; Hüttner, E.; Obe, G.: Zytogenetische Analysen an menschlichen Populationen (Human Population Monitoring: HPM), Bundesgesundhbl., 3/94, 118–119 (1994). Speit, G.; Dennog, C.; Lampl, L.: Biological significance of DNA damage induced by hyperbaric oxygen, Mutagenesis, 13(1), 85–87 (1998). Sram, R. J.; Rossner, P.; Peltonen, K.; Podrazilova, K.; Mrackova, G.; Demopoulos, N. A.; Stephanou, G.; Vlachodimitropoulos, D.; Darroudi, F.; Tates, A. D.: Chromosomal aberrations, sister-chromatid exchanges, cells with high frequency of SCE, micronuclei and comet assay parameters in 1,3 butadiene-exposed workers, Mutation Res., 419, 145–154 (1998). Tice, R.: The single cell gel / comet assay: a microgel electrophoretic technique for the detection of DNA damage and repair in individual cells, in: Phillips, D. H.; Venitt, S. (eds.): Environmental Mutagenesis, pp. 315–339, BIOS Scientific Publishers Ltd., Oxford (1995). Valverde, M.; Lopez, C. M.; Lopez, I.; Sanchez, I.; Fortoul, T.; Ostrosky-Wegman, P.; Rojas, E.: DNA damage in leukocytes and buccal and nasal epithelial cells of individuals exposed to air pollution in Mexico City, Environ. Mol. Mutagen., 30, 147– 152 (1997). Vodicka, P.; Bastlova, T.; Vodickova, L.; Peterkova, K.; Lambert, B.; Hemminki, K.: Biomarkers of styrene exposure in lamination workers: levels of O6-guanine DNA adducts, DNA strand breaks and mutant frequencies in the hypoxanthine guanine phosphoribosyltransferase gene in T-lymphocytes, Carcinogenesis, 16, 1473–1481 (1995).
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6
Immunology
6.1
Immunoglobulins as Markers of Long-term Exposure to Allergenic Substances Hans Drexler *
Allergic diseases caused by exposure to hazardous substances are a problem that even at the modern workplace is not given sufficient consideration. It is estimated, for example, that with an asthma prevalence in the general population of 5 %, about 0.2 % to 0.5 % of persons suffer from occupationally-induced asthma (Blanc & Toren 1999, Kogevinas et al. 1999). The annual incidence of occupationally-caused dermatitis on the hands, depending on the activity is given as 0.5 to 1.9 per 1000 full-time employees (Diepgen & Coenraads 1999). At least some of these cases of disease are of immunological pathogenesis. Although over the past decades it has been possible to reduce the number of cases of occupational diseases by monitoring the exposure to toxic substances and by the consistent use of threshold limit values, allergens present at the workplace have often remained unconsidered. In neither the definition of the MAK value, nor in that of the BAT value is the allergenic potential of hazardous substances at the workplace considered in the setting of the threshold limit values (DFG 1999). Although there is no doubt that both the induction of an allergic reaction and the triggering of the reaction is governed by a dose-response relationship, it is at present not possible to define safe threshold limit values for contact with allergens (Drexler 1997). Such threshold limit values cannot at present be evaluated as, among other things, most of the case-reports available were drawn up without suitable quantification of the exposure. In studies in which the exposure was determined, it could be shown that a reduction in the allergen concentration causes a decrease in the number of cases of sensitization and the number of symptoms (Grieshaber 1997, Drexler et al. 1999). It was also evident that the protection of health is not guaranteed to the extent intended in the definitions of the toxicologically grounded MAK and BAT values (DFG 1999). With allergic diseases it must be differentiated between the humoral (IgE, IgG, IgM) and the cell-mediated pathomechanisms. The possible use *
Institut für Arbeits-, Sozial- und Umweltmedizin, Universität Erlangen-Nürnberg, Schillerstr. 25/29, 91054 Erlangen Biological Monitoring: Prospects in Occupational and Environmental Medicine. Deutsche Forschungsgemeinschaft (DFG) Copyright © 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-27795-7
6.1 Immunoglobulins as Markers of Long-term Exposure of immunoglobulins is probably limited mainly to the humorally mediated allergies. Of occupational-medical interest are the allergies of immediate type (type I allergy) and the type III allergies caused by immune complexes. In these allergies, the allergen-presenting cell induces a TH2 response (T helper cells of subtype 2). In the frequent type I allergies, interleukin-4 is regarded as the decisive cytokine; it is responsible for the switching-over of the B lymphocyte antibody production from IgG to IgE. The IgE antibodies then produced are directly involved in the inflammatory reaction initiated by contact with the allergen. The process of induction (sensitization) and provocation (triggering) was, and still is, often considered purely qualitatively and thus attributed more to the properties of the substance than to the amount of allergens acting. Only comparatively few allergological publications also describe the exposure exactly. The reason for this is evidently the difficulty in exactly quantifying allergens. The allergens are mostly proteins of natural origin with a high molecular weight, which are present in the air in concentrations in the ng/m3 range. The resulting analytical problems are apparent. In this concentration range the analytical results are affected increasingly by temporal and spatial inhomogeneity of the substance in the workplace air. There is much that indicates short-term peak concentrations are more relevant for sensitization than allergen doses. Individual factors also play an important role (when, for example, the natural barrier function of the skin and mucous membranes is impaired and allergens therefore have easier access to the immunocompetent cells). Allergens can be detected most easily in materials and in dust. In view of the arguments listed above, however, the relevance of such determinations must be questioned. Also monitoring of the air is problematical. Investigations of biological materials would therefore certainly be of great use, if it were possible to find suitable parameters. In this context it would seem to be particularly sensible to differentiate between exposure monitoring (determination of the concentration in the body or target organ), effect monitoring (determination of biochemical or biological effects) and response monitoring (evaluation of the adverse effects). In the case of allergic diseases, classic (systemic) exposure monitoring has so far not been able to detect the high molecular weight proteins of biological origin. It is possible, however, to detect various haptens of low molecular weight, i. e. compounds that can trigger an allergic reaction only after binding with endogenous proteins. These are found almost exclusively after workplace exposures (e. g. to diisocyanates, acid anhydrides, platinum salts). These compounds therefore represent interesting models for understanding the dose-response relationship of allergic diseases. There are only relatively few groups of low molecular weight compounds which, as a result of their ability to bind to specific endogenous macromolecules, have allergenic potential. If this binding is passive, the detection of haemoglobin or albumin adducts must still be described as 141
6 Immunology systemic exposure monitoring of the compartment >Proteins<. It is wellknown that allergenic substances induce the formation of specific IgE antibodies in only some of the persons exposed to them. In persons already sensitized, these antibodies and also the results of skin tests represent, by definition, parameters of critical biological responses which are associated with the adverse effects. Antibodies of IgG type against immunogenic xenobiotics seem to be detectable, however, in almost all exposed persons after exposure to sufficient concentrations for a sufficient length of time, and would be termed effect parameters in biological monitoring. To be able to use the advantages offered by biological monitoring even if the prerequisites for evaluating BAT values (or EKA values in the case of carcinogenic substances) are not fulfilled, a concept is at present being dawn up by the DFG which also in these cases will allow health and safety measures at the workplace to be monitored using the results of biological monitoring. In addition to the monitoring of carcinogens, this concept should also be suitable for monitoring allergenic substances. Among the allergens that seem suitable for verifying this concept are the group of acid anhydrides. Acid anhydrides are low molecular weight allergens with a high sensitization potential (Venables 1989, Welinder et al. 1990, Nielsen et al. 1992, Nielsen et al. 1994, Drexler 1994, Baur et al. 1995, van Tongeren et al. 1998). These compounds are used mainly as hardeners for epoxy resin and powdered paint hardeners; usually it is not just one single derivate that is used, but a mixture of different derivatives (e. g. phthalic anhydride [PA], hexahydrophthalic anhydride [HHPA], methyltetrahydrophthalic anhydride [MTHPA], methylhexahydrophthalic anhydride [MHHPA], trimellitic anhydride [TMA]). With unfavourable exposure conditions, incidences of sensitization of up to 50 % have been described. Also under relatively favourable exposure conditions the prevalence of immediate-type sensitizations in cross-sectional studies is around 20 %. Acid anhydrides can be detected both in the air at the workplace and by biological monitoring (metabolites in urine, protein adducts). In addition, specific immunoglobulins of IgG type can be detected as biological effect parameters, and in sensitized persons immunoglobulins of IgE type as critical response parameters. Also the skin prick test with free acid anhydrides (Drexler et al. 1993) and their protein conjugates (Nielsen et al 1992) produces positive results after sensitization has occurred, and can be used as a response parameter. In field studies with the aim to evaluate the sensitization potential of different acid anhydrides the question was posed whether, for example, biological monitoring allows the evaluation of the risk after exposure to allergens and whether the findings of biological monitoring correspond in particular with the risk of sensitization. A total of 219 persons were investigated from three industrial plants with exposure to acid anhydrides. The investigation included, in addition to the analysis of the workplace air, a physical examination, a skin prick test with protein conjugates of the acid anhydrides and a serum and urine analysis. Specific immunoglobulin E 142
6.1 Immunoglobulins as Markers of Long-term Exposure and immunoglobulin G antibodies (Welinder et al. 1990) against HHPA conjugates and MTHPA conjugates were determined in serum. In the urine samples the corresponding dicarbonic acids of the allergens were analysed (Jönsson & Lindh 1996). In agreement with data in the literature, incidences of sensitization of 21 % to 35 % were found for MTHPA concentrations in the air of up to 58.5 lg/m3. The concentration of the metabolites in urine was greatest in the employees from the plant with the highest incidence of sensitization (incidence of sensitization 35 %; median: 346 nmol/mmol creatinine) and lowest in the employees with the lowest incidence of sensitization (incidence of sensitization 21 %; median: 10.1 nmol/mmol creatinine). The levels of specific immunoglobulin E against MTHPA in the serum of the sensitized employees (specific IgE >0.3) followed the same pattern as the concentrations of urinary metabolites. There were no differences in the levels of dicarbonic acids excreted in the urine of sensitized persons and those not sensitized. No correlation could be found between the dicarbonic acids in urine and specific IgE. The specific IgG in serum correlated both with the metabolites of MTHPA in urine and with specific IgE. Unlike specific IgE, specific IgG is not regarded as a response parameter but as a parameter for long-term exposure (Kim et al. 1997, Park et al. 1999). Specific immunoglobulin G could therefore be a suitable parameter for biological monitoring with exposure to allergenic substances at the workplace. The statistically significant correlation of the specific IgG level of the exposed persons with the metabolite concentration in urine seems to confirm this. If specific IgG can be used as a parameter for long-term exposure, a correlation with specific IgE would indicate that with increasing long-term exposure also the intensity of sensitization, in other the words the response, increases. It must be clarified whether this relationship is really a correlation, and not caused, for example, by the fact that the formation of antibodies is promoted as a matter of course by the sensitization. Two arguments speak, however, against a general stimulation of the formation of antibodies. Firstly, it is not biologically plausible that the formation of both immunoglobulin classes is stimulated at the same time. Interleukin-4 (IL4) is thought to be responsible for the switching-over of the plasma cells and the consequent change in antibody production from IgG to IgE. It is therefore unlikely that the production of both antibodies is stimulated by one stimulus. Secondly, as to be expected, the IgE antibody levels differ in atopic persons and those who are not atopic (atopia is, by definition, the disturbed regulation of the immune system which allows the easier formation of IgE, and it is hereditary). With specific IgG, however, this difference was not found. This also does not seem to be the result of selection bias (for example, when atopic persons, who become sensitized more quickly, leave the workplace as a result of sensitization), as the IgG levels of sensitized persons and those not sensitized did not reveal any differences between atopic persons and those not atopic. 143
6 Immunology Even though the possible pathogenic meaning of IgG antibodies is still a matter of some controversy, these antibodies have been used successfully as markers of exposure to diisocyanates, enzymes, dust mites, grain dust and fungi (Kim et al. 1997, Zentner et al. 1997, Smith et al. 1998, Johanning et al. 1999, Park et al. 1999). Prospects Despite the social-medical-epidemiological importance of allergic diseases, little effort has been made to validly determine exposure. The IgG response of the body with long-term exposure to allergens could become a valuable parameter for this. First, however, analytical practice must be standardized. Only then can the method be validated for specific allergens. It must be asscertained for which allergens the IgG level is related to the exposure and whether increased long-term exposure represents a risk factor for sensitization. Low molecular weight allergens are suitable model substances, as these can be detected reliably and precisely both in the workplace air and in biological materials. If sufficient data can be collected, it is conceivable that innovative concepts for health and safety at the workplace could be developed. The IgG concentration would not be suitable as an individual ceiling level, comparable with a BAT value, but perhaps occupational hygiene could be better evaluated than is possible at present for allergic diseases with a concept such as that of the American BEI values. References Baur, X.; Czuppon, A. B.; Rauluk, I.; Zimmermann, F. B.; Schmitt, B.; Egen-Korthaus, M.; Tenkhoff, N.; Degens, P. O.: A clinical and immunological study on 92 workers occupationally exposed to anhydrides, Int. Arch. Occup. Environ. Health, 67, 395–403 (1995). Blanc, P. D.; Toren, K.: How much adult asthma can be attributed to occupational factors? Amer. J. Med., 107, 580–587 (1999). DFG (Deutsche Forschungsgemeinschaft): MAK und BAT-Werte-Liste 1999, Wiley VCH Verlag, Weinheim (1999). Diepgen, T.; Coenraads, L. P. J.: The epidemiology of occupational contact dermatitis, Int. Arch. Occup. Environ. Health, 72, 8, 496–506 (1999). Drexler, H.; Schaller, K.-H.; Nielsen, J.; Weber, A.; Weihrauch, M.; Welinder, H.; Skerfving, S.: Efficiency of hygienic measures in workers sensitised to acid anhydrides and the influence of selection bias on the results, Occup. Environ. Med.; 56, 202–205 (1999). Drexler, H.; Weber, A.; Letzel, S.; Kraus, G.; Schaller, K.-H.; Lehnert, G.: Detection and clinical relevance of a type I allergy with occupational exposure to hexahydrophthalic anhydride and methyltetrahydrophthalic anhydride, Int. Arch. Occup. Environ. Health, 65, 279–283 (1994). Drexler, H.; Schaller, K.-H.; Weber, A.; Letzel, S.; Lehnert, G.: Skin prick test with solutions of acid anhydrides in acetone, Int. Arch. Allergy Immunol., 100, 251–255 (1993). Drexler, H.: Präventionsstrategien bei allergischem Berufsasthma, Allergologie, 20, 168–173 (1997). Grieshaber, R.: Umsetzung von Forschungsergebnissen für die Prävention im Backgewerbe, in: Borsch-Galetke, E.; Struwe, F. (eds.): Dokumentationsband über die 37.
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6.1 Immunoglobulins as Markers of Long-term Exposure Jahrestagung der Deutschen Gesellschaft für Arbeits- und Umweltmedizin e. V.; Rind Druck, Fulda, 631–637 (1997). Johanning, E.; Landsbergis, P.; Gareis, M.; Yang, C. S.; Olmsted, E.: Clinical experience and results of a sentinel health investigation related to indoor fungal exposure, Environmental Health Perspectives, 107 (Suppl. 3), 489–494 (1999). Jönsson, B. A. G.; Lindh, C. H.: Determination of hexahydrophthalic acid and methylhexahydrophthalic acid in urine using gas chromatography and negative ion chemical ionisation mass spectrometry, Chromatographia, 42, 647–652 (1996). Kim, H.; Kim, Y. D.; Choi, J.: Seroimmunological characteristics of Korean workers exposed to toluene diisocyanate, Environmental Research, 75, 1–6 (1997). Kogevinas, M.; Maria Anto, J.; Sunyer, J.; Aurelio, T.; Kromhout, H.; Burney, P.: European Community Respiratory Health Survey Study Group: Occupational asthma in Europe and other industrialised areas: A population-based study, Lancet, 353, 1750– 1754 (1999). Nielsen, J.; Welinder, H.; Ottosson, H.; Bensryd, I.; Vege, P.: Nasal challenge shows pathogeneic relevance of specific IgE serum antibodies for nasal symptoms caused by hexahydrophthalic anhydride, Clin. Exp. Allergy, 24, 440–449 (1994). Nielsen, J.; Welinder, H.; Horstmann, V.; Skerfving, S.: Allergy to methyltetrahydrophthalic anhydride in epoxy resin workers, Br. J. Ind. Med., 49, 769–775 (1992). Park, H.-S.; Suh, C.-H.; Nahm, D.-H.; Kim, H.-Y.: Presence of specific IgG antibody to grain dust does not go with respiratory symptoms, Journal of Korean Medical Science, 14 (1), 39–44 (1999). Smith, A. M.; Yamaguchi, H.; Platts-Mills, T. A. E.; Fu, S. M.: Prevalence of IgG anti-Der p 2 antibodies in children from high and low antigen exposure groups: Relationship of IgG and subclass antibody responses to exposure and allergic symptoms, Clinical Immunology & Immunopathology, 86 (1), 102–109 (1998). Van Tongeren, M. J. A.; Barker, R. D.; Gardiner, K.; Harris, J. M.; Venables, K. M.; Harringtron, J. M.; Newman Taylor, A. J.: Retrospective exposure assessment for a cohort study into respiratory effects of acid anhydrides, Occup. Environ. Med., 55, 692–696 (1998). Venables, K. M.: Low molecular weight chemicals, hypersensitivity, and direct toxicity: the acid anhydrides, Brit. J. Ind. Med.; 46, 222–232 (1989). Welinder, H.; Nielsen, J.; Gustavsson, C.; Bensryd, I.; Skerfving, S.: Specific antibodies to methyltetrahydrophthalic anhydride in exposed workers, Clin. Exp. Allergy, 20, 639–645 (1990). Zentner, A.; Jeep, S.; Wahl, R.; Kunkel, G.; Kleine-Tebbe, J.: Multiple IgE-mediated sensitizations to enzymes after occupational exposure: Evaluation by skin prick test, RAST, and immunoblot, Allergy, 52, 928–934 (1997).
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6.2
Immunological Effects of Polymorphic Key Enzymes Jürgen Lewalter *
6.2.1 Basic Facts The immune system is enormously important for maintaining the integrity of the body. In addition to defence against attack by pathogens and toxic chemicals, this includes the “repair” of damaged tissue and the elimination of altered cells (for example, tumour cells and aged cells). These complex tasks require a finely regulated system. The main pillar of the immune response is the induction of an inflammatory reaction as a result of the activation of immune cells via receptors on their surface. The cellular carriers of the phylogenetically older immune system (macrophages, granulocytes, mast cells etc.) have a set of so-called “pattern recognition receptors” with a relatively wide specificity for ubiquitous chemical structures, above all those expressed by micro-organisms (for example, endotoxin, mannose, CG-rich DNA, RNA, phosphorylcholine), and the many chemical substances used at the workplace. The entry of such compounds into tissue leads to the activation of all the immunocompetent cells in the area, as shown below (Figure 1) (Baenkler 1992, Bauer 1991, Keller 1987, Lewalter 1994, Reinke et al. 1999, Schleicher 1997). Humoral recognition mediators (complement, C-reactive protein [CRP] etc.) make this system even more effective. The stimulation of these cells via their pattern recognition receptors does not lead to direct activation (increased phagocytosis and improved killing function), but rather to activation of the surrounding cells (e. g. endothelium) via the released cytokines and their inclusion in the inflammatory reaction (paracrine function). Factors which may have an effect on the system are shown in Figure 2 (Neumann 1992, Schleicher 1997). The release of large amounts of cytokines has general effects (endocrine function) and activates systemic inflammatory systems (synthesis of acute phase proteins, change-over to a catabolic metabolic condition, induction of fever and other neuro-endocrine reactions, induced haematopoiesis etc.) and even “systemic inflammatory response syndrome” (SIRS). *
146
BAYER AG, Institut für Biologisches Monitoring, 51368 Leverkusen
6.2 Immunological Effects of Polymorphic Key Enzymes
Figure 1: Properties and performance of the immune system (Playfair & Baron 1995).
Figure 2: Simplified diagram of the development of cellular and humoral immunity showing the sites of action of immunosuppressive substances (Neumann 1992).
147
6 Immunology In addition, to out-manoeuvre the many “escape” mechanisms of the micro-organisms, in the course of evolution the specific, adaptive immune system developed; the lymphocytes are its cellular carrier. Instead of the multi-reactive “pattern recognition receptors”, they have billions of different, clonally distributed, highly specific antigen receptors, which allow the recognition of practically all chemical structures that could theoretically be formed in nature. As by nature there are only few cells (clones) of a given specificity in the body, a useful effector response presupposes the clonal expansion of the stimulated clones after contact with the microbial stimulus or other allergenic stimuli. For this reason this specific immune system reacts markedly slower (days) than the unspecific immune system (hours). On the effector side, the specific immune system, however, uses many of the unspecific inflammation reactions. The complex relationships are described briefly in Figure 3 (Baenkler 1992, Schleicher 1997, Vorlaender 1980). Acute inflammatory reactions improve the defence against pathogens and also aid wound healing and tissue repair. However, acute inflammatory reactions always involve a risk of self destruction, avoidance of which calls for complex regulation. In addition to the auto-regulative turning off of the activated inflammatory cells (apoptosis, “exhaustion”) the inflammatory reaction is controlled by regulatory cytokines of autocrine, paracrine and endocrine function (for example IL-10, TGF-b) and by the neuro-endocrine system (hypothalamic/hypophyseal/adrenal system, catecholamines, endorphins). This system, tried and tested and further developed over millions of years, can get out of control during intensive medical care and occupational-medical disorders, mainly after intoxications.
" Figure 3: Hypersensitivity syndrome (Schleicher 1997). Damage as a result of an inadequate reaction: • Excessive and unnecessary immune reaction to harmless antigens: hypersensitivity; allergy – xenogeneic: against antigens foreign to the species – classic allergy; – infection-induced processes – allogeneic: against antigens foreign to the individual – rejection of transplantations – immunofoetopathy – allogeneic infertility – autologous: against endogenous antigens – auto-aggression, auto-allergy • Insufficient reaction or no immune reaction to pathogenic antigens: immunodeficiency, defective immunity – as a result of anomaly or deletion: state of “primary” deficiency – as a result of loss or impediment: state of “secondary” deficiency • Uncontrolled monoclonal proliferation: malignant immunoproliferation
148
6.2 Immunological Effects of Polymorphic Key Enzymes
149
6 Immunology Over the past few years a main focus of attention has been excessive reactions of the inflammatory system. Immune factors such as TNFa and IL-1 are thought to play a decisive role in the pathogenesis of systemic inflammatory reactions (SIRS) and sepsis. While numerous body functions (liver, kidneys, blood coagulation, heart, blood pressure) are subject to strict monitoring in occupational-medical prophylaxis, it is hardly ever taken into consideration that as a result of anaphylactoid events or multi-organ failure also the immune system, an “organ” essential to life, can fail. In general, evaluation of its function is limited to determination of the only moderately useful CRP (C-reactive protein) (Reinke et al. 1999, Vorlaender 1980). In the fields of occupational and environmental medicine, the following humoral and cellular mechanisms of immunological competence are differentiated: • Immunostimulation: stimulation of the antigen-induced immune defence system; adverse effects: sensitization etc. • Immune tolerance: suppression of the antigen-induced immune defence system; adverse effects: persistent infections etc.
6.2.2 Selection of Diagnostic Parameters for Immunity Which parameters of immunity should be recorded during occupationalmedical surveillance? In the literature numerous parameters have been described as useful for diagnosis/prognosis. The parameters described below are regarded as useful and above all fulfil modern standardization requirements – unfortunately still a handicap of many immunological methods. Naturally, for reasons of cost, there is always a demand for the (screening) parameter – but it is illusory to expect to be able to evaluate the complex functions of the immune system with one single parameter (this is also not possible for functions of other organs). When selecting parameters, above all the questions they are intended to answer plays an important role (Vorlaender 1980). The immune response resulting from exogenous and endogenous induction is a complex process. As can be seen in the following diagram (Figure 4), numerous immunocompetent cells are involved in the immune response as are multifunctional mediators (cytokines) (Baenkler 1992, Dean et al. 1991, Kirchner et al. 1993). " Figure 4: Development, interaction and cells of the immune system (Dean & Murray 1993).
150
6.2 Immunological Effects of Polymorphic Key Enzymes
151
6 Immunology The immune system is divided into a cellular and a humoral part, both of which can react specifically and unspecifically to induction by antigens. The T and B lymphocytes represent the specific cellular part of the immune system, the macrophages and natural killer cells the unspecific cellular part. Unlike the sensitive markers of cellular defence, which are difficult to determine, the parameters of humoral defence in plasma can be analysed using largely standardized methods (Bauer 1991, Dean et al. 1991, Vorlaender 1980). The humoral immune response is mediated by specific serum proteins. The antibodies of immunoglobulin classes G (IgG), A, M and E (IgE) are representative of the specific part, the complement system of the unspecific part. In addition to these markers of humoral immune defence, the labile cytokines and stable soluble cytokine receptors, early indicators of immunomodulation, are integral to the cellular defence. They are involved in all immunological cell activation, inhibition, differentiation and maturation processes. They have no enzyme activity and, as a result of their high, and usually only transient biological effectiveness, require sensitive regulation. This regulation can be induced, at least in part, by inhibition of the expression of their specific, and usually long-lived receptors, and also via an autocrine mechanism. Exposure to sensitizing substances at the workplace can lead to modulation of the immune system; the disturbed immune tolerance can be detected as increased antinuclear antibody titers. Finally, macrophages are involved in unspecific immune defence via mediators and also indirectly in the specific immune defence against workplace substances (Baenkler 1992, Vorlaender 1980).
6.2.3 Evaluation of Immunological Competence Below, after an explanation of the molecular biological background to immune reactions, examples of the immunological processes that lead to obstructive diseases of the respiratory passages are discussed. It is known from reports from the BG (Berufsgenossenschaften = German liability insurance associations) that persons handling allergenic and toxic-irritative substances can develop obstructive diseases of the respiratory passages (Table 1, Schleicher 1997). Despite intensive occupational-medical health monitoring, obstructive diseases of the respiratory passages are still among the most frequent occupational diseases resulting from contact with substances at the workplace. In the meantime, the mechanisms of induction of obstructive respiratory diseases are largely understood (Diller 1990, 1991, 1997). The following obstructive diseases of the respiratory passages are differentiated: 152
4301 4302
4302
Welding fumes
Total number of investigations: 4,638,128.
1301
Aromatic nitro and amino compounds 44,037
17,546
23,883 ?
54,868
207
90
29 647
176 207
213
99
24 713
146 213
Occupational diseases Number of persons Reported occupational diseases acc. list no. (appendix examined in 1996 1, BKV) 1995 1996
Isocyanates – airway obstruction, alveolitis 1315 – skin disease 5101
Obstructive diseases of the respiratory passages – allergenic substances – toxic-irritative substances
BG guideline
59 [28.5]
33 [36.7]
15 [51.7] 153 [23.6]
54 [30.7] 59 [28.5]
1995
57 [26.8]
36 [36.4]
17 [70.8] 127 [17.8]
54 [37.0] 57 [26.8]
1996
Recognized occupational diseases [ %]
Table 1: Guidelines for preventive medical examinations issued by the Employers’ Liability Insurance Association.
6.2 Immunological Effects of Polymorphic Key Enzymes
153
6 Immunology • unspecific bronchial hyperreactivity (UBH), • specific bronchial hypersensitivity (G23); allergenic substances (IgEmediated; BKV A4301); toxic-irritative substances (BKV A4302), • hypersensitivity of the airways to isocyanates (alveolitis, G27; BKV A1315), immunological-humoral mechanisms (IgE, IgG), immunologicalcellular mechanisms (T lymphocytes). In field studies (Table 2) a threshold concentration was found for the triggering of sensitization by substances at the workplace (Porter et al. 1975). Experience with isocyanates can be summarized as follows: • Dose–response relationship for the induction of bronchial hypersensitivity (occupational asthma). • Sensitization thresholds have been observed for the toxic-irritative forms and also for the allergic forms in the induction phase, and for the provocation phase (Vandenplas et al. 1993). • The immunological sensitization thresholds for persons exposed repeatedly to isocyanates are about 20 ppb (Diller 1990). • Fundamentally, a single massive high-level exposure, perhaps even via the skin, is sufficient for sensitization or induction of specific bronchial hypersensitivity.
Table 2: Dose-response relationship for the induction by TDI of pulmonary sensitization in employees of a large chemical plant (Porter 1975). Observation period
Average concentration of TDI (ppb)
1957 1958 1959 1960 1961 1962 1963 1964 1965 1966 1967 1968 1969 1970 1971 1972 1973 1974
60 ppb 60 ppb 60 ppb 60 ppb 60 ppb 60 ppb 60 ppb 60 ppb 60 ppb 60 ppb 60 ppb 60 ppb 60 ppb 50 ppb 50 ppb 20 ppb 20 ppb 4 ppb
154
Incidence of isocyanateasthma cases per year 4 3 3 1 continuous reduction 2 3 in the height of exposure peaks 1 3 1 1 1 1 2 1 2 0 0 0 t
6.2 Immunological Effects of Polymorphic Key Enzymes Immediate type (type 1) IgE-mediated sensitization is triggered primarily by allergens or haptens, i. e. in particular by adducts formed by xenobiotics with endogenous proteins. In the meantime, case reports and field studies with molecular biological analyses have demonstrated that the levels of protein adducts formed with workplace substances depend mainly on enzyme deficiencies in the metabolism involved (Lewalter & Neumann 1996, 1998). Medical evaluation of occupational and environmental risks must take into account the genetic polymorphisms of key enzymes of xenobiotic metabolism, for which the frequencies of at least 2 alleles are over 1 % (WHO recommendation, 1993): hydrolases (phase I, detoxifying) • cholinesterases (ChE, AChE) – organophosphate metabolism • carboxylesterases (CE) – pyrethroid metabolism oxygenases (phase I, toxifying) • cytochrome P450 monooxygenases – CYP 1A1: metabolism of aliphatics, olefins and aromatics – CYP 2E1: metabolism of solvents transferases (phase II, detoxifying) • N-acetyltransferases (NAT-1, NAT-2) – metabolism of amino aromatics • glutathione-S-transferases (GST a, l, p, t) – metabolism of ethylene oxide, acrylonitrile, styrene/styrene oxide, alkyl halides, isocyanates • glucuronidases (UDPG) – phenol metabolism During the IgE-specific metabolism of typical workplace substances the formation of adducts with human serum albumin (HSA) is influenced e. g.
Figure 5: IgE-relevant metabolism.
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6 Immunology by the following polymorphic enzymes (Figure 5) (Lewalter & Neumann 1996, 1998).
6.2.4 Aim of the Study Presented below are our first results obtained on the basis of the molecular biological concept for the prevention of obstructive diseases of the respiratory passages. a) The diagnostic significance of IgE-antibodies induced by adduct-antigens in the clarification of disorders ranging from disturbances in wellbeing to obstructive diseases of the respiratory passages. b) Evidence of the dependence of concentration thresholds for sensitization of individuals with toxic-irritative or allergenic substances on the indi-
Figure 6: Isocyanate haptens.
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6.2 Immunological Effects of Polymorphic Key Enzymes vidual polymorphisms of enzymes essential in the metabolism of the substance. Improper handling of isocyanates can lead to obstructive respiratory diseases. The sensitizing allergens can be formed via the following reaction cascades (Figure 6) (Lewalter 1998). The effects on health observed in persons handling isocyanates are not induced only by the isocyanates themselves, but can be caused also by the solvents used and other confounding factors (Lewalter 1998). Potential symptoms of adverse effects on health caused by isocyanates and confounding factors are: in the case of isocyanates (aromatic monoisocyanates and diisocyanates): • primary irritation of the skin and mucous membranes and sensitization • allergic reaction in sensitive persons in the case of (thio) carbamates (isocyanates and (sulf)hydr(ox)yl groups): • headaches, visual disturbances, vomiting, cyanosis, spasms, miosis, cramps, respiratory arrest, etc. • more rapid increase and decrease in cholinesterase inhibition than with organophosphates • effects on kidney functions in the case of carbamides (isocyanates and amino groups): • increased Met-Hb values • moderate irritation of the skin and mucous membranes • sensitization of sensitive persons • mobility disorders in the case of solvents: • increased activity of liver enzymes in plasma • neurological dysfunction and in the case of alcohol: • increased activity of liver enzymes in plasma • neurological dysfunction. The following case report (Table 3) makes it clear that not only the amount of HSA adducts formed short-term, but in particular how long they are present (bioavailability) in the plasma of the patient can be responsible for his later sensitization (memory effect; RAST values >2.0 RU). Generally the natural biological half-life of HSA (19 days) is significantly shortened by the formation of adducts with workplace substances, as can be seen in the course of the HSA adduct curve in Figure 7 (Lewalter & Neumann 1996, 1998).
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6 Immunology Table 3: Evaluation of risks following intoxication with p-fluorophenylisocyanate (p-FPI). Time of examination
Before intoxication Intoxication 1 day later 5 months later 1 year later
Lung function
IgE levels
HSA adducts before following shift [ng p-FPI/l blood]
HSA recovery [ %]
[total PU]
[p-FPI-spe- at the end cific RU] of the shift [ng p-FPI/l blood]
normal
119. 5
1.5
310
230
26
normal normal normal reduced
117.3 136.6 128.1 135.8
1.4 1.4 1.9 2.1
100,000 900 <100 280
40,000 570 <100 210
60 37 0 25
PU = PRIST unit. RU = RAST unit.
Figure 7: Evaluation of the short-term and persistent amounts of workplace substances in Hb adducts.
To prevent sensitization to substances used at the workplace the following points should be observed in practice: • HSA adducts with xenobiotics have significantly shorter biological half-lives than does free HSA. • The regeneration of less than 50 % free HSA from HSA adducts within up to 16 hours without contact with the substance is regarded as the individual susceptibility marker for IgE-mediated sensitization. 158
normal
11 [5.2]
73 (0.5–1130) 183 (57–1980) 201 (64–230) 71 (0.5–910) 265 (48–2340) 186 (57–1840)
[Total PU]
IgE levels
1. 2 (0.7–1.9) 3.1 (2.4–7.6) 2.8 (2.1–8.1) 1.1 (0.5–1.9) 3.8 (2.2–8.5) 4.5 (2.5–7.9)
[MDI-specific RU]
430 (<10–1320) 780 (120–4300) 850 (90–3870) 390 (<10–890) 910 (150–4800) 870 (130–3100) 170 (<10–620) 630 (95–3700) 680 (84–2950) 130 (<10–340) 790 (130–4100) 750 (110–2900) 18
13
67
20
19
60
1.7 (<1.0–2.5) 3.4 (1.7–5.9) 5.2 (1.5–8.3) 1.5 (<1.0–2.3) 4.7 (1.5–7.3) 4.1 (1.8–6.9)
HSA recovery 4-ADP [ng/l blood] rate * End of the At the beginning [%] shift of the next shift [ng MDA/l bl.] [ng MDA/l blood]
HSA adducts after 3 workdays
* Percentage of HSA released during the 16-hour recovery period (exposure-free period).
normal
limited
normal
limited
17 [8.1]
183 [86.7] 25 [11.8] 3 [1.4]
normal
183 [86.7]
1995
1996
Lung function
Years Persons N [ %]
Table 4: Biological monitoring of workers exposed to MDI in 1995 and 1996. The average concentration in the air at the workplace in 1995 was 24 lg/m3 and 1996 17 lg/m3.
6.2 Immunological Effects of Polymorphic Key Enzymes
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6 Immunology By taking into consideration the regeneration of free HSA during occupational-medical surveillance of persons handling isocyanates according to G27, beginning obstructive diseases of the respiratory passages were detected early in 11 symptom-free persons exposed to MDI and the later effects markedly reduced by prompt avoidance of contact with isocyanates (Table 4) (Lewalter 1998). Since 1996 handling of isocyanates in industry is permitted only for workers with HSA recovery rates above 50 %. As a result, no new obstructive diseases of the respiratory passages have been observed. Further guidelines for the prevention of disease caused by isocyanates are listed below (Diller 1990, Diller 1991, Diller 1997). • The allergen (asthmogen) concentration must always remain below the sensitization threshold. • In addition, the skin must be well protected. • Adjuvant effects of substances in the workplace air must be prevented. • The formation of IgE as a result of cigarette smoking must be avoided (anti-smoking campaign). • Atopic persons are at greater risk from high molecular weight allergens than non-atopic persons. • Depending on the extent of atopia and the level of exposure to the allergen, special gas masks, strict health monitoring or avoidance of contact with the substance are necessary (Diller 1997). It must be borne in mind that the sensitization threshold itself can also be influenced by numerous factors (Diller 1997): • endogenous disposition (atopic persons) (Gubian & de Zotti 1994), • smoking habits (smokers‘ asthma) (Newman-Taylor 1995), • viral infections of the respiratory tract (inhibition of cellular tolerance mechanisms) (Holt 1989), • adjuvants such as solvents, amines, inert dust, ozone etc. (concentration-dependent, with significant NOEL) (Goren & Hellmann 1988), • previous sensitizations increase the prevalence of current sensitizations (Kersten & Wahl 1994).
6.2.5 Immunomodulation as a Result of Exposure to Substances at the Workplace Protein adducts can be formed during metabolism not only of isocyanates, but also of other hazardous substances; the extent of this adduct-formation depends on the polymorphism of the enzymes involved (Table 5). 160
6.2 Immunological Effects of Polymorphic Key Enzymes Table 5: Correlation between the amount of 3,4-dichloroaniline excreted in the urine and the formation of protein adducts after the intoxication of plant operators with 3,4dichlorophenylisocyanate. Subject
Urine
Haemoglo- HSA bin total free amine [lg adduct/l amine [lg/g crea- blood] [lg/l] tinine]
Acetylator IgE PRIST IgE RAST phenotype [PU] [RU]
1 2 3 4
40 100 170 250
40 2.3 5.7 230
5 510 40 Normal values 10 persons <1.0 <1.0 10 persons <1.0 <1.0
20 1 4 70
15.8 9.5 8.3 37.5
slow fast fast slow
10
4.3
fast
<0.1 <0.1
fast slow
<0.1 <0.1
3.9 130 1.7 28 1.2 44 5.2 * 480 (symptoms!) 31 1.5
25 47
1.1 1.4
PRIST value ≥ 150 PU: positive result; RAST value <2.0 RU: negative result; RAST value 2.0–2.9 RU: borderline result; RAST value ≥ 3.0 RU: positive result; HSA: human serum albumin; * validated in RAST inhibition test.
Figure 8: Carbamide adducts.
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6 Immunology Table 6: Dependence of the Met-Hb and 4-ADP-HSA adduct background levels on G6-PDH in 292 fast acetylators without industrial contact with amino aromatics. Non-smokers G-6-PDH (%) [U]
Met-Hb [%]
HSA adducts Total IgE [ng 4-ADP/l blood] [PU]
282 (97) 7 (2) 3 (1)
0.7 (0.1–1.1) 1.3 (0.9–2.7) 2.1 (1.4–4.3)
1.3 (0.5–2.1) 3.5 (2.4–5.1) 7.3 (5.5–9.8)
130 ± 10 70 ± 8 0
IgE RAST [RU]
38 (1–72) 1.1 (0.9–1.2) 55 (8–97) 1.3 (1.1–1.7) 81 (34–112) 1.6 (1.4–1.9)
Figure 9: Comparison of specific biomarkers in strong and weak chromium(VI) reducers exposed to dichromate concentrations up to half the TRK value, and in not exposed controls.
In slow acetylators, in addition to increased amounts of adducts, also significantly increased IgE RAST values are observed (Lewalter 1994). The molecular biological explanation of these effects is as follows in Figure 8. The metabolism of amino aromatics is influenced not only by the NAT1 and NAT-2 polymorphism, but also by that of G-6-PDH (Table 6) (Lewalter & Neumann 1996, 1998). Also exposure to Cr(VI) can activate the immunological defence system (Figure 9). Depending on the individual Cr(VI) reducing status, significant IgE induction is observed (Lewalter 1994, Lewalter & Neumann 1996, 1998). The Cr(VI) reducing status moreover modulates the proliferation and differentiation of B and T lymphocytes via the production of interleukin 2 (Table 7). 162
6.2 Immunological Effects of Polymorphic Key Enzymes Table 7: Inhibition of the proliferation of T lymphocytes and their release of interleukin 2 by sodium dichromate in the blood of strong and weak chromium(VI) reducers. Phytohaemagglutinin stimulation
lM Cr(VI) per sample
% T cell proliferation Cr(VI) reducers
% IL-2 production Cr(VI) reducers
strong
weak
strong
Normal status – –
–
–
Mitogen stimulation + – + 0.1 + 10 + 100
100 103 98 0.3
100 74 34 0
weak
34
20
100 86 55 34
100 38 21 13
Table 8: The excretion of 8-hydroxydeoxyguanosine (8-HO-dG) after contact with devices that emit “ozone” (concentration in the air: up to 0.05 ppm). Persons
Urine samples
8-HO-dG [nmol/g creatinine]
81 women 25 men
324 101
17.9 (6.1–34.0) 25.3 (8.6–42.7)
Controls 50 women 50 men
50 50
7.9 (5.1–9.0) 9.2 (8.5–10.6)
Total IgE [kPU/l] 95 (21–168) 72 (34–91) <23 (<1–45) <20 (<1–38)
IL-2 [lg/l] 163 (115–430) 151 (104–310) 189 (125–640) 180 (135–450)
Similar effects are also induced by exposure to ozone; the production of interleukin 2 is evidently inhibited to a greater extent in men than in women (Table 8) (Lewalter 1994, Lewalter & Neumann 1996, 1998). Around 15 percent of Europeans suffer as a result of polymorphic CYP 2E1 from alcohol intolerance induced by the accumulation of the metabolic intermediate, acetaldehyde (Figure 10) (Lewalter 1994, Lewalter & Neumann 1996, 1998). The acetaldehyde formed as a metabolic intermediate inhibits various methylating enzymes and this causes not only an increase in the level of genotoxic O6-methylguanosine in the cells but, via increased HSA methylation, also significant IgE induction, the extent of which can be determined by means of the specific RAST test (Table 9).
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Figure 10: The interindividual variability of alcohol metabolism. Table 9: Consequences of alcohol-induced O6-MeGT inhibition on the extent of HSA and DNA methylation and the level of IgE (average values). Ethanol in blood [‰]
Smokers (N = 21) (e. g. hydrazineinduced methylation) Non-smokers (N = 27) (e. g. endogenous methylation)
0 87 0.8 (0.2–1.5) 112
0.7 2.5
0 0.8 (0.3–1.4)
0.4 1.3
164
Human serum albumin [N-methylaspar tic acid ng/l blood]
Lymphocytes IgE RAST [O6-methyl- [RU] guanosine lmol/mol guanosine]
Persons
65 93
Symptoms (subjective)
1.2 (1.0–1.4) none 1.9 (1.3–3.7) unspecific, general feeling of well-being impaired 0.8 (0.5–1.0) none 1.6 (1.1–2.3) unspecific, general feeling of well-being impaired
6.2 Immunological Effects of Polymorphic Key Enzymes Table 10: Immunodiagnostic parameters. Parameter Material
Function
Biomarker
IL-2
Plasma
Proliferation and differentiation of T cells, B cells and thymocytes Release of cytokines from T cells (CD4 cells) Activation of cytotoxic T cells Regulation of lymphocyte function
Indicator of individual immunomodulation
IL-6
Plasma
Induction of the differentiation of B cells into antibody-producing plasma cells Promotion of the production of the antibodies IgM, IgG and IgA Increase in the phagocytosis activity of monocytes and macrophages Induction of acute phase proteins in hepatocytes Together with IL-1, effects on the proliferation and cytokine production of T helper cells Together with IL-2, effects on the division and differentiation of cytotoxic T cells Together with IL-3, stimulation of stem cell proliferation
Induction of the antigen-specific immune response as a result of B cell stimulation Inflammatory reactions
TNFa (tumour necrosis factor)
Plasma
Increase in monocyte phagocytosis and cytotoxicity Increase in the expression of endothelial cells and granulocytes Amplification of the expression of MHC class 1 molecules on fibroblasts and endothelial cells Together with IL-1, support of the activation of antigen-stimulated T cells and the regulation of antibody production Induction of cytokine formation Necrosis in tumours
Essential for intact immune response Evidence of inflammatory and anti-carcinogenic effects of the immune system Checking the functioning of the immune system
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6.2.6 Evaluation of Immunological Competence As the immune system is essential for the elimination of pathogens and xenobiotics that have entered the organism, the evaluation of its ability to function is of great prognostic importance. It has recently become possible to carry out such functional tests, for example the determination of IgE and IgG according to a standard procedure to yield generally comparable results (Baenkler 1992, Bauer 1991, Dean et al. 1991, Lewalter 1994, Lewalter & Neumann 1996, 1998). Validation of the specific IgE RAST tests has to date not been possible as the allergens are not commercially available. Therefore every positive result obtained in the IgE RAST test should be verified by a specific inhibition test (Karol 1981, Karol 1992, Lewalter 1994, Vorlaender 1980). In addition to these generally known immunological biomarkers, a few other valuable parameters and their most important indications are listed in Table 10 (Baenkler 1992, Schleicher 1997, Vorlaender 1980). As a result of the increasing diagnostic importance of these immune parameters for monitoring the functioning of the immune system, the relevant test procedures must be validated according to the analytical criteria of the DFG.
6.2.7 Summary and Prospects Substance-specific IgE antibodies can be determined and quantified in the RAST. Generally, correlations are observed between the amount and the half-life of HSA adducts, the polymorphism of enzymes essential in the metabolism of the particular substance, positive results in the RAST and the clinical symptoms. To prevent hypersensitivity of the bronchial system being induced immunologically by both humoral (IgE, IgG) and cellular (T lymphocyte) mechanisms, it has been suggested that thresholds of about 20 ppb (isocyanates) be observed. Significantly increased IgE levels without verified exposure to xenobiotics can often be interpreted as the consequence of enzyme deficiencies. The test procedures used to determine the immune parameters must be validated according to the analytical criteria of the “Deutsche Forschungsgemeinschaft” (DFG).
166
6.2 Immunological Effects of Polymorphic Key Enzymes References Baenkler, H.-W.: Checkliste Immunologie, Georg Thieme Verlag, Stuttgart, 1–243 (1992). Bauer, P. C.: Obstruktive Atemwegserkrankungen durch allergisierende Stoffe, in: Konietzko, J.; Dupuis, H. (eds.): Handbuch der Arbeitsmedizin, 4. Erg. Lfg. 3/91, Kap. IV-5.4, S. 1–25 (1991). Dean, J. H.; Murray, M. J.: Toxic responses of the immune system, in: Amdur, M. O.; Doull, J.; Klaasen, C. D. (eds.): Casarett and Doull‘s Toxikologie: the basic science of poisons, 4. Edition, Pergamon Press, New York, 282–333 (1991). Diller, W. E.: Arbeitsmedizinische Gesichtspunkte bei der Begutachtung des “Isocyanat-Asthmas”, Arbeitsmed. Sozialmed. Präventivmed., 26, 393–398 (1991). Diller, W. E.: Quantitative Noxenabhängigkeit beim Berufsasthma, Allergologie, 13, 140–144 (1990). Diller, W. E.: Umwelteinflüsse bei der Induktion des Berufsasthmas aus der Sicht des Arbeitsmediziners, Zbl. Arbeitsmed., 47, 206–209 (1997). Goren, S.; Hellmann, W.: Prevalence of respiratory symptoms and diseases in schoolchildren living in polluted and in a low polluted area in Israel, Environ. Res., 45, 28– 35 (1988). Gubian, F.; De Zotti, R.: Smoking occupation, and allergic lung disease, Proc. Ann. Meeting Europ. Respir. Soc., Nizza, 82 (1994). Holt, P. G.: Environmental pollutants as co-factors in IgE production, Current Opinion in Immunology, 1, 643–646 (1989). Karol, M. H.: Occupational asthma and allergic reaction to inhaled compounds, in: Miller, K.; Türk, J.; Nicklin, S. (eds.): principles and practice of immunotoxicology, Blackwell Scientific Publication, London, 228–241 (1992). Karol, M. H.: Survey of industrial workers for antibodies to toluene diisocyanate, J. Occup. Med., 23, 741–747 (1981). Keller, R.: Immunologie und Immunpathologie: Eine Einführung, Gerorg Thieme Verlag, Stuttgart, 3. Aufl., 3–408 (1987). Kersten, W.; van Wahl, P. G.: Allergische Atemwegserkrankungen in der holzverarbeitenden Industrie, Allergologie, 17, 55–60 (1994). Kirchner, H.; Kruse, A.; Neustock, P.; Rink, L.: Cytokine und Interferone: Botenstoffe des Immunsystems, Spektrum Akademischer Verlag, Heidelberg, 11–295 (1993). Kucharz, E. J.; Sierakowski, S. J.: Immunotoxicity of chromium compounds: effects of sodium dichromate on the T cell activation in vitro, Arhiv za higijenu rada i toksikol, 38, 239–242 (1987). Lewalter, J.; Neumann, H.-G.: Biologische Arbeitsstoff-Toleranzwerte (Biomonitoring) Teil VIII: Bewertung der Hintergrundbelastungen bei beruflich nicht-exponierten Personen, Arbeitsmed. Sozialmed. Umweltmed., 31, 418–432 (1996); Teil XII: Die Bedeutung der individuellen Empfindlichkeit beim Biomonitoring, Arbeitsmed. Sozialmed. Umweltmed., 33, 352–364 (1998); Teil XIII: Die Bedeutung von Referenzwerten für die Bewertung von Fremdstoffbelastungen, Arbeitsmed. Sozialmed. Umweltmed., 33, 388–393 (1998). Lewalter, J.; Steinmann-Steiner-Haldenstätt, W.: Untersuchung der molekularbiologischen Konsequenzen des 4.4'-Methylenbiphenyldiisocyanat (MDI)-Umgangs, in: Kessel, R. (ed.), Verhandlungen der Deutschen Gesellschaft für Arbeitsmedizin und Umweltmedizin e.V.: 34. Jahrestagung, Wiesbaden, Gentner-Verlag, Stuttgart, 329– 334 (1994). Lewalter, J.: Monitoring und immunologische Bedeutung von Schadstoffaddukten, Berichte der österreichischen Gesellschaft für klinische Chemie (öGKC), 17, 9–18 (1994).
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6 Immunology Lewalter, J.: Immunologische Aspekte bei der Verarbeitung isocyanathaltiger Produkte im Bereich Tiefbau, in: Dokumentation der 4. Erfurter Tage, Monade-Verlag, Leipzig, 183–200 (1998). Neumann, H.-G.: Immunsuppressiva, in: Forth, W.; Henschler, D.; Rummel, W.; Starke, K. (eds.): Allgemeine und spezielle Pharmakologie und Toxikologie: Entstehung und Behandlung von Tumoren, BI Wissenschaftsverlag, Mannheim, 6. Aufl., 744–746 (1992). Newman-Taylor, A. J.: Environmental determinants of asthma, Lancet, 345, 296–299 (1995). Playfair, J. H. L.; Baron, D.: Immunologie, Blackwell Wissenschaftsverlag, Berlin, Wien (1995). Porter, C. V.; Higgins, R. L.; Scheel, L. D. A: Retrospective study of clinical, physiological and immunologic changes in workers exposed to toluene diisocyanates, Am. Ind. Hyg. Assoc. J., 36, 159–165 (1975). Reinke, P.; Döcke, W.-D.; Kox, W.; Zuckermann, H.; Volk, H.-D.: Neue Entwicklungen in der Immundiagnostik intensivmedizinischer Patienten, Dtsch. Med. Wschr., 124, 1527–1529 (1999). Schleicher, P.: Grundzüge der Immundiagnostik und -therapie, Hippokrates Verlag, Stuttgart, 2. Aufl., 11–206 (1997) Van den Plas, O.; Cartier, A.; Gliezzo, H.; Malo, J.-L.: Response to isocyanates, Am. Rev. Respir. Dis., 148, 226–235 (1993). Vorlaender, K. O.: Diagnostik unter Verwendung immunologischer Methoden, Georg Thieme Verlag, Stuttgart, 169–310 (1980).
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Epidemiology
7.1
Evaluation of Exposure in Epidemiological Studies Kurt Ulm *
7.1.1 Summary The evaluation of exposure is still one of the weak points of epidemiological studies. In occupational medicine, data for the concentrations of hazardous substances in the air are often lacking. The exposure levels are, therefore, estimated. A consequence of this imprecise evaluation of the exposure is the reduced correlation between the dose and the effects. Another problem is the adequate description of the exposure using a suitable parameter. The evaluation of the external exposure by means of biological monitoring has, at least in some situations, brought about an improvement. But even here it must be decided which sampling times are appropriate and which parameters for the exposure levels must be defined. Examples are presented below.
7.1.2 Introduction The aim of epidemiological studies is to investigate the relationships between a certain factor and a disease. In many studies the exposure to a factor is often categorized only by yes or no. This applies e.g. for the factor smoking, and even for substances used at the workplace. The exposed group is therefore a mixture of very different persons. There is usually a minimum requirement, e.g. exposure for over 1 year, for the exposure to a substance at the workplace to actually constitute exposure. The result, usually represented in the form of the relative risk, then depends on the distribution of the exposure levels. If most of the workers are exposed to low levels of the substance the relative risk will be about 1.0. In such a *
Institut für Medizinische Statistik und Epidemiologie, Technische Universität München, Ismaninger Str. 22, 81675 München
Biological Monitoring: Prospects in Occupational and Environmental Medicine. Deutsche Forschungsgemeinschaft (DFG) Copyright © 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-27795-7
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7 Epidemiology case it is unclear whether there is no relationship, or whether, as a result of the low exposure, no effects can be detected. An example of this is exposure to trichloroethylene. There are several cohort studies available in which the mortality of workers exposed to trichloroethylene was compared with that in the general population used as a surrogate for a control group of not exposed persons. The results differ considerably. The relative risks are between 1.0 (Antilla et al. 1995, Axelson et al. 1994) and around or above 8.0 (Henschler et al. 1995, Vamvakas et al. 1998). An explanation for this discrepancy could be the level of exposure. In the studies without an increased risk, the exposure was below 20 ppm, while in the studies with a markedly increased risk the values for exposure were probably above 1000 ppm. However, there are no data available. Therefore, the exposure levels in these studies can only be estimated.
7.1.3 Determining the Exposure The evaluation of exposure is one of the weak points of epidemiology. As a result of the long latency periods before chronic diseases become manifest, the levels of exposure in the earliest years are of decisive importance. Usually for this period there are, however, no reliable data available. In such cases often a job exposure matrix is drawn up (e.g. Checkoway et al. 1989). Here, relative exposures, also called scores or grades, are attributed to the various activities. These scores are usually allocated by calendar year(s). These scores are then used to try to describe the exposure for each worker. One approach is so-called cumulative exposure, in which the scores are multiplied with the corresponding duration of the activity and these values added together. In the evaluation, this cumulative exposure is then sub-divided into different intervals. An example is an analysis of exposure to TCDD related to the occurrence of coronary heart disease (Steenland et al. 1999). The cumulative exposure was divided into 7 classes. The results of the analysis are shown in Table 1. Only a slight increase in the risk with increasing exposure could be found. One of the most important questions in such analyses is the method of quantifying the exposure. Does the cumulative evaluation describe the exposure to an adequate extent? As an example, a study is analysed in which a relationship between sulfuric acid and laryngeal carcinomas was investigated (Soskolne et al. 1984). In this case-control study 43 patients with a laryngeal carcinoma and 151 controls were investigated. The exposure to sulfuric acid was evaluated qualitatively on the basis of 3 criteria: the concentration, the frequency of exposure and the reliability of the determination (Soskolne et al. 1992). All 3 criteria were graded from 0 to 3. On the basis of these 3 criteria five parameters, cumulative ex170
7.1 Evaluation of Exposure in Epidemiological Studies Table 1: Analysis of the dose-response relationship between TCDD and the occurrence of coronary heart disease (ICD 9; 410–414) (acc. Steenland et al. 1999; n = 3538 workers). Cumulative exposure
<19 19 – <139 139 – <581 581 – <1650 1650 – <5740 5740 – <20200 ≥ 20200 Total
Observed Expected numnumber of ber of deaths deaths
Standard mortality rate (observed/ expected)
95 % confidence interval
29 39 45 42 48 44 43
31.18 39.00 42.86 43.30 43.64 36.67 33.59
0.93 1.00 1.05 0.97 1.10 1.20 1.28
0.62–1.34 0.71–1.37 0.77–1.40 0.70–1.31 0.81–1.46 0.87–1.61 0.93–1.72
290
270.24
1.07
0.95–1.20
posure (CEI), average exposure (MG), maximum exposure (HG), timeweighted average (TWA), and exposure duration (TTE) were calculated. The two parameters MG and TWA differ in their denominator. For the MG, the cumulative exposure is divided by the duration of exposure, for the TWA by the whole period of employment. All 5 parameters were subdivided into 3 classes (no or low, moderate, high). Table 2 shows the results of the analyses of these 5 parameters. Alcohol, smoking habits and some accompanying diseases were taken into consideration in the calculation of the odds ratios. For 2 of the 5 parameters there is a clear positive relationship (MG and HG). For the other 3 parameters no influence or a negative influence of sulfuric acid was found. Here the question is evident: which is the “right” parameter? The choice can vary from case to case. The great differences in some cases are explained by the controls selected. The duration of employment was one of the criteria. For this reason, all parameters which directly include the duration cannot be used for the evaluation. The two parameters MG (= mean grade) and HG (= high grade) are therefore better suited for evaluating the relationship between sulfuric acid and laryngeal carcinomas. There are, however, other studies, in which the analysis of the different parameters leads to similar results (e.g. Ulm et al. 1999). Situations are conceivable in which the maximum or average exposure, or even the cumulative exposure are suitable. At present, however, there are no known selection criteria. Problematical in these analyses, above all with the use of the cumulative exposure, is the grouping of the workers in the different classes. During his working life, a worker belongs to various exposure classes. A worker from the highest exposure category, for example, spends part of his time 171
7 Epidemiology Table 2: Odds ratios (OR) and 95% confidence intervals (= 95% CI) for the different parameters for investigating a relationship between exposure to sulfuric acid and the occurrence of tumours of the upper respiratory tract (Suarez-Almazor et al. 1992). OR a
95% CI
25 94 32
0.58 0.70
0.22–1.50 0.22–2.18
4 24 15
32 92 27
2.89 5.20 b
0.74–11.27 1.23 – 22.09
HG no or low moderate high
3 37 3
27 113 11
4.69 b 2.40
1.03–21.33 0.29–20.15
TWA no or low moderate high
10 24 9
26 92 33
0.67 0.59
0.27–1.67 0.19–1.80
TTE no or low moderate high
9 27 7
27 89 35
1.15 0.55
0.43–3.10 0.17–1.74
Parameter
Cases
CEI no or low moderate high
11 22 10
MG no or low moderate high
a b
Controls
Comparison with the lowest exposure category. Statistically significant (p<0.05).
in every category. An increase in exposure can only take place if he is healthy enough for further employment. The higher exposure categories therefore represent a selection, which makes it more difficult to evaluate a dose-response relationship. There are evaluation methods that try to take this into account, but, as a result of their complexity, they are still rarely used (Robins et al. 1987). The determination of exposure levels is a first improvement towards describing the exposure. These values are usually the results of static determinations. These static determinations usually apply to a work area and still do not describe the exposure of a particular worker precisely enough. Personal sampling represents a marked improvement. This is the best way of determining external exposure. The more exactly the exposure can be described, the better its relationship to the effects can be shown. Personal sampling is, however, very complicated and is not carried out routinely. This procedure has, therefore, only found limited use to date for determin172
7.1 Evaluation of Exposure in Epidemiological Studies ing the exposure. Examples are e.g. with exposure to radon and electromagnetic fields. All the approaches mentioned are suitable for describing the external exposure. It is, however, not clear whether this really reflects the body burden. A few studies therefore attempt to correlate the internal exposure with the effects.
7.1.4 Evaluation of Exposure via Biological Monitoring In a few situations the internal exposure can be used as a measure of external exposure. An example of this is exposure to trichloroethylene. In the Scandinavian countries, workers exposed to trichloroethylene have been offered biological monitoring by the factories since 1955. The circumstances for the analysis are therefore particularly favourable. The determined values cover a long period and describe not only the conditions found today. Yet even here the same problems occur when the values have to be grouped together in a suitable way. Axelson et al. (1994), for example, report that both the number of determinations and the time they were made vary considerably. For this reason two parameters are used: the average and the maximum concentration. In a similar study from Finland (Antilla et al. 1995), the average concentration is used as the parameter. This study shows the high variability between the determined values, which in some cases reach values of over 10,000 lmol/l. The median is between 80 and 90 lmol/l. The problem of how to combine all the values in a meaningful way is, however, also apparent in these two studies.
7.1.5 Consequences The aim must be to evaluate the exposure as exactly as possible. Exposure is understood to be both the external exposure and particularly the internal exposure. The more precisely the exposure can be quantified, the clearer its relationship to a disease can be shown. If the exposure can be evaluated exactly, it will hopefully be possible to derive more precise threshold limit values. This applies above all for the internal exposure. In particular it must be borne in mind at what intervals determinations should be made. Furthermore, it must be decided which parameters are suitable for describing the exposure. In occupational medicine there is already much experience in these areas, but particularly in the field of dose monitoring there is still plenty that needs researching.
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7 Epidemiology References Antilla, A.; Pukkala, E.; Sallmen, M.; Hernberg, S.; Hemminki, K.: Cancer incidence among Finnish workers exposed to halogenated hydrocarbons. J. Occup. Med. 37: 797–806 (1995). Axelson, O.; Selden, A.; Andersson, K.; Hogstedt, C.: Updated and expanded Swedish cohort study on trichloroethylene and cancer risk, J. Occup. Med., 36: 556–562 (1994). Checkoway, H.; Pearce, Neil, E.; Crawford-Brown, Douglas, J.: Research Methods in Occupational Epidemiology. Oxford University Press (Monographs in Epidemiology and Biostatistics), Vol. 13, 1989. Henschler, D.; Vamvakas, S.; Lammert, M.; Dekant, W.; Kraus, B.; Thomas, B.; Ulm, K.: Increased incidence of renal cell tumors in a cohort of cardboard workers exposed to trichlorethene. Arch. Toxicol. 69, 291–299 (1995). Robins, J.: A graphical approach to the identification and estimation of causal parameters in mortality studies with sustained exposure periods. J. Chronic. Dis. 40 (suppl. 2), 139S–161S (1987). Soskolne, C. et al.: Occupational exposure to sulfuric acid associated with laryngeal cancer, southern Ontatio, Canada. Scand. J. Work. Environ, Health, 18, 225–232 (1992). Soskolne, D. L.; Zeighami, E. A.; Hanis, N. M.; Kupper, L. L.; Herrmann, N.; Amsel, J.; Mausner, J. S.; Stellman, J. M.: Laryngeal cancer and occupational exposure to sulfuric acid. Am. J. Epid. 120, 358–369 (1984). Steenland, et al.: Cancer, heart disease and diabetes in workers exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin. JNCI, 91, 779–786 (1999). Suarez-Almazor, M. E.; Soskolne, C. L.; Fung, K.; Jhangri, G. S.: Empirical assessment of the effect of different summary worklife exposure measures on the estimation of risk in case-referent studies of occupational cancer. Scand. J. Work Environ. Health, 18, 233–241 (1992). Ulm, K.; Waschulzik, B.; Ehnes, H.; Guldner, K.; Thomasson, B.; Schwebig, A.; Nuß, H.: Silica dust and lung cancer in the German stone, quarrying, and ceramics industries: results of a case-control study. Thorax, 54, 347–351 (1999). Vamvakas, S.; Brüning, T.; Thomasson, B.; Lammert, M.; Baumüller, A.; Bolt, H. M.; Dekant, W.; Birner, G.; Henschler, D.; Ulm, K.: Renal cell cancer correlated with occupational exposure to trichlorethene. J. Cancer Res. Clin. Oncol., 124, 374–382 (1998).
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7.2
Possibilities and Limitations of the Molecular Epidemiology of Workplace Exposures Kurt Straif *
Already at the beginning of the last century, laboratory tests were used in surveys. An early example was the use of the Wasserman test to describe the frequency of syphilis in an American city (Williams 1920). Even in the early phase of these “seroprevalence studies” or “seroepidemiology”, not only new possibilities but also new problems became apparent, such as the social stigmatization and discrimination of persons with (potentially false) positive test results (Roy 1996). Since the end of the 1980s there has been a considerable increase in the use of biomarkers in epidemiological studies (Hulka 1990 a). This development has been facilitated by current developments in laboratory technology and the methodological limitations of classical epidemiology when studying only slightly increased risks. In particular the rapid progress in laboratory technology in the meantime allows numerous biomarkers to be investigated quickly and cheaply even in large study collectives. These developments have led to the creation of the term “molecular epidemiology”.
7.2.1 Strengths and Weaknesses of Classical Epidemiology The strengths of classical epidemiology are demonstrated well by the archetype of epidemiological studies, the investigation of the cholera epidemic in London by John Snow in 1855. At the height of the great scientific debate between supporters of the miasma theory and supporters of the contagion theory, Snow, using epidemiological methods, was able to detect the cause of this epidemic – without identifying the then still unknown cholera bacillus – and by removing the pump handle from the contaminated drinking water pump was able to prevent the cholera epidemic from spreading further (Snow 1855). *
Institut für Epidemiologie und Sozialmedizin, Universität Münster, Domagkstr. 3, 48129 Münster
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7 Epidemiology In the criteria for the evaluation of carcinogenic substances, national and international epidemiological results in man are regarded as decisive for classification of the substance in category 1, i.e. for classification as a certain human carcinogen (IARC 1992, DFG 1999) – among other things because the interspecies extrapolation of the results of laboratory experiments or experiments with animals (which is associated with a certain degree of uncertainty) is not necessary for epidemiological studies. Critical points to remember in the evaluation of the validity of epidemiological studies are the various ways in which the results can potentially be distorted. A differentiation is made between • distortion as a result of selection (e.g. the “healthy worker effect” when comparing the mortality in an exposed cohort with the mortality in the general population); • distortion as a result of non-systematic or systematic misclassification (e.g. as a result of evaluating the exposure via the surrogate of the work area or obtaining the cause of death from death certificates); • distortion as a result of confounding (e.g. as a result of different smoking habits in exposed persons and persons not exposed).
Figure 1: True and observed risk relationship in a closed cohort as a function of sensitivity (SE) and specificity (SP) of the diagnostic test.
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7.2 Possibilities and Limitations of the Molecular Epidemiology Figure 1 shows an example of the effects of non-systematic misclassification of the variables in a closed cohort. A 10 percent reduction in the sensitivity and specificity of the diagnostic instrument has the effect that a relative risk of merely about 2.5 is observed in the study instead of the true relative risk of six. In addition to the validity, the precision of the effect estimator plays an important role in the evaluation of epidemiological studies. In particular for rare forms of cancer in small, highly exposed collectives, true increases in the risk may not be reflected in statistically significant changes in the risk estimators, as the statistical power of these studies is often insufficient. After checking the validity and the precision of individual epidemiological studies, further criteria must be included in order to evaluate whether an observed statistical relationship is a causal relationship (Evans 1995). An important criteria is the question of the consistency of the results of different epidemiological studies of different design and in different populations. While, for example, epidemiological studies in Caucasian populations find a relationship between exposure to aromatic amines and cancer of the bladder, epidemiological studies in Asian populations cannot detect such a relationship; this can be seen as evidence of an inconsistency in the results and the limited validity of some studies. Assuming the evaluated studies to be of sufficient validity and precision, the apparent inconsistency in the results can, however, also be explained by an effect modification, e.g. as a result of the different susceptibility of different ethnic groups.
7.2.2 Strengths and Weaknesses of Molecular Epidemiology The paradisal era of epidemiology, in which study results with large relative risks could be plucked from the trees like ripe fruit, is a thing of the past. The relative risks expected in current and future epidemiological studies are usually below 2.0, if not even below 1.5. This does not mean that the risks are of little importance to health. For example, the relationship between exposure to radon in the home and lung cancer shows that also low relative risks can have a high attributable risk with a sufficiently high prevalence of exposure (BEIR VI 1999). The multi-factor genesis of many chronic diseases and probable gene-environment interactions are an additional challenge for epidemiology. To obtain meaningful study results, in particular the critical points for validity must be minimized. Opening the “black box” (Figure 2) could provide some potentially successful approaches. The continuum between external exposure and clinical diagnosis is represented here as transition between categories of markers. It is in principle conceivable that the various biomarkers be investigated in the target tissue (for the target variable “lung cancer” therefore in bronchial cells) or 177
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Figure 2: The transition from classical to molecular epidemiology.
in surrogate cells (e.g. in peripheral leukocytes). It is decisive here to consider the availability of target tissue and the meaningfulness of investigations in surrogate cells, which depends among other things on the exposure investigated, the specific biomarker, and the half-life of the biomarker in surrogate tissue. Taking into account biomarkers for the internal dose, the biologically effective dose, early biological effects, functional or morphological changes, and susceptibility could potentially contribute the following to epidemiology: • the misclassification of exposure would be reduced by determination of the individual internal dose, • the misclassification of the target variables would be reduced by the molecular biological definition of various subtypes of diseases with potentially different aetiology, • earlier stages of disease would be identified and thus the duration of cohort studies could potentially be shortened, • pathomechanisms could be detected, • genetically determined susceptibility and effect modification could be better taken into account: in particular for diseases with a low penetrance, the methods of traditional epidemiology (such as the differentiation of different ethnic groups or the collection of the family history of diseases as a surrogate for genetically determined susceptibility) can be optimized by a genetic definition of susceptibility. Taking advantage of the potential of molecular epidemiology requires intensive interdisciplinary co-operation between epidemiology and toxicology, so that both important methodological standards of toxicology (e.g. laboratory standards) and also of traditional epidemiology (study design, 178
7.2 Possibilities and Limitations of the Molecular Epidemiology avoidance of systematic and non-systematic errors, consideration of statistical models) are taken into account adequately. Insufficient knowledge of the specific methods of the other scientific disciplines can otherwise easily lead to false results or the premature causal interpretation of statistical relationships. A prominent historical example of this was the hope invested in the determination of the level of CEA in serum for the early recognition of colorectal tumours (Ransohoff & Feinstein 1978). As reported in a publication of Bogardus et al., even current molecular epidemiological studies in renowned journals are found to have considerable methodological shortcomings. In the 1995 issues of the “Lancet”, “British Medical Journal”, “New England Journal of Medicine” and “Journal of the American Medical Association” the authors identified a total of 40 molecular-epidemiological articles in which more than ten patients were investigated and analytical conclusions were drawn. The following methodological aspects were tested in these articles: • Reliability and validity of the tests used (repeat tests in a subgroup or confirmation of the test results using another procedure) • Objectivity (investigator unaware of the patient‘s status) • Case definition (description of demographic details of the case group and clear criteria for target variables) • Adequate range of cases • Definition of the control group (inclusion criteria) • Adequate control group • Quantitative description of the results (confidence interval or p value). The results of the investigation are shown in Table 1. Only about a third of the studies investigated fulfilled the first two criteria, reliability and validity of the tests used and objectivity of the investigator.
Table 1: Number and percentage (%) of articles a judged to be satisfactory according to 7 methodological standards Methodological standards
Satisfactory articles Number (%)
Reliability of the test Objectivity Case definition Adequate range of cases Definition of the control group Adequate control group Quantitative description of the results
15 13 31 35 28 35 36
a
(37.5) (32.5) (77.5) (87.5) (70.0) (87.5) (90.0)
40 articles were investigated from the 1995 issues of the Lancet, New England Journal of Medicine, JAMA and BMJ, modified acc. Bogardus et al. (1999).
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7 Epidemiology In nearly a third of the studies the control group was inadequately described. If the authors had not classed another eight articles with an analytical approach as purely descriptive, as no control groups were investigated in these studies, their conclusions would have been even more critical.
7.2.3 Short Description of Biomarkers according to Various Classification Systems Biomarkers can be classified as shown in Figure 2 according to their function in the continuum between exposure and disease or according to their disease group. Table 2 classifies different biomarkers according to the former classification and lists reviews of the various markers. Table 3 shows the classification of biomarkers according to disease groups and lists reviews of the various markers. Table 2: Classification of biomarkers according to their function: examples and reviews Markers for
Examples
Reviews
Internal dose chemical substances, biological agents
Lead in blood Urinary mutagenicity Hepatitis serology
Coggon and Friesen 1997 Rynard 1990 Munoz and Bosch 1997
Biologically effective dose
Protein and DNA products
Wild and Pisani 1997, Goldring 2000
Early biological effects
Sister chromatid exchange, micronuclei, chromosomal aberrations
Tucker et al. 1997, Wilcosky and Rynard 1990, Vine 1990, Schwartz 1990 a
Changed structure Mutation in somatic cells, oncogenes Albertini and Hayes 1997, or function and tumour suppressor genes Schwartz 1990 b Disease
Tumour markers
Zhang et al. 1997
Susceptibility
Ah receptor gene Cytochrome P4501A1 Cytochrome P4501A2 Cytochrome P4502A subfamily Cytochrome P4502D6 Glutathione-S-transferase N-Acetyltransferases Further P450 cytochromes and other polymorphisms DNA repair
Garte and Sogawa 1999 Kawajiri 1999 Landi et al. 1999 Raunio et al. 1999 Wolf and Smith 1999 Strange and Fryer 1999 Hirvonen 1999 Stubbins and Wolf 1999
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Vineis and Malats 1999 Vine and McFarland 1990
7.2 Possibilities and Limitations of the Molecular Epidemiology Table 3: Reviews of biomarkers according to disease groups. Disease group
References
Infectious diseases Harrison and Griffin 1993 Cancer Perera and Santella 1993 Diseases of the immune system Vogt Jr. and Schulte 1993 Diseases of the nervous system Griffith and Garry 1993 Diseases of the cardio-vascular system Schulte et al. 1993 a Diseases of the respiratory organs Tockman et al. 1993 Liver diseases Tamburro and Wong 1993 Diseases of the urinary and genital organs Hemstreet Ill et al. 1993 Diseases of the skeleton/muscles/connective tissue Mastin et al. 1993 Hereditary diseases Khoury and Dorman 1993 Reproductive disturbances Lemasters and Schulte 1993
Depending on the specific questions asked and the study design, a particular biomarker can have a completely different epidemiological meaning – as an exposure variable, target variable or confounder. For example, in an investigation of the health risks represented by occupational exposure to polycyclic aromatic hydrocarbons (PAHs), specific DNA adducts can be defined as exposure variables, while lung cancer is the target variable. In the same context these DNA adducts can, however, also be regarded as target variables, if the relationship between PAH exposure and DNA adduct formation is to be investigated. It would also be conceivable in a study of the relationship between exposure to asbestos and lung cancer to regard PAH-specific DNA adducts as an indicator of smoking habits, in other words as confounders. In every concrete epidemiological study the exact function of the biomarker used should be clarified: in the example of the DNA adducts as exposure variables, the question should be asked whether the adducts should reflect as exactly as possible the individual biologically effective dose or serve as an indicator of workplace exposure. In the second case, factors which influence the individual formation of DNA adducts, e.g. genetically determined polymorphisms of PAH metabolism or different individual DNA repair capacities, would be regarded as confounders, while in the first case, at least theoretically, the DNA adducts reflect the individual, biologically effective dose with fewer misclassifications than determination of the hazardous substance at the workplace – as different individual susceptibilities are already taken into account as potential confounders.
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7.2.4
The Planning and Implementation of Molecular-Epidemiological Studies
Due to the limited space, descriptions of specific aspects of the planning and implementation of molecular-epidemiological studies must remain cursory; interested readers can find more detailed descriptions of specific aspects in the literature listed.
7.2.4.1 Laboratory Studies and Transitional Studies When planning molecular-epidemiological studies, the first thing to consider is the selection of the most suitable markers for the questions to be answered (Schulte & Perera 1993, Wilcosky 1990, Hulka 1990 b). In transitional studies, which include the development, characterization and evaluation of biomarkers, the biomarker to be investigated is usually the target variable (Schulte & Perera 1993, Harris 1991). In addition to the variability in laboratory data (Vineis et al. 1993, Vineis 1997) and the establishment of suitable quality controls (Wild & Pisani 1997, Gompertz 1997) also aspects of biological variability, short-term or long-term intraindividual and interindividual variability must be characterized (Vineis 1997, Hulka 1990 b). An overview of specific aspects which should be taken into consideration when setting up a specimen bank can be found in Winn & Gunter (1993). In an epidemiological study, the sampling, further processing, stability and storage of the biological material, and in particular also its transportation, must be carefully checked and planned (Landi & Caporaso 1997). Embedded reliability studies, depending on the questions to be answered and the study design, can illustrate particular individual critical steps, such as e.g. processing or storage of the samples or laboratory variability between different investigators, different laboratories or during the course of the study. The inclusion and analysis of standardized samples can provide evidence of specific problem areas in quality control. Alternatively, depending on the specific design of the reliability studies, they can quantify, quasi integratively, the total variability of all critical steps. Evidence of the effects of biomarker measurement errors in molecular-epidemiological studies can be found in White (1997) and Rothman et al. (1999).
7.2.4.2 Suitability of Certain Biomarkers for Different Study Designs Criteria for the suitability of certain biomarkers for different study designs can be found in several reviews (Schulte et al. 1993 b, Boffetta & Pearce 1999, Brennan 1999, Hunter 1997, Rothman et al. 1995). Case-control studies illustrate the design-specific advantages and disadvantages of molecular-epidemiological studies: in classical epidemiologi182
7.2 Possibilities and Limitations of the Molecular Epidemiology cal case-control studies, for methodological reasons, in particular for reducing possible selection effects, often a population-related case-control study is preferable. In molecular-epidemiological case-control studies, however, often hospital-based case-control studies are preferred: in view of the necessary sampling of biological material (blood, urine) in molecular-epidemiological studies, with hospital-based controls greater participation can be expected than with population-related controls. In addition, the necessary logistics, i.e. sampling, preparation and storage of the samples, can with population-related controls in some cases cause irresolvable problems, while with hospital-based controls the necessary sampling of biological materials can often be integrated elegantly into the usual sampling routine and rapid processing of the samples – without long transport routes in between – can usually be guaranteed also with relatively little extra effort. For cancer and other chronic diseases with longer latency periods, the molecular-biological determination of exposure in case-control studies is often problematical, as the exposure markers available today often determine only the current exposure, but cannot provide any information about the relevant exposure ten or more years ago. Exposures which can change over the course of time, such as e.g. smoking habits, can be determined more exactly with standardized questionnaires than with the molecularbiological markers available today. On the other hand, exposure to human papilloma virus (HPV) as a risk factor for cervical carcinomas can be detected even in case-control studies more exactly via virus serology than indirectly via standardized questionnaires about the frequency of sexual intercourse, partner changes etc. Early morphological or functional changes are, as long as there is no suitable register, not suitable as the target variables of case-control studies. On the other hand, genetically determined markers of susceptibility can be used validly and efficiently in case-control studies. Methodological considerations about the use of biomarkers in cohort studies can be found in a review by Hunter (1997). Specific particularities of non-traditional epidemiological study designs, such as case-case studies investigating gene-environment interaction (Khoury & Flanders 1996, Andrieu & Goldstein 1998) or family-based linkage studies cannot be taken into account here.
7.2.4.3 Statistical Analysis of Molecular-Epidemiological Studies Suitable statistical tests must be used. Errors often observed are, for example, that tests which presuppose normal distribution of the determined values are used for data which is not distributed normally or that statistical tests which presuppose that the individual observations are independent of one another are used with repeated determinations of biomarkers in a collective, in other words with serially correlated data which is not statistically independent (Stover Hertzberg & Russek-Cohen 1993). 183
7 Epidemiology The correlation coefficients frequently presented in molecular-epidemiological studies are not a suitable substitute for the effect estimators used in epidemiology, such as the Odds ratio or relative risk (Greenland et al. 1991). Standardization or adjustment of the results for classical risk factors and potential confounders is often neglected as a result of a lack of data or the small number of cases involved. On the other hand, with multivariate analysis the potential multi-colinearity of various variables must be taken into account. The fact that various intermediate steps on the way from exposure to disease are investigated (which leads to the inclusion of intermediate variables in the analysis), can result in the analytically problematical situation that a variable is both an intermediate variable and a confounder (Robins et al. 1992). The possibility of investigating an ever increasing number of biomarkers quickly and cheaply in a single study – in some cases without a clear hypothesis to be researched – will intensify the discussion of the question of the statistically correct method of treatment of multiple comparisons and the problem of publication bias.
7.2.4.4 Estimation of the Cohort Size of Molecular-Epidemiological Studies The enormous costs in some cases or the large number of personnel needed for the laboratory analysis of biomarkers often leads to compromises in the cohort size. On the other hand, particularly the investigation of polymorphisms, which is often only meaningful for investigating geneenvironment interaction, or the investigation of gene-gene interactions, call for a particularly large collective to achieve sufficient statistical power (Hulka 1990b, Hwang et al. 1994, Cuzick 1999).
7.2.4.5 Interpretation and Communication of the Results of Molecular-Epidemiological Studies The protection of personal data is of particular importance in molecularepidemiological studies. Which investigations may be carried out in stored biological material without the additional written consent of the patient? If during a study a new susceptibility marker is identified, how should the individual test result be communicated to the test person (Schulte 1993)? Such, possibly even provisional, results can lead to considerable disruption in a patient‘s private and family life, and to discrimination when taking out health and life insurance and at the workplace (Hainaut & Vahakangas 1999, Schulte et al. 1999).
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7.2.5 Summary This article gives an overview of specific aspects of molecular epidemiology. There are two recurring themes. Molecular epidemiology has the potential to generate important new insights; inherent problems specific to molecular epidemiology must, however, be carefully taken into account. Recommendations for methodological standards: 1. Evaluation of biomarkers in transitional studies: • short-term and long-term, intraindividual and interindividual variability, • background levels, • drawing up of specific check-sheets for individual biomarkers (sampling, processing, storage, confounders). 2. Observation of “good epidemiological practice”: • study design, • minimization of bias and confounding, • adequate statistical models, • sufficient statistical power, • quality controls: handling of the samples, blind investigations, monitoring of experimental drift, reliability and validity studies. 3.
Storage of histological sections and tumour blocks.
4. Natural history of disease • storage of healthy tissue, benign lesions, premalignant changes, possibly material of the contra-lateral organ. 5. Simultaneous investigation of various biomarkers in individual collaborative studies with the exchange of standards and biological samples: • chemical-specific and general markers, • markers of current and historical exposure, • markers for early clinical effects and susceptibility. Recommendations for research projects: 1.
selection of biomarkers on the basis of biological relevance,
2.
setting up of specimen banks from clearly defined cohorts,
3.
validation of exposure-specific mutation patterns,
4. investigation of model populations (e.g. cancer patients with highdose chemotherapy treatment and a high risk of secondary tumours), 185
7 Epidemiology 5. parallel studies with man and animals using the same biological markers to optimize inter-species extrapolation: pathomechanisms, exposure responses. Intensive interdisciplinary collaboration between geneticists, toxicologists and epidemiologists is an important prerequisite for the success of molecular-epidemiological projects. Let me finish with the words of Geoffrey Rose: ”Just as epidemiologists need to look inside the black box, so the molecular biologists need to look outside it” (Rose 1990). References Albertini, R. J.; Hayes, R. B.: Somatic cell mutations in cancer epidemiology, in: Application of biomarkers in cancer epidemiology, Lyon: IARC scientific publication no. 142, 159–184 (1997). Andrieu, N.; Goldstein, A. M.: Epidemiologic and genetic approaches in the study of gene-environment interaction: an overview of available methods. Epidemiol. Rev., 20, 137–147 (1998). Beir, V. I.: Health risks of exposure to radon. Washington: National Academy of Sciences (1999). Boffetta, P.; Pearce, N.: Chapter 10. Epidemiological studies on genetic polymorphism: study design issues and measures of occurrence and association, in: Metabolic polymorphisms and susceptibility to cancer. Lyon: IARC scientific publication no. 148, 89– 96 (1999). Brennan, P.: Design and analysis issues in case-control studies addressing genetic susceptibility, in: Metabolic polymorphisms and susceptibility to cancer. Lyon: IARC scientific publication no. 148, 123–132 (1999). Coggon, D.; Friesen, M. D.: Markers of internal dose: chemical agents, in: Application of biomarkers in cancer epidemiology. Lyon: IARC scientific publication no. 142, 95– 101 (1997). Cuzick, J.: Interaction, subgroup analysis and sample size, in: Metabolic polymorphisms and susceptibility to cancer. Lyon: IARC scientific publication no. 148, 109–121 (1999). DFG: MAK- und BAT-Werte-Liste (1999): Senatskommission zur Prüfung gesundheitsschädlicher Arbeitsstoffe, Mitteilung 35. Weinheim: Wiley-VCH Verlag GmbH (1999). Evans, A. S.: Causation and disease: a chronological journey. The Thomas Parran Lecture, 1978. Am. J. Epidemiol., 142, 1126–1135 (1995). Garte, S.; Sogawa, K.: Ah receptor gene polymorphisms and human cancer susceptibility, in: Metabolic polymorphisms and susceptibility to cancer. Lyon: IARC scientific publication no. 148, 149–157 (1999). Goldring, J. M.: Protein and DNA adducts, in: Biological markers in epidemiology, edited by Hulka, B. S. et al. New York Oxford: Oxford University Press, 78–104 (2000). Gompertz, D.: Quality control of biomarker measurement in epidemiology. In: Application of biomarkers in cancer epidemiology. Lyon: IARC scientific publication no. 142, 215–222 (1997). Greenland, S.; Maclure, M.; Schlesselman, J. J.; Poole, C.; Morgenstern, H.: Standardized regression coefficients: a further critique and review of some alternatives. Epidemiology, 2, 387–392 (1991). Griffith, J. D.; Garry, V. F.: Neurologic disease, in: Molecular epidemiology: principles and practices, edited by Schulte, P. A.; Perera, F. San Diego, New York, Boston, London, Sydney, Tokyo, Toronto: Academic Press, 497–516 (1993).
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Summary Jürgen Angerer * and Helmut Greim **
The term biological monitoring is understood to mean the determination of hazardous substances, their metabolites, their adducts with protein and DNA or other parameters of intermediate metabolism in body fluids. Biological monitoring is for occupational and environmental medicine a valuable way of protecting the individual against the effects of hazardous substances at the workplace (protection of the individual against disease). After its early beginnings in the 1930s, biological monitoring has been used in occupational and environmental medicine since the mid 1960s on an ever increasing basis. The progress in biological monitoring is closely linked with the corresponding progress in the analytical techniques. Germany used biological monitoring earlier and subsequently more consistently than other countries for the prevention of damage to the health from chemical substances. Since 1975 the DFG Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area has promoted the development of suitable, valid and tested analytical methods for biological monitoring. In the meantime a collection of analytical methods has been produced that is like no other world-wide. In 1979 the DFG commission was the first to begin to evaluate tolerable threshold limit values for the parameters of biological monitoring in the form of BAT values (Biological Tolerance Values for Occupational Exposures) and EKA correlations (Exposure Equivalents for Carcinogenic Substances). The USA then followed our example. In 1984 the German Society of Occupational Medicine and Environmental Medicine began to check the quality of the results of biological monitoring in intercomparison programmes. Today the most extensive intercomparison programmes for occupational-medical and environmental-medical, toxicological investigations are organized in Germany. In Germany it was not only the basis for biological monitoring that was created. Unlike in other countries, biological monitoring forms part of ** Institut und Poliklinik für Arbeits-, Sozial- und Umweltmedizin, Universität Erlangen-Nürnberg, Schillerstr. 25, 91054 Erlangen ** Technische Universität München, Institut für Toxikologie und Umwelthygiene, Hohenbachernstraße 15–17, 85354 Freising-Weihenstephan Biological Monitoring: Prospects in Occupational and Environmental Medicine. Deutsche Forschungsgemeinschaft (DFG) Copyright © 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-27795-7
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8 Summary the legislation on health and safety at work. For this reason we also have the greatest practical experience of the use of biological monitoring in occupational and environmental medicine. Whether Germany can hold on to its leading role in this field now depends on whether it is possible to use and develop the new possibilities offered by effect monitoring. For a long time biological monitoring was limited mainly to dose monitoring, in other words to the detection of the hazardous substance and its metabolites in biological materials. Today there are other possibilities available. For example biochemical effect monitoring, i. e. the determination of e. g. products of the reaction of mutagenic substances with protein and with DNA. We speak of biological effect monitoring when the first effects on the human organism of exposure to hazardous substances are detected. Such biological effects can be changes in enzyme activity after exposure to hazardous substances, early effects on the kidneys, lungs, nervous system etc. but also the chromosome structure and chromosome count, micronuclei or sister chromatid exchange. As far as the diagnostic validity of immune parameters in relationship with exposure to hazardous substances is concerned, we are still at the beginning of a possible development. The measurable parameters of biological monitoring (dose and effect monitoring) are modulated by the interindividually different ways of making a substance more and less toxic. Enzyme polymorphisms are the reason for the differences in the metabolic performance of xenobiotic-metabolizing enzymes. The metabolic performance is also dependent on the induction and also the inhibition of enzymes. By determining hazardous substances and their metabolites in body fluids, the dose of the substance taken up by the individual can be evaluated specifically and sensitively (internal exposure, dose monitoring). Thanks to the methods of instrumental analysis available today, it is possible to detect many substances down to the concentration ranges relevant to environmental medicine. The spectrum of parameters today includes metals, organic solvents, persistent and non-persistent pesticides, aromatic amines and aromatic nitro compounds, polycondensated aromatic hydrocarbons etc. Nevertheless, this parameter spectrum must be continuously extended. For example, for many pesticides there are no suitable methods of dose monitoring. In this context also the metabolism of many organic substances, such as e. g. pesticides, must be clarified in man. The aim here is to find those metabolites which are closest to the health risk. With the help of metabolic profiling, in addition to the ultimate toxic metabolite, also interindividually different susceptibilities should be recognizable. In biochemical effect monitoring we have learnt to determine the products of the reaction of different groups of mutagenic substances with haemoglobin (so-called haemoglobin adducts). These include, above all, alkylating substances, aromatic amines, aromatic nitro compounds, PAHs and various individual substances, such as dimethylformamide, benzoquinone etc. Haemoglobin adducts are regarded today from a toxicological point of view as a surrogate for the products of the reaction of mutagenic substances with DNA. 192
8 Summary It is already possible to carry out first quantitative estimations of the cancer risk on the basis of the haemoglobin adduct concentration in blood. This is possible on a group basis, for example for aromatic amines, for whom the cancer risk can be estimated from the amount of the dose taken up which is bound to haemoglobin. This is based on the adduct concentration which in experiments with animals led to a 50% cancer risk (TD50). The possibilities of determining haemoglobin adducts must be extended beyond the substance groups listed above. The adduct levels also reflect the interindividually different enzyme polymorphisms, which themselves also have a great influence on the individual cancer risk. The effects of enzyme systems on the toxicity of substances can be detected globally, but quantitatively in the adduct level. If one assumes that the binding of mutagenic substances to DNA is an early stage in the process of chemical mutagenesis and carcinogenesis, the evaluation of DNA adducts is of great occupational-medical and environmental-medical importance. That increased DNA adduct levels reflect not only increased exposure to hazardous substances, but also the interindividually different cancer risk, was shown using the example of occupational exposure to PAHs or that resulting from tobacco smoking. The determination of DNA adducts of carcinogenic substances is therefore an excellent instrument for recognizing and minimizing the cancer risk to the individual. Of the methods used so far for determining DNA adducts, 32Ppostlabelling is the most sensitive. However, this procedure has its limitations; it does not allow the substance-specific detection of the substances in question. This is due not least to the low chromatographic resolution offered by thin-layer chromatography. The combination of 32P-postlabelling and high-pressure liquid chromatography is an improvement, as this chromatographic method has a better resolution. Such procedures have been used successfully e. g. to determine the adducts of PAHs and of heterocyclic amines. Nevertheless, the detection limits possible with this method are evidently not low enough for it to be used to determine DNA adducts in peripheral lymphocytes. When investigating exposed groups of persons it must be checked whether there is a relationship between the DNA adduct concentration and the occurrence of tumours. The knowledge of such a relationship could be used for the specific prevention of cancer. A special case among the DNA adducts are the nucleosides caused by oxidative stress. Unlike the DNA adducts, they are, however, unspecific to the causative agent. They can be used in the prevention of disease if at the same time the exposure to the hazardous substances is specifically determined. In this way also the diagnostic meaningfulness of oxidatively changed nucleosides must be validated. Indispensable for both dose monitoring and the determination of protein and DNA adducts is progress in the sector of instrumental chemical analysis. ICP-MS should be included in the determination of metals. This method is suitable, for example, for the determination of refractory ele193
8 Summary ments such as tungsten, which plays a great role in the formation of hardmetal fibrosis. New separation techniques, coupled with atomic absorption or ICP-MS, will allow metal species to be determined which, as a result of their different toxicity, are of great occupational-medical and environmental-medical importance. The possibilities offered by the combination LC/ MS/MS is the prerequisite for the determination of protein adducts, and above all DNA adducts, specifically and sensitively. To be able to detect DNA adducts in the range of the necessary detection limit, new enrichment phases, column arrangements etc. are needed. This requires the use of tailor-made chromatographic phases such as restricted access material (RAM) or molecular imprinting (MIP). Interindividually different susceptibility leads to different health risks for the same exposures. Enzyme polymorphisms are therefore of great importance in the prevention and evaluation of health risks caused by exposure to hazardous substances. For example, persons with low N-acetyltransferase activity have a greater risk of developing bladder carcinomas after exposure to aromatic amines than fast acetylators. In addition to the acetyltransferases, above all cytochrome P 450-dependent oxidases, glutathione transferases and sulfatases are investigated in occupational medicine and environmental medicine. While today the various genotypes can be determined relatively easily with the PCR technique, much more complicated methods are necessary for the determination of the phenotype. The procedures for the determination of the genotype have been constantly improved. The determination of the phenotype is, however, imperative for evaluating the actual extent of enzyme activity. Research is therefore necessary above all into the factors which modulate the genetically determined enzyme status (e. g. induction, inhibition etc.). The development of practicable and standardized methods of biological monitoring for evaluating the individual enzyme activity is therefore necessary. As different enzyme systems, depending on the substrate, can have more toxic or less toxic effects, it must be discovered, for example, for the sulfotransferases which substances are made more toxic or less toxic by which sulfotransferases. In addition, it must be investigated whether other polymorphisms are present and this knowledge used to evaluate relationships between the activities of the enzymes and the health risks. For the prevention of disease, risk groups must be identified. Above all, the importance of enzyme polymorphism in the evaluation of the individual case must be investigated. Cytogenetic parameters, such as chromosomal aberrations, cytogenetic indicator effects, such as sister chromatid exchange and micronuclei, and procedures for detecting chromosomal rearrangements, such as chromosome in-situ suppression hybridization (CISS = chromosome painting), or even molecular-genetic techniques, such as the comet assay, provide ways of quantifying the effects of mutagenic substances on the germ plasm. Compared to the classical cytogenetic parameters mentioned earlier, chromosome painting offers the advantage of being able to detect long-lived 194
8 Summary reciprocal translocations, which are of great importance for the prevention of adverse effects on the health. The comet assay has the advantage that it is particularly practicable and can detect the widest spectrum of parameters for DNA damage of different genesis. It must, however, be borne in mind that the comet assay can only detect short-term previous exposure and is generally to be regarded as a dose parameter. It must also be noted that the comet assay is influenced by many peripheral factors. The comet assay is of greater diagnostic validity, for example, with the use of certain endonucleases, which allow the damage to be evaluated specifically. The comet assay can also be used for the determination of the repair capacity. Common to all other genetic parameters is that to date they have not been systematically integrated in the biological monitoring of hazardous substances at the workplace or in the environment, so that their diagnostic potential in this area could be neither evaluated nor exploited. With regard to the prevention of damage to the health, this is lamentable. Also allergens are evidently subject to a dose-response relationship. As a result of a lack of data in the fields of occupational medicine, occupational hygiene and environmental medicine, little use could be made of this fact to date. On the basis of a dose-response relationship, threshold limit values could, however, be defined with which the health of workers exposed to allergenic substances could be effectively protected. The reason this is not possible at present is that when sensitizing effects occur the external and internal exposure to the allergen is usually not quantitatively determined. With exposure to substances of high molecular weight it is also not possible, for purely analytical reasons. It is possible, however, to determine haptens of low molecular weight in the air and in body fluids with the methods available today. Examples of this are acid anhydrides, platinum salts, isocyanates etc. With sensitization to such substances it should be possible to use antibodies of IgG type as part of biological monitoring as a marker of exposure or of an effect. To be able to answer this question it must be investigated whether there is a relationship between the level of exposure to the allergen and the level of IgG. It must be clarified above all whether the level of exposure is a risk factor for sensitization. On the basis of such investigations it should be possible to derive threshold limit values for allergens with which the risk of disease can be reduced. During such investigations it should also be tested whether substance-specific IgE antibodies are suitable as biological effect markers. IgE antibodies can be analytically determined today using e. g. the RAST. It is immediately clear that biochemical effects, and to an even greater extent biological effects, are closer to the ultimate damaging principle and thus the effects of the toxic substance than dose monitoring. This means the predictive value of biological monitoring parameters increases from dose monitoring to effect monitoring. Problematical is, however, that at the same time the substance specificity decreases towards effect monitoring. With many effect parameters, such as chromosomal aberrations or DNA strand breaks, it can no longer be seen whether they occurred as a result 195
8 Summary of exposure to polyaromatic hydrocarbons or to aromatic amines etc. It is the other way round with dose monitoring. Here the hazardous substance can be determined specifically, but without providing any direct information about the effects. The task of epidemiology is to investigate relationships between exposure to hazardous substances, biochemical and biological effects and the occurrence of damage to the health. It must also be clarified which role the sensitivity of the employees plays in the development of diseases. The evaluation of the exposure has always been one of the weak points of epidemiological studies. Often there are not even determinations available of the chemical substances in the workplace air or in other environmentallyrelevant media. The description of the exposure situation according to the yes/no principle or with scores has proved to be unsuitable in many studies. Even rarer are epidemiological studies in which the internal exposure or the resulting body burden are determined. Studies in which data for dose monitoring, effect monitoring and if possible effects on the health can be linked using epidemiological methods, are therefore urgently needed. With such a relationship the advantages and disadvantages of the individual biological monitoring parameters can be exploited or compensated for. By identifying the parameter with the greatest diagnostic validity it should be possible not only to improve the prevention of damage to the health, but at the same time to limit the effort needed. By determining the dose of the hazardous substance taken up or the resulting biochemical and biological effects, epidemiological investigations could provide more precise information about the effects on the health of exposed persons. With the use of epidemiological methods in biological monitoring it should be possible to clarify pathomechanisms, to recognize the early stages of diseases and also to include the genetically determined susceptibility in the estimation of the risk. However, if epidemiological methods are to be used in biological monitoring, the methodological shortcomings often observed (validity of the test, power of the study, selection of the control group etc.) must be excluded. The prerequisite for successful research in the whole field of biological monitoring are analytical results which are comparable i. e. from laboratory to laboratory. This means that conditions must be created which lead to the minimization of possible influences on the analytical result. In the pre-analytical phase, which begins with the preparation of the test persons and ends with the preparation of the biological samples in the laboratory, all the conditions for sampling, storage of the samples, sampling time etc. must be standardized. The analytical phase, which takes place in the laboratory, is accompanied by an effective internal and external quality control system. Important prerequisites are, for example, reference substances and control materials. The availability of reference substances represents an ever growing problem for biological monitoring. Chemists involved in the preparation of samples should therefore be included in the research in this 196
8 Summary area. Detailed descriptions of the complete analytical procedures, so-called standard operating procedures (SOPs), lead to the further reduction of possible influences on the analytical result. As far as the quality control of the analytical procedure in this field is concerned, the most progress has been made in dose monitoring (see above). In the case of the biochemical effect markers and biological effect markers, however, we are still at the beginning of such a development. Such activities must therefore be included in appropriate research projects. The validity and reproducibility of all analytical procedures used in such studies must be checked according to the criteria of the DFG commission. Research in the field of biological monitoring calls for the collaboration of the different disciplines such as in particular toxicology, occupational medicine, environmental medicine, allergiology, human genetics, analytical and synthetic chemistry, epidemiology etc. As in these disciplines only a small number of highly specialized scientists work in the sector of exposure to hazardous substances, collaboration at a national level is necessary, as this cannot be achieved within one university. Today and in the future, the aim of biological monitoring is to improve the prevention of diseases caused by hazardous substances and at the same time to allow fundamental progress to be made in the different disciplines, such as in particular toxicology, occupational and environmental medicine, allergology, chemistry and epidemiology. In addition, relationships must be established between the parameters of biological monitoring and early indicators of disease. To this end, exposed persons and groups from the general population must be investigated. Epidemiological expertise is required during the planning of the studies and the evaluation of the results. In this context, the following can be regarded as biological monitoring parameters: • • • • • • •
the concentration of the substance and its metabolites in body fluids, products of intermediary metabolism, protein and DNA adducts, genotypes and phenotypes, immunological parameters, cytogenetic parameters, early toxic effects, e. g. on the kidneys, nervous system, lungs, etc.
For this purpose • Analytical procedures must be drawn up and validated with which further biological monitoring parameters can be determined. This applies not only to the whole area of dose monitoring, but above all also to effect monitoring, and immunological and susceptibility parameters. • Progress must be made in the preparation of the biological material and enrichment. 197
8 Summary • New methods of instrumental analysis must be integrated into biological monitoring. This applies above all to ICP-MS and LC/MS/MS, but also to techniques such as capillary electrophoresis/MS etc. It must be guaranteed in all studies that the data collected are comparable. This is the prerequisite for deducing threshold limit values. To achieve this aim • Existing biological monitoring procedures must be validated and their reproducibility checked, if this has not already been done by the DFG commission. This applies in particular for “new” biological monitoring parameters. • Standardization must be carried out in the pre-analytical phase. This applies to the preparation of the test person for sampling, the sampling procedure, storage of the samples etc. • Reference substances must be synthesized. • Measures of internal and external quality control must be carried out based on appropriate control and reference materials.
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Authors and Participants
Prof. Dr. J. Angerer Institut für Arbeits-, Sozial- und Umweltmedizin der Universität Erlangen-Nürnberg Schillerstraße 25/29 91054 Erlangen 09131/852-6131 Fax: 09131/852-6131
[email protected] Prof. Dr. H. Bartsch Deutsches Krebsforschungszentrum Institut für Toxikologie und Chemotherapie Im Neuenheimer Feld 280 69120 Heidelberg 06221/42-3301 Fax: 42 33 59
[email protected] Prof. Dr. Dr. H. M. Bolt Institut für Arbeitsphysiologie Abt. Toxikologie und Arbeitsmedizin der Universität Dortmund Ardeystraße 67 44139 Dortmund 02 31/10 84-3 48 Fax: 02 31/10 84-3 08
[email protected]
Prof. Dr. H. Drexler Institut für Arbeits-, Sozial- und Umweltmedizin der Universität Erlangen-Nürnberg Schillerstraße 25/29 91054 Erlangen 09131/852-2312 Fax: 09131/852-2317
[email protected] Prof. Dr. E. Gebhart Univ.-Inst. f. Humangenetik Schwabachanlage 10 91054 Erlangen 09131/8522351 Fax: 0 91 31/20 92 97 Egebhart@humgenet. uni-erlangen.de Prof. Dr. H. R. Glatt Deutsches Institut für Ernährungsforschung Abt. Ernährungstoxikologie Arthur-Scheunert-Allee 114–116 14558 Bergholz-Rehbrücke 033200/88-321 Fax: 033200/88-426
[email protected]
Biological Monitoring: Prospects in Occupational and Environmental Medicine. Deutsche Forschungsgemeinschaft (DFG) Copyright © 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-27795-7
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9 Authors and Participants PD Dr. K. Golka Institut für Arbeitsphysiologie an der Universität Dortmund Ardeystraße 67 44139 Dortmund 02 31/10 84-3 44 Fax: 02 31/10 84-3 08
[email protected] Prof. Dr. H. Greim Technische Universität München Institut für Toxikologie und Umwelthygiene Hohenbachernstraße 15–17 85354 Freising-Weihenstephan 0 81 61/71 56 00 Fax: 0 81 61/71 56 18
[email protected] Prof. Dr. E. Hallier Georg-August-Universität Göttingen Zentrum Umwelt- und Arbeitsmedizin Abteilung Arbeits- und Sozialmedizin Waldweg 37 37073 Göttingen 05 51/39-49 50 Fax: 39-61 84
[email protected] Prof. Dr. D. Henschler Institut für Toxikologie der Universität Würzburg Versbacher Str. 9 97078 Würzburg 09 31/2 01-39 84 Fax: 09 31/2 01 39 84
[email protected]
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Dr. Beate Konze-Thomas Deutsche Forschungsgemeinschaft Kennedyallee 40 53175 Bonn 02 28-885-22 43 Fax : 02 28-885-27 28
[email protected] Dr. J. Lewalter BAYER AG WV-LE ärztliche Abteilung Institut für Biologisches Monitoring Gebäude L 9 51368 Leverkusen 0214/30-65679 Fax: 0214/30-21307 Zentr.: 0214/30-0
[email protected] Prof. Dr. W. K. Lutz Institut für Toxikologie der Universität Würzburg Versbacher Str. 9 97078 Würzburg 09 31/2 01-48402 Fax: 09 31/20 14 84 46
[email protected] Apotheker M. Maisch Institut für Toxikologie der Universität Würzburg Versbacher Str. 9 97078 Würzburg Dr. M. Müller Georg-August-Universität Göttingen Abteilung für Arbeits- und Sozialmedizin Waldweg 37 37073 Göttingen 05 51/39-80 44 Fax: 39-6184; 05 51/39-4950
[email protected]
9 Authors and Participants Prof. Dr. G. Obe Universität Essen 45117 Essen 02 01/1 83-33 88 Sekr. 1 83-28 66
[email protected] PD Dr. W. Pfau Universitätsklinikum HamburgEppendorf Institut für klinische und experimentelle Toxikologie Vogt-Kölln-Str. 30 22527 Hamburg 0 40/42 83 2127
[email protected] Prof. Dr. A. W. Rettenmeier Universitätsklinikum der GH Essen Institut für Hygiene und Arbeitsmedizin Hufelandstr. 55 45147 Essen 02 01/7 23-45 74 02 01/7 23-45 75 Fax: 02 01/7 23-59 11
[email protected] Prof. Dr. G. Sabbioni Walther-Straub-Institut für Pharmakologie und Toxikologie Ludwig-Maximilian-Universität München Nussbaumstr. 26 D-80336 München Tel./Fax: 089/51 60 72 65
[email protected] Prof. Dr. G. Speit Universitätsklinikum Ulm Abteilung Humangenetik Albert-Einstein-Allee 11 89070 Ulm 0731/50 02 34 29 Fax: 07 31/50 02 34 38
[email protected]
Dr. B. Spiegelhalder Deutsches Krebsforschungszentrum Institut für Toxikologie und Chemotherapie Im Neuenheimer Feld 280 69120 Heidelberg 0 62 21/42-52 33 05 Fax: 0 62 21/42-33 08
[email protected] PD Dr. K. Straif, M.P.H. Institut für Epidemiologie und Sozialmedizin der Universität Münster Domagkstraße 3 48129 Münster 02 51/83 55-3 98 Fax: 02 51/83 55-3 00
[email protected] PD Dr. Ricarda Thier Institut für Arbeitsphysiologie Universität Dortmund Ardeystraße 67 44139 Dortmund 02 31/10 84-3 56 Fax: 02 31/10 84-4 03
[email protected] Prof. Dr. K. Ulm Institut für Medizinische Statistik und Epidemiologie der TU München Ismaninger Str. 22 81675 München 0 89/41 40-43 23 Fax: 41 40-43 20
[email protected] Prof. Dr. M. Wilhelm Ruhr-Universität Institut für Hygiene, Sozial- und Umweltmedizin Universitätsstr. 50 44801 Bochum 02 34/3 22-23 65 Fax: 02 34/32 14-199
[email protected] 201
