Dimethylnitrosamine-induced DNA damage and toxic cell death in cultured mouse hepatocytes.
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.
Kamendulis
L M
LM
Toxicology Program, College of Pharmacy, University of New Mexico, Albuquerque 87131-1066, USA.
Corcoran
G B
GB
D000477:Alkylating Agents / Q000633:toxicity*; D000704:Analysis of Variance; D000818:Animals; D016923:Cell Death; D002478:Cells, Cultured; D004247:DNA / Q000187:drug effects*; D011088:DNA Ligases / Q000378:metabolism; D004128:Dimethylnitrosamine / Q000633:toxicity*; D008099:Liver / Q000166:cytology; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D009153:Mutagens / Q000633:toxicity*; D051381:Rats
D000477:Alkylating Agents; D009153:Mutagens; D004247:DNA; D011088:DNA Ligases; D004128:Dimethylnitrosamine