Acetaminophen metabolism and cytotoxicity in PC12 cells transfected with cytochrome P4502E1
Acetaminophen metabolism and cytotoxicity in PC12 cells transfected with cytochrome P4502E1
复制标题
DOI：
10.1007/s001090050137
复制
发表时间：
1997-07
期刊：
影响因子：
--
通讯作者：
A. Hołownia;J. Mapoles;J. Menez;J. Braszko
中科院分区：
文献类型：
--
作者：
A. Hołownia;J. Mapoles;J. Menez;J. Braszko
虽然许多研究证实了代谢物在对乙酰氨基酚的细胞毒性中的重要作用，但其确切机制仍不清楚。对乙酰氨基酚通过微粒体酶代谢。细胞色素P4502 E1（CYP 2 E1）介导的N-羟基化导致形成N-乙酰基-苯醌亚胺，这是一种高度反应性的中间体。我们研究了对乙酰氨基酚暴露的PC 12细胞和经基因工程改造表达人CYP 2 E1的PC 12细胞系中与坏死和凋亡过程相关的生化参数。[3 H]胸苷掺入试验和蛋白质测定均显示暴露于药物的两种细胞系均出现剂量和时间相关的显著生长迟缓。这在CYP 2 E1转染细胞中更为明显。此外，对乙酰氨基酚的细胞毒性作用增加，如培养基中的乳酸脱氢酶活性所证明的。随机寡核苷酸引物合成试验和酶联免疫吸附试验均显示两种细胞系中存在显著的DNA片段化，转染细胞中DNA片段化程度更高，约占细胞总DNA的11%。这些结果通过流式细胞术和细胞核的显微镜检查得到证实。细胞内钙水平仅在转染细胞中增加，当给予5 mM对乙酰氨基酚48 h时，增加约3倍。这些结果表明对乙酰氨基酚通过细胞凋亡、坏死和生长迟缓产生细胞毒性作用。虽然确切的机制仍然不清楚，但似乎DNA片段化和凋亡级联反应代表了急性细胞死亡中的初步生化事件，并且CYP 2 E1的对乙酰氨基酚生物转化刺激了该途径。
Although a number of studies confirm the important role of metabolites in the cytotoxicity of acetaminophen, its precise mechanisms remain unknown. Acetaminophen is metabolized by microsomal enzymes. Cytochrome P4502E1 (CYP2E1) mediated N-hydroxylation results in the formation ofN-acetyl-benzo-quinoneimine, a highly reactive intermediate. We examined biochemical parameters related to necrotic and apoptotic processes in acetaminophen-exposed PC12 cells is and in a PC12 cell line genetically engineered to express human CYP2E1. Both the [3H]thymidine incorporation test and the protein assay uniformly showed dose- and time-related significant growth retardation in both cell lines exposed to the drug. This was more evident in CYP2E1-transfected cells. Moreover, the cytotoxic effect of acetaminophen was increased as evidenced by lactate dehydrogenase activity in the culture medium. Both random oligonucleotide primed synthesis assay and enzyme-linked immunosorbent assay revealed significant DNA fragmentation in both cell lines, which was greater in transfected cells, reaching about 11% of total cellular DNA. These results were confirmed by flow cytometry and microscopic examination of cell nuclei. Intracellular calcium levels were increased only in transfected cells, approximately threefold when 5 mM acetaminophen was administered for 48 h. These results indicate the cytotoxic effects of acetaminophen via apoptosis, necrosis, and growth retardation. While the precise mechanism remains obscure, it seems that DNA fragmentation and apoptotic cascade represent a preliminary biochemical event in acute cell death, and that acetaminophen biotransformation by CYP2E1 stimulates this pathway.