Carcinogenesisvol.il no.2 pp.213 —218, 1990
Characterkaitnoffli off oxMattive amd ratacttive metabolism in vitro off mitrofflMorainiitlhemes by rat liver enzymes
Maria Antonietta Belisario1, Rita Pecce, Rossella Delia Morte, Amalia R.Arena, Angelo Cecinato2, Paolo Ciccioli2 and Norma Staiano Dipartimento di Biochimica e Biotecnologie Mediche, Universita di Napoli, Via S.Pansini n. 5, 80131 Napoli and 2Istituto di Inquinamento Atmosferico, Consiglio Nazionale delle Ricerche, Monte Rotondo, Roma, Italy 'To whom reprint requests should be sent
Introduction Nitrated polycyclic aromatic hydrocarbons (nitro-PAHs*) have been detected in the environment as products of incomplete combustion processes, fossils fuels, cigarette smoke, diesel emissions, coal tar and charcoal barbeques (1-4). They can also be formed in the atmosphere from the reaction of polycyclic aromatic hydrocarbons with traces of nitrogen oxides (5 — 8). Many of these nitro-PAHs have been shown to be highly mutagenic and carcinogenic in rodents (2,9-14). Much attention has been devoted to nitrofluoranthenes (NFs) since a larger amount of 2-nitrofluoranthene (2-NF) than 1-nitropyrene (1-NP) •Abbreviations: nitro-PAHs, nitrated polycyclic aromatic hydrocarbons; NFs, nitrofluoranthenes; cyt. P450, cytochrome P450; 3-NH2F, 3-aminofluoranthene; DCM, dichloromethane; PB, phenobarbital; 3-MC, 3-methylcholanthrene; DMSO, dimethylsulphoxide; FMN, flavin mononucleotide; cyt. c, cytochrome c; SKF 525-A, 2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride © Oxford University Press
Materials and methods Chemicals 3-Aminofluoranthene (3-NH2F), 1-nitropyrene, menadione (2-methyl-1,4-naphtochinon), 2-hydroxypirimidine, dicumarol, yV-methylnicotinamide and acetaldehyde were obtained from Aldrich-Chemie (FRG); hypoxanthine, allopurinol and NAD(P)H were purchased from Sigma Chemical Co. (St Louis, MO), SKF 525-A from Smith, Kline and French, Inc. (UK). All other reagents were of analytical grade. Preparation of NFs Fluoranthene (2.5 mg) dissolved in dichloromethane (DCM) was added to acetic anhydride (500 /tl) containing 100 /il concentrated nitric acid. The reaction was performed in a refrigerated (+4°C) water bath. After 1 h, 1 ml distilled water plus 500 ii\ DCM were added to reaction mixture. The organic phase was chromatographed on basic allumine (60-230 mesh) manufactured by Carlo Erba (Italy). The N-esane eluate was discarded and the DCM fraction containing NFs was concentrated and chromatographed by HPLC using an Erbasil analytical silica column (10 jjm, 0.4 x 25 cm) to separate single isomers (21,22). This procedure was repeated several times in order to obtain ample material for bioassay. Quantitative evaluation of purified isomers was performed by HRGC — FID on a gas-chromatograph Carlo Erba 5300 with a FID 40 detector, using a capillary DBS-silica column (25 m x 320 /tm x 0.3 fim). A solution containing a known amount of 1-nitropyrene was used as reference. Each isomer was analysed by El MS at 70 eV: 247 |M| + , 217 |M-N0| + , 201 |M-NO2| + , 200 |M-HNO2| + , 189 |M-NO-CO| + . A 35% yield of NF isomers was obtained by nitrating fluoranthene in these experimental conditions. The yields of each isomer were 1-NF 4.5%; 7-NF 6.0%; 3-NF 53.2% and 8-NF 36.3%. Purity of each isomer used in biological tests were: 1-NF > 99.9%; 7-NF 99.2%; 3-NF 98.5% (presence of 8-NF 1.35%) and 8-NF 99.7%. Preparation of subcellular fractions from rat liver Fresh livers from male Sprague-Dawley rats weighing 150-160 g each were perfused thoroughly with cold 0.9% NaCl and homogenized with 3 ml/g tissue of 0.15 M KC1/0.05 M Tris-HCl buffer pH 7.4. Homogenates were centrifuged for 10 min at 12 000 g and the supernatant 1 h at 105 000 g. The supernatant (S-105 fraction or cytosol) was divided into small aliquots, frozen on dry ice and stored at -80°C. The microsomal pellets were resuspended in 0.15 M KCI/0.05 M Tris-HCl buffer and centrifuged for 1 h at 105 000 g. Microsomes
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Nitrofluoranthenes (NFs) are mutagenic and carcinogenic environmental pollutants found in incomplete combustion products and urban air participate. We have studied both oxidative and reductive metabolism in vitro of different NF isomers mediated by subcellular rat liver fractions. Under aerobic conditions only ring hydroxylation of NFs by rat liver microsomes occurred and the isomeric position of the nitro group affected both the amount and the type of phenolic metabolites formed. Liver microsomes from 3-methylcholanthrene-induced rats were most effective in giving ring hydroxylated 7- and 8-nitrofluoranthene, whereas liver microsomes from phenobarbital-pretreated rats were the most active in metabolizing 1- and 3-nitrofluoranthene. Under anaerobic conditions, only reduction of NFs mediated by both cytosolic and microsomal rat liver enzymes occurred. Cofactor requirements and inhibition experiments indicated that the reductase activity in rat liver cytosolic fractions could be ascribed to DT-diaphorase, aldehyde oxidase and/or other unknown enzymes. The microsomal reductase activity was inhibited by oxygen, carbon monoxide, 2-diethylaminoethyl2,2-diphenylvalerate hydrochloride and n-octylamine, and slightly by cytochrome c; flavin mononucleotide greatly enhanced this activity. 3-Nitrofluoranthene microsomal nitroreductase activity was increased by phenobarbital rat pretreatment and this increment correlated well with the content of cytochrome P450. These results indicate a participation of cytochrome P45Q in the reductive metabolism of NFs by rat liver microsomes.
was found in environmental paniculate matter (1,7-8). Although NF genotoxicity has been studied extensively, few investigations have been made on their metabolism in mammals. It has been shown that 3-nitrofluoranthene (3-NF) and mixtures of NFs produce a significant induction of hepatic and pulmonary aryl hydrocarbon hydroxylase and 7-ethoxyresorufin-O-deethylase activities in rats (15). Khan etal. (15) demonstrated that NFs induce cytochrome P450 (cyt. P450) isozymes c and d, using highly specific monoclonal antibodies. Phenolic metabolites have been identified as products of in vitro hepatic microsomal metabolism of 2-NF and 3-NF (10). Wide species variability has been observed in the oxidative metabolism of 3-NF (16). In this study we examined some characteristics of both oxidative and reductive metabolism in vitro of 1-, 3-, 7- and 8-NF, using subcellular rat liver fractions. Knowing that microsomal cyt. P450 system and cytosolic enzymes such as DTdiaphorase, xanthine oxidase and aldehyde oxidase are involved in the reductive metabolism of some nitroaromatic compounds (17-20), we attempted to evaluate their involvement in NF reduction. We present data on induction and inhibition experiments showing the contribution of cyt. P450 form(s) in the reductive metabolism of NFs.
M.A.Belisario et at. suspended in 0.1 M sodium phosphate buffer, pH 7.4 (containing 30% v/v glycerol) were divided into small aliquots, frozen on dry ice and stored at —80°C. Microsomal and cytosolic protein determination was performed according to Lowry (23) using spectrophotometrically checked bovine serum albumin solution as standard. Quantitative evaluation of cyt. P450 in microsomes was performed according to Omura and Sato (24). In induction experiments 80 mg/kg body weight phenobarbital (PB) sodium salt was given i.p. to rats for 3 consecutive days, 40 mg/kg body weight 3-methylcholanthrene (3-MC) was given i.p. for 2 consecutive days. Animals were killed 24 h after the last injection.
Assay of NF oxidative metabolism A standard incubation mixture contained 0.1 M sodium potassium phosphate buffer (pH = 7.4), 2 mM NADPH, 10 mM MgCl2, 0.1 mM NF isomer dissolved in DMSO and 0.25 mg of microsomal proteins in a final volume of 0.5 ml. The
1-NF
Results Oxidative metabolism of NFs NFs were incubated under aerobic conditions with liver microsomes from control and induced rats. HPLC analysis of reaction products revealed no fluorescent peaks, indicating that no amino derivatives had formed. Incubation of NF isomers with liver microsomes gave metabolites tentatively identified as phenolic derivatives; we are currently characterizing them using mass spectrometry and NMR. An almost quantitative recovery of unmetabolized NF isomers from incubation mixtures was achieved with the extraction procedure described in Materials and methods. The absence of large non covalent binding of NFs to microsomal proteins was ascertained in standardization experiments using heat-inactivated microsomal proteins (unpublished data). Therefore the decrease in peak areas at the unmetabolized NF isomer's retention time allowed us to evaluate the nmoles of metabolized chemical. Figure 1 reports the time-dependent metabolization rate of NFs incubated with liver microsomes from control and induced rats. NF isomers were metabolized from uninduced rat liver microsomes to a different extent in the following order: 8-NF = 1-NF > 7-NF > 3-NF. Treatment of rats with the inducers 3-MC and PB produced different patterns of NF metabolization rates: 7-NF and 8-NF were better transformed by 3-MC-induced
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Fig. 1. Time-dependent metabolization rates of NF isomers under aerobic conditions. Each isomer (0.1 mM) was incubated with liver microsomes (0.25 mg) from control (O), PB (A)- or 3-MC (O)-induced rats, in the presence of NADPH. The extraction procedure of unmetabolized NFs and metabolites and HPLC anlaysis were performed as described in Materials and methods.
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Assay of NF reduction A typical reaction mixture (final volume 0.5 ml) contained 0.1 M sodium potassium phosphate buffer (pH 7.4), different amounts of microsomal or cytosolic proteins, 2 mM NAD(P)H or other cofactors (hypoxanthine, acetaldehyde, etc.), 10 raM MgCl2 and 0.1 mM NF isomer dissolved in dimethylsulphoxide (DMSO). Incubation tubes were sealed with rubber stoppers and all mixtures except NAD(P)H were bubbled for 2 min with nitrogen gas passed through a deoxygenizer system. When necessary, carbon monoxide gas was used instead of nitrogen gas. Reactions were carried out in duplicate at 37°C under a nitrogen atmosphere in a water shaking bath and stopped with an equal volume of cold ethanol just before addition of substrate (time 0) or after different times of incubation. Mixtures were extracted three times with toluene:ethylacetate (1:1 v/v); the organic phase was evaporated under a nitrogen stream, dried residues were dissolved in methanol and analysed by HPLC. Analyses were performed on a Varian liquid chromatograph (Varian Instruments, Co) using a varian Qg Amino Tag analytical column (0.46 x 15 cm). A constant flow of 1.0 ml/min and a 15-min linear gradient from 70 to 100% methanol in water was used. Peaks were monitored simultaneously by UV absorbance at 254 nm and fluorescence (X exc. = 340 nm, X emiss. = 520 nm) and areas were calculated by Vista printer plotter (Varian Instruments). .
reaction was started by adding NADPH solution. Incubation was carried out in duplicate tubes at 37°C in a water shaking bath and stopped at different times with an equal volume of cold ethanol. The mixtures were extracted and the products formed were analysed by the procedure described above.
Oxidative and reductive metabolism of NFs by liver enzymes
rat liver microsomes while 1-NF and 3-NF were better metabolized by PB-induced rat liver microsomes. Moreover both 3-MC- and PB-induced microsomes showed a higher affinity for 8-NF than for the other isomers. We noted not only quantitative but also qualitative differences between liver microsomes from control and variously induced rats used as metabolizing systems, as shown in Figure 2. Control and PB-induced microsomes formed a greater amount of 3-NF metabolites corresponding to peaks a and d, while with 3-MC microsomes the metabolite related to peak c became the most abundant. Analogous differences were observed when the other isomers were used as substrates of variously induced rat liver microsomes (data not shown).
E Table I. Nitroreduction rates of NF isomers mediated by subcellular liver fractions from uninduced rats, in the presence of NADPH SO CM
NF amino-derivative formed (nmol/mg protein/min)
Compound
Microsomes8
Cytosola
Characterkaitnoffli off oxMattive amd ratacttive metabolism in vitro off mitrofflMorainiitlhemes by rat liver enzymes
Maria Antonietta Belisario1, Rita Pecce, Rossella Delia Morte, Amalia R.Arena, Angelo Cecinato2, Paolo Ciccioli2 and Norma Staiano Dipartimento di Biochimica e Biotecnologie Mediche, Universita di Napoli, Via S.Pansini n. 5, 80131 Napoli and 2Istituto di Inquinamento Atmosferico, Consiglio Nazionale delle Ricerche, Monte Rotondo, Roma, Italy 'To whom reprint requests should be sent
Introduction Nitrated polycyclic aromatic hydrocarbons (nitro-PAHs*) have been detected in the environment as products of incomplete combustion processes, fossils fuels, cigarette smoke, diesel emissions, coal tar and charcoal barbeques (1-4). They can also be formed in the atmosphere from the reaction of polycyclic aromatic hydrocarbons with traces of nitrogen oxides (5 — 8). Many of these nitro-PAHs have been shown to be highly mutagenic and carcinogenic in rodents (2,9-14). Much attention has been devoted to nitrofluoranthenes (NFs) since a larger amount of 2-nitrofluoranthene (2-NF) than 1-nitropyrene (1-NP) •Abbreviations: nitro-PAHs, nitrated polycyclic aromatic hydrocarbons; NFs, nitrofluoranthenes; cyt. P450, cytochrome P450; 3-NH2F, 3-aminofluoranthene; DCM, dichloromethane; PB, phenobarbital; 3-MC, 3-methylcholanthrene; DMSO, dimethylsulphoxide; FMN, flavin mononucleotide; cyt. c, cytochrome c; SKF 525-A, 2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride © Oxford University Press
Materials and methods Chemicals 3-Aminofluoranthene (3-NH2F), 1-nitropyrene, menadione (2-methyl-1,4-naphtochinon), 2-hydroxypirimidine, dicumarol, yV-methylnicotinamide and acetaldehyde were obtained from Aldrich-Chemie (FRG); hypoxanthine, allopurinol and NAD(P)H were purchased from Sigma Chemical Co. (St Louis, MO), SKF 525-A from Smith, Kline and French, Inc. (UK). All other reagents were of analytical grade. Preparation of NFs Fluoranthene (2.5 mg) dissolved in dichloromethane (DCM) was added to acetic anhydride (500 /tl) containing 100 /il concentrated nitric acid. The reaction was performed in a refrigerated (+4°C) water bath. After 1 h, 1 ml distilled water plus 500 ii\ DCM were added to reaction mixture. The organic phase was chromatographed on basic allumine (60-230 mesh) manufactured by Carlo Erba (Italy). The N-esane eluate was discarded and the DCM fraction containing NFs was concentrated and chromatographed by HPLC using an Erbasil analytical silica column (10 jjm, 0.4 x 25 cm) to separate single isomers (21,22). This procedure was repeated several times in order to obtain ample material for bioassay. Quantitative evaluation of purified isomers was performed by HRGC — FID on a gas-chromatograph Carlo Erba 5300 with a FID 40 detector, using a capillary DBS-silica column (25 m x 320 /tm x 0.3 fim). A solution containing a known amount of 1-nitropyrene was used as reference. Each isomer was analysed by El MS at 70 eV: 247 |M| + , 217 |M-N0| + , 201 |M-NO2| + , 200 |M-HNO2| + , 189 |M-NO-CO| + . A 35% yield of NF isomers was obtained by nitrating fluoranthene in these experimental conditions. The yields of each isomer were 1-NF 4.5%; 7-NF 6.0%; 3-NF 53.2% and 8-NF 36.3%. Purity of each isomer used in biological tests were: 1-NF > 99.9%; 7-NF 99.2%; 3-NF 98.5% (presence of 8-NF 1.35%) and 8-NF 99.7%. Preparation of subcellular fractions from rat liver Fresh livers from male Sprague-Dawley rats weighing 150-160 g each were perfused thoroughly with cold 0.9% NaCl and homogenized with 3 ml/g tissue of 0.15 M KC1/0.05 M Tris-HCl buffer pH 7.4. Homogenates were centrifuged for 10 min at 12 000 g and the supernatant 1 h at 105 000 g. The supernatant (S-105 fraction or cytosol) was divided into small aliquots, frozen on dry ice and stored at -80°C. The microsomal pellets were resuspended in 0.15 M KCI/0.05 M Tris-HCl buffer and centrifuged for 1 h at 105 000 g. Microsomes
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Nitrofluoranthenes (NFs) are mutagenic and carcinogenic environmental pollutants found in incomplete combustion products and urban air participate. We have studied both oxidative and reductive metabolism in vitro of different NF isomers mediated by subcellular rat liver fractions. Under aerobic conditions only ring hydroxylation of NFs by rat liver microsomes occurred and the isomeric position of the nitro group affected both the amount and the type of phenolic metabolites formed. Liver microsomes from 3-methylcholanthrene-induced rats were most effective in giving ring hydroxylated 7- and 8-nitrofluoranthene, whereas liver microsomes from phenobarbital-pretreated rats were the most active in metabolizing 1- and 3-nitrofluoranthene. Under anaerobic conditions, only reduction of NFs mediated by both cytosolic and microsomal rat liver enzymes occurred. Cofactor requirements and inhibition experiments indicated that the reductase activity in rat liver cytosolic fractions could be ascribed to DT-diaphorase, aldehyde oxidase and/or other unknown enzymes. The microsomal reductase activity was inhibited by oxygen, carbon monoxide, 2-diethylaminoethyl2,2-diphenylvalerate hydrochloride and n-octylamine, and slightly by cytochrome c; flavin mononucleotide greatly enhanced this activity. 3-Nitrofluoranthene microsomal nitroreductase activity was increased by phenobarbital rat pretreatment and this increment correlated well with the content of cytochrome P450. These results indicate a participation of cytochrome P45Q in the reductive metabolism of NFs by rat liver microsomes.
was found in environmental paniculate matter (1,7-8). Although NF genotoxicity has been studied extensively, few investigations have been made on their metabolism in mammals. It has been shown that 3-nitrofluoranthene (3-NF) and mixtures of NFs produce a significant induction of hepatic and pulmonary aryl hydrocarbon hydroxylase and 7-ethoxyresorufin-O-deethylase activities in rats (15). Khan etal. (15) demonstrated that NFs induce cytochrome P450 (cyt. P450) isozymes c and d, using highly specific monoclonal antibodies. Phenolic metabolites have been identified as products of in vitro hepatic microsomal metabolism of 2-NF and 3-NF (10). Wide species variability has been observed in the oxidative metabolism of 3-NF (16). In this study we examined some characteristics of both oxidative and reductive metabolism in vitro of 1-, 3-, 7- and 8-NF, using subcellular rat liver fractions. Knowing that microsomal cyt. P450 system and cytosolic enzymes such as DTdiaphorase, xanthine oxidase and aldehyde oxidase are involved in the reductive metabolism of some nitroaromatic compounds (17-20), we attempted to evaluate their involvement in NF reduction. We present data on induction and inhibition experiments showing the contribution of cyt. P450 form(s) in the reductive metabolism of NFs.
M.A.Belisario et at. suspended in 0.1 M sodium phosphate buffer, pH 7.4 (containing 30% v/v glycerol) were divided into small aliquots, frozen on dry ice and stored at —80°C. Microsomal and cytosolic protein determination was performed according to Lowry (23) using spectrophotometrically checked bovine serum albumin solution as standard. Quantitative evaluation of cyt. P450 in microsomes was performed according to Omura and Sato (24). In induction experiments 80 mg/kg body weight phenobarbital (PB) sodium salt was given i.p. to rats for 3 consecutive days, 40 mg/kg body weight 3-methylcholanthrene (3-MC) was given i.p. for 2 consecutive days. Animals were killed 24 h after the last injection.
Assay of NF oxidative metabolism A standard incubation mixture contained 0.1 M sodium potassium phosphate buffer (pH = 7.4), 2 mM NADPH, 10 mM MgCl2, 0.1 mM NF isomer dissolved in DMSO and 0.25 mg of microsomal proteins in a final volume of 0.5 ml. The
1-NF
Results Oxidative metabolism of NFs NFs were incubated under aerobic conditions with liver microsomes from control and induced rats. HPLC analysis of reaction products revealed no fluorescent peaks, indicating that no amino derivatives had formed. Incubation of NF isomers with liver microsomes gave metabolites tentatively identified as phenolic derivatives; we are currently characterizing them using mass spectrometry and NMR. An almost quantitative recovery of unmetabolized NF isomers from incubation mixtures was achieved with the extraction procedure described in Materials and methods. The absence of large non covalent binding of NFs to microsomal proteins was ascertained in standardization experiments using heat-inactivated microsomal proteins (unpublished data). Therefore the decrease in peak areas at the unmetabolized NF isomer's retention time allowed us to evaluate the nmoles of metabolized chemical. Figure 1 reports the time-dependent metabolization rate of NFs incubated with liver microsomes from control and induced rats. NF isomers were metabolized from uninduced rat liver microsomes to a different extent in the following order: 8-NF = 1-NF > 7-NF > 3-NF. Treatment of rats with the inducers 3-MC and PB produced different patterns of NF metabolization rates: 7-NF and 8-NF were better transformed by 3-MC-induced
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Fig. 1. Time-dependent metabolization rates of NF isomers under aerobic conditions. Each isomer (0.1 mM) was incubated with liver microsomes (0.25 mg) from control (O), PB (A)- or 3-MC (O)-induced rats, in the presence of NADPH. The extraction procedure of unmetabolized NFs and metabolites and HPLC anlaysis were performed as described in Materials and methods.
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Assay of NF reduction A typical reaction mixture (final volume 0.5 ml) contained 0.1 M sodium potassium phosphate buffer (pH 7.4), different amounts of microsomal or cytosolic proteins, 2 mM NAD(P)H or other cofactors (hypoxanthine, acetaldehyde, etc.), 10 raM MgCl2 and 0.1 mM NF isomer dissolved in dimethylsulphoxide (DMSO). Incubation tubes were sealed with rubber stoppers and all mixtures except NAD(P)H were bubbled for 2 min with nitrogen gas passed through a deoxygenizer system. When necessary, carbon monoxide gas was used instead of nitrogen gas. Reactions were carried out in duplicate at 37°C under a nitrogen atmosphere in a water shaking bath and stopped with an equal volume of cold ethanol just before addition of substrate (time 0) or after different times of incubation. Mixtures were extracted three times with toluene:ethylacetate (1:1 v/v); the organic phase was evaporated under a nitrogen stream, dried residues were dissolved in methanol and analysed by HPLC. Analyses were performed on a Varian liquid chromatograph (Varian Instruments, Co) using a varian Qg Amino Tag analytical column (0.46 x 15 cm). A constant flow of 1.0 ml/min and a 15-min linear gradient from 70 to 100% methanol in water was used. Peaks were monitored simultaneously by UV absorbance at 254 nm and fluorescence (X exc. = 340 nm, X emiss. = 520 nm) and areas were calculated by Vista printer plotter (Varian Instruments). .
reaction was started by adding NADPH solution. Incubation was carried out in duplicate tubes at 37°C in a water shaking bath and stopped at different times with an equal volume of cold ethanol. The mixtures were extracted and the products formed were analysed by the procedure described above.
Oxidative and reductive metabolism of NFs by liver enzymes
rat liver microsomes while 1-NF and 3-NF were better metabolized by PB-induced rat liver microsomes. Moreover both 3-MC- and PB-induced microsomes showed a higher affinity for 8-NF than for the other isomers. We noted not only quantitative but also qualitative differences between liver microsomes from control and variously induced rats used as metabolizing systems, as shown in Figure 2. Control and PB-induced microsomes formed a greater amount of 3-NF metabolites corresponding to peaks a and d, while with 3-MC microsomes the metabolite related to peak c became the most abundant. Analogous differences were observed when the other isomers were used as substrates of variously induced rat liver microsomes (data not shown).
E Table I. Nitroreduction rates of NF isomers mediated by subcellular liver fractions from uninduced rats, in the presence of NADPH SO CM
NF amino-derivative formed (nmol/mg protein/min)
Compound
Microsomes8
Cytosola
