Induction of chromosome aberrations in vitro by phenolphthalein: mechanistic studies

Phenolphthalein induces tumors in rodents but because it is negative in ass ays for mutation in Salmonella and in mammalian cells, for DNA adducts and for DNA strand breaks, its primary mechanism does not seem to be DNA damage . Chromosome aberration (Ab) induction by phenolphthalein in vitro is assoc iated with marked cytotoxicity. At very high doses, phenolphthalein induces weak increases in micronuclei (MN) in mouse bone marrow; a larger response is seen with chronic treatment. All this suggests genotoxicity is a second ary effect that may not occur at lower doses. In heterozygous TSG-p53(R) mi ce, phenolphthalein induces lymphomas and also MN, many with kinetochores ( K), implying chromosome loss. Induction of aneuploidy would be compatible w ith the loss of the normal p53 gene seen in the lymphomas. Here we address some of the postulated mechanisms of genotoxicity in vitro, including metabolic activation, inhibition of thymidylate synthetase, cyto toxicity, oxidative stress, DNA damage and aneuploidy. We show clearly that phenolphthalein does not require metabolic activation by S9 to induce Abs. Inhibition of thymidylate synthetase is an unlikely mechanism, since thymi dine did not prevent Ab induction by phenolphthalein. Phenolphthalein drama tically inhibited DNA synthesis, in common with many non-DNA reactive chemi cals that induce Abs at cytotoxic doses. Phenolphthalein strongly enhances levels of intracellular oxygen radicals (ROS). The radical scavenger DMSO s uppresses phenolphthalein-induced toxicity and Abs whereas H2O2 potentiates them, suggesting a role for peroxidative activation. Phenolphthalein did n ot produce DNA strand breaks in rat hepatocytes or DNA adducts in Chinese h amster ovary (CHO) cells. All the evidence points to an indirect mechanism for Abs that is unlikely to operate at low doses of phenolphthalein. We als o found that phenolphthalein induces mitotic abnormalities and MN with kine tochores in vitro. These are also enhanced by H2O2 and suppressed by DMSO. Our findings suggest that induction of Abs in vitro is a high-dose effect i n oxidatively stressed cells and may thus have a threshold. There may be mo re than one mechanism operating in vitro and in vivo, possibly indirect gen otoxicity at high doses and also chromosome loss, both of which would likel y have a threshold. (C) 2000 Published by Elsevier Science B.V.