Devi B G, Henderson G I, Frosto T A, Schenker S
Department of Medicine, University of Texas Health Science Center, San Antonio 78284-7878.
Hepatology. 1993 Sep;18(3):648-59.
Studies have shown that ethanol at moderate concentrations inhibits epidermal growth factor-dependent replication of fetal rat hepatocytes in culture. This may account for the growth/development impairment associated with fetal alcohol syndrome and decreased liver regeneration in alcoholic liver disease. In this study, we further define the mechanism(s) of the negative impact of ethanol on fetal rat hepatocytes and provide evidence that ethanol-induced injury to these cells is associated with membrane damage caused by lipid peroxidation, altered cell glutathione homeostasis and deranged mitochondrial structure and function. Exposure of fetal rat hepatocyte replication to ethanol (2 mg/ml) promptly resulted in blockade of replication, as indicated by a 40% reduction in DNA synthesis (p < 0.05). Assessment of cell injury on the basis of lactate dehydrogenase and ALT leakage indicated a statistically significant but not appreciable effect, whereas 51Cr leakage was more substantially increased (p < 0.05). Within 6 hr of ethanol exposure, superoxide radical levels increased more than twofold (p < 0.05). We noted a 56% increase in levels of diene conjugates, a 131% increase in malonaldehyde concentration and a 66% increase in fluorescent products of lipid peroxidation (all p < 0.05). Glutathione levels were decreased to 47% below control values (p < 0.05). Electron microscopic studies illustrated a slight disruption of mitochondrial structure (enlargement of mitochondria and dilation of cristae). This disruption was accompanied by mitochondrial swelling (increased permeability), altered mitochondrial membrane potential (a 16% decrease in rhodamine uptake), a 28% decrease in succinate dehydrogenase activity and a 30% decrease in cellular ATP level (p < 0.05). Pretreatment of fetal rat hepatocytes with 0.1 mmol/L N-acetylcysteine or S-adenosylmethionine for 24 hr prevented the ethanol-induced reduction of ATP and glutathione levels, essentially restored cell replication, ameliorated 51Cr leakage and decreased malonaldehyde and diene conjugate levels to 41% to 65% and 25% above control values, respectively. Pretreatment with 0.1 mmol/L vitamin E fully normalized malonaldehyde and diene conjugate levels and 51Cr leakage but failed to improve ATP levels or to increase significantly cell replication and glutathione levels. Concomitant administration of glutathione precursors with ethanol, rather than pretreatment, did not alter the impaired cell replication. Thus our data underscore the importance of cellular glutathione and ATP in preventing ethanol-induced decreases in fetal cell replication and suggest that alleviation of cellular lipid peroxidation alone is not sufficient to prevent this abnormality in fetal rat hepatocyte function.
研究表明，中等浓度的乙醇会抑制培养的胎鼠肝细胞中表皮生长因子依赖性复制。这可能解释了与胎儿酒精综合征相关的生长/发育障碍以及酒精性肝病中肝脏再生能力下降的原因。在本研究中，我们进一步明确了乙醇对胎鼠肝细胞产生负面影响的机制，并提供证据表明乙醇诱导的这些细胞损伤与脂质过氧化引起的膜损伤、细胞内谷胱甘肽稳态改变以及线粒体结构和功能紊乱有关。胎鼠肝细胞复制暴露于乙醇（2mg/ml）后，DNA合成立即减少40%（p<0.05），这表明复制迅速被阻断。基于乳酸脱氢酶和谷丙转氨酶泄漏对细胞损伤进行评估，结果显示具有统计学意义但影响不明显，而51Cr泄漏则显著增加（p<0.05）。乙醇暴露6小时内，超氧自由基水平增加了两倍多（p<0.05）。我们发现二烯共轭物水平增加了56%，丙二醛浓度增加了131%，脂质过氧化荧光产物增加了66%（所有p<0.05）。谷胱甘肽水平降至对照值以下47%（p<0.05）。电子显微镜研究显示线粒体结构略有破坏（线粒体肿大和嵴扩张）。这种破坏伴随着线粒体肿胀（通透性增加）、线粒体膜电位改变（罗丹明摄取减少16%）、琥珀酸脱氢酶活性降低28%以及细胞ATP水平降低30%（p<0.05）。用0.1mmol/L的N-乙酰半胱氨酸或S-腺苷甲硫氨酸对胎鼠肝细胞进行24小时预处理，可防止乙醇诱导的ATP和谷胱甘肽水平降低，基本恢复细胞复制，改善51Cr泄漏，并使丙二醛和二烯共轭物水平分别降至比对照值高41%至65%和25%。用0.1mmol/L维生素E预处理可使丙二醛和二烯共轭物水平以及51Cr泄漏完全恢复正常，但未能提高ATP水平，也未显著增加细胞复制和谷胱甘肽水平。乙醇与谷胱甘肽前体同时给药而非预处理，并未改变受损的细胞复制。因此，我们的数据强调了细胞内谷胱甘肽和ATP在预防乙醇诱导的胎儿细胞复制减少方面的重要性，并表明仅减轻细胞脂质过氧化不足以预防胎鼠肝细胞功能的这种异常。
