IMMUNOCYTOCHEMICAL EVIDENCE FOR A NUCLEAR AND A CYTOPLASMIC O-6-METHYLGUANINE REPAIR MECHANISM IN CULTURED RAT HEPATOCYTES

The localization of DNA and RNA adducts was studied at the ultrastruct ural level using antibodies directed against O-6-metG and the protein A-gold technique. Primary rat hepatocyte cultures were exposed for 2-2 4 h to 5 mM N-nitrosodimethylamine (NDMA) or 0.1 mM N-methyl-N'-nitro- N-nitrosoguanidine (MNNG). In both cases, the O-6-metG immunoreactive sites were concentrated in the nucleus and in the rough endoplasmic re ticulum (RER) rich cytoplasmic regions. The highest gold labeling dens ity measured was observed at 2 h of NDMA or MNNG treatment. However, a fter a 24-h exposure, very little labeling was observed in both the nu clear and the cytoplasmic compartments. The rate of disappearance of i mmunoreactive sites was faster in the cytoplasm than in the nucleus. U ntreated control preparations showed no specific immunogold labeling. Furthermore, when cells were exposed first to NDMA and MNNG for a few hours and then to culture medium containing no genotoxin, and subseque ntly were reexposed to NDMA or MNNG for a few hours, very little label ing of both the nuclear and cytoplasmic compartments was observed. Con trol preparations without a second genotoxin exposure showed a normal labeling pattern. Control preparations without genotoxin showed no gol d labeling. Our results provide evidence for the existence of a cytopl asmic O-6-metG repair mechanism that behaves like its nuclear counterp art.