Alessandro F. Casini

Lipid peroxidation and antioxidant systems in the liver injury produced by glutathione depleting agents

The mechanisms of the liver damage produced by three glutathione (GSH) depleting agents, bromobenzene, allyl alcohol and diethylmaleate, was investigated. The change in the antioxidant systems represented by alpha-tocopherol (vitamin E) and ascorbic acid were studied under conditions of severe GSH depletion. With each toxin liver necrosis was accompanied by lipid peroxidation that developed only after severe depletion of GSH. The hepatic level of vitamin E was decreased whenever extensive lipid peroxidation developed. In the case of bromobenzene intoxication, vitamin E decreased before the onset of lipid peroxidation. Changes in levels of the ascorbic and dehydroascorbic acid indicated a redox cycling of vitamin C with the oxidative stress induced by all the three agents. Such a change of the redox state of vitamin C (increase of the oxidized over the reduced form) may be an index of oxidative stress preceding lipid peroxidation in the case of bromobenzene. In the other cases, such a change is likely to be a consequence of lipid peroxidation. Experiments carried out with vitamin E deficient or supplemented diets indicated that the pathological phenomena occurring as a consequence of GSH depletion depend on hepatic levels of vitamin E. In vitamin E deficient animals, lipid peroxidation and liver necrosis appeared earlier than in animals fed the control diet. Animals fed a vitamin E supplemented diet had an hepatic vitamin E level double that obtained with a commercial pellet diet. In such animals, bromobenzene and allyl alcohol had only limited toxicity and diethylmaleate none in spite of comparable hepatic GSH depletion. Thus, vitamin E may largely modulate the expression of the toxicity by GSH depleting agents.

Extraction and partial characterization of dialysable products originating from the peroxidation of liver microsomal lipids and inhibiting microsomal glucose 6-phosphatase activity

Abstract The dialysate obtained from a system containing actively peroxidizing liver microsomes shows inhibitory effects on glucose 6-phosphatase activity of freshly prepared liver microsomes (test system). The inhibitory factors were recovered in extracts obtained from the dialysate with ethyl ether or ethyl acetate. The extraction procedure completely removed the inhibitory activity from the dialysate. A partial separation of the products present in the dialysate extract was obtained by thin-layer chromatography. When the lipid materials eluted from individual bands or groups of bands were tested for inhibitory activity, it was found that various Chromatographic bands contained various degrees of inhibitory activity, and that the highest inhibitory activity occurs in a well resolved band that is stained yellow by a N , N -dimethyl- p -phenylene-diamine reagent. Additional studies indicated that this band contains most of the carbonyl functional groups detectable in the unfractionated dialysate extract, while it contains peroxide functional groups in trace amounts only. The peroxide functional groups present in the unfractionated dialysate extract were found to occur in various Chromatographic bands without a well defined relationship with the toxicological activity. It is concluded that lipoperoxidation products highly active in inhibiting microsomal glucose 6-phosphatase activity are fatty aldehydes or other carbonyl compounds, probably provided with a relatively long carbon chain.

Glutathione depletion: Its effects on other antioxidant systems and hepatocellular damage

1. The mechanisms of the liver damage produced by three glutathione (GSH)-depleting agents, bromobenzene, allyl alcohol and diethyl maleate, were investigated. 2. With each toxin liver necrosis was accompanied by lipid peroxidation that developed only after severe depletion of GSH. 3. Changes in antioxidant systems by alpha-tocopherol (vitamin E) and ascorbic acid were studied. A decrease in the hepatic level of vitamin E, and a change in the redox state of vitamin C (increase in oxidized over reduced form) were evident whenever extensive lipid peroxidation developed. However, in the case of bromobenzene intoxication these alterations preceded lipid peroxidation, and may be an index of oxidative stress leading to subsequent membrane damage. 4. Experiments carried out with vitamin E-deficient or supplemented diets indicated that pathological phenomena occurring as a consequence of GSH depletion depend on hepatic levels of vitamin E. In vitamin E-deficient animals, lipid peroxidation and liver necrosis appeared earlier than in animals fed the control diet. In animals fed a vitamin E-supplemented diet, bromobenzene and allyl alcohol had only limited toxicity, and diethyl maleate none, in spite of similar hepatic GSH depletion. Thus, vitamin E may largely modulate the expression of toxicity by GSH-depleting agents.

Lipid Peroxidation, Protein Thiols and Calcium Homeostasis in Bromobenzene-Induced Liver Damage

The mechanisms of bromobenzene hepatotoxicity in vivo were studied in mice. The relationships among glutathione (GSH) depletion, lipid peroxidation, loss of protein thiols, disturbed calcium homeostasis and liver necrosis were investigated. Liver necrosis (as estimated by the serum glutamate-pyruvate transaminase (SGPT) level) appeared between 9 and 12 hr and increased at 18 hr. Lipid peroxidation which was already detectable at 6 hr in some animals, increased thereafter showing a good correlation with the severity of liver necrosis. Despite a quite fast depletion of hepatic GSH, a significant decrease in protein thiols could be observed at 12-18 hr only. Loss of protein thiols in both whole liver and subcellular fractions (microsomes and mitochondria) was correlated with lipid peroxidation. Also a good inverse correlation was seen between lipid peroxidation and the calcium sequestration activity of liver microsomes and mitochondria. The treatment of mice with desferrioxamine (DFO) after bromobenzene-intoxication completely prevented lipid peroxidation, loss of protein thiols and liver necrosis in the animals sacrificed 15 hr after poisoning. When, however, the animals were examined at 24 hr, although the general correlation between lipid peroxidation and liver necrosis was held, in some animals (about 30% of the survivors) elevation of SGPT was observed in the virtual absence of lipid peroxidation. It seems likely therefore that the liver damage seen during the first phase of bromobenzene-intoxication is strictly related to lipid peroxidation. It is, however, possible that in some animals in which for some reason lipid peroxidation does not develop, another mechanism of liver necrosis unrelated to lipid peroxidation occurs at later times.

Determination of 4-hydroxynonenal by high-performance liquid chromatography with electrochemical detection

Abstract4-Hydroxy-trans-2-nonenal (HNE) is a highly reactive product of lipid peroxidation originating from the breakdown of phospholipid-bound polyunsaturated fatty acids of cellular membranes. Despite its biological relevance, this aldehyde is only occasionally determined due to the complexity of previously described procedures. Here we present a simple and very sensitive method for the detection of HNE in biological samples. The method is based on the measurement of the 2,4-dinitrophenylhydrazone (DNPH) of the aldehyde by electrochemical detection after separation by reverse-phase high-performance liquid chromatography (HPLC). The greater sensitivity of this procedure as compared to the ultraviolet detection method commonly employed to measure DNPH derivatives of aldehydes after HPLC will allow the detection of HNE below the pmol level. The detection of HNE is highly reproducible even in normal tissues and cells. Increased amounts of HNE were detected in the livers of animals intoxicated with prooxidant agents such as carbon tetrachloride, bromotrichloromethane or bromobenzene. An exponential increase in HNE (and in malondialdehyde) was measured in peroxidizing liver microsomes (in the NADPH/Fe-dependent system). The method is also suitable for the study of very small samples, since HNE could be detected in approximately 1 million cultured cells (polyoma virus-transformed baby hamster kidney fibroblasts); the level rose after exposure of the cells to a Fe3+/ADP prooxidant system.

Studies on the relationships between carbon tetrachloride-induced alterations of liver microsomal lipids and impairment of glucose-6-phosphatase activity

Abstract When liver microsomes were incubated with NADPH under anaerobic conditions either in the presence or in the absence of EDTA, no substantial amount of malonic dialdehyde (MDA) was formed and no substantial decrease in glucose-6-phosphatase (G-6-Pase) activity was seen. The addition of CCl4 to these in vitro systems had minor effects on both MDA production and the enzyme activity. When, on the contrary, the incubation was carried out under aerobic conditions in the absence of EDTA, a marked production of MDA occurred and G-6-Pase activity was almost completely destroyed. These results confirm the findings of Glende et al. [(1976) Biochem. Pharmacol.25, 2163–2170]. The presence of EDTA in the aerobic system reduced both the extent of lipoperoxidation and the decline in enzyme activity. The addition of CCl4 to these systems resulted in some increase in both MDA formation and G-6-Pase inactivation. The relationships between various steps of the lipid peroxidation process and the inactivation of G-6-Pase were then studied. It was observed that when the incubation was carried out under anaerobic conditions with carbon tetrachloride, the binding of CCl4 free radicals to microsomal lipids (as evidenced by the g.l.c. analysis of fatty acid methyl esters with an electron-capture detector, ECD) was qualitatively identical to that observed after CCl4 poisoning in vivo. Also, the diene conjugation absorption detected in the samples incubated in vitro was similar to that observed in the in vivo situation. Furthermore, when the fatty acid methyl esters of the lipids of liver microsomes incubated as above were analyzed by thin-layer chromatography, a typical spot (“D” spot), previously reported to occur in the fatty acid methyl esters of liver microsomal lipids of CCl4-poisoned rats, was observed. As in the in vivo situation, the lipids recovered from this spot showed the absorption of conjugated dienes. On the other hand, when the incubation was carried out aerobically in the absence of EDTA either with CCl4 or without it, the g.l.c. analysis of the fatty acid methyl esters of microsomal lipids showed ECD responses which are probably due to the interaction of oxygen with unsaturated fatty acids. Ultraviolet spectra characteristic of peroxidized lipids were also found in this experimental condition. The activity of G-6-Pase, depressed by the aerobic incubation, could not be restored by the addition of different phospholipid fractions (phosphatidylcholine or phosphatidylethanolamine), denoting that during the incubation some irreversible damage to the enzyme activity occurs. The damage does not seem to be related to the alterations of the molecular structure of the membrane lipids (the binding of chlorinated radicals and the presence of conjugated dienes) since these alterations are also present after the anaerobic incubation, which does not cause a decrease in the G-6-Pase activity. Since the only experimental condition which produces an extensive loss of the enzyme activity is aerobic incubation, in which a large MDA formation occurs, it is concluded that some product evolved during the peroxidative breakdown of unsaturated lipids is responsible for the G-6-Pase inactivation.

Protection by ascorbic acid against oxidative injury of isolated hepatocytes

1. The ability of ascorbic acid to protect from prooxidant-induced toxic injury was investigated in isolated, intact rat hepatocytes, whose ascorbic acid content had been restored by means of exogenous supplementation. 2. Ascorbate-supplemented and ascorbate-non-supplemented cells in suspension were treated with a series of different prooxidants (allyl alcohol, diethyl maleate, carbon tetrachloride, menadione), and the development of lipid peroxidation and cell injury was evaluated. 3. With allyl alcohol and diethyl maleate, ascorbic acid was able to protect cells from both lipid peroxidation and cell injury. The same protection was offered by ascorbate also in hepatocytes obtained from vitamin E-deficient animals. 4. With carbon tetrachloride, ascorbate supplementation did not affect the initial steps of lipid peroxidation, but nevertheless provided a marked protection against lipid peroxidation and cell injury at later times of incubation. The protection was unaffected by the vitamin E content of cells. 5. With menadione, a toxin which does not induce lipid peroxidation, ascorbic acid did not protect cells against injury. 6. It is concluded that ascorbic acid can act as an efficient antioxidant in isolated rat liver cells, with protection against cell injury. The antioxidant effect appears primarily to involve membrane lipids, and can be independent from the cellular content of vitamin E, thus suggesting that ascorbic acid can play a direct and independent role in the intact cell, in addition to its synergistic interaction with vitamin E described in other models.

Early alterations induced by carbon tetrachloride in the lipids of the membranes of the endoplasmic reticulum of the liver cell I. Separation and partial characterization of altered lipids

Abstract Alterations induced by carbon tetrachloride poisoning in fatty acids of liver microsomal lipids were studied. Thin layer chromatography of fatty acid methyl esters prepared from liver microsomal lipids, revealed, in the CCl 4 -treated rats, the presence of a component (the “D” spot) with an R f value lower than that of the methyl esters. The lipids recovered from this component showed a marked diene conjugation absorption when examined spectrophotometrically over the UV range, while the lipids recovered from the spot of the methyl esters showed no absorption of conjugated dienes. Studies carried out with labelled carbon tetrachloride indicated that compounds present in the “D” spot contained 28% of 14 C applied to the chromatoplate. The spot of the methyl esters (the “M” spot) contained 42% of 14 C applied to the chromatoplate. However, specific activity of the “D” spot was about 1000 times greater than specific activity of the “M” spot. The lipids recovered from either the “D” spot or the spot of the methyl esters were analyzed separately by gas-liquid chromatography (GLC) with an electron capture detector (ECD). It was found that the lipids recovered from the “D” spot showed no response, while those recovered from the spot of the methyl esters exhibited the response of the ECD, which was similar to that observed with the unfractionated fatty acid methyl esters. The lack of the response of the ECD for compounds in the “D” spot appears to be due to the fact that they cannot be eluted from the column. On the basis of the analytical results, it can be postulated that the “D” spot contains compounds formed by a chain termination addition reaction of free radicals derived from CCl 4 (probably trichloromethyl free radicals) to fatty acid free radicals containing conjugated dienes. On the other hand, the spot of the methyl esters appears to contain also, together with unmodified fatty acids, the fatty acids in which a simple addition of CCl 4 free radicals to double bonds has occurred.

Binding of products originating from the peroxidaton of liver microsomal lipids to the non-lipid constituents of the microsomal membrane

The binding of products derived from the peroxidation of liver microsomal lipids to the non-lipid constituents of the microsomes was studied. To this end arachidonic acid labelled with tritium at the positions of the double bonds was given to rats and allowed to incorporate into the membrane lipids of the liver cell. When liver microsomes containing labelled arachidonic acid were incubated aerobically in the NADPH-dependent system, a marked production of malonic dialdehyde (MDA) occurred and, concomitantly, there was a consistent release of radioactivity from the microsomes into the incubation medium. The addition of EDTA to the incubation medium prevented, to a large extent, both the MDA formation and the release of radioactivity. Chromatographic studies showed that the bulk of the radioactivity released from the incubated microsomes is not MDA. In the incubated microsomes, the radioactivity decreased in total lipids, while it increased by about 15 times in the non-lipoidal residue. A similar increase in radioactivity was seen in microsomal protein, while no increase was observed in microsomal RNA (the radioactivity was negligible in both the incubated and the non-incubated samples). It seems therefore that products originating from lipoperoxidation of arachidonic acid covalently bind to the microsomal protein. In order to investigate whether alterations similar to those observed in the in vitro peroxidation of liver microsomes could be detected in the in vivo intoxication with carbon tetrachloride, rats given labelled arachidonic acid as above, were poisoned with CCl4. Sixty minutes after poisoning, the radioactivity present in the microsomal lipids was generally lower in the intoxicated rats than in the controls, while the labelling of the non-lipoidal residue and of the protein was higher in the CCl4-poisoned rats.

Early alterations induced by carbon tetrachloride in the lipids of the membranes of the endoplasmic reticulum of the liver cell. II. Distribution of the alterations in the various lipid fractions.

Abstract The distribution of carbon tetrachloride-induced alterations of membrane lipids in various fractions of liver microsomal lipids was studied. The chromatographic spot (referred to as the “D” spot in the previous paper [1]) which has been shown to contain the compounds responsible for the diene conjugation absorption [1], was found in the fatty acid methyl esters prepared from the fraction containing phosphatidylethanolamine (PE) and also in those obtained from the fraction containing phosphatidylserine (PS) and phosphatidylinositol (PI). The absorption of conjugated dienes was very marked in PE and less intense in PS and PI. The fatty acid methyl esters prepared from the fraction containing phosphatidylcholine (PC) showed no presence of the “D” spot and minimal absorption of conjugated dienes. A decrease in arachidonic acid content was found in the fraction containing PE, while no change in content of this fatty acid was found in the fraction containing PC. Results similar to those observed for PC were also found for neutral lipids (NL). Analysis of the fatty acid methyl esters of the various lipid fractions by gas-liquid chromatography (GLC) with an electron capture detector (ECD) gave a qualitative index of the free radical attack by CCl4 metabolites. Quantitative estimation was attained by study of the irreversible binding of 14C from 14CCl4 to the various lipid fractions. It was found that the fraction containing PS had the highest specific activity, while the fraction containing PC had the lowest specific activity of all the phospholipids. Thin layer chromatography (TLC) of the fraction containing PS revealed that only 11% of the radioactivity was associated with the pure PS moiety, while the remainder was associated with uncharacterized lipids (probably oxidation products). The possible relevance of the alterations induced by carbon tetrachloride in the various phospholipid fractions of liver microsomes to functional changes is discussed.