Environmental carcinogens - American Chemical Society

has been a legitimate concern about environmental mutagensand carcin- ogens. Although useful as a first approxi- mation of potential exposure, the de-...
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John S. Wishnok

gen may have been-absorbed through the skin o r inhaled). Rigorous risk assessment requires measurement of the chemical effects resulting from exposure, knowledge of the dose-response relationship, and an understanding of the mechanisms of mutagenesis for each substance being considered. These measurements ultimately depend on the availability of suitable analytical methods, especially for quantitation of the actual active metabolites arising from the environmental substances. It is particularly challenging to make such measurements in human experiments, because the analyte levels are determined by fortuitous exposure a n d t h e samples typically are limited to urine or small amounts of blood. Although different classes of mutagens react by specific mechanisms, the majority-after metabolic activation-appear to initiate their effects by reacting at electron-rich sites on DNA. When DNA alkylation and subsequent mutation occur, along with a series of subsequent conditions that repair the mutation, car1126 A

cinogenesis may result. The development of sensitive ana-

1960s that some components of the diet, such as aflatoxins in poorly

ducts appear to provide the most reliable indication of exposure, because their existence is direct evidence of n wtive forms of the mutapal, relative adduct relative risk ( I ) . ion we will be ly with research s Division of Toxerunners, the De-

and target organs are strongly dependent on the compound’s structure (3). These observations led many researchers to assess a variety of nitrite - preserved foods for evidence of nitrosamine formation. Although secondary amines are common i n food, natural dietary nitrite is rare. Nitrate, however, is plentiful in the

diet and can be reduced to nitrite by bacteria, potentially leading to nitrosamine formation (4). Some experiments were carried out to determine if nitrite could react in the mouth or the stomach with co-ingested amines formed amines

routinely to quantita N-nitrosamines and nitr I n MIT’s Department of N and Food Science and later in the Department of Applied Biological Sciences and the Division of Toxicology, the use of MS to detect and monitor mutagens grew out of earlier work in which MS was used primarily to identify flavoring agents. The shift in emphasis from food science to toxicology was partly the result of discoveries in the 1950s and early

ANALYTICAL CHEMISTRY, VOL. 64, NO. 23,DECEMBER 1,1992

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and often have been carried out with GC/MS. A major advantage of MS is t h a t nonradioactive s t a b l e isotopomers can be used safely and ethically in humans. Such experiments typically involve measurement of [l5N/I4N]nitrate ratios in urinary nit r a t e following administration of [15N]nitrate (8)or labeled precursors such as 15NH, (9).The nitrate is derivatized by conversion to nitrobenzene, which separates well on polar GC columns (Scheme 1) (7).

compound nitric oxide. When morpholine was added to the culture medium, N-nitrosomorpholine was p r o d u c e d ; i n t h e p r e s e n c e of [15N] arginine, the nitroso nitrogen i n t h e N-nitrosomorpholine contained the label. In other words, the n i t r o s a t i o n r e a c t i o n proceeded through the same intermediatenitric oxide-as the nitrate-producing reaction (11). Similar results were obtained in vivo with rats (12). Nitrosamine formation is believed

HZSO

Nitrobenzene

Scheme '

Following the discovery that nitrate was produced endogenously, the next objective was to define the biochemical details of this process. An accidental observation, t h a t strikingly elevated nitrate excretion in one volunteer coincided with a n intestinal infection, directed attention toward t h e immune system. Subsequently it was discovered that a major cell type responsible for nitrate production was the macrophage

(10). 15N labeling experiments with macrophage-like cell lines in culture demonstrated t h a t t h e nitrogen source was one of the two equivalent guanido nitrogens of L-arginine and that the major intermediate in the synthesis was the s i m d e inorganic

to proceed via the oxidation of NO to NO, and then N20, as well as (to some extent) N,O,, which can react with secondary amines to yield Nnitrosamines at a physiological pH (13).These transformations are summarized in Scheme 2. In addition to the formation of N-nitroso compounds, NO may damage DNA via nitrosative deamination of nucleic acids in the presence of 0,. These alterations in DNA could lead to mutations that are important in activating human oncogenes (14, 15). The relevance of these pathways to humans was demonstrated by a n experiment in which two volunteers ingested [l'N]arginine. Two individuals consumed low-nitrate diets for two days. Urine was collected for two

28

5N] arginir

Scheme 2

24-h periods. At the beginning of the second 24-h period, each drank a solution containing labeled arginine. Increased levels of [15N]nitrate were detected in urine samples collected the next day (Table I [161). The top numbers were obtained from urine analyses from the first 24-h period (baseline levels). The bottom numbers were obtained from the second 24-h period. During and after these studies, a s t a r t l i n g number of endogenous sources and functions of nitric oxide were discovered. In addition to NO synthesis by stimulated macrophages, NO production by endothelial cells (as endothelial-derived relaxing factor, EDRF) activates a heme - depen dent guanylate cyclase, which leads to vascular smooth muscle relaxation. The role of NO in intracellular signaling is known to occur across species lines i n cells from many sources, including placental and pulmonary vessels, and in the perfused heart. NO is also produced by hepatocytes, neutrophils, and cells in the brain, where it functions as a neurotransmitter. These topics are summarized and discussed in several extensive and complementary reviews (17-19). Most analyses of NO production have been indirect; that is, they have involved quantitation of ultimate nitrite/nitrate concentrations (20)or measurement of related physiological effects such as muscle relaxation (21). Direct measurements of NO have been carried out with thermal

ompouna:

Source: Reference 16 tmoles/day

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ANALYTICAL APPROACH energy analysis (TEA) (21) or with GUMS (22).In a recent paper we describe a membrane mass spectrometer inlet for direct NO quantitation in either gases or liquids that can monitor NO production at concentrations as low as 1.4 pM in real time (23). Figure 1 shows t h e doseresponse characteristics of the inlet in both phases. A recent electrochemical microprobe method appears to have greater sensitivity (24). Mutagen-protein adducts The formation of altered DNA by a n active carcinogen metabolite is a principal event in the complex processes leading to clinical cancer.

Quantitation of chemically altered DNA in individuals is a challenge because of problems such as low adduct levels; efficient repair of the damage; dilution of adduct levels by cell division; and the difficulty of sampling human DNA, especially in the wide variety of target organs. Stimulated largely by suggestions from Lars Ehrenberg and co-workers (25), many cancer epidemiologists have focused attention on protein adducts with the assumption that levels of carcinogen-protein adducts will reflect levels of carcinogen-DNA adducts. In other words, the electrophilic intermediates that react with nucleophilic sites on DNA will also

Figure 1. Uose-response curves Tor the NO membrane mass spectrometer inlet. The gas-phase response is obtained by placing the inlet in an argon atmosphere and injecting known volumes of NO directly into the flask. The liquid-phase response is obtained with the inlet below the surface of water while NO is injected into the headspace. (Adapted from Reference 23 with permission from John Wiley and Sons, Ltd.) e . /---

Figure 2. Accumulation of Hb adduct following administration of '-%-labeled 4-aminobiphenyl to rats. (Adapted from Reference 1 with permission from Oxford University Press.)

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react with nucleophilic sites on other molecules such as serum albumin or hemoglobin (Hb). Ideally, relative levels of protein adducts would be proportional to levels of DNA adducts and might therefore be a n indication of relative individual risk. Although this relationship has been demonstrated in some cases (for recent examples, see References 26 and 27), it has not been shown to be universal. However, much research shows that protein adducts are good measures of relative exposure to active forms of carcinogens. Carcinogen-protein adducts are not directly related to carcinogenesis, but they offer several advantages as monitors for exposure to active carcinogen metabolites. It is relatively easy, for example, to obtain large amounts of human protein. In addition, many proteins undergo a fairly slow turnover and can therefore provide a n integral of exposure over weeks or months of exposure (1). For example, Hb has a lifetime of approximately 120 days in humans and can reflect cumulative exposure to reactive mutagens over a fourmonth period. This is illustrated by a dosing experiment with rats (Figure 2). The adduct levels build up to a steady s t a t e significantly higher than what would be expected from a single dose, and then they decay at essentially the same rate as that for red blood cell turnover when the dosing is stopped (1).In addition, Hb is present in the blood in comparatively large quantities and sampling is routine and minimally invasive. Aromatic amines Two groups of compounds for which Hb adducts have been quantitated are aromatic amines and polycyclic aromatic hydrocarbons (PAHs). Aromatic amines, many of which are human and animal carcinogens, form covalent adducts with Hb that can be analyzed in a straightforward manner. The reaction of aromatic amines with Hb proceeds via cytochrome P450 oxidation to hydroxylamines, followed by oxyhemoglobin- mediated formation of aromatic C-nitroso com pounds. The covalent adducts form by reaction of the nitroso compounds with Hb cysteines, followed by rearrangement to sulfinamides. Typical adduct levels are in the range of a few picomoles per gram of Hb. Quantitation of these levels would be expected to be extremely difficult if done on the adducted Hb itself. The actual quantitation, however, is done on small organic molecules; the sulfin-

amide reaction products can hydrolyze to release t h e original free amines, which are derivatized with pentafluoropropionic a n h y d r i d e (PFPA) (Figure 3) and quantitated by GC/MS with negative -ion chemical ionization (NICI) (Figure 4). The NICI mass spectra of these compounds typically show a single large peak corresponding to a nominal loss of HF from the derivative, as illustrated in Figure 4 for the PFPA derivative of 4- aminobiphenyl (28). Single-ion monitoring (SIM) analysis is done with these ions. The method is sensitive and reasonably selective. Because only a single ion is usually monitored in quantitative runs, it is important to confirm the identities of analytes by using pooled samples to obtain complete mass spectra. The most extensively studied

Oxyhemoglol

member of this class is 4-aminobiphenyl. This compound is a potent human bladder carcinogen found in tobacco smoke and is also a bladder carcinogen for several animal spe cies. I t is believed that the carcinogenicity of 4 - aminobiphenyl results from hepatocyte oxidation and subsequent hydrolysis of N-hydroxy-4aminobiphenyl to yield a n electro philic nitrenium ion that binds to DNA (29). Hemoglobin-aminobiphenyl a d duct levels are consistently higher in smokers than in nonsmokers. Involuntary exposure to tobacco smoke, or “passive smoking,” produces elevated levels of carcinogens such as 4-aminobiphenyl and 3 - aminobiphenyl. Adduct levels in individuals who quit smoking decline at the expected rate, based on the lifetime of human hemoglobin. Elevated levels of 4 -aminobiphenyl-Hb adducts were found in the fetal blood of children of pregnant smokers, demonstrating that tobacco smoke carcinogens can cross human placental membranes. Some of this work is described in Reference 1; more recent results will be summarized in a forthcoming review (30). As noted above, this analytical method works well for Hb adducts arising from most aromatic amines and has also been used successfully for other types of Hb adducts, such as those arising from tobacco - specific nitrosamines (31).In addition to the results described above for aminobi phenyl, the mean adduct levels of o-

and $-toluidine and 2-naphthylamine were higher in smokers than in nonsmokers. Adducts of 2-, 3-, and 4-ethylaniline; 2,3-, 2,4-, 2,5-, and 2,6-dimethylaniline; and 3-aminobiphenyl have also been detected in smokers’ blood (28).Figure 5 shows a representative set of SIM data indicating the presence of Hb adducts of several aromatic amines in the blood of a smoker. Polycyclic aromatic hydrocarbons Epoxides and diol epoxides are the resulting carcinogenic metabolites of many PAHs, which are found in coal tar, tobacco smoke, and diesel exhaust. Formation of the activated metabolites is catalyzed by cytochrome P450 (32). Figure 6 provides an overview of the metabolism of benzo[alpyrene (BAP),a typical carcinogenic PAH (33).The reaction between the benzylic carbon of the epoxide and a nucleophilic site in DNA presumably is the initial carcinogenic event. As with other DNA alkylators, reactions also occur with proteins, leading to stable adducts that are potentially useful as dosimeters for exposure to the active forms of the carcinogen. For example, both human and mouse Hb form carboxylate esters with anti- benzo[a]pyrene diol epoxide that release benzo[a]pyrene tetrols when hydrolyzed. Quantitation of these tetrols forms the basis for PAH dosimetry. In one study (33), 10 reactive me-

Figure 3. Summary of aminobiphenylHb adduct formation and derivatization.

Figure 4. NlCl mass spectrum of 4-aminobiphenyl-PFP.

(Adapted from Reference 28 with permission from John Wiley and Sons, Ltd.)

The ion at m/z= 295 (M - 20) represents loss of HF from the molecule. An HP 5987 gas chromatograph/ mass spectrometer was used with methane as the moderating gas.

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ANALYTICAL APPROACH tabolites of five PAHs and styrene were investigated to determine the common occurrence of ester adduct

formation with human Hb. Polycyclic aromatic alcohols were determined by subjecting the Hb to enzymatic

Figure 5. SIM data of lower molecular weight aromatic amine-Hb adducts in a smoker’s blood. Adduct concentrations ranged from about 10 pg/g Hb for 2-naphthylamine to about 2 ng/g Hb for aniline; the 4-aminobiphenyl adduct concentrationwas about 230 pg/g Hb. (Adapted from Reference 28 with permission from John Wiley and Sons, Ltd.)

P450

,8-Dihydrodio

‘01s

’01s

Figure 6. Summary of benzo[a]pyrene metabolism. Hydrolysis of the hemoglobin adducts releases tetrols, which can be determined by GC/NICI-MS as tetrakis(trimethylsily1)derivatives. (Adapted with permissionfrom Reference 33.)

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proteolysis followed by immunoaffinity chromatography. GC/MS was used for detection and quantitation of tetrakis(trimethylsily1) (TMS) derivatives (Figures 7 and 8). Analysis of blood from several different human populations showed t h a t Hb adducts of anti-benzoralpyrene diol epoxide were the most prevalent. These results suggested t h a t alkylation of carboxylic acid groups on Hb to form esters is a general reaction for epoxide and diol epoxide metabolites of PAHs. Hydrolysis of t h e e s t e r adducts t o t h e corresponding alcohols thus appears well suited for the quantitation of PAH-protein adducts in humans. These methods have all been developed specifically for the quantitation of adducts known or suspected to be present in groups of people who have been exposed to carcinogens. Identification of completely unknown adducts is substantially more difficult, largely because of the much lower sensitivity of MS in the scan mode than in the SIM mode; obtaining full spectra for qualitative analysis is often impossible. Nonetheless, we have been successful in identifying a Hb adduct arising from chrysene (35).The presence of a phenanthrene chromophore in a n unidentified peak of the HPLC chromatogram from a Hb fraction that had been partially purified by immunoaffinity chromatography was inferred from fluorescence spectra. When we compared our results with the known standards, the spectrum of the unknown compound resembled that of chrysene- 1,2,3,4-tetrahydrotetrol. The identification was sup ported by HPLC and GC retention times, the latter obtained by GC/MS in the NICI/SIM mode at m/z = 422, which is a characteristic ion for chrysene tetrahydrotetrols and for related PAH metabolites (35). DNA adducts There is a large amount of research on the detection and, to some extent, the characterization of carcinogenDNA adducts, especially with 32Ppostlabeling techniques (for a recent review, see Reference 36). As noted in the introduction, however, generally it is more difficult to quantitate carcinogen-DNA adducts than carcinogen-protein adducts in humans. The reasons for this, in addition to low adduct levels in general, include dilution of adducted DNA by cell division and the difficulty of obtaining sufficient sample for analysis, particularly in target tissue. However,

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ANALYTICAL APPROACH quantitation has been a long-term goal in cancer epidemiology (37)and has been performed successfully in several cases. Repair of methylated DNA, for example, leads to 3-methyladenine and 7-methylguanine in the urine at levels high enough for quantitation by GC/MS (38).This problem will almost certainly yield to the increasing sensitivity and sophistication of analytical techniques-for example, mass spectrometric developments such as matrix-assisted laser desorption, electrospray, FT- ICR spectrometry, and quadrupole ion traps with extended mass ranges. Analytical techniques The analytical techniques underlying various projects are often straightforward, especially with standards and in vitro samples. Major problems have been encountered during sample preparation; in many samples, it has been difficult to develop cleanup methods that allow quantitation of compounds present at low levels in complex matrices. Sample preparation is especially significant with human samples because there is no possibility of increasing the individual's exposure to carcinogens or, in many cases, of obtaining additional sample material.

We used an HP 5971 mass spectrometer equipped with a massselective detector as well as H P 5987 and 5989 GC/MS instruments. Most carcinogen-protein adduct quantitation is carried out on fluorinated derivatives using NICI with methane as the moderating gas. Electron ionization (EI) and positive ion chemical ionization (PICI) are often comparably sensitive when tested with standards, but NICI has generally given the best combination of sensitivity and selectivity when working with low levels of adducts in biological matrices (28,32, 39). For E1 runs (e.g., for 15N incorporation in urinary nitrate), the autotune routines generally are adequate. In NICI experiments, however, manual tuning and frequent source cleaning usually are required. (For a good discussion of NICI tuning for maximum sensitivity, see Reference 40.) 15N incorporation into urinary nitrate Our group (7) and others (41)have carried out urinary analyses by converting the nitrate into nitrobenzene, which can be readily analyzed by GC/ MS. Urine is first treated with zinc sulfate and silver sulfate to precipitate protein, followed by treatment

with benzene and sulfuric acid; this process is the well-known acid-catalyzed nitration of benzene. The product is extracted with toluene, benzene, or ethyl acetate and then dried and injected into the gas chromatograph, using a relatively polar column such as Supelcowax or DB-17. Recovery of nitrobenzene is unimportant if the ratio of 15N to 14N is the only thing of interest. The addition of a n internal standard allows this method to be used to quantitate total nitrate excretion. Nitrosation in vivo or in cell culture This process can be carried out by adding morpholine to the cells, extracting the resulting N-nitrosomorpholine with CH,Cl,, drying with Na,S04, and t h e n analyzing t h e extract directly by GC/TEA or-if a labeled precursor is used-by GCI MS. Nitrosation of morpholine i n vivo is followed by measuring a nitrosomorpholine metabolite, N- nitro sohydroxyethylglycine (NHEG), in the urine. The pH of the urine is adjusted to 7, and N-nitrosopipecolic acid is added as a n internal standard. Cleanup is achieved by using ion - exchange columns followed by extraction of the NHEG into ethyl acetate. The organic extracts a r e dried and the solvent removed under vacuum. The NHEG is converted into the bis(tert-butyldimethylsilyl) derivative and analyzed by GC/MS using a DB- 17 column (12,42). Nitric oxide Headspace NO analysis has been achieved by using several methods, including TEA, GC/MS, and membrane inlet MS, and by indirect forma-

1

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in)

Figure 7. Mass spectra of tetrakis(trimethylsily1)derivative of benzo[a]pyrene tetrol.

Figure 8. Selected-ion chromatogram at m/z = 446.0 from a sample of hydrolyzed Hb from an Italian smoker's blood suggesting the presence of up to three benzojalpyrene tetrols.

(a) NlCl and (b) El mass spectra using an HP 5989 gas chromatograph/massspectrometer with methane as the moderating gas. The major ion in the NlCl spectrum corresponds to loss of (CH,), Si-O-Si-(CH,),, M-162. (Adapted with permission from Reference 34.)

The lower trace at m/z = 448.0 is for the I4C-labeled internal standard. (Adapted with permission from Reference 34.)

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ANALYrICAL APPROACH tion of nitrosating agents such as N,O,. For TEA analysis, the gas chromatograph was used only as an injector. The column was replaced with a short length of uncoated wide-bore fused-silica capillary, and a dry-ice cold trap was placed between the column exit and the TEA inlet fitting. The headspace was then sampled directly with a gas-tight syringe (43). In some bacterial experiments, a solution of 1,2-diaminonaphthalene in CH,Cl, was placed in a test tube inside a sealed (serum cap) vessel containing cells grown anaerobically in the presence of nitrate. The amine thus was physically separated from t h e culture medium. Nitrite was added to the medium, followed in some cases by oxygen. In the absence of oxygen, no change was observed in the 1,2-diaminonaphthalene. If oxygen was added, however, the nitrosation product could be detected in the test tube (44). This experiment gave us a major clue that NO was being formed by the bacteria, because the nitrosation in the test tube could only have occ u r r e d via a n i t r o s a t i n g a g e n t formed by reaction of a substance with oxygen in the headspace of the culture medium. This agent then dissolved in the CH,Cl,; N,O, seemed a reasonable nitrosating species, and NO therefore was deemed a good candidate for the bacterial reduction product. The membrane inlet (23) consists of a short length of Silastic tubing fitted into a Swagelok tee that is connected directly to t h e mass spectrometer via the tuning probe. 4-Aminoblphenyl-Hb adducts Although this analysis originally was developed for 4-aminobiphenyl, i t appears to be applicable to Hb adducts of aromatic amines (28) and can be done routinely with 10 mL of whole blood. The blood is centrifuged to harvest red blood cells, which are washed with phosphate-buffered saline (PBS).The cells are lysed with water and toluene and then centrifuged again. The resulting aqueous phase, containing the Hb, is dialyzed against distilled water. The concentration of Hb is determined, and internal standard (4’-fluoro-4aminobiphenyl or perdeuterio-4aminobiphenyl) is added. The 4-aminobiphenyl is then cleaved from the Hb by hydrolysis with 0.1 M NaOH. The amine is carefully extracted with double-distilled hexane, and the ext r a c t is dried over Na,SO, and MgSO,. Following derivatization with pentafluoropropionic anhydride/ 1134 A

trimethylamine, the solvent is removed and the (4-aminobipheny1)pentafluoropropionic anhydride derivative is reconstituted in hexane or heptane. Quantitation is achieved with GC/MS on a polar column (e.g., Supelcowax) using NICI. PAH adducts The general technique is analogous in concept to that used for the aminobiphenyl-Hb adducts; the Hb is isolated, and the covalent adducts are released by hydrolysis and then analyzed by GC/MS (33, 39, 45). There are, however, several differences. Metabolites, for example, rather than the hydrocarbons themselves, are released when hydrolyzed; the key step in the cleanup is immunoaffinity chromatography; and TMS derivatives rather than pentafluoropropionyl d e r i v a t i v e s a r e used for GC/MS quantitation. Hb is collected and measured, as described above. The internal standard for BAP tetrol is the ‘*C isotopomer (?)-[7-l4C] -Y- 7,t-8,t-9,c- 10-tetrahydrobenzo[a]pyrene prepared from a commercially available 14C diol epoxide. After adding internal standard, the Hb is hydrolyzed in 0.3 N HC1 at 75 “C and then cooled, taken to pH 10-11, and extracted with ethyl acetate. The organic extracts are dried and redissolved in PBS, and this solution is applied to an immunoaffinity column t h a t recognizes BAP tetrols. The tetrols are eluted with methanol: water. The eluate is dried thoroughly, and the tetrols are derivatized in the gas chromatograph injector by coinjection with Tri-si1 ‘Z’ (Pierce Chemical Co.). The mass spectrometer is operated in the NICI mode; methane is the moderating gas. The GC column is DB- 17. In this ANALYTICAL APPROACH we have demonstrated that it is possible to do routine quantitative analysis of in vivo (including human) responses to known or potential carcinogens using unmodified commercially available GC/MS equipment. Researchers in this field must incorpor a t e multidisciplinary experience from t h e t h e fields of analytical chemistry, biochemistry, organic chemistry, and epidemiology to ensure that the results are meaningful. We a r e planning to extend our studies to investigate cultural or racial differences in protein adduct levels following dietary or environmental exposure to carcinogens, effects of illnesses on the arginine-nitric oxide pathway, and the direct effects of nitric oxide on human cells.

ANALYTICAL CHEMISTRY, VOL. 64, NO. 23, DECEMBER 1, 1992

The research conducted a t MIT was initiated and nurtured primarily by Steven R. Tannenbaum. The macrophage-arginine-nitric oxide pathway was elucidated by Michael Marietta of the University of Michigan and his co-workers. Paul Skipper has been a major contributor to the protein adduct projects. W. G. Stillwell’s analytical skills are evident in virtually all of the GC/MS experiments. The experimental work itself was done mostly by the excellent graduate students, postdocs, and visiting scientists who have co-authored the references. We thank the National Cancer Institute (Grant (2-4267311,the Department of Health and Human Services and the National In(Grant l-SlO-RR01901), stitute of Environmental Health Sciences (Grants ES02109 and ES05622)for support. Thanks to Alan Schein for help with the manuscript. The contents of this manuscript are based solely on the opinions of the author and do not necessarily represent the official views of the National Cancer Institute.

References (1) Skipper, P. L.; Tannenbaum, S. R. Carcinogenesis 1990,11, 507-18. (2) Busby, W. F., Jr.; Wogan, G. N. In Chemical Carcinogens, 2nd ed.; Searle, C. E., Ed.; ACS Monograph 182; American Chemical Society: Washington, DC, 1984; pp. 945-1136. (3) Preussmann, R.; Eisenbrand, G. In Chemical Carcino ens, 2nd ed.; Searle, C. E., Ed.; ACS onograph 182; American Chemical Society: Washington, DC, 1984. DD. 829-68. (4) S a d e r , J. Hoppe Seylers Z. Physiol. Chem. 1968,349,429-32. (5) Tannenbaum, S. R.; Archer, M. C.; Wishnok, J. S.; Bishop, W. W. I. Nut. Cancer Inst. 1978,60,25i-53. (6) Green, L. C.; Wagner, D. A.; Ruiz de Luzuriaga, K.; Istfan, N.; Young, V. R.; Tannenbaum, S. R. Proc. Natl. Acad. Sci. USA 1981, 78, 7764-68. (7) Green, L.C.; Wagner, D.A.; Glogowski, J.; Skipper, P. L.; Wishnok, J. S.; Tannenbaum, S. R. Anal. Biochem. 1982,126, 131-38. ( 8 ) Wagner, D. A.; Schultz, D. S.; Deen, W. M.; Young, V. R.; Tannenbaum, S. R. Cancer Res. 1983,43,1921-25. (9) Wagner, D. A.; Young, V. R.; Tannenbaum, S. R. Proc. Natl. Acad. Sci. USA 1983,80,4518-21. (10) Stuehr, D. J.; Marletta, M. A. PYOC. Natl. Acad. Sci. USA 1985, 82,7738-42. (11) Marletta, M. A. Chem. Res. Toxicol. 1989,1, 249-57. (12) Leaf, C. D.; Wishnok, J. S.; Tannenbaum, S. R. Carcinogenesis 1991, 12, 537-39. (13) Leaf, C. D.; Wishnok, J. S.; Tannenbaum, S. R. Cancer Surveys 1989, 8(3). 323-34. (14) Nguyen, T.; Brunson, D.; Crespi, C. L.; Penman, B. W.; Wishnok, J. S.; Tannenbaum, S. R. PYOC.Natl. Acad. Sci. USA 1992,89,3030-34. (15) Wink, D. A.; Kasprzak, K. S.; Maragos, C. M.; Elespuru, R. K.; Misra, M.; Dunams, T. M.; Cebula, T. A.; Koch, W. H.; Andrews, A. W.; Allen, J. S.; Keefer, L. K. Science 1991, 254, 100103. (16) Leaf, C. D.; Wishnok, J. S.; Tannenbaum, S. R. Biochem. Biophys. Res. Commun. 1989,163,1032-37. (17) Snyder, S. H.; Bredt, D. S. Sci. Am. 1992,266,68 -77. (18) Lancaster, J. R., Jr. Am. Sci. 1992,

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(19)Moncada, S.;Palmer, R.M.J.; Higgs, E. A. Pharm. Rev. 1991,43,109-42. (20)Iyengar, R.; Stuehr, D. J.; Marletta, M. A. Proc. Natl. Acad. Sci. USA 1987,84, 6369-73. (21)Palmer, R.M.J.; Ashton, D. S.; Moncada, S. Nature 1988,333,664-66. (22)Marletta, M. A.; Yoon, P. S.; Iyengar, R.; Leaf, C. D.; Wishnok, J. S. Biochemist v 1988,27,8706-11. (23)Lewis, R. S.;Deen, W. M.; Tannenbaum, S. R.; Wishnok, J. S. J. Biol. Mass Spectrom., in press. (24)Malinski, T.; Taha, Z.Nature 1992, 358,676-78. (25)Osterman-Golkar, S.;Ehrenberg, L.; Segerback, D.; Hallstrom, I. Mutat. Res. 1976,34,1-10. (26)Umemoto, A.; Monden, Y.; Grivas, S.; Yamashita, K.; Sugimura, T. Carcinogenesis 1992,13,1025-30. (27)Talaska, G.; Schamer, M.; Skipper, P.; Tannenbaum, S. R.; Caporaso, N.; Unruh, L.; Kadlubar, F. F.; Bartsch, H.; Malaveille, C.; Vineis, P. Cancer Epidemiology, Biomarken, and Prevention 1991,1, 61-66. (28)Stillwell, W. G.; Bryant, M. S.; Wishnok, J. S. Biomed. Environ. Mass Spectrom. 1987,14,221-27. (29)Kadlubar, F. F.; Dooley, K. L.; Teitel, C. H.; Roberts, D. W.; Benson, R. W.; Butler, M. A.; Bailey, J . R.; Young, J. F.; Skipper, P. L.; Tannenbaum, S. R. Cancer Res. 1991,51,4371-77. (30) Skipper, P. L.; Tannenbaum, S. R. Environ. Health Perspect., in press. (31)Carmella, S. G.; Hecht, S. S. Cancer

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Res. 1987,47,2626-30. (32) Osborne, M. R.; Crosby, N. T. I n Cambridge Monographs on Cancer Research: Benzopyrenes; Osborne, M.R.; Crosby, N.T., Eds.; Cambridge University Press: New York, 1987. (33)Day, B. W.; Naylor, S.; Gan, L-S.; Sahali, Y.; Nguyen, T. T.; Skipper, P. L.; Wishnok, J. S.; Tannenbaum, S. R. J. Chromatogr. Biomed. Appl. 1991, 562, 563-71. (34) Day, B. W.; Taghizadeh, K.; Skipper, P. L.; Wishnok, J. S.; Tannenbaum, S. R.; Caporaso, N. E.; Vineis, P. Proceedings of the AACR Meeting; American Assn. for Cancer Research: Houston, 1991. (35)Day, B. W.; Skipper, P. L.; Wishnok, J. S.; Coghlin, J.; Hammond, S. K.; Gann, P.; Tannenbaum, S. R. Chem. Res. Toxicol. 1990,3,340-43. (36)Beach, A. C.; Gupta, R. C. Carcinogenesis 1992,13,1053-74. (37)Shuker, D.E.G.; Farmer, P. B. Chem. Res. Toxicol. 1992,50,450-60. (38)Shuker, D.E.G.; Bailey, E.; Parry, A.; Lamb, J.; Farmer, P. B. Carcinogenesis 1987,8,959-62. (39)Skipper, P.L.;Naylor, S.; Gan, L-S.; Day, B. W.; Pastorelli, R.; Tannenbaum, S. R. Chem. Res. Toxicol. 1989,2,280-81. (40)Abdel-Baky, S.; Giese, R. W. Anal. Chem. 1991,63,2986-89. (41)Dull, B. J.; Hotchkiss, J. H. Toxicol. Lett. 1984,23,79-89. (42)Hecht, S . S.;Morrison, J. B. Cancer Res. 1984,44,2873-77. (43)Wishnok, J. S.;Marletta, M. A., unpublished work.

(44)Wishnok, J. S.;Ralt, D.; Tannenbaum, S. R., unpublished work. (45)Day, B. W.; Gan, L-S.; Sahali, Y.; Nguyen, T. T.; Skipper, P. L.; Wishnok, J. S.; Tannenbaum, S. R. Cancer Res. 1990,50,4611-18. 80,248-59.

John S. Wishnok is a principal research scientist and director of mass spectrometry at the Massachusetts Institute of Technology’s Division of Toxicology. Wishnok received a B.A. degree in chemistryfiom the College of Wooster (Ohio) and earned an M.A.T. degree and a Ph.D. in chemistry fromBrown University. He then spent two years as a postdoctoral fellow with Paul Schleyer at Princeton University, after which he taught organic chemistry at Boston University f i r four years. He has been at MIT since 1974.

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