Causes of a Hangover

=Mechanisms of Hangover=

Twenty percent of ethanol is absorbed in the stomach and 80% in the upper small intestine.

Peak blood ethanol concentrations usually occur within 30-90 minutes of ingestion.

When ethanol, an amphipathic molecule, enters the body, it distributes itself in total body water at equilibrium, and its extracellular and intracellular concentrations are approximately equal (1).

Accord- ingly, breath alcohol concentration (BrAC) more accu- rately reflects the level of CNS exposure than similar mea- surements of venous blood during the early (ascending and immediately post-peak) portions of the blood alcohol con- centration (BAC) curve. These conditions may prevail for as long as 2 hr after ethanol c o n ~ u m p t i o

After the oral administration of ethanol and before equilibration among all tissue and extracellular compartments is reached, the concentrations of ethanol in the brain and arterial blood are substantially higher than those in muscle and peripheral veins.

At equilib- rium, extracellular and intracellular (adjusted for water content) concentrations are approximately equal

Unchanged ethanol is eliminated in small quantities (approximately 5% of orally absorbed ethanol) by the kidneys (0.5–2%), lungs (1.6–6%) and skin (<0.5%) [4].

During ethanol elim- ination, concentrations of ethanol in the liver are in the millimolar range, whereas acetaldehyde concentrations are micromolar.

BAC reached undetectable levels within 3 to 3.5 hr after a dose of 0.3 g/kg, given before or after a meal.

The variance in peak BAC and elimination rate within monozygotic twin pairs was significantly less than that within dizygotic twin pairs or that among all twin pairs. From these data, they calculated that 62% of the variability in peak BAC and 49% of the variability in elimination rate are genetically determined. Twelve percent of the variance in peak BAC was due to differences in prior drinking experience, and the combined contributions of age, weight, adiposity, and lung volume were <lo%.

was 55 mg – dl-’ – hr-’, compared with 34 mg dl-’ – hr-’ when ethanol was given after the meal (the AUC value provides an indication of level of exposure over time).

In the first study, within-subject coefficient of variation for peak BAC (Cm,) and area under the blood alcohol curve (AUC) was similar to between-subject variation both in the fasted (21 to 24%) and the fed (34 to 44%) states

In the second study, ethanol doses of 0.15, 0.3, and 0.6 gikg were given 1 hr after a meal. Mean C,,, values of 13, 34, and 87 mg% were attained, but the ranges of interin- dividual variation were considerable: about 5 to 22, 14 to 62, and 50 to 115 mg%, respectively.

In a recent study of 150 subjects,60 it was observed that ethanol elimination from blood varied as much as 4-fold (8 to 36 mg/dl/hr). Elimi- nation rates in moderate drinkers were lower (8 to 25 mg/dl/hr) than in heavy drinkers. In alcoholics, rates of 36 to 40 mg/dl/hr were observed.

Kopun and Propping40 had reported in their study that the coefficient of variation for repeated measures on single subjects was 10% or less, but the range for all 80 subjects varied approximately 3-fold from 58 to 148 mg/kg/hr.


Therefore, endogenous ethanol may be produced in the gastrointestinal tract and blood, especially in patients with high carbohydrate intake and under cimetidine therapy, which allows bacterial overgrowth by increasing gastric pH [106-108].

Indeed, it is clear that the elderly exhibit higher blood ethanol concentrations, and ethanol metabolism is reduced with age due to lower activities of ADH, CYP2E1 and ALDH [111, 112].

When subjective responses to ethanol were measured in some studies, the pleasant effects of ethanol were more pronounced in males than femaleslgl

Food, specifically carbohydrates (including glucose and fructose), can increase ethanol metabolism as much as 80%, on the average.76 However, a great deal of individual variation was observed, ranging from a 13% decrease to a 300% increase in the 10 subjects given fructose.77

Eating a meal also increases the rate of ethanol elimination.7

Data demonstrate that peak systemic ethanol concentrations are likely to be <10 mg% when the drink is taken immediately after a meal high in carbohydrate and/or fat.

It was found that the high carbohydrate meal had the largest effect, followed by the high fat meal and the high protein meal. Compared with ethanol given in the fasted state, carbohydrate decreased overall alcohol absorption by 96%, compared with 90% for fat and 75% for protein. Peak BACs were: fasting, 18 mg%; protein, 8 mg%; fat, 3 mg%; and carbohydrate, <2 mg%.

Sedman et al.73s tudied the relative effects of carbohydrate, fat, and protein as liquid meals, when a 0.46 g/kg dose of ethanol was given orally. It was found that the simultaneous administration of ethanol and the liquid meals retarded ethanol absorption in the order of fat > carbohydrate > protein.

For the 0.15, 0.3, and 0.6 gikg doses, the mean AUCs were 10, 53, and 255 mg * dl-‘ * hr-‘, respectively.

Peak BACs and AUCs were higher with beer than with whiskey in the prandial and postprandial state, whereas the opposite was true in the preprandial state. Wine and sherry yielded peak BACs intermediate between those for beer and whiskey

The reverse rela- tionship when food is in the stomach may be due to the greater ability to retain a smaller liquid volume (i.e., whis- key rather than beer).

It is also possible that the slowing of gastric emptying by food results in increased retention of the ingested ethanol in the stomach, thereby increasing the contribution of the ADH in this organ to first-pass metabolism.

It is notable that, at the lower doses, ethanol bioavailability, as measured by the AUC, was disproportionately low, owing to the dispropor- tionately large contribution of first-pass metabolism.

The mean C,, (peak BAC) when 0.3 g of ethanolkg was given before the meal was 40 mg%, compared with 21 mg% when given after the meal.

Body water content also changes with the menstrual cycle, edema, ascites and re- tained fluid in the gastrointestinal tract.

It has been demonstrated that humans, as well as other ani— mals, can develop tolerance to the effects of ethanol during the time that a single dose of ethanol is present in the body (“acute tolerance”).

Modern genetic studies have made it clear, for example, that the offspring of alcoholic biological parents may be significantly more resistant to the acute intoxicating effects of alcohol than the offspring of nonal- coholic parents, even when the two groups of offspring have had similar experience with

In the gastric mucosa (and not in the small intestine), 5-10% of ethanol is metabolized by alcohol dehydrogenase isoform (or isozyme) 7 (ADH7) and this is called gastric first pass metabolism of ethanol [3, 4].

ethanol is primarily bioactivated (92-95%) by cytosolic (especially hepatic) ADH1B into acetaldehyde,

High levels of class I ADH isozymes are present in the liver (especially in the centrolobular area) and adrenals, with lower levels in the kidneys, lungs, blood vessels (in the case of ADH1B), gastric mucosa (in the case of ADH1C), and other tissues, but not in the brain or placenta.

Indeed, ADH1B2 (formerly known as ADH22) is known as “atypical” ADH, and is responsible for the unusually rapid metabolism (40 times more active than the enzyme encoded by the ADH1B1 allele) of ethanol to acetaldehyde in up to 90% of the East Asian population, whereas only 10% of Caucasians express this allele.

Indeed, since individuals with the ADH1B2 allele produce relevant amounts of acetaldehyde and experience its toxic effects (e.g., flushing, tachycardia, diaphoresis, nausea and vomiting), they tend to avoid ethanol consumption.

Indeed, among the human ADH forms, ADH7 has the highest activity toward ethanol [14].

[ADH7] is the main ADH expressed in human gastric mucosa (also ADH1C and ADH3) and other cells in the upper gastrointestinal tract (e.g., esophagus, pharynx, gingiva, mouth, and tongue), and in the cornea [14].

In contrast to the other ADHs, ADH7 is not expressed in the adult human liver [9].

[ADH7s] contribution to ethanol metabolism is no more than 5-10% in vivo but may be significantly influenced by a number of factors [15] (Table 2).

Besides producing endogenous ethanol, microbial ADHs also metabolize ingested ethanol to acetaldehyde in the oral cavity, stomach and large intestine.

The ADH reaction is the rate-limiting step of ethanol metabolism, but the rate of this reaction is inhib- ited by elevated concentrations of acetaldehyde and NADH.4

Finally, it is important to mention that acetaldehyde may be present in alcoholic drinks (especially spirits) as a result of the action of yeast and bacteria, and also autooxidation [52].

The equilibrium of the ADH reaction at pH 7.3 favors the reduction of aldehyde to alcohol.

For ethanol oxidation to occur, the steady-state concentration of acetaldehyde and of the reduced coenzyme NADH must be kept low.

====MEOS (CYP2E1)====
At high blood ethanol levels, the microsomal ethanol-oxidizing system (CYP2E1 isoform, located in the smooth endoplasmic reticulum) also has an important role.

CYP2E1 utilizes the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor, and also requires O2, which is reduced to water during catalysis.

The Km of CYP2E1 for ethanol is 10 mM, which is roughly one order of magnitude higher than that of ADH1.

Numerous genetic polymorphisms in the CYP2E1 gene have been identified but none gives rise to a poor- or ultra-metabolizer phenotype [26].

From [CYP2E1] activity, ROS are produced even in the absence of substrate, similarly to CYP1A2 (the other high-spin enzyme) [30-32].

CYP2E1 also metabolizes a large number of volatile halogenated alkanes (e.g., chloroform, carbon tetrachloride, vinyl chloride, and many more), halogenated anesthetics (e.g., halothane, enflurane, and others), small aromatic and nitrogen aromatic compounds (e.g., benzene, styrene, toluene, pyridine, pyrazole, and others), alkanes (ethane-hexane), other alcohols besides ethanol (e.g., primary aliphatic alcohols such as methanol and propanol, secondary alcohols such as isopropanol and tertiary alcohols such as t-butanol), cocaine, the colon carcinogen azoxymethane, and numerous mutagenic/carcinogenic nitrosamines [26].

CYP2E1 is expressed in the liver (i.e., the smooth endoplasmic reticulum of the centrolobular area) and several extrahepatic tissues, such as the kidneys, pancreas, brain, lungs, intestine, nasal epithelium, bone marrow, and lymphocytes.

The CYP2E1-catalyzed metabolism of ethanol produces an unstable intermediate (i.e., a gem-diol) that decomposes to acetaldehyde.

CYP2E1 indirectly catalyzes the formation of a radical species from ethanol itself (i.e., 1 (or )-hydroxyethyl radical – CH3HOH), which also contributes to oxidative damage [36].

a) NADPH-oxidase activity of CYP2E1 is responsible for the formation of O2.-and H2O2. The interaction of transition metals (e.g., Fe2+ and Cu+) with H2O2 produces HO. that, by reacting (scavenging) with ethanol, may give rise to less reactive hydroxyethyl free radicals;

Males exhibit higher activity of CYP2E1 and this isoenzyme is induced by fasting and prolonged starvation [39-41].

We reported a significant induction of cytochrome P450-2E1 activity, which was associated with increased levels of reactive oxygen species (ROS) and nitric oxide (NO) production in human neurons after exposure to 17.5 mM ethanol (EtOH) [17].

A major function of P450-catalyzed reactions is to convert a compound into a more polar metabolite that can be easily excreted directly by the organism or conjugated by phase II enzymes into more polar excretable metabolites.

For P450s to function catalytically, flavoprotein reductases such as NADPHcytochrome P450 reductase, adrenodoxin, and adrenodoxin reductase, are necessary to transfer electrons from NADPH or NADH to reduce the heme from the ferric redox state to the ferrous state.

It is important to recognize that oxygen activation by P450, necessary for the enzyme’s catalytic function, can also result in the production of reactive oxygen species (ROS).

Small amounts of the superoxide anion radical (O2U−) can be produced from decay of the oxygenated P450 complex, whereas hydrogen peroxide (H2O2) can form from either dismutation of O2U− or decay of the peroxy P450 complex [8–10].

macrophages and neutrophils contain an NADPH oxidase which produces ROS to destroy foreign organisms

In addition to these in vivo studies, in vitro studies with hepatocytes also showed that ethanol can produce oxidative stress and hepatocyte toxicity.

The Km for ethanol oxidation by MEOS (about 10 mM) was about an order of magnitude greater than the Km for ethanol by alcohol dehydrogenase.

Acetaldehyde is the product resulting from ethanol oxidation by MEOS, and it is clear that MEOS represents a minor pathway of ethanol oxidation, probably accounting for less than 10% of the liver capacity to oxidize ethanol [41].

Ethanol-inducible P450s have been isolated from many species and although several P450s may be induced by ethanol, the major inducible P450 is now referred to as CYP2E1.

Procarcinogens including nitrosamines and azo compounds are effective substrates for CYP2E1,

CYP2E1, relative to several other P450 enzymes, displays high NADPH oxidase activity, as it seems to be poorly coupled with NADPH-cytochrome P450 reductase [51,52].

Interestingly, acetaldehyde is also a substrate for CYP2E1 and is oxidized to acetate; thus CYP2E1 can, at least theoretically, catalyze the oxidation of ethanol to acetate [57]. However, this oxidation is likely to be negligible in the presence of ethanol, the substrate which generates acetaldehyde [58].

Detection of the 1-hydroxyethyl radical in the bile after administration of ethanol to rodents has been a most valuable assay for determining ethanol-induced radical formation and oxidant stress in vivo [26,59].

CYP2E1 is expressed mainly in the hepatocytes of the liver; however, significant amounts are also found in the Kupffer cells [61] and hepatocyte and Kupffer cell CYP2E1 is inducible, e.g., by ethanol.

CYP2E1 is mainly found in the liver but significant amounts are also found in most organs, including the brain [60].

CYP2E1, like other xenobiotic-metabolizing P450s, is mainly located in the membrane of the endoplasmic reticulum (ER).

CYP2E1 has also been detected in other cellular compartments such as the plasma membrane [62–64].

CYP2E1 was shown to be transported out of the ER to the Golgi apparatus, with subsequent transfer to the plasma membrane [68,69].

Ingelman-Sundberg and co-workers, and Avadhani and coworkers, have shown that CYP2E1 is also present in the mitochondria [70–75].

It is not clear what regulates either the phosphorylation or the aminoterminal truncation which directs CYP2E1 to the mitochondria.

The mitochondrial CYP2E1 is catalytically active with typical substrates but requires, as do the other mitochondrial P450s, adrenodoxin and adrenodoxin reductase (and NADPH) as electron donors [70,73].

The mitochondrial CYP2E1 was present at about 30% of the level of the microsomal CYP2E1 under basal conditions

Ethanol can be oxidized by other P450s in addition to CYP2E1, notably CYPs 3A and 1A, and ethanol treatment can elevate the levels of these CYPs [79,80].

Catalase is of minor importance (responsible for approximately 5%) in the metabolism of ethanol.

Thus, the capacity of [catalase] pathway is limited due to the low levels of H2O2 and, like CYP2E1, is also associated with the generation of ROS.

Nevertheless, under physiological conditions, catalase seems to play an insignificant role [46]. Indeed, it is estimated that <5% of an ethanol dose is metabolized through this pathway [4, 46].

These results suggest that the source of ROS may be associated with XOX activation and mitochondrial leakage.

Besides CY2E1 and ALDH, two metalloflavoproteins, xanthine oxidase and aldehyde oxidase, have also proved to oxidize acetaldehyde present in tissues [61, 62].

The oxidation of ethanol by ADH and ALDH leads to the formation of acetic acid. Elevated acetate blood levels have been proposed as an indicator of alcoholism [53].

Acetaldehyde is subsequently detoxified to acetate, mainly by the mitochondrial enzyme aldehyde dehydrogenase (ALDH2) [5].

ALDHs (especially ALDH2 but also ALDH1B1 and ALDH1A) oxidize acetaldehyde to acetic acid [1, 49]. This pathway generally uses NAD+ as the cofactor that is reduced to NADH

ALDH enzymes are involved in the oxidation of other aldehydes, such as those formed from allyl alcohol, carbon tetrachloride, cyclophosphamide, and ifosfamide [46].

The ALDH reaction is essentially irreversible in the direc- tion of acetate formation, and the high catalytic efficiency of ALDH2 and a high rate of NADH reoxidation to NADC by mitochondria enable the metabolism of ethanol to pro- ceed at reasonable rates in the liver.

We found no significant conversion of ethanol into acetaldehyde in brain.

This implies that NO generated by neurons diffuses into glial cells and reacts with superoxide to form highly reactive peroxynitrite.

Ninety percent of ethanol is metabolized in the liver after multiple passages but the lungs (especially via CYP2E1) also contribute to metabolism.

However, this oxidative metabolism of ethanol cannot account for cellular damage in organs such as brain, heart or pancreas, as this metabolic pathway is minimal or absent (1) and these tissues are free of substantial acetaldehyde production (2).

These findings indicate that chronic alcohol ingestion preferentially modulates iNOS protein level in neurons but not in astrocytes or microglia, validating our recent findings that EtOH/Ach exposure increased the level of iNOS protein in cultured primary human neurons [17].

The significance of the present findings is that chronic alcohol feeding activated ROS- and RNS-generating pathways in specific brain cell types.

In one study where 14C-labeled ethanol was used to observe ethanol metabolism in monkeys (44), Mushahwar and colleagues found that some of the glutamate and glutammine in the brain tissue was originated from the 14C-labeled ethanol (14). Because they did not find 14C-labeled acetaldehyde or acetate, their results cannot be taken as evidence that ethanol is broken down directly in the brain.

We found that 13C labels from [1-13C]ethanol are readily incorporated into glutamate, glutamine and aspartate in brain, suggesting that ethanol and/or its metabolites (e.g., acetaldehyde, acetate) are metabolized by brain tissues (see Fig 2).

However, neither AcHC1 (207.0 ppm) nor [1-13C]acetate (182.6 ppm) metabolized from [1-13C]ethanol were detected in the brain 13C MRS spectra.

This similarity can be explained by the conversion of ethanol into acetate in the liver and the subsequent recirculation of acetate into the brain.

Using results obtained from isolated, perfused rat brain, Mukherji and colleagues concluded that ethanol itself cannot be metabolized by brain directly, although they simultaneously found that acetaldehyde can be broken down by the brain (2).

It has also been suggested that acetaldehyde produced in the brain (where ADH is inactive) due to metabolism of ethanol by catalase plays a role in the development of tolerance and in the positive reinforcing actions of ethanol [48].

ALDH2 is expressed at the highest levels in the liver, although it can also be found in other tissues such as kidney, skeletal, cardiac muscle, and mammary tissues.

ALDH1B1 (mitochondrial) and 1A1 (cytosolic) presented higher Km than ALDH2.

Acetaldehyde itself does not seem to be able to penetrate the blood-brain barrier (BBB) (6,7); furthermore, blood levels of acetaldehyde are kept extremely low by the powerful hepatic enzyme systems (6), and even when blood acetaldehyde concentrations are high, acetaldehyde can be converted into acetate by enzymes located on the BBB (e.g. aldehyde dehydrogenase (ALDH)) (8,9).

in the gastrointestinal tract, where high levels of acetaldehyde can be generated as a result of ethanol metabolism by microorganisms [20].

These findings indicate that ethanol metabolism and subsequent reactive oxidant production by both NADPH oxidase (NOX) and inducible nitric oxide synthase (iNOS) significantly contribute to oxidative stress in the central nervous system (CNS).

We conclude that iNOS induction led to accumulation of NO and peroxynitrite formation thereby promoting the transduction of oxidative stress and neuronal degeneration during chronic ethanol consumption.

Recently, we demonstrated that generation of these oxidative products associated with alcohol metabolism is due to the activation of ROS/ RNS producing enzymes by Ach, the major metabolite of EtOH [1, 3, 4].

Porasuphatana et al. [44] demonstrated that, in the presence of l-arginine, NOS1 (neuronal) and NOS2 (inducible) can metabolize ethanol to a 1hydroxyethyl radical.

H2O2 in the presence of transition metals (e.g., Fe2+ and Cu+) can lead to the formation of HO., radicals that are responsible for attacking another molecule of acethaldehyde giving rise to methyl carbonyl radical (CH3=O) [64, 65].

Less than 2% of the ethanol ingested is metabolized through [EtG and EtS] nonoxidative route [67].

b) Direct one-electron oxidation of ethanol by O2.- at the catalytic site of the enzyme may also account for the formation of ethanol-derived radical species as a result of interaction of ethanol with the ferric cytochrome P450-oxygen complex (CYP2E1Fe3+O2-).

Authors have also observed that once [1-hydroxyethyl radical] is formed, it is metabolized to acetaldehyde.

EtG and EtS are direct, non-volatile metabolites of ethanol with relevant clinical and forensic applications [66].

More recently, Deng et al. [102] also proved the formation of EtN in vivo after ethanol administration.

Authors hypothesized that EtP may originate from ethanolysis of endogenous phosphate esters.

The same is true for EtN, which was found in vivo when ethanol and tobacco were consumed together [99, 100].

FAEEs are also minor lipophilic nonoxidative products of ethanol metabolism [87].

Triglycerides, lipoproteins and phospholipids (i.e., myristate, palmitate, oleate and stearate) have also been reported to contribute to FAEEs formation [68].

Based on these results, authors suggested that EtN is formed as a consequence of ethanol nitrosation by NO, most likely by peroxynitrite (ONOO), a potent oxidizing formed by the reaction of NO and O2.- [101]

When present, ethanol acts as a co-substrate in the transphosphatidylation reaction catalyzed by the enzyme phospholipase D (PLD), which inserts ethanol in place of choline in phosphatidylcholine [78-80].

PLD has a high Km for ethanol, and therefore the reaction would be most important predominantly in high circulating ethanol concentrations.

PEth is not a single molecule but a group of abnormal phospholipid homologues. It is formed by a glycerol molecule with two fatty acid chains (typically containing 14 to 22 carbon atoms with 0 to 6 double bonds) in the sn-1 and sn-2 position and with phosphoethanol as the head group [82].

At least 48 homologues of PEth have been identified in blood [82]. The structure of the most common PEth homologue is 16:0/18:1 (1-palmitoyl-2-oleosyl-snglycero-3-phosphoethanol).

Compared with its formation, the elimination half-life of PEth from human blood (mainly in erythrocytes) is slow (approximately 4 days) [7, 83].

Moreover, PEth concentration is highly correlated with the amount of ingested ethanol [83].

One of the most prominent feature of FAEE formation is that these compounds may accumulate in concentrations as high as 100 µM in the myocardium, and that their half-life is approximately 16 hours; thus, they may persist at potentially toxic levels, long after ethanol has been fully cleared from the blood (7).

Thus, the esterification of free fatty acids in response to an ethanol load may represent part of a toxic cycle (9).

FAEE are largely associated with mitochondrial membranes, where a lipase exists that cleaves FAEE to free fatty acids, which are also known uncouplers of oxidative phosphorylation;

Fatty acid ethyl ester synthase (FAEES) activity has been recognized in different organs, such as human heart, brain, pancreas, and liver and has been demonstrated to be a glutathione (GSH) transferase (5).


Thus, while acetate is generally not considered a toxic compound, it can itself have important effects on the body, including increased portal blood flow in the liver and central nervous system depression, and seems to play a role in hangovers [58, 59].

Nevertheless, most acetate enters the systemic circulation and is then taken by several tissues such as heart, skeletal muscle, and brain, which have a high concentration of mitochondrial acetylCoA synthetase isoform (ACS II) [55].

The principal isoform of acetyl-CoA synthetase (ACS I) in the liver is a cytosolic enzyme that generates acetyl-coenzyme A, which is used for fatty acid and cholesterol synthesis

CoA is then rapidly oxidized to carbon dioxide and water in the Krebs cycle, or is used as a substrate for protein acetylation [56]. AMP is also produced from acetyl-CoA synthetase activity and is further hydrolyzed by 5’nucleotidase to adenosine, which is a powerful physiological vasodilator [57].

However, acetate in the liver is oxidized at a much lower rate than the oxidation of ethanol itself (28).

Thus, after the administration of ethanol, the main role of the brain is simply to use the ethanol product acetate originated from extracerebral tissues.

Most of the acetate produced leaves the liver, since the need to oxidize the NADH generated by ADH and ALDH uses 70% to 75% of the oxidative capacity of the organ [54].

The CAMP generating signal transduction system may be altered in its activity by moderate levels of ethanol and may participate in certain acute actions of ethanol, but it may also participate in the neuroadaptive consequences (tolerance) of ethanol intake.

Elevated levels of FAEEs are present in blood soon after ethanol exposure and may remain detectable for 24 hours to four days (especially in heavy drinkers) due to its accumulation in several organs and consequent long half-life (approximately 16 hours) [7, 88, 89].

It has been shown that FAEEs stimulates the Ca2+release from the endoplasmic reticulum, possibly by activating IP3 receptors and fatty acids (released from the deesterification of FAEEs by intracellular hydrolases) predominantly inhibit mitochondrial ATP synthesis, which would inhibit both Ca2+ uptake into the endoplasmic reticulum and Ca2+ extrusion across the cytoplasm membrane [93, 94].

Interestingly, early postmortem studies shown that FAEEs are present in the pancreas, at higher concentrations than in any other organ, of patients intoxicated with alcohol at the time of death [96].

Carboxyl ester lipase and glutathione S-transferase have FAEEs synthase activity [87].

Fatty acid ethyl esters have been shown to uncouple oxidative phosphorylation in mitochondria in vivo (9), increase lysosomal fragility (22), and decrease incorporation of thymidine into DNA and methionine into new proteins (23).

Moreover, FAEE have been shown to be present in human serum many hours after ethanol ingestion and (24), which suggests that their half-life in tissue may be much longer than that of ethanol itself or its oxidation products.

Our data support the hypothesis that fatty acid ethyl esters are cytotoxic mediators involved in the production of alcohol-dependent cellular damage (28–29).

even low concentrations of FAEE have been demonstrated to inhibit the hepatic enzyme triacylglycerole lipase as well as protein synthesis,

FAEE have disordering effects on membranes, exceeding that of ethanol by approximately an order of magnitude;

Even at pharmacologically relevant concentrations, and particularly in the concentration range of 10 to 20 mM, the effect of ethanol on the “fluidity” of the membrane bulk lipids is very small, or undetectable (1).

The molecular pathology of ethanol is almost certainly multi-factorial, but one of its major effects is to alter the composition of lipids in various cells throughout the body.

However, it is not clear whether [lipid concentration] alterations are related to any pharmacological effects of ethanol, per se, or to its oxidative metabolism, which produces oxidants and free radical-mediated reactions.

It was also apparent that alcohol caused severe oxidative stress in gastric tissue manifested as stimulated lipid peroxidation via increasing MDA content and reduction of gastric GSH content.

We and others have demonstrated the biochemical and molecular mechanisms of alcohol-induced oxidative production as contributing factors to neuronal degeneration after alcohol-induced breaching of brain microvascular endothelial cells [15,16], brain astrocytes [17,18], and cultured microglia [19].

These findings support the notion that oxidant generation is a cause of neuroinflammation and neuronal degeneration in alcoholics.

Ethanol administration significantly increased the levels of lipid hydroperoxides in all tissues studied.

The SOD activity was decreased by 65%, while that of GST was reduced by about 58.5% compared to the control values (Table 1). The CAT activity was unchanged after alcohol intake.

Alcohol also decreased the gastric level of GSH by about 52%.

Alcohol administration markedly stimulated lipid peroxidation in gastric tissues, and the MDA content was elevated by about 125.5% compared to control animals (Table 1).

Nevertheless, our findings indicate that simultaneous induction of SOD and catalase during alcohol-induced stress is an acute adaptive defense response to counteract the oxidative damage within the endothelium.

Our data demonstrate that catalase could act as an adaptive response to alcohol insults for the shortterm only, but not in chronic conditions.

Treatment of endothelial cells with 20 mM EtOH (about 0.11% v/v) resulted in a linear induction of cellular SOD activity for up to 24 hrs, followed by a gradual decline below basal levels in 240 hr (Fig. 4A).

These data suggest that EtOH/Ach activated calciumindependent iNOS, leading to enhanced NO levels in brain endothelium.

hBECs exposure to 20 mM EtOH resulted in significant increase of ROS, and pretreatment of hBECs with 4MP significantly inhibited the EtOH-elicited increase in ROS levels, suggesting the involvement of EtOH metabolism in ROS generation (Fig. 1A).

We showed that the underlying mechanism of alcohol-elicited BBB oxidative damage is due to activation of NADPH oxidase (NOX) and inducible nitric oxide synthase (iNOS) by acetaldehyde (Ach) and subsequent ROS/RNS generation.

During its catalytic cycle CYP2E1 is prone to uncoupling, so that, during the metabolism of ethanol, approximately 50% of the cycles result in the production of superoxide anion (O2.-) and related ROS instead of metabolite formation [33, 34].

Indeed, ethanol stimulates the conversion of xanthine dehydrogenase to O2.-producing oxidase [63].

A variety of enzymatic and nonenzymatic mechanisms have evolved to protect cells against ROS, including the superoxide dismutases, which remove O2U−; catalase and the glutathione (GSH) peroxidase system, which remove H2O2; glutathione transferases, which can remove reactive intermediates and lipid aldehydes; metallothioneins, heme oxygenase, and thioredoxin, which remove various ROS; ceruloplasmin and ferritin, which help remove metals such as iron, which promote oxidative stress reactions; and nonenzymatic, low-molecular-weight antioxidants such as GSH itself, vitamin E, ascorbate (vitamin C), vitamin A, ubiquinone, uric acid, and bilirubin [22,23].

Oxidative stress or toxicity by ROS reflects a balance between the rates of production of ROS and the rates of removal of ROS plus repair of damaged cellular macromolecules.

In addition, ROS at low concentrations, especially H2O2, may be important in signal transduction mechanisms in cells and thus be involved in cellular physiology and metabolism [25].

The ability of acute and chronic ethanol treatment to increase production of reactive oxygen species and enhance peroxidation of lipids, protein, and DNA has been demonstrated in a variety of systems, cells, and species, including humans.

Again, many of these pathways are not exclusive of one another and it is likely that several, indeed many, systems contribute to the ability of alcohol to induce a state of oxidative stress

Addition of iron, known to generate ·OH and promote oxidative stress, to these diets exacerbated the liver injury [31].

Importantly, addition of antioxidants such as vitamin E, ebselen, superoxide dismutase, and GSH precursors prevented the alcohol-induced liver injury [28]

A rather moderate ethanol consumption promoted oxidative stress and liver injury in SOD1-knockout mice, indicating that compromised antioxidant defense promotes alcohol liver injury.

In our laboratory, we found that microsomes isolated from rats fed ethanol chronically were about two- to threefold more reactive in generating superoxide radical and H2O2 and, in the presence of ferric complexes, in generating hydroxyl radical and undergoing lipid peroxidation compared to microsomes from pair-fed controls [53–56].

===Glucose Transport===
We recently demonstrated that alcohol affects BBB integrity, which may potentially cause neurodegeneration by limiting the glucose supply to the CNS [22].

Taken together, our results suggest that EtOH impairs the glucose uptake by inhibiting the expression of GLUT in human astrocytes and neurons.

Concentrations up to 100 mM did not cause neuronal toxicity at 24 hr exposure (data not shown), however glucose uptake was decreased in a dose-dependent manner, starting at a concentration of 5 mM EtOH (Figure 4A).

However, the significant decrease in glucose uptake was noted from 50 mM to 500 mM EtOH

In 1994, William-Hemby and Porrino reported that acute administration of low doses of ethanol increased glucose utilization in specific brain regions, while high doses of ethanol decreases [17].

In this study, we hypothesized that ethanol (EtOH)-mediated disruption in glucose uptake would deprive energy for human astrocytes and neurons inducing neurotoxicity and neuronal degeneration.

Our results showed that exposure of brain endothelial cells to EtOH or Ach increased catalase activity for 1–2 hrs, after which its activity declines to basal control levels (Fig. 3A–D) where it remained up to 68 hrs treatment (data not shown).

Ethanol administration led to a significant increase in the GSH S-transferase activity which (compared to the control group) was approximately 8-fold in the brain, 5-fold in the heart, 2-fold in the kidney and 3-fold in the liver respectively.

Unlike the other two major pathways, a 4-10-fold increase of CYP2E1 expression has been associated with ethanol abuse [21, 22].

Ethanol is not capable of inducing its own metabolism via ADH.

Ethanol itself induces the activity of CYP2E1, and therefore also influences the metabolism of other xenobiotics (e.g., paracetamol and cocaine).

The mechanism of [CYP2E1] induction was, therefore, suggested to be at the level of protein degradation.

From in vivo data of CYP2E1 turnover in rats chronically treated with acetone [103] and in vitro hepatocyte culture systems [104,105], exogenous CYP2E1 inducers such as acetone, ethanol, imidazole, and 4-methylpyrazole (4-MP) were shown to increase CYP2E1 by protein stabilization.

In addition, CYP2E1 protein stabilization seemed dependent on blood ethanol or acetone concentration.

Similarly, McGhee et al. [112] reported the half-life of CYP2E1 in a hepatoma cell line to be 1.8 h in the absence of ethanol and 45 h in the presence of ethanol.

It is clear that a major level of regulation of CYP2E1 formation seems to be posttranscriptional as various substrates and ligands increase the content of CYP2E1 by protection against rapid degradation by intracellular proteolytic pathways.

Roberts [113] provided evidence for a role of the proteasome in the degradation of several cytochrome P450s, including CYP2E1.

Importantly, Bardag-Gorce et al. [118] showed that the rapid loss of CYP2E1, which occurs in vivo after the ethanol inducer is withdrawn, could be blocked by the proteasome inhibitor PS-341, thus establishing the critical role of the proteasome in regulating CYP2E1 turnover in vivo.

In these models, large increases in microsomal lipid peroxidation have been observed and the ethanol-induced liver pathology has been shown to correlate with CYP2E1 levels and elevated lipid peroxidation [29,30,119,120].

A strong association between dietary carbohydrate, enhanced CYP2E1 induction, and hepatic necrosis was observed. No liver injury was found if carbohydrate levels were elevated [128].

They concluded that CYP2E1 induction by chronic ethanol treatment was responsible for the decrease in proteasome activity and accumulation of oxidized proteins in the liver. They speculated the pathology found in the CYP2E1 knockouts by Kono et al. [135] may be due to upregulation of NADPH-cytochrome P450 reductase and other CYPs such as CYP3A and 4A [137] (see below).

Ethanol increases levels of CYP2E1, largely by a posttranscriptional mechanism involving enzyme stabilization against degradation.

They concluded that CYP2E1 induction by chronic ethanol treatment was responsible for the decrease in proteasome activity and accumulation of oxidized proteins in the liver.

These results show that obesity contributes to oxidative/nitrosative stress and liver injury and that induction of CYP2E1 may synergize with high fat in obesity to promote liver cell injury.

Actually, natural agents inhibiting CYP2E1, including diallyl sulfide (from garlic) mentioned above, phenylethyl isothiocyanate and sulforaphane (present in cruciferous vegetables), and bergamottin (found in the essential oils of grapefruit and certain oranges) have been proposed as possible candidates for minimizing the ethanol-induced hepatotoxicity [225].

Regulation of CYP2E1 protein levels is complex, with transcriptional, translational, and posttranscriptional effects observed;

CYP2E1 levels were elevated about three- to fivefold in the liver microsomes after rats were fed the Lieber–DeCarli diet for 4 weeks.

the increased activity in these microsomes was due to CYP2E1.

Importantly, ethanol was shown to elevate the levels of mitochondrial CYP2E1 in addition to the well-known increase in microsomal CYP2E1 [78].

CYP2E1 can also be induced under a variety of metabolic or nutritional conditions.

Somewhat paradoxically, in rats levels of CYP2E1 were also increased by fasting and by prolonged starvation [85,86].

For example, CYP2E1 levels were elevated in chronically obese, overfed rats and in rats fed a high-fat diet [84].

In this respect, it is interesting that alcohol-induced liver damage is magnified in diets with very low levels of carbohydrate and high levels of fat [94].

Testosterone increased renal but not hepatic CYP2E1 levels [97].

The carbohydrate content of the diet influences CYP2E1 levels, as a low-carbohydrate diet increased the extent of induction of MEOS by ethanol [92] and high-fat/low-carbohydrate diets resulted in the highest levels of CYP2E1 induced by ethanol [93].

CYP2E1 is regulated by multiple, distinct regulatory mechanisms [83,98,99].

CYP2E1 is not transcriptionally activated by an acute bolus dose or chronic administration of ethanol, acetone, or other exogenous inducing agents.

====Ethanol consumption does increase miR-122 in healthy humans (2x), but the increase is very small compared to acetaminophen toxicity (100-10,000x).====
Conclusion: miR-122 increased with moderate ethanol consumption, but the fold change was modest.

Their mean serum ethanol concentration was 113 mg/dl (95% confidence interval [CI] 91–135 mg/dl) after consuming ethanol.

Upon fasting or induced diabetes, the mRNA for CYP2E1 is increased several fold due to posttranscriptional mRNA stabilization [100].

Although elevation of CYP2E1 mRNA levels has been reported [101], most investigators have found little induction or a slight reduction of CYP2E1 mRNA levels after ethanol administration [81,83,102].

If one is to summarize currently available data on the acute neurochemical effects of ethanol at concentrations up to 20 mM, data indicate that a discrete set of neurotransmitter systems, which can be Characterized as receptor-gated ion Channels, is important in mediating the effects of such concentrations of ethanol (i.e., the GABAA receptors, the NMDA and possibly other glutamate receptors, the nicotinic cholinergic receptors, and the 5-HT3 receptors).

it has been widely demonstrated that the stimulatory, sedative, anxiolytic, and reinforcing effects of ethanol occur within different and relatively narrow dose rangesg0

These studies also indicate a general consensus that the most sensitive measure of ethanol’s action in a human is the self-report of feeling “intoxicated” (i.e., the interoceptive perception of ethanol’s actions). This effect occurs at doses as low as 0.25 g/kg, with corresponding blood alcohol concentrations of approximately l0 to 30 mg% (2 to 7 mM).

In our experiments, we found that neuronal cells were more sensitive to EtOH exposure than astrocytes.

Our findings revealed that dopaminergic neurons, mostly localized in the substantia nigra region, exhibited degeneration after ethanol administration (Figs. 2D–F).

Application of absolute alcohol by gastric gavage induced marked damage to the gastric mucosa that was obvious by macroscopic examination.

Depletion of non-protein sulfhydryls concentrations [15], modulation of nitric oxide system [35], reduction of mucosal blood flow [18], and autonomic nervous system regulation [36], have been suggested to be involved in the development of gastric lesions.

Intake of absolute alcohol induced severe and extensive macroscopic gastric mucosal damage characterised by elongated haemorrhagic lesions confined mainly to the gastric corpus and running parallel to the long axis of the stomach that had the highest ulcer scoring rate (Fig. 1).

===Exogenous Interactions===
With some compounds, e.g., carbon tetrachloride or acetaminophen, metabolism by P450 can give rise to toxic metabolites which damage cells.

In fact, these two properties explain the ability of ethanol to inhibit the metabolism of certain substrates when it is present, i.e., ethanol and the substrate compete for oxidation by CYP2E1, and to increase the metabolism of substrates when it is no longer present to compete, but had already elevated the levels of the CYP2E1 catalyst.

A variety of heterocyclic compounds such as imidazole, pyrazole, 4methylpyrazole, thiazole, and isoniazid have been shown to elevate CYP2E1 levels, as do solvents such as dimethyl sulfoxide, various alcohols, benzene, and acetone [81–83].

After administration of ethanol, acetone, or pyrazole to rats, Song et al. found that CYP2E1 mRNA levels did not increase [81].

Conversely, ethanol consumption in excess has been implicated in more than 60 types of diseases, and it is a contributing cause in 200 others [1, 2].

insulin, which lowers them [89,95].

Because CYP2E1 can generate ROS during its catalytic circle, and its levels are elevated by chronic treatment with ethanol, CYP2E1 has been suggested to be a major contributor to ethanol-induced oxidant stress and to ethanol-induced liver injury.

It was concluded that diet is an important factor in toxicity mediated by ethanol because of modulation of the levels of CYP2E1 [128].

However, micro- and macrovesicular steatosis, occasional inflammatory foci, and a threefold increase in transaminase levels were observed in a nutritionally adequate ethanol-containing liquid diet with a carbohydrate content of 5.5%; no changes were found if the level of carbohydrate was elevated to 11% [94,129].

On the other hand, studies by Thurman and colleagues have presented powerful support for a role for endotoxin, activation of Kupffer cells, and cytokines such as TNFα in the alcoholinduced liver injury found in the intragastric infusion model [133,134]. They suggested that CYP2E1 may not play a role in alcohol liver injury based upon studies with gadolinium chloride or CYP2E1-knockout mice [135,136].

Bradford et al. [139], using CYP2E1 and NADPH oxidase-knockout mice, concluded that CYP2E1 was required for ethanol induction of oxidative stress to DNA, whereas NADPH oxidase was required for ethanol-induced liver injury.

As mentioned earlier, it is likely that several mechanisms contribute to alcoholinduced liver injury and that ethanol-induced oxidant stress is likely to arise from several sources, including CYP2E1, mitochondria, and activated Kupffer cells.

Damage to mitochondria by CYP2E1-derived oxidants seems to be an early event in the overall pathway of cellular injury.

We found that the levels of GSH and several antioxidant enzymes, such as glutathione S-transferase (GST), catalase, and heme oxygenase1, were upregulated in the CYP2E1-expressing cells.

This upregulation was prevented by antioxidants, suggesting that ROS generated by CYP2E1 were responsible for the transcriptional activation of these antioxidant genes.

We observed similar results in HepG2 cells expressing CYP2E1, as the half-life of human CYP2E1 was about 3–6 h in the absence of substrate or ligand and was elevated in the presence of various substrates and ligands [150].

We believe that the upregulation of these antioxidant genes reflects an adaptive mechanism to remove CYP2E1-derived oxidants.

CYP2E1, a loosely coupled enzyme, generates ROS such as O2U− and H2O2 during its catalytic cycle.

In the presence of iron, which is increased after ethanol treatment, more powerful oxidants including UOH, ferryl species, and 1-hydroxyethyl radical are produced.

Initially, the liver cells respond to the CYP2E1-related oxidative stress by transcriptionally inducing various antioxidant enzymes via their antioxidant response elements.

Ultimately, these protective Y. Lu, A.I. Cederbaum / Free Radical Biology & Medicine 44 (2008) 723–738 729

mechanisms are overwhelmed and the cells become sensitive to the CYP2E1-generated oxidants.

These various oxidants can promote toxicity by protein oxidation and enzyme inactivation, oxidative damage to the DNA, and disturbing cell membranes via lipid peroxidation and production of reactive lipid aldehydes, such as malondialdehyde (MDA) and 4-hydroxynonenal. Mitochondria seem to be among the critical cellular organelles damaged by CYP2E1-derived oxidants.

A decrease in ΔΨm, likely due to the mitochondrial membrane permeability transition, causes the release of proapoptotic factors, resulting in apoptosis.

Some CYP2E1-derived ROS, such as H2O2, LOOH, and HNE, are diffusible and may exit hepatocytes and enter other liver cell types such as stellate cells and stimulate these cells to produce collagen and elicit a fibrotic response [160,161].

We believe that the linkage between CYP2E1-derived oxidative stress, mitochondrial injury, and GSH homeostasis contributes to the toxic actions of ethanol on the liver.

CYP2E1 is the major factor in CCl4 hepatotoxicity [179].

CYP2E1 plays a critical role in catabolism of acetone after fasting [180].

Formation of benzene metabolites such as hydroquinone, catechol, and phenol were lowered more than 90% with microsomes from CYP2E1-knockout mice compared to microsomes from controls [181].

CYP2E1-knockout mice were highly resistant to liver toxicity compared to wild-type mice treated with acetaminophen [49].

The authors concluded that CYP2E1 but not NADPH oxidase is required for the ethanol induction of oxidative stress to DNA [in mice with 4-week alcohol treatment]and thus CYP2E1 may play a key role in ethanol-associated hepatocarcinogenesis [139].

On the other hand, as mentioned above, studies by Thurman and colleagues suggest that CYP2E1 may not play a role in alcohol-induced liver injury [135].

Taken as a whole, these and other studies clearly implicate TNFα as a major risk factor for the development of alcoholic liver injury.

These results suggest that increased oxidant stress from CYP2E1 may sensitize isolated hepatocytes to TNFα-induced toxicity

Based on such studies, we hypothesize [212,213] that increased production of ROS by CYP2E1 may prime or sensitize the liver to LPS/TNFα, and such interactions may be important in alcohol-induced liver injury.

Thus, although CYP2E1 contributes to the pathogenesis of NASH, it is not unique among P450 enzymes in promoting oxidant stress, as some CYP4A enzymes can serve as alternative initiators of oxidant stress in the liver.

Thus CYP4A can mediate lipid peroxidation as an alternative pathway when CYP2E1 is absent [219].

Peroxynitrite (ONOO−), formed by the rapid reaction between NO and O2U−, has been shown to nitrate free and protein-associated tyrosine residues and produce nitrotyrosine; therefore, either decreased NO production by 1400W or a decline in O2U− production by CMZ prevented 3-nitrotyrosine formation [223].

Alcohol-induced liver injury is probably a multifactorial process involving several mechanisms.

Some of the major proposed systems require more detail about mechanism, e.g., how ethanol-derived NADH, by itself or when reoxidized in the mitochondrial respiratory chain, produces ROS.

The role of CYP2E1 in the toxic effects of ethanol requires further study as this remains a controversial issue.

At present, acetone and some fatty acids (ω-1 hydroxylase activity) seem to be physiological substrates for CYP2E1, but further studies should be carried out because altered metabolism of such putative endogenous substrates, if any, could contribute to the cellular actions associated with CYP2E1.

CYP2E1-nutritional interactions require further study, especially interactions with pro-oxidants, such as iron; polyunsaturated fatty acids; or reagents that lower oxidant defenses, e.g., GSH levels.

Moreover, these findings could result in the development of more effective and selective new medications capable of blocking the actions of CYP2E1 and ROS and, consequently, the toxic effects of alcohol.

===Lu_Cederbaum_2008_CYP2E1 and oxidative liver injury by alcohol.pdf===

J Dinis-Oliveira_2016_Oxidative and non-oxidative metabolomics of ethanol.pdf

===McCrae et al_2016_Ethanol consumption produces a small increase in circulating miR-122 in healthy.pdf===
Serum miR-122 increased from a mean of 71.3 million (95% CI 29.3–113.2 million) to 139.1 million (95% CI 62.6–215.7 million) copies/ ml (2.2-fold increase).

As increases with acetaminophen toxicity are 100to 10 000-fold, moderate ethanol intoxication is unlikely to confound the use of this biomarker of hepatotoxicity.

There was a 2.2-fold increase of miR-122 from a mean value of 71.3 (95% confidence interval [CI] 29.3–113.2 million) to 139.1 million (95% CI 62.6– 215.7 million) copies/ml (Fig. 1; p ¼0.006).

Xiang_Shen_2011_In vivo detection of intermediary metabolic products of [1-13C]ethanol in the.pdf

===Eckardt_File_Gessa_Grant_Guerri_Hoffman_Kalant_Koob_Li_Tabakoff_1998_Effects of Moderate Alcohol Consumption on the Central Nervous System.pdf===


We will review the neurochemical and behavioral effects of these brain alcohol levels, keeping in mind that animals such as mice may be more resistant to certain effects of alcohol,

Intakes of from 2.7 to 82 g of alcohol/day (<1 drink to about 6 drinks/day) have been con— sidered “moderate” (Table 1).