Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas 66160, USA.
Toxicol Sci. 2010 Oct;117(2):515-23. doi: 10.1093/toxsci/kfq208. Epub 2010 Jul 8.
Oxidative stress and mitochondrial dysfunction play an important role in acetaminophen (APAP)-induced hepatocyte cell death. However, exact mechanisms involved in the process are controversial, in part, because of the disparity in findings between in vitro and in vivo studies. A major difference in this context is the oxygen tension, with cells in culture being exposed to 21% oxygen, whereas those in the liver experience a gradient from 3 to 9% oxygen. To determine if oxygen tensions could modulate hepatocyte responses to APAP, primary mouse hepatocytes were treated with 5mM APAP for up to 15 h under various oxygen tensions and mitochondrial dysfunction (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide inner salt assay, 5,5',6,6'-tetrachloro-1,1,3,3-tetraethylbenzimidazolylcarbocyanine iodide [JC-1] fluorescence ratio) and cell death (lactate dehydrogenase release) was evaluated. Mitochondrial reactive oxygen and reactive nitrogen species were measured using Mitosox Red or dihydrorhodamine fluorescence and nitrotyrosine staining, respectively. Exposure of hepatocytes to 5mM APAP at 21% O(2) resulted in mitochondrial oxidant stress formation, deterioration of mitochondrial function, and loss of membrane potential as early as 6 h and massive cell death at 15 h. Culture of cells at 10% O(2) resulted in no increase in mitochondrial oxidant stress and better preserved mitochondrial function at 6 h and significant protection against cell death at 15 h. Furthermore, dihydrorhodamine fluorescence was significantly attenuated at 10% oxygen. Cells cultured at 5% oxygen were also protected but showed evidence of hypoxia (accumulation of lactate and nuclear translocation of hypoxia-inducing factor-1α). These results suggest that oxygen tension can modulate hepatocyte responses to APAP, with low physiological levels (10%) decreasing mitochondrial oxidant stress and delaying hepatocyte cell death.
氧化应激和线粒体功能障碍在乙酰氨基酚（APAP）诱导的肝细胞死亡中起着重要作用。然而，由于体外和体内研究结果存在差异，确切的作用机制仍存在争议。在这种情况下的一个主要区别是氧气张力，培养中的细胞暴露于 21%的氧气中，而肝脏中的细胞经历从 3%到 9%氧气的梯度。为了确定氧气张力是否可以调节肝细胞对 APAP 的反应，将原代小鼠肝细胞在不同氧气张力和线粒体功能障碍（2,3-双[2-甲氧基-4-硝基-5-磺苯基]-2H-四唑-5-羧基苯胺内盐测定法，5,5'，6,6'-四氯-1,1,3,3-四乙基苯并咪唑基羰氰化物碘化物[JC-1]荧光比）和细胞死亡（乳酸脱氢酶释放）下用 5mM APAP 处理长达 15 小时。使用 Mitosox Red 或二氢罗丹明荧光分别测量线粒体活性氧和活性氮物种，并用硝基酪氨酸染色。将肝细胞暴露于 21%O(2)中的 5mM APAP 会导致线粒体氧化剂应激形成、线粒体功能恶化和膜电位丧失，最早在 6 小时出现，15 小时出现大量细胞死亡。在 10%O(2)下培养细胞不会增加线粒体氧化剂应激，在 6 小时时更好地保留线粒体功能，并在 15 小时时显著防止细胞死亡。此外，在 10%氧气下二氢罗丹明荧光显著减弱。在 5%氧气下培养的细胞也受到保护，但显示出缺氧的证据（乳酸积累和缺氧诱导因子-1α的核易位）。这些结果表明，氧气张力可以调节肝细胞对 APAP 的反应，低生理水平（10%）可减少线粒体氧化剂应激并延迟肝细胞死亡。
