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.     

Preliminary studies on the ability of Drosophila microsomal preparations to activate mutagens and carcinogens

Subcellular fractions from Drosophila melanogaster, known to have several xenobiotic-metabolizing enzymatic activities, were investigated with respect to their ability to biotransform compounds that require metabolic activation before exerting mutagenic effects. Nitrofurazone, dimethylnitrosamine, cyclophosphamide and 2-acetylaminofluorene were activated to mutagens upon incubation with Drosophila microsomes or 20000 x g supernatant: mutagenicity was observed in Chinese hamster ovary cells, Escherichia coli strains 343/113/R-9 and 343/113/uvrB, and Salmonella typhimurium TA1538. Under the conditions used, microsomal preparations of Drosophila were not able to activate benzo[a]pyrene to a mutagen for Salmonella typhimurium TA98. The spectrum of mutagenic effects observed shows some correlation with the known mutagenicity of these compounds in vivo in Drosophila melanogaster. Drosophila microsomes appeared to be at least as active as rat-liver microsomes when compared in this type of mutagenicity testing.     

Metabolic activation of N-acetylbenzidine and N,N'-diacetylbenzidine to mutagens, using isolated hepatocytes and the 9000 X g liver supernatant from rat, hamster and guinea pig

The metabolic activation of MABZ and DABZ, forming products mutagenic towards Salmonella typhimurium TA1538, was studied with isolated hepatocytes from rat, hamster and guinea pig and the S9 fraction (9000 X g supernatant) prepared from these hepatocytes. Special attention was given to the influence of acetyl-CoA, the cofactor for N-acetylation, on the mutagenicity of these arylamides. The rat and guinea pig S9 preparation activated MABZ as well as DABZ to a much higher degree than the intact hepatocytes of these animal species. Addition of acetyl-CoA to the S9 preparation decreased the mutagenicity of MABZ and DABZ. On the contrary, for the hamster the mutagenicity of MABZ and DABZ appeared to be lower with the S9 preparation than with intact hepatocytes. Addition of acetyl-CoA to the S9 here increased the mutagenic activity of these arylamides. In the presence of intact hepatocytes obvious interspecies differences were observed in the activation of MABZ and DABZ. DABZ was far more effectively activated by hamster hepatocytes than by rat hepatocytes. This was not found with MABZ. Both substrates were poorly activated by guinea pig hepatocytes.     

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.     

The mutation of V79 cells by N-nitrosobis(2-oxopropyl)amine activated by pancreas acinar and duct tissue from Syrian hamsters and MRC-Wistar rats

The mutagenicity of N-nitrosobis(2-oxopropyl)amine was measured in the V79 assay using homogenates of acinar cells and duct tissue from the pancreases of Syrian hamsters and MRC-Wistar rats as the activating systems. Mutations at the sodium/potassium ATPase and hypoxanthine:guanine phosphoribosyltransferase loci were measured by resistance to ouabain and 6-thioguanine (TG). The order of effectiveness in generating mutagens from BOP was hamster duct, hamster acinar, rat duct, rat acinar. These data show extensive differences in BOP activation by hamster acinar and duct tissue.     

Effect of cigarette smoke on the mutagenic activation of environmental carcinogens by rodent liver.

In order to assess the effect of cigarette smoke (CS) on metabolic enzymes, male hamsters and rats were exposed for two weeks to smoke produced in a Hamburg type II smoking machine. The livers were then used for Ames liquid incubation and western immunoblot assays. Mutagenic activities of seven heterocyclic amines (HCAs) in Salmonella typhimurium TA98 in the presence of rat or hamster liver S9 were elevated up to 3.7 times above controls (including sham smoke control). Enhancement of mutagenic activities of PhIP and aflatoxin B(1) was observed only in CS-exposed hamster, whereas no significant alteration of mutagenicity was observed with 2-aminofluorene, benzo[a]pyrene, and 3'-hydroxymethyl-N, N-dimethyl-4-aminoazobenzene in strain TA98 or with six N-nitrosodialkylamines in strain TA100. 7,8-Benzoflavone and/or furafylline considerably inhibited the mutagenic activation of IQ and Trp-P-1 in the presence of liver S9 from untreated hamsters and sham smoke- or CS-exposed hamsters and rats, indicating the predominant involvement of hamster cytochrome P450 (CYP) 1A enzymes in the metabolic activation of HCAs. In addition, the data suggest that CS-exposure may selectively induce hepatic CYP1A1/1A2 isoforms. Western immunoblot analyses of liver microsomes using anti-rat CYP antibodies revealed that CS-exposure increased the levels of hamster CYP1A2 (3.9-fold) and rat CYP1A2 (3.0-fold) and CYP1A1, without significant change in the levels of CYP2E1 and CYP2B and 3A isoforms in each species. The presently observed selective induction of HCA activation and CYP isozymes due to CS supports the idea that CS may contribute to enhancing effects on initiation by carcinogens which are metabolically activated by hepatic CYP1A1/1A2. In conjunction with results observed for smokers, the present findings indicate that the hamster is a good animal for studies with CS, and that cigarette smoking in combination with intake of heating protein-rich foods as a life style may markedly contribute to the human carcinogenesis by HCAs.     

Comparative genotoxicity studies of the flame retardant tris(2,3-dibromopropyl)phosphate and possible metabolites

Tris(2,3-dibromopropyl)phosphate (Tris-BP) was activated to mutagens in the Salmonella/microsome quantitative test system. Liver microsomes from rats pretreated with phenobarbital (PB) increased the mutagenicity of 0.05 mM Tris-BP to 186% of the activity obtained with liver microsomes from untreated rats. The addition of 0.02 mM Tris-BP to V79 Chinese hamster cells co-incubated with liver microsomes from PB-pretreated rats increased the number of mutants by a factor of 9.7. Tris-BP also caused genotoxic and cytotoxic responses in primary monolayers of rat hepatocytes. The relative increase in unscheduled DNA synthesis after treatment with 0.05 mM Tris-BP was 2.3-fold as measured by scintillation counting of radiolabelled thymidine incorporated into DNA of isolated nuclei. The use of hepatocytes isolated from PB-pretreated rats reduced the increases in DNA repair synthesis relatively to that in control cells. Monolayers of hepatocytes from untreated rats co-cultured with Salmonella typhimurium TA100 activated Tris-BP to mutagenic intermediates which were released into the culture medium. The studies with the V79 and liver-cell systems indicate that the reactive intermediates formed from Tris-BP are sufficiently stable and lipophilic to traverse the various membranes from the site of generation to the respective cellular targets. The relative degree of genotoxic responses of bis(2,3-dibromopropyl)phosphate, 2,3-dibromopropylphosphate, tris(2,3-bromopropyl)phosphate, tris(2-bromopropyl)phosphate and 2,3-dibromopropanol in the systems studied did not indicate that these compounds were proximate or ultimate reactive metabolites of Tris-BP in liver-derived activation systems.     

Condensed tannins induce micronuclei in cultured V79 Chinese hamster cells

The tannins, delphinidin and procyanidin were isolated from flowers of white clover (Trifolium repens) and the leaves of Arnot Bristly Locust (Robina fertilis) respectively, and tested for mutagenic properties in a range of systems. There was no evidence for either compound causing significant levels of frameshift or base-pair mutagenesis in bacterial mutagenicity assays, although both were weakly positive in a bacterial DNA-repair test. Both compounds very slightly increased the frequency of petite mutagenesis in Saccharomyces cerevisiae strain D5. In V79 Chinese hamster cells, both were efficient inducers of micronuclei. In each of these test systems, increasing the potential of the compound for metabolic activation by addition of 'S9' mix had little effect on toxicity or mutagenicity of either tannin. It would seem that potential chromosome-breaking activity of condensed tannins could represent a carcinogenic hazard for animals grazing on pastures of white clover in flower. It may also have wider implications for human carcinogenesis by some, if not all, condensed tannins.     

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.     

Species difference in the metabolic activation of phenacetin by rat and hamster liver microsomes

Phenacetin is mutagenic in Salmonella typhimurium TA 100 when liver 9,000 X g supernatant fractions from PCB-treated hamsters instead of rats are used. A mechanism of the species difference in phenacetin mutagenicity was investigated. By high-performance liquid chromatography analysis, it was found that phenacetin is activated to direct-acting mutagens through N-hydroxylation and deacetylation by hamster liver microsomes. Although no significant species difference was observed in N-hydroxylation, rates of deacetylation were 9 to 150 times higher in hamsters than in rats. The results indicate that the marked species difference in phenacetin mutagenicity is due to the difference in deacetylation activity between rat and hamster liver microsomes.     

Mutagenic activities of cyclophosphamide (NSC-26271) and its main metabolites in Salmonella typhimurium, human peripheral lymphocytes and Chinese hamster ovary cells

Cyclophosphamide (CPA) and its main metabolites were analyzed with respect to their mutagenic activities in Salmonella, human peripheral lymphocytes (PL), and Chinese hamster ovary (CHO) cells. In Salmonella, the compounds were activated with S9 mix from rat livers, which were unstimulated or stimulated with Aroclor 1254 or phenobarbital. For the enzyme inducers the following order of efficiency was found for all test compounds except carboxyphosphamide: phenobarbital greater than Aroclor 1254 greater than non-induced. The most potent mutagens in all 3 test systems were 4-OH-CPA, PAM and nor-HN2. S9 mix transforms 4-OH-CPA to strong mutagenic compounds in the Salmonella assay. All metabolites tested in the Salmonella assay were activated by S9 mix to higher mutagenic potential.     

Mutagenicity of benzotrichloride and related compounds.

Benzotrichloride (BTC), benzal chloride (BDC), benzyl chloride (BC) and benzoyl chloride (BOC) were surveyed for their mutagenicity in microbial systems such as rec-assay using Bacillus subtilis and reversion assays using E. coli WP2 and Ames Salmonella TA strains with or without metabolic activation in vitro. BTC and BDC required metabolic activation for their mutagenic activities in several strains of E. coli and Salmonella. The mutagenic metabolites of these compounds may not have been produced by hydrolysis. BC was weakly mutagenic without metabolic activation. Only BOC exhibited no mutagenic activity in the detection procedures used. The mutagenic metabolite of BTC might be very unstable under our experimental conditions. The strain E. coli WP2 try hcr was more sensitive than E. coli B/r WP2 try (hcr+) with regard to the mutagenicity of BTC.     

Studies on chemical carcinogens and mutagens. XXIX. Mutagenic N-trimethylsilylmethyl-N-nitrosourea as a biological alkylating agent equivalent to N-methyl-N-nitrosourea (MNU)

N-Trimethylsilylmethyl-N-nitrosourea (TMS-MNU), a silicon analogue of N-neopentyl-N-nitrosourea (neoPNU), was assayed for mutagenicity and/or cytotoxicity on a series of E. coli B tester strains, S. typhimurium TA100, Chinese hamster V79, and cultured murine leukemia L1210 cells. All the biological activities demonstrated in this study reveal that this nitrosourea is a biological methylating agent equivalent to N-methyl-N-nitrosourea (MNU) but definitely distinguished from all the other alkylnitrosoureas examined so far, including neoPNU (the carbon analogue of TMS-MNU). The proposed molecular mechanism is that trimethylsilylmethanediazohydroxide is produced by hydrolytic activation of TMS-MNU and undergoes a nucleophilic cleavage of the Si--CH2 chemical bond at a high rate to form methanediazohydroxide (the reactive intermediate of MNU) which, in turn, methylates the informational biopolymer leading to mutagenesis.     

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.     

Mutagenicity of some substituted 1-phenyl-3,3-dimethyltriazenes. I. The salmonella/mammalian microsome assay and repair test

The mutagenic activity and related biological properties of Br-, Cl-, NO2- and CH3-derivatives of 1-(phenyl)-3,3-dimethyltriazene were investigated in Salmonella/microsome assays with standard and preincubation metabolic activation and in the repair test using Salmonella and E. coli B/r. In the repair test, the CH3-derivative was slightly positive in the E. coli recA and uvrA repair system, the NO2-derivative had a killing effect on Salmonella typhimurium uvrB-deficient strains. In Salmonella mutagenicity assays, all tested triazene derivatives reverted frameshift tester strains, especially TA1537. The highest number of frameshift mutations was induced by the CH3-derivative in the presence of a standard metabolic activation system; direct mutagenicity of this derivative was weak, reaching about the same level of activity as seen after preincubation. The only test compound that induced mutations of the base-substitution type was the NO2-derivative; this derivative showed the highest mutagenicity when activated by preincubation.     

Mutagenicity of mono-, di- and tri-nitropyrenes in Chinese hamster ovary cells

1-Nitropyrene (1-NP), 1,3-dinitropyrene (1,3-DNP), 1-6-dinitropyrene (1,6-DNP), 1,8-dinitropyrene (1,8-DNP) and 1,3,6-trinitropyrene (1,3,6-TNP) were tested for mutagenicity in cultured Chinese hamster ovary (CHO) cells. Mutation at the hypoxanthine-guanine phosphoribosyl transferase gene locus was quantified. While 1-NP and 1,3-DNP had only marginal direct-acting mutagenicity, 1,6-DNP, 1,8-DNP and 1,3,6-TNP showed definite mutagenicity, with specific mutagenic activities of 8.1, 21 and 54 mutants/10(6) survivors/micrograms . ml-1 respectively. The mutagenicity of 1-NP increased with increasing concentrations of Aroclor-1254 induced liver homogenate (S9) in the treatment medium. However, S9 at all concentrations tested decreased the mutagenicity of 1,6-DNP and 1,8-DNP. S9 at low concentrations enhanced the mutagenicity of 1,3-DNP and 1,3,6-TNP and that at high concentrations decreased their mutagenicity. The positive mutagenic response of the nitropyrenes suggests that they are potentially carcinogenic, and that further research into their possible human health risk should be performed.     

Effect of dipyridoimidazole pretreatment on mutagen activation in rats

Rats were pretreated with a single oral dose of different mutagenic fractions obtained from glutamic acid pyrolysate: Glu-P-2 (2-amino-dipyrido[1,2-a:3',2'-d]imidazole), Glu-P-3 (3-amino-4,6-dimethyldipyrido[1,2-a:3',2'-d]imidazole), the tar residue and a basic extract (B2). The liver S9 fractions of these animals were used to investigate the mutagenic activation of 3 promutagens (2-aminoanthracene, Glu-P-2 and Glu-P-3) in Salmonella typhimurium strain TA1538. Different factors were analyzed; influence of the structure of the compounds administered, doses, time interval between pretreatment and sacrifice and sex of the rats. Interpretation of the hepatic induction effects was complicated, however, by the fact that simple oral pretreatment with the solvents (DMSO or ethanol) enhances the activation of the substrates tested for mutagenicity. A dose-effect relationship was found between 2-AA mutagenic activation and Glu-P-2 pretreatment. Glu-P-3 induced the activation of 2-AA more than did Glu-P-2, in the male as in the female. The mutagenicity of 2-AA activated with S9 from male rats was found to be optimal after 24 h pretreatment with 20 mg Glu-P-2/kg b.w. The mutagenicity of Glu-P-2 was poorly influenced by the different pretreatments applied to either the males or the females, whereas some dose effect was found in the autoinduction of Glu-P-2 mutagenicity. Compared to Glu-P-2, the mutagenicity of Glu-P-3 was increased at higher levels when tested with S9 from males pretreated with the same compound, but no differences were observed between males and females.     

Metabolic activation of 10-aza-substituted benzo[a]pyrene by cytochrome P450 1A2 in human liver microsomes

We previously reported that 10-azabenzo[a]pyrene (10-azaBaP), a 10-aza-analog of BaP and an environmental carcinogen, showed greater mutagenicity than BaP in the Ames test using pooled human liver S9. To investigate the cytochrome P450 (CYP) isoform involved in the activation of 10-azaBaP to the genotoxic form, the mutagenicity of 10-azaBaP using nine individual donors' and pooled human liver microsome preparations was compared with each CYP activity. Induced revertants by 2.5 nmol per plate 10-azaBaP with 0.5 mg per plate human liver microsomal protein showed a large inter-individual variation (42-fold) among the nine donors. The number of induced revertants highly correlated with the CYP1A2-selective catalytic activity from each microsome preparation, and no correlation was observed with other CYP isoform-selective catalytic activities. Moreover, recombinant human CYP1A2 contributed to the mutagenicity of 10-azaBaP more markedly than recombinant human CYP1A1. These results suggest that CYP1A2 may be the principal enzyme responsible for the metabolic activation of 10-azaBaP in human liver microsomes. With regard to the proposal that BaP may be activated by human CYP1A1, our results suggest that the nitrogen-substitution at position-10 of BaP may cause the CYP enzyme-specificity in metabolic activation to change from CYP1A1 to CYP1A2.     

Enhancement of UV-induced mutagenesis and sister-chromatid exchanges by nickel ions in V79 cells: evidence for inhibition of DNA repair

With regard to contradictory results concerning the mutagenicity of nickel compounds in short-term assays, especially in bacterial test systems, Chinese hamster V79 cells were used to measure mutagenicity, comutagenicity and the induction of sister-chromatid exchanges (SCEs) by NiCl2. We confirmed the induction of mutations at the HGPRT locus as well as SCEs. In addition, NiCl2 shows a pronounced comutagenic effect towards UV. When using confluent cultures or resting cells due to serum deprivation, where more time is given for repair processes, the comutagenic effect is higher compared to logarithmically growing cells (10 and 4 times, respectively, compared to twice). Hence, we attribute this enhancement in mutagenicity to inhibition of DNA repair. Also the increase in induced SCEs after combined treatment with UV and NiCl2 supports this thesis. Furthermore, NiCl2 enhances the cyto-toxicity of cis-DDP about 12-fold. Since no comutagenic effect is observed in combination with MMS, we suggest that the inhibition of DNA repair by Ni(II) applies to all DNA changes that are repaired by the 'long-patch' excision repair system. This inhibition may occur via replacement of other divalent metal ions essential in repair and regulation processes.