digitonin and allyl-alcohol

The present study compares the actions of the hepatotoxic agents allyl alcohol, acetaminophen, and carbon tetrachloride on energy metabolism in freshly isolated and cryopreserved rat hepatocytes. After 30 min incubation of freshly isolated hepatocytes at 37 degrees C to allow metabolic equilibration, hepatocytes were supplemented with cryoprotectants and cooled in a stepwise manner to liquid nitrogen temperature. Hepatocytes stored in liquid nitrogen for 2 weeks to 6 months were thawed and centrifuged through Percoll to remove damaged cells. Despite similarities in energy status as indexed by ATP content and the rate of urea synthesis in freshly isolated and cryopreserved hepatocytes, cryopreserved hepatocytes were more sensitive to hepatotoxicants. All three hepatotoxicants caused ATP and rates of urea synthesis to decline to a greater extent in cryopreserved than in freshly isolated hepatocytes. Rates of oxygen uptake were higher in cryopreserved cells than in freshly isolated hepatocytes and declined in cryopreserved cells but not in freshly isolated cells during the initial period of incubation. Rates of mitochondrial respiration stimulated with site-specific substrates were comparable in freshly isolated and cryopreserved cells permeabilized with digitonin. Allyl alcohol and acetaminophen inhibited site-specific respiration to the same extent in both groups of cells. Collectively, these results suggest that increased sensitivity to hepatotoxic agents and elevated oxygen consumption in cryopreserved hepatocytes recovered after storage in liquid nitrogen are related to higher demand for energy in these cells rather than to permanent injury caused by cryopreservation and irreversible uncoupling of oxidative phosphorylation.

Topics: 1-Propanol; Acetaminophen; Adenosine Triphosphate; Animals; Carbon Tetrachloride; Cell Separation; Cryopreservation; Digitonin; Energy Metabolism; In Vitro Techniques; Liver; Male; Oxygen Consumption; Propanols; Rats; Rats, Sprague-Dawley; Urea

The mechanism of the periportal (p.p.) toxicity of allyl alcohol (AlOH) was investigated in p.p. and perivenous (p.v.) hepatocytes isolated by digitonin-collagenase perfusion. The distinct origin of the cell preparations was confirmed by the p.p./p.v. ratios of alanine aminotransferase (p.p./p.v. = 1.8), lactate dehydrogenase (1.3) and glutamine synthetase (0.10). The activity of alcohol dehydrogenase (ADH) was not markedly different in p.p. and p.v. cells. Both types of cells oxidized AlOH at a high but equal rate of about 3 mumol/(min.g cells). Concomitantly with rapid oxidation of 0.7 mM AlOH, glutathione (GSH) was depleted by about 95% and its secretion was completely inhibited in both cell types. Although the GSH content was partially restored during a subsequent 3-h incubation, cellular ATP and K+ content gradually decreased and the leakage of lactate dehydrogenase increased in both types of cells. However, the p.p. cells tended to resist AlOH in vitro better, probably due to their 26% higher GSH content after preincubation with L-methionine. Altering the partial pressure of oxygen in physiological range had no effect on the toxicity of AlOH. The results are contrary to the suggestions that the p.p. location of AlOH liver injury is caused by higher ADH activity or higher oxygen tension in the p.p. zone. Rather, the regiospecificity of the injury may be due to rapid uptake and oxidation of AlOH in the p.p. region.

Topics: 1-Propanol; Adenosine Triphosphate; Alcohol Dehydrogenase; Animals; Digitonin; Glutathione; In Vitro Techniques; Liver; Microbial Collagenase; Oxidation-Reduction; Oxygen; Propanols; Rats