Subcellular localization of a glutathione-dependent dehydroascorbate reductase within specific rat brain regions.

Recently, we described the occurrence of a dehydroascorbate reductase within the rat CNS. This enzyme regenerates ascorbate after it is oxidized during normal aerobic metabolism. In this work, we describe the neuronal compartmentalization of the enzyme, using transmission electron microscopy of those brain areas in which the enzyme was most densely present when observed under light microscopy. In parallel biochemical studies, we performed immunoblotting and measured the enzyme activity of the cytoplasm and different nuclear fractions. Given the abundance of ascorbate in the caudate-putamen, we focused mostly on the occurrence of dehydroascorbate reductase at the striatal subcellular level. We also studied cerebellar Purkinje cells, hippocampal CA3 pyramidal cells and giant neurons in the magnocellular part of the red nucleus. In addition to neurons, immunolabeling was found in striatal endothelial cells, in the basal membrane of blood vessels and in perivascular astrocytes. In neuronal cytosol, the enzyme was observed in a peri-nuclear position and on the nuclear membrane. In addition, in both the striatum and the cerebellum, we found the enzyme within myelin sheets. Dehydroascorbate reductase was also present in the nucleus of neurons, as further indicated by measuring enzyme activity and by immunoblotting selected nuclear fractions. Immunocytochemical labeling confirmed that the protein was present in isolated pure nuclear fractions. Given the great amount of free radicals which are constantly generated in the CNS, the discovery of a new enzyme with antioxidant properties which translocates into neuronal nuclei appears to be a potential starting point to develop alternative strategies in neuroprotection.

Localization of a glutathione-dependent dehydroascorbate reductase within the central nervous system of the rat.

In this study, we describe for the first time the occurrence, within the central nervous system of the rat, of a dehydroascorbate reductase analogous to the one we recently described in the liver. Dehydroascorbate reductase plays a pivotal role in regenerating ascorbic acid from its oxidation product, dehydroascorbate. In a first set of experiments, we showed that a dehydroascorbate reductase activity is present in brain cytosol; immunoblotting analysis confirmed the presence of an immunoreactive cytosolic protein in selected brain areas. Immunotitration showed that approximately 65% of dehydroascorbate reductase activity of brain cytosol which was recovered in the ammonium sulphate fraction can be attributed to this enzyme. Using immunohistochemistry, we found that a variety of brain areas expresses the enzyme. Immunoreactivity was confined to the gray matter. Amongst the several brain regions, the cerebellum appears to be the most densely stained. The enzyme was also abundant in the hippocampus and the olfactory cortex. The lesion of norepinephrine terminals following systemic administration of DSP-4 markedly decreased immunoreactivity in the cerebellum. Apart from the possible co-localization of the enzyme with norepinephrine, the relative content of dehydroascorbate reductase in different brain regions might be crucial in conditioning regional sensitivity to free radical-induced brain damage. Given the scarcity of protective mechanisms demonstrated in the brain, the discovery of a new enzyme with antioxidant properties might represent a starting-point to increase our knowledge about the antioxidant mechanisms operating in several central nervous system disorders.

Molecular cloning and functional expression of rat liver glutathione-dependent dehydroascorbate reductase.

We have isolated a cDNA clone for a novel glutathione-dependent dehydroascorbate reductase from a rat liver cDNA library in lambdagt11 by immunoscreening. The authenticity of the clone was confirmed as follows: first, the antibody that had been purified through affinity for the protein expressed by the cloned lambdagt11 phage recognized only the enzyme in a crude extract from rat liver; and second, two internal amino acid sequences of purified enzyme were identified in the protein sequence predicted from the cDNA. The predicted protein consists of 213 amino acids with a molecular weight of 24,929, which is smaller by approximately 3,000 than the value obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. This discrepancy of the molecular weight was explained by post-translational modification because the recombinant protein expressed by a mammalian system (Chinese hamster ovary cells) was of the same size as rat liver enzyme but larger than the protein expressed by a bacterial system (Escherichia coli). Chinese hamster ovary cells, originally devoid of glutathione-dependent dehydroascorbate reductase activity, was made to elicit the enzyme activity (1.5 nmol/min/mg of cytosolic protein) by expression of the recombinant protein. Additionally, the cells expressing the enzyme were found to accumulate 1.7 times as much ascorbate as the parental cells after incubation with dehydroascorbate. This result points to the importance of the dehydroascorbic acid reductase in maintaining a high concentration of ascorbate in the cell.

Localization of a GSH-dependent dehydroascorbate reductase in rat tissues and subcellular fractions.

A novel GSH-dependent dehydroascorbate (DHA) reductase from rat liver cytosol has been recently purified and partially characterized in our laboratory. A further characterization study has been carried out in order to determine intracellular and tissue distribution of the enzyme. A modified purification method, yielding a threefold increase in enzyme activity recovery, has been used. Polyclonal antibodies were obtained in rabbits and specific anti-DHA reductase IgG were purified by affinity chromatography employing the homogeneous enzyme as ligand. Immunoblotting analysis of subcellular fractions showed the exclusively cytosolic location of the enzyme. Immunotitration experiments, performed in order to determine the percentage of cytosolic DHA reductase activity ascribable to our enzyme, revealed that purified enzyme activity was completely titrable, while only 70% of DHA reducing activity was titrable in liver cytosol preparation. When immunoblotting analysis was employed to determine tissue distribution of the enzyme, liver, intestinal mucosa, kidney, adrenals, submaxillary gland, testis, and pancreas appeared most endowed with the enzyme, and lower levels were observed in all the other tissues examined. Immunohistochemical studies showed clear zonal distributions in kidney and intestinal tract and overall homogeneous patterns in the other tissues.

Purification of NADPH-dependent dehydroascorbate reductase from rat liver and its identification with 3 alpha-hydroxysteroid dehydrogenase.

Rat liver cytosol has been found to reduce dehydroascorbic acid (DHAA) to ascorbic acid in the presence of NADPH. The enzyme responsible for such activity has been purified by ammonium sulphate fractionation, DEAE-Sepharose, Sephadex G-100 SF and Reactive Red column chromatography, with an overall recovery of 27%. SDS/PAGE of the purified enzyme showed one single protein band with an M(r) of 37,500. A similar value (36,800) was found by gel filtration on a Sephadex G-100 SF column. The results indicate that the enzyme is a homogeneous monomer. The Km for DHAA was 4.6 mM and the Vmax. was 1.55 units/mg of protein; for NADPH Km and Vmax. were 4.3 microM and 1.10 units/mg of protein respectively. The optimum pH was around 6.2. Several typical substrates and inhibitors of the aldo-keto reductase superfamily have been tested. The strong inhibition of DHAA reductase effected by steroidal and non-steroidal anti-inflammatory drugs, together with the ability to reduce 5 alpha-androstane-3,17-dione strongly, suggest the possibility that DHAA reductase corresponds to 3 alpha-hydroxysteroid dehydrogenase. Microsequence analysis performed on the electro-transferred enzyme band shows that the N-terminus is blocked. Internal primary structure data were obtained from CNBr-derived fragments and definitely proved the identity of NADPH-dependent DHAA reductase with 3 alpha-hydroxysteroid dehydrogenase.

Purification and characterization of glutathione-dependent dehydroascorbate reductase from rat liver.

GSH-dependent enzymic reduction of dehydroascorbic acid to ascorbic acid has been studied in rat liver cytosol. After gel filtration of cytosol on Sephadex G-100 SF, dehydroascorbate reductase activity was recovered in two distinct peaks, one corresponding to glutaredoxin (an enzyme already known for its dehydroascorbate reductase activity) and another, much larger one, corresponding to a novel enzyme different from glutaredoxin. The latter was purified to apparent homogeneity. The purification process involved (NH4)2SO4 fractionation, followed by DEAE-Sepharose, Sephadex G-100 SF and Reactive Red chromatography. SDS/PAGE of the purified enzyme in either the presence or absence of 2-mercaptoethanol demonstrated a single protein band of M(r) 31,000. The M(r) determined by both Sephadex G-100 SF chromatography and h.p.l.c. was found to be approx. 48,000. H.p.l.c. of the denatured enzyme gave an M(r) value identical with that obtained by SDS/PAGE (31,000). The apparent Km for dehydroascorbate was 245 microM and the Vmax. was 1.9 mumol/min per mg of protein; for GSH they were 2.8 mM and 4.5 mumol/min per mg of protein respectively. The optimal pH range was 7.5-8.0. Microsequence analysis of the electro-transferred enzyme band showed that the N-terminus is blocked. Data on internal primary structure were obtained from CNBr-and N-chlorosuccinimide-derived fragments. No significative sequence similarity was found to any of the protein sequences contained in the Protein Identification Resource database.

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.

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

4-Hydroxy-trans-2-nonenal (HNE) is a highly reactive product of lipid peroxidation originating from the break-down 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.

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.

Early mitochondrial disfunction in bromobenzene treated mice: a possible factor of liver injury.

The membrane potential of liver mitochondria isolated from bromobenzene treated mice was studied. Specifically, the efficiency of the energy-transducing mitochondrial membrane was measured during the phase between the occurrence of a massive loss of hepatic GSH, after 2-3 hr of bromobenzene intoxication, and the appearance of lipid peroxidation and cell death (12-15 hr after treatment). Partial uncoupling of oxidative phosphorylation was observed in mitochondria during the early period of intoxication (3-9 hr). These anomalies in oxidative metabolism did not result in irreversible damage to the mitochondrial inner membrane. The possibility that phenolic metabolites of bromobenzene are responsible for the uncoupling effects was examined. Orto- and especially para-bromphenol reproduced the alterations of mitochondrial function when added to normal mitochondria at concentrations comparable to those found in the livers of the intoxicated animals. Since the concentration of the bromophenols (especially p-bromophenol) largely increases after the intoxication times as tested here, mitochondrial uncoupling may represent a mechanism of liver damage acting synergistically with or even independently of other factors such as oxidative stress and lipid peroxidation.

Evidence for the participation of activated oxygen species and the resulting peroxidation of lipids in the killing of cultured hepatocytes by aryl halides.

Primary cultures of rat hepatocytes were used to explore the mechanisms of the toxicity of aryl halides. The sensitivity of the hepatocytes to chloro-, bromo-, and iodobenzene was enhanced by inhibition of glutathione reductase with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). In each case, the increased cell killing depended on the metabolism of the toxicant, a result shown by the protective effect of SKF-525A, an inhibitor of mixed function oxidation. BCNU decreased the metabolism of [14C]bromobenzene and the covalent binding of its metabolites by 20%. Chelation by deferoxamine of a cellular source of ferric iron prevented the cell killing in the presence or absence of BCNU. Deferoxamine had no effect on the metabolism or the covalent binding of [14C]bromobenzene. Similarly, the antioxidant N,N'-diphenyl-p-phenylenediamine (DPPD) reduced the cell killing and had no effect on the metabolism of [14C]bromobenzene. Thus, the toxicity of the three aryl halides was manipulated in ways that modify the sensitivity of hepatocytes to an oxidative stress, and the changes in cell killing occurred without parallel changes in the metabolism of [14C]bromobenzene or the covalent binding of its metabolites.

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.

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.

Histochemical detection of lipid peroxidation in the liver of bromobenzene-poisoned mice.

The possibility of detecting lipid peroxidation histochemically was investigated in liver tissue in vivo, in conditions in which the process has been demonstrated by biochemical methods. The technique was based on the detection of aldehyde functions with the use of the Schiff's reagent. The study was carried out on bromobenzene-intoxicated mice, which generally exhibit levels of lipid peroxidation considerably higher than those observed in the case of other hepatotoxins. Liver sections from control animals were unstainable by the reagent, while sections from bromobenzene-poisoned mice showed a purple stain of various intensity, unhomogeneously distributed, sometimes with a mediolobular localization. Microphotometric measurements were performed at 565 nm by means of a computer-controlled microscope photometer. The ratios of Schiff-positive area relative to total section area, as well as the total extinctions referred to 100 sq mu of the sections, showed a high correlation with the corresponding hepatic contents of malonic dialdehyde, chosen as biochemical index of lipid peroxidation. In vitro studies in which liver sections were incubated in the presence of NADPH-Fe2+, showed a Schiff positivity which increased with the incubation time, confirming the reliability of the histochemical method. Another procedure, based on the use of 2-OH-3-naphtoic acid hydrazide coupled with fast blue B, was also developed and proved to be possibly more sensitive than Schiff's reagent in the detection of lipid peroxidation in liver tissue.

Measurement of lipid peroxidation in vivo: a comparison of different procedures.

A study was undertaken to investigate whether some of the methods commonly used to detect lipid peroxidation of cellular membranes in vivo correlate with each other. The study was performed with the livers of bromobenzene-intoxicated mice, in which lipid peroxidation develops when the depletion of glutathione (GSH) reaches a threshold value. The methods tested and compared were the following: i) measurement of the malondialdehyde (MDA) content of the liver; ii) detection of diene conjugation absorption in liver phospholipids; iii) measurement of the loss of polyunsaturated fatty acids in liver phospholipids; and iv) determination of carbonyl functions formed in acyl residues of membrane phospholipids as a result of the peroxidative breakdown of phospholipid fatty acids. Correlations among the values obtained with these methods showed high statistical significances, indicating that the procedures measure lipid peroxidation in vivo with comparable reliability. Analogously, the four methods appeared also to correlate when applied to in vitro microsomal lipid peroxidation.

Lipid peroxidation and cellular damage in extrahepatic tissues of bromobenzene-intoxicated mice.

The mechanisms of bromobenzene toxicity in extrahepatic tissues of mice were studied. Kidney, lung, heart and brain were examined. As observed in this as well as in a previous report for the liver, bromobenzene intoxication caused a progressive decrease in the glutathione content of all the tissues examined. Cellular damage (as assessed by both biochemical determinations and histologic observations) appeared after 6 hours in the case of the kidney and the heart and after 15 hours in the case of the lung. Lipid peroxidation (as assessed by the tissue content of malonic dialdehyde, a parameter correlating with both the diene conjugation absorption and the amount of carbonyl functions in cellular phospholipids) was found to occur at the same times at which cellular damage was observed or even before. As in the case of bromobenzene-induced liver injury, when the individual values for cell damage obtained at 15-20 hours were plotted against the corresponding glutathione contents, a severe cellular damage was generally observed when the glutathione levels reached a threshold value (3.0-0.5 nmol/mg protein). Such a glutathione threshold was also observed for the onset of lipid peroxidation. Glutathione depletion and lipid peroxidation are therefore general phenomena occurring not only in the liver but in all the tissues as a consequence of bromobenzene poisoning. The possibility that lipid peroxidation is the cause of bromobenzene-induced damage to liver and extrahepatic tissues is discussed.