Inhibition Of Ethanol Metabolism Research Articles

Role of ethanol-derived acetaldehyde in operant oral self-administration of ethanol in rats.

The role of ethanol-derived acetaldehyde has not been examined yet on performance in a model of operant oral self-administration. However, previous studies reported that an acetaldehyde-sequestering agent, D-penicillamine (DP) and an inhibitor of catalase-mediated acetaldehyde production, 3-amino-1,2,4-triazole (3-AT) reduce voluntary ethanol consumption. The aim of our investigation was to evaluate the effects of DP and 3-AT on acquisition and maintenance of oral operant ethanol self-administration. Using operant chambers, rats learned to nose poke in order to receive ethanol solution (5-10 % v/v) under an FR1 schedule of reinforcement in which discrete light and tone cues were presented during ethanol delivery. DP and 3-AT impair the acquisition of ethanol self-administration, whereas its maintenance is not affected neither by drug given alone for both 10 or 5 % ethanol nor by drugs association for 5 % ethanol. Moreover, when the concentration of ethanol was diminished from 10 to 5 %, rats increased the rate of self-administration behaviour. These findings suggest that brain acetaldehyde plays a critical role during acquisition of operant self-administration in ethanol-naïve rats. In contrast, during the maintenance phase, acetaldehyde could contribute to ethanol self-administration by a combined mechanism: On one hand, its lack (by DP or 3-AT) might result in further ethanol-seeking and taking and, on the other, inhibition of ethanol metabolism (by 3-AT) might release an action of the un-metabolised fraction of ethanol that does not overall result in compromising maintenance of ethanol self-administration.

Yeast Stress, Aging, and Death

Yeast Stress, Aging, and Death

Acetaldehyde-Mediated Neurotoxicity: Relevance to Fetal Alcohol Spectrum Disorders

Ethanol-induced neuro-developmental abnormalities are associated with impaired insulin and IGF signaling, and increased oxidative stress in CNS neurons. We examined the roles of ethanol and its principal toxic metabolite, acetaldehyde, as mediators of impaired insulin/IGF signaling and oxidative injury in immature cerebellar neurons. Cultures were exposed to 3.5 mM acetaldehyde or 50 mM ethanol ± 4-methylpyrazole (4-MP), an inhibitor of ethanol metabolism, and viability, mitochondrial function, oxidative stress, DNA damage, and insulin responsiveness were measured 48 hours later. Acetaldehyde or ethanol increased neuronal death and levels of 8-OHdG and 4-HNE, and reduced mitochondrial function. Ethanol inhibited insulin responsiveness, whereas acetaldehyde did not. 4-MP abated ethanol-induced oxidative stress and mitochondrial dysfunction, but failed to restore insulin responsiveness. Furthermore, alcohol and aldehyde metabolizing enzyme genes were inhibited by prenatal ethanol exposure; this effect was mediated by acetaldehyde and not ethanol + 4MP. These findings suggest that brain insulin resistance in prenatal alcohol exposure is caused by direct effects of ethanol, whereas oxidative stress induced neuronal injury is likely mediated by ethanol and its toxic metabolites. Moreover, the adverse effects of prenatal ethanol exposure on brain development may be exacerbated by down-regulation of genes needed for metabolism and detoxification of alcohol in the brain.

Exposure of precision-cut rat liver slices to ethanol accelerates fibrogenesis

Ethanol metabolism in the liver induces oxidative stress and altered cytokine production preceding myofibroblast activation and fibrogenic responses. The purpose of this study was to determine how ethanol affects the fibrogenic response in precision-cut liver slices (PCLS). PCLS were obtained from chow-fed male Wistar rats (200-300 g) and were cultured up to 96 h in medium, 25 mM ethanol, or 25 mM ethanol and 0.5 mM 4-methylpyrazole (4-MP), an inhibitor of ethanol metabolism. Slices from every time point (24, 48, 72, and 96 h) were examined for glutathione (GSH) levels, lipid peroxidation [thiobarbituric acid-reactive substance (TBARS) assay], cytokine production (ELISA and RT-PCR), and myofibroblast activation [immunoblotting and immunohistochemistry for smooth muscle actin (SMA) and collagen]. Treatment of PCLS with 25 mM ethanol induced significant oxidative stress within 24 h, including depletion of cellular GSH and increased lipid peroxidation compared with controls (P < 0.05). Ethanol treatment also elicited a significant and sustained increase in interleukin-6 (IL-6) production (P < 0.05). Importantly, ethanol treatment accelerates a fibrogenic response after 48 h, represented by significant increases in SMA and collagen 1alpha(I) production (P < 0.05). These ethanol-induced effects were prevented by the addition of 4-MP. Ethanol metabolism induces oxidative stress (GSH depletion and increased lipid peroxidation) and sustained IL-6 expression in rat PCLS. These phenomena precede and coincide with myofibroblast activation, which occurs within 48 h of treatment. These results indicate the PCLS can be used as in vitro model for studying multicellular interactions during the early stages of ethanol-induced liver injury and fibrogenesis.

Acetaldehyde elicits ERK phosphorylation in the rat nucleus accumbens and extended amygdala

Recent advances suggest that acetaldehyde mediates some of the neurobiological properties of ethanol. In a recent study, we have shown that ethanol elicits the phosphorylation of extracellular signal-regulated kinase (pERK) in the nucleus accumbens and extended amygdala, via a dopamine D(1) receptor-mediated mechanism. The aim of this study was to determine whether acetaldehyde and ethanol-derived acetaldehyde elicit the activation of ERK in the nucleus accumbens and extended amygdala. The effects of acetaldehyde (10 and 20 mg/kg) and ethanol (1 g/kg), administered to rats intragastrically, were assessed by pERK peroxidase immunohistochemistry. To establish the role of ethanol-derived acetaldehyde, the alcohol dehydrogenase inhibitor, 4-methylpyrazole (90 mg/kg), and the acetaldehyde-sequestering agent, D-penicillamine (50 mg/kg), were administered before ethanol. Acetaldehyde increased pERK immunoreactivity in the nucleus accumbens and extended amygdala. Inhibition of ethanol metabolism and sequestration of newly synthesized acetaldehyde completely prevented ERK activation by ethanol. In addition, to establish the role of D(1) receptors stimulation in acetaldehyde-elicited ERK phosphorylation, we studied the effect of the D(1) receptor antagonist, SCH 39166. Pretreatment with the D(1) receptor antagonist (50 μg/kg) fully prevented acetaldehyde-elicited ERK activation. Overall, these results indicate that ethanol activates ERK by means of its metabolic conversion into acetaldehyde and strengthen the view that acetaldehyde is a centrally acting compound with a pharmacological profile similar to ethanol.

Alcohol-Induced Interactive Phosphorylation of Src and Toll-like Receptor Regulates the Secretion of Inflammatory Mediators by Human Astrocytes

Secretion of pro-inflammatory molecules by astrocytes after alcohol treatment was shown to be associated with neuroinflammation. We hypothesized that activation of cytosolic phospholipase A2 (cPLA2) and cyclooxygenase (COX-2) by ethanol in astrocytes enhanced the secretion of inflammatory agents via the interactive tyrosine phosphorylation of toll-like receptor 4 (TLR4) and Src kinase. To test this hypothesis, we treated primary human astrocytes with 20mM ethanol for 48h at 37°C. Ethanol exposure elevated cytochrome P450-2E1 activity, reactive oxygen species levels, and secretion of prostaglandin E2 (PGE2) in these cells. Secretion of PGE2 was associated with induction of cPLA2 activity and protein content as well as COX-2 protein level in a Src phosphorylation-dependent manner that occurred by enhanced transcription. Immunoprecipitation and Western blot analyses indicated that the interactive tyrosine phosphorylation of TLR4-Src complex at the cell membrane triggered the activation of cPLA2 and COX-2 in the cytoplasm through a Src signaling intermediate. Inhibition of ethanol metabolism, blockage of Src activity, or inactivation of TLR4 prevented the activation of cPLA2 and COX-2 as well as diminished PGE2 production, suggesting that interactive phosphorylation of TLR4-Src regulated the pro-inflammatory response in astrocytes. Experiments with small interfering RNA knockdown of TLR4 in human astrocytes confirmed that silencing expression also abolished the interactive phosphorylation of both TLR4 and Src in the presence of ethanol.

Acetate, the key modulator of inflammatory responses in acute alcoholic hepatitis

Acute alcoholic hepatitis is characterized by disproportionate macrophage inflammatory cytokine responses to bacterial lipopolysaccharide. Lack of knowledge of the underlying mechanism has limited progress toward effective therapy. We postulated a novel mechanism by which ethanol increases histone acetylation, increasing proinflammatory gene transcription and cytokine synthesis. Cytokine responses to lipopolysaccharide in a human macrophage cell line cultured in 86 mM ethanol, 1 mM acetate, and normal media were measured by multiplex immunoassay. Changes in histone acetylation were determined by immunofluorescence microscopy and chromatin immunoprecipitation on presentation. The effect of ethanol and acetate on acetyl-coenzyme A (acetyl-coA) synthetases, which convert acetate to acetyl-coA, the substrate for histone acetylation, was determined by quantitative reverse-transcription polymerase chain reaction and immunoblotting. Knockdown of acetyl-coA synthetases by short hairpin RNA (shRNA) was used to determine their role in ethanol's enhancement of the inflammatory cytokine response. Ethanol-exposed macrophages developed enhanced interleukin 6 (IL6), IL8, and tumor necrosis factor alpha responses to lipopolysaccharide with time-dependent increases in histone acetylation that could be prevented by inhibition of ethanol metabolism. Chromatin immunoprecipitation confirmed increased histone acetylation at promoter regions of specific cytokine genes. The effect of ethanol was reproduced by incubation with acetate, the principal hepatic metabolite of ethanol, and both ethanol and acetate reduced histone deacetylase activity and up-regulated acetyl-coA synthetases. Knockdown of the acetyl-coA synthetases abrogated the effect of ethanol on cytokine production. Synthesis of metabolically available acetyl-coA from acetate is critical to the increased acetylation of proinflammatory gene histones and consequent enhancement of the inflammatory response in ethanol-exposed macrophages. This mechanism is a potential therapeutic target in acute alcoholic hepatitis.

Ethanol-mediated expression of connective tissue growth factor (CCN2) in mouse pancreatic stellate cells

Activated pancreatic stellate cells (PSC) play a central role in the pathogenesis of pancreatic fibrosis, a common feature of chronic pancreatitis which is often caused by excessive alcohol consumption. In view of the central role of connective tissue growth factor (CCN2) in fibrosis, we investigated the mechanisms by which CCN2 is regulated in PSC following their exposure to ethanol or acetaldehyde. Primary cultures of PSC from Balb/c mice were treated with 0–50 mM ethanol or 0–200 μM acetaldehyde in the presence or absence of 4-methylpyrazole (4MP; an inhibitor of alcohol dehydrogenase), diallyl sulfide (DAS; an inhibitor of cytochrome P4502E1) or anti-oxidant catalase or vitamin D. CCN2 production, assessed by reverse-transcriptase polymerase chain reaction to measure CCN2 mRNA levels or by fluorescence activated cell sorting to assess CCN2 protein, was enhanced in a dose-dependent manner by ethanol or acetaldehyde. In the presence of 4MP, DAS, or the anti-oxidants vitamin D or catalase, there was a substantial decrease in the ability of ethanol to stimulate CCN2 mRNA expression and a concomitant decrease in CCN2-positive PSC. Accumulation of reactive oxygen species in PSC after exposure to ethanol was verified by loading the cells with dichlorofluorescin diacetate and showing that there was a stimulation of its oxidized fluorescent product, the latter of which was diminished in the presence of catalase or vitamin D. These results show the production of acetaldehyde and oxidant stress in mouse PSC are the cause of increased CCN2 mRNA and protein production after exposure of the cells to ethanol. The potential therapeutic effects of inhibitors of ethanol metabolism or anti-oxidants in alcoholic pancreatitis may arise in part through their ability to attenuate CCN2 production by PSC.

An in vitro method of alcoholic liver injury using precision-cut liver slices from rats

Alcohol abuse results in liver injury, but investigations into the mechanism(s) for this injury have been hampered by the lack of appropriate in vitro culture models in which to conduct in depth and specific studies. In order to overcome these shortcomings, we have developed the use of precision-cut liver slices (PCLS) as an in vitro culture model in which to investigate how ethanol causes alcohol-induced liver injury. In these studies, it was shown that the PCLS retained excellent viability as determined by lactate dehydrogenase and adenosine triphosphate (ATP) levels over a 96-h period of incubation. More importantly, the major enzymes of ethanol detoxification; alcohol dehydrogenase, aldehyde dehydrogenase, and cytochrome P4502E1, remained active and PCLS readily metabolized ethanol and produced acetaldehyde. Within 24 h and continuing up to 96 h the PCLS developed fatty livers and demonstrated an increase in the redox state. These PCLS secreted albumin, and albumin secretion was decreased by ethanol treatment. All of these impairments were reversed following the addition of 4-methylpyrazole, which is an inhibitor of ethanol metabolism. Therefore, this model system appears to mimic the ethanol-induced changes in the liver that have been previously reported in human and animal studies, and may be a useful model for the study of alcoholic liver disease.

Acute ethanol exposure disrupts VEGF receptor cell signaling in endothelial cells

Physiological angiogenesis is regulated by various factors, including signaling through vascular endothelial growth factor (VEGF) receptors. We previously reported that a single dose of ethanol (1.4 g/kg), yielding a blood alcohol concentration of 100 mg/dl, significantly impairs angiogenesis in murine wounds, despite adequate levels of VEGF, suggesting direct effects of ethanol on endothelial cell signaling (40). To examine the mechanism by which ethanol influences angiogenesis in wounds, we employed two different in vitro angiogenesis assays to determine whether acute ethanol exposure (100 mg/dl) would have long-lasting effects on VEGF-induced capillary network formation. Ethanol exposure resulted in reduced VEGF-induced cord formation on collagen and reduced capillary network structure on Matrigel in vitro. In addition, ethanol exposure decreased expression of endothelial VEGF receptor-2, as well as VEGF receptor-2 phosphorylation in vitro. Inhibition of ethanol metabolism by 4-methylpyrazole partially abrogated the effect of ethanol on endothelial cell cord formation. However, mice treated with t-butanol, an alcohol not metabolized by alcohol dehydrogenase, exhibited no change in wound vascularity. These results suggest that products of ethanol metabolism are important factors in the development of ethanol-induced changes in endothelial cell responsiveness to VEGF. In vivo, ethanol exposure caused both decreased angiogenesis and increased hypoxia in wounds. Moreover, in vitro experiments demonstrated a direct effect of ethanol on the response to hypoxia in endothelial cells, as ethanol diminished nuclear hypoxia-inducible factor-1alpha protein levels. Together, the data establish that acute ethanol exposure significantly impairs angiogenesis and suggest that this effect is mediated by changes in endothelial cell responsiveness to both VEGF and hypoxia.

Differential Phosphorylation of Translation Initiation Regulators 4EBP1, S6k1, and Erk 1/2 Following Inhibition of Alcohol Metabolism in Mouse Heart

Acute alcohol intoxication leads to an inhibition of protein synthesis in heart that results in part through altered phosphorylation of protein factors controlling mRNA translation initiation. The purpose of the present set of experiments was designed to examine the effects of inhibitors of ethanol metabolism on the phosphorylation of 4E-binding protein (4EBP1) and S6k1(Thr(389)), two factors regulating mRNA translation initiation. Phosphorylation of 4E-BP1, S6k1(Thr(389)), and Erk 1/2 was reduced 2 h following IP injection of alcohol. Pretreatment with 4-methylpyrazole (4-MP), an inhibitor of alcohol dehydrogenase (ADH), did not attenuate the ethanol-induced decrease in phosphorylation of 4EBP1 and S6k1(Thr(389)). In contrast, 4-MP prevented the decrease in Erk 1/2 phosphorylation observed with acute ethanol intoxication. Pretreatment with cyanamide, an inhibitor of aldehyde dehydrogenase, did not attenuate the ethanol-induced decrease in phosphorylation S6k1(Thr(389)), but partially prevented the ethanol-induced lowering of 4EBP1 phosphorylation. The studies indicate that modulation of ethanol metabolism through inhibition of ADH or aldehyde dehydrogenase leads to preferential modulation of the phosphorylation of distinct myocardial signaling systems involved in regulating protein synthesis.

Stabilization of IGFBP-1 mRNA by ethanol in hepatoma cells involves the JNK pathway

Insulin-like growth factor-binding protein-1 (IGFBP-1) modulates cell growth and metabolism in a variety of physiopathological conditions. The aim of this study was to determine the molecular mechanisms involved in IGFBP-1 upregulation by ethanol. We studied IGFBP-1 regulation by ethanol at the protein, mRNA and gene promoter levels in the human hepatocarcinoma cell line, HepG2, which does not express significantly ethanol-metabolizing enzymes. Ethanol (35-150mM) induced the IGFBP-1 mRNA and protein up to 5-fold in a dose-dependent manner. A similar effect was observed using primary cultures of human hepatocytes. Various inhibitors of ethanol metabolism and the antioxidant N-acetylcysteine did not prevent ethanol effects. While ethanol did not modify the IGFBP-1 gene promoter activity, it elicited a 2- to 3-fold increase in IGFBP-1 mRNA half-life and this stabilization required the 5' and the 3' untranslated mRNA region. Ethanol triggered a rapid activation of c-Jun N-terminal Kinase (JNK) in HepG2 cells and IGFBP-1 induction was significantly decreased by a specific inhibitor of JNK. This study reveals a novel pathway of gene regulation by alcohol which involves the activation of JNK and the consequent mRNA stabilization.

Mechanism of superoxide anion production by hepatic sinusoidal endothelial cells and Kupffer cells during short-term ethanol perfusion in the rat.

The aim of this study was to clarify the candidate cells for and the mechanism of superoxide anion (O2*-) release into the hepatic sinusoids during short-term exposure to ethanol. The rat liver was perfused continuously with ethanol (a substrate for alcohol dehydrogenase) or tert-buthanol (not a substrate for alcohol dehydrogenase) for 20 min at a final concentration of 40 mM. In order to detect O2*- production, MCLA (2-methyl-6-[p-methoxyphenyl]-3,7-dihydroimidazo[1,2-a]pyrazin-3-one), a Cypridina luciferin analogue, was simultaneously infused and MCLA-enhanced chemiluminescence was measured. The effects of gadolinium chloride (GdCL3) (a suppressor of Kupffer cells (KCs)), staurosporine (ST) (an inhibitor of serine-threonine kinases, including protein kinase C), diphenyleneiodonium chloride (DPI) (an inhibitor of NADPH oxidase), ibuprofen (IB) (an inhibitor of cyclooxygenase) and 4-methylpyrazole (4MP) (an inhibitor of ethanol metabolism) on the ethanol-induced chemiluminescence were also evaluated. Sites where O2*- could be released were determined by histochemical detection of nitro blue tetrazolium reduction. Both ethanol and tert-buthanol rapidly caused O2*- release. GdCL3 suppressed the ethanol-induced O2*- release by 61%. Staurosporine and DPI, but neither IB nor 4-MP, also significantly inhibited the ethanol-induced O2*- release. In the histochemical examination, ethanol-stimulated liver showed blue formazan precipitate on both sinusoidal endothelial cells (SECs) and Kupffer cells (KCs), whereas the GdCl3-pretreated liver had the precipitate only on SECs. This study shows that ethanol itself stimulates both SECs and KCs to release O2*- via activation of NADPH oxidase probably involving protein kinase C (PKC).

Effects of Alcohol and Oxidative Stress on Liver Pathology: The Role of the Mitochondrion

This article represents the proceedings of a symposium at the 2001 Research Society on Alcoholism meeting in Montreal, Canada. The chairs were Alan Cahill and Carol C. Cunningham. The presentations were (1) Mitochondrial regulation of ethanol‐induced hepatocyte apoptosis: possible involvement of proapoptotic Bcl‐2 family protein Bax, by Masayuki Adachi and Hiromasa Ishii; (2) Effects of ethanol on mitochondrial reactive oxygen species production and oxidative protein modification, by Shannon M. Bailey; (3) Acute ethanol binges elicit widespread oxidative mitochondrial DNA damage and depletion: protective effects of antioxidants and inhibitors of ethanol metabolism, by Bernard Fromenty; and (4) Effects of chronic ethanol consumption upon hepatic mtDNA oxidative modification and depletion, by Alan Cahill and Adrian Davies.

Effects of Alcohol and Oxidative Stress on Liver Pathology: The Role of the Mitochondrion

This article represents the proceedings of a symposium at the 2001 Research Society on Alcoholism meeting in Montreal, Canada. The chairs were Alan Cahill and Carol C. Cunningham. The presentations were (1) Mitochondrial regulation of ethanol-induced hepatocyte apoptosis: possible involvement of proapoptotic Bcl-2 family protein Bax, by Masayuki Adachi and Hiromasa Ishii; (2) Effects of ethanol on mitochondrial reactive oxygen species production and oxidative protein modification, by Shannon M. Bailey; (3) Acute ethanol binges elicit widespread oxidative mitochondrial DNA damage and depletion: protective effects of antioxidants and inhibitors of ethanol metabolism, by Bernard Fromenty; and (4) Effects of chronic ethanol consumption upon hepatic mtDNA oxidative modification and depletion, by Alan Cahill and Adrian Davies.

Metabolic fate of [14C]-ethanol into endothelial cell phospholipids including platelet-activating factor, sphingomyelin and phosphatidylethanol.

The metabolic fate of ethanol into the phospholipid pool of calf pulmonary artery endothelial cells was studied. [14C]-ethanol was incorporated into various endothelial cell phospholipids including phosphatidylethanol (PEth), which may represent a substantial fraction in microdomains of membrane phospholipids. The incorporation into phospholipids was reduced in the presence of pyrazole and cyanamide, inhibitors of ethanol metabolism. Wortmannin, the phosphatidylinositol 3-kinase inhibitor, increased [14C]-PEth formation. [3H]-acetate was also incorporated into endothelial cell phospholipids but in a different pattern. Distribution of [3H]-acetate and [14C]-ethanol into the fatty acyl moiety versus the glycerophosphoryl backbone of the phospholipids was also different. Stimulation of the endothelial cells with ATP increased [3H]-acetate incorporation into platelet-activating factor (PAF) and ethanol decreased it. Ethanol exposure increased ATP-stimulated [3H]-acetate incorporation into sphingomyelin. However, ATP had no effect on the incorporation of [14C]-ethanol into any phospholipids. The results suggest that the two precursors contribute to a separate acetate pool and that the sphingomyelin cycle may be sensitized in ethanol-treated cells. Thus, metabolic conversions of ethanol into lipids and the effect of ethanol on specific lipid mediators, e.g PAF, PEth and sphingomyelin, may be critical determinants in the altered responses of the endothelium in alcoholism.

Ethanol alters angiotensin II stimulated mitogen activated protein kinase in hepatocytes: agonist selectivity and ethanol metabolic independence

Angiotensin II activated mitogen-activated protein kinase (MAPK) (p42 and p44) in rat hepatocytes exposed to ethanol and the relevance of ethanol metabolism on this activation was investigated. Hepatocytes, isolated from rat liver, were treated with or without ethanol for 24 h. Angiotensin II, vasopressin, insulin, serum and epinephrine significantly increased hepatocyte MAPK activity. Platelet activating factor (PAF), tumor necrosis factor-α (TNF-α), and insulin-like growth factor-1 (IGF-1) had little effect on MAPK activation. Interestingly, among the above agonists, which activated hepatocyte MAPK, ethanol exposure potentiated only angiotensin II and epinephrine-stimulated MAPK. Thus, potentiation of MAPK by ethanol exhibited agonist selectivity. In contrast to several other cells, there was prevalence of p42 over p44 MAPK band in hepatocytes. Angiotensin II treatment caused a rapid activation (peak 5 min) of MAPK followed by a decrease to basal levels in 30 min. Exposure with 100 mM ethanol potentiated the angiotensin II stimulated MAPK activity. This potentiation was partially blocked by pertussis toxin suggesting it to be a G-protein-dependent event. Treatment of the hepatocytes with pyrazole (an inhibitor of ethanol metabolism) or acetaldehyde (an ethanol metabolite) had no effect on potentiation. Thus, ethanol potentiation of hepatocyte MAPK is agonist-selective and independent of ethanol metabolism.

An alcoholic binge causes massive degradation of hepatic mitochondrial DNA in mice

Background & Aims: Ethanol causes oxidative stress in the hepatic mitochondria of experimental animals and mitochondrial DNA deletions in alcoholics. We postulated that ethanol intoxication may cause mitochondrial DNA strand breaks. Methods: Effects of an intragastric dose of ethanol (5 g/kg) on hepatic mitochondrial DNA levels, structure, and synthesis were determined by slot blot hybridization, Southern blot hybridization, and in vivo [ 3H]thymidine incorporation, respectively. Results: Two hours after ethanol administration, ethane exhalation (an index of lipid peroxidation) increased by 133%, although hepatic lipids were unchanged. Mitochondrial DNA was depleted by 51%. Its supercoiled form disappeared, whereas linearized forms increased. Long polymerase chain reaction evidenced lesions blocking polymerase progress on the mitochondrial genome. Mitochondrial transcripts decreased. Subsequently, [ 3H]thymidine incorporation into mitochondrial DNA increased, and mitochondrial DNA levels were restored. In contrast, nuclear DNA was not fragmented and its [ 3H]thymidine incorporation was unchanged. Liver ultrastructure only showed inconstant mitochondrial lesions. Ethanol-induced mitochondrial DNA depletion was prevented by 4-methylpyrazole, an inhibitor of ethanol metabolism, and attenuated by melatonin, an antioxidant. Conclusions: After an alcoholic binge, ethanol metabolism causes oxidative stress and hepatic mitochondrial DNA degradation in mice. DNA strand breaks may be involved in the development of mitochondrial DNA deletions in alcoholics. GASTROENTEROLOGY 1999;117:181-190

Inhibition of gap junction intercellular communications of cultured rat hepatocytes by ethanol: role of ethanol metabolism

Background/Aims: In a previous study, we reported that in cultured rat hepatocytes, ethanol inhibits intercellular communication which is known to play a central role in the regulation of cell growth and differentiation. This work was designed to find out if ethanol exerts a direct action on cell membranes, comparable to other long-chain (C6–C9) alcohols, or an indirect action. Methods: Intercellular communication was measured on short-term cultured rat hepatocytes by the fluorescent Lucifer-Yellow CH transfer method. Intracellular pH was measured by spectrofluorimetry and membrane expression of connexin 32 by indirect immunofluorescence. Results: Under our conditions, ethanol (20 mM) inhibited intercellular communication of hepatocytes to the same extent as did octanol at 1 mM. Immunofluorescence semi-quantitative studies of connexin 32 suggested that the observed inhibition was not related to a decrease in the number of gap junction plaques. In contrast with those of octanol, the inhibitory effects of ethanol appeared to be indirect because the inhibition of ethanol metabolism by 4-methyl pyrazole abolished its effects on intercellular communication, while 4-methyl pyrazole did not influence the effects of octanol. Acetaldehyde, the main metabolite of ethanol was without effect on gap junctions. Conclusions: This suggests that the inhibition of intercellular communication induced by ethanol may be included among the consequences of intermediary cell metabolism disturbances indirectly due to ethanol oxidation. This may be one of the mechanisms by which ethanol metabolism exerts a hepatotoxic and possibly carcinogenic action.

Inhibition of ethanol metabolism by fructose in alcohol dehydrogenase-deficient deer mice in vivo

The purpose of this work was to compare the roles of a newly described mitochondrial dehydrogenase and catalase in ethanol elimination in deer mice deficient in alcohol dehydrogenase (ADH −). Fructose was used because of its well-known ability to stimulate dehydrogenase-dependent ethanol metabolism. Rates of ethanol metabolism in vivo were decreased significantly by about 60% in a dose-dependent manner by fructose in deer mice fed an ethanol-containing or a corn oil control diet. In addition, rates of metabolism of methanol, a selective substrate for catalase in rodents, were similar to rates of ethanol elimination and were decreased from 6.9 ± 1.0 to 1.7 ± 0.5 mmol/kg/h by fructose, supporting the hypothesis that catalase and not a mitochondrial dehydrogenase predominates in ethanol oxidation in ADH − deer mice. Glycolate, a substrate for peroxisomal H 2O 2 generation, reversed the inhibition of alcohol metabolism by fructose completely, indicating that fructose did not inhibit catalase directly. As expected, the ATP ADP ratio was decreased by fructose significantly from 4.2 ± 0.4 to 2.4 ± 0.4 in deer mouse livers. These data are consistent with the hypothesis that fructose decreases catalasedependent ethanol metabolism in vivo by inhibiting hepatic H 2O 2 generation.