Envir onmental cancer risk, nutrition and individual susceptibility
Edited by
Peter B. Farmer, Jean M. Emeny
iomarkers of carcinogen
exposure and early effects
ISBN 83-88261-78-9
B
ISBN 978-83-88261-78-1
Biomarkers of car
cinogen exposur
e and early effects
e te r B. F a rme r, J ean M. Emen y
Biomarkers of carcinogen
exposure
and early effects
.of the potential of diet to prevent cancer and of the ways by which heredity can affect individual susceptibility to carcinogens, with the ultimate aim of reducing the cancer burden in Europe. ECNIS is coordinated by Prof. Konrad Rydzyƒski, The Nofer Institute of Occupational Medicine, Êw. Teresy 8, 91-348 ¸ódê, Poland.
This review has been prepared as part of ECNIS Work Package 6: Development and Validation of Biomarkers of Exposure and Bioindicators of Disease for Use in Epidemiology.
© ECNIS, 2006
All rights reserved. No part of this book may be reproduced in any form without the permission of the publisher.
Editors: Prof. Peter B. Farmer and Dr Jean M. Emeny University of Leicester University Road Leicester LE1 7RH Tel.: +44 (0)116 223 1823 Fax: +44 (0)116 223 1840 Website: http://www.ecnis.org ISBN 83-88261-78-9 ISBN 978-83-88261-78-1
Cover design: Beata Grabska
Make-up editor: Katarzyna Rogowska
Published by The Nofer Institute of Occupational Medicine, Êw. Teresy 8, 91-348 ¸ódê, Poland
Tel.: +48 (0) 42 6314504 Fax: +48 (0) 42 6568331 E-mail: ecnis@ecnis.org
Executive summary . . . 7
1. Introduction . . . 13
2. Sensitivity and specificity of techniques for the identification of biomarkers . . . 17
2.1. DNA adducts . . . 17
2.1.1. Introduction . . . 17
2.1.2. 32P-Postlabelling . . . 18
2.1.3. Mass spectrometric detection of DNA adducts . . . 24
2.1.4. Other techniques to detect DNA adducts . . . 30
2.2. Protein adducts . . . 40
2.2.1. Introduction . . . 40
2.2.2. Nature of adducts . . . 40
2.2.3. Analysis of protein adducts . . . 41
2.2.4. Mass spectrometric approaches . . . 42
2.2.5. Future analytical requirements . . . 43
2.2.6. Conclusion . . . 43
2.3. Chromosomal damage . . . 47
2.3.1. Chromosomal aberrations . . . 48
2.3.2. Micronuclei . . . 51
3. Oxidative damage . . . 63
3.1. Biomarkers of DNA base oxidation . . . 63
3.1.1. Mutagenic and carcinogenic properties of DNA base derivatives . . . 63
3.1.2. Urinary measurement of oxidatively modified bases and nucleosides . . . 66
3.1.3. Sources of urinary lesions . . . 67
4. Specific biomarkers related to food . . . 83
4.1. Biomarkers for intake of heterocyclic aromatic amines . . . 83
4.1.1. Introduction . . . 83
4.1.2. Parent compounds and metabolites in urinary samples . . . 85
4.1.3. Blood protein and DNA adducts . . . 90
4.1.4. Hair . . . 94
4.1.5. HCA biomarkers and cancer risk . . . 95
4.2. Polycyclic aromatic hydrocarbons in food . . . 99
4.2.1. Introduction . . . 99
4.2.2. Methods of analysis . . . 99
4.2.3. Uncooked food . . . 99
4.2.4. Processed food . . . 100
4.2.5. Cooked food . . . 100
4.2.6. PAHs in the total human diet . . . 101
4.2.7. Diet versus other sources of human exposure . . . 101
4.2.8. Biomarkers of human exposure to PAHs . . . 102
4.2.9. Conclusions . . . 103
4.3. Biomarkers of N-nitroso compounds . . . 106
4.3.1. Biomarkers of NNOC carcinogenesis . . . 106
4.3.2. Biomarkers of tobacco-specific nitrosamines . . . 110
4.3.3. Biomarkers for exposure to NNK and NNN . . . 111
4.4. Acrylamide . . . 119
4.4.1. Introduction . . . 119
4.4.2. Acrylamide biomarkers . . . 120
4.4.3. Conclusions . . . 123
4.5. Alcohol biomarkers . . . 127
4.6. Aflatoxins and other mycotoxins . . . 129
4.6.1. Introduction . . . 129
4.6.2. Aflatoxins: occurrence and health effects . . . 130
4.6.3. Metabolism of aflatoxins: activation and detoxication of AFB1 . . . 130
4.6.4. Biomarkers of aflatoxins . . . 131
6. Correlations among biomarkers . . . 143
6.1. Introduction . . . 143
6.2. Study populations and types of exposure . . . 143
6.2.1. Occupational populations . . . 143
6.2.2. General population/healthy volunteers . . . 144
6.2.3. Medicinal exposure . . . 144
6.2.4. Cancer patients . . . 144
6.3. Most frequent biomarkers used in the studies evaluated here . . . 144
6.4. Correlation between levels of DNA adducts in human samples from a methodological point of view . . . 145
6.4.1. Common DNA adduct structures — different methods . . . 145
6.4.2. Different DNA adduct structures — common methods . . . 146
6.5. Correlation between levels of the same DNA adduct in various tissues . . . 146
6.5.1. DNA adducts in normal target and surrogate tissues . . . 146
6.5.2. DNA adduct levels in tumour and normal tissues . . . 147
6.6. Correlation between different biomarkers of human genotoxic exposure . . . 148
6.6.1. DNA adducts and protein adducts . . . 148
6.6.2. Different urinary metabolites and urinary mutagenicity . . . 148
6.6.3. DNA adducts, urinary PAH metabolites and urinary mutagenicity . . . 149
6.6.4. Protein adducts, urinary 1-hydroxypyrene and urinary mutagenicity . . . 150
6.6.5. DNA adducts, protein adducts and urinary cotinine . . . 150
6.7. Correlations among multiple biomarkers monitoring genotoxic exposure and effect . . . 150
6.8. Preliminary conclusions of the literature review . . . 152
7. Conclusions . . . 161
A major objective of ECNIS is to develop and validate novel biomarkers of exposure, effect and susceptibility and to employ them in the pursuit of greater understanding of environ-mental cancer risks and their modulation. A second important objective is to facilitate the integration of biomarkers into the framework of environmental cancer risk assessment.
The purpose of this review is to summarise the current situation regarding the types and uses of biomarkers of exposure and effect for the main classes of food-derived genotoxic carcinogens, and to consider some aspects of the intercomparison between these biomarkers. The biomarkers of exposure and early effects of carcinogens that have been most extensively developed are those for genotoxic agents and for compounds that generate hydroxyl radicals and other reactive radical species, and it is on these that this review is mostly concentrated.
The carcinogenic process can be monitored at several points between the initial exposure to the compound or reactive radical and the ultimate health effect. For all carcinogens, determination of the compound in the external medium in which it is contained, together with an estimate of human intake of the medium, allows us to calcu-late the level of compound received by the individual (‘external exposure dose’). The regulatory procedures used for controlling human exposures to carcinogens are largely based on limits on the amount/concentration in the external medium. A more accurate procedure for determining uptake of these carcinogens in the exposed individual is the measurement of the compound or its metabolites in a tissue or body fluid, yielding the ‘internal dose’. The ‘biologically effective dose’ of carcinogens can be elucidated by determination of the interaction products of the compounds with their site of toxicolo-gical action, which in the case of genotoxic carcinogens signifies the measurement of co-valently bound adducts with DNA. Protein adducts, although not being directly involved in the carcinogenic process, are also widely used as measures of biologically effective dose. DNA damage also occurs in the case of exposure to reactive radical species, and this can be measured for example as oxidised DNA bases, etheno or malondialdehyde adducts.
There is a continuous transition from biomarkers of exposure to biomarkers of effect, i.e. some biomarkers of effect also contribute to some degree as biomarkers of exposure and vice versa. Susceptibility factors, such as genetic polymorphism for metabolic activation, detoxification, DNA repair etc, may affect biomarkers of both exposure and effect.
The approaches described for the determination of DNA adducts mostly reach sensitivity limits in the range of 1 adduct/107–109nucleotides and are thus applicable for
studies of environmental exposure to genotoxins. Availability of DNA may be a limiting feature and the kind of sample required will determine how a method is used in human
biomonitoring studies. Protein adducts are generally stable and are therefore very suitable for use as biomarkers of exposure. The sensitivity of the mass spectral approaches for these assays has been shown to be sufficient for detection of adducts at low pmol/g protein levels. However, there is a lack of a screening method for characterisation of exposures to complex mixtures and no really high throughput analytical methods, preferable for large-scale human molecular epidemiological studies, exist.
Structural chromosomal aberrations in peripheral blood lymphocytes have been widely used in occupational and environmental settings as a biomarker of early effects of geno-toxic carcinogens. The predictivity of chromosomal aberrations as a biomarker for increased cancer risk may depend on the composition of the cohorts included in the study. The use of micronuclei as a measure of chromosomal damage has become a standard assay in both genetic toxicology testing and human biomonitoring studies. Analysis of re-sults from European cohorts indicated that subjects with cancer had a significant increase in frequency of micronuclei.
Free radical attack upon DNA generates, among other changes, modified bases.
The amount of 8-oxo-7,8-dihydroguanine, one of the most critical lesions in human lympho-cytes, was found to be 1.2/106. Gua by a mass spectrometric approach. Spurious oxidation
occurring during sample preparation is the major problem in measurement of oxidised ba-ses in DNA. Exposure to ambient air particles and benzene is associated with higher levels of 8-oxo-7,8-dihydroguanine and other oxidatively modified bases. Studies of the effects of antioxidant supplements and antioxidant-rich food have given contradictory results. New techniques allowing the simultaneous determination of 8-oxo-7,8-dihydro-2’-deoxy-guanosine, 8-oxo-7,8-dihydroguanine and 5-methylhydroxy-uracil in urine samples have revealed that the combined amount of these compounds excreted into urine of healthy human subjects corresponds to about 2800 oxidative modifications of guanine per cell per day. Cohort studies are required to assess whether an increased level of biomarkers of oxidative DNA damage is associated with an increased risk of developing cancer.
Lipid peroxidation resulting from chronic inflammatory processes can result
in production of excess reactive oxygen and reactive nitrogen species and DNA-reactive aldehydes, including 4-hydroxy-2-nonenal, malonaldehyde, acrolein, and crotonaldehyde. Alkylation of DNA bases by these reactive electrophiles is thought to contribute to the mutagenic and carcinogenic effects associated with oxidative stress-induced lipid peroxi-dation. The specific and sensitive methods developed for the detection of these poten-tially mutagenic adducts in human tissues and in urine have been applied to the analysis of DNA from a variety of normal and disease-related samples.
The mutagenic and carcinogenic heterocyclic aromatic amines are formed from precur-sors in meat and fish at temperatures exceeding 130°C and bind covalently to DNA after metabolic activation. Heterocyclic aromatic amines in urine have short half-lives but could be used to validate intake as estimated by questionnaires. Blood protein adducts and DNA adducts from various tissues have also been analysed. No epidemiological studies have yet been conducted in humans that have examined the association between hetero-cyclic aromatic amine exposure assessed by means of any biomarker and the risk of cancer.
Polycyclic aromatic hydrocarbons, formed by the incomplete combustion
of organic matter, are ubiquitous contaminants of the environment. There have been two main approaches to measurement of polycyclic aromatic hydrocarbons in complex matrices such as food: determination of around 15–20 polycyclic aromatic hydrocarbons, including carcinogenic compounds, or measurement of benzo[a]pyrene as a surrogate for all polycyclic aromatic hydrocarbons. The first approach gives a truer picture of the overall burden of these compounds in food; however, benzo[a]pyrene, because of its carcinogenic potency in experimental animals, represents a biologically significant measure. Most types of food contain measurable levels of polycyclic aromatic hydro-carbons and dietary exposure can be a significant effect in studies designed to determine occupational exposure or exposure due to urban pollution.
N-nitroso compounds, and especially alkyl nitrosamines, are well known
experimental carcinogens. Nitrosamines are present in significant quantities in tobacco smoke, while dimethylnitrosamine is also found in nitrate- or nitrite-treated foods. N-nitroso com-pounds can be formed endogenously at significant levels. Most epidemiological studies attempting to associate exposure to N-nitroso compounds and various human cancers have been inconclusive. The main problem was the ina-dequacy of methods for estimation not only of external exposure but, more importantly, of endo-genous exposure to N-nitroso compounds.
Large-scale molecular epidemiological studies to determine the carcinogenic risk associated with the widespread presence in human DNA of O6-methylguanine, which
plays an important role in mutagenesis, carcinogenesis and cytotoxicity by methylating agents, are lacking due to the lack of high throughput, high sensitivity assays for this adduct. The quantitatively most important DNA alkylation lesion N-7-methylguanine is not directly premutagenic, but can undergo spontaneous depurination to form muta-genic apurinic sites. N-7-Methylguanine accumulates more in tissues from smokers than nonsmokers, indicating that this biomarker could be used as an internal dosimeter for exposure to nitrosamines.
Extensive research on tobacco-specific nitrosamines has failed to provide conclusive evidence of their role in human cancer, despite their being potent rodent carcinogens. The urinary 4-(nitrosomethylamino)-1-(3-pyridyl)-1-butanone metabolites 4-(methylnitro-samino)-1-(3-pyridyl)-1-butanol and its glucuronide are absolutely specific for tobacco exposure.
Significant amounts of acrylamide can occur in certain food items high in carbo-hydrates and amino acids after heating. Acrylamide has been classified by IARC as “pro-bably carcinogenic to humans” and the EU has classified it as a “Category 2 carcinogen and cate-gory 2 mutagen”. The adduct of acrylamide itself to the N-terminal valine in haemoglobin and to some extent the corresponding adduct of glycidamide have been applied in human studies to assess exposure. Epidemiological investigations have not shown an increased risk from dietary exposure but larger studies of populations with more varied diets are needed; in addition data from intervention studies and on urinary metabolites and cytogenetic effects would be useful.
Biomarkers of alcohol may potentially be used to address the limitations of questionnaires and interviews in exposure assessment in epidemiological studies of alco-hol as a risk factor for cancer. The available biomarkers differ in sensitivity, specificity, ease of assay, and the time period that they reflect and none alone is ideal; combinations of various markers may allow for finer assessment of alcohol exposures in the future.
Mycotoxins are ubiquitous toxic secondary metabolites of a number of species
of moulds, and occur in foods and animal feeds. Naturally occurring aflatoxins are a cause of hepato-cellular carcinoma. Most European countries have imposed limits for aflatoxin B1 in foods and for aflatoxin M1 in milk. Urinary aflatoxin B1-N-7-guanine is an excellent biomarker for studies of acute exposure but does not reflect chronic intake of aflatoxin. Aflatoxin B1-albumin adducts are currently the most widely used biomarkers of aflatoxin in epidemiological studies. Assessment of functional polymorphisms in CYP3A4 and in other enzymes involved in the activation and detoxification of aflatoxin B1 may be used as markers of susceptibility to aflatoxins. The codon 249 mutation in p53 must be used cautiously as a marker of exposure to aflatoxin until evidence has been obtained from studies measuring both aflatoxin B1 adducts and mutations in the same individuals.
In addition to reacting with DNA bases, many genotoxic agents react with the oxygen of the internucleotide phosphodiester linkages to form phosphotriester adducts. Several studies have shown that phosphotriester lesions have a half-life that exceeds that of any other DNA alkylation product. The relative abundance of phosphotriesters depends upon the alkylating character of the agent. The role of phosphotriester adducts in carcinogenesis is unknown. Mutation resulting from phosphotriester formation has not been fully studied, but such adducts may influence cellular function by altering the binding of pro-teins to DNA.
An extensive literature survey on correlation of biomarkers resulted in the following preliminary conclusions:
• No significant correlation between individual pairs of DNA adducts was found in a number of the 32P-postlabelling and immunoassay studies, indicating limited
over-lapping of the substrate specificity of the different DNA adduct methods.
• Attempts to show correlations between a group of related DNA adduct structures and a chemically specific single DNA adduct structure by using the same type of methodo-logy gave controversial results. These suggest the co-existence of both closely linked and independent metabolic activation pathways for complex mixtures of xenobiotics, and may also reflect differences in the kinetics of DNA adduct formation and eli-mination.
• A larger number of studies revealed a positive correlation between DNA adduct levels in target and surrogate tissues than did not find a correlation. Correlation may depend on exposure dose and the metabolic capacity of the corresponding tissues.
• In the majority of the studies there was a positive correlation between DNA adduct levels in tumour and normal tissues, suggesting similarities in the xenobiotic activation/elimination processes of the tumour and normal tissues. However, the levels of DNA adducts found suggested organ specificity.
• There was a positive correlation for different urinary metabolites and urinary mutagenicity in most of the studies.
• No correlation between DNA adducts and urinary polycyclic aromatic hydrocarbon metabolites was generally found, but stratification by genotype suggested that correlation might be present.
• Correlation was more probable between structurally specified protein adducts and urinary polycyclic aromatic hydrocarbon metabolite 1-hydroxypyrene than with less specific xenobiotic-protein structures.
• Stratification of the study population for confounding factors, such as smoking status, may reveal hidden correlations.
• Cytogenetic biomarker studies also gave complex results. Examples of both positive correlation and lack of correlation with exposure markers were found.
Molecular epidemiological studies of cancer that include exposure biomarkers have greatly increased in number in recent years, the rationale being to measure the biologically relevant aspect(s) of exposure. However, the use of biomarkers to measure exposure is not a panacea. Most biomarker-based studies, of both prospective and retrospective design, rely on a single biological sample. Most exposures in cancer epidemiology are time-related variables, and both carcinogenesis models and empirical evidence strongly point towards the importance of induction and latency periods in cancer onset, the need to separate the role of duration and intensity of exposure, and the decrease in effect after cessation of exposure. While most biomarkers measure recent exposure, it is possible to apply them in the measurement of temporal changes, e.g. by collecting repeated samples from subjects enrolled in prospective studies or from a sample of the original cohort. Another important parameter is the in vivo lifetime of the biomarker after it has been generated by a carcinogen exposure.
Biomarker-based epidemiological results are subject to the methodological problems affecting all types of observational studies, namely bias and confounding. A further problem in the measurement of exposure biomarkers in retrospective studies is their possible dependence on the disease process. With the increasing use of biomarkers of dose and effect of carcinogens and the possible input of these data into the regulatory area, it is essential that the methodology be standardised and wherever possible internationally accepted protocols be established for this.
The need to validate exposure biomarkers before their application in population-based studies arises from the variability in biomarker-based measurements of exposure, which can be due to interindividual sources (e.g. differences between exposed and unexposed individuals, usually the component of variability of primary interest), intraindividual sources (e.g. variability in hormonal levels) and observer sources, including measurement error. This is the domain of so-called transitional studies, which aim to characterise the biomarker itself rather than the underlying biological phenomenon. This is the area in which most work is needed in the near future: efforts such as the ECNIS Network of Excellence will be instrumental in providing a logical framework for the development and validation of biomarkers of exposure with the ultimate goal of their application in molecular epidemiological studies.
Peter B. Farmer
University of Leicester, Leicester, UK
A biomarker is any substance, structure or process that can be measured in the body or its products and influence or predict the incidence of outcome or disease. Biomarkers can be classified into markers of exposure, effect and susceptibility1. The biomarkers
of exposure and early effects of carcinogens that have been most extensively developed are those for genotoxic agents and for compounds that generate hydroxyl radicals and other reactive radical species, and it is on these that this review is mostly concentrated. As illustrated in Fig. 1.1., the carcinogenic process can be monitored at several points between the initial exposure to the compound or reactive radical and the ultimate health effect.
Fig. 1.1. Potential monitoring points in the carcinogenic process.
For all carcinogens, determination of the compound in the external medium in which it is contained (e.g. air, water, food, medicine), together with an estimate of human intake of the medium, allows us to calculate the level of compound received by the individual
(‘external exposure dose’). To date, the regulatory procedures used for controlling human exposures to carcinogens are largely based on limits on the amount/concentration in the external medium, or (for some genotoxic carcinogens) on requiring this to be as low as is reasonably possible. A more accurate procedure for determining uptake of these carcinogens in the exposed individual is the measurement of the compound or of its metabolites in a tissue or body fluid. This yields what is designated the ‘internal dose’ or ‘body burden’ of the toxic compound. Such measurements may also give additional information, such as revealing the existence of other sources of exposure to the compound (including endogenous production), or the existence of genetic polymorphism for meta-bolic enzymes.
The ‘biologically effective dose’, sometimes known as the ‘tissue dose’, of carcinogens can be elucidated by determination of the interaction products of the compounds with their site of toxicological action, which in the case of genotoxic carcinogens signifies the measurement of covalently bound adducts with DNA. Protein adducts, although not being directly involved in the carcinogenic process, are also widely used as measures of biologically effective dose. These may also provide the opportunity to detect exposure to a genotoxic species a considerable time after its occurrence.
DNA damage also occurs in the case of exposure to reactive radical species, such as the hydroxyl radical. Direct interaction of DNA with the radical may result in the formation of oxidised DNA bases, but adducts may also be formed by indirect processes such as the reaction of lipid peroxidation products with DNA, e.g. etheno- or malondialdehyde adducts. Measurement of DNA adducts or other DNA damage indicates the amount of compound that has reached, in its reactive form, the target site, and might therefore be considered as an indication of the extent of the primary event in the initiation stage of the multi-stage process of chemical carcinogenesis. In view of the complexity of the later stages of the carcinogenic process, one cannot generalise how effective adduct mea-surements might be as a basis for risk estimates. However, they are considered to be a closer estimate of risk than measures of external dose or internal dose, as they are directly associated with mutation and other biological effects.
The biological effects derived from carcinogen exposure can be determined for example by measurement of mutations in DNA, cytogenetic alterations, promoter methylation and later as markers of altered structure and function of the cell including for example precancerous lesions, apoptosis, differentiation, circulating tumour cells and other circulating tumour markers.
Although in Fig. 1.1. biomarkers of exposure are indicated as measures of ‘external dose’ and ‘internal dose’, there is a continuous transition from biomarkers of exposure to biomarkers of effect, i.e. some biomarkers of effect also contribute to some degree as biomarkers of exposure and vice versa. Susceptibility factors, such as genetic polymorphism for metabolic activation, detoxification, DNA repair etc, may affect biomarkers of both exposure and effect.
With the increasing use of biomarkers of dose and effect of carcinogens and the pos-sible input of these data into the regulatory area, it is essential that the methodology
be standardised and wherever possible internationally accepted protocols established for this. This has occurred to a certain extent for some biomarkers of external exposure, internal dose and for some cytogenetic determinations, such as the measurement of chromosome aberrations. However, despite the extensive development of biomarker techniques for determining biologically effective dose, many of these techniques have not been fully validated, and further work on this is needed. Another aspect which needs standardisation (and which has on occasions been overlooked) is the collection and storage procedures for the biological samples. Prior to use of the biomarker one should establish the stability of the biomarker during the sampling procedure, any sample work-up and storage conditions, and then develop appropriate protocols to ensure that the biomarker is stable, or its rate of loss accurately known, or standardised for, in the period between biological sample collection and analysis.
Many of the newer procedures for determination of biomarkers of internal dose, biologically effective dose and early effects are highly complex and developed only within a few laboratories that have the specialised expertise and equipment. Sample throughput from some methods (e.g. 32P-postlabelling) is insufficient for large epidemiological studies,
and ECNIS partners believe that there is overall a need for more high-throughput and possibly automated procedures for biomarkers of exposure and early effect.
Another important parameter, which should always be considered before embarking on a human biomarker measurement, is the in vivo lifetime of the biomarker after it has been generated by a carcinogen exposure. There is a very large variation in this lifetime according to the compound and according to which biomarker is used2. In some cases the
measurement of a metabolite in plasma or urine reflects exposure only a few hours previously (e.g. benzene in blood, mandelic acid in urine as a marker of styrene exposure). In other cases the excretion half-lives of some compounds are measured in years (e.g. biphenyls) and thus detection of exposure that has occurred a long time before the biomarker measurement is possible. Similarly some DNA adducts are repaired within hours, but for others, e.g. phosphotriesters, there are no higher eukaryotic repair systems known. Protein adducts are also not repaired and, assuming that they are chemically stable, should have the same lifetime as the protein, e.g. 120 days for globin. Some DNA adducts are excreted in urine after repair and determination of these will represent exposure to the carcinogen over the previous day or two. Chromosomal aberrations and induction of micronuclei, and the existence of specific mutational effects, may indicate that exposure to a genotoxic species has occurred but determining from these markers the exact time of exposure is not easily possible. However, these may be considered as longer lifetime biomarkers.
Thus the choice of biomarker that should be used for a study in a human population will depend in part on whether the purpose is to detect an acute or a chronic exposure,
2 Farmer PB. Exposure biomarkers for the study of toxicological impact on carcinogenic processes. In: Buffler P,
Rice J, Bird M, Boffetta P, editors. Mechanisms of carcinogenesis. Contributions of molecular epidemiology. IARC Scientific Publication No. 157. Lyon: IARC Press; 2004. p. 71–90.
on the availability of suitable biological tissues and, importantly, on ethical permission being obtained for the study. In the case of food the exposure to carcinogenic components is often chronic and at a low level, and long lifetime biomarkers may often be preferred. The absence of sufficient long lifetime biomarkers for biologically effective dose has been noted by the ECNIS partners.
If the biomarker is going to be used in the risk assessment process, the relationship between the biomarker and response is of critical importance. Knowledge of the nature of the biomarker-exposure relationship is also important if one wishes to control the exposure scenario on the basis of a biomarker measurement. Considerable attention is now being paid to the question of whether or not there is a threshold for biological effects resulting from DNA adducts. Also the scientific community has yet to agree on whether there is a level of DNA adducts that may be acceptable in human DNA because the risk level associated with it is so low as to be of little or no concern. Thus the study of the relationships of biomarkers with later biological effects at low exposure levels is of critical importance.
Human samples for biomonitoring purposes should wherever possible be collected in a non-invasive fashion. Urine may be suitable for measuring biomarkers of internal dose, such as for example 1-hydroxypyrene as a marker of polycyclic aromatic hydrocarbon uptake or S-phenylmercapturic acid as a biomarker for benzene. Urine also contains some alkylated DNA bases (e.g. N-7-alkylguanines, N-3-alkyladenines) which are in part due to DNA adduct repair, and are thus biomarkers of biologically effective dose. These can be used as a monitor after exposure to an alkylating carcinogen during the previous 1–2 days. Buccal cells also present another moderately non-invasive way to acquire DNA, but have been little used to date. Alternatively, DNA is obtained most commonly from lymphocytes, although placenta, sperm, cervix or lung have also been used as DNA sources. Critically important when one is collecting samples for biomarker measurements are the ethical considerations. These are considered in ECNIS Work Package 12, Socio-ethical Impact of Biomarker Use.
Not all carcinogens fall into the categories described above. Biomarkers for non-geno-toxic carcinogens, which by definition do not cause direct DNA damage, can be either measures of external exposure dose, internal exposure dose or of biological effect but markers of biologically effective dose such as DNA adducts are clearly not appropriate. Measurements of oxidative DNA damage may, however, be a useful biomarker for some non-genotoxic agents which produce reactive oxygen species.
The purpose of this review is to summarise the current situation regarding the types and uses of biomarkers of exposure and effects for the main classes of food-derived genotoxic carcinogens, and to consider some aspects of the intercomparison between these biomarkers. A second review will consider in more detail the state of validation of biomarkers of exposure and early effects, and their applicability in molecular epidemiology.
of techniques for
the identification of biomarkers
2.1. DNA adducts
2.1.1. Introduction
Peter B. Farmer
University of Leicester, Leicester, UK
The measurement of DNA adducts is one of the most important biomarkers for exposure to genotoxic carcinogens, as it gives an indication of the biologically effective dose of the carcinogen that has reached the tissue DNA under study. Additionally the extent of DNA adduct formation may be indicative of the risk associated with the exposure. For example, the pioneering study by Ross et al. (1992) [1] showed that adduct formation from dietary intake of aflatoxin B1 was statistically related to the subsequent incidence of hepato-cellular carcinoma. A recent meta-analysis by Peluso et al. (2005) [2] showed that leuko-cyte DNA adducts were associated with the subsequent risk of lung cancer, the associa-tion being stronger in never-smokers.
The assay methods available for DNA adducts vary greatly in their sensitivity and selectivity, the amount of DNA that is required, and the window of exposure for which the methods can be applied (see Section 1; Farmer, 2004 [3]). Sensitivities achieved are normally in the range 1 adduct/109nucleotides to 1 adduct/108nucleotides, sufficient
to give useful information on human environmental or dietary exposures. Although many of these methods have been validated in animal models where single compound exposures have been used, human exposure is to complex mixtures of genotoxic compounds from a variety of sources, and the challenge for the successful use of these biomarkers is therefore much greater. Some of the most commonly used assays [e.g. 32P-postlabelling (Section 2.1.2.), and some immunoassays (Section 2.1.4.)] are
capable of measuring mixtures of DNA adducts, and others [e.g. mass spectrometry (Section 2.1.3.)] hold particular advantage for individual adduct determination. Comparison of biomarker methods for a particular adduct is only possible if one knows the efficiency of recovery of the adduct from DNA and the accuracy of each quantitative procedure, which is not always the case. Endeavours to undertake extensive procedures for prepurification of DNA to isolate a specific adduct normally require large amounts of DNA and are therefore not amenable to the screening of large numbers of biological
samples. There is no perfect approach for the measurement of DNA adducts in humans, and the procedures should be selected on a ‘case-by-case’ basis. An understanding of the nature of the exposure and the chemistry of the adduct in question is essential before choosing the experimental approach for adduct quantification.
References
1. Ross RK, Yuan J-M, Yu MC, Wogan GN, Qian G-S, Tu J-T, et al. Urinary aflatoxin biomarkers and risk of hepatocellular carcinoma. Lancet 1992;339:943–6.
2. Peluso M, Munnia A, Hoek G, Krzyzanowski M, Veglia F, Airoldi L, et al. DNA adducts and lung cancer risk: A prospective study. Cancer Res 2005;65:8042–8.
3. Farmer PB. Exposure biomarkers for the study of toxicological impact on carcinogenic processes. In: Buffler P, Rice J, Bird M, Boffetta P, editors. Mechanisms of carcinogenesis. Contributions of molecular epidemiology. IARC Scientific Publication No. 157. Lyon: IARC Press; 2004. p. 71–90.
2.1.2. 32P-Postlabelling
David Phillips
Institute of Cancer Research, London, UK
The 32P-postlabelling assay has been in use for over 20 years [1]. It is a highly sensitive
technique whereby modified bases in DNA are tagged with a radioactive group and detected by means of their radioactive decay. The use of a high specific activity radioactive isotope, namely phosphorus-32, permits the assay to detect DNA adducts formed in human tissues as a result of either occupational or environmental exposure. The method has been widely used and reviewed [2–5].
For most practical purposes the assay consists of four main steps. (i) DNA digestion; (ii) a procedure that isolates or selects the adducts for preferential labelling; (iii) polynucleotide kinase (PNK)-mediated phosphorylation of the adducts using [γ-32P]ATP; and (iv) chromatographic resolution of the labelled adducts and their detection
and quantitation by means of their 32P-decay.
Polynucleotide kinase will mediate the phosphorylation of the 5’-position on deoxyribose provided the 3’-position is phosphorylated, i.e. if the substrate is a nucleotide or polynucleotide. Thus stage 1, DNA digestion, usually involves enzymatic digestion with micrococcal nuclease and spleen phosphodiesterase. This generates nucleoside 3’-phosphates. After postlabelling, 3’,5’-bisphosphates, containing the radiolabel, are generated. These can be resolved and detected in this form; alternatively further digestion with nuclease P1 converts them to labelled nucleoside 5’-monophosphates [6]. A different digestion method involves using nuclease P1 and prostatic acid phosphatase, which
results in normal nucleosides and adducted dinucleotides due to the resistance of modified nucleotides to complete digestion [7]. After postlabelling, further digestion by venom phosphodiesterase cleaves the phosphodiester bond to generate the labelled adduct as a modified nucleoside 5’-monophosphate.
Strategies that avoid the labelling of non-adducted nucleotides can greatly enhance the sensitivity of the method. The two most commonly used are butanol extraction [8], whereby adducted nucleotides are selectively isolated by solvent extraction and then subjected to 32P-labelling, and nuclease P1 digestion [9], whereby normal nucleoside
3’-monophosphates are converted to nucleosides (which are not substrates for PNK) under conditions in which some, but not all, classes of adducts are resistant to digestion (and thus remain substrates for the labelling reaction).
Other procedures for sensitivity enhancement include extraction of adducts prior to labelling using solid-phase adsorption including high performance liquid chromato-graphy (HPLC) [10], small cartridge columns [11,12] or immunoaffinity columns containing antibodies to the adducts of interest [13,14].
Resolution and detection of adducts is commonly carried out by thin-layer chromatography (TLC) because: (i) this is sensitive, allowing visualisation of adducts as radioactive spots or ‘zones’ using autoradiography or an equivalent electronic radioactivity imaging system; (ii) many samples can be analysed in parallel; and (iii) cross-contamination of samples can be avoided. Nevertheless, the procedure is time consuming, labour intensive and has low resolution. Alternatively, HPLC separation can be used [15], although this requires samples to be analysed in series, it is less sensitive and great care is needed to avoid cross-contamination. There is, at present, considerable scope for improving the assay through the coordinated development of higher throughput, semi-automated procedures. Such developments would enhance considerably the application of the technique to large-scale human biomonitoring studies (see below).
The sensitivity with which different DNA adducts can be detected by 32P-postlabelling is variable, but optimally limits of detection as low as 1 adduct
in 1010nucleotides can be achieved in a sample of around 10 µg. More routinely, limits
of sensitivity of at least 1 in 109nucleotides are achieved for bulky adducts.
Biomonitoring studies using 32P-postlabelling are now very numerous. Many of them
reveal complex patterns of DNA adducts that are broadly characteristic of the patterns formed in experimental systems by complex mixtures of bulky carcinogens, such as polycyclic aromatic hydrocarbons (PAHs), although the exact nature of much of the DNA damage that is being detected by 32P-postlabelling in these circumstances is still
relatively uncharacterised. The definition of what constitutes a ‘bulky adduct’ is also open to variable interpretation, but in general it is taken to include aromatic moieties with two or more aromatic rings, and some large extended non-aromatic or aliphatic structures. Quantitative differences between environmentally exposed and unexposed groups have been noted in many studies [16], as have differences between the adduct levels detected in many tissues of smokers relative to non-smokers [17]. In many cases,
the potential of the technique is restricted by its being so labour intensive, inhibiting its application to truly large-scale human biomonitoring studies.
There have been a few small-scale interlaboratory trials to compare 32P-postlabelling
results between laboratories [18–20] and one larger-scale trial [21]. Although some external adduct standards have been prepared [21,22], these are not used routinely to normalise results obtained with human samples. There is undoubtedly a need for more standardised protocols to allow comparisons between results obtained in different laboratories, a process that was started in the large interlaboratory trial, but by no means completed [21]. Nevertheless, 32P-postlabelling analysis is the most versatile
of the methods available for monitoring DNA adduct formation in humans and it has the potential to provide significant insight into the etiology of human cancer, as well as in monitoring human exposure to known environmental sources of carcinogens.
References
1. Randerath K, Randerath E. Postlabelling methods — an historical review. In: Phillips DH, Castegnaro M, Bartsch, H, editors. Postlabelling methods for detection of DNA adducts. Lyon: International Agency for Research on Cancer; 1993. p. 3–9.
2. Beach AC, Gupta RC. Human biomonitoring and the 32P-postlabelling assay. Carcinogenesis
1992;13:1053–74.
3. Strickland PT, Routledge MN, Dipple A. Methodologies for measuring carcinogen adducts in humans. Cancer Epidemiol Biom Prev 1993,2:607–19.
4. Randerath K, Randerath E. 32P-Postlabelling methods for DNA adduct detection: overview
and critical evaluation. Drug Metab Rev 1994;26:67–85.
5. Phillips DH. Detection of DNA modifications by the 32P-postlabelling assay. Mutat Res
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6. Pfau W, Brockstedt U, Söhren K, Marquardt H. 32P-Post-labelling analysis of DNA adducts
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7. Randerath K, Randerath E, Danna TF, van Golen KL, Putman KL. A new sensitive
32P-postlabeling assay based on the specific enzymatic conversion of bulky DNA lesions to
radio-labeled dinucleotides and nucleoside 5’-monophosphates. Carcinogenesis 1998;10:1231–9. 8. Gupta RC. Enhanced sensitivity of 32P-postlabeling analysis of aromatic carcinogen: DNA
adducts. Cancer Res 1985;45:5656–62.
9. Reddy MV, Randerath K. Nuclease P1-mediated enhancement of sensitivity of 32P-postlabeling
test for structurally diverse DNA adducts. Carcinogenesis 1986;7:1543–51.
10. Dunn BP, San RHC. HPLC enrichment of hydrophobic DNA adducts for enhanced sensitivity of 32P-postlabeling analysis. Carcinogenesis 1988;9:1055–60.
11. Turesky RJ, Markovic J. DNA adduct formation of the food carcinogen 2-amino-3-methylimi-dazo[4,5-f]quinoline at the C-8 and N2atoms of guanine. Chem Res Toxicol 1994;7:752–61.
12. Phillips DH, Carmichael PL, Hewer A, Cole KJ, Hardcastle IR, Poon GK, et al. Activation of tamoxifen and its metabolite α-hydroxytamoxifen to DNA-binding products: comparisons between human, rat and mouse hepatocytes. Carcinogenesis 1996;17:88–94.
13. Cooper DP, Griffin KA, Povey AC. Immunoaffinity purification combined with 32P-postlabelling for
the detection of O6-methylguanine in DNA from human tissues. Carcinogenesis 1992;13:469–75.
14. Nair J, Barbin A, Guichard Y, Bartsch H. 1,N6-Ethenodeoxyadenosine and 3,N4
-ethenodeoxy-cytidine in liver DNA from humans and untreated rodents detected by immuno-affinity/32P-postlabelling. Carcinogenesis 1995;16:613–7.
15. Möller L, Zeisig M, Vodicka P. Optimization of an HPLC method for analyses of 32P-postlabeled
DNA adducts. Carcinogenesis 1993;14:1343–8.
16. Phillips DH. Macromolecular adducts as biomarkers of human exposure to polycyclic aromatic hydrocarbons. In: Luch A, editor. The carcinogenic effects of polycyclic aromatic hydrocarbons. London: Imperial College Press; 2005; p. 137–69.
17. Phillips DH. Smoking-related DNA and protein adducts in human tissues. Carcino-genesis 2002;23:1979–2004.
18. Hemminki K, Grzybowska E, Chorazy M, Twardowska-Saucha K, Sroczynski JW, Putman KL, et al. DNA adducts in humans environmentally exposed to aromatic compounds in an industrial area of Poland. Carcinogenesis 1990;11:1229–31.
19. Savela K, Hemminki K, Hewer A, Phillips DH, Putman KL, Randerath K. Interlaboratory comparison of the 32P-postlabelling assay for aromatic DNA adducts in white blood cells of iron
foundry workers. Mutat Res 1989;224:485–92.
20. Phillips DH, Castegnaro M. Results of an interlaboratory trial of 32P-postlabelling. In:
Phillips DH, Castegnaro M, Bartsch H, editors. Postlabelling methods for detection of DNA damage. Lyon: IARC; 1993, p. 35–49.
21. Phillips DH, Castegnaro M, on behalf of trial participants. Standardization and validation of DNA adduct postlabelling methods: report of interlaboratory trials and production of recommended protocols. Mutagenesis 1999;14:301–15.
22. Baan RA, Steenwinkel M-JST, van Asten S, Roggeband R, van Delft JHM. The use of benzo[a]pyrene diolepoxide-modified DNA standards for adduct quantification in 32
P-postla-belling to assess exposure to polycyclic aromatic hydrocarbons: application in a biomonitoring study. Mutat Res 1997;378:41–50.
Addendum
32P-HPLC
Lennart Möller
Leocordia AB, Selagarden, Stallarholmen, Sweden
32P-HPLC was developed in the early 1990s and proved to be somewhat less sensitive
than 32P-TLC with autoradiography. However, 32P-HPLC is usually faster, with better
separation, versatility and reproducibility compared with 32P-TLC [1]. 32P-HPLC, using
only HPLC for separation and online detection, in contrast to variants using TLC for pre-separation and radioactivity measurement on collected eluent fractions, has since been used to analyse many kinds of DNA-adduct samples, from in vitro experiments [2-4], in vivo experiments [5,6] and from humans, e.g. after exposure to
endogenous factors [7] and alkylation [8,9], specific environmental factors like ultra-violet radiation [10] and food-derived heterocyclic amines [11,12], more general en-vironmental exposures [13–15], specific occupational factors like 1,3-butadiene [16,17] and propylene oxide [18], more general occupational factors [19–22], and medical exposure such as from tamoxifen [23].
In addition there are studies in press that show DNA-adduct fingerprint changes in normal colonic epithelium during the disease process from controls to polyp patients to colon cancer cases [Möller et al. unpublished]. Further, application of 32P-HPLC
to human tissues showed different levels and patterns in different tissues of indivi-duals. The pancreas showed very high levels, suggesting a link to diabetes [Möller et al. unpublished]. Provocation of human lymphocytes with mutagens revealed a strong seasonal variation within the same individuals which was linked to a seasonal variation in CYP450 [Möller et al. unpublished]. Human lung tissue in different countries showed higher DNA-adduct levels than liver tissue, suggesting a connection with air pollutants [Möller et al. unpublished].
Reproducibility of retention times with the 32P-HPLC method is very good, enabling
comparison between chromatograms run many years apart by using endogenous peaks visible in all chromatograms for calibration. Quantitative reproducibility is somewhat less efficient owing to variations in enzyme batches and the [32P]ATP used for postlabelling.
References
1. Eriksson HL, Zeisig M, Ekstrom LG, Moller L. 32P-postlabeling of DNA adducts arising from
complex mixtures: HPLC versus TLC separation applied to adducts from petroleum products. Arch Toxicol 2004;78:174–81.
2. Möller L, Zeisig M, Vodicka P. Optimization of an HPLC method for analyses of 32P-postlabeled
DNA adducts. Carcinogenesis 1993;14:1343–8.
3. Zeisig M, Moller L. 32P-Postlabeling high-performance liquid chromatographic improvements
to characterize DNA adduct stereoisomers from benzo[a]pyrene and benzo[c]phenanthrene, and to separate DNA adducts from 7,12-dimethylbenz[a]anthracene. J Chromatogr B Biomed Sci Appl 1997;691:341–50.
4. Zeisig M, Hofer T, Cadet J, Moller L. 32P-postlabeling high-performance liquid chromatography
(32P-HPLC) adapted for analysis of 8-hydroxy-2’-deoxyguanosine. Carcinogenesis 1999;20:1241–5.
5. Möller L, Zeisig M. DNA adduct formation after oral administration of 2-nitrofluorene and N-acetyl-2-aminofluorene, analyzed by 32P-TLC and 32P-HPLC. Carcinogenesis 1993;14:53–9.
6. Möller L., Zeisig M, Midtvedt T, Gustafsson JÅ. Intestinal microflora enhances formation of DNA adducts following administration of 2-NF and 2-AAF. Carcinogenesis 1994;15:857–61. 7. Yang K, Fang JL, Hemminki K. Abundant lipophilic DNA adducts in human tissues. Mutat
Res 1998;422:285–95.
8. Kim DY, Cho MH, Yang HK, Hemminki K, Kim JP, Jang JJ, et al. Detection of methylation damage in DNA of gastric cancer tissues using 32
P-postlabelling assay. Jpn J Cancer Res 1999;90:1104–8.
9. Zhao C, Hemminki K. The in vivo levels of DNA alkylation products in human lymphocytes are not age dependent: an assay of 7-methyl- and 7-(2-hydroxyethyl)-guanine DNA adducts. Carcinogenesis 2002;23:307–10.
10. Bykov VJ, Hemminki K. UV-induced photoproducts in human skin explants analysed by TLC and HPLC-radioactivity detection. Carcinogenesis 1995;16:3015–9.
11. Banaszewski J, Szmeja Z, Szyfter W, Szyfter K, Baranczewski P, et al. Analysis of aromatic DNA adducts in laryngeal biopsies. Eur Arch Otorhinolaryngol 2000;257:149–53.
12. Baranczewski P, Möller L. Relationship between content and activity of cytochrome P450 and induction of heterocyclic amine DNA adducts in human liver samples in vivo and in vitro. Cancer Epidemiol Biomarkers Prev 2004;13:1071–8.
13. Möller L, Grzybowska E, Zeisig M, Cimander B, Hemminki K, Chorazy M. Seasonal variation of DNA adduct pattern in human lymphocytes analyzed by 32P-HPLC.
Carcino-genesis 1996;17:61–6.
14. Carlberg CE, Möller L, Paakki P, Kantola M, Stockmann H, Purkunen R, et al. DNA adducts in human placenta as biomarkers for environmental pollution, analysed by the 32P-HPLC
method. Biomarkers 2000;5:182–92.
15. Jaloszynski P, Jaruga P, Olinski R, Biczysko W, Szyfter W, Nagy E, et al. Oxidative DNA base modifications and polycyclic aromatic hydrocarbon DNA adducts in squamous cell carcinoma of larynx. Free Radic Res 2003;37:231–40.
16. Zhao C, Koskinen M, Hemminki K. 32P-postlabelling of N6-adenine adducts of epoxybutanediol in vivo after 1,3-butadiene exposure. Toxicol Lett 1998;102–103:591–4.
17. Zhao C, Vodicka P, Sram RJ, Hemminki K. Human DNA adducts of 1,3-butadiene, an important environmental carcinogen. Carcinogenesis 2000;21:107–11.
18. Czene K, Osterman-Golkar S, Yun X, Li G, Zhao F, Perez HL, et al. Analysis of DNA and hemo-globin adducts and sister chromatid exchanges in a human population occupationally exposed to propylene oxide: a pilot study. Cancer Epidemiol Biomarkers Prev 2002;11:315–8.
19. Carstensen U, Yang K, Levin JO, Ostman C, Nilsson T, Hemminki K, et al. Genotoxic exposures of potroom workers. Scand J Work Environ Health 1999;25:24–32.
20. Plna K, Osterman-Golkar S, Nogradi E, Segerback D. 32P-post-labelling of
7-(3-chloro-2-hydroxy-propyl)guanine in white blood cells of workers occupationally exposed to epichlorohydrin. Carcinogenesis 2000;21:275–80.
21. Sram RJ, Binkova B. Molecular epidemiology studies on occupational and environmental exposure to mutagens and carcinogens, 1997–1999. Environ Health Perspect 2000;108 (Suppl 1):57–70.
22. Tuominen R, Baranczewski P, Warholm M, Hagmar L, Möller L, Rannug A. Susceptibility factors and DNA adducts in peripheral blood mononuclear cells of aluminium smelter workers exposed to polycyclic aromatic hydrocarbons. Arch Toxicol 2002;76:178–86.
23. Hemminki K, Rajaniemi H, Lindahl B, Moberger B. Tamoxifen-induced DNA adducts in endometrial samples from breast cancer patients. Cancer Res 1996;56:4374–7.
2.1.3. Mass spectrometric detection of DNA adducts
Raj Singh
University of Leicester, Leicester, UK
The use of mass spectrometry for the detection of DNA adducts has been recently reviewed by Singh and Farmer, with particular reference to liquid chromato-graphy-electrospray ionization-mass spectrometry (ESI-LC-MS) [1]. Further compre-hensive reviews describing the application of liquid chromatography coupled to mass spectrometry for the study of DNA adducts have been published by Esmans et al. (1998) Andrews et al. (1999), Doerge et al. (2002), Koc and Swenberg (2002) and Watson
et al. (2003) [2–6]. Traditionally the role of mass spectrometry in the determination
of DNA adducts has been limited to providing information for the identification of new DNA adducts, or for the structural characterisation of DNA adduct standards that have been utilised to determine adduct levels by other detection methods, such as 32P-postlabelling, described in Section 2.1.2. [7]. However, in recent times, owing
to technological advances in instrumentation, mass spectrometry has become a viable method for the quantitation of DNA adduct levels in animal and human samples obtained after exposure to exogenous and endogenous genotoxic compounds.
Accelerator mass spectrometry, which measures isotope ratios, represents the most sensitive analytical method so far available for detecting DNA adducts, with a limit of de-tection that may be as low as 1 adduct per 1012unmodified DNA bases [8]. For example,
2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) DNA adducts at a level of 30–130 adducts per 1012unmodified DNA bases have been detected in normal human
colon tissue after exposure to dietary-relevant doses of the carcinogen [9]. The main limitation of the technique is that it depends on the presence of an isotope such as 14C
or 3H in the molecule of interest, which means that at present its application is restricted
to experimental systems where labelled compounds may be used. Numerous DNA adducts have been studied using gas chromatography-electron capture-negative chemical ionization-mass spectrometry (GC-EC-NCI-MS), examples of which include malondi-aldehyde-guanine, N-7-(2-hydroxyethyl)guanine, N2,3-ethenoguanine, 1,N2
-etheno-guanine and 1,N6-ethenoadenine [10–13]. Gas chromatography-mass spectrometry has
also been used to study DNA adducts that are derived from carcinogens normally present in tobacco smoke, such as 4-aminobiphenyl (4-ABP) and 4-(methylnitrosoamine)-1-(3--pyridyl)-1-butanone (NNK), as well as in the diet, such as PhIP [14–16]. Furthermore GC-MS has been used to demonstrate the presence of benzo[a]pyrene DNA adducts in human placental tissue [17]. The main advantage of gas chromatography is greater peak resolution compared with conventional liquid chromatography. The disadvantage is that only non-polar/volatile compounds can be analysed, and in the case of the majority of DNA adducts, which are non-volatile and/or polar, there is a requirement for derivatization at high temperature prior to analysis. The development of the electro-spray ionization source has meant that polar compounds can be directly analysed
without the need for derivatization, allowing the coupling of liquid chromatography to the mass spectrometer.
The process of electrospray ionization occurs at atmospheric pressure, resulting in negligible dissociation of the molecule ion into fragment ions, and is termed a soft ioniza-tion technique. Comprehensive reviews explaining the electrospray ionizaioniza-tion process in detail have been published by Gaskell (1997) and by Bruins (1998) [18,19]. Electrospray ionization is the main choice of ionization technique for studying DNA adducts, although atmospheric pressure chemical ionization has also been employed for their detection, albeit to a much lesser extent [20,21]. The most common design of instrument used for DNA adduct analysis is a triple quadrupole which allows for tandem mass spectrometry (MS/MS) to be performed. The mass spectrometer consists of the first (Q1) and third (Q3) quadrupoles which are the mass analysers (filters) separated by the second (q2) quadrupole called the collision cell. Analyte ions undergo collision induced dissociation (CID) in the collision cell, which is filled with an inert gas (normally argon) [1]. Time of flight instruments coupled with a quadrupole analyser (Q-TOF) have also been used to detect DNA adducts. The use of TOF instruments has been limited to providing high resolution data (accurate mass data) for the characterisation of DNA adducts, for example the adducts formed in the lung tissue of mice following exposure to asphalt fumes [22].
The two main detection methods used for determination of DNA adducts are single ion monitoring (SIM) and selected reaction monitoring (SRM). For SIM the first quadrupole (Q1) is set to pass only the ions of interest to the detector. In contrast SRM involves the selection of a precursor ion of the compound of interest in the first quadrupole (Q1), which undergoes transition in the collision cell (q2) by CID to a specific product ion, which is then determined by the third quadrupole (Q3). Opti-mum sensitivity and specificity for the quantitative analysis of DNA adducts is obtained by using SRM where a unique and abundant product ion is detected following CID [1]. Another tandem mass spectrometry detection mode is constant neutral loss (CNL), which is used primarily to screen for specific compounds that share a common CID pathway with the loss of a specific neutral moiety or functional group. CNL is applicable to the study of 2’-deoxynucleoside DNA adducts since they share a common CID pathway resulting in the loss of 2’-deoxyribose as a neutral moiety [23,24]. In terms of the analysis of DNA adducts by LC-MS the method of analysis can be applied to adducted purine or pyrimidine bases, 2’-deoxynucleosides as well as 2’-deoxy-nucleotides. Numerous studies have been published of the detection and quantification of DNA adducts ranging from small or polar adducts to bulky or non-polar adducts using ESI-LC-MS, further details of which can be found in the review by Singh and Farmer [1]. The main advantage mass spectrometry has to offer compared with other analytical methods is that it provides information about the structural identity of the compound under investigation, thus confirming that the correct compound is being studied. Accurate quantitation of DNA adduct levels is achieved by the use of stable isotope internal standards. These standards are identical in every respect chromatographically to the DNA adduct of interest but differ in mass and hence can only be differentiated
by mass spectrometry. The stable isotope internal standards are normally added at the start of the procedure, thus accounting for any losses during sample manipulation and preparation. The stable isotope internal standards also account for any variations in the response of the mass spectrometer such as suppression of ionization due to matrix effects [1,25]. The typical detection limits of ESI-LC-MS that have been quoted for various DNA adducts range from 0.5–5 adducts per 108unmodified DNA bases. Analyses are typically
performed using 50–100 µg of DNA [26–28]. The results obtained using ESI-LC-MS have been compared with results obtained using other detection techniques such as 32
P-post-labelling and immunoassays. For example, Beland et al. [29] compared ESI-LC-MS,
32P-postlabelling and dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA)
for the determination of 4-ABP adduct levels in calf thymus DNA that was reacted in
vitro, and in the liver DNA of mice treated with radiolabelled [3
H]N-hydroxy-4--aminobiphenyl. The results showed that 32P-postlabelling underestimated adduct levels
(3–5% relative level), DELFIA overestimated adduct levels (260–550% relative level) and that ESI-LC-MS gave levels of adducts that agreed most closely with those obtained for the specific binding of the 3H-radiolabel to the mouse liver DNA. Furthermore the authors
found that the LC-MS method provided the most accurate and precise results following statistical evaluation of the data when compared with the other two methods [29].
Further gains in the sensitivity of LC-MS for the detection of DNA adducts, particularly for the levels of adducts found in human samples, can be achieved by using capillary and nanocapillary LC (to replace the currently employed narrow bore and micro-bore LC) coupled to micro- or nanoelectrospray ionization mass spectrometry [30,31]. For example Ricicki et al. used capillary LC coupled to microelectrospray ionization-mass spectrometry for the quantitation of 4-ABP DNA adducts in human pancreas tissue using only 13.3 µg of the processed DNA sample for the analysis [32]. The coupling of capillary electrophoresis to mass spectrometry to study DNA adducts is reviewed in [33].
Mass spectrometry can also provide information regarding the site of the adducts within the DNA sequence. For example LC-ESI-MS/MS has been used to provide sequence information for numerous model studies where oligonucleotides have been adducted with genotoxic carcinogens in vitro [34,35]. The technique of matrix assisted laser desorption ionization (MALDI) has been used for DNA sequencing for the detection of mutations. Due to its wide mass range and ease of automation for high speed sample throughput a wide variety of approaches have been developed for genotyping [single nucleotide polymorphism (SNP) detection] and mutation detection using MALDI-MS. The use of mass spectrometry for genotyping has been reviewed by Jackson et al. (2000) and Edwards et al. (2005) [36,37].
Recently a LC-MS approach has been developed for the detection of mutations in short DNA fragments. Referred to as short oligonucleotide mass analysis (SOMA), this procedure involves polymerase chain reaction (PCR) amplification of the area of interest of the gene with primers containing a sequence for the restriction endonuclease BpmI, and digestion of the PCR product with BpmI, liberating short (< 20-mer) oligonucleotides
that can be analysed by LC-ESI-MS/MS (or MALDI-TOF) [38]. This approach has been used for the detection of base changes in codon 249 of the p53 gene, the hepatitis B virus and codon 12 of the K-ras gene [39–41].
The technological advances in mass spectrometry are still on-going, for example improvements in instrumentation have resulted in increased efficiency of ionization, transmission and detection of ions. There seems to be no doubt that in future mass spectrometry will take on a larger role in DNA adduct determinations in human studies, which is highlighted by the increasing number of publications employing LC-MS for the detection of DNA adducts in human populations exposed to genotoxic carcinogens.
References
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