Effects of carbon tetrachloride on isolated rat liver cells: stimulation of lipid peroxidation and inhibitory action of free-radical scavengers [proceedings

Abstract

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