Constitutive and Aroclor 1254-induced hepatic glutathione S-transferase, peroxidase and reductase activities in genetically inbred mice

1. Constitutive and Aroclor 1254-induced hepatic glutathione (GSH) S-transferases, GSH peroxidase and GSH reductase activities were determined in 12 strains of 8-10 week-old inbred male mice. 2. The constitutive GSH S-transferase activity varied from 2.5 (SJL/JCR) to 8.9 (C57BL/6N) mumol/min/mg protein and the corresponding values for the Aroclor 1254-treated mice were in the range of 7.1-23.0 mumol/min/mg protein. Aroclor 1254 significantly induced GSH S-transferase activity in all mice, however, significant interstrain differences were found in inducibility. 3. Aroclor 1254-treatment caused a 4.2-fold induction of GSH S-transferase in NFS/NCR but only a 1.4-fold increase in AKR/NCR mice. Aroclor 1254 significantly induced GSH reductase in all strains studied while GSH peroxidase activity decreased in these mice. 4. The range of hepatic GSH levels in control and Aroclor 1254-treated mice was relatively narrow for both groups (6.59-11.25 microM/g wet tissue).     

Potentiation of sodium chromate(VI)-induced chromosomal aberrations and mutation by vitamin B2 in Chinese hamster V79 cells.

The effect of vitamin B2, which is capable of reducing chromium(VI) to chromium(V), on chromosomal aberrations and mutation caused by Na2CrO4 was investigated in Chinese hamster V79 cells. Pretreatment with 200 microM vitamin B2 (riboflavin) for 24 h prior to exposure to Na2CrO4 (2.5-5 microM) resulted in an increase of metal-induced chromosomal aberrations and mutation at the HGPRT locus. These and other previous studies suggest that vitamin B2 enhances the clastogenic and mutagenic action of chromate compounds, through its ability to directly reduce chromium(VI) in cells.     

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.     

T-2毒素遗传毒性作用的初步研究

T-2毒素是一种可由三线镰刀菌,拟支孢镰刀菌、梨孢镰刀菌等产生的单端孢霉素,与食物中毒性白细胞缺乏症(Alimentary toxicaleukia,ATA)的发生有关。据报道,T-2毒素可以诱导人的成纤维细胞非程序DNA合成增高,引起地鼠骨髓细胞和人外周血淋巴细胞染色体畸变,导致大鼠淋巴细胞的DNA单链断裂,还可以使人胎儿食管上皮     

Comparison of metabolic systems required to activate pro-mutagens/carcinogens in vitro for sister-chromatid exchange studies

Irradiated Syrian hamster fetal cells (feeder layer) and rat-liver homogenate (S9 mix) were used to compare their capacity to metabolize 3 known promutagens/carcinogens; BaP, 3-MC and DMBA. DNA-damaging potential was determined by the induction of SCE in V79 target cells. The S9 mix (1/20th strength) was toxic to the target cells and reduced the mitotic index by half with an exposure time of 2.5 h. The feeder layer was not toxic to the target cells and, therefore, was included for the duration of the Expt. The test chemicals elicited a dose-response with both activating systems. At similar concentrations of the test chemicals, the cells grown on the feeder layer showed a greater number of SCEs as compared to those activated by the S9 mix.     

Protective effect of vitamin E against chromosomal aberrations and mutation induced by sodium chromate in Chinese hamster V79 cells

The effect of vitamin E on chromosomal aberrations and mutation caused by Na2CrO4 was investigated in Chinese hamster V79 cells. Pretreatment with 25 microM alpha-tocopherol succinate (vitamin E) for 24 h prior to chromate exposure (2.5-5 microM) resulted in a decrease of metal-induced chromosomal aberrations. Na2CrO4 (2.5-7.5 microM) induced mutations at the HGPRT locus, but only within a very limited concentration range. This mutagenic response could also be suppressed by pretreatment with vitamin E. These results suggest that vitamin E can protect cells from the clastogenic and mutagenic action of chromate compounds, possibly through its ability to scavenge chromium(V) and/or free radicals.     

Absence of genotoxic activity of penbutolol in bacterial and mammalian cell screening systems

The genotoxic potential of the beta-adrenergic blocker penbutolol was assessed using the Ames and HGPRT tests, unscheduled DNA synthesis (UDS) and alkaline elution assays. In the Ames test, penbutolol was tested for cytotoxicity and genotoxic activity in concentration ranges of 0.8-500 micrograms/plate and 0.1-125 micrograms/ml in the HGPRT, UDS and alkaline elution assays. In the Ames test penbutolol showed significant toxicity above 500 micrograms/plate. In the mammalian cells (V79) used for the HGPRT test and A459 cells used for alkaline elution and UDS assays, penbutolol was cytotoxic at concentrations above 30 micrograms/ml. In another series of experiments, male Wistar rats were treated i.p. with penbutolol (1, 10 and 100 mg/kg) and after 2 h liver nuclei were isolated and formation of single DNA-strand breaks was measured. The results of the present study demonstrate the absence of genotoxic activity of penbutolol in the 5 strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA1538) and in the strain of Escherichia coli WP2 uvrA in the presence or absence of metabolic activation. In V79 cells, penbutolol showed no mutagenic effects at the HGPRT locus in the presence or absence of metabolic activation. Additionally, no significant incorporation of [3H]thymidine into the DNA in the UDS test or formation of DNA-strand breaks in the alkaline elution assay was detected in the non-toxic concentration range of penbutolol with or without metabolic activation. Furthermore, penbutolol did not cause DNA damage in liver nuclei isolated from penbutolol-treated rats.     

Micronucleus formation in cultured human keratinocytes following exposure to mitomycin C and cyclophosphamide

A method is described to investigate the induction of micronuclei in cultured human keratinocytes after short-term exposure to known clastogenic agents. The cytokinesis-block method was applied to facilitate the scoring of micronucleated cells. Mitomycin C, a direct-acting compound, caused a 5-20-fold increase in micronuclei over the controls at the highest concentration tested (1 microgram/ml). Cyclophosphamide, an agent requiring metabolic activation, did not induce the formation of micronuclei in cultured keratinocytes. However, after pretreatment of the keratinocyte cultures with Aroclor 1254 for 72 h, exposure to cyclophosphamide resulted in a 3-fold increase in micronucleus frequency over the controls. No cytogenetic effect of Aroclor 1254 was observed in control experiments.     

Mutagenic studies on the hair dye 2-(2',4'-diaminophenoxy)ethanol with different genetic systems

A new hair-dye coupler, 2-(2',4'-diaminophenoxy)ethanol was analyzed for its potential mutagenic activity in different genotoxic assays, namely gene reverse mutations in Salmonella typhimurium, forward mutations in the yeast Schizosaccharomyces pombe, and in the V79 Chinese hamster cell line grown in vitro (HGPRT forward mutation system). Two other genetic test systems, measuring the mitotic gene conversion in Saccharomyces cerevisiae (strain D4) and the unscheduled DNA-repair synthesis in a HeLa cell line grown in vitro, were also used. 2,4-Diaminoanisole, a mutagenic/carcinogenic structurally related hair-dye coupler, and a group of well-known mutagens, namely methyl methanesulfonate, ethyl methanesulfonate, cychlophosphamide, hycanthone and N-nitrosodimethylamine, were used as positive controls. The new aromatic amine, 2-(2',4'-diaminophenoxy)ethanol, was negative in all the assays performed, under the same treatment conditions as in the case of all the positive controls.     

High sensitivity of Chinese hamster epithelial liver cells to toxic analogues of purines

The resistance of Chinese hamster epithelial liver cells (CHEL) and Chinese hamster fibroblasts (V79) towards toxic purine analogues has been determined. The liver cells are more sensitive than fibroblasts to 6-thioguanine (6-TG), 8-azaguanine (8-AZ) and 2,6-diaminopurine (DAP). The hypoxanthine-guanine (HGPRT) and adenine phosphoribosyl transferase (APRT) activities of extracts of CHEL cells were lower than those of corresponding extracts of V79. The level of 5'-nucleotidase was about 5-fold higher in the epithelial cells. It appears that HGPRT and APRT activities of extracts of liver epithelial cells are masked or reduced by 5'-nucleotidase activity and other inhibitors. The significance of these findings is discussed.     

Heritable hypersensitivity to induced mutagenesis in the progeny of cell populations exposed to UVC (254 nm)

The ability of mutagens to transform benign papillomas to malignancy in the mouse skin model of multistage carcinogenesis [Hennings et al. Nature 303, 67-68 (1983)] suggests that multiple events may underlie carcinogenic progression, and that mutagenic exposures separated by time can act synergistically. Such synergism may result from initial mutagenic exposure which induces heritable sensitivity to subsequent mutagenic exposures. For example, progeny of X-irradiated V79 cells are hypersensitive to subsequent mutation induced by psoralen plus long-wave ultraviolet light, PUVA [Frank and Williams, Science 216, 307-308 (1982)]. In the present studies 100 to 200 surviving clones of short-wave ultraviolet light (UVC) irradiated V79 cells were assayed for mutation at two loci. Cultures derived from these cells were found to be hypermutable at the hypoxanthine guanine phosphoribosyl transferase (HGPRT) locus following exposure to PUVA, but showed mutant frequencies similar to control cells following UVC challenge at the HGPRT and ATPase loci.     

Kinetics of chromosomal aberrations and first mitosis division in human lymphocytes exposed to mitomycin C

In order to understand the relationship between the chromosomal damage detectable at the first mitosis after mutagen treatment and the induced mitotic delay we studied the time pattern of both mitotic indices and chromosomal aberration frequencies in human lymphocytes treated in G1 with mitomycin C (2.5 microM) and cultured in vitro in the presence of 5-bromo-2'-deoxyuridine. Mitotic delay was observed in treated cells cultured for 81 h. At this point an increase in the frequency of chromosomal aberrations is evident and a higher proportion of abnormal cells enters mitosis, the long delay being due to the extensiveness of DNA damage. The importance of cell cycle progression for the detection of the maximal amount of induced chromosomal damage is discussed.     

A multiple end-point approach to evaluation of cytotoxicity and genotoxicity of erythrosine (FD and C Red No. 3) in a V79 hepatocyte-mediated mutation assay

V79 Chinese hamster lung cells were used to evaluate in vitro the cytotoxicity and genotoxicity of erythrosine (2', 4', 5', 7'-tetraiodofluorescein disodium salt; FD and C Red No. 3), a color additive used widely in foods, drugs and cosmetics. Erythrosine reduced colony size at 200 micrograms/ml and was lethal to 90% or more of the cells at 400 micrograms/ml. At dose levels of 100, 200 and 300 micrograms/ml of medium, erythrosine was non-mutagenic to V79 cells at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and sodium, potassium ATPase (Na+, K+ -ATPase) gene loci and did not increase the frequency of sister-chromatid exchanges with or without rat hepatocyte-mediated activation. Erythrosine at 300 micrograms/ml, unlike lower dose levels, produced an increase in micronucleus frequency in the absence of hepatocytes. An erythrosine dose-related increase in the mitotic frequency was due to an increase in the number of first mitoses at the expense of later cell divisions. Hepatocytes moderated the effect of erythrosine treatment on micronucleus frequency, mitotic frequency and MII/MI ratio. These results demonstrate the advantage of a multiple end-point approach to the evaluation of cytotoxicity and genotoxicity within a single-assay system.     

Induction of mutation in V79 Chinese hamster cells by tetrachlorohydroquinone, a metabolite of pentachlorophenol

Tetrachlorohydroquinone (TCHQ) and tetrachlorocatechol (TCC), two metabolites of the environmental mutagen and carcinogen pentachlorophenol, were tested without exogenous activation in V79 Chinese hamster cells for the induction of mutation at the hypoxanthine phosphoribosyl transferase (HPRT) locus to 6-thioguanine resistance (TGr) and at the Na/K-ATPase locus to ouabain resistance (OuaR). Treatment was for 24 h at 37 degrees C. TCHQ produced statistically significant increases in the frequency of TGr mutants. The lowest observed effective dose (LOED) was 20 microM, where the relative cloning efficiency was 63%. The relationship between the dose of TCHQ and the frequency of TGr mutants was approximately linear over the range of 0-60 microM with an estimated slope (+/- 95% confidence limits) of 1.1 +/- 0.3 mutants per 10(6) clonable cells per microM. At the highest tested dose of TCHQ, 60 microM, the relative cloning efficiency was reduced to 7%. In contrast to TCHQ, TCC was unable to induce TGr mutants at doses up to 120 microM. The relative cloning efficiency at this dose was 5%. Both TCHQ and TCC were unable to induce OuaR mutants. The results suggest that TCHQ is at least partly responsible for the genotoxic activity of pentachlorophenol. TCHQ can produce reactive oxygen species, which may cause large genetic damage such as deletions, resulting in mutation to TGr but not to OuaR.     

Mutagenicity evaluation of HC Blue No. 1 and HC Blue No. 2. III. Effects in the Salmonella typhimurium/Escherichia coli reversion assay and the mouse lymphoma L5178Y TK+/- forward mutation assay

The hair-dye ingredients, HC Blue No. 1 (HCB1) and HC Blue No. 2 (HCB2), were tested for the induction of bacterial mutation using Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100; and Escherichia coli strains WP2uvrA-. In addition, both dyes were evaluated in the mouse lymphoma L5178Y TK+/- assay (MLA) for the potential to induce forward mutation. A liver homogenate (S9) prepared from Aroclor 1254-induced male Fischer 344 rats was used to provide a means for metabolic activation. HCB1 was not mutagenic in the Ames assay, but was weakly mutagenic in the MLA only in the presence of metabolic activation. In contrast, HCB2 was a strong mutagen in the Ames assay in tester strain TA98 both in the presence and absence of metabolic activation. A positive response was also noted with HCB2 in the MLA, both in the presence and absence of metabolic activation. Negative findings from the Ames assay of this study agree with other published results where an identical lot of HCB1 was used. Using the same lot, a weak positive result was observed in the MLA, however, the activation requirements and magnitude of the response were different from that of a lot evaluated by the NTP. In contrast, HCB2 appears to be both a bacterial and mammalian cell mutagen independent of lot variability.     

Survival and mutagenesis of bacterial plasmids with localized carcinogen adducts

Induction of 6-thioguanine (TG) resistance by chemical mutagens was examined in a line of cells derived from a human epithelial teratocarcinoma cell clone. The cells, designated as P3 cells, have a stable diploid karyotype with 46(XX) chromosomes, including a translocation between chromosomes 15 and 20. Efficient recovery of TG-resistant mutants induced by the direct-acting mutagens: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG); 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10 -tetrahydrobenzo[a]pyrene (BPDE); and benzo[a]pyrene (B[a]P); activated in a cell-mediated assay, required an expression time of 7 days and a saturation density of 2 X 10(4) cells/60-mm petri dish. The TG-resistant mutant cells induced by MNNG and BPDE maintained their resistant phenotype 4-6 weeks after isolation. This mutant phenotype was associated with a more than 10-fold reduction in hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity relative to that of the parental P3 cell line, which was shown to catalyze the formation of 4.6 pmoles inosine-5'-monophosphate (IMP)/min/microgram protein. Induction of TG resistance was also observed in P3 cells cocultivated in a cell-mediated assay with human breast carcinoma cells, which are capable of polycyclic aromatic hydrocarbon (PAH) metabolism, after treatment with the carcinogenic PAHs: B[a]P, chrysene, 7,12-dimethylbenz[a]anthracene (DMBA), and 3-methylcholanthrene (MCA). The degree of mutant induction in this assay was related to the carcinogenic potency of these PAHs in experimental animals. The most potent mutagen was DMBA, followed in decreasing order by MCA, B[a]P, and chrysene. DMBA, at 0.4 microM, increased the frequency of mutants for TG resistance from 2 for the control to about 200 TG-resistant mutants/10(6) colony-forming cells (CFC). Benzo[e]pyrene (B[e]P) and pyrene, which are not carcinogenic, were not effective in the assay. None of the PAHs was mutagenic in the P3 cells cultivated in the absence of the PAH-metabolizing cells. These results indicate that the P3 cells can be useful for the study of mutagenesis at the HGPRT locus by direct-acting chemical mutagens, as well as by chemicals activated in a cell-mediated assay.     

Comparative genotoxicity of 3 procarcinogens in V79 cells as related to glutathione S-transferase activity of hepatocytes from untreated rats and those fed 2% butylated hydroxyanisole

When 7,12-dimethylbenz[a]anthracene (DMBA) and aflatoxin B1 (AFB1) were activated by hepatocytes from Fischer 344 rats fed a diet containing 2% butylated hydroxyanisole (BHA), frequencies of mutation to 6-thioguanine resistance (TGR) at the HGPRTase gene locus and to ouabain resistance (OuR) at the Na+,K(+)-ATPase gene locus in V79 cells were 30-70% less than those obtained with hepatocytes from untreated controls. A difference in the mutation frequency did not occur when dimethylnitrosamine (DMN) was activated by BHA induced- rather than control-hepatocytes. Analysis of hepatocytes from rats fed 2% BHA showed a small (1.5-fold), but significant, increase in glutathione levels over that in the controls but no change in activity of cytochrome P450. Cytosolic glutathione S-transferase (GST) activity was increased 2-3-fold in hepatocytes from rats fed the 2% BHA diet. These results suggest that mutagenic response to DMBA and AFB1 is reduced, at least in part, because of BHA-induction of hepatocyte GST activity; while activation of DMN can occur by pathway(s) unaffected by BHA-induction of these liver enzymes. In contrast to mutation frequencies, significant differences between BHA- and control-activation in the production of sister-chromatid exchange (SCE) and micronucleus formation (MN) were not detected with any of the genotoxins. It was concluded that the mechanism(s) by which SCE and MN occur are likely unrelated to the capacity of BHA to induced activity of hepatic enzymes, e.g. the GSH S-transferases, that directly or indirectly affect mutation end-points.     

The effects of dietary selenium on the biotransformation of 7,21-dimethylbenz[a]anthracene

The influence of dietary selenium on the mutagenic activation of 7,12-dimethylbenz[a]anthracene (DMBA) by rat liver S9 was studied using the Ames test. Rats received supplemental selenium, as sodium selenite, in the drinking water or in the diet. All rats additionally received 0, 20, 50, 100, or 500 mg Aroclor 1254 per kg body weight. Revertant counts decreased 72 and 31% at the 20- and 100-mg/kg induction levels, respectively, with S9 preparations from rats given selenium supplementation, compared to controls. No significant effects of selenium on S9 preparations was observed in rats treated with 500 mg/kg Aroclor. Preparations of S9 from rats receiving 2.5 ppm Se in their diet produced 46, 84 and 70% less revertants than controls at the 20-, 50- and 100-mg/kg induction levels. Increasing the selenium concentration in the diet to 5 ppm reduced the revertant counts by 71, 68 and 65%, at the 20-, 50- and 100-mg/kg induction level of Aroclor, respectively. Dietary selenium supplementation was shown to decrease the mutagenic activation of DMBA by liver microsomes. These studies indicate that in vivo selenium supplementation may reduce susceptibility to the action of various carcinogens.     

Comparison of three different in vitro mutation assays used for the investigation of cytochrome P450-mediated mutagenicity of nitro-polycyclic aromatic hydrocarbons

Three different in vitro mutation assays were used to investigate the involvement of cytochrome P450 enzymes in the activation of the nitro-polycyclic aromatic hydrocarbons (nitroPAHs) 1-nitropyrene and 2-nitrofluorene and their reduced metabolites amino-polycyclic aromatic hydrocarbons (aminoPAHs) 1-aminopyrene and 2-aminofluorene. Mutagenicity was investigated at the HPRT locus in Chinese hamster V79 cells with (V79-NH) or without (V79-MZ) endogenous acetyltransferase activity, stably expressing human cytochrome P450 cDNAs; in NIH/3T3 control or stably expressing human CYP1A2 cells, in combination with a shuttle vector containing a reporter gene; and in Salmonella typhimurium TA98, by inhibition of cytochrome P450 enzymes in rat liver S9 mix.Both the HPRT assay and the Ames test did not show any involvement of CYP3A in the activation of 1-nitropyrene to a mutagenic metabolite. In addition, a clear involvement of CYP1A2 in the activation of the nitroPAH 1-nitropyrene was demonstrated in both mutation assays using eukaryotic cells. However, no activation of 1-nitropyrene was seen in the eukaryotic cell lines when expressing only CYP1A2 (V79-MZ1A2) or acetyltransferase (V79-NH, 3T3-LNCX). The reduced metabolite of 1-nitropyrene, 1-aminopyrene, was also shown to be activated to a mutagenic metabolite by CYP1A2, using 3T3-1A2 cells in combination with a shuttle vector, and the Amestest in combination with the specific CYP1A2 inhibitor furafylline. No clear involvement of cytochrome P450 could be demonstrated for activation of 2-nitrofluorene to a mutagenic metabolite, whereas a role for CYP1A2 in the bioactivation of 2-aminofluorene is suggested.In the present study, we have demonstrated the complementary value of the three in vitro mutation assays in the examination of promutagen activation pathways.