The relation between chemically induced sister-chromatid exchanges and chromatid breakage.

Studies of classical chromosome aberrations and sister-chromatid exchanges (SCES) suggest independent mechanisms for the two events despite some common features. Examination of chromosome breakage caused by X-rays, visible light, and viruses has shown that few chromatid breaks are accompanied by SCEs at the sites of breaks. No similar observations were available for chemically induced breaks, but it has been reported that rat chromosomes exposed to dimethylbenzanthracene (DMBA) contained a preponderance of both aberrations and SCEs in certain specific regions, implicating a common process in their formation. These conclusions were drawn from a comparison of breaks induced in vivo with SCEs induced in vitro. However, we used 7 chemical mutagens to induce both chromatid breaks and SCEs in "harlequin" chromosomes of cultured rat and Chinese hamster ovary (CHO) cells and found that 25% of the 914 breaks scored were associated with SCEs. The proportion of breaks accompanied by SCEs is related to the overall SCE frequency and falls into the range predicted on the basis that breaks and SCEs occur independently. The reported association between sites for SCEs and aberrations also reflects secondary factors, such as induction of SCEs and aberrations during DNA synthesis in late replicating regions of the chromosomes.     

Factors influencing the frequency of mitomycin C-induced sister-chromatid exchanges in 5-bromodeoxyuridine-substituted human lymphocytes in culture.

Newly developed techniques for the detection of sister-chromatid exchanges (SCE) require the substitution of 5-bromodeoxyuridine (BrdU) for thymidine in DNA. We investigated the possibility of interactions between BrdU and one mutagen--carcinogen, mitomycin C (MMC) for the induction of both chromosomal aberrations and SCE in human peripheral lymphocytes in culture. No effect on aberration yield was found. Neither comparisons between the yields of SCE by BrdU substitution and differential staining and those detected by tritiated thymidine incorporation and autoradiography nor between the yields of SCE for different levels of BrdU incorporation provided any evidence of synergism. It was found, however, that MMC persists in cultures and continues to increase SCE frequencies for about 30 h. It was also observed that some MMC-induced DNA lesions persist long enough so that some of those present prior to S phase of the first cell cycle cause additional SCE in the third cycle.     

The effects of high and low fluoride diets on the frequencies of sister chromatid exchanges

A recent report suggests that fluoride has mutagenic activity in mice. To examine the potential clastogenic effect of ingested fluoride, we examined the frequencies of baseline SCE and mitomycin C induced SCE as well as baseline chromosomal aberrations and cell-cycle kinetics in mice raised on high and low fluoride diets. The lack of significant differences in any of these parameters between the two groups of animals indicates that dietary fluoride is not clastogenic and supports the continued use of water fluoridation.     

The effects of high and low fluoride diets on the frequencies of sister chromatid exchanges

A recent report suggests that fluoride has mutagenic activity in mice. To examine the potential clastogenic effect of ingested fluoride, we examined the frequencies of baseline SCE and mitomycin C induced SCE as well as baseline chromsomal aberrations and cell-cycle kinetics in mice raised on high and low fluoride diets. The lack of significant differences in any of these parameters between the two groups of animals indicates that dietary fluoride is not clastogenic and supports the continued use of water fluoridation.     

Dependency of the yield of sister-chromatid exchanges induced by alkylating agents on fixation time. Possible involvement of secondary lesions in sister-chromatid exchange induction

Chinese hamster V79 cells were pulse-treated (for 60 min) with various mutagens three, two or one cell cycles before fixation (treatment variants A, B and C, respectively) and the frequencies of induced SCEs were analysed and compared. The degree of increase in frequency of SCEs with dose in the treatment variants depended on the mutagen used. For the methylating agents MNU, MNNG and DMPNU, high yields of SCEs were obtained in the treatment variants A and B, and there was no difference in the efficiency with which these agents induced SCEs in these treatment variants. In the treatment variant C, however, no SCEs were induced with mutagen doses yielding a linear increase in SCE frequency in treatment variants A and B. A slight increase in SCE frequency in treatment variant C was observed only when relatively high doses of MNU or MNNG were applied. Like the above agents, EMS, ENU and MMS induced more SCEs in treatment variants A and B than in C, but for these agents treatment variant B was most effective and SCEs were induced over the entire dose range, also in treatment variant C. As opposed to the methylating and ethylating agents, MMC induced SCEs with high efficiency when treatment occurred one or two generations prior to fixation. There was no difference in SCE frequency between these treatment variants. MMC was completely ineffective for the induction of SCEs when treatment occurred three generations before fixation. The unexpectedly low SCE frequencies induced by the methylating and ethylating agents when treatment occurred one generation before fixation were not due to the exposure of cells to BrdU prior to mutagen treatment. From the results obtained, it is concluded that DNA methylation and ethylation lesions give rise to SCEs only with very low probability during the replication cycle after the lesion's induction, and that subsequent lesions produced during or after replication of the methylated or ethylated template (secondary lesions) are of prime importance for SCE formation after alkylation. For MMC, however, primary lesions seem to be most important for SCE induction.     

Effect of tumor promoter 12-O-tetradecanoyl-phorbol 13-acetate on induction of sister-chromatid exchanges in Chinese hamster V79 cells treated with mutagens

The effect of a tumor promoter, 12-O-tetradecanoyl-phorbol 13-acetate (TPA) alone and in combination with mitomycin C (MMC) or cyclophosphamide (CPP) on the induction of sister-chromatid exchanges (SCE) in Chinese hamster V79 cells was investigated. TPA alone at various doses and durations caused no increase of SCE frequency. MMC either at the dose of 0.03 or 0.003 μg/ml alone or in combination with TPA (2 μg/ml) all caused a significant increase of SCE frequencies. There was no difference in SCE frequencies between the cultures treated with MMC alone at 0.03 μg/ml and those treated with MMC plus TPA. However, cultures treated with MMC at 0.003 μg/ml plus TPA had significantly and consistently higher SCE frequencies than those treated with MMC alone at all durations. Treatment of CPP at 1 μg/ml activated by S9 mix caused significant increase of SCE frequencies. Surprisingly, the cultures treated with CPP, S9 mix plus TPA (2 μg/ml) caused a drastic reduction of SCE frequencies as compared to those treated with CPP and S9 mix only at all durations. These results indicate that TPA alone had no effect on SCE in V79 cells. TPA enhanced the SCE induction in V79 cells treated with MMC at a low dose, i.e. 0.003 μg/ml, but it inhibited SCE induction in cultures treated with the indirect mutagen CPP. Thus, TPA has no direct effect on genetic materials but it may indirectly alter the effects of a mutagen.     

Effects of 3-aminobenzamide on Chinese hamster cells treated with thymidine analogues and DNA-damaging agents. Chromosomal aberrations, mutations and cell-cycle progression

The nuclear enzyme, poly(ADP-ribose) synthetase is involved in the repair of damaged DNA. We report here the results obtained with 3-aminobenzamide (3AB), an inhibitor of this enzyme, on induced biological effects. 3AB increases the frequency of chromosomal aberrations induced by DMS, EMS, ENU, bleomycin and CldUrd. The magnitude of the effect is dependent on the type of chemical used, the combinations with DMS and EMS being the most potent ones. No potentiation was observed after treatment of cells with MMC. Mutation frequencies were determined on the HPRT locus and showed that 3AB did not increase the frequency of gene mutations induced by EMS, ENU and CldUrd. Cell-cycle progression is affected when cells are grown in medium containing CldUrd and 3AB, primarily when the inhibitor is present during the second cell cycle when substituted DNA becomes replicated. The extent of the effect depends on the amount of analogue incorporated and is independent of the presence of the analogue in the medium during the second cell cycle. Analysis of chromosomal aberrations in delayed G2 cells with the aid of the premature chromosome-condensation technique revealed numerous aberrations after incorporation of CldUrd and treatment with 3AB.     

DNA crosslinking, sister-chromatid exchange and specific-locus mutations

Chinese hamster ovary cells were treated with the DNA-crosslinking chemicals, mitomycin C (MMC) and porfiromycin (POR), and their monofunctional derivative decarbamoyl mitomycin C (DCMMC). After exposure, the cells were studied for the induction of sister-chromatid exchanges (SCEs) and mutations at the hypoxanthine phosphoribosyltransferase and adenine phosphoribosyltransferase loci. The frequency of SCEs varied significantly in successive sampling intervals, requiring the weighting of each interval by the percentage of second-division mitosis in that interval to obtain the mean SCE frequency for each dose. All 3 compounds were potent inducers of SCEs but weakly mutagenic. All 3 chemicals by concentration were approximately equally effective in inducing SCEs or mutations. When the induced SCEs and mutations were compared at equal levels of survival, DCMMC was slightly more effective than MMC or POR in inducing SCEs and somewhat less mutagenic. These results indicate that the DNA interstrand crosslink is not the major lesion responsible for the induction of SCE or mutation by these compounds.     

Mutagenicity testing in mammalian cells: I. Derivation of a chinese hamster ovary cell line heterozygous for the adenine phosphoribosyltransferase and thymidine kinase loci

As a first step in the development of a multiple-marker, mammalian cell mutagenesis assay system, we have isolated a Chinese hamster ovary (CHO) cell line that is heterozygous for both the adenine phosphoribosyltransferase (aprt) and thymidine kinase (tk) loci. Presumptive aprt^+/? heterozygotes with intermediate levels of APRT activity were selected from unmutagenized CHO cell populations on the basis of resistance to low concentrations of the adenine analog, 8-azaadenine. A functional aprt^+/? heterozygote with ～50% wild-type APRT activity was subsequently used to derive sublines that were also heterozygous for the tk locus. Biochemical and genetic characterization of one such subline, CHO-AT3-2, indicated that it was indeed heterozygous at both the aprt and tk loci. CHO-AT3-2 cells permitted single-step selection of mutants resistant to 8-azaadenine or 5-fluorodeoxyuridine, allowing quantitation and direct comparison of mutation induction at the autosomal aprt or tk loci, as well as in the gene involved in ouabain resistance or at the X-linked, hypoxanthine-guanine phosphoribosyltransferase (hgprt) locus. Significant dose-dependent increases in mutation frequency were observed for all 4 genetic markers after treatment of CHO-AT3-2 cells with ethyl methanesulfonate.