Paolo Clot

Modulation of experimental alcohol‐induced liver disease by cytochrome P450 2E1 inhibitors

This study was done to determine if a relationship exists between CYP2E1 induction by ethanol, lipid peroxidation, and liver pathology in experimental alcohol-induced liver disease in the rat. Rats were fed ethanol with or without diallyl sulfide (DAS) or phenethyl isothiocyanate (PIC) intragastrically for 1 month. CYP2E1 induction by ethanol was correlated with lipid peroxidation, liver microsomal CYP2E1 hydroxylation of paranitrophenol, and the liver pathology score using the data from the PIC-fed rats. Some of the data from the ethanol and DAS-fed rats were not included here because they have been reported elsewhere. Microsomal CYP2E1 protein levels induction by ethanol was decreased by PIC ingestion. Similarly, PIC reduced the increase microsomal reduced form of nicotinamide-adenine dinucleotide (NADPH)-dependent lipid peroxidation and p-nitrophenol hydroxylase (PNPH) activity, induced by ethanol feeding. The lipid peroxidation was reduced to below control levels; however, the pathology score was partially but not significantly reduced by isothiocyanate feeding. CYP2E1 messenger RNA (mRNA) was decreased by both inhibitors of CYP2E1. Immunohistochemical staining of liver for CYP2E1 protein showed that the lobular distribution of the isozyme changed from the centrilobular to a diffuse pattern, with an increase in the periportal region when the CYP2E1 inhibitors were fed with ethanol, and that this change correlated with the change in the distribution of fat in the lobule. The data support the idea that there is a link between CYP2E1 induction by ethanol and the early phase of ethanol-induced liver injury in this rat model. This link may involve lipid peroxidation, but other factors related to CYP2E1 induction must also be involved.

Detection of antibodies against proteins modified by hydroxyethyl free radicals in patients with alcoholic cirrhosis

BACKGROUND/AIMS We have previously shown that hydroxyethyl free radicals produced during cytochrome P4502E1-mediated oxidation of ethanol covalently bind to microsomal proteins. The present study examined whether alkylation of proteins by hydroxyethyl radicals induces an immunologic response in alcoholic patients. METHODS A microplate enzyme-linked immunosorbent assay was developed using as antigen human serum albumin or bovine fibrinogen reacted with chemically produced hydroxyethyl radicals. RESULTS This assay showed that the sera of alcoholic cirrhotics contained both immunoglobulin (Ig) Gs and IgAs that recognized proteins modified by hydroxyethyl radicals, whereas practically no reaction was observed in the sera of healthy controls or cirrhotics without evidence of alcohol abuse. The reactivity of the sera from alcoholic patients was not influenced by the protein to which hydroxyethyl radicals were bound. The sera of alcoholic cirrhotics also contained antibodies directed against acetaldehyde-modified albumin. However, the reaction of alcoholic sera with hydroxyethyl radical epitopes was not inhibited by increasing concentrations of acetaldehyde-modified albumin produced under either reducing or nonreducing conditions. CONCLUSIONS The results indicate that a new group of antigens that do not cross-react with antibodies against acetaldehyde-derived epitopes is formed by the alkylation of protein by hydroxyethyl radicals and is involved in the development of immunologic reactions in alcoholic patients.

Cytochrome P4502E1 inducibility and hydroxyethyl radical formation among alcoholics

BACKGROUND/AIMS Animal studies have shown that the induction of cytochrome P4502E1 (CYP2E1) modulates oxidative damage induced by ethanol. Since CYP2E1 activity varies substantially in humans, we have investigated whether differences in CYP2E1 activity might influence the formation of hydroxyethyl free radicals and the stimulation of lipid peroxidation among alcohol abusers. METHODS Chlorzoxazone oxidation, an index of CYP2E1 activity, and the levels of antibodies reacting with hydroxyethyl radical and malonyldialdehyde protein adducts were investigated in 51 alcoholic patients. RESULTS We observed that in 40 out of 51 (78%) alcoholics, chlorzoxazone oxidation was increased over the control levels, consistently with CYP2E1 induction by ethanol. However, in the remaining 22% of the patients, in spite of a similar alcohol intake, chlorzoxazone oxidation was within the control range, indicating a lack of CYP2E1 inducibility. IgG reacting with hydroxyethyl free radical-protein adducts were absent in subjects without CYP2E1 induction, while they were significantly increased in alcoholics with induced CYP2E1 activity. IgG against malonyldialdehyde protein-adducts were increased in all patients, irrespective of CYP2E1 inducibility. Moreover, chlorzoxazone oxidation was significantly lower in alcoholics without clinical and biochemical signs of liver disease as compared to patients with alcoholic liver disease. CONCLUSIONS These results indicate that CYP2E1 activity greatly influences the formation of hydroxyethyl radicals in humans, and suggest a possible role of CYP2E1 in the development of alcoholic liver disease.

Specificity of autoantibodies against oxidized LDL as an additional marker for atherosclerotic risk.

BACKGROUND LDL oxidation is a crucial step in the development and progression of atherosclerotic lesions. The detection of an increase in the anti-oxidized LDL antibody titre may thus represent a biological marker of enhanced LDL oxidation in vivo. METHODS The occurrence of anti-oxidized LDL autoantibodies was investigated in control patients, in patients with atherosclerotic coronary artery disease, in those without clinically relevant signs of atherosclerosis, but considered at risk, and in patients with chronic alcohol-related liver disease. RESULTS Anti-oxidized LDL autoantibodies were present in the plasma of the majority of patients with overt coronary atherosclerosis. An increased antibody titre can also be detected well before the onset of clinically relevant signs of the atherosclerotic disease in patients classically considered at risk, indicating the occurrence of in-vivo LDL oxidation during atherosclerosis development. The specificity of molecular targets (LDL) for oxidative modifications is supported by the demonstration that anti-oxidized LDL autoantibodies are absent in the plasma of alcoholic patients who exhibit a marked increase in biological markers of oxidative stress but do not classically develop atherosclerosis. CONCLUSION These data demonstrate that the occurrence of anti-oxidized LDL autoantibodies could be specifically related to the promotion and progression of atherosclerosis and is not a simple epiphenomenon of any oxidative process occurring in vivo.

Plasma membrane hydroxyethyl radical adducts cause antibody-dependent cytotoxicity in rat hepatocytes exposed to alcohol.

BACKGROUND & AIMS We reported previously that patients with alcoholic liver disease (ALD) have circulating immunoglobulins reacting with cytochrome P4502E1 (CYP2E1) complexed with hydroxyethyl free radicals. The aim of this study was to investigate whether hydroxyethyl radical adducts are present on the plasma membranes of ethanol-treated hepatocytes and their role in antibody-dependent cytotoxicity. METHODS Immunofluorescence confocal laser microscopy, Western blotting, and antibody-dependent cell-mediated cytotoxicity assay were used. RESULTS Isolated rat hepatocytes incubated in vitro with ethanol or obtained from ethanol-treated animals showed strong surface fluorescence when exposed to rabbit anti-hydroxyethyl radical serum or sera from patients with ALD. No surface fluorescence was evident on control hepatocytes or after scavenging hydroxyethyl radicals with 4-pyridyl-1-oxide-t-butyl nitrone. The presence of CYP2E1-hydroxyethyl radical adducts on hepatocyte plasma membranes was shown by Western blot and by immunofluorescence using double staining for human and rabbit anti-CYP2E1 immunoglobulin G. Cytotoxicity was observed in ethanol-treated hepatocytes incubated with immunoglobulin G from patients with ALD and normal human blood mononuclear cells. This effect was blocked by preabsorbing the sera with human albumin complexed with hydroxyethyl radicals, which also eliminated the antibody reaction with the plasma membranes. CONCLUSIONS Hydroxyethyl radicals bound to CYP2E1 on hepatocyte plasma membranes can target immune reactions triggered by alcohol abuse.

Lipid Peroxidation, CYP2E1 and Arachidonic Acid Metabolism in Alcoholic Liver Disease in Rats

The role of cytochrome P450 metabolism of fatty acids and lipid peroxidation in the alterations of the fatty acid composition of the liver and liver pathology was investigated. The CYP2E1 inhibitors partially prevented CYP2E1 induction by ethanol and completely blocked lipid peroxidation. However, the liver pathology induced by ethanol was only partially prevented as was the decrease in arachidonic acid in total liver lipid, triglycerides and cholesterol esters. This means that liver peroxidation induced by ethanol can not completely account for the liver pathology or the decrease in arachidonic acid caused by ethanol. Lauric acid omega-1 hydroxidation by the liver microsomes in vitro was increased by ethanol and partially blocked by CYP2E1 inhibitors. However, although ethanol feeding increased the total hydroxidation and epoxidation of arachidonic acid, these were not inhibited by CYP2E1 inhibitors. Thus the ethanol-induced arachidonic acid depletion is not likely due to CYP2E1 metabolism of arachidonic acid, since the severity of liver pathology correlated negatively with the decrease in arachidonic acid in the ethanol-fed rats. The increase in its metabolism by microsomes and decrease in synthesis may be an important mechanism of ethanol-induced liver injury.

Free radical activation of acetaldehyde and its role in protein alkylation

The formation of carbon centered free radicals, identified as methylcarbonyl species, was observed using ESR spectroscopy and the spin trapping agent 4‐pyridyl‐1‐oxide‐N‐t‐butyl nitrone (4‐POBN) during the oxidation of acetaldehyde by xanthine oxidase. The reaction was dependent upon the presence of OH• radicals and was inhibited by the addition of superoxide dismutase, catalase or OH• radical scavengers. The generation of methylcarbonyl radicals was associated with a doubling of stable acetaldehyde adducts with serum albumin, and 4‐POBN or superoxide dismutase and catalase, completely blocked this effect. Thus, methylcarbonyl radicals contributed to acetaldehyde‐mediated protein alkylation which is involved in causing toxic as well as immunological reactions ascribed to acetaldehyde.

Oxidative Damage and Human Alcoholic Liver Diseases. Experimental and Clinical Evidence

An increasing number of studies have shown that hepatic lipid peroxidation and depletion of the liver content of antioxidants is evident in experimental animals exposed to ethanol, suggesting the possible involvement of oxidative damage in the pathogenesis of some of the toxic effects of alcohol. Recent findings demonstrate that hydroxyethyl radicals are generated during ethanol metabolism by the alcohol-inducible form of cytochrome P-450 (CYP2E1) and that human liver microsomes are similary capable of producing ethanol-derived radicals. These intermediates, along with reactive oxygen species, also produced by CYP2E1, can be regarded as possible causes of the stimulation of lipid peroxidation, detectable in liver biopsies and in the blood of patients suffering from alcohol-related liver diseases.

Possible Role of Free Radical Intermediates in Hepatotoxicity of Hydrazine Derivatives

Hydrazine derivatives constitute a wide group of compounds and have found application in industry, agriculture, and (as therapeutical agents) medicine. In spite of their widely spread use, several hydrazine derivatives arc known to exert hepatotoxic effects and are carcinogenic. Free radical species are produced during the hepatic biotransformation ofalkylhydrazines by both rat and humans liver microsomes. Cytochrome P-450 dependent monoxygenase system is responsible for the production of these reactive species and specific cytochrome P-450 isoenzymes appear to catalyze the formation of free radical intermediates. Free radicals generated during the metabolism of alkylhydrazines are capable of inducing oxidative stress in isolated hepatocytes and might contribute to the development of cell injury.

Activation of alkylhydrazines to free radical intermediates by ethanol-inducible cytochrome P-4502E1 (CYP2E1)

Electron spin resonance (EPR) spectroscopy analysis using the spin trapping agent 4-pyridyl-1-oxide-t-butyl nitrone (4-POBN) was used to measure the formation of free radical intermediates during NADPH-dependent oxidation of 1-methyl-, 1-ethyl-, and 1-isopropylhydrazine in rat liver microsomes and in reconstituted enzyme systems. The experiments in microsomes revealed that the specific activation of the hydrazines, as measured by the EPR signal intensities, was about two-fold higher, when expressed per nmol of P-450, in microsomes from rats treated with ethanol (EtOH) as compared to membranes isolated from either phenobarbital (PB)-, beta-naphthoflavone (beta-NF)-treated or control rats. Furthermore, kinetic experiments revealed that EtOH-microsomes had an apparent affinity for 1-ethylhydrazine about one order of magnitude higher than PB-microsomes. In reconstituted vesicular systems composed of phospholipids, NADPH cytochrome P-450 reductase and P-450, the intensities of EPR signals produced by the formation of the methyl-, ethyl- and isopropyl-free radicals, were 3- to 5-fold more intense in membrane vesicles containing ethanol-inducible CYP2E1 than phenobarbital-inducible CYP2B1. By contrast, CYP1A2, CYP2B4 and CYP2C4 were inefficient catalysts of radical formation. Desferrioxamine, catalase and superoxide dismutase did not influence the extent of ethyl radicals formed in EtOH-microsomes, indicating that hydroxyl radicals are not involved in the CYP2E1-dependent activation of 1-ethylhydrazine. Addition of cytochrome b5, an efficient donor of the second electron to P-450 and hence an inhibitor of the formation of the oxy-cytochrome P-450 complex, increased to be consistent with the results, did not influence the amount of ethyl radicals trapped. In liver microsomes from untreated rats selective substrates of CYP2E1, such as diethyl-dithiocarbamate and p-nitrophenol, as well as anti-CYP2E1-IgG, inhibited the free radical formation from 1-ethylhydrazine by about 60%. The anti-CYP2E1 IgG used significantly inhibited ethyl radical production also in human liver microsomes incubated with 1-ethylhydrazine and 4-POBN. Taken together, these results indicate that CYP2E1, as compared to other rat liver cytochromes P-450, is an efficient catalyst of transformation of alkylhydrazines to free radical intermediates, a finding that might be of importance in the development of the toxicity of these compounds.