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Revista médica de Chile

versión impresa ISSN 0034-9887

Rev. méd. Chile v.130 n.6 Santiago jun. 2002 

Rev Méd Chile 2002; 130: 681-690

Alcohol-induced liver disease:
From molecular damage to treatment

José C Fernández Checa1, Stefano Bellentani2,
Claudio Tiribelli2,3

Although the interaction between alcohol and the liver has been the subject of intensive investigation since many years, several uncertainties remain to be solved. Good examples of what we need to learn are: The real number of patients with alcohol-induced liver disease (AILD), the dose of alcohol "safe" for the liver, the genetic predisposition to the damage or, on the other side of the coin, protecting from the damage. Rather recently, however, part of these questions started to be clarified, thus permitting a better definition of the role of each of these factors in AILD. In parallel to the clinical approach to AILD, the unveiling of the molecular and biochemical mechanisms involved in AILD has progressed and proved to be important in both a better understanding of the disease and, more important, in a more rational treatment of these disorders. This review will focus on what we currently know of AILD in clinical, biochemical and molecular terms and what we need to address in the future (Rev Med Chile 2002; 130: 681-690).
(Key Words: Alcohol dehydrogenase; Alcohol-induced disorders; Cytokines; Liver diseases, alcoholic; Tumor necrosis factor)

Enfermedad hepática alcohólica: desde
el daño molecular a su tratamiento

Aunque la interacción entre el alcohol y el hígado ha sido un tema de intensa investigación desde hace muchos años, varias incertidumbres requieren todavía ser resueltas. Buenos ejemplos de lo que necesitamos aprender son: el número real de pacientes con enfermedad hepática alcohólica (EHA), la dosis "segura" de alcohol (que no dañaría el hígado), la predisposición genética para el daño hepático o, en la otra cara de la moneda, que protege de este daño. Sin embargo, recientemente comenzaron a aclararse algunas de estas preguntas, permitiendo una mejor definición del rol de cada uno de estos factores en la patogenia de la EHA. Paralelamente con el enfoque clínico de la EHA, la pesquisa de los mecanismos moleculares y bioquímicos involucrados en la EHA ha progresado, demostrando su importancia tanto para una mejor comprensión de la enfermedad como para diseñar su tratamiento más racional. Esta revisión actualiza los conocimientos clínicos, bioquímicos y moleculares sobre la EHA y bosqueja lo que debemos seguir investigando.

Manuscrito preparado por invitación de los Editores de la Revista.
Recibido el 23 de enero de 2002. El Dr. Tiribelli es miembro del
Comité Asesor Internacional de la Revista Médica de Chile.
1 Liver Unit, Instituto Malalties Digestives, Hospital Clinic i Provincial,
Instituto Investigaciones Biomédicas IDIBAPS, Consejo Superior
Investigaciones Científicas, Barcelona, Spain
2 Fondo Studio Malattie de Fegato-ONLUS, Trieste, Italy
3 CSF, Department BBCM, University of Trieste, Trieste, Italy.

The correlation between the ingestion of large amounts of alcohol and liver damage has been recognized since centuries. However, only recently has been clearly established the relationship between ethanol consumed and risk of cirrhosis, and the ultimate expression of alcohol-induced liver disease (AILD)1-3.


The true prevalence of AILD is not fully understood. In the Dionysos Study, AILD was observed in less that 5% of the population of Northern Italy but cirrhosis was found only in about 1%4,5 (Table 1). This figure is about one tenth of what was observed in autoptic studies performed over 25 years (about 90,000 autopsies) where cirrhosis was found in about 10%6. Cirrhosis was found to be associated with HCV infection in about 50%7 thus leaving to "pure" alcoholic damage something ranging between 40 and 50% of the series. Therefore, we should conclude that the clinical diagnosis of cirrhosis is usually done in 1 out of 5 cases (20%) indicating that what we observe in clinical practice is just a tiny portion of the problem ("the tip of the iceberg"). This is particularly important as it indicates that almost all the series reported are underestimating the true dimension of AILD.


One of the key questions in AILD was the definition of a dose above which AILD ensues. Dose-response curves between lifetime alcohol ingested and the relative risk of cirrhosis have been reported8,9 and there is concordance that the risk of developing cirrhosis increases exponentially with the amount of alcohol ingested throughout the subject’s lifetime10.

On the other hand, the risk threshold, that is the amount of alcohol ingested which separates the populations with near zero risk of cirrhosis from those where the risk is much greater, varies from series to series with substantial differences in the characteristics of the population studied as well as in study design (retrospective, prospective, case-control or cohort)9,10. In a cohort study like the Dionysos study4,11 the risk threshold for having AILD has been found to be 30 g of alcohol per day, and the risk for liver cirrhosis was directly proportional to the amount of alcohol consumed both every day and during lifetime (Figure 1). In spite of the axiom "No alcohol, no AILD", the epidemiological data correlating alcohol consumption and risk of AILD suggest that alcohol consumption might not be the only determinant of this disease. Clinical observations commonly suggest a wide individual susceptibility to AILD. Although alcohol consumption above the risk level is a condition sine qua non for the development of cirrhosis in a given population, only a small fraction of the heavy drinkers, ranging from 6 to 30%, develops the disease11,12. Thus, one is forced to postulate that the relation between alcohol consumption and cirrhosis is a multifunctional phenomenon, involving the interplay of other factors.

Figure 1. Alcohol consumption (g/day) and risk (expressed as Odd Ratio: OR) for liver cirrhosis. Data from the Dionysos study (ref. 11).


The search for other potential factors has been extensive but inconclusive, probably because only one factor (usually gender) has been studied at any given time13. Moreover, it has been impossible to determine if more than one factor acting simultaneously predisposes the heavy drinker to the various forms of AILD (steatosis, steatohepatitis, cirrhosis). Among the most investigated aspects in understanding the different susceptibility to AILD is the possible genetic involvement. Several studies linking human leucocyte antigens (HLA) with AILD concluded that none of the HLA phenotypes so far investigated in Caucasians can be shown to be significantly more common in AILD than in controls14. Other genes, like those encoding for alcohol dehydrogenase (ADH2, ADH3) and aldehyde dehydrogenase (AILDH2), as well as for the microsomal ethanol oxidation system [cytochrome P4502E1 (CYP2E1)] have been the subject of intensive investigations over the last years, with contradictory results. Studies performed in Japanese or Chinese populations indicated that the allele ADH2*2 of the ADH2 gene15, the allele AILDH2*2 of the AILDH2 gene16 and the allele C2 of the CYP 2E1 gene17 were more frequently associated with AILD. However, other investigations failed to confirm these findings18,19, possibly because they were performed in Caucasians. The effects of genetic variation in ADH2 and ADH3 on AILD showed that ADH3 variations seem to decrease significantly the risk of alcohol dependence up to 2-3 fold and to increase 2 fold the risk of AILD among alcoholics20. These conflicting results were mainly related to the rather small number of patients, their derivation from a selected series and not from an open population, and the difficulty of a precise definition of the disease. The Dionysos Study helped in shading some light on this complicated issue. The distribution of 9 different polymorphisms in 3 genes involved in alcohol metabolism (ADH2, ADH3, and CYP2E1) was investigated among the drinkers reporting comparable high amount of ethanol intake (more than 120 g/day for more than 10 years) but differing for the presence or absence of AILD. In the inhabitants of Campogalliano, the C2 allele in the promoter region of the CYP2E1 gene had a frequency significantly higher in heavy drinkers with AILD as compared with healthy heavy drinkers. This association became even higher in heavy drinkers with cirrhosis. In Cormons, whose inhabitants have different genetic derivation, a prominent association between AILD and homozygosity for allele ADH3*2 of ADH3 was observed, with a prevalence of 31% and 7% in heavy drinkers with or without AILD, respectively21. These results indicate that, depending on allelic frequencies of the population, the presence of either at least one allele C2 of cytochrome P4502E1 (in Campogalliano) or of the homozygosity for the ADH3*2 allele (in Cormons) are predisposing factors for the development of AILD. Heterozygosity for allele C2 of CYP2E1 and homozygosity for allele ADH3*2 of ADH3 seem to be independent risk factors for AILD in alcohol abusers, and the relative contribution of these genes to AILD is dependent on their allelic frequencies in the population screened. Accordingly, heavy drinkers not having either of these two genotypes were 3 and 4 times more protected from developing AILD, respectively. The identification of two genetic polymorphisms predisposing to AILD reinforces the notion that AILD is a multigenic disorder. Whether this genetic background may be extrapolated to a wider Caucasian population is still undefined and needs further studies.


One of the main problems in the study of AILD is the lack of a reliable animal model which prevents the unraveling of the mechanisms related to the development and the progression of the damage. Recent data suggest that the immortalized liver cell line (HepG2 cells) may be useful in assessing the mechanisms by which ethanol initiates the cascade ending to liver disease. Admittedly this model may be useful in the study of the first steps of the long chain of reactions leading to permanent and irreversible alteration in humans. When HepG2 cells were exposed to ethanol, a dose dependent reduction in cell viability was observed; no damage was found below 60 mM ethanol pointing to a threshold effect. Most interesting was the observation that ethanol was able to induce ultrastructural alterations (enlargement of the smooth endoplasmic reticulum, swelling of mitochondria and loss of cristae, and lipid accumulation in the cytoplasm) similar to those observed in humans22. These alterations were associated with an increased secretion of different cytokines such as IL1, IL6 and in particular TNF-a23 suggesting a role of cytokines in AILD. Of notice was the observation that the toxic effects of ethanol were mainly related to the production of TNF-a as indicated by the almost complete prevention when cells were added with anti- TNF-a antibody. Ethanol treatment was followed by a progressive reduction on the content of glutathione (GSH) in mitochondria which was prevented by bile acids (UDCA or TUDCA) with the abolishment of the damage23,24. These data confirm the role of cytokines observed in humans with AILD25-27 and suggest a potential beneficial effect of bile acid treatment.

Additional studies with HepG2 treated in vitro with acetaldehyde showed activation of transcription factors NF-kB and AP-1 by a mechanism independent of the generation of reactive oxygen species involving activation of protein kinase C28,29. These data identify a new role for acetaldehyde as an inflammatory agent leading to induction of cytokines and chemokines through activation of NF-kB. Collectively these studies illustrate the usefulness of in vitro models of AILD to discern and identify the putative players mediating the disturbing cellular effects of ethanol.


Alcohol-induced liver disease (AILD) is the result of a complex interplay of factors that cooperate in the functional failure of the liver to undertake its multiple tasks. As reported above, one of the reasons for the limited progress in elucidating the molecular pathways and mechanisms underlying the ethanol-induced liver damage has been the lack of convenient experimental models. Although malnutrition was first thought to act as an important driving factor conditioning the disease, with the development of experimental animal models, it was first recognized that AILD results from the dose and time dependent consumption of ethanol. The development of experimental models represented a significant advance in the characterization of individual factors that contribute to the progress of this condition that culminates in cirrhosis. In this section we will briefly overview the most significant experimental models developed to test the role of ethanol in AILD.

Lieber-De Carli liquid diet. More than four decades ago, an experimental model of chronic ethanol consumption incorporated in a liquid-diet balanced formula was developed30. In this setting, ethanol constitutes 36% of calories in the diet, isocalloricaly replaced by carbohydrates in the control diet. This pioneering study described the occurrence of steatosis induced by chronic ethanol consumption in rats despite the fact that the animals were well nourished, thus indicating that malnutrition was not required for the damaging effects of ethanol in the liver. With this model, though, the animals developed only the initial stage of liver steatosis, regardless of the duration of the feeding regimen. Despite the inability to reproduce other stages of the disease (inflammation, necrosis, fibrosis), this model has been very useful to test the role of ethanol on liver function, from altered lipid metabolism to energy production and mitochondrial function, to protein secretion31. In particular, this model has defined important morphological and functional alterations in mitochondria induced by ethanol intake describing an overall decreased ATP level in animals treated chronically with ethanol. The reduced ATP level results from a lower rate of ATP synthesis rather than from a decreased efficiency in the synthesis of ATP, since there is little or no change in the resting state respiration32. One of the major drawbacks of this model is that the amount of ethanol consumed per day is not sufficient to recruit the action of factors known to contribute to the progression of AILD, including endotoxin increase subsequent to intestinal permeabilization and the stimulation of Küpffer cells.

Intragastric enteral alcohol feeding model. A further development in this research area was the description of an intragastric enteral alcohol feeding model, by Tsukamoto and French. The intragastric infusion of a liquid diet containing alcohol over several months resulted in a time-dependent development of ethanol-induced liver damage including steatosis, inflammation, necrosis and fibrosis in the rat33,34. This model has been particularly useful to examine the temporal relationship of biochemical changes and their impact on liver dysfunction. One of the earliest changes was a progressive and selective depletion of mitochondrial GSH with the sparing of cytosolic GSH levels35. The depletion of mitochondrial GSH induced by alcohol intake preceded signs of lipid peroxidation, and the onset of inflammation and fibrosis detected after 9 and 16 weeks of ethanol feeding. These findings paralleled those described earlier with the Lieber-DeCarli model, pointing to the mitochondrial pool of GSH as a critical sensitization factor for the deleterious effects of alcohol36,37. Furthermore, using this model it has been shown that the inactivation of Küpffer cells with gadolinium chloride prevented early alcohol-induced liver damage, and that intestinal sterilization with antibiotics or decreasing endotoxin with lactobacillus prevented alcohol-induced liver injury38,39. In addition to the critical role of these factors in the initiation and progression of AILD, TNF-a has been pointed to as an-other key player in the initiation and progression of the disease and the early parenchymal damage induced by ethanol intake. TNF-a levels have been reported increased in the plasma of both patients and experimental models and the presence of antibodies against TNF-a ameliorates AILD in the intragastric enteral feeding model. In line with this conclusion is the observation that knockout mice deficient in the receptor for TNF-a, TNFR1 (p55) do not develop signs of AILD except for steatosis40. Thus, increased susceptibility of the liver towards TNF-a by chronic ethanol exposure stands as a primary factor contributing to the recruitment of additional players that cooperate in the induction of liver damage due to ethanol intake. While TNF-a stimulates the generation of toxic reactive oxygen species from mitochondrial complex III in parenchymal cells, it also induces the expression of factors for neutrophil chemotaxis (IL-8/CINC, MIP, MIP-2) and the expression of intracellular adhesion molecule-1, leading to microcirculatory disturbances, effects mediated by an activation of transcription factor NF-kB. The sensitivity to and regulation of NF-kB activation by TNF-a is in part controlled by the availability of GSH in the mitochondria36,41 and hence, the recovery and regulation of mitochondrial GSH emerges as a potential therapeutic target in AILD. In this regard, S-adenosyl-L-methionine (SAMe) or tauroursodeoxycholic acid (TUDCA) have been reported to prevent TNF-a-induced cell death in hepatocytes from chronic ethanol-fed rats42,43.

Experimental model of AILD in female rats. Al-though the intragastric enteral feeding model has been very useful to identify some of the factors involved in the pathogenesis of AILD, this experimental model is time-consuming, expensive and complex, requiring surgery, all limiting its use. To overcome this problem, it has recently been described a new, simpler rat model of early alcohol-induced liver injury44. Female rats respond to chronic ethanol intake with enhanced endotoxin levels exhibiting greater susceptibility to ethanol than male rats. Female rats were fed ad libitum an adequate liquid diet containing 32, 43 and 16% of calories as fat, carbohydrate and protein respectively, and were given a single dose of ethanol (5g/kg body weight intragastrically) every 24 hours for 6-8 weeks. The pathological alterations resembled that of the surgery-requiring Tsukamoto-French model33 including steatosis, inflammation and necrosis with the important exception that surgery was not required. The simplicity and the similarity of biochemical and pathological changes achieved with this experimental scheme to those of the enteral feeding model, made this new approach attractive to identify the chain of changes that mediate AILD. An important point derived from this model is that rats develop liver pathology due to ethanol intake despite the fact that carbohydrate contents were adequate. This contrasts with previous findings in which a low carbohydrate content potentiated ethanol-induced liver damage45.

Acute vs chronic synergism between endotoxin and ethanol in AILD. As alluded above, endotoxin (LPS) has been identified as an important factor in the pathogenesis of AILD acting as a potent inflammation trigger and stimulating the release of toxic mediators including reactive oxygen species, thus establishing a correlation between endotoxin levels and severity of AILD in patients and in the intragastric enteral model46,47. In previous studies, endotoxin was given as a bolus to rats fed Lieber-De Carli ethanol liquid diet for 6 weeks resulting in hepatocellular necrosis and inflammation48. Furthermore, under these circumstances there was a significant generation of TNF-a subsequent to the acute endotoxin exposure and chronic ethanol feeding, and the enhanced overproduction of TNF-a by LPS mediated the onset of necrosis and neutrophil infiltration. Since acute exposure to endotoxin represents a fundamentally different situation than that of alcoholics with chronically elevated endotoxin levels, a new approach has been recently described in which chronic ethanol-fed rats are infused chronically with endotoxin49. Under these conditions it was found that the combination of ethanol plus endotoxin attenuated rather than enhanced the generation of pro-inflammatory cytokines, eg. TNF-a and IL-1. These of IL-10, IL-4 and decreased expression of TGF-b 1 and CD14. Collectively, these findings were interpreted as an adaptation to the chronic exposure to LPS.


Although all the metabolic pathways of ethanol in mammalian cells are extramitochondrial, these are intimately linked with mitochondrial metabolism, since ethanol metabolism both oxidative and nonoxidative affects mitochondrial energy metabolism. Quantitatively, the liver is the major organ involved in the metabolic disposal of ethanol. Ethanol oxidation in the liver is catalyzed predominantly by a class I, pyrazole-sensitive alcohol dehydrogenase (ADH) in the cytosol of which different isoenzymes have been described50. This enzyme has a Km for ethanol in the sub to low millimolar range and saturates at ethanol concentrations that are readily achieved physiologically. Other isoforms for ADH have been also described, displaying a wide distribution, al-though their contribution to ethanol oxidation in vivo is unknown51. In addition to the ADH, the liver oxidizes ethanol through a cytochrome P450-linked pathway, which utilizes NADPH and molecular oxygen and is localized in the endoplasmic reticulum, which is referred in the literature as MEOS52. After a great deal of controversy centered on the contribution of this pathway in the overall rate of ethanol oxidation, it became clear that its quantitative significance is small in normal conditions but increases with prolonged intake of ethanol due to the selective induction by ethanol of a specific high Km cytochrome P450 isoform called P4502E1 which uses ethanol as a preferred substrate53. Ethanol oxidation also occurs in peroxisomes, mediated by catalase. The catalase-dependent pathway in the liver is limited by the supply of hydrogen peroxide, so that reactions that produce hydrogen peroxide in peroxisomes greatly enhance the rate of ethanol oxidation through this pathway. One of the most physiologically relevant processes that generate hydrogen peroxide is the (-oxidation of long chain fatty acids54. All oxidative routes for ethanol metabolism result in the formation of acetaldehyde. This metabolite is further metabolized to acetate, primarily by a low Km aldehyde dehydrogenase localized in the mitochondria. This enzyme is found in many tissues but is most abundant in the liver, where the rate of acetaldehyde formation is highest. The impairment of this activity, which is responsible for maintaining the acetaldehyde concentrations at very low levels, in selective population of Orientals is responsible for the aversion of these individuals to keep drinking to the accumulation of acetaldehyde. The impairment of the low Km acetaldehyde dehydrogenase in mitochondria from perivenous hepatocytes indicates the existence of an acetaldehyde gradient along the liver acinus being greater in the perivenous zone of the liver, the area where most of the ethanol-induced liver injury is seen in both alcoholic patients and experimental animal models55,56. Sequential acetaldehyde production, lipid peroxidation and fibrogenesis develop in a micropig model of alcohol-induced liver disease56-58. Nonoxidative pathways of ethanol oxidation include the formation of ethyl esters of long-chain fatty acids, mediated by isoenzymes of the glutathione S-transferases and by a cholesterol esterase59. These compounds have been reported to affect mitochondrial function acting as uncouplers, with a decreased state 3 respiration and increased state 4 respiration60. These fatty acid ethyl esters are detected in alcoholic patients in several tissues affected by long-term alcohol abuse61. Thus, both the oxidative and non-oxidative metabolism of alcohol generate the intermediates that initiate a cascade of events critical for the development of the illness including the targeting of mitochondria and subsequent dysfunction.

Several recognized factors known to contribute to the development of the illness are listed in Figure 2. An altered cellular redox potential reflected in the decreased NAD/NADH ratio and the generation of acetaldehyde, an extremely reactive chemical intermediate, due to the continuous oxidation of alcohol are potential mediators of alcohol-induced deleterious effects62. The former induce several metabolic disturbances, whereas the latter can react with nucleophilic moieties of proteins that can result in their inactivation or formation of stable acetaldehyde-protein adducts. The generation of antibodies against these adducts mediate the autoimmune injury in advanced states of the disease and have been described in the livers of alcoholic patients. Furthermore, the oxidative alcohol metabolism results in an overproduction of reactive oxygen species (ROS) than along with the depletion of antioxidant stores causes oxidative stress, a condition that is thought to mediate some of the damaging effects of ethanol. In addition to these factors, there is an important genetic component that may determine why some individuals develop the disease while others do not despite continued consumption of alcohol.

Figure 2. Metabolic events linking oxidative stress induced by alcohol to liver damage.

Hallmarks of alcohol-induced liver damage are structural and functional alterations of mitochondria. It was described more than 3 decades ago, both in human and animal models, that ethanol results in striking morphological alterations of hepatocellular mitochondria. Although the structural changes include reduced number of cristae and paracrystalline inclusions, one of the most prevalent changes induced by chronic alcohol intake is the enlargement in size of mitochondria resulting in the appearance of giant structures that have been named "megamitochondria". Indeed, these giant structures were first detected in alcoholic patients upon examination of liver histology by light microscopy revealing the presence of the megamitochondria that are clearly distinguishable from Mallory bodies63. Similar structural changes in mitochondria evoked by chronic ethanol intake are described in experimental animal models of the disease which have profound consequences for the mitochondrial dysfunction induced by prolonged periods of ethanol consumption64-67. One of the most dramatic consequences of continued alcohol intake is the impaired oxidative phosphorylation and consequent hepatocellular ATP production. Thus, the overall decreased ATP levels observed in animals treated chronically with ethanol results from a lower rate of ATP synthesis rather than from a decreased efficiency in the synthesis of ATP since there is little or no change in the resting state respiration indicating a lack of uncoupling by ethanol feeding.


As indicated above, the molecular, genetic and environmental mechanisms by which ethanol ingestion is associated with liver damage are still not fully unraveled in spite of the longstanding observation that increased alcohol consumption may be associated with liver disease. What is still missing is the linkage between the biochemical alterations induced by ethanol in different, rather simple in vivo and in vitro models and the much more complex human model where the relative contribution of inadequate alcohol intake is hard to be defined. However some important breakthroughs have been recently obtained, and these should be the basis of stringent clinical trials. AILD results of the interplay of numerous factors that recruit a variety of cellular and molecular mechanisms whereby ethanol afflicts liver function. The identification of individual factors and/or mechanisms that predispose or enhance the susceptibility to ethanol’s toxic liver consequences may be of relevance in designing new therapeutical protocols. In this regard, mitochondrial dysfunction has been recognized as an early and vital target of ethanol effects, in particular those mediating the selective depletion of GSH due to impaired transport of mitochondrial GSH. Restoration of the mitochondrial GSH pool depleted by chronic ethanol intake may thus stand as a potential therapeutic target in the management of AILD, and may actually determine the reported usefulness of hepatoprotective agents, including SAMe and TUDCA in the treatment of AILD. The elucidation of the mechanisms involved in the impairment of the mitochondrial transport of GSH by ethanol may open new therapeutical strategies to prevent its depletion by ethanol. This new approach, together with a genetic analysis, should provide important information on which subjects may be candidates to AILD and the most efficient treatment to prevent it.


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The work reported in this review was supported in part by the Research Center for Liver and Pancreatic Diseases, P50 AA11999, funded by the US National Institute on Alcohol Abuse and Alcoholism (JCFC), Plan Nacional de I+D, grants SAF 99-0138 and SAF01-2118 (JCFC), from the Ministry of Education (Rome, Italy) (CT) and grants from Fondo Studio Malattie Fegato-ONLUS (FCRT00/01) (CT and SB).

Correspondencia a: Claudio Tiribelli, MD. CSF - Dept. BBCM, University of Trieste. Via Giorgeri 1. 34127 Trieste - Italy. Phone +39-040-399 4927. Fax +39-040-399 4924. E-mail:

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