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.

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.

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.

Chrysin modulates ethanol metabolism in Wistar rats: a promising role against organ toxicities.

AIMS: Hepato- and nephro-protective efficacy of chrysin was investigated against sequential increase of ethanol intake on the alteration of alcohol metabolizing enzymes-alcohol dehydrogenase (ADH), cytochrome P450 2E1 (CYP 2E1), xanthine oxidase (XO) and oxidant/anti-oxidant status. METHODS: Thirty female Wistar rats segregated into five groups, each with six animals, were put to different doses. Group I as control followed by Group II, III and IV were treated with ethanol (5,8,10 and 12 g/kg body weight per week respectively) for 4 weeks. While Group III and IV were administered with chrysin at 20 mg (D1) and 40 mg/kg body weight (D2), respectively, prior to ethanol administration. Group V was given only chrysin (D2). Various oxidative stress and ethanol metabolizing enzymes were estimated in hepatic and renal tissues. RESULTS: Ethanol administration significantly induced CYP 2E1, ADH and XO in liver and kidneys, respectively, along with an enhancement in levels of malondialdehyde and serum alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen, creatinine and lactate dehydrogenase when compared with the control group and this enhancement is significantly normalized with chrysin administration. Oxidative stress markers: reduced glutathione, glutathione peroxidase, catalase and glutathione reductase were significantly (P < 0.001) depleted in the ethanol-treated group, while chrysin administration significantly restored all of these. Only chrysin administration did not show any adverse effect. CONCLUSION: Results demonstrate that chrysin administration prevents the liver and kidney of Wistar rats against oxidative damage during chronic ethanol consumption by inhibiting the activities of ADH, CYP 2E1, XO and catalase.

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.

Opioid peptides derived from food proteins suppress aggregation and promote reactivation of partly unfolded stressed proteins.

A new view of the opioid peptides is presented. The potential of small peptides derived from precursor food proteins, to bind to partly unfolded stressed proteins to prevent their irreversible aggregation and inactivation has been demonstrated in various in vitro test systems: dithiothreitol-induced aggregation of alpha-lactalbumin (LA), heat-induced aggregation of alcohol dehydrogenase (ADH), and aggregation and inactivation of bovine erythrocyte carbonic anhydrase (CA) in the process of its refolding after removal of stress conditions. Using dynamic light scattering (DLS), turbidimetry, fluorescence, and circular dichroism measurements protective effects of the synthetic opioid peptides: exorphin C from wheat gluten (Tyr-Pro-Ile-Ser-Leu), rubiscolin-5 from spinach ribulose-bisphosphate-carboxylase/oxygenase (Rubisco) (Tyr-Pro-Leu-Asp-Leu), and hemorphin-6 from bovine hemoglobin (Tyr-Pro-Trp-Thr-Gln-Arg) have been revealed. We have demonstrated the concentration-dependent suppression of light scattering intensity of aggregates of LA and ADH in the presence of the peptides, the population of nanoparticles with higher hydrodynamic radii being shifted to the lower ones, accompanied by an increase in the lag period of aggregation. The presence of the peptides in the refolding solution was shown to assist reactivation of CA and enhance the yield of the CA soluble protein. The results suggest that bioactive food protein fragments may be regarded as exogenous supplements to the endogenous defense mechanisms of the human organism under stress conditions.

Kinetics of ethanol oxidation catalyzed by yeast alcohol dehydrogenase in aqueous solutions of sodium dodecylsulfate.

A study has been made of the effect of sodium dodecylsufate (SDS) addition on the oxidation of ethanol catalyzed by yeast alcohol dehydrogenase. Experiments were performed at pH = 8.1 and SDS concentrations employed were below and above the surfactant critical micelle concentration (CMC). The double reciprocal plots obtained in the absence and in the presence of the surfactant were compatible with a sequential bi-bi ordered mechanism. In the presence of the surfactant the initial reaction rates were consistently lower than in pure buffer at all the surfactant concentrations considered (0.5-50 mM). This effect is mainly due to an increase in the dissociation constant of beta-NAD(+) which reaches its maximum value (7,100 +/- 1,700 microM) at the CMC. Above the CMC the effect of the surfactant is mainly due to an increase in the Michaels constants of the alcohol, with values of 41 +/- 1 mM for 15 mM SDS and 50 +/- 1 mM for 50 mM SDS. The catalytic rate constant was found to be practically independent of the presence of the surfactant in the range of concentrations considered (up to 50 mM).

Effect of low temperature on ethanolic fermentation in rice seedlings.

Rice seedlings (Oryza sativa L.) were incubated at 5-30 degrees C for 48 h and the effect of temperature on ethanolic fermentation in the seedlings was investigated in terms of low-temperature adaptation. Activities of alcohol dehydrogenase (ADH, EC 1.1.1.1) and pyruvate decarboxylase (PDC, EC 4.1.1.1) in roots and shoots of the seedlings were low at temperatures of 20-30 degrees C, whereas temperatures of 5, 7.5 and 10 degrees C significantly increased ADH and PDC activities in the roots and shoots. Temperatures of 5-10 degrees C also increased ethanol concentrations in the roots and shoots. The ethanol concentrations in the roots and shoots at 7.5 degrees C were 16- and 12-times greater than those in the roots and shoots at 25 degrees C, respectively. These results indicate that low temperatures (5-10 degrees C) induced ethanolic fermentation in the roots and shoots of the seedlings. Ethanol is known to prevent lipid degradation in plant membrane, and increased membrane-lipid fluidization. In addition, an ADH inhibitor, 4-methylpyrazole, decreased low-temperature tolerance in roots and shoots of rice seedlings and this reduction in the tolerance was recovered by exogenous applied ethanol. Therefore, production of ethanol by ethanolic fermentation may lead to low-temperature adaptation in rice plants by altering the physical properties of membrane lipids.

[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.

Physiologic and genomic analyses of nutrition-ethanol interactions during gestation: Implications for fetal ethanol toxicity.

Nutrition-ethanol (EtOH) interactions during gestation remain unclear primarily due to the lack of appropriate rodent models. In the present report we utilize total enteral nutrition (TEN) to specifically understand the roles of nutrition and caloric intake in EtOH-induced fetal toxicity. Time-impregnated rats were intragastrically fed either control or diets containing EtOH (8-14 g/kg/day) at a recommended caloric intake for pregnant rats or rats 30% undernourished, from gestation day (GD) 6-20. Decreased fetal weight and litter size (P < 0.05) and increased full litter resorptions (33% vs. 0%), were observed in undernourished dams compared to adequately fed rats given the same dose of EtOH, while undernutrition alone did not produce any fetal toxicity. Undernutrition led to impairment of EtOH metabolism, increased blood EtOH concentrations (160%), and decreased maternal hepatic ADH1 mRNA, protein, and activity. Microarray analyses of maternal hepatic gene expression on GD15 revealed that 369 genes were altered by EtOH in the presence of undernutrition, as compared to only 37 genes by EtOH per se (+/-2-fold, P < 0.05). Hierarchical clustering and gene ontology analysis revealed that stress and external stimulus responses, transcriptional regulation, cellular homeostasis, and protein metabolism were affected uniquely in the EtOH-under-nutrition group, but not by EtOH alone. Microarray data were confirmed using real-time RT-PCR. Undernourished EtOH-fed animals had 2-fold lower IGF-1 mRNA and 10-fold lower serum IGF-1 protein levels compared to undernourished controls (P < 0.0005). Examination of maternal GH signaling via STAT5a and -5b revealed significant reduction in both gene and protein expression produced by both EtOH and undernutrition. However, despite significantly elevated fetal BECs, fetal IGF-1 mRNA and protein were not affected by EtOH or EtOH-undernutrition combinations. Our data suggest that undernutrition potentiates the fetal toxicity of EtOH in part by disrupting maternal GH-IGF-1, signaling thereby decreasing maternal uterine capacity and placental growth.

Kavalactones fail to inhibit alcohol dehydrogenase in vitro.

In recent years, Kava kava (Piper methysticum, Forst. f., Piperaceae), a folkloric beverage and popular herbal remedy, has been implicated in a number of liver failure cases. Many hypotheses as to the mechanism of its hepatotoxicity, for example interactions with other co-ingested medication, have been postulated. This present study investigated whether pharmacokinetic interactions between kava constituents and alcohol via alcohol dehydrogenase (ADH) inhibition by individual kavalactones might explain its claimed hepatotoxic effects. Four kavalactones, (+/-)-kavain, methysticin, yangonin and desmethoxyyangonin, fail to inhibit ADH in vitro at 1, 10 or 100 microM concentrations.

Uncoupler of oxidative phosphorylation prolongs the lifespan of Drosophila.

The effect of a moderate ("soft") uncoupling of mitochondria on the lifespan and some parameters of biological age of Drosophila melanogaster strain Oregon was studied. Addition of the uncoupler 2,4-dinitrophenol (DNP) to the nutritional mixture of larvae significantly increased the average lifespan of the flies without changing their maximal lifespan. DNP significantly increased the rate of oxygen consumption by isolated mitochondria and tissue homogenates of the flies in state 4 (of Chance). DNP also decreased the activity of alcohol dehydrogenase (a parameter of flies' biological age) in the tissue homogenates, especially on octanol as the reaction substrate. However, being deprived of food the DNP-treated flies displayed a markedly decreased viability as compared to the control flies. On the whole, the results suggest that "soft" uncoupling of mitochondria may increase the lifespan.

Alcohol-induced oxidative stress in brain endothelial cells causes blood-brain barrier dysfunction.

Brain microvascular endothelial cells (BMVEC) connected by tight junctions (TJ) form a tight monolayer at the blood-brain barrier (BBB). We investigated the idea that BBB dysfunction seen in alcohol abuse is associated with oxidative stress stemming from ethanol (EtOH) metabolism in BMVEC. Exposure to EtOH induced catalytic activity/expression of EtOH-metabolizing enzymes, which paralleled enhanced generation of reactive oxygen species (ROS). EtOH-mediated oxidative stress led to activation of myosin light chain (MLC) kinase, phosphorylation of MLC and TJ proteins, decreased BBB integrity, and enhanced monocyte migration across BBB. Acetaldehyde or ROS donors mimicked changes induced by EtOH in BMVEC. Thus, oxidative stress resulting from alcohol metabolism in BMVEC can lead to BBB breakdown in alcohol abuse, serving as an aggravating factor in neuroinflammatory disorders.

Retinoic acid negatively regulates GDNF and neurturin receptor expression and responsiveness in embryonic chicken sympathetic neurons.

Neurotrophins and members of the GDNF family influence the generation, differentiation and survival of sympathetic neurons during development. Neurotrophin receptor expression and responsiveness has previously been shown to be regulated by all-trans retinoic acid (RA) in embryonic chicken sympathetic neurons. To determine if RA also regulates responsiveness to GDNF family members and expression of their receptors, we studied the effect of treating cultures of these neurons at a stage when they survive in response to GDNF and neurturin. RA caused a dose-dependent decrease in the survival response to both GDNF and neurturin. Transcripts for the ligand-specifying receptors for GDNF and neurturin, GFRalpha-1 and GFRalpha-2, as well as the common signal transducing receptor Ret were all down-regulated by RA treatment in a dose-dependent manner. Transcripts for all three retinoic acid receptors RaRalpha, RaRbeta and RaRgamma as well as the enzymes involved in RA synthesis, Adh-1 and RALDH-2, RALDH-3, were present in both sympathetic targets and neurons. Studies with retinoic acid receptor agonists and antagonists revealed that the effects of RA on receptor expression were mediated mainly by RaRalpha. These findings implicate RA in regulating the actions of members of the GDNF family on developing sympathetic neurons.

Chaperone-like activity of beta-casein.

The caseins are major components of milk for most mammals and are secreted as large colloidal aggregates termed micelles. They have less ordered secondary and tertiary structures in comparison with typical globular proteins. In this work, beta-casein, a member of the casein family, has been demonstrated to exhibit chaperone-like activity, being able to suppress the thermal and chemical aggregation of such substrate proteins as insulin, lysozyme, alcohol dehydrogenase, and catalase by forming stable complexes with the denaturing substrate proteins. Meanwhile, beta-casein was found to not only prevent aggregation of the substrate proteins, but also solubilize the protein aggregates already formed. Data also show that beta-casein exhibits a higher chaperone-like activity than alpha-casein, likely due to the difference in the number of proline residues present and/or in the extent of exposed hydrophobic surfaces. The implications for their in vivo functions of the caseins, based on their exhibiting such in vitro chaperone-like activities, are discussed.