Down-regulation of the expression of alcohol dehydrogenase 4 and CYP2E1 by the combination of α-endosulfan and dioxin in HepaRG human cells.

Pesticides and other persistent organic pollutants are considered as risk factors for liver diseases. We treated the human hepatic cell line HepaRG with both 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) and the organochlorine pesticide, α-endosulfan, to evaluate their combined impact on the expression of hepatic genes involved in alcohol metabolism. We show that the combination of the two pollutants (25nM TCDD and 10µM α-endosulfan) led to marked decreases in the amounts of both the mRNA (up to 90%) and protein (up to 60%) of ADH4 and CYP2E1. Similar results were obtained following 24h or 8days of treatment with lower concentrations of these pollutants. Experiments with siRNA and AHR agonists and antagonist demonstrated that the genomic AHR/ARNT pathway is necessary for the dioxin effect. The PXR, CAR and estrogen receptor alpha transcription factors were not modulators of the effects of α-endosulfan, as assessed by siRNA transfection. In another human hepatic cell line, HepG2, TCDD decreased the expression of ADH4 and CYP2E1 mRNAs whereas α-endosulfan had no effect on these genes. Our results demonstrate that exposure to a mixture of pollutants may deregulate hepatic metabolism.

Toxicology and the biological role of methanol and ethanol: Current view.

BACKGROUND: Alcohol variants such as ethanol and methanol are simple organic compounds widely used in foods, pharmaceuticals, chemical synthesis, etc. Both are becoming an emerging health problem; abuse of ethanol containing beverages can lead to disparate health problems and methanol is highly toxic and unfit for consumption. METHODS AND RESULTS: This review summarizes the basic knowledge about ethanol and methanol toxicity, the effect mechanism on the body, the current care of poisoned individuals and the implication of alcohols in the development of diseases. Alcohol related dementia, stroke, metabolic syndrome and hepatitis are discussed as well. Besides ethanol, methanol toxicity and its biodegradation pathways are addressed. CONCLUSIONS: The impact of ethanol and methanol on the body is shown as case reports, along with a discussion on the possible implication of alcohol in Alzheimer's disease and antidotal therapy for methanol poisoning. The role of ethanol in cancer and degenerative disorders seems to be underestimated given the current knowledge. Treatment in case of poisoning is another issue that remains unresolved even though effective protocols and drugs exist.

Diosmin protects against ethanol-induced hepatic injury via alleviation of inflammation and regulation of TNF-α and NF-κB activation.

The present investigation was designed to evaluate the efficacy of diosmin against ethanol-induced hepatotoxicity in rats by modulating various mechanisms including ethanol metabolizing enzymes, generation of free radicals, imbalance in oxidant-antioxidant status, oxidative damage to membrane lipids, activation of transcription factors and elevation in inflammatory markers involved in ethanol-induced hepatic damage. Diosmin is a flavone glycoside, having anti-inflammatory and anti-cancer properties. Thirty female Wistar rats segregated in five groups, each with six animals. Group I as control followed by Group II, III and IV were treated with ethanol for 28 days. While groups III and IV were administered with diosmin at 10 mg/kg b wt (D1) and 20 mg/kg b wt (D2) respectively prior to ethanol administration. Group V was given only higher dose of diosmin. In ethanol-treated group, ethanol metabolizing enzymes viz., CYP 450 2E1 and alcohol dehydrogenase (ADH) significantly increased by 77.82% and 32.32% in liver tissues respectively as compared with control group and this enhancement is significantly normalized with diosmin administration. Diosmin administration (D1 & D2) significantly (p < 0.001) attenuates oxidative stress markers i.e., LPO, GSH, GPx, GR and XO by 90.77 & 137.55%, 17.18 & 25%, 37.3 & 49.86%, 21.63 & 44.9% and 56.14 &77.19% respectively. Serum ALT, AST and LDH significantly increased by 102.03, 116.91 and 45.20% in ethanol-treated group as compared with control group. Group III and IV animals showed significant reduction in the serum toxicity markers. Diosmin further alleviated ethanol-induced NF-κB activation, enhanced expression of TNF-α, COX-2 and iNOS. Findings from the present study permit us to conclude that diosmin alleviates alcoholic liver injury via modulating ethanol metabolizing pathway, inhibition of oxidative stress markers and suppression of inflammatory markers. This may represent a novel protective strategy against ethanol-induced liver diseases.

In vitro evidence for chronic alcohol and high glucose mediated increased oxidative stress and hepatotoxicity.

BACKGROUND: Hyperglycemia or alcoholism can lead to impaired liver functions. Cytochrome P450 2E1 (CYP2E1) is elevated in hyperglycemia or alcoholism and plays a critical role in generating oxidative stress in the cell. METHODS: In the present study, we have used VL-17A cells that overexpress the alcohol metabolizing enzymes [alcohol dehydrogenase (ADH) and CYP2E1] to investigate the toxicity due to ethanol (EtOH) plus high glucose. Toxicity was assessed through viability assay and amount of acetaldehyde adduct formation. Oxidative stress parameters included measuring reactive oxygen species (ROS) levels and malondialdehyde adduct formation. Apoptosis was determined through caspase-3 activity, Annexin V- Propidium iodide staining, and changes in mitochondrial membrane potential. The effects of antioxidants and specific inhibitors of ADH and CYP2E1 on cell viability and ROS levels were also studied. RESULTS: When present together, EtOH plus high glucose-treated VL-17A cells exhibited greater oxidative stress and toxicity than other groups. Apoptosis was observed in liver cells treated with the toxins, and the EtOH plus high glucose-treated VL-17A cells exhibited apoptosis to the largest extent. A distinct and graded increase in CYP2E1 level occurred in the different groups of VL-17A cells. Further, antioxidants or inhibitors of ADH and CYP2E1 were effective in decreasing the observed oxidative stress and toxicity. CONCLUSIONS: The combined oxidative insult due to alcohol plus high glucose leads to greater liver injury, which may prove to be a timely warning for the injurious effects of alcohol consumption in diabetics.

Gene-selective histone H3 acetylation in the absence of increase in global histone acetylation in liver of rats chronically fed alcohol.

AIMS: The aim of this study was to determine the effect of chronic ethanol feeding on acetylation of histone H3 at lysine 9 (H3-Lys9) at promoter and coding regions of genes for class I alcohol dehydrogenase (ADH I), inducible nitric oxide synthase (iNOS), Bax, p21, c-met and hepatocyte growth factor in the rat liver. METHODS: Rats were fed ethanol-containing liquid diet (5%, w/v) for 1-4 weeks. The global level of acetylation of H3-Lys9 in the liver was examined by western blot analysis. The levels of mRNA for various genes were measured by real-time reverse transcriptase-polymerase chain reaction. The association of acetylated histone H3-Lys9 with the different regions of genes was monitored by chromatin immunoprecipitation assay. RESULTS: Chronic ethanol treatment increased mRNA expression of genes for iNOS, c-jun and ADH 1. Chronic ethanol treatment did not cause increase in global acetylation of H3-Lys9, but significantly increased the association of acetylated histone H3-Lys9 in the ADH I gene, both in promoter and in coding regions. In contrast, chronic ethanol treatment did not significantly increase the association of acetylated histone H3-Lys9 with iNOS and c-jun genes. CONCLUSION: Chronic ethanol exposure increased the gene-selective association of acetylated H3-Lys9 in the absence of global histone acetylation. Thus, not all genes expressed by ethanol are linked to transcription via histone H3 acetylation at Lys9.

S-adenosylmethionine decreases the peak blood alcohol levels 3 h after an acute bolus of ethanol by inducing alcohol metabolizing enzymes in the liver.

INTRODUCTION: An alcohol bolus causes the blood alcohol level (BAL) to peak at 1-2 h post ingestion. The ethanol elimination rate is regulated by alcohol metabolizing enzymes, primarily alcohol dehydrogenase (ADH1), acetaldehyde dehydrogenase (ALDH), and cytochrome P450 (CYP2E1). Recently, S-adenosylmethionine (SAMe) was found to reduce acute BALs 3 h after an alcohol bolus. The question, then, was: what is the mechanism involved in this reduction of BAL by feeding SAMe? To answer this question, we investigated the changes in ethanol metabolizing enzymes and the epigenetic changes that regulate the expression of these enzymes during acute binge drinking and chronic drinking. METHODS: Rats were fed a bolus of ethanol with or without SAMe, and were sacrificed at 3 h or 12 h after the bolus. RESULTS: RT-PCR and Western blot analyses showed that SAMe significantly induced ADH1 levels in the 3 h liver samples. However, SAMe did not affect the changes in ADH1 protein levels 12 h post bolus. Since SAMe is a methyl donor, it was postulated that the ADH1 gene expression up regulation at 3 h was due to a histone modification induced by methylation from methyl transferases. Dimethylated histone 3 lysine 4 (H3K4me2), a modification responsible for gene expression activation, was found to be significantly increased by SAMe at 3 h post bolus. CONCLUSION: These results correlated with the low BAL found at 3 h post bolus, and support the concept that SAMe increased the gene expression to increase the elimination rate of ethanol in binge drinking by increasing H3K4me2.

[Studies on the effect of extracts of several Chinese herbal medicines and other medicines on alcohol dehydrogenase activity].

OBJECTIVE: To study the effects of water and alcohol extracts of several Chinese herbal medicines and other medicines on alcohol dehydrogenase activity in order to provide enzymology basis on new medicine. METHODS: Water or alcohol extracts of Chinese herbal medicine and other medicine were tested on the effects of alcohol dehydrogenase activity by Valle and Hoch method. RESULTS: Among them, 8 were found to have the effect of activation on alcohol dehydrogenase. They were water extracts of Amomum kravanh and Pueraria flowers, the alcohol extracts of Pueraria flowers, compound hepatcare Chinese medicine and compound Pueraria medicine, L-cysteine, notoginseng saponin. Others had inhibiting action. CONCLUSION: To decrease alcohol concentration in the body through activating the activity of ADH may be one of the mechanisms for some traditional Chinese herbal medicine in neutralizing the effect of alcohol drink.

Relevance of Frank's solvent classification as typically aqueous and typically non-aqueous to activities of firefly luciferase, alcohol dehydrogenase, and alpha-chymotrypsin in aqueous binaries.

Effects of cosolvent concentration on activity of fire fly luciferase, alpha-chymotrypsin, and alcohol dehydrogenase from baker's yeast (Saccharomyces cerevisiae) have been studied for several solvents with varying hydrophobicities (logP from +1.0 to -1.65) and polarities (dielectric constant from 7.4 to 109). The inhibitory effect of the cosolvent is examined in light of Frank's classification of solvents into 'typically aqueous (TA)' and 'typically non-aqueous (TNA).' The solvent concentration at which the enzyme activity decreases to half, the C(50) values, for TA solvents such as 1-cyclohexyl-2-pyrrolidinone, 2-butoxyethanol, 1-methyl-2-pyrrolidinone, tetrahydrofuran, t-butanol, and ethanol correlate quite well with their critical hydrophobic interaction concentration, rather than logP, while those for TNA solvents such as acetonitrile, dimethyl formamide, formamide, and dimethyl sulfoxide correlate well with logP. The interactions of TA solvents with proteins appear to be governed mainly by hydrophobic interactions while both hydrophobic and hydrophilic interactions play important role in case of TNA solvents.

Human choriocarcinoma cell line JEG-3 produces and secretes active retinoids from retinol.

Vitamin A (retinol) and its active derivatives (the retinoids) are essential for growth and development of the mammalian fetus. Maternally-derived retinol has to pass through the placenta to reach the developing fetus. Despite its apparent importance, little is known about placental metabolism of retinol, and particularly placental production and/or secretion of active retinoids. It has been previously considered that retinoids are recruited from the uterine environment to influence placental development and function during gestation. We have studied retinoid metabolism in the human choriocarcinoma cell line JEG-3 and demonstrate, for the first time, that active retinoids are produced endogenously by the JEG-3 cell line from retinol. These retinoids induce gene expression from a retinoic acid-responsive enhancer element reporter plasmid and modulate placental transglutaminase activity. Furthermore, retinoids are secreted from JEG-3, as shown by the activation of retinoic acid-responsive beta lacZ reporter cells grown in conditioned media. These results suggest that there could be an active role for trophoblast-derived retinoids during human development.

Use of cultured cells in assessing ethanol toxicity and ethanol-related metabolism.

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Terrence M. Donohue, Jr, and Dahn L. Clemens. The presentations were (1) Characterization of single and double recombinant hepatoma cells that express ethanol-metabolizing enzymes, by Terrence M. Donohue, Jr; (2) Inhibition of cell growth by ethanol metabolism, by Dahn L. Clemens; (3) Use of transfected HeLa cells to study the genesis of alcoholic fatty liver, by Andrea Galli and David Crabb; (4) CYP2E1-mediated oxidative stress induces COL1A2 mRNA in hepatic stellate cells and in a coculture system of HepG2 and stellate cells, by Natalia Nieto; (5) Transforming growth factor-alpha secreted from ethanol-exposed hepatocytes contributes to development of alcoholic hepatic fibrosis, by Junji Kato; and (6) Effect of ethanol on Fas-dependent caspase-3 activation and apoptosis in CD4+ T cells, by Shirish S. Barve.

Toxicokinetics of ethylene glycol during fomepizole therapy: implications for management. For the Methylpyrazole for Toxic Alcohols Study Group.

STUDY OBJECTIVE: The elimination kinetics of ethylene glycol (EG) in human subjects treated with fomepizole (4-methylpyrazole) were analyzed to establish the efficacy of alcohol dehydrogenase (ADH) inhibition and to characterize elimination pathways. METHODS: Drug concentration data from patients enrolled in the EG arm of the Methylpyrazole for Toxic Alcohols trial, a prospective, multicenter, open-label trial of fomepizole, were analyzed and compared with published estimates. RESULTS: In 19 patients analyzed (EG concentrations of 3.5 to 211 mg/dL), elimination was first order during fomepizole monotherapy (half-life of 19.7+/-1.3 hours) and was not affected by the presence of ethanol. The elimination rate was significantly faster (half-life of <8.6+/-1.1 hours, P <.001) in the absence of fomepizole and ethanol. EG elimination by the kidneys was directly proportional to remaining renal function as estimated by creatinine clearance, with a fractional excretion of 25.5%+/-9.4%. Renal elimination and hemodialysis were the only significant routes of EG elimination as long as fomepizole concentrations were maintained well above 10 micromol/L (EG/fomepizole molar ratio, <100:1). All patients with normal serum creatinine concentrations at the initiation of fomepizole treatment had rapid rates of renal elimination (half-life of 16.8+/-0.8 hours). CONCLUSION: At doses used, fomepizole effectively inhibits ADH-mediated metabolism of EG. Serum creatinine concentration at presentation and creatinine clearance can be used to predict EG elimination during fomepizole therapy and can help determine which patients will require hemodialysis to expedite EG elimination. An absolute EG concentration above 50 mg/dL should no longer be used as an independent criterion for hemodialysis in patients treated with fomepizole.

Gastric and intestinal ethanol toxicity in the rat. Effect on glutathione level and role of alcohol and acetaldehyde metabolisms.

BACKGROUND/AIMS: This study investigated the dose- and time-dependent effect of ethanol on gastric and intestinal glutathione and protein oxidative state in the rat. METHODS: Rats received 1 or 4 g/kg of 25% ethanol solution orally or isocaloric glucose. Some rats received diethylmaleate, cimetidine or cyanamide before ethanol (1 g/kg). Glutathione, carbonyl proteins and histological damage were evaluated in the gastric and intestinal mucosa 6 hours after treatment. RESULTS: An increase in glutathione was observed 2 to 6 hours after 1 g/kg of ethanol both in the gastric and intestinal mucosa, whereas 4 g/kg decreased glutathione. The rise in glutathione after ethanol was associated with increased levels of its oxidized form; however, the total/oxidized ratio was significantly decreased only in the intestinal tract. Diethylmaleate depleted mucosal glutathione, while the subsequent ingestion of ethanol increased it. Unlike stomach, intestine showed a significant increase in carbonyl proteins and marked histological lesions after ethanol ingestion. Cimetidine and cyanamide inhibited by 50% the activity of alcohol dehydrogenase and by 80% aldehyde dehydrogenase, respectively, in the gastric and intestinal mucosa. Cyanamide significantly enhanced ethanol-induced protein oxidation and mucosal injury in the stomach. No such effect was observed in the intestine. CONCLUSIONS: The increase of glutathione after ingestion of low amounts of ethanol appears to be an adaptive mechanism against ethanol toxicity. Depletion of glutathione increased protein oxidation and the extent of histological damage in ethanol-treated rats. At gastric level, the effects of ethanol are exaggerated by the inhibition of acetaldehyde metabolism; while intestinal damages appear to be ascribed to ethanol itself.

In vitro stabilization and in vivo solubilization of foreign proteins by the beta subunit of a chaperonin from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1.

The gene encoding the beta subunit of a molecular chaperonin from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1 (cpkB) was cloned, sequenced, and expressed in Escherichia coli. The cpkB gene is composed of 1,641 nucleotides, encoding a protein (546 amino acids) with a molecular mass of 59,140 Da. The enhancing effect of CpkB on enzyme stability was examined by using Saccharomyces cerevisiae alcohol dehydrogenase (ADH). Purified recombinant CpkB prevents thermal denaturation and enhances thermostability of ADH. CpkB requires ATP for its chaperonin function at a low CpkB concentration; however, CpkB functions without ATP when present in excess. In vivo chaperonin function for the solubilization of insoluble proteins was also studied by coexpressing CpkB and CobQ (cobryic acid synthase), indicating that CpkB is useful for solubilizing the insoluble proteins in vivo. These results suggest that the beta subunit plays a major role in chaperonin activity and is functional without the alpha subunit.

Human gastric alcohol dehydrogenase: in vitro characteristics and effect of cimetidine.

The presence of at least two types of alcohol dehydrogenase has been demonstrated in surgical specimens from the human stomach. One isoenzyme has a Km of approximately 1-2 mM for ethanol comparable to that of class I alcohol dehydrogenase isoenzyme as defined for the liver. This isoenzyme can also be detected by immunohistology using a polyclonal rabbit antibody against human liver class I alcohol dehydrogenase. The other isoenzyme of alcohol dehydrogenase has a much lower affinity to ethanol (greater than 300 mM), but with activities that become significant at ethanol concentrations of more than 100 mM commonly present in the human stomach. Cimetidine was found to be a noncompetitive inhibitor of gastric alcohol dehydrogenase at concentrations as low as 1 mM in vitro. Since the human gastric alcohol dehydrogenase is responsible for the first-pass metabolism of ethanol, its inhibition by cimetidine may explain the reduced first-pass metabolism of alcohol which is associated with elevated ethanol blood concentrations seen after cimetidine therapy.

Effects of cannabis and tobacco on the enzymes of alcohol metabolism in the rat.

The effects of cannabis and tobacco on the enzymes of ethanol metabolism were studied in the Wistar rat. The activities of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (AlDH) were measured in the liver and the brain, after treatment with an extract of cannabis resin, with an extract of tobacco leaves, or with nicotine. A condensate of cannabis resin extract was collected in a smoking machine, using a tobacco cigarette as the vehicle. Unsmoked or smoked cannabis extracts were dissolved in olive oil and were given i.p. (twice daily, for 7 days). In both cases, a similar dose level was used in terms of starting material (raw cannabis resin), estimated at about 100 mg/kg body weight. Control animals were treated either with olive oil, or with the same amount of smoked condensate obtained from a reference cigarette. Nicotine was dissolved in olive oil and it was given i.p. (10 micrograms/kg, twice daily for 7 days). An extract of unsmoked tobacco was dissolved in olive oil and was given with the same schedule, at a dose which was estimated to correspond to about 10 micrograms nicotine/kg b.w. All groups of animals received an additional i.p. injection on day 8, one hour before sacrifice. Our results showed that unsmoked cannabis inhibited the hepatic activities of the microchondrial AlDH (low-Km and high-Km), the hepatic low-Km cytosolic AlDH (p less than 0.001), and the low-Km mitochondrial AlDH of the brain (p less than 0.001). Administration of smoked cannabis to the animals inhibited the hepatic mitochondrial low-Km AlDH (p less than 0.001), but it did not influence the brain enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)