Evaluation of the genotoxicity of domoic acid in a hepatocyte-mediated assay with V79 Chinese hamster lung cells

Domoic acid, a recognized neurotoxin derived from contaminated samples of the blue mussel (Mytilus edulis L.), was analyzed for mutagenicity at 2 loci and for 2 cytogenetic parameters in a hepatocyte-mediated assay with V79 Chinese hamster lung fibroblasts. Genetic end-points measured were: mutation to 6-thioguanine resistance at the HGPRTase locus; mutation to ouabain resistance at the Na+,K+-ATPase locus; sister-chromatid exchange (SCE) and micronucleus frequency (MN). None of these genetic end-points was significantly affected by exposure to domoic acid at dose levels of 27.2 and 54.4 micrograms/ml with or without activation by freshly isolated rat liver hepatocytes. It was concluded that, within the limits of the test system employed, domoic acid was non-genotoxic to V79 cells.     

Combined mutagenicity of methyl methanesulfonate and ethyl methanesulfonate in Chinese hamster V79 cells.

The combined effects of methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) on the induction of 6-thioguanine (6TG)-resistant mutants and chromosome aberrations were examined in Chinese hamster V79 cells. Cells were simultaneously treated with EMS at a concentration of D20 and MMS at various concentrations for 3, 6 or 9 h. In other experiments cells were simultaneously treated with MMS at a concentration of D20 and EMS at various concentrations for 3, 6 or 9 h. The mathematical analysis of the combined effects of both chemicals for cell killing (cytotoxicity) and 6TG-resistant mutations indicates that synergistic interactions were observed for both cell killing and mutations induced by MMS and EMS. The frequency of chromosome aberrations induced by simultaneous treatment with MMS at a concentration of D20 and EMS at various concentrations for 3 h was additive. However, the frequency of chromosome aberrations induced by EMS at a concentration of D20 and MMS at various concentrations for 3 h was not significantly different from those induced by MMS alone.     

Lack of mutagenicity of synthetic pyrethroids in Salmonella typhimurium strains and in V79 Chinese hamster cells

Seven pyrethroids, i.e., cypermethrin, permethrin, deltamethrin, bioresmethrin, resmethrin, cismethrin and fenvalerate, were not found to be mutagenic in (a) Salmonella typhimurium strains TA100 or TA98 in the presence or absence of a rat liver activation system using the plate incorporation assay and fluctuation tests, or (b) V79 Chinese hamster cells in the presence or absence of hepatocytes.     

Toxicity and mutagenicity of 6 anti-cancer drugs in Chinese hamster V79 cells co-cultured with rat hepatocytes

Toxicity and induction of 6-thioguanine-resistant mutants in Chinese hamster V79 cells, co-cultured with or without isolated rate hepatocytes, by 6 anti-cancer drugs (cyclophosphamide, adriamycin, methotrexate, cytosine arabinoside, 6-mercaptopurine and vincristine) were studied. The effect of hepatocyte density on the cloning efficiency and recovery of mutants was found using dimethylnitrosamine as a positive control. In the absence of hepatocytes, this compound was neither toxic nor mutagenic to V79 cells, but in their presence it was highly mutagenic and extremely toxic. The cloning efficiency and mutation frequency of control (untreated) cells was unaffected by hepatocyte density. All the drugs were toxic to V79 cells, although different responses were found for certain of them depending upon whether hepatocytes were present or not. Cyclophosphamide and adriamycin were clearly mutagenic, and 6-mercaptopurine only weakly so. A slight mutagenic effect was seen for cytosine arabinoside, but both methotrexate and vincristine were negative. Here also, the presence or absence of hepatocytes was important.     

Genotoxicity of 1-nitronaphthalene in Chinese hamster V79 cells

1-Nitronaphthalene (1-NN) has been identified in the U.S. National Toxicology Program as a non-carcinogen showing some evidence of in vitro genotoxicity. We tested this compound in Chinese hamster V79 cells at 20-80 micrograms/ml with two endpoints: sister-chromatid exchange (SCE) and thioguanine resistance (TGR), with 5 repeat experiments. The SCE values in the presence of rat or hamster hepatocytes were consistently above the 95% and usually the 99% upper confidence limits for the corresponding control. Without hepatocyte activation, the control upper confidence limits were not exceeded except in one experiment in which the control SCE value was unusually low. TGR was scored both as proportion of plates with mutant colonies and as number of mutant colonies per plate. In 2 of 5 experiments, these values exceeded control 95% or 99% upper confidence limits; on the other hand, these values were substantially lower than those of the positive controls, dimethylbenz[a]anthracene (2.6 micrograms/ml) with activation and ethyl methanesulfonate (155 microgram/ml), which is direct-acting. For TGR, activation of 1-NN by either rat or hamster hepatocytes produced inconsistent results. Overall we would consider this compound to be a weak genotoxin, to which a cancer bioassay would be expected to be relatively insensitive.     

Mutagenicity of hydrogen peroxide in V79 Chinese hamster cells

Hydrogen peroxide (H2O2) was investigated for its potential to induce gene mutations in V79 Chinese hamster cells. Exposure of 2-3 X 10(6) cells/100-mm dish to 0.5-4.0 mM H2O2 for 1 h resulted in a concentration-dependent increase in the frequency of 6-thioguanine-resistant clones. At 4 mM H2O2 the mutation frequency was increased about 6-fold above that in controls and survival of the cells was reduced by 50%. Cytotoxicity was markedly increased at lower cell densities. When only 100-200 cells/100-mm dish were exposed to H2O2 for 1 h, 50% were killed at an H2O2 concentration as low as 60 microM. The results show that mutagenicity of H2O2 in mammalian cells in vitro has escaped attention previously because the concentrations tested were too low, presumably because the likely toxicity of H2O2 to V79 cells treated at high cell densities was overestimated.     

Mutagenicity of 4-hydroxynonenal in V79 Chinese hamster cells

4-Hydroxynonenal (HNE), a major product of the peroxidation of liver microsomal lipids, was examined for mutagenic activity at the hypoxanthine-guanine phosphoribosyltransferase locus in V79 Chinese hamster lung cells. At concentrations ranging from 10 to 45 microM, HNE induced a dose-dependent increase in the number of mutations to 6-thioguanine resistance, which reached the level of 4.7X baseline at the highest concentration tested.     

Genotoxic effects of paracetamol in V79 Chinese hamster cells

Paracetamol was studied for possible genotoxic effects in V79 Chinese hamster cells. Paracetamol (0.5 mM for 30 min) reduced the rate of DNA synthesis in exponentially growing V79 cells to about 50% of control. A further decrease in the DNA synthesis was seen during the first 30 min after termination of paracetamol exposure. Paracetamol (3 and 10 mM for 2 h) caused a small increase in DNA single-strand breaks, as measured by the alkaline elution technique. After 16 h elution, the amount of DNA retained on the filters was 79 and 70% of controls in cells treated with 3 and 10 mM paracetamol respectively. No indication of DNA damage was seen in measuring the effect of paracetamol (0.25-10 mM for 2 h) on unscheduled DNA synthesis in growth-arrested cultures of V79 cells. At the highest concentrations (3 and 10 mM paracetamol), decreased unscheduled DNA synthesis was observed. Also UV-induced DNA-repair synthesis was inhibited by 3 and 10 mM paracetamol. DNA-repair synthesis was, however, inhibited at a much higher concentration than that inhibiting replicative DNA synthesis. The number of sister-chromatid exchanges (SCE) increased in a dose-dependent manner on 2 h exposure to paracetamol from 1 mM to 10 mM. At the highest dose tested (10 mM), the number of SCE increased to 3 times the control value. Co-culturing the V79 cells with freshly isolated mouse hepatocytes had no further effect on the paracetamol induced sister-chromatid exchanges. The present study indicates that paracetamol may cause DNA damage in V79 cells without any external metabolic activation system added.     

Biphenyl and fluorinated derivatives: liver enzyme-mediated mutagenicity detected in Salmonella typhimurium and Chinese hamster V79 cells.

Hepatocarcinogenic polychlorinated and polybrominated biphenyls usually show negative results in in vitro mutagenicity assays. Problems in their testing result from their low water solubility and their slow rate of metabolism. We therefore investigated better soluble model compounds, namely biphenyl and its 3 possible monofluorinated derivatives. In the direct test, these compounds proved to be nonmutagenic in Salmonella typhimurium TA98 and TA100 (reversion to histidine prototrophy) and in Chinese hamster V79 cells (acquisition of resistance to 6-thioguanine). However, when the exposure was carried out in the presence of NADPH-fortified postmitochondrial fraction of liver homogenate from Aroclor 1254-treated rats, all 4 compounds showed mutagenic activity in V79 cells. 3-Fluorobiphenyl produced strong mutagenic effects in S. typhimurium TA100 as well, whereas the other biphenyls were inactive. In strain TA98, 3- and 4-fluorobiphenyl showed mutagenic activity. This mutagenicity was enhanced in the presence of 1,1,1-trichloropropene 2,3-oxide, an inhibitor of microsomal epoxide hydrolase, thus suggesting that epoxides may be active metabolites.     

Differences between survival, mutagenicity and DNA replication in MMS- and MNU-treated V79 hamster cells

After treatment with methyl methanesulfonate (MMS) or N-methyl-N-nitrosourea (MNU), the mutagenicity and survival of Chinese hamster V79 cells were investigated, as well as the inhibition of daughter DNA synthesis and, using the DNA unwinding technique and hydroxylapatite chromatography, the character of the newly synthesized DNA was studied. It was found that different cytotoxicity and mutagenicity of MMS and MNU was accompanied by different types of DNA synthesis inhibition. The treatment with the former compound resulted in a longer inhibition of DNA synthesis, while the treatment with the latter showed that as early as 2 h after exposure the percentage of nascent DNA increased. Shortly after the exposure to both alkylating agents, the newly synthesized DNA contained a higher number of gaps than control DNA, in dependence on the concentration used. During culturing after treatment, the character of nascent DNA in MMS-treated cells gradually returned to that of control DNA, while MNU-treated cells, for the whole time of our study, synthesized DNA with a larger number of gaps than control DNA. We suggest that the character of nascent daughter DNA reflects the occurrence of lesions in parental DNA. These are repaired within a shorter time in MMS- than in MNU-treated cells. The long-term persistence of lesions in the DNA of MNU-treated cells might be one of the factors responsible not only for the higher cytotoxic but also for the many times higher mutagenic effect of this alkylating agent.     

Recovery of thioguanine-resistant cells from Chinese hamster V79 spheroids

Multicell spheroids may prove useful in evaluting the interactions of mutagens with cells exposed in a tissue-like environment. However, direct comparisons among populations of Chinese hamster V79 spheroids of different sizes or with monolayers are complicated by the observation that as spheroids enlarge, the fraction of mutant cells resistant to 6-thioguanine (TG^r) gradually decreases from about 5 in 10⁵ to less than 1 in 10⁵. There appear to be at least 2 explanations for these observations. First, TG^r cells grow less well as spheroids than do 6-thioguanine-sensitive (TG^s) cells. Second, the clonal nature of spheroid growth means that small samples fo spheroids are likely to contain fewer pre-existing TG^r cells.     

Elimination of MNU-induced mutational lesions in V79 Chinese hamster cells

Studies were performed on the non-linear dose response for gene mutations induced by low doses of monofunctional methylating agents in V79 Chinese hamster cells. When treatment with methylnitrosourea was applied at the beginning of the S phase in synchronized cells, a linear dose-response curve was obtained, whereas application of the dose after gene replication resulted in a strong reduction of the number of induced mutations. Additional time for repair resulted in reduced dose response of MNU, indicating that an error-free repair process operates on methylated DNA in V79 Chinese hamster cells.     

Mutagenic characterization of cholesterol epoxides in Chinese hamster V79 cells

The uptake, metabolism and alkylating properties of the diastereomeric cholesterol epoxides were studied using Chinese hamster lung fibroblasts (V79 cells). Specific emphasis is given to the comparative cyto- and geno-toxic effects of cholesterol 5 beta,6 beta-epoxide (beta CE) and cholesterol 5 alpha,6 alpha-epoxide (alpha CE) and data are provided for the first time indicating that beta CE can induce more 6-thioguanine-resistant cells than alpha CE. Cholesterol 5 beta,6 beta-epoxide induced colonies of cells resistant to 6-thioguanine at 2-3-fold the frequencies observed with the alpha-isomer, but neither compound produced ouabain-resistant colonies. The cytotoxicity (LD50) of alpha CE was estimated to be 45-50 microM whereas beta CE displayed an LD50 of 25-29 microM. Inhibition of DNA synthesis (IC50) was observed over the same dose ranges as the LD50 for each epoxide isomer. The epoxides were assimilated by cells to an equal extent, however, beta CE was metabolized to cholestane 3 beta,5 alpha-6 beta-triol twice as rapidly as the alpha-isomer. Both epoxides reacted with 4-(4'-nitrobenzyl)-pyridine to a similar extent, and with identical nucleophilic selectivity at pH 7.4, but their alkylating activity was estimated on this basis to be two orders of magnitude less than methyl methanesulfonate. Binding experiments with the DNA or cultured V79 cells or with calf-thymus DNA indicated that interactions were noncovalent and DNA binding did not correlate with the potency of the epoxides to induce the 6-thioguanine-resistant phenotype. Our results could be interpreted as indicating that both cholesterol epoxide isomers are weak mutagens or that they might induce some epigenetic event repressing the hypoxanthine guanine-phosphoribosyltransferase gene. The similarity of the epoxides' alkylating activity and their DNA-binding properties are inconsistent with their different potencies in inducing the 6-thioguanine-resistant phenotype, suggesting that the mechanism leading to this phenotype is not necessarily the result of DNA alkylation.     

Mutagenicity of some intercalating agents in Chinese hamster V79 cells

ICR 170 and ICR 191, but not 9-aminoacridine or chloroquine, induced both 6-thioguanine- and, to a smaller extent, ouabain-resistance in Chinese hamster V79 cells. These results indicate that covalent binding to DNA is necessary for intercalating agents to induce mutation in this cell line, and that this assay can distinguish potential carcinogens from non-carcinogenic analogues of this chemical type. The induction of ouabain-resistance by both ICR 170 and ICR 191 indicates that these frameshift mutagens induce base-pair substitution to some extent in V79 cells.     

Genotoxicity of 2,4,6-trichlorophenol in V79 Chinese hamster cells.

The genotoxicity of the rodent carcinogen 2,4,6-trichlorophenol (TCP) was studied without exogenous metabolic activation in V79 Chinese hamster cells. TCP did not induce mutation at the hprt locus to 6-thioguanine resistance or structural chromosome aberrations. However, it produced statistically significant, dose-related increases in hyperdiploidy and micronuclei. From these results it appears that TCP causes chromosome malsegregation as its major mode of genotoxic action.