[show abstract][hide abstract] ABSTRACT: Chronic ethanol (EtOH) drinking produces neuronal alterations within the limbic system. To investigate changes in protein expression levels associated with EtOH drinking, inbred alcohol-preferring (iP) rats were given one of three EtOH access conditions in their home-cages: continuous ethanol (CE: 24h/day, 7days/week access to EtOH), multiple scheduled access (MSA: four 1-h sessions during the dark cycle/day, 5 days/week) to EtOH, or remained EtOH-naïve. Both MSA and CE groups consumed between 6 and 6.5g of EtOH/kg/day after the 3rd week of access. On the first day of EtOH access for the seventh week, access was terminated at the end of the fourth MSA session for MSA rats and the corresponding time point (2300h) for CE rats. Ten h later, the rats were decapitated, brains extracted, the nucleus accumbens (NAcc) and amygdala (AMYG) microdissected, and protein isolated for 2-dimensional gel electrophoretic analyses. In the NAcc, MSA altered expression levels for 12 of the 14 identified proteins, compared with controls, with six of these proteins altered by CE access, as well. In the AMYG, CE access changed expression levels for 22 of the 27 identified proteins, compared with controls, with 8 of these proteins altered by MSA, as well. The proteins could be grouped into functional categories of chaperones, cytoskeleton, intracellular communication, membrane transport, metabolism, energy production, or neurotransmission. Overall, it appears that EtOH drinking and the conditions under which EtOH is consumed, differentially affect protein expression levels between the NAcc and AMYG. This may reflect differences in neuroanatomical and/or functional characteristics associated with EtOH self-administration and possibly withdrawal, between these two brain structures.
[show abstract][hide abstract] ABSTRACT: The aldehyde dehydrogenase 2 (ALDH2) promoter contains a nuclear receptor response element (NRRE) that represents an overlapping direct repeat-1 (DR-1) and -5 (DR-5) element. Because DR-1 elements are preferred binding sites for peroxisome proliferator-activated receptors (PPARs), we tested the hypothesis that PPARs regulate ALDH2 expression.
We examined the ability of PPAR isoforms to bind to the ALDH2 NRRE in electrophoretic mobility shift assays, their ability to activate the transcription of promoter-reporter constructs containing this NRRE, the effect of PPAR ligands on ALDH2 expression in liver, and the role of the PPARalpha on the expression of ALDH2 by using PPARalpha-null mice.
In vitro translated PPARs bound the ALDH NRRE with high affinity. Mutation of the NRRE indicated that binding was mediated by the DR-1 element. Cotransfection of PPAR expression plasmids showed that PPARalpha had no effect on expression of heterologous promoter constructs containing the NRRE. PPARgamma slightly induced expression, whereas PPARdelta repressed basal activity of the promoter and blocked induction by hepatocyte nuclear factor 4. Treatment of rats with the PPAR ligand clofibrate repressed expression of ALDH2 in rats fed either stock rodent chow or a low-protein diet. Consistent with the transfection data, expression of ALDH2 protein was not different in PPARalpha-null mice. Treatment of the mice with the PPARalpha agonist WY14643 slightly decreased the level of ALDH2 protein in both wild-type and PPARalpha-null mice, suggesting that the effect of WY14643 was not mediated by the receptor.
These data indicate that ALDH2 is not part of the battery of lipid metabolizing enzymes and proteins regulated by PPARalpha
Alcoholism Clinical and Experimental Research 08/2001; 25(7):945-52. · 3.42 Impact Factor
[show abstract][hide abstract] ABSTRACT: Research has substantiated the role of several mechanisms responsible for alcohol-induced hepatotoxicity. These mechanisms include: oxidative stress and lipid peroxidation; immunogenic processes initiated by formation of protein adducts of acetaldehyde, other aldehydes and 1-hydroxyethyl radicals; and activation of Kupffer cells by endotoxin and subsequent cascade of events that involved cytokines, chemokines, and adhesion molecules. Increasing evidence implicates enhanced intestinal permeability caused by alcohol ingestion as the culprit that leads to endotoxemia. While oxidative stress is important, the principal source of reactive oxygen species that causes alcohol-induced liver injury is hotly debated. Potential sources may include cytochrome P450IIE1, activated Kupffer cells, and mitochondrial electron transfer chain. Apoptosis is likely an important pathway that culminates in hepatocyte cell death. Abstinence, corticosteroids, and enteral nutrition remain the cornerstones in the treatment of alcoholic hepatitis. The efficacies of medications such as S-adenosylmethionine and pentoxifylline will need further confirmation by additional randomized trials before they can be recommended as standard therapies for alcoholic hepatitis.
Current Opinion in Gastroenterology 06/2001; 17(3):211-20. · 4.10 Impact Factor
[show abstract][hide abstract] ABSTRACT: This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Hirokazu Yokoyama and David Crabb. The presentations were (1) Roles of vitamin A, retinoic acid, and retinoid receptors in the expression of liver ALDH2, by J. Pinaire, R. Hasanadka, M. Fang, and David W. Crabb; (2) Alcohol, vitamin A, and beta-carotene: Adverse interactions, by M. A. Leo and Charles S. Lieber; (3) Retinoic acid, hepatic stellate cells, and Kupffer cells, by Hidekazu Tsukamoto, K. Motomura, T. Miyahara, and M. Ohata; (4) Retinoid storage and metabolism in liver, by William Bosron, S. Sanghani, and N. Kedishvili; (5) Characterization of oxidation pathway from retinol to retinoic acid in esophageal mucosa, by Haruko Shiraishi, Hirokazu Yokoyama, Michiko Miyagi, and Hiromasa Ishii; and (6) Ethanol in an inhibitor of the cytosolic oxidation of retinol in the liver and the large intestine of rats as well as in the human colon mucosa, by Ina Bergheim, Ina Menzl, Alexandr Parlesak, and Christiane Bode.
Alcoholism Clinical and Experimental Research 06/2001; 25(5 Suppl ISBRA):207S-217S. · 3.42 Impact Factor
[show abstract][hide abstract] ABSTRACT: Fatty acids are ligands for the peroxisome proliferator-activated receptor alpha (PPAR alpha). Fatty acid levels are increased in liver during the metabolism of ethanol and might be expected to activate PPAR alpha. However, ethanol inhibited PPAR alpha activation of a reporter gene in H4IIEC3 hepatoma cells expressing alcohol-metabolizing enzymes but not in CV-1 cells, which lack these enzymes. Ethanol also reduced the ability of the PPAR alpha ligand WY14,643 to activate reporter constructs in the hepatoma cells or cultured rat hepatocytes. This effect of ethanol was abolished by the alcohol dehydrogenase inhibitor 4-methylpyrazole and augmented by the aldehyde dehydrogenase inhibitor cyanamide, indicating that acetaldehyde was responsible for the action of ethanol. PPAR alpha/retinoid X receptor extracted from hepatoma cells exposed to ethanol or acetaldehyde bound poorly to an oligonucleotide containing peroxisome proliferator response elements. This effect was also blocked by 4-methylpyrazole and augmented by cyanamide. Furthermore, in vitro translated PPAR alpha exposed to acetaldehyde failed to bind DNA. Thus, ethanol metabolism blocks transcriptional activation by PPAR alpha, in part due to impairment of its ability to bind DNA. This effect of ethanol may promote the development of alcoholic fatty liver and other hepatic consequences of alcohol abuse.
Journal of Biological Chemistry 02/2001; 276(1):68-75. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: This manuscript represents the proceedings of a symposium at the 2000 RSA Meeting in Denver, Colorado. The organizer/chair was Ting-Kai Li. The presentations were: (1) Introduction to the Symposium, by Ting-Kai Li; (2) ALDH2 polymorphism and alcohol metabolism, by Shih-Jiun Yin; (3) ALDH2 promoter polymorphism and alcohol metabolism, by David W. Crabb; (4) Use of BrAC clamping to estimate alcohol elimination rates: Application to studies of the influence of genetic and environmental determinants, by Sean O'Connor; and (5) Effect of food and food composition on alcohol elimination rates as determined by clamping, by Vijay A. Ramchandani.
Alcoholism Clinical and Experimental Research 02/2001; 25(1):136-44. · 3.42 Impact Factor
[show abstract][hide abstract] ABSTRACT: Vitamin A has been suggested to regulate the expression of liver alcohol dehydrogenase (ADH) in humans. There are few studies on the ability of retinoic acid to affect ADH expression in vivo and none on its effects on alcohol metabolic rate.
Male Sprague Dawley rats were used for isolation of hepatocytes or were rendered vitamin A deficient by feeding a deficient diet for 7 weeks. ADH, retinoic acid receptor beta, and retinoid X receptor alpha protein levels were analyzed by Western blotting. Alcohol elimination rate was determined by following blood alcohol levels after administering a 1.5 g/kg dose of ethanol intraperitoneally.
Retinoic acid had no effect on ADH protein in cultured hepatocytes. In the vitamin A deficient rats, retinol was not detectable in serum or liver at the time animals were killed. ADH and retinoid X receptor alpha protein levels were unchanged in the deficient group compared with a vitamin A sufficient control group, whereas retinoic acid receptor beta levels increased 40%. The deficient rats had a reduced volume of distribution of alcohol, but this largely was accounted for by their smaller body size. The alcohol elimination rates were lower in the deficient animals, but this was accounted for by reduced body and liver weights.
Severe vitamin A deficiency did not alter liver ADH protein expression or rates of alcohol elimination when expressed per gram of body or liver weight.
Alcoholism Clinical and Experimental Research 01/2001; 24(12):1759-64. · 3.42 Impact Factor
[show abstract][hide abstract] ABSTRACT: The understanding of how alcohol damages the liver has expanded substantially over the last decade. In particular, the genetics of alcoholism, the genesis of fatty liver, the role of oxidant stress, interactions between endotoxin and the Kupffer cell, and the factors that control activation of the hepatic stellate cell (HSC) have been the focus of a great deal of research. Genetic mechanisms for increasing the risk of alcoholism include alterations in alcohol metabolizing enzymes as well as neurobiological differences between individuals. The development of fatty liver may involve both redox forces, oxidative stress, and alterations in peroxisome proliferator activated receptor function. Oxidative stress is now known to involve both microsomal and mitochondrial systems. Recent studies implicate stimulation of Kupffer cells by portal vein endotoxin as a cause of release of cytokines and chemokines, hepatocyte hyper-metabolism, and activation of HSC. These actions appear to be in part gender-dependent and may explain the susceptibility of women to alcoholic liver disease. Activation of HSC underlies liver fibrosis and cirrhosis of all types; control of this activation might permit control of the progression of fibrosis. These advances suggest a number of new approaches as therapy for alcoholic liver injury.
The Keio Journal of Medicine 01/2000; 48(4):184-8.
[show abstract][hide abstract] ABSTRACT: The strong protective effect of the ALDH2*2 mutation on risk of alcoholism suggests that other mutations that reduce mitochondrial aldehyde dehydrogenase (ALDH) activity in the liver might also deter drinking. This study describes a polymorphic locus found in the promoter of the ALDH2 gene that affects expression of reporter constructs.
Polymerase chain reaction (PCR)-based sequencing was used to search for polymorphisms. The ability of the promoter variants to bind transcription factors apolipoprotein A regulatory protein 1 (ARP-1) and chicken ovalbumin upstream promoter-transcription factor (COUP-TF) was tested in gel retardation assays using in vitro synthesized transcription factors. The variant promoters were tested for transcriptional activity using a heterologous promoter system and transient transfection assays.
A common polymorphism (A or G) in the human ALDH2 promoter region was found at -361 base pair (bp) from the translation start site. This polymorphism was found at different frequencies in African Americans, Caucasians, and Asians. The polymorphism occurs adjacent to the core binding motif for the transcription factors COUP-TF and ARP-1. Competition and binding affinity determinations did not show differences in the ability of these two sequences to bind the factors. Reporter genes containing these elements upstream of a basal thymidine kinase promoter had similar activity when transfected into a fibroblast (CV-1) cell line. However, the reporter containing the G allele was more active than that containing the A allele in hepatoma (H4IIEC3) cells.
The -361 bp A/G polymorphism is common in all racial groups tested. The G allele was more active than the A allele in a transfection assay. The basis for this difference is not known. If the differences in activity of the promoter constructs were paralleled by differences in ALDH2 enzyme activity in the liver, this polymorphism could affect risk of alcoholism.
Alcoholism Clinical and Experimental Research 07/1999; 23(6):963-8. · 3.42 Impact Factor
[show abstract][hide abstract] ABSTRACT: Many medications can interact with alcohol, thereby altering the metabolism or effects of alcohol and/or the medication. Some of these interactions can occur even at moderate drinking levels and result in adverse health effects for the drinker. Two types of alcohol-medication interactions exist: (1) pharmacokinetic interactions, in which alcohol interferes with the metabolism of the medication, and (2) pharmacodynamic interactions, in which alcohol enhances the effects of the medication, particularly in the central nervous system (e.g., sedation). Pharmacokinetic interactions generally occur in the liver, where both alcohol and many medications are metabolized, frequently by the same enzymes. Numerous classes of prescription medications can interact with alcohol, including antibiotics, antidepressants, antihistamines, barbiturates, benzodiazepines, histamine H2 receptor antagonists, muscle relaxants, nonnarcotic pain medications and anti-inflammatory agents, opioids, and warfarin. In addition, many over-the-counter and herbal medications can cause negative effects when taken with alcohol.
Alcohol research & health: the journal of the National Institute on Alcohol Abuse and Alcoholism 02/1999; 23(1):40-54. · 0.58 Impact Factor
[show abstract][hide abstract] ABSTRACT: During a series of transfection experiments, the pRSV-luc plasmid used as an internal control was found to be sensitive to cotransfection with expression vectors for several members of the steroid/thyroid/retinoid superfamily of nuclear receptors. Therefore, a survey of the effect of these expression vectors on the activity of four reporter plasmids was conducted. In CV-1 cells, the activity of pRSV-luc, which contains the P. pyralis luciferase gene, was repressed by co-transfection of PPARalpha and ARP-1 and was activated by COUP-TFI. Expression of pSV40-luc, containing the same luciferase gene, was repressed by PPARalpha and HNF-4 and activated by both COUP-TFI and ARP-1. All four of these expression vectors reduced the expression of the pRL-TK plasmid, which contains the luciferase gene from Renilla reniformis. RXR expression vectors had no effect on luciferase activity in CV-1 cells but induced luciferase activity in H4IIEC3 hepatoma cells. This activation was blocked by the addition of ligand, 9-cis retinoic acid. pSV2-CAT, which contains the chloramphenicol acetyltransferase gene, was insensitive to all receptor expression vectors tested. Both the P. pyralis and R. reniformis luciferase genes appear to contain sequences that render them responsive to steroid/thyroid/retinoid nuclear receptors.
The Journal of Steroid Biochemistry and Molecular Biology 01/1999; 70(4-6):197-201. · 3.98 Impact Factor
[show abstract][hide abstract] ABSTRACT: Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is expressed in a tissue-specific fashion with high levels in liver, heart, kidney, and muscle, and low levels in most other tissues. The ALDH2 promoter was found to bind nuclear proteins at a pair of adjacent sites approximately 300 bp upstream from the translation start site, each of which was contacted at motifs containing the hexamer A/GGGTCA. The 3' site was shown to bind in vitro translated HNF-4. It was also shown by electrophoretic mobility shift assay utilizing antibodies against nuclear factors and rat liver nuclear extracts to be bound by hepatocyte nuclear factor 4 (HNF-4), chicken ovalbumin upstream promoter transcription factor I and II, and retinoid X receptors. A reporter construct containing four copies of this promoter element was activated by co-transfection of an HNF-4 expression plasmid in COS-1 and hepatoma cell lines. These results suggest that the tissue specificity of ALDH2 expression is in part determined by its activation by HNF-4.
Biochimica et Biophysica Acta 09/1998; 1399(2-3):181-6. · 4.66 Impact Factor