Eric A. Glende

[40] Spectrophotometric detection of lipid conjugated dienes

Publisher Summary Like many other substances, naturally occurring lipids exhibit simple end absorption in ultraviolet light as the wavelength is lowered toward 200 nm. The spectra of a variety of organic molecules containing conjugated dienes, however, are characterized by intense absorption, the so-called K band, which may range, with respect to peak absorption, from 215 to 250 nm, depending on nearby substituent groups. Ultraviolet spectrophometric detection of conjugated dienes has been used for many years in the food industry for the detection of autoxidized lipids. The method appears to have been applied for the first time to the problem of liver cell lipid peroxidation of toxigenic origin in 1966 and has been widely used since. For a variety of pathological processes, the question has been raised whether peroxidative decomposition of membrane lipids has occurred in vivo . A second principle of the method recognizes that for whole-animal studies involving possible lipid peroxidation, the fraction of endogenous lipids actually peroxidized may not only be low, but the process of lipid peroxidation may be confined to a particular subcellular structure.

Pathological mechanisms in carbon tetrachloride hepatotoxicity.

Liver cell injury induced by carbon tetrachloride involves initially the metabolism of carbon tetrachloride to trichloromethyl free-radical by the mixed function oxidase system of the endoplasmic reticulum. It is postulated that secondary mechanisms link carbon tetrachloride metabolism to the widespread disturbances in hepatocyte function. These secondary mechanisms could involve the generation of toxic products arising directly from carbon tetrachloride metabolism or from peroxidative degeneration of membrane lipids. The possible involvement of radical species such as trichloromethyl (.CCl3), trichloromethylperoxy (.OOCCl3), and chlorine (.Cl) free radicals, as well as phosgene and aldehydic products of lipid peroxidation, as toxic intermediates is discussed. Data do not support the view that an increase in cytosolic free calcium is important in the toxic action of carbon tetrachloride or bromotrichloromethane. In addition, carbon tetrachloride-induced inhibition of very low density lipoprotein secretion by hepatocytes is not a result of elevated levels of cytosolic free calcium.

Critical role of lipid peroxidation in carbon tetrachloride-induced loss of aminopyrine demethylase, cytochrome P-450 and glucose 6-phosphatase

Abstract Liver microsomal glucose 6-phosphatase. cytochrome P-450 and aminopyrine demethylase all decrease rapidly in the CCl 4 -poisoned rat. It has been suggested that such enzyme loss may be due to direct attack on enzyme catalytic sites by free radical cleavage products of CCl 4 metabolism. An alternate view has favored the suggestion that peroxidative decomposition of lipids is an intermediate link between initial homolytic cleavage of the CCl 3 -Cl bond and eventual loss of these enzymes. We have subjected these two ideas to a critical test. In an anaerobic system in vitro containing liver microsomes supplemented with an NADPH-generating system, and in the presence of EDTA, all added CCl 4 is metabolized in 90 min. About one-third appears as CHCl 3 amd most of the remainder is covalently bound to microsomal lipids and proteins. In this anaerobic system in vitro there is no evolution of malonic dialdehyde. During the period of conversion of CCl 4 to CHCl 3 , when extensive binding of 14 C from 14 CCl 4 is also taking place, there was no detectable loss of either glucose 6-phosphatase or cytochrome P-450, and aminopyrine demethylase activity decreased minimally. In the same system, under aerobic conditions and without EDTA, there is vigorous lipid peroxidation and all three of these enzyme systems decrease markedly. These experiments demonstrate conclusively that CCl 3 -Cl bond cleavage and covalent binding of products of CCl 4 metabolism do not constitute a mechanism for loss of microsomal glucose 6-phosphatase, cytochrome P-450 or aminopyrine demethylase for the particular anaerobic conditions employed in vitro . By extension they suggest, but do not prove, that covalent binding of CCl 4 carbon probably does not constitute a mechanism for CCl 4 -induced loss of these enzymes in vivo . Rather, these experiments support the view that lipid peroxidation is an obligatory link between initial CCl 3 -Cl bond cleavage and loss of these enzymes.

Carbon tetrachloride-induced protection against carbon tetrachloride toxicity. The role of the liver microsomal drug-metabolizing system.

Abstract Intragastric administration to rats of a small non-lethal dose of carbon tetrachloride produces a dramatic protective action against subsequently administered large and ordinarily lethal doses of the same liver poison. Resistance to the lethal effect of carbon tetrachloride sets in by 12 hr and is fully developed by 24 hr. Protection lasts for 3–5 days after which it gradually subsides. Activity of liver microsomal aminopyrine demethylase and cytochrome P-450 concentration were followed throughout the course of development of the protection phenomenon. Activity of aminopyrine demethylase and cytochrome P-450 concentration began to fall immediately after administration of the small initial dose of carbon tetrachloride. At 24 hr, when protection was complete, the activity of this enzyme system was about one-fourth of control levels. The depression lasted for 4 days, which was followed by gradual recovery. The exact parallelism between depression of liver microsomal mixed function oxidase activity and resistance to the lethal effects of carbon tetrachloride affords strong evidence for the view that metabolism of carbon tetrachloride by the liver microsomal drug-metabolizing system is a necessary prerequisite for the toxicity of this liver poison.

Destruction of liver microsomal calcium pump activity by carbon tetrachloride and bromotrichloromethane

Abstract Disturbed cellular calcium homeostatis has been observed during carbon tetrachloride (CCl4) poisoning, with large alterations in calcium content occurring 8 hr after administration. Moore el at. [10] have shown that the hepatic smooth endoplasmic reticulum can sequester calcium and that this ability is decreased severely within 30 min after CCl4 administration to rats. It was suggested that disturbed endoplasmic reticulum calcium pump activity may have a critical role in the expression of CCl4 hepatotoxicity. We examined the effect of bromotrichloromethane (BrCCl3) and CCl4 metabolism on the calcium pump of Fe2+-free rat liver microsomes. It was determined that severe deficits in calcium uptake can be correlated with minimal lipid peroxidation induced by these agents. At a given level of lipid peroxidation, calcium uptake was affected more severely than were the activities of the microsomal enzymes glucose-6-phosphatase and aminopyrine demethylase. Calcium uptake was increased 7-fold by the presence of 5 mM ATP in incubations prior to assay of calcium sequestration. Lipid peroxidation induced by BrCCl3-NADPH was accompanied by leakage of calcium from calcium-loaded microsomes. These results strengthen the possibility that disturbances in intracellular calcium homeostasis may be a key event in liver injury induced by BrCCl3 and CCl4.

Activation of phospholipase A2 by carbon tetrachloride in isolated rat hepatocytes

Freshly isolated rat hepatocytes were exposed to carbon tetrachloride (CCl4) for periods up to 4 hr. Phospholipase A2 activity of these preparations was determined by measuring either the release of [3H]arachidonic acid from cellular phospholipids prelabeled with [3H]arachidonic acid or by measuring the formation of [14C]lysophosphatidylethanolamine from cellular lipids prelabeled with [14C]ethanolamine. Through the use of hexane-partition extraction and thin-layer chromatographic analysis of hepatocyte lipid extracts it was found that CCl4 stimulated phospholipase A2 activity in a dose- and time-dependent manner. Carbon tetrachloride at concentrations of 0.23 to 1.3 mM produced a 1.4- to 5.3-fold increase in phospholipase activity which was initiated within 30-60 min of incubation at 37 degrees. The role of phospholipase activation as a secondary mechanism of CCl4-induced hepatocyte injury is discussed.

Hepatotoxicity of bromotrichloromethane— bond dissociation energy and lipoperoxidation

Abstract Bromotrichloromethane (BrCCl 3 ) is much more potent than CCl 4 or CHCl 3 as a liver poison. Bond dissociation energies for cleavage of H-CCl 3 , Cl-CCl 3 , and Br-CCl 3 are in the order H-CCl 3 > Cl-CCl 3 > Br-CCl 3 . A low bond dissociation energy implies a greater tendency of the given bond to cleave homolytically. In vitro , BrCCl 3 is 200 times more potent than CCl 4 in promoting peroxidative decomposition of rat liver microsomal lipids. CHCl 3 has virtually no potency as a pro-oxidant in vitro . BrCCl 3 produces in rats about three times the degree of liver microsomal lipid peroxidation than does CCl 4 , at equivalent doses. CHCl 3 does not produce lipid peroxidation in vivo . Administration of BrCCl 3 in very low doses (0.025 ml/kg of body weight) produces a dramatic fall in liver microsomal cytochrome P-450. Rats with low levels of cytochrome P-450 due to prior administration of BrCCl 3 are completely resistant to large, and otherwise lethal doses of CCl 4 . A lethality study is presented which suggests that BrCCl 3 may have extrahepatic sites of action.

Destruction of microsomal cytochrome P-450 and glucose-6-phosphatase by lipids extracted from peroxidized microsomes

Abstract Lipids were extracted from peroxidized and nonperoxidized rat liver microsomes. The lipids were dried down under O 2 -free nitrogen, and then suspended by sonication in aqueous media suitable for further testing. Lipids extracted from peroxidized microsomes destroyed 61% of the cytochrome P -450 content and 73% of the glucose-6-phosphatase activity of fresh microsomes during 90 min of incubation at 38°C. Lipids isolated from nonperoxidized microsomes had no such destructive qualities. These results suggest that when cytochrome P -450 and glucose-6-phosphatase decrease during the peroxidation of liver endoplasmic reticulum, toxic lipids may play an important role in the mechanism of destruction.

On the mechanism of carbon tetrachloride toxicity—Coincidence of loss of drug-metabolizing activity with peroxidation of microsomal lipid

Abstract Lipoperoxidation in vitro of liver microsome preparations results in a coincidental loss of drug metabolism measured as aminopyrine demethylase activity. NADPH-cytochrome c reductase activity, on the contrary, increased as peroxidative damage to the membrane progressed. These results suggest the effect of lipoperoxidation to be remote from the flavoprotein stage of the microsomal electron transport system. The addition of carbon tetrachloride, in amounts equivalent to that found after dosage, in vivo , to microsomal preparations protected from lipid peroxidation did not alter the enzymic action toward aminopyrine demethylation. Carbon tetrachloride administration is known to induce microsomal lipid peroxidation as well as depress the microsomal drug-metabolizing system. The fact that aminopyrine demethylase has been shown to be very sensitive to lipoperoxidative decomposition of the microsomal membrane supports the contention that carbon tetrachloride-induced inhibition of drug metabolism is a direct result of lipoperoxidative damage to the endoplasmic reticulum.

Biochemical basis for the in vitro pro-oxidant action of carbon tetrachloride

Abstract The occurrence of TPNH-linked liver microsomal lipid peroxidation has been confirmed in the present study. Cytochrome c and ferricyanide were effective inhibitors in vitro of malonaldehyde production in hepatic microsomes and microsome-supernatant fractions, but only when added at the beginning of the incubation. Although malonaldehyde formation ceased when EDTA was added to a microsome-supernatant preparation undergoing vigorous lipid peroxidation, cytochrome c or ferricyanide, when added at a similar time, did not affect the further course of lipid peroxidation. A pro-oxidant action of carbon tetrachloride which required TPNH as a cofactor was demonstrated in washed liver microsome preparations. Addition of carbon tetrachloride produced an increase in malonaldehyde formation in this system. When a TPNH-generating system was added to the microsome-supernatant fraction of rat liver, malonaldehyde production was depressed, and in this supplemented microsome-supernatant system carbon tetrachloride exhibited a marked pro-oxidant action. Addition of reduced glutathione to a microsome-supernatant system supplemented with a TPNH-generating system resulted in a further suppression of spontaneous lipid peroxidation, and in this system a maximal pro-oxidant effect of added carbon tetrachloride was observed. Benzene and heptane did not exhibit pro-oxidant actions in hepatic microsome-supernatant fractions supplemented with TPNH. The role of the microsomal electron transport system in the necrogenic action of carbon tetrachloride is briefly discussed. Alteratons of this system may explain why certain substances protect the liver from carbon tetrachloride-induced damage as well as why other materials potentiate the effects of the haloalkane.