Shen W, Kamendulis L M, Ray S D, Corcoran G B
Toxicology Program, College of Pharmacy, University of New Mexico, Albuquerque 87131-1066.
Toxicol Appl Pharmacol. 1992 Jan;112(1):32-40. doi: 10.1016/0041-008x(92)90276-x.
Hepatotoxic alkylation of mouse liver cells by acetaminophen is characterized by an early loss of ion regulation, accumulation of Ca2+ in the nucleus, and fragmentation of DNA in vitro and in vivo. Acetaminophen-induced DNA cleavage is accompanied by the formation of a "ladder" of DNA fragments characteristic of Ca(2+)-mediated endonuclease activation. These events unfold well in advance of cytotoxicity and the development of necrosis. The present study utilized cultured mouse hepatocytes and mechanistic probes to test whether DNA fragmentation and cell death might be related in a "cause-and-effect" manner. Cells were isolated by collagenase perfusion, cultured in Williams' E medium for 22-26 hr, and exposed to acetaminophen. Aurintricarboxylic acid, a general Ca(2+)-endonuclease inhibitor, and EGTA, a chelator of Ca2+ required for endonuclease activation, significantly decreased DNA fragmentation at 6 and 12 hr and virtually abolished cytotoxicity. N-Acetylcysteine also eliminated DNA fragmentation and cytotoxicity. 3-Aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase-stimulated DNA repair, failed to alter the amount of DNA fragmentation at 6 hr but substantially increased acetaminophen cytotoxicity in hepatocytes at 12 hr. With the exception of when DNA repair was inhibited by 3-aminobenzamide, Ca2+ accumulation in the nucleus, DNA fragmentation, and hepatocyte death varied consistently and predictably with one another. Collectively, these findings suggest that unrepaired damage to DNA contributes to acetaminophen-induced cell death in vivo and may play a role in necrosis in vivo.
对乙酰氨基酚对小鼠肝细胞的肝毒性烷基化作用的特征是早期离子调节丧失、细胞核中Ca2+积累以及体外和体内DNA片段化。对乙酰氨基酚诱导的DNA切割伴随着Ca(2+)介导的核酸内切酶激活所特有的DNA片段“梯形”的形成。这些事件在细胞毒性和坏死发展之前就已充分显现。本研究利用培养的小鼠肝细胞和机制性探针来测试DNA片段化与细胞死亡是否可能以“因果”方式相关。通过胶原酶灌注分离细胞，在Williams' E培养基中培养22 - 26小时，然后暴露于对乙酰氨基酚。金精三羧酸，一种通用的Ca(2+) - 核酸内切酶抑制剂，以及EGTA，一种核酸内切酶激活所需的Ca2+螯合剂，在6小时和12小时时显著降低了DNA片段化，并几乎消除了细胞毒性。N - 乙酰半胱氨酸也消除了DNA片段化和细胞毒性。3 - 氨基苯甲酰胺，一种聚(ADP - 核糖)聚合酶刺激的DNA修复抑制剂，在6小时时未能改变DNA片段化的量，但在12小时时显著增加了对乙酰氨基酚对肝细胞的细胞毒性。除了3 - 氨基苯甲酰胺抑制DNA修复的情况外，细胞核中Ca2+积累、DNA片段化和肝细胞死亡彼此之间始终且可预测地变化。总体而言，这些发现表明未修复的DNA损伤导致了对乙酰氨基酚在体内诱导的细胞死亡，并且可能在体内坏死中起作用。
