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Use of biomarkers — new frontiers in occupational toxicology and epidemiology
Toxicology Letters 102]103 Ž1998. 581]589
Use of biomarkers } new frontiers in occupational toxicology and epidemiology Harri VainioU Unit of Chemopre¨ ention, International Agency for Research on Cancer, 150 cours Albert Thomas, 69372 Lyon, France
Abstract The incorporation of biomarkers into occupational toxicology and epidemiology some 25 years ago marked a turning-point for the discipline. The advances in molecular biology have provided new tools. At first, the major interest was in biomarkers of exposure, borrowing concepts from pharmacology, then it moved from the external estimates of exposure to internal measures of dose, and ultimately, to markers of target dose. Concerted efforts to measure carcinogens at the molecular level, e.g. DNA adducts, occupied a substantial fraction of the biomarkers work. In parallel, more quantitative and more sensitive end-points for etiological studies were sought earlier. Again, with advancing techniques in cytogenetics, extensive studies were conducted on such markers as sister chromatid exchanges ŽSCEs., micronuclei and other changes deemed to represent genomic damage. However, these types of end-points were quite unspecific for application to new hazards of uncertain human carcinogenic potential. Recent work focusing on more specific early-effect markers such as certain oncogenes and tumour-suppressor genes have substantial promise as shown by work with aflatoxins and vinyl chloride. Such studies have also enhanced mechanistic insight. The advances in molecular genetics have led to an upsurge in interest in most susceptibility factors, and identification of polymorphisms of various enzymes has become possible. Ongoing search for ‘ultra-high risk’ individuals may be fruitful, but probably only relevant to a small segment of potentially exposed populations. Factors associated with a small differential risk, however theoretically or mechanistically important, offer only little practical use. Q 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Metabolic polymorphisms; Individual susceptibility; Molecular epidemiology
1. Introduction The techniques of molecular biology have revolutionized the research opportunities in toxicology and epidemiology. Until recently, most occupational epidemiological studies were limited
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to the possible causative associations between two types of event: exposure to a potential causative agent Ži.e. specific occupational exposures. and disease outcome in terms of clinically apparent disease. Molecular epidemiology combines the tools of standard epidemiology Žsuch as case histories, questionnaires and monitoring of exposure. with the sensitive laboratory techniques of molecular biology ŽPerera, 1987.. It has the ad-
0378-4274r98r$ - see front matter Q 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0378-4274Ž98.00252-5
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vantage of being directly relevant to human risk. It also has the potential to give early warning by flagging the early effects of exposure and increased susceptibility, thus signalling opportunities to arrest disease through timely intervention ŽPerera, 1996.. A quieter revolution is underway in toxicology that has now entered the molecular age. This revolution has enormous potential also for disease prevention. The key enzymes that activate or inactivate the xenotoxicants are being cloned and characterized. They show remarkable polymorphic variation in humans and the common laboratory animals. This means that a given toxicant may be vastly more toxic in one individual than in another. Several types of biomarkers have been developed that make it possible to monitor some of the events in the multistage process precisely. These biomarkers can be divided into specific categories: internal dose, biologically effective dose, early biological effects and susceptibility ŽHulka et al., 1990.. Examples of markers in each of these categories are discussed briefly below. 2. Biomarkers of internal dose In conventional epidemiological studies of cancer, exposure is usually assessed at the level of the external source; that is, levels of substances in ambient air or water are analyzed or assessments are made on the basis of e.g. monitoring badges and dietary history. These approaches have limited precision and reliability. Fortunately, sensitive analytical procedures and immunoassays now make it possible to measure the amount of a potential carcinogen or its metabolites in cells, tissues and body fluids such as saliva, blood, urine and faeces. Such biomarkers of internal dose take into account individual differences in absorption or bioaccumulation of the compound in question and indicate its actual level in the body and in specific tissues or compartments. Examples of biomarkers of internal dose include continine in urine or serum resulting from exposure to cigarette smoke, 1-hydroxypyrene in urine resulting from exposure to polynuclear aromatic com-
pounds and aflatoxin M 1 in urine resulting from dietary exposure. An alternative method, which is not specific to individual chemicals, is Ames’ Salmonella typhimurium mutagenesis assay, which measures the presence of mutagens in body fluids Že.g. urine, saliva and nipple aspirate. that reflect exposure to cigarette smoke or other genotoxic environmental and dietary agents ŽVainio et al., 1984.. 3. DNA and protein adducts During the last decade or so, assays have been developed to ascertain the ‘biologically effective dose’ of a compound by measuring the amount of the compound that has actually reacted with critical cellular macromolecules, usually DNA, or an established surrogate target, for example, proteins in blood ŽPhillips and Farmer, 1995.. The rationale for measuring carcinogen]DNA adducts is that a number of chemical carcinogens, or their metabolites, exert their biological effects by binding covalently to cellular DNA, thereby inducing DNA damage and possibly mutations in critical cellular genes ŽFarmer, 1994.. Carcinogen]DNA adducts in target tissues are a more relevant marker than internal dose, because the former reflect not only individual differences in absorption and distribution but also differences in the metabolism Žactivation versus detoxification. of the chemical in question and the extent of repair of DNA damage. Unfortunately, in many studies in humans, DNA from the target tissue is not readily accessible and surrogate tissues are often used Že.g. peripheral blood cells. and the carcinogen]DNA adducts generally reflect recent exposure rather than that in the distant past. Studies of carcinogen]DNA adducts in peripheral blood cells suggest that they are rapidly removed after exposure ceases ŽHemminki, 1997.. Assays for protein adducts have been used increasingly to monitor exposure. Although proteins are not thought to be the critical targets of carcinogenesis, the extent to which they are modified can be a useful indicator of biologically effective dose. For instance, 4-aminobiphenyl] haemoglobin adducts have been measured in re-
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lation to tobacco smoking. A comparison of adducts in haemoglobin from controls and from smokers using blond and those using black tobacco demonstrated significant differences between the three groups ŽBryant et al., 1987; Vineis et al., 1994.. It should be emphasized, however, that as the quantitative relationship between levels of protein adducts and DNA adducts varies for different compounds and in different tissues and proteins, assays for protein adducts may not necessarily predict the extent of DNA modification in a given target tissue. Despite these limitations, these assays have proven to be highly informative about exposure to certain chemicals. Because of their abundance in blood, both haemoglobin Žextracted from red blood cells. and serum albumin have been used for such assays. Protein adducts provide information on relatively recent exposure, because the life span of red blood cells is about 4 months and the half-life of serum albumin is about 21 days. Several studies have demonstrated the feasibility of measuring carcinogen]protein adducts in humans. Its ultimate use for risk assessment remains to be determined. 4. Mutations and cytogenetic effects Biomarkers of mutation and cytogenetic effects are markers of the very early responses of cells to carcinogen-induced damage. These effects can be measured in target tissues or in a more convenient surrogate, for example, peripheral white blood cells. The biomarkers include DNA singlestrand breaks, various cytogenetic effects, including chromosomal aberrations, sister chromatid exchanges and micronuclei, and somatic mutations. Since these markers are not chemical- or exposure-specific, extensive information on other environmental and lifestyle factors Že.g. cigarette smoking habits, alcohol intake, exposure to radiation, drugs and viral infections . that might affect the results of these assays must be determined ŽAlbertini and O’Neill, 1995.. The measurement of chromosomal aberrations in peripheral lymphocytes has been used extensively as a sensitive monitor of exposure to ionizing radiation. Chromosomal aberrations can also
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be monitored after exposure to chemicals such as styrene oxide, alkylating cytostatic drugs and vinyl chloride ŽSorsa et al., 1992.. Micronuclei are small amounts of DNA found in cytoplasm that were not incorporated into daughter nuclei during mitosis, because of direct damage to the chromosomes or to the spindle apparatus. Micronuclei are easy to score using recently developed staining techniques. Micronuclei can also be monitored in exfoliated cells, such as oral mucosal cells, which can be sampled noninvasively ŽNorppa, 1997.. Sister chromatid exchange is considered to be a sensitive, simple-to-score cytogenetic end-point. The lesion that leads to the formation of these changes can persist in the cells for days, months or even years. Increased levels of sister chromatid exchange in peripheral blood cells have been found to result from exposure to cigarette smoke, certain factors in the work place and certain drugs ŽSorsa et al., 1992.. In some studies, multiple markers have been used. For instance, in ethylene oxide-exposed workers, the most sensitive indicator of recent exposure was the level of hydroxyethyl valine adducts; however, such adducts reflect only exposure during the few months before blood sampling. In contrast hprt mutations, which were found at an elevated frequency in ethylene oxide-exposed workers in comparison with controls, may indicate exposure that occurred years previously ŽMayer et al., 1991; Tates et al., 1991.. In a study on styrene-exposed workers, one of the cytogenetic end-points Žcells with high SCE frequency. turned out to be the most sensitive ŽTates et al., 1994.. 5. Growth factors and oncoproteins Numerous studies have shown aberrant expression of genes that encode proteins involved in cellular signal transduction, including growth factors and protein products of oncogenes and tumour suppressor genes Žfor a recent review, see Brandt-Rauf, 1997.. Such aberrant expression can be seen as a quantitative difference from normal Ži.e. overexpression of a wild-type protein. andror a qualitative difference Že.g. expression of the
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mutant form of a protein.. Increased expression of these proteins and expression of mutant forms are therefore potential biomarkers of susceptibility for the development of cancer ŽPerera et al., 1992; Brandt-Rauf, 1997.. Since growth factors are actively secreted from cells, they are logical targets for detection in blood. Platelet-derived growth factor, for instance, was detected in 59 breast cancer patients and only nine normal female controls; the levels in the controls were below the limit of detection of the assay, but 13 of 41 stage-4 cancer patients had levels more than twice the lower limit of detection of the assay ŽAriad et al., 1991.. Several other growth factors have been measured in plasma or serum from patients with various cancers. In a study of banked serum samples from asbestosis patients, transforming growth factor a levels were elevated in 13 of 36 Ž36%. patients who subsequently developed cancer, in 27 of 71 Ž38%. asbestosis patients who had not yet developed cancer and in none of 10 controls without asbestosis. Several patients had had elevated serum levels years before the detection of clinical disease ŽPartanen et al., 1995.. Growth factors are also probably important in other proliferative diseases, such as fibroproliferative disorders. For example, elevated levels of transforming growth factor b in the plasma of patients receiving chemotherapy in conjunction with bone-marrow transplantation for cancer have been shown to be predictive of subsequent liver and lung fibrosis ŽAnscher et al., 1993.. Similarly, elevated serum levels of platelet-derived growth factor were reported in 25 of 45 Ž56%. pneumoconiosis patients, and the elevated levels were more frequent in patients in whom the fibrotic disease progressed ŽBrandt-Rauf et al., 1992.. The ras oncogene encodes a 21-kDa membrane-associated G protein Žp21. that is involved in growth signal transduction ŽBarbacid, 1987.. The ras oncogene is activated in carcinogenesis either by overexpression of p21 or by expression of mutated forms of p21. Elevated serum levels of p21 were found in seven of 18 Ž39%. pneumoconiosis patients who later developed cancer Žfive of which were lung cancers . and in only two of 28 Ž7%. patients who did not develop cancer. Six of
the seven cancer patients had had elevated serum p21 levels before clinical diagnosis Žaverage, 16.3 months., again suggesting that this protein may be a biomarker of early malignant disease ŽBrandt-Rauf et al., 1992.. Mutant p21 protein has also been detected in blood. Asp-13 mutant p21 was studied by immunoblotting the sera of patients with angiosarcoma of the liver and individuals with heavy exposure to vinyl chloride ŽDeVivo et al., 1993; Brandt-Rauf et al., 1995.. Tumour tissue from four of five Ž80%. cases of angiosarcoma of the liver was found to contain the mutant ras gene, and the mutant p21 was expressed in tumour tissue and serum. The serum of eight of nine Ž89%. individuals with vinyl chloride-associated non-malignant angiomatous lesions of the liver and 22r45 Ž49%. who had had heavy exposure to vinyl chloride but had no detectable liver lesions also contained mutant p21, whereas none was found in the serum of 28 age-, sex- and racematched controls. Stratification of this small cohort by years of exposure to vinyl chloride showed a significant linear trend in the occurrence of mutant p21 in serum with increasing duration of exposure. Since increased exposure to this chemical is associated with increased cancer risk, mutant p21 in serum may be a biomarker of early carcinogenic change in vinyl chlorideexposed people. In another cohort of vinyl chloride-exposed workers, nine patients with angiosarcoma of the liver were found to have antibodies to p53 in their serum; in three cases, the antibodies were detectable before clinical detection of disease Žaverage, 8 years. ŽTrivers et al., 1995.. These examples suggest that the expression of genes involved in growth signal transduction may be a convenient biomarker for monitoring cancer. Some of these oncoproteins andror growth factors may not only distinguish between diseased and non-diseased states but could also be used for early detection of the disease process. 6. Markers of metabolism and detoxification The principles of multistage carcinogenesis predicate that factors other than exposure to spe-
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cific causative agents, will also influence the probability of tumour development in an individual. Such individual susceptibility factors may be acquired Ži.e. exogenous or environmental. or inherited Ži.e. genetic.. Individual susceptibility to cancer may result from differences in the metabolism of carcinogenic chemicals Župtake, activation and detoxification., in DNA repair, in inherited or acquired alterations in proto-oncogenes or tumour suppressor genes, in mutational status, in hormonal factors and in immunological factors. The superfamily of cytochrome P450 enzymes catalyses the oxidation of a large number of endogenous Že.g. hormones and fatty acids. and exogenous Že.g. polycyclic aromatic hydrocarbons, aromatic amines and mycotoxins. chemicals. Many of the P450 genes are known to exist in variant forms that have different activities. Because a number of carcinogens require metabolic activation before they can bind to DNA, individuals with an elevated metabolic capacity to activate specific carcinogens may be at an increased risk of cancer ŽGuengerich, 1994; Nebert et al., 1996.. Certain genes, such as cytochrome P450 CYP2D6, carry mutations in introns or at intronrexon boundaries that result in frameshift mutations and inactivate the gene. In addition, certain polymorphisms result in single amino acid changes that lead to altered protein function. Many studies have been performed on the association between polymorphic expression of CYP2D6 and the incidences of various types of cancer Žfor reviews, see Smith et al. Ž1995. and Nebert et al. Ž1996..; however, its role in determining cancer susceptibility remains unclear. The combined results of several studies carried out in various parts of the world suggest that the ‘poor metabolizer’ CYP2D6 genotype is associated with a moderately decreased risk for lung cancer ŽWolf et al., 1994.. The human CYP1A gene locus consists of CYP1A1, which is expressed predominantly in extrahepatic tissues such as the lung, and the liverspecific CYP1A2 ŽAnttila et al., 1992.. The substrates for and inducers of CYP1A1 include polynuclear aromatic compounds, whereas CYP1A2 is active in the metabolism of nitrosamines and aryl amines. CYP1A1 is inducible by, e.g. tobacco
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smoke, and its inducibility has been associated with a higher risk for lung cancer in smokers ŽMcLemore et al., 1990; Anttila et al., 1992.. Individuals vary widely in the extent to which CYP1A1 is induced by, e.g. tobacco smoking, 10% of Caucasians being highly inducible ŽNebert, 1991.. A combination of homozygous mutated CYP1A1 and GSTM1 null genotype was associated, at a similar or even lower smoking dose, with a stronger increase of DNA adducts in lungs ŽRojas et al., 1998.. The CYP1A1 ‘high inducibility’ phenotype ŽMspI RFLP. has been associated with the incidence of lung cancer in a Japanese population ŽHayashi et al., 1991., but a similar correlation was not found in Caucasians ŽTefre et al., 1991; Hirvonen et al., 1992.. CYP1A2 metabolizes aromatic amine procarcinogens, including tobacco-specific nitrosamines. Although no genetic polymorphism has yet been characterized in the CYP1A2 gene, considerable interindividual variation both in the level of expression in human liver ŽIkeya et al., 1989. and in the rate of metabolism of CYP1A2 substrates Že.g. caffeine and aromatic amines. has been reported ŽIlett et al., 1993.. Phenotyping with caffeine as a metabolic probe suggests a trimodal distribution of CYP1A2 phenotypes, representing slow, intermediate and rapid metabolizers ŽButler et al., 1992; Tang et al., 1994.. While ‘phase-I’ enzymes are involved in the activation of chemicals carcinogens, ‘phase-II’ enzymes Že.g. epoxide hydrolase, glutathione Stransferase ŽGST., sulfotransferase, glucuronosyl transferase . are involved mainly in detoxification of the metabolites of chemicals by conjugating them with glutathione, glucuronide or sulfate to produce hydrophilic products that are readily excreted. The balance between phase-I and phase-II enzymes determines the molecular dose of carcinogens, thereby substantially influencing the potential cancer risk. GSTs are a family of proteins which generally detoxify carcinogenic electrophilic substances by conjugating them with glutathione. There are four classes of dimeric cytosolic GSTs } a ŽGSTA., m ŽGSTM., p ŽGSTP. and u ŽGSTT.. Each has been shown to be polymorphic, although the pathological consequences of this variation re-
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mains largely unclear ŽKetterer et al., 1993.. The possibility that GSTM1 is a marker for susceptibility to smoking-related cancers was first tested in lung cancer cases by Seidegard ˚ et al. Ž1986.. Numerous subsequent studies on smoking-related cancers have had somewhat conflicting results, and the influence of the GSTM1 null genotype on risk remains unclear. Recent studies on the null polymorphism of the u class GSTT1 suggest that it is a potentially important susceptibility factor in cancer as it metabolizes such carcinogens as ethylene oxide and methylene chloride. GSTT1 also conjugates many epoxides, suggesting that individuals with null genotypes at GSTM1 and GSTT1 might be at high risk for smoking-related cancers. Many attempts have been made to relate the acetylation phenotype to cancer susceptibility. In humans, the arylamine N-acetyltransferases, NAT1 and NAT2, catalyse the acetylation of a range of arylamines and hydrazines ŽGrant, 1993.. NAT2 is assumed to be responsible for the classical acetylator polymorphism first identified in isoniazid-treated patients with tuberculosis: ‘slow’ acetylators inactivate the drug more slowly than ‘fast’ acetylators ŽPrice-Evans et al., 1960.. Four studies have addressed populations known to have been exposed to aromatic amines in the workplace, two showed a clear preponderance of slow acetylators among Caucasian bladder cancer patients Ž96% and 71%, respectively; Cartwright et al., 1982; Risch et al., 1994., and of the studies which failed to do so, one was too small ŽMiller and Cosgriff, 1983. and the other addressed Chinese subjects ŽHayes et al., 1993.. It has been reported that fast acetylators are at increased risk for colorectal cancer ŽRoberts-Thomson et al., 1996., and recent studies have suggested that this is particularly the case for individuals who also carry a mutation in the non-coding region of NAT1 ŽBell et al., 1995.. The importance of NAT1 in bladder cancer is corroborated by the recent findings that NAT1 protein is expressed in both well-differentiated bladder tumours and exfoliated cells collected from urine ŽStacey et al., 1996.; NAT2 protein, however, is unlikely to be expressed in bladder epithelium. Attempts have also been made to correlate exposure with risk by identifying arylamine adducts in haemoglobin of
slow and fast acetylators ŽVineis et al., 1994.. At low levels of exposure to tobacco smoke, slow acetylators had a risk for bladder cancer due to environmental exposures to aromatic amines that was about twice as high as that of fast acetylators ŽVineis and Caporaso, 1995.. 7. Future directions in use of biomarkers The use of biomarkers in studies of human cancer has increased markedly over the last two decades. Although most studies have been of a ‘transitional’ nature ŽHulka et al., 1990., smallscale and of limited design, they have demonstrated the huge potential of such studies in humans Žfor a recent review, see Perera, 1996.. Biological markers of exposure are already wellintegrated into epidemiological settings, as they indicate exposure via multiple routes, from multiple sources and of different patterns Žpast, current, intermittent and continuous.. Their use should enhance the possibilities for cancer prevention, e.g. by allowing better determination of acceptable levels of exposure. It will also contribute to our understanding of how cancer develops. As individual genetic traits can also influence cancer development, molecular epidemiologists also seek biomarkers of increased inborn and acquired susceptibility. Biomarkers of response and of susceptibility must be validated for their specificity, sensitivity and predictive value in order to ensure that they can distinguish individuals who are truly ‘at risk’ from those who are not. The increasing use of biomarkers in human studies will bring together cancer researchers interested in prevention and those interested in therapy. These new bridges between the fields of basic cancer research, causation, prevention and therapy will eventually be beneficial to all parties. Molecular characterization of cancers should improve the current system of staging and determining prognosis, which are based on the extent of disease spread as detected by conventional examination and imaging. The specific genetic and biochemical abnormalities that can define aggressive tumours may give rise to new staging systems based on intrinsic tumour characteristics. Studies
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on the prognostic value of molecular changes have been reported occasionally, and many other such studies are currently under way. For instance, factors that have been reported to confer a poor prognosis for lung cancer cases include activating ras mutations ŽSlebos et al., 1990; Mitsudomi et al., 1991., ErbB2 expression ŽKern et al., 1990. and the presence of antigens associated with proliferation, whereas tumours that express bcl2 may have a favourable prognosis ŽPezzella et al., 1993.. Mixed results have been obtained with regard to some markers; for example, patients with lung tumours expressing p53 protein may have either better or worse survival Žfor a review, see Greenblatt and Harris, 1995.. Biomarkers might be used to identify early premalignant lesions. Mutations in the ras and p53 genes were detected retrospectively in exfoliated bladder, bronchial and colonic cells from patients whose tumours were known to contain mutations ŽSidransky et al., 1991, 1992; Mao et al., 1994; Villa et al., 1996.. The use of biomarkers as surrogate end-points may also be used in the evaluation of chemopreventive agents. Molecular epidemiology could be used in risk assessment. An individual’s risk for developing cancer depends on both inherited and environmental factors; exposure to specific carcinogens is clearly related to increased cancer risk. Individual susceptibility varies greatly, however, and may be the factor that determines who will develop cancer. Molecular targets also open promising avenues for cancer therapy. The hypothesis that tumours containing wild-type p53 protein are more responsive to ionizing radiation and DNA damaging drugs is supported by clinicopathological inferences ŽFisher, 1994.. The presence of a mutated p53 gene in human chronic lymphocytic leukaemia in vivo has been correlated with a poor response to chemotherapy ŽEl Rouby et al., 1993.. 8. Conclusions Use of biomarkers in occupational toxicology and epidemiological research supplies information on various stages of the multistep process of putative disease. It allows the identification of
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biological signs, or biomarkers, that may indicate an increased risk for disease. Markers of exposure have an established position in environmental and occupational health; some reflect progression along the causal pathway to disease, and others reflect innate or acquired susceptibility to the effects of etiological agents. If unequivocal markers of effects and markers of susceptibility to disease could be developed, the identification of individualsrgroups at increased risk would then be helpful to the field of preventive medicine. References Albertini, R.J., O’Neill, J.P., 1995. Human monitoring for somatic mutation in humans. In: Phillips, D.N., Venitt, S. ŽEds.., Environmental Mutagenesis. Bios Scientific Publishers, Oxford, pp. 341]366. Anscher, M.S., Peters, W.P., Reisenbichler, H., Petros, W.P., Jirtle, R.L., 1993. Transforming growth factor b as a predictor of liver and lung fibrosis after autologous bone marrow transplantation for advanced breast cancer. N. Engl. J. Med. 328, 1592]1598. Anttila, S., Vainio, H., Hietanen, E., et al., 1992. Immunohistochemical detection of pulmonary cytochrome P4501A and metabolic activities associated with P4501A1 and P4501A2 isozymes in lung cancer patients. Environ. Health Perspect. 98, 179]182. Ariad, S., Seymour, L., Bezwoda, W.R., 1991. Platelet-derived growth factor ŽPDGF. in plasma or breast cancer patients: correlation with stage and rate of progression. Breast Cancer Res. Treat. 20, 11]17. Barbacid, M., 1987. Ras genes. Annu. Rev. Biochem. 56, 779]827. Bell, D.A., Stephens, E.A., Castriano, T., et al., 1995. Polyadenylation polymorphism in the acetyltransferase 1 gene ŽNAT1. increases risk of colorectal cancer. Cancer Res. 55, 3537]3542. Brandt-Rauf, P.W., 1997. Biomarkers of gene expression: growth factors and oncoprotein. Environ. Health Perspect. 105, 807]816. Brandt-Rauf, P.W., Smith, S., Hemminki, K., et al., 1992. Serum oncoproteins and growth factors in asbestosis and silicosis patients. Int. J. Cancer 50, 881]885. Brandt-Rauf, P.W., Marion, M.J., DeVivo, I., 1995. Mutant p21 protein as a biomarker of chemical carcinogenesis in humans. In: Mendelsohn, M.L., Peters, J.P., Normandy, M.J. ŽEds.., Biomarkers and Occupational Health: Progress and Prospectives. Joseph Henry Press, Washington, DC, pp. 163]173. Bryant, M.S., Skipper, P.L., Tannenbaum, S.R., 1987. Hemoglobin adducts of 4-aminobiphenyl in smokers and nonsmokers. Cancer Res. 47, 602]608. Butler, M.A., Lang, N.P., Young, J.F., et al., 1992. Determina-
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Ketterer, B., Taylor, J., Meyer, D., et al., 1993. Some functions of glutathione transferases. In: Tew, K., Mannewik, B., Mantle, T.J., Pickett, C.B., Hayes, J.D. ŽEds.., Structure and Function of Glutathione Transferases. CRC Press, Boca Raton, pp. 15]27. Mao, L., Hruban, R.H., Boyle, J.O., Tockman, M., Sidransky, D., 1994. Detection of oncogene mutations in sputum precedes diagnosis of lung cancer. Cancer Res. 54, 1634]1637. Mayer, J., Warburton, D., Jeffrey, A.M., et al., 1991. Biologic markers in ethylene oxide-exposed workers and controls. Mutat. Res. 248, 163]176. McLemore, T.L., Adelberg, S., Liu, M.C., et al., 1990. Expression of CYP1A1 gene in patients with lung cancer: evidence for cigarette smoke-induced expression of normal lung tissue and for pulmonary carcinomas. J. Natl. Cancer Inst. 82, 1333]1339. Miller, M.E., Cosgriff, J.M., 1983. Acetylator phenotype in human bladder cancer. J. Urol. 130, 65]66. Mitsudomi, T., Steinberg, S.M., Oie, H.K., et al., 1991. ras Gene mutations in non-small cell lung cancers are associated with shortened survival irrespective of treatment intent. Cancer Res. 51, 4999]5002. Nebert, D.W., 1991. Role of genetics and drug metabolism in human cancer risk. Mutat. Res. 247, 267]281. Nebert, D.W., McKinnon, R.A., Puga, A., 1996. Human drugmetabolizing enzyme polymorphisms: effects on risk of toxicity and cancer. DNA Cell Biol. 15, 273]280. Norppa, H., 1997. Cytogenetic markers of susceptibility: influence of polymorphic carcinogen-metabolizing enzymes. Environ. Health Perspect. 105, 829]836. Partanen, R., Koskinen, H., Oksa, P., et al., 1995. Serum oncoproteins in asbestosis patients. Clin. Chem. 41, 1844]1847. Perera, F., 1987. The potential usefulness of biological markers in risk assessment. Environ. Health Perspect. 76, 141]145. Perera, F.P., 1996. Molecular epidemiology: insights into cancer susceptibility, risk assessment and prevention. J. Natl. Cancer Inst. 88, 5496]5509. Perera, F.P., Hemminki, K., Gryzbowska, E., et al., 1992. Molecular and genetic damage in humans from environmental pollution in Poland. Nature 360, 256]258. Pezzella, F., Turley, H., Kuzu, I., et al., 1993. bcl-2 protein in non-small cell lung carcinoma. N. Engl. J. Med. 329, 690]694. Phillips, D.N., Farmer, P.B., 1995. Protein and DNA adducts as biomarkers of exposure to environmental mutagens. In: Phillips, D.N., Venitt, S. ŽEds.., Environmental Mutagenesis. Bios Scientific Publishers, Oxford, pp. 367]396. Price-Evans, D.A., Maly, K.A., McKussick, V.A., 1960. Genetic control of isoniazid metabolism in man. Br. Med. J. ii, 485]491. Roberts-Thomson, I.C., Ryan, P., Khoo, K.K., Hart, W.J., McMichael, A.J., Butler, R.N., 1996. Diet, acetylator phenotype, and risk of colorectal neoplasia. Lancet 347, 1372]1374. Rojas, M., Alexandrov, K., Cascorbi, I., et al., 1998. High
H. Vainio r Toxicology Letters 102]103 (1998) 581]589 benzow axpyrene diol-epoxide DNA adduct levels in lung and blood cells from individuals with combined CYP1A1 MspIrMspI-GSTM1U 0 r U 0 genotypes. Pharmacogenetics 8, 109]118. Risch, A., Wallace, D.M.A., Bathers, S., Sim, E., 1994. Slow N-acetylation is a susceptibility factor in occupational and smoking related bladder cancer. Hum. Mol. Genet. 4, 231]236. Seidegard, ˚ J., Pero, R.W., Miller, D.G., Beattie, E.J., 1986. A glutathione transferase in human leucocytes as a marker for the susceptibility to lung cancer. Carcinogenesis 7, 751]753. Sidransky, D., Von Eschenbach, A., Tsai, Y.C., et al., 1991. Identification of p53 gene mutations in bladder cancers and urine samples. Science 252, 706]709. Sidransky, D., Tokino, T., Hamilton, S.R., et al., 1992. Identification of ras oncogene mutations in the stool of patients with curable colorectal tumors. Science 256, 102]105. Slebos, R.J., Kibbelaar, R.E., Dalesio, O., et al., 1990. K-ras oncogene activation as a prognostic marker in adenocarcinoma of the lung. N. Engl. J. Med. 323, 561]565. Smith, G., Stanley, L.A., Sim, E., Strange, R.C., Wolf, C.R., 1995. Metabolic polymorphisms and cancer susceptibility. Cancer Surv. 25, 27]65. Sorsa, M., Wilbourn, J., Vainio, H., 1992. Human cytogenetic damage as a predictor of cancer risk. In: Vainio, H., Magee, P.N., McGregor, D.B., McMichael, A.J. ŽEds.., Mechanisms of Carcinogenesis in Risk Identification. International Agency for Research on Cancer, Lyon, pp. 543]554. Stacey, M., Thygesen, P., Stanley, L., Matas, N., Risch, A., Sim, E., 1996. Arylamine N-acetyltransferase as a potential biomarker in bladder cancer: fluorescent in situ hybridisation and immunochemistry studies. Biomarkers 1, 55]61. Tates, A.D., Grummt, T., Tornqvist, M., et al., 1991. Biological and chemical monitoring of occupational exposure to ethylene oxide. Mutat. Res. 250, 483]497. Tates, A.D., Grummt, T., van Dam, F.J., et al., 1994. Measure-
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ment of frequencies of HPRT mutants, chromosomal aberrations, micronuclei, sister-chromatid-exchanges and cells with high frequencies of SCEs in styrenerdichloromethane-exposed workers. Mutat. Res. 313, 249]262. Tang, B.K., Zhou, Y., Kalow, W., 1994. Caffeine as a probe for CYPIA2 activity: potential influence of renal factors on urinary phenotype trait measurements. Pharmacogenetics 4, 117]124. Tefre, T., Rydborg, D., Haugen, A., et al., 1991. Human CYP1A1 Žcytochrome P450. gene: lack of association between the MspI restriction fragment length polymorphism and the incidence of lung cancer in a Norwegian population. Pharmacogenetics 1, 20]25. Trivers, G.E., Cawley, H.L., DeBenedetti, C.M., et al., 1995. Anti-p53 antibodies in sera of workers occupationally exposed to vinyl chloride. J. Natl. Cancer Inst. 87, 1400]1407. Vainio, H., Sorsa, M., Falck, K., 1984. Bacterial urinary assay in monitoring exposure to mutagens. In: Berlin, A., Hemminki, K., Draper, M., Vainio, H. ŽEds.., IARC Scientific Publications no. 59, Lyon, pp. 247]258. Villa, E., Dugani, A., Rebecchi, A.M., et al., 1996. Identification of subjects at risk for colorectal carcinoma through a test based on K-ras determination in the stool. Gastroenterology 110, 1346]1353. Vineis, P., Caporaso, N., 1995. Tobacco and cancer: epidemiology and the laboratory. Environ. Health Perspect. 103, 156]160. Vineis, P., Bartsch, H., Caporaso, N., et al., 1994. Geneticallybased N-acetyltransferase metabolic polymorphism and low-level environmental exposure to carcinogens. Nature 369, 154]156. Wolf, C.R., Smith, C.A.D., Forman, D., 1994. Metabolic polymorphisms in carcinogen metabolizing enzymes and cancer susceptibility. In: Ponder, B.A.J. ŽEd.., Genetics of Malignant Disease ŽBritish Medical Bulletin 50.. Churchill Livingstone, Edinburgh, pp. 718]731.
Use of biomarkers } new frontiers in occupational toxicology and epidemiology Harri VainioU Unit of Chemopre¨ ention, International Agency for Research on Cancer, 150 cours Albert Thomas, 69372 Lyon, France
Abstract The incorporation of biomarkers into occupational toxicology and epidemiology some 25 years ago marked a turning-point for the discipline. The advances in molecular biology have provided new tools. At first, the major interest was in biomarkers of exposure, borrowing concepts from pharmacology, then it moved from the external estimates of exposure to internal measures of dose, and ultimately, to markers of target dose. Concerted efforts to measure carcinogens at the molecular level, e.g. DNA adducts, occupied a substantial fraction of the biomarkers work. In parallel, more quantitative and more sensitive end-points for etiological studies were sought earlier. Again, with advancing techniques in cytogenetics, extensive studies were conducted on such markers as sister chromatid exchanges ŽSCEs., micronuclei and other changes deemed to represent genomic damage. However, these types of end-points were quite unspecific for application to new hazards of uncertain human carcinogenic potential. Recent work focusing on more specific early-effect markers such as certain oncogenes and tumour-suppressor genes have substantial promise as shown by work with aflatoxins and vinyl chloride. Such studies have also enhanced mechanistic insight. The advances in molecular genetics have led to an upsurge in interest in most susceptibility factors, and identification of polymorphisms of various enzymes has become possible. Ongoing search for ‘ultra-high risk’ individuals may be fruitful, but probably only relevant to a small segment of potentially exposed populations. Factors associated with a small differential risk, however theoretically or mechanistically important, offer only little practical use. Q 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Metabolic polymorphisms; Individual susceptibility; Molecular epidemiology
1. Introduction The techniques of molecular biology have revolutionized the research opportunities in toxicology and epidemiology. Until recently, most occupational epidemiological studies were limited
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E-mail: [email protected]
to the possible causative associations between two types of event: exposure to a potential causative agent Ži.e. specific occupational exposures. and disease outcome in terms of clinically apparent disease. Molecular epidemiology combines the tools of standard epidemiology Žsuch as case histories, questionnaires and monitoring of exposure. with the sensitive laboratory techniques of molecular biology ŽPerera, 1987.. It has the ad-
0378-4274r98r$ - see front matter Q 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0378-4274Ž98.00252-5
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vantage of being directly relevant to human risk. It also has the potential to give early warning by flagging the early effects of exposure and increased susceptibility, thus signalling opportunities to arrest disease through timely intervention ŽPerera, 1996.. A quieter revolution is underway in toxicology that has now entered the molecular age. This revolution has enormous potential also for disease prevention. The key enzymes that activate or inactivate the xenotoxicants are being cloned and characterized. They show remarkable polymorphic variation in humans and the common laboratory animals. This means that a given toxicant may be vastly more toxic in one individual than in another. Several types of biomarkers have been developed that make it possible to monitor some of the events in the multistage process precisely. These biomarkers can be divided into specific categories: internal dose, biologically effective dose, early biological effects and susceptibility ŽHulka et al., 1990.. Examples of markers in each of these categories are discussed briefly below. 2. Biomarkers of internal dose In conventional epidemiological studies of cancer, exposure is usually assessed at the level of the external source; that is, levels of substances in ambient air or water are analyzed or assessments are made on the basis of e.g. monitoring badges and dietary history. These approaches have limited precision and reliability. Fortunately, sensitive analytical procedures and immunoassays now make it possible to measure the amount of a potential carcinogen or its metabolites in cells, tissues and body fluids such as saliva, blood, urine and faeces. Such biomarkers of internal dose take into account individual differences in absorption or bioaccumulation of the compound in question and indicate its actual level in the body and in specific tissues or compartments. Examples of biomarkers of internal dose include continine in urine or serum resulting from exposure to cigarette smoke, 1-hydroxypyrene in urine resulting from exposure to polynuclear aromatic com-
pounds and aflatoxin M 1 in urine resulting from dietary exposure. An alternative method, which is not specific to individual chemicals, is Ames’ Salmonella typhimurium mutagenesis assay, which measures the presence of mutagens in body fluids Že.g. urine, saliva and nipple aspirate. that reflect exposure to cigarette smoke or other genotoxic environmental and dietary agents ŽVainio et al., 1984.. 3. DNA and protein adducts During the last decade or so, assays have been developed to ascertain the ‘biologically effective dose’ of a compound by measuring the amount of the compound that has actually reacted with critical cellular macromolecules, usually DNA, or an established surrogate target, for example, proteins in blood ŽPhillips and Farmer, 1995.. The rationale for measuring carcinogen]DNA adducts is that a number of chemical carcinogens, or their metabolites, exert their biological effects by binding covalently to cellular DNA, thereby inducing DNA damage and possibly mutations in critical cellular genes ŽFarmer, 1994.. Carcinogen]DNA adducts in target tissues are a more relevant marker than internal dose, because the former reflect not only individual differences in absorption and distribution but also differences in the metabolism Žactivation versus detoxification. of the chemical in question and the extent of repair of DNA damage. Unfortunately, in many studies in humans, DNA from the target tissue is not readily accessible and surrogate tissues are often used Že.g. peripheral blood cells. and the carcinogen]DNA adducts generally reflect recent exposure rather than that in the distant past. Studies of carcinogen]DNA adducts in peripheral blood cells suggest that they are rapidly removed after exposure ceases ŽHemminki, 1997.. Assays for protein adducts have been used increasingly to monitor exposure. Although proteins are not thought to be the critical targets of carcinogenesis, the extent to which they are modified can be a useful indicator of biologically effective dose. For instance, 4-aminobiphenyl] haemoglobin adducts have been measured in re-
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lation to tobacco smoking. A comparison of adducts in haemoglobin from controls and from smokers using blond and those using black tobacco demonstrated significant differences between the three groups ŽBryant et al., 1987; Vineis et al., 1994.. It should be emphasized, however, that as the quantitative relationship between levels of protein adducts and DNA adducts varies for different compounds and in different tissues and proteins, assays for protein adducts may not necessarily predict the extent of DNA modification in a given target tissue. Despite these limitations, these assays have proven to be highly informative about exposure to certain chemicals. Because of their abundance in blood, both haemoglobin Žextracted from red blood cells. and serum albumin have been used for such assays. Protein adducts provide information on relatively recent exposure, because the life span of red blood cells is about 4 months and the half-life of serum albumin is about 21 days. Several studies have demonstrated the feasibility of measuring carcinogen]protein adducts in humans. Its ultimate use for risk assessment remains to be determined. 4. Mutations and cytogenetic effects Biomarkers of mutation and cytogenetic effects are markers of the very early responses of cells to carcinogen-induced damage. These effects can be measured in target tissues or in a more convenient surrogate, for example, peripheral white blood cells. The biomarkers include DNA singlestrand breaks, various cytogenetic effects, including chromosomal aberrations, sister chromatid exchanges and micronuclei, and somatic mutations. Since these markers are not chemical- or exposure-specific, extensive information on other environmental and lifestyle factors Že.g. cigarette smoking habits, alcohol intake, exposure to radiation, drugs and viral infections . that might affect the results of these assays must be determined ŽAlbertini and O’Neill, 1995.. The measurement of chromosomal aberrations in peripheral lymphocytes has been used extensively as a sensitive monitor of exposure to ionizing radiation. Chromosomal aberrations can also
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be monitored after exposure to chemicals such as styrene oxide, alkylating cytostatic drugs and vinyl chloride ŽSorsa et al., 1992.. Micronuclei are small amounts of DNA found in cytoplasm that were not incorporated into daughter nuclei during mitosis, because of direct damage to the chromosomes or to the spindle apparatus. Micronuclei are easy to score using recently developed staining techniques. Micronuclei can also be monitored in exfoliated cells, such as oral mucosal cells, which can be sampled noninvasively ŽNorppa, 1997.. Sister chromatid exchange is considered to be a sensitive, simple-to-score cytogenetic end-point. The lesion that leads to the formation of these changes can persist in the cells for days, months or even years. Increased levels of sister chromatid exchange in peripheral blood cells have been found to result from exposure to cigarette smoke, certain factors in the work place and certain drugs ŽSorsa et al., 1992.. In some studies, multiple markers have been used. For instance, in ethylene oxide-exposed workers, the most sensitive indicator of recent exposure was the level of hydroxyethyl valine adducts; however, such adducts reflect only exposure during the few months before blood sampling. In contrast hprt mutations, which were found at an elevated frequency in ethylene oxide-exposed workers in comparison with controls, may indicate exposure that occurred years previously ŽMayer et al., 1991; Tates et al., 1991.. In a study on styrene-exposed workers, one of the cytogenetic end-points Žcells with high SCE frequency. turned out to be the most sensitive ŽTates et al., 1994.. 5. Growth factors and oncoproteins Numerous studies have shown aberrant expression of genes that encode proteins involved in cellular signal transduction, including growth factors and protein products of oncogenes and tumour suppressor genes Žfor a recent review, see Brandt-Rauf, 1997.. Such aberrant expression can be seen as a quantitative difference from normal Ži.e. overexpression of a wild-type protein. andror a qualitative difference Že.g. expression of the
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mutant form of a protein.. Increased expression of these proteins and expression of mutant forms are therefore potential biomarkers of susceptibility for the development of cancer ŽPerera et al., 1992; Brandt-Rauf, 1997.. Since growth factors are actively secreted from cells, they are logical targets for detection in blood. Platelet-derived growth factor, for instance, was detected in 59 breast cancer patients and only nine normal female controls; the levels in the controls were below the limit of detection of the assay, but 13 of 41 stage-4 cancer patients had levels more than twice the lower limit of detection of the assay ŽAriad et al., 1991.. Several other growth factors have been measured in plasma or serum from patients with various cancers. In a study of banked serum samples from asbestosis patients, transforming growth factor a levels were elevated in 13 of 36 Ž36%. patients who subsequently developed cancer, in 27 of 71 Ž38%. asbestosis patients who had not yet developed cancer and in none of 10 controls without asbestosis. Several patients had had elevated serum levels years before the detection of clinical disease ŽPartanen et al., 1995.. Growth factors are also probably important in other proliferative diseases, such as fibroproliferative disorders. For example, elevated levels of transforming growth factor b in the plasma of patients receiving chemotherapy in conjunction with bone-marrow transplantation for cancer have been shown to be predictive of subsequent liver and lung fibrosis ŽAnscher et al., 1993.. Similarly, elevated serum levels of platelet-derived growth factor were reported in 25 of 45 Ž56%. pneumoconiosis patients, and the elevated levels were more frequent in patients in whom the fibrotic disease progressed ŽBrandt-Rauf et al., 1992.. The ras oncogene encodes a 21-kDa membrane-associated G protein Žp21. that is involved in growth signal transduction ŽBarbacid, 1987.. The ras oncogene is activated in carcinogenesis either by overexpression of p21 or by expression of mutated forms of p21. Elevated serum levels of p21 were found in seven of 18 Ž39%. pneumoconiosis patients who later developed cancer Žfive of which were lung cancers . and in only two of 28 Ž7%. patients who did not develop cancer. Six of
the seven cancer patients had had elevated serum p21 levels before clinical diagnosis Žaverage, 16.3 months., again suggesting that this protein may be a biomarker of early malignant disease ŽBrandt-Rauf et al., 1992.. Mutant p21 protein has also been detected in blood. Asp-13 mutant p21 was studied by immunoblotting the sera of patients with angiosarcoma of the liver and individuals with heavy exposure to vinyl chloride ŽDeVivo et al., 1993; Brandt-Rauf et al., 1995.. Tumour tissue from four of five Ž80%. cases of angiosarcoma of the liver was found to contain the mutant ras gene, and the mutant p21 was expressed in tumour tissue and serum. The serum of eight of nine Ž89%. individuals with vinyl chloride-associated non-malignant angiomatous lesions of the liver and 22r45 Ž49%. who had had heavy exposure to vinyl chloride but had no detectable liver lesions also contained mutant p21, whereas none was found in the serum of 28 age-, sex- and racematched controls. Stratification of this small cohort by years of exposure to vinyl chloride showed a significant linear trend in the occurrence of mutant p21 in serum with increasing duration of exposure. Since increased exposure to this chemical is associated with increased cancer risk, mutant p21 in serum may be a biomarker of early carcinogenic change in vinyl chlorideexposed people. In another cohort of vinyl chloride-exposed workers, nine patients with angiosarcoma of the liver were found to have antibodies to p53 in their serum; in three cases, the antibodies were detectable before clinical detection of disease Žaverage, 8 years. ŽTrivers et al., 1995.. These examples suggest that the expression of genes involved in growth signal transduction may be a convenient biomarker for monitoring cancer. Some of these oncoproteins andror growth factors may not only distinguish between diseased and non-diseased states but could also be used for early detection of the disease process. 6. Markers of metabolism and detoxification The principles of multistage carcinogenesis predicate that factors other than exposure to spe-
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cific causative agents, will also influence the probability of tumour development in an individual. Such individual susceptibility factors may be acquired Ži.e. exogenous or environmental. or inherited Ži.e. genetic.. Individual susceptibility to cancer may result from differences in the metabolism of carcinogenic chemicals Župtake, activation and detoxification., in DNA repair, in inherited or acquired alterations in proto-oncogenes or tumour suppressor genes, in mutational status, in hormonal factors and in immunological factors. The superfamily of cytochrome P450 enzymes catalyses the oxidation of a large number of endogenous Že.g. hormones and fatty acids. and exogenous Že.g. polycyclic aromatic hydrocarbons, aromatic amines and mycotoxins. chemicals. Many of the P450 genes are known to exist in variant forms that have different activities. Because a number of carcinogens require metabolic activation before they can bind to DNA, individuals with an elevated metabolic capacity to activate specific carcinogens may be at an increased risk of cancer ŽGuengerich, 1994; Nebert et al., 1996.. Certain genes, such as cytochrome P450 CYP2D6, carry mutations in introns or at intronrexon boundaries that result in frameshift mutations and inactivate the gene. In addition, certain polymorphisms result in single amino acid changes that lead to altered protein function. Many studies have been performed on the association between polymorphic expression of CYP2D6 and the incidences of various types of cancer Žfor reviews, see Smith et al. Ž1995. and Nebert et al. Ž1996..; however, its role in determining cancer susceptibility remains unclear. The combined results of several studies carried out in various parts of the world suggest that the ‘poor metabolizer’ CYP2D6 genotype is associated with a moderately decreased risk for lung cancer ŽWolf et al., 1994.. The human CYP1A gene locus consists of CYP1A1, which is expressed predominantly in extrahepatic tissues such as the lung, and the liverspecific CYP1A2 ŽAnttila et al., 1992.. The substrates for and inducers of CYP1A1 include polynuclear aromatic compounds, whereas CYP1A2 is active in the metabolism of nitrosamines and aryl amines. CYP1A1 is inducible by, e.g. tobacco
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smoke, and its inducibility has been associated with a higher risk for lung cancer in smokers ŽMcLemore et al., 1990; Anttila et al., 1992.. Individuals vary widely in the extent to which CYP1A1 is induced by, e.g. tobacco smoking, 10% of Caucasians being highly inducible ŽNebert, 1991.. A combination of homozygous mutated CYP1A1 and GSTM1 null genotype was associated, at a similar or even lower smoking dose, with a stronger increase of DNA adducts in lungs ŽRojas et al., 1998.. The CYP1A1 ‘high inducibility’ phenotype ŽMspI RFLP. has been associated with the incidence of lung cancer in a Japanese population ŽHayashi et al., 1991., but a similar correlation was not found in Caucasians ŽTefre et al., 1991; Hirvonen et al., 1992.. CYP1A2 metabolizes aromatic amine procarcinogens, including tobacco-specific nitrosamines. Although no genetic polymorphism has yet been characterized in the CYP1A2 gene, considerable interindividual variation both in the level of expression in human liver ŽIkeya et al., 1989. and in the rate of metabolism of CYP1A2 substrates Že.g. caffeine and aromatic amines. has been reported ŽIlett et al., 1993.. Phenotyping with caffeine as a metabolic probe suggests a trimodal distribution of CYP1A2 phenotypes, representing slow, intermediate and rapid metabolizers ŽButler et al., 1992; Tang et al., 1994.. While ‘phase-I’ enzymes are involved in the activation of chemicals carcinogens, ‘phase-II’ enzymes Že.g. epoxide hydrolase, glutathione Stransferase ŽGST., sulfotransferase, glucuronosyl transferase . are involved mainly in detoxification of the metabolites of chemicals by conjugating them with glutathione, glucuronide or sulfate to produce hydrophilic products that are readily excreted. The balance between phase-I and phase-II enzymes determines the molecular dose of carcinogens, thereby substantially influencing the potential cancer risk. GSTs are a family of proteins which generally detoxify carcinogenic electrophilic substances by conjugating them with glutathione. There are four classes of dimeric cytosolic GSTs } a ŽGSTA., m ŽGSTM., p ŽGSTP. and u ŽGSTT.. Each has been shown to be polymorphic, although the pathological consequences of this variation re-
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mains largely unclear ŽKetterer et al., 1993.. The possibility that GSTM1 is a marker for susceptibility to smoking-related cancers was first tested in lung cancer cases by Seidegard ˚ et al. Ž1986.. Numerous subsequent studies on smoking-related cancers have had somewhat conflicting results, and the influence of the GSTM1 null genotype on risk remains unclear. Recent studies on the null polymorphism of the u class GSTT1 suggest that it is a potentially important susceptibility factor in cancer as it metabolizes such carcinogens as ethylene oxide and methylene chloride. GSTT1 also conjugates many epoxides, suggesting that individuals with null genotypes at GSTM1 and GSTT1 might be at high risk for smoking-related cancers. Many attempts have been made to relate the acetylation phenotype to cancer susceptibility. In humans, the arylamine N-acetyltransferases, NAT1 and NAT2, catalyse the acetylation of a range of arylamines and hydrazines ŽGrant, 1993.. NAT2 is assumed to be responsible for the classical acetylator polymorphism first identified in isoniazid-treated patients with tuberculosis: ‘slow’ acetylators inactivate the drug more slowly than ‘fast’ acetylators ŽPrice-Evans et al., 1960.. Four studies have addressed populations known to have been exposed to aromatic amines in the workplace, two showed a clear preponderance of slow acetylators among Caucasian bladder cancer patients Ž96% and 71%, respectively; Cartwright et al., 1982; Risch et al., 1994., and of the studies which failed to do so, one was too small ŽMiller and Cosgriff, 1983. and the other addressed Chinese subjects ŽHayes et al., 1993.. It has been reported that fast acetylators are at increased risk for colorectal cancer ŽRoberts-Thomson et al., 1996., and recent studies have suggested that this is particularly the case for individuals who also carry a mutation in the non-coding region of NAT1 ŽBell et al., 1995.. The importance of NAT1 in bladder cancer is corroborated by the recent findings that NAT1 protein is expressed in both well-differentiated bladder tumours and exfoliated cells collected from urine ŽStacey et al., 1996.; NAT2 protein, however, is unlikely to be expressed in bladder epithelium. Attempts have also been made to correlate exposure with risk by identifying arylamine adducts in haemoglobin of
slow and fast acetylators ŽVineis et al., 1994.. At low levels of exposure to tobacco smoke, slow acetylators had a risk for bladder cancer due to environmental exposures to aromatic amines that was about twice as high as that of fast acetylators ŽVineis and Caporaso, 1995.. 7. Future directions in use of biomarkers The use of biomarkers in studies of human cancer has increased markedly over the last two decades. Although most studies have been of a ‘transitional’ nature ŽHulka et al., 1990., smallscale and of limited design, they have demonstrated the huge potential of such studies in humans Žfor a recent review, see Perera, 1996.. Biological markers of exposure are already wellintegrated into epidemiological settings, as they indicate exposure via multiple routes, from multiple sources and of different patterns Žpast, current, intermittent and continuous.. Their use should enhance the possibilities for cancer prevention, e.g. by allowing better determination of acceptable levels of exposure. It will also contribute to our understanding of how cancer develops. As individual genetic traits can also influence cancer development, molecular epidemiologists also seek biomarkers of increased inborn and acquired susceptibility. Biomarkers of response and of susceptibility must be validated for their specificity, sensitivity and predictive value in order to ensure that they can distinguish individuals who are truly ‘at risk’ from those who are not. The increasing use of biomarkers in human studies will bring together cancer researchers interested in prevention and those interested in therapy. These new bridges between the fields of basic cancer research, causation, prevention and therapy will eventually be beneficial to all parties. Molecular characterization of cancers should improve the current system of staging and determining prognosis, which are based on the extent of disease spread as detected by conventional examination and imaging. The specific genetic and biochemical abnormalities that can define aggressive tumours may give rise to new staging systems based on intrinsic tumour characteristics. Studies
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on the prognostic value of molecular changes have been reported occasionally, and many other such studies are currently under way. For instance, factors that have been reported to confer a poor prognosis for lung cancer cases include activating ras mutations ŽSlebos et al., 1990; Mitsudomi et al., 1991., ErbB2 expression ŽKern et al., 1990. and the presence of antigens associated with proliferation, whereas tumours that express bcl2 may have a favourable prognosis ŽPezzella et al., 1993.. Mixed results have been obtained with regard to some markers; for example, patients with lung tumours expressing p53 protein may have either better or worse survival Žfor a review, see Greenblatt and Harris, 1995.. Biomarkers might be used to identify early premalignant lesions. Mutations in the ras and p53 genes were detected retrospectively in exfoliated bladder, bronchial and colonic cells from patients whose tumours were known to contain mutations ŽSidransky et al., 1991, 1992; Mao et al., 1994; Villa et al., 1996.. The use of biomarkers as surrogate end-points may also be used in the evaluation of chemopreventive agents. Molecular epidemiology could be used in risk assessment. An individual’s risk for developing cancer depends on both inherited and environmental factors; exposure to specific carcinogens is clearly related to increased cancer risk. Individual susceptibility varies greatly, however, and may be the factor that determines who will develop cancer. Molecular targets also open promising avenues for cancer therapy. The hypothesis that tumours containing wild-type p53 protein are more responsive to ionizing radiation and DNA damaging drugs is supported by clinicopathological inferences ŽFisher, 1994.. The presence of a mutated p53 gene in human chronic lymphocytic leukaemia in vivo has been correlated with a poor response to chemotherapy ŽEl Rouby et al., 1993.. 8. Conclusions Use of biomarkers in occupational toxicology and epidemiological research supplies information on various stages of the multistep process of putative disease. It allows the identification of
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