© 1992 Oxford University Press
research-article |
Colloidal gold ultraimmunocytochemical localization of DNA and RNA adducts in rat hepatocytes
Département des Sciences Biologiques Université du Québec à Montréal Montréal, Québec, Canada H3C 3P8
1INRS-Santé Université du Québec à Montréal Montréal, Québec, Canada H3C 3P8
2Département de Chimie, and TOXEN, Université du Québec à Montréal Montréal, Québec, Canada H3C 3P8
The localization of DNA and RNA adducts was studied at the ultrastructural level using antibodies directed against O6-methylguanine (O6-metG) and the protein A—gold technique. Primary rat hepatocyte cultures were exposed for 2 h to 5 mM N-nitrosodimethylamine (NDMA). In NDMA-treated cells, the O6-metG-induced immunoreactive sites do not appear at random but seem to be concentrated in the nucleus, and in the cytoplasm, in areas rich in rough endoplasmic reticulum (RER) elements. Mitochondria were not significantly labelled. Untreated control preparations showed no specific immunogold labelling. After RNase digestion of ultrathin sections obtained from cells exposed to NDMA and subsequent immunogold labelling, most of the immunolabelling in the cytoplasm had disappeared, and that over the nucleus had only been slightly reduced, as compared to undigested specimens from NDMA-treated cultures. After similar digestion with DNase, a strong reduction of the labelling of the nucleus was observed, but labelling of the cytoplasm was practically unaffected by this enzymatic treatment, as compared to what was observed in undigested preparations of NDMA-treated hepatocytes. The results provide evidence of preferential formation of O6-metG at the DNA and RNA levels, in the nucleus and cytoplasm RER, respectively. Furthermore, this study demonstrates the applicability of the high-resolution protein A—gold technique for ultrastructural detection of nucleic acid adducts in NDMA-treated hepatocytes using affinity-purified anti-O6-metG polyclonal antibodies.