Chronic exposure to
dimethylnitrosamine produces hepatic
tumors through recurrent
DNA alkylation, whereas acute exposure can cause liver
necrosis through mechanisms that remain largely unknown. Our laboratory recently demonstrated that DNA fragmentation occurs early on and may be a causal event in
dimethylnitrosamine-induced
necrosis in liver. A challenge to interpreting these results is that up to 30% of liver cells are non-parenchymal and could account for the observed DNA fragmentation. 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 fragmented fraction that was not sedimented at 27,000 x g, and qualitatively from
agarose gel electrophoresis. Cellular response to DNA damage was 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 [3H]
adenine.
Dimethylnitrosamine produced a twofold increase in [3H]
adenine release by 6 h and LDH release at 36 h. DNA fragmentation and
DNA ligase activity increased by as early as 1 h. The Ca(2+)-
endonuclease inhibitor
aurintricarboxylic acid and the Ca2+
chelator ethylenediamine tetraacetic
acid (
EDTA) prevented DNA fragmentation through 6 h and virtually abolished cytotoxicity through 30 h.
DNA ligase induction was strongly associated with DNA fragmentation. Early increases in DNA fragmentation and
DNA ligase were highly correlated with later toxic cell death. Such results strongly suggest that
dimethylnitrosamine-induced fragmentation of
DNA in target parenchymal cells is a causal factor in the toxic death of these liver cells.