Sodium arsenite enhances the cytotoxicity, clastogenicity, and 6-thioguanine-resistant mutagenicity of ultraviolet light in Chinese hamster ovary cells

Cytotoxicity, chromosome aberrations, and mutations to 6-thioguanine resistance were synergistically increased by incubating the ultraviolet light (UV)-irradiated Chinese hamster ovary (CHO) cells in medium containing sodium arsenite. However, the frequencies of sister-chromatid exchanges and mutations to ouabain resistance induced by UV were not synergistically increased by sodium arsenite. The synergistic effect of sodium arsenite on UV-induced chromosome aberrations varied with cell-harvesting time and decreased with increasing time intervals between UV and sodium arsenite treatments.     

Pesticide clastogenicity in Chinese hamster ovary cells

Paraquat, alachlor, butachlor, phorate and monocrotophos, several of the most extensively used pesticides in Taiwan, were investigated for their clastogenicity using chromosome aberration (CAb) induction in Chinese hamster ovary (CHO) cells. Significance levels of the binomial trend analysis and binomial mutagenicity data test were two criteria for the summary judgement of the pesticide clastogenicity. Except for phorate, all pesticides tested were clastogenic to CHO cells in the absence of in vitro metabolic activation by S9. 5 microliters/ml rat-liver extract, S9, were used as the source of in vitro metabolic activation. 3 different outcomes were found after the addition of S9. Paraquat: significant decrease in induced CAbs. Monocrotophos: concomitant occurrence of decreased cytotoxicity and increased clastogenicity. Alachlor, butachlor and phorate: increased cytotoxicities with no sign of enhancement in clastogenicity.     

Single-strand breaks in DNA of hamster and human cells exposed to methyl methanesulfonate and ultraviolet light

The number of single-strand breaks produced in DNA after exposure to UV light or to methyl methanesulfonate (MMS) was additive when cells were exposed to both agents in close succession. Repair of the damage from either agent was partially inhibited by cytosine arabinoside, resulting in higher break frequencies under all conditions of exposure. Exposure to both agents followed by growth in cytosine arabinoside resulted in break frequencies that were approximately the same as the sum of those from each agent individually. These findings contrast with previous results in which pyrimidine dimer excision and repair replication after exposure to UV light were inhibited by MMS. These observations are not due to cell permeability changes after alkylation, but can be explained if the complex of excision-repair proteins is only partially inactivated by alkylation. Initial incisions to start repair would still occur but only limited amounts of repair replication would ensue without actual removal of the pyrimidine dimers.     

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.     

Methylisobutylxanthine, a potent inhibitor of cAMP phosphodiesterase, enhances the sensitivity of Chinese hamster ovary cells to mitomycin C

The effect of 1-methyl-3-isobutylxanthine (MIX) on the sensitivity of CHO cells to mitomycin C (MC) was examined. Treatment of cells with MIX before MC-exposure greatly enhanced the cellular sensitivity to MC, with no effect on plating efficiency. Treatment with MIX after MC-exposure had only a slight effect. The sensitivity to MC was enhanced, biphasically, with increases in the period of pretreatment with MIX. The sensitivity of cells to MC fluctuated through the cell cycle, in that they were most sensitive to MC in G1 and resistant in the G2/M phase. Flow cytometric studies revealed a partial accumulation of cells in G1 (12% increase) at 16 h after MIX treatment. This means that the enhanced sensitivity with MIX pretreatment may partly depend on the accumulation of the cells in G1 and which are sensitive to MC. However, other cellular processes may also be involved, since the accumulation in G1 cannot explain all of the sensitization.     

Plasmid-dosage effects on ultraviolet, visible light and methyl methanesulfonate mutagenesis in Salmonella typhimurium

Salmonella typhimurium LT2 strains bearing plasmids pKM101, R64 or pColIb-P9 demonstrated enhanced UV survival when compared with strains not bearing plasmids. A strain of S. typhimurium bearing both pKM101 and pColIb-P9 survived UV irradiation slightly better than either of the single-plasmid strains. Spontaneous reversion of the hisG46 and trpE8 missense alleles was enhanced in each single-plasmid strain, and for the dual-plasmid strain containing pKM101 and pColIb-P9 enhancement represented a near additivity of the response seen for the single-plasmid strains. Following exposure to UV or visible-light irradiation, reversion of hisG46 and trpE8 was also enhanced in each single-plasmid strain, but quantitatively greater in the dual-plasmid strain and was equal to or slightly greater than additive the responses of the single-plasmid strains. In contrast to visible-light irradiation, UV exposure resulted in two phenotypic Trp+-revertant classes. One Trp+ class, having normal colony size (2.0 mm) and similar in number to His+ revertants, was comprised of intragenic revertants of trpE8, while the predominant Trp+ class, having smaller colony size (0.8 mm), represented intergenic suppressor revertants, illuminating the differences in mutation and/or repair specificity for UV and visible-light exposure. Methyl methane-sulfonate (MMS)-induced reversion of hisG46 was similar in effect to that seen with UV or visible-light irradiation. Plasmids pKM101 or pColIb-P9 enhanced the frequency of hisG46 reversion, while a more than additive response was seen in a strain with both plasmids. Furthermore, MMS-induced reversion of hisG46 was also observed to be greatest in a strain bearing plasmid R64 (incompatibility group I alpha) and pKM101, when compared with single-plasmid strains bearing either R64 or pKM101.     

Effects of vanillin on sister-chromatid exchanges and chromosome aberrations induced by mitomycin C in cultured Chinese hamster ovary cells

Effects of vanillin on the induction of sister-chromatid exchanges (SCEs) and structural chromosome aberrations by mitomycin C (MMC) were investigated in cultured Chinese hamster ovary cells. Vanillin induced neither SCEs nor chromosome aberrations by itself. However, an obvious increase in the frequency of SCEs was observed when MMC-treated cells were cultured in the presence of vanillin. The effect of vanillin was S-phase-dependent. On the contrary, the frequency of cells with chromosome aberrations was significantly decreased by the post-treatment with vanillin at G2 phase.     

Effect of ultraviolet light on thymidine incorporation, DNA chain elongation and replicon initiation in wild-type and excision-deficient Chinese hamster ovary cells

Wild-type Chinese hamster ovary cells (AA8) and five excision-deficient clones derived from the AA8 line (UV-4, UV-5, UV-20, UV-24 and UV-41) were exposed to ultraviolet light and then analyzed for their ability to incorporate [3H]thymidine and to initiate as well as elongate replicon-sized DNA fragments. After exposure to ultraviolet light, all cell lines exhibited a depression in the rate of thymidine incorporation. For exposures of 4.0 J/m2 or higher the wild-type cells recovered normal rates of thymidine incorporation within a few hours, while none of the excision-deficient lines exhibited complete recovery. For fluences below 4.0 J/m2 all but the UV-5 line exhibited at least some recovery. The ability to elongate DNA chains appeared to correlate with the thymidine incorporation data, with the UV-5 line exhibiting the strongest blockage of DNA chain elongation, the AA8 line exhibiting the least blockage, and the UV-20 line exhibiting an intermediate response. All cell lines exhibited a decrease in the distance between replication origins, thus supporting models which propose that exposure to ultraviolet light results in the use of alternative sites for the initiation of replication.     

3-Aminobenzamide synergistically increases sister-chromatid exchanges in cells exposed to methyl methanesulfonate but not to ultraviolet light

3-Aminobenzamide, an inhibitor of poly(ADP-ribose) synthesis, increased baseline sister-chromatid exchange (SCE) frequencies and acted synergistically with the alkylating agent methyl methanesulfonate to induce exchanges in Chinese hamster ovary and SV40-transformed human (GM637) cells. In contrast, 3-aminobenzamide did not affect the frequency of ultraviolet light-induced SCEs. Our data suggest that, in these 2 cell types, synthesis of poly(ADP-ribose) is more important in damage and repair after exposure to an alkylating agent than after exposure to ultraviolet light.     

DNA base sequence changes induced by ultraviolet light mutagenesis of a gene on a chromosome in Chinese hamster ovary cells

The DNA base sequence changes induced by mutagenesis with ultraviolet light have been determined in a gene on a chromosome of cultured Chinese hamster ovary (CHO) cells. The gene was the Escherichia coli gpt gene, of which a single copy was stably incorporated and expressed in the CHO cell genome. The cells were irradiated with ultraviolet light and gpt- colonies were selected by resistance to 6-thioguanine. The gpt gene was amplified from chromosomal DNA by use of the polymerase chain reaction (PCR), and the amplified DNA sequenced directly by the dideoxy method. Of the 58 sequenced mutants of independent origin 53 were base change mutations. Forty-one base substitutions were single base changes, ten had two adjacent (or tandem) base changes, and one had two base changes separated by a single base-pair. Only one mutant had a multiple base change mutation with two or more well separated base changes. In contrast much higher levels of such mutations were reported in ultraviolet mutagenesis of genes on a shuttle vector in primate cells. Two deletions of a single base-pair were observed and three deletions ranging from 6 to 37 base-pairs. The mutation spectrum in the gpt gene had similarities to the ultraviolet mutation spectra for several genes in prokaryotes, which suggests similarities in mutational mechanisms in prokaryotes and eukaryotes.     

Reverse transformation of Chinese hamster ovary cells by methyl xanthines : Structure-function relationships

Using a number of drugs that increase cellular cAMP levels, alterations in the amount of cell surface fibronectin and other transformation parameters were studied in Chinese hamster ovary (CHO) cells. The drugs include db-cAMP, different methylxanthines (theophylline, aminophylline, methyl isobutyl xanthine (MIX), caffeine and theobromine), papaverine and cholera toxin. Methylxanthines that have a methyl group at the seventh position lack reverse transforming potential; those that lack a methyl group at the seventh position induced reverse transformation in CHO cells, causing an increase in surface fibronectin, cell substratum adhesive strength and anchorage dependence for growth. Further, as methyl xanthines are substituted in other positions different from the seventh position, the more efficient they become in restoring normal phenotypic properties; the later agents also induced low saturation density via a cytostatic state causing accumulation of cells in the S and G2 phases of the cycle in contrast to the G1 arrest of normal cells at low saturation density. db-cAMP and cholera toxin induced cell elongation but like caffeine and theobromine, did not induce surface fibronectin. The non-methylxanthine phosphodiesterase inhibitor papaverine induced neither cell elongation nor surface fibronectin but produced a cytostatic effect similar to aminophylline and MIX. These studies suggest that the reverse transformation properties fall into two groups: (a) Differentiation-related properties including cell morphology, parallel alignment and surface matrix fibronectin, etc.; (b) cell cycle-related properties-low saturation density, cell arrest at G1 phase and anchorage-dependent growth. Phosphodiesterase inhibitors reversibly eliminate indefinite division potential of CHO cells by inducing a cytostatic situation and not by inducing a G1-specific arrest.     

The nature of ultraviolet light-induced mutations at the heterozygous aprt locus in Chinese hamster ovary cells

A system for studying mutational specificity at a heterozygous locus in Chinese hamster ovary (CHO) cells is described. The strategy employed is based on restriction fragment analysis and DNA sequencing of enzymatically amplified mutant adenine phosphoribosyltransferase (aprt) alleles. We have demonstrated the usefulness of this approach through the characterization of a collection of aprt- mutants with respect to the role played by loss of heterozygosity events in ultraviolet light (UV) induced mutagenesis. A similar strategy has also been applied to speculate on the identity of the premutational lesion responsible for a UV-induced mutational hotspot at the aprt locus.     

Effects of an inhibitor and a mimic of superoxide dismutase on bleomycin mutagenesis in Chinese hamster ovary cells

We have investigated the roles of reactive oxygen species (ROS) in bleomycin (BLM)-induced gene mutations in Chinese hamster ovary (CHO) cells using a superoxide dismutase (SOD) inhibitor, triethylenetetramine (TRIEN), and a SOD mimic, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), to lower and increase intracellular “SOD activity”, respectively. Pretreatment of CHO cells with TRIEN (1 mM) for 1 h enhanced the mutagenic response of BLM (5–50 μg/ml, 1 h treatment) in the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in CHO cell clone K1-BH4 (CHO/HPRT assay) and the xanthine-guanine phosphoribosyltransferase (gpt) gene in a CHO-K1 cell derivative AS52 (AS52/GPT assay). Pretreatment with TEMPOL (1 mM) for 1 h decreased the BLM (20–100 μg/ml, 1 h treatment) mutagenicity in the AS52/GPT assay. The mutagenic response of BLM appears to be modulated by the intracellular level of ‘SOD activity’ and hence the intracellular level of ROS. These data provide further evidence for the involvement of ROS in bleomycin mutagenesis in mammalian cells.     

Differences in sensitivity between human, mouse and Chinese hamster cells to killing by monochromatic ultraviolet light

Irradiation of human (IMR-91), mouse (10T1/2) and Chinese hamster (V79) fibroblasts with monochromatic ultraviolet light (u.v.) in the far-, mid-, and near-u.v. regions resulted in cell-strain-specific changes in sensitivity as a function of the wavelength used. The data suggested cell-strain-specific action spectra for cell killing by ultraviolet light that did not correlate with the ability of examined cells to excise pyrimidine dimers.     

Microsome-mediated clastogenicity of butylated hydroxyanisole (BHA) in cultured Chinese hamster ovary cells: the possible role of reactive oxygen species

Butylated hydroxyanisole (BHA) was found to induce chromosome aberrations in Chinese hamster ovary (CHO) cells in the presence of Aroclor-induced rat-liver S9. The effects were more marked when washed microsomes were employed and chromosome damage was considerably reduced in the presence of catalase, suggesting that hydrogen peroxide was involved. Stimulation of H2O2 production by BHA in S9 or microsome incubation mixtures was demonstrated using the catalase-mediated production of formaldehyde from methanol. One of the major microsomal metabolites of BHA, tert.-butyl hydroquinone (t-BHQ), which autoxidises in solution producing H2O2 also induced extensive catalase-sensitive chromosome damage in the absence of metabolic activation. These observations suggest that extracellular generation of reactive oxygen species may be implicated in the mechanism of BHA clastogenicity in vitro. However, chromosome damage was not completely abolished by catalase and the end product of t-BHQ oxidation, tert.-butyl quinone, was also weakly clastogenic, suggesting that intracellular effects of quinone metabolites may also be involved in the clastogenicity of BHA.