reductive metabolism of aromatic nitro compounds …tosol, exhibited significant nitroreductase...

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

Reductive Metabolism of Aromatic Nitro Compounds Including Carcinogens byRabbit Liver Preparations1

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

Reductive metabolism of aromatic nitro compounds was examined with rabbit liver preparations. Under anaerobic conditions, carcinogenic 2-nitrofluorene, 4-nitrobiphenyl, and 1-nitro-

naphthalene were reduced to the corresponding hydroxylaminesand amines, whereas the carcinogenic 1-nitropyrene was re

duced only to the corresponding amine by liver cytosol in thepresence of 2-hydroxypyrimidine, an electron donor of aldehyde

oxidase. These metabolites were identified unequivocally bycomparing their mass spectra and thin-layer Chromatographie

behaviors with those of the authentic samples.Both liver microsomes and cytosol catalyzed the reduction of

these aromatic nitro compounds in varying degrees. The micro-

somes required reduced pyridine nucleotides for occurrence ofthe nitroreductase activities. In this case, reduced nicotinamideadenine dinucleotide phosphate was more effective than reducednicotinamide adenine dinucleotide as an electron donor. Thecytosol by itself exhibited some nitroreductase activities, whichwere markedly enhanced by addition of an electron donor ofaldehyde oxidase, i.e., A/1-methylnicotinamide or 2-hydroxypyr

imidine. The full activities of the cytosol with the electron donorwere much higher than those of the microsomes with the reducedpyridine nucleotide. Purified liver aldehyde oxidase, like the cytosol, exhibited significant nitroreductase activities in the presence of its electron donor.

These results indicated that cytosolic aldehyde oxidase functions as a major enzyme responsible for the reduction of aromaticnitro compounds including carcinogens in rabbit liver.

INTRODUCTION

Certain aromatic nitro compounds have been proved to becarcinogenic (1-5), some of which have been noticed as important environmental pollutants (5-10). The mechanism of carcin-

ogenicity is thought to be mediated by the metabolic reductionof these nitro compounds to reactive hydroxylamine intermediates (11-15). However, previous works showed that the anaerobic incubation of carcinogenic 1-nitropyrene (16-20), 2-nitrofluorene (18, 21), 4-nitrobiphenyl (15), 2-nitronaphthalene (15), and1-nitronaphthalene (15) with mammalian liver preparations led to

the formation of the corresponding amines, with no evidence forthe formation of the corresponding hydroxylamines.

The reduction of aromatic nitro compounds occurs with micro-

Received 7/22/85; revised 11/18/85; accepted 11/19/85.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 inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported in part by a grant-in-aid from the Ministry of

Education, Science, and Culture, Japan.2To whom requests for reprints should be addressed.3 Present address: Faculty of Pharmaceutical Sciences, Setsunan University,

45-1, Nagaotogecho, Hirakata-shi, Osaka 573-01, Japan.

somal and cytosolic fractions of mammalian livers (15, 18, 20,22-24). Previous studies showed that a cytochrome P-450 sys

tem (23, 25) and xanthine oxidase (23) are involved in thereduction of nitrobenzene orp-nitrobenzoic acid catalyzed by rat

liver microsomes and cytosol, respectively. In a preliminary communication (18), recently, we demonstrated that a microsomalcytochrome P-450 system and cytosolic aldehyde oxidase ofrabbit liver catalyze the reduction of 1-nitropyrene and 2-nitroflu

orene to the corresponding amines. More recently, the participation of cytochrome P-450 in nitroreduction was further confirmed with a reconstituted cytochrome P-450 system from rat

liver microsomes (20).The present study shows the first example of enzymatic

formation of hydroxylamine derivatives from 2-nitrofluorene, 4-nitrobiphenyl, and 1-nitronaphthalene. In addition, the study pro

vides the first evidence that aldehyde oxidase functions as amajor liver enzyme responsible for the reduction of aromatic nitrocompounds including carcinogens.

MATERIALS AND METHODS

Chemicals. 2-Nitrofluorene, 1-aminopyrene, 2-acetylaminofluorene,A/'-methylnicotinamide chloride, 2-hydroxypyrimidine hydrochloride, di-

phenylamine, phenothiazine, and benzamide were purchased from TokyoChemical Industry Company, Ltd. 2-Aminofluorene, xanthine, menadi-

one, sodium arsenite, and potassium cyanide were obtained from NakafaiChemical, Ltd. NADPH, NADH, and chlorpromazine were obtained fromSigma Chemical Company. 4-Nitrobiphenyl and 4-aminobiphenyl werepurchased from Aldrich Chemical Company. 1-Nitronaphthalene and

bovine serum albumin were purchased from Katayama Chemical IndustryCompany, Ltd. 1-Aminonaphthalene was obtained from Ishizu Pharmaceutical Company, Ltd., and p-nitrobenzoic acid was from Yoneyama

Chemical Industries, respectively. Cyproheptadine was donated by Nippon Merck-Banyu Company, Ltd.

1-Nitropyrene (mp 148Â°C)was synthesized by the method of Ristagnoand Shine (26), 2-hydroxylaminofluorene (mp 169-173Â°C) by that ofPoirier eÃal. (27), W-acetoxy-2-acetylaminofluorene (mp 176-178Â°C) by

that of Gutmann and Erickson (28), 1-hydroxylaminonaphthalene (mp76-78Â°C) by that of WillstÃ¤tter and Kubli (29), and 4-hydroxylaminobi-phenyl (mp 178-180Â°C) by the method of MÃ¤heref al. (30), respectively.

Enzymes. Bovine liver catalase was purchased from Sigma ChemicalCompany. Male albino rabbits weighing 2.0-2.5 kg were used in this

study. Rabbit liver aldehyde oxidase was purified by the method ofRajagopalan ef a/. (31). Rabbit liver microsomes and cytosol wereprepared as follows. The liver was homogenized in 4 volumes of 1.15%KCI, the homogenate was centrifugea for 20 min at 9,000 x g, and the9,000 x g supernatant fraction was centrifuged for 60 min at 105,000 xg. The microsomal fraction was washed by resuspension in the KCIsolution and resedimentation for 60 min at 105,000 x g.

Isolation of Metabolites. The incubation mixture consisted of 12 //molof a nitro compound, 120 (Â¿molof 2-hydroxypyrimidine, and liver cytosol

(equivalent to 12 g of liver) in a final volume of 150 ml of 0.1 M phosphatebuffer (pH 7.4). The nitro compound was placed in the side arm of theThunberg apparatus separate from the rest of the incubation mixturewhich was placed in the main tube. The incubation was carried out

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REDUCTIVE METABOLISM OF AROMATIC NITRO COMPOUNDS

anaerobically as follows. The tube was gassed for 5 min with nitrogen,which was passed through a deoxygenizing solution consisting of 0.5%sodium dithionite and 0.05% sodium 2-anthraquinonesulfonate in 0.4%

NaOH, evacuated with an aspirator for 10 min, and again gassed withthe nitrogen. After the tube was tightly closed, the reaction was startedby mixing a nitro compound of the side arm and all other components ofthe body together, and it was continued for 30 min at 37Â°C. After

incubation, the mixture was extracted once with an equal volume of ethylacetate, the ethyl acetate extract was evaporated to dry ness in avacuum, and the residue was subjected to TLC4. TLC was conducted

on silica gel plates (Kieselgel 60 GF25<; Merck; 0.25 mm thick) anddeveloped in benzene:acetone (7:3), and the chromatograms were visualized under UV light (254 nm). Metabolites were eluted with methanolfrom the TLC plates and subjected to mass spectrometric study. Massspectra were taken with a Shimadzu 7000 mass spectrometer at anionizing voltage of 70 eV. These procedures were quickly conducted ina nitrogen atmosphere and in the dark as much as possible, to precludethe loss of the hydroxylamine derivatives formed. The assay of nitrored-

uctase activity described below was also performed under such precautions.

Assay of Nitroreductase Activity. The incubation mixture consistedof 0.2 iimol of a nitro compound, 1 ^mol of an electron donor, and livermicrosomes (equivalent to 1 g of liver) or liver cytosol (equivalent to 0.2g of liver) in a final volume of 2.5 ml of 0.1 M phosphate buffer (pH 7.4).When aldehyde oxidase (0.1 mg of protein) was used as an enzymesource, 1.25 mg of bovine serum albumin and 12 Â¿igof catalase werefurther added to this incubation mixture. Prior to incubation, a Thunbergtube was gassed for 3 min with deoxygenated nitrogen, evacuated for5 min, and again gassed with the nitrogen. The reaction was started bymixing the solution of the side arm and the body together and continuedfor 30 min at 37Â°Cas described above. The incubation mixture containing

a boiled liver preparation was used as a control.In the case of 2-nitrofluorene, its metabolites, i.e., 2-hydroxylaminoflu-

orene and 2-aminofluorene, were determined as their acetyl derivatives.

The incubation mixture, after adding 0.2 jumol of diphenylamine as aninternal standard, was extracted once with 10 ml of ethyl acetate, andto the ethyl acetate extract was added 0.5 ml of acetic anhydride. Thereaction mixture was allowed to stand for 3 h at room temperature underan atmosphere of nitrogen, washed with NaHCO3-saturated solution and

water, successively, and then evaporated to dryness in a vacuum. Theresidue was dissolved in 50 Â¿ilof methanol and subjected to HPLC.HPLC was performed in a Toyo Soda HLC-803A high-pressure liquidChromatograph equipped with a UV-8 UV absorption detector. Theinstrument was fitted with a 30-cm x 4-mm inner diameter LS-410Kreversed-phase column (Toyo Soda). The mobile phase was 0.1 M

KHÃ¼POvCHsCN(1:1). The Chromatograph was operated at a flow rateof 1 ml/min at ambient temperature and at a wavelength of 254 nm. 2-Hydroxylaminofluorene- or 2-aminofluorene-spiked buffer samples were

extracted and acetylated to provide a linear standard curve. In theexperiment concerning the effect of some chemicals on 2-nitrofluorenereductase and aldehyde oxidase activities of cytosol (Table 3), the 2-

nitrofluorene that disappeared during incubation was determined by gasChromatographie analysis as follows. The incubation mixture, after adding 0.5 ftmo\ of phenothiazine as an internal standard, was extractedonce with 5 ml of ethyl acetate, and the ethyl acetate extract wasevaporated to dryness in a vacuum. The residue was dissolved in 50 n\of methanol and then applied to a Hitachi Model 163 gas Chromatographequipped with a flame ionizing detector and a 2-m x 3-mm inner diameter

glass column packed with 3% Dexsil 400 GC on Chromosorb W. Theoperation conditions were as follows: injection port and detector temperature, 260Â°C; column temperature, 210Â°C.

In the cases of 4-nitrobiphenyl and 1-nitronaphthalene, the incubation

mixture, after adding the internal standard described above, 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 in50 iil of methanol and then applied to a Gilson 1B high-pressure liquid

Chromatograph equipped with a M & S Variactor 311 UV absorptiondetector. The instrument was fitted with a 15-cm x 4.6-mm inner diameter

M & S da column. The Chromatograph was also operated at ambienttemperature and at a wavelength of 254 nm. In the determination of 4-

nitrobiphenyl metabolites, the mobile phase was 0.1 M KH2PO4:CH3CN(3:2), and the flow rate was 1 ml/min.

In the case of 1-nitropyrene, the incubation mixture, after adding 0.31

,<mol of cyproheptadine as an internal standard, was adjusted to pH 10with N NaOH and extracted once with 5 ml of n-heptane. The n-heptane

extract was evaporated to dryness in a vacuum, and the residue, afterbeing dissolved in 50 Â¿tlof methanol, was applied to a Shimadzu GC-

4CM gas Chromatograph equipped with a hydrogen flame ionizing detector. The instrument was fitted with a 2-m x 3-mm inner diameter glass

column packed with 3% Dexsil 400 GC on Chromosorb W. The operatingconditions were as follows: injection port and detector temperature,310Â°C;column temperature, 260Â°C.

Under the assay conditions, 94% of 2-nitrofluorene, 85% of 4-nitrobiphenyl, and 73% of 1-nitronaphthalene that disappeared were recovered

as the corresponding hydroxylamines and amines, respectively. In thecase of 1-nitropyrene, 93% of the compound that disappeared was

exclusively recovered as the corresponding amine. The recoveries ofauthentic hydroxylamine derivatives added to 0.1 M phosphate buffer(pH 7.4) were 87% for 2-hydroxylaminofluorene, 85% for 4-hydroxyla-minobiphenyl, and 76% for 1-hydroxylaminonaphthalene, respectively,at the end of 30-min incubation.

Assay of Aldehyde Oxidase. The assay was performed by themethod of Felsted ef al. (32), measuring the increase in absorbance at300 nm, which accompanies the oxidation of N1-methylnicotinamide to

the 2- and 4-pyridones.

Determination of Protein. Protein was determined by the method ofLowry ef al. (33) with bovine serum albumin as a standard.

RESULTS

When aromatic nitro compounds were incubated with rabbitliver cytosol in the presence of 2-hydroxypyrimidine, an electrondonor of aldehyde oxidase, their metabolites were isolated fromthe incubation mixtures as described in "Materials and Methods."

Table 1 shows Rt values of these metabolites in TLC.As shown in Figs. 1 to 3, mass spectra of the 2-nitrofluorene

metabolite 1, 4-nitrobiphenyl metabolite 1, and 1-nitronaphtha

lene metabolite 1 gave molecular ions at m/z 197,185, and 159,suggesting that these metabolites are the corresponding hydroxylamine derivatives. In addition, the spectra showed that fragment ions at m/z M+-2 (195, 183, and 157) are attributable to

the loss of 2 hydrogen atoms from molecular ions to form thecorresponding nitroso derivatives, and base peaks at m/z M+-

16 (181,169, and 143) result from the loss of one oxygen atomfrom molecular ions to form the corresponding amine derivatives.The mass spectra and the Rf values in TLC of these metabolites

Table 1R, values of reductive metabolites ot aromatic nitro compounds in thin-layer

chromatography

TLC was conducted on silica gel plates developed in benzene:acetone (7:3) asdescribed in "Materials and Methods."

4The abbreviations used are: TLC, thin-layer chromatography; HPLC, high-

pressure liquid chromatography.

MetaboliteR,2-Nitrofluorene

metabolite 12-Nitrofluorene metabolite 24-Nitrobiphenyl metabolite 14-Nitrobiphenyl metabolite 21-Nitronaphthalene metabolite 11-Nitronaphthalene metabolite 21-Nitropyrene metabolite0.17

0.320.290.450.330.500.30


1090




were completely identical to those of authentic hydroxylaminederivatives. Based on these facts, 2-nitrofluorene metabolite 1,4-nitrobiphenyl metabolite 1, and 1-nitronaphthalene metabolite1 were identified as 2-hydroxylaminofluorene, 4-hydroxylamino-biphenyl, and 1-hydroxylaminonaphthalene, respectively.

On the other hand, the 2-nitrofluorene metabolite 2, 4-nitrobiphenyl metabolite 2, 1-nitronaphthalene metabolite 2, and 1-

nitropyrene metabolite showed mass spectra with molecular ionsat m/z 181,169,143, and 217. These metabolites were identifiedas 2-aminofluorene, 4-aminobiphenyl, 1-aminonaphthalene, and

100

so

100

50

a)JÃœL-iei195(Ml,9,i

so 100 150 200(m/z)

^-Â©â€¢NHOHULJ_j-181 ,95CM')

1971

50 100 150 200 (m/z)

Fig. 1. Mass spectra of 2-nitrofluorene metabolite 1 (a) and authentic 2-hydroxylaminofluorene (b).

100

50

a),1.-169183185Ã•M')L

50 100 ISO 200(m/z)

100

5Â»cc

50

b) ,-)-Â©-NHOH1

J I-169183I

185(M')i

IL50 no ISO 200 (m/z)

Fig. 2. Mass spectra of 4-nitrobiphenyl metabolite 1 (a) and authentic 4-hydrox-

ylaminobiphenyl (b).

1-aminopyrene, respectively, by comparison with the authenticsamples of their mass spectra and thin-layer Chromatographie

behavior (data not shown).Rabbit liver microsomes and cytosol can catalyze the reduction

of 2-nitrofluorene, 4-nitrobiphenyl, and 1-nitronaphthalene to the

corresponding hydroxylamines and amines in varying degrees(Table 2). In microsomes, NADPH was more effective than NADHas an electron donor. The NADPH-linked activities were partly

inhibited by carbon monoxide, indicating the involvement ofcytochrome P-450 in the microsomal nitroreduction (data notshown). On the other hand, cytosol by itself exhibited somenitroreductase activities, which were markedly enhanced byaddition of electron donors of aldehyde oxidase, such as A/1-

methylnicotinamide and 2-hydroxypyrimidine. NADPH, NADH,

and xanthine had only a little effect on the cytosolic nitroreductase activities. The full activities of cytosol supplemented withthe electron donor of aldehyde oxidase were much higher thanthose of microsomes supplemented with the reduced pyridinenucleotide. Such cytosolic nitroreductase activity appeared to bedue to aldehyde oxidase, considering the result of electron donorrequirement.

To support this concept, the ability of some chemicals to inhibit2-nitrofluorene reducÃaseand aldehyde oxidase activities of cytosol was examined. As shown in Table 3, both activities weresimilarly susceptible to inhibition by all of these chemicals, indicating the involvement of aldehyde oxidase in the cytosolicnitroreduction.

100

so

a)Ã

\i i L i-

143157ÃŒ159IWI

'

50 100 150 200(m/z)

I50b)NHOH[Ã”tÃ´lJ

J 1 til-

143157I59IM*)Â£

50 100 150 200(m/z)Fig. 3. Mass spectra of 1-nitronaphthalene metabolite 1 (a) and authentic 1-

hydroxylaminonaphthalene (D).

Table 2

Reduction of aromatic nitro compounds by rabbit liver fractionsMetabolites 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(nmol/30 min/gliver)FractionMicrosomesNoneNADPHNADHCytosol

NoneNADPHNADHXanthineN

' -Methylnicotinamide

2-HydroxypyrimidineHydroxylamine

formed82.812.319.684.012.015.675.82.7

112.9Amine

formed"8.0109.125.912.084.277.615.0586.4

324.44-Nitrobiphenyl

(nmol/30 min/gliver)Hydroxylamine

formed012.818.14.029.225.610.868.0

88.2Amine

formed065.631.748.883.274.452.8724.8

391.21-Nitronaphthalene

(nmol/30 min/gliver)Hydroxylamineformed02.31.50.20.40.40.20.50.6Amine

formed089.928.33.051.853.265.4615.0

428.8aDetermined as N-acetoxy-2-acetylaminofluorene.


1091




Table 4 shows the ability of purified rabbit liver aldehydeoxidase to reduce the aromatic nitro compounds to the corresponding hydroxylamines and amines. The enzyme, like cytosoldescribed above, exhibited significant nitroreductase activities inthe presence of its own electron donor, but slightly in thepresence of xanthine, NADPH, or NADH.

Similar results were obtained with the reduction of 1-nitropy-

rene by rabbit liver preparations. In this case, however, thecorresponding hydroxylamine could not be detected as its metabolite. The microsomal nitroreductase activity was dependenton NADPH or NADH, while the cytosolic nitroreductase activitywas markedly enhanced by addition of A/1-methylnicotinamide or

2-hydroxypyrimidine (Table 5). Purified rabbit liver aldehyde oxidase supplemented with its electron donor showed a significantactivity towards the nitro compound (Table 6).

From these facts, we concluded that cytosolic aldehyde oxidase is involved in the reduction of aromatic nitro compoundsincluding carcinogens as a major liver enzyme.

p-dinitrobenzene (36), and p-nitrobenzenesulfonamide (36). The

present paper is the first description that enzymatic formation ofhydroxylamine derivatives is observed with aromatic nitro compounds which possess 2 or more rings in the molecules. Theprecautions described in "Materials and Methods" made the

identification of these unstable metabolites possible.On the other hand, 1-nitropyrene, which is the predominant

nitropolycyclic aromatic hydrocarbon in environmental samples,was exclusively reduced to the corresponding amine by rabbitliver preparations. In a preliminary study, however, when thenitro compound was anaerobically incubated with hamster livercytosol and a NADH-generating system in 0.1 Mphosphate buffer

(pH 7.4), 2 metabolites were isolated from the incubation mixtureas their acetyl derivatives. One of them showed the mass spectrum with a molecular ion at m/z 317 (C2oH1503N,attributable toA/-acetoxy-1-acetylaminopyrene), accompanying fragment ionsat m/z 275 (Ci8H1302N, W-hydroxy-1-acetylaminopyrene), m/z259 (C18H13ON, 1-acetylaminopyrene), m/z 233 (Ci6H,,ON, 1-

DISCUSSION

A great deal of attention has been focused on the metabolicconversion of carcinogenic aromatic nitro compounds to thecorresponding hydroxylamines as a potential activation pathway(11-15). However, the range of previous aromatic nitro compounds described to give hydroxylamine derivatives during enzymatic reduction was limited to nitrobenzene (23) and its derivatives, such as 2,4,6-trinitrotoluene (34), p-nitrobenzoic acid (24),methyl p-nitrobenzoate (35, 36), p-nitroacetophenone (35, 36),

Table 3Effect of some chemicals on nitroreductaseand aldehyde oxidase activities of

rabbit liver cytosol2-Nitrofluorene disappearancewas determined from its peak area in gas chro-

matography which was performed as described in "Materials and Methods." Each

valuerepresents the mean of 4 experiments. Retention time of the nitro compoundin the gas chromatography was 14 min.

% ofcontrolAdditionNone

(control)MenadioneChlorpromazineSodium arsenitePotassium cyanideConcentration(M)5x

IO"45x 10-"1 x 10-1

2.5 x 10-3Nitroreductase

activity(2-nitrc-fluorene

disappeared)"10015

81823Aldehyde

oxidaseactivity100

00

1118

Table 5Reduction of 1-nitropyreneby rabbit liver fractions

1-Aminopyreneformation was determined from its peak area in gas chromatography which was performed as described in "Materials and Methods." Each value

represents the mean of 4 experiments. Retention time of the metabolite in the gaschromatography was 14 min.

FractionMicrosomesNoneNADPHNADHCylosolNoneNADPHNADHXanthineW'-Methylnicotinamide2-Hydroxypyrimidine1

-Aminoypreneformed

(nmol/30 min/gliver)0.3148.121.099.8111.2159.183.11483.31021.4

Table 6Reduction of 1-nitropyreneby rabbit liver aldehyde oxidase

As regards determination of 1-aminopyreneformation, see the legend of Table5. Each value represents the mean of 4 experiments.

8 The incubation was carried out for 30 min in the presenceof W-methylnicotin-

amide.

AdditionNone

W1-Methylnicotinamide2-HydroxypyrimidineXanthineNADPHNADH1

-Aminopyreneformed(nmol/30 min/mgprotein)01203.5

1131.210.29.5

13.3

Table 4Reduction 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(nmol/30 min/mg protein)

4-Nitrobiphenyl(nmol/30 min/mg protein)

1-Nitronaphthalene(nmol/30 min/mg protein)

AdditionNone

N'-Methylnicotinamide2-HydroxypyrimidineXanthineNADPHNADHHydroxylamine

formed80

253.230.314.414.415.6Amine

formed"0

1185.6392.4

15.613.213.2Hydroxylamine

formed0

163.8129.6

8.132.416.2Amine

formed0720.9

540.010.83.45.4Hydroxylamine

formed0

10.212.8000Amine

formed0

937.6814.326.419.621.3

8 Determinedas N-acetoxy-2-acetylaminofluorene.0 Determinedas 2-acetylaminofluorene.


1092




hydroxylaminopyrene), and m/z 217 (CieHnN, 1-aminopyrene).The other was identified as 1-acetylaminopyrene by comparisonwith the authentic sample. These facts strongly suggest that 1-

nitropyrene is convertible not only to the corresponding amine,but also to the corresponding hydroxylamine by mammalian liverenzymes under certain conditions.

Cytosolic aldehyde oxidase rather than microsomal cyto-chrome P-450 appears to be mainly responsible for the reduction

of aromatic nitro compounds including carcinogens in rabbit liver.Participation of the molybdenum-containing flavoenzyme in nitro-

reduction was first demonstrated by Wolpert ef al, (37) withheterocyclic nitro compounds, such as 5-nitro-2-furaldehydesemicarbazone (nitrofurazone) and 4-nitroquinoline /V-oxide. Re

cently, we found that the enzyme in the presence of its electrondonor can also catalyze the reduction of sulfoxide (38, 39),nitrosamines (40, 41), hydroxamic acids (42, 43), azo dyes (44),and A/-oxides (45,46). These facts indicate that the enzyme may

play a role in the reductive metabolism of aromatic nitro compounds in mammalian livers.

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1986;46:1089-1093. Cancer Res Kiyoshi Tatsumi, Shigeyuki Kitamura and Noriko Narai Carcinogens by Rabbit Liver PreparationsReductive Metabolism of Aromatic Nitro Compounds Including

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