Ghatineh, Simin; (1991) Studies on the biochemical basis of hydrazine toxicity in isolated rat hepatocytes. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Isolated hepatocytes in suspension and in primary monolayer culture were used for study of the metabolism and toxicity of the hepatotoxin, hydrazine. Dye uptake and the leakage of the cytosolic enzyme LDH were used as measures of cytotoxicity in vitro. Only 16mM and higher concentrations were cytotoxic to isolated hepatocytes in suspension. Hydrazine caused a dose-dependent depletion of ATP both in isolated hepatocytes and in rat liver in vivo. The depletion in isolated hepatocytes occured at a non-cytotoxic concentration, and the depletion in liver occured without lethality suggesting that ATP depletion may underlie the hepatotoxicity of hydrazine. The ATP depletion may be related to fatty liver caused by hydrazine. In isolated hepatocytes in suspension non-cytotoxic concentrations of hydrazine effected other biochemical parameters such as GSH, GSSG and NADPH. Hydrazine increased cellular triglycerides in hepatocytes in suspension and caused a parallel decrease in the medium. Therefore, inhibition of triglyceride secretion from the liver may be responsible for hydrazine-induced fatty liver. Hydrazine appeared to be more cytotoxic to hepatocytes in suspension than in primary culture, as measured by LDH leakage and ATP levels. This difference could be due to the loss of metabolising enzymes such as cytochrome P450 in primary culture. However, hydrazine inhibited protein synthesis in cultured hepatocytes at a concentration far below that which had measurable effects on cell viability and ATP levels. The inhibition of protein synthesis is probably involved in the toxicity of hydrazine (reduced lipoprotein synthesis may be responsible for fatty accumulation in hepatocytes). Using 1H and 15N NMR in hepatocytes, various metabolites of hydrazine including some acetylated species were detected. Metabolic inhibitors or low temperatures (4°C), reduced the levels of hydrazine inside the cells, suggesting that hydrazine entry into cells may be energy-dependent. Hydrazine levels in cultured hepatocytes were higher than those in suspension, but in both systems the concentration of hydrazine inside the cells was lower than that outside. Both metabolic inhibitors KCN and DNP increased hydrazine-induced cytotoxicity and ATP depletion. Cytochrome P450 has been implicated in the metabolism of hydrazine. The effects of single and repeated administration of hydrazine on the activity of different forms of this enzyme showed that hydrazine when administered repeatedly increased some of these activities.

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