Maria A. Leo

Alcohol and the Liver

The mortality rates for cirrhosis vary greatly from country to country. For example, in 1972 the World Health Organization reported mortality rates of 7.5/100,000 in Finland and 57.2/100,000 in France. The corresponding per-capita alcohol consumptions were 5.1 and 16.8 liters of absolute alcohol (International Statistics on Alcoholic Beverages: Production Trade and Consumption 1950–1972, 1977). Whereas deaths from other causes such as cardiovascular diseases decreased in the period from 1950 to 1974 by 2% in the United States, deaths from cirrhosis climbed 71.7%. Since then, mortality from cirrhosis has leveled off or even decreased in the United States. The reduction, however, pertains primarily to nonalcoholic cirrhosis (Liver Cirrhosis Mortality in the United States, 1988). In Canada, cirrhosis has been the most rapidly increasing cause of death in the population over 25 years of age, followed by lung and bronchial cancer and suicide (Schmidt, 1977), and it is now the fifth major cause of death for men in the productive years from 25 to 64 (Rankin, 1977). The concentration of mortality of alcoholic cirrhosis to a rather young population group also occurs in the United States. In Baltimore, it was the fifth leading cause of death for persons 25 to 44 years of age (Kuller et al., 1969) and the fourth leading cause of death for those 25 to 64 years of age in New York City (New York City; Summary of Vital Statistics, 1984).

Phosphatidylcholine Protects Against Fibrosis and Cirrhosis in the Baboon

BACKGROUND/AIMS Polyunsaturated soybean lecithin (55%-60% phosphatidylcholine [PC]) protects against fibrosis in alcohol-fed baboons. The present study was undertaken to determine whether PC is the active agent. METHODS Virtually pure PC (equivalent to that contained in the lecithin) was administered for up to 6.5 years with or without alcohol, and the results were compared with those of unsupplemented groups. RESULTS Control livers remained normal, whereas 10 of 12 baboons fed alcohol without PC developed septal fibrosis or cirrhosis with transformation of 81% +/- 3% of the hepatic lipocytes to collagen-producing transitional cells. By contrast, none of the eight animals fed alcohol with PC developed septal fibrosis or cirrhosis, and only 48% +/- 9% of their lipocytes were transformed, indicating that PC was indeed the protective compound. Ethanol feeding also resulted in decreased liver phospholipids and PC, and both were corrected by the supplementation. Furthermore, PC stimulated collagenase activity in cultured lipocytes. This PC consisted of several species, mainly dilinoleoylphosphatidylcholine (40%-52%) and palmitoyl-linoleoylphosphatidylcholine (23%-24%). Only dilinoleoylphosphatidylcholine duplicated the effect of the PC on collagenase. Other species of PC, phosphatidylethanolamine, free fatty acids, or choline were without effect. CONCLUSIONS PC prevents alcohol-induced fibrosis and cirrhosis in nonhuman primates, and dilinoleoylphosphatidylcholine appears to be the active species, possibly by promoting collagen breakdown.

Silymarin retards the progression of alcohol-induced hepatic fibrosis in baboons.

Goal/Background Hepatoprotective effects of silymarin in patients with alcoholic liver disease are controversial. For strict control, this was assessed in non-human primates. Study Twelve baboons were fed alcohol with or without silymarin for 3 years with a nutritionally adequate diet. Results Silymarin opposed the alcohol-induced oxidative stress (assessed by plasma 4-hydroxynonenal) and the rise in liver lipids and circulating ALT. Alcohol also increased hepatic collagen type I by 50% over the 3 years with a significant rise in mRNA for &agr;1 (I) procollagen, both prevented by silymarin. There were corresponding morphologic changes: at 36 months, 2 of 6 animals fed alcohol had cirrhosis and 2 septal fibrosis, with perivenular fibrosis in 2, whereas with alcohol + silymarin, there was only 1 cirrhosis and 1 septal fibrosis, with perivenular fibrosis in 2, and virtually no lesions in the remaining 2. Conclusions Silymarin retards the development of alcohol-induced hepatic fibrosis in baboons, consistent with several positive clinical trials. The negative outcome observed in other trials possibly reflects poor compliance resulting in irregular or low silymarin intake. Thus, in view of the innocuity of silymarin, it might be advisable in future clinical studies to insure the controlled administration of sufficient amounts of silymarin.

Effect of chronic alcohol consumption on Hepatic SIRT1 and PGC-1α in rats

The nuclear genes, NAD-dependent deacetylase Sirtuis 1 (SIRT1) and the peroxisome proliferator-activated receptor-gamma coactivator1alpha (PGC-1alpha) are regulators of energy metabolism. Here, we studied the role of alcohol consumption in expression of these sensing molecules. Alcohol significantly reduced hepatic SIRT1 mRNA by 50% and PGC-1alpha mRNA by 46% and it significantly inhibited the protein expression of SIRT1 and PGC-1alpha, while the transcription factor PPAR-gamma remained unchanged. However, when the lipid composition of the alcohol diet was changed by replacing long-chain triglycerides (LCT) with medium chain triglycerides (MCT), SIRT1 and PGC-1alpha mRNA were restored to near control levels. This study demonstrates that alcohol reduces key energy sensing proteins and that replacement of LCT by MCT affects the transcription of these genes. Since there is a pathophysiological link between SIRT1 and PGC-1alpha and mitochondrial energy, the implication of the study is that mitochondrial dysfunction due to alcohol abuse can be treated by dietary modifications.

Gene expression profiling of alcoholic liver disease in the baboon (Papio hamadryas) and human liver

The molecular pathogenesis of alcoholic liver disease (ALD) is not well understood. Gene expression profiling has the potential to identify new pathways and altered molecules in ALD. Gene expression profiles of ALD in a baboon model and humans were compared using DNA arrays. Reverse transcriptase-polymerase chain reaction and immunohistochemistry were used for downstream analysis of array results. cDNA array analysis revealed differential expression of several novel genes and pathways in addition to genes known to be involved in ALD pathogenesis. Overall gene expression profiles were similar in both species, with a majority of genes involved with fibrogenesis and xenobiotic metabolism, as well as inflammation, oxidant stress, and cell signaling. Genes associated with stellate cell activation (collagens, matrix metalloproteinases, tissue inhibitors of matrix metalloproteinase) were up-regulated in humans. Decreased expression of several metallothioneins was unexpected. Fourteen molecules related to the annexin family were up-regulated, including annexin A1 and A2. Immunofluorescence revealed a marked overexpression of annexin A2 in proliferating bile duct cells, hepatocyte cell surface, and selective co-localization with CD14-positive cells in human ALD. The gene expression profile of ALD is dominated by alcohol metabolism and inflammation and differs from other liver diseases. Annexins may play a role in the progression of fibrosis in ALD.

Desmin distinguishes cultured fat-storing cells from myofibroblasts, smooth muscle cells and fibroblasts in the rat

To differentiate cultured rat liver myofibroblasts, fat-storing cells, aortic smooth muscle cells and skin fibroblasts from each other, desmin and vimentin stainings were undertaken by indirect immunofluorescence using monoclonal antibodies. In myofibroblasts, the reaction with antibodies to vimentin was positive but that with antibodies to desmin was virtually negative. In primary cultures as well as subsequent passage of fat-storing cells, reactions with antibodies to both desmin and vimentin were positive. In primary culture of smooth muscle cells, both reactions were positive, but in the first passage, smooth muscle cells lost the reactivity with antibodies to desmin. Fibroblasts showed a positive reaction with antibodies to vimentin and a negative one with antibodies to desmin. Thus, immunohistochemistry of intermediate filaments allows for the differentiation between fat-storing cells, which are desmin- and vimentin-positive, and myofibroblasts or fibroblasts, which are desmin-negative but vimentin-positive. Smooth muscle cells are also vimentin-positive and become desmin-negative after the first passage.

Ethanol metabolism in vivo by the microsomal ethanol-oxidizing system in deermice lacking alcohol dehydrogenase (ADH)

To assess the importance of non-ADH ethanol metabolism, ADH-negative and ADH-positive deermice were fed liquid diets containing ethanol or isocaloric carbohydrate for 2-4 weeks. Blood ethanol disappearance rate increased significantly after chronic ethanol feeding in both strains. Although at low ethanol concentrations (between 5 and 10 mM) there was no significant difference between ethanol-fed and pair-fed control animals, at high ethanol concentrations (between 40 and 70 mM) blood ethanol elimination rates were increased significantly after chronic ethanol feeding in both ADH-positive and ADH-negative animals. There was no significant effect of the catalase inhibitor 3-amino-1,2,4-triazole on the ethanol elimination/rates in both strains. Whereas catalase and ADH activities were not altered after chronic ethanol treatment, the activity of the microsomal ethanol-oxidizing system (MEOS) was enhanced three to four times in both strains, and microsomal cytochrome P-450 content was also increased significantly. When MEOS activity was expressed per cytochrome P-450 content, it was higher in ADH-negative than in ADH-positive animals, and it increased after ethanol administration. When microsomal proteins were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, ethanol-fed animals had a distinct band which reflected the increase in microsomal cytochrome P-450 content and seemed to reflect a unique form of cytochrome P-450 induced by ethanol. Thus, despite the absence of the ADH pathway, a large amount of ethanol was metabolized by MEOS in ADH-negative deermice; this was associated with increased blood ethanol elimination rates, enhanced MEOS activity, and quantitative and qualitative changes of cytochrome P-450.

Metabolism and metabolic effects of ethanol, including interaction with drugs, carcinogens and nutrition

Different pathways of alcohol metabolism, the alcohol dehydrogenase pathway, the microsomal ethanol-oxidizing system and the catalase pathway are discussed. Alcohol consumption leads to accelerated ethanol metabolism by different mechanisms including an increased microsomal function. Microsomal induction leads to interactions of ethanol with drugs, hepatotoxic agents, steroids, vitamins and to an increased activation of mutagens/carcinogens. A number of ethanol-related complications may be explained by the production of its first metabolite, acetaldehyde, such as alterations of mitochondria, increased lipid peroxidation and microtubular alterations with its adverse effects on various cellular activities, including disturbances of cell division. Nutritional factors in alcoholics such as malnutrition are discussed especially with respect to its possible relation to cancer.

Interaction of drugs and retinol

In liver microsomes of ethanol-fed rats, retinol competitively inhibited the hydroxylation of aniline, the demethylation of dimethylnitrosamine, and the oxidation of ethanol to acetaldehyde, whereas the inhibition of benzphetamine demethylation was of the mixed type in microsomes of phenobarbital-treated, ethanol-treated or control rats. Conversely, benzphetamine exerted a striking inhibition of the 4-hydroxylation of retinol in microsomes of phenobarbital-treated rats. At the concentration used, ethanol (100 mM) and dimethylnitrosamine (10 mM) had no such effect. In vivo administration of phenobarbital resulted in a 9-fold increase in the Vmax of the microsomal retinol 4-hydroxylase activity, with a 3-fold increase of the Km, whereas ethanol feeding resulted in a doubling of the Vmax with no significant change in the Km. The induction of this microsomal retinol-metabolizing system may contribute to the hepatic vitamin A depletion that has been reported previously after either ethanol or drug administration. Conversely, the observed inhibition, by retinol, of microsomal drug metabolism, including the demethylation of dimethylnitrosamine, may be of significance with regards to the interaction of retinol with carcinogenesis.

Lycopene attenuates alcoholic apoptosis in HepG2 cells expressing CYP2E1.

To test the hypothesis that ethanol-induced hepatic apoptosis is secondary to the oxidative stress generated by cytochrome P4502E1 (CYP2E1), we assessed the effects of the carotenoid lycopene, a potent antioxidant extracted from tomatoes, on oxidative stress and apoptosis in HepG2 cells overexpressing CYP2E1 (2E1 cells). These were exposed for 5 days to 100mM ethanol and 10 microM lycopene or an equal volume of placebo (vehicle). Ethanol significantly increased apoptosis measured by flow cytometry and by TUNEL assay. This was accompanied by an ethanol-induced oxidative stress: hydrogen peroxide production was significantly increased and mitochondrial GSH was strikingly decreased. Both were restored by lycopene, with a significant decrease in apoptosis. The placebo had no protective effect. In conclusion, Lycopene opposes the ethanol-induced oxidative stress and apoptosis in 2E1 cells. The parallelism between these effects suggests a causal link. Furthermore, these beneficial effects and the innocuity of lycopene now justify an in vivo trial.