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Folate deficiency, methionine metabolism, and alcoholic liver disease
Alcohol 27 (2002) 169–172
Folate deficiency, methionine metabolism, and alcoholic liver disease Charles H. Halsted*, Jesus A. Villanueva, Angela M. Devlin University of California, Davis, Davis, CA 95616, USA Received 4 February 2002; received in revised form 22 March 2002; accepted 22 March 2002
Abstract Methionine metabolism is regulated by folate, and both folate deficiency and abnormal hepatic methionine metabolism are recognized features of alcoholic liver disease (ALD). Previously, histological features of ALD were induced in castrated male micropigs fed diets containing ethanol at 40% of kilocalories for 12 months, whereas in male micropigs fed the same diets for 12 months abnormal methionine metabolism and hepatocellular apoptosis developed. Folate deficiency may promote the development of ALD by accentuating abnormal methionine metabolism. Intact male micropigs received eucaloric diets that were folate sufficient, folate deficient, or each containing 40% of kilocalories as ethanol for 14 weeks. Folate deficiency alone reduced hepatic folates by one half, and ethanol feeding alone reduced methionine synthase, S-adenosylmethionine (SAM), and glutathione (GSH) levels and elevated plasma malondialdehyde (MDA) levels. The combined regimen elevated plasma homocysteine, hepatic S-adenosylhomocysteine (SAH), urinary 8-hydroxy-2-deoxyguanosine (oxy8dG), an index of DNA oxidation, and serum aspartate aminotransferase (AST) levels. Terminal hepatic histopathologic characteristics included typical features of steatonecrosis and focal inflammation in pigs fed the combined diet, with no changes in the other groups. Hepatic SAM levels correlated with those of GSH, whereas urinary oxy8dG and plasma MDA levels correlated with the SAM:SAH ratio and to hepatic GSH. The results demonstrate the linkage of abnormal methionine metabolism to products of DNA and lipid oxidation and to liver injury. The finding of steatonecrosis and focal inflammation only in the combined diet group supports the suggestion that folate deficiency promotes and folate sufficiency protects against the early onset of methionine cycle–mediated ALD. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Folate; Methionine; Alcohol; Liver injury
1. Introduction Alcohol provides 7.1 kcal/g, and when consumed in excess it affects the availability and hepatic metabolism of many nutrients (Lieber, 1995). Folate deficiency is the most common micronutrient deficiency in individuals who are chronically dependent on alcohol, and it occurs in up to 80% of those at risk for developing alcoholic liver disease (ALD) (Eichner & Hillman, 1971; Leevy et al., 1965; Wu et al., 1975). Folate deficiency in persons who are chronically dependent on excessive amounts of alcohol results from poor diet and combinations of decreased folate absorption and hepatic uptake, and increased renal excretion (Halsted et al., 1971; Herbert et al., 1963; McMartin et al., 1989; Romero et al., 1981; Russell et al., 1983; Tamura & Halsted, 1983; Tamura et al., 1981).
Abnormal hepatic methionine metabolism is regulated by dietary folate and is integral to the development of ALD. 5-Methyltetrahydrofolate (5-MTHF) and homocysteine are substrates with co-factor vitamin B12 for methionine synthase (MS) in the production of endogenous methionine, which is a substrate for methionine adenosyltransferase (MAT1) in the daily hepatic production of 6-8 / of S-adenosylmethionine (SAM). S-adenosylmethionine regulates a number of methionine cycle pathways that are perturbed in ALD, including the generation of glutathione (GSH) from homocysteine (Lu, 1998; Tsukamoto & Lu, 2001). The ratio of SAM to its product S-adenosylhomocysteine (SAH) is considered a comprehensive measure of inhibition of functional SAM activity (Mato et al., 1994). 2. Summary of published work
* Corresponding author. Division of Clinical Nutrition, TB156, School of Medicine, One Shields Avenue, University of California, Davis, Davis, CA 95616, USA. Tel.: 1-530-752-6778; fax: 1-530-752-3470. E-mail address: [email protected] (C.H. Halsted). Editor: T.R. Jerrells
We developed an animal model of ALD in the micropig, a species that consumes ethanol voluntarily in the diet. In our original studies, castrated male Yucatan micropigs were fed 40% of kilocalories as ethanol or cornstarch control and all essential nutrients, including an excess of folic acid.
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Among the ethanol-fed micropigs, we observed typical features of alcoholic liver injury after 12 months of feeding and cirrhosis after 21 months, together with the accumulation of protein adducts of the alcohol metabolite acetaldehyde and the lipid oxidant product malondialdehyde (MDA) (Halsted et al., 1993; Niemela et al., 1995). There were no histological changes in a subsequent study of intact and uncastrated micropigs fed the same diets for 12 months, which we ascribed to changes in testosterone secretion (Halsted et al., 1996; Niemela et al., 1999). However, 12 months of feeding ethanol at 40% of kilocalories reduced the activity of hepatic MS and the SAM:SAH ratio, while causing DNA nucleotide imbalance and increasing hepatocellular apoptosis (Halsted et al., 1996). 3. Summary of ongoing work Our ongoing work tests the hypothesis that folate deficiency promotes both abnormal hepatic methionine metabolism and the development of ALD. This hypothesis is based on evidence for the frequent association of folate deficiency in chronic alcoholism, prior evidence for disturbed methionine metabolism in ALD, and the essential role of folate in hepatic methionine metabolism. Twenty-four intact male micropigs were grouped to receive four different diets for 14 weeks. The diets were identical to those used in prior studies, containing 90 kcal per kilogram of body weight per day as polyunsaturated corn oil at 33% of kilocalories, protein as vitamin-free casein at 2 g per kilogram of body weight per day, and cornstarch as carbohydrate, or the same diet substituting ethanol for cornstarch at 40% of kilocalories. Methionine, choline, and all essential minerals and vitamins were provided with or without folic acid at 14.5 g per kilogram of body weight in accord with the established requirements of growing swine (Subcommittee on Swine Nutrition, Committee on Animal Nutrition, National Research Council, 1998). The U.C. Davis Animal Welfare Committee approved the feeding protocol and all experimental studies. Animals were housed in individual kennels at the University of California, Davis Animal Resources Center facilities, which are approved by the National Institutes of Health, and animal care followed the standards and procedures outlined in the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council, 1996). All data described below reached statistical significance according to multivariate analysis. During the 14 weeks of the experiment, micropigs in the control group gained more than twice the weight of each of the other three groups. Mean plasma homocysteine levels were increased in all three experimental groups from week 6 onward and were maximal in animals fed the combined folate-deficient and ethanol diet over those in the control group. Terminal hepatic folate levels were decreased by one half in groups fed the folate-deficient diets with or without ethanol. Methionine synthase activity and hepatic methionine, SAM, and GSH levels were decreased in animals in
both ethanol-fed groups, whereas SAH levels were increased and the SAM:SAH ratio was decreased in animals fed the combined diet. Plasma MDA levels were increased in ethanol-fed animals, whereas urinary 8-hydroxy-2-deoxyguanosine (oxy8dG), a measure of DNA oxidation, and serum aspartate aminotransferase (AST) levels were increased in animals fed the combined diet. Hepatic SAM levels correlated with hepatic GSH levels, whereas urinary oxy8dG and plasma MDA levels correlated with the SAM:SAH ratio and to hepatic GSH levels. Livers from 5 of 6 animals in the combined group demonstrated moderate to marked intralobular steatohepatitis; there were no changes in the other groups. In summary, the findings showed both separate and combined effects of folate deficiency and ethanol feeding on the hepatic methionine cycle. The findings that the combined diet enhanced plasma homocysteine levels, reduced the SAM:SAH ratio, and increased both urinary oxy8dG and plasma AST levels together with characteristic histopathologic findings support the suggestion that folate deficiency increases the risk of oxidative liver injury in chronic alcoholism and supports the concept that perturbations in the methionine cycle contribute to the pathogenesis of ALD. 4. Summary of published literature Results of studies with other experimental animal models have demonstrated many effects of ethanol feeding on the hepatic methionine cycle. For example, reduced hepatic MS activity and SAM levels were found in rodent models by three independent groups (Barak et al., 1987; Finkelstein et al., 1974; Trimble et al., 1993). The intragastric ethanol-fed rat model demonstrated decreased hepatic levels of methionine and SAM, together with decreased hepatic MAT1 (Lu et al., 2000). The administration of SAM attenuated decreased GSH levels and the experimental development of ALD in ethanolfed baboons, as well as improved the clinical status of patients with varied degrees of ALD (Lieber et al., 1990; Mato et al., 1999). S-adenosylmethionine promotes the fluidity of the mitochondrial membrane and the transport of intracellular GSH to its mitochondrial site of activity (Colell et al., 1997). S-adenosylmethionine is reduced through free radical modification of cysteine residue 121 in MAT (Avila et al., 1998), whereas conversely, GSH maintains MAT1 activity (Corrales et al., 1991). Hepatic SAM levels were markedly reduced, and GSH levels were increased, in a recently described mouse MAT1A knockout model, in which hepatic steatosis developed together with an enhancement of gene expression of many inflammatory markers in these mice (Lu et al., 2001). Although results of previous studies with ethanol-fed rodents and micropigs showed altered hepatic methionine metabolism together with nucleotide imbalance, DNA global hypomethylation and strand breaks, and hepatocellular apoptosis (Halsted et al., 1996; Lu et al., 2000), the dietary induction of folate, methionine, and choline deficiency promoted hepatic nucleotide imbalance, DNA strand breaks, and apoptosis in rat liver (James et al., 1997).
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5. Future directions Future work should be directed at defining the mechanisms for induction of oxidative liver injury by ethanol and folate deficiency, both singly and in combination. Potential mechanisms include SAM-regulated effects on oxidative defense, enhancement of hepatocellular apoptosis through the effects of both ethanol and folate deficiency on DNA integrity, and potential effects of both factors on the cytokineregulated inflammatory response. Results of prior work support each or all of these potential mechanisms. In addition to the chronic effects of ethanol on SAM and antioxidative GSH activity in the rat and baboon (Colell et al., 1997; Corrales et al., 1991; Lieber et al., 1990; Lu et al., 2000) and independently observed effects of ethanol feeding, folate deficiency, or both on DNA integrity and hepatocellular apoptosis (Halsted et al., 1996; James et al., 1997; Lu et al., 2000), recent study findings support the suggestion that cellular homocysteine triggers a proinflammatory response that may be mediated through nuclear factor-kappa B (NF-B). Pertinent data in this regard include findings that hepatic NF-B activation is associated with oxidation and precedes the inflammatory response in ethanol-fed rats (Jokelainen et al., 2001; Nanji et al., 1999) and that homocysteine elevation stimulates reactive oxygen species (ROS) production and activates NF-B in HepG2 cells cultured in folate-deficient media (Chern et al., 2001). Acknowledgments This work was supported by grants DK-35747 and DK45301 from the National Institutes of Health. We are indebted to S. Jill James and S. Melnyk, National Toxicological Research Center, for assays of methionine metabolites, and to L. Wallock and M. Shigenaga, Children’s Hospital of Oakland Research Institute, for assays of urinary oxy8dG and plasma MDA. References Avila, M. A., Corrales, F. J., Ruiz, F., Sanchez-Gongora, E., Mingorance, J., Carretero, M. V., & Mato, J. M. (1998). Specific interaction of methionine adenosyltransferase with free radicals. Biofactors 8, 27–32. Barak, A. J., Beckenhauer, H. C., Tuma, D. J., & Badakhsh, S. (1987). Effects of prolonged ethanol feeding on methionine metabolism in rat liver. Biochem Cell Biol 65, 230–233. Chern, C. L., Huang, R. F., Chen, Y. H., Cheng, J. T., & Liu, T. Z. (2001). Folate deficiency–induced oxidative stress and apoptosis are mediated via homocysteine-dependent overproduction of hydrogen peroxide and enhanced activation of NF-kappaB in human Hep G2 cells. Biomed Pharmacother 55, 434–442. Colell, A., García-Ruiz, C., Morales, A., Ballesta, A., Ookhtens, M., Rodés, J., Kaplowitz, N., & Fernández-Checa, J. C. (1997). Transport of reduced glutathione in hepatic mitochondria and mitoplasts from ethanol-treated rats: effect of membrane physical properties and S-adenosyl-L-methionine. Hepatology 26, 699–708. Corrales, F., Ochoa, P., Rivas, C., Martin-Lomas, M., Mato, J. M., & Pajares, M. A. (1991). Inhibition of glutathione synthesis in the liver leads to S-adenosyl-L-methionine synthetase reduction. Hepatology 14, 528–533.
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Eichner, E. R., & Hillman, R. S. (1971). The evolution of anemia in alcoholic patients. Am J Med 50, 218–232. Finkelstein, J. D., Cello, J. P., & Kyle, W. E. (1974). Ethanol-induced changes in methionine metabolism in rat liver. Biochem Biophys Res Commun 61, 525–531. Halsted, C. H., Robles, E. A., & Mezey, E. (1971). Decreased jejunal uptake of labeled folic acid (3 H-PGA) in alcoholic patients: roles of alcohol and nutrition. N Engl J Med 285, 701–706. Halsted, C. H., Villanueva, J., Chandler, C. J., Ruebner, B., Munn, R. J., Parkkila, S., & Niemela, O. (1993). Centrilobular distribution of acetaldehyde and collagen in the ethanol-fed micropig. Hepatology 18, 954– 960. Halsted, C. H., Villanueva, J., Chandler, C. J., Stabler, S. P., Allen, R. H., Muskhelishvili, L., James, S. J., & Poirier, L. (1996). Ethanol feeding of micropigs alters methionine metabolism and increases hepatocellular apoptosis and proliferation. Hepatology 23, 497–505. Herbert, V., Zalusky, R., & Davidson, C. S. (1963). Correlation of folate deficiency with alcoholism and associated macrocytosis, anemia, and liver disease. Ann Intern Med 58, 977–988. Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council. (1996). Guide for the Care and Use of Laboratory Animals. Washington, DC: National Academy Press. James, S. J., Miller, B. J., Basnakian, A. G., Pogribny, I. P., Pogribna, M., & Muskhelishvili, L. (1997). Apoptosis and proliferation under conditions of deoxynucleotide pool imbalance in liver of folate/methyl deficient rats. Carcinogenesis 18, 287–293. Jokelainen, K., Reinke, L. A., & Nanji, A. A. (2001). Nf-kappab activation is associated with free radical generation and endotoxemia and precedes pathological liver injury in experimental alcoholic liver disease. Cytokine 16, 36–39. Leevy, C. M., Cardi, L., Frank, O., Gellene, R., & Baker, H. (1965). Incidence and significance of hypovitaminemia in a randomly selected municipal hospital population. Am J Clin Nutr 17, 259–271. Lieber, C. S. (1995). Medical disorders of alcoholism. N Engl J Med 333, 1058–1065. Lieber, C. S., Casini, A., DeCarli, L. M., Kim, C. I., Lowe, N., Sasaki, R., & Leo, M. A. (1990). S-adenosyl-L-methionine attenuates alcoholinduced liver injury in the baboon. Hepatology 11, 165–172. Lu, S. C. (1998). Methionine adenosyltransferase and liver disease: it’s all about SAM [editorial; comment]. Gastroenterology 114, 403–407. Lu, S. C., Alvarez, L., Huang, Z. Z., Chen, L., An, W., Corrales, F. J., Avila, M. A., Kanel, G., & Mato, J. M. (2001). Methionine adenosyltransferase 1A knockout mice are predisposed to liver injury and exhibit increased expression of genes involved in proliferation. Proc Natl Acad Sci U S A 98, 5560–5565. Lu, S. C., Huang, Z. Z., Yang, H., Mato, J. M., Avila, M. A., & Tsukamoto, H. (2000). Changes in methionine adenosyltransferase and S-adenosylmethionine homeostasis in alcoholic rat liver. Am J Physiol Gastrointest Liver Physiol 279, G178–G185. Mato, J. M., Alvarez, L., Ortiz, P., Mingorance, J., Durán, C., & Pajares, M. A. (1994). S-adenosyl-L-methionine synthetase and methionine metabolism deficiencies in cirrhosis. Adv Exp Med Biol 368, 113–117. Mato, J. M., Camara, J., Fernandez de Paz, J., Caballeria, L., Coll, S., Caballero, A., Garcia-Buey, L., Beltran, J., Benita, V., Caballeria, J., Sola, R., Moreno-Otero, R., Barrao, F., Martin-Duce, A., Correa, J. A., Pares, A., Barrao, E., Garcia-Magaz, I., Puerta, J. L., Moreno, J., Boissard, G., Ortiz, P., & Rodes, J. (1999). S-adenosylmethionine in alcoholic liver cirrhosis: a randomized, placebo-controlled, double-blind, multicenter clinical trial. J Hepatol 30, 1081–1089. McMartin, K. E., Collins, T. D., Eisenga, B. H., Fortney, T., Bates, W. R., & Bairnsfather, L. (1989). Effects of chronic ethanol and diet treatment on urinary folate excretion and development of folate deficiency in the rat. J Nutr 119, 1490–1497. Nanji, A. A., Jokelainen, K., Rahemtulla, A., Miao, L., Fogt, F., Matsumoto, H., Tahan, S. R., & Su, G. L. (1999). Activation of nuclear factor kappa B and cytokine imbalance in experimental alcoholic liver disease in the rat. Hepatology 30, 934–943.
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Niemela, O., Parkkila, S., Pasanen, M., Viitala, K., Villanueva, J. A., & Halsted, C. H. (1999). Induction of cytochrome P450 enzymes and generation of protein–aldehyde adducts are associated with sex-dependent sensitivity to alcohol-induced liver disease in micropigs. Hepatology 30, 1011–1017. Niemela, O., Parkkila, S., Yla-Herttuala, S., Villanueva, J., Ruebner, B., & Halsted, C. H. (1995). Sequential acetaldehyde production, lipid peroxidation, and fibrogenesis in micropig model of alcohol-induced liver disease. Hepatology 22, 1208–1214. Romero, J. J., Tamura, T., & Halsted, C. H. (1981). Intestinal absorption of [3H]folic acid in the chronic alcoholic monkey. Gastroenterology 80, 99–102. Russell, R. M., Rosenberg, I. H., Wilson, P. D., Iber, F. L., Oaks, E. B., Giovetti, A. C., Otradovec, C. L., Karwoski, P. A., & Press, A. W. (1983). Increased urinary excretion and prolonged turnover time of folic acid during ethanol ingestion. Am J Clin Nutr 38, 64–70.
Subcommittee on Swine Nutrition, Committee on Animal Nutrition, National Research Council. (1998). Nutrient Requirements of Swine: 10th Revised Edition. Washington, DC: Board on Agriculture and Natural Resources. Tamura, T., & Halsted, C. H. (1983). Folate turnover in chronically alcoholic monkeys. J Lab Clin Med 101, 623–628. Tamura, T., Romero, J. J., Watson, J. E., Gong, E. J., & Halsted, C. H. (1981). Hepatic folate metabolism in the chronic alcoholic monkey. J Lab Clin Med 97, 654–661. Trimble, K. C., Molloy, A. M., Scott, J. M., & Weir, D. G. (1993). The effect of ethanol on one-carbon metabolism: increased methionine catabolism and lipotrope methyl-group wastage. Hepatology 18, 984–989. Tsukamoto, H., & Lu, S. C. (2001). Current concepts in the pathogenesis of alcoholic liver injury. FASEB J 15, 1335–1349. Wu, A., Chanarin, I., Slavin, G., & Levi, A. J. (1975). Folate deficiency in the alcoholic—its relationship to clinical and haematological abnormalities, liver disease and folate stores. Br J Haematol 29, 469–478.
Folate deficiency, methionine metabolism, and alcoholic liver disease Charles H. Halsted*, Jesus A. Villanueva, Angela M. Devlin University of California, Davis, Davis, CA 95616, USA Received 4 February 2002; received in revised form 22 March 2002; accepted 22 March 2002
Abstract Methionine metabolism is regulated by folate, and both folate deficiency and abnormal hepatic methionine metabolism are recognized features of alcoholic liver disease (ALD). Previously, histological features of ALD were induced in castrated male micropigs fed diets containing ethanol at 40% of kilocalories for 12 months, whereas in male micropigs fed the same diets for 12 months abnormal methionine metabolism and hepatocellular apoptosis developed. Folate deficiency may promote the development of ALD by accentuating abnormal methionine metabolism. Intact male micropigs received eucaloric diets that were folate sufficient, folate deficient, or each containing 40% of kilocalories as ethanol for 14 weeks. Folate deficiency alone reduced hepatic folates by one half, and ethanol feeding alone reduced methionine synthase, S-adenosylmethionine (SAM), and glutathione (GSH) levels and elevated plasma malondialdehyde (MDA) levels. The combined regimen elevated plasma homocysteine, hepatic S-adenosylhomocysteine (SAH), urinary 8-hydroxy-2-deoxyguanosine (oxy8dG), an index of DNA oxidation, and serum aspartate aminotransferase (AST) levels. Terminal hepatic histopathologic characteristics included typical features of steatonecrosis and focal inflammation in pigs fed the combined diet, with no changes in the other groups. Hepatic SAM levels correlated with those of GSH, whereas urinary oxy8dG and plasma MDA levels correlated with the SAM:SAH ratio and to hepatic GSH. The results demonstrate the linkage of abnormal methionine metabolism to products of DNA and lipid oxidation and to liver injury. The finding of steatonecrosis and focal inflammation only in the combined diet group supports the suggestion that folate deficiency promotes and folate sufficiency protects against the early onset of methionine cycle–mediated ALD. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Folate; Methionine; Alcohol; Liver injury
1. Introduction Alcohol provides 7.1 kcal/g, and when consumed in excess it affects the availability and hepatic metabolism of many nutrients (Lieber, 1995). Folate deficiency is the most common micronutrient deficiency in individuals who are chronically dependent on alcohol, and it occurs in up to 80% of those at risk for developing alcoholic liver disease (ALD) (Eichner & Hillman, 1971; Leevy et al., 1965; Wu et al., 1975). Folate deficiency in persons who are chronically dependent on excessive amounts of alcohol results from poor diet and combinations of decreased folate absorption and hepatic uptake, and increased renal excretion (Halsted et al., 1971; Herbert et al., 1963; McMartin et al., 1989; Romero et al., 1981; Russell et al., 1983; Tamura & Halsted, 1983; Tamura et al., 1981).
Abnormal hepatic methionine metabolism is regulated by dietary folate and is integral to the development of ALD. 5-Methyltetrahydrofolate (5-MTHF) and homocysteine are substrates with co-factor vitamin B12 for methionine synthase (MS) in the production of endogenous methionine, which is a substrate for methionine adenosyltransferase (MAT1) in the daily hepatic production of 6-8 / of S-adenosylmethionine (SAM). S-adenosylmethionine regulates a number of methionine cycle pathways that are perturbed in ALD, including the generation of glutathione (GSH) from homocysteine (Lu, 1998; Tsukamoto & Lu, 2001). The ratio of SAM to its product S-adenosylhomocysteine (SAH) is considered a comprehensive measure of inhibition of functional SAM activity (Mato et al., 1994). 2. Summary of published work
* Corresponding author. Division of Clinical Nutrition, TB156, School of Medicine, One Shields Avenue, University of California, Davis, Davis, CA 95616, USA. Tel.: 1-530-752-6778; fax: 1-530-752-3470. E-mail address: [email protected] (C.H. Halsted). Editor: T.R. Jerrells
We developed an animal model of ALD in the micropig, a species that consumes ethanol voluntarily in the diet. In our original studies, castrated male Yucatan micropigs were fed 40% of kilocalories as ethanol or cornstarch control and all essential nutrients, including an excess of folic acid.
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Among the ethanol-fed micropigs, we observed typical features of alcoholic liver injury after 12 months of feeding and cirrhosis after 21 months, together with the accumulation of protein adducts of the alcohol metabolite acetaldehyde and the lipid oxidant product malondialdehyde (MDA) (Halsted et al., 1993; Niemela et al., 1995). There were no histological changes in a subsequent study of intact and uncastrated micropigs fed the same diets for 12 months, which we ascribed to changes in testosterone secretion (Halsted et al., 1996; Niemela et al., 1999). However, 12 months of feeding ethanol at 40% of kilocalories reduced the activity of hepatic MS and the SAM:SAH ratio, while causing DNA nucleotide imbalance and increasing hepatocellular apoptosis (Halsted et al., 1996). 3. Summary of ongoing work Our ongoing work tests the hypothesis that folate deficiency promotes both abnormal hepatic methionine metabolism and the development of ALD. This hypothesis is based on evidence for the frequent association of folate deficiency in chronic alcoholism, prior evidence for disturbed methionine metabolism in ALD, and the essential role of folate in hepatic methionine metabolism. Twenty-four intact male micropigs were grouped to receive four different diets for 14 weeks. The diets were identical to those used in prior studies, containing 90 kcal per kilogram of body weight per day as polyunsaturated corn oil at 33% of kilocalories, protein as vitamin-free casein at 2 g per kilogram of body weight per day, and cornstarch as carbohydrate, or the same diet substituting ethanol for cornstarch at 40% of kilocalories. Methionine, choline, and all essential minerals and vitamins were provided with or without folic acid at 14.5 g per kilogram of body weight in accord with the established requirements of growing swine (Subcommittee on Swine Nutrition, Committee on Animal Nutrition, National Research Council, 1998). The U.C. Davis Animal Welfare Committee approved the feeding protocol and all experimental studies. Animals were housed in individual kennels at the University of California, Davis Animal Resources Center facilities, which are approved by the National Institutes of Health, and animal care followed the standards and procedures outlined in the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council, 1996). All data described below reached statistical significance according to multivariate analysis. During the 14 weeks of the experiment, micropigs in the control group gained more than twice the weight of each of the other three groups. Mean plasma homocysteine levels were increased in all three experimental groups from week 6 onward and were maximal in animals fed the combined folate-deficient and ethanol diet over those in the control group. Terminal hepatic folate levels were decreased by one half in groups fed the folate-deficient diets with or without ethanol. Methionine synthase activity and hepatic methionine, SAM, and GSH levels were decreased in animals in
both ethanol-fed groups, whereas SAH levels were increased and the SAM:SAH ratio was decreased in animals fed the combined diet. Plasma MDA levels were increased in ethanol-fed animals, whereas urinary 8-hydroxy-2-deoxyguanosine (oxy8dG), a measure of DNA oxidation, and serum aspartate aminotransferase (AST) levels were increased in animals fed the combined diet. Hepatic SAM levels correlated with hepatic GSH levels, whereas urinary oxy8dG and plasma MDA levels correlated with the SAM:SAH ratio and to hepatic GSH levels. Livers from 5 of 6 animals in the combined group demonstrated moderate to marked intralobular steatohepatitis; there were no changes in the other groups. In summary, the findings showed both separate and combined effects of folate deficiency and ethanol feeding on the hepatic methionine cycle. The findings that the combined diet enhanced plasma homocysteine levels, reduced the SAM:SAH ratio, and increased both urinary oxy8dG and plasma AST levels together with characteristic histopathologic findings support the suggestion that folate deficiency increases the risk of oxidative liver injury in chronic alcoholism and supports the concept that perturbations in the methionine cycle contribute to the pathogenesis of ALD. 4. Summary of published literature Results of studies with other experimental animal models have demonstrated many effects of ethanol feeding on the hepatic methionine cycle. For example, reduced hepatic MS activity and SAM levels were found in rodent models by three independent groups (Barak et al., 1987; Finkelstein et al., 1974; Trimble et al., 1993). The intragastric ethanol-fed rat model demonstrated decreased hepatic levels of methionine and SAM, together with decreased hepatic MAT1 (Lu et al., 2000). The administration of SAM attenuated decreased GSH levels and the experimental development of ALD in ethanolfed baboons, as well as improved the clinical status of patients with varied degrees of ALD (Lieber et al., 1990; Mato et al., 1999). S-adenosylmethionine promotes the fluidity of the mitochondrial membrane and the transport of intracellular GSH to its mitochondrial site of activity (Colell et al., 1997). S-adenosylmethionine is reduced through free radical modification of cysteine residue 121 in MAT (Avila et al., 1998), whereas conversely, GSH maintains MAT1 activity (Corrales et al., 1991). Hepatic SAM levels were markedly reduced, and GSH levels were increased, in a recently described mouse MAT1A knockout model, in which hepatic steatosis developed together with an enhancement of gene expression of many inflammatory markers in these mice (Lu et al., 2001). Although results of previous studies with ethanol-fed rodents and micropigs showed altered hepatic methionine metabolism together with nucleotide imbalance, DNA global hypomethylation and strand breaks, and hepatocellular apoptosis (Halsted et al., 1996; Lu et al., 2000), the dietary induction of folate, methionine, and choline deficiency promoted hepatic nucleotide imbalance, DNA strand breaks, and apoptosis in rat liver (James et al., 1997).
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5. Future directions Future work should be directed at defining the mechanisms for induction of oxidative liver injury by ethanol and folate deficiency, both singly and in combination. Potential mechanisms include SAM-regulated effects on oxidative defense, enhancement of hepatocellular apoptosis through the effects of both ethanol and folate deficiency on DNA integrity, and potential effects of both factors on the cytokineregulated inflammatory response. Results of prior work support each or all of these potential mechanisms. In addition to the chronic effects of ethanol on SAM and antioxidative GSH activity in the rat and baboon (Colell et al., 1997; Corrales et al., 1991; Lieber et al., 1990; Lu et al., 2000) and independently observed effects of ethanol feeding, folate deficiency, or both on DNA integrity and hepatocellular apoptosis (Halsted et al., 1996; James et al., 1997; Lu et al., 2000), recent study findings support the suggestion that cellular homocysteine triggers a proinflammatory response that may be mediated through nuclear factor-kappa B (NF-B). Pertinent data in this regard include findings that hepatic NF-B activation is associated with oxidation and precedes the inflammatory response in ethanol-fed rats (Jokelainen et al., 2001; Nanji et al., 1999) and that homocysteine elevation stimulates reactive oxygen species (ROS) production and activates NF-B in HepG2 cells cultured in folate-deficient media (Chern et al., 2001). Acknowledgments This work was supported by grants DK-35747 and DK45301 from the National Institutes of Health. We are indebted to S. Jill James and S. Melnyk, National Toxicological Research Center, for assays of methionine metabolites, and to L. Wallock and M. Shigenaga, Children’s Hospital of Oakland Research Institute, for assays of urinary oxy8dG and plasma MDA. References Avila, M. A., Corrales, F. J., Ruiz, F., Sanchez-Gongora, E., Mingorance, J., Carretero, M. V., & Mato, J. M. (1998). Specific interaction of methionine adenosyltransferase with free radicals. Biofactors 8, 27–32. Barak, A. J., Beckenhauer, H. C., Tuma, D. J., & Badakhsh, S. (1987). Effects of prolonged ethanol feeding on methionine metabolism in rat liver. Biochem Cell Biol 65, 230–233. Chern, C. L., Huang, R. F., Chen, Y. H., Cheng, J. T., & Liu, T. Z. (2001). Folate deficiency–induced oxidative stress and apoptosis are mediated via homocysteine-dependent overproduction of hydrogen peroxide and enhanced activation of NF-kappaB in human Hep G2 cells. Biomed Pharmacother 55, 434–442. Colell, A., García-Ruiz, C., Morales, A., Ballesta, A., Ookhtens, M., Rodés, J., Kaplowitz, N., & Fernández-Checa, J. C. (1997). Transport of reduced glutathione in hepatic mitochondria and mitoplasts from ethanol-treated rats: effect of membrane physical properties and S-adenosyl-L-methionine. Hepatology 26, 699–708. Corrales, F., Ochoa, P., Rivas, C., Martin-Lomas, M., Mato, J. M., & Pajares, M. A. (1991). Inhibition of glutathione synthesis in the liver leads to S-adenosyl-L-methionine synthetase reduction. Hepatology 14, 528–533.
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