Kamendulis L M, Corcoran G B
Toxicology Program, College of Pharmacy, University of New Mexico, Albuquerque 87131-1066, USA.
J Toxicol Environ Health. 1995 Sep;46(1):31-46. doi: 10.1080/15287399509532016.
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.
长期接触二甲基亚硝胺会通过反复的DNA烷基化作用引发肝肿瘤，而急性接触则可能通过在很大程度上仍不清楚的机制导致肝坏死。我们实验室最近证明，DNA片段化在早期就会发生，并且可能是二甲基亚硝胺诱导肝坏死的一个因果事件。解释这些结果面临的一个挑战是，高达30%的肝细胞是非实质细胞，这可能是观察到的DNA片段化的原因。在本研究中，我们研究了二甲基亚硝胺是否会在培养的小鼠肝细胞中诱导早期基因组DNA片段化。从雄性ICR小鼠分离的肝实质细胞在Williams E培养基中培养。DNA损伤通过在27,000×g下未沉淀的片段化部分进行定量评估，并通过琼脂糖凝胶电泳进行定性评估。通过测量DNA修复酶DNA连接酶的诱导来评估细胞对DNA损伤的反应。通过[3H]腺嘌呤预标记的细胞中乳酸脱氢酶（LDH）或腺嘌呤核苷酸的释放来估计毒性细胞死亡。二甲基亚硝胺在6小时时使[3H]腺嘌呤释放增加两倍，在36小时时使LDH释放增加。DNA片段化和DNA连接酶活性早在1小时就增加。Ca(2+) - 核酸内切酶抑制剂金精三羧酸和Ca2+螯合剂乙二胺四乙酸（EDTA）在6小时内阻止了DNA片段化，并在30小时内几乎消除了细胞毒性。DNA连接酶的诱导与DNA片段化密切相关。DNA片段化和DNA连接酶的早期增加与后期毒性细胞死亡高度相关。这些结果强烈表明，二甲基亚硝胺诱导的靶实质细胞中DNA片段化是这些肝细胞毒性死亡的一个因果因素。
