Chem. Res. Toxicol. 1992,5, 450-460
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Invited Review Relevance of Urinary DNA Adducts as Markers of Carcinogen Exposure David E. G. Shuker**+ and Peter B.Farmer1 Unit of Environmental Carcinogens and Host Factors, International Agency for Research on Cancer, 150 Cours Albert Thomas, 69372 Lyon Cedex 08, France, and Medical Research Council Toxicology Unit, Medical Research Council Laboratories, Woodmansterne Road, Carshalton, Surrey SM5 4EF, U.K. Received June 5,1992
Introduction In the rapidly expanding area of molecular epidemiology, the precise determination of individual exposure to carcinogens is regarded as a desirable goal. Whether or not it will be possible to determine individual risk of developing cancer as the result of measuring exposure to carcinogens is open to question; however, it is clear that groups of people a t elevated risk of developing cancer may be identified using molecular markers of carcinogen exposure. This is an extension of the use of existing tools of cancer epidemiology where carcinogen exposure is established by a variety of methods (for example, using questionnaires or by personal sampling). However, these approaches rarely, if ever, address such questions as the biologically effective internal dose or even the identity of the active component(s) in complex mixtures such as diet or tobacco smoke. The interest in using molecular markers is that their specificity and sensitivity in detecting carcinogen exposure introduces greater statistical power to resolve relatively small differences in risk which would otherwise be undetectable using existing methodology. Current approaches to the determination of human exposure to carcinogens are based on the idea that many of them are genotoxic and that initiation of cells and other changes, acting via formation of mutagenic lesions in DNA, are crucial (but not individually sufficient) steps in multistage carcinogenesis (11. The large majority of genotoxic carcinogens appear to act through electrophilic intermediates which react with nucleophilic sites on DNA, giving rise to characteristic adducts (2). These adducts can be detected and quantified by a number of methods [reviewed,for example, by Phillips ( 3 ) ]in DNA extracted from a variety of sources including target tissues (obtained at necropsy or during surgery) or from nontarget tissues, such as nucleated blood cells. These approaches have unequivocally demonstrated human exposure to a range of alkylating agents arising from food [e.g., aflatoxin B1 (AFB1;l 4 ) ] , occupation [e.g., benzo* Author for correspondence. +
International Agency for Research on Cancer.
t Medical Research Council Laboratories.
1Abbreviations: AFB1, aflatoxin B1; AFBI-Gua, 8,9-dihydro-8-(N7guanyl)-9-hydroxy-AFBI;AP, aminopyrine; AP-de, hexadeuterated aminopyrine; B[a]P, benzo[a]pyrene; DIC, 4(5)-(3,3-dimethyl-I-triazeno)imidazole5(4)-carboxamide; EO, ethyleneoxide; Fapy, 5-formamido-4,6diaminopyrimidine; dG, 2’-deoxyguanosine; G, guanosine; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; ip injection, intraperitoneal injection; MNNG, N-methyl-N’-nitro-N-nitrosoguanidine; MNU, N methyl-N-nitrosourea; NDMA, N-nitrosodimethylamine; NMCA, N -
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[alpyrene (B[alP; 5)1,and lifestyle [e.g., tobaccosmoking (611. In addition, the use of alkylating medicinal drugs is a unique example of human exposure to carcinogens at known doses (e.g., cis-platin; 7). Blood protein adducts to these same exposures have been exploited as surrogate markers of DNA damage (4,6,8). All of these methods require tissue or blood, which in many cases limits the number of samples available for analysis and may not provide information on whole-body dose of carcinogen. It is from this standpoint that the idea of using noninvasive methods to determine human exposure to carcinogens, based on the analysis of urinary DNA adducts, has been developed. The presence of modified nucleic acid bases in human urine has been known since 1898when Kriiger and Salomon fractionated 10 OOO L of urine and obtained several grams of 7-methylguanine (7-MeGua; 9). A more detailed study by Weissmann et al. (10, 1 0 , using purification by precipitation of the purine fraction by silver nitrate, followed by ion exchange and paper chromatography, confirmed the presence of 7-MeGua (ca. 5.9 mg/day) and also revealed 1-methylguanine (1-MeGua,ca. 0.6 mg/day), W-methylguanine (0.5 mg/day), 1-methylhypoxanthine (0.4 mg/day), 8-hydroxy-7-methylguanine (1.6 mg/day), and several methylxanthines. Labeling studies by Mandel et al. (12) showed that the methyl group of urinary methylpurines was derived from methionine. Numerous studies have shown that tRNA contains various methylated nucleoside bases and that normal turnover of tRNA combined with the inability of purine salvage pathways to catabolize methylated purines explains the presence of relatively high levels of these products in normal human urine (13). In experimental animals, CraddGik and Magee (14) also showed that administration of methylating carcinogens such as N-nitrosodimethylamine (NDMA) resulted in the increased excretion of 7-MeGua which was in part derived from DNA methylation in the target organ, methyl-N-nitrosocyclohexylamine; MMS, methyl methanesulfonate; MSMS, tandem maas spectrometry; uurABC, repair enzyme complex in bacteria responsible for excision repair; OB-MedG, OB-methyl-2’-deoxyguanosine;OB-MeGua,OB-methylguanine;I-MeAde, N1-methyladenine; 1-MeGua, N1-methylguanine; 7-alkdG, IV7-alkyl-2’-deoxyguaosine;7alkG, N7-alkylguanosine; 7-alkGua, IV7-alkylguanine; 7-BPDEGua, N7(7,8,9-trihydroxy-7,8,9,lO-tetrahydroben~alpyren-l0-yl~guaine; 7-BPGua, N7-(benzo[alpyren-6-yl)guanine; 7-EtGua, N7-ethylguanine; 7HOEtAde, N3-(2-hydroxyethyl)adenine;7-HOEtGua, M-(2-hydroxyethy1)guanine; 7-MedG, M-methyl-2’-deoxyguanosine;7-MeGua, N7methylguanine; 7-PhGua, N7-phenylguanine; 3-alkAde,N3-alkyladenine; 3-BzAde,N3-benzyladenine;g-EtAde, N3-ethyladenine; g-HOEtAde,N3(2-hydroxyethy1)adenine; 3-MedA, N3-methyl-2’-deoxyadenosine;3MeAde, N3-methyladenine; 3-MeGua, N3-methylguanine.
0 1992 American Chemical Society
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Invited Reviews
\+ /
sugar
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neutral pH
cytosine
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guanine
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Figure 2. Depurination and ring-opening of hT-methylguanine nucleosides.
thymine
- adenine
Figure 1. Sites of modification by alkylating agents in DNA. liver. This latter observation formed the basis for much subsequent work on the excretion of carcinogen-derived DNA adducts. The application of techniques such as stable isotope labeling and immunochemical purification and detection has considerably increased the possibilities for analysis of urinary DNA adducts. It is therefore timely to review the literature in this area and to evaluate critically the relevance of these measures of carcinogen exposure. The organizationof this review is by class of carcinogenic agent-alkylating and arylating agents (although, in fact, most polycyclicaromatic hydrocarbon adducts are, strictly speaking, alkyl adducts), considered separately, and mycotoxins (almost exclusively aflatoxins). First, however, it is important to examine the overall rationale for the use of excreted adducts of these classes of compounds and to describe the relationship between the formation of DNA adducts and their repair and subsequent excretion.
The Origins of Urinary DNA Adducts The Formation of DNA Adducts. Alkylating agents react with DNA at most, if not all, of the nucleophilic nitrogen and oxygen centers (Figure 1). Highly reactive electrophiles such as the methyldiazonium ion (CH3Nz+) show little discrimination and are known as SN1-typealkylating agents (15). In contrast, less reactive S~2-type agents such as methyl methanesulfonate (MMS) give adducts predominately at nitrogen centers with very little reaction at oxygen. For arylating agents, there are other factors which influence the course of reaction with DNA. For example, with a bulky adduct like B[alP, steric hindrance and intercalation result in preferential formation of *-guanine adducts (16). From the point of view of noninvasive measures of DNA alkylation, it is important to consider the relative contribution of alkylation of other nucleic acids or precursors as sources of urinary adducts. N-Methyl-N'-nitro-Nnitrosoguanidine (MNNG) and N-methyl-N-nitrosourea (MNU) react in vitro with various forms of RNA to give primarily 7-MeGua (80-90%) along with small amounts of other adducts: 06-methylguanine (06-MeGua, 3-4 % ),
N1-methyladenine (l-MeAde, 2-4 % ), and N3-methyladenine (3-MeAde, ca. 1%)(17, 18). Similar patterns of adduct formation were obtained in liver ribosomal RNA (whichcomprises >80 7% of total RNA) of rats treated with NDMA (19). There was apparently no repair of methyl adducts in liver RNA in either rats or Syrian golden hamsters. 7-MeGua in rat liver RNA, as a result of methylating agent exposures, disappeared with the same kinetics of labeled RNA itself (t1p = 5 days; 20). Adduct ratios did not alter over a 4-day period in hamster liver RNA following treatment with NDMA, suggesting that even minor adducts such as 06-MeGua and N3-methylguanine (3-MeGua) were not repaired (21). It has also been suggested that deoxynucleotide pools may be s i g nificant targets for adduct formation (22). However, the pool size is normally very small compared to DNA, even in S-phase, and the profile of adducts formed by deoxynucleotides is different from those of the same bases in DNA (23). It therefore seems unlikely that either RNA or deoxynucleotide pools are significant sources of urinary alkyl adducts in short-term (several days) monitoring studies. Chemical Stability of DNA Adducts. (A)N7-Alkylguanines (7-AlkGua). Alkylation of 2'-deoxyguanosine (dG) or guanosine (G) a t N7 gives the corresponding "7alkyl-2'-deoxyguanosine (7-alkdG) or "7-alkylguanosine (7-alkG)which have positive charges on the imidazolering (24). 7-AlkdG are relatively unstable and undergo spontaneous depurination to give the corresponding 7-alkGua. As the pH is raised, a competing hydrolysis reaction takes place and results in opening of the imidazole ring, presumably via attack of hydroxide at C-8, to give the (Fapy) derivcorresponding 5-formamido-4,6-pyrimidine ative (Figure 2). The structure of the alkyl group [for a series of "7-(2-X-ethyl)Gl has a negligible effect on the rate of depurination whereas ring-opening is faster with more electronegative X (25). Similar observations were made for "7-@-X-benzyl)G (26). The lability of N7-methyl-2'-deoxyguosine (7-MedG) in methylated DNA has been demonstrated in a number of in vitro systems. With Tz phage DNA which had been methylated with [WIMMS, the half-life for the loss of 7-MeGua was 6 days (24). Margison et al. (27) observed a similar rate of loss of 7-MeGua when DNA removed from livers of rats treated with NDMA was incubated in phosphate buffer at pH 7.8. Interestingly, the t1p (at pH 6.9)for depurination of N7-methyl-2'-deoxyguanosine5'monophosphate itself was much shorter (16.4h; 24). (B)N3-Alkyladenines (3-AlkAde). 3-AlkAde, like 7-alkGua, are spontaneously depurinated from DNA. The
452 Chem. Res. Toxicol.,
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) I
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Shuker and Farmer
I
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Figure 3. Depurinationand ring-opening of N3-methyladenine nucleosides. average tl/2 for this process, from a variety of in vitro experiments with alkylated DNA, is 26 h (28,29). N3Methyl-2’-deoxyadenosine[3-MedA, t1p 35 min (30)l was much less stable than the adduct in DNA ( t l p 24 h; 15). Fujii et al. (30)observed pyrimidine ring-opening of 3MedA under alkaline conditions, but it occurred much less rapidly than depurination (Figure 3). (C) N7-(AFBI)-guanine(AFBI-Gua). The rate of spontaneous depurination of AFB1-Gua in DNA in vitro (t1p > 100 h) is similar to that of 7-MeGua (31). Enzymatic Repair of Purine Adducts. In early experimental studies on the formation and stability of alkyl adducts in vivo it rapidly became apparent that alkylpurine adducts were removed from DNA much faster than could be explained by the known rates of spontaneous hydrolysis (29).Extensive studies in bacteria and rodents have established the presence of a number of DNA repair enzymes including alkyltransferases (which repair O-alkyl adducts by removal of the alkyl group) and glycosylases (which catalyze the cleavage of the glycosidic bond, thus liberating the free alkylpurines; 32). Bacteria possess at least two distinct alkylpurine-DNA glycosylases(3-MeAde glycosylases I and 11; 33) whereas rodents and mammals appear to have only the equivalent of 3-MeAde glycosylase 11,which has a broad substrate specificity and removes 3-MeAde, 7-MeGua, and 3-MeGua as well as higher homologues (34,35).The human 3-MeAde glycosylasegene has recently been cloned and expressed in bacteria, enabling the purification of the enzyme to homogeneity (36,37).Den Engelse et al. (38) studied the stability of methyl- and ethyl-DNA adducts in rat liver and reported apparent half-lives for 3-Me- and 3-EtAde (6.5and 7.5 h) and 7-Me- and 7-EtGua (29h and 6.1days) which are, for the most part, indicative of active repair. AFB1-Gua is actively removed from DNA in rat liver (31)and human fibroblasts (39).The activity responsible for this depurination in bacteria has been identified as the uvrABC excision repair system (40). The N7-guanine adduct of benzo[alpyrene diol epoxide (BPDE) in DNA is reported to be unstable (tlp 3 h; 41)and is rapidly excreted in vivo (42). A recently identified 7-BP-Gua adduct is also reported to undergo rapid spontaneous depurination in DNA (43). Excretion of Excised DNA Adducts. The presence of alkylpurines in urine suggested that they were resistant to catabolic enzymes which operate in the purine salvage pathways. A number of experimental studies provided indirect evidence that 7-MeGua (14)and 3-MeAde (44) were excreted unchanged in urine. Recent studies in rats showed that PH1-3-MeAde was essentially quantitatively
excreted irrespective of the route of administration (45). A similar observation was also made in the case of [14C]7-(carboxymethyl)guanine (46). The metabolism of a series of deuterium-labeled 3alkAde was studied in human volunteers, following oral administration, and showed that 3-Me-, 3-Et-, and 3-HOEtAde were rapidly excreted unchanged whereas the low recovery of N3-benzyladenine (3-BzAde) indicated that some metabolism was taking place to as yet unidentified products (47).2 The metabolism of AFBl-M-Gua has not been studied directly, but it is known that 30-4074 of the total AFB1Gua adducts in the liver are excreted as this adduct (48). The main DNA adduct of cis-platin, cis-[Pt(NH3)2(dGpdG)], is excreted in urine following administration of the preformed adduct by intraperitoneal (ip) injection. The adduct was unstable in urine (49). The currently available data on urinary excretion of excised DNA adducts is summarized in Table I (note that, where values for percent excretion are given, these refer to the percentage of formed DNA adduct which is excreted and not the percentage of the dose of administered corresponding alkylating agent which forms the adduct, which is normally much less than 1% 1. Background Levels of Urinary Adducts. A number of early studies established the presence of background levels of various alkylpurines in human and animal urine (10,11,50).In most cases these compounds are derived from tRNA turnover (12)and are unrelated to alkylating agent exposure. However,the use of increasingly sensitive analytical techniques such as GC-MS or tandem mass spectrometry (MS-MS) has revealed the presence of very low levels of adducts which either are present in foodstuffs and are thus an artifact or, more importantly, are related to continuous endogenous alkylating agent exposure. Lawley (29)proposed that 3-MeAde may be a useful urinary marker of methylating agent exposure because it, unlike 7-MeGua, was not present as a modified base in tRNA. As discussed later, recent studies showed that 3-MeAde was present in both human and animal urine due to its presence in foodstuffs (51a),possibly as a result of the use of methyl bromide as a fumigant (51b). Recent studies using MS-MS and gas chromatographytandem mass spectrometry (GC-MS-MS) have established the presence of 7-(2-hydroxyethyl)guanine (7HOEtGua) in human urine and allowed quantification of W-methyl- and W,W-dimethylguanine (0.35and 0.07 mg/ day re~pectively).~ The presence of W,W-dimethylguanine in human urine (probably from RNA) had previously been demonstrated by paper chromatography (52)and GC-MS (53). A urinary constituent of molecular weight corresponding to an ethylated guanine (179) was shown by deuterium exchange studies and MS-MS to be Nzethylguanine (54)rather than 7-EtGua. The analysis of human urine using an immunoaffinity cleanup step selective for 3-alkAde as a group followed by separation and quantification by GC-MS revealed the presence of low levels of 3-HOEtAde (ca. 10 nmol/day) and 3-EtAde (95 5% of the AFBl residues Benzene and Related Compounds. Norpoth et al. (97) bound to DNA (4). AFBl binds to rat liver DNA in a found that "7-phenylguanine (7-PhGua) was excreted in linear dose-dependent manner, even at low doses (106), rats treated with benzene by intraperitoneal injection. 7and good correlations have been observed between urinary PhGua was identified by mass spectrometry and comexcretion of AFB1-Gua and both administered dose of parison with authentic material (98). Approximately 1% AFBl and AFB1-DNA in rat liver DNA (48). of the administered dose of benzene was excreted as 7Urinary AFB1-Guaappears to be a very good marker of PhGua over the 5 days following administration, and a carcinogenic risk in animal models. In studies on the rather unusual, unexplained, biphasic excretion curve was inhibitory effect of ethoxyquin on preneoplastic hepatic observed. Interestingly, 7-PhGua was also found to be lesions (7-glutamyl transpeptidase-positive foci), reduced excreted (0.04% of the administered dose over 7 days posturinary excretion of AFB1-Gua appeared to correlate with dosing) as the only detectable guanine adduct following lowered initiallevels of AFB1-Guain liver DNA (107,108). administration of chlorobenzene to phenobarbital-treated Administration of the antischistosomal drug oltipraz 15rats (99). The absence of the expected ortho, meta, or (2-pyrazinyl)-4-methyl-1,2-dithiole-3-thionel or the parent para isomers of AV-(chlorophenyl)guanine adducts was compound, 1,2-dithiole-3-thione,to rats given a carcinoconfirmed by comparison with authentic compounds. genic regime of AFBl results in dramatically reduced tumor (2) Benzo[a]pyrene (B[a]P). Autrup and Seremet yields (109,lI 0). This phenomenon was accompanied by (42) isolated a urinary guanine adduct following admina reduction in the initial levels of excretion of AFB1-Gua istration of B[alP to rats and identified it as 7-(7,8,9trihydroxy-7,8,9,10-tetrahydrobenzo[alpyrene-lO-y1)- (which mirrored the reduced levels of AFB1-DNA in liver in the 24-h period immediately following AFBl adminguanine (7-BPDEGua) (Figure 7). The excretion of 7istration). However, the levels of total urinary AFBl BPDEGua was complete within 72 h postdosing and metabolites remained unchanged, thus indicating that corresponded to between 0.06% and 0.15% of the adshort-term molecular dosimetry based on the excreted ministered dose of B[alP (10-100 pg). In view of the DNA adduct (which accounts for less than 1%of the hydrophobic nature of BPDEGua adducts it is perhaps not surprising that Tierney et al. (100) reported the administered dose of AFB1) is indeed a valid predictive presence of such adducts in feces following topical marker of ultimate cancer risk. The mechanism of inhibition by 1,2-dithiole-3-thiones appears to involve treatment of B[alP to mouse skin; however, the fecal adinduction of glutathione S-transferases, which leads to duct was tentatively identified as the N7-BPDE-deoxyguanosine adduct and not the free base as found in urine. more efficient trapping of electrophilic intermediates.
Invited Reviews
(B) Human Exposures. Autrup et al. (111)detected AFB1-Gua in urine samples collected from subjects in an area of Kenya where food samples were contaminated with AFBl and where the incidence of liver cancer is high. The identity of urinary AFB1-Gua was confirmed by fluorescence spectrophotometry and comparison with an authentic sample. In a subsequent study a regional variation in urinary AFB1-Gua was observed, but only moderate correlations with liver cancer risk were obtained (113). The analysisof the large numbers of urine samples which are required in order to carry out molecular epidemiological studies on the role of AFBl in liver cancer has been greatly expedited by the development of monoclonal antibodybased immunoaffinity columns which allow rapid purification of AFB1-Gua prior to quantitative analysis (73, 112). Several recent studies of human populations with exposure to dietary AFBl and elevated liver cancer risk illustrate the value of urinary AFB1-Gua as an informative biomarker. In samples collected from subjects living in Guangxi province, in the People’s Republic of China, a good correlation was found between dietary AFBl and AFB1-Gua over a l-week period. A somewhat poorer correlation was observed when daily intake and the following day’s excretion were used. Both of these correlations were superior to those obtained when urinary excretion of AFBl metabolites, either as a total or individually,was used (114). Areasonably good correlation was found between dietary AFBl intake and total urinary AFBl metabolites in subjects from The Gambia, but a better correlation was obtained using AFB1-Gua. Interestingly, no differencewas detected in AFB1-Gua excretion between hepatitis surface antigen positive and negative carriers for the same dietary intake of AFBl (115). In a case-control study in Shanghai, subjects with liver cancer were more likely than controls to have detectable levels of urinary AFBl metabolites, including AFB1-Gua (RR = 4.9, 95% CI = 1.5-16.3) (116). Summary and Prospects The available literature, which has been considered in this review, suggeststhat urinary DNA adducts can provide valuable information about human exposure to alkylating carcinogens. In many cases the received dose of carcinogen may be unknown, particularly if a major part of it is derived from endogenous processes [as may well be the case for some N-nitroso compounds (117)l. As a result of efficient DNA repair or chemical depurination of the major adducts of many alkylating carcinogens, alkylated nucleic acid bases are excreted in urine. These urinary adducts either are largely derived from modifications in the target organ (for example, liver in the case of AFB1) or represent an integrated measure of whole-body dose for carcinogens which show organotropism but which form adducts in many, if not all, tissues (for example, ethylene oxide; 118). The extensive studies which have been carried out on AFBl exposure in both experimental animals and humans provide a valuable paradigm for the exploitation of urinary DNA adducts in the rapidly developing field of molecular epidemiology. Early experimental studies demonstrated a clear relationship between the excretion of AFB1-Gua and adduct formation in the liver. A convincing demonstration of the link between urinary adduct excretion and biological outcome (both preneoplastic liver foci and hepatocellular carcinoma) in experimental animals was
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made using inhibitors of carcinogenesis based on the dithiolethiones. Not only was excretion of AFB1-Gua a good predictor of carcinogenic outcome following AFBl treatment but its reduction in inhibition experiments predicted the consequent reduction in tumors. This overall approach was borne out in a case-control study in China in which elevated levels of urinary AFB1-Gua (as well as some other AFBl metabolites) were associated with risk of developing hepatocellular carcinoma (116). A logical extension of these studies is to study the excretion of AFB1-Gua in subjects taking part in intervention studies aimed at either reducing exposure to AFBl or evaluating the effectiveness of chemopreventive agents. This illustrates the use of urinary adducts as short-term indicators of carcinogenic risk. The situation for many carcinogens is not so clear-cut as that for AFB1, but the field of excreted DNA adducts is still relatively unexplored and gives every indication of developing rapidly. The suspected human leukemogen, ethylene oxide (EO),has been extensively studied and the dosimetric and biomonitoring approach based on its protein adducts by Ehrenberg and co-workers (8)is widely used (119). However, experimentalstudies (118)indicated that EO formed DNA adducts and that these adducts were repaired, to more or less the same extent, in all tissues. Recent results by Skopek et al. (120) on the mutational spectra induced by EO in exon 3 of the hprt locus in rats indicate that apurinic sites induced by depurination of 7-HOEtGua and 3-HOEtAde may be important mutagenic lesions and suggested that excreted hydroxyethylated bases may be a direct measure of this phenomenon. The use of alkylating chemotherapeutic drugs offers a unique opportunity to study human exposure to welldefined doses of known or suspected carcinogens. As survival of cancer patients increases, the risk of developing iatrogenic second cancers also increases, and it would be of great benefit to reduce this risk while maintaining or even improving therapeutic efficacy. In view of the ease of obtaining blood samples from cancer patients there have been a number of studies on leucocyte DNA adducts arising from alkylating agent use (most notably with cis-platin; 7). Nonetheless, the studies which have been carried out using urinary adducts suggest that this technique can provide interesting results by noninvasive means. For example, the use of MNU in chemotherapy demonstrated that urinary 3-MeAde was a good indicator of internal dose (see above) and showed that there was wide interindividual variation in adduct formation for the same dose, which may have consequences not only for clinical response but also for the risk of second cancer in patients who survive long enough after treatment. Urine collectionfrom healthy, nonhospitalized subjects is easy, and it is relatively safe to handle compared to other biological fluids. The apparent lack of cultural barriers to its collection makes it amenable, in practical terms, to ready incorporation into molecular epidemiological studies. The resistance to the use of urinary markers is most often related to their intrinsic lack of long-term exposure information compared to, for example, blood protein adducts or stable DNA adducts. However, with an increasing tendency toward the conduct of prospective epidemiological studies with the aim of detecting previously unknown risk factors, this apparent shortcoming of urinary DNA adducts is less relevant. In
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addition, the analytical potential of currently available techniques for the identification and quantification of urinary DNA adducts provides a powerful tool for the molecular epidemiologist to evaluate human exposure to carcinogenic alkylating agent exposures. Acknowledgment. The work from our laboratories which is described in this review would not have been possible without the enthusiastic and talented contributions of many collaborators and colleagues, including technicians, students, and postdoctoral fellows. The comments and suggestions of Drs. Virginie Prevost and Linda Shuker on early drafts are gratefully acknowledged. Dr. Stephen Naylor is thanked for his contributions to the genesis of this review. D.E.G.S. acknowledges support from the US. National Cancer Institute (1988-1991, CA48473).
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