Clastogenic Damage Research Articles

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.

The role of specific DNA adducts in the induction of micronuclei by N-hydroxy-2-acetylaminofluorene in rat liver in vivo.

N-Hydroxy-2-acetylaminofluorene (N-OH-AAF) was administered i.p. to male Wistar rats 17 h after partial hepatectomy. Hepatocytes were analyzed for the presence of micronuclei 7 h, 1, 2, 3 and 4 days after injection. N-OH-AAF treatment resulted in a high frequency of micronucleated hepatocytes at days 3 and 4 (19.5% and 19.6% respectively). The frequency of micronucleated hepatocytes was not increased above control values when hepatocytes were isolated as early as 7 h, 1 or 2 days after injection. Pretreatment with the sulfotransferase inhibitor pentachlorophenol (PCP) 45 min before injection of N-OH-AAF almost completely prevented the formation of micronuclei by N-OH-AAF. Parallel biochemical studies indicated that inhibition of sulfation of N-OH-AAF by PCP pretreatment prevented the formation of the N-acetylated DNA adducts N-deoxyguanosin-8-yl-AAF and 3-deoxyguanosin-N2-yl-AAF by approximately 85%. Total adduct formation to DNA was, however, not lowered because of an increase in the formation of the deacetylated adduct, N-deoxyguanosin-8-yl-AAF. The lower frequency of micronucleated hepatocytes observed in the group pretreated with PCP, did not result from less proliferative activity in this group as compared to the group treated with N-OH-AAF alone. Therefore, the decrease in the formation of micronuclei indicates that PCP prevents the clastogenic damage caused by N-OH-AAF. It is concluded that the clastogenicity of N-OH-AAF in rat liver is related to the formation of N-acetylated DNA adducts (i.e. N-deoxyguanosin-8-yl-AAF and/or 3-deoxyguanosin-N2-yl-AAF) and is not related to the formation of the deacetylated DNA adduct N-deoxyguanosin-8-yl-AF.

Association of Marginal Folate Depletion With Increased Human Chromosomal Damage In Vivo: Demonstration by Analysis of Micronucleated Erythrocytes

Recent studies have demonstrated that in the absence of spleen function, frequencies of micronuclei (Howell-Jolly bodies) in peripheral blood rbcs can be used to measure in vivo cytogenetic damage. Among 20 subjects studied greater than or equal to 6 months after splenectomy, 1 had a frequency of micronucleated rbcs more than an order of magnitude higher than rates for the others. Initial data suggested that this subject was mildly folate-depleted, and a therapeutic trial with folate rapidly reduced the frequency of micronucleated rbcs to normal values. These observations suggest a need to evaluate further the contribution of mild levels of folate depletion to spontaneous chromosomal damage. The approach used here provides a sensitive index of clastogenic damage and offers unique opportunities for investigating the determinants of cytogenetic damage in humans.

Tests for urethane induction of germ-cell mutations and germ-cell killing in the mouse

Urethane, a chemical that has given varied results in mutagenesis assays, was tested in the mouse specific-locus test, and its effect on germ-cell survival was explored. Altogether 32 828 offspring were observed from successive weekly matings of males exposed to the maximum tolerated i.p. dose of 1750 mg urethane/kg. The combined data rule out (at the 5% significance level) an induced mutation rate greater than 1.7 times the historical control rate. For spermatogonial stem cells alone, the multiple ruled out is 3.2, and for poststem-cell stages, 3.5. Litter sizes from successive conceptions made in any of the first 7 weeks give no indication of induced dominant lethality, confirming results of past dominant-lethal assays. That urethane (or an active metabolite) reaches germ cells is indicated by SCE induction in spermatogonia demonstrated by other investigators. Cytotoxic effects in spermatogonia are suggested by our finding of a slight reduction in numbers of certain types of spermatogonia in seminiferous tubule cross-sections and of a borderline decrease in the number of litters conceived during the 8th and 9th posttreatment weeks. The negative results for induction of gene mutations as well as clastogenic damage are at variance with Nomura's reports of dominant effects (F 1 cancers and malformations) produced by urethane.

DNA synthesis, mitotic index, drug-metabolising systems and cytogenetic analysis in regenerating rat liver: Comparison with bone marrow test after ‘in vivo’ treatment with cyclophosphamide

Rat-liver cells can be used to reveal “in vivo” clastogenic activity of indirect mutagens, provided that they are stimulated to divide by partial hepatectomy. In order to characterize the rat-liver metabolic capacity in such experimental conditions, several biochemical parameters were measured during the first 54–66 h of liver regeneration in Sprague-Dawley male rats, subjected to a partial hepatectomy. The levels of cytochrome P-450, the activities of styrene monooxygenase, epoxide hydrolase and glutathione- S-epoxide transferase were chosen as markers. All the enzymatic activities and the level of cytochrome P-450 decreased during the first 12 h after the hepatectomy to about 50% of the activities of the sham-operated rats considered as controls. Subsequent recovery of the metabolic capacity was not observed. DNA synthesis and the mitotic index were measured to find the most suitable time for metaphase analysis. DNA synthesis and the number of metaphases were maximal at, respectively, 22–25 and 28–31 h after partial removal of the liver. The sensitivity to clastogenic damage induced by “in vivo” treatment with cyclophosphamide (CPA) was assayed in regenerating liver cells by chromosome-aberration analysis. Different doses, ranging from 5 to 30 mg/kg b.w., were given i.p. to the rats 17 h before or 7 h after partial hepatectomy. Liver cells were collected 31 h after surgery. Clastogenic damage was greater when the drug was administered to the animals after the hepatectomy (24 h of exposure) than before (48 h of exposure). The sensitivity to CPA-induced damage was compared with a bone marrow cell test carried out on non-hepatectomized rats treated in the same way. The results indicated that in these conditions regenerating liver cells are more sensitive than bone marrow cells to the induction of chromosome aberrations by CPA.

Cytogenetic damage induced in human lymphocytes by adriamycin and vincristine: A comparison between micronucleus and chromosomal aberration assays

A quantitative comparison was made between the induction of micronuclei and of chromosomal aberrations in human lymphocytes using a clastogen (adriamycin) and a non-disjunctional agent (vincristine) as model compounds. Cytochalasin B, an inhibitor of cytokinesis, was used to make it possible to evaluate the frequency of micronuclei only in cells that had divided after the treatment. The results showed that for the assessment of clastogenic damage due to adriamycin, the sensitivity of the micronucleus assay was comparable to that of conventional chromosomal analysis. For the non-disjunctional effect of vincristine, the micronucleus assay was much more sensitive than conventional chromosomal analysis.

Exogenous glutathione induces sister chromatid exchanges, clastogenicity and endoreduplication in V79-E Chinese hamster cells.

Glutathione (GSH) dissolved in Eagle's MEM and added to cultures of V79-E cells in concentrations between 2.5 X 10(-4) and 10(-3) moles/l for 1 h induces a dose-dependent cell cycle delay, sister chromatid exchanges and clastogenic damage. 7-8% of the metaphases showed endoreduplication at a recovery phase of 25 and 30 h after treatment with 10(-3) moles/l GSH. Higher concentrations were lethal. The highest tolerated dose corresponds to the intracellular GSH level in V79-E cells. In the same range of concentrations, glutathione disulfide was inactive. Endoreduplication induction by GSH is G2-phase specific and endoreduplication metaphases show a reduced occurrence of single SCEs when extrapolated to the diploid complement. The adverse effects of GSH are independent of the presence of serum in the culture fluid but completely abolished when the treatment is performed in Hank's solution instead of MEM. The mechanism of genotoxicity of exogenous GSH is discussed but, at present, no pertinent explanation can be given.

Genetic and molecular mechanisms of the in vitro transformation of Syrian hamster embryo cells by the carcinogen N-ethyl-N-nitrosourea. I. Correlation of morphological transformation and enhanced fibrinolytic activity to gene mutation, chromosomal alterations and lethality.

The role of chromosomal alterations, as opposed to gene mutations, in the origin of early stages of the in vitro transformation of Syrian hamster embryo (SHE) cells was investigated. For that purpose we compared the rates at which SHE cells recover from potential tumorigenic, mutagenic, clastogenic and cytotoxic damage if they are held in confluence in low serum medium for 3 or 6 days following a single treatment of N-ethyl-N-nitrosourea (ENU) before the cells are allowed to divide and to express this damage. The results show: (i) that frequencies of gene mutations remain constant; (ii) frequencies of sister chromatid exchange (SCE) and cytotoxicity decrease with very similar kinetics; and (iii) frequencies of chromatid aberrations and micronuclei decrease rapidly in the first 3 days, but slowly or not at all between days 3 and 6. Thus, all the mutational damage and a small fraction of the clastogenic damage still persist after 6 days confluent holding. From the two early stages of in vitro transformation studied, morphological transformation and enhanced fibrinolytic activity, the former shows similar kinetic behaviour as chromatid aberrations and micronuclei, whereas the kinetics of the latter correspond with those of gene mutations. Neither is correlated to SCE or cytotoxicity. Our results suggest that chromosomal alterations can play a major role in induction of morphological transformation of SHE cells. Insofar as enhanced fibrinolytic activity is due to a genetic change, gene mutations can be responsible. Our observations further indicate that different types of ENU-induced DNA lesions are involved in gene mutations, SCE and cytotoxicity, and clastogenic damage. We have reported the results of experiments analysing these relationships in another paper.

Genetic and cytogenetic effects of 1,3-dimethyl-3-phenyl-1-nitroso-urea in Salmonella typhimurium and Chinese hamster V79 cells

1,3-Dimethyl-3-phenyl-1-nitrosourea (DMPNU) shows no spontaneous decomposition in aqueous buffered solution (pH 7.0) at 37 degrees C over a measuring period of 10 days, but it is directly genotoxic in all assays applied. This compound induces base substitutions in Salmonella typhimurium (especially in the plasmid harbouring strain TA 100), gene mutations at the HGPRT+ locus of Chinese hamster V79 cells, and is a very potent inducer of clastogenic damage and sister chromatid exchanges in V79 cells. Data on gene mutation induction by 1-methyl-1-nitrosourea are included for comparison. The findings on DMPNU are discussed in comparison with trialkylnitrosoureas. In contrast to these latter compounds, DMPNU is active without addition of S9 mix in vitro. It is assumed that its genotoxic activity is due to an intracellular catalytic degradation favoured by the electron-withdrawing effect of the phenyl group.

The heritable translocation assay: its relationship to assessment of genetic risk for future generations.

The Heritable Translocation Assay (HTA) is an in vivo mammalian mutation test which measures specific clastogenic damage. It can be subjected to rigorous statistical scrutiny and detects a type of heritable genetic damage that is known to contribute to the human disease burden. Because of these factors the HTA is one of the major tests of the battery used in regulatory evaluation of potential environmental mutagens. Although tests for detecting inherited translocations have been developed and improved over the past 40 years, further emphasis on several statistical considerations affecting the precision and reliability of HTA animal model data applied to regulatory hazard evaluation and risk assessment seems warranted. In this paper we have presented a brief history and the basic methodology for the HTA. Statistical concepts related to the design and implementation of HTA studies, such as the incorporation of false classification error probabilities in determination of required sample size and use of the single sample binomial versus Fisher's exact test for analysis of data, hory and the basic methodology for the HTA. Statistical concepts related to the design and implementation of HTA studies, such as the incorporation of false classification error probabilities in determination of required sample size and use of the single sample binomial versus Fisher's exact test for analysis of data, hory and the basic methodology for the HTA. Statistical concepts related to the design and implementation of HTA studies, such as the incorporation of false classification error probabilities in determination of required sample size and use of the single sample binomial versus Fisher's exact test for analysis of data, have been examined in detail. We have also suggested a simplified method of extrapolating HTA animal data to man similar to that used with carcinogenesis data and propose other developmental needs and perspectives for application of this assay to regulatory assessment of environmental mutagens.