Effect of metabolic activation on the cytotoxicity and mutagenicity of 1,2-dibromoethane in the CHO/HGPRT system

Abstract

When ethylene dibromide (EtBr 2) was assayed with the Chinese hamster ovary/hypoxanthine-guanine phosphoribosyl transferase (CHO/HGPRT) system coupled with a rat liver metabolic activation system (S9), which contains Ca 2+ (Ca, Mg−S9), the cytotoxicity of EtBr 2 was greatly increased over that obtained when NADP was omitted from the Ca, Mg−S9 or when EtBr 2 was assayed as a direct-acting agent. However, on a molar basis, the mutagenicity of EtBr 2 remained unaffected. The omission of Ca 2+ from the Ca, Mg−S9 metabolic activation system (Mg−S9), with either the addition or omission of NADP, caused approximately a 2-fold decrease in the mutagenicity of EtBr 2 when compared to the results obtained by using the Ca, Mg−S9 system. The cytotoxicity of EtBr 2 was further increased when a purified microsomal fraction, prepared from the S9 fraction, was used in the presence of Ca 2+. In the absence of this calcium ion, this metabolic activation system was extremely cytotoxic Chinese hamster ovary cells even without the presence of a mutagen or promutagen. The cytotoxicity of EtBr 2 in the following assay systems decreased in this order: Ca, Mg-microsomes > Ca, Mg−S9 > Mg−S9 > direct-acting agent ⩾ Ca, Mg−S9 without NADP ⩾ Mg−S9 without NADP. Cytotoxicity appears to be NADP-dependent. Although the mutagenic activity of EtBr 2 (mutants/10 6 cells/mM) was not dependent on the presence of NADP in the S9 system, the mutant yield (number of mutants that could be induced) was higher in its absence. Addition of reduced glutathione to Mg−S9 without NADP increased the mutagenicity of EtBr 2 to values that did not exceed those obtained when EtBr 2 was tested as a direct-acting agent. On a molar basis, ethyl methanesulfonate (EMS) is less cytotoxic but equally as mutagenic as EtBr 2. However the mutant yield of EMS was higher than that of EtBr 2. Inclusion of Ca, Mg−S9 in the assay system had no effect on the biological activities of EMS.