Immunocytochemical evidence for a nuclear and a cytoplasmic O⁶‐methylguanine repair mechanism in cultured rat hepatocytes

Abstract

The localization of DNA and RNA adducts was studied at the ultrastructural level using antibodies directed against O⁶ ‐metG and the protein A‐gold technique. Primary rat hepatocyte cultures were exposed for 2–24 h to 5 mM N‐nitrosodimethylamine (NDMA) or 0.1 mM N‐methyl‐H'‐nitro‐N‐nitrosoguanidine (MNNG). In both cases, the O⁶ ‐metG immunoreactive sites were concentrated in the nucleus and in the rough endoplasmic reticulum (RER) rich cytoplasmic regions. The highest gold labeling density measured was observed at 2 h of NDMA or MNNC treatment. However, after a 24‐h exposure, very little labeling was observed in both the nuclear and the cytoplasmic compartments. The rate of disappearance of immunoreactive sites was faster in the cytoplasm than in the nucleus. Untreated control preparations showed no specific immunogold labeling. Furthermore, when cells were exposed first to NDMA and MNNC for a few hours and then to culture medium containing no genotoxin, and subsequently were reexposed to NDMA or MNNC for a few hours, very little labeling of both the nuclear and cytoplasmic compartments was observed. Control preparations without a second genotoxin exposure showed a normal labeling pattern. Control preparations without genotoxin showed no gold labeling. Our results provide evidence for the existence of a cytoplasmic O⁶ ‐metG repair mechanism that behaves like its nuclear counterpart.