A conjugate of doxorubicin and glutathione via glutaraldehyde (GSH-DXR) inhibited glutathione S-transferase (GST) activity of rat hepatoma AH66 cells, and treatment of the cells with GSH-DXR induced caspase-3 activation and DNA fragmentation. After treatment of AH66 cells with 0.1 M GSH-DXR, GST-P (placental type of rat GST isozymes) mRNA and its protein increased transiently and then decreased thereafter compared with the levels in nontreated cells. Caspase-3 activation and DNA fragmentation were induced following the suppression of GST-P expression by treatment with GSH-DXR. When the cells were treated with 100 M ethacrynic acid (ECA), an inhibitor of GST, DNA fragmentation and caspase-3 activation were observed. In contrast, treatment of AH66 cells with a low concentration of ECA (1 M) that showed little inhibition of GST activity induced slight, but significantly enhanced expression and activity of GST-P, and consequent prevention of DXR-and GSH-DXR-induced DNA fragmentation. Overexpression of GST-(placental type of human GST isozymes) by transfection of GST-sense cDNA into AH66 cells decreased sensitivities to DXR and GSH-DXR, and the suppression of GST-P by transfection of the antisense cDNA into the cells increased drug sensitivity. On the other hand, there was little change in drug sensitivity caused by overexpression of site-directedly mutated GST-P in which the active-site residue Tyr39 was replaced with His (W39H) or the substrate-binding site residue Cys48 was replaced with Ser (C48S) by transfection of those cDNAs into AH66 cells. These results suggested that the suppression of GST-P in AH66 cells treated with GSH-DXR must play an important role in the induction of apoptosis.

Alcohol abuse can induce brain atrophy, but it only occurs in some alcoholics. To investigate whether genetic polymorphism of alcohol-metabolizing enzymes [including alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH)] was related to alcoholic brain atrophy, we determined restriction fragment-length polymorphisms of the ADH2 and ALDH2 genes in 77 male alcoholics. Computed tomography was used to determine the severity of brain atrophy. Digestion with MaeIII and MboII after polymerase chain reaction amplification showed that the ADH2(1) gene frequency was significantly higher in patients with brain atrophy than in those without brain atrophy (chi 2 = 9.274, p < 0.01), whereas no significant association was observed between brain atrophy and the ALDH2 gene Multivariate analysis (including age, total alcohol intake, liver cirrhosis, and ADH2 genotype) showed that the ADH2(1)/ADH2(1) genotype was associated with alcoholic brain atrophy. These findings suggest that the ADH2(1) allele may be associated with alcoholic brain atrophy.

Abstract. It was confirmed that CD147 (Emmprin) was expressed on the cell surface of carcinoma cells. For the purpose of studying the efficacy of a CD147-targeting agent on CD147-expressing carcinoma cells, we investigated the effect of a conjugate of glutathione-doxorubicin (GSH-DXR) encapsulated in an anti-CD147 antibody-labeled liposome (aCD147ab-liposome) in terms of specific accumulation and cytotoxicity in CD147-expressing human carcinoma cells. Expression of CD147 was not observed in many normal human tissues. However, slight expression of CD147 in kidney, prostate and breast tissues was observed. By contrast, high-level expression of CD147 in all carcinoma cells such as A431, PC3 and Ishikawa cell lines was confirmed by fluorescent microscopy and Western blot analysis. Specific accumulation of the aCD147ab-liposome in the abovedescribed CD147-expressing cells was observed. GSH-DXR encapsulated in an aCD147ab-liposome expressed specific cytotoxicity against these carcinoma cells. These results suggested that target chemotherapy of GSH-DXR encapsulated in an aCD147ab-liposome on CD147-expressing carcinoma cells was effective.

Background: Alcohol abuse can induce testicular atrophy, but it only occurs in some alcoholics. Alcohol dehydrogenase (ADH) is located principally on the Leydig cells.Methods: To investigate whether genetic polymorphism of alcohol dehydrogenase (ADH) 2 and aldehyde dehydrogenase (ALDH) 2 was related to alcoholic testicular atrophy, we determined restriction fragment-length polymorphisms of the ADH2 and ALDH2 genes in 43 Japanese male alcoholics and 50 healthy subjects. An orchidometer was used to determine the testicular size.Results: Less than 16 ml in testicular size was defined as testicular atrophy. Testicular atrophy was found in 24 (55.8%) cases out of 43 alcoholics. Digestion with MaeIII and MboII after polymerase chain reaction amplification showed that the ADH2 1 allele frequency was significantly higher in patients with testicular atrophy than in those without testicular atrophy ( 2 ϭ 4.665, p ϭ 0.031), whereas no significant association was observed between testicular atrophy and the ALDH2 gene.Conclusions: The ADH2 1 allele may be associated with alcoholic testicular atrophy.

Background: A carbohydrate-deficient transferrin (CDT) is the most useful marker of alcohol abuse; however, the mechanism of production and the pathophysiologic roles of CDT remain obscure. The effects of alcohol and its metabolites on growth and proliferation, transferrin synthesis, and phosphomannomutase enzyme activity in a human hepatoblastoma, HepG2, were examined.Methods: HepG2 cells were treated with either ethanol at 80 mM or acetaldehyde at 400 M. Transferrin secreted by the cells was prepared from conditioned culture medium by single-step immunoaffinity column chromatography using a goat-specific antibody against human transferrin. Phosphomannomutase and some related enzyme activities in the cell extracts were determined. Reverse transcription-polymerase chain reaction analysis of phosphomannomutase mRNA expression was also determined in HepG2 cultured with or without acetaldehyde (400 M).Results: HepG2 cells usually synthesized and secreted transferrin with three separated bands: main broad bands estimated to be 78 to 82 kDa, 75 kDa, and 72 kDa. The last two bands were compatible with part or the entire N-glycans-deficient transferrin (CDT) from alcoholic liver damage. Increased secretion of CDT from HepG2 correlated well with the extent of growth retardation to the level of confluent cell density. The activity of phosphomannomutase also decreased with prolongation of cellular doubling time. Furthermore, acetaldehyde treatment at 400 M accelerated the inhibitory effect of cell growth compared with nontreated cells, and this condition facilitated CDT secretion from HepG2 cells. Determination of the enzyme activity and mRNA expression indicated that acetaldehyde showed competitive type inhibition of phosphomannomutase activity but not suppression of phosphomannomutase gene expression.Conclusions: By culturing HepG2 cells with acetaldehyde containing media, growth inhibitiondependent increase of CDT showed good correlation with reduced enzyme activity of phosphomannomutase. Acetaldehyde facilitated growth retardation, inhibition of phosphomannomutase activity, and increased secretion of CDT. The HepG2 cell line is useful as an in vitro model to investigate the pathophysiologic state of alcoholic liver damage and mechanisms of production as well as the physiologic role of CDT.