Jenner, Andrew Michael; (1992) Studies on the metabolism and toxicity of hydrazine. Doctoral thesis (Ph.D.), University College London.

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Abstract

ABSTRACT Hydrazine is extensively utilised in industry and is a m inor metabolite of the clinically used drugs isoniazid and hydralazine. It is toxic, carcinogenic and mutagenic, but the metabolism and biochemical mechanisms of toxicity are not yet fu lly understood. Isolated rat liver microsomes incubated w ith both 2.0 mM and 0.2 m M hydrazine in the presence of NADPH and oxygen at 37°C resulted in the disappearance of hydrazine, which was demonstrated to be due to both enzymatic and chemical oxidation. Boiled microsomes increased the proportion of chemical disappearance whereas incubating the microsomes on ice effectively eliminated it. Further in vitro microsomal studies therefore incorporated samples incubated on ice as controls, allow ing the microsomal enzymatic metabolism of hydrazine to be calculated. Absence of NADPH and oxygen markedly reduced microsomal hydrazine metabolism, as did the presence of each of the cytochrome P450 inhibitors carbon monoxide, piperonyl butoxide and metyrapone, thus indicating that microsomal hydrazine metabolism is catalysed by cytochrome P450. Methimazole, an inhibitor of flavin monooxygenase, also diminished hydrazine metabolism, whereas N ADH in the presence of NADPH, but not alone, increased metabolism. Microsomes prepared from either p-naphthoflavone, acetone or isoniazid pretreated rats did not show significantly increased hydrazine metabolism compared to control microsomes per g protein. However phenobarbitone pretreatment did increase metabolism. Hydrazine metabolism was 20-70% lower in human microsomes prepared from 3 individuals compared to control rats. The dose response for hydrazine hepatotoxicity in vivo, as manifested by triglyceride increase and depletion of ATP and glutathione (GSH), was measured in control rats 6 hr after an i.p. dose. This was then compared to animals which had been pretreated w ith various inhibitors and inducers of cytochrome P450. Pretreatment w ith the inhibitor piperonyl butoxide resulted in an increase in hepatotoxicity, while induction by phenobarbitone (inducer of P450IIB) or p-naphthoflavone (inducer of P450IA) decreased hepatotoxicity. In contrast, acetone or isoniazid (inducers of II PREVIEW x ic /v iixj/ iaiu u ^ u u ii vauovu an 111^1 gaov 111 iic |/a iu iU A iu ijr, i n c 11 i u u u ia u u ii u i hydrazine hepatotoxicity by such pretreatments indicates that different isozymes of cytochrome P450 catalyse the metabolic transformation of hydrazine toxicity by various mechanisms. 6 hr after an acute i.p. hydrazine dose, certain dose related alterations in hepatic microsomal enzyme activity were measured, including a depletion in ethoxyresorufin O-deethylase and p-nitrophenol hydroxylase activity. Repeated adm inistration of 0.78 mM hydrazine in drinking water (2.5 m g.kg^.day'1) had a significant effect on several hepatic biochemical parameters and microsomal enzyme activities after 1,5 and 10 days. This indicated hydrazine to be a probable inducer of cytochrome P450IIE1. Hepatic biochemical parameters and activities of microsomal enzymes were virtually unchanged after repeated administration of 65 pM hydrazine in drinking water (0.25 m g.kg'1.day’1) for 5 or 10 days. In the presence of over 5 pM hydrazine, ATP synthesis in isolated mitochondria was inhibited. Inhibition up to 100 pM hydrazine was found to be dose related and reached a maximum 20-30% inhibition of control. However, above this concentration further inhibition did not occur. Hydrazine was also found to be metabolised by isolated mitochondria, which was not significantly decreased in the presence of either the monoamine oxidase A inhibitor, clorgyline or B inhibitor, pargyline.

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