DIMETHYLNITROSAMINE-INDUCED DNA-DAMAGE AND TOXIC CELL-DEATH IN CULTURED MOUSE HEPATOCYTES

Chronic exposure to dimethylnitrosamine produces hepatic tumors throug h recurrent DNA alkylation, whereas acute exposure can cause liver nec rosis through mechanisms that remain largely unknown. Our laboratory r ecently demonstrated that DNA fragmentation occurs early on and may be a causal event in dimethylnitrosamine-induced necrosis in liver. A ch allenge to interpreting these results is that vp to 30% of liver cells are nonparenchymal and could account for the observed DNA fragmentati on. In the present study, we have examined whether dimethylnitrosamine induces early genomic DNA fragmentation in cultured mouse hepatocytes . Hepatic parenchymal cells isolated from male ICR mice were cultured in Williams E medium. DNA damage was assessed quantitatively as a frag mented fraction that was not sedimented at 27,000 x g, and qualitative ly from agarose gel electrophoresis. Cellular response to DNA damage w as assessed by measuring induction of the DNA repair enzyme DNA ligase . Toxic cell death was estimated from release of lactate dehydrogenase (LDH) or adenine nucleotides from cells prelabeled with [H-3]adenine. Dimethylnitrosamine produced a twofold increase in [H-3]adenine relea se by 6 h and LDH release at 36 h. DNA fragmentation and DNA ligase ac tivity increased by as early as 1 h. The Ca2+-endonuclease inhibitor a urintricarboxylic acid and the Ca2+ chelator ethylenediamine tetraacet ic acid (EDTA) prevented DNA fragmentation through 6 h and virtually a bolished cytotoxicity through 30 h. DNA ligase induction was strongly associated with DNA fragmentation. Early increases in DNA fragmentatio n and DNA ligase were highly correlated with later toxic cell death. S uch results strongly suggest that dimethylnitrosamine-induced fragment ation of DNA in target parenchymal cells is a causal factor in the tox ic death of these liver cells.