Chromosome aberrations in Chinese hamster and human cells: a comparison using compounds with various genotoxicity profiles.

Chromosome aberrations (Cabs) can be induced in vitro by non-DNA damaging compounds, often associated with cytotoxicity and DNA synthesis inhibition, and under conditions that would not be relevant in vivo. Such misleading positive results are reported both in Chinese hamster cell lines and in human peripheral blood lymphocytes (HL). We assessed the response of HL to compounds with varied genetic toxicity profiles, all of which induced Cabs in CHO cells Seven of 10 compounds were negative or equivocal in HL. Results in purified lymphocytes for four verified that the difference was not due to the presence of blood in cultures. Two compounds that were weakly positive in the Ames test and one that induced DNA adducts were negative or equivocal in the HL assay; their overall mutagenic potential in vivo is not clear. Of four Ames-negative compounds, three of which inhibited DNA synthesis in CHO cells, three were negative and one was equivocal in the HL assay. A potent Cab inducer, which also induced micronuclei in vivo (but was negative in the Ames test) was clearly positive in the HL assay. Two compounds were clearly positive in HL only when the mitotic indices (MI) were below 50% of control. These are genotoxic in other assays but our evidence suggests that Cab induction is related more to toxicity than to primary DNA damage. For this limited set of 10 compounds, HL were more likely than CHO cells to give negative or equivocal results. It is likely that more stringent checkpoint controls in human cells prevent damaged cells reaching mitosis, and may also influence the reported greater sensitivity to induction of aneuploidy and polyploidy of normal rodent compared with human cells. In the studies reported here, two strong inducers of polyploidy in CHO cells gave weaker increases in HL. Human lymphocytes have disadvantages as a routine screening assay (finding donors, known individual variability, increased time required and the inadequacy of the MI as a toxicity measure), but may be useful in follow-up testing to assess weight of evidence about genotoxic risk to humans, for compounds that are positive in the Chinese hamster cell Cabs assays.

Population doubling: a simple and more accurate estimation of cell growth suppression in the in vitro assay for chromosomal aberrations that reduces irrelevant positive results.

International guidelines for cytotoxicity limits for the in vitro chromosomal aberration assay require reductions in cell growth of greater than 50%. This sets no upper limit on toxicity and there is concern about the number of false or irrelevant results obtained in the aberration assay, i.e., positive results at toxic dose levels only, with no evidence for primary DNA damaging ability and with negative results in the other genotoxicity tests. We have previously proposed that no truly genotoxic compound would be missed if the toxicity of the highest dose did not exceed 50%. Cell growth measured by cell counts as a percentage of controls can underestimate toxicity. For example, if we seed half a million cells per culture, and the controls double to 1 million during the experiment, a culture that truly has no growth will still have a cell count 50% of the control. Measurement of population doublings (PDs) more accurately assesses cell growth. To assess the use of PD in dose selection, we examined previous data from this lab and data from new experiments with "true," primary DNA damaging clastogens, and with clastogens, including drugs, thought to act indirectly, through cytotoxicity-associated mechanisms. We compared aberration results where the highest doses scored were based on 50% reductions in final cell counts with results obtained when the highest doses were based on PD. The PD method allows detection of true clastogens, including those that are active in a range with some toxicity, and reduces the number of toxicity-related "false"-positive results.