Damage of rat liver microsomal mixed function oxidase system by carbon tetrachloride. In vivo study with selective inhibitor of lipid peroxidation.

Pretreatment of rats with 4-[4-N-sodium-N-(5-ethyl-1-thia-3,4-diazol-2-yl)sulfophenylamin o]-5- methoxy-1,2-benzoquinone (Q) before carbon tetrachloride intoxication inhibited lipid peroxidation by 85% but did not prevent cytochrome P-450 destruction, decrease of hydroxylase activity, and loss of the capability to bioactivate carbon tetrachloride in rat liver microsomes. Also no influence of Q on 10-day lethality was found. We conclude that covalent binding of free radical products of metabolic cleavage to various cellular structures is apparently the main damage factor of carbon tetrachloride hepatotoxicity rather than lipid peroxidation.     

Reduced glutathione protection against rat liver microsomal injury by carbon tetrachloride. Dependence on O2.

Rat liver microsomal membranes contain a reduced-glutathione-dependent protein(s) that inhibits lipid peroxidation in the ascorbate/iron microsomal lipid peroxidation system. It appears to exert its protective effect by scavenging free radicals. The present work was carried out to assess the effect of this reduced-glutathione-dependent mechanism on carbon tetrachloride-induced microsomal injury and on carbon tetrachloride metabolism because they are known to involve free radicals. Rat liver microsomes were incubated at 37 degrees C with NADPH, EDTA and carbon tetrachloride. The addition of 1 mM-reduced glutathione (GSH) markedly inhibited lipid peroxidation and glucose 6-phosphatase inactivation and, to a lesser extent, inhibited cytochrome P-450 destruction. GSH also inhibited covalent binding of [14C]carbon tetrachloride-derived 14C to microsomal protein. These results indicate that a GSH-dependent mechanism functions to protect the microsomal membrane against free-radical injury in the carbon tetrachloride system as well as in the iron-based systems. Under anaerobic conditions, GSH had no effect on chloroform formation, carbon tetrachloride-induced destruction of cytochrome P-450 or covalent binding of [14C]carbon tetrachloride-derived 14C to microsomal protein. Thus, the GSH protective mechanism appears to be O2-dependent. This suggests that it may be specific for O2-based free radicals. This O2-dependent GSH protective mechanism may partly underlie the observed protection of hyperbaric O2 against carbon tetrachloride-induced lipid peroxidation and hepatotoxicity.     

Purification of phospholipid methyltransferase from rat liver microsomal fraction.

Phospholipid methyltransferase, the enzyme that converts phosphatidylethanolamine into phosphatidylcholine with S-adenosyl-L-methionine as the methyl donor, was purified to apparent homogeneity from rat liver microsomal fraction. When analysed by SDS/polyacrylamide-gel electrophoresis only one protein, with molecular mass about 50 kDa, is detected. This protein could be phosphorylated at a single site by incubation with [alpha-32P]ATP and the catalytic subunit of cyclic AMP-dependent protein kinase. A less-purified preparation of the enzyme is mainly composed of two proteins, with molecular masses about 50 kDa and 25 kDa, the 50 kDa form being phosphorylated at the same site as the homogeneous enzyme. After purification of both proteins by electro-elution, the 25 kDa protein forms a dimer and migrates on SDS/polyacrylamide-gel electrophoresis with molecular mass about 50 kDa. Peptide maps of purified 25 kDa and 50 kDa proteins are identical, indicating that both proteins are formed by the same polypeptide chain(s). It is concluded that rat liver phospholipid methyltransferase can exist in two forms, as a monomer of 25 kDa and as a dimer of 50 kDa. The dimer can be phosphorylated by cyclic AMP-dependent protein kinase.     

Spin-trapping studies on the free-radical products formed by metabolic activation of carbon tetrachloride in rat liver microsomal fractions isolated hepatocytes and in vivo in the rat.

1. The metabolic activation of carbon tetrachloride to free-radical intermediates is an important step in the sequence of disturbances leading to the acute liver injury produced by this toxic agent. Electron-spin-resonance (e.s.r.) spin-trapping techniques were used to characterize the free-radical species involved. 2. Spin trapping was applied to the activation of carbon tetrachloride by liver microsomal fractions in the presence of NADPH, and by isolated intact rat hepatocytes. The results obtained with the spin trap N-benzylidene-2-methylpropylamine N-oxide ('phenyl t-butyl nitrone') (PBN) and [13C]carbon tetrachloride provide unequivocal evidence for the formation and trapping of the trichloromethyl free radical in these systems. 3. With the spin trap 2-methyl-2-nitrosopropane, however, the major free-radical species trapped are unsaturated lipid radicals produced by the initiating reaction of lipid peroxidation. 4. Although pulse radiolysis and other evidence support the very rapid formation of the trichloromethyl peroxy radical from the trichloromethyl radical and oxygen, no clear evidence for the trapping of the peroxy radical was obtainable. 5. The effects of a number of free-radical scavengers and metabolic inhibitors on the formation of the PBN-trichloromethyl radical adduct were studied, as were the influences of changing the concentration of PBN and incubation time. 6. High concentrations of the spin traps used were found to have significant effects on cytochrome P-450-mediated reactions; this requires caution in interpreting results of experiments done in the presence of PBN at concentrations greater than 50 mM.     

Calcium inhibition of rat liver microsomal calcium-dependent ATPase

Measurement of the inward rate of Ca2+ transport by rat liver microsomes under conditions of varying free intravesicular Ca2+ (1 microM to 5 mM) revealed that inward transport rate is maximum at low intravesicular Ca2+, and that transport rate decreases with an apparent inhibition constant of about 250-350 microM as intravesicular Ca2+ accumulates. This relationship is confirmed by measurement of Ca2+-dependent ATPase activity; activity is greatest when intravesicular Ca2+ is 1 microM, is lower when intravesicular Ca2+ is 60 microM, and is minimum when intravesicular Ca2+ is 5 mM. Unexpectedly, the ratio of Ca2+ transport rate to Ca2+-dependent ATP hydrolysis rate appears to be significantly greater than 2:1.     

Effect of carbon tetrachloride administration on the synthesis of triglycerides and phospholipids in rat liver

In vivo incorporation of choline-methyl-(14)C into liver lecithin and its biosynthetic precursors was studied in CCl(4)-treated rats. Radioactivity in cytidine diphosphoryl (CDP-)choline and lecithin was reduced to one-third of control levels, whereas that of phosphorylcholine was increased to 4.7 times control levels. Incorporation of phosphorylcholine-(32)P into lecithin by homogenates prepared from livers of CCl(4)-treated animals was reduced, but conversion of CDP-choline-(32)P to lecithin by the isolated microsomal fraction did not show any significant depression. A block in the synthesis of CDP-choline is indicated. The in vivo utilization of methionine for lecithin synthesis was not affected. After intravenous injection of palmitic acid-1-(14)C, radioactivity of triglycerides from microsomal and mitochondrial fractions was markedly lower than the controls, whereas radioactivity of triglycerides in the soluble fraction was greatly increased. Radioactivity of diglycerides changed from 0.5% of total lipids in the control to 10% of total lipids in CCl(4)-treated animals. Incorporation of palmitic acid into phospholipids was also suppressed. The results demonstrate that synthesis of both phospholipids and triglycerides is inhibited in rats 4-5 hr after CCl(4) administration.     

The effect of a one-time administration of phenobarbital and carbon tetrachloride on comuton activity in the rat liver

Infusion of phenobarbital and CCl4 was found to induce comuton control of mitochondrial respiration in a liver of starved rats. Comuton regulation of liver mitochondria respiration can be activated either by increase in liver activity or by damage caused by CCl4. The comuton regulation is directly induced by disturbance of energetic homeostasis of liver cells.     

High-performance liquid chromatographic determination of phospholipid peroxidation products of rat liver after carbon tetrachloride administration

A method to detect and determine phospholipid peroxidation products in a biological system was developed using reversed-phase high performance liquid chromatography and normal-phase HPLC. Reversed-phase HPLC could separate phosphatidylcholine (PC) hydroperoxides and phosphatidylethanolamine (PE) hydroperoxides of rat liver from the respective phospholipids. A linear relationship was observed between these hydroperoxides and their peak areas on the chromatogram. In the experiment with rats administered CCl4, reversed-phase HPLC gave prominent, large peaks attributable to the peroxidation of phospholipids, and the peroxide level of the liver phospholipids was tentatively determined. Normal-phase HPLC analysis confirmed that both PC and PE in the liver phospholipids were peroxidized after CCl4 treatment. Neither the thiobarbituric acid value of the liver homogenate nor the fatty acid composition of the liver phospholipid fraction showed any significant difference between CCl4-treated and control rats. It is concluded that normal-phase HPLC and reversed-phase HPLC can complement each other to serve as a direct and sensitive method for the determination of lipid peroxide levels in a biological source. However, it was difficult to distinguish phospholipid hydroperoxides from their hydroxy derivatives.     

Heterogeneous activation of protein kinase C during rat liver regeneration induced by carbon tetrachloride administration

During rat liver regeneration induced by carbon tetrachloride administration, the protein kinase C alpha subspecies was activated in a heterogeneous fashion, a higher number of hepatocytes expressing the protein kinase C alpha subspecies being detected in the pericentral zone than in the periportal zone. This zonal heterogeneity became maximal at 24 h after the treatment. The distribution of hepatocytes expressing the protein kinase C alpha subspecies was roughly coincident with that of hepatocytes exhibiting DNA synthesis. These results suggest that protein kinase C may play a crucial role in liver regeneration.     

Binding of inositol trisphosphate by a liver microsomal fraction.

To test the hypothesis that inositol trisphosphate (InsP3) mediates adaptation and excitation in invertebrate photoreceptors, we measured its formation on a rapid time scale in squid retinas. For squid, excitation and adaption occurs within 0.1 and 1-2 s respectively. We could detect an elevation in InsP3 within 200 ms of a bright flash. This increase is about 240% over dark basal levels and is maintained for at least 2 min after a flash. The increase probably occurs in the photoreceptors, which are the only neurons in squid retinas. Analysis by h.p.l.c. indicates that the light-regulated isomer is Ins(1,4,5)P3, which is formed by the hydrolysis of phosphatidylinositol bisphosphate (PtdInsP2).     

Effect of sodium valproate on subcellular fraction enzymes in rat liver

Previous observations that valproic acid (VPA) causes hepatic damage prompted us to investigate the effect of large doses of the drug (0.6, 1.2 and 1.8 mmol/kg/day) on a number of liver enzymes located on different subcellular fractions. In mitochondria, glutamate dehydrogenase, aspartate aminotransferase and ornithine carbamoyltransferase were significantly increased (1.8 mmol/kg/day). In microsomes, gamma-glutamyltransferase activity increased significantly (1.8 mmol/kg) and cytochrome P-450 content decreased significantly (1.2 and 1.8 mmol/kg). In cytosol, both aspartate and alanine aminotransferase activities were increased at all dose levels. These results indicate that VPA induces dose-dependent changes in some liver enzyme activities.     

A note on the stability of conjugated diene absorption of rat liver microsomal lipids after carbon tetrachloride poisoning

Liver microsomal lipid peroxidation has been observed in fatal human CCl(4) poisoning, in rats with fatty livers induced by CCl(4) or by yellow phosphorus, and in mice poisoned with 1,1,2,2-tetrachloroethane. These observations suggest the possibility that other instances of toxic liver injury may involve lipid peroxidation. Cases of acute, fatal, toxic liver injury (e.g., from halothane anesthesia) are not likely to occur at or near laboratories equipped to determine whether any lipid peroxidation might have taken place. The data presented indicate that rat livers may be stored frozen for at least 7 days with no demonstrable diminution in CCl(4)-induced conjugated diene absorption of liver microsomal lipids.     

Mechanism of in vivo carbon tetrachloride-induced liver microsomal cytochrome P-450 destruction.

Prior administration of aminotriazole (3-amino-1,2,4-triazole) or pyrazole to rats resulted in a significant prevention of the CCl₄-induced decrease in the liver microsomal P-450 content. In A/J mice the CCl₄ activation and P-450 destruction occurred in absolute absence of lipid peroxidation as determined by uv absorption. The data suggest that P-450 destruction is mainly mediated by direct attack of CCl₄ metabolites rather than by CCl₄-induced lipid peroxidation.     

Lipid peroxidation inactivates rat liver microsomal glycerol-3-phosphate acyl transferase. Effect of iron and copper salts and carbon tetrachloride

Lipid peroxidation is known to affect the activity of several enzymes including microsomal enzymes such as glucose-6-phosphatase; but its effect on the enzymes of lipid biosynthesis has not been investigated. Glycerol-3-phosphate acyltransferase (GPAT) represents the first committed step and probably the rate limiting step in glycerolipid synthesis and thus may be a good candidate for study. Rat liver microsomal GPAT was assayed after preincubating the microsomes under conditions known to induce peroxidation. In 30 min, 10 microM Fe2+ can diminish the activity by as much as 80%. The inactivating effect can be blocked to different extents by several antioxidants, while ascorbic acid enhances it. These effects, along with the concomitant measurement of lipid peroxidation, indicate that microsomal GPAT activity is inactivated by lipid peroxidation in a sensitive and rapid fashion. This is further confirmed by the inactivating effect of carbon tetrachloride, which is known to induce lipid peroxidation in microsomes. Fe3+ also inactivates the enzyme, but at a higher concentration. Copper salts inactivate GPAT by a mechanism apparently different from that of iron. The mechanism might involve a direct sulfhydryl modification by copper and lipid peroxidation apparently different from that induced by iron. It is suggested that the inactivation of GPAT by lipid peroxidation could accelerate the process of membrane disintegration caused by lipid peroxidation in pathological conditions involving free radical-mediated tissue injury.