Alan Cahill

S-adenosyl-L-methionine co-administration prevents the ethanol-elicited dissociation of hepatic mitochondrial ribosomes in male rats

BACKGROUND Chronic ethanol feeding to male rats has been shown to result in decreased mitochondrial translation, depressed respiratory complex levels and mitochondrial respiration rates. In addition, ethanol consumption has been shown to result in an increased dissociation of mitoribosomes. S-adenosyl-L-methionine (SAM) is required for the assembly and subsequent stability of mitoribosomes and is depleted during chronic ethanol feeding. The ability of dietary SAM co-administration to prevent these ethanol-elicited lesions was investigated. METHODS Male Sprague-Dawley rats were fed a nutritionally adequate liquid diet with ethanol comprising 36% of the calories according to a pair-fed design for 28 days. For some animals, SAM was supplemented in the diet at 200 mg/l. Liver mitochondria were prepared and mitoribosomes isolated. Respiration rates, ATP levels, respiratory complex levels, and the extent of mitoribosome dissociation were determined. RESULTS Twenty-eight days of ethanol feeding were found to result in decreased SAM content, depressed respiration, and increased mitoribosome dissociation. No changes in mitochondrial protein content; levels of respiratory complexes I, III, and V; complex I activities; and ATP levels were detected. Co-administration of SAM in the diet was found to prevent ethanol-induced SAM depletion, respiration decreases and mitoribosome dissociation. CONCLUSIONS Taken together, these findings suggest (1) that mitoribosome dissociation precedes respiratory complex depressions in alcoholic animals and (2) that dietary supplementation of SAM prevents some of the early mitochondrial lesions associated with chronic ethanol consumption.

Effects of alcohol and oxidative stress on liver pathology: the role of the mitochondrion.

This article represents the proceedings of a symposium at the 2001 Research Society on Alcoholism meeting in Montreal, Canada. The chairs were Alan Cahill and Carol C. Cunningham. The presentations were (1) Mitochondrial regulation of ethanol-induced hepatocyte apoptosis: possible involvement of proapoptotic Bcl-2 family protein Bax, by Masayuki Adachi and Hiromasa Ishii; (2) Effects of ethanol on mitochondrial reactive oxygen species production and oxidative protein modification, by Shannon M. Bailey; (3) Acute ethanol binges elicit widespread oxidative mitochondrial DNA damage and depletion: protective effects of antioxidants and inhibitors of ethanol metabolism, by Bernard Fromenty; and (4) Effects of chronic ethanol consumption upon hepatic mtDNA oxidative modification and depletion, by Alan Cahill and Adrian Davies.

Effects of chronic ethanol feeding on the protein composition of mitochondrial ribosomes

Chronic ethanol feeding has been shown to decrease the number of functionally active mitochondrial ribosomes by 55%. In this work, 55S mitochondrial ribosomes were isolated from rat liver and their constitutive proteins characterized by two‐dimensional polyacrylamide gel electrophoresis and quantified by densitometry. A total of 86 proteins were found to be associated with the mitochondrial ribosome. This compares with 70 isolated from cytoplasmic ribosomes. In addition, mitochondrial ribosomal proteins were found to be significantly less basic than their cytoplasmic counterparts. Chronic ethanol feeding was found to significantly decrease the levels of a number of constitutive proteins of the mitochondrial ribosome when compared to those isolated from pair‐fed controls. Sucrose density gradient analyses revealed a significant decrease in the number of intact 55S ribosomes. It is suggested that ethanol‐elicited alterations in specific constitutive proteins of the mitochondrial ribosome may lead to impaired assembly of the monosome and that this may result in lower levels of those displaying functional activity.

Metabolism of 3-amino-1,2,4-benzotriazine-1,4-dioxide (SR 4233) by purified DT-diaphorase under aerobic and anaerobic conditions

Purified DT-diaphorase [NAD(P)H (quinone acceptor) oxidoreductase (EC.1.6.99.2)] from Walker cells was used to investigate the reductive metabolism of 3-amino-1,2,4-benzotriazine-1,4-dioxide (SR 4233) under aerobic and anaerobic conditions. In the presence of NADPH, under aerobic conditions, HPLC analysis showed the four-electron reduction product 3-amino-1,2,4-benzotriazine (SR 4330) was the major reaction product. In contrast, anaerobically, the 2-electron reduction product 3-amino-1,2,4-benzotriazine-1-oxide (SR 4317) was the predominant metabolite. Anaerobic reduction of SR 4233 to the known metabolites SR 4317 and SR 4330, catalyzed by DT-diaphorase, was 3-fold higher than reduction under aerobic conditions. Anaerobically, approximately half of the substrate utilized could not be accounted for by the formation of known products. Aerobically, the majority of the SR 4233 lost could be accounted for by its conversion to SR 4317 and SR 4330. In Walker cells incubated with SR 4233 anaerobically, SR 4317 was the major metabolite formed. Dicoumarol (100 microM) had little effect on the rate of formation of this metabolite in this cell line or in a rat liver epithelial derived (JBJ) cell line. Dicoumarol did however partially reduce the induction of unscheduled DNA synthesis caused by SR 4233 in Walker cells but not in JB1 cells, suggesting the action of dicoumarol may be specific to Walker cells. It is concluded that DT-diaphorase plays only a minor role in the overall reduction of SR 4233 in the two cell lines studied.

Chronic ethanol feeding causes depression of mitochondrial elongation factor Tu in the rat liver: implications for the mitochondrial ribosome

Chronic ethanol feeding is known to negatively impact hepatic energy metabolism. Previous studies have indicated that the underlying lesion responsible for this may lie at the level of the mitoribosome. The aim of this study was to characterize the structure of the hepatic mitoribosome in alcoholic male rats and their isocalorically paired controls. Our experiments revealed that chronic ethanol feeding resulted in a significant depletion of both structural (death-associated protein 3) and functional [elongation factor thermo unstable (EF-Tu)] mitoribosomal proteins. In addition, significant increases were found in nucleotide elongation factor thermo stable (EF-Ts) and structural mitochondrial ribosomal protein L12 (MRPL12). The increase in MRPL12 was found to correlate with an increase in the levels of the 39S large mitoribosomal subunit. These changes were accompanied by decreased levels of nuclear- and mitochondrially encoded respiratory subunits, decreased amounts of intact respiratory complexes, decreased hepatic ATP levels, and depressed mitochondrial translation. Mathematical modeling of ethanol-mediated changes in EF-Tu and EF-Ts using prederived kinetic data predicted that the ethanol-mediated decrease in EF-Tu levels could completely account for the impaired mitochondrial protein synthesis. In conclusion, chronic ethanol feeding results in a depletion of mitochondrial EF-Tu levels within the liver that is mathematically predicted to be responsible for the impaired mitochondrial protein synthesis seen in alcoholic animals.

Alcoholic Liver Disease and the Mitochondrial Ribosome: Methods of Analysis

Chronic alcohol consumption has been shown to severely compromise mitochondrial protein synthesis. Hepatic mitochondria isolated from alcoholic animals contain decreased levels of respiratory complexes and display depressed respiration rates when compared to pair-fed controls. One underlying mechanism for this involves ethanol-elicited alterations in the structural and functional integrity of the mitochondrial ribosome. Ethanol feeding results in ribosomal changes that include decreased sedimentation rates, larger hydrodynamic volumes, increased levels of unassociated subunits and changes in the levels of specific ribosomal proteins. The methods presented in this chapter detail how to isolate mitochondrial ribosomes, determine ribosomal activity, separate ribosomes into nucleic acid and protein, and perform two-dimensional nonequilibrium pH gradient electrophoretic polyacrylamide gel electrophoresis to separate and subsequently identify mitochondrial ribosomal proteins.

Acute lesions in rats caused by 3-amino-1,2,4-benzotriazine-1,4-dioxide (SR 4233) or nitromin: a comparison with rates of reduction in microsomal systems from target organs

Pathological lesions to male Fischer rats were investigated 24 h after the administration of 3-amino-1,2,4-benzotriazine-1,4-dioxide (SR 4233) or nitromin, two compounds which need to undergo bioreductive activation in order to exert their toxic effects. Although SR 4233 reduction leads to a putative free radical species while with nitromin a bifunctional alkylating agent is formed, in both instances, the bone marrow was a major target organ. However, the response of other organs to these compounds differed. SR 4233 caused lesions to the olfactory epithelium, liver, kidney and thymus. Nitromin caused focal haemorrhages on the intestine, which were reduced in germ-free rats. Rates of reduction of SR 4233 or nitromin were determined under anaerobic conditions using microsomal preparations from target tissues. With SR 4233 as a substrate, reductase activities were highest in the olfactory epithelium, 6 fold higher than in the liver. SR 4233 reductase activities generally correlated with those of NADPH: cytochrome c reductase or the concentration of cytochrome P-450 reductase protein in the affected organs while with nitromin, there appeared to be no such relationship. The present results support the concept that the expression of pathological damage in vivo is a multifactorial process and does not directly correlate with initial rates of reduction of either drug determined in vitro.