Carolyn L. Doerr

Micronucleus, chromosome aberration, and small-colony TK mutant analysis to quantitate chromosomal damage in L5178Y mouse lymphoma cells☆

In testing the hypothesis that the small-colony thymidine kinase-deficient mutants of L5178Y/TK+/- -3.7.2C mouse lymphoma cells represent an estimate of the clastogenicity of test chemicals, we have been performing gross aberration analysis. The present study was initiated to determine if the cytokinesis block method of micronucleus analysis could be performed in mouse lymphoma cells and to compare 3 different endpoints of clastogenicity: the number of metaphases with aberrations, number of binucleates with micronuclei, and small-colony TK mutant frequency. In this study, 12 compounds having varying clastogenic potencies were evaluated. As would be expected, the 3 endpoints vary in the relative magnitude of the quantitated response. This difference likely results from the types of clastogenic damage detected by each endpoint. Of the 3 endpoints tested, only the small-colony TK mutant frequency measures events compatible with long-term cell survival.

Mutagenicity and clastogenicity of teniposide (VM-26) in L5178Y/TK +/- -3.7.2C mouse lymphoma cells.

The antitumor drug teniposide (VM-26) is a potent inducer of DNA breaks (Long et al., Cancer Res., (1985) 45, 3106), but it is only weakly mutagenic at the hprt locus in CHO cells (Singh and Gupta, Cancer Res., (1983) 43, 577). In the present study, the mutagenic and clastogenic activities of teniposide were evaluated in L5178Y/TK +/- -3.7.2C mouse lymphoma cells. Although teniposide is a weak mutagen at the hprt locus, it is a potent mutagen at the tk locus, with as little as 0.5 ng/ml producing 220 TK mutants/10(6) survivors at 96% survival (background = 100/10(6) survivors). This same dose of teniposide induced 38 aberrations per 100 metaphases (background = 7/100 cells). At 7 ng/ml, teniposide induced approximately 2700 TK mutants/10(6) survivors at approximately 10% survival. At the highest dose sampled for aberration analysis (5 ng/ml), teniposide induced 44 aberrations/100 cells. Most of the aberrations were chromosomal rather than chromatid events. As expected for a compound acting primarily by a clastogenic mechanism, most of the TK mutants were small colonies. Thus, teniposide is a potent clastogen, and it is a potent mutagen at the tk locus but not at the hprt locus. These results support the hypothesis that the location of the target gene affects the ability of the assay to detect both intragenic events and events causing functional multilocus effects. Thus, a heterozygous locus (like tk) but not a functionally hemizygous locus (like hprt) may permit the detection of mutagens that act primarily by a clastogenic mechanism. Because teniposide induces topoisomerase II-associated DNA breaks, and because there is evidence that teniposide may not interact directly with DNA, we discuss the possibility that the potent clastogenic/mutagenic activity of teniposide may be mediated by topoisomerase II.

Genotoxicity of γ-irradiation in L5178Y mouse lymphoma cells☆

Abstract The ability of γ-irradiation to induce gene mutation at the thymidine kinase locus and gross chromosome aberrations in L5178Y TK+/− 3.7.2C mouse lymphoma cells was evaluated. Positive results were obtained for both end-points. The majority of mutants were found to be small-colony mutants which correlated with the induction of gross chromosome aberrations.

Cytogenetic studies of mice exposed to styrene by inhalation

The data for the in vivo genotoxicity of styrene (STY) are equivocal. To evaluate the clastogenicity and sister-chromatid exchange (SCE)-inducing potential of STY in vivo under carefully controlled conditions, B6C3F1 female mice were exposed by inhalation for 6 h/day for 14 consecutive days to either 0, 125, 250 or 500 ppm STY. One day after the final exposure, peripheral blood, spleen, and lungs were removed and cells were cultured for the analysis of micronucleus (MN) induction using the cytochalasin B-block method, chromosome breakage, and SCE induction. Peripheral blood smears were also made for scoring MN in erythrocytes. There was a significant concentration-related elevation of SCE frequency in lymphocytes from the spleen and the peripheral blood as well as in cells from the lung. However, no statistically significant concentration-related increases were found in the frequency of chromosome aberrations in the cultured splenocytes or lung cells, and no significant increases in MN frequencies were observed in binucleated splenocytes or normochromatic erythrocytes in peripheral blood smears.

Inhalation studies of the genotoxicity of trichloroethylene to rodents

Trichloroethylene (TCE) (CAS No. 79-01-6) is an industrial solvent used in degreasing, dry cleaning, and numerous other medical and industrial processes. Controlled inhalation studies were performed using male C57BL/6 mice and CD rats to determine if TCE can induce cytogenetic damage in vivo. Animals were exposed in groups of five to target concentrations of either 0, 5, 500, or 5000 ppm TCE for 6 h. Tissue samples were taken between 18 and 19 h post exposure. Peripheral blood lymphocytes (PBLs) in rats and splenocytes in mice were cultured and analyzed for the induction of sister-chromatid exchanges, chromosome aberrations, and micronuclei (MN) in cytochalasin B-blocked binucleated cells. Bone marrow polychromatic erythrocytes (PCEs) were analyzed for MN. The only positive response observed was for MN in rat bone marrow PCEs. TCE caused a statistically significant increase in MN at all concentrations, inducing an approximate fourfold increase over control levels at 5000 ppm. TCE was also cytotoxic in rats, causing a significant concentration-related decrease in the ratio of PCEs/normochromatic erythrocytes. This study indicates that there may be species-specific cytogenetic effects attributed to TCE inhalation exposure. In follow-up studies, CD rats were exposed for 6 h/day over 4 consecutive days to either 0, 5, 50 or 500 ppm TCE. No statistically significant concentration-related increases in cytogenetic damage were observed. While the MN frequencies in the 4-day study were comparable to those at the equivalent concentrations in the 1-day study, they were not significantly elevated due to an unusually high MN frequency in the controls. A subsequent replication of the 1-day 5000 ppm TCE exposure with rats again showed a highly significant increase in MN frequencies compared to concurrent controls.

Mutagenicity and clastogenicity of proflavin in L5178Y/TK+/−−3.7.2.C cells

We evaluated the ability of proflavin to induce specific-locus mutations at the heterozygous thymidine kinase (tk) locus of L5178Y/TK +/- -3.7.2C mouse lymphoma cells, which appears to permit the recovery of mutants due to single-gene and chromosomal mutations. Proflavin was highly mutagenic at the tk locus, producing 724-965 TK mutants/10(6) survivors (background = 56-85/10(6); survival = 29-32%). Most of the mutants were small colonies, which suggested that proflavin may induce chromosomal mutations. The potent clastogenicity of proflavin was confirmed by cytogenetic analysis for chromosomal aberrations. At the highest dose analyzed (1.5 micrograms/ml), proflavin produced 82 aberrations/100 metaphaes (background = 2/100). The large-colony TK mutant frequency produced by proflavin (48-109/10(6) survivors; background = 23/10(6); survival = 57-61%) was similar to published HPRT mutant frequencies produces by proflavin in L5178Y and CHO cells (50-100/10(6) survivors; background = 2-50/10(6); survival = 50-62%). These results lead to the conclusion that proflavin is a potent clastogen and induces a high frequency of small-colony TK mutants; however, it induces a low frequency of HPRT mutants and a low frequency of large-colony TK mutants.

Mutagenicity and clastogenicity of adriamycin in L5178Y/TK+/−-3.7.2C mouse lymphoma cells

Adriamycin was found to be both mutagenic and clastogenic to L5178Y/TK(+/-)-3.7.2C mouse lymphoma cells. A dose of only 5 ng/ml (survival = 62% or 67%) gave an induced TK mutant frequency of 307 or 296 per 10(6) survivors in two separate experiments. This dose was also clastogenic, inducing 20 chromosome aberrations/100 cells analyzed. The majority of the mutants were small-colony mutants, indicating that adriamycin likely acts primarily by a clastogenic mechanism.

Mutagenicity of actinomycin D in mammalian cells due to clastogenic effects.

Actinomycin D was clastogenic and mutagenic in L5178Y/TK +/- -3.7.2C mouse lymphoma cells. The majority of the mutants were small colonies, indicating that actinomycin D acts primarily by a clastogenic mechanism.

Genotoxicity of three pyridine compounds to L5178Y mouse lymphoma cells

The L5178Y mouse lymphoma assay was used to examine the potential mutagenicity of three halogenated pyridine compounds. Position effects of the halogen moiety and the role of metabolic activation were analyzed based on induced mutant frequency, gross chromosome aberrations, and micronuclei. Without activation, 2-chloropyridine, 3-chloropyridine, and 2-chloro-5-trifluoromethylpyridine produced a small increase in mutant frequency; only the 2-chloropyridine activity was significantly increased with activation. All three compounds were also clastogenic as demonstrated by increases in chromosome aberrations and micronuclei (except for 2-chloro-5-trifluoromethylpyridine which did not induce micronuclei either with or without activation).

Genotoxicity of 2-amino-6-N-hydroxyadenine (AHA) to mouse lymphoma and CHO cells.

2-Amino-6-N-hydroxyadenine (AHA) treated L5178Y/TK (+/-)-3.7.2C mouse lymphoma cells were evaluated for mutations at the tk, hgprt, and Na+/K+ ATPase loci, as well as for gross chromosome aberrations and induction of micronuclei. In addition, AHA was evaluated for its ability to induce HGPRT mutants in CHO cells. AHA was found to induce mutations at all evaluated loci and in both cell types. The TK mutants were primarily large colonies although a few small colonies were also induced, particularly at the higher concentrations. Preliminary cytogenetic analysis of AHA-treated mouse lymphoma cells indicated that some gross aberrations but not micronuclei were induced. The 20 small-colony TK mutants evaluated by banded karyotype indicate that only a small fraction (2 of 20) showed chromosome 11 abnormalities. From these studies, it appears that AHA may be one of a very few chemicals that is capable of inducing multi-locus point mutations, with only slight clastogenic activity. Particularly at the higher concentrations, some of the mutants may contain multi-locus point mutations that result in slow growth.