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BACKGROUND: Glutathione-S-transferase A4-4 (GSTA4) is a key enzyme for removal of toxic lipid peroxidation products such as 4-hydroxynonenal (4-HNE). In the current study, we examined the potential role of GSTA4 on protein carbonylation and progression of alcoholic liver disease (ALD) by examining the development of liver injury in male wild type SV/J mice (WT) and SV/J mice lacking functional GSTA4 (GSTA4(−/−) mice). METHODS: Adult male WT and GSTA4(−/−) mice were fed chow (N = 10-12) or high fat Lieber-DeCarli liquid diets containing up to 28% calories as ethanol (EtOH) (N = 18-20) for 116 days. At the end of the study, half of the ethanol-fed mice were acutely challenged with an ethanol binge (3 g/kg given intragastrically) 12 hours before sacrifice. Carbonylation of liver proteins was assessed by immunohistochemical staining for 4-HNE adduction and by comprehensive liquid chromatography tandem mass spectrometry, LC-MS/MS, of purified carbonylated proteins. RESULTS: Chronic EtOH intake significantly increased hepatic 4-HNE adduction and protein carbonylation, including carbonylation of ribosomal proteins. EtOH intake also resulted in steatosis and increased serum ALTs. Hepatic infiltration with B-cells, T-cells, and neutrophils and mRNA expression of pro-inflammatory cytokines TNFα and IFNγ was modest in WT mice. However, an ethanol binge increased hepatic necrosis, hepatic cell proliferation and expression of TNFα mRNA (P

Background We previously reported that acute functional tolerance (AFT) to the hypnotic effects of alcohol was significantly correlated with drinking in the dark (DID) in the LXS recombinant inbred panel, but only in mice that had been pretreated with alcohol. Here, we have conducted quantitative trait locus (QTL) mapping for AFT. DNA sequencing of the progenitor ILS and ISS strains and microarray analyses were also conducted to identify candidate genes and functional correlates. Methods LXS mice were given either saline or alcohol (5 g/kg) on day 1 and then tested for loss of righting reflex AFT on day 2. QTLs were mapped using standard procedures. Two microarray analyses from brain were conducted: (i) naive LXS mice and (ii) an alcohol treatment time course in the ILS and ISS. The full genomes of the ILS and ISS were sequenced to a depth of approximately 30×. Results A significant QTL for AFT in the alcohol pretreatment group was mapped to distal chromosome 4; numerous suggestive QTLs were also mapped. Preference drinking and DID have previously been mapped to the chromosome 4 locus. The credible interval of the significant chromosome 4 QTL spanned 23 Mb and included 716 annotated genes of which 150 had at least 1 nonsynonymous single nucleotide polymorphism or small indel that differed between the ILS and ISS; expression of 48 of the genes was cis-regulated. Enrichment analysis indicated broad functional categories underlying AFT, including proteolysis, transcription regulation, chromatin modification, protein kinase activity, and apoptosis. Conclusions The chromosome 4 QTL is a key region containing possibly pleiotropic genes for AFT and drinking behavior. Given that the region contains many viable candidates and a large number of the genes in the interval fall into 1 or more of the enriched functional categories, we postulate that many genes of varying effect size contribute to the observed QTL effect.

Background: Proper ascertainment of the history of alcohol consumption by an individual is an important component of medical diagnosis of disease and influences the implementation of appropriate treatment strategies that include prescription of medication, as well as intervention for the negative physical and social consequences of hazardous/harmful levels of alcohol consumption. Biological (biometric) diagnostic tests that provide information on current and past quantity and frequency of alcohol consumption by an individual, prior to onset of organ damage, continue to be sought. Methods: Platelet monoamine oxidase B (MAO-B) protein was quantitated in 2 populations of subjects who had histories of different levels of alcohol consumption. Levels were assayed by immunoblotting or by ELISA. The development and evaluation of the new ELISA-based measure of platelet MAO-B protein levels is described. Results: One subject population constituted a nontreatment-seeking, cross-sectional subject sample, and the other population was a longitudinally followed, hospitalized group of subjects. An algorithm combining measures of platelet MAO-B protein with the plasma levels of carbohydrate-deficient transferrin (CDT) and with liver enzymes (aspartate aminotransferase or γ-glutamyltransferase [GGT]) can detect hazardous/harmful alcohol use (HHAU) with the highest sensitivity and specificity in the cross-sectional nontreatment-seeking population. In the treatment-seeking population, low MAO-B protein levels at admission are associated with heavy drinking prior to admission, and these protein levels increase over a period of abstinence from alcohol. Conclusions: The platelet MAO-B protein measurement is particularly effective for male alcohol consumers. The combined use of MAO-B protein measures together with measures of CDT and GGT does, however, improve the diagnostic utility of both markers for ascertaining HHAU in women. Furthermore, measurement of changes in platelet MAO-B protein levels during treatment for alcohol dependence may help monitor the success of the treatment program. Language: en

Background: Differences in ethanol metabolizing enzymes expressed in brain have been suggested to contribute to the significant differences in ethanol (alcohol) preference between inbred C57BL/6 and DBA/2 mouse strains. Methods: We have utilized 2 different platforms of oligonucleotide microarray technology (CodeLink UniSet I BioArray from G.E. Healthcare and MG U74A v2.0 from Affymetrix) to simultaneously assess expression of alcohol and acetaldehyde metabolizing enzymes in the whole brain of naive (no exposure to alcohol) C57BL/6 and DBA/2 mice. Results: There were no significant differences between the 2 strains of mice in gene expression intensity for alcohol dehydrogenases (ADH), catalase, and a number of the cytochrome P450 family of genes, which can be involved in ethanol catabolism. However, significantly higher expression of mRNA for aldehyde dehydrogenase 2 (ALDH2), an isoform mainly responsible for the catabolism of acetaldehyde, was observed in whole brains of DBA/2 mice with both platforms. Aldehyde dehydrogenase 2 protein was also higher in DBA/2 brain. Expression of aldehyde dehydrogenase 1A1 (ALDH1A1) mRNA was found to be higher in brains of DBA/2 mice, when measured with the CodeLink platform, but not when measured with Affymetrix arrays or quantitative reverse transcriptase–real-time polymerase chain reaction (qRT-PCR). The ALDH1A1 protein, however, reflected the results obtained with the CodeLink arrays and was higher in DBA/2 brain, compared with brains of C57BL/6 mice. In contrast, the expression intensity for the aldehyde dehydrogenase 7A1 (ALDH7A1) mRNA and protein was significantly higher in C57BL/6 mice than DBA/2 mice. These expression differences are consistent with more rapid metabolism of acetaldehyde in brains of DBA/2 mice. Conclusions: The use of 2 different microarray platforms provides important cross-validation of many results, and some discrepancies can be resolved with qRT-PCR and immunoblotting. The expression differences that were validated may affect alcohol/aldehyde metabolism in brain and/or alcohol preference in the 2 strains of mice.