Cytogenetic and molecular characterization of the mutagenicity of chlorambucil in V79 cells

Chlorambucil (CBC) is used as a chemotherapeutic agent and immunosuppressant. Recently, it could be shown that CBC is considerably more effective than radiation or any chemical investigated to date in inducing high yields of germ-line mutations that appear to be multilocus deletions or other structural changes. We therefore reinvestigated the in vitro genotoxic effects of CBC in V79 cells and characterized induced sister-chromatid exchanges (SCEs), chromosome aberrations and gene mutations by means of cytogenetic and molecular methods. CBC effectively induced chromosome aberrations and SCEs in a dose-dependent manner. The chromosome aberrations found after a 14-h treatment were mainly chromatid-type aberrations. 3-Aminobenzamide (3AB) did not influence the incidence of CBC-induced SCEs and chromosome aberrations. Combined treatment with CBC and caffeine (CAF) strongly increased the frequency of aberrations, but had no effect on the yield of SCEs. CAF at lower concentrations enhanced the production of chromatid breaks and exchange figures while higher concentrations (10(-3) M) caused multiple breaks and pulverised mitoses. Mutations at the hprt locus were induced in a narrow range of CBC concentrations (10(-5) M-2 x 10(-5) M) and the mutagenic effect was accompanied by strong cytotoxicity. The CBC-induced gene mutation frequency was not increased after CAF treatment. The molecular analysis of CBC-induced mutations by Southern hybridization and PCR demonstrated that CBC predominantly produced small alterations but not deletions or gross structural alterations in the hprt gene of V79 cells. For the first time, these results reveal striking differences in the mutagenic action of an alkylating agent in cultivated cells compared to germ-line cells at the molecular level.