Comparative effectiveness in the induction of sister chromatid exchanges and chromosome aberrations

The dose curves for 5 chemicals were studied to compare the efficiency of induction of SCEs and chromosomal aberrations by "polycentric" mutagens. The number of SCEs was found to increase linearly with the dose while that of chromosomal aberrations--nonlinearly. The efficiency of SCEs induction by these mutagens was found to be 25-50 times as high as in the induction of chromosomal aberrations. Division of alkylating mutagens into "monocentric" and "polycentric" is shown to be useful. It reflects their different efficiency in damaging one or simultaneously two DNA strands. The correlation between SCEs and formation of aberrations of the chromatid type is stated.     

Antagonization by cycloheximide of ethyl methanesulfonate-induced 6-thioguanine-resistant mutation and sister-chromatid exchanges

We studied the effect of cycloheximide (CH) on the induction of mutation and sister-chromatid exchanges (SCEs) in ethyl methanesulfonate (EMS)-treated Chinese hamster cells. At 10^?6 M, CH strongly antagonized the induction of mutation and SCEs and cell survival increased. This suggests that protein synthesis is essential for the induction of mutation as well as SCEs. Results of experiments in which CH treatment preceded or followed exposure to mutagens were similar with respect to the response curves obtained for mutation and SCEs.     

Comparison of the levels of chromosome aberration and sister chromatid exchange induced by chemical mutagens in vitro

Dose dependencies of the induction of sister chromatid exchanges (SCEs) and chromosome aberrations were studied under in vivo exposure of mouse bone marrow cells to 5 alkylating agents. The efficacy of the induction of SCEs for all the substances was 20 to 60 times higher than that of the induction of chromosome aberrations. It was demonstrated that SCEs induced by chemical mutagens in vivo and in vitro are more sensitive tests than chromosome aberrations.     

The influence of incorporated bromodeoxyuridine on mutagenicity testing by sister chromatid exchange induction inVicia faba root tip cells

The induction of sister chromatid exchanges (SCEs) inVicia faba root-tip cells after short-term (2 h) and long-term (24 h) treatments with alkylating agents (N-methyl-N-nitrosourea, ethyl methanesulphonate) and maleic hydrazide was studied. The primary roots were treated with mutagens before or after 5-bromodeoxyuridine (BrdU) incorporation into DNA and the influence of mutagen application on SCE induction in the cells with non- and BrdU-substituted chromosomal DNA. On the contrary, application of maleic hydrazide after the incorporation of BrdU into DNA strongly increased the rate of SCEs. The lowest limit concentrations of mutagens capable of significantly increasing SCE frequency in the cells with non-substituted DNA after the long-term treatment were estimated.     

Correlation of the sister chromatid exchange level with chromosome aberrations induced by chemical mutagens in vivo

The data on the dose dependencies of the induction of sister chromatid exchanges (SCE) and chromosomal aberrations during exposure of mouse bone marrow cells in vivo to 5 alkylating substances are provided. The efficacy of SCE induction was found to be higher than that of chromosomal aberrations. It was established that SCE induced by chemical mutagens in vivo and in vitro are more sensitive and stable tests than chromosomal aberrations.     

Chromosomal instability in mutagen-sensitive mutants isolated from mouse lymphoma L5178Y cells. II. Abnormal induction of sister-chromatid exchanges and chromosomal aberrations by mutagens in an ionizing radiation-sensitive mutant (M10) and an alkylating agent-sensitive mutant (MS1)

To determine the mutual relationships between cell survival and induction of sister-chromatid exchanges (SCEs) as well as chromosomal aberrations (CAs), mutagen-induced SCEs and CAs were analyzed in an ionizing radiation-sensitive mutant (M10) and an alkylating agent-sensitive mutant (MS 1) isolated from mouse lymphoma L5178Y cells. The levels of CA induction in both mutants strictly corresponded to the sensitivity to lethal effects of mutagens, except that caffeine-induced CAs in M10 are considerably lower than those in L5178Y. The results clearly indicate that except for caffeine-induced CAs in M10, mutagen-induced lethal lesions are responsible for CA induction. In contrast, SCE induction in mutants was complicated. In M10, hypersensitive to killing by gamma-rays, methyl methanesulfonate (MMS), and 4-nitroquinoline 1-oxide (4NQO), but not sensitive to UV or caffeine, the frequency of SCEs induced by gamma-rays was barely higher than that in L5178Y, and the frequencies of MMS- and UV-induced SCEs were similar to those in L5178Y, but 4NQO- and caffeine-induced SCEs were markedly lower than those in L5178Y. MS 1, which is hypersensitive to MMS and caffeine, but not sensitive to UV or 4NQO, responded to caffeine with an enhanced frequency of SCEs and had a normal frequency of MMS-induced SCEs, but a reduced frequency of UV- and 4NQO-induced SCEs. Thus, susceptibility to SCE induction by mutagens is not necessarily correlated with sensitivity of mutants to cell killing and/or CA induction by mutagens. Furthermore, the spontaneous levels of SCEs are lower in M10 and higher in MS 1 than that in L5178Y (Tsuji et al., 1987). Based on these results, we speculate that M10 may be partially defective in the processes for the formation of SCEs caused by mutagens. On the other hand, MS 1 may modify SCE formation-related lesions induced by UV and 4NQO to some repair intermediates that do not cause SCE formation. In addition, MMS-induced lethal lesions in MS 1 may not be responsible for SCE induction whereas caffeine-induced lethal lesions are closely correlated with SCE induction. Thus, the lesions or mechanisms involved in SCE production are in part different from those responsible for cell lethality or CA production.     

Bimodal distribution of sensitivity to SCE induction by diepoxybutane in human lymphocytes. I. Correlation with chromosomal aberrations

We carried out a cross-sectional analysis of sister-chromatid exchanges (SCEs) and chromosomal aberrations induced by diepoxybutane (DEB) in lymphocyte cultures from 58 normal blood donors. DEB-induced SCE frequencies were measured in all subjects and chromosomal aberrations in 18. Analysis of variance was used to assess the contributions of exposure to organic solvents, age, smoking history, alcohol and coffee consumption, and red and white blood cell counts to variations in DEB-induced SCEs. In 10 individuals, the epoxide-detoxifying enzyme, glutathione (GSH)-S-transferase mu, was also measured. We observed a bimodal distribution of DEB-induced SCEs in the study population. Approx. 24% of the individuals were twice as sensitive to the induction of SCEs by DEB as the remaining 76%. Lymphocytes from persons sensitive to SCE induction by DEB contained a 4.4-fold increase in the number of DEB-induced chromatid deletions and exchanges. Within sensitive and resistant groups, significant interindividual variations in DEB-induced SCE frequencies were noted. Cigarette smoking was weakly associated with lower SCE frequencies within each group. Genetic deficiency in GSH-S-transferase mu was not correlated with increased sensitivity to SCE induction by DEB. Sensitivity to induction of SCEs by DEB can be rapidly determined and may be a marker of sensitivity to the induction of genotoxicity by certain classes of mutagens.     

小鼠体内脾脏,骨髓及精原细胞姐妹染色单体交换的比较研究

本文对几种化学诱变剂诱发小鼠体内脾脏、骨髓和精原细胞的SCE进行了比较研究,同时分析了几类常见化合物在小鼠脾脏细胞中诱发SCE的活力。结果显示诱变剂在脾脏细胞中诱发SCE比骨髓和精原细胞敏感。几类化合物都能显著地诱发小鼠脾脏SCE的增加,与对照相比差异显著(P<0.05)或极显著(P<0.01),说明利用小鼠脾脏细胞检测环境诱变物是相当灵敏的。     

Cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6. III. Induction of cell killing, chromosomal aberrations and sister-chromatid exchanges by bleomycin, mono- and bi-functional alkylating agents

Two X-ray-sensitive mutants of CHO-K1 cells, xrs 5 and xrs 6, were characterised with regard to their responses to genotoxic chemicals, namely bleomycin, MMS, EMS, MMC and DEB for induction of cell killing, chromosomal aberrations and SCEs at different stages of the cell cycle. In addition, induction of mutations at the HPRT and Na+/K+ ATPase (Oua) loci was evaluated after treatment with X-rays and MMS. Xrs 5 and xrs 6 cells were more sensitive than wild-type CHO-K1 to the cell killing effect of bleomycin (3 and 13 times respectively) and for induction of chromosomal aberrations (3 and 4.5 times). In these mutants a higher sensitivity for induction of chromosomal aberrations to MMS, EMS, MMC and DEB was observed (1.5-3.5 times). The mutants also showed increased sensitivity for cell killing effects of mono- and bi-functional alkylating agents (1.7-2.5 times). The high cell killing effect of X-rays in these mutants was accompanied by a slight increase in the frequency of HPRT mutation. The xrs mutants were also more sensitive to MMS for the increased frequency of TGr and Ouar mutants when compared to wild-type CHO-K1 cells. Though bleomycin is known to be a poor inducer of SCEs, an increase in the frequency of SCEs in xrs 6 cells (doubling at 1.2 micrograms/ml) was found in comparison to no significant increase in xrs 5 or CHO-K1 cells. The induced frequency of SCEs in all cell types increased in a similar way after the treatment with mono- or bi-functional alkylating agents. MMS treatment of G2-phase cells yielded a higher frequency of chromatid breaks in the mutants in a dose-dependent manner compared to no effect in wild-type CHO-K1 cells. Treatment of synchronised mutant cells at G1 stage with bleomycin resulted in both chromosome- and chromatid-type aberrations (similar to the response to X-ray treatment) in contrast to the induction of only chromosome-type aberrations in wild-type CHO-K1 cells. The frequency of chromosomal aberrations chromosome and chromatid types) also increased with MMC treatment in G1 cells of xrs mutants. DEB treatment of G1 cells induced mainly chromatid-type aberrations in all cell types. The possible reasons for the increased sensitivity of xrs mutants to the chemical mutagens studied are discussed and the results are compared to cells derived from radiosensitive ataxia telangiectasia patients.     

Studies on the influence of photoreactivation on the frequencies of UV-induced chromosomal aberrations, sister-chromatid exchanges and pyrimidine dimers in chicken embryonic fibroblasts

Chicken embryonic fibroblasts, which possess photoreactivating enzyme were used to study the influence of photoreactivating light on the induction of pyrimidine dimers, sister-chromatid exchanges (SCEs) and chromosomal aberrations by 254 nm UV. While photoreactivation (PR) efficiently removed most of the induced dimers (75-95%), the frequencies of SCEs and chromosomal aberrations were reduced only by about 30-65%, in parallel experiments. Since pyrimidine dimers are the only photoreactivable photolesions known, the reduction in the frequencies of SCEs and chromosomal aberrations on PR has been interpreted as due to disappearance of pyrimidine dimers, implying that these lesions are the primary events responsible for the induction of the biological end points studied. The possible reasons for the lack of quantitative relationship between the frequencies of dimers and the frequencies of SCEs and chromosomal aberrations are discussed.     

Induction and persistence of micronuclei, sister-chromatid exchanges and chromosomal aberrations in splenocytes and bone-marrow cells of rats exposed to ethylene oxide

Studies on the induction and persistence of ethylene oxide (EO) induced chromosomal alterations in rat bone-marrow cells and splenocytes following in vivo exposure were carried out. Rats were exposed to ethylene oxide either chronically by inhalation (50-200ppm, 4 weeks, 5 days/week, 6h/day) or acutely by intraperitoneal injection (i.p.) at dose levels of 50-100ppm.Spontaneous- and induced-frequencies of micronuclei (MN), sister-chromatid exchanges (SCEs) and chromosomal aberrations were determined in rat bone-marrow cells, and in splenocytes following in vitro mitogen stimulation. Unstable chromosomal aberrations were studied in whole genome using standard Giemsa staining technique and fluorescence in situ hybridisation using probe for chromosome #2 was employed to detect chromosome translocations.Following chronic exposure, the cytogenetic analyses were carried out at days 5 and 21 in rat splenocytes, to study the induction and persistence of sister-chromatid exchanges. Following chronic exposure, ethylene oxide was effective in inducing SCEs, and markedly cells with high frequency SCEs were observed and they in-part persisted until day 21 post-exposure. However, no significant effect was observed in rat splenocytes for induction of MN and chromosomal aberrations. Following acute exposure, both SCEs and MN were increased significantly in rat bone-marrow cells as well as splenocytes.In conclusion, this study indicates that ethylene oxide at the concentrations employed by intraperitoneal injection or inhalation in adult rats is mutagenic and can induce both SCEs and MN.     

Sister chromatid exchange (SCE) and structural chromosome aberration in mutagenicity testing

Summary Data from previous studies published on the induction by mutagens of sister chromatid exchanges (SCEs) and structural chromosome damage were compared qualitatively and quantitatively. Although a good correlation between the incidence of both cytogenetic phenomena has been pointed out in many previous publications, about 30% of the agents for which comparable data were available yielded non-corresponding qualitative results concerning both indicator effects. However, even in the group with good qualitative agreement distinct quantitative differences indicated different molecular mechanisms of the formation of SCEs and breaks. Additional information supporting the importance of these differences for the validity of both indicator systems has been derived from the results obtained using strong clastogens exhibiting a low or no SCE-inducing activity and vice versa, from special observations on chromosomal breakage syndromes, and from studies on the action of known co- and anti-clastogens on SCE-induction by chemical mutagens. As a result, it has been suggested that the SCE-technique should be considered as a valuable additional method for cytogenetic mutagenicity testing, which, however, is not adequate to replace the classical methods of analysis of structural chromosome damage.     

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.     

Genotoxic effects of o-phenylphenol metabolites in CHO-K1 cells

The effects of microsomal activation and/or deactivation on the induction of chromosomal aberrations and sister-chromatid exchanges (SCEs) in cultured Chinese hamster ovary cells (CHO-K1 cells) by o-phenylphenol (OPP) were studied, and concurrently the metabolites were determined. After a 3-h incubation in the presence of 15% S9 mix (45 microliters/ml of S9), OPP (25-150 micrograms/ml) dose-independent SCEs and chromosomal aberrations were induced, while the amount of phenylhydroquinone (PHQ) metabolite produced from OPP did not increase linearly in the higher doses. The maximum induction of chromosomal aberrations was 18% at the 150 micrograms/ml dose, and of SCEs 13.8/cell at 75 micrograms/ml. The corresponding control values were 3% and 5.8/cell. The lowest dose required to induce SCEs in the presence of S9 mix was 25 micrograms/ml. Changing the percent of S9 mix (0-50%) while holding the OPP dose constant (100 micrograms/ml) produced a correlation between SCEs and the production of PHQ. PHQ caused cytogenetic effects both with and without S9 mix, however, in the absence of S9 mix it was more lethal and was oxidized to phenylbenzoquinone (PBQ). These results suggest that the enhanced cytogenetic effects of OPP by the addition of S9 mix correlated with the amount of PHQ produced or with the further oxides of PHQ such as phenylsemiquinone and/or PBQ which are capable of being produced from PHQ spontaneously or by the mixed-function oxidase system.     

Cytogenetical characterisation of Chinese hamster 43-3B transferants with the amplified or non-amplified human DNA repair gene ERCC-1

A comparative study on the biological responses to different mutagens (UV, 4NQO, MMC, MMS and EMS) was made on CHO wild-type cells (CHO-9), its UV-hypersensitive mutant 43-3B, and 2 types of its transferants, i.e., one containing a few copies of the human repair gene ERCC-1 and the other having more than 100 copies of ERCC-1 (due to gene amplification). Cell survival, chromosomal aberrations and SCEs were used as biological end-points. The spontaneous frequency of chromosomal aberrations in the transferants was less than found in 43-3B mutant cells, but still 2-3 times higher than in wild-type CHO cells. The spontaneous frequency of SCEs in the transferants was less than in 43-3B and similar to that of wild-type cells. The induction of SCEs by all tested agents in transferants was similar to that found in CHO-9 cells, while the mutant is known to respond with higher frequencies. ERCC-1 also bestowed resistance to MMS and EMS on the mutant to induction of chromosomal aberrations and cell killing to levels comparable with those of the wild-type strain. On the other hand ERCC-1 could not completely regain the repair proficiency against cell killing and induction of chromosomal aberrations by UV or MMC to the wild-type level. These results suggest that the ERCC-1 corrects the repair defect in CHO mutant cells, but it is unable to rectify fully the defect; probable reasons for this are discussed. However, amplified transferants (having more than 100 copies of the ERCC-1 gene) restored the impaired repair function in 43-3B to UV-, MMC- or 4NQO-induced DNA damage better than non-amplified transferants with a few copies of the ERCC-1. This difference may be due to the high amount of gene product involved in the excision repair process in the amplified cells.     

Genotoxic effects of sodium arsenite on human cells.

The effects of sodium arsenite (SA) were studied either alone or in combination with X-rays in peripheral blood lymphocytes, and with short-wave ultraviolet (UV) radiation in primary human fibroblast culture systems. It was found that SA (i) inhibited the cell cycle progression of phytohaemagglutinin (PHA)-responsive lymphocytes, (ii) induced chromatid-type aberrations and sister-chromatid exchanges (SCEs) as a function of concentration and (iii) potentiated the X-ray- and UV-induced chromosomal damage. Our results suggest that SA interferes with the DNA repair process, presumably by inhibiting the ligase activity. This accounted for an increase in the DNA replication-dependent processes, chromatid aberrations and SCEs and synergistic enhancement of the X-ray- and UV-induced chromosomal damage. This ability of arsenite may be responsible for its comutagenic properties with different types of mutagens and hence its carcinogenicity.     

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.     

Chemical induction of streptomycin-resistant mutations in Escherichia coli. Dose and mutagenic effects of dichlorvos and methyl methanesulfonate

A procedure for the quantitative determination of induced streptomycin-resistant mutants in E. coli was applied to study and compare mutation induction by the organophosphate dichlorvos and by methyl methanesulfonate (MMS). Both compounds increased the frequency of mutants even under conditions where no inactivation of cell was observed. Mutation induction by these agents as a function of both concentration and exposure time was measured. The dose-response curves found with both mutagens were non-linear; atp higher doses more mutants were induced per unit dose than at lower doses. Possible relationships between dose-effect curves and the chemical nature of alkylating mutagenic agents are discussed.     

Modulating effects of beta-naphthoflavone on the induction of SCEs by model compounds with emphasis on benzo[a]pyrene

The inducing capability of the synthetic flavonol beta-naphthoflavone (beta-NF) on cytochrome P-450 content was studied in primary chick embryo hepatocytes. In addition, the modulating effects of pretreatment with beta-NF on the induction of sister-chromatid exchanges (SCEs) in V79 cells by mutagens from different chemical classes were investigated in a co-cultivation system consisting of primary chick embryo hepatocytes and V79 Chinese hamster cells. Finally, the effects of pretreatment on benzo[a]pyrene (B(a)P) metabolism were studied in more detail. Pretreatment of cultured primary chick embryo hepatocytes with beta-NF resulted in a large increase in cytochrome P-450 content (a 2.8-fold increase after 31 h). Pretreatment with beta-NF had no effect on the level of SCEs induced by N-nitroso-dimethylamine (NDMA) and 2-aminoanthracene (2AA). Pretreatment with beta-NF resulted in a decrease in B(a)P-induced SCEs. This inhibitory potential was positively related to the beta-NF dose. However, there was an inverse relationship between the inhibitory action of beta-NF and the dose of B(a)P, at higher doses less inhibition was observed. When beta-NF was applied simultaneously with B(a)P the percentage of decrease was about the same as for pretreatment. Pretreatment with beta-NF followed by simultaneous application of beta-NF and B(a)P did not result in larger effects. In addition, subcellular fractions were prepared from chick embryos pretreated with beta-NF in ovo. The use of the S9 fraction resulted in a large decrease (80%) in the induction of SCEs in V79 cells by B(a)P whereas the use of the microsomal fraction resulted in a 70% increase in SCE induction compared with non-pretreated microsomes. Pretreatment with beta-NF in ovo gave rise to a large increase in aryl hydrocarbon hydroxylase (AHH) activity in the hepatic microsomal fraction. Increases were observed in the formation of all B(a)P metabolites. In particular the formation of the proximate carcinogenic and mutagenic metabolite B(a)P-7,8 dihydrodiol was increased 7-fold. The data strongly suggest that the inhibitory effects of pretreatment of cultured primary chick embryo hepatocytes with beta-NF cannot be ascribed to its inducing capabilities but instead seem to be due to the formation of an intracellular pool of beta-NF which acts as a competitive inhibitor for B(a)P metabolism.     

Differences in the induction of SCEs between human whole blood cultures and purified lymphocyte cultures and the effect of an S9 mix

Studies for SCE induction are frequently performed on human blood cultures. Either whole blood cultures (WBC) or purified lymphocyte cultures (PLC) are employed. However, it has been shown that fundamental differences with respect to metabolic activity exist between these two systems. In order to further characterize the whole blood culture and the purified lymphocyte culture, differently acting substances were studied comparatively with and without an Aroclor-1254-induced S9 mix. Treatment with ethyl methanesulfonate (EMS), a direct mutagen, produced distinct SCE induction in both systems. Cyclophosphamide (CP) and benzo[a]pyrene (BP), two indirect mutagens, also led to a significant increase of SCEs both in WBC and PLC without S9 mix. Only with CP was this effect more pronounced after addition of S9 mix. Sodium selenite (Na2SeO3), which induced SCEs in WBC, did not show this effect in the PLC. After S9 mix was added to purified lymphocytes, an increase of SCEs by sodium selenite was observed as in WBC. H2O2, a radical former, led to SCE induction in purified lymphocytes but not in the whole blood culture. By adding S9 mix, a distinct reduction of the SCEs induced by H2O2 was established. These results show that human lymphocytes can metabolize indirect mutagens and that it should be kept in mind when using S9 mix that, besides mixed-function oxygenases, it also contains enzymes which influence the SCE-inducing effects of substances.