Reductive Metabolism of Aromatic Nitro Compounds Including Carcinogens by Rabbit Liver Preparations1

[CANCER RESEARCH 46, 1089-1093, March 1986]

Kiyoshi Tatsumi,2 Shigeyuki Kitamura, and Noriko Narai3

Institute of Pharmaceutical Sciences, Hiroshima University School of Medicine, 1-2-3, Kasumi, Minami-ku, Hiroshima 734, Japan

ABSTRACT somal and cytosolic fractions of mammalian livers (15, 18, 20, 22-24). Previous studies showed that a cytochrome P-450 sys Reductive metabolism of aromatic nitro compounds was ex tem (23, 25) and xanthine oxidase (23) are involved in the amined with rabbit liver preparations. Under anaerobic condi reduction of nitrobenzene orp-nitrobenzoic acid catalyzed by rat tions, carcinogenic 2-nitrofluorene, 4-nitrobiphenyl, and 1-nitro- liver microsomes and cytosol, respectively. In a preliminary com naphthalene were reduced to the corresponding hydroxylamines munication (18), recently, we demonstrated that a microsomal and amines, whereas the carcinogenic 1-nitropyrene was re cytochrome P-450 system and cytosolic aldehyde oxidase of duced only to the corresponding amine by liver cytosol in the rabbit liver catalyze the reduction of 1-nitropyrene and 2-nitroflu presence of 2-hydroxypyrimidine, an electron donor of aldehyde orene to the corresponding amines. More recently, the partici oxidase. These metabolites were identified unequivocally by pation of cytochrome P-450 in nitroreduction was further con comparing their mass spectra and thin-layer Chromatographie firmed with a reconstituted cytochrome P-450 system from rat behaviors with those of the authentic samples. liver microsomes (20). Both liver microsomes and cytosol catalyzed the reduction of The present study shows the first example of enzymatic these aromatic nitro compounds in varying degrees. The micro- formation of hydroxylamine derivatives from 2-nitrofluorene, 4- somes required reduced pyridine nucleotides for occurrence of nitrobiphenyl, and 1-nitronaphthalene. In addition, the study pro the nitroreductase activities. In this case, reduced nicotinamide vides the first evidence that aldehyde oxidase functions as a adenine dinucleotide phosphate was more effective than reduced major liver enzyme responsible for the reduction of aromatic nitro nicotinamide adenine dinucleotide as an electron donor. The compounds including carcinogens. cytosol by itself exhibited some nitroreductase activities, which were markedly enhanced by addition of an electron donor of aldehyde oxidase, i.e., A/1-methylnicotinamide or 2-hydroxypyr MATERIALS AND METHODS Chemicals. 2-Nitrofluorene, 1-aminopyrene, 2-acetylaminofluorene, imidine. The full activities of the cytosol with the electron donor A/'-methylnicotinamide chloride, 2-hydroxypyrimidine hydrochloride, di- were much higher than those of the microsomes with the reduced phenylamine, phenothiazine, and benzamide were purchased from Tokyo pyridine nucleotide. Purified liver aldehyde oxidase, like the cy Chemical Industry Company, Ltd. 2-Aminofluorene, xanthine, menadi- tosol, exhibited significant nitroreductase activities in the pres one, sodium arsenite, and potassium cyanide were obtained from Nakafai ence of its electron donor. Chemical, Ltd. NADPH, NADH, and chlorpromazine were obtained from These results indicated that cytosolic aldehyde oxidase func Sigma Chemical Company. 4-Nitrobiphenyl and 4-aminobiphenyl were tions as a major enzyme responsible for the reduction of aromatic purchased from Aldrich Chemical Company. 1-Nitronaphthalene and nitro compounds including carcinogens in rabbit liver. bovine serum albumin were purchased from Katayama Chemical Industry Company, Ltd. 1-Aminonaphthalene was obtained from Ishizu Pharma ceutical Company, Ltd., and p-nitrobenzoic acid was from Yoneyama INTRODUCTION Chemical Industries, respectively. Cyproheptadine was donated by Nip pon Merck-Banyu Company, Ltd. Certain aromatic nitro compounds have been proved to be 1-Nitropyrene (mp 148Â°C)was synthesized by the method of Ristagno carcinogenic (1-5), some of which have been noticed as impor and Shine (26), 2-hydroxylaminofluorene (mp 169-173Â°C) by that of tant environmental pollutants (5-10). The mechanism of carcin- Poirier eÃal. (27), W-acetoxy-2-acetylaminofluorene (mp 176-178Â°C) by ogenicity is thought to be mediated by the metabolic reduction that of Gutmann and Erickson (28), 1-hydroxylaminonaphthalene (mp of these nitro compounds to reactive hydroxylamine intermedi 76-78Â°C) by that of WillstÃ¤tter and Kubli (29), and 4-hydroxylaminobi- ates (11-15). However, previous works showed that the anaer phenyl (mp 178-180Â°C) by the method of MÃ¤heref al. (30), respectively. obic incubation of carcinogenic 1-nitropyrene (16-20), 2-nitroflu Enzymes. Bovine liver catalase was purchased from Sigma Chemical orene (18, 21), 4-nitrobiphenyl (15), 2-nitronaphthalene (15), and Company. Male albino rabbits weighing 2.0-2.5 kg were used in this 1-nitronaphthalene (15) with mammalian liver preparations led to study. Rabbit liver aldehyde oxidase was purified by the method of the formation of the corresponding amines, with no evidence for Rajagopalan ef a/. (31). Rabbit liver microsomes and cytosol were prepared as follows. The liver was homogenized in 4 volumes of 1.15% the formation of the corresponding hydroxylamines. The reduction of aromatic nitro compounds occurs with micro- KCI, the homogenate was centrifugea for 20 min at 9,000 x g, and the 9,000 x g supernatant fraction was centrifuged for 60 min at 105,000 x g. The microsomal fraction was washed by resuspension in the KCI Received 7/22/85; revised 11/18/85; accepted 11/19/85. solution and resedimentation for 60 min at 105,000 x g. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in Isolation of Metabolites. The incubation mixture consisted of 12 //mol accordance with 18 U.S.C. Section 1734 solely to indicate this fact. of a nitro compound, 120 (Â¿molof2-hydroxypyrimidine, and liver cytosol 1This work was supported in part by a grant-in-aid from the Ministry of (equivalent to 12 g of liver) in a final volume of 150 ml of 0.1 M phosphate Education, Science, and Culture, Japan. 2 To whom requests for reprints should be addressed. buffer (pH 7.4). The nitro compound was placed in the side arm of the 3 Present address: Faculty of Pharmaceutical Sciences, Setsunan University, Thunberg apparatus separate from the rest of the incubation mixture 45-1, Nagaotogecho, Hirakata-shi, Osaka 573-01, Japan. which was placed in the main tube. The incubation was carried out

CANCER RESEARCH VOL. 46 MARCH 1986 1089

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1986 American Association for Cancer Research. REDUCTIVE METABOLISM OF AROMATIC NITRO COMPOUNDS anaerobically as follows. The tube was gassed for 5 min with nitrogen, was evaporated to dryness in a vacuum. The residue was dissolved in which was passed through a deoxygenizing solution consisting of 0.5% 50 iil of methanol and then applied to a Gilson 1B high-pressure liquid sodium dithionite and 0.05% sodium 2-anthraquinonesulfonate in 0.4% Chromatograph equipped with a M & S Variactor 311 UV absorption NaOH, evacuated with an aspirator for 10 min, and again gassed with detector. The instrument was fitted with a 15-cm x 4.6-mm inner diameter the nitrogen. After the tube was tightly closed, the reaction was started M & S da column. The Chromatograph was also operated at ambient by mixing a nitro compound of the side arm and all other components of temperature and at a wavelength of 254 nm. In the determination of 4- the body together, and it was continued for 30 min at 37Â°C. After nitrobiphenyl metabolites, the mobile phase was 0.1 M KH2PO4:CH3CN incubation, the mixture was extracted once with an equal volume of ethyl (3:2), and the flow rate was 1 ml/min. acetate, the ethyl acetate extract was evaporated to dry ness in a In the case of 1-nitropyrene, the incubation mixture, after adding 0.31 vacuum, and the residue was subjected to TLC4. TLC was conducted ,benzene:acetone (7:3), and the chromatograms were vis extract was evaporated to dryness in a vacuum, and the residue, after ualized under UV light (254 nm). Metabolites were eluted with methanol being dissolved in 50 Â¿tlof methanol, was applied to a Shimadzu GC- from the TLC plates and subjected to mass spectrometric study. Mass 4CM gas Chromatograph equipped with a hydrogen flame ionizing de spectra were taken with a Shimadzu 7000 mass spectrometer at an tector. The instrument was fitted with a 2-m x 3-mm inner diameter glass ionizing voltage of 70 eV. These procedures were quickly conducted in column packed with 3% Dexsil 400 GC on Chromosorb W. The operating a nitrogen atmosphere and in the dark as much as possible, to preclude conditions were as follows: injection port and detector temperature, the loss of the hydroxylamine derivatives formed. The assay of nitrored- 310Â°C; column temperature, 260Â°C. uctase activity described below was also performed under such precau Under the assay conditions, 94% of 2-nitrofluorene, 85% of 4-nitrobi tions. phenyl, and 73% of 1-nitronaphthalene that disappeared were recovered Assay of Nitroreductase Activity. The incubation mixture consisted as the corresponding hydroxylamines and amines, respectively. In the of 0.2 iimol of a nitro compound, 1 ^mol of an electron donor, and liver case of 1-nitropyrene, 93% of the compound that disappeared was microsomes (equivalent to 1 g of liver) or liver cytosol (equivalent to 0.2 exclusively recovered as the corresponding amine. The recoveries of g of liver) in a final volume of 2.5 ml of 0.1 M phosphate buffer (pH 7.4). authentic hydroxylamine derivatives added to 0.1 M phosphate buffer When aldehyde oxidase (0.1 mg of protein) was used as an enzyme (pH 7.4) were 87% for 2-hydroxylaminofluorene, 85% for 4-hydroxyla- source, 1.25 mg of bovine serum albumin and 12 Â¿igof catalase were minobiphenyl, and 76% for 1-hydroxylaminonaphthalene, respectively, further added to this incubation mixture. Prior to incubation, a Thunberg at the end of 30-min incubation. tube was gassed for 3 min with deoxygenated nitrogen, evacuated for Assay of Aldehyde Oxidase. The assay was performed by the 5 min, and again gassed with the nitrogen. The reaction was started by method of Felsted ef al. (32), measuring the increase in absorbance at mixing the solution of the side arm and the body together and continued 300 nm, which accompanies the oxidation of N1-methylnicotinamide to for 30 min at 37Â°Cas described above. The incubation mixture containing the 2- and 4-pyridones. a boiled liver preparation was used as a control. Determination of Protein. Protein was determined by the method of In the case of 2-nitrofluorene, its metabolites, i.e., 2-hydroxylaminoflu- Lowry ef al. (33) with bovine serum albumin as a standard. orene and 2-aminofluorene, were determined as their acetyl derivatives. The incubation mixture, after adding 0.2 jumol of diphenylamine as an RESULTS internal standard, was extracted once with 10 ml of ethyl acetate, and to the ethyl acetate extract was added 0.5 ml of acetic anhydride. The When aromatic nitro compounds were incubated with rabbit reaction mixture was allowed to stand for 3 h at room temperature under liver cytosol in the presence of 2-hydroxypyrimidine, an electron an atmosphere of nitrogen, washed with NaHCO3-saturated solution and donor of aldehyde oxidase, their metabolites were isolated from water, successively, and then evaporated to dryness in a vacuum. The the incubation mixtures as described in "Materials and Methods." residue was dissolved in 50 Â¿ilof methanol and subjected to HPLC. Table 1 shows Rt values of these metabolites in TLC. HPLC was performed in a Toyo Soda HLC-803A high-pressure liquid As shown in Figs. 1 to 3, mass spectra of the 2-nitrofluorene Chromatograph equipped with a UV-8 UV absorption detector. The metabolite 1, 4-nitrobiphenyl metabolite 1, and 1-nitronaphtha instrument was fitted with a 30-cm x 4-mm inner diameter LS-410K reversed-phase column (Toyo Soda). The mobile phase was 0.1 M lene metabolite 1 gave molecular ions at m/z 197,185, and 159, KHÃ¼POvCHsCN(1:1). The Chromatograph was operated at a flow rate suggesting that these metabolites are the corresponding hydrox of 1 ml/min at ambient temperature and at a wavelength of 254 nm. 2- ylamine derivatives. In addition, the spectra showed that frag Hydroxylaminofluorene- or 2-aminofluorene-spiked buffer samples were ment ions at m/z M+-2 (195, 183, and 157) are attributable to extracted and acetylated to provide a linear standard curve. In the the loss of 2 hydrogen atoms from molecular ions to form the experiment concerning the effect of some chemicals on 2-nitrofluorene corresponding nitroso derivatives, and base peaks at m/z M+- reductase and aldehyde oxidase activities of cytosol (Table 3), the 2- 16 (181,169, and 143) result from the loss of one oxygen atom nitrofluorene that disappeared during incubation was determined by gas from molecular ions to form the corresponding amine derivatives. Chromatographie analysis as follows. The incubation mixture, after add The mass spectra and the Rf values in TLC of these metabolites ing 0.5 ftmo\ of phenothiazine as an internal standard, was extracted once with 5 ml of ethyl acetate, and the ethyl acetate extract was evaporated to dryness in a vacuum. The residue was dissolved in 50 n\ Table 1 R, values of reductive metabolites ot aromatic nitro compounds in thin-layer of methanol and then applied to a Hitachi Model 163 gas Chromatograph equipped with a flame ionizing detector and a 2-m x 3-mm inner diameter chromatography TLC was conducted on silica gel plates developed in benzene:acetone (7:3) as glass column packed with 3% Dexsil 400 GC on Chromosorb W. The described in "Materials and Methods." operation conditions were as follows: injection port and detector tem perature, 260Â°C; column temperature, 210Â°C. R,2-NitrofluoreneMetabolite In the cases of 4-nitrobiphenyl and 1-nitronaphthalene, the incubation metabolite 1 2-Nitrofluorene metabolite 2 0.32 mixture, after adding the internal standard described above, was ex 4-Nitrobiphenyl metabolite 1 0.29 tracted once with 5 ml of ethyl acetate, and the ethyl acetate extract 4-Nitrobiphenyl metabolite 2 0.45 1-Nitronaphthalene metabolite 1 0.33 4 The abbreviations used are: TLC, thin-layer chromatography; HPLC, high- 1-Nitronaphthalene metabolite 2 0.50 1-Nitropyrene metabolite0.17 pressure liquid chromatography. 0.30

CANCER RESEARCH VOL. 46 MARCH 1986 1090

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1986 American Association for Cancer Research. REDUCTIVE METABOLISM OF AROMATIC NITRO COMPOUNDS were completely identical to those of authentic hydroxylamine 1-aminopyrene, respectively, by comparison with the authentic derivatives. Based on these facts, 2-nitrofluorene metabolite 1, samples of their mass spectra and thin-layer Chromatographie 4-nitrobiphenyl metabolite 1, and 1-nitronaphthalene metabolite behavior (data not shown). 1 were identified as 2-hydroxylaminofluorene, 4-hydroxylamino- Rabbit liver microsomes and cytosol can catalyze the reduction biphenyl, and 1-hydroxylaminonaphthalene, respectively. of 2-nitrofluorene, 4-nitrobiphenyl, and 1-nitronaphthalene to the On the other hand, the 2-nitrofluorene metabolite 2, 4-nitrobi corresponding hydroxylamines and amines in varying degrees phenyl metabolite 2, 1-nitronaphthalene metabolite 2, and 1- (Table 2). In microsomes, NADPH was more effective than NADH nitropyrene metabolite showed mass spectra with molecular ions as an electron donor. The NADPH-linked activities were partly at m/z 181,169,143, and 217. These metabolites were identified inhibited by carbon monoxide, indicating the involvement of as 2-aminofluorene, 4-aminobiphenyl, 1-aminonaphthalene, and cytochrome P-450 in the microsomal nitroreduction (data not shown). On the other hand, cytosol by itself exhibited some 100 nitroreductase activities, which were markedly enhanced by a)JÃœL-iei195(Ml,9,i addition of electron donors of aldehyde oxidase, such as A/1- so methylnicotinamide and 2-hydroxypyrimidine. NADPH, NADH, and xanthine had only a little effect on the cytosolic nitroreduc tase activities. The full activities of cytosol supplemented with so 100 150 200(m/z) the electron donor of aldehyde oxidase were much higher than 100 those of microsomes supplemented with the reduced pyridine nucleotide. Such cytosolic nitroreductase activity appeared to be ^-Â©â€¢NHOHULJ_j-181 ,95CM') due to aldehyde oxidase, considering the result of electron donor 50 1971 requirement. To support this concept, the ability of some chemicals to inhibit 2-nitrofluorene reducÃaseand aldehyde oxidase activities of cy 50 100 150 200 (m/z) tosol was examined. As shown in Table 3, both activities were Fig. 1. Mass spectra of 2-nitrofluorene metabolite 1 (a) and authentic 2-hydrox similarly susceptible to inhibition by all of these chemicals, indi ylaminofluorene (b). cating the involvement of aldehyde oxidase in the cytosolic nitroreduction. 100 a),1.-169183185Ã•M')L 100 a)Ã 143157ÃŒ159IWI 50 so

' 50 100 ISO 200(m/z) \i i L i- 50 100 150 200(m/z) 100 b) ,-)-Â©-NHOH1 5 NHOH[Ã”tÃ´lJ 143157I59IM*)Â£ Â» cc I50b) 50

185(M')i

J I-169183I IL J 1 til- 50 no ISO 200 (m/z) 50 100 150 200(m/z) Fig. 2. Mass spectra of 4-nitrobiphenyl metabolite 1 (a) and authentic 4-hydrox- Fig. 3. Mass spectra of 1-nitronaphthalene metabolite 1 (a) and authentic 1- ylaminobiphenyl (b). hydroxylaminonaphthalene (D).

Table 2 Reduction of aromatic nitro compounds by rabbit liver fractions Metabolites formed were determined from their peak areas in HPLC which was performed as described in "Materials and Methods." Each value represents the mean of 4 experiments. Elution times of the metabolites in the HPLC were as follows: W-acetoxy-2-acetylaminofluorene, 11 min; 2-acetylaminofluorene, 8 min; 4- hydroxylaminobiphenyl, 5 min; 4-aminobiphenyl, 7 min; 1-hydroxylaminonaphthalene, 8 min; and 1-aminonaphthalene, 12 min. 2-Nitrofluorene liver)FractionMicrosomesNoneNADPHNADHCytosol(nmol/30 min/g liver)Hydroxylamine(nmol/30 min/g liver)Hydroxylamineformed02.31.50.20.40.40.20.50.6Amine(nmol/30 min/g

formed82.812.319.684.012.015.675.82.7formed"8.0109.125.912.084.277.615.0586.4 formed012.818.14.029.225.610.868.0formed065.631.748.883.274.452.8724.8 formed089.928.33.051.853.265.4615.0

NoneNADPHNADHXanthineN

' -Methylnicotinamide 428.8a 2-HydroxypyrimidineHydroxylamine 112.9Amine 324.44-Nitrobiphenyl 88.2Amine 391.21-Nitronaphthalene Determined as N-acetoxy-2-acetylaminofluorene.

CANCER RESEARCH VOL. 46 MARCH 1986 1091

Table 4 shows the ability of purified rabbit liver aldehyde p-dinitrobenzene (36), and p-nitrobenzenesulfonamide (36). The oxidase to reduce the aromatic nitro compounds to the corre present paper is the first description that enzymatic formation of sponding hydroxylamines and amines. The enzyme, like cytosol hydroxylamine derivatives is observed with aromatic nitro com described above, exhibited significant nitroreductase activities in pounds which possess 2 or more rings in the molecules. The precautions described in "Materials and Methods" made the the presence of its own electron donor, but slightly in the presence of xanthine, NADPH, or NADH. identification of these unstable metabolites possible. Similar results were obtained with the reduction of 1-nitropy- On the other hand, 1-nitropyrene, which is the predominant rene by rabbit liver preparations. In this case, however, the nitropolycyclic aromatic hydrocarbon in environmental samples, corresponding hydroxylamine could not be detected as its me was exclusively reduced to the corresponding amine by rabbit tabolite. The microsomal nitroreductase activity was dependent liver preparations. In a preliminary study, however, when the on NADPH or NADH, while the cytosolic nitroreductase activity nitro compound was anaerobically incubated with hamster liver was markedly enhanced by addition of A/1-methylnicotinamide or cytosol and a NADH-generating system in 0.1 Mphosphate buffer 2-hydroxypyrimidine (Table 5). Purified rabbit liver aldehyde oxi (pH 7.4), 2 metabolites were isolated from the incubation mixture dase supplemented with its electron donor showed a significant as their acetyl derivatives. One of them showed the mass spec activity towards the nitro compound (Table 6). trum with a molecular ion at m/z 317 (C2oH1503N,attributable to From these facts, we concluded that cytosolic aldehyde oxi A/-acetoxy-1-acetylaminopyrene), accompanying fragment ions dase is involved in the reduction of aromatic nitro compounds at m/z 275 (Ci8H1302N, W-hydroxy-1-acetylaminopyrene), m/z including carcinogens as a major liver enzyme. 259 (C18H13ON, 1-acetylaminopyrene), m/z 233 (Ci6H,,ON, 1-

DISCUSSION Table 5 Reduction of 1-nitropyreneby rabbit liver fractions A great deal of attention has been focused on the metabolic 1-Aminopyreneformation was determinedfrom its peak area in gas chromatog raphy which was performed as described in "Materials and Methods." Each value conversion of carcinogenic aromatic nitro compounds to the represents the mean of 4 experiments. Retention time of the metabolite in the gas corresponding hydroxylamines as a potential activation pathway chromatography was 14 min. (11-15). However, the range of previous aromatic nitro com -Aminoyprene pounds described to give hydroxylamine derivatives during en formed zymatic reduction was limited to nitrobenzene (23) and its deriv FractionMicrosomesNoneNADPHNADHCylosolNoneNADPHNADHXanthineW'-Methylnicotinamide2-Hydroxypyrimidine1(nmol/30 min/gliver)0.3148.121.099.8111.2159.183.11483.31021.4 atives, such as 2,4,6-trinitrotoluene (34), p-nitrobenzoic acid (24), methyl p-nitrobenzoate (35, 36), p-nitroacetophenone (35, 36),

Table 3 Effect of some chemicals on nitroreductaseand aldehyde oxidase activities of rabbit liver cytosol 2-Nitrofluorene disappearancewas determined from its peak area in gas chromatography which was performed as described in "Materials and Methods." Each valuerepresents the mean of 4 experiments. Retention time of the nitro compound in the gas chromatography was 14 min. controlAdditionNone % of Table 6 Reduction of 1-nitropyreneby rabbit liver aldehyde oxidase activity As regards determination of 1-aminopyreneformation, see the legend of Table (2-nitrc- 5. Each value represents the mean of 4 experiments. fluorene oxidase disappeared)"10015activity100 -Aminopyreneformed AdditionNone (nmol/30 min/mgprotein)01203.5 (control) Menadione IO"4 0 5x 10-" W1-Methylnicotinamide Chlorpromazine 8 0 Sodium arsenite 1 x 10-1 18 11 2-Hydroxypyrimidine 1131.2 2.5 x 10-3Nitroreductase Potassium cyanideConcentration(M)5x 23Aldehyde 18 Xanthine 10.2 8 The incubation was carried out for 30 min in the presenceof W-methylnicotin- NADPH 9.5 NADH1 13.3 amide.

Table 4 Reduction of aromatic nitro compounds by rabbit liver aldehyde oxidase As regards determination of metabolites formed, see the legend of Table 2. Each value represents the meanof 4 experiments. 2-Nitrofluorene 4-Nitrobiphenyl 1-Nitronaphthalene (nmol/30 min/mg protein) (nmol/30 min/mg protein) (nmol/30 min/mg protein)

formed80 formed"0 AdditionNone formed0 formed0720.9 formed0 formed0

N'-Methylnicotinamide 253.2 1185.6 163.8 10.2 937.6 2-Hydroxypyrimidine 30.3 392.4 129.6 540.0 12.8 814.3 Xanthine 14.4 15.6 8.1 10.8 0 26.4 NADPH 14.4 13.2 32.4 3.4 0 19.6 NADHHydroxylamine 15.6Amine 13.2Hydroxylamine 16.2Amine 5.4Hydroxylamine 0Amine 21.3 8 Determinedas N-acetoxy-2-acetylaminofluorene. 0 Determinedas 2-acetylaminofluorene.

CANCER RESEARCH VOL. 46 MARCH 1986 1092

hydroxylaminopyrene), and m/z 217 (CieHnN, 1-aminopyrene). 18. Kitamura, S., Narai, N., Hayashi, M., and Tatsumi, K. Rabbit liver enzymes responsible for reduction of nitropolycyclic aromatic hydrocarbons. Chem. The other was identified as 1-acetylaminopyrene by comparison Pharm. Bull. (Tokyo), 37: 776-779, 1983. with the authentic sample. These facts strongly suggest that 1- 19. Nachtman, J. P., and Wei, E. T. Evidence for enzymatic reduction of 1- nitropyrene by rat liver fractions. Experientia (Basel), 38: 837-838,1982. nitropyrene is convertible not only to the corresponding amine, 20. Saito, K., Kamataki, T., and Kato, R. Participation of cytochrome P-450 in but also to the corresponding hydroxylamine by mammalian liver reductive metabolism of 1-nitropyrene by rat liver microsomes. Cancer Res., enzymes under certain conditions. 44: 3169-3173,1984. Cytosolic aldehyde oxidase rather than microsomal cyto- 21. Uehleke, H., and Nestel, K. Hydroxylaminobiphenyl and nitrosobiphenyl: bio logical oxidation products of 4-aminobiphenyl and reduction metabolites of 4- chrome P-450 appears to be mainly responsible for the reduction nitrobiphenyl. Naunyn-Schmiedebergs Arch. Exp. Pathol. Pharmakol., 257: of aromatic nitro compounds including carcinogens in rabbit liver. 151-171,1967. 22. Fouts, J. R., and Brodie, B. B. The enzymatic reduction of chloramphenicol, Participation of the molybdenum-containing flavoenzyme in nitro- p-nitrobenzoic acid, and other aromatic nitro compounds in mammals. J. reduction was first demonstrated by Wolpert ef al, (37) with Pharmacol. Exp. Then, 779: 197-207, 1957. heterocyclic nitro compounds, such as 5-nitro-2-furaldehyde 23. Harada, N., and Omura, T. Participation of cytochrome P-450 in the reduction of nitro compounds by rat liver microsomes. J. Biochem., 87: 1539-1554, semicarbazone (nitrofurazone) and 4-nitroquinoline /V-oxide. Re 1980. cently, we found that the enzyme in the presence of its electron 24. Kato, R., Oshima, T., and Takanaka, A. Studies on the mechanism of nitro reduction by rat liver. Mol. Pharmacol., 5: 487-498, 1969. donor can also catalyze the reduction of sulfoxide (38, 39), 25. Gillette, J. R., Kamm, J. J., and Sasame, H. A. Mechanism of p-nitrobenzoate nitrosamines (40, 41), hydroxamic acids (42, 43), azo dyes (44), reduction in liver: the possible role of cytochrome P-450 in liver microsomes. and A/-oxides (45,46). These facts indicate that the enzyme may Mol. Pharmacol., 4: 541-548, 1968. 26. Ristagno, C. V., and Shine, W. J. Ion radicals. XX. Aromatic nitration. A new play a role in the reductive metabolism of aromatic nitro com preparation of 3-nitroperylene and 1-nitropyrene. J. Am. Chem. Soc., 93: pounds in mammalian livers. 1811-1812,1971. 27. Poirier, L. A., Miller, J. A., and Miller, E. C. 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CANCER RESEARCH VOL. 46 MARCH 1986

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1986 American Association for Cancer Research. Reductive Metabolism of Aromatic Nitro Compounds Including Carcinogens by Rabbit Liver Preparations

Kiyoshi Tatsumi, Shigeyuki Kitamura and Noriko Narai

Cancer Res 1986;46:1089-1093.

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