Urinary markers for measuring exposure to endogenous and exogenous alkylating agents and precursors.

Noninvasive methodologies for measuring carcinogen exposure in humans, based on the use of urinary markers, are being developed and validated for use in molecular epidemiological studies. A range of 3-alkyladenines can be determined in urine samples by an immunoaffinity purification-GC/MS approach [3-methyladenine, 3-ethyladenine, 3-(2-hydroxyethyl)adenine, and 3-benzyladenine]. Using this method, recent results in human subjects suggest that urinary 3-alkyladenines are potentially useful markers of alkylating agent exposure, particularly where the backgrounds of such adducts are much lower than 3-methyladenine. Urinary excretion of S-benzylmercapturic acid has been studied in experimental animals as a marker of exposure to benzylating agents such as N-nitroso-methylbenzylamine. 3-Nitrotyrosine (NTyr) is formed in vivo in tissue or blood proteins after exposure to nitrosating and/or nitrating agents such as tetranitromethane. After turnover of proteins, NTyr is released and excreted in urine as metabolites 3-nitro-4-hydroxy-phenylacetic acid and 3-nitro-4-hydroxyphenylacetic acid, which are determined by GC with a thermal energy analyzer. The sensitivity and specificity, combined with ease of use, of these noninvasive biomonitoring approaches means that they may be readily incorporated into molecular epidemiological studies in which exposure to nitrosating and alkylating agents may be important risk factors.


Introduction
In the rapidly expanding field ofmolecular epidemiology, the precise determination of individual exposure to carcinogens is regarded as a desirable goal. It would, nevertheless, be a mistake to translate this objective into one of the determination of individual cancer risk [although this remains a popular idea (1)].
In contrast, it is clear that groups of people at high risk of developing a particular cancer may be identified using molecular markers of carcinogen exposure. A number of methods are available to determine human exposure to carcinogens and are based on the idea that initiation, via formation of mutagenic lesions in DNA, is a crucial (but not sufficient) step in multistage carcinogenesis (2). Thus, characteristic adducts can be determined in DNA extracted from a variety ofsources inducing target tissues (obtained during surgery or at necropsy), or from nucleated blood cells such as lymphocytes. This approach has unequivocally demonstrated human exposure to a range of agents arising from food [e.g., aflatoxin B1 (3)], occupation [e.g., benzo[aJpyrene (4)], and lifestyle [e.g., tobacco smoking (5)].
Blood protein adducts to these same exposures have been exploited as surrogate markers of DNA damage (3,5). All ofthese methods require tissue or blood samples which may limit the numbers of samples available for analysis. In addition, levels of adducts in nontarget tissues may not necessarily be good indicators oftarget tissue exposure or even ofwhole-body burden. It is from this standpoint that the idea of using noninvasive methods based on urinary analysis has been developed. Of the many types of DNA damage arising from carcinogen exposure, DNA alkylation has been extensively studied and biomonitoring methods based on this phenomenon form the main focus of this review.
As mentioned above, measures of human exposure to agents resulting in characteristic DNA damage are among the most important types of information to obtain, and it is crucial to show that any indirect method (such as urinary analysis) can give relevant information. Work ty Craddock and Magee (6) showed that exposure ofrats, whose DNA was labeled with 14C, to 3H-labeled dimethylnitrosamine (NDMA) resulted in urinary excretion of doubly labeled N7-methylguanine (N7-MeGua). It was shown, unambiguously, that most of this urinary adduct was derived from liver, which is the major target organ for NDMA carcinogenesis. More recently, Gombar et al. (7)  able at doses where in situ methylation was undetectable due to practical problems in obtaining sufficient DNA from liver for adduct analysis at low levels of modification (whereas urinary N7-MeGua was derived from the whole organ and possibly other sites). Bennett et al. (8) showed in rats that urinary excretion of aflatoxin B,-guanine adduct correlated well with levels of adduct in the liver (which is the target organ). These results suggested that determination of excreted DNA adducts (or alkylpurines) that arise as a consequence of DNA repair mediated by DNA glycosylases (9) form the basis of noninvasive methods for determining of DNA damage following human exposure to alkylating carcinogens.
In a broader context, it may be useful to obtain information on the whole-body burden of alkylating agents and/or precursors. Many alkylating carcinogens react with glutathione (GSH) in vivo and give rise to mercapturic acids (MAs), which in many cases represent the major urinary metabolite (10). SN2 alkylating agents (such as alkyl halides, epoxides, and alkylsulfonates) can give up to 50% of the administered dose as MAs, which has led to much interest in the use of MAs as urinary markers of exposure to SN2 alkylating agents (11). In contrast,SN1 alkylating agents such as alkyldiazonium ions (the presumed active metabolites of N-alkyl-N-nitroso compounds) tend to give much lower yields although fewer of these compounds have been studied in this respect [e.g., dimethylnitrosamine (12), Nnitroso-di-n-butylamine (13), and N-nitroso-N-methylbenzyl- From the point ofview of human biomonitoring to alkylating carcinogens, mercapturic acids offer the possibility of noninvasively measuring the biological effective dose of alkylating species. There are indications, however, that the use of mercapturic acids must be approached with care. Many studies in humans have made use of methods to determine total urinary thioethers (which include MAs), and in many cases no differences could be detected between controls and exposed subjects with wide variation in individual levels (11). Aringer and Lidums (14) have also drawn attention to the problems of dietary confounding in the total urinary thioether method. As a consequence, it has been recognized that the determination of individual mercapturic acids is not only desirable because of sensitivity, but also adds to the specificity of the method, as in the case of acrylonitrile (15), benzene (16) and ethylene oxide (17). Individual MAs can be determined by a number of methods including GC (15), HPLC-electrochemical detection (18), HPLCfluorescence of derivatives (17), and GC-MS (19).
Endogenous formation of N-nitroso compounds has been shown to occur in humans, using N-nitrosamino acids excreted in urine as a marker (20,21). Carcinogenic N-nitroso compounds appear to be formed mainly in the stomach by the reaction of certain nitrogen-containing compounds with nitrite under acidic conditions. However, endogenous nitrosation may also occur in other organs such as the lung (22) and skin (23) after exposure to oxides of nitrogen and in inflamed or infected tissues by activated macrophages and bacteria (24,25), although such reactions have not yet been demonstrated to occur in tissues in vivo.
We are currently developing and validating noninvasive methodologies, based on the use of urinary markers, for even-tual use in human biomonitoring studies. This approach is divided into three parts: a) an assessment of DNA damage by measurement of excreted alkylpurines b) the determination of specific urinary mercapturic acids, and c) the evaluation of precursors, e.g., endogenous nitrosation potential using 3nitrotyrosine metabolites. Recent results in these areas are reviewed in this paper.

Urinary Alkylpurines
Alkylation at N-3 of adenine is a major route of DNA-adduct formation for many alkylating carcinogens (26). The resulting 3-alkyldeoxyadenosines are unstable and rapidly depurinate either spontaneously or via the action of specific DNA glycosylases to give the corresponding 3-alkyladenines (3-alkAde) (9).
We have recently shown that 3-methyladenine (3-MeAde) can be rapidly quantitated in human urine by immunochemical and/or GC-MS methods (27,28). In a recent study of cancer patients receiving methylnitrosourea (MNU, at a total single dose of 300 or 600 mg) as part ofa combination chemotherapy, 24-hr urine samples were collected from each patient immediately before and after MNU administration. Analysis of urinary 3-MeAde showed, in every case, increased excretion of this marker of methylation after treatment. Overall, a dose-dependent excretion of 3-MeAde was observed, and preliminary experiments suggest some correlation between methyl adducts (7-methylguanine and O6-methylguanine) in lymphocyte DNA and urinary 3-MeAde excretion (Shuker et al., manuscript in preparation).
The use of3-MeAde as a marker of methylating agent exposure to low levels of methylating agents is complicated by a relatively high urinary background. However, recent studies have indicated that simple dietary manipulation can virtually eliminate this background (27). In an experiment designed to examine the effect of cigarette smoking on urinary methyl adduct excretion, three healthy volunteers (current or ex-smokers) agreed to collect 24-hr urine samples for 10 consecutive days. During this period, they consumed normal diets, and smoked if they wished, on days 1, 2, 9, and 10. On days 3-8 they consumed a balanced liquid diet and bottled water, which was shown to contain a very low amount of preformed 3-MeAde (27). On days 5 and 6, the subjects were allowed to smoke their normal brand of cigarette ad libitum. Urines were analyzed for 3-MeAde levels, and the results are shown in Figure 1. It is clear that the dietary control rapidly lowers and stabilizes urinary 3-MeAde excretion (interestingly, a slight excess of excretion over intake is always observed) and that cigarette smoking markedly increases levels of this marker. In view ofthe apparent ubiquitous occurrence of preformed 3-MeAde, an analysis of tobacco smoke (from each brand used) was undertaken, and a small amount was detected. However, in no case did this background account for more than 10% of the observed increase. The rapid rise and fall of urinary 3-MeAde, is consistent with endogenous DNA methylation and rapid repair by tobacco smoke constituents, such as tobaccospecific nitrosamines (Prevost and Shuker, manuscript in preparation).

Specific Mercapturic Acids
Measures of alkylating agent exposure based on total urinary mercapturic acids are often confounded by the presence of natural background levels. In contrast, the determination of individual mercapturic acids should be characteristic of a particular exposure.
In view of interest in the role of N-nitroso-N-methylbenzylamine (NMBzA) as a possible etiological agent in esophageal cancer in China, the utility of S-benzylmercapturic acid (SBzMA) as a marker ofbenzylation was investigated. The excretion of SBzMA in the urine ofrats treated with NMBzA was determined by GC-MS using d7-SBzMA as an intenal standard.
The amount ofurinary SBzMA varied with the dose ofNMBzA (up to 5 mg/kg) and with rat strain. For the three strains investigated, most of a 2.5 mg/kg dose of SBzMA was excreted within 24 hr. Comparison ofthe levels of SBzMA excreted by rats treated with equivalent doses of either NMBzA or benzaldehyde indicates that urinary SBzMA is derived mainly from benzylating species resulting from the hydroxylation of the methyl group of NMBzA (30). In view of the success of immunochemical methods, in particular, immunoaffinity purification, in the area ofurinary DNA adducts, current work is aimed at preparing antibodies against mercapturic acids either individually (e.g., SBzMA) or as a group.
Endogenous Nitrosation 3-Nitrotyrosine (NTyr) in tissue or blood proteins was evaluated as a possible exposure marker for exogenous and endogenous nitrosating or nitrating agents. A sensitive and selective method for analyzing NTyr by gas chromatography with a thermal energy analyzer (GC-TEA) was developed. Using this method, kinetic studies were carried out. It was found that free and protein-bound tyrosine residues easily react with nitrating/nitrosating agents to yield NTyr. NTyr formation in vivo showed a dose dependent increase in NTyr in both plasma proteins and hemoglobin obtained from rats 24 hr after IP injection of various doses (0.5-2.5 ymole/rat) oftetranitromethane. Major urinary metabolites of NTyr, given orally to rats, were isolated and identified by GC-MS as 3-nitro-4-hydroxyphenylacetic acid (NHPA) and 3-nitro-4-hydroxyphenyllactic acid (NHPL). About 44 and 5 % of the oral dose of NTyr (100 lsg/rat) was excreted as NHPA and NHPL, respectively. Eleven 24-hr human urine samples were analyzed for NHPA by GC-TEA after ethyl acetate extraction and HPLC purification: quantities ranging from 0 to 7.9 Ag/24 hr (mean ± SD, 2.8 ± 2.3, n = 11) were detected (detection limit 0.2 ug/L). Thus, NTyr in proteins or its metabolites in urine can be readily analyzed by GC-TEA as a marker for endogenous nitrosation and nitration (31).
Initial attempts to prepare antibodies to NTyr in order to prepare immunoaffinity columns were not particularly successful, and an approach using novel haptenic forms of NTyr is currently being pursued.

Discussion
The methods described here are undergoing continuous refinement, particularly in the area of specificity, as well as being thoroughly validated. A criticism that may be leveled against the use of urinary markers such as excreted adducts is that they may be derived from total nucleic acid alkylation (i.e., DNA and RNA) because only the modified purine base is excreted. Although an experimental approach involving the use of stable isotope labeled DNA in vivo is being developed to directly address this question (Shuker, unpublished data), it is nonetheless interesting to consider the published information about RNA alkylation and repair and compare it to that on DNA alkylation. N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and Nmethyl-N-nitrosourea (MNU) react in vitro with various forms ofRNA to give primarily 7-methylguanine (7-MeGua; 80-90%) along with small amounts ofother adducts [06-MeGua, 3-4%; I-MeAde, 2-4%; 3-MeAde, -1% (32,33)]. Similar patterns of adduct formation were obtained in liver ribosomal RNA (which makes up > 80% oftotal RNA) ofrats treated with NDMA (34). Interestingly, there is apparently no repair of methyl adducts in liver RNA in either rats or Syrian golden hamsters. 7-Methylguanine in rat liver RNA, as a result of methylating agent exposure, disappears with the same kinetics as labeled RNA It112 = 5 days (35)]. Adduct ratios did not alter over a 4-day period in hamster liver RNA after treatment with NDMA, suggesting that even minor products such as 06-MeGua and 3-MeGua were not repaired (36). In comparison, various adducts in rat liver DNA after treatment with NDMA underwent active and sometimes rapid repair (adducts t /2: 7-MeGua, 29 hr; 06-MeGua, 21 hr; 3-MeAde, 6.5 hr), with similar results being obtained for ethyl adducts after treatment with N-ethyl-Nnitrosourea (37). Overall, these results suggest that increases in urinary alkylated purine bases at short time periods following alkylating agent exposure are likely to be mainly ofDNA origin.
It is clear, therefore, that noninvasive methods for measuring DNA damage based on the urinary excretion ofrepaired adducts is both technologically feasible, based on recent advances in analytical methodology, and scientifically valid, based on the published literature. However, a direct experimental validation is underway. The measurement of specific urinary mercapturic acids offers the potential of sensitive detection ofexposure, particularly for alkylating agents for which this is a major pathway of excretion, such as acrylamide.
Endogenous nitrosation can arise from endogenous sources of nitrosating agents such as macrophage-mediated synthesis of nitric oxide or exogenous sources such as nitrogen oxides (NO,) from cigarette smoke or urban pollution. Measurements of nitrotyrosine and its metabolites offer a possibility to quantitate this factor and are being used, particularly in studies involving urban pollution. Each ofthe three approaches described in this paper have the potential to convey information about various aspects of human exposure to alkylating carcinogens. It is clear, however, that a combination of these approaches is more powerful than when they are applied individually. For example, DNA adducts arising from preformed tobacco-specific nitrosamines can be readily determined using urinary adducts, but it will be interesting, in addition, to assess the role of endogenous nitrosation of tobacco alkaloids, using 3-nitrotyrosine, as this may be a major contributing pathway.
This manuscript was presented at the Conference on Biomonitoring and Susceptibility Markers in Human Cancer: Applications in Molecular Epidemiology and Risk Assessment that was held in Kailua-Kona, Hawaii, 26 October-l November 1991.
The technical assistance of Liliane Garren and Isabelle Brouet is gratefully acknowledged. Partial funding support from the U.S. National Cancer Institute is gratefully acknowledged (CA 48473).