Short-term tests for carcinogens and mutagens

Interactions between effects of environmental chemicals and natural stressors: A review

https://mmbr.asm.org/content/mmbr/52/1/70.full.pdf

Deoxyribonucleic acid repair in the yeast Saccharomyces cerevisiae.

A literature review was conducted using original papers published during 1964-1985 on the in vitro clastogenicity of chemical substances. Results of tests on 951 chemical substances were abstracted from over 240 reports to form the database. The evaluation of these data relied on each author's original conclusion on a positive or negative outcome. Of these 951 substances, 447 (47%) were consistently positive either with or without activation; 417 (44%) were negative in the direct test but not tested with metabolic activation systems; 4 were negative but tested only with activation; and 30 (3%) were clearly negative both with and without activation. The remaining 53 substances gave variable results when tested under different experimental protocols or in different cell types, but were positive in at least one test. Although discrepant results were found associated with some cell types, the addition of metabolic activation systems tended to eliminate such variability. No one cell appeared to be superior in response to all clastogens. For screening purposes, the choice of cell may thus depend more on the general usefulness and reliability of a cell type than on a strong response to a particular chemical. However, the use of a suitable metabolic activation system does appear to be of critical importance. The concentration at which clastogenic effects were detected varied extensively for different test substances, ranging from a minimum of 4.3 X 10(-8) to 6.9 X 10(2) mM. Possible mechanisms of action for substances active at only high levels are discussed, but no satisfactory explanation is available at this time. The relevance of tests conducted at concentrations high enough to alter significantly the osmolarity and other culture conditions is considered, and caution urged in the interpretation of test results obtained under physiologically stressful conditions. The clastogenic potential was compared quantitatively using an index of effective concentration (D20) and one which estimates the number of cells with exchange aberrations expected per mg/ml (TR) for data obtained by using a uniform protocol and cultures of Chinese hamster lung (CHL) cells. Both values were distributed over a wide range, demonstrating the variety of genotoxic potential in chemicals. In general, a substance which was active at only high concentrations produced fewer exchange-type aberrations. In vivo activity, as measured by tumourigenic effect and formation of micronuclei in bone marrow, tended to be greater for substances with a D20 below 10(-2) mg/ml and a TR value over 10(3).(ABSTRACT TRUNCATED AT 400 WORDS)

A comparative analysis of data on the clastogenicity of 951 chemical substances tested in mammalian cell cultures

This report presents an assessment made by the U.S. Environmental Protection Agency Gene-Tox Program's Work Group on mammalian cytogenetics of the clastogenic effects of chemicals in in vivo and in vitro mammalian cell assays. This assessment is based on information provided by the Environmental Mutagen Information Center, Oak Ridge National Laboratory, with the proviso that the experimental protocol used in these papers was adjudged to be acceptable by standards outlined by the Work Group. Some data were accepted as "qualitative only" because the protocol used was fairly close to that proposed as suitable. Using these criteria, 177 papers were selected for review. 6 assays were reviewed: bone marrow (32 papers, 31 chemicals), spermatogonial (10 papers, 10 chemicals), spermatocyte (25 papers, 25 chemicals), oocyte or early embryo (18 papers, 19 chemicals), in vitro cell culture (30 papers, 66 chemicals), and leukocyte (66 papers, 53 chemicals). Each assay was considered separately, and comparisons were then made between them for their similarities or differences in producing a positive or negative clastogenic effect of a particular chemical or chemical class. A large proportion of the available cytogenetic data was not suitable for inclusion in the final data base because of poor experimental design or unsatisfactory reporting of the information. It was not possible to recommend any one assay for determining potential clastogenicity because each had its own particular advantages and limitations and provided unique information. For demonstrating in vivo effects, the bone-marrow assay is probably the simplest and most economical. If only in vitro exposures were considered, leukocytes or cultured mammalian cell lines would be suitable. However, there are advantages to using leukocytes because they are a synchronous population, at least through their cell division, and because of the ready availability of human cells. In general, there was good agreement between clastogenicity and carcinogenicity.

Mammalian in vivo and in vitro cytogenetic assays: a report of the U.S. EPA's gene-tox program.

Mammalian in vivo cytogenetic assays. Analysis of chromosome aberrations in bone marrow cells.

A variety of in vivo mammalian test models are available for screening of chemicals for mutagenicity at the chromosomal level. These models have been grouped into those focusing on somatic cell effects and those dealing with germ cell effects. An analysis of available literature indicates that 76 compounds have been tested from chromosome effects in both somatic and germ cells. Of these, concordant results (positive-positive or negative-negative) were obtained with 58 compounds. Of the remaining 18 compounds with discordant results, all were positive in somatic cells, but negative in germ cell assays. These results suggest an inherent relative insensitivity of germ cells themselves to mutagenic chemicals. In the context of screening for safety evaluation purposes, this analysis suggests that a negative somatic-cell response can be taken as highly predictive of negative results in a germ cell assessment.

Comparison of somatic and germ cell models for cytogenetic screening

In recent years, evidence has accumulated that suggests that mammalian topoisomerase may play a role in the formation of spontaneous or chemically induced sister chromatid exchange (SCE). In microbial systems, nalidixic acid is known to disrupt the function of a topoisomerase-like enzyme, DNA gyrase. To explore the possible relationship to topoisomerase function and SCE formation in mammalian cells, an analog of nalidixic acid with potent topoisomerase II inhibitory activity was selected for examination in a variety of genetic toxicology assays. This analog, CP-67,015, proved to be a positive direct-acting mutagen in the L5178Y/TK+/-, CHO/HGPRT, and V79/HGPRT systems. However, no gene mutational activity was observed using the Ames test in direct plate, mouse and rat metabolic activation, and mouse urine tests. In vitro cytogenetic studies showed strong clastogenic activity in human lymphocytes and in CHO cells. Compound-induced chromosome damage was also observed in vivo in mouse bone marrow cells. Surprisingly, SCE studies in vitro in human lymphocytes or CHO cells showed only slight increases, even at levels producing severe chromosome breakage. Mouse bone marrow showed no significant elevation of SCE following parenteral treatment with CP-67,015. These results, taken together, demonstrate that CP-67,015 is a direct-acting mutagen in mammalian cells with both gene and chromosomal level effects. The relative ineffectiveness in producing SCEs suggests that CP-67,015 may interfere with a DNA replicative/repair process, perhaps by alteration of one or more DNA polymerase activities. This suggestion is based in part on the known effect of the analog nalidixic acid on DNA gyrase in microbial cells and on topoisomerase in mammalian cells. The profile of genetic activity of CP-67,015, coupled with its inhibitory effect on topoisomerase function, gives rise to a model for SCE formation that is based on anomalies of topoisomerase activity during DNA synthesis.

Genetic profile of a nalidixic acid analog: a model for the mechanism of sister chromatid exchange induction.

Cytogenetic studies have been performed to determine the effect of 6-mercaptopurine (6-MP) on mammalian chromosomes in vivo . An elevated level of chromosome breakage is present in mouse bone marrow cells within 12 h after oral or parenteral dosing with 200 mg/kg. Damage is most severe at 24 h after oral dosing and at 72 h following intraperitoneal injection. A dose-response curve was constructed using oral doses of 10 to 200 mg/kg. The no-effect dose level lies within the 10–25 mg/kg range when administered as a single dose. In subacute studies, positive results have been obtained with as little as 2.5 mg/kg/day administered orally for 5 days.

Mutagenicity studies with 6-mercaptopurine: I. Cytogenetic activity in vivo

Published reports have suggested the possible association of mutagenic or teratogenic properties of some benzimidazole analogs with induced spindle disruption and consequent mitotic arrest. Studies were conducted to explore this correlation. Seven benzimidazole analogs were evaluated in a human lymphocyte system for ability to block mitosis at metaphase. Confirming the previously reported results, four compounds, mebendazole, parbendazole, cambendazole, and fenbendazole, caused metaphase accumulation, which we found to be dose- and time-related. Minimum effective dose for mebendazole and cambendazole was 10 microgram/ml; for parbendazole, 1 microgram/ml; and for fenbendazole, 100 microgram/ml. The drug-induced mitotic arrest is qualitatively and quantitatively similar to that produced by colcemid. No activity was observed with three other compounds, benzimidazole, thiabendazole, and oxfendazole when tested at 100 microgram/ml. Presence of absence of mitotic effects is correlated with reported teratogenicity with five out of the seven analogs. This suggests some utility of mitotic assessments for predicting teratogenicity.

Henry E. Holden

Papers

Mammalian in vivo cytogenetic assays. Analysis of chromosome aberrations in bone marrow cells.

Comparison of somatic and germ cell models for cytogenetic screening

Genetic profile of a nalidixic acid analog: a model for the mechanism of sister chromatid exchange induction.

Mutagenicity studies with 6-mercaptopurine: I. Cytogenetic activity in vivo

Mitotic arrest by benzimidazole analogs in human lymphocyte cultures