Metabolic mechanisms of methanol/formaldehyde in isolated rat hepatocytes: Carbonyl-metabolizing enzymes versus oxidative stress
Metabolic mechanisms of methanol/formaldehyde in isolated rat hepatocytes: Carbonyl-metabolizing enzymes versus oxidative stress
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DOI：
10.1016/j.cbi.2011.01.017
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发表时间：
2011-05-30
影响因子：
5.1
通讯作者：
O'Brien, Peter J.
中科院分区：
文献类型：
--
作者：
MacAllister, Stephanie L.;Choi, Joanna;O'Brien, Peter J.
甲醇（CH3OH）是一种常见的工业溶剂，通过几种酶和自由基途径代谢成有毒化合物。确定哪个过程能最好地增强或阻止ch3oh诱导的细胞毒性，有助于深入了解急性ch3oh诱导的肝毒性的分子基础。所研究的代谢途径包括：1)分离的肝细胞系统和2)无细胞系统。加速细胞毒性机制筛选（ACMS）技术表明，在95% O-2浓度下，CH3OH对大鼠肝细胞几乎没有毒性，即使在2m浓度下也是如此，而在1% O-2浓度下，50 mM是估计的LC50（2小时），估计是肝脏小叶中心区域的生理浓度，也是乙醇毒性的目标区域。细胞毒性归因于由ADH1催化的CH3OH代谢引起的NADH水平升高，导致还原性应激，使铁蛋白中的亚铁离子减少并释放，引起氧活化。先前发现乙醇在1%的浓度下也有类似的细胞毒性机制。在95%的浓度下，添加无毒浓度的Fe(II)/H2O2是增加1 M CH3OH诱导的肝细胞毒性的最有效药物，细胞毒性和ROS形成增加了3倍。铁螯合剂、去铁胺、NADH氧化剂和ATP生成剂（如果糖）也能保护肝细胞，减少ROS的形成和细胞毒性。当CH3OH被Fenton-like系统Fe(II)/H2O2氧化时，甲醛诱导的肝细胞蛋白羰基化（HCHO）也增加了约4倍，并与细胞毒性增加相关。在无细胞牛血清白蛋白系统中。Fe(II)/H2O2也增加了CH3OH氧化和HCHO蛋白羰基化。无毒的亚铁和H2O2生成系统增加了hcho诱导的细胞毒性和肝细胞蛋白羰基化。此外，ADH1和ALDH2抑制剂或gsh耗尽肝细胞显著增加HCHO的细胞毒性。肝细胞中HCHO浓度升高与HCHO诱导的蛋白羰基化增加相关。这些结果表明，1% O-2的CH3OH参与了芬顿系统的激活，形成HCHO。然而，在较高的02水平下，通过Fe(II)/H2O2产生的自由基可氧化CH3OH/HCHO形成促氧化自由基，通过蛋白质羰基化和ROS形成导致氧化应激增加，最终导致细胞死亡。2011爱思唯尔爱尔兰有限公司版权所有。
Methanol (CH3OH), a common industrial solvent, is metabolized to toxic compounds by several enzymatic as well as free radical pathways. Identifying which process best enhances or prevents CH3OH-induced cytotoxicity could provide insight into the molecular basis for acute CH3OH-induced hepatoxicity. Metabolic pathways studied include those found in 1) an isolated hepatocyte system and 2) cell-free systems. Accelerated Cytotoxicity Mechanism Screening (ACMS) techniques demonstrated that CH3OH had little toxicity towards rat hepatocytes in 95% O-2, even at 2 M concentration, whereas 50 mM was the estimated LC50 (2 h) in 1% O-2, estimated to be the physiological concentration in the centrilobular region of the liver and also the target region for ethanol toxicity. Cytotoxicity was attributed to increased NADH levels caused by CH3OH metabolism, catalyzed by ADH1, resulting in reductive stress, which reduced and released ferrous iron from Ferritin causing oxygen activation. A similar cytotoxic mechanism at 1% 02 was previous found for ethanol. With 95% 02, the addition of Fe(II)/H2O2, at non-toxic concentrations were the most effective agents for increasing hepatocyte toxicity induced by 1 M CH3OH, with a 3-fold increase in cytotoxicity and ROS formation. Iron chelators, desferoxamine, and NADH oxidizers and ATP generators, e.g. fructose, also protected hepatocytes and decreased ROS formation and cytotoxicity. Hepatocyte protein carbonylation induced by formaldehyde (HCHO) formation was also increased about 4-fold, when CH3OH was oxidized by the Fenton-like system, Fe(II)/H2O2, and correlated with increased cytotoxicity. In a cell-free bovine serum albumin system. Fe(II)/H2O2 also increased CH3OH oxidation as well as HCHO protein carbonylation. Nontoxic ferrous iron and a H2O2 generating system increased HCHO-induced cytotoxicity and hepatocyte protein carbonylation. In addition, HCHO cytotoxicity was markedly increased by ADH1 and ALDH2 inhibitors or GSH-depleted hepatocytes. Increased HCHO concentration levels correlated with increased HCHO-induced protein carbonylation in hepatocytes. These results suggest that CH3OH at 1% O-2 involves activation of the Fenton system to form HCHO. However, at higher 02 levels, radicals generated through Fe(II)/H2O2 can oxidize CH3OH/HCHO to form pro-oxidant radicals and lead to increased oxidative stress through protein carbonylation and ROS formation which ultimately causes cell death. (C) 2011 Elsevier Ireland Ltd. All rights reserved.