Hepatic binding proteins translocating azo dye carcinogen metabolites from cytoplasm into nucleus in rats

Abstract

When liver cytosol prepared from rats administered [14C]-3′-Methyl-N,N-dimethyl-4-aminoazobenzene was subjected to Sephadex gel chromatography, four peaks of radioactivity containing proteins (Peak-I–IV) and one peak devoid of protein (Peak-V) were obtained. Translocation of azo dye metabolites from these various cytosolic fractions into nucleus was studied in an in vitro system and a maximum of about 10% of the radioactivity associated with a particular cytosolic fraction (Peak-II) could translocate into the nuclei. Radioactivity (%) translocated did not increase upon addition of excess nuclei. Passage of this protein fraction through an immobilized protease column reduced the azo dye metabolite translocation by 65%, concomitant with the degradation of proteins. Translocation was not observed with protein-free metabolites extracted from this cytosolic fraction; addition of proteins corresponding to peak-II from normal rat liver cytosol significantly restored the metabolite translocation. This observation suggests that specific cytosolic proteins are involved in the translocation of azo dye carcinogen metabolites from liver cytoplasm into the nucleus. When the liver cytosolic proteins corresponding to this fraction (Peak-II) were iodinated with 125I-iodine and incubated with purified nuclei, translocation of three specific proteins into nucleus was observed as seen by SDS–PAGE and fluorography of nuclear proteins. Covalent binding of azo dye metabolites to DNA was not observed when cytosolic peak-II fraction containing azo dye metabolites was incubated with isolated liver DNA instead of liver nuclei. This suggests that the interaction of azo dye metabolites with nuclear macromolecules necessitate further prior processing which actually may occur in the nucleus.