Oxygen-dependent effect of microsomes on the binding of doxorubicin to rat hepatic nuclear DNA.
Doxorubicin is metabolically activated by microsomal NADPH-dependent cytochrome P450 reductase as well as by intact nuclei forming semiquinone free radicals, which reoxidize to doxorubicin in the presence of oxygen. Nuclear activated doxorubicin became bound to DNA in a time-dependent fashion. The addition of microsomal protein to intact nuclei increased the amount of doxorubicin bound to DNA under aerobic conditions. In contrast, inclusion of microsomes virtually abolished DNA binding under anaerobic conditions. Disruption of the nuclear membrane by sonification increased the amount of drug bound to DNA, indicating that the nuclear envelope serves as a partial barrier to the diffusion of microsomal DNA-directed intermediates. The data indicate that under aerobic conditions metabolites produced by microsomes either traverse the nuclear membrane and bind to DNA or act indirectly by disrupting the nuclear membrane. In contrast, inhibition of DNA binding by doxorubicin under anaerobic conditions suggests either that the microsomal metabolites do not diffuse across the nuclear membrane or that the metabolites are not capable of binding to DNA. The decreased diffusibility of the anaerobic metabolites may represent either the generation of metabolites having lower diffusion constants or the formation of highly reactive intermediates which preferentially bind in the immediate vicinity to the site of generation on the microsomal surface. In conclusion, it appears that, under aerobic conditions, metabolic activation of doxorubicin on the surface of the endoplasmic reticulum can contribute to the amount of the drug which becomes closely associated with nuclear DNA. Consequently, the microsomal drug-metabolizing system may be important in affecting the therapeutic or cytotoxic properties of the drug.