Tsutsumi M, Lasker J M, Shimizu M, Rosman A S, Lieber C S
Section of Liver Disease, Bronx Veterans Administration Medical Center, New York 10468.
Hepatology. 1989 Oct;10(4):437-46. doi: 10.1002/hep.1840100407.
Perivenular hepatocytes are the first cells within the liver lobule to display signs of toxicity following long-term alcohol use. In an attempt to explain this phenomenon, we have examined the hepatic intralobular distribution in rats and man of P450IIE1, a P-450 isozyme that not only oxidizes ethanol but is also inducible by this agent. Frozen liver sections and microsomes were prepared from male Sprague-Dawley rats pair-fed liquid diets containing 36% of total calories as either ethanol or carbohydrate (control) for 10 to 21 days. Frozen sections or microsomes were also prepared from liver biopsy samples obtained from 17 male patients with diverse drinking histories. Immunohistochemical staining was performed using the peroxidase-antiperoxidase method after liver sections were reacted with monospecific antibody (IgG) directed against human P450IIE1. Immunoreaction intensity was blindly rated in order to provide a semiquantitative assessment of P450IIE1 levels in perivenular, midzonal and periportal hepatocytes. At low applied anti-P450IIE1 IgG concentrations (2.5 micrograms per ml), P450IIE1 immunostaining was observed exclusively within the perivenular area in sections from all ethanol-treated rats, whereas no visible immunoreaction was found in sections from their pair-fed controls. At higher applied antibody concentrations (15 micrograms per ml), panlobular antigen immunostaining was observed in five of the six ethanol-treated animals, and P450IIE1 could now also be detected in perivenular hepatocytes from the control rats. In accordance with these immunohistochemical findings, protein blotting with anti-P450IIE1 IgG revealed a 7.5-fold increase in liver microsomal P450IIE1 content in ethanol-treated animals when compared to their pair-fed controls. With human liver, perivenular P450IIE1 immunostaining was observed only in biopsy sections obtained from recently drinking alcoholics (abstinence period of 1 day) when limiting concentrations (5 micrograms per ml) of the primary antibody were used. Increasing the applied anti-P450IIE1 IgG concentration to 15 micrograms per ml resulted in perivenular staining of the immunogen in liver sections from abstinent alcoholics (abstinence period of 4 to 8 days) and nondrinkers as well. Immunoblot analysis of human liver microsomes disclosed that the hepatic microsomal P450IIE1 content in recently drinking alcoholics was 4-fold higher than that found in nondrinkers. Our results show that, in both rats and in man, P450IIE1 is normally localized within the perivenular region, or zone 3, of the liver lobule, and that induction of P450IIE1 by prolonged alcohol consumption occurs primarily within the same acinar regi
长期饮酒后，肝小叶内的肝血窦周围肝细胞是最先出现毒性迹象的细胞。为了解释这一现象，我们研究了大鼠和人类肝脏小叶内P450IIE1的分布，P450IIE1是一种细胞色素P-450同工酶，不仅能氧化乙醇，还可被乙醇诱导。从雄性Sprague-Dawley大鼠制备冷冻肝切片和微粒体，这些大鼠成对喂养含36%总热量的液体饮食，其中一种饮食的热量来源为乙醇，另一种为碳水化合物（对照），持续10至21天。还从17名有不同饮酒史的男性患者的肝活检样本制备了冷冻切片或微粒体。肝切片与针对人P450IIE1的单特异性抗体（IgG）反应后，采用过氧化物酶-抗过氧化物酶法进行免疫组织化学染色。为了对肝血窦周围、中区和汇管区肝细胞中P450IIE1水平进行半定量评估，对免疫反应强度进行了盲法评分。在低浓度抗P450IIE1 IgG（每毫升2.5微克）时，在所有经乙醇处理的大鼠切片中，仅在肝血窦周围区域观察到P450IIE1免疫染色，而在其成对喂养的对照大鼠切片中未发现可见的免疫反应。在较高抗体浓度（每毫升15微克）时，在6只经乙醇处理的动物中有5只观察到全小叶抗原免疫染色，此时在对照大鼠的肝血窦周围肝细胞中也能检测到P450IIE1。根据这些免疫组织化学结果，用抗P450IIE1 IgG进行蛋白质印迹分析显示，与成对喂养的对照相比，经乙醇处理的动物肝脏微粒体中P450IIE1含量增加了7.5倍。对于人类肝脏，当使用限量浓度（每毫升5微克）的一抗时，仅在最近饮酒的酗酒者（戒酒1天）的活检切片中观察到肝血窦周围P450IIE1免疫染色。将抗P450IIE1 IgG浓度提高到每毫升15微克时，戒酒的酗酒者（戒酒4至8天）和不饮酒者的肝切片中免疫原也出现了肝血窦周围染色。对人类肝脏微粒体的免疫印迹分析表明，最近饮酒的酗酒者肝脏微粒体中P450IIE1含量比不饮酒者高4倍。我们的结果表明，在大鼠和人类中，P450IIE1通常定位于肝小叶的肝血窦周围区域或3区，长期饮酒对P450IIE1的诱导主要发生在同一腺泡区域。
