Reactive nitrogen species in acetaminophen-induced mitochondrial damage and toxicity in mouse hepatocytes: a cautionary note on the impact of cell culture conditions.
Reactive nitrogen species in acetaminophen-induced mitochondrial damage and toxicity in mouse hepatocytes: a cautionary note on the impact of cell culture conditions.
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对乙酰氨基酚诱导的小鼠肝细胞线粒体损伤和毒性中的活性氮物种：关于细胞培养条件影响的警告。
DOI：
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发表时间：
2010
影响因子：
4.1
通讯作者：
A. Ramachandran
中科院分区：
文献类型：
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作者：
H. Jaeschke;Hui‐min Yan;A. Ramachandran
致编辑：我们怀着极大的兴趣阅读了Burke等人最近发表的论文（1）。然而，根据本文提交后发表的研究结果，我们认为实验条件可能对这些细胞培养实验中获得的结果产生了显著影响。首先，作者似乎能够通过短期暴露于1 mM对乙酰氨基酚（APAP）（1）迅速引发严重损伤，这与许多其他研究组报告的情况不同，其中连续暴露于5 - 2-5 mM APAP用于获得更延迟的肝细胞损伤（2 - 5）。这些意外结果的一个可能解释是作者使用了悬浮液中的肝细胞（1）。作者观察到，早在悬浮液中的肝细胞中1 mM APAP暴露后3 - 4 h，酶释放就急剧增加（4 h时比基线增加约> 20倍）（1）。相比之下，在接触5 mM APAP后，贴壁细胞中酶释放的类似时间过程在3 h时没有导致任何显著的酶释放，并且在6 h时仅显示出2倍的增加（2）。事实上，当我们直接比较贴壁细胞与悬浮细胞中的APAP毒性时，我们观察到悬浮细胞中的细胞死亡高4 - 5倍，与所用APAP的剂量无关（Ramachandran，A.，Yan，H. - M.，Jaeschke，H.，未出版的作品）。因此，悬浮液中的细胞通常比贴壁细胞更容易受到APAP暴露的影响。作者实验的另一个警告是使用极高的氧气浓度，即，在95%氧气下孵育细胞（1）。体内完整肝脏中的肝细胞在血窦中的氧浓度在3 - 9%之间（6）。在最近的一项研究中，我们证明，即使在室内空气（21%氧气）下培养细胞，与在10%氧气下培养的细胞相比，也会导致加速和更高的线粒体氧化应激和过氧亚硝酸盐形成，从而导致加速和更高的细胞死亡（7）。有趣的是，线粒体氧化应激和过氧亚硝酸盐形成的差异似乎在基线条件下不存在，而仅在APAP暴露后存在（7）。此外，这些差异有助于这样一个事实，即亲脂性抗氧化剂如维生素E仅在细胞在21%的氧气下培养时才能部分保护细胞，而在10%或更低的生理氧气水平下培养细胞时则不能。这些体外观察结果与体内实验一致，其中维生素E不能防止APAP过量（8）。因此，我们的数据表明，在21%氧气下培养时，将细胞暴露于APAP会增强线粒体氧化应激和过氧亚硝酸盐的形成。总之，这些最近的发现可以解释Burke等人报告的观察结果。在这些体外研究中，通过将细胞保持在悬浮液中诱导的严重应激和由APAP触发的初始线粒体功能障碍通过在这些实验中使用的高氧条件（95%氧）进一步放大。因此，这些肝细胞对低浓度APAP的明显高敏感性实际上是三重打击的结果，即，药物毒性、高氧和细胞应激。其次，作者得出结论，"这些数据与活性氮物质（RNS）引起线粒体损伤以及线粒体损伤诱导额外RNS的假设一致......"（一）.然而，他们工作的表1中的结果表明，线粒体膜通透性转换（MPT）抑制剂环孢素A消除了二氯二氢荧光素荧光和硝基酪氨酸蛋白加合物（1）。因此，可以得出结论，在这些实验条件下，几乎所有的氧化剂应力和过氧亚硝酸根形成都发生在MPT孔打开之后。这与过氧亚硝酸盐导致MPT的假设不一致。然而，该实验系统中的观察结果与许多不同的体外和体内结果不一致。首先，APAP过量后的氧化应激在GSH耗尽后立即开始，但远在细胞死亡之前（2），这与MPT相关（3）。许多体内研究表明，清除过氧亚硝酸盐可有效防止细胞坏死（9 - 13）。最重要的是，最近的一项研究表明，APAP诱导的肝损伤和DNA损伤在亲环素D缺陷小鼠中被完全预防，但氧化应激和过氧亚硝酸盐形成仅部分减少（14）。因此，体内数据明确支持以下结论：活性氧物质（ROS）和过氧亚硝酸盐的形成发生在MPT孔打开之前，并且至少部分参与触发MPT（14）。
To the Editor: We read with great interest the recent paper by Burke et al. (1). However, on the basis of findings published after the submission of this manuscript we think that the experimental conditions may have significantly influenced the results obtained in these cell culture experiments. First, the authors appear to be able to quite rapidly trigger severe injury with short-term exposure to 1 mM acetaminophen (APAP) (1), which is different from what has been reported by a number of other groups where continuous exposure to 5-25 mM APAP was used to get a much more delayed hepatocellular injury (2-5). One likely explanation for these unexpected results could be the fact that the authors used hepatocytes in suspension (1). The authors observed a very steep increase in enzyme release as early as 3-4 h after 1 mM APAP exposure in hepatocytes in suspension (approximately >20-fold increase over baseline at 4 h) (1). In contrast, a similar time course of enzyme release in adherent cells after exposure to 5 mM APAP did not results in any significant enzyme release at 3 h and showed only a 2-fold increase at 6 h (2). In fact, when we directly compared APAP toxicity in adherent versus suspended cells we observed 4-5-fold higher cell death in suspended cells independent of the dose of APAP used (Ramachandran, A., Yan, H.-M., Jaeschke, H., unpublished work). Thus, cells in suspension are generally more susceptible to APAP exposure than adherent cells. Another caveat of the authors’ experiments is the use of extremely high oxygen concentrations, i.e., incubation of cells under 95% oxygen (1). Hepatocytes in the intact liver in vivo experience oxygen concentrations in sinusoids between 3-9% (6). In a recent study, we demonstrated that even culturing cells under room air (21% oxygen) leads to an accelerated and higher mitochondrial oxidant stress and peroxynitrite formation causing accelerated and higher cell death compared to cells cultured under 10% oxygen (7). Interestingly, the differences in mitochondrial oxidant stress and peroxynitrite formation do not appear to exist under baseline conditions but only after APAP exposure (7). Furthermore, these differences contribute to the fact that lipophilic antioxidants such as vitamin E partially protect only when cells are cultured under 21% oxygen but not when cells are cultured under more physiological oxygen levels of 10% or lower (7). These in vitro observations are consistent with in vivo experiments where vitamin E did not protect against APAP overdose (8). Thus, our data suggest that exposing cells to APAP when cultured under 21% oxygen enhances the mitochondrial oxidant stress and peroxynitrite formation. Together, these more recent findings may explain the observations reported by Burke et al. (1). In these in vitro studies, the severe stress induced by keeping the cells in suspension and the initial mitochondrial dysfunction triggered by APAP is further exaggerated by the hyperoxic conditions (95% oxygen) used throughout these experiments. Thus, the apparently high susceptibility of these hepatocytes to low concentrations of APAP is actually the result of a triple hit, i.e., drug toxicity, hyperoxia, and cellular stress by preventing adherence. Second, the authors conclude that “the data are consistent with the hypothesis that reactive nitrogen species (RNS) cause mitochondrial damage and that mitochondrial damage induces additional RNS...” (1). However, the results in Table 1 of their work show that the mitochondrial membrane permeability transition (MPT) inhibitor cyclosporine A eliminates dichlorodihydrofluorescein fluorescence and nitrotyrosine protein adducts (1). Thus, one would conclude that virtually all of the oxidant stress and peroxynitrite formation under these experimental conditions occurred after the MPT pore opening. This would be inconsistent with the assumption that peroxynitrite causes the MPT. However, the observations in this experimental system do not agree with a number of different in vitro and in vivo findings. First, the oxidant stress after APAP overdose starts right after GSH depletion but well before cell death (2), which correlates with the MPT (3). A number of in vivo studies showed that scavenging peroxynitrite effectively prevented cell necrosis (9-13). Most importantly, a recent study demonstrated that APAP-induced liver injury and DNA damage was completely prevented in cyclophilin D-deficient mice but that there was only a partial reduction of the oxidant stress and peroxynitrite formation (14). Thus, in vivo data clearly support the conclusion that the formation of reactive oxygen species (ROS) and peroxynitrite occurs prior to the MPT pore opening and are at least in part involved in triggering the MPT (14).
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影响因子：
3.8
作者：
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通讯作者：
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影响因子：
3.8
作者：
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影响因子：
3.9
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影响因子：
3.8
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3.8
作者：
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