Effects of carbon tetrachloride on isolated rat liver cells: stimulation of lipid peroxidation and inhibitory action of free-radical scavengers [proceedings].
Effects of carbon tetrachloride on isolated rat liver cells: stimulation of lipid peroxidation and inhibitory action of free-radical scavengers [proceedings].
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四氯化碳对离体大鼠肝细胞的影响：刺激脂质过氧化和自由基清除剂的抑制作用[论文集]。
DOI：
10.1042/bst0060589
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发表时间：
1978
影响因子：
3.9
通讯作者：
E. Gravela
中科院分区：
文献类型：
--
作者：
G. Poli;M. Chiono;T. Slater;M. Dianzani;E. Gravela
四氯化碳对大鼠肝脏毒性作用的生化机制已被广泛研究（参考文献，见Recknagel, 1967; Slater, 1972）。最早的形态学紊乱与内质网有关；体外微粒体部分的生化研究强调了四氯化碳破坏这种重要肝细胞结构的许多酶活性的速度，四氯化碳对肝脏的许多破坏性影响需要四氯化碳的初步代谢或“激活”，以形成一种高活性的中间体，可能是三氯甲基自由基（CC13*）。肝内质网产生CC&'的一个后果是刺激脂质过氧化，通常通过硫代巴比妥酸反应物质的形成来测量（见Slater， 1972）。在肝微粒体中，四氯化碳对脂质过氧化的刺激依赖于NADPH的供应，受到多种自由基清除剂的抑制（Slater & Sawyer, 1971c），并且不受对氯脲苯甲酸盐或一氧化碳浓度的影响，后者会强烈抑制药物代谢（Slater & Sawyer, 19716）。这样的结果导致了这样的建议（Slater & Sawyer, 19716）：四氯化碳是通过解离电子捕获代谢的，涉及nadph -黄蛋白，可能还有非血红素铁/硫醇蛋白，而不是细胞色素P-450。然而，这些先前的研究都是用肝微粒体悬浮液进行的，可能会认为内质网的破坏产生这些悬浮液可能导致四氯化碳还原的途径是人为的。在这项研究中，我们重复了先前用肝微粒体悬浮液获得的结果，但这次使用的是分离的全肝细胞，其中内质网的结构完整性得到了维持。雄性白化Wistar大鼠来自英国肯特郡马盖特查尔斯河（Charles River），体重250-3008；各组饲喂C.R.M.X.日粮（c.h ill Group, Poole, Dorset， U.K.）和随意饮水。肝细胞是通过对Gravela ctal描述的方法进行小修改而分离出来的。977(1)。由于与硫代巴比妥酸反应的物质只有在细胞悬浮在生理盐水介质中才能检测到，因此我们使用以下培养基：60m~-NaC1/40m~-KC1/5Om~-Hepes缓冲液（p H 7）。4) / 2 m~ MgSOJl m~ C a C l, / 1 m~ N a, H P 0， / 5 m~-葡萄糖/0.58m~-氨基酸混合物。用血细胞计测定细胞数；采用台盼蓝排除法常规评估细胞活力。细胞悬浮液样品（3mI，每个2 × lo7个细胞）在37°C的黑暗环境中，有或没有四氯化碳（2.5~1/3ml）在封闭系统中孵育60年（见图1；E. Gravela， G. Poli， E. Albano和M. U. Dianzani，未发表的作品）。用三氯乙酸沉淀蛋白质后，在细胞悬液上测定硫代巴比妥酸反应物质（Slater & Sawyer, 1 7 1 ~）。用Slater & Sawyer（19716）描述的方法测定细胞匀浆中的氨基芘代谢。细胞色素P-450在全细胞中测定，如Kupfer & Orrenius（1970）所述，使用91升的毫摩尔消光系数。mmolz1.cm——”。异丙胺和化合物skf525a是英国埃塞克斯郡达格南郡的May和Baker以及英国赫茨郡韦尔文花园城的Smith、Kline和French赠送的礼物。分别是美国和英国。图1给出了一些代谢抑制剂和自由基清除剂对硫代巴比妥酸阳性产物产生影响的结果
The biochemical mechanisms underlying the toxic effects of carbon tetrachloride on rat liver have been studied extensively (for references, see Recknagel, 1967; Slater, 1972). The earliest morphological disturbances described have concerned the endoplasmic reticulum; biochemical studies in vitro with microsomal fractions have emphasized the rapidity with which carbon tetrachloride damages many of the enzymic activities of this important hepatocellular structure, Many of the damaging effects of carbon tetrachloride on the liver require a preliminary metabolism or 'activation' of carbon tetrachloride t o a highly reactive intermediate that is probably the trichloromethyl radical (CC13*). One consequence of the production of CC&' in the liver endoplasmic reticulum is a stimulation of lipid peroxidation, which is usually measured by the formation of thiobarbituric acidreactive substances (see Slater, 1972). In liver microsomal fractions, the stirnulation of lipid peroxidation by carbon tetrachloride is dependent on a supply of NADPH, is inhibited by a variety of free-radical scavengers (Slater & Sawyer, 1971c), and is not decreased by concentrations of p-chloromercuribenzoate or CO that strongly inhibit drug metabolism (Slater & Sawyer, 19716). Such results have led to the suggestion (Slater & Sawyer, 19716) that carbon tetrachloride is metabolized through dissociative electron capture involving the NADPH-flavoprotein and, possibly, a non-haem iron/ thiol protein rather than cytochrome P-450. However, these previous studies were all done with liver microsomal suspensions, and it might be argued that the disruption of endoplasmic reticulum to produce these suspensions may result in pathways of carbon tetrachloride reduction that are artifacts. In this study we have repeated results obtained previously with liver microsomal suspensions, but this time using isolated whole liver cells, where the structural integrity of the endoplasmic reticulum is maintained. Male albino Wistar rats were used from Charles River, Margate, Kent, U.K. (body wt. 250-3008); they were fed on diet C.R.M.X. (C. Hill Group, Poole, Dorset, U.K.) and water ad libitum. Liver cells were isolated by a small modification of the method described by Gravela ctal. (1 977). Since substances that react with thiobarbituric acid were detectable only when cells were suspended in saline media, we used the following incubation medium: 60m~-NaC1/40m~-KC1/5Om~-Hepes buffer ( p H 7 . 4 ) / 2 m ~ MgSOJl m ~ C a C l , / l m ~ N a , H P 0 , / 5 m~-glucose/0.58m~-amino acid mixture. Cell numbers were measured with a haemocytometer; cell viability was routinely assessed by the Trypan Blue-exclusion procedure. Samples ofcell suspension (3mI, 2 x lo7 cells each) were incubated for 60niin a t 37°C in thedark, withorwithout carbon tetrachloride(2.5~1/3ml)inaclosedsystem(seelegend t o Fig. 1 ; E. Gravela, G. Poli, E. Albano and M. U. Dianzani, unpublished work). Thiobarbituric acid-reacting material was measured (Slater & Sawyer, 1 9 7 1 ~ ) on cell suspensions after protein precipitation with trichloroacetic acid. Aminopyrene metabolism was measured in the cell homogenate by the method described by Slater & Sawyer (19716). Cytochrome P-450 was determined in whole cells as described by Kupfer & Orrenius (1970) by using a millimolar extinction coefficient of 91 litre. mmolz1.cm-'. Promethazine and compound S K F 525A were gifts from May and Baker, Dagenham, Essex, U.K. and Smith, Kline and French, Welwyn Garden City, Herts., U.K., respectively. Fig. 1 gives some of the results obtained for the effects of a number of metabolic inhibitors and free-radical scavengers on the production of thiobarbituric acid-positive