Nonoxidative Ethanol Metabolism Research Articles

Alcohol Promotes Lipogenesis in Sebocytes-Implications for Acne.

The oral consumption of alcohol (ethanol) has a long tradition in humans and is an integral part of many cultures. The causal relationship between ethanol consumption and numerous diseases is well known. In addition to the well-described harmful effects on the liver and pancreas, there is also evidence that ethanol abuse triggers pathological skin conditions, including acne. In the present study, we addressed this issue by investigating the effect of ethanol on the energy metabolism in human SZ95 sebocytes, with particular focus on qualitative and quantitative lipogenesis. It was found that ethanol is a strong trigger for lipogenesis, with moderate effects on cell proliferation and toxicity. We identified the non-oxidative metabolism of ethanol, which produced fatty acid ethyl esters (FAEEs), as relevant for the lipogenic effect-the oxidative metabolism of ethanol does not contribute to lipogenesis. Correspondingly, using the Seahorse extracellular flux analyzer, we found an inhibition of the mitochondrial oxygen consumption rate as a measure of mitochondrial ATP production by ethanol. The ATP production rate from glycolysis was not affected. These data corroborate that ethanol-induced lipogenesis is independent from oxygen. In sum, our results give a causal explanation for the prevalence of acne in heavy drinkers, confirming that alcoholism should be considered as a systemic disease. Moreover, the identification of key factors driving ethanol-dependent lipogenesis may also be relevant in the treatment of acne vulgaris.

Recent Advances in Understanding the Complexity of Alcohol-Induced Pancreatic Dysfunction and Pancreatitis Development.

Chronic excessive alcohol use is a well-recognized risk factor for pancreatic dysfunction and pancreatitis development. Evidence from in vivo and in vitro studies indicates that the detrimental effects of alcohol on the pancreas are from the direct toxic effects of metabolites and byproducts of ethanol metabolism such as reactive oxygen species. Pancreatic dysfunction and pancreatitis development are now increasingly thought to be multifactorial conditions, where alcohol, genetics, lifestyle, and infectious agents may determine the initiation and course of the disease. In this review, we first highlight the role of nonoxidative ethanol metabolism in the generation and accumulation of fatty acid ethyl esters (FAEEs) that cause multi-organellar dysfunction in the pancreas which ultimately leads to pancreatitis development. Further, we discuss how alcohol-mediated altered autophagy leads to the development of pancreatitis. We also provide insights into how alcohol interactions with other co-morbidities such as smoking or viral infections may negatively affect exocrine and endocrine pancreatic function. Finally, we present potential strategies to ameliorate organellar dysfunction which could attenuate pancreatic dysfunction and pancreatitis severity.

Non-oxidative ethanol metabolism in human hepatic cells in vitro: Involvement of uridine diphospho-glucuronosyltransferase 1A9 in ethylglucuronide production

Ethanol is the most frequently psychoactive substance used in the world, leading to major public health problems with several millions of deaths attributed to alcohol consumption each year. Metabolism of ethanol occurs mainly in the liver via the predominant oxidative metabolism pathway involving phase I enzymes including alcohol dehydrogenases (ADH), cytochrome P450 (CYP) 2E1 and catalase. In a lesser extent, an alternative non-oxidative pathway also contributes to the metabolism of ethanol, which involves the uridine diphospho-glucuronosyltransferase (UGT) and sulfotransferase (SULT) phase II enzymes. Using liquid chromatography-high resolution mass spectrometry, ethylglucuronide (EtG) and ethylsulfate (EtS) produced respectively by UGT and SULT conjugation and detected in various biological samples are direct markers of alcohol consumption. We report herein the efficient non-oxidative metabolic pathway of ethanol in human differentiated HepaRG cells compared to primary human hepatocytes (HH). We showed dose- and time-dependent production of EtS and EtG after ethanol (25 or 50 mM) treatment in culture media of differentiated HepaRG cells and HH and a significant induction of CYP2E1 mRNA expression upon acute ethanol exposure in HepaRG cells. These differentiated hepatoma cells thus represent a suitable in vitro human liver cell model to explore ethanol metabolism and more particularly EtG and EtS production. In addition, using recombinant HepG2 cells expressing different UGT1A genes, we found that UGT1A9 was the major UGT involved in ethanol glucuronidation.

Should phosphatidylethanol be currently analysed using whole blood, dried blood spots or both?

Phosphatidylethanol (PEth) are phospholipids produced through non-oxidative ethanol metabolism. They accumulate in red blood cells and have been traditionally analysed in whole blood as potential biomarkers for moderate to long-term alcohol consumption. More recently, their analysis in dried blood spots has been gaining favour, namely, due to the ease in sampling, transport and storage conditions required. This paper aims at providing a short comparative review between analysing PEth in whole blood and dried blood spots and the potential pitfalls that researchers may face when setting up PEth testing for clinical use.

P-166 - Docosahexaenoic acid inhibits IL-6 and IL-1β expression by suppressing production of mitochondrial reactive oxygen species in alcoholic pancreatitis

High alcohol intake is a major risk factor for severe acute pancreatitis which may lead to multi-organ dysfunction and premature death of the individual. Non-oxidative metabolism of ethanol resulting in the formation of fatty acid ethyl esters (FAEEs) is associated with severe acute pancreatitis. Several lines of evidence indicate that oxidative stress plays an important role in the development of alcoholic pancreatitis. Docosahexaenoic acid (DHA) is a powerful antioxidant that protects cells against oxidative stress in vivo and in vitro. The present study was undertaken to investigate the anti-inflammatory effects and mechanisms of DHA on alcoholic pancreatitis induced by ethanol and palmitoleic acid (POA) in pancreatic acinar AR42J cells. In present study, the treatment of ethanol and POA induced expression of proinflammatory cytokines such as IL-6 and IL-1f3 in AR42J cells. The production of mitochondrial reactive oxygen species (ROS) was induced by ethanol and POA. DHA suppressed mitochondrial ROS production and expression of IL-1f3 and IL-6 in ethanol/POA-stimulated AR42J cells. In conclusion, DHA showed anti-inflammatory effect through inhibition of mitochondrial ROS production in ethanol and POA-stimulated pancreatic acinar cells.

Nonoxidative ethanol metabolism in humans-from biomarkers to bioactive lipids.

Ethanol is a widely used psychoactive drug whose chronic abuse is associated with organ dysfunction and disease. Although the prevalent metabolic fate of ethanol in the human body is oxidation a smaller fraction undergoes nonoxidative metabolism yielding ethyl glucuronide, ethyl sulfate, phosphatidylethanol and fatty acid ethyl esters. Nonoxidative ethanol metabolites persist in tissues and body fluids for much longer than ethanol itself and represent biomarkers for the assessment of ethanol intake in clinical and forensic settings. Of note, the nonoxidative reaction of ethanol with phospholipids and fatty acids yields bioactive compounds that affect cellular signaling pathways and organelle function and may contribute to ethanol toxicity. Thus, despite low quantitative contributions of nonoxidative pathways to overall ethanol metabolism the resultant ethanol metabolites have important biological implications. In this review we summarize the current knowledge about the enzymatic formation of nonoxidative ethanol metabolites in humans and discuss the implications of nonoxidative ethanol metabolites as biomarkers of ethanol intake and mediators of ethanol toxicity. © 2016 IUBMB Life, 68(12):916–923, 2016

Monoacylglycerol Lipases Act as Evolutionarily Conserved Regulators of Non-oxidative Ethanol Metabolism

Fatty acid ethyl esters (FAEEs) are non-oxidative metabolites of ethanol that accumulate in human tissues upon ethanol intake. Although FAEEs are considered as toxic metabolites causing cellular dysfunction and tissue damage, the enzymology of FAEE metabolism remains poorly understood. In this study, we used a biochemical screen in Saccharomyces cerevisiae to identify and characterize putative hydrolases involved in FAEE catabolism. We found that Yju3p, the functional orthologue of mammalian monoacylglycerol lipase (MGL), contributes >90% of cellular FAEE hydrolase activity, and its loss leads to the accumulation of FAEE. Heterologous expression of mammalian MGL in yju3Δ mutants restored cellular FAEE hydrolase activity and FAEE catabolism. Moreover, overexpression or pharmacological inhibition of MGL in mouse AML-12 hepatocytes decreased or increased FAEE levels, respectively. FAEEs were transiently incorporated into lipid droplets (LDs) and both Yju3p and MGL co-localized with these organelles. We conclude that the storage of FAEE in inert LDs and their mobilization by LD-resident FAEE hydrolases facilitate a controlled metabolism of these potentially toxic lipid metabolites.

The role of fat and alcohol in acute pancreatitis: A dangerous liaison.

Excessive alcohol consumption is a major trigger for severe acute pancreatitis which may lead to multi-organ dysfunction and premature death of the individual. Hyperlipidaemia is a risk factor for both acute and chronic pancreatitis and the role of fatty acids in mediating damage has received increasing attention in recent years. In the pancreas ethanol is metabolised by both oxidative and non-oxidative pathways. The latter, predominant route generates fatty acid ethyl esters (FAEEs) from fatty acid substrates via the action of diverse enzymes called FAEE synthases, including carboxylester lipase an enzyme synthesized and secreted by the acinar cells. Inhibition of the oxidative pathway promotes formation of FAEEs which induce sustained elevations of cytosolic calcium leading to inhibition of mitochondrial function, loss of ATP and necrosis of isolated pancreatic acinar cells. Furthermore, FAEEs undergo hydrolysis in the mitochondria releasing free fatty acids that exert toxic effects. Our recent work has shown that pharmacological inhibition of carboxylester lipase ameliorated detrimental effects of non-oxidative ethanol metabolism in isolated pancreatic acinar cells invitro and in a new invivo experimental model of alcoholic acute pancreatitis, revealing a specific enzyme target for ethanol-induced injury. Strategies that prevent FAEE synthesis, protect mitochondria, reduce calcium overload or sustain calcium homeostasis by ATP provision may provide promising therapeutic avenues for the treatment of alcoholic acute pancreatitis.

A comparison of the prevalence of prenatal alcohol exposure obtained via maternal self-reports versus meconium testing: a systematic literature review and meta-analysis.

BackgroundMaternal self-reports, used for the detection of prenatal alcohol exposure (PAE), may lack validity, necessitating the use of an objective biomarker. The detection of fatty acid ethyl esters (products of non-oxidative ethanol metabolism) in meconium has been established as a novel biomarker of PAE. The purpose of the current study was to compare the prevalence of PAE as reported via maternal self-reports with the results of meconium testing, and to quantify the disparity between these two methods.MethodsA systematic literature search for studies reporting on the prevalence of PAE, using maternal self-reports in combination with meconium testing, was conducted using multiple electronic bibliographic databases. Pooled prevalence estimates and 95% confidence intervals (CI) were calculated based on eight studies, using the Mantel-Haenszel method, assuming a random effects model. A random effects meta-regression was performed to test for a difference.ResultsThe pooled prevalence of PAE as measured by meconium testing was 4.26 (95% CI: 1.34-13.57) times the pooled prevalence of PAE as measured by maternal self-reports. Large variations across the studies in regard to the difference between estimates obtained from maternal self-reports and those obtained from meconium testing were observed.ConclusionsIf maternal self-reports are the sole information source upon which health care professionals rely, a number of infants who were prenatally exposed to alcohol are not being recognized as such. However, further research is needed in order to validate existing biomarkers, as well as discover new biomarkers, for the detection of PAE.

Alcohol oxidizing enzymes and ethanol-induced cytotoxicity in rat pancreatic acinar AR42J cells

Alcoholic chronic pancreatitis (ACP) is a serious inflammatory disease causing significant morbidity and mortality. Due to lack of a suitable animal model, the underlying mechanism of ACP is poorly understood. Chronic alcohol abuse inhibits alcohol dehydrogenase (ADH) and facilitates nonoxidative metabolism of ethanol to fatty acid ethyl esters (FAEEs) in the pancreas frequently damaged during chronic ethanol abuse. Earlier, we reported a concentration-dependent formation of FAEEs and cytotoxicity in ethanol-treated rat pancreatic tumor (AR42J) cells, which express high FAEE synthase activity as compared to ADH and cytochrome P450 2E1. Therefore, the present study was undertaken to investigate the role of various ethanol oxidizing enzymes in ethanol-induced pancreatic acinar cell injury. Confluent AR42J cells were pre-treated with inhibitors of ADH class I and II [4-methylpyrazole (MP)] or class I, II, and III [1,10-phenanthroline (PT)], cytochrome P450 2E1 (trans-1,2-dichloroethylene) or catalase (sodium azide) followed by incubation with 800 mg% ethanol at 37°C for 6 h. Ethanol metabolism, cell viability, cytotoxicity (apoptosis and necrosis), cell proliferation status, and formation of FAEEs in AR42J cells were measured. The cell viability and cell proliferation rate were significantly reduced in cells pretreated with 1,10-PT + ethanol followed by those with 4-MP + ethanol. In situ formation of FAEEs was twofold greater in cells incubated with 1,10-PT + ethanol and ∼1.5-fold in those treated with 4-MP + ethanol vs. respective controls. However, cells treated with inhibitors of cytochrome P450 2E1 or catalase in combination of ethanol showed no significant changes either for FAEE formation, cell death or proliferation rate. Therefore, an impaired ADH class I-III catalyzed oxidation of ethanol appears to be a key contributing factor in ethanol-induced pancreatic injury via formation of nonoxidative metabolites of ethanol.

Potential in vitro Protective Effect of Quercetin, Catechin, Caffeic Acid and Phytic Acid against Ethanol-Induced Oxidative Stress in SK-Hep-1 Cells

Phytochemicals have been known to exhibit potent antioxidant activity. This study examined cytoprotective effects of phytochemicals including quercetin, catechin, caffeic acid, and phytic acid against oxidative damage in SK-Hep-1 cells induced by the oxidative and non-oxidative metabolism of ethanol. Exposure of the cells to excess ethanol resulted in a significant increase in cytotoxicity, reactive oxygen species (ROS) production, lipid hydroperoxide (LPO), and antioxidant enzyme activity. Excess ethanol also caused a reduction in mitochondrial membrane potential (MMP) and the quantity of reduced glutathione (GSH). Co-treatment of cells with ethanol and quercetin, catechin, caffeic acid and phytic acid significantly inhibited oxidative ethanol metabolism-induced cytotoxicity by blocking ROS production. When the cells were treated with ethanol after pretreatment of 4-methylpyrazole (4-MP), increased cytotoxicity, ROS production, antioxidant enzyme activity, and loss of MMP were observed. The addition of quercetin, catechin, caffeic acid and phytic acid to these cells showed suppression of non-oxidative ethanol metabolism-induced cytotoxicity, similar to oxidative ethanol metabolism. These results suggest that quercetin, catechin, caffeic acid and phytic acid have protective effects against ethanol metabolism-induced oxidative insult in SK-Hep-1 cells by blocking ROS production and elevating antioxidant potentials.

Determination of maternal-fetal biomarkers of prenatal exposure to ethanol: A review

The deleterious effects exerted by prenatal ethanol exposure include physical, mental, behavioural and/or learning disabilities that are included in the term fetal alcohol spectrum disorder (FASD). Objective assessment of exposure to ethanol at both prenatal and postnatal stages is essential for early prevention and intervention. Since pregnant women tend to underreport alcohol drinking by questionnaires, a number of biological markers have been proposed and evaluated for their capability to highlight gestational drinking behaviour. These biomarkers include classical biomarkers (albeit indirect) of alcohol-induced pathology (mean corpuscular volume (MCV), gamma glutamyltransferase (GGT), aspartate aminotransferase (AST) and alanine aminotransferase (ALT)) acetaldehyde-derived conjugates, and finally derivatives of non-oxidative ethanol metabolism (fatty acid ethyl esters (FAEEs), ethyl glucuronide (EtG), ethyl sulphate (EtS) and phosphaditylethanol (PEth)). Since ethanol itself and acetaldehyde are only measured few hours after ethanol intake in conventional matrices such as blood, urine and sweat, they are only useful to detect recent ethanol exposure. In the past few years, the non-oxidative ethanol metabolites have received increasing attention because of their specificity and in some case wide time-window of detection in non-conventional matrices from the pregnant mother (oral fluid and hair) and fetus-newborn (neonatal hair, meconium, placenta and umbilical cord). This article reviews bioanalytical procedures for the determination of these markers of ethanol consumption during pregnancy and related prenatal exposure. In addition, clinical toxicological applications of these procedures are presented and discussed.

Modulation of Intestinal Epithelial Barrier Function by Fatty Acid Ethyl Esters in a Three Dimensional (3D) Epithelial Cell Culture Model: Role of Nonoxidative Ethanol Metabolism

Modulation of Intestinal Epithelial Barrier Function by Fatty Acid Ethyl Esters in a Three Dimensional (3D) Epithelial Cell Culture Model: Role of Nonoxidative Ethanol Metabolism

Prevalence of Fetal Ethanol Exposure in a Regional Population-Based Sample by Meconium Analysis of Fatty Acid Ethyl Esters

Challenges in identifying children exposed prenatally to ethanol necessitate the development of a biomarker for neonates at risk for fetal alcohol spectrum disorder. Meconium fatty acid ethyl esters (FAEE), products of nonoxidative ethanol metabolism, have been established as a novel biomarker of fetal ethanol exposure. We present the first application of this biomarker to a population-based sample in Canada. Six-hundred eighty-two meconium specimens were anonymously collected in the region of Grey Bruce, Ontario, Canada. Meconium FAEE were extracted by liquid-liquid and solid-phase extraction and analyzed by gas chromatography with flame-ionization detection confirmed by gas chromatography with mass spectrometry. We measured ethyl palmitate (E16:0), ethyl palmitoleate (E16:1), ethyl stearate (E18:0), ethyl oleate (E18:1), ethyl linoleate (E18:2), ethyl linolenate (E18:3), and ethyl arachidonate (E20:4). Seventeen of 682 meconium samples tested positive for significant prenatal ethanol exposure (>2.0 nmol/g). FAEE analysis detected fivefold more ethanol-exposed pregnancies than standard postpartum questionnaires in this population (2.5% versus 0.5%) (P < 0.001). The prevalence of ethanol-exposed pregnancies was consistent with Centers for Disease Control and Prevention estimates of "frequent" prenatal drinking and previously published estimates of fetal alcohol spectrum disorder disease prevalence in the general North American population. The FAEE concentrations of negative (95% confidence interval, 0.38-0.49 nmol/g) versus positive (95% confidence interval, 7.74-151.28 nmol/g) samples were distinct, further demonstrating the specificity of this biomarker in determining significant prenatal ethanol exposure. Meconium FAEE analysis demonstrates a fivefold increase in sensitivity over currently used methods of self-report-based screening in Ontario for the detection of ethanol-exposed pregnancies in a clinical setting.

Zinc inhibits ethanol-induced HepG2 cell apoptosis

Alcohol consumption produces a variety of metabolic alterations in liver cells, associated with ethanol oxidation and with nonoxidative metabolism of ethanol, among others apoptosis of hepatocytes. As zinc is known as a potent antioxidant and an inhibitor of cell apoptosis, the aim of this paper was to investigate whether zinc supplementation could inhibit ethanol-induced HepG2 apoptosis, and whether this inhibition was connected with attenuation of oxidative stress and modulation of FasR/FasL system expression. The results indicated that zinc supplementation significantly inhibited ethanol-induced HepG2 cell apoptosis (measured by cytochrome c release from mitochondria and caspase-3 activation) by attenuation of reactive oxygen species (ROS) production, increase in the cellular level of GSH, inhibition of ethanol-induced sFasR and FasL overexpression and caspase-8 activation. These results indicate that zinc can inhibit ethanol-induced hepatocyte apoptosis by several independent mechanisms, among others by an indirect antioxidative effect and probably by inhibition of caspase-8 and caspase-9 activation.

Metabolic basis of ethanol-induced hepatic and pancreatic injury in hepatic alcohol dehydrogenase deficient deer mice

Alcoholic liver disease (ALD) and alcoholic pancreatitis (AP) are major diseases causing high mortality and morbidity among chronic alcohol abusers. Neutral lipid accumulation (steatosis) is an early stage of ALD or AP and progresses to inflammation and other advanced stages of diseases in a subset of chronic alcohol abusers. However, the mechanisms of alcoholic steatosis leading to ALD and AP are not well understood. Chronic alcohol abuse impairs hepatic alcohol dehydrogenase (ADH, a major enzyme involved in ethanol oxidative metabolism) and facilitates nonoxidative metabolism of ethanol to fatty acid ethyl esters (FAEEs, nonoxidative metabolites of ethanol). These esters are implicated in the pathogenesis of various alcoholic diseases and shown to cause hepatocellular and pancreatitis-like injury. Ethanol exposure is known to increase synthesis of FAEEs by several-fold in the livers and pancreata of rats pretreated with hepatic ADH inhibitor. Therefore, studies were undertaken to evaluate hepatocellular and pancreatic injury in hepatic ADH-deficient (ADH −) deer mice versus ADH-normal (ADH +) deer mice fed ethanol (4% wt/vol) via Lieber-DeCarli liquid diet for 60 days. A significant mortality was found in ethanol-fed ADH − deer mice (11 out of 18) versus ADH + deer mice (1 out of 16); most of the deaths occurred during the first 2 weeks of ethanol exposure. The surviving animals, sacrificed at the end of 60th day, showed distinct changes in hepatic and pancreatic histology and several-fold increases in nonoxidative metabolism of ethanol in ethanol-fed ADH − versus ADH + deer mice. Extensive vacuolization with displacement or absence of nucleus in some hepatocytes, and significant increase in hepatic neutral lipids were found in ethanol-fed ADH − versus ADH + deer mice. Ultrastructural changes showed perinuclear space, edema, presence of apoptotic bodies and disintegration, and/or dilatation of endoplasmic reticulum (ER) in the pancreata of ethanol-fed ADH − deer mice. FAEE levels were significantly higher in ADH − versus ADH + deer mice, approximately four-fold increases in the livers and seven-fold increases in the pancreata. Ethyl esters of oleic, linoleic, and arachidonic acids were the major FAEEs detected in ethanol-fed groups. The role of FAEEs in pancreatic lysosomal fragility is reflected by higher activity of cathepsin B (five-fold) in ethanol-fed ADH − versus ADH + deer mice. Although the present studies clearly indicate a metabolic basis of ethanol-induced hepatic and pancreatic injury, detailed dose- and time-dependent toxicity studies in this ADH − deer mouse model could reveal further a better understanding of mechanism(s) of ethanol-induced hepatic and pancreatic injuries.

Increased Plasma Fatty Acid Ethyl Ester Levels Following Inhibition of Oxidative Metabolism of Ethanol by 4‐Methylpyrazole Treatment in Human Subjects

Recent experimental evidence suggests that fatty acid ethyl esters (FAEE), nonoxidative metabolites of ethanol, mediate ethanol-induced organ damage. A direct association between pancreas-specific toxicity and increased levels of FAEE following inhibition of the oxidative metabolism of ethanol by 4-methylpyrazole (4-MP) has previously been shown in studies with rats. We obtained plasma samples from 32 healthy human volunteers who drank ethanol following 4-MP or placebo ingestion to determine whether in vivo inhibition of oxidative metabolism of ethanol causes a shift to nonoxidative metabolism of ethanol and the subsequent production of increased levels of FAEE. Plasma FAEE were isolated by solid-phase extraction and quantified by gas chromatography-mass spectrometry (GC-MS). Plasma FAEE levels in subjects receiving 4-MP treatment before ethanol consumption were elevated compared with plasma FAEE concentrations taken from control subjects who received a placebo before ethanol ingestion. Increased FAEE levels in the 4-MP treatment group occurred after peak blood ethanol, and peak FAEE levels were achieved. There was a correlation between the blood ethanol and the plasma FAEE levels, and the correlation persisted in the presence or absence of 4-MP. The peak FAEE values were greater in men than in women, with or without 4-MP treatment. Our results indicate that the in vivo inhibition of the oxidative metabolism of ethanol using 4-MP results in an increased circulating concentration of FAEE, products of the nonoxidative metabolism of ethanol.

Metabolic basis of ethanol-induced cytotoxicity in recombinant HepG2 cells: Role of nonoxidative metabolism

Chronic alcohol abuse, a major health problem, causes liver and pancreatic diseases and is known to impair hepatic alcohol dehydrogenase (ADH). Hepatic ADH-catalyzed oxidation of ethanol is a major pathway for the ethanol disposition in the body. Hepatic microsomal cytochrome P450 (CYP2E1), induced in chronic alcohol abuse, is also reported to oxidize ethanol. However, impaired hepatic ADH activity in a rat model is known to facilitate a nonoxidative metabolism resulting in formation of nonoxidative metabolites of ethanol such as fatty acid ethyl esters (FAEEs) via a nonoxidative pathway catalyzed by FAEE synthase. Therefore, the metabolic basis of ethanol-induced cytotoxicity was determined in HepG2 cells and recombinant HepG2 cells transfected with ADH (VA-13), CYP2E1 (E47) or ADH + CYP2E1 (VL-17A). Western blot analysis shows ADH deficiency in HepG2 and E47 cells, compared to ADH-overexpressed VA-13 and VL-17A cells. Attached HepG2 cells and the recombinant cells were incubated with ethanol, and nonoxidative metabolism of ethanol was determined by measuring the formation of FAEEs. Significantly higher levels of FAEEs were synthesized in HepG2 and E47 cells than in VA-13 and VL-17A cells at all concentrations of ethanol (100-800 mg%) incubated for 6 h (optimal time for the synthesis of FAEEs) in cell culture. These results suggest that ADH-catalyzed oxidative metabolism of ethanol is the major mechanism of its disposition, regardless of CYP2E1 overexpression. On the other hand, diminished ADH activity facilitates nonoxidative metabolism of ethanol to FAEEs as found in E47 cells, regardless of CYP2E1 overexpression. Therefore, CYP2E1-mediated oxidation of ethanol could be a minor mechanism of ethanol disposition. Further studies conducted only in HepG2 and VA-13 cells showed lower ethanol disposition and ATP concentration and higher accumulation of neutral lipids and cytotoxicity (apoptosis) in HepG2 cells than in VA-13 cells. The apoptosis observed in HepG2 vs. VA-13 cells incubated with ethanol appears to be mediated by release of mitochondrial cytochrome c via activation of caspase-9 and caspase-3. These results strongly support our hypothesis that diminished hepatic ADH activity facilitates nonoxidative metabolism of ethanol and the products of ethanol nonoxidative metabolism cause apoptosis in HepG2 cells via intrinsic pathway.

Fatty acid ethyl esters: markers of alcohol abuse and alcoholism

Chronic alcoholism, which is associated with hepatic, pancreatic, and myocardial diseases, is one of the major health problems in the United States with high morbidity and mortality. Many individuals who abuse alcohol chronically die even before reaching the clinical stage of the disease. Reliable biomarkers of the diseases induced by chronic alcohol abuse, as well as for alcoholism, currently are not available. In the current study, we measured plasma concentrations of fatty acid ethyl esters [(FAEEs), nonoxidative metabolites of ethanol] in 39 patients with a detectable concentration of alcohol in their blood samples. In turn, we determined the relation of FAEE concentrations with blood alcohol concentration (BAC). Of 39 patients in whom we evaluated this relation, only five had a history of chronic alcohol abuse, and six had a history of acute alcohol abuse. Patients' age ranged from 25 to 71 years. Within this age range, greater concentrations of FAEEs were found in the plasma samples obtained from patients in the 41- to 50-year age group. There were no sex-related differences in BAC, nor in FAEE concentrations. Thirteen patients had a BAC greater than 300 mg%. For 11 patients, the BAC ranged between 200 and 299 mg%, and, for 12 patients, the BAC ranged between 100 and 199 mg%. In comparison with findings for patients with a BAC that ranged between 100 and 299 mg%, the FAEE concentrations were approximately twofold higher in patients with a BAC greater than 300 mg%. Ethyl palmitate and ethyl oleate were the main FAEEs detected in most patients. In general, FAEE concentrations increased with increasing BAC. However, in comparison with patients with a history of acute alcohol abuse, a greater increase in total FAEE concentrations was observed in patients with a history of chronic alcohol abuse (4,250 ng/ml and 15,086 ng/ml, respectively). Fatty acid ethyl esters were either detected in trace amounts or not detectable in the plasma of control subjects with no known alcohol ingestion. These results support our hypothesis that nonoxidative metabolism of ethanol to FAEEs is an important pathway of ethanol disposition during chronic alcohol abuse, and that FAEE concentrations can be a more reliable biomarker of chronic alcohol abuse than a history of acute alcohol abuse.

Phosphatidylethanol in post-mortem blood as a marker of previous heavy drinking.

Phosphatidylethanol (PEth) is an ethanol-phospholipid adduct, formed via non-oxidative metabolism of ethanol. PEth was measured in femoral blood from 85 consecutive forensic autopsies and was detected in 35 of the cases at concentrations ranging from 0.8 to 22.0 micromol/l. Of the PEth positive cases, 12 did not have significant levels of ethanol in the blood. Two cases (both suicides involving hanging) had detectable ethanol, but no PEth present in the blood. We conclude that measurements of PEth provide indications of previous alcohol abuse in cases where this may not otherwise be evident.