Main pathway for mutagenic activation of 2-acetylaminofluorene by guinea pig liver homogenates.

The activation pathway of 2-acetylaminofluorene (AAF) to N-hydroxy-2-amino-fluorene (N-OH-AF), a potent mutagen to $\text{Salmonella}$, by guinea pig liver postmitochondrial supernatant fraction (S-9 fraction) was studied. 2-Aminofluorene (AF), as well as N-hydroxy-2-acetylaminofluorene (N-OH-AAF, Takeishi et al., Mutation Res. in press), was detected as a metabolite of AAF. The mutagenicities of AF and N-OH-AAF comparable to that of AAF were inhibited by antiserum against NADPH-cytochrome $\text{c}$ reductase and by paraoxon, respectively. These data indicate that in the mutagenic activation of AAF, N-OH-AF can be produced by both N-hydroxylation of AF and deacetylation of N-OH-AAF. Furthermore, the data on the relative contribution of paraoxon-sensitive activation pathway to mutagenicities of AAF and N-OH-AAF led to a conclusion that deacetylation of AAF followed by N-hydroxylation to produce N-OH-AF is the main pathway for the mutagenic activation of AAF by guinea pig liver S-9 fraction.     

Modulation of genotoxic and cytotoxic effects of aromatic amines in monolayers of rat hepatocytes

Cultured rat hepatocytes exposed to 2-acetylaminofl uorene (AAF), 2-aminofl uorene (AF) or N-hydroxy-2-acetylaminofluorene (N-OH-AFF) for 3 hrs resulted in an increase in DNA repair measured as unscheduled DNA synthesis, with N-OH-AAF > AAF > AF. Cytotoxic effects were only seen with N-OH-AAF above 10^–6 M. -Naphthof avone increased the unscheduled DNA synthesis and cytotoxic effects of N-OH-AAF, whereas it decreased DNA repair and the covalent binding of AAF to cellular proteins. In contrast, very little effects of paraoxon were seen on the repair synthesis elicited by AAF, AF or N-OH-AAF. The addition of ascorbate reduced the covalent binding of AAF, the DNA repair synthesis caused by AAF and N-OH-AAF, and the cytotoxic effects of N-OH-AAF. The addition of pentachlorophenol or salicylamide all resulted in similar effects as ascorbate, through reduction of sulfation. Galactosamine, an inhibitor of glucuronidation, and the nucleophile GSH caused no or only minor effects of the activation of AAF, AF or N-OH-AAF as judged from the endpoints tested. These results are consistent with an arylnitrenium ion, a sulfate ester or a free radical as the arylamine metabolite causing cellular DNA damage, whereas the sulfate ester or a radical intermediate may be responsible for the cytotoxic effects of N-OH-AAF.Abbreviations AAF 2-acetylaminofluorene - AF 2-aminofluorene - N-OH-AAF N-hydroxy-2-acetylaminofluorene - cytochrome P-450 a collective term for all forms of the cytochrome P-450 polysubstrate monooxygenase - DMSO dimethyl sulfoxide - HU hydroxyurea - S-9 9000 g supernatants - LDH lactate dehydrogenase - UDS unscheduled DNA synthesis - ANF -naphthoflavone - GSH glutathione - PCP pentachlorophenol - MET metyrapone - PAR paraoxon - DEM dimethylmaleate     

Mutagenicity-enhancing effect of quercetin on the active metabolites of 2-acetylaminofluorene with mammalian metabolic activation systems

The effects of quercetin on the mutagenicity of 2-acetylaminofluorene (AAF) and its 3 active metabolites, N-hydroxy-AAF (N-OH-AAF), aminofluorene (AF) and N-acetoxy-AAF(N-OAc-AAF) were investigated. The mutagenicity assays were carried out with Salmonella typhimurium TA98, and S9, microsomes and cytosol were used as metabolic activation systems. In the presence of S9, quercetin enhanced the mutagenicity of AAF, N-OH-AAF, AF and N-OAc-AAF by 6.9-, 4.3-, 3.6- and 3.9-fold, respectively. Quercetin enhanced the mutagenicity of these substrates with microsomes, whereas it depressed the mutagenicity of these substrates with cytosol. From these results, it seemed probable that quercetin promotes the N-hydroxylation and deacetylation in the microsomes, whereas it inhibits the deacetylation in the cytosol. It was shown that in the metabolism of AAF and its metabolites, quercetin modulates the balance between the mutagenicity activation and inactivation processes, which is catalysed by the enzymes in the microsomes and cytosol, and causes enhancement of the mutagenicity of AAF.     

Bacterial mutagenesis and host cell DNA damage by chemical carcinogens in the Salmonella/hepatocyte system

Coincubation of isolated and intact rat hepatocytes and Salmonella typhimurium, (Salmonella/hepatocyte system) strain TA 98 was employed to determine both bacterial mutagenicity and DNA damage in the hepatocytes as measured by alkaline elution, following treatment with 2-acetylaminofluorene (AAF), 2-aminofluorene (AF) and N-hydroxy-2-acetylaminofluorene (N-OH-AAF). Both the mutagenicity and the rate of DNA elution were dose-dependent for all three compounds. N-OH-AAF was 5 times more mutagenic and caused 80–100 times more DNA damage in the hepatocytes than AAF and AF when compared on a molar basis. The Salmonella/hepatocyte system may provide a more comprehensive evaluation of the potential genotoxic effect of chemicals than the currently used microbial mutagenesis sytems.     

Effects of harman and norharman on the metabolism and genotoxicity of 2-acetylaminofluorene in cultured rat hepatocytes

Monolayers of rat hepatocytes metabolize 0.25 m M 2-acetylaminofluorene (AAF) to various ether-extractable, water-soluble as well as covalently bound products. The major ether-extractable metabolite formed is 2-aminofuorene (AF), followed by 7-OH-AAF and 9-OH-AAF. Pretreatment of rats with the inducer Aroclor 1254 (PCB) increased the metabolism of AAF and caused an increased DNA repair synthesis in hepatocytes exposed to AAF or AF. With N-OH-AAF, a decreased genotoxic response in PCB-treated cells compared to control cells was seen. The addition of harman and norharman decreased the metabolism of AAF to ether-extractable metabolites, water-soluble metabolites and metabolites covalently bound to macromolecules. In contrast, the DNA-repair synthesis caused by the same concentrations of AAF was increased by harman. One explanation for this apparent discrepancy could be that the aromatic amines changed the metabolism of harman and norharman in such a way that these compounds were converted into genotoxic metabolites.Abbreviations AAF 2-acetylaminofluorene - AF 2-aminofluorene - DMSO dimethylsulfoxide - HPLC high performance liquid chromatography - N-OH-AAF N-ydroxy-2-acetylaminofluorene - PCB polychlorinated biphenyls, Aroclor 1254 - TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin - TdR thymidine - Trp-P-1 3-amino-1,4dimethyl-5H-pyrido(4,3b)indole - Trp-P-2 3-amino-l-methyl-5H-pyrido(4,3b)indole - UDS unscheduled DNA synthesis     

The importance of 2-aminofluorene in the mutagenic activation of 2-acetylaminofluorene

The mutagenic activation of 2-acetylaminofluorene (AAF) and its derivatives N-hydroxy-AAF and 2-aminofluorene (AF) by pulmonary and hepatic microsomal fractions from untreated rabbits was investigated using Salmonella strain TA98. The mutagenicity of AAF in the presence of hepatic microsomes followed typical saturation kinetics. However, in the presence of pulmonary microsomes, the mutagenic activity increased linearly with increasing substrate concentration and approximated that obtained with low concentrations of AF. N-Hydroxy-AAF was 1/10th as mutagenic as AF in the presence of pulmonary microsomes, but 2-2.5 times more mutagenic than AF in the presence of hepatic microsomes. The activation of AAF by both fractions was completely inhibited by the deacetylase inhibitor paraoxon. Although AAF does not appear to be a substrate for cytochrome P450 form 5, antibodies to this form inhibited the activation of AAF by pulmonary and hepatic microsomes by 90% and 60%, respectively. These results indicate that the mutagenic activation of AAF by these fractions primarily involves deacetylation to AF, followed by cytochrome P450 form 5-mediated activation of AF.     

Induction of -2 frameshift mutations by 2-nitrofluorene, N-hydroxyacetylaminofluorene, and N-2-acetylaminofluorene in reversion assays in Escherichia coli strains differing in permeability and acetyltransferase activity

The mutagenicity of 2-nitrofluorene (NF), N-hydroxyacetylaminofluorene (N-OH-AAF), and N-2-acetylaminofluorene (AAF) was measured in strains of Escherichia coli that contain a lacZ allele that reverts by -2 frameshift mutations from CG(5) to CG(4). Mutagenesis was compared in a strain having wild-type permeability and metabolism, a strain with increased permeability caused by a lipopolysaccharide-defective (LPS(d)) mutation, a strain with N- and O-acetyltransferase (NAT/OAT) activity conferred by the Salmonella nat gene on plasmid pYG219, and a strain carrying both an LPS(d) mutation and pYG219. The LPS(d) mutation facilitated the measurement of mutagenicity but was not absolutely required, in that lower levels of mutagenicity were detected in LPS(+) strains. The NAT/OAT activity conferred by pYG219 strongly potentiated the mutagenicity of NF and N-OH-AAF. Surprisingly, AAF was mutagenic in the NAT/OAT LPS(d) strain without an exogenous P450 metabolic activation system. Its activity may be ascribable to the detection of a directly mutagenic impurity by the highly sensitive strain or to a low level of metabolic activation by the bacteria under the assay conditions. The findings add to the evidence that the lacZ allele derived from E. coli strain CC109 is an effective indicator of -2 frameshift mutagenesis and that strains expressing high levels of NAT/OAT activity are highly sensitive in monitoring the mutagenicity of nitroarenes and aromatic amides.     

The effect of quercetin, a mutagenicity-enhancing agent, on the metabolism of 2-acetylaminofluorene with mammalian metabolic activation systems

The effect of quercetin as the comutagen on 2-acetylaminofluorene (AAF) was investigated. AAF was metabolized with mammalian metabolic systems (S9 mix) in the presence or absence of quercetin in vitro, and its metabolites were determined by high-performance liquid chromatography. In the presence of quercetin, the total metabolic rate of AAF decreased compared with that in the absence of quercetin, whereas the formation of N-hydroxy-AAF (N-OH-AAF) and 2-aminofluorene (AF) increased. Since the main metabolic pathway of AAF is aryl-hydroxylation, it is suggested that the decrease of total metabolic rate of AAF is due to the inhibition of aryl-hydroxylation by quercetin. From these results, it seems probable that the comutagenic effect of quercetin on AAF is due to the inhibition of aryl-hydroxylation (the detoxifying pathway) and the promotion of N-hydroxylation and deacetylation (the activating pathway) in the AAF metabolism with S9 mix.     

Peptization of the Gel of Konjac Mannan

The addition of 1,8-pyrenequinone into the assay system containing rat liver homogenates (S-9) caused an approximately 10-fold increase in the mutagenicity of 2-acetylaminofluorene (AAF) in the current Salmonella reversion assay system. Since no chemical reaction between 1,8-pyrenequinone and AAF was observed, the in vitro effects of 1,8-pyrenequinone on the metabolisms of AAF with S-9 mix were studied. The enhancement of mutagenicity by 1,8-pyrenequinone was not dependent on the dose of NADPH under the present assay condition. The mutagenicity of AAF was increased approximately 4-fold by the addition of 1,8-pyrenequinone into microsomes, whereas it remained at the spontaneous level in the presence of cytosol. However, by reconstituting microsomes with cytosol, the mutagenicity enhancing activity was recovered to the original level. Since 1,8-pyrenequinone inhibited the AAF hydroxylase activity, chemical analysis of the incubation mixture of AAF was tried. This indicated that a higher amount of unmetabolized AAF remained and higher amounts of 2-aminofluorene and N-hydroxy-2-acetylaminofluorene were accumulated in the presence of 1,8-pyrenequinone compared with those in the absence of 1,8-pyrenequinone. From these results, it seems probable that 1,8-pyrenequinone inhibits C-hydroxylation (the detoxifying pathway) and promotes N-hydroxylation (the activating pathway) as well as deacetylation in the AAF metabolism.     

Differential effects induced by N-hydroxy-2-acetylaminofluorene and N-hydroxy-2-aminofluorene in DNA excision-repair-deficient Chinese hamster cells

The direct-acting cytotoxic properties of N-hydroxy-2-acetylaminofluorene (N-OH-AAF) and N-hydroxy-2-aminofluorene (N-OH-AF) have been determined in repair-proficient (AA8-4) and repair-deficient (UV-5) Chinese hamster ovary cells. Cytotoxicity comparisons indicate that UV-5 cells are considerably more sensitive to exposure to N-OH-AAF than is the parental AA8-4 cell line, i.e., concentrations needed to obtain a D37 for survival of AA8-4 is greater than 5-fold higher than for UV-5. Mutation analysis at the HGPRT locus also indicates the increased sensitivity of UV-5 cells to N-OH-AAF as witnessed by an enhanced induction of 6-thioguanine-resistant colonies at equitoxic doses. Conversely, N-OH-AAF, did not induce a 'UV-mimetic' response when comparing genotoxicity between these two cell lines. Our data coupled with previously published model-building and adduct removal studies (Broyde and Hingerty, 1983; Fuchs and Daune, 1974; Grunberger and Weinstein, 1976; Yamasaki et al., 1977) suggest that the minor DNA adduct species, N-(2'-deoxyguanosin-8-yl)-2-acetylaminofluorene, may be responsible for the hypermutagenicity witnessed in DNA excision-repair-deficient cells treated with N-OH-AAF.     

Glutathione and glutathione S-transferases in the salmonella mammalian-microsome mutagenicity test

Levels of the tripeptide glutathione (GSH) and the activity of glutathione S-transferases were investigated in S9 fractions of rats and mice and in Salmonella typhymurium tester strains TA1535, TA100, TA1538 and TA98. The S9 and Salmonella typhimurium tester strains had high levels of glutathione. Compared with S9, the activity of GSH S-transferases was lower in the bacteria. However, electrophiles such as 1-chloro-2,4-dinitrobenzene (CDNB), diethyl maleate and styrene oxide were effectively bound to bacterial GSH.

The mutagenicity of the direct mutagen CDNB was drastically lowered in presence of S9 fractions but not in presence of microsomes. A comparable decrease was obtained when microsomal supernatant, which contains GSH and GSH S-transferases, was added to the microsomes. Addition of GSH in excess completely abolished mutagenicity of CDNB. These results demonstrate that the conjugation of electrophiles with GSH mediated by the S9 fraction or the bacterial tester strains represents an important detoxication mechanism which may influence the results obtained with the Salmonella typhimurium mammalian-microsome mutagenicity test.     

A comparison of the inhibition of deacetylase in primary cultures of rat and human hepatocytes effecting metabolism and DNA-binding of 2-acetylaminofluorene

The metabolism of 2-acetylaminofluorene (AAF) in primary cultures of rat and human hepatocytes was investigated to determine if the activation of this well-studied chemical carcinogen proceeds via similar routes of metabolism between species. The total level of AAF metabolite(s) bound to hepatocellular DNA was determined in the presence of deacetylase inhibitors, diethyl(p-nitrophenyl) phosphate (paraoxon) or bis(p-nitrophenyl) phosphate (BPNPP). These compounds are known to inhibit deacetylase and to decrease the mutagenicity of AAF. Experiments with rat and human hepatocytes demonstrated inhibition in the deacetylation of AAF (5×10⁻⁴ M) with paraoxon or BPNPP. The BPNPP (5×10⁻⁴ M inhibited 99% of the AF formation in the human hepatocytes and 88% inhibition in the rat hepatocytes. Paraoxon at 10⁻⁴ M demonstrated a 98% inhibition of deacetylation with humans and a 92% inhibition with rats. The rat hepatocytes also showed a 53% decrease in DNA binding in the presence of paraoxon. In contrast with human hepatocytes, while paraoxon decreased the AF metabolite by > 97%, there was no change in total DNA binding.     

Catechin inhibition of mutagenesis and alteration of DNA binding of 2-acetyl-aminofluorene in rat hepatocytes

Bioflavonoids are naturally occurring plant products that have demonstrated inhibitory effects on chemically induced carcinogenesis or mutagenesis. The chemoprotective effects are either direct scavenging of reactive molecules or indirect effects, such as enzyme activity alteration. Exposure of cultures of isolated rat hepatocytes to catechin (0.01-1.0 mM), a plant phenolic flavonoid, and subsequent addition of 2-acetylaminofluorene (AAF) resulted in an enhanced binding of AAF metabolites to hepatocellular DNA. Incubations of hepatocytes with catechin and S. typhimurium demonstrated no mutagenicity of catechin. At 1.0 and 5.0 mM concentrations of catechin with AAF and 30-min incubation with hepatocytes prior to plating there was inhibition of AAF-induced mutagenicity. However, at 0.5 mM of catechin there was a significant enhancement in mutagenicity. The increase in DNA binding of AAF in the cultures of hepatocytes is due to the alteration of metabolism by exposure to catechin. Catechin increases both N-hydroxylation and deacetylation pathways in the hepatocytes producing increases in N-hydroxy-AAF and aminofluorene. Both of these metabolites are important in AAF intermediates binding with DNA. The short-term incubation of catechin, AAF, hepatocytes, and S. typhimurium in the mutagenesis assay is not sufficient for induction of metabolic pathways. However, previously reported inhibition of detoxification pathways and/or scavenging of the proximate carcinogen can occur to alter mutagenesis in a dose-dependent manner.     

The effect of quercetin on the mutagenicity of 2-acetylaminofluorene and benzo[alpha]pyrene in Salmonella typhimurium strains

The comutagenic and desmutagenic effect of quercetin on the mutagenicity of typical mutagens e.g. 2-acetylaminofluorene (AAF), 4-nitroquinoline-1-oxide (4NQO) and benzo[alpha]pyrene (B[a]P), in Salmonella typhimurium TA98, TA100 and TA98/1,8 DNP6 were examined. In the mixed application of AAF with quercetin in the presence of mammalian metabolic activation system (S9 mix), the numbers of revertants in TA98 increased by as much 2.2-5.0-fold compared with the sum of those in the separate applications of AAF and quercetin. A 1.4-2.7-fold increase was observed in TA100. Quercetin did not affect the mutagenicity of 4NQO, and depressed that of B[a]P. Dose-response curves for mutagenicity of quercetin with or without AAF (5 micrograms/plate) were examined. The results suggest that quercetin, present in a molarity of up to 1.5 times that of AAF, is apparently effective in enhancing the mutagenicity of AAF, because a linear dose-response curve was observed in the range of 0-5 micrograms/plate quercetin with AAF although quercetin alone was not mutagenic in the same range. Dose-response curves for mutagenicity of quercetin with or without 5 micrograms/plate B[a]P did not increase compared with that for quercetin alone. The mutagenicity of the mixed application of B[a]P with quercetin was reduced to about 60% of the sum of separate application at doses ranging from 25 to 100 micrograms/plate of quercetin. Since enhancement and depression of mutagenicity by quercetin were observed for indirect mutagens, AAF and B[a]P, respectively, in the presence of S9 mix, quercetin may affect the metabolic pathway of these mutagens.     

Mutagenic activity of selected aromatic amines and polycyclic hydrocarbons in Drosophila melanogaster

With the use of a series of wild-type and repair-deficient strains and appropriate application procedures, it is possible to demonstrate that carcinogenic aromatic amines and polycyclic hydrocarbons are mutagens in Drosophila. We have shown evidence that AAF, N-OH-AAF, AcO-AAF, BP, DAS and DMBA produce recessive lethals when fed to or injected into adult males. Mutagenic activity was also observed when male larvae were exposed to AAF, BP, DMBA, 3-MC or NA. DA was not mutagenic in the recessive lethal assay under the conditions of the test. DMBA can now be considered as a potent mutagen for Drosophila, although demonstration of its activity depends upon the choice of the treatment procedure and the strain selected. One of the questions concerning the action of aromatic amines and polycyclic hydrocarbons is how their genetic effectiveness in Drosophila can be enhanced. The observation that none of several enzyme inducers (PB, BF, AC, 3-MC) increased their mutagenicity may be interpreted in terms of a more efficient metabolic activation or deactivation. This assumes that active metabolite(s) did not reach the testis in doses sufficient for mutation induction. It also appears that, since the problems pertaining to mutagenicity in Drosophila of aromatic hydrocarbons are obviously a matter of metabolism, the use of repair-deficient strains is no longer an attractive proposal for their elucidation. The present investigation shows that, with weak mutagens, usage of strains mei-9Li or y mei-9a mei-4lD5 does not improve the sensitivity of the recessive lethal method or the test for chromosomal loss. As an alternative, in our opinion more attention should be devoted to possible differences in metabolism between somatic and gonadal tissue. We feel strongly that somatic assay systems might be particularly valuable as a complement to recessive lethal tests on the germ line.     

Modulation of the cytotoxicity and mutagenicity of benzo[a]pyrene and benzo[a]pyrene 7,8-diol by glutathione and glutathione S-transferases in mammalian cells (CHO/HGPRT assay)

Biologically reactive metabolites of benzo[a]pyrene (BP) and benzo[a]-pyrene 7,8-diol (BP-diol), formed by the mixed-function oxidase (MFO) system, are substrates for conjugation and detoxication by glutathione (GSH) when catalyzed by glutathione S-transferases (GSHT). We have investigated the detoxication of BP- and BP-diol-induced cytotoxicity and mutagenicity with GSH by supplementing the S9 mix used in the Chinese hamster ovary cells/hypoxanthine-guanine phosphoribosyltransferase (CHO/HGPRT) assay with GSH (6.5 mM) or GSH plus GSHT. The addition of GSH to the S9 mix resulted in a reduction of BP- and BP-diol induced cytotoxicity. GSH plus GSHT eliminated BP-induced cytotoxicity and reduced the mutagenicity of BP. GSH inhibited the mutagenicity at low (essentially non-lethal) concentrations of BP-diol, but did not do so at toxic concentrations. GSH plus GSHT inhibited the cytotoxicity and mutagenicity of BP-diol at concentrations not affected by GSH alone. These studies indicate that biochemical mechanisms of detoxication can affect the biological activity of a carcinogen, such as BP or BP-diol as profoundly as bioactivation by the MFO system.     

Studies on liver mitochondrial 5′-endonuclease activity in rats fed carcinogenic amines and on their binding to mitochondrial proteins

Mitochondrial 5′-endonuclease activity has been determined at regular time intervals in the livers of rats fed a diet containing 0.09% 2-aminofluorene (AF), 0.09% 2-acetylaminofluorene (AAF) or 0.06% N,N-dimethyl-4-aminoazobenzene (DAB). The results obtained indicate that the 5'-endonuclease activity was not affected significantly.The quantity of AF, AAF or DAB bound to liver homogenate and mitochondrial fraction proteins has also been measured at regular time intervals. The amount of AF and AAF bound to homogenate proteins after 4 weeks of carcinogen feeding is about 60-fold higher than that of DAB. The binding of the AF compounds to the mitochondrial fraction proteins is comparatively more important, reaching a level 300-fold higher than that of DAB. The amount of AF residues bound per mg of mitochondrial fraction proteins is higher than that of the homogenate while that of rats fed DAB is smaller. The present results suggest that no relation can be established between the total amount of these carcinogens bound to liver cellular proteins in vivo and their potential carcinogenic effect.     

Inhibition of acetyl-coenzyme A dependent activation of N-hydroxyarylamines by phenolic compounds, pentachlorophenol and 1-nitro-2-naphthol

Pentachlorophenol (PCP) and 1-nitro-2-naphthol were found to be potent inhibitors of enzymatic acetyl-CoA dependent activation, which is suggested as proceeding through direct O-acetylation, of N-hydroxyarylamines to tRNA binding by liver cytosolic enzymes from hamsters and rats. IC₅₀ values of PCP for the activation of 2-hydroxyamino-6-methyldipyrido[1,2-a:3′,2′-d]imidazole (N-OH-Glu-P-1), 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (N-OH-Trp-P-2) and N-hydroxy-2-aminofluorene (N-OH-AF) were 20, 25 and 17 μM, respectively, in hamster cytosol system. Similar inhibition was observed with rat liver cytosol (IC₅₀ values of PCP and 1-nitro-2-naphthol were 13 and 12 μM, respectively, for the binding of N-OH-Glu-P-1). PCP is known as an inhibitor of sulfotransferase; however, another potent inhibitor of sulfotransferase, 2,6-dichloro-4-nitrophenol, did not inhibit the acetyl-CoA dependent binding. Antibiotic thiolactomycin, which inhibits bacterial O-acetyltransferase, did not affect the activation by hamster and rat cytosol, indicating the difference in property between bacterial and mammalian enzymes. The kinetic data obtained with hamster cytosol suggested the competitive inhibition of PCP with substrate, N-OH-Glu-P-1, and non-competitive inhibition with acetyl-CoA. In addition to the O-acetylation, PCP and 1-nitro-2-naphthol also inhibited N-acetylation of arylamines and N, O-acetyltransfer reaction of N-hydroxy-2-acetylaminofluorene (N-OH-AAF) by hamster cytosol. IC₅₀ values for these two types of acetyltransfer reactions, however, were slightly higher than those observed for acetyl-CoA dependent activations of N-hydroxyarylamines.     

Oxidative transformation of 2-acetylaminofluorene by a chemical model for cytochrome P450: A water-insoluble porphyrin and tert-butyl hydroperoxide

Oxidation of 2-acetylaminofluorene (AAF), a carcinogen, by a chemical model for cytochrome P450 was investigated to identify an active mutagen and elucidate the oxidation pathway. The oxidation system consisted of a water-insoluble tetrakis(pentafluorophenyl)porphyrinatoiron(III) chloride and tert-butyl hydroperoxide. The mutagen derived from AAF by the chemical model was 2-nitro-9-fluorenone (NO(2)=FO), which was mutagenic in Salmonella typhimurium TA1538. AAF was oxidized initially at position 9 of the fluorene carbon by the chemical model forming 2-acetylamino-9-fluorenol (AAF-OH), and then oxidized further to 2-acetylamino-9-fluorenone (AAF=O) as a major product. Initial oxidation of the nitrogen formed 2-nitrofluorene (NO(2)F), and further oxidation yielded 2-nitro-9-fluorenol (NO(2)F-OH) as a minor product. These products, AAF-OH, AAF=O, NO(2)F, and NO(2)F-OH, and their presumable common intermediate, N-hydroxy-2-acetylaminofluorene, were oxidized by the chemical model, and the formation of NO(2)F=O was determined. These results showed that NO(2)F=O was the mutagen derived from AAF in the presence of the chemical model and was formed via oxidation of N-OH-AAF, NO(2)F, and NO(2)F-OH. These results may lead to a new metabolic pathway of AAF.