Mutagenicity of benzidine and 4-aminobiphenyl after metabolic activation with isolated hepatocytes and liver 9000 × g supernatant from rat,hamster and guinea pig

The mutagenicity of benzidine and 4-aminobiphenyl towards Salmonella typhimurium strain TA1538 was measured in the presence of isolated hepatocytes from rat, hamster and guinea pig. The mutagenic potency of these compounds was also assayed with S9 (9000 × g supernatant) prepared from disrupted hepatocytes of these aryl amines was investigated.For all 3 animal species it was found that the mutagenicity of benzidine is higher with intact hepatocytes than with S9 prepared from disrupted hepatocytes. Addition of acetyl coenzyme A to the S9 fraction increased the mutagenicity of benzidine. In contrast to benzidine, the mutagenicity of 4-aminobiphenyl appeared to be lower with hepatocytes than with S9. Addition of acetyl coenzyme A to the S9 fraction decreased the mutagenicity of 4-aminobiphenyl.The mutagenic potency of 4-aminobiphenyl was almost equal in the presence of the liver preparations from the 3 different species, whereas obvious species differences were seen with benzidine.     

Mutagenicity towards Salmonella typhimurium of some known genotoxic agents, activated by isolated hepatocytes of monkey (Macaca fascicularis). Comparison with isolated human hepatocytes

This paper describes some striking differences between isolated human and monkey hepatocytes in their capacity to activate some known genotoxic agents into products mutagenic towards Salmonella typhimurium. Isolated monkey hepatocytes, in contrast to human hepatocytes, appeared to activate benzidine (BZ), N-acetylbenzidine (MABZ), N,N'-diacetylbenzidine (DABZ), 2-aminofluorene (2-AF) and 2-acetylaminofluorene (2-AAF) poorly. With monkey hepatocytes BZ was slightly more mutagenic than DABZ, whereas with human hepatocytes DABZ was more active than BZ. N-Nitrosodimethylamine (DMN) and N-nitrosodiethylamine (DEN) were also found to be poorly mutagenic when activated by monkey hepatocytes, unlike the human hepatocytes. However, the polycyclic arylhydrocarbons benzo[a]pyrene (B[a]P) and 7,12-dimethylbenzanthracene (7,12-DMBA) were highly active in the presence of monkey hepatocytes, unlike the human hepatocytes. A metabolic study showed that monkey liver preparations seem to possess a higher monooxygenase activity towards B[a]P than human liver preparations.     

Microsome- and hepatocyte-mediated mutagenicity of hydroxyurea and related aliphatic hydroxamic acids in V79 Chinese hamster cells

The potential of N-hydroxyurea to induce gene mutations in V79 Chinese hamster cells was investigated. Upon metabolic activation by liver microsomes from phenobarbital-treated rats or by isolated rat hepatocytes co-cultured with the V79 cells, hydroxyurea caused a concentration-dependent increase in the frequency of HGPRT-deficient mutants. Hydroxyurea was not mutagenic in the absence of metabolic activation. Addition of catalase inhibited microsome-mediated mutagenicity, indicating that hydrogen peroxide was involved in the formation of the mutagenic DNA lesion. Acetohydroxamic acid and N-hydroxyurethane also induced hepatocyte-mediated mutagenicity, suggesting that the potential to elicit metabolism-dependent mutagenicity may be a common property of aliphatic hydroxamic acids.     

Metabolic activation of polycyclic aromatic hydrocarbons to mutagens in the Ames test by various animal species including man

6 polycyclic aromatic hydrocarbons were assayed for mutagenicity in the Ames test, in the presence of hepatic post-mitochondrial preparations isolated from the mouse, rat, hamster, pig and man. Benzo[a]pyrene, dibenzo[a,i]pyrene and benz[a]anthracene gave a positive mutagenic response only in the presence of activation systems derived from the hamster. With the exception of the pig, activation systems derived from all animal species could convert 3-methylcholanthrene to mutagens, the hamster being the most efficient. With the exception of the rat and pig, all animal species activated 7,12-dimethylbenz[a]-anthracene to mutagens, the human preparation being the most effective followed by the hamster and mouse. Dibenz[a,h]anthracene was not activated by any of the hepatic preparations. It is concluded that, among the animal species studied the hamster is generally the most efficient in activating polycyclic aromatic hydrocarbons to mutagens in the Ames test.     

Application of modified Salmonella/microsome prescreen to petroleum-derived complex mixtures and polynuclear aromatic hydrocarbons (PAH)

In some cases, the Salmonella mutagenicity assay may fail to predict the carcinogenic potential of PAH (and of complex mixtures containing PAH) because of nonoptimal in vitro metabolic activation parameters. In this study, 7 petroleum-derived complex mixtures, as well as a number of individual PAH which were representative constituents of such mixtures, were tested in a Salmonella prescreen using quadrant plates with rat or hamster S9 at concentrations approximately 2-8 times those used in the standard assay. Some PAH (perylene, quinoline, benzo[b]chrysene, phenanthrene, anthracene) were optimally activated to mutagens by S9 at 400 microliters/plate. Rat S9 was similar to hamster S9 for most tested PAH, but anthracene and quinoline mutagenicity was enhanced by hamster S9. All 7 complex mixtures were more mutagenic with 200-400 microliters/plate S9; rat was generally slightly more efficient than hamster. Modifying this assay to include a prescreen using a range of S9 concentrations (and perhaps from species other than rat) may improve prediction of the potential carcinogenicity of complex petroleum-derived mixtures.     

Activation of mutagens by hepatocytes and liver 9000 X g supernatant from human origin in the Salmonella typhimurium mutagenicity assay. Comparison with rat liver preparations

The mutagenicity of 10 known genotoxic compounds, of several chemical classes, was measured in Salmonella typhimurium mutagenicity assays comprising isolated human hepatocytes or human liver 9000 X g supernatant (S9) from 4 different individuals, as activating system. The mutagenic activity of several compounds as determined with the Salmonella/hepatocyte suspension assay showed obvious differences when compared with the values obtained in the Salmonella/S9 plate assay. For instance, the mutagenic activity of BZ, DMN and DEN appeared to be much higher in the hepatocyte assay than in the S9 assay. However, 2-AF and 2-AAF were activated more effectively into mutagens in the S9 assay than in the hepatocyte assay. 2-AF was slightly more mutagenic than 2-AAF in the hepatocyte assay, whereas it was far more mutagenic than 2-AAF in the S9 assay. DMN was found more mutagenic than DEN in the hepatocyte assay, whereas in the S9 assay DEN appeared to be slightly more mutagenic. Furthermore, great interindividual differences in the metabolic activation of certain compounds, e.g. BZ and DMN, were observed in the hepatocyte suspension assay, whereas these variations were less evident in the S9 plate assay. Comparison of the mutagenicity data obtained with the human liver preparations, with those obtained with rat liver preparations, showed great interspecies differences in the capacity to activate certain chemicals into mutagens. The use of human liver preparations, in particular isolated human hepatocytes, may be of great value in studies on inter- and intraspecies variations in metabolic activation of genotoxic agents.     

Evaluation of genotoxicity of N-nitrosodibenzylamine in Chinese hamster V79 cells and in Salmonella

Health concerns have arisen due to the formation of N-nitrosodibenzylamine (NDBzA; CAS No. 5336-53-8) in pork processed in a new type of rubber netting. In view of the potent carcinogenicity of related nitrosamines (e.g. N-nitroso-n-dibutylamine and N-nitrosodiethylamine), NDBzA was evaluated for genotoxicity in vitro in both Chinese hamster V79 cells and in Salmonella. In V79 cells, concentrations up to 25 micrograms/ml were tested with and without activation by rat or hamster hepatocytes. Significant elevation of SCE frequency was seen only at 25 micrograms/ml in the presence of uninduced hamster hepatocytes. Mutation to 6-thioguanine resistance was observed at 25 micrograms/ml, in the absence of hepatocytes and in the presence of induced (Aroclor 1254) or uninduced hamster hepatocytes, but not with rat hepatocytes. With uninduced rat hepatocytes, a small but significant (p less than 0.05) increase in the mutation frequency was seen with 10 micrograms/ml NDBzA. In the Salmonella assay, using a pre-incubation protocol and concentrations up to 1000 micrograms/ml, NDBzA was negative in strain TA98, and in TA100 with rat S9, but was positive at the highest dose in TA100 with hamster S9, and more strongly with Aroclor 1254-induced hamster S9. When activated by uninduced rat or hamster hepatocytes, as opposed to S9, NDBzA was negative with all tester strains. Hamster hepatocytes activated more than rat in the V79 studies, and hamster S9 was more strongly activating in the Salmonella assay. These results indicate that NDBzA is weakly mutagenic to both Salmonella and V79 cells.     

Mutagenicity of the coccidiostat diaveridine in the Salmonella/mammalian microsome assay

The coccidiostat diaveridine was tested for mutagenicity in the Salmonella/microsome assay with tester strains TA100 and TA98. This compound was not mutagenic in either tester strain in the presence and absence of rat S9 mix, but was found to be mutagenic in strain TA100 after metabolic activation with hamster S9 mix.     

The genotoxicity of lucidin,a natural component of Rubia tinctorum L., and lucidinethylether,a component of ethanolic Rubia extracts

The genotoxic activity of lucidin (1,3-dihydroxy-2-hydroxymethyl-9,10-anthraquinone), a natural component of Rubia tinctorum L., was tested in a battery of short-term tests. The compound was mutagenic in five Salmonella typhimurium strains without metabolic activation, but the mutagenicity was increased after addition of rat liver S9 mix. In V79 cells, lucidin was mutagenic at the hypoxanthine-guanine phosphoribosyl transferase gene locus and active at inducing DNA single-strand breaks and DNA protein cross-links as assayed by the alkaline elution method. Lucidin also induced DNA repair synthesis in primary rat hepatocytes and transformed C3HI M2-mouse fibroblasts in culture. We also investigated lucidinethylether, which is formed from lucidin by extraction of madder roots with boiling ethanol. This compound was also mutagenic in Salmonella, but only after addition of rat liver S9 mix. Lucidinethylether was weakly mutagenic to V79 cells which were cocultivated with rat hepatocytes. The compound did not induce DNA repair synthesis in hepatocytes from untreated rats, but positive results were obtained when hepatocytes from rats pretreated with phenobarbital were used. We conclude that lucidin and its derivatives are genotoxic.Abbreviations DMBA 7,12-dimethylbenz(a)anthracene - HA hydroxyanthraquinones - LUE lucidinethylether - PRH primary rat hepatocytes - UDS unscheduled DNA synthesis     

Hepatocyte-mediated mutagenicity of mononitrobenzo[a]pyrenes in Salmonella typhimurium strains

The mononitro-substituted isomers of benzo[a]pyrene (B[a]P), 1-, 3- and 6-nitrobenzo[a]pyrene (NB[a]P), are environmental pollutants and are metabolized to mutagens in Salmonella by rat-liver homogenate postmitochondrial supernatant (S9) fractions. In this study, activation of these compounds to mutagens was investigated using the hepatocyte-mediated Salmonella mutagenicity assay. Hepatocytes from rats treated with Aroclor 1254 activated both 3-NB[a]P and 1-NB[a]P to mutagens, while 6-NB[a]P was not mutagenic. The positive mutagenicity responses were functions of both the chemical dose and the hepatocyte concentration. By using a nitroreductase-deficient strain (TA98NR) and a transesterificase-deficient strain (TA98/1,8-DNP6), it was verified that the direct-acting mutagenicities of 1- and 3-NB[a]P primarily were due to metabolic processes involving nitroreduction while the S9- and hepatocyte-mediated mutagenicity responses were also dependent on transesterification. When compared with the mutagenic responses produced with S9, the mutations induced by 1- and 3-NB[a]P in the presence of hepatocytes were relatively more dependent upon nitroreductase metabolism and less on transesterification. Thus, intact hepatocytes were capable of activating 1- and 3-NB[a]P to mutagenic metabolites and some of these metabolites appeared to be different from those produced by S9.     

The spectrum of enzymes involved in activation of 2-aminoanthracene varies with the metabolic system applied

The aim of this study was to estimate the involvement of cytochrome P450s (CYPs) in the metabolic activation of 2-aminoanthracene (2AA) by use of metabolic systems such as liver S9 or hepatocytes from untreated and beta-naphthoflavone (BNF)- or phenobarbital (PB)-treated rats. Metabolic activation was determined in the Salmonella reverse mutation assay (Ames test). Unexpectedly, both enzyme inducers, BNF and PB, significantly decreased the mutagenicity of 2AA activated by S9 fractions. 2AA mutagenicity was detected in the presence of cytochrome P450 inhibitors such as alpha-naphthoflavone (ANF), clotrimazole and N-benzylimidazole to study the contribution of CYP isoenzymes to the activation process. ANF significantly decreased the activation of 2AA by S9 from untreated rats. In contrast, ANF significantly increased the metabolic activation of 2AA by S9 from BNF- and PB-treated rats. The enhanced mutagenicity was not altered by co-incubation with clotrimazole and ANF. Pre-incubation of 2AA in the presence of N-benzylimidazole significantly increased the activation of 2AA by S9 from BNF- and PB-treated rats, which suggests that CYPs play minor role in 2AA metabolic activation by rat liver S9 fractions. In contrast with the results described above, BNF treatment of rats significantly enhanced the activation of 2AA by hepatocytes. ANF attenuated the extent of this activation suggesting that different enzymes play a major role in the activation processes in these metabolic systems. Our results indicate that identification of mutagenic hazard by use of the Ames test may depend on the metabolic system applied.     

Retinol (vitamin A) inhibits the mutagenicity of o-aminoazotoluene activated by liver microsomes from several species in the Ames test

Retinol (vitamin A) has earlier been shown to inhibit the mutagenicity of o-aminoazotoluene (OAAT) in the Salmonella/microsome assay when OAAT is activated with S9 from Sprague-Dawley rats. The results presented in this paper confirm this and also show that S9 from mice, hamsters and gerbils activates OAAT to mutagenic metabolites detected by Salmonella typhimurium TA100. However, S9 from rabbits is inactive. The S9 fraction from rabbits also shows a low aryl hydrocarbon hydroxylase (AHH) activity. The AHH activity or protein content of the microsomal fraction cannot be used to predict the activating capacity of S9 from the other species. Retinol, added in vitro, inhibits the mutagenic effect of OAAT activated by mouse, gerbil or hamster S9. The strongest inhibition is observed with hamster S9 while the inhibition of mouse and gerbil S9 is lower but still higher than in the rat.     

Mutagenicity of N-arylacetohydroxamic acids and their O-glucosides derived from chlorinated 4-nitrobiphenyl ethers

The mutagenic activity of N-arylacetohydroxamic acids, their O-acetates, their O-glucosides, and N-arylhydroxylamines, derived from chlorinated 4-nitrobiphenyl ethers (CNBs), was tested in the Salmonella reversion assay. N-Arylhydroxylamines were mutagenic by themselves; however, other compounds containing an N-acetyl group showed mutagenic activity in the presence of guinea pig liver S9. The mutagenic activation of the glucosides of N-arylacetohydroxamic acids was caused by Ms but not by S10.5, whereas their aglycones, N-arylacetohydroxamic acids, were activated to mutagens by both the fractions. The mutagenic activation of these compounds was inhibited by bis(p-nitrophenyl)phosphate, which indicates that enzymatic deacetylation is a crucial step in the mutagenic activation. Analysis of metabolites of the O-glucosides of N-arylacetohydroxamic acids by h.p.l.c. indicates that the corresponding deacetylated O-glucosides are primary metabolites, which decomposed to amino and azoxy (via hydroxylamine) derivatives, and that the deacetylating activity of S9 locates exclusively in Ms.     

Mutagenicity of derivatives and metabolites of 1-nitropyrene: activation by rat liver S9 and bacterial enzymes

The mutagenicity and activation requirements of purified synthetic derivatives and potential metabolites of 1-nitropyrene have been characterized in the Ames plate incorporation assay with the Salmonella tester strains TA98, TA98NR and TA98/1,8-DNP6, in the presence or absence of exogenous metabolic activation provided by Aroclor-induced rat liver S9. All the compounds tested (1-aminopyrene, N-acetyl-1-aminopyrene, N-hydroxy-N-acetyl-1-aminopyrene, 3-hydroxy-1-nitropyrene, 6-hydroxy-1-nitropyrene, and 8-hydroxy-1-nitropyrene) exhibited mutagenic activity under one or more assay conditions. 1-Nitropyrene was metabolized to 3-hydroxy-1-nitropyrene, 6- or 8-hydroxy-1-nitropyrene, 1-aminopyrene, N-acetyl-1-aminopyrene and other unidentified products (including some bound to protein) by an S9 preparation analogous to that used for exogenous metabolic activation in the Ames assay. 1-Nitropyrene and 3-hydroxy-1-nitropyrene were activated primarily by the 'classical' nitroreductase, while the other compounds, particularly in the presence of S9 metabolic activation, were dependent on transesterification for expression of their mutagenicity.     

Species heterogeneity of hepatic alpha 1-adrenoceptors: alpha 1A-, alpha 1B- and alpha 1C-subtypes.

alpha 1-Adrenergic activation stimulated phosphorylase and phosphoinositide turnover in hepatocytes from guinea pigs, rats and rabbits. Chlorethylclonidine inhibited these effects in rat and rabbit cells but not in guinea pig hepatocytes; low concentrations of 5-methyl urapidil blocked the alpha 1 actions in guinea pig and rabbit liver cells, but not in rat hepatocytes. Binding competition experiments also showed high affinity for 5-methyl urapidil in liver membranes from guinea pigs and rabbits and low affinity in those from rats. The data indicated that guinea pig hepatocytes express alpha 1A-, rat hepatocytes alpha 1B- and rabbit hepatocytes alpha 1C- adrenoceptors. This was confirmed by Northern analysis using receptor subtype-selective probes.     

Mechanisms of butylated hydroxytoluene-mediated modulation of 2-acetylaminofluorene mutagenicity in rat and human hepatocyte/Salmonella assays

Salmonella typhimurium (TA98) mutagenesis assays were used to study the influence of the antioxidant butylated hydroxytoluene (BHT) on 2-acetylaminofluorene (2-AAF) mutagenesis, in search of the mechanism of the anticarcinogenic effects of BHT. Rats pre-treated with BHT in the diet (0.5% w/w for 10 days) provided hepatocytes and hepatocyte S9 which were more efficient in the activation of 2-AAF than were similar preparations from control rats. The increased release of mutagens from hepatocytes might explain the reported increase in the incidence of bladder tumours in BHT-treated rats. In contrast, the mutagenic activity of 2-AAF was inhibited by the in vitro addition of BHT into incubations where human or rat liver S9 and intact hepatocytes were used for metabolic activation. Both competitive and un-competitive inhibition by BHT of 7-ethoxycoumarin O-deethylation was observed in hepatocytes which suggested that the antimutagenic activity may be mediated by one or more mechanisms of cytochrome P-450 inhibition. BHT inhibition of the mutagenicity of N-OH 2-AAF and of rat urinary metabolites of 2-AAF indicated that effects other than those mediated by cytochrome P-450 also occur e.g. scavenging of reactive metabolites. It was concluded that BHT-modulation of 2-AAF metabolic activation and mutagenesis (which may relate to BHT-protection against hepatocarcinogenicity) involves multiple mechanisms.     

Dependence on intact cells for the in vitro activation of dimethylnitrosamine to an immunosuppressive form

The in vitro activation of dimethylnitrosamine (DMN) to an immunosuppressive form was studied utilizing liver-enzyme fractions and intact hepatocytes. The N-demethylation of DMN by mouse S9 and microsome preparations was confirmed by determination of formaldehyde generation. S9 fractions from both phenobarbital(PB)- and isopropanol(iso)-pretreated mice displayed significantly greater demethylase activity than uninduced S9 fractions. However, when incubated with spleen cells, neither S9 preparation was capable of activating DMN to a form capable of suppressing antibody responses by recovered spleen cells. In contrast, the positive control, cyclophosphamide, was activated to a markedly immunosuppressive form. S9 fractions failed to activate DMN to an immunosuppressive form regardless of S9 concentration, time of preincubation, or rocking speed. Liver microsomes from PB-pretreated mice displayed significantly greater N-demethylase activity than S9 fractions yet were unable to activate DMN to an immunosuppressive form. In contrast, the addition of DMN to mixed cultures of mouse hepatocytes and mouse spleen cells resulted in activation of DMN and marked suppression of antibody responses. The separation of spleen cells from the hepatocyte monolayer by an agar layer less than 1 mm thick resulted in complete reversal of the immunosuppressive effect of DMN. Unlike the metabolism of DMN to a mutagenic form, the in vitro activation of DMN to an immunosuppressive form was therefore dependent on intact cells. Furthermore, the activation by intact hepatocytes was shown to be dependent on cell-cell contact or close proximity of activating and target cells.     

Azo reduction of trypan blue to a known carcinogen by a cell-free extract of a human intestinal anaerobe.

The azo reductase activity of a cell-free extract of Fusobacterium sp. 2 is characterized using trypan blue as a substrate. Either chemical reduction of this dye with sodium hydrosulfite or reduction by the cell-free extract produces a mutagenic product, o-tolidine. The o-tolidine is mutagenic in the Ames Salmonella/mammalian-microsome mutagenicity test when activated by a rat liver S9 preparation.     

Evidence for an acetoxyarylamine as the ultimate mutagenic reactive intermediate of the carcinogenic aromatic amine 2,4-diaminotoluene

2,4-Diaminotoluene (2,4-DAT) is a mutagenic and hepatocarcinogenic aromatic amine, requiring metabolic activation. We have found that the mutagenic potency of 2,4-DAT in Salmonella TA98 is similar when activated by either Aroclor-1254-induced rat primary hepatocytes or 9000 x g supernatant. Previous work has demonstrated that 2,4-DAT is activated by cytochrome P450. The present report describes an investigation of the role of acetyltransferase in 2,4-DAT activation. Substitution of TA98 with the acetyltransferase-deficient strain TA98/1,8-DNP6 resulted in an approximately 90% decrease in the mutagenic potency for 2,4-DAT using S9 activation. The newly engineered acetyltransferase-enhanced Salmonella tester strain YG1024 (TA98(pYG219] demonstrated greatly enhanced sensitivity to the mutagenicity of 2,4-DAT. Inhibition of O-acetyltransferase activity, either with the selective acetyltransferase inhibitor thiolactomycin, or by competitive inhibition with an alternative substrate for the enzyme, reduced the mutagenicity of 2,4-DAT in this acetyltransferase-enhanced bacterial strain. From these data we conclude that following 2,4-DAT activation by N-hydroxylation by cytochrome P450, the resulting hydroxylamino intermediate is further activated in the bacteria via O-acetylation to form the ultimate reactive intermediate, which is postulated to be 4-acetoxyamino-2-aminotoluene.     

Activation of aromatic amines to mutagens by various animal species including man

2-Acetylaminofluorene, 2-aminofluorene, 4-aminobiphenyl, 2-naphthylamine, 2-aminoanthracene and benzidine were assayed for mutagenicity in the Ames test in the presence of hepatic microsomal preparations derived from mouse, hamster, rat, pig and man. Prior to each mutagenicity assay all activation systems were fully characterized with respect to mono-oxygenase and mixed-function amine oxidase activities. All compounds were metabolically activated to mutagens by all activation systems, but with markedly different efficiencies, hamster being the only species which readily activated all amines. The hamster also exhibited the highest ethoxyresorufin O-deethylase and dimethylaniline N-oxidase activities.