Oxidative stress in the development of human ischemic hepatitis during circulatory shock.

An increasing number of studies support the involvement of free radical-mediated oxidative reactions in the pathogenesis of tissue injury following ischemia reperfusion. In particular, a condition of oxidative stress is evident in patients with circulatory shock, a disease process often complicated by progressive organ failure sustained by inflammatory reactions. In all shock patients without signs of organ failure, a consistent increase of intermediate and final products of lipid peroxidation (lipid peroxides and aldehydes respectively) was observed. Impairment of the redox equilibrium in the tissues of these patients was confirmed by a significant reduction of glutathione and vitamin E hematic concentrations. Moreover, a selective increase of plasma aldehyde-protein adducts, actual proof of oxidative damage of macromolecules, is only present in the shock patients who, in addition, show hepatic cytolysis (ischemic hepatitis) as estimated by plasma levels of LDH5 isoenzyme. Aldehyde adducts well mark the progression of the disease towards multiple organ failure. Finally, the good statistical correlation between aldehyde-modified proteins and LDH5, as well as their distinct behaviour in control and ischemic hepatitis, support the involvement of oxidative damage in the expression and worsening of circulatory shock.

Oxidative damage and transforming growth factor beta 1 expression in pretumoral and tumoral lesions of human intestine.

The aim of this study was to evaluate a possible relationship between oxidative stress and transforming growth factor beta 1 (TGF beta 1) expression in human colon adenocarcinoma. Crohn's disease, an inflammatory pathology of the intestine often regarded to as precancerous, was also examined. Indices of impaired redox balance were monitored in blood and in bioptic samples from 10 adult patients with adenocarcinoma of the colon and from five patients with Crohn's disease. On tissue samples TGF beta 1 mRNA expression was also determined. Ten healthy adults provided normal reference values for plasma indices of oxidative stress, and normal tissue distant from the lesions was used for comparative analysis. Fluorescent adducts with plasma proteins of malonaldehyde (MDA) and 4-hydroxynonenal (HNE) were significantly lower than controls in the plasma from cancer patients and significantly higher in the plasma from Crohn's patients. In adenocarcinoma biopsies, susceptibility to lipid peroxidation processes and TGF beta 1 expression were below the relative control; in Crohn's disease, lipid peroxidation and cytokine expression were both above the relative control. The findings obtained suggest the existence of an association between oxidative damage and fibrogenic cytokine expression in the human intestine. Further studies are needed to conclusively prove the correlation between the two events.

In vivo potentiation of 1,2-dibromoethane hepatotoxicity by ethanol through inactivation of glutathione-s-transferase.

In the rat, a single ethanol (EtOH) pretreatment (2.5 g/kg b.w., per os) was able to strongly enhance the cytotoxicity of 1,2-dibromoethane (DBE)(87 mg/kg b.w., per os). The principal metabolic routes of DBE involve both oxidative and conjugative transformations. Microsomal cytochrome P450 content and dimethyl nitrosamine demethylase activity were not changed, while a significant loss of cytosolic total GSH-transferase was observed in rats killed 6 h after EtOH pretreatment. Pretreatment with methylpyrazole, an inhibitor of alcohol-dehydrogenase prevented the effects provoked by ethanol. The major EtOH metabolite, acetaldehyde. seemed thus to play a fundamental role in the mechanism responsible for the potentiation of DBE toxicity mediated by EtOH. To further support this hypothesis, disulfiram (75 mg/kg b.w.), an inhibitor of aldehyde dehydrogenase, was given i.p. to rats. When DBE was administered to disulfiram- and EtOH-pretreated rats, a marked increase of liver cytolysis was shown and cytosolic GSH-transferase activity was further inhibited if compared to that induced by EtOH treatment alone. The results are consistent with the hypothesis that EtOH given to rats increases DBE liver toxicity because its major metabolite, acetaldehyde, reduces the DBE conjugates to GSH transferase, with consequent shift of DBE metabolism to the oxidative route and accumulation of reactive oxidative intermediates no longer effectively conjugated with GSH.

Biogenic 4-hydroxy-2-nonenal activates transcription factor AP-1 but not NF-kappa B in cells of the macrophage lineage.

A large spectrum of pro-oxidant agents, including molecules with lipoperoxidative effect, can modulate gene expression through modification of the DNA binding activity of the transcription factors activator protein 1 (AP-1) and nuclear factor kappa B (NF-kappa B). In this study the effect on these redox-sensitive factors by 4-hydroxy-2-nonenal (HNE), a major aldehydic product of lipid peroxidation, was examined in two cell lines of the macrophage type. Incubation in the presence of microM concentrations of the aldehyde led to a rapid increase of AP-1 binding with a transient maximum 30 min from HNE addition to the culture medium in both cell lines. On the contrary, HNE did not stimulate nuclear translocation of NF-kappa B. The diverging effect of HNE on the two transcription factors is likely related to the demonstrated differential activation pathway of AP-1 and NF-kappa B in macrophages. The HNE-induced activation of AP-1 suggests the aldehyde's involvement in the regulatory mechanisms of cell proliferation and differentiation.

Rat hepatocytes in single cell suspension: a still suitable system for lipid peroxidation studies.

The investigation of toxic compounds which need a previous metabolic activation often gets advantage by the employment of cell suspensions, in most of the cases rat hepatocytes. In particular, this model system allows the characterization of lipid peroxidation kinetics, the evaluation of its pathogenetic role in the induction of cell death and the analysis of the interaction among different prooxidant compounds in bringing about both oxidative damage and cytolysis.

Rat hepatocytes in single cell suspension: A still suitable system for lipid peroxidation studies.

The investigation of toxic compounds which need a previous metabolic activation often gets advantage by the employment of cell suspensions, in most of the cases rat hepatocytes. In particular, this model system allows the characterization of lipid peroxidation kinetics, the evaluation of its pathogenetic role in the induction of cell death and the analysis of the interaction among different prooxidant compounds in bringing about both oxidative damage and cytolysis.

Change of liver metabolism of 1,2-dibromoethane during simultaneous treatment with carbon tetrachloride.

The combination of carbon tetrachloride (CCl4) and 1,2-dibromoethane (DBE) in isolated rat hepatocytes led to a significant potentiation of both lipid peroxidation and of plasma membrane damage observed after a single treatment with CCl4. Such a synergistic effect appeared to be related to the CCl4-induced shift of DBE metabolism from the cytosolic conjugation with glutathione towards the microsomal transformation into toxic intermediates. In fact, CCl4 significantly inactivated hepatocyte total GSH-transferase, i.e. the DBE detoxification pathway. Furthermore, while the microsomal metabolism of CCl4 was not affected by the simultaneous presence of DBE, the amount of DBE reactive metabolites covalently bound to hepatocyte protein was significantly enhanced in the presence of CCl4.

Metabolism of 4-hydroxy-2-nonenal and aging.

Hepatocytes isolated from male Wistar rats of 2-3 and 20-24 months of age were compared as regards concentration and metabolism of 4-hydroxy-2-nonenal, one of the major aldehyde products of n-6 fatty acid oxidative breakdown. A significant accumulation of fluorescent 4-hydroxy-2-nonenal-membrane lipid adducts was found in the cells from the old rats. The mechanism mainly responsible for such aldehyde accumulation was shown to be the impairment of its enzymatic metabolism. In fact, while endogenous, that is non-stimulated, aldehyde production was not impaired, the reductive pathway of 4-hydroxy-2-nonenal metabolism in hepatocytes from old rats was strongly depressed. The decrease of 4-hydroxy-2-nonenal consumption with age was confirmed in homogenates from liver and kidney, while there were no differences between young and old animals in heart, lung or brain.

Ethanol-induced potentiation of rat hepatocyte damage due to 1,2-dibromoethane.

Low amounts of 1,2-dibromoethane (DBE), not able per se to exert pro-oxidant and cytotoxic activity on rat hepatocyte suspensions, become effective when administered with carbon tetrachloride (CCl4), due to impairment of the glutathione transferase detoxication pathway by CCl4. Treatment of rats with a single dose of ethanol (2.5 g/kg body wt) 2 h before liver cell isolation potentiates the effect of DBE alone on both malonaldehyde formation and lactate dehydrogenase release by the hepatocyte. The potentiation of the DBE effects by ethanol may be through a series of mechanisms, such as a strong inactivation of hepatocyte glutathione transferase similar to that caused by CCl4, an increased basal level of lipid peroxidation and a significant loss of total glutathione.

The lipid peroxidation end product 4-hydroxy-2,3-nonenal up-regulates transforming growth factor beta1 expression in the macrophage lineage: a link between oxidative injury and fibrosclerosis.

An increasing number of reports underscore the frequent association of fibrosclerotic diseases of lung, liver, arterial wall, brain, etc., with the accumulation of oxidatively modified lipids and proteins. A cause-and-effect relationship has been proposed between cellular oxidative damage and increased fibrogenesis based on the fact that experimental treatment with antioxidants either prevents or quenches the fibrotic process. With some peculiarities in the different organs, fibrosclerosis is essentially the result of the interaction of macrophages and extracellular matrix-producing cells. The cross-talk is mediated by fibrogenic cytokines, among which the most important appears to be transforming growth factor beta1 (TGF-beta1). This report describes treatment of different types of macrophage, of both human and murine origin, with 4-hydroxy-2,3-nonenal (HNE) a major aldehyde end product of membrane lipid oxidation found consistently to induce both mRNA expression and synthesis of TGF-beta1. Since increased HNE levels have been demostrated in the cirrhotic liver and in the oxidatively modified low-density human lipoproteins associated with atherosclerosis, the up-regulation of macrophage TGF-beta1 by HNE appears to be involved in the pathogenesis of these and similar diseases characterized by fibrosclerosis.

Acetaldehyde involvement in ethanol-induced potentiation of rat hepatocyte damage due to the carcinogen 1,2-dibromoethane.

Previous experiments with hepatocytes isolated from ethanol-treated rats showed that alcohol potentiates the toxic action of 1,2-dibromoethane (DBE) by inhibiting its metabolism via glutathione-S-transferase. The aim of this study was to investigate whether acetaldehyde, the main product of ethanol metabolism, may be responsible for such inactivation. By pretreatment with 4-methylpyrazole, an inhibitor of acetaldehyde formation, the ethanol inactivation of glutathione transferase was actually prevented. As a consequence of this protective action, 4-methylpyrazole also prevented the high basal lipid peroxidation and the potentiated DBE toxicity observed in hepatocytes from ethanol-dosed animals. Finally, the inactivation of glutathione-S-transferase by concentrations of acetaldehyde likely to occur in the ethanol-intoxicated animal was confirmed in an in vitro model by direct aldehyde addition to hepatocyte suspensions.

Nuclear factor kB is activated by arachidonic acid but not by eicosapentaenoic acid.

The omega-6 arachidonic acid supplementation of the human promonocytic cell line U937 strongly stimulates the nuclear translocation of the transcription factor NF-kB. Inhibitors of arachidonate oxidative metabolism prevent NF-kB activation, indirectly indicating a role for prostaglandin and leukotriene metabolites in the genesis of this phenomenon. Of note, omega-3 eicosapentaenoic acid does not exert any effect on NF-kB DNA binding. In subsequent experiments, prostaglandin E2 consistently showed the ability to activate NF-kB in U937 promonocytic cells, as well as in J774 macrophages. NF-kB activation by arachidonate, together with the lack of effect by eicosapentaenoic acid, suggests a way to modulate the expression of certain genes by means of a suitable dietary n-6/n-3 fatty acid ratio.