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DRUG METABOLISM REVIEWS, 22(2&3), 209-268 (1990)
METABOLISM OF NITRO=POLYCYCLIC AROMATIC HYDROCARBONS* PETER P. FU National Center for Toxicological Research Jefferson, Arkansas 72079
I. 11.
INTRODUCTION ...................................
210
GENERAL CONSIDERATION OF METABOLISM OF NITRO-PAHS ....................................... A. Enzyme Systems and Metabolic Pathways . . . . . . . . . . . . . B. Enzyme Induction by Nitro-PAHS.. . . . . . . . . . . . . . . . . . .
211 211 214
111. MICROBIAL METABOLISM . . . . . . . . . . . . . . . . . . . . . . . . . 215 A. Salmonella typhimurium . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215 B. Human and Rodent Intestinal Microflora.. . . . . . . . . . . . . . 217 IV. MAMMALIAN METABOLISM.. . . . . . . . . . . . . . . . . . . . . . . A. 4-Nitrobiphenyl, 1-Nitronaphthalene, 2-Nitronaphthalene, and 5-Nitroacenaphthalene . . . . . . . . . . . . . . . . . . . . . . . . . . B. 2-Nitrofluorene. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. 2-Nitrofluoranthene and 3-Nitrofluoranthene . . . . . . . . . . . D. 2-Nitroanthracene and 9-Nitroanthracene . . . . . . . . . . . . . . E. 1-Nitropyrene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. 4-Nitropyrene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. 1,3-, 1,6-, and 1,8-Dinitropyrene . . . . . . . . . . . . . . . . . . . . .
.
220 220 221 224 226 230 235 237
*This paper was refereed by Douglas Rickert, Ph.D., Department of Drug Metabolism, Glaxo Inc., 5 Moore Drive, Research Triangle Park, NC 27709; and Shen K. Yang, Ph.D., Department of Pharmacology, F. Edward Herbert School of Medicine, Uniformed Services University of Health Sciences, Bethesda, MD 20814-4799. 209 Copyright 8 1990 by Marcel Dekker, Inc.
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210
FU H. 6-Nitrochrysene ................................... I. 6-Nitro-5-Methylchrysene ........................... J. 7-Nitrobenz[a]anthracene............................ K. 3-Nitroperylene ................................... L. 1- and 3-Nitrobenzo[a]pyrene ....................... M. 6-Nitrobenzo[a]pyrene ............................. N. 1- and 3-Nitrobenzo[e]pyrene ....................... 0. 1,6-Dinitrobenzo[e]pyrene ..........................
238 240 24 1 243 243 247 249 25 1
V. EFFECT OF NITRO SUBSTITUTION AND ORIENTATION ..................................... A. Metabolism ...................................... B. Mutagenicity in Salmonella typhimurium . . . . . . . . . . . . . . C. Tumorigenicity ....................................
251 25 1 253 255
VI. CONCLUSION AND PERSPECTIVE. . . . . . . . . . . . . . . . . . .
256
References..........................................
257
I. INTRODUCTION Nitro-polycyclic aromatic hydrocarbons (nitro-PAHs) are a class of compounds with one or more nitro substituents attached to a polycyclic aromatic hydrocarbon. Since the first synthesis of 1-nitropyrene [ 11 and 6-nitrochrysene [2] in the late 19th century, a large number of nitro-PAHs have been prepared [3]. It was not until 1950 that a nitro-PAH, 2-nitrofluorene, was found to be tumorigenic [4]. In 1975 Ames and co-workers observed that several nitro-PAHs were mutagenic in the Salmonella typhimurium assay without requiring an exogenous enzymatic activation system [5]. In 1978 Pitts et al. [6] reported that mutagenic nitro-PAHs were found in model atmospheres containing trace quantities of PAHs, nitrogen oxide, and nitric acid. In the same year Wang et al. [7] found that the urban air particulates contained direct-acting bacterial mutagens, which they suggested were nitro-PAHs. Since then, nitro-PAHs have been recognized as a class of chemicals which are widespread environmental contaminants detected from different environmental sources, including diesel and airplane emissions [&14], combustion emissions from kerosene heaters and gas fuel and liquified petroleum [ 151, airborne particulates [16-191, coal fly ash [20, 211, and food [22]; and their presence has been suggested in cigarette smoke [23]. In the environment, nitro-PAHs may be formed either during the combustion process or from atmospheric reactions of PAHs and nitrogen oxides, both of which are abundant environmental contaminants [6, 24-28]. It has been
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METABOLISM OF NITRO-PAHS
21 1
suggested that the atmosphere nitration of PAHs involves radical reactions and that, if nitro-PAHs are formed during combustion, an ionic mechanism is involved and that the predominant nitrating species is the nitronium (NO:) ion [14, 29-32]. Nitro-PAHS have been shown to exhibit a large variety of biological activities [ 14, 33-35]. These biological activities include: induction of mutations in bacteria and eukaryotic cells [22, 331; neoplastic transformation of cultured normal human diploid fibroblasts, Syrian hamster, and BALB/c 3T3 cells [35-371; induction of DNA strand breaks [38]; induction of DNA repair [39]; induction of sister chromatid exchanges [40]; and induction of chromosomal aberrations [41, 421. Nitro-PAHs have also been demonstrated to bind cellular DNA in bacteria [4343] and mammalian cells [49-531, to inhibit preferentially the growth of repair-deficient bacteria [53], to have recombinogenic activity in yeast [54, 551, and to induce tumors in experimental animals [ 14,33-35,56,57]. Because nitro-PAHs are widespread environmental contaminants and are genotoxic, these compounds may pose a health risk to humans. Several reviews have been published recently on the mutagenicity and tumorigenicity of nitro-PAHs [11, 14, 33-35, 581. In view of the fact that nitro-PAHs require metabolism in order to exert their biological activities, including tumorigenicity [ 11, 14, 33-35, 581, it is relevant to understand the metabolic fate of these compounds. A review concerning their metabolic activation by Beland et al. 1341 and another review discussing the effects of the nitro functional group on metabolism by Fu et al. [35] have been previously reported. The present review describes metabolism of nitro-PAHs in a detailed manner. The nitroarenes derived from one benzyl ring, such as nitrobenzene, nitrotoluene, etc., are not included in the discussion. An excellent review concerning the metabolism of one-benzo-ring nitroarenes has recently been published by Rickert [59]. 11. GENERAL CONSIDERATION OF METABOLISM OF
NITRO-PAHS A. Enzyme Systems and Metabolic Pathways Metabolism of nitro-PAHs is much more complicated than that of PAHs. While metabolism of PAHs involves only oxidation as the first biotransformation. which is followed by hydrolysis and/or conjugation reactions, the initial metabolism of nitro-PAHs can proceed through either oxidation of the aromatic ring system (ring oxidation) or reduction of the nitro functional group (nitro reduction) [34, 351. Although reductive metabolism of nitro-PAHs has been studied since 1950. oxidative metabolism of nitro-PAHs has been examined
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212
FU
only recently. In most instances, these studies have been conducted in vitro employing liver microsomes or homogenates as enzyme sources. In these enzymatic systems, only phase I metabolites are normally formed, which simplifies the analysis of the metabolism results. Theoretically, any enzymatic system which can metabolize PAHs should be capable of metabolizing nitro-PAHs to the ring-oxidized metabolites. The phase I ring-oxidized metabolites so far identified from aerobic metabolism of nitro-PAHs include oxides, phenolic compounds, trans-dihydrodiols, tetrahydrotetrols, hydroquinones, quinones, benzylic alcohols, and ketones [34, 351. The types of the ring-oxidized metabolites formed are similar to those obtained from metabolism of PAHs [60]. The bacterial mutagenicity of the ring-oxidized metabolites [34, 611 and the structures of the DNA adducts formed in vivo [35] provide evidence that both ring oxidation and ring oxidation followed by nitro reduction can be involved in the metabolic activation of nitro-PAHs [62-66]. Both bacterial and mammalian enzymatic systems have been demonstrated to be capable of reductive metabolism of nitro-PAHs under anaerobic or hypoxic incubation conditions [33, 341. In mammalian systems, reductive enzymes are present in both hepatic cytosol and microsomes. The hepatic microsomal fraction contains the reduced form of cytochrome P-450 and NADPH-cytochrome P-450 reductase, while the cytosolic enzymes include xanthine oxidase, aldehyde oxidase, and DT-diaphorase [33, 34, 45, 67, 681. The reduction of a nitro-PAH proceeds through three consequent steps: (i) the formation of the corresponding nitroso-PAH; (ii) the further reduction to the N-hydroxyamino-PAH; and (iii) final reduction to the amino-PAH. Although each of these steps is formally a two-electron process, the reduction mechanism may involve one- and/or two-electron transfers. Of these reduced metabolites, the nitroso and N-hydroxyamino metabolites are potential electrophiles; however, only the latter derivative appears to bind DNA [46]. Thus, nitro-PAHs share common electrophilic intermediates with aromatic amines, except that nitro-PAHs are formed from reduction, while with aromatic amines, they arise from oxidation. Certain N-hydroxyamino-PAHs are also further activated through 0-esterification [47, 69-72]. While the Ames Salmonella typhimurium reversion assay represents a simple way to bioassay nitro-PAHs and their metabolites, this assay can also provide information regarding their metabolic activation pathways. This is due to the availability not only of the normal tester, TA98, but also of two additional strains with altered metabolic capabilities, TA98NR and TA98/1,8-DNP6. Strain TA98 contains a full complement of nitroreductases (consisting of one major component and two minor components) and an N-hydroxyarylamine 0-acetyltransferase. TA98NR is similar to TA98, except that the major component of nitroreductase activity is missing. In contrast, the N-hydroxyarylamine 0-acetyltransferase activity is lacking in TA98/1,8-DNP6[33, 44,
213
METABOLISM OF NITRO-PAHS
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Nilroreduclion
Esterificalion
P-450 02
HNOR
Nitroreduction
OH
Esterification
OH
RingOxidized Metabolite
FIG. 1. Metabolic activation pathways of nitro-PAHs leading to mutation.
69, 70, 731. Thus, the combined use of these three strains for bioassay allows the determination of whether or not nitroreductase and acetyltransferase are involved in the metabolic activation of the nitro-PAHs tested. It is worthwhile to note that reduction of nitro-PAHs to the corresponding amino-PAHs may not always be a detoxification pathway, as aromatic amines (amino-PAHs) are a class of chemical carcinogens and some of those derived from nitro-PAHs are known to be tumorigenic [62]. Therefore, it is still important to determine whether nitro reduction of nitro-PAHs to the corresponding aromatic amines and their subsequent N-oxidation is a significant activation pathway [74]. Based on the nitro-PAHs studied, there exist at least five metabolic activation pathways through which mutations are induced in Salmonella typhimurium and/or through which DNA binding occurs [34,45, 47, 63-67]. These are: (1) nitro reduction; (2) nitro reduction followed by esterification;(3) ring oxidation; (4) ring oxidation followed by nitro reduction; and ( 5 ) ring oxidation followed by nitro reduction and esterification (Fig. 1).
FU
214
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B. Enzyme Induction by Nitro-PAHs
Metabolizing enzymes, particularly the hepatic microsomal enzymes, are inducible by a variety of foreign compounds [60], and modulation of these enzymes can affect metabolism leading to detoxification and activation of carcinogenic compounds. Nitro-PAHs have been demonstrated to be able to induce hepatic enzyme activity in a similar manner. Asokan et al. [75] examined the effect of a single topical application of two pure nitro-PAHs, 1-nitropyrene and 3-nitrofluoranthene, and several nitro-PAH mixtures on cutaneous and hepatic drug and carcinogen metabolism in neonatal rats. Significant induction of aryl hydrocarbon hydroxylase, 7-ethoxyresorufin 0-deethylase, and 7ethoxycoumarin 0-deethylase activities in both skin and liver was observed. Among the nitro-PAHs studied, 1-nitropyrene exhibited the least activity in inducing these enzymes. It was suggested that the nitropyrenes were inducers of the 3-methylcholanthrene type [75, 761. Chou et al. [77] examined the induction of rat hepatic microsomal enzymes by the environmental nitro-PAHs 1-nitropyrene; 1,3-, 1,6-, and 1,8-dinitropyrene; 6-nitrochrysene; 7-nitrobenz[a]anthracene; 3-nitrofluoranthene; and 1-, 3-, and 6-nitrobenzo[a]pyrene. None of these nitro compounds increased the cytochrome P-450 content more than twofold. However, 1,8-dinitropyrene, 6-nitrochrysene, and 1- and 3nitrobenzo[a]pyrene significantly increased aryl hydrocarbon hydroxylase activity, while 6-nitrobenzo[a]pyrene caused a significant decrease. The induction of 7-ethoxycoumarin 0-deethylase activity paralleled that found with aryl hydrocarbon hydroxylase. Both of these activities were increased 2 to 6 times above that observed with their parent PAHs. In general, the nitro-PAHs did not cause significant increases in aminopyrine N-demethylase, epoxide hydrolase, or NADPH-cytochrome c reductase. 1-Nitropyrene reductase activity was induced by each of the compounds with the exception of 6-nitrobenzo[a]pyrene. Djuric et al. [78] studied the induction of hepatic cytosolic nitroreductases in rats. Pretreatment of rats with either 1-nitropyrene or 1,6-dinitropyrene resulted in a 2- to 3-fold increase in cytosolic nitro reduction, and this was paralleled by a 2- to 3-fold increase in the binding of 1,6-dinitropyrene to DNA in vitro. Induction of rat hepatic phase I1 conjugation enzymes by several nitro-PAHs was studied by Pegram and Chou [79]. Intraperitoneal injection of rats with 1-nitropyrene, 1-nitrobenzo[a]pyrene, 3-nitrobenzo[a]pyrene, and 6-nitrochrysene significantly increased the activities of UDP-glucuronyltransferase and cytosolic glutathione S-transferases. However, the activity of sulfotransferases was not increased.
METABOLISM OF NITRO-PAHS
215
111. MICROBIAL METABOLISM
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A. Salmonella typhimurium Although numerous papers have described the mutagenicity of nitro-PAHs using the Ames Salmonella typhimurium assay [ 14,33-351, use of Salmonella for studying metabolic activation has been less studied [43-48, 80, 811. 1-Nitropyrene is the most abundant nitro-PAH found in many environmental sources [82] and has been the prototype used for the study of the carcinogenicity of nitro-PAHs. 1-Nitropyrene is a moderately potent direct-acting mutagen tested in Salmonella typhimurium TA98. Addition of S9 activation enzymes has been reported to both increase and decrease its mutagenic activity [67,83-86]. In the nitroreductase-deficient strain, TA98NR, the mutagenicity of 1nitropyrene decreases [67, 711. These data suggest that both reductive and oxidative metabolic pathways may be involved in the metabolic activation of 1-nitropyrene in Salmonella. Although 1-nitropyrene is mutagenic in bacteria, it is at best a very weak mutagen in eukaryotic cells [49, 50, 67, 71, 87, 881. This limited activity has been ascribed to these cells being deficient in nitroreductase activity [49, 50, 71, 881. By comparison, the partially reduced intermediate, 1-nitrosopyrene, is a potent mutagen in these assays as well as in bacteria [46,49,50,54,71,85]. Messier et al. [43] and Quilliam et al. [80] first reported that exposure of ['HI 1-nitropyrene to Salmonella resulted in the formation of 1-aminopyrene and N-acetyl- 1-aminopyrene. The nitro reduction of 1-nitropyrene proceeded quite slowly, but the formation of l-nitropyreneDNA adducts increased in parallel. A subsequent study by Howard et al. suggested that N-(deoxyguanosin-8-yl)-l-aminopyrenewas the major adduct [45] (Fig. 2). When the nitroreductase-deficient strain TA98NR was used, the 1-aminopyrene and DNA adduct formation greatly decreased. These results indicate that the N-(deoxyguanosin-8-y1)- 1-aminopyrene adduct induces mutations in Salmonella [43]. The same adduct has also been determined as the major DNA adduct formed from incubation of 1-nitropyrene with Salmonella strain TA1538, and a strong correlation was found between the extent of DNA binding and the induced histidine reversion frequency [45]. Evidence has been presented that 1-nitrosopyrene is an intermediate in the metabolic activation of 1-nitropyrene to a mutagen in Salmonella typhimurium TA1538 [46]. Metabolism of l&dinitropyrene with Salmonella typhimurium TA98 and TA1538 resulted in the formation of 1-amino-8-nitropyrene and 1,8-diaminopyrene, both of which exhibited much weaker mutagenicity than the 1,8-dinitropyrene substrate [44,80]. Heflich et al. [47] reported that incubation of l&dinitropyrene with the strain TA1538 resulted in the l-amino-8nitropyrene as the major metabolite, together with 1-acetylamino-8-nitropy-
FU
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216
DNA
1
FIG. 2. Reductive metabolism and DNA adduct formation of 1-nitropyrene by Salmonella typhimurium. NPz
FIG. 3. Reductive metabolism and DNA adduct formation of 1,B-dinitropyrene by Salmonella iyphimurium.
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METABOLISM OF NITRO-PAHS
217
rene, N,N'-diacetyl-lS-diaminopyrene, and l&diaminopyrene as the minor metabolites. The major DNA adduct was identified as N-(deoxyguanosin-8y1)-1-amino-8-nitropyrene (Fig. 3). When this adduct was formed at a level of one DNA adduct per lo5nucleotides, it resulted in ca. 23 revertants/106viable cells. At the same level of binding, the major DNA adduct of 1-nitropyrene resulted in 17 revertants/lO"viable cells. These results indicate that the mutation frequency induced by same quantity of DNA adduct cannot account for the tremendous difference in mutagenicity of these two compounds [47]. Incubation of 6-nitrochrysene with Salmonella typhimurium TAlOO in the presence of S9 from the liver of rats pretreated with Aroclor afforded 6-nitrochrysene trans-1,2-dihydrodiol as the predominant metabolite, and 6-nitrochrysene trans-9,lO-dihydrodiol and 1,2-dihydroxy-6-nitrochryseneas the minor metabolites (Fig. 4) [48]. When incubated in the absence of the S9 activation enzymes, three DNA adducts were identified (Fig. 4): N-(deoxyinosin-8-yl)-6-aminochrysene,5-(deoxyguanosin-N2-yl)-6-aminochrysene,and N-(deoxyguanosin-~-yI)-6-aminochrysene. These three adducts were identical to those obtained from incubation of N-hydroxy-6-aminochrysenewith calf thymus DNA.
B. Human and Rodent Intestinal Microflora Intestinal microflora may play a critical role in the metabolic activation and detoxification of certain nitro-PAHs in animals and humans [89]. Consequently, metabolism of nitro-PAHs by isolated human and rodent intestinal microflora has been studied [90-971. Comparison of the metabolite and DNA adduct patterns between the conventional and germ-free animals treated with nitro-PAHs has been a convenient approach to study the role of intestinal microflora of animals in the metabolic activation of nitro-PAHs in vivo [91, 98-1 011. Kinouchi et al. [lo21 reported that gut flora were able to catalyze the reduction of 1-nitropyrene to 1-aminopyrene. Metabolism of 1-nitropyrene by rat and human intestinal microflora, anaerobic bacterial suspensions from human and rat feces, and by purified intestinal bacteria has subsequently been reported [91-941; however, in each of these cases, neither 1-nitrosopyrene nor the biologically active N-hydroxy- 1-aminopyrene has been detected. Therefore, the role of intestinal bacteria in the metabolic activation of 1-nitropyrene is not clear, although activation of 1-aminopyrene by subsequent N-oxidation to N-hydroxy-1-aminopyrene in the liver is possible. Anaerobic bacterial suspensions from human and rat feces and intestinal contents, and pure cultures of anaerobic bacteria metabolized 6-nitrobenzo[a]pyrene to 6-aminobenzo[a]pyrene [93]. Reinvestigation by using human intestinal microflora confirmed that
218
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FU
I
NHOH
J
Nn,
+ I dA
FIG. 4. Metabolism and DNA adduct formation of 6-nitrochrysene by Salmonella typhmurium. The ring-oxidized metabolites were formed only when S9 activation vvrere incorported in the incubation. activatic3n enzymes were
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METABOLISM OF NITRO-PAHS
219
DRUG METABOLISM REVIEWS, 22(2&3), 209-268 (1990)
METABOLISM OF NITRO=POLYCYCLIC AROMATIC HYDROCARBONS* PETER P. FU National Center for Toxicological Research Jefferson, Arkansas 72079
I. 11.
INTRODUCTION ...................................
210
GENERAL CONSIDERATION OF METABOLISM OF NITRO-PAHS ....................................... A. Enzyme Systems and Metabolic Pathways . . . . . . . . . . . . . B. Enzyme Induction by Nitro-PAHS.. . . . . . . . . . . . . . . . . . .
211 211 214
111. MICROBIAL METABOLISM . . . . . . . . . . . . . . . . . . . . . . . . . 215 A. Salmonella typhimurium . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215 B. Human and Rodent Intestinal Microflora.. . . . . . . . . . . . . . 217 IV. MAMMALIAN METABOLISM.. . . . . . . . . . . . . . . . . . . . . . . A. 4-Nitrobiphenyl, 1-Nitronaphthalene, 2-Nitronaphthalene, and 5-Nitroacenaphthalene . . . . . . . . . . . . . . . . . . . . . . . . . . B. 2-Nitrofluorene. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. 2-Nitrofluoranthene and 3-Nitrofluoranthene . . . . . . . . . . . D. 2-Nitroanthracene and 9-Nitroanthracene . . . . . . . . . . . . . . E. 1-Nitropyrene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. 4-Nitropyrene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. 1,3-, 1,6-, and 1,8-Dinitropyrene . . . . . . . . . . . . . . . . . . . . .
.
220 220 221 224 226 230 235 237
*This paper was refereed by Douglas Rickert, Ph.D., Department of Drug Metabolism, Glaxo Inc., 5 Moore Drive, Research Triangle Park, NC 27709; and Shen K. Yang, Ph.D., Department of Pharmacology, F. Edward Herbert School of Medicine, Uniformed Services University of Health Sciences, Bethesda, MD 20814-4799. 209 Copyright 8 1990 by Marcel Dekker, Inc.
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210
FU H. 6-Nitrochrysene ................................... I. 6-Nitro-5-Methylchrysene ........................... J. 7-Nitrobenz[a]anthracene............................ K. 3-Nitroperylene ................................... L. 1- and 3-Nitrobenzo[a]pyrene ....................... M. 6-Nitrobenzo[a]pyrene ............................. N. 1- and 3-Nitrobenzo[e]pyrene ....................... 0. 1,6-Dinitrobenzo[e]pyrene ..........................
238 240 24 1 243 243 247 249 25 1
V. EFFECT OF NITRO SUBSTITUTION AND ORIENTATION ..................................... A. Metabolism ...................................... B. Mutagenicity in Salmonella typhimurium . . . . . . . . . . . . . . C. Tumorigenicity ....................................
251 25 1 253 255
VI. CONCLUSION AND PERSPECTIVE. . . . . . . . . . . . . . . . . . .
256
References..........................................
257
I. INTRODUCTION Nitro-polycyclic aromatic hydrocarbons (nitro-PAHs) are a class of compounds with one or more nitro substituents attached to a polycyclic aromatic hydrocarbon. Since the first synthesis of 1-nitropyrene [ 11 and 6-nitrochrysene [2] in the late 19th century, a large number of nitro-PAHs have been prepared [3]. It was not until 1950 that a nitro-PAH, 2-nitrofluorene, was found to be tumorigenic [4]. In 1975 Ames and co-workers observed that several nitro-PAHs were mutagenic in the Salmonella typhimurium assay without requiring an exogenous enzymatic activation system [5]. In 1978 Pitts et al. [6] reported that mutagenic nitro-PAHs were found in model atmospheres containing trace quantities of PAHs, nitrogen oxide, and nitric acid. In the same year Wang et al. [7] found that the urban air particulates contained direct-acting bacterial mutagens, which they suggested were nitro-PAHs. Since then, nitro-PAHs have been recognized as a class of chemicals which are widespread environmental contaminants detected from different environmental sources, including diesel and airplane emissions [&14], combustion emissions from kerosene heaters and gas fuel and liquified petroleum [ 151, airborne particulates [16-191, coal fly ash [20, 211, and food [22]; and their presence has been suggested in cigarette smoke [23]. In the environment, nitro-PAHs may be formed either during the combustion process or from atmospheric reactions of PAHs and nitrogen oxides, both of which are abundant environmental contaminants [6, 24-28]. It has been
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METABOLISM OF NITRO-PAHS
21 1
suggested that the atmosphere nitration of PAHs involves radical reactions and that, if nitro-PAHs are formed during combustion, an ionic mechanism is involved and that the predominant nitrating species is the nitronium (NO:) ion [14, 29-32]. Nitro-PAHS have been shown to exhibit a large variety of biological activities [ 14, 33-35]. These biological activities include: induction of mutations in bacteria and eukaryotic cells [22, 331; neoplastic transformation of cultured normal human diploid fibroblasts, Syrian hamster, and BALB/c 3T3 cells [35-371; induction of DNA strand breaks [38]; induction of DNA repair [39]; induction of sister chromatid exchanges [40]; and induction of chromosomal aberrations [41, 421. Nitro-PAHs have also been demonstrated to bind cellular DNA in bacteria [4343] and mammalian cells [49-531, to inhibit preferentially the growth of repair-deficient bacteria [53], to have recombinogenic activity in yeast [54, 551, and to induce tumors in experimental animals [ 14,33-35,56,57]. Because nitro-PAHs are widespread environmental contaminants and are genotoxic, these compounds may pose a health risk to humans. Several reviews have been published recently on the mutagenicity and tumorigenicity of nitro-PAHs [11, 14, 33-35, 581. In view of the fact that nitro-PAHs require metabolism in order to exert their biological activities, including tumorigenicity [ 11, 14, 33-35, 581, it is relevant to understand the metabolic fate of these compounds. A review concerning their metabolic activation by Beland et al. 1341 and another review discussing the effects of the nitro functional group on metabolism by Fu et al. [35] have been previously reported. The present review describes metabolism of nitro-PAHs in a detailed manner. The nitroarenes derived from one benzyl ring, such as nitrobenzene, nitrotoluene, etc., are not included in the discussion. An excellent review concerning the metabolism of one-benzo-ring nitroarenes has recently been published by Rickert [59]. 11. GENERAL CONSIDERATION OF METABOLISM OF
NITRO-PAHS A. Enzyme Systems and Metabolic Pathways Metabolism of nitro-PAHs is much more complicated than that of PAHs. While metabolism of PAHs involves only oxidation as the first biotransformation. which is followed by hydrolysis and/or conjugation reactions, the initial metabolism of nitro-PAHs can proceed through either oxidation of the aromatic ring system (ring oxidation) or reduction of the nitro functional group (nitro reduction) [34, 351. Although reductive metabolism of nitro-PAHs has been studied since 1950. oxidative metabolism of nitro-PAHs has been examined
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only recently. In most instances, these studies have been conducted in vitro employing liver microsomes or homogenates as enzyme sources. In these enzymatic systems, only phase I metabolites are normally formed, which simplifies the analysis of the metabolism results. Theoretically, any enzymatic system which can metabolize PAHs should be capable of metabolizing nitro-PAHs to the ring-oxidized metabolites. The phase I ring-oxidized metabolites so far identified from aerobic metabolism of nitro-PAHs include oxides, phenolic compounds, trans-dihydrodiols, tetrahydrotetrols, hydroquinones, quinones, benzylic alcohols, and ketones [34, 351. The types of the ring-oxidized metabolites formed are similar to those obtained from metabolism of PAHs [60]. The bacterial mutagenicity of the ring-oxidized metabolites [34, 611 and the structures of the DNA adducts formed in vivo [35] provide evidence that both ring oxidation and ring oxidation followed by nitro reduction can be involved in the metabolic activation of nitro-PAHs [62-66]. Both bacterial and mammalian enzymatic systems have been demonstrated to be capable of reductive metabolism of nitro-PAHs under anaerobic or hypoxic incubation conditions [33, 341. In mammalian systems, reductive enzymes are present in both hepatic cytosol and microsomes. The hepatic microsomal fraction contains the reduced form of cytochrome P-450 and NADPH-cytochrome P-450 reductase, while the cytosolic enzymes include xanthine oxidase, aldehyde oxidase, and DT-diaphorase [33, 34, 45, 67, 681. The reduction of a nitro-PAH proceeds through three consequent steps: (i) the formation of the corresponding nitroso-PAH; (ii) the further reduction to the N-hydroxyamino-PAH; and (iii) final reduction to the amino-PAH. Although each of these steps is formally a two-electron process, the reduction mechanism may involve one- and/or two-electron transfers. Of these reduced metabolites, the nitroso and N-hydroxyamino metabolites are potential electrophiles; however, only the latter derivative appears to bind DNA [46]. Thus, nitro-PAHs share common electrophilic intermediates with aromatic amines, except that nitro-PAHs are formed from reduction, while with aromatic amines, they arise from oxidation. Certain N-hydroxyamino-PAHs are also further activated through 0-esterification [47, 69-72]. While the Ames Salmonella typhimurium reversion assay represents a simple way to bioassay nitro-PAHs and their metabolites, this assay can also provide information regarding their metabolic activation pathways. This is due to the availability not only of the normal tester, TA98, but also of two additional strains with altered metabolic capabilities, TA98NR and TA98/1,8-DNP6. Strain TA98 contains a full complement of nitroreductases (consisting of one major component and two minor components) and an N-hydroxyarylamine 0-acetyltransferase. TA98NR is similar to TA98, except that the major component of nitroreductase activity is missing. In contrast, the N-hydroxyarylamine 0-acetyltransferase activity is lacking in TA98/1,8-DNP6[33, 44,
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Nilroreduclion
Esterificalion
P-450 02
HNOR
Nitroreduction
OH
Esterification
OH
RingOxidized Metabolite
FIG. 1. Metabolic activation pathways of nitro-PAHs leading to mutation.
69, 70, 731. Thus, the combined use of these three strains for bioassay allows the determination of whether or not nitroreductase and acetyltransferase are involved in the metabolic activation of the nitro-PAHs tested. It is worthwhile to note that reduction of nitro-PAHs to the corresponding amino-PAHs may not always be a detoxification pathway, as aromatic amines (amino-PAHs) are a class of chemical carcinogens and some of those derived from nitro-PAHs are known to be tumorigenic [62]. Therefore, it is still important to determine whether nitro reduction of nitro-PAHs to the corresponding aromatic amines and their subsequent N-oxidation is a significant activation pathway [74]. Based on the nitro-PAHs studied, there exist at least five metabolic activation pathways through which mutations are induced in Salmonella typhimurium and/or through which DNA binding occurs [34,45, 47, 63-67]. These are: (1) nitro reduction; (2) nitro reduction followed by esterification;(3) ring oxidation; (4) ring oxidation followed by nitro reduction; and ( 5 ) ring oxidation followed by nitro reduction and esterification (Fig. 1).
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B. Enzyme Induction by Nitro-PAHs
Metabolizing enzymes, particularly the hepatic microsomal enzymes, are inducible by a variety of foreign compounds [60], and modulation of these enzymes can affect metabolism leading to detoxification and activation of carcinogenic compounds. Nitro-PAHs have been demonstrated to be able to induce hepatic enzyme activity in a similar manner. Asokan et al. [75] examined the effect of a single topical application of two pure nitro-PAHs, 1-nitropyrene and 3-nitrofluoranthene, and several nitro-PAH mixtures on cutaneous and hepatic drug and carcinogen metabolism in neonatal rats. Significant induction of aryl hydrocarbon hydroxylase, 7-ethoxyresorufin 0-deethylase, and 7ethoxycoumarin 0-deethylase activities in both skin and liver was observed. Among the nitro-PAHs studied, 1-nitropyrene exhibited the least activity in inducing these enzymes. It was suggested that the nitropyrenes were inducers of the 3-methylcholanthrene type [75, 761. Chou et al. [77] examined the induction of rat hepatic microsomal enzymes by the environmental nitro-PAHs 1-nitropyrene; 1,3-, 1,6-, and 1,8-dinitropyrene; 6-nitrochrysene; 7-nitrobenz[a]anthracene; 3-nitrofluoranthene; and 1-, 3-, and 6-nitrobenzo[a]pyrene. None of these nitro compounds increased the cytochrome P-450 content more than twofold. However, 1,8-dinitropyrene, 6-nitrochrysene, and 1- and 3nitrobenzo[a]pyrene significantly increased aryl hydrocarbon hydroxylase activity, while 6-nitrobenzo[a]pyrene caused a significant decrease. The induction of 7-ethoxycoumarin 0-deethylase activity paralleled that found with aryl hydrocarbon hydroxylase. Both of these activities were increased 2 to 6 times above that observed with their parent PAHs. In general, the nitro-PAHs did not cause significant increases in aminopyrine N-demethylase, epoxide hydrolase, or NADPH-cytochrome c reductase. 1-Nitropyrene reductase activity was induced by each of the compounds with the exception of 6-nitrobenzo[a]pyrene. Djuric et al. [78] studied the induction of hepatic cytosolic nitroreductases in rats. Pretreatment of rats with either 1-nitropyrene or 1,6-dinitropyrene resulted in a 2- to 3-fold increase in cytosolic nitro reduction, and this was paralleled by a 2- to 3-fold increase in the binding of 1,6-dinitropyrene to DNA in vitro. Induction of rat hepatic phase I1 conjugation enzymes by several nitro-PAHs was studied by Pegram and Chou [79]. Intraperitoneal injection of rats with 1-nitropyrene, 1-nitrobenzo[a]pyrene, 3-nitrobenzo[a]pyrene, and 6-nitrochrysene significantly increased the activities of UDP-glucuronyltransferase and cytosolic glutathione S-transferases. However, the activity of sulfotransferases was not increased.
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111. MICROBIAL METABOLISM
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A. Salmonella typhimurium Although numerous papers have described the mutagenicity of nitro-PAHs using the Ames Salmonella typhimurium assay [ 14,33-351, use of Salmonella for studying metabolic activation has been less studied [43-48, 80, 811. 1-Nitropyrene is the most abundant nitro-PAH found in many environmental sources [82] and has been the prototype used for the study of the carcinogenicity of nitro-PAHs. 1-Nitropyrene is a moderately potent direct-acting mutagen tested in Salmonella typhimurium TA98. Addition of S9 activation enzymes has been reported to both increase and decrease its mutagenic activity [67,83-86]. In the nitroreductase-deficient strain, TA98NR, the mutagenicity of 1nitropyrene decreases [67, 711. These data suggest that both reductive and oxidative metabolic pathways may be involved in the metabolic activation of 1-nitropyrene in Salmonella. Although 1-nitropyrene is mutagenic in bacteria, it is at best a very weak mutagen in eukaryotic cells [49, 50, 67, 71, 87, 881. This limited activity has been ascribed to these cells being deficient in nitroreductase activity [49, 50, 71, 881. By comparison, the partially reduced intermediate, 1-nitrosopyrene, is a potent mutagen in these assays as well as in bacteria [46,49,50,54,71,85]. Messier et al. [43] and Quilliam et al. [80] first reported that exposure of ['HI 1-nitropyrene to Salmonella resulted in the formation of 1-aminopyrene and N-acetyl- 1-aminopyrene. The nitro reduction of 1-nitropyrene proceeded quite slowly, but the formation of l-nitropyreneDNA adducts increased in parallel. A subsequent study by Howard et al. suggested that N-(deoxyguanosin-8-yl)-l-aminopyrenewas the major adduct [45] (Fig. 2). When the nitroreductase-deficient strain TA98NR was used, the 1-aminopyrene and DNA adduct formation greatly decreased. These results indicate that the N-(deoxyguanosin-8-y1)- 1-aminopyrene adduct induces mutations in Salmonella [43]. The same adduct has also been determined as the major DNA adduct formed from incubation of 1-nitropyrene with Salmonella strain TA1538, and a strong correlation was found between the extent of DNA binding and the induced histidine reversion frequency [45]. Evidence has been presented that 1-nitrosopyrene is an intermediate in the metabolic activation of 1-nitropyrene to a mutagen in Salmonella typhimurium TA1538 [46]. Metabolism of l&dinitropyrene with Salmonella typhimurium TA98 and TA1538 resulted in the formation of 1-amino-8-nitropyrene and 1,8-diaminopyrene, both of which exhibited much weaker mutagenicity than the 1,8-dinitropyrene substrate [44,80]. Heflich et al. [47] reported that incubation of l&dinitropyrene with the strain TA1538 resulted in the l-amino-8nitropyrene as the major metabolite, together with 1-acetylamino-8-nitropy-
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DNA
1
FIG. 2. Reductive metabolism and DNA adduct formation of 1-nitropyrene by Salmonella typhimurium. NPz
FIG. 3. Reductive metabolism and DNA adduct formation of 1,B-dinitropyrene by Salmonella iyphimurium.
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rene, N,N'-diacetyl-lS-diaminopyrene, and l&diaminopyrene as the minor metabolites. The major DNA adduct was identified as N-(deoxyguanosin-8y1)-1-amino-8-nitropyrene (Fig. 3). When this adduct was formed at a level of one DNA adduct per lo5nucleotides, it resulted in ca. 23 revertants/106viable cells. At the same level of binding, the major DNA adduct of 1-nitropyrene resulted in 17 revertants/lO"viable cells. These results indicate that the mutation frequency induced by same quantity of DNA adduct cannot account for the tremendous difference in mutagenicity of these two compounds [47]. Incubation of 6-nitrochrysene with Salmonella typhimurium TAlOO in the presence of S9 from the liver of rats pretreated with Aroclor afforded 6-nitrochrysene trans-1,2-dihydrodiol as the predominant metabolite, and 6-nitrochrysene trans-9,lO-dihydrodiol and 1,2-dihydroxy-6-nitrochryseneas the minor metabolites (Fig. 4) [48]. When incubated in the absence of the S9 activation enzymes, three DNA adducts were identified (Fig. 4): N-(deoxyinosin-8-yl)-6-aminochrysene,5-(deoxyguanosin-N2-yl)-6-aminochrysene,and N-(deoxyguanosin-~-yI)-6-aminochrysene. These three adducts were identical to those obtained from incubation of N-hydroxy-6-aminochrysenewith calf thymus DNA.
B. Human and Rodent Intestinal Microflora Intestinal microflora may play a critical role in the metabolic activation and detoxification of certain nitro-PAHs in animals and humans [89]. Consequently, metabolism of nitro-PAHs by isolated human and rodent intestinal microflora has been studied [90-971. Comparison of the metabolite and DNA adduct patterns between the conventional and germ-free animals treated with nitro-PAHs has been a convenient approach to study the role of intestinal microflora of animals in the metabolic activation of nitro-PAHs in vivo [91, 98-1 011. Kinouchi et al. [lo21 reported that gut flora were able to catalyze the reduction of 1-nitropyrene to 1-aminopyrene. Metabolism of 1-nitropyrene by rat and human intestinal microflora, anaerobic bacterial suspensions from human and rat feces, and by purified intestinal bacteria has subsequently been reported [91-941; however, in each of these cases, neither 1-nitrosopyrene nor the biologically active N-hydroxy- 1-aminopyrene has been detected. Therefore, the role of intestinal bacteria in the metabolic activation of 1-nitropyrene is not clear, although activation of 1-aminopyrene by subsequent N-oxidation to N-hydroxy-1-aminopyrene in the liver is possible. Anaerobic bacterial suspensions from human and rat feces and intestinal contents, and pure cultures of anaerobic bacteria metabolized 6-nitrobenzo[a]pyrene to 6-aminobenzo[a]pyrene [93]. Reinvestigation by using human intestinal microflora confirmed that
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I
NHOH
J
Nn,
+ I dA
FIG. 4. Metabolism and DNA adduct formation of 6-nitrochrysene by Salmonella typhmurium. The ring-oxidized metabolites were formed only when S9 activation vvrere incorported in the incubation. activatic3n enzymes were
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