**8. Ethanol metabolism**

Ethanol is absorbed rapidly in the gastrointestinal tract; the surface of greatest adsorption is the first portion of the small intestine with 70%; 20% is absorbed in the stomach, and the remainder, in the colon. Diverse factors can cause the increase in absorption speed, such as gastric emptying, ingestion without food, ethanol dilution (maximum absorption occurs at a 20% concentration), and carbonation. Under optimal conditions, 80‒90% of the ingested dose is completely absorbed within 60 minutes. Similarly, there are factors that can delay ethanol absorption (from 2-6 hours), including high concentrations of the latter, the presence of food, the co-existence of gastrointestinal diseases, the administration of drugs, and individual variations (Goldfrank et al., 2002).

Once ethanol is absorbed, it is distributed to all of the tissues, being concentrated in greatest proportion in brain, blood, eye, and cerebrospinal fluid, crossing the feto-placentary and hematocephalic barrier (Téllez & Cote, 2006). Gender difference is a factor that modifies the distributed ethanol volume; this is due to its hydrosolubility and to that it is not distributed in body fats, which explains why in females this parameter is found diminished compared with males.

Ethanol is eliminated mainly (> 90%) by the liver through the enzymatic oxidation pathway; 5-10% is excreted without changes by the kidneys, lungs, and in sweat (Goldfrank et al., 2002). The liver is the primary site of ethanol metabolism through the following three different enzymatic systems:

#### **8.1 Alcohol dehydrogenases (ADH)**

Alcohol dehydrogenases (ADH) are cytoplasmic enzymes with numerous isoforms in the liver of humans, with high specificity for ethanol as substrate; these are codified by three separate genes designated as *ADH1*, *ADH2*, and *ADH3*; these genes translate into peptide subunits denominated alpha, beta, and gamma. Variations in ADH isoforms can explain the significance in alcohol elimination levels among ethnic groups (Feldman et al., 2000).

This enzyme utilizes Nicotinamide adenin dinucleotide (NAD+) as a Hydrogen (H+) receiver for oxidizing ethanol into acetaldehyde. In this process, H+ is transferred from the

a complex that impedes the entrance of toxins into the interior of liver cells and, on the other hand, metabolically stimulates hepatic cells, in addition to activating RNA biosythesis of the ribosomes, stimulating protein formation. In a study published by Sandoval et al., in 2008, the authors observed that silymarin's protector effect on hepatic cells in rats when they employed this as a comparison factor on measuring liver weight/animal weight % (hepatomegaly), their values always being less that those of other groups administered with other possibly antioxidant substances; no significant difference was observed between the silymarin group and the silymarin-alcohol group, thus demonstrating the protection of silymarin. On the other hand, silymarin diminishes Kupffer cell activity and the production of glutathione, also inhibiting its oxidation. Participation has also been shown in the increase of protein synthesis in the hepatocyte on stimulating polymerase I RNA activity. Silymarin reduces collagen accumulation by 30% in biliary fibrosis induced in rat (Boigk, 1997). An assay in humans reported a slight increase in the survival of persons with cirrhotic

Ethanol is absorbed rapidly in the gastrointestinal tract; the surface of greatest adsorption is the first portion of the small intestine with 70%; 20% is absorbed in the stomach, and the remainder, in the colon. Diverse factors can cause the increase in absorption speed, such as gastric emptying, ingestion without food, ethanol dilution (maximum absorption occurs at a 20% concentration), and carbonation. Under optimal conditions, 80‒90% of the ingested dose is completely absorbed within 60 minutes. Similarly, there are factors that can delay ethanol absorption (from 2-6 hours), including high concentrations of the latter, the presence of food, the co-existence of gastrointestinal diseases, the administration of drugs, and

Once ethanol is absorbed, it is distributed to all of the tissues, being concentrated in greatest proportion in brain, blood, eye, and cerebrospinal fluid, crossing the feto-placentary and hematocephalic barrier (Téllez & Cote, 2006). Gender difference is a factor that modifies the distributed ethanol volume; this is due to its hydrosolubility and to that it is not distributed in body fats, which explains why in females this parameter is found diminished compared

Ethanol is eliminated mainly (> 90%) by the liver through the enzymatic oxidation pathway; 5-10% is excreted without changes by the kidneys, lungs, and in sweat (Goldfrank et al., 2002). The liver is the primary site of ethanol metabolism through the following three

Alcohol dehydrogenases (ADH) are cytoplasmic enzymes with numerous isoforms in the liver of humans, with high specificity for ethanol as substrate; these are codified by three separate genes designated as *ADH1*, *ADH2*, and *ADH3*; these genes translate into peptide subunits denominated alpha, beta, and gamma. Variations in ADH isoforms can explain the

This enzyme utilizes Nicotinamide adenin dinucleotide (NAD+) as a Hydrogen (H+) receiver for oxidizing ethanol into acetaldehyde. In this process, H+ is transferred from the

significance in alcohol elimination levels among ethnic groups (Feldman et al., 2000).

alcoholism compared with untreated controls (Ferenci, 1989).

**8. Ethanol metabolism** 

with males.

different enzymatic systems:

**8.1 Alcohol dehydrogenases (ADH)** 

individual variations (Goldfrank et al., 2002).

substrate (ethanol) to the co-factor (NAD+), converting it into its reduced form, NADH; likewise, H+ acetaldehyde is transferred from acetaldehyde to NAD+. Later, the acetaldehyde oxidizes into acetate by means of the reduced Aldehyde-dehydrogenase enzyme (ALDH). Under normal conditions, acetate is converted into acetyl coenzyme A (acetyl-CoA), which enters the Krebs cycle and is metabolized into carbon dioxide and water (Goldfrank et al., 2002).

#### **8.2 Microsomal ethanol oxidation system (MEOS)**

This system is localized at hepatocyte smooth reticulum cisterns; this cytochrome P-450 2E1 dependent enzymatic system contributes to 5‒10% of ethanol oxidation in moderate drinkers, but its activity increases significantly in chronic drinkers by up to 25% (Roldán et al., 2003).

A critical component of MEOS is Cytochrome P-450 2E1 (CYP2E1); this enzyme catalyzes not only ethanol oxidation, but also the metabolism of other substances such as paracetamol, the barbiturates, the haloalkalines, and the nitrosamines, among others (Feldman et al., 2000).

CYP2E1 utilizes Nicotinamide adenin dinucleotide phosphate (NADP+) as a (H+) receiver for oxidizing ethanol into acetaldehyde. In this process, H+ is transferred from the substrate (ethanol) to the co-factor (NADP+), converting this into its reduced form, NADH; Similarly, the acetaldehyde is as the H+ transfers from acetaldehyde to NADP+ (Goldfrank et al., 2002).

While MEOS participation is more active in the ethanol metabolism of chronic than in occasional drinkers and its relative contribution in comparison with ADH is difficult to determine, notwithstanding this, MEOS is important in the pathogeny of ethanol consumption-associated hepatic lesions because oxidation of this CYP2E1-mediated substance produces reactive oxygen intermediaries as subproducts (Feldman et al., 2000).

#### **8.3 Catalase system**

This presents in the peroxisomes and utilizes Hydrogen peroxide (H2O2) for ethanol oxidation; its contribution is minimal (Roldán et al., 2003). This system exists in a tight relationship with the reduced-oxidase-glutathione system and, like MEOS, is induced by chronic ethanol consumption. Ethanol oxidizes into acetaldehyde, utilizing H2O2 as coenzyme; this metabolite continues the same course as for converting into acetate through the ALDH enzyme (Morales-González et al., 2001; Morales-González et al., 1998).

Any of the three ethanol pathways transforms it into acetaldehyde, which afterward is oxidized into acetate by the Aldehyde-dehydrogenase (ALDH) enzyme. Aldehyde is a highly reactive compound and is potentially toxic for the hepatocyte.

#### **9. Hepatic regeneration**

Hepatic regeneration (HR) is a process arising throughout evolution to protect animals from the catastrophic results of hepatic necrosis caused by the effect of the toxins of plants that serve them as food; this extraordinary process has been the object of the curiosity of scientists of all times. In ancient Greece, the myth of the chained Prometheus in Caucasus mountains of the Caucasus while an eagle daily devoured his entrails, which regenerated

The Protective Effect of Antioxidants in Alcohol Liver Damage 111

alterations derived from its chronic consumption, from the Central nervous system (CNS) as

There are several mechanisms involved in ethanol consumption-related liver damage; the former results from the effects of alcohol dehydrogenase (ADH) mediated by the excessive generation of NADH and acetaldehyde, which generates the formation of FR (Lieber, 2004). Acetaldehyde increases the production of alkanes and causes an imbalance in potential cytosolic redox on altering the NAD+/NADH relationship, as occurs in the mitochondria; this redox alteration favors the production of lipoperoxides, which increase damage to the

Acetaldehyde inactivates the enzymes, diminishing DNA repair, antibody production, and glutathione depletion, and increasing mitochondrial toxicity, endangering oxygen

Another probable mechanism of hepatic damage associated with chronic ethanol consumption is the increase in the synthesis of fatty acids and triglycerides and a decrease of the oxidation of the former, generating hyperlipidemias that leads to the development of fatty liver, in addition to inhibiting fatty acids utilization and the availability of precursors,

Previous studies have suggested that Kupffer cells are involved in hepatic damage caused by ethanol consumption; this is due to that there are reports that ethanol alters the functions of these cells, such as phagocytosis, bactericide activity, and the production of inflammatory cytokines such as Tumor growth factor alpha (TNF)-α, Interleukin 1 (IL-1), and IL-6, among others, which result in hepatic cell toxicity (Thurman et al., 1998). It has been reported that TNF-α and IL-1 inhibit protein synthesis in hepatocytes in rat, in addition to stimulating neutrophil migration and activation, as well as protease induction and FR release (Thurman,

Cytokines and chemokines originated by the Kupffer cells employ autoparacrine as well as paracrine effects that initiate the defensive response in the liver, but that also promote the infiltration of inflammatory leukocytes and activate the oxidative attack response, accompanied by strong damage originating from degrading cytokines and proteins. Cellular infiltration of activated neutrophils produces oxygen FR and secretes other toxic mediators; additionally, these can increase the inflammatory response, causing damage and cell death

Ethanol consumption induces changes in the mitochondrial membrane, such as Mitochondrial permeability transition (MPT), which is associated with loss of mitochondrial energy, mitochondrial matrix inflammation, and external membrane rupture; this is accompanied by the release of numerous proapoptotic factors; in addition, TNF-α activates different hepatic cell cascades, resulting in the stimulation of mitotic genes such as *p38* of Mitogen-activated protein kinases (MAPK) and the Jun N-terminal kinase (JNK), which affect mitochondrial sensitivity to the proapoptotic stimulus as follows: JNK by the phosphorylation of proapoptotic proteins such as Bcl-2 and Bcl-XL, and *p38* MAPK by re-

Acute ethanol consumption can produce a hypermetabolic state in the liver that is characterized by increase in mitochondrial respiration, which is driven by the great demand

enforcing the effects of the Bcl-2 proapoptotic Bax Protein family.

well as from the peripheral nervous system (Díaz et al., 2002).

cell (Gutiérrez-Salinas & Morales-González, 2004).

1998).

(Thurman, 1998).

utilization, and increasing collagen synthesis (Lieber, 2000).

which stimulate the hepatic synthesis of triglycerides (Téllez & Cote, 2006).

during the night, is based on the recognition from those times of hepatic regeneration (Michalopoulos & DeFrances, 1997).

Several terms such as replacement of lost parts, restitution, or repair have been employed to designate "regeneration", which is indicative of the active cell proliferation that invariably precedes differentiation. After partial liver extraction, cell proliferation does not occur at the level of the incision, new lobules do not develop instead of the removed part; ascribing to cell migration and differentiation results in the formation of new lobules, and the liver's preoperative weight is rapidly restored (Higgins & Anderson, 1931).

The liver in humans as well as in rodents possess a noteworthy capacity of regenerative response to several stimuli, including massive destruction of hepatic tissue by toxins, viral agents, or by surgical desertion; liver regeneration depends on the ability of the hepatocytes to be submitted to cell division, which is strictly controlled by intra- and extrahepatic factors (Gutiérrez-Salinas et al., 1999).

HG is a physiological process that includes hypertrophy (increase in cell size or in protein content in the replicative phase) and hyperplasia (increase in cell number), the latter governed to a greater degree by functional than by anatomical needs (Palmes & Spiegel, 2004). Studies with hepatic resections in large animals (dogs and primates) and in humans have established that the regenerative response is proportional to the amount of the liver removed (Michalopoulos & DeFrances, 1997).

Partial hepatectomy (PH) is the best studied animal hepatic regeneration model and was proposed in 1931 by Higgins and Anderson and consists of the surgical extraction of 70% of hepatic mass; hepatic regeneration is induced at an important stage when all of the hepatocytes are virtually found in phase G0 of the cell cycle. Stimulated by PH, all of the hepatocytes in synchronized fashion enter into the cell cycle; initiation of maximum Deoxyribonucleic acid (DNA) synthesis takes place 24 hours after PH and 7‒14 days after the removed hepatic mass is restored (Palmes & Spiegel, 2004).

In contrast with other regenerating tissues (bone marrow and skin) hepatic regeneration does not depend on a small population of progenitor cells; liver regeneration after PH is carried out by the proliferation of all of the existing mature call populations that comprise the intact organ; this includes the hepatocytes, biliary endothelial cells, fenestrated endothelial cells, Kupffer cells and Ito cells; all of these cells divide during hepatic proliferation, hepatocytes the first to do this. Cell proliferation kinetics differs slightly from one species to another; the first DNA synthesis peak in hepatocytes occurs at 24 hours, with a second peak between 36 and 48 hours; when three quarters of the hepatic tissue is removed, restoration of the original number of hepatocytes theoretically requires 1.66 cell cycles per residual hepatocyte (Michalopoulos & DeFrances, 1997).

#### **10. Ethanol-derived hepatic damage**

The liver is the main target organ of ethanol toxicity; it has been demonstrated experimentally that chronic ethanol ingestion leads to an increase of lipid peroxidation products and a decrease of antioxidant factors such as glutathione (GSH) and derived enzymes. Likewise, oxidative stress has been related as the main factor implicated in

during the night, is based on the recognition from those times of hepatic regeneration

Several terms such as replacement of lost parts, restitution, or repair have been employed to designate "regeneration", which is indicative of the active cell proliferation that invariably precedes differentiation. After partial liver extraction, cell proliferation does not occur at the level of the incision, new lobules do not develop instead of the removed part; ascribing to cell migration and differentiation results in the formation of new lobules, and the liver's

The liver in humans as well as in rodents possess a noteworthy capacity of regenerative response to several stimuli, including massive destruction of hepatic tissue by toxins, viral agents, or by surgical desertion; liver regeneration depends on the ability of the hepatocytes to be submitted to cell division, which is strictly controlled by intra- and extrahepatic factors

HG is a physiological process that includes hypertrophy (increase in cell size or in protein content in the replicative phase) and hyperplasia (increase in cell number), the latter governed to a greater degree by functional than by anatomical needs (Palmes & Spiegel, 2004). Studies with hepatic resections in large animals (dogs and primates) and in humans have established that the regenerative response is proportional to the amount of the liver

Partial hepatectomy (PH) is the best studied animal hepatic regeneration model and was proposed in 1931 by Higgins and Anderson and consists of the surgical extraction of 70% of hepatic mass; hepatic regeneration is induced at an important stage when all of the hepatocytes are virtually found in phase G0 of the cell cycle. Stimulated by PH, all of the hepatocytes in synchronized fashion enter into the cell cycle; initiation of maximum Deoxyribonucleic acid (DNA) synthesis takes place 24 hours after PH and 7‒14 days after

In contrast with other regenerating tissues (bone marrow and skin) hepatic regeneration does not depend on a small population of progenitor cells; liver regeneration after PH is carried out by the proliferation of all of the existing mature call populations that comprise the intact organ; this includes the hepatocytes, biliary endothelial cells, fenestrated endothelial cells, Kupffer cells and Ito cells; all of these cells divide during hepatic proliferation, hepatocytes the first to do this. Cell proliferation kinetics differs slightly from one species to another; the first DNA synthesis peak in hepatocytes occurs at 24 hours, with a second peak between 36 and 48 hours; when three quarters of the hepatic tissue is removed, restoration of the original number of hepatocytes theoretically requires 1.66 cell

The liver is the main target organ of ethanol toxicity; it has been demonstrated experimentally that chronic ethanol ingestion leads to an increase of lipid peroxidation products and a decrease of antioxidant factors such as glutathione (GSH) and derived enzymes. Likewise, oxidative stress has been related as the main factor implicated in

preoperative weight is rapidly restored (Higgins & Anderson, 1931).

(Michalopoulos & DeFrances, 1997).

(Gutiérrez-Salinas et al., 1999).

removed (Michalopoulos & DeFrances, 1997).

the removed hepatic mass is restored (Palmes & Spiegel, 2004).

cycles per residual hepatocyte (Michalopoulos & DeFrances, 1997).

**10. Ethanol-derived hepatic damage** 

alterations derived from its chronic consumption, from the Central nervous system (CNS) as well as from the peripheral nervous system (Díaz et al., 2002).

There are several mechanisms involved in ethanol consumption-related liver damage; the former results from the effects of alcohol dehydrogenase (ADH) mediated by the excessive generation of NADH and acetaldehyde, which generates the formation of FR (Lieber, 2004). Acetaldehyde increases the production of alkanes and causes an imbalance in potential cytosolic redox on altering the NAD+/NADH relationship, as occurs in the mitochondria; this redox alteration favors the production of lipoperoxides, which increase damage to the cell (Gutiérrez-Salinas & Morales-González, 2004).

Acetaldehyde inactivates the enzymes, diminishing DNA repair, antibody production, and glutathione depletion, and increasing mitochondrial toxicity, endangering oxygen utilization, and increasing collagen synthesis (Lieber, 2000).

Another probable mechanism of hepatic damage associated with chronic ethanol consumption is the increase in the synthesis of fatty acids and triglycerides and a decrease of the oxidation of the former, generating hyperlipidemias that leads to the development of fatty liver, in addition to inhibiting fatty acids utilization and the availability of precursors, which stimulate the hepatic synthesis of triglycerides (Téllez & Cote, 2006).

Previous studies have suggested that Kupffer cells are involved in hepatic damage caused by ethanol consumption; this is due to that there are reports that ethanol alters the functions of these cells, such as phagocytosis, bactericide activity, and the production of inflammatory cytokines such as Tumor growth factor alpha (TNF)-α, Interleukin 1 (IL-1), and IL-6, among others, which result in hepatic cell toxicity (Thurman et al., 1998). It has been reported that TNF-α and IL-1 inhibit protein synthesis in hepatocytes in rat, in addition to stimulating neutrophil migration and activation, as well as protease induction and FR release (Thurman, 1998).

Cytokines and chemokines originated by the Kupffer cells employ autoparacrine as well as paracrine effects that initiate the defensive response in the liver, but that also promote the infiltration of inflammatory leukocytes and activate the oxidative attack response, accompanied by strong damage originating from degrading cytokines and proteins. Cellular infiltration of activated neutrophils produces oxygen FR and secretes other toxic mediators; additionally, these can increase the inflammatory response, causing damage and cell death (Thurman, 1998).

Ethanol consumption induces changes in the mitochondrial membrane, such as Mitochondrial permeability transition (MPT), which is associated with loss of mitochondrial energy, mitochondrial matrix inflammation, and external membrane rupture; this is accompanied by the release of numerous proapoptotic factors; in addition, TNF-α activates different hepatic cell cascades, resulting in the stimulation of mitotic genes such as *p38* of Mitogen-activated protein kinases (MAPK) and the Jun N-terminal kinase (JNK), which affect mitochondrial sensitivity to the proapoptotic stimulus as follows: JNK by the phosphorylation of proapoptotic proteins such as Bcl-2 and Bcl-XL, and *p38* MAPK by reenforcing the effects of the Bcl-2 proapoptotic Bax Protein family.

Acute ethanol consumption can produce a hypermetabolic state in the liver that is characterized by increase in mitochondrial respiration, which is driven by the great demand

The Protective Effect of Antioxidants in Alcohol Liver Damage 113

liver activity on transcriptional levels, interfering with RNA synthesis in the nucleus

Investigations that have been conducted have demonstrated that the acute as well as the chronic ethanol administration jeopardize the incorporation of thymidine into the DNA of hepatocytes of rats on which PH had been performed with or without diminution of the DNA contents, in addition to reporting that chronic consumption of this substance inhibits regeneration 24 hours after the PH due to delay in the induction of ornithine

It has been suggested that damage cause by FR as the product of ethanol consumption occurs at the early phase of HR; on the other hand, a transcending increase has been reported in mitochondrial lipoperoxidation of the liver in rats after PH. In the same study, a diminution was also observed in the early HR phase of mitochondrial glutathione levels

One factor that suggested that ethanol causes cell damage due to its hepatic metabolism is the excessive generation of FR, which can be the result of a state denominated oxidative stress; this is because any of ethanol's metabolic pathways, principally MEOS, is made up of chemicals oxido-reduction reactions, which produce highly unstable molecules called

1. Hydrogen abstraction, in which FR interact with another molecule that acts as the donor of an atom of H+. As a result, FR bind to H+ and become more stable, while the

2. Addiction. Because the FR binds to a more stable molecule, which converts a receiver

3. Termination. In this, two FR react between themselves to form a more stable

4. Disproportion. This consists of two LR that are identical to each other react between themselves. In this reaction, one FR acts as an electron donor and the other, as an

Free radicals are chemical species that possess an unpaired electron in their last layer, which allows these to react with a high number of molecules of all types, first oxidizing these and afterward attacking their structures. If lipids (polyunsaturated fatty acids) are involved, they damage the structures rich in the latter, such as cell membranes and lipoproteins (Rodríguez et al., 2001). Within this generic concept, the partially reduced forms of oxygen are denominated Reactive oxygen species (ROS). This is a collective term that includes not only

The oxidating mechanism of FR is intimately linked to their origin, which follows a sequence of chain reactions; in these reactions, a very reactive molecule is capable of reacting with another, non-reactive molecule, inducing in the latter the formation of a FR

electron receiver. In this manner, they become two more stable molecules.

oxygen free radicals, but also some reactive non-radical oxygen derivatives.

ready to initiate a new neutrophilic attack, and so on successively.

‒

), Hydrogen peroxide

Reactive oxygen species (ROS), such as the superoxide anion (O2

(H2O2), and the hydroxyl radical (OH•) (Nanji & French, 2003). FR can perform four main reactions (Wu & Cederbaum, 2003):

(Morales-González et al., 2001; Morales-González et al., 1999).

carbamoyltransferase activity (Yoshida et al., 1997).

(Guerrieri et al., 1998).

**12. Ethanol and free radicals** 

donor is converted into a FR.

molecule into a FR.

compound.

for NADH reoxidation produced during ethanol metabolism by cytosolic ADH (Adachi & Ishii, 2002); in addition, it alters hepatic microcirculation by stimulating endothelial-1 production (Thurman, 1998); similarly, the acetaldehyde generated by ethanol metabolism causes hypoxia on chemically reacting with free sulfate groups such as glutathione, in such as way as to alters the reaction of this metabolite, which activates the xantine oxidase and xantine dehydrogenase enzymes, in order to finally diminish the NAD+/NADH equilibrium (Gutiérrez-Salinas & Morales-González, 2004).

Depletion of antioxidant levels, above all that of hepatic glutathione, caused by acute as well as by chronic ethanol consumption, increases oxidative stress, which induces changes in the mitochondrial membrane, such as diminution of the mitochondrial membrane potential in hepatocytes and MPT, both inhibited by the antioxidants or by an ADH inhibitor (Adachi & Ishii 2002).
