DT-diaphorase activity and the cytotoxicity of quinones in C3H/10T1/2 mouse embryo cells

Abstract

A permanent mouse fibroblast cell line derived from C3H mouse embryos, C3H/10T1/2 C18, was used to study the cytotoxicity of some model quinones under conditions in which DT-diaphorase (EC 1.6.99.2) activity was induced or inhibited. Sudan III [1-[[4-(phenylazo)phenyl]azo]-2-naphthalenol] and 3-methylcholanthrene (MCA), but not butylated hydroxyanisole (BHA), induced DT-diaphorase in a concentration-dependent manner. Induction of DT-diaphorase activity was dependent upon new RNA and protein synthesis, as shown by experiments employing actinomycin D and cycloheximide respectively. Induction of DT-diaphorase by Sudan III or MCA was associated with protection against the cytotoxicity of quinones as measured by a colony survival assay. When control and induced cells were also exposed to dicoumarol, a specific and potent inhibitor of DT-diaphorase, the cytotoxicity of the quinones in both control and induced cells was enhanced markedly. The results support the hypothesis that DT-diaphorase competes with one-electron quinone-reducing enzymes (such as cytochrome P-450 reductase) which generate auto-oxidizable semiquinones and forms more stable hydroquinones as an initial step in the detoxification of quinones in 10T1/2 cells.