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.     

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.     

Critical importance of microsome concentration in mutagenesis assay with V79 Chinese hamster cells.

For optimum mutagensis in V79 Chinese hamster cells, the amount of liver postmitochondrial fraction in the assay was found to be of critical importance, depending on the chemicals being tested. Benzo[a]pyrene (BP) required lower (1-5%) concentrations of the liver 15 000 X g supernatant (S15) from methylcholanthrene pretreated rats for a maximum induction of cytotoxicity and mutagenicity, as determined by 8-azaguanine- and ouabain-resistance. A sharp peak of mutagenicity and cytotoxicity was induced by 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (7,8-diol BP) at a concentration of 1% of the S15 fraction. Little or no response was induced by these compounds with the S15 concentrations of more than 10%. Similarly, aflatoxin B1 induced a sharp peak of mutagenicity and cytotoxicity at a concentration of 2% of the liver S15 fraction from Aroclor-pretreated rats. Under the same condition, non-carcinogenic aflatoxin G2 did not induce cytotoxicity and mutagenicity. Analysis of BP metabolites by high-pressure liquid chromatography indicates that with the 30% S15 fraction, more than 80% of BP was metabolized during the first 15 min, while with the 2% S15 fraction, 7,8-diol BP increased continuously throughout the 120-min incubation period, suggesting a strong metabolic competition to rapidly remove BP and 7,8-diol BP with a high concentration of the S15. In contrast with these compounds, N-nitrosodimethylamine induced mutagenicity and cytotoxicity which increased linearly in proportion to the increasing amount of the S15 fraction from phenobarbitone- and Aroclor-pretreated rats. Various nitrosamines with different lipophilicity were examined at a high (30%) and low (2%) concentration of the S15 fraction from Aroclor-pretreated rats, in which ratios of mutation frequencies at 30% and 2% correlated inversely with lipophilicity of the compound. This result suggests that the lipid solubility of test compounds may be one factor which determines the concentration of post-mitochondrial supernatant for optimum mutagenesis.     

Sodium butyrate affects the cytotoxic and mutagenic response of V79 Chinese hamster cells to the genotoxic agents, daunorubicin and U.V. radiation

It has been suggested that conditions which lead to modifications in the chromatin structure could be responsible for an increased accessibility of DNA to genotoxic agents in eukaryotic cells. With this in mind, the cytotoxic and mutagenic activity of the anthracycline antibiotic, daunorubicin, and of UV radiation was assayed on V79 Chinese hamster cells pretreated or not with 5 mM sodium butyrate, an agent known to induce modifications in the chromatin structure: this treatment in fact proved to induce the hyperacetylation of the core histones, and moreover to enhance the cytotoxic response of the cells to both daunorubicin and UV radiation and the mutagenic response to daunorubicin.     

The elimination of mutagenic lesions induced by alkylating agents and UV in V79 Chinese hamster cells

The mutagenicity of MNU, EMS, BMS and UV light was compared by analyzing the dose-response curve just before and after the replicative process of the HGPRT gene in synchronized V79 Chinese hamster cells. This system makes it possible to compare a 10-h period for repair of different mutagenic lesions with no time for repair. Additional time for repair in synchronized V79 cells resulted in a reduced response for MNU and UV, but not for EMS and BMS. This result suggests that an error-free repair process operates on mutagenic lesions in methylated DNA and on thymine dimers, but not on ethylated and butylated DNA. Based on these results, it is concluded that the repair capacity of V79 cells to remove mutagenic lesions is characterized as low for UV, moderate for MNU and not detectable for the mutagenic lesions induced by EMS and BMS.     

Inhibition of DNA synthesis and cytotoxic effects of some DNA topoisomerase II and gyrase inhibitors in Chinese hamster V79 cells

In this study, some DNA topoisomerase II and gyrase inhibitors have been identified as inhibitors of polymerization of deoxyribonucleotides [novobiocin (NVB), nalidixic acid (NDA), oxolinic acid (OXA)], or inhibitors of replicon initiation and DNA-chain elongation [etoposide (VP-16), teniposide (VM-26), 4'-(9-acridinylamino)methansulfon-m-anisidine (m-AMSA), ellipticine (ELT)]. The inhibitors of deoxyribonucleotide polymerization produced a significant (greater than 85%) suppression of [3H]thymidine incorporation into V79 cells within 20 min of treatment, followed by a rapid recovery of DNA synthesis, and reduced cell killing. In contrast, the inhibitors of replicon initiation and DNA-chain elongation needed about 60 min to induce a partial, but irreversible inhibition of DNA replication, associated with extensive cell killing.     

The effect of thymidine on the induction of micronuclei by alkylating agents in V79 Chinese hamster cells

Incubation in thymidine-containing medium resulted in increased lethality and micronucleus frequency in V79 cells treated with ethyl nitrosourea (ENU), methyl nitrosourea (MNU) and ethyl methanesulphonate (EMS) but not with methyl methanesulfonate (MMS). Thymidine had no effect in ENU treated HeLa cells. In V79 cells, the presence of thymidine during post-treatment DNA replication was necessary for the effect. It is suggested that the increase in chromosome damage was the result of an increased O⁶-alkylguanine-thymine mispairing in cells which are defective in the repair of O⁶-alkylguanine. Treatment of V79 cells with O⁶-ethylguanine resulted in increased production of both micronuclei and polyploid cells. These effects might be explained by spindle dysfunction caused by the alkylated guanine.     

Cytogenetic effects of 3,4-dichloroaniline in human lymphocytes and V79 Chinese hamster cells

3,4-Dichloroaniline (3,4-DCA), an intermediate in various chemical syntheses, has been detected as an environmental contaminant in surface waters and in the effluents from dye-manufacturing plants. Tested for clastogenicity in human lymphocytes in vitro the compound was inactive in the chromosome aberration assay yet exhibited a positive sister-chromatid exchange response in the presence of a mammalian metabolic activation system. Exposure of V79 Chinese hamster cells to 3,4-DCA caused a concentration-dependent increase in the incidence of spindle disturbances, predominantly of the initial c-mitotic type. The results indicate that 3,4-DCA might induce aneuploidy in mammalian cells by interaction with the mitotic apparatus.     

Activation of N-acetoxy- and N-hydroxy-2-acetylaminofluorene to mutagenic and cytotoxic metabolites by V79 Chinese hamster cells

N-Acetoxy-2-acetylaminofluorene (AAAF) and N-hydroxy-2-acetylaminofluorene (OH-AAF) are mutagenic to V79 cells, causing the induction of 6-thioguanine-resistant clones, and are cytotoxic. The presence of the deacetylase inhibitor, paraoxon, drastically reduces both the mutagenic and cytotoxic effects. This strongly suggests that deacetylated metabolites are the major active species. Furthermore, when Salmonella typhimurium TA98 is used as target organism, addition of homogenate of V79 cells strongly potentiates the mutagenicity of OH-AAF. To our knowledge, this is the first report demonstrating a significant biological effect due to the metabolism of a mutagen by V79 cells.     

V79 Chinese hamster cells deacetylate Trans-N-acetoxy-4-acetylaminostilbene and Trans-N-hydroxy-4-acetylaminostilbene to mutagenic and cytotoxic metabolites

The N-acetoxy and N-hydroxy derivatives of trans-4-acetylamino-stilbene (AAS) were demonstrated to induce gene mutations at the hgprt locus and to be cytotoxic in V79 cells. These cells deacetylated AAS. Paraoxon inhibited the deacetylation of AAS by more than 99% and reduced the mutagenicicity and cytotoxicity of N-hydroxy-ASS and N-acetoxy-AAS to about one-tenth. Hence, deacetylated metabolites, formed by the target cells, were important for the observed biological effects.Abbreviations AAF 2-acetylaminofluorene - AAS trans-4-acetylaminostilbene - AS trans-4-aminostilbene     

2-Chlorobenzylidene malonitrile (CS) causes spindle disturbances in V79 Chinese hamster cells

Exposure of V79 Chinese hamster cells to 2-chlorobenzylidene malonitrile (CS), a chemical used as a sensory irritant for riot control, caused a concentration-dependent increase in the incidence of spindle disturbances. A C-mitotic effect with the appearance of C-metaphases, a metaphase block and the concomitant disappearance of ana-telophase figures were observed after a 3-h treatment. The results indicate that CS might induce aneuploidy in mammalian cells by interacting with the mitotic apparatus.     

Clastogenicity of 2-chlorobenzylidene malonitrile (CS) in V79 Chinese hamster cells.

The clastogenicity of the sensory irritant 2-chlorobenzylidene malonitrile (CS) to V79 Chinese hamster cells was investigated at various exposure conditions. CS efficiently induced chromatid-type aberrations in a dose-dependent manner provided the cells could run through at least one or two S-phases during a 20-h exposure over a 3-h exposure followed by a 20-h recovery period (cell cycle time 8-10 h). The induction of SCEs indicates an S-dependent mechanism. The hydrolysis products o-chlorobenzaldehyde and malonitrile were inactive in these experiments.     

Biological effects of incorporation of O6-methyldeoxyguanosine into Chinese hamster V79 cells

Analysis of the biological effects of specific DNA alkylations by simple alkylating agents is complicated by the variety of sites involved. It is, therefore, of value to be able to incorporate into cellular DNA nucleosides alkylated in a single position, e.g., O⁶-methyldeoxyguanosine. Such cellular incorporation is particularly difficult to achieve because this nucleoside is rapidly demethylated by adenosine deaminase. We have attempted to achieve such incorporation into the DNA of V79 cells by using coformycin, an inhibitor of adenosine deaminase, and by forcing the cells to depend on exogenous purines by the use of medium containing aminopterin. The DNA of V79 cells exposed to O⁶-methyl-[8-³H]deoxyguanosine (2.4 μM, sp. act. 14 500 Ci/mole) showed an incorporation level of 4 × 10⁻⁸ nucleotides. When 1000-fold higher concentrations were employed (3–15 mM, sp. act. 1.6 Ci/mole), significant cytotoxicity and inhibition of DNA synthesis was observed. However, because it was not economically feasible to administer high specific activity O⁶-methyldeoxyguanosine to the cells at these concentrations, we could not determine the amount of labeled nucleoside incorporated into DNA. Examination of the frequency of 6-thioguanine-resistant cells in these treated populations showed no significant increase above the background level. Comparison of the cytotoxic effect of O⁶-methyldeoxyguanosine with deoxyadenosine showed that the toxicity induced by O⁶-methyldeoxyguanosine could have resulted from mimicry of deoxyadenosine, rather than by incorporation of the alkylated nucleoside itself.     

Further studies on tetracycline-induced mutation in V79 Chinese hamster cells

The interaction between ultraviolet light and tetracycline in producing cell killing and mutation has been studied in V79 Chinese hamster cells. It has been established that these agents act independently of each other. Cycloheximide altered the response to tetracycline in the fractionation experiment: when cycloheximide was not present, fractionation of TC treatment resulted in a higher mutation yield but no change in survival level; in the presence of cycloheximide, however, mutation was greatly reduced but survival increased. The results were taken to indicate that for tetracycline action to take place, de novo protein synthesis during tetracycline treatment was necessary. Caffeine had no influence on tetracycline-induced lethality or mutagenicity. This observation was considered to suggest that tetracycline did not affect cellular repair processes.     

Biological effects of incorporation of O-methyldeoxyguanosine into Chinese hamster V79 cells

Analysis of the biological effects of specific DNA alkylations by simple alkylating agents is complicated by the variety of sites involved. It is, therefore, of value to be able to incorporate into cellular DNA nucleosides alkylated in a single position, e.g., O⁶-methyldeoxyguanosine. Such cellular incorporation is particularly difficult to achieve because this nucleoside is rapidly demethylated by adenosine deaminase. We have attempted to achieve such incorporation into the DNA of V79 cells by using coformycin, an inhibitor of adenosine deaminase, and by forcing the cells to depend on exogenous purines by the use of medium containing aminopterin. The DNA of V79 cells exposed to O⁶-methyl-[8-³H]deoxyguanosine (2.4 μM, sp. act. 14 500 Ci/mole) showed an incorporation level of 4 × 10⁻⁸ nucleotides. When 1000-fold higher concentrations were employed (3–15 mM, sp. act. 1.6 Ci/mole), significant cytotoxicity and inhibition of DNA synthesis was observed. However, because it was not economically feasible to administer high specific activity O⁶-methyldeoxyguanosine to the cells at these concentrations, we could not determine the amount of labeled nucleoside incorporated into DNA. Examination of the frequency of 6-thioguanine-resistant cells in these treated populations showed no significant increase above the background level. Comparison of the cytotoxic effect of O⁶-methyldeoxyguanosine with deoxyadenosine showed that the toxicity induced by O⁶-methyldeoxyguanosine could have resulted from mimicry of deoxyadenosine, rather than by incorporation of the alkylated nucleoside itself.