Archives ot
Toxicology
Arch Toxicol (1990) 64:49-53
9 Springer-Verlag 1990
Course of ATP depletion in hydrazine hepatotoxicity Nicholas E. Preece, Simin Ghatineh, and John A. Timbrell
Toxicology Unit, School of Pharmacy, University of London, 29/39 Brunswick Square, London WCIN lAX, UK Received May 2, 1989/Received after revision July 17, 198P/Accepted July 18, 1989
Abstract. The effect of hydrazine on ATP levels has been investigated in rats in vivo and in hepatocytes in vitro. Hydrazine was found to cause a dose-dependent depletion of hepatic ATP in vivo 3 h after dosing. In isolated hepatocytes in vitro hydrazine also caused a concentration-dependent depletion of ATP which preceded cytotoxicity as indicated by loss of cell viability. The ATP depletion in isolated hepatocytes was also significant at a concentration of hydrazine which was not cytotoxic. Attempts to determine hepatic ATP depletion in vivo over time using topical 31p N M R were confounded by the effects of the thiopentobarbitone used to anaesthetise the animals. This was found to ameliorate the effects of hydrazine on ATP depletion but potentiate the lethality of hydrazine. Consequently, although ATP depletion was detected in some hydrazine-treated animals, this was only observed in animals which subsequently died. The results indicate that ATP depletion may underlie the hepatotoxicity of hydrazine.
Key words: ATP - Hepatocytes 3tP-NMR - Thiopentobarbitone
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Hydrazine
-
Introduction
Hydrazine is a widely used chemical in industry and is also a minor metabolite of various drugs such as isoniazid and hydralazine. Hydrazine is toxic, the target organs being the liver and central nervous system. It causes many biochemical perturbations including hypoglycaemia and lacticaciduria. The hepatotoxicity is manifested as fatty liver which is a dose-related response, and which can be detected within 30 min of dosing using electron microscopy (Scales and Timbrell 1982). The mechanism underlying the accumulation of triglycerides causing the fatty liver is, however, currently unknown but seems to be at least partly due to a failure of secretion. Some studies however have postulated inhibition of protein synthesis (Lopez-Mendoza and Villa-Trevino 1971) or increased synthesis of diglycerides (Lamb and Banks 1979; Haghighi and Honarjou 1987) as mechanisms. Inhibition of protein synthesis has been reported to occur within 1 h of hydrazine administration yet was found to be increased after 24h. The key
Offprint requests to: N. E. Preece
enzyme which regulates triglyceride synthesis is phosphatidate phosphohydrolase, which cleaves phosphatidic acid to give the 1,2-diglyceride, and an increase in the activity of this enzyme follows the administration of hydrazine. One mechanism for the production of fatty liver is depletion of ATP as described for ethionine (Farber 1967). In a previous study of various toxic compounds, hydrazine was reported to perturb ATP levels in isolated cells in association with inhibition of urea synthesis and loss of cell viability (Story et al. 1983). Phenylhydrazine has also been shown to affect ATP levels in erythrocytes (Magnani et al. 1988). Consequently, the current study set out to determine if hydrazine causes a dose-dependent depletion of ATP in vivo and in vitro using a sensitive luminescence assay and also using topical 31p N M R for determination in the whole animal. Materials and methods
Hepatocyte experiments. Liver parenchymal cells were isolated from male Sprauge-Dawley rats (approx. 250 g) by collagenase perfusion (Moldeus et ai. 1978). They were subsequently incubated in rotating flasks at 37~ in Krebs-Henseleit buffer (containing varying concentrations of hydrazine, in an atmosphere of 95% 02/5% CO2) at a cell density of 2.25 • 106 cells/ml. Aliquots were taken at hourly intervals up to 4 h and the ceils were gently sedimented by centrifugation and resuspended in fresh buffer. Cell viability was estimated in a sample of each aliquot by the Trypan Blue method. This involved calculating the fraction of the total number of cells (approx. 100) on a haematometer slide able to exclude a 0.2% solution of Trypan Blue in 0.9% saline. The cells in the remainder of the aliquot were rapidly vortexed in 10% TCA to maintain endogenous ATP levels. Animal experiments. Rats were given doses of hydrazine (10, 20, 32, 40, 50 or 60 mg/kg i.p.) and killd 3 h later. Control animals were also always run on the same day and were dosed with saline at the same time. Liver samples were then rapidly freeze-clamped in liquid nitrogen and later pulverised in ice cold 10% TCA for ATP determination. Some animals were given an injection of either saline or 0.23 mmol sodium thiopentobarbitone/kg 1 h before the hydrazine treatment. A TP determination. ATP was measured by luciferaselinked bioluminescence (Stanley and Williams 1969) in
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Toxicology
Arch Toxicol (1990) 64:49-53
9 Springer-Verlag 1990
Course of ATP depletion in hydrazine hepatotoxicity Nicholas E. Preece, Simin Ghatineh, and John A. Timbrell
Toxicology Unit, School of Pharmacy, University of London, 29/39 Brunswick Square, London WCIN lAX, UK Received May 2, 1989/Received after revision July 17, 198P/Accepted July 18, 1989
Abstract. The effect of hydrazine on ATP levels has been investigated in rats in vivo and in hepatocytes in vitro. Hydrazine was found to cause a dose-dependent depletion of hepatic ATP in vivo 3 h after dosing. In isolated hepatocytes in vitro hydrazine also caused a concentration-dependent depletion of ATP which preceded cytotoxicity as indicated by loss of cell viability. The ATP depletion in isolated hepatocytes was also significant at a concentration of hydrazine which was not cytotoxic. Attempts to determine hepatic ATP depletion in vivo over time using topical 31p N M R were confounded by the effects of the thiopentobarbitone used to anaesthetise the animals. This was found to ameliorate the effects of hydrazine on ATP depletion but potentiate the lethality of hydrazine. Consequently, although ATP depletion was detected in some hydrazine-treated animals, this was only observed in animals which subsequently died. The results indicate that ATP depletion may underlie the hepatotoxicity of hydrazine.
Key words: ATP - Hepatocytes 3tP-NMR - Thiopentobarbitone
--
Hydrazine
-
Introduction
Hydrazine is a widely used chemical in industry and is also a minor metabolite of various drugs such as isoniazid and hydralazine. Hydrazine is toxic, the target organs being the liver and central nervous system. It causes many biochemical perturbations including hypoglycaemia and lacticaciduria. The hepatotoxicity is manifested as fatty liver which is a dose-related response, and which can be detected within 30 min of dosing using electron microscopy (Scales and Timbrell 1982). The mechanism underlying the accumulation of triglycerides causing the fatty liver is, however, currently unknown but seems to be at least partly due to a failure of secretion. Some studies however have postulated inhibition of protein synthesis (Lopez-Mendoza and Villa-Trevino 1971) or increased synthesis of diglycerides (Lamb and Banks 1979; Haghighi and Honarjou 1987) as mechanisms. Inhibition of protein synthesis has been reported to occur within 1 h of hydrazine administration yet was found to be increased after 24h. The key
Offprint requests to: N. E. Preece
enzyme which regulates triglyceride synthesis is phosphatidate phosphohydrolase, which cleaves phosphatidic acid to give the 1,2-diglyceride, and an increase in the activity of this enzyme follows the administration of hydrazine. One mechanism for the production of fatty liver is depletion of ATP as described for ethionine (Farber 1967). In a previous study of various toxic compounds, hydrazine was reported to perturb ATP levels in isolated cells in association with inhibition of urea synthesis and loss of cell viability (Story et al. 1983). Phenylhydrazine has also been shown to affect ATP levels in erythrocytes (Magnani et al. 1988). Consequently, the current study set out to determine if hydrazine causes a dose-dependent depletion of ATP in vivo and in vitro using a sensitive luminescence assay and also using topical 31p N M R for determination in the whole animal. Materials and methods
Hepatocyte experiments. Liver parenchymal cells were isolated from male Sprauge-Dawley rats (approx. 250 g) by collagenase perfusion (Moldeus et ai. 1978). They were subsequently incubated in rotating flasks at 37~ in Krebs-Henseleit buffer (containing varying concentrations of hydrazine, in an atmosphere of 95% 02/5% CO2) at a cell density of 2.25 • 106 cells/ml. Aliquots were taken at hourly intervals up to 4 h and the ceils were gently sedimented by centrifugation and resuspended in fresh buffer. Cell viability was estimated in a sample of each aliquot by the Trypan Blue method. This involved calculating the fraction of the total number of cells (approx. 100) on a haematometer slide able to exclude a 0.2% solution of Trypan Blue in 0.9% saline. The cells in the remainder of the aliquot were rapidly vortexed in 10% TCA to maintain endogenous ATP levels. Animal experiments. Rats were given doses of hydrazine (10, 20, 32, 40, 50 or 60 mg/kg i.p.) and killd 3 h later. Control animals were also always run on the same day and were dosed with saline at the same time. Liver samples were then rapidly freeze-clamped in liquid nitrogen and later pulverised in ice cold 10% TCA for ATP determination. Some animals were given an injection of either saline or 0.23 mmol sodium thiopentobarbitone/kg 1 h before the hydrazine treatment. A TP determination. ATP was measured by luciferaselinked bioluminescence (Stanley and Williams 1969) in
50 100-
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