Studies on the Disposition and Metabolism of Hydrazine in Rats in vivo Nicholas E.
Preece,* Simin Ghatineh
Toxicology Unit, School of Pharmacy, University
and John A. Timbrell of
London, Brunswick Square, London WC1N
1AX, UK 1 Rats were given various doses of hydrazine orally and their plasma and liver hydrazine levels were determined (at various times up to 270 min after dosing) by gas chromatography/ mass
spectrometry.
2 The increase in the peak plasma level and in the area under the plasma concentrationtime curve (AUC) were not directly proportional to the dose. 3 The ratio of plasma to liver hydrazine varied with dose suggesting saturation of an uptake mechanism might be occurring. 4 In a separate experiment hydrazine was still detectable in the plasma and liver 24 h after dosing with hydrazine i.p. 5 Rats were given the same doses of hydrazine and urine was collected for 24 h after dosing and assayed for hydrazine and acetylhydrazine. The proportion of hydrazine and acetylhydrazine excreted declined with dose. 6 Liver samples were taken for histopathological examination 96 h after dosing. Only after the highest dose (81 mg kg ) was there evidence of fatty liver, 96 h after a single dose, and a -1 reduction in both liver and body weight. Introduction
Hydrazine is a simple compound used extensively in the manufacture of many industrial products including plastic blowing agents, growth retardants and pharmaceuticals, and it is also used in rocket propellants. It has been shown to be a 2 metabolite of two important drugs, isoniazid1,2 has been itself and hydralazine,3 and hydrazine used in the treatment of weight loss in terminal cancer patients.4 Hydrazine is, however, a toxic compound causing a fatty liver5-9 and, in some cases, liver
necrosisl° and disturbances of the
system.ll It may also cause various organs,12,13 but only after the repeated administration of toxic doses. 13 An understanding of its metabolism and toxicity is therefore important. The disposition of hydrazine has been previously investigated using 15N2 hydrazine in rats 14 and unlabelled hydrazine in mice.15 Plasma and urinary hydrazine were measured after i.p. dosing in both species and the plasma profile showed two phases in both studies. 14,15 central
nervous
tumours in
*
Present address: Department of J.A. Timbrell.
Biophysics,
The
Certain of the urinary metabolites of hydrazine have been characterized by various authors’4-’9 and these previous metabolic studies have revealed that hydrazine is acetylated first to mono- and then to diacetyl-hydrazine, although these metabolites represent only a very small proportion of the dose. Hydrazine has also been shown to be metabolized to a significant extent to nitrogen which can be detected in expired air 14,15 and to form hydrazones.15,19 However, a significant proportion of the dose has not been accounted for. The effect of dose on the disposition and metabolism of hydrazine has not been studied and, in particular, the relationship of the liver hydrazine concentration to that of the plasma. Therefore the aim of this study was to examine the metabolism and disposition of toxic and nontoxic doses of hydrazine and to compare liver and plasma levels of the compound after the administration of these doses. We were particularly interested to discover the concentration of hydra-
Royal College
of
Surgeons.
Correspondence:
Downloaded from het.sagepub.com at EMORY UNIV on August 11, 2015
122
zine in the liver after toxic and non-toxic doses, to compare the results with in-vitro toxicity data and to determine how long hydrazine remains detectable in the liver.
Materials and methods
Male, Sprague Dawley rats (200 g), obtained from Olac Ltd, (Bicester, UK) were used. lsN2_hydrazine sulphate (doubly labelled; 98.6% g atom) was purchased from MSD Isotopes Ltd., Montreal, Canada. Hydrazine sulphate and pentafluorobenzaldehyde were obtained from Sigma Chemical Co. Ltd (Poole, Dorset, UK). Determination of time course of plasma and liver hydrazine levels For each dose level, rats’2 were dosed orally by stomach tube with hydrazine hydrate in distilled water. This was carried out at staggered intervals starting at 11.00 a.m. Animals were deprived of food for the 24 h prior to dosing. The doses administered were 4.7, 14.1, 42.2 and 126 mg kg hydrazine hydrate in water (3, 9, 27 and 81 mg kg hydrazine free base; 1 ml dosing solution kg body weight-1. These doses were devised as follows: 3 mg kg-1 is the acute dose below which it is known from our studies there is no detectable toxic effect on the liver measured as an accumulation of triglycerides. The doses were then increased by a factor of three to give a maximum dose (81 mg kg-1) which is approximately the maximum tolerated dose. At specified times after dosing (10, 30, 90, 270 min) each rat was anaesthetized with ether, blood was taken from the vena cava into a heparinized springe, transferred into a heparinized tube, centrifuged (2000 rpm) and plasma removed. The liver was removed from the animal, weighed and 5 g homogenized in 20 ml methanol/water (50/50; v/v). The homogenate was centrifuged and the supernatant was removed. The internal standard for the chromatography/mass spectroscopy assay (1 N2 hydrazine sulphate) was added to aliquots of supernatant and plasma immediately as described below. Control plasma samples were obtained from non-dosed control rats.
~as
Determination of hydrazine in plasma or liver. Internal standard solution, (15N2 hydrazine sulphate; 100 pm ; 0.1 ml) was added to plasma or liver supernatant samples (0.9 ml or 4.9 ml, respectively). Protein was precipitated in plasma or liver supemantant samples with HE1/ ammonium sulphate (0.2 ml 1 M HCI plus 2.3 ml saturated ammonium sulphate) and the sample vortexed (30 s) and centrifuged (20 min; 2500 g).
The supernatant was removed, an equal volume of citrate buffer (0.5 M; pH 6) added, the mixture vortexed and allowed to stand. The mixture was extracted with dichloromethane (20 ml), the aqueous layer removed and pentafluorobenzaldehyde solution (1 g 5 mi-1 methanol; 0.1I ml) added. The mixture was vortexed, left to stand for 30 min and extracted with chloroform (5 ml). The chloroform extract was separated and used for gas chromatography/mass spectroscopy assay. A standard curve was also prepared using spiked samples. Hydrazine concentration was determined by gas chromatography/mass spectrometry, with selected ion monitoring for the pentafluorobenzaldehyde azine molecular ion as described elsewhere.2° The ratio of 14Nr to ’SN2-azine was determined and used to calculate the concentration of 14 N2-hydrazine from the standard curve. A second study was carried out in which rats were dosed with ’SN2-hydrazine hydrate prepared from ’SN2-hydrazine sulphate (1.88 mmol kg-1 or 2.5 mmol kg-’; i.p.) and plasma and urine collected 4 or 24 h after dosing. methods were as described above except that N2-hydrazine and also acetylhydrazine were added to plasma or liver extracts as internal standards. The levels of hydrazine and acetylhydrazine were determined by gas chromatography/mass spectrometry essentially as described above.
Assay
Determination of toxicity and effect of dose of hydrazine on urinary metabolites Male, Sprague Dawley rats, (220 g) were dosed orally with hydrazine hydrate in water (0, 3, 9, 27, 81 mg kg~~ hydrazine as free base; 1 ml dosing solution kg body weight-1), three rats per dose. The animals were housed in metabolism cages designed for the separate collection of urine and faeces. Rats were deprived of food for the 24 h prior to dosing. Urine was collected daily over ice. Water intake and body weight were monitored daily for 4 d. At the termination of the study each rat was killed by exsanguination while anaesthetized with ether. The major organs (liver, testes, heart, kidneys, spleen) were taken from all rats and weighed. A section of liver was removed for histology and fixed in phosphate buffered formalin. Liver sections were embedded in paraffin wax for sectioning for subsequent staining with haematoxylin and eosin and frozen sections were cut for Oil Red 0 staining. All urine samples were stored frozen (-80°C) until analysis. Urine was analysed for hydrazine and acetylhydrazine by gas chromatography using a method to be fully described elsewhere. The area under the curve (AUCo_2?o) for both liver and plasma hydrazine concentrations was
Downloaded from het.sagepub.com at EMORY UNIV on August 11, 2015
123
determined by the trapezoidal method, calculated from individual data points.
Results Plasma and liver levels of hydrazine The levels of hydrazine determined in plasma and liver at various times after dosing are shown in Figures la-ld. These data show that the liver hydrazine concentration is initially higher than that in the plasma after doses of 3 and 9 mg kg 1, but lower after doses of 27 and 81 mg kg 1. In confirmation of this, when the ratio of the liver to plasma hydrazine concentrations is averaged over the whole experiment and plotted against dose (Figure 2) it can be seen that the ratio is greater than one at the lowest doses, but decreases to less than one at the highest dose. When the plasma and liver concentrations of hydrazine at 30 min are plotted against the dose (Figure 3) it can be seen that the liver concen-
Figure 1 Plasma and liver levels of d. 81 mg kg-1.
hydrazine
tration reaches a plateau whereas the plasma concentration increases in a linear fashion. The liver concentration measured at 10 min shows a similar profile (data not shown). However, consideration of the AUC for hydrazine in liver and plasma (Figure 4) indicates that there is not a proportionate increase in either the liver or plasma level of hydrazine with increasing dose level. There are insufficient data points to calculate kinetic parameters for each dose. The analysis of plasma and liver samples from animals dosed with 15N2-hydrazine indicated that hydrazine was still detectable 24 h after dosing, but more significantly that there was a five-fold greater amount in the liver than in the plasma at this time after both doses (Table 1). No acetylhydrazine was detected in plasma 24 h after
dosing. Gas chromatographic analysis of rat urine collected 0-24 h after dosing with various single doses of hydrazine showed that both hydrazine
after various doses.
a.
3 mg
kg-’; b. 9 mg kg-I; c. 27 mg kg-1;
Downloaded from het.sagepub.com at EMORY UNIV on August 11, 2015
124
Table 1 Liver and plasma concentrations of after two doses of 15N2-hydrazine.
hydrazine
Rats were dosed with 15N2-hydrazine sulphate. Results are means ± s.e. from three rats.
Figure 2 Ratio of liver hydrazine to plasma hydrazine concentration plotted against dose of hydrazine. The values are the mean ratios of liver:plasma hydrazine measured at 10, 30 90 and 270 min after dosing, averaged for each dose.
and acetylhydrazine could be detected. The excretion of hydrazine and acetylhydrazine were both found to be dose dependent (Table 2). The effects of single doses of hydrazine on the body weights of animals over a period of 4 d are shown in Figure 5. It can be seen that in all animals there is a significant gain in weight except in those receiving the highest dose of hydrazine (81 mg kg~1). These animals clearly lost weight after dosing and had a significantly lower weight compared with the controls. The animals which had received the highest dose of hydrazine also drank more water and produced more urine in the first 24 h (Table 3), but then drank less water than the other animals. Over the whole course of the study these animals drank significantly less water than control animals (97 vs 125 ml, P 0.036) and the ratio of urine:water was significantly higher for Day 1 when compared with the control animals (Table 3). This was mainly due to an increase in urine output and was maintained for 4 d. The animals which had received 27 mg kg of hydrazine also seemed to =
Figure 3 Plasma and liver levels of hydrazine measured at 30 min after dosing with hydrazine plotted against nmoles ml-’
g ’.
the dose.
Hydrazine concentration:
Figure 4 hydrazine
Area under the curve (AU Co-270) for in liver and plasma after various doses.
or
Figure 5 Effect
body weight.
Downloaded from het.sagepub.com at EMORY UNIV on August 11, 2015
P
of various
Preece,* Simin Ghatineh
Toxicology Unit, School of Pharmacy, University
and John A. Timbrell of
London, Brunswick Square, London WC1N
1AX, UK 1 Rats were given various doses of hydrazine orally and their plasma and liver hydrazine levels were determined (at various times up to 270 min after dosing) by gas chromatography/ mass
spectrometry.
2 The increase in the peak plasma level and in the area under the plasma concentrationtime curve (AUC) were not directly proportional to the dose. 3 The ratio of plasma to liver hydrazine varied with dose suggesting saturation of an uptake mechanism might be occurring. 4 In a separate experiment hydrazine was still detectable in the plasma and liver 24 h after dosing with hydrazine i.p. 5 Rats were given the same doses of hydrazine and urine was collected for 24 h after dosing and assayed for hydrazine and acetylhydrazine. The proportion of hydrazine and acetylhydrazine excreted declined with dose. 6 Liver samples were taken for histopathological examination 96 h after dosing. Only after the highest dose (81 mg kg ) was there evidence of fatty liver, 96 h after a single dose, and a -1 reduction in both liver and body weight. Introduction
Hydrazine is a simple compound used extensively in the manufacture of many industrial products including plastic blowing agents, growth retardants and pharmaceuticals, and it is also used in rocket propellants. It has been shown to be a 2 metabolite of two important drugs, isoniazid1,2 has been itself and hydralazine,3 and hydrazine used in the treatment of weight loss in terminal cancer patients.4 Hydrazine is, however, a toxic compound causing a fatty liver5-9 and, in some cases, liver
necrosisl° and disturbances of the
system.ll It may also cause various organs,12,13 but only after the repeated administration of toxic doses. 13 An understanding of its metabolism and toxicity is therefore important. The disposition of hydrazine has been previously investigated using 15N2 hydrazine in rats 14 and unlabelled hydrazine in mice.15 Plasma and urinary hydrazine were measured after i.p. dosing in both species and the plasma profile showed two phases in both studies. 14,15 central
nervous
tumours in
*
Present address: Department of J.A. Timbrell.
Biophysics,
The
Certain of the urinary metabolites of hydrazine have been characterized by various authors’4-’9 and these previous metabolic studies have revealed that hydrazine is acetylated first to mono- and then to diacetyl-hydrazine, although these metabolites represent only a very small proportion of the dose. Hydrazine has also been shown to be metabolized to a significant extent to nitrogen which can be detected in expired air 14,15 and to form hydrazones.15,19 However, a significant proportion of the dose has not been accounted for. The effect of dose on the disposition and metabolism of hydrazine has not been studied and, in particular, the relationship of the liver hydrazine concentration to that of the plasma. Therefore the aim of this study was to examine the metabolism and disposition of toxic and nontoxic doses of hydrazine and to compare liver and plasma levels of the compound after the administration of these doses. We were particularly interested to discover the concentration of hydra-
Royal College
of
Surgeons.
Correspondence:
Downloaded from het.sagepub.com at EMORY UNIV on August 11, 2015
122
zine in the liver after toxic and non-toxic doses, to compare the results with in-vitro toxicity data and to determine how long hydrazine remains detectable in the liver.
Materials and methods
Male, Sprague Dawley rats (200 g), obtained from Olac Ltd, (Bicester, UK) were used. lsN2_hydrazine sulphate (doubly labelled; 98.6% g atom) was purchased from MSD Isotopes Ltd., Montreal, Canada. Hydrazine sulphate and pentafluorobenzaldehyde were obtained from Sigma Chemical Co. Ltd (Poole, Dorset, UK). Determination of time course of plasma and liver hydrazine levels For each dose level, rats’2 were dosed orally by stomach tube with hydrazine hydrate in distilled water. This was carried out at staggered intervals starting at 11.00 a.m. Animals were deprived of food for the 24 h prior to dosing. The doses administered were 4.7, 14.1, 42.2 and 126 mg kg hydrazine hydrate in water (3, 9, 27 and 81 mg kg hydrazine free base; 1 ml dosing solution kg body weight-1. These doses were devised as follows: 3 mg kg-1 is the acute dose below which it is known from our studies there is no detectable toxic effect on the liver measured as an accumulation of triglycerides. The doses were then increased by a factor of three to give a maximum dose (81 mg kg-1) which is approximately the maximum tolerated dose. At specified times after dosing (10, 30, 90, 270 min) each rat was anaesthetized with ether, blood was taken from the vena cava into a heparinized springe, transferred into a heparinized tube, centrifuged (2000 rpm) and plasma removed. The liver was removed from the animal, weighed and 5 g homogenized in 20 ml methanol/water (50/50; v/v). The homogenate was centrifuged and the supernatant was removed. The internal standard for the chromatography/mass spectroscopy assay (1 N2 hydrazine sulphate) was added to aliquots of supernatant and plasma immediately as described below. Control plasma samples were obtained from non-dosed control rats.
~as
Determination of hydrazine in plasma or liver. Internal standard solution, (15N2 hydrazine sulphate; 100 pm ; 0.1 ml) was added to plasma or liver supernatant samples (0.9 ml or 4.9 ml, respectively). Protein was precipitated in plasma or liver supemantant samples with HE1/ ammonium sulphate (0.2 ml 1 M HCI plus 2.3 ml saturated ammonium sulphate) and the sample vortexed (30 s) and centrifuged (20 min; 2500 g).
The supernatant was removed, an equal volume of citrate buffer (0.5 M; pH 6) added, the mixture vortexed and allowed to stand. The mixture was extracted with dichloromethane (20 ml), the aqueous layer removed and pentafluorobenzaldehyde solution (1 g 5 mi-1 methanol; 0.1I ml) added. The mixture was vortexed, left to stand for 30 min and extracted with chloroform (5 ml). The chloroform extract was separated and used for gas chromatography/mass spectroscopy assay. A standard curve was also prepared using spiked samples. Hydrazine concentration was determined by gas chromatography/mass spectrometry, with selected ion monitoring for the pentafluorobenzaldehyde azine molecular ion as described elsewhere.2° The ratio of 14Nr to ’SN2-azine was determined and used to calculate the concentration of 14 N2-hydrazine from the standard curve. A second study was carried out in which rats were dosed with ’SN2-hydrazine hydrate prepared from ’SN2-hydrazine sulphate (1.88 mmol kg-1 or 2.5 mmol kg-’; i.p.) and plasma and urine collected 4 or 24 h after dosing. methods were as described above except that N2-hydrazine and also acetylhydrazine were added to plasma or liver extracts as internal standards. The levels of hydrazine and acetylhydrazine were determined by gas chromatography/mass spectrometry essentially as described above.
Assay
Determination of toxicity and effect of dose of hydrazine on urinary metabolites Male, Sprague Dawley rats, (220 g) were dosed orally with hydrazine hydrate in water (0, 3, 9, 27, 81 mg kg~~ hydrazine as free base; 1 ml dosing solution kg body weight-1), three rats per dose. The animals were housed in metabolism cages designed for the separate collection of urine and faeces. Rats were deprived of food for the 24 h prior to dosing. Urine was collected daily over ice. Water intake and body weight were monitored daily for 4 d. At the termination of the study each rat was killed by exsanguination while anaesthetized with ether. The major organs (liver, testes, heart, kidneys, spleen) were taken from all rats and weighed. A section of liver was removed for histology and fixed in phosphate buffered formalin. Liver sections were embedded in paraffin wax for sectioning for subsequent staining with haematoxylin and eosin and frozen sections were cut for Oil Red 0 staining. All urine samples were stored frozen (-80°C) until analysis. Urine was analysed for hydrazine and acetylhydrazine by gas chromatography using a method to be fully described elsewhere. The area under the curve (AUCo_2?o) for both liver and plasma hydrazine concentrations was
Downloaded from het.sagepub.com at EMORY UNIV on August 11, 2015
123
determined by the trapezoidal method, calculated from individual data points.
Results Plasma and liver levels of hydrazine The levels of hydrazine determined in plasma and liver at various times after dosing are shown in Figures la-ld. These data show that the liver hydrazine concentration is initially higher than that in the plasma after doses of 3 and 9 mg kg 1, but lower after doses of 27 and 81 mg kg 1. In confirmation of this, when the ratio of the liver to plasma hydrazine concentrations is averaged over the whole experiment and plotted against dose (Figure 2) it can be seen that the ratio is greater than one at the lowest doses, but decreases to less than one at the highest dose. When the plasma and liver concentrations of hydrazine at 30 min are plotted against the dose (Figure 3) it can be seen that the liver concen-
Figure 1 Plasma and liver levels of d. 81 mg kg-1.
hydrazine
tration reaches a plateau whereas the plasma concentration increases in a linear fashion. The liver concentration measured at 10 min shows a similar profile (data not shown). However, consideration of the AUC for hydrazine in liver and plasma (Figure 4) indicates that there is not a proportionate increase in either the liver or plasma level of hydrazine with increasing dose level. There are insufficient data points to calculate kinetic parameters for each dose. The analysis of plasma and liver samples from animals dosed with 15N2-hydrazine indicated that hydrazine was still detectable 24 h after dosing, but more significantly that there was a five-fold greater amount in the liver than in the plasma at this time after both doses (Table 1). No acetylhydrazine was detected in plasma 24 h after
dosing. Gas chromatographic analysis of rat urine collected 0-24 h after dosing with various single doses of hydrazine showed that both hydrazine
after various doses.
a.
3 mg
kg-’; b. 9 mg kg-I; c. 27 mg kg-1;
Downloaded from het.sagepub.com at EMORY UNIV on August 11, 2015
124
Table 1 Liver and plasma concentrations of after two doses of 15N2-hydrazine.
hydrazine
Rats were dosed with 15N2-hydrazine sulphate. Results are means ± s.e. from three rats.
Figure 2 Ratio of liver hydrazine to plasma hydrazine concentration plotted against dose of hydrazine. The values are the mean ratios of liver:plasma hydrazine measured at 10, 30 90 and 270 min after dosing, averaged for each dose.
and acetylhydrazine could be detected. The excretion of hydrazine and acetylhydrazine were both found to be dose dependent (Table 2). The effects of single doses of hydrazine on the body weights of animals over a period of 4 d are shown in Figure 5. It can be seen that in all animals there is a significant gain in weight except in those receiving the highest dose of hydrazine (81 mg kg~1). These animals clearly lost weight after dosing and had a significantly lower weight compared with the controls. The animals which had received the highest dose of hydrazine also drank more water and produced more urine in the first 24 h (Table 3), but then drank less water than the other animals. Over the whole course of the study these animals drank significantly less water than control animals (97 vs 125 ml, P 0.036) and the ratio of urine:water was significantly higher for Day 1 when compared with the control animals (Table 3). This was mainly due to an increase in urine output and was maintained for 4 d. The animals which had received 27 mg kg of hydrazine also seemed to =
Figure 3 Plasma and liver levels of hydrazine measured at 30 min after dosing with hydrazine plotted against nmoles ml-’
g ’.
the dose.
Hydrazine concentration:
Figure 4 hydrazine
Area under the curve (AU Co-270) for in liver and plasma after various doses.
or
Figure 5 Effect
body weight.
Downloaded from het.sagepub.com at EMORY UNIV on August 11, 2015
P
of various
