Mutagenic characterization of cholesterol epoxides in Chinese hamster V79 cells

Abstract

The uptake, metabolism and alkylating properties of the diastereomeric cholesterol epoxides were studied using Chinese hamster lung fibroblasts (V79 cells). Specific emphasis is given to the comparative cyto- and geno-toxic effects of cholesterol 5β,6β-epoxide (βCE) and cholesterol 5α,6α-epoxide (αCE) and data are provided for the first time indicating that βCE can induce more 6-thioguanine-resistant cells than αCE. Cholesterol 5β,6β-epoxide induced colonies of cells resistant to 6-thioguanine at 2–3-fold the frequencies observed with the α-isomer, but neither compound produced ouabain-resistant colonies. The cytotoxicity (LD 50) of αCE was estimated to be 45–50 μM whereas βCE displayed an LD 50 of 25–29 μM. Inhibition of DNA synthesis (IC 50) was observed over the same dose ranges as the LD 50 for each epoxide isomer. The epoxides were assimilated by cells to an equal extent, however, βCE was metabolized to cholestane 3β,5α-6β-triol twice as rapidly as the α-isomer. Both epoxides reacted with 4-(4′-nitrobenzyl)-pyridine to a similar extent, and with identical nucleophilic selectivity at pH 7.4, but their alkylating activity was estimated on this basis to be two orders of magnitude less than methyl methanesulfonate. Binding experiments with the DNA or cultured V79 cells or with calf-thymus DNA indicated that interactions were noncovalent and DNA binding did not correlate with the potency of the epoxides to induce the 6-thioguanine-resistant phenotype. Our results could be interpreted as indicating that both cholesterol epoxide isomers are weak mutagens or that they might induce some epigenetic event repressing the hypoxanthine guanine-phosphoribosyltransferase gene. The similarity of the epoxides' alkylating activity and their DNA-binding properties are inconsistent with their different potencies in inducing the 6-thioguanine-resistant phenotype, suggesting that the mechanism leading to this phenotype is not necessarily the result of DNA alkylation.