Metabolic and Mechanistic Studies on Hydrazine Hepatotoxicity in The

Home , Gyromitrin

TOXICOLOGY DEPARTMENT

THE SCHOOL OF PHARMACY

UNIVERSITY OF LONDON

METABOLIC AND MECHANISTIC

STUDIES ON HYDRAZINE

HEPATOTOXICITY IN THE RAT

A Thesis Submitted by

Jane Delaney

to the University o f London

for examination for the degree of

Doctor of Philosophy

7996 ProQuest Number: 10104259

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Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author.

ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 ABSTRACT

Hydrazine, a chemical with numerous applications in industry, is carcinogenic and acutely toxic to the central nervous system and liver. Metabolism and mechanisms of toxicity are not yet fully understood.

Previous workers have observed increased acute hydrazine hepatotoxicity in cytochrome P4502E 1-induced rats (isoniazid pretreated). In contrast apparent induction of this isoenzyme by pretreatment of rats with hydrazine protected against hydrazine-induced cytotoxicity in isolated hepatocytes but had little effect in vivo. These results may indicate that P4502E1 catalyses a detoxication pathway for hydrazine which plays only a minor role in vivo. Cytochromes P4501A1/2 and P4502B1/2 appear to catalyse detoxication pathways as a reduction in their activities correlates with increased hydrazine toxicity.

A further study failed to clarify the induction of P4502E1 by hydrazine as, despite a dose dependent increase in p-nitrophenol hydroxylase activity, there was no apparent increase in enzyme protein, determined by immunohistochemical staining. This could explain the discrepancy between isoniazid and hydrazine pretreatments with regard to acute hydrazine toxicity.

There is conflicting data regarding the effect of hydrazine on protein synthesis. Incorporation of ^H-leucine into protein in vitro in isolated ra t hepatocytes was reduced 2 hours after exposure to 0.5mM hydrazine and 1 hour after higher concentrations. In vivo there was some evidence of protein synthesis inhibition 3 hours after 30 and 60mg/Kg hydrazine administration, followed by stimulation 24 hours post dose. The mechanism of action is currently unknown.

Fatty liver is the major acute toxic manifestation in liver after exposure to hydrazine and appears to be unrelated to hydrazine metabolism. Microsomal phosphatidate phosphohydrolase activity and triglyceride content (NMR analysis) were raised 24 hours after hydrazine dosing, consistent with increased lipid synthesis. Raised serum triglycerides and control levels of phosphatidylcholine, required for VLDL synthesis, tend to suggest normal triglyceride secretion at this time, however there may have been a transient drop in VLDL production, due to decreased protein synthesis 3 hours post dose, which may have facilitated the accumulation of fat. Phosphatidylinositol was also raised but the reason for this is unknown.

11 ACKNOWLEDGEMENT

I would like to express my gratitude to my supervisor, Professor John Timbrell, for his advice and guidance (and not forgetting the occasional piece of Kit-Kat!) over the last 3 years.

I would also like to thank everyone that suffered along with me throughout my ordeal, including all my fellow Tox. students, Adrian Rogers for processing many late orders, Dave McCarthy for his help on both light and electron microscopes, and Dr. Cathy Waterfield on whose expertise I constantly relied. Thanks also to Professor Gibbons and Mire Zloh, from the chemistry department, for their assistance in generating and interpretting NMR spectra.

Special thanks must go to Alka for her friendship, and to Ian for his patience, and support. PhD life would have been much harder without them.

Finally, I would like to thank my parents who have given me the best opportunities in life. Without their continuous support and encouragement I would never have got this far! It is to them that I dedicate this thesis.

Ill CONTENTS

ABSTRACT...... i

ACKNOWLEDGEMENT...... iii

CONTENTS ...... iv

LIST OF FIGURES ...... viii

LIST OF TABLES ...... xi

LIST OF PHOTOMICROGRAPHS...... xii

LIST OF ELECTRONMICROGRAPHS ...... xii

ABBREVIATIONS...... xiii

CHAPTER 1 ...... 1

GENERAL INTRODUCTION ...... 1 1.1 IN TR O D U C TIO N ...... 1 1.2 PHYSICAL AND CHEMICAL PROPERTIES ...... 1 1.3 A PPLIC A TIO N S...... 2 1.4 METABOLISM OF HYDRAZINE AND ITS DERIVATIVES . 4 1.4.1 Absorbance, Distribution and Urinary Excretion of Hydrazine ...... 4 1.4.2 Metabolism of the Parent Compound ...... 5 1.5 INTERACTION OF HYDRAZINE COMPOUNDS WITH METABOLIC SYSTEMS ...... 10 1.5.1 Oxyhaemoglobin ...... 10 1.5.2 Haem-containing Enzymes ...... 10 1.5.3 Flavin-containing Enzymes ...... 11 1.6 TOXICITY OF HYDRAZINE...... 11 1.7 BIOCHEMICAL EFFECTS OF HYDRAZINE ...... 13 1.7.1 CNS Disturbances ...... 13 1.7.2 Carbohydrate Metabolism ...... 15 1.7.3 Lipid M etabolism ...... 16 1.7.3.1 Source of precursors for lipid synthesis .... 16 1.7.3.2 Synthesis of Liver L ipids ...... 17 1.7.3.3 Mechanism for the development of fatty l i v e r...... 18 1.7.3.4 The Effect of Hydrazine on Liver Lipids .... 20 1.7.4 Protein Metabolism ...... 23 1.7.5 Urea Cycle ...... 26 1.7.6 Mitochondrial E ffects ...... 28 1.7.7 Depletion of Reduced Glutathione ...... 30 1.8 MUTAGENICITY AND CARCINOGENICITY OF HYDRAZINE AND ITS DERIVATIVES...... 33

IV 1.9 ANTITUMOUR ACTIVITY OF HYDRAZINES ...... 37 1.10 TERATOGENICITY ...... 38 1.11 AIM OF STUDY ...... 39

CHAPTER 2 ...... 40

MATERIALS AND METHODS ...... 40 2.1 CHEMICALS ...... 40 2.2 IN VIVO S T U D IE S ...... 41 2.2.1 Animal Husbandry ...... 41 2.2.2 Autopsy Procedure ...... 41 2.2.3 Isolation of rat liver microsomes...... 42 2.2.4 Total Cytochrome-P450 ...... 42 2.2.5 p-NitrophenoI Hydroxylase ...... 43 2.2.6 Ethoxyresorufin o-deethylase (EROD)/ Pentoxyresorufin o-depentylase (PROD) ...... 44 2.2.7 Determination of Protein ...... 45 2.2.8 Adenosine Triphosphate (ATP) ...... 45 2.2.9 Total Non-Protein Sulphydryls (TNPSH) ...... 46 2.2.10 Oxidised Glutathione (GSSG) ...... 46 2.2.11 Liver Triglyceride Analysis ...... 47 2.2.12 Serum Clinical Chemistry ...... 47 2.3 IN VITRO ST U D IE S ...... 48 2.3.1 Animal Husbandry ...... 48 2.3.2 Preparation of Isolated Hepatocytes ...... 48 2.3.3 Trypan Blue Dye Exclusion ...... 49 2.3.4 Lactate Dehydrogenase (LDH) ...... 50 2.3.5 Reduced Glutathione (GSH) ...... 50

CHAPTER 3 ...... 51

THE EFFECT OF INDUCTION OR INHIBITION OF CYTOCHROME- P450 ON HYDRAZINE TOXICITY 7 W /T R 0 ...... 51 3.1 INTRODUCTION ...... 51 3.2 MATERIALS AND METHODS ...... 52 3.2.1 Animal Pretreatm ent ...... 52 3.2.2 Hepatocyte Preparation ...... 52 3.2.3 Biochemical Determinations ...... 53 3.2.4 Statistical Analysis ...... 53 3.3 RESULTS ...... 53 3.3.1 The Toxicity of Hydrazine in Hepatocytes Isolated from Untreated Rats ...... 53 3.3.2 The Effect of Induction and Inhibition of Cytochrome P4502E1 on Hydrazine Toxicity ...... 56 3.4 DISCUSSION ...... 64 CHAPTER 4 ...... 69

THE EFFECT OF REPEATED EXPOSURE TO HYDRAZINE ON CYTOCHROME P450 AND LIVER TOXICITY/AT y /y O ...... 69 4.1 INTRODUCTION ...... 69 4.2 METHODS ...... 71 4.2.1 Animal Husbandry ...... 71 4.2.2 The Hepatic Effects of Subchronic Exposure to a Range of Hydrazine Doses: Study 1 ...... 71 4.2.3 The Effect of Hepatic Cytochrome P4502E1 Induction on Acute Hydrazine Toxicity: Study 2 ...... 72 4.2.4 Light Microscopy ...... 72 4.2.5 Electron Microscopy ...... 73 4.2.6 Statistical Analysis ...... 73 4.3 RESULTS ...... 74 4.3.1 The Hepatic Effects of Subchronic Exposure to a Range of Hydrazine Doses: Study 1 ...... 74 4.3.2 The Effect of Induction of Cytochrome P4502E1 on Acute Hydrazine Toxicity: Study 2 ...... 84 4.4 DISCUSSION ...... 89

CHAPTER 5 ...... 95

THE EFFECT OF HYDRAZINE ON HEPATIC PROTEIN SYNTHESIS IN VrVO ANT> IN VITRO ...... 95 5.1 INTRODUCTION ...... 95 5.2 METHODS ...... 97 5.2.1 The Effect of Hydrazine on Protein Synthesis in Isolated Rat Hepatocytes In V itr o...... 97 5.2.2 The Effect of Hydrazine on Hepatic Protein Synthesis in the Rat In Vivo ...... 98 5.2.2.1 Animal Husbandry ...... 98 5.2.2.2 Dose Response Experiment ...... 98 5.2.2.3 Time Course Experim ent ...... 98 5.2.3 Estimation of Serum Protein Synthesis ...... 99 5.2.4 Estimation of Hepatic Protein Synthesis ...... 99 5.2.4.1 Acid (TCA) soluble proteins: ...... 99 5.2.4.2 Acid (TCA) precipitable proteins: ...... 100 5.2.5 Serum Ammonia ...... 100 5.2.6 Preparation of samples for total RNA and DNA c o n te n t ...... 101 5.2.7 Estimation of Total RNA Using the Orcinol Method . 101 5.2.8 Estimation of Total Liver DNA ...... 102 5.3 Statistical Analysis ...... 102 5.3 RESULTS...... 102 5.3.1 The Effect of Hydrazine on Protein Synthesis in Isolated Hepatocytes In V itro ...... 102 5.3.2 The Effect of Hydrazine on Protein Synthesis in Rat Liver In Vivo ...... 105

VI 5.3.2.1 Study 1 Dose Response Experiment 105 5.3.2.2 Study 2 Time Course Experim ent ...... 108 5.4 DISCUSSION ...... 116

CHAPTER 6 ...... 121

THE EFFECT OF AN ACUTE DOSE OF HYDRAZINE ON LIVER LIPIDS...... 121 6.1 INTRODUCTION ...... 121 6.2 METHODS ...... 123 6.2.1 Treatment Regime ...... 123 6.2.2 Phosphatidate Phosphohydrolase Activity ...... 123 6.2.3 Determination of Liver Lipids ...... 124 6.2.3.1 Extraction of Lipids for Proton NM R 124 6.2.3.2 Separation of Different Classes of Lipids . . . 125 6.2.3.3 Proton NMR Spectroscopy ...... 126 6.3 RESULTS...... 126 6.4 DISCUSSION ...... 131

CHAPTER 7 ...... 135

FINAL DISCUSSION ...... 135 7.1 THE ROLE OF CYTOCHROMES P450 IN HYDRAZINE TOXICITY ...... 135 7.1.1 Modulation of the Activities of Certain Cytochrome P450 Isoenzymes on Acute Hydrazine Toxicity In Vivo and In V itro ...... 135 7.1.2 Induction of Cytochrome P4502E1 by Repeated Exposure to Hydrazine ...... 136 7.2 THE EFFECT OF HYDRAZINE ON LIVER LIPIDS ...... 138 7.3 THE EFFECT OF HYDRAZINE ON PROTEIN SYNTHESIS ...... 139 7.4 GENERAL CONCLUSIONS ...... 140 7.5 FUTURE STUDIES ...... 141 7.5.1 Verification of Involvement of Cytochrome P4502E1 in Hydrazine Metabolism ...... 141 7.5.2 Further Investigations into Hydrazine-induced Fatty Liver ...... 142 7.5.3 Further Investigations into the Effects of Hydrazine on Protein Synthesis ...... 142

REFERENCES ...... 144

APPENDICES...... 173

PUBLICATIONS...... 183

Vll LIST OF FIGURES

Figure 1.1 T h e Structure of Hydrazine and Some of its Therapeutic Derivatives

Figure 1.2 The Metabolic Pathways of Hydrazine

Figure 1.3 The Pathways of Fat Metabolism

Figure 1.4 The Pathways of Glycerolipid Synthesis

Figure 1.5 The U rea Cycle

Figure 2.1 The Equipment Utilised For Hepatocyte Isolation

Figure 3.1 The Effect of a Range of Hydrazine Concentrations on Cell Viability and LDH Leakage in Hepatocytes Isolated From Control Rats

Figure 3.2 The Effect of a Range of Hydrazine Concentrations on GSH Depletion in Hepatocytes Isolated From Control Rats

Figure 3.3 The Effect of a Range of Hydrazine Concentrations on ATP Depletion in Hepatocytes Isolated From Control Rats

Figure 3.4 The Effect of Various Pretreatments on Hydrazine Induced Cell Death in Isolated Rat Hepatocytes

Figure 3.5 The Effect of Various Pretreatments on Hydrazine Induced LDH Leakage in Isolated Rat Hepatocytes

Figure 3.6 The Effect of Various Pretreatments on Hydrazine Induced GSH Depletion in Isolated Rat Hepatocytes

Figure 3.7 The Effect of Various Pretreatments on Hydrazine Induced ATP Depletion in Isolated Rat Hepatocytes

Figure 3.8 Correlation Between p-Nitrophenol Hydroxylase Activity (P4502E1) and Cell Viability in Hepatocytes Exposed to Hydrazine

Figure 3.9 Correlation Between EROD Activity (P4501A1/2) and Cell Viability in Hepatocytes Exposed to Hydrazine

Figure 3.10 Correlation Between PROD Activity (P4502B1/2) and Cell Viability in Hepatocytes Exposed to Hydrazine

Figure 4.1 The Effect of Repeated Exposure to a Range of Hydrazine Doses on Body Weight in Rats

vm Figure 4.2 The Effect of Repeated Exposure to a Range of Hydrazine Doses on Food Intake in Rats

Figure 4.3 The Effect of Repeated Exposure to a Range of Hydrazine Doses on Water Intake in Rats Figure 4.4 The Effect of Repeated Exposure to a Range of Hydrazine Doses on Hepatic Reduced Glutathione Content in Rats

Figure 4.5 The Effect of Repeated Exposure to a Range of Hydrazine Doses on Hepatic Oxidised Glutathione Content in Rats

Figure 4.6 The Effect of Repeated Exposure to a Range of Hydrazine Doses on Hepatic ATP Content in Rats

Figure 4.7 The Effect of Repeated Exposure to a Range of Hydrazine Doses on Hepatic Triglyceride Content in Rats

Figure 4.8 The Effect of Repeated and Acute Exposure of Rats to Hydrazine on Relative Liver Weight

Figure 4.9 The Effect of Repeated and Acute Exposure of Rats to Hydrazine on Hepatic Triglyceride Content

Figure 5.1 Inhibition of Protein Synthesis in Control Rat Hepatocytes after Exposure to a Range of Hydrazine Doses for 3 Hours

Figure 5.2 The Relationship Between Protein Synthesis and Glutathione Content in Isolated Rat Hepatocytes Exposed to Hydrazine

Figure 5.3 The Relationship Between Protein Synthesis and ATP Content in Isolated Rat Hepatocytes Exposed to Hydrazine

Figure 5.4 Incorporation of %-Leucine into Hepatic and Serum Proteins 3 Hours After Administration of a Range of Hydrazine Doses to Rats In Vivo

Figure 5.5 The Time-Dependent Depletion of Hepatic GSH after Administration of 60mg/Kg Hydrazine to Rats In Vivo

Figure 5.6 The Time-Dependent Depletion of Hepatic ATP after Administration of 60mg/Kg Hydrazine to Rats In Vivo

Figure 5.7 Incorporation of^^C-Leucine into Acid Precipitable Proteins 0.5-24 Hours After Administration of 60mg/Kg Hydrazine to Rats In Vivo

Figure 5.8 Incorporation of ^^C-Leucine into Acid Soluble Proteins 0.5-24 Hours After Administration of 60mg/Kg Hydrazine to Rats In Vivo

Figure 5.9 Incorporation of ^^C-Leucine into Serum Proteins 0.5-24 Hours After Administration of 60mg/Kg Hydrazine to Rats In Vivo

IX Figure 5.10 Time-Dependent Fluctuation of Serum Ammonia 0.5-24 Hours After Administration of 60mg/Kg Hydrazine to Rats In Vivo LIST OF TABLES

Table 3.1 The Effect of Various Pretreatments on Cytochrome P450 Activities in Isolated Rat Hepatocytes

Table 3.2 Correlation Between Cytochrome P450 Activities and Biochemical Parameters in Isolated Rat Hepatocytes

Table 4.1 The Effect of Repeated Exposure of Rats to 2.5, 5, and lOmg/Kg Hydrazine on Body and Liver Weight

Table 4.2 The Effect of Repeated Exposure of Rats to 2.5, 5, and lOmg/Kg Hydrazine on Hepatic Microsomal Enzyme Activities

Table 4.3 The Effect of an Acute Dose of Hydrazine on Microsomal Enzyme Activities in Control and Hydrazine Pretreated Rats

Table 4.4 The Influence of Hydrazine Pretreatment on the Biochemical Effects of a Subsequent Acute Dose of Hydrazine

Table 5.1 The Effect of a Range of Hydrazine Doses on Body and Liver Weights and Parameters of Liver Toxicity 3 Hours Post Dose

Table 5.2 The Effect of a Range of Hydrazine Doses on Liver DNA, RNA, and Protein 3 Hours Post Dose

Table 5.3 The Effect of a Range of Hydrazine Doses on the Protein Content of Liver and Serum 3 Hours Post Dose

Table 5.4 The Effect of 60mg/Kg Hydrazine on Body and Liver Weights and Parameters of Liver Over a 24 Hour Exposure Period

Table 5.5 The Effect of 60mg/Kg Hydrazine on Liver DNA, RNA, and Protein Over a 24 Hour Exposure Period

Table 5.6 The Effect of 60mg/Kg Hydrazine on the Protein Content of Liver and Serum Over a 24 Hour Exposure Period

Table 6.1 Alterations to Lipid Parameters in Liver and Serum 24 Hours After an Acute Dose of Hydrazine

Table 6.2 Lipids Extracted From the Livers of Rats Dosed 24 Hours Previously with 30mg/Kg Hydrazine. Peak Areas Obtained by NMR Analysis of Chloroform Extracts

XI LIST OF PHOTOMICROGRAPHS

Photomicrograph 4.1 Distribution of Cytochrome P4502E1 Within The Liver Lobule

Photomicrograph 4.2a H+E Staining of Liver Taken From a Control Rat

Photomicrograph 4.2b H+E Staining of Liver Taken From a Rat Dosed With lOmg/Kg Hydrazine For 10 Days

Photomicrograph 4.3a Oil Red O Staining of Liver Taken From a Control Rat

Photomicrograph 4.3b Oil Red 0 Staining of Liver Taken From a Rat Dosed with 30mg/Kg Hydrazine 24 Hours Previously

LIST OF ELECTRONMICROGRAPHS

Electronmicrograph 4.1a Transmission EM of Liver Taken From a Control Rat

Electronmicrograph 4.1b Transmission EM of Liver Taken From a Rat Dosed With lOmg/Kg Hydrazine For 10 Days

Xll ABBREVIATIONS

ADP adenosine diphosphate ATP adenosine triphosphate CDP cytidyl diphosphate CO carbon monoxide OTP cytidyl triphosphate DAG diacylglycerol DEDC diethyldithiocarbamate DNA deoxyribonucleic acid DTNB dithiobis-(2-nitrobenzoic acid) DW deionised water EROD ethoxyresorufin o-deethylase FADHg flavin adenine dinucleotide (reduced form) GABA gamma-amino butyric acid GABA-T gamma-amino butyric acid transaminase GAD gamma-amino butyric acid decarboxylase GSH reduced glutathione GSSG oxidised glutathione H+E haematoxylin and eosin LDH lactate dehydrogenase MAO monoamine oxidase NADH nicotinamide adenine dinucleotide (reduced form) NADPH nicotinamide adenine dinucleotide phosphate (reduced form) NE FA non-esterified fatty acids NEM N-ethy Imaleimide NMR nuclear magnetic resonance (spectroscopy) OPT o-phthaldialdehyde PAP phosphatidate phosphohydrolase PC phosphatidylcholine PGA perchloric acid PE phosphatidylethanolamine PEPCK phosphoenolpyruvate carboxykinase PI phosphatidylinositol PL phospholipids PLP pyridoxal phosphate PROD pentoxyresorufin o-depentylase RNA ribonucleic acid mRNA messenger ribonucleic acid SDS sodium dodecyl sulphate SER smooth endoplasmic reticulum SLE systemic lupus eryhtematosus SSA sulphosalicylic acid TCA trichloroacetic acid TG triglycerides THOPC l,4,5,6-tetrahydro-6-oxo-3-pyridazine carboxylic acid TNF tumour necrosis factor TNPSH total non-protein sulphydryls VLDL very low density lipoprotein

Xlll (DIHâlPÏME ©NU

GENERAL INTRODUCTION

1.1 INTRODUCTION

A proportion of the human population is exposed to hydrazine due to its widespread use in pharmaceutical and agricultural chemicals and in numerous industrial processes. Hydrazine also occurs in nature in tobacco (Liu et al, 1974) and some species of edible mushroom, for example Gyromitra esculenta (Braun et al, 1981) as well as being produced in soil by the reduction of nitrogen by bacteria such as Azotobacter chroococcum (Dilworth & Eady, 1991).

The true risk associated with long-term exposure in humans is unknown as data are scarce and often inconclusive. Although many reports have detailed the toxicity associated with this compound, the metabolism and mechanisms of action of hydrazine and/or its metaboHtes have not been fully elucidated. It is therefore of great importance to continue to study the effects of hydrazine on biological systems in order to increase our knowledge which may ultimately allow more accurate prediction of human risk.

1.2 PHYSICAL AND CHEMICAL PROPERTIES

Hydrazine is the simplest diamine with the chemical formula NgH^. In the anhydrous form it is a fuming, hygroscopic, colourless liquid with an ammoniacal odour. Hydrazine decomposes on heating (boiling point 113.5 °C) to form ammonia, hydrogen and nitrogen and may be explosive if this reaction is catalysed by certain metals (Schiessel, 1980). Hydrazine readily dissolves in polar solvents such as alcohols and forms a basic solution in water. In aqueous solution increases in pH, temperature and impurities such as metals (Gaunt & Wetton, 1966) or organic pollutants (Slonim & Gisclard, 1976) will enhance the degradation of hydrazine. Although the major products of hydrazine degradation in water are nitrogen gas and water itself hydrogen peroxide and ammonia have also been detected (Ellis et aly 1960).

Hydrazine is very chemically reactive and forms hydrazones with the carbonyl groups of ketones, azines with aldehydes and hydrazides with acids. Hydrazine compounds with a free amino group are the most potent nucleophiles. Hydrazine also undergoes many oxidation-reduction reactions. In the presence of strong reducing agents hydrazine is converted to ammonia. Oxidation of hydrazine leads to the formation of an unstable diazene which decomposes to nitrogen gas (Moloney & Prough, 1983).

1.3 APPLICATIONS

The multiple applications for hydrazine arise predominantly because of its chemical reactivity with inorganic and organic compounds. Some of these uses are listed below.

Hydrazine can be explosive at high temperature and in the presence of oxygen and this forms the basis of its use as a fuel and an auxiliary power source in military aircraft and as explosives.

Hydrazine is used as an antioxidant in hot water heating systems due to its ability to react with and thus remove dissolved oxygen. It is also an anticorrosion agent as it reduces ferric oxide (rust).

The reducing capacity of hydrazine and its ability to clean metal surfaces and prevent oxidation allows its use in the deposition of metal on glass and plastic surfaces. Hydrazine is also used in the production of noble metal catalysts, as a soldering flux and as a means of retrieving metals, such as mercury, from waste water in metal processing.

Some hydrazine derivatives are drugs such as isoniazid, an antitubercular agent, hydralazine, an antihypertensive agent, phenelzine, an antidepressant and procarbazine, used in combination therapy for the treatment of Hodgkin’s disease. A number of therapeutic hydrazine compounds are illustrated in Figure 1.1. Other hydrazine compounds are insecticides, fungicides and plant growth regulators (maleic acid hydrazide).

Figure 1.1 The Structure of Hydrazine and Some of its Therapeutic Derivatives

Hydrazine H2NNH2

CONHNH2

Isoniazid

NHNH2

Hydralazine

Procarbazine CH3NHNHCH2—<\ />—C0NHCH(CH3)2

CH2CH2NHNH2 Phenelzine 1.4 METABOLISM OF HYDRAZINE AND ITS DERIVATIVES

1.4.1 Absorbance, Distribution and Urinary Excretion of Hydrazine

When applied topically to shaved canine skin hydrazine was detected in plasma after 30 seconds. Hydrazine concentration continued to rise (10-180 minutes) in a dose-dependent manner (3-15mmol/Kg) then gradually declined (Smith & Clark, 1972). In contrast intraperitoneal administration to rats resulted in an almost immediate peak plasma hydrazine concentration followed by a phase of rapid removal (tV^ 0.7 hour) over the first 4 hours. Thereafter plasma hydrazine concentration declined more slowly {W2 26.9 hours) (Springer et al, 1981). Similar results were obtained in the mouse (Nelson & Gordon, 1982)

Subcutaneous or intravenous administration of hydrazine to rats and mice resulted in maximal tissue distribution within 0.5-2 hours (Dambrauskas & Cornish, 1964; Kaneo et al, 1984). Determination of hydrazine concentration in individual organs 2 hours post-dose revealed that the kidneys contained by far the greatest amount followed by spleen, lungs, heart, liver, stomach, intestines, muscle and brain (Dambrauskas & Cornish, 1964; Nelson & Gordon, 1982). All tissues displayed similar elimination half lives and contained approximately the same concentration of hydrazine after 8 hours (Kaneo et al, 1984). By 24 hours almost complete elimination had occurred (Nelson & Gordon, 1982).

The results of several studies indicate that uptake of hydrazine into tissues may be an active, saturable process. Dambrauskas and coworkers reported that 30 minutes after administration of various doses of hydrazine the amount metabolised per gram of tissue was not proportional to the dose, for example 25pg of 40pg/g hydrazine was metabolised compared to 41pg of a lOOpg/g dose (Dambrauskas & Cornish, 1964). The authors suggested that binding sites on individual tissues had become saturated at the higher doses of hydrazine. This hypothesis was reaffirmed in a later study in which decreased liveriplasma ratio was obtained with increasing hydrazine dose. However twenty-four hours post dose liver levels were 5 times that of plasma suggesting that hydrazine had been sequestered within this organ (Preece et al, 1992). Finally the results from a study in vitro demonstrated that the uptake of hydrazine into isolated rat hepatocytes was reduced when the cells were incubated at 2°C or in the presence of metabolic inhibitors indicative of an active uptake process (Ghatineh & Timbrell, 1990b).

Urinary excretion of hydrazine is inversely proportional to dose (Dambrauskas & Cornish, 1964; Preece et oZ, 1992). McKennis and coworkers detected 50% of a 0.47mmol/Kg dose of hydrazine in the urine of dogs 48 hours post dose and identified the metabolites as unchanged hydrazine, labile conjugates and acetylated derivatives (McKennis et aZ, 1955). Excretion of hydrazino metabolites 48 hours after administration of Immol/Kg dose to rats and mice were 50% and 40% respectively, 25% and 15% of which was unchanged hydrazine (Springer et al, 1981; Nelson & Gordon, 1982). Data fi'om other studies reveal 24% unchanged hydrazine (29% total hydrazine metabolites) excreted after a 0.31mmol/Kg dose (Kaneo et al, 1984) and 10% unchanged hydrazine (14% total hydrazine metabolites) excreted after a 0.16mmol/Kg dose (Wright & Timbrell, 1978).

1.4.2 Metabolism of the Parent Compound

Approximately 20-35% of a dose of hydrazine is expired as nitrogen gas (Springer et al, 1981; Nelson & Gordon, 1982) due to oxidation of hydrazine predominantly by oxyhaemoglobin in the blood but also by metabolism by microsomal enzymes (Nelson & Gordon, 1982). In both cases nitrogen is generated due to the breakdown of a diazene intermediate (Kondo, 1977).

Acétylation of hydrazine is a minor pathway resulting in the excretion of only 2-5% of the original dose as mono- or diacetylhydrazine (Wright & Timbrell, 1978; Noda et al, 1985a). Diacetylhydrazine is non-toxic and does not undergo further metabolism (McKennis & Yard, 1959). A chemical reaction between hydrazine and ketoacids leads to the production of pyruvic acid and oxoglutaric acid hydrazones (Moloney & Prough, 1983). Cyclisation of the latter metabolite generates l,4,5,6-tetrahydro-6-oxo-3-pyridazine carboxylic acid (THOPC) (Nelson & Gordon, 1982).

More recent studies using proton (^H) and ^®N-NMR have confirmed and expanded knowledge of hydrazine metabolism. Proton NMR detected the acetylated products and THOPC in the urine of rats (Sanins et uZ, 1992). Urinary analysis by ^^N-NMR identified unchanged hydrazine, and confirmed the presence of mono- and diacetylhydrazine, THOPC and pyruvate hydrazone (Preece et al, 1991). For the first time ammonia and urea were identified as metabolites of hydrazine, inferring enzymatic or chemical cleavage of the N-N bond in vivo. Carbazic acid, the product of the reaction between hydrazine and carbon dioxide, was also detected (Preece et al, 1991). The hydrazine metabolites identified to date are shown in Figure 1.2.

Figure 1.2 The Metabolic Pathways of Hydrazine

Ng Nitrogen Gas

NH=NH Diazene A

CH3-G-NHNH2 NH2NH2 -► NH3 — "► NH2-G-NH2

Acetyihydrazine Hydrazine Ammonia Urea

O 0 GOGH II II I Pyruvate GH3-G-NHNH-G-GH3 C=N—NH2 O Hydrazone CH3 Diacetylhydrazine II / ^ \ HN CH2 I I N CH2 THOPG

I COOH Proton NMR also detected changes in the urinary output of certain organic molecules, such as increased lactate, taurine, 13-alanine and methylamine (Sanins et al, 1992) and decreased 3-hydroxybutyrate, 2-oxoglutarate and citrate (Sanins et al, 1990; Sanins et al, 1992; Ghatineh et al, 1992) which reflect alterations to intermediary metabolism induced by hydrazine.

Rat liver microsomes metabolise hydrazine in an oxygen and NADPH- dependent manner (Noda et al, 1985b; Timbrell et al, 1982; Jenner & Timbrell, 1995) with the formation of a hydrazyl radical (Matsuki et al, 1991) and ultimately generation of nitrogen gas (Nelson, 1982). There is some evidence to suggest that NADPH-cytochrome P450 reductase may catalyse the first step in hydrazine degradation/metabolism leading to the conversion of hydrazine to the hydrazyl radical ( NHNHg) (Noda et al, 1988). Further oxidation of this radical would generate diimide (diazene, HNNH), characteristic difference spectra for which have been identified during cytochrome-P450 mediated metabolism of hydrazine in microsomes and isolated rat hepatocytes (Noda et al, 1985; Noda et al, 1987).

Acetyihydrazine is also oxidised by microsomal enzymes. Metabolism of acetyihydrazine by this route is thought to proceed via AT-hydroxy- acetylhydrazine (Timbrell et al, 1980), dehydration of which yields the diazene (Morike et al, 1996), a metabolite identified during the metabolism of other hydrazides (Moloney et al, 1984; Erikson & Prough, 1986). Fragmentation of the diazene generates nitrogen gas (Morike et al, 1996) and acetyl free radicals (Albano et al, 1985; Albano & Tomasi, 1987) which ultimately decompose to carbon dioxide (Wright & Timbrell, 1978; Lauterberg et al, 1985).

Much of the present knowledge concerning hydrazine metabolism has focused on cytochrome-P450, although the specific isoenzymes involved are still to be elucidated. Non-specific inhibition of cytochrome-P450 by pretreatment of animals with piperonyl butoxide was shown to reduce the rate of hydrazine metabolism by microsomes in vitro (Jenner & Timbrell, 1995) and increase hepatotoxicity in vivo, manifested as increased triglyceride accumulation (Timbrell et al, 1982; Jenner & Timbrell, 1994a). In comparison pretreatment of rats with phenobarbitone increased the rate of microsomal metabolism of hydrazine (Jenner & Timbrell, 1995) and reduced the toxicity in vivo (Timbrell et al, 1982; Jenner & Timbrell, 1994a). Phenobarbitone pretreatment also enhanced the eHmination rate of hydrazine from plasma and decreased the amount of unchanged hydrazine in the urine of rats (Noda et al, 1985a).

In comparison induction of cytochromes P4501A1 or P4502E1 by pretreatment of rats with B-naphthoflavone and acetone or isoniazid, respectively, did not appear to alter the rate of hydrazine metabolism by microsomes (Jenner & Timbrell, 1995) yet the former decreased and the latter increased hydrazine hepatotoxicity in vivo (Jenner & Timbrell, 1994a).

Taken as a whole these data suggest that hydrazine itself may be toxic and that overall metabolism via cytochromes P450 constitutes a detoxification pathway. However individual isoenzymes, such as P4501A1/2 and P4502B1/2, may catalyse the detoxification of hydrazine whereas others, such as P4502E1, may metaboHse hydrazine to toxic intermediates. If this is the case the relative induction state of these individual isoenzymes would be an important factor in determining the extent of hydrazine toxicity.

Cytochrome P4502E1 has been associated with the toxicity of many structurally unrelated, low molecular weight compounds, such as halogenated hydrocarbons including carbon tetrachloride (Johansson & Ingelman-Sundberg, 1985; Lindros et al, 1990; Raucy, 1993), trichloroethylene (Guengerich et al, 1991), ethanol (French et al, 1993; Morimoto et al, 1993) and N- nitrosomethylaniline (Quan et al, 1992). This isoenzyme is responsible for 50- 60% free radical formation from alkyhydrazines, the polarity of the hydrazine determining substrate specificity (Albano et al, 1995). In addition inhibition of enzyme activity can suppress hydrazine derived radical formation in control microsomes (Albano et al, 1993).

Disulfiram, an inhibitor of cytochrome P4502E1 (Lauriault et al, 1992), has been shown to abolish the carcinogenic action of 1,2-dimethylhydrazine (Wattenberg, 1975) by blocking the conversion of azomethane to azoxymethane

8 from which a carcinogenic carbonium ion is generated (Fiala et al, 1977). It is therefore possible that cytochrome P4502E1 may indeed metabolise the parent hydrazine to a toxic metabolite. As repeated exposure to even very low doses of hydrazine has been shown to induce cytochrome P4502E1 (Akin & Norred, 1978; Ghatineh et al, 1990c; Jenner & Timbrell, 1994b) it seems plausible that hydrazine may induce its own metabolism via this route.

Although cytochrome P450 catalyses the major route of metabolism of hydrazine (Tomasi et al, 1987) there are other enzyme systems which participate. The reduced rate of disappearance of hydrazine from microsome preparations in the presence of methimazole indicates that flavin-containing mono-oxygenases, such as amine oxidase, also participate in hydrazine metabolism (Jenner & Timbrell, 1995). In fact microsomal amine oxidase is the primary route of metabolism (65-75%) for 1,1-disubstituted hydrazines (Prough et al, 1981). Mitochondrial monoamine oxidase metabolises mono- and disubstituted hydrazines and to a lesser extent hydrazides (Erikson & Prough, 1986) and thus possibly hydrazine itself.

Peroxidase-mediated metabolism of hydrazine derivatives has been reported (Sinha, 1984; Gamberini & Lei te, 1993; Mahy et al, 1994; Van Der Walt et al, 1994) with little or no data available regarding the parent compound. Nevertheless myeloperoxidase, in circulating neutrophils, metabolises mono- and disubstituted hydrazines (Gamberini & Lei te, 1993) and hydrazides (Van Der W alt et al, 1994). Substituted hydrazines are also metabolised by horse radish peroxidase and prostaglandin synthetase in the presence of hydrogen peroxide giving rise to carbon centred radicals (Sinha, 1984; Auguste et al, 1985; Goria-Gatti et al, 1992).

A novel pathway in the metabolism of hydrazine compounds has recently been published. Phenelzine, benzylhydrazine and hydralazine are methylated by an enzyme present in bovine adrenal medulla however unsubstituted hydrazine, iproniazid, phenylhydrazine and isonicotinyl-2-isopropylhydrazine are not (Yu et al, 1991). Twenty-five per cent of a hydrazine dose is yet to be accounted for (Springer et al, 1981; Nelson & Gordon, 1982).

1.5 INTERACTION OF HYDRAZINE COMPOUNDS WITH METABOLIC SYSTEMS

1.5.1 Oxyhaemoglobin

During the autoxidation of hydrazine by oxyhaemoglobin, superoxide anions and hydrogen peroxide are generated, in addition to diazene, ultimately leading to Heinz body formation, lipid peroxidation and red cell haemolysis (Jain & Hochstein, 1979). Oxidation of phenylhydrazine, which is a more potent haemolysing agent than hydrazine itself, generates phenyldiazene and subsequently phenyl radicals (Misra & Fridovich, 1976; Goldberg & Stern, 1977) which covalently bind to both the globin and haem moieties of haemoglobin to form A^-phenylprotoporphyrin IX (Moloney & Prough, 1983).

1.5.2 Haem-containing Enzymes

Hepatic microsomal metabolism of hydrazines proceeds via radical formation (Kalyanaraman & Sinha, 1985), production of which can be influenced by induction or inhibition of specific cytochrome-P450 isoenzymes (Albano et al, 1993; Albano et al, 1995). As a result of radical generation inactivation and/or destruction of the cytochrome P450 enzyme has been reported (Jonen et al, 1982; Ortiz de Montillano et al, 1983; Moloney et al, 1984; Ortiz de Montillano & Watanabe, 1987).

Monoalkyl hydrazines, especially those with aromatic groups, procarbazine and iproniazid decrease the amount of CO-reactive P450 and cause haem destruction while hydrazides, isoniazid and acetyihydrazine, 1,1-disubstituted hydrazines and hydrazine itself cause loss of CO-reactive P450 alone (Jonen et al, 1982; Moloney et al, 1984). Loss of CO-reactive P450 results from the

10 formation of an iron-porphyrin-diazenyl complex (Mansuy et al, 1982; Battioni et al, 1983; Moloney et al, 1984), as previously identified in oxyhaemoglobin, degradation of which can lead to haem destruction due to alkylation of the porphyrin prosthetic group (Moloney et al, 1984). In the case of phenelzine the complex is N-phenylethylprotoporphyrin IX (Ortiz de Montillano et al, 1983).

Peroxidases are also haem containing enzymes which may be inactivated during hydrazine metabolism as a result of alkyl- or aryl-Fe(III) complex formation. The former adduct causes haem destruction (Mahy et al, 1994).

1.5.3 Flavin-containing Enzymes

Mitochondrial MAO has been shown to oxidise monosubstituted hydrazines to unstable diazenes and di-substituted hydrazines to stable azo- derivatives (Erikson & Prough, 1986). The former class of hydrazines irreversibly inhibit the enzyme at high concentrations due to reductive alkylation of the flavin moiety (Patek & Hellerman, 1974) whereas the latter class of compounds exhibit competitive inhibition of enzyme activity alone (Erikson & Prough, 1986).

1.6 TOXICITY OF HYDRAZINE

The manifestations of acute hydrazine toxicity have been studied in a number of animal species including monkeys, dogs, cats, rabbits, guinea pigs, rats and mice and were found to be comparable (Toth, 1988). Convulsions, which precede death, have been reported to occur in dogs (Witkin, 1955), rats and monkeys (Patrick & Back, 1965) exposed to hydrazine. Fat accumulation, predominantly in the periportal and midzonal regions of liver but also in other organs such as kidney and heart, is the major toxic effect (Comstock et al, 1954; Amenta & Johnston, 1962; Reinhardt & Dinman, 1965; Warren et al, 1984) which is maximal 24 hours post dose and returns to normal after 72 hours (Amenta & Johnston, 1962). Serum clinical chemistry data suggests that

11 no significant hepatic injury occurs (Warren et aZ, 1984) although liver necrosis has been reported in one study (Patrick & Back, 1965).

Other manifestations of hydrazine poisoning are restlessness and lethargy, muscular tremors, weight loss, vomiting, irritation to skin and eyes, lung effects such as pulmonary oedema, emphysema and irregular respiration, drop in blood pressure, cardiac arrhythmia and irregular pulse, alterations in blood levels of haemoglobin, glucose, lactate and pyruvate (Comstock et al, 1954; Toth, 1988).

Exposure of humans to hydrazine has been reported to cause similar effects to those stated above (Toth, 1988). Evaluation of hepatic function in 140 missile propellant handlers did not reveal significant liver disease although fatty liver was noted in 3 workers (King et al, 1969). Many of the reported cases involving hydrazine poisoning in humans arise due to inhalation and spillages on the skin, which result in eczema and contact dermatitis (Hovding, 1967; Toth, 1988). Chronic occupational exposure to hydrazine resulted in the death of one worker from a combination of lesions in the lungs (inflammation and pneumonia), kidneys (tubular necrosis and nephritis), liver (focal necrosis and degeneration) and heart (degeneration of muscle fibres) (Sontaniemi et al, 1987).

Exposure to hydrazine derivatives during drug therapy has also resulted in a number of toxic side effects. Treatment with hydralazine, isoniazid and procainamide has been associated with a syndrome similar to that of systemic lupus erythematosus (SLE) (Lunde et aZ,1977; Batchelor et al, 1980; Tanaka et al, 1982). Hydrazine itself, a minor metabolite of hydralazine and isoniazid (Noda et al, 1978; Noda et al, 1979; Blair et a/,1985), may be the causative agent as exposure to this chemical has also been reported to cause an SLE-like syndrome (Reidenberg et al, 1983).

Isoniazid, used in the treatment of tuberculosis, causes liver damage (Black et al, 1975) however the role of hydrazine has not been fully substantiated. Many patients receiving isoniazid incur mild liver damage which resolves with

12 prolonged treatm ent (Mitchell et al y 1975). Despite the fact that hydrazine has been detected in the blood of both adults (Blair et aZ, 1985) and children

(Donald et al, 1994) receiving isoniazid, no relationship between hydrazine formation and liver dysfunction has been found. However administration of antitubercular drugs to patients already exhibiting liver disease resulted in increased serum levels of isoniazid, due to the prolonged half-life of the drug, (Acocella, 1972) and also higher serum hydrazine (Gent et oZ, 1992; Woo et oZ, 1992). In an isolated case this culminated in hepatic necrosis and death (Woo et aly 1992). Hydrazine itself has not been found to cause any overt liver dysfunction, assessed by measurement of liver specific enzymes in serum, in chronically exposed humans (King et aly 1969) and primates (Warren et aly 1984) and thus the existence of liver disease or previous liver injury may be partly responsible for rendering individuals more susceptible to the toxicity of hydrazine and its derivatives.

1.7 BIOCHEMICAL EFFECTS OF HYDRAZINE

1.7.1 CNS Disturbances

Many of the early studies investigating the effects of hydrazine utilised lethality as the toxicological endpoint and convulsions were noted to be a prerequisite to death (Witkin, 1955; McKennis et aly 1955). Non-lethal doses of hydrazine also depressed normal behaviour including spontaneous motor activity (Pradhan & Ziecheck, 1971) and caused lethargy, weakness and tremors (Patrick & Back, 1965) in experimental animals. Hydrazine induced severe hypoglycaemia but this did not always coincide with the onset of convulsions suggesting that low levels of glucose in the brain were not responsible (Fortney, 1966). However high levels of circulating ammonia following hydrazine administration were thought to be a contributory factor (Floyd, 1980).

Studies on rat brain after hydrazine administration revealed elevated levels of GABA, an inhibitory neurotransmitter, as a result of GABA-transaminase

13 (GABA-T) inhibition (Medina, 1963). Several studies have verified that a free hydrazine group is required for GABA-T inhibition (McKenna, 1992; Yamada, 1993) which occurs due to the formation of hydrazones with pyridoxal phosphate (PLP) (Cornish, 1968; Castagné et al, 1987), the prosthetic group required for the activity of transaminases and other enzymes. Administration of pyridoxine, a precursor to PLP, prior to hydrazine prevented lethality (Dubnick, 1960; Toth & Erikson, 1977) hut pyridoxal and PLP exacerbated the toxicity as the hydrazones formed were more toxic than the parent hydrazine (Dubnick, 1960; Medina, 1963).

Some convulsant hydrazines, for example unsymmetrical dimethylhydrazine and isoniazid, decreased brain GABA content as a result of glutamic acid decarboxylase (GAD) inhibition (Wood & Peesker, 1974; Matsuyama et al, 1983), the enzyme responsible for GABA synthesis. Hydrazine itself also inhibited GAD (Medina, 1963) but had a greater effect on GABA-T thus causing a net increase in GABA concentration (Perry et al, 1981).

Consequently there appears to be a role for GABA in the aetiology of hydrazine induced seizures however there is no correlation with whole brain content and the onset of these convulsions (Wood & Peesker, 1974). The activity of GAD appears to be the major contributory factor determining the function of the GABA system as administration of pyridoxine can reduce GAD inhibition and prevent seizures regardless of GABA content (Wood & Peesker, 1974).

Hydrazine has been shown to increase postsynaptic GABA content which caused down-regulation of GABA^ receptors (Wood & Davies, 1991). In contrast isoniazid decreased pre- and post-synaptic GABA content, the latter causing up-regulation of GABA^^ receptors (Wood & Davies, 1991). Although hydrazine and isoniazid differ in their mode of action the modulation of GABA content at the synapse could result in increased excitability in the brain as in both instances the response to GABA at the synapse is reduced.

Hydrazines are also monoamine oxidase (MAO) inhibitors and thus suppress the degradation of other neurotransmitters such as catecholamines, dopamine

14 and 5-hydroxytryptamine (Roth, 1979; McMannus et al, 1992). However 85% MAO inhibition is required before the content of the above mentioned neurotransmitters is altered (McMannus et al, 1992). GABA content on the other hand is significantly reduced by approximately 20-30% inhibition of GAD (Wood & Peesker, 1974) suggesting that this would be the first to be affected by exposure to hydrazine and thus is most likely to be involved in the aetiology of seizures.

1.7.2 Carbohydrate Metabolism

In dogs hydrazine caused rapid hypoglycaemia and elevation of plasma lactate and pyruvate which occurred irrespective of initial plasma glucose concentration but was delayed if liver glycogen content was high (Fortney, 1966). Similar changes were observed in the rat and it was additionally noted that incorporation of labelled pyruvate, alanine and aspartate into glucose was inhibited by 75% (Fortney et al, 1967).

Prior to entry into the gluconeogenic pathway amino acids are transaminated. As hydrazine was reported to cause widespread elevation of amino acids in plasma, liver, brain and muscle of rats (Cornish & Wilson, 1968; Banks, 1970) and in plasma and urine of dogs (Korty & Coe, 1968), as well as inhibiting a number of transaminases, such as brain GABA-transaminase (Medina, 1963), liver ornithine ketoacid transaminase (Roberge, 1971), and liver aspartate aminotransferase (Stein et al, 1971), it was concluded that suppression of gluconeogenesis by hydrazine was a result of transaminase inhibition.

Glucose production from lactate was almost completely inhibited in the presence of hydrazine whereas gluconeogenesis was unaffected using fhictose, propionate and dihydroxyacetone as substrates (Haeckel & Oellerich, 1977). In addition in vivo in the fasted rat hydrazine also caused accumulation of citrate, malate and oxaloacetate in the face of reduced levels of phosphoenolpyruvate. These results indicated a metabolic block which was unrelated to transaminase inhibition. Indeed the activity of

15 phosphoenolpyruvate carboxykinase (PEPCK), the enzyme responsible for catalysing the conversion of oxaloacetate to phosphoenolpyruvate, was increased in vivo (Ray et al, 1970; Silverstein et al, 1989) but was non- competitively inhibited in vitro (Ray et al, 1970). Silverstein and coworkers suggested that hydrazine may transiently inhibit PEPCK activity in vivo eliciting a drop in blood glucose which in turn would initiate a hormonal response (Silverstein et al, 1989). This was in agreement with the findings of Ray and coworkers who noted that hydrocortisone exacerbated the accumulation of citrate and malate, which are converted back to oxaloacetate in the cytosol, but that administration of a high concentration of glucose could suppress this (Ray et al, 1970).

Hydrazine not only inhibited hepatic gluconeogenesis but also renal gluconeogenesis (Suzuki et al, 1975). In addition hepatic glycolysis was diminished, exemplified by reduced expiration of from ^'^C-glucose (Amenta & Dominguez, 1965) as was glycogen synthesis at stages prior to and beyond the formation of glucose-6-phosphate (Back et al, 1978; Silverstein et al, 1989).

Conclusions Hydrazine inhibits several pathways of carbohydrate metabolism. Gluconeogenesis is suppressed due to a block in amino acid transamination and inhibition of PEPCK. Glycolysis and glycogen synthesis are also impaired.

1.7.3 Lipid Metabolism

1.7.3.1 Source of precursors for lipid svnthesis

Free fatty acids for glycerolipid synthesis can be derived fi"om the diet, adipose tissue or de novo synthesis in the liver. Fat in the diet is absorbed firom the gut and combined with apoproteins to form chylomicrons which are ultimately released into the blood. In the capillaries of adipose tissue and liver,

16 triglycerides are hydrolysed from chylomicrons by lipoprotein lipases, the resultant fatty acids and glycerol diffusing into neighbouring cells.

Release of fatty acids from adipose tissue is mediated by certain hormones in response to a fall in plasma glucose concentration. Catecholamines and glucocorticoids activate tissue specific lipases while thyroid hormones indirectly stimulate hydrolysis of triglycerides by increasing the metabolic rate of all cells. Fatty acids from this source are transported in the blood to the liver bound to albumin.

Once in the liver free fatty acids are esterified to triglycerides, incorporated into phospholipids or some are utilised for the production of cholesterol. Phospholipids predominantly remain within the cell whereas triglycerides are secreted, coupled to very low density lipoprotein (VLDL), and transported either to adipose tissue for storage or to other peripheral tissues for utilisation as an energy fuel. The processes described above are summarised in Figure 1.3.

Figure 1.3 The Pathways of Lipid Metabolism

Chylomicrons Synthesis of Hydrolysis 7 phospholipids

Secretion as VLDL U polysis Pool of hepatic Adipose tissue Synthesis of free fatty acids triglycerides ^ Hydrolysis by lysosomal lipase

Intrahepatic P-Oxidation in synthesis mitochondria

1.7.3.2 Svnthesis of Liver Lipids

Phosphatidate (diacylglycerol 3-phosphate) is the common precursor in triglyceride and phospholipid synthesis. In the synthesis of triglycerides, phosphatidylcholine (PC) and phospatidylethanolamine (PE) phosphatidate is

17 hydrolysed to diacylglycerol (DAG), a reaction catalysed by phosphatidate phosphohydrolase (PAP). Triglycerides are then synthesised by estérification of a third fatty acid to DAG, a reaction catalysed by diacylglycerol acyltransferase, and PC/PE are formed by the transfer of CDP- choline/ethanolamine to DAG, a reaction catalysed by diacylglycerol choline/ethanolamine phosphotransferase (Figure 1.4).

For the production of phosphatidylinositol (PI), phosphatidylglycerol (PG) and cardiolipin phosphatidate is activated by CTP to form CDP-diacylglycerol after which inositol or glycerol are added. Cardiolipin is formed from phosphatidylglycerol (Figure 1.4)(Bell & Coleman, 1980).

1.7.3.3 Mechanism for the development of fatty liver

Free fatty acids are not released fi"om the liver eind so must be utilised or degraded. Triglyceride synthesis is stimulated in the presence of high levels of precursors which can arise due to increased uptake of fatty acids from extrahepatic tissues, stimulated lipogenesis within the liver itself and impairment of fatty acid oxidation. Excess triglycerides must be secreted or fatty liver will ensue. Very low density lipoproteins (VLDL) are the carrier molecules into which endogenously produced triglyceride are incorporated. Impairment of protein and phospholipid synthesis or lipid peroxidation may reduce the availability of functionally active VLDL thus limiting the secretory capacity of the liver. Failure of coupling of triglyceride with VLDL and also diminished secretion for different reasons than those mentioned above would lead to fat accumulation.

18 Figure 1.4 The Pathways of Glycerolipid Synthesis

GLYCEROL 3 -P T " "S' DIHYDROXYACETONE - P FATTY ACID a tV L ■ Coâ NAO* NAOM L — acyl - C o A C04

COP- monoacylglycerol I-ACYLGLYCEROL 3 - P I-ACYL - DIHYROXYACETONE - P ARAC m iOONOY l - Co a Fa t t y a ci O COA NAOP* NAOPM

COP - D iacylglycerol e t h e r lipids

PHOSPHATIDYL P hosphatidyl - ethanolamine

GLYCEROL - P INOSITOL ATP

, ADP

CHOLINE - p ETHANOLAMINE - P to PHOSPHATIDYL CDP - ethanolamine

Gl y c e r o l dis MOn O- ACYLGlYCEROL- F

CARDIOLIPIN

a OE s OS tl h CimO ANOL AMINE

s - a o e n o s y l ^ PHOSPHATIDYL- SERINE

Enzymes of Glycerolipid Synthesis: 1 Phosphatidate Phosphohydrolase 2 Diacylglycerol Acyltransferase 3 Diacylglycerol cholinephosphotransferase 4 Diacylglycerol ethanolaminephosphotransferase 5 Phosphatidate cytidy 1-transferase 6 Phosphatidylinositol synthase 1.7.3.4 The Effect of Hydrazine on Liver Lipids

Triglyceride synthesis is partly regulated by circulating glucocorticoids (Knox et alf 1979). An increase in the glucocorticoidiinsulin ratio in response to low blood sugar leads to mobihsation of fatty acids from adipose tissue, elevated PAP activity (Jennings et al, 1981; Lawson et al, 1982) and promotion of estérification of fatty acids to triglyceride (Glenny & Brindley, 1978).

Hydrazine has been reported to invoke an early rise in plasma corticosterone (Cooling et al, 1979; Silverstein et al, 1991), which persisted for up to 12 hours (Haghighiet al, 1985). In addition an abrupt rise in plasma free fatty acids, enhanced influx of these fatty acids into the liver (Trout, 1965; Amenta & Dominguez, 1965) and a time-dependent rise in liver triglycerides coincident with elevated PAP activity (Lamb & Banks, 1979; Haghighi & Honaijou, 1987) also followed hydrazine exposure. These lipid changes occurred in the face of normal blood glucose concentration but were enhanced during hypoglycaemia (Trout, 1966) suggesting that hydrazine may directly or indirectly mediate a hormonal response.

Other hormones such as glucagon, thyroid hormones, insulin and catecholamines also regulate lipid generating pathways (Glenny & Brindley, 1978; Boyd et al, 1981; Abumrad et al, 1988). Plasma glucagon and insulin are not altered by hydrazine (Cooling et al, 1979; Silverstein et al, 1991) however thyroid hormone is reduced (Silverstein et al, 1991). This latter effect is due to irreversible inhibition of thyroid iodide peroxidase (Hidaka et al, 1971), a crucial enzyme in the synthesis of thyroid hormones. The effect of hydrazine on circulating catecholamine levels is unknown, however increased adrenal gland concentration of these hormones has been observed (Haghighi & Honaijou, 1987).

The involvement of adrenal hormones was confirmed in adrenalectomised rats by abrogation of hydrazine-induced fatty liver (Haghighi et al, 1985; Haghighi & Honaijou, 1987). As adrenalin inhibits PAP activity (Haghighi et al, 1990) it stands to reason that corticosterone is the likely effector of the above

2 0 mentioned alterations in fat metabolism.

Free fatty acids have the ability to modulate triglyceride synthesis in the short term causing the translocation of PAP from the cytoplasm to endoplasmic reticulum (Cascales et al, 1984; Butterwith et al, 1985) where the majority of phosphatidate is generated (Mok & McMurray, 1990). Fatty acids also stimulate diacylglycerol acyltransferase activity (Haagsman & Van Golde, 1981), the only enzyme unique to triglyceride synthesis (Bell & Coleman, 1980). CTP:phosphocholine cytidyltransferase activity is also activated by free fatty acids in the same manner as PAP (Pelech et al, 1983; Vance & Pelech, 1984; Asiedu et al, 1992) indicating coordinated regulation of synthesis of TG and PL (Butterwith et al, 1985; Skorve et al, 1990). However triglyceride synthesis exceeds that of phospholipids possibly due to increased specificity of PL synthesising enzymes for certain fatty acids (Sundler et al, 1974).

It is clear that hydrazine promotes hepatic biosynthesis of TG from adipose- derived fatty acids but hydrazine also caused enhanced lipogenesis in the livers of treated rats (Marshall et al, 1983). Increased arterial lactate (Fortney, 1966; M arshall et al, 1983) was also noted and as this is a major precursor for hepatic lipogenesis (Boyd et al, 1981) there may be direct stimulation of de novo fatty acid synthesis with subsequent triglyceride production as a result of hydrazine intoxication.

Alternatively high levels of lactate indirectly stimulate triglyceride synthesis due to enhancement of the citric acid cycle, possibly due to energy generation which is utilised in fatty acid estérification (Henly & Berry, 1991). Elevated levels of malate and citrate are detected when the citric acid cycle is boosted (Henly & Berry, 1991), and the accumulation of these compounds has been observed after hydrazine administration (Ray et al, 1970). On the contrary acetate oxidation via the citric acid cycle has been reported to be unchanged by hydrazine exposure implying a lack of effect (Amenta, 1963; Amenta & Johnston, 1963). A contribution from this cycle in the generation of hydrazine- induced fatty liver cannot be totally disregarded but has yet to be proven.

2 1 The fact that hepatic triglyceride content was so much greater then serum triglyceride concentration after hydrazine exposure led some authors to suggest that secretion of lipids from the liver was impaired (Clark et al, 1970; Haghighi et al, 1985). However other authors found no defect (Trout, 1966) or a small transitory defect (Amenta & Dominguez, 1965).

If diminished secretion is a contributory cause to hydrazine-induced fatty liver there are several possible mechanisms by which this could occur. As previously mentioned triglycerides are transported from the liver incorporated into VLDL, assembly of which could be disrupted by any of the following: inhibition of synthesis of phosphatidylcholine which is an integral part of the VLDL molecule; inhibition of protein synthesis; and peroxidation of VLDL lipid.

Unfortunately the literature contains contradictory reports as to the ability of hydrazine to induce any of these effects. For example hepatic phospholipid content was reduced in one study (Lamb & Banks, 1979) but increased in others (Clark et al, 1970; Haghighiet al, 1985). Similarly lipid peroxidation has been reported by some authors (DiLuzio et al, 1973; DiLuzio & Stege, 1977) but not others (Preece & Timbrell, 1989) and likewise with protein synthesis (Amenta & Johnston, 1963; Banks, 1970; Lopez-Mendoza & Villa-Trevino, 1971).

Conclusions Hydrazine appears to cause fatty degeneration in the liver by stimulating triglyceride synthesis, an effect which may be mediated by hormones (glucocorticoids). Increased availability of fatty acids arises due to mobilisation from adipose tissue and from enhanced hepatic lipogenesis. Impaired secretion of triglycerides does not appear to contribute to the accumulation of fat although a definite conclusion cannot be made.

2 2 1.7.4 Protein Metabolism

There are several threads of evidence which suggest that hydrazine induces protein synthesis in the livers of treated animals. Firstly the incorporation of exogenously added radiolabelled amino acids into hepatic protein was reported to be enhanced in vitro in liver slices taken from rats dosed 5 hours previously (Amenta & Johnston, 1963) and in vivo in rats treated with hydrazine 24 hours before (Banks, 1970). Hepatic protein content correlated with RNA content at all times (Banks, 1970).

Secondly polyamines, intracellular levels of which increase prior to DNA, RNA or protein synthesis (Morgan, 1987), were raised in the livers of animals treated with hydrazine (Banks & Hubbard, 1975), as was the activity of ornithine decarboxylase (Springer et al, 1980; Rogers et al, 1988), a key enzyme in polyamine synthesis. Putrescine was raised between 2-24 hours and spermidine content was maximal at 24 hours post dose (Banks & Hubbard, 1975) in close temporal sequence with hepatic DNA concentration which was raised between 4-24 hours post dose (Banks et al, 1967).

Further evidence of the effect of hydrazine on the synthetic machinery of the cell is the increased activity of guanylate cyclase (Vesely & Levey, 1977; Vesely et al, 1978), an enzyme involved in both DNA (Seifart, 1974) and protein synthesis (Watson, 1973).

Taken together these data imply that hydrazine may first influence the synthesis of DNA which in turn influences protein biosynthesis. As several authors have noted increased levels of plasma (Korty & Coe, 1968; Cornish & Wilson, 1968) and liver (Simonsen & Roberts, 1967; Banks, 1970) amino acids following hydrazine administration, these building blocks are readily available for protein synthesis.

However, in contrast to the above, inhibition of protein synthesis has been noted to occur in vivo in rats immediately after hydrazine injection with maximal inhibition occurring after 1 hour (Lôpez-Mendoza & Villa-Trevino,

23 1971). The rate of radiolabelled leucine or glycine incorporation had increased to 50% of control after 3.5 hours and was maintained at this level for a further 5 hours (Lôpez-Mendoza & Villa-Trevino, 1971). The reasons for this discrepancy is unknown.

In vitro in isolated rat hepatocytes, either cultured or in suspension, the incorporation of radiolabelled leucine was significantly reduced by a concentration of hydrazine as low as 0.5mM (Ghatineh & Timbrell, 1990a; Delaney & Timbrell, 1995). The discrepancy between in vivo and in vitro data is possibly less surprising as the normal physiological control mechanisms are lacking in the latter system.

It is well known that amino acid deprivation increases the rate of protein degradation in a number of biological systems including rat fibroblasts (Poole 6 Wibo, 1973) and bacteria (Nath & Koch, 1971). Addition of amino acids is capable of restoring synthesis (Knowles & Ballard, 1976). In isolated hepatocytes incubated in an amino acid unsupplemented medium, protein degradation proceeds at approximately ten times the rate of protein synthesis (Seglen, 1977; Seglen, 1978).

Perfusion of livers from fed rats with amino acid deprived medium results initially in inhibition of protein synthesis which later recovers due to supply of amino acids from enhanced degradation of endogenous protein (Flaim et al, 1982a). This reduction in protein synthesis in response to decreased nutrient supply is due to inhibition of peptide chain initiation (Flaim et al, 1982b, Cox et al, 1988). Thus the discrepancy between in vivo and in vitro results may initially be due to amino acid deprivation in the latter system, however there is no recovery of protein synthesis during hydrazine exposure and so other mechanisms must be involved.

There are many compounds which modulate protein degradation via lysosomes (DeDuve et al, 1974; Seglen et al, 1979), which carry out both basal protein breakdown in the well nourished state and enhanced breakdown (long-lived proteins) induced by hormonal or nutritional deprivation (Mortimore & Poso,

24 1984). Weak bases such as ammonia readily cross biological membranes and accumulate in lysosomes (DeDuve et al, 1974). As a result intralysosomal pH increases culminating in the inactivation of enzymes which normally operate under acidic conditions (Okhuma & Poole, 1978). Substances with a pK of around 8 are the most efficient at entering via permeation (DeDuve et al, 1974). Indeed hydrazine has a pK of 7.93 (Hadler & Cook, 1978).

The inhibition of protein degradation would lead to inhibition of protein synthesis in the absence of exogenous amino adds as has been demonstrated with ammonia (Seglen, 1978; Seglen et al, 1979). Many diamines also inhibit protein synthesis in isolated hepatocytes by an unknown mechanism which occurs irrespective of protein degradation and the presence/absence of amino adds (Seglen et al, 1980c). Hydrazine itself is a simple diamine and can also be metabolised to ammonia (Preece et al, 1991) thus the mechanism of action could be similar to any of those described above. However hydrazine inhibited protein synthesis in WB344 rat liver epithelial cells and 743X male Chinese hamster lung cells cultured in supplemented medium (Pravecek et al, 1994) suggesting that hydrazine may be acting in a similar manner to diamines.

It is therefore quite feasible that the mechanisms of action of hydrazine on protein synthesis in vivo and in vitro are completely different and thus a comparison cannot be made.

Conclusions Hydrazine may stimulate protein synthesis in vivo as a result of increased DNA synthesis. The activities of several transaminases are inhibited thus amino adds are readily available for incorporation into proteins. Hydrazine irreversibly inhibits protein synthesis in vitro but the underlying mechanism has not yet been established.

25 1.7.5 Urea Cycle

Hydrazine exposure has been associated with elevated blood ammonia concentration without concomitant changes in urea (Floyd, 1980) and with elevated plasma and tissue amino acids including citrulline, arginine and ornithine which are associated with the urea cycle (Simonsen & Roberts, 1967; Korty & Coe, 1968; Banks, 1970). Prior administration of arginine and glutamate prevented or diminished hydrazine-induced toxicity in vivo (Roberts et al, 1965) suggesting urea cycle disruption. Indeed urea synthesis was inhibited by hydrazine in vitro in rat liver homogenate but stimulated in vivo after administration for several days (Roberge et al, 1971).

The first stage in the degradation of amino acids is the transfer of the a-amino group to an a-ketoacid, a reaction catalysed by specific transaminases. The product glutamate is then deaminated to yield ammonium ions which enter the urea cycle incorporated in carbamoyl-phosphate. The urea cycle is summarised in Figure 1.5.

Figure 1.5 The Urea Cycle

TCA Cycle A

H 20 Fumarate Arginine Arginase

Arginosuccinase UREA

Arginosuccinate OOrnithine r n ith in e ------Polyamine Synthesis

Carbamoyl Phosphate Arginosuccinate Synthetase Ornithine A Carbanioyl Citrulline Transcarbamoylase Phosphate Synthetase

Aspartate NH4 + + C02

26 In vitro hydrazine can replace ammonia in the carbamoyl-phosphate synthetase reaction giving rise to a new product, N-aminocarbamoyl-phosphate, which is not a substrate for ornithine transcarbamoylase and thus inhibits citrulline formation (McKinley et al, 1967). This reaction does not appear to be of importance in vivo as citrulline accumulates (Simonsen & Roberts, 1967; Roberge et al, 1971). Hydrazine does not stimulate carbamoyl-phosphate synthetase activity (Roberge et al, 1971) but may increase the content of its allosteric activator, iV-acetylglutamate, due to elevated levels of glutamate and ammonia (Simonsen & Roberts, 1967; Korty & Coe, 1968; Banks, 1970). Enhanced breakdown of excess amino acids to glutamate and subsequent dehydrogenation to release ammonia is enhanced by the action of glucocorticoids. This has been reported to be the mode of action of hydrazinosuccinate (Yamada et al, 1988) and may also apply to hydrazine itself.

The activities of urea cycle enzymes were unaffected by hydrazine with the exception of arginosuccinase which was stimulated (Roberge et al, 1971). Addition of arginine caused further accumulation of ornithine and citrulline in hydrazine treated rats (Simonsen & Roberts, 1967) suggesting that the rate limiting step becomes the condensation reaction of citrulline with aspartate. This may arise due to limited supply of aspartate, as although this is raised after hydrazine treatment 2-fold (Korty & Coe, 1968; Banks, 1970) citrulline is increased 20-fold (Simonsen & Roberts, 1967).

Conclusions Hydrazine may transiently disrupt the urea cycle in vivo resulting in elevated plasma ammonia concentration. However elevated ammonia could also arise as a direct result of excess amino acid degradation, competition of ammonia with hydrazine for a-ketoglutarate and glutamine and possibly the breakdown of hydrazine itself to ammonia.

27 1.7.6 Mitochondrial Effects

Although mitochondrial respiration and ATP synthesis have been reported to be stimulated in vivo 24 hours after hydrazine exposure (Higgins & Banks, 1971) much of the literature focuses on the early depletion of hepatic ATP in vivo and in vitro (Preece et at, 1990; Ghatineh et al, 1992) which occurs in a time and dose-dependent manner. As this depletion of ATP occurs prior to cell death in isolated hepatocytes in vitro (Ghatineh et a f 1992) it would appear that this is a cause and not a consequence of hydrazine toxicity.

The majority of cellular ATP is generated in mitochondria from acetyl CoA derived from pyruvate and fatty acids. Acetyl CoA is oxidised in the citric acid cycle generating high-energy electrons, carried by NADH and FADHg, that pass along the respiratory chain. In turn the respiratory chain creates an electrochemical proton gradient across the inner mitochondrial membrane which drives ATP synthesis.

Acute exposure to hydrazine (60mg/Kg) has been shown to damage mitochondria in vivo, evident as swelling of these organelles 30 minutes post dose followed by loss of matrical structure and disruption of cristae (Scales & Timbrell, 1982). Several pieces of evidence suggest that hydrazine also disrupts the citric acid cycle. These include the reduction of succinate dehydrogenase activity and depletion of pyridine nucleotides in isolated rat hepatocytes in vitro (Ghatineh et al, 1992) and the limitation of the availability of a-ketoacids in vivo as a result of hydrazone formation (Nelson & Gordon, 1982; Preece et al, 1991). However these are possibly early, transient effects as citric acid cycle intermediates have been reported to accumulate in the livers of hydrazine treated rats (Ray et al, 1970).

Several hydrazine compounds such as carbonyl cyanide p- trifluoromethoxvphenvlhvdrazone and the 1. l'-diphenvl-2-picrvlhvdrazvl radical and its hydrazine homologue are uncouplers of oxidative phosphorylation depleting ATP by stimulating respiration (ADP-dependent), enhancing ATP hydrolysis and thus collapsing the proton electrochemical gradient across the

28 mitochondrial membrane (Coleman, 1971; Niemen et al, 1990). Hydrazine itself uncouples oxidative phosphorylation in vitro by preventing inorganic phosphate from binding to ADP (Hadler & Cook, 1978). Hydrazine also interacts with cytochrome c oxidase, the terminal enzyme in cellular respiration (Kubota & Yoshikawa, 1993) thus potentially dissipating the intramitochondrial membrane potential.

Loss of this intramitochondrial membrane potential leads to the cessation of ATP synthesis (Carini, 1992) unless ATP is generated by alternative pathways such as glycolysis (Wu et al, 1990). Addition of fhictose to the incubation medium afforded some protection against hydrazine-induced ATP depletion in isolated rat hepatocytes in vitro (Kerai & Timbrell, in press) suggesting that inhibition of glycolysis by hydrazine (Amenta & Dominguez, 1965) contributes to its toxicity.

Free fatty acids also uncouple oxidative phosphorylation by inhibiting ATP synthesis and interacting directly with the ATPase pump without effect on the electrochemical gradient (Rottenberg & Hashimoto, 1986). An early response to hydrazine intoxication in vivo is a rise in free fatty acids (Clark et al, 1970).

Hydrazine also induces physical alterations to mitochondria. Chronic treatment of rats with hydrazine (1% in the diet for 3-7 days) leads to the formation of megamitochondria (Wakabayashi et al, 1987). Membrane constituents and chemical properties are altered in such a way as to suggest membrane fusion as the mode of formation of these organelles (Adachi et al, 1994). The initial cause of the membrane alterations is still unknown however suppression of the formation of megamitochondria by coadministration with a-tocopherol suggests free radical mediated lipid peroxidation (Antosiewitz et al, 1994). Induction of lipid peroxidation by hydrazine has not been categorically proven as the literature contains several conflicting reports (Diluzio et al, 1973; Preece & Timbrell, 1989). Nonetheless this could also be the mechanism of mitochondrial damage after acute dosing with hydrazine as free radicals have been reported to be generated during hydrazine metabolism (Kalyanaraman & Sinha, 1985; Noda et al, 1987) in addition to depletion of

29 cellular glutathione (Timbrell et al, 1982; Noda et al, 1987).

It must be noted that administration of steroids such as cortisone can lead to the production of megamitocbondria, again possibly by fusion of adjacent mitochondria (Weiner et al, 1968) and can uncouple oxidative phosphorylation (Kimberg et al, 1968). As hydrazine has been shown to elicit an early rise of plasma corticosterone (Cooling et al, 1979) hormonal intervention cannot be ruled out.

Conclusions Hydrazine causes both physical and functional alterations to mitochondria. Although the reasons for the formation of megamitocbondria remain elusive, the enhancement of ATP hydrolysis possibly occurs in response to increased cellular demand for energy. The similarity of the effects of corticosterone and hydrazine on mitochondria may indicate the mediation of a hormonal response.

1.7.7 Depletion of Reduced Glutathione

Reduced glutathione is an effective cellular defence against chemical injury and in concert with other systems, such as superoxide dismutase, protects against oxidative stress (Munday et al, 1989; Reed, 1990). The majority of cellular GSH is cytosolic (85-95%), the remainder being mitochondrial (Redegeld et al, 1992). Loss of cellular GSH has been implicated in the toxicity of many compounds including pairaquat (Palmeira et al, 1994), acetaminophen (Moore et al, 1985; Lauriault et al, 1991), polycyclic aromatic hydrocarbons (Hallberg & Rydstrom, 1989), and ethanol (Fernandez-Checa et al, 1991).

Hydrazine causes a time and dose dependent depletion of GSH in vitro in isolated rat hepatocytes (Albano & Tomasi, 1987; Noda et al, 1987; Ghatineh et al, 1992) and in vivo (Timbrell et al, 1982; Jenner & Timbrell, 1994a). The mechanism of this depletion is at present unknown.

30 Glutathione would be depleted by its interaction with and thus scavenging of free radicals generated during metabolism of certain compounds, indeed this bas been demonstrated during cytochrome P450 catalysed oxidation of alkylbydrazines (Albano et al, 1993). However there was apparently no depletion of GSH during microsomal metabolism of hydrazine itself (Jenner & Timbrell, 1995) despite the fact that free radicals have been identified (Noda et al, 1985b).

Although some hydrazines have been reported to cause oxidative stress (Albano et al, 1993) there is no such evidence concerning the parent compound, in fact the literature contains conflicting data with respect to lipid peroxidation (DiLuzio et al, 1973; Preece & Timbrell, 1989). In addition no significant increase in the concentration of oxidised glutathione has been observed after hydrazine administration (Noda et al, 1987; Ghatineh et al, 1992).

Thus if GSH depletion is not a result of conjugation with reactive intermediates generated during hydrazine metabolism or of oxidation, then a reduction in its synthesis is possible. In support of this hydrazine has been reported to decrease the hepatic content of both GSH and GSSG in vivo (Timbrell et al, 1982). Glutathione synthesis is dependent on ATP (Meister & Anderson, 1983) which is also depleted by hydrazine (Preece et al, 1990; G hatineh et al, 1992; Jenner & Timbrell, 1994a). As depletion of GSH has been recorded in concert with normal ATP levels (Jenner & Timbrell, 1994a) these events possibly occur independently. A further possibility is the removal of gamma-glutamyl amino acids, which are essential for GSH synthesis, as a result of the enzymic conversion of hydrazine and glutamine to gamma- glutamylhydrazine (McKennis et al, 1961). Hydrazine also interacts with many enzymes (Moloney & Prough, 1983) and may therefore inhibit those involved in glutathione synthesis.

Prior depletion of GSH with diethyl maleate did not alter hydrazine toxicity in vivo (Timbrell et al, 1982) suggesting that GSH depletion may be a consequence and not a cause of hydrazine intoxication. Indeed in vitro GSH depletion is a later event, but one which precedes cell death (Ghatineh et al,

31 1992). Total loss of cellular GSH to 5-10% control is not associated with cell death unless accompanied or followed by ATP depletion (Redegeld et al, 1992). As this is the case with hydrazine the combination of effects may be jointly responsible for the cytotoxicity.

Nevertheless alteration of thiol status may be detrimental to the normal functioning of cells as GSH is involved in regulating many processes such as enzyme function, mitochondrial function, protein synthesis and plasma membrane integrity, to name a few (Kosower & Kosower, 1969; Meister & Anderson, 1983; Reed, 1990). In vivo lipid peroxidation and tissue necrosis are causally related to GSH levels (Maellaro et al, 1990) and in vitro cell viability is directly correlated with the level of protein thiols, which are maintained in the reduced state by GSH and other antioxidants such as a-tocopherol (Pascoe et al, 1987; Reed et al, 1990).

Diamide, or Kosower's reagent, is a long-term thiol oxidising agent which maintains low cellular GSH and leads to delay or inhibition of cell growth and changes in membrane integrity (Kosower & Kosower, 1969). Hydrazine, itself a simple diamide, decreased population growth of several different cell lines in culture (Siemens et al, 1980; Pravecek et al, 1994), inhibited protein synthesis and compromised membrane integrity as illustrated by LDH leakage (Ghatineh & Timbrell, 1990; Delaney & Timbrell, 1995). These similarities may imply that a decrease in GSH, possibly by oxidation to GSSG, may play a role in hydrazine toxicity.

Thiol status also controls calcium homeostasis (Jewell et al, 1982) loss of which can result in impairment of cell signalling, mitochondrial dysfunction and activation of catabolic enzymes culminating in cell death (Nicotera et al, 1992). Low cytosolic calcium is maintained by the extrusion of Ca^^ by translocases (thiol-containing ATPases) in the plasma membrane and by sequestration in endoplasmic reticulum and mitochondria (Nicotera et al, 1992).

Maintenance of mitochondrial pyridine nucleotides and GSH in the reduced state are essential for the retention of Ca^^ in this organelle (Beatrice, 1985;

32 Lehninger, 1978; Bellomo, 1982). The former is promoted by succinate oxidation (Lehninger et aly 1978). Indeed hydrazine inhibits succinate dehydrogenase activity and depletes pyridine nucleotides in vitro in isolated rat hepatocytes (Ghatineh et aly 1992). In addition any factor which depletes NADPH, which is required in the reduction of GSSG back to GSH, will lead to

GSSG accumulation (Eklow et aly 1981). In the case of hydrazine a small but insignificant rise in GSSG was observed in vitro (Ghatineh et al, 1992). Thus hydrazine-induced depletion of GSH may lead to a cascade of events which culminate in cell death.

Conclusion Hydrazine-induced GSH depletion may be a consequence of hydrazine toxicity and not a cause. The fact that there are similarities regarding the manifestations of cytotoxicity induced by hydrazine and by other thiol depleting agents indicate some role for GSH depletion in the aetiology of hydrazine toxicity. However taken as a whole the data suggest that a combination of effects, such as GSH and ATP depletion, may be required to induce cell death.

1.8 MUTAGENICITY AND CARCINOGENICITY OF HYDRAZINE AND ITS DERIVATIVES

Of the many hydrazine derivatives tested, both naturally occurring and synthetic, most have been reported to cause tumour formation in long-term animal studies (Toth, 1980). These compounds increased the incidence of lung and liver tumours in mice and rats (Severi & Biancifiori, 1968; Biancifiori,

1970), hepatocellular tumours in hamsters (Bosan et aly 1987) and enhanced the development of Brown-Pearce tumours and papillomas of the bronchial mucosa in rabbits (Bianciferi & Severi, 1966). Other tumours such as nasal, breast, thyroid, colon and stomach have also been identified in rats and hamsters after chronic inhalation of hydrazine (Vemot et aly 1985).

33 As hydrazine did not induce tumour formation at the site of administration, except in the case of inhalation where nasal and lung tumours arose (Vernot et al, 1985), it was proposed that metabolic activation or reaction with tissue specific components were deemed necessary for carcinogenic action (Balo, 1979). In accordance with this the most abundant cancers induced by hydrazines arise in liver, lung and blood vessels where metabolism is known to occur (Erikson & Prough, 1986; Netto, 1988; Misra & Fridovich, 1976).

Hydrazine causes mutations in E. coli (Noda et al, 1986), S. typhimurium (McCann et al, 1975; Bhide et al, 1984) and other in vitro systems (Kimball, 1977; Levi et al, 1986). Despite the general consensus that hydrazine is also mutagenic in mammalian systems both in vitro (Brookes et al, 1981) and in vivo (EC book on carcinogens) many negative results have been recorded.

Administration of single, very high doses of hydrazine sulphate (up to 400mg/Kg) to transgenic mice did not result in mutations in the lacZ gene in liver, lung or bone marrow (Douglas et al, 1995) intimating that repeated exposure to hydrazine may be necessary to induce mutations in vivo or th at hydrazine is a non-genotoxic carcinogen. Administration of hydrazine and 1,1- dimethylhydrazine to Swiss mice increased the incidence of pulmonary tumours which were known to naturally arise in this species (Roe et al, 1967). Also isoniazid increased the incidence of colon adenocarcinomas in 1,2-DMH treated rats whereas isoniazid treatment alone did not induce this type of tumour (Gershbein & Rao, 1992). These two studies may indicate a promoter role for hydrazines as genetic mutations may already be present in these animals.

High doses of hydrazine in vitro directly interact with DNA causing pyrimidine ring cleavage with subsequent loss of a base (Kimball, 1977) or modification of bases such as thymine (Kimball, 1977) and cytosine (Cashmore, 1978). Nitrogen and carbon centred radicals formed during oxyhaemoglobin mediated metabolism of hydrazines also damage DNA (Runge-Morris et al, 1994). Administration of hydrazine in vivo results in aberrant méthylation of cytosine to 5-methylcytosine (Barrows & Shank, 1981; Leakakos & Shank, 1994) and guanine at the N^ and O® positions (Barrows & Shank, 1981; Bosan al, 1987).

34 These adducts can be detected 5-24 hours after acute dosing (60-90mg/Kg) (Barrows & Shank, 1981) and after chronic dosing (lOmg/Kg over 2 years) (Bosan et al, 1987). N^-methylguanine was by far the most prevalent adduct in the livers of dosed animals, the concentration being approximately 15-30 fold greater than 0^-methylguanine (Becker et al, 1981). Alkylation of liver DNA in CBA mice exposed to hydrazine in vivo correlated with tumour production in these animals (Quintero-Ruiz et al, 1981).

The methylating agent, formaldehyde hydrazone, the product of the reaction between hydrazine and endogenous formaldehyde (Bosan et al, 1986) caused adduct formation in the presence of 89 supernatant in vitro (Lambert & Shank, 1988). Catalase, abundant in this liver fraction, was found to metabolise formaldehyde hydrazone to yield methyl radicals (Gomes & Augusto, 1991). Hydroxyl radicals, also capable of damaging DNA (Breen & Murphy, 1995), were produced during oxidation of hydrazine itself by catalase (Gomes & Augusto, 1991).

Hydrazine derivatives also give rise to the same methylguanine adducts as the parent compound. Hydralazine is mutagenic in mammalian and bacterial systems in vitro with or without S9 (McQueen et al, 1993) and can cause méthylation of guanine at the N^ position in vivo, albeit to a lesser extent than hydrazine (Matthison et al, 1994). Hydralazine does react with formaldehyde to produce a hydrazone but this appears to have no effect on methylating efficacy. In this particular case catalase can inhibit DNA damage indicating the importance of hydrogen peroxide in this reaction (Yamamoto & Kawanishi, 1991). Méthylation of liver guanine in vivo has been detected after acute administration of other therapeutic hydrazine derivatives such as isoniazid, procarbazine, phenylhydrazine and nialamide (Matthison et al, 1994). No O®- methylguanine was detected under the experimental conditions used (Matthison et al, 1994).

Treatment with 1,2-dimethylhydrazine, which specifically induces colon cancer in rodents, causes the appearance of 0^-methylguanine adducts predominantly in the distal colon coincident with a reduction in the activity of the repair

35 enzyme 0®-alkylguanine DNA alkyltransferase and increased cell proliferation (Jacoby et al, 1993). Many of the carcinomas induced by this hydrazine derivative have been reported to contain K-ms mutations manifested as G to A transitions (Llor, 1991). This specific mutation is consistent with the production of 0^-methylguanine adducts (Mitra et al, 1989). Méthylation of cytosine and/or guanine bases or specific mutations in response to unsubstituted hydrazine caused the deletion of restriction sites (5’-CCGG-3’) located at or near to specific genes in liver DNA of neonatal rats (Leakakos & Shank, 1994). The authors suggested that hydrazine-induced DNA damage was not a random response and that the lack of base changes in some of the genes investigated, such as H-ms, may have been due to rapid repair (Leakakos & Shank, 1994).

In addition the formation of N^-methylguanines can lead to secondary lesions such as apurinic sites which are also associated with GC—>TA base substitutions in E. coli (Laval et al, 1990). Hydralazine causes GC—>TA mutations in the Salmonella strain TAIOO (Lemke & McQueen, 1995).

The earlier carcinogenicity studies used doses of hydrazine that fell within the toxic range. Recent reassessment of hydrazine carcinogenicity using maximal doses of 5mg/Kg administered over the lifespan of the animals revealed no evidence of induced levels of tumour formation (Steinhoff et al, 1990). These authors suggested hydrazine carcinogenicity was linked with toxicity. The results obtained in the following studies are in agreement with this. Lifetime exposure to lOmg/Kg hydrazine in the rat caused tumour development in the liver where high levels of aberrant méthylation coincided with cytotoxicity (Bosan et al, 1987). In the neonatal (Leakakos, 1994) and adult rat (Barrows & Shank, 1981) 0^-methylguanines were detected only after acute doses of hydrazine that were hepatotoxic.

Despite the wealth of evidence that hydrazine is a cancer causing agent the risk to man has not been established. The outcome of one study in which 427 workers exposed to approximately 1-lOppm or possibly more hydrazine over a period of 6 months to 2 years was that there was no obvious risk as the

36 observed mortality from all cancers was close to that expected irrespective of the level of exposure (Wald et al, 1984). Reassessment of these same workers 10 years later found no significant excess in the overall incidence of cancer although a small increase in deaths from lung and digestive tract tumours was noted (Morris et al, 1995).

Interpretation of human epidemiological data regarding hydrazine is difficult for two reasons. Firstly there are very few studies in which workers were exposed solely to hydrazine and thus any increases in cancer rates cemnot be directly attributed to this compound. Secondly the number of individuals exposed solely to hydrazine are too small to allow accurate conclusions to be made (Toth, 1994). Thus more occupational and epidemiological data must be collected before a true risk assessment for man can be carried out.

1.9 ANTITUMOUR ACTIVITY OF HYDRAZINES

Several hydrazines, despite being carcinogenic, also display antineoplastic activity. Hydrazine and benzylhydrazine although extremely toxic selectively inhibit Ehrlich ascites carcinoma in mice (Beisler et al, 1977). Cyanoacetylated hydrazides were active against sarcoma 180, Ehrlich carcinoma and Nemeth Kellner lymphoma in mice but had no effect on leukaemias (Fiszer- Maliszewska et u/,1987). Procarbazine is probably the best known hydrazine antitumour agent and it displays activity against a range of neoplasms in laboratory animals but is used clinically in the treatment of Hodgkin’s lymphoma (Tweedie et al, 1987).

Hydrazine sulphate has been utilised in many clinical trials involving terminal cancer patients who exhibit cachexia/weight loss, a condition which arises due to the excessive nutritional demands of the tumour(s). Eventually extreme weight loss reduces the ability of the patient to fight the disease and to withstand the treatments. Due to its ability to inhibit gluconeogenesis hydrazine increased the appetite and calorific intake in such patients stabilizing or increasing body weight (Chlebowski et al, 1987; 1990) due to

37 improved glucose tolerance and reduced glucose production (Chlebowski et al, 1984). Amino acid utilisation was also diminished thus reducing muscle wasting (Tayek et al, 1987). In addition tumour regression, reduction of neoplastic-associated disorders such as jaundice, decreased pain and improved treatment responsiveness were noted (Gold, 1975; 1987).

Another aspect of cancer is the immune response which causes the release of tumour necrosis factor (TNF)/cachectin which is also involved in the wasting process in neoplastic states (Beutler et al, 1986). Cachectin release can be suppressed by prior administration of glucocorticoids but not if activation of host reticuloendothelial system has already been initiated (Beutler et al, 1986). Indeed hydrazine has been shown to inhibit the release of TNF in mice via a pituitary-dependent response mediated by corticosterone (Silverstein, 1991). In addition the activity of TNF, namely abolition of cytolytic activity and potentiation of antiviral activity, was modulated in vitro (Hughes, 1989). It is apparent that the anticancer activity of hydrazine involves several different mechanisms.

1.10 TERATOGENICITY

Hydrazine and many of its derivatives are teratogenic in a number of animal species (Lee & Aleyassine, 1970; Toth, 1993). In rats teratogenicity of hydrazine itself was manifested as decreased litter size and poor survival of new born pups (Lee & Aleyassine, 1970) as well as numerous fetal abnormalities including decreased birth weight and skeletal and somatic abnormalities (Toth, 1993). The most common abnormalities caused by a range of hydrazines in several animal species (Xenopus, chicken, fish, rat and mouse) include skull, vertebrae, limb and sternum malformations and heart and brain defects (Toth, 1993). The rabbit appears to be relatively "immune" to the teratogenic effects of these compounds (Toth, 1993).

Although there have been reports suggesting that therapeutic hydrazines hydralazine, isoniazid and procarbazine are teratogenic in humans the data is

38 inconclusive as these drugs are generally used as adjunct therapy. However the similarity of certain lesions found both in animal and human offspring, such as reduced birth weight, limb malformations and brain disorders (Toth, 1993) should not be ignored.

1.11 AIM OF STUDY

Although there are some data indicating a role for cytochrome P450 in the hepatic metabolism of hydrazine, the involvement of specific isoenzymes is not fully understood. The first aim of this project was to advance existing knowledge in this area by modulating hydrazine toxicity, in vivo and in vitro, by inducing or inhibiting these isoenzymes.

There are conflicting reports in the literature regarding the effect of hydrazine on protein synthesis. Inhibition of this pathway can lead to fat accumulation in the liver, the major toxic manifestation of hydrazine. Thus both protein synthesis and triglyceride accumulation were studied in an attempt to clarify the mechanisms by which hydrazine generates these lesions.

39 MATERIALS AND METHODS

2.1 CHEMICALS

Acetaldehyde (A.C.S. reagent), adenosine 5’-triphosphate (disodium salt), albumin (bovine fraction V), ascorbic acid, collagenase (from Clostridium histolyticum, type I), deuterated chloroform (99.8 atom % D), deuterated m ethyl alcohol (99.8 atom % D; containing 0.03% TMS), dicumarol (crystalline), diethyldithiocarbamate (sodium salt), diphenylamine (free base), 5,5’-dithiobis- (2-nitrobenzoic acid), dithiothreitol, ethoxyresorufin, N-ethylmaleimide, firefly lantern extract, glutathione (reduced form), glutathione (oxidised form), glutathione reductase (stock 250units/ml), glycerol, HEPES, hydrazine hydrate, isonicotinic acid hydrazide (isoniazid), L-leucine, molybdate (ammonium salt), NADH (yeast grade III, disodium salt), NADPH (tetrasodium salt), niacinamide, p-nitrocatechol (crystalline), p-nitrophenol (spectrophotometer grade, crystalline), orcinol, osmium tetroxide, pentoxyresorufin, L-a phosphatidate, o-phthaldialdehyde, pyruvic acid (sodium salt, type II crystalline), resorufin, sodium arsenite, sodium dithionite, sodium m-periodate, sucrose, 2 -vinylpyridine were obtained from Sigma Chemical Company (Poole, Dorset, UK). Acetone, chloroform (reagent grade), chromotropic acid (sodium salt), Folin & Ciocalteau’s phenol reagent, sodium cacodylate were obtained from BDH Ltd. (Poole, Dorset, UK). L[3,4,5-^H(N)]-leucine (37MBq, ImCi/ml), L[^"^C]-leucine (37MBq, ImCi/ml) were obtained from DuPont (NEN) (Stevenage, Herts, UK). Araldite CY212 Epoxy resin, benzyldimethylamine (BDMA), dodecenyl succinic anhydride (DDSA), and glutaraldehyde (25%) were obtained from Agar Scientific (Stamstead, Essex, UK). Formaldehyde (10.5%) was purchased from Pioneer Research Chemicads Ltd. (Colchester, Essex, UK). Hexane amd methanol (HPLC grade) were obtained from Rathburn Chemicals Ltd. (Walkerbum, Scotland, UK).

40 2.2 /iVV/VO STUDIES

2.2.1 Animal Husbandry

Male Sprague Dawley rats (200-300g) were housed individually in metabolism cages and allowed to acclimatise for 2-3 days prior to treatment. During this time food and water was given ad libitum and was replaced daily. The environment of the animal room was maintained at 20°C ± 2 with a 12 hour light/dark cycle.

During treatment periods body weight, food consumption and water intake of all animals were monitored daily. Any animal showing overt signs of toxicity was removed from the study and sacrificed.

2.2.2 Autopsy Procedure

Prior to autopsy each animal was weighed and anaesthetised with diethylether. Throughout the procedure an ether hood was placed over the nose to prevent resuscitation.

The abdomen was opened and the viscera displaced to expose the abdominal aorta via which the animal was exsanguinated using a 10ml syringe and 23G needle. The blood was immediately transferred to microtainer tubes (Becton and Dickinson) and allowed to clot at room temperature for a minimum of 30 minutes. The tubes were then spun at 13,500 rpm for 2 minutes and the serum frozen at -80°C until required for clinical chemistry analysis.

The liver (and other organs if required) was excised, blotted and the initial weight recorded. A liver sHce approximately 1cm thick was taken from the median lobe and put into 10.5% formaldehyde for fixation and subsequent processing for examination under the light microscope. A small amount of liver was cut into cubes approximately 1mm thick and put in 2.5% glutaraldehyde in O.IM cacodylate huffer for preparation and future examination under the

41 electron microscope. Slices of liver for ATP, GSH and GSSG estimations were homogenised into preweighed tubes containing the appropriate acid (ice-cold) and immediately frozen at -80°C. Finally, the remaining liver was reweighed and either processed for microsomes or frozen at -80°C for analysis at a later date.

2.2.3 Preparation of rat liver microsomes

Male Sprague Dawley rats (200-300g) were anaesthetised with diethylether and exsanguinated via the abdominal aorta. The liver was rapidly removed, blotted to remove excess blood, weighed and then washed in ice-cold buffer (0.25M sucrose buffer containing lOmM tris and ImM EDTA, pH 7.4). The liver was then scissor chopped in 4 volumes of fresh buffer and homogenised using an Ystral tissue homogeniser.

An aliquot of the crude homogenate was frozen at -80°C for analysis at a later date but the majority was spun at 10,000g for 20 minutes in an MSE Europa 50 ultracentrifuge (4°C). The pellet was discarded and the supernatant (post- mitochondrial fraction) was centrifuged at 100,000g for 70 minutes. The resultant supernatant (cytosol) was retained and frozen at -80°C for analysis at a later date. The microsomal pellet was washed by resuspending in phosphate buffer (O.IM, pH 7.4) using a hand held Potter-Elvehjem homogeniser and then centrifuged again as described above. Finally the washed microsomal pellet was weighed and resuspended in 4 volumes of phosphate buffer (O.IM containing 20% glycerol, pH 7.4), aliquoted and frozen at -80°C until required.

2.2.4 Total Cytochrome-P450

Detection of cytochrome-P450 relies on the reduction of the haem iron which then binds carbon monoxide to give a characteristic absorption spectrum (maximal absorbance at 450nm) as described by Omura and Sato (1964).

42 A microsomal sample was diluted 10-fold (final concentration 1.5mg protein/ml) in phosphate buffer (O.IM, pH7.4) and added to two matched glass cuvettes. The samples were mixed by inversion and placed in the sample and reference cells of the spectrophotometer. After adjusting the absorbance to zero a baseline reading was taken by scanning between 400-500nm. A few grains of sodium dithionite were then added to both samples. Carbon monoxide (2 bubbles/ second for 30 seconds) was bubbled through the test sample only and the reduced spectrum was rescanned as described above.

Total cytochrome-P450 content was calculated using the absorbance difference (450-490nm) and the extinction coefficient (91mM‘^cm‘^) for this wavelength couple. Values were expressed as nmol cytochrome-P450/mg microsomal protein.

2.2.5 p-Nitrophenol Hydroxylase p-Nitrophenol hydroxylase activity was measured using the methods of Reinke & Moyer (1985) and Koop (1986). p-Nitrophenol is a substrate for the ethanol- inducible cytochrome-P4502El. The method relies on the formation of p- nitrocatechol, which can be detected spectrophotometrically after total ionization under alkaline conditions.

The reaction mixture (total volume 2ml) containing microsomal protein (1.5mg/ml), 150pM substrate (60pl of 5mM p-nitrophenol) and phosphate buffer (O.IM Na-K salts, pH 7.4) was equilibrated at 37°C prior to initiation of the reaction by the addition of 500pM NADPH (40pl, 25mM). A blank consisting of denatured microsomes (1.5mg/ml; heated at 70-100°C for 10 minutes) was prepared for each sample and treated as above.

The reaction was allowed to proceed for 15 minutes after which it was terminated by addition of PC A (0.5ml; 0.5M). After mixing and holding on ice for 5-10 minutes the tubes were spun (2,500 rpm for 5 minutes) and the p- nitrocatechol formed determined spectrally at 510nm in supernatant ( 1ml) after

43 the addition of NaOH (0.1ml; lOM). Enzyme activity was calculated with reference to a p-nitrocatechol standard curve (G- 6 G|iM, final volume 2ml) and expressed as nmol p-nitrocatechol formed/min/mg microsomal protein.

2.2.6 Ethoxyresorufin o-deethylase (EROD) I Pentoxyresorufin o-depentylase (PROD)

Ethoxyresorufin and pentoxyresorufin are highly specific substrates for B- naphthoflavone (P45G1A1) and phenobarbitone (P45G2B1/2) inducible cytochromes-P45G respectively. The activity of the above enzymes was determined in microsomes using the method of Burke et al (1985) and in hepatocytes using the method of Lubet et al (1985).

The reaction mixture (total volume 2 ml) containing microsomal protein (15Gpg/ml), 5pM substrate (IGpl of ImM EROD/PROD) and phosphate buffer (G.IM Na-K salts, pH 7.6) was mixed by inversion and equilibrated at 37°C for 1 minute. A baseline reading was taken for several seconds by monitoring the fluorescence at 53Gnm excitation and 585nm emission (default slit size IGnm) after which the reaction was started by the addition of 25GpM NADPH (IGpl, 5GmM). The increase in fluorescence was followed for 3-5 minutes or until a linear gradient was recorded.

EROD/PROD activity in hepatocytes was determined as above but using 2GGpl of a hepatocyte homogenate, obtained by sonicating an aliquot of IxlGVml hepatocyte suspension. In addition IGpM dicumarol (IGpl of 2mM stock solution) was added to the reaction mixture (total volume 2ml).

Enzyme activity was calculated from the average fluorescence given by stepwise additions of IGpM resorufin (5pl) to phosphate buffer (total volume 2 ml) and expressed as pmol resorufin formed/min/mg microsomal protein.

44 2.2.7 Determination of Protein

Tissue or cellular protein was assessed using the method of Lowry et al (1951). The method combines the biuret and Folin-Ciocalteu reactions. In the former a coloured chelate is formed between peptide bonds and Cu(II) ions in alkaline medium. The latter reaction relies on the reduction of phosphotungstic- phosphomolybdic acid by tyrosine and tryptophan residues to give a blue complex. Sample and buffer preparation are described in Appendix I.

Alkaline copper reagent (5ml; see Appendix I) was added to sample/standard (0.5ml; duplicates), the tubes mixed and allowed to stand for 10 minutes at room temperature. Folin and Ciocalteau’s Phenol Reagent (0.5ml; 1:1 with water) was then added and the tubes immediately vortexed. After 30 minutes absorbance was read at 750nm against a water blank. Protein content of samples was calculated with reference to a bovine serum albumin standard curve (O-lOOpg/ml, final volume of 0.5ml).

2.2.8 Adenosine Triphosphate (ATP)

Tissue or cellular ATP was determined using the method of Stanley and Williams (1969). The assay depends on the detection of bioluminescence produced by luciferase extract of firefly tails in the presence of ATP. The amount of light emitted is proportional to the amount of ATP present. Sample preparation is outlined in Appendix II.

An aliquot of acid supernatant ( lOpl) was added to buffer (2ml; see Appendix II) in 12x75mm plastic tubes and vortexed to ensure the TCA was thoroughly mixed. The reaction was initiated with luciferase (lOOpl), immediately vortexed for 2-3 seconds and the tube placed in the cell holder of the luminometer. The photon count was initiated after 15 seconds. ATP content of test samples was calculated with reference to a standard curve (0-40pM ATP in 10% TCA) and expressed as pmol/g tissue or nmol/10^ cells.

45 2.2.9 Total Non-Protein Sulphydryls (TNPSH)

The estimation of tissue TNPSH, which comprises approximately 95% reduced glutathione, was carried out using the method of Ellman (1961). This assay is based on the reduction of DTNB by -SH groups to yield a yellow coloured complex. Sample preparation is outlined in Appendix III.

Phosphate buffer (0.25ml; O.IM, pH7.4) was added to an equal volume of acid supernatant (duplicates) and mixed. This was followed by addition of phosphate buffer (4.5ml; O.IM, pH8). DTNB (50pl; 39.6mg in 10ml buffer pH7.4) was then added, the tubes vortexed and allowed to stand for 15 minutes at room temperature. The absorbance was read at 412nm against a water blank. TNPSH levels were calculated using a GSH standard curve (0- ImM in a final volume of 0.25ml SSA) and expressed as pmol/g tissue.

2.2.10 Oxidised Glutathione (GSSG)

Tissue oxidised glutathione was determined using the method of Griffith (1980). The method involves masking of liver GSH by derivatisation with 2- vinylpyridine followed by reduction of GSSG in the presence of NADPH and glutathione reductase. The resultant GSH is reacted with DTNB to form a coloured complex. Sample preparation is outlined in Appendix III.

Acid supernatant (84pl) was added to an eppendorf containing O.IM Tris/5mM EGTA buffer (336pl; pH7.4) and 2-vinylpyridine ( 6 pl). The tubes were vortexed and incubated at 30°C for 20 minutes. NADPH (0.7ml; 6.3mg/25ml buffer), DTNB (0.1ml; 23.8mg/10ml buffer) and sample (142pl) were then added to a small volume plastic cuvette, mixed and preincubated for 1 minute at 30°C. After adjusting the spectrophotometer to zero glutathione reductase (lOpl; 50 unit/ml) was added to the cuvette. The sample was mixed by inversion and the change in absorbance followed at 412nm for 1 minute. GSSG content was calculated using a standard curve (0- lOOpM in SSA) and expressed as nmol/g tissue.

46 2.2.11 Liver Triglyceride Analysis

Liver triglyceride content was determined using the method of Butler et al (1961). This multistep procedure involves: a) the extraction and hydrolysis of triglycerides; b) oxidation of the resultant glycerol with sodium m-periodate to generate formic acid and formaldehyde; and c) the reaction between formaldehyde and chromotropic acid to yield a coloured complex. The preparation of buffers and tissue samples is outlined in Appendix IV.

Alcoholic KOH (0.5ml) was pipetted into 3 tubes (saponified samples) and 95% ethanol (0.5ml) into 2 tubes (unsaponified samples) all of which were incubated at 60°C for 20 minutes. The samples were neutralised with HgSO^ (0.5ml; O.IM) and boiled for 20 minutes to evaporate the alcohol. The tubes were left to cool in ice-water.

Sodium m-periodate (lOOpl; 0.05M), sodium arsenite (200pl; IM) and finally chromotropic acid (5ml; see Appendix IV) were added sequentially at 10 minutes intervals and the samples boiled for 30 minutes in the dark. Absorbance was read at 570nm against a water blank and tissue triglyceride content calculated from a triglyceride standard curve (O-O.lmg/ml chloroform).

2.2.12 Serum Clinical Chemistry

A range of clinical chemistry assays were carried out at Glaxo Research and Development (Ware, Herts) on a Hitachi 705 autoanalyser using the appropriate kits supplied by Boehringer. Assays included: alanine transaminase; aspartate transaminase; alkaline phosphatase; total protein; albumin; glucose; blood urea nitrogen; creatinine; triglycerides; cholesterol; non-esterified fatty acids; total bilirubin.

47 2.3 IN VITRO STUDIES

2.3.1 Animal Husbandry

Male Sprague Dawley rats (200-300g) were used for these studies. Prior to isolation of hepatocytes the animals were housed 4/cage and allowed food and water ad libitum. The room was light and temperature regulated as previously described.

2.3.2 Preparation of Isolated Hepatocytes

Hepatocytes were isolated by collagenase perfusion using the method of Moldeus et al (1978). The procedure involves perfusion of the liver with: a) buffer containing EDTA which chelates calcium and thus leads to cleavage of calcium-dependent cell-cell interactions; and b) with collagenase which dissolves intercellular collagen. The equipment used for the isolation procedure is shown in Figure 2.1 and preparation of buffers outlined in Appendix V.

Figure 2.1 The Equipment Utilised For The Isolation of Rat Hepatocytes

Direction of Flow of Perfusate

Bubble Trap

Regulator Clip

To Cannula Support Unit ^ z— Manometer

Liver To Carbogen

W a te I Bath Set at 37 C

48 A rat was anaesthetised using diethylether and the abdomen opened by means of a V-shaped transverse incision. The viscera were displaced and the large hepatic portal vein identified. An incision was made in the portal vein and the cannula inserted and secured by means of the ligature. The cannula clip was then opened to begin the perfusion with Hank I (preequilibrated to 37°C). After release from surrounding connective tissue the flow rate of the buffer was adjusted to approximately 2 drops/second.

After 5-10 minutes the liver was transferred to a beaker containing Hank Il/collagenase and was perfused until the liver showed signs of digestion (10-15 minutes). After draining Hank II the liver was detached from the canula and transferred to K+H/Alb (50ml). The capsule was broken using a fork and the cells released by gentle shaking.

The crude hepatocyte suspension was filtered through nylon mesh, to remove connective tissue and clumps of cells, and subsequently transferred to large volume plastic tubes and centrifuged at 50g for 1 minute. The supernatant was aspirated and the cells gently resuspended in fresh K+H buffer and centrifuged at 50g for 2 minutes. This wash step was repeated. The washed hepatocytes were finally resuspended in K+H buffer, the volume measured and the cells transferred to a glass conical flask. The total number of cells and their viability was assessed using Trypan blue dye exclusion.

2.3.3 Trypan Blue Dye Exclusion

An aliquot of hepatocyte suspension (50pl) was added to trypan blue (450pl; 0.4% trypan blue in 0.9% NaCl). Samples were gently mixed to ensure the cells were fully resuspended prior to pipetting into the haemocytometer. Viability, determined by counting the num ber of cells which did not take up the dye (live cells), was expressed as a percentage of total cell number. Initial cell viability was usually in the range of 80-95% and total yield 200-300x10® cells.

49 2.3.4 Lactate Dehydrogenase (LDH)

The appearance of lactate dehydrogenase in the incubation medium is a marker of irreversible damage to the plasma membrane and the amount of leakage correlates well with cell viability assessed by trypan blue dye exclusion (Tyson & Green, 1987). The activity of this enzyme in external medium was determined using the method of Bergmeyer et al (1965). Preparation of solutions and samples are outlined in Appendix VI.

Cell supernatant (lOOpl) was added to a cuvette containing phosphate/pyruvate buffer (2.8ml; 3.75mg pyruvate/lOOml) and NADH (lOOpl; 6 .6 6 mg in 1ml phosphate buffer). The cuvette contents were immediately mixed and the reaction followed for 1 minute at 340nm. LDH activity was assessed by the change in absorbance. The activity in supernatant was expressed as a percentage of the total (cells and supernatant at time zero).

2.3.5 Reduced Glutathione (GSH)

Reduced glutathione content in isolated hepatocytes was determined using the method of Hissin and Hilf (1976). The assay is based on the reaction of OPT with GSH, at pH 8 , to yield a highly fluorescent product. Preparation of buffers and samples is outlined in Appendix VII.

An aliquot of acid supernatant (75pl) was added to phosphate buffer (2.775ml; see Appendix VII). o-Phthaldialdehyde (150pl; Img/lml methanol) was then added, the tubes vortexed and allowed to stand at room temperature for 25 minutes. The fluorescence was read at 350nm excitation and 420nm emission. Gluathione content of hepatocytes was determined using a standard curve (0- 750ng, final volume of 75pl) and expressed as nmol GSH/10^ cells.

50 THE EFFECT OF INDUCTION OR INHIBITION OF CYTOCHROME-P450 ON HYDRAZINE TOXICITY/iV VITRO

3.1 INTRODUCTION

Hepatic cytochrome-P450 has been implicated in hydrazine metabolism but the isoenzymes involved are still to be fully elucidated. Several studies in vivo and in vitro have been carried out to determine the effect of induction and inhibition of cytochromes P450 on hydrazine toxicity.

Pretreatment of rats with phenobarbitone prior to hydrazine administration resulted in reduced plasma concentration and urinary excretion of hydrazine (Noda et al, 1985) and diminished hepatic lipid accumulation (Timbrell et al, 1982) compared to that seen in control rats. In contrast pretreatment of rats with piperonyl butoxide, a non-specific inhibitor of cytochrome P450, exacerbated hydrazine toxicity (Timbrell et al, 1982; Jenner & Timbrell, 1994). These same pretreatments increased and decreased respectively the disappearance of hydrazine from isolated rat hepatocytes (Noda et al, 1987).

Induction of cytochrome P4502E1 has received much attention over the last decade due to its metabolism of many carcinogens or procarcinogens to reactive intermediates (Guengerich et al, 1991; Raucy et al, 1993). Indeed this isoenzyme has been linked with the tumourigenic effect of 1,2 - dimethylhydrazine (Wattenberg, 1975). Hydrazine itself is carcinogenic in animal species (Toth, 1980) and repeated exposure to rats results in induction of cytochrome P4502E1 (Akin & Norred, 1978; Jenner & Timbrell, 1994a). In addition induction of cytochrome P4502E1 by acetone and isoniazid has been shown to increase the acute toxicity of hydrazine in vivo, manifested as increased hepatic lipid accumulation (Jenner & Timbrell, 1994a).

51 It is apparent that modulation of the activity of specific cytochrome P450 isoenzymes by pretreatment with inducers or inhibitors alters hydrazine toxicity. As cytochrome P4502E1 is to date the only isoenzyme to be associated with increased hydrazine toxicity in vivo, it was of particular interest to study the effect of induction or inhibition of this isoenzyme on the toxicity of hydrazine using isolated rat hepatocytes in vitro.

The toxicity of a range of hydrazine concentrations was assessed first in control hepatocytes and then in hepatocytes isolated firom rats that had been pretreated with compounds known to modulate cytochrome P4502E1 activity. The toxicity between groups was compared and correlated to microsomal enzyme activities.

3.2 MATERIALS AND METHODS

3.2.1 Animal Pretreatment

Male Sprague Dawley rats (200-300g) were pretreated with acetone (20% v/v in distilled water (DW), 5ml/Kg by gavage 24 hours prior to isolation), diethyldithiocarbamate (750mg/Kg, i.p. in DW, 24 hours prior to isolation), isoniazid (0.1% w/v [approximately 125mg/Kg/day] in drinking water for 20 days,) or hydrazine (2.5mg/Kg in drinking water for 10 days). Control animals received no pretreatment. All animals were allowed food and water ad libitum up to the time of autopsy.

3.2.2 Hepatocyte Preparation

Hepatocytes were isolated by collagenase perfusion according to the method of Moldeus et al (1978) as previously described in Chapter 2 . Cells were subsequently incubated in rotating siliconised flasks in Krebs-Henseleit buffer containing 0.1-20mM concentrations of hydrazine for the control experiment and 8 -2 0 mM for the induction/inhibition experiment. Cells were incubated at

52 37°C, under an atmosphere of 95% 0

3.2.3 Biochemical Determinations

Viability was assessed by trypan blue dye exclusion and leakage of lactate dehydrogenase (LDH) into the incubation medium (Bergmeyer et al, 1965). Reduced glutathione (GSH) was measured in trichloroacetic acid (TCA) extracts of hepatocytes using the fluorometric method of Hissin and Hilf (1976). ATP was measured in TCA extracts of hepatocytes by luciferase-linked bioluminescence (Stanley and Williams, 1969).

EROD (an indicator of P4501A1/2) and PROD (an indicator of P4502B1/2) activities were assessed using a modified version of the methods of Lubet et al (1985) and Burke et al (1985). p-Nitrophenol hydroxylase (an indicator of P4502E1) activity was determined using the spectrophotometric method of Reinke & Moyer (1985) and Koop (1986).

3.2.4 Statistical Analysis

Statistical analysis was assessed using Dunnetts t-test to compare pretreated with control values.

3.3 RESULTS

3.3.1 The Toxicity of Hydrazine in Hepatocytes Isolated from Untreated Rats

Hepatocytes isolated from untreated rats were exposed to a range of hydrazine concentrations (0.1-20mM). Hydrazine caused a concentration and time

53 dependent loss of viability, assessed by trypan blue dye exclusion and LDH leakage. The threshold concentration for cytotoxicity (loss of viability) was 12mM hydrazine with 16 and 20mM hydrazine inducing significant cell death after 3 hours of incubation (Figure 3.1).

Reduced glutathione and ATP were also depleted in a time and concentration dependent manner. Glutathione was depleted without recovery after 1 hour by 4-20mM hydrazine (Figures 3.2). After 2 hours of incubation 0.1 and ImM hydrazine significantly reduced glutathione levels but this became insignificant after 3 hours due to a drop in control levels (Figure 3.2).

Depletion of ATP occurred slightly later than GSH being reduced by 16 and 20mM hydrazine after 1 hour (Figure 3.3) and 4-20mM after 2 hours (Figure 3.3). The lowest doses (0.1-lmM) had no effect on this parameter at any time (Figure 3.3).

Figure 3.1 The Effect of a Range of Hydrazine Concentrations on Cell Viability and LDH Leakage in Hepatocytes Isolated from Control Rats

120 r 90 Viability □ LDH leakage ^ "@100 - 75 o _ 80 - 60 5" 7K-D> 0» 60 - 45 CO(» X 40 - 30 ** o 0» 20 - 15

0 0 0.1 1 10 100 Concentration of hydrazine (mM)

Isolated control rat hepatocytes incubated (at a density of lxlO®/ml) with hydrazine (0.1, 0,5, 1, 4, 8,12,16 and 20mM). Viability at 3 hours assessed by trypan blue dye exclusion and LDH leakage. Values are means ± SEM; n = 4 repetitions; *p<0.05, **p<0.01 significantly different from controls.

54 Figure 3.2 The Effect of a Range of Hydrazine Concentrations on GSH Depletion in Hepatocytes Isolated from Control Rats

1 hour ^ 120 1 2 hours # 100 - 3 hours □ 3«> fO > 80 - <0 ** c 60 - ** 40 - «* ** X(/) O ** 20 - **

0 5 10 15 20

Concentration of hydrazine (mM)

Figure 3.3 The Effect of a Range of Hydrazine Concentrations on ATP Depletion in Hepatoc>’tes Isolated from Control Rats

1 2 0 -I 1 hour ^ 2 hours o 100 - 3 hours □ 3«> «0 > 80 - (O

** QL *♦ < ** 20 - ** **

0 5 10 15 20

Concentration of hydrazine (mM)

Isolated control rat hepatocytes incubated (at a density of lxlO®/ml) with hydrazine (0.1, 0.5, 1, 4, 8, 12, 16 and 20mM). Toxicity at 3 hours assessed by GSH and ATP depletion (Figures 3.2 and 3.3 respectively). Values are means ± SEM; n = 4 repetitions; *p<0.05, **p<0.01 significantly different from controls.

55 3.3.2 The Effect of Induction and Inhibition of Cytochrome P4502E1 on Hydrazine Toxicity

As discussed in the previous section the lower concentrations of hydrazine (0.1- 4mM) caused limited toxicity and thus further studies were performed utilising the higher concentrations (8-20mM) to determine whether induction or inhibition of cytochrome P450 could shift the threshold of hydrazine cytotoxicity in isolated rat hepatocytes.

Comparable to the response in cells from untreated rats 16 and 20mM hydrazine caused significant loss of viability after 3 hours in hepatocytes from acetone, isoniazid and hydrazine pretreated rats compared to the relevant treatment controls. These concentrations of hydrazine became cytotoxic after 2 hours in cells from DEDC pretreated rats as did 12mM after 3 hours.

When making comparisons between the control and pre treatment groups, acetone and hydrazine pretreatments increased the threshold dose, manifested as a significant reduction in the extent of cell death after 2 hours, whereas DEDC pretreatment increased the toxicity of all concentrations of hydrazine after 3 hours (Figure 3.4). There was no significant difference between isoniazid pretreated and untreated cells, although there was a trend towards increased toxicity in the former group Figure 3.4).

In cells from acetone pretreated rats exposure to 8-20mM hydrazine did not cause a significant rise in LDH leakage. Similarly in cells from hydrazine pretreated rats LDH leakage was minimal, although 20mM hydrazine did cause significant leakage after 3 hours compared to the relevant hydrazine pretreated control. DEDC pretreatment clearly shifted hydrazine toxicity with 12mM hydrazine causing LDH leakage after 1 hour (data not shown).

Acetone, hydrazine and isoniazid pretreatments did not significantly alter the degree of LDH leakage compared to that seen in the untreated group whereas DEDC pretreatment increased it (Figure 3.5).

56 In cells isolated from untreated, DEDC, isoniazid and hydrazine pretreated rats 8-20mM hydrazine significantly depleted GSH after 1 hour compared to the pretreatment control while GSH depletion in cells from acetone pretreated animals became significant after exposure to 8-20mM hydrazine after 2 hours (data not shown). DEDC pretreatment caused the greatest depletion in response to hydrazine whereas isoniazid pretreatment did not appear to alter GSH depletion compared to cells from untreated rats (Figure 3.6). Both acetone and hydrazine pretreatments protected against GSH depletion the former becoming significant after 3 hours and the latter after 1 hour (Figure 3.6).

In cells from both hydrazine and isoniazid pretreated rats all concentrations of hydrazine caused significant ATP depletion after 1 hour, compared to the relevant pretreatment control. As with other parameters measured pretreatment of animals with DEDC increased the susceptibility of the cells to hydrazine, 12-20mM hydrazine causing significant ATP depletion after 1 hour compared to the DEDC control. Acetone pretreatment protected against hydrazine toxicity, ATP depletion occurring after 2 hours with 12, 16 and 20mM hydrazine compared to the acetone control. In comparison to the effect in cells from untreated rats, acetone and hydrazine pretreatment decreased while DEDC pretreatment increased hydrazine-induced ATP depletion (Figure 3.7). Overall the degree of ATP depletion in cells from isoniazid pretreated rats was similar to that seen in cells from untreated rats (Figure 3.7).

The effects of the pretreatments on the activity of specific isoenzymes of cytochrome P450 are illustrated in Table 3.1. Most of the parameters of cytotoxicity measured correlated with the activities of the cytochrome P450 isoenzymes, such that the greater the activity the less the toxicity and vice versa (Table 3.2). Figures 3.8-3.10 illustrate the correlations obtained for cell viability and enzyme activity.

57 Figure 3.4 The Effect of Various Pretreatments on Hydrazine-induced Cell Death in Isolated Rat Hepatocytes

Pretreatment: 100 -1 Control ■ «> DEDC A 80 - «0 Acetone o > Isoniazid A [w 60 - Hydrazine ♦ c X >•

10 20 -

0 5 10 15 20 Concentration of hydrazine (mM)

Figure 3.5 The Effect of Various Pre treatments on Hydrazine-induced LDH Leakage in Isolated Rat Hepatocytes

Pretreatment: 120 -1 Control 100 - DEDC Acetone fO 80 - Isoniazid A o Hydrazine ♦ 60 -

X 40 -

20 -

0 5 10 15 20

Concentration of hydrazine (mM)

Isolated hepatocytes from control; DEDC; acetone; isoniazid; and hydrazine pretreated rats incubated (at a density of lxlO®/ml) with hydrazine (8,12, 16 and 20mM). Viability at 3 hours assessed by trypan blue dye exclusion (Figure 3.4) and LDH leakage (Figure 3,5). Values are means ± SEM; n = 4 repetitions; *p<0.05, **p<0.01 significantly different from controls.

58 Figure 3.6 The Effect of Various Pretreatments on Hydrazine-induced GSH Depletion in Isolated Rat Hepatocytes

Pretreatment: 120 Control ■ DEDC ^ 100 30) Acetone o •0 > 80 isoniazid A 10 Hydrazine ♦ 'c 60 ** 40 ** X(/) a 20

0 5 10 15 20 Concentration of hydrazine (mM)

Figure 3.7 The Effect of Various Pretreatments on Hydrazine-induced ATP Depletion in Isolated Rat Hepatocytes

Pretreatment: 140

Control ■ 120 DEDC ^ Acetone o 100 Isoniazid A 80 Hydrazine ♦ « 60 Q. I- 40 < 20 j. 0 0 5 10 15 20 Concentration of hydrazine ImM)

Isolated hepatocytes from control; DEDC; acetone; isoniazid; and hydrazine pretreated rats incubated (at a density of lxlO®/ml) with hydrazine (8, 12, 16 and 20mM). Toxicity at 3 hours assessed by GSH and ATP depletion (Figures 3.6 and 3.7 respectively). Values are means ± SEM; n = 4 repetitions; *p<0.05, **p<0.01 significantly different from controls.

59 Table 3.1 The Effect of Various Pretreatments on Cytochrome P450 Activities in Isolated Rat Hepatocytes

Animal Ethoxyresorufin Pentoxyresorufin p-Nitrophenol Pretreatment o-Deethylase o-Depentylase Hydroxylase (pmol/min/ 10® cells) (pmol/min/ 10® cells) (nmol/min/ 10® cells)

None (4) 79.9 ± 2.4 61.7 ± 4.7 0.57 ± 0.02

DEDC (4) 26.8 ± 2 .6 **( 21.7 ± 2.2**^ 0.08 ± O.OU a o Acetone (3;4) 119.1 ± 19.7* 67.3 ± 14.8 0.80 ± 0.17

Isoniazid (3) 65.3 ± 11.5 31.4 ± 3.1**^ 2.73 ± 0.29**^

Hydrazine (4) 65.6 ± 2.2^ 51.7 ± 1.2 0.95 ± 0.06^

Results are expressed as means ± S.E.M.; number of repetitions () Statistical significance: *p < 0.05 and **p < 0.01 Dunnett’s t-Test; ^p < 0.05 Unpaired t-Test Figure 3.8 Correlation (Linear Regression) Between p-Nitrophenol Hydroxylase Activity (P4502E1) and Cell Viability in Hepatocytes Exposed to Hydrazine

Isoniazid pretreatment ^ 3.5 -1 Other pretreatments • « = 3.0 - | s 2.5 - !i 2.0 - 1.5 -

1.0 -

0.5 -

0 0 10 20 30 40 50 60 70 80 90 % Viability

Figure 3.9 Correlation (Linear Regression) Between EROD Activity (P4501A1/2) and Cell Viability in Hepatocytes Exposed to Hydrazine

All Pretreatments # M 160 -1 o o 140 - "b 120 -

o Ê 80 - CL 60 -

B 40 - a CO Û 20- O CC 0-1 UJ 20 30 40 50 60 70 80 90

X Viability

Each data point represents the activity of either p-nitrophenol hydroxylase or EROD in cells prior to hydrazine exposure compared to the loss of cell viability (trypan blue dye exclusion) after incubation with 12mM hydrazine for 3 hours (obtained from separate hepatocyte isolations). Data from untreated, DEDC, acetone, isoniazid and hydrazine pretreatments.

61 Figure 3.10 Correlation (Linear Regression) Between PROD Activity (P4502B1/2) and Cell Viability in Hepatocytes Exposed to Hydrazine

= 100 All pretreatments • o u «D O 80 - c E 60 - o E CL 40 - > • •

Ü - ID 20 Q O CC Q. 20 30 40 50 60 70 80 90

X Viability

Each data point represents the activity of PROD in cells prior to hydrazine exposure compared to the loss of cell viability (trypan blue dye exclusion) after incubation with 12mM hydrazine for 3 hours (obtained from separate hepatocyte isolations). Data from untreated, DEDC, acetone, isoniazid and hydrazine pretreatments.

62 Tables 3.2a/b Correlations Between Cytochrome P450 Activities and Biochemical Parameters in Isolated Rat Hepatocytes Exposed to 12mM Hydrazine

Table 3.2a Linear Regression Analysis

Biochemical/ EROD PROD pNPH pNPH Toxicological +INH -INH Parameter Viability r=0.602 r=0.727 r=0.017 r=0.795 (% initial) p<0.01 p<0.001 p=0.76 p<0.001 LDH Leakage r=0.544 r=0.594 r= -0.233 r=0.661 (% total) p<0.05 p=0.01 p=0.34 p<0.01 ATP r=0.656 r=0.646 r=0.152 r=0.736 (% initial) p<0.01 p<0.01 p=0.54 p<0.01 GSH r=0.459 r=0.529 r=0.133 r=0.717 (% initial) p=0.06 p<0.05 p=0.59 p<0.01

As illustrated in figures 3.8-3.10, these data appear to represent two distinct populations. Further analysis was therefore carried out using the Spearman Rank test. The resultant correlations (Table 3.2b) were generally in agreement with those obtained using linear regression (Table 3.2a).

Table 3.2b Spearman-Rank Correlation Analysis

Biochemical/ EROD PROD pNPH Toxicological -INH Parameter Viability r,=0.326 r =0.656 r=0.730 (% initial) p=0.22 p<0.01 p=0.001 LDH Leakage r,=-0.450 r=-0.592 r=-0.575 (% total) p=0.06 p=0.01 p<0.05 ATP r =0.500 r=0.561 r=0.717 (% initial) p=0.04 p<0.05 p<0.01 GSH r^=0.230 r,=0.402 r=0.772 (% initial) p=0.41 p=0.10 p<0.001 pNPH = p-nitrophenol hydroxylase; INH = isoniazid; r = correlation coefficient from linear regression; r^ = correlation coefficient from Spearman-Rank analysis; p = statistically significant correlation if less than 0.05.

63 3.4 DISCUSSION

The results obtained in this study regarding the toxicity of hydrazine in hepatocytes isolated from untreated rats are similar to those previously described (Preece et al, 1990; Ghatineh et al, 1992). In the present study loss of cell viability occurred 3 hours after incubation with 16-20mM hydrazine (Figure 3.1). However other manifestations of toxicity, namely depletion of ATP and GSH, induced by these same concentrations of hydrazine were evident after 1 hour (Figures 3.2 and 3.3).

Hydrazine-induced ATP and GSH depletion was concentration and time dependent and occurred at concentrations which were not cytotoxic, as previously described (Timbrell et al, 1982; Preece et al, 1990). The lowest concentration to have an effect on ATP and GSH was 4mM hydrazine. A concentration of ImM hydrazine has been reported to deplete GSH in isolated rat hepatocytes after 30-60 minutes of incubation (Noda et al, 1987). Indeed GSH was depleted by this concentration of hydrazine in the present study, but this was a transient response which was evident only after 2 hours of incubation (Figure 3.2).

In the present study GSH was depleted prior to ATP in contrast to a previous report (Ghatineh et al, 1992). These authors proposed that GSH synthesis was impaired as a result of reduced levels of ATP, which is required at several stages (Meister & Anderson, 1983). The present data suggest that, at least during the initial period of incubation, depletion of GSH is independent of ATP. Mitochondrial damage (Scales & Timbrell, 1982; Wakayabashi et al, 1987) or disturbances of intermediary metabolism (Moloney and Prough, 1983) may disrupt maintenance of both these compounds.

Hydrazine is considerably less toxic in vitro th an in vivo. The maximum liver concentration of hydrazine attained after administration of a dose of 81mg/Kg body weight to rats was 0 .2mM hydrazine (Preece et al, 1992). Furthermore in vivo administration of a dose of hydrazine as low as lOmg/Kg caused GSH depletion (Timbrell et al, 1982), and 20mg/Kg caused ATP depletion and lipid

64 accumulation (Timbrell et al, 1982; Preece et al, 1990).

The reason for the discrepancy between in vivo and in vitro systems is currently unknown. One possible explanation is reduced uptake of hydrazine into cells in vitro. However this seems unlikely as a study carried out using isolated rat hepatocytes demonstrated active uptake of hydrazine which could be blocked by incubation a) with metabolic inhibitors or b) at low temperature (Ghatineh & Timbrell, 1990b). A further possibility is that hydrazine is metabolised in vivo by other cell types in the liver, such as Kupffer cells, which are discarded during the process of hepatocyte isolation.

The pretreatment of animals to manipulate the activities of cytochromes P4501A1/2, P4502B1/2 and P4502E1 clearly influenced hydrazine toxicity in vitro. Pretreatment with DEDC increased the toxicity whereas pretreatment with acetone and hydrazine decreased the toxicity. Isoniazid pretreatment slightly, but not significantly, increased hydrazine-induced cell death.

Enzyme activities generally correlated with loss of cell viability (Figures 3.8- 3.10) and the depletion of ATP and GSH (Table 3.2). The results suggest that hydrazine is detoxified by cytochromes P4501A1/2, P4502B1/2 and P4502E1 as the lower the activity the higher the toxicity. The correlations obtained for cytochrome P4502E1 were only accomplished in the absence of isoniazid data whose points were clearly isolated from the rest. This may reflect interference of hydrazine metabolism by isoniazid which will be discussed later.

DEDC pretreatment inhibited all of the isoenzymes measured (Table 3.1) however this dosing regime should inhibit cytochromes P4502E1 and P4502B1/2 alone (Lauriault et al, 1992). Carbon disulphide, a metabolite of DEDC inhibits P4501A1/2 as well as those mentioned above (Lauriault et al, 1992) and thus it is possible that this compound may have been formed in vivo. Nevertheless DEDC pretreatment increased hydrazine toxicity in vitro. This correlates with the situation in vivo where non-specific inhibition of cytochrome P450 with piperonyl butoxide exacerbated hydrazine toxicity, manifested as accumulation of triglycerides and increased depletion of ATP in the liver

65 (Timbrell et al, 1982; Jenner and Timbrell, 1994).

Induction of cytochromes P4502B1/2 and P4501A1 by pretreatment with phenobarbitone and B-naphthoflavone respectively reduced the in vivo hepatotoxicity of an acute dose of hydrazine whereas induction of cytochrome P4502E1 by acetone or isoniazid increased hydrazine toxicity, manifested as enhanced triglyceride accumulation without significant effect on ATP or GSH (Jenner and Timbrell, 1994).

Acetone pre treatment protected against hydrazine toxicity in vitro. Acetone is classically known to induce cytochromes P4502B1/2 and P4502E1 after acute dosing (Ronis & Ingelman-Sundberg, 1989) however induction of cytochrome P4501A1/2 has also been reported after administration of acetone (15mmol/Kg) for 3 days (B arnett et al, 1992). Although the activity of all three isoenzymes was increased by acetone pretreatment only cytochrome P4501A1/2 was significantly raised in the present study (Table 3.1). In accordance with in vivo data induction of this isoenzyme afforded protection against hydrazine cytotoxicity and depletion of ATP and GSH despite the different inducing agents (Jenner & Timbrell, 1994). The discrepancy between in vivo and in vitro results with respect to acetone may therefore be solely attributable to the pattern of enzyme induction.

Hydrazine and isoniazid pretreatment both induced cytochrome P4502E1 (2- fbld and 5-fold respectively) and inhibited cytochromes P4501A1/2 (significant with hydrazine only) and P4502B1/2 (significant with isoniazid only) (Table 3.1). Despite qualitatively similar changes in enzyme activity the former protected against hydrazine toxicity and the latter had no significant effect, although there was a trend towards increased cell death. The results obtained from isoniazid treatment are in agreement with those obtained in vivo (Jenner & Timbrell, 1994).

The situation with isoniazid may be complex. Isoniazid may induce and subsequently bind to cytochrome P4502E1 therefore blocking hydrazine metabolism. This phenomenon was observed during coadministration studies

66 in man where oxidative metabolism of chlorzoxazone and paracetamol were reduced in the presence of isoniazid but gradually increased after isoniazid withdrawal (Zand et al, 1993). The hepatocyte isolation procedure should wash out any isoniazid remaining in the liver after the last exposure however it is possible that a proportion of the dose may remain bound to the enzyme. This would have resulted in an apparent inhibition of p-nitrophenol hydroxylase activity but this was not the case (Table 3.1). A further possibility for the increased amount of cell death in hepatocytes from isoniazid pretreated rats is exposure of hepatocytes additionally to residual hydrazine, which is a metabolite of isoniazid (Timbrell & Wright, 1979; Peretti et al, 1987). This too is doubtful as the amount of hydrazine present would be extremely small and thus unlikely to exacerbate the toxicity of a subsequent acute dose of hydrazine.

The negative correlation of cytochrome P4502E1 activity with loss of cellular viability and ATP and GSH depletion seem to suggest that this isoenzyme catalyses a detoxication pathway with regard to hydrazine (Table 3.2). Thus isoniazid pretreatment ought to have exerted a greater protective influence than hydrazine pretreatment as the magnitude of induction of cytochrome P4502E1 was 5-fold and 2-fold respectively (Table 3.1). However this was not the case and as such one might speculate that cytochrome P4502E1 does not play a major role in hydrazine metabolism.

Another possibility is that hydrazine and isoniazid do not induce the same cytochrome P450 isoenzyme, despite the increase in p-nitrophenol hydroxylase activity. However this seems unlikely as the hydrazine moiety of the isoniazid molecule plays an important role in the selective induction of cytochrome P4502E1 and the magnitude of induction by this compound (Park et al, 1993). As such one might expect hydrazine itself to induce the same enzyme.

The difference between the two pretreatments with regard to hydrazine cytotoxicity may be a result of inhibition of cytochromes P4502B1/2 and P4501A1, which have been shown to be detoxication pathways in vivo (Timbrell et al, 1982; Jenner & Timbrell, 1994b). In the present study hydrazine

67 pretreatment inhibited the activity of cytochrome P4502B1/2 by 16% whereas isoniazid pretreatment inhibited the activity by 50% (Table 3.1). Both pretreatments inhibited the activity of cytochrome P4501A1 equally (Table 3.1). If the former isoenzyme represents a major detoxication pathway of hydrazine inhibition of enzyme activity would be expected to result in greater toxicity, as is the case after isoniazid pretreatment.

3.5 CONCLUSION

In agreement with results obtained in vivo the induction of cytochromes P4502B1/2 and P4501A1 appear to be protective against hydrazine toxicity and thus catalyse detoxification pathways. According to the data obtained in the present study, cytochrome P4502E1 may also catalyse a detoxification pathway. However if cytochrome P4502E1 catalyses a "toxic pathway", as inferred by results obtained from in vivo studies (Jenner & Timbrell, 1994), the extent of induction of this enzyme in relation to the activities of the detoxification enzymes may determine the outcome. The data accrued to date regarding the role of cytochrome P4502E1 in hydrazine metabolism is confusing and as such its participation has not yet been elucidated.

As general inhibition of cytochrome P450 increased hydrazine cytotoxicity in isolated rat hepatocytes, the parent compound is either inherently toxic, is metabolised by other enzymes/enzyme systems, or is non-enzymically converted to a reactive intermediate which then causes damage. Overall the data infer that cytochromes P450 may be detoxication pathways.

68 THE EFFECT OF REPEATED EXPOSURE TO HYDRAZINE ON CYTOCHROME P450 AND LIVER TOXICITY/ivy/vo

4.1 INTRODUCTION

Repeat doses of hydrazine in vivo induce cytochrome P4502E1 (Akin & Norred, 1978; Jenner and Timbrell, 1994), an enzyme well characterised for metabolising small, polar compounds (Koop, 1992). The list of substrates for this enzyme is extensive and includes a range of structurally unrelated compounds such as isoniazid, benzene, nitrosamines, acetone, pyridine and halogenated hydrocarbons. Many of these substrates are also inducers (Raucy et al, 1993).

The physiological state of the animal can regulate the expression of this enzyme. For example in untreated diabetes and starvation cytochrome P4502E1 is elevated possibly in response to increased ketone levels, including acetone (Schenkman et al, 1989). There is also developmental regulation as seen in the neonatal rat, which has no detectable cytochrome P4502E1 until 2 days post-partum after which levels rise to a maximum after 14 days (Wu & Cederbaum, 1993).

The mechanisms of enzyme induction appear to be dependent on the inducer and the dosing regime employed. Acute doses of ethanol and acetone have been reported to increase the amount of cytochrome P4502E1 protein and enzyme activity without increases in mRNA suggesting stabilisation of the enzyme protein (Song et al, 1986). This occurs when a ligand binds and protects the protein from cAMP-dependent phosphorylation (Eliasson et al, 1990) and subsequent lysosomal degradation (Ronis & Ingelman-Sundberg, 1989). The pattern of induction of cytochrome P4502E1 by isoniazid was

69 similar to that seen above but was prevented by cyclobeximide, an inhibitor of protein synthesis, suggesting increased efficiency of mRNA translation (Parke et al, 1993). However induction of cytochrome P4502E1 with sulphur or nitrogen heterocyclic compounds was accompanied by decreased mRNA levels interpreted to be due to rapid utilisation and turnover of preexisting mRNA (Kim & Novak, 1993).

More recent studies have provided evidence of transcriptional activation of the cytochrome P4502E1 gene after exposure of cultured rabbit hepatocytes to acetone for 6-24 hours (Kraner et al, 1993). In rats receiving continuous intragastric infusion of ethanol, cyclical increases in blood alcohol levels were observed in concert with induction of cytochrome P4502E1. Levels of mRNA were increased, via gene transcription, only when blood alcohol concentration was high (Martin et al, 1993). The above findings suggest that at low inducer concentrations the prominent mechanisms of induction are protein stabilisation and/or increased efficiency of mRNA translation but at high concentrations gene transcription and/or stabilisation of mRNA and subsequent translation may be more important.

Oxidative metabolism of substrates by cytochrome P4502E1 gives rise to hydrogen peroxide and superoxide anions (Ekstrom & Ingelman-Sundberg, 1989) and possibly other reactive intermediates, as is the case with ethanol where carbon-centred radicals are generated (Albano et al, 1991). The addition of hydrazine to the list of substrates of cytochrome P4502E1 is premature but if hydrazine is metabolised by this enzyme there is the possibility of radical production and potential tissue damage. Therefore a study was carried out in which rats were subchronically exposed to a range of hydrazine doses in order to assess the magnitude of cytochrome P4502E1 induction and production of liver toxicity, if any.

Induction of cytochrome P4502E1 by acetone and isoniazid has been shown to increase the acute hepatotoxicity of hydrazine in vivo, increasing lipid accumulation without significant enhancement of ATP and GSH depletion compared to control animals (Jenner & Timbrell, 1994). Induction of this

70 isoenzyme has also been shown to increase the toxicity of other chemicals such as carbon tetrachloride (Lindros et al, 1990), acetaminophen (Koop et al, 1982), dimethylnitrosamine (Lauriault et al, 1992) and N-nitrosomethylaniline (Quan et al, 1992).

With respect to hydrazine, the information gained from in vitro and in vivo studies to date is conflicting with certain inducers of cytochrome P4502E1 increasing and others decreasing hydrazine toxicity (Jenner & Timbrell, 1994b; Delaney & Timbrell, 1995). Thus a role for this isoenzyme in hydrazine metabolism and toxicity has not yet been determined. In an attempt to resolve this, a study was undertaken in vivo in rats whereby cytochrome P4502E1 was induced by repeated administration of hydrazine and the toxicity of a subsequent acute dose of hydrazine assessed.

4.2 METHODS

4.2.1 Animal Husbandry

Rats were weighed, randomly allocated to treatment groups and housed individually in metabolism cages. Animals were acclimatised for 3 days to a 12 hour light/dark cycle and ambient room temperature of 20±3°C. During this time food and water were given ad libitum.

4.2.2 The Hepatic Effects of Subchronic Exposure to a Range of Hydrazine Doses: Study 1

Hydrazine, 2.5, 5 and lOmg/Kg, was administered in the drinking water for 10 days to those animals allocated to treatment groups. Dosing solutions were made fresh daily and adjusted according to the amount of water consumed in order to ensure accurate dosage. The average hydrazine intake for each dose group was calculated to be 2.3, 4.5 and 8.7 mg/Kg respectively. During this treatment period food and water were given ad libitum to dosed animals. As

71 hydrazine is known to reduce the appetite, each treated animal was assigned a pair-fed control which received the same amount of food as its dosed counterpart. These controls were allowed unlimited access to water.

Food and water intake was monitored on a daily basis. Animals were also observed daily for signs of distress or excessive weight loss. Any animal showing overt signs of toxicity was removed from the study immediately.

4.2.3 The Effect of Hepatic Cytochrome P4502E1 Induction on Acute Hydrazine Toxicity: Study 2

Rats were exposed to 2.5mg/Kg hydrazine in the drinking water for 10 days as described above. Each animal was assigned a pair-fed control. At the end of the pretreatment period half of the rats were given an acute dose of 30mg/Kg hydrazine i.p. and the rest received saline. Food was withdrawn immediately after dosing and all animals were culled 24 hours later.

At autopsy the liver was excised, weighed and examined for any abnormal features. Samples were then taken and processed for histology (light and electron microscopy), preparation of microsomes and biochemical determinations (ATP, GSH, GSSG and triglycerides).

4.2.4 Light Microscopy

A shce (approximately 1cm thick) of the median lobe of the liver was placed in 10.5% formaldehyde for fixation. After the appropriate fixation time samples were either frozen and sectioned on a cryostat for subsequent staining with Oil Red O (determination of triglycerides) or were dehydrated and embedded in wax for staining with haematoxylin and eosin (determination of cellular/tissue damage). These samples were processed by Glaxo Research and Development, Ware, Herts.

72 Wax embedded samples were also sent to the Armstrong Laboratory (Wright- Patterson Air Force Base, Ohio, USA) for localisation of cytochrome P4502E1 using an immunohistochemical staining technique. The assay was performed on a fully automated Techmate 1000 using Biotek solutions (Santa Barbara, Ca).

4.2.5 Electron Microscopy

Liver taken at autopsy was cut into small cubes (1mm) and fixed in 2.5% glutaraldehyde in O.IM cacodylate buffer for 24 hours at 4°C. The samples were then washed in 0. IM cacodylate buffer for 30 minutes then counterfixed in osmium tetroxide (0.5%) for 2-4 hours at 4°C. After fixation the samples were dehydrated with increasing concentrations of alcohol (70% methanol for 30 minutes; 100% methanol (x3) for 40 minutes; 100% acetone (x2) for 15 minutes) and then immersed in resin (CY212 20ml: DDSA 20ml: BDMA 0.8ml) and left overnight at room temperature to allow infiltration. Finally selected blocks were tranferred into moulds containing fresh resin which was polymerised by heating at 60°C for 48 hours.

Sections (60-80nm, silver interference colour) were cut on an ultramicrotome using a diamond knife and subsequently stained with uranyl acetate (nucleic acid and protein) and lead citrate (cellular and tissue compounds) for examination on a Phillips 201 transmission electron microscope.

4.2.6 Statistical Analysis

All data were expressed as mean ± SD. The number of animals per treatment group was 4 unless otherwise stated. Statistical significance was determined using Student’s (unpaired) t-Test.

73 4.3 RESULTS

4.3.1 The Hepatic Effects of Subchronic Exposure to a Range of Hydrazine Doses: Study 1

Body weight gain in the control animals closely paralleled that of their dosed partners as a result of pair feeding (Figure 4.1). Exposure to the lowest dose of hydrazine had no apparent effect on appetite however body weight gain was reduced immediately after the onset of exposure to the higher doses. This was especially apparent in the high dose group where no weight gain was seen for four days, after which only small increases occurred (Figure 4.1). This slowing of growth in dosed animals occurred concurrently with reduced food and water intake (Figures 4.2 and 4.3). At the end of the pretreatment period there was an apparent dose related weight loss, although this was only significantly reflected in the pair-fed controls (Table 4.1).

When comparing absolute liver weight no difference could be found between dosed animals and their pair-fed controls (Table 4.1). However when expressed as a percent of body weight, the livers taken from animals dosed with 5 and 10 mg/Kg hydrazine were larger than those taken from the relevant controls, although this was only significant in the former group (Table 4.1).

When making intergroup comparisons it was found that the livers from the animals in the lOmg/Kg group (dosed and controls) were smaller than those from the other dose groups (Table 4.1). But when expressed as percentage liver:body weight only the dosed animals which received 5mg/Kg showed a dose related increase compared to those from 2.5mg/Kg group (Table 4.1).

The effects of repeated exposure to hydrazine on microsomal enzyme activities are illustrated in Table 4.2. Total cytochrome P450 levels were unaffected by all treatments but there was a dose dependent increase in p-nitrophenol hydroxylase activity. Subjective viewing of immunohistological slides suggested that there was no dose-related increase in P4502E1 protein, which was localised around the central veins of the liver (Photomicrograph 4.1).

74 Photomicrograph 4.1 Distribution of Cytochrome P4502E1 within the Liver Lobule

Immunohistochemical staining of liver taken from a control rat, showing pericentral localisation of cytochrome P4502E1. Magnification xlO; CV = central vein; PT = portal tract.

Figure 4.1 The Effect of Repeated Exposure to a Range of Hydrazine Doses on Body Weight in Rats

Hydrazine: 300 2.5mg/Kg ^ 275 5mg/Kg • ^ lOmg/Kg ■ ^ 250 o> 'o> 225

•O>. o 200 CO

175

150 3 -2 -1 0 1 2 3 4 5 6 7 8 9 10

Days of Treatment

Male Sprague Dawley rats exposed to 2.5, 5 and lOmg/Kg hydrazine in the drinking water for 10 days. Control rats were given the same amount of food as dosed counterparts; open symbols. Values are means ± SD; n = 4, except in the highest dose group (and controls) where n=3.

75 Figure 4.2 The Effect of Repeated Exposure to a Range of Hydrazine Doses on Food Intake in Rats

Hydrazine: 30 2.5mg/Kg ^ 5mg/Kg • lOmg/Kg ■ cn

® 20 (0 c T3 O O 11 - Dose

2 -1 01 2 3 4 5 6 7 8 9 10

Days of Treatment

Figure 4.3 The Effect of Repeated Exposure to a Range of Hydrazine Doses on Water Intake in Rats

Hydrazine: 50 - 2.5mg/Kg ^ 45 - 5mg/Kg • 40 - lOmg/Kg ■ E 35 - 30 - io S 25 - 0) 20 - ra 15 - b b * * 10 - b«-aa b * * * aa 5 -

0 - I 1------1------1------1------1------1------1------1------1------1 I I -2 -1 01 23456789 10

Days of Treatment

Male Sprague Dawley rats exposed to 2.5, 5 and lOmg/Kg hydrazine in the drinking water for 10 days. Control rats were given the same amount of food as dosed counterparts; thus data not shown in Figure 4.2; open symbols in Figure 4.3. Values are means ± SD; n = 4, except in the highest dose group (and controls) where n=3; *p<0.05, **p<0.01, ***p<0.001 compared to 2.5mg/Kg dosed; “p<0.05, “ p<0.01, ““p<0.001 compared to 5mg/Kg dosed; ^p<0.05, %<0.01, ^^^p<0.001 compared to own control.

76 Table 4.1 The Effect of Repeated Exposure to 2.5, 5 or lOmg/Kg Hydrazine on Body and Liver Weight

Treatment Body Weight (g) Liver Weight (g) LiveriBody Weight (%)

Control (4) 270±12 11.09±1.43 4.1±0.4 2.5mg/Kg Hydrazine (4) 254±28 10.32±1.40 4.0±0.1

Control (4) 245±14* 9.67±0.67 3.9±0.1 5mg/Kg Hydrazine (4) 246±17 10.54±0.66 4.3±0.2'=

Control (3) 225±13" 8.31±0.09*^ 3.7±0.2 lOmg/Kg Hydrazine (3) 221±10 9.08±0.83^ 4.1±0.2

Statistical analysis using Student’s t-test. Data expressed as mean ± S.D.; ( ) number of animals/group * p<0.05; ** p<0.01 compared to Control (2.5mg/Kg) ® p<0.05 compared to Control (5mg/Kg) ^ p<0.05 compared to Dosed (5mg/Kg) Table 4.2 The Effect of Repeated Exposure to 2.5, 5 and lOmg/Kg Hydrazine on Hepatic Microsomal Enzyme Activities

TREATMENT Total P450 p-Nitrophenol EROD PROD (nmol/mg protein) Hydroxylase (pmol/min/mg (pmol/min/mg (nmol/min/mg protein) protein) protein)

CONTROL (4) 0.85±0.11 0.37±0.11 209±18 39±9 2.5mg/Kg/Day (4) 0.95±0.17 0.90±0.06" 235±33 48±5

00 CONTROL (4) 0.59±0.13 0.39±0.19 204±4 61±5 5mg/Kg/Day (4) 0.74±0.06 1.28±0.36"' 163±43 52±10

CONTROL (3) 0.79±0.21 0.39±0.09 224±37 72±14 lOmg/Kg/Day (3) 0.81±0.16 1.97±0.67"* 147±7 ' 48±4 *

Data expressed as mean ± S.D.; ( ) number of animals/group Statistical analysis using Student’s t-test. * p<0.05; ** p<0.01; *** p<0.001 compared to relevant control Liver toxicity was assessed by measuring ATP, GSH, GSSG (oxidised glutathione) and triglycerides. Depletion of GSH and accununulation of GSSG appeared to be dose dependent. Liver levels of GSH remained constant in controls but were significantly lower in animals that received 5 and lOmg/Kg compared to those that received 2.5mg/Kg (Figures 4.4). The reverse was observed for GSSG (Figure 4.5). Changes in both of these parameters correlated with p-nitrophenol hydroxylase activity (GSH r=-0.636, p=0.001; GSSG r=0.662, p<0.001).

There was no dose related effect on ATP, but some depletion was was evident after exposure to 5mg/Kg hydrazine only (Figure 4.6). The apparent dose related effect on liver lipids was not due to hydrazine as the triglyceride content in the livers of treated and control animals were very similar (Figure 4.7). However there was a significant correlation between liver lipid content and food intake (r=0.724; p<0.001) with the animals eating the least having lower triglycerides. There was no correlation between liver lipids and microsomal enzyme activity (data not shown).

Examination of histological shdes stained with haematoxylin and eosin (H-i-E) revealed no treatment related pathological changes (Photomicrographs 4.2a/b). Glycogen was apparent in midzonal to periportal areas and appeared to be related to dietary intake, the lower the food intake the greater the storage.

Examination of tissue sections under the electron microscope revealed that livers taken from dosed rats showed an apparent increase in the number of mitochondria (not quantitated) when compared to controls. These organelles were highly active as illustrated by the clarity of the internal membranes (cristae) (Electronmicrographs 4.1a/b). Smooth endoplasmic reticulum did not appear to be proliferated as one might expect from a cytochrome P450 inducing agent (Electronmicrographs 4.1a/b).

79 Figure 4.4 The Effect of Repeated Exposure to a Range of Hydrazine Doses on Hepatic GSH

Control 7.0 -1 Dosed 6.0 -

«> _> 5.0 -

O) 4.0 - "5 E 3 . 3.0 - X ifi 2.0 - 0 1.0 -

0 -

Figure 4.5 The Effect of Repeated Exposure to a Range of Hydrazine Doses on Hepatic GSSG

360 1 Control 1 _l 330 - Dosed H i 300 - 0) 270 - > — 240 - 0» 210 - "o E 180 - c 150 - 0 120 - CO CO 90 - 0 60 - 30 - 0 -

Figures 4.4 and 4.5 illustrate the effects of exposure of male Sprague Dawley rats to 2.5, 5 and 10 mg/Kg hydrazine in the drinking water for 10 days. Values are means ± SD; n = 4, except in the highest dose group (and controls) where n=3; “®p<0.01 compared to own pair-fed control; *p<0.05, **p<0.01 compared to 2.5mg/Kg dosed.

80 Figure 4.6 The Effect of Repeated Exposure to a Range of Hydrazine Doses on Hepatic ATP

2.0 1 Control CZU Dosed ■ ■ 1,8 - 1,6 - o 1,4 - O) 1,2 - o 1,0 - E E 0,8 - CL 0,6 - h- < 0,4 - 0,2 - 0 -

Figure 4.7 The Effect of Repeated Exposure to a Range of Hydrazine Doses on Hepatic Triglycerides

Control □ □ Dosed mm

2.5 5 10

Figures 4,6 and 4,7 illustrate the effects of exposure of male Sprague Dawley rats to 2,5, 5 and 10 mg/Kg hydrazine in the drinking water for 10 days. Values are means ± SD; n = 4, except in the highest dose group (and controls) where n=3; ®p<0,05 compared to own pair-fed control; *p<0.05, *"p<0.01 compared to pair-fed controls of 2.5mg/Kg dose group.

81 H+E Staining of Liver Slices Taken From a Control and Hydrazine Treated Rat

Photomicrograph 4.2a

Photomicrograph 4.2b

o CV

a) pair-fed control rat; b) rats dosed with lOmg/Kg hydrazine/day for 10 days in the drinking water; Magnification xlO; G = glycogen, MZ = midzonal area, CV = central vein, PT = portal tract.

82 Transmission Electron Micrographs of Liver Taken From Control and Dosed Rats

Electrorunicrograph 4.1a

Electronmicrograph 4.1b

a) pair-fed control rat; b) rats dosed with lOmg/Kg hydrazine/day for 10 days in the drinking water; Magnification xll,200; M = mitochondion; ER = endoplasmic reticulum; N = nucleus; L = lysosome; BC = bile canaliculus. I = inclusion in mitochondrion

83 4.3.2 The Effect of Induction of Cytochrome P4502E1 on Acute Hydrazine Toxicity: Study 2

The second study involved pretreatment of rats with 2.5mg/Kg hydrazine for 10 days followed by an acute dose of 30mg/Kg hydrazine (threshold dose in vivo, Timbrell et at, 1982). These doses of hydrazine were chosen so that a direct comparison with in vitro data (Chapter 3) could be made.

As expected from Study 1 (Table 4.2), pretreatment of rats with 2.5mg/Kg hydrazine did not alter total cytochrome P450 content or the activities of EROD and PROD compared to pair-fed controls (Table 4.3). Unlike the previous study, however, in which p-nitrophenol hydroxylase activity was doubled after pretreatment with 2.5mg/Kg hydrazine (Table 4.2), the pretreatment had no significant effect in this study (Table 4.3). This discrepancy may be due to the 24 hour starvation period endured by all rats at the end of this second study. This may also explain the fact that the activity of p-nitrophenol hydroxylase in the control livers from rats in Study 2 was approximately 3-fold higher than in the controls firom Study 1 (Tables 4.2 and 4.3), as starvation is known to induce this enzyme.

An acute dose of hydrazine did not alter total cytochrome P450 content or PROD activity in control or pretreated rats but reduced EROD activity equally in both groups (Table 4.3). Acute hydrazine dosing induced a small but insignificant rise in p-nitrophenol hydroxylase activity in control and pretreated rats (Table 4.3).

In agreement with the data obtained in study 1, there was no pre treatment effect on body or liver weight (data not shown). Acute dosing with hydrazine, however, caused an increase in liver weight, when expressed in absolute (data not shown) or relative terms (Figure 4.8).

84 Table 4.3 The Effect of an Acute Dose of Hydrazine on Microsomal Enzyme Activities in Control and Hydrazine Pretreated Rats

TREATMENT Total P450 p-Nitrophenol EROD PROD (nmol/mg protein) Hydroxylase (pmoPmin/mg (pmoPmin/mg (nmoPmin/mg protein) protein) protein)

CONTROL (4) 0.88±0.09 0.96±0.22 227±58 45±12 00 cn 4-30mg/Kg ACUTE (4) 0.70±0.14 1.52±0.33 140±19" 36±7

2.5mg/Kg/Day (4) 0.77±0.07 1.44±0.11 212±46 50±14 -k 30mg/Kg ACUTE (4) 0.74±0.10 1.91±0.48 122±20" 32±3

Data expressed as mean ± S.D.; () number of animals/group Statistical significance: * p<0.05; ** p<0.01 using Student’s t-test Hydrazine pretreatment, again as expected, failed to alter liver levels of triglycerides (Figure 4.9), ATP, GSH and GSSG (Table 4.4). When measured 24 hours post dose, there was no significant alteration in the levels of ATP, GSH or GSSG in the livers of animals that received 30mg/Kg hydrazine (Table 4.4). The only manifestation of acute toxicity still evident at this time was a 4-fold increase in liver triglycerides in both control and pretreated animals (Figure 4.9). The fatty liver induced by acute hydrazine exposure is illustrated by Oil red O staining of tissue slices (photomicrographs 4.3a/b).

Table 4.4 The Influence of Hydrazine Pretreatment on the Biochemical Effects of a Subsequent Acute Dose of Hydrazine

TREATMENT ATP (pmol/g GSH (pmol/g GSSG (nmol/g liver) liver) liver)

CONTROL (4) 0.81±0.11 5.45±0.92 61.48±16.50 +30mg/Kg 0.68±0.13 4.30±0.70 44.69±15.95 ACU TE (4)

2.5mg/Kg/Day (4) 0.75±0.20 5.51±0.77 69.72±20.91 4-30mg/Kg 0.62±0.09 4.22±0.80 50.81±18.63 ACU TE (4)

Data expressed as mean ± S.D.; () number of rats/treatment group

When attempting to associate certain acute toxicological changes with enzyme activity it was found that there were no correlations with p-nitrophenol hydroxylase activity. On the other hand liver triglycerides (r=-0.733; p=0.001), ATP (r=0.571; p=0.021) and GSH (r=0.663; p=0.005) levels correlated with EROD activity and triglycerides (r=-0.641; p=0.007) and ATP (r=0.572; p=0.020) with PROD activity. In each case the lower the enzyme activity the greater the toxicity and vice versa.

86 Figure 4.8 The Effect of Repeated and Acute Exposure to Hydrazine on Relative Liver Weight

Control I— I 5.0 Acute Dosed ■■ 4.5 « 4.0 3.5 2» 5 3.0 >* 2.5 ■o o 2.0 m L!a> 1.5 > 1.0 0.5 0 C OH H HH

Figure 4.9 The Effect of Repeated and Acute Exposure to Hydrazine on Hepatic Triglycerides

Control I I 120 1 Acute Dose ■■ 0) 100 >

o> 80 - CD E M 60 - 0> 12 oV 40 - O) Z 20 H X

C CH H HH

Figures 4.8 and 4.9 illustrate the effects of exposure of male Sprague Dawley rats to 2.5mg/Kg hydrazine in the drinking water for 10 days followed by an acute dose of 30mg/Kg hydrazine (ip). C = control; H = hydrazine pretreatment; CH = acute dose of hydrazine; HH = hydrazine pretreatment followed by acute dose. Values are means ± SD; n = 4; *p<0.05, ***p<0.01 compared to own control.

87 The Effect of an Acute Dose of Hydrazine on Liver Lipids Illustrated by Oil Red O Staining of Liver Sections

Photomicrograph 4.3a i' y -, - '

' ' ' - • ^ . V ' iviz > " » ... < • ^LD

' ■* ' * .

cv » * . V 4

Photomicrograph 4.3b

- , - cv

p . ; n - ' r ' y - . - ■- ^r. " '-L- ‘ . ^

. « a # ' : ; ; ' « , \r

Liver taken 24 hours after dosing control rats with a) saline (ip); b) 30mg/Kg hydrazine (ip). Magnification xlO; LD = lipid droplets; CV = central vein; P = portal artery; MZ = midzonal area.

88 4.4 DISCUSSION

Chronic dosing with hydrazine reduced both food and water intake in animals receiving 5 and lOmg/Kg/day (Figures 4.2/3), resulting in an apparent dose dependent weight loss (Table 4.1). In anticipation of such an effect control animals were pair fed to ensure that any toxicity seen was due to hydrazine alone.

There was an increase in liver weight only in animals that received the middle dose of hydrazine (Table 4.1). Acute dosing with hydrazine is well documented to increase liver size as a result of fat accumulation (Amenta & Dominguez, 1965; Clark et al, 1970; Timbrell et al, 1982) however repeated exposure had no such effect on hepatic triglycerides, in fact a reduction was noted (Figure 4.7). Adaptive changes in response to xenobiotic insult, such as enzyme induction and proliferation of organelles, can cause liver enlargement. Hepatocyte hypertrophy can occur as a result of proliferation of the smooth endoplasmic reticulum (SER) in response to chemical inducers of cytochrome P450, such as phenobarbitone. In the present study total cytochrome P450 content was unaffected by subchronic exposure to the doses of hydrazine employed (Table 4.2) and the amount of SER appeared to be similar to controls (Electronmicrographs 4.1a/b).

Hydrazine-induced mitochondrial effects are well documented (Scales & Timbrell, 1982; Wakabayashi et al, 1987) and may be responsible for the liver enlargement observed in the animals repeatedly dosed with 5mg/Kg hydrazine. Damage to mitochondria or disruption of normal function may account for the diminished hepatic ATP content in these animals (Figure 4.6). Indeed many of the mitochondria in electronmicrograph (4.1b) exhibit disrupted cristae, which is well characterised after hydrazine administration (Scales & Timbrell, 1982), and others contain inclusions, both of which are indicative of damage.

Subchronic exposure to the doses of hydrazine used in this study did not produce any overt hepatic toxicity. Histologically there were no treatment related lesions (Photomicrographs 4.2a/b). Induction of cytochrome P4502E1

89 is often associated with centrilobular fibrosis and/or necrosis as a result of free radical damage (Lindros et al, 1990; French et al, 1993). Necrotic lesions caused by exposure to hydrazine are extremely rare but have been observed in primates (Patrick & Back, 1965), which are much more sensitive to hydrazine than the rat. In order to produce these lesions in the rat a longer exposure period may have been necessary.

As previously stated, the concentration of hepatic ATP in rats dosed with 5mg/Kg was reduced compared to the pair-fed controls whereas ATP in animals from the other dose groups was similar to that seen in pair-fed controls (Figure 4.6). The lack of effect on this parameter by the highest dose may be related to the reduced food intake in these animals as there was also a small but insignificant drop in hepatic ATP in the pair-fed controls (Figure 4.6).

Subchronic exposure to hydrazine did not change the hepatic content of total cytochrome P450 (Table 4.2). There was a dose-dependent increase in the activity of p-nitrophenol hydroxylase but a reduction in the activities of EROD and PROD (Table 4.2). A similar pattern of induction is seen after pretreatment with isoniazid, a hydrazine derivative, which also induces cytochrome P4502E1 without altering total P450 content (Rice & Talcott, 1979; Ryan et al, 1985).

The increase in p-nitrophenol hydroxylase activity, representative of induction of cytochrome P4502E1, has been previously documented with respect to hydrazine (Jenner & Timbrell, 1994a). Induction of this isoenzyme by 4/5-fold is usually a result of post-translational regulation of enzyme protein whereas higher levels of induction usually involve gene transcription (Longo & Ingelman-Sundberg, 1993). As the highest dose of hydrazine in the present study caused a 4-fold induction of p-nitrophenol hydroxylase activity (Table 4.2) the former mechanism is more likely. F urther work is required to confirm this.

Cytochrome P4502E1 is distributed throughout centrilobular regions of liver (Forkert et al, 1991) and although there was positive immunostaining for cytochrome P4502E1 protein in this region (Photomicrograph 4.1), following

90 hydrazine administration there was apparently no increase in protein concentration (data not shown). This was the result of a subjective examination of histological slides (Appendix IX) but should this be correct, hydrazine may be inducing other isoenzymes of cytochrome P450 which also exhibit p-nitrophenol hydroxylase activity. However there are two lines of evidence which may support induction of cytochrome P4502E1 by hydrazine: 1) the specificity of cytochrome P4502E1 induction by isoniazid is known to be dependent on the hydrazine side-chain (Parke et al, 1993); 2) this particular isoenzyme is known to metabolise akylhydrazines (Albano et al, 1995). One way to verify this would be to carry out Western blots of microsomal samples taken from hydrazine treated rats. This would give a true picture of the inductive pattern of hydrazine.

Depletion of hepatic ATP by subchronic exposure to hydrazine did not correlate with increased cytochrome P4502E1 activity suggesting that alterations to these biochemical parameters by hydrazine may occur independently. However the dose dependent depletion of GSH and concomitant elevation of GSSG, indicative of oxidative stress, was highly correlated to p-nitrophenol hydroxylase activity (both p<0.001). It must be noted that the elevation of GSSG is not equal to the depletion of GSH (Figures 4.4 & 4.5). Two possible explanations for this are a) the rapid excretion of GSSG in bile (Eklow et al, 1981; 1984), and b) reduced GSH synthesis in concert with some oxidation to GSSG.

Oxidative stress has not been verified with respect to hydrazine. Although it has been established that hydrazine is metabolised to hydrazine radicals by microsomes prepared from phénobarbital treated rats (Noda et al, 1985), there is no evidence that metabolism of hydrazine by microsomes prepared from cytochrome P4502E 1-induced rats generates radicals. Also, based on acute administration, hydrazine has not been proven to cause lipid peroxidation (DiLuzio et al, 1973; DiLuzio & Stege, 1977; Preece & Timbrell, 1989).

However oxidative stress is well characterised to occur during cytochrome P4502E 1-mediated metabolism of substrates, which generates both oxygen and

91 compound related radicals (Koop, 1992; Albano et al, 1991; Raucy et al, 1989; Johansen & Ingelman-Sundberg, 1985). Indeed cytochrome P4502E1 can catalyse NADPH-dependent reduction of molecular oxygen in the presence or absence of substrates ultimately giving rise to peroxide (Koop, 1992). Thus induction of this enzyme alone, with or without hydrazine as a substrate, may be sufficient to cause oxidative stress in these animals.

The observation that subchronic exposure to hydrazine causes oxidative stress, possibly as a result of cytochrome P4502E1 induction, may suggest that hydrazine is a substrate for this isoenzyme. If this were the case one might expect stimulated cytochrome P4502E1 activity to influence the toxicity of a subsequent acute dose of hydrazine. Unfortunately induction of cytochrome P4502E1 by pretreatment of rats with 2.5mg/Kg hydrazine for 10 days had little effect. There are several possibilities for this: a) The magnitude of induction was not sufficient to have any effect. This is highly likely as in this particular study the increase in p-nitrophenol hydroxylase activity was not significantly greater than the control (Table 4.3). b) The time at which toxicity was examined (24 hours post dose) was too late, allowing recovery of certain parameters such as ATP and GSH which may have been depleted hours earlier. Again this is highly plausible as although ATP and GSH levels were not significantly different from the controls at this time the mean values were lower. c) Cytochrome P4502E1 does not metabolise hydrazine. Chemical toxins that are substrates for cytochrome P4502E1 tend to predispose the centrilobular (perivenous) region of the liver to toxicity (Lindros et al, 1990; Koop, 1992; Raucy et al, 1993) but the only notable lesion in animals given an acute dose of hydrazine was the accumulation of lipid in midzonal and/or periportal regions (Photomicrographs 4.5a/b). This observation cannot be used as evidence against hydrazine being a substrate for cytochrome P4502E1 as the production of fatty liver by ethanol and acetone is periportal and is unrelated to the activity/induction state of this isoenzyme (Forkert et al, 1991; Leiber et

92 aly 1988). Although, to my knowledge, no studies have been performed to determine which cell types are most affected with respect to hydrazine-induced ATP and GSH depletion, one might find that perivenous hepatocytes are more prone to toxicity.

The fact that none of the biochemical parameters measured in relation to acute toxicity correlated with p-nitrophenol hydroxylase activity may suggest that hydrazine itself, or a metabolite generated by a different metabolic pathway was responsible. A repeat of the study but with greater induction of the enzyme and assessment of toxicity at an earlier time point may provide more information.

The activities of EROD and PROD were reduced by both repeated and acute exposure to hydrazine (Table 4.2/3). Significant correlations between the levels of ATP, GSH and triglyceride and EROD activity were obtained in the latter study. The activity of PROD was also correlated with ATP and triglyceride levels. The animals showing the lowest activities manifested greater toxicity and vice versa. This data at least is in agreement with the in vitro data presented earlier and with other published in vivo work (Jenner & Timbrell, 1994a) which suggests that cytochromes P4502B1/2 and P4501A1/2 detoxify hydrazine.

4.5 CONCLUSIONS

A role for cytochrome P4502E1 in hydrazine metabolism has still not been unequivocally established. Chronic treatment with hydrazine induced cytochrome P4502E1 in a dose-dependent manner. Animals treated with the highest dose of hydrazine and thus exhibiting the greatest increase in p- nitrophenol hydroxylase activity also showed signs of oxidative stress. This may indicate that cytochrome P4502E1 does indeed metabolise hydrazine, with the generation of free radicals, but that this only becomes important, in relation to toxicity, with high enzyme activity. Indeed a small induction of cytochrome P4502E1 was not sufficient to modulate the toxicity of a

93 subsequent hydrazine dose.

It was observed however that alterations in the levels of ATP, GSH and triglycerides after acute hydrazine exposure correlated with the activities of cytochromes P4501A1/2 and P4502B1/2 suggesting that these are detoxication pathways. This is in agreement with in vitro data presented in the previous chapter.

94 THE EFFECT OF HYDRAZINE ON HEPATIC PROTEIN SYNTHESIS IN VIVO AND IN VITRO

5.1 INTRODUCTION

Protein synthesis is a very complicated series of events beginning with transcription of single stranded DNA and the subsequent translocation of the resultant messenger RNA (mRNA) from the nucleus into the cytoplasm. It is here that the mRNA associates with ribosomes and the protein sequence is translated by transfer RNA (tRNA). Translation is by far the most complicated process and comprises three distinct phases, a) initiation, b) elongation and c) termination of the peptide chain.

There are numerous stages at which protein synthesis can be regulated/ modulated by endogenous and exogenous compounds. For example, initiation of protein synthesis, which starts with the formation of a ternary complex consisting of initiator tRNA, eukaryotic initiation factor (eIF-2) and guanine triphosphate (GTP) (Pain, 1986), is inhibited by calcium-mobilising hormones, such as adrenalin and vasopressin, and compounds such as ethionine by inactivation of eIF-2 (Kimball and Jefferson, 1990; Brostrom et nZ, 1985; Lyon and Kisilevsky, 1990). Cycloheximide inhibits initiation and elongation of the peptide chain by preventing translocation and binding of aminoacyl-tRNA to the P-site of the ribosome (Obrig et al, 1971). Puromycin, due to close structural similarity to aminoacyl-tRNA, causes premature termination of the peptide chain (Nathans, 1964).

Hydrazine is a compound which has been reported by some authors to stimulate protein synthesis in vivo (Amenta & Johnston, 1963; Banks, 1970) and by others to inhibit it (Lopez-Mendoza & Villa-Trevino, 1971). Although the mode of action has not been fully identified, stimulation of protein synthesis in vivo was thought to result from an early elevation of hepatic DNA

95 (Banks et al, 1967). No explanation was offered with respect to protein synthesis inhibition. Hydrazine also inhibited protein synthesis in vitro in cultured rat hepatocytes (Ghatineh & Timbrell, 1990).

The discrepancy between these data is as yet unexplained. For this reason a study was undertaken in an attempt to clarify the effect of hydrazine on protein synthesis using both in vivo and in vitro systems.

In the liver there are several classes of cellular protein: a) long-lived proteins which constitute the majority of cellular proteins and which have a half-life of up to 40 hours; b) short-lived secretory proteins which have a half-life of approximately 10 minutes (Solheim and Seglen, 1980); and c) abnormal proteins which are broken down even more rapidly them secretory proteins (Hershko and Ciechanover, 1982).

There are several methods to measure protein synthesis in vivo. In the present study the "flooding dose" technique was utilised which allows the assessment of both short- and long-lived proteins. Radiolabel (20-50pCi) is administered in a solution containing a high concentration of "cold" amino acid, which ensures rapid distribution and equilibration of radiolabel in tissues. Incorporation of radiolabel into protein is then monitored after approximately 10-15 minutes (Hasselgren et al, 1988). However estimation of protein synthesis within this time frame is heavily influenced by the synthesis of short term proteins and may imply an overall synthetic rate which is falsely high (Hasselgren et al, 1988).

A similar protocol was followed to measure protein synthesis in vitro. Hepatocytes incubated in an unsupplemented medium are in a state of high protein turnover where degradation exceeds synthesis by approximately 10-fold (Seglen, 1977). A flooding dose of ^H-leucine thus combats the effects of isotope dilution, which occurs as proteins are degraded (Seglen, 1976).

96 5.2 METHODS

5.2.1 The Effect of Hydrazine on Protein Synthesis in Isolated Rat Hepatocytes In Vitro

Hepatocytes were isolated by collagenase perfusion as previously described in Chapter 2. The stock suspension was diluted with K+H buffer to give a cell density of IxlOVml. Protein synthesis in these hepatocytes was carried out using the method of Seglen (1976).

An aliquot ( 10ml) of this suspension was preincubated at 37°C for 30 minutes after which radiolabelled ^H-leucine ( 1ml; lpCi/10® cells in 5mM "cold" leucine) was added. This was gently mixed by swirling and immediately a sample ( 1ml) was taken and centrifuged to pellet the cells. The supernatant was retained for measurement of LDH leakage. The pellet was resuspended in 10% TCA (1ml) for later analysis of incorporation of radiolabel into cellular protein. Samples were also taken for ATP and GSH estimations.

The remaining suspension was then dosed with a range of hydrazine concentrations and incubated [37°C, 95% 0/5% COg] for up to 3 hours in amino acid unsupplemented K+H buffer. Samples were taken at hourly intervals and processed as above.

On the day of analysis the TCA samples were thawed, centrifuged and the acid aspirated. The pellet was washed (x5) by resuspending in fresh ice-cold aliquots of TCA (1ml). After the final wash the pellet was resuspended in IM NaOH (1ml). Aliquots (400pl, duplicates) of this protein digest were then pipetted into a vial with scintillant (4ml), the samples mixed and subsequently counted for 15 minutes each on a Beckman Scintillation Counter.

97 5.2.2 The Effect of Hydrazine on Hepatic Protein Synthesis in the Rat In Vivo

5.2.2.1 Animal Husbandry

Animals were randomly allocated to treatment groups and housed 4/cage. Food and w ater was given ad libitum. After acclimatisation for 3 days in a light and temperature controlled room the animals were dosed as follows. Food was withdrawn after dosing but free access to water was allowed.

5.2.2.2 Dose Response Experiment

Rats (4/group) received saline (controls), 10, 30 or 60mg/Kg body weight hydrazine free base {ip) and immediately after 50pCi ^H-leucine. After 3 hours the animals were sacrificed and liver and blood taken for analysis.

5.2.2.3 Time Course Experiment

Rats (4/group) received saline (controls) or 60mg/Kg body weight hydrazine free base {ip) and were sacrificed 0.5, 1.5, 6, 12 or 24 hours after the initial injections. The dosing regime did not allow administration of radiolabel 10 minutes prior to death, the time course over which incorporation is usually assessed using this technique, instead animals were given a dose of 25pCi leucine 1.5 hours prior to death except those animals in the 0.5 hour dose group which received the radiolabel immediately after hydrazine. This may be advantageous as synthesis of long-term proteins may be more accurately represented.

98 5.2.S Estimation of Serum Protein Synthesis

Absolute ethanol ( 1ml) was added to an aliquot of serum (0.3ml) to precipitate protein which was pelleted by centrifugation (2,500 rpm for 5 minutes) and washed (x3) by resuspension in 75% EtOH (4ml) to remove any unbound leucine.

The final wash was decanted and the pellet resuspended in IM NaOH (2ml). Aliquots (200pl, duplicates) of the digest were pipetted into scintillation vials with scintillant (4ml). The samples were vortexed thoroughly and left in the dark for 24 hours to allow any chemiluminescence to dissipate. The samples were then counted for 15 minutes each, reading against a NaOH blank.

5.2.4 Estimation of Hepatic Protein Synthesis

5.2.4.1 Acid (TCA) soluble proteins:

These proteins are reputed to be short-lived secretory proteins (tV4 10 minutes or possibly less) newly synthesised in the hepatocyte (Solheim and Seglen, 1980), abnormal proteins destined for degradation or remnants of protein degradation (Hershko & Ciechanover, 1982).

The procedure for monitoring synthesis of these proteins was the same as described above for serum proteins. A known amount of liver (approximately Ig) was homogenised in 10% TCA (4ml) and the samples centrifuged (2,500rpm for 5 minutes). An aliquot of the acid supernatant was taken and absolute ethanol added (2.5ml:7.5ml respectively).

The samples were then spun and the pellet washed (x3, 4ml 75% ethanol). The final wash was decanted and the pellet (very fragile) resuspended in IM NaOH (1ml). Aliquots of the digest (400pl, duplicates) were pipetted into scintillation vials and counted.

99 5.2.4.2 Acid (TCA) precipitable proteins:

These proteins constitute the bulk of hepatic protein and are long-lived {W2 up to 40 hours) (Hershko & Ciechanover, 1982).

Liver (approximately 300mg) was homogenised into 10% TCA ( 1ml), the samples spun (2,500rpm, 5 minutes) and the supernatant decanted. The pellet was washed (x5) with 10% TCA (2ml). The washed pellet was then resuspended in IM NaOH (5ml) and aliqouts (200pl, duplicates) counted as described above.

The remainder of the NaOH digests from all of the above samples were appropriately diluted with distilled water and analysed for protein content using the method of Lowry (1951).

5.2.5 Serum Ammonia

Measurement of this parameter was carried out using a SIGMA diagnostic kit (catalogue number 171-A). The principle of the method is illustrated below:

2-oxoglutarate + NH 3 + NADPH glutamate + NADP

The reaction is catalysed by glutamate dehydrogenase. The decrease in absorbance at 340nm, due to oxidation of NADPH, is proportional to ammonia concentration.

Serum ( 0 .1ml) or ammonia solution (5pg/ml; 0.1ml) were added to buffer ( 1ml; containing 3.4mM 2-oxoglurarate and 0.23mM NADPH) in a cuvette and mixed. In the blank water replaced serum. The samples were equilibrated at 30°C for 3 minutes and the initial absorbance read, versus water, at 340nm. The reaction was then initiated by addition of glutamate dehydrogenase solution (1200U/ml; lOpl) and the final absorbance noted 5 minutes later. Serum ammonia was calculated from the absorbance change.

1 0 0 NB. Hydrazine forms a hydrazone with 2-oxoglutarate thus potentially depleting levels of this substrate. At the concentrations of hydrazine expected in the serum after dosing with 60mg/Kg {ip) there is no interference with the assay.

5.2.6 Preparation of samples for total RNA and DNA content

Liver samples were prepared using the method of Choo and coworkers (1992).

Liver (300mg) was homogenised in 2% perchloric acid (PCA, 10ml). The samples were then centrifuged (2,500rpm, 10 minutes) and the supernatant discarded. The pellet was washed with 2% PCA (10ml, x2) by resuspension and centrifugation and incubated in 0.3M NaOH (5ml, 37°C) to dissolve the protein.

An aliquot of the digest (2.5ml) was then taken and 20% PCA (1ml) added. This mixture was centrifuged (2,500rpm for 10 minutes) and the supernatant retained for RNA estimation. The resultant pellet was resuspended in 8 % PCA (5ml) and heated (70°C, 45 minutes). The samples were cooled in ice-water and centrifuged (2,500rpm, 10-15 minutes). The supernatant was retained for total DNA estimation.

An aliquot of the NaOH digest was analysed for protein content using the method of Lowry (1951).

5.2.7 Estimation of Total RNA Using the Orcinol Method

Orcinol reagent (3ml; 1ml of 1% orcinol: 40ml conc. HCl: 0 .1ml 10% FeClg.OHgO) was added to standard (0-50pg RNA/ml) or test samples (0.5ml, duplicates), mixed and heated at 100°C for 25 minutes. A marble was placed on top of every tube to prevent evaporation. After cooling on ice the absorbance was read at 660nm against a water blank using plastic cuvettes.

1 0 1 5.2.8 Estimation of Total Liver DNA

Liver DNA was measured using the method of Burton (1956). The preparation of the working diphenylamine reagent is outlined in Appendix VIII.

The test samples were diluted (x4) with deionised water. An aliquot of test/standard (O-lOOpg DNA/ml) (1ml, duplicates) was added to working diphenylamine reagent (2ml) and mixed. All tubes were then incubated at 30°C for 16-20 hours following which the absorbance was read at 600nm against a water blank.

5.3 Statistical Analysis

All data for in vitro studies is expressed as mean ± SEM. Data from cells dosed with hydrazine was compared to that obtained in controls using a Dunnett's t-Test.

D ata for in vivo studies is expressed as mean ± SD. Data obtained from dosed animals was compared to that obtained in controls using Unpaired t-Test.

5.3 RESULTS

5.3.1 The Effect of Hydrazine on Protein Synthesis in Isolated Hepatocytes In Vitro

The lowest concentration of hydrazine to reduce the incorporation of label into protein was 0.5mM, inhibition becoming significant after 2 hours (Figure 5.1). Higher concentrations of hydrazine (4-20mM) inhibited protein synthesis within 1 hour (Figure 5.1). Protein synthesis inhibition was maximal after 1 hour for all high doses (8-20mM) and 2 hours vdth low doses (0.5-4mM) (Figure 5.1).

1 0 2 Inhibition of protein synthesis occurred prior to loss of cell viability, which was observed after exposure to 16 and 20mM hydrazine for 3 hours (Figure 3.1) and depletion of GSH and ATP, GSH depletion became evident after 1 hour with Q.5-2GmM and after 2 hours with O.lmM hydrazine (Figure 3.2). ATP depletion followed with 12-20mM depleting after 1 hour and 4-8mM depleting after 2 hours (Figure 3.3).

There appears to be some correlation between protein synthesis and the cellular content of both GSH and ATP (Figures 5.2 and 5.3) such that the incorporation of radiolabelled leucine into protein was greater at higher concentrations of cellular GSH and ATP, and vice versa.

Figure 5.1 Inhibition of Protein Synthesis in Control Rat Hepatocytes in Suspension after Exposure to a Range of Hydrazine Doses for 3 Hours

125 1 hour A 2 hours • o 3 hours □ § 100 o

Otn ** 50

CO c ** ** « 25 ** O ** &

0 5 10 2015

Concentration of hydrazine (mM)

Figure 5.1 represents the incorporation of ^H-leucine (IpCi/lxlO® cells) into cellular protein after incubation of hepatocytes (IxlOVml) with hydrazine (0.1, 0.5, 1, 4, 8, 12, 16 and 20mM) for up to 3 hours. Values are means ± SEM; n=4 repetitions; *p < 0.05 and **p<0.01 Dunnett’s t-Test.

103 Figure 5.2 The Relationship Between Protein Synthesis and GSH Content in Isolated Rat Hepatocytes Exposed to Hydrazine

140 130 -

120 - c o 110 - u 100 X 90 H 80 70 - 60 - oo 50 40 - . • c • • • • ’© 30 - o 20 - QÜ 10 - 0 I— I— I— I— I— I— I— I— I I I I 0 10 20 30 40 50 60 70 80 90 100 110

GSH (% control)

Figure 5.3 The Relationship Between Protein Synthesis and ATP Content in Isolated Rat Hepatocytes Exposed to Hydrazine

120

110 -

100 - o u 90 - 80 - 70 - 60 - c 50 - >• CO 40 - 30 - V o 20 - OL 10 -

0 - 1------1 1 1 1 1 1 1 1 1 1 1 1 1 0 10 20 30 40 50 60 70 80 90 100110120130

ATP (% control)

Each data point represents the incorporation of ®H-leucine into protein compared to cellular content of GSH (Figure 5.2) and ATP (Figure 5.3) after exposure of hepatocytes to hydrazine (0.1, 0.5,1, 4, 8, 12,16, 20mM) for up to 3 hours. Data from 4 separate hepatocyte isolations.

104 5.3.2 The Effect of Hydrazine on Protein Synthesis in Rat Liver In Vivo

5.3.2.1 Study 1 Dose Response Experiment

After 3 hours of exposure to a range of hydrazine doses there was no change in liver or body weight (Table 5.1). Reduced and oxidised glutathione were also unchanged but ATP was significantly reduced by all doses (Table 5.1).

Liver DNA was estimated to be in the range of 1.6-1.8mg/g liver and was unchanged by treatment with hydrazine as was protein content ( 188-2 llmg/g liver). Although the content of RNA (8.73-10.35mg/g liver) was not significantly different from controls there was an apparent dose related effect, with animals exposed to 30 and 60mg/Kg hydrazine having lower RNA levels. This reduction in RNA content was reflected in decreased RNAiDNA and raised proteiniRNA ratios, the latter of which is significantly different from controls (Table 5.2). Expression of data as a ratio with DNA allows interpretation on a cellular level as DNA content should reflect cell number.

Unfortunately the serum samples for all of these animals were haemolysed. An estimate of radiolabel incorporation was determined but the accuracy of these results must be in doubt as haemolysis interferes with colour quenching during scintillation counting.

Exposure to hydrazine failed to alter the content of serum protein and hepatic acid precipitable proteins but decreased, in a dose dependent manner, the content of hepatic acid soluble protein (Table 5.3). There was no significant effect on the incorporation of radiolabelled leucine into any of these proteins 3 hours after dosing with hydrazine (Figure 5.4).

105 Table 5.1 The Effect of a Range of Hydrazine Doses on Body and Liver Weight and Parameters of Liver Toxicity 3 Hours Post Dose

T reatm ent Body Liver ATP GSH GSSG Group Weight Weight pmol/g pmol/g nmol/g (g) (g)

Control 245±15 10.4±0.5 3.32±1.18 4.52±0.64 120±17

lOmg/Kg 246±11 10.5±0.5 0.94±0.21' 4.29±0.54 124±17

30mg/Kg 244±12 10.6±0.6 0.95±0.23* 4.54±0.73 121±18

60mg/Kg 246±6 10.5±1.1 0.68±0.14* 4.92±0.62 138±12

Mean ± SD; 60mg/Kg group n=3 others n=4; * p<0.05 unpaired t-test

Table 5.2 The Effect of a Range of Hydrazine Doses on Liver DNA, RNA and Protein 3 Hours Post-Dose

T reatm ent RNAiDNA ProteiniDNA ProteiniRNA Group (mg/mg) (mg/mg) (mg/mg)

Control 6.2±0.9 114.9±6.4 19.0±2.2

lOmg/Kg 6 .1±1.1 116.5±18.4 19.4±1.0

30mg/Kg 5.4±0.3 120.6±9.1 22.3±l.r

60mg/Kg 4.9±1.2 117.5±25.8 24.2±0.7'

Mean ± SD, 60mg/Kg group n=3 others n=4; * p<0.05 unpaired t-test

106 Table 5.3 The Effect of a Range of Hydrazine Doses on The Protein Content of Liver and Serum 3 Hours Post-Dose

Treatm ent Serum Protein Acid Ppt Acid Sol Group (mg/ml) Protein Protein (pg/g (mg/g liver) liver)

Control 32.7±3.6 172.5±12.7 255±23

lOmg/Kg 35.4±5.3 170.7±7.4 202±17"

30mg/Kg 36.2±3.0 169.7±7.3 194±29*

60mg/Kg 40.3±11.9 180.0±4.5 i5 3 ± e r

Mean ± SD; 60mg/Kg group n=3 others n=4; * p<0.05; **p<0.01 unpaired t-Test

Figure 5.4 Incorporation of %-Leucine into Hepatic and Serum Proteins 3 Hours After Administration of a Range Of Hydrazine Doses to Rats In Vivo

8000 Protein: Serum c0) 7000 TCA precipitated ■■■ A)3 "c• 6000 TCA soluble 5000 •IOl c o> 4000 •S E 3000

2000 1o 1 1000

0 CON 10 30 60

Dose Hydrazine (mM)

Figure 5.4 illustrates the incorporation of ^H-Leucine (50pCi) into hepatic acid precipitable and acid soluble protein and serum protein in rats 3 hours after dosing with 10, 30 and 60mg/Kg hydrazine Up). Values are means ± SD; n=4 except in top dose group where n=3.

107 5.3.2.2 Study 2 Time Course Experiment

Throughout the experiment body weight was similar in both groups of animals. Liver weight remained unchanged until 6 hours after dosing with hydrazine at which time there was a significant increase in size in the treated group (Table 5.4). Inspection of the livers at autopsy revealed mottled and yellow discolouration characteristic of fat infiltration.

Hepatic GSH was significantly depleted at 6 and 12 hours by hydrazine (Figure 5.5) without any concomitant increases in oxidised glutathione (data not shown). In addition there was rapid depletion of ATP after 1.5 hours (Figure 5.6).

The level of total DNA was unaffected by treatment but when expressed as DNA/unit weight of liver there was a significant reduction at 6 (con 2.16±0.13; hz 1.75±0.17mg/g; p<0.01) and 24 hours (con 2.23±0.12; hz 1.63±0.06mg/g; p<0.001). The reason for this apparent decrease in DNA content is enlargement of the liver, probably as a result of lipid accumulation. Thus the DNA is essentially "diluted" by the infiux of triglycerides. As both RNA and protein are also under the same dilutional influences, these data were expressed as a ratio with DNA.

Total RNA was significantly raised at 24 hours (con 43.63± 17.85; hz 71.15±10.32 mg/liver; p<0.05) as was total protein (con 1.67±0.09;hz 2.01±0.15 g/liver; p<0.001) thus RNAiDNA and proteiniDNA ratios were higher than controls at this time. On the other hand proteiniRNA ratio was not altered suggesting that protein content was raised as a result of an increase in RNA (Table 5.5). Overall these data suggest that protein synthesis was stimulated 24 hours after dosing with hydrazine.

In agreement with those data obtained above the synthesis of acid precipitable proteins was increased in dosed animals 24 hours post-dose, illustrated by a significant increase in incorporation of ^^C-leucine at this time (Figure 5.7).

108 Inspection of data obtained for acid soluble proteins revealed variations both in the content of these proteins and the pattern of synthesis (Table 5.6; Figure 5.8). The protein content in dosed animals was reduced at all times but was significantly reduced at 6 , 12 and 24 hours (Table 5.6). In contrast, there was a trend indicative of increased incorporation of ^"^C-leucine into these proteins in dosed rats, but at no time was this statistically significant (Figure 5.8).

Incorporation of ^"^C-leucine into serum protein also appeared to fluctuate throughout the 24 hour period but at no time was there a significant difference between dosed and control animals (Figure 5.9). Serum proteins were maintained at steady state concentration at all times and there was no treatment related effect (Table 5.6).

In isolated hepatocytes in vitro ammonia has been shown to inhibit protein synthesis indirectly by inhibiting protein degradation and thus limiting the supply of amino acids (Seglen & Gordon, 1980). As hydrazine is structurally similar to ammonia and indeed can be metabolised to ammonia (Preece et al^ 1991) it is possible that these compounds have a similar mode of action. To test this hypothesis in vivo serum ammonia was measured at each time point after hydrazine administration and temporal changes compared to those of protein synthesis.

Initially serum ammonia levels were higher than controls, although not significantly, however after 6 hours levels fell significantly below controls (Figure 5.10). It was observed that serum urea levels increased after 6 hours perhaps indicating removal of ammonia from the circulation (data not shown). There was no correlation between serum ammonia levels and protein synthesis in rats in vivo (data not shown).

109 Table 5.4 The Effect of 60mg/Kg Hydrazine on Body and Liver Weight Over 24 Hour Exposure Period

Time of Exposure Treatment Group Body Weight (g) Liver W eight (g) LiveriBody Weight (h) (%)

0.5 Control 243±28 10.97±1.36 4.51±0.29 Hydrazine 245±20 11.27±0.78 4.61±0.18

1.5 Control 226±12 10.72±0.36 4.75±0.17 Hydrazine 217±23 9.94±1.87 4.55±0.39

6 Control 225±7 8.23±0.26 3.66±0.05 Hydrazine 225±7 9.38±0.63* 4.18±0.19'

12 Control 225±16 7.89±0.60 3.51±0.08 Hydrazine 224±12 9.79±1.15* 4.37±0.35'

24 Control 216±13 7.79±0.73 3.61±0.18 Hydrazine 206±17 10.58±1.06" 5.12±0.10"*

Mean ± SD; n=4 except in 0.5h control group where n=3 Statistical Significance: * p<0.05; ** p< 0 .01; *** p< 0.001 unpaired t-test Figure 5.5 The Time-Dependent Depletion of Hepatic GSH After Administration of 60mg/Kg Hydrazine to Rats In Vivo

Control 60mg/Kg Hydrazine 5 - o> > 4. - I o> I •

2 - X(O O 1 -

0 0.5 1.5 6 12 24

Time (hours)

Figure 5.6 The Time-Dependent Depletion of Hepatic ATP After Administration of 60mg/Kg Hydrazine to Rats In Vivo

3 -I Control Hydrazine 60mg/Kg « “ 2 0» o 3E o_ 1 - H < I

Time (hours)

Figures 5.5 and 5.6 illustrate the depletion of hepatic GSH and ATP depletion, respectively, in rats in vivo after exposure to 60mg/Kg hydrazine (ip) for 0.5, 1.5, 6, 12, and 24 hours. Values are means ± SD; n=4 except in 0.5h control group where n=3 rats/treatment group; * p<0.05 and ** p<0.01 unpaired t-Test.

Ill Table 5.5 The Effect of 60mg/Kg Hydrazine on Liver DNA, RNA and Protein Over 24 Hours

Time of Exposure Treatment Group RNAiDNA ProteiniDNA ProteiniRNA (h) (mg/mg) (mg/mg) (mg/mg)

0.5 Control 2.76±0.99 118.63±17.37 46.17±15.67 Hydrazine 3.75±0.65 121.40±5.35 33.17±6.36

1.5 Control 3.45±0.57 112.14±19.55 32.74±4.80 Hydrazine 3.42±0.26 119.71±5.59 35.14±2.94

6 Control 3.24±0.15 94.28±10.35 36.06±16.61 bO Hydrazine 3.57±0.73 107.45±14.56 30.30±2.91

12 Control 3.76±0.54 107.51±7.72 29.08±5.07 Hydrazine 3.12±0.36 99.76+10.63 32.20±5.28

24 Control 2.50±0.97 96.56±2.06 35.99±12.07 Hydrazine 4.15±0.64' 116.96±7.89* 28.64±4.16

Mean ± SD; n=4 except 0.5h control group where n=3 Statistical Significance: * p<0.05 unpaired t-test Table 5.6 The Effect of 60mg/Kg Hydrazine on the Protein Content of Liver and Serum Over 24 Hours

Time of Exposure Treatment Group Serum Protein Acid Ppt Protein Acid Sol Protein (h) (mg/ml) (mg/g liver) (pg/g liver)

0.5 Control 48.9±7.1 168±3 190±23 Hydrazine 41.2±3.5 170±6 143±36

1.5 Control 45.6±4.2 171±1 182±22 Hydrazine 47.2±1.4 178±12 142±47

6 Control 44.9±4.0 187±17 76±32 CO Hydrazine 44.6±4.6 164±10 25±6*

12 Control 49.0±2.5 196±7 75±12 Hydrazine 47.9±3.7 166±8*** 38±12"

24 Control 45.6±3.4 188±10 138±28 Hydrazine 46.9±4.0 180±6 48±21**

Mean ± SD; n=4 except 0.5h control group where n=3 Figure 5.7 Incorporation of ^'^C-Leucine into Acid Precipitable Protein 0.5-24 Hours After Administration of 60mg/Kg Hydrazine to Rats In Vivo

^ 6000 -I Control [ = 1 60mg/Kg Hydrazine 0 5000 3000 OC d. L .

2000 1 3 o o> 1000 H Q_ O 0 0.5 1.5 6 12 24

Time (hours)

F igure 5.8 Incorporation of ^‘^C-Leucine into Acid Soluble Protein 0.5-24 Hours After Administration of 60mg/Kg Hydrazine to Rats In Vivo

2000 Control c 60mg/Kg Hydrazine .il 1750 3 Q. 0) 1500 _J 0> 1250

n 1000 0 T J 1 5 750 a. o> 500 8 l — CL 250 O 0 0.5 1.5 6 12 24

Time (hours)

The incorporation of ^'’C-leucine (25pCi) into hepatic acid precipitable (Figure 5.7) and acid soluble proteins (Figure 5.8) after exposure of rats in vivo to 60mg/Kg hydrazine (ip) for 0.5, 1.5, 6, 12 and 24 hours. Values are means ± SD; n=4 except in 0.5h control group where n=3 rats/treatment group; ’ p<0.05 unpaired t-Test.

114 Figure 5.9 Incorporation of ^'^C-Leucine into Serum Protein 0.5-24 Hours After Administration of 60mg/Kg Hydrazine to Rats In Vivo

-rr 3000 -I Control o c c a> 60mg/Kg Hydrazine o o c o. o o> 3 0> E 2000 - —1 1 Z Ü a. # o c c o (/) 0 1.5 6 12

Time (hours)

Figure 5.10 The Time-dependent Effect on Serum Ammonia 0.5-24 Hours After Administration of OOmg/Kg Hydrazine to Rats In Vivo

Control o 4,0 -I Hydrazine 60mg/Kg • E O) 3.0 - 3

1 1.0 - 0) ^ 0.5 -

0 5 10 20 2515 Time (hours)

The effect of exposure of rats in vivo to 60mg/Kg hydrazine {ip) for 0.5, 1.5, 6, 12 and 24 hours on: the incorporation of ^^C-leucine (25pCi in Ipmol/lOOg) into serum protein (Figure 5.9); and serum ammonia (Figure 5.10). Values are means ± SD; n=4 except in 0.5h control group where n=3 rats/treatment group; * p<0.05 and ** p<0.01 unpaired t-Test.

115 5.4 DISCUSSION

Protein synthesis is a very sensitive marker of the metabolic competence of the liver. In vitro in isolated hepatocytes protein synthesis is usually altered by lower concentrations of cytotoxin and at an earlier time than other indicators of cell toxicity/death. Inhibition can result from defective amino acid transport, impaired mitochondrial function and altered protein catabolism as well as effects on RNA and DNA function.

Hydrazine, at a concentration as low as 0.5mM, has been reported to inhibit protein synthesis in hepatocyte cultures (Ghatineh & Timbrell, 1990). This result was reproduced in the present study using isolated rat hepatocytes in suspension (Figure 5.1).

Amino acid deprivation ensues when hepatocytes are incubated in unsupplemented medium resulting in the inhibition of protein synthesis as a result of inhibition of peptide chain initiation (Flaim et al, 1982b; Cox et al, 1988). Under these conditions protein degradation is promoted (Scomik, 1984; Poole & Wibo, 1973; Nath & Roch, 1971; Flaim et al, 1982a), which ultimately provides the necessary amino acids to allow recovery of the synthetic process. In response to hydrazine, protein synthesis in hepatocytes in the present study (incubated in amino add deprived medium) proceeded at a slower rate than in control cells (data not shown) but did not recover to control levels. This could be due to inhibition of protein degradation.

However as hydrazine appears capable of inhibiting protein synthesis in hepatocytes cultured in amino acid-containing medium (Ghatineh & Timbrell, 1990; Pravaceket al, 1994) this may suggest that hydrazine inhibits protein synthesis by an amino add independent mechanism. If the primary target site of hydrazine is the plasma membrane, as has been suggested (Siemens et al, 1980), it is possible that amino add transport mechanisms were disrupted in the hepatocyte cultures, limiting the entry of external amino adds into the hepatocytes. However preliminary data suggest that entry of different amino adds is not affected by hydrazine. Diamines, which, independently, inhibit

116 both protein synthesis and degradation, are thought to exert their actions at the plasma membrane as these compounds are not internalised (Seglen & Gordon, 1980). Hydrazine, as the simplest diamine, may have a similar mode of action.

Weak hases such as ammonia reversibly inhibit lysosomal protein degradation and subsequently protein synthesis when incuhated with isolated hepatocytes in amino acid deficient medium (Seglen & Gordon, 1980). Ammonia, at a concentration of lOmM inhibits protein synthesis by 60% after 1 hour (Seglen & Gordon, 1980). Concentrations higher than lOmM do not have any additional effect. Similarly 12mM hydrazine inhibited protein synthesis hy approximately 60% of control after 1 hour (Figure 5.1) and higher doses did not exert any further effect. It is plausible that hydrazine, like ammonia, inhibits protein synthesis indirectly as a result of inhibition of lysosomal protein degradation.

Inhibition of protein synthesis occurred either in the absence of or prior to ATP depletion and cell death (Figure 3.1 and 3.3) but in a similar temporal manner to that of GSH depletion (Figure 3.2). Thus if the mechanism of action of hydrazine is amino acid independent, inhibition of protein synthesis may be related in some way to cellular GSH levels. Comparison of the data revealed that the rate of protein synthesis varied considerably at high levels of GSH but was inhibited to approximately 30% control when GSH levels were reduced helow approximately 80% control (Figure 5.2). A similar graph was obtained when plotting ATP levels versus protein synthesis (Figure 5.3). These data suggest a causal relationship may exist between protein synthesis inhibition and ATP and GSH depletion, as has been demonstrated for ethionine (Glaser & Mager, 1974; Lyon and Kisilevsky, 1990).

Depletion of GSH can be accompanied by an increase in GSSG, which is a recognised inhibitor of initiation of protein synthesis with levels as minute as 50-60nmol/L capable of inhibition (Kosower et al, 1972). An attempt to measure GSSG in hepatocytes was unsuccessful in the present study. However a rise in GSSG, although statistically insignificant, has been reported to occur

117 after hydrazine administration in vitro along with a decrease in NADPH, the cofactor required for GSSG reduction (Ghatineh et al, 1992).

In vivo there was no obvious inhibition of protein synthesis, but there was an increase in protein synthesis in dosed animals after 24 hours (Figure 5.7). Depletion of ATP was dose-dependent (Table 5.1) and occurred almost immediately (Figure 5.6) whereas depletion of GSH became evident 6 hours post-dose (Figure 5.5). There was no detectable increase in GSSG (data not shown) and previous reports suggest that the GSHiGSSG ratio is not altered by hydrazine (Timbrell et al, 1982). Thus if hepatocytes in vitro are more susceptible to hydrazine induced generation of GSSG than their counterparts in the intact liver this might explain why there is strong inhibition of protein synthesis in this system and not in vivo.

Although there was no significant inhibition of protein synthesis by hydrazine in vivo, illustrated by the lack of effect on incorporation of ^H-leucine into several types of protein (Figure 5.4), liver RNA was reduced after 3 hours of exposure to 30 and 60mg/Kg hydrazine (Table 5.2). Later, at 24 hours post dose, protein synthesis was clearly stimulated, illustrated not only by increased radiolabel incorporation (Figure 5.7) but also by elevated RNA and protein levels relative to DNA (Table 5.5), in accordance with data published by Banks (1970). At no time was total hepatic DNA content altered by treatment with hydrazine (see pages 105 & 108). This is in contrast to th a t reported by Banks and coworkers (1967) who noted increased liver DNA 4 hours post dose. This result led these authors to suggest that hydrazine initially alters DNA metabolism which in turn promotes increased RNA and protein synthesis.

Hydrazine inhibits gluconeogenesis in the liver (Fortney, 1967; Ray al, 1970). In such situations when amino acids are required in the production of glucose endogenous proteins are broken down; indeed hydrazine increases the amino acid content in several tissues and plasma (Cornish & Wilson, 1968; Korty & Coe, 1968; Banks, 1970. When protein catabolism is stimulated protein synthesis is reduced and RNA content drops (Hirsch & Hiatt, 1966). The data obtained after exposure to hydrazine for 3 hours may reflect this situation.

118 However amino acids accumulate in the liver as hydrazine limits the entry of these amino acids into the gluconeogenic pathway as a result of transaminase inhibition (Fortney et aly 1967). High amino acid concentrations normally stimulate protein synthesis but only if transaminases or other pyridoxal phosphate-dependent enzymes are active (Seglen et aly 1980). Thus when the concentration of hydrazine in the liver is high presumably more pyridoxal phosphate would be removed by the formation of a hydrazone but as hydrazine levels gradually diminish the latter would be replenished, allowing reactivation of transaminases. This may explain why protein synthesis was not stimulated for many hours after hydrazine dosing (Figure 5.7).

In an attempt to identify the fraction of protein that was affected by hydrazine, the concentration of, and radiolabel incorporation into serum proteins and hepatic acid soluble proteins were also investigated. Serum proteins are predominantly synthesised in the liver and thus a block in hepatic protein synthesis might be reflected in serum. However at no time was serum total protein content or radiolabel incorporation significantly altered (Table 5.6; Figure 5.9).

Hepatic acid soluble proteins are thought to be regulatory proteins, degradation products and/or damaged/abnormal proteins. The content of these proteins was reduced almost immediately in a dose (Table 5.3) and time-dependent manner by hydrazine (Table 5.6) and thus if the acid soluble proteins were synthetic products, enhanced utilisation or altered disposition in response to hydrazine could decrease their hepatic content.

If the acid soluble proteins were abnormal or damaged proteins one might expect the content of these to rise as hydrazine is known to damage both plasma membrane and other cellular proteins (Mortensen & Novak, 1991), possibly by cleaving peptide bonds at specific sites within a protein (Miyatake et aly 1994). However hydrazine also promotes enhanced non-lysosomal degradation of these proteins (Mortensen & Novak, 1991) thus removing them from the system.

119 The final possible identity of these proteins are degradation products, a reduction in content being consistent with inhibition of degradative pathways. As hydrazine stimulates non-lysosomal pathways (Mortensen & Novak, 1991) the major lysosomal route would have to be inhibited. This would explain why the incorporation of radiolabelled leucine was unaltered but the protein content was decreased. As has been discussed with reference to cells in vitro amm onia inhibits lysosomal protein degradation (Seglen et al, 1979). In vivo serum ammonia levels were raised 30 minutes after dosing with hydrazine, although not significantly, after which there was a drop to below control level (Figure 5.10). Unfortunately there was no correlation between serum ammonia concentration and protein synthesis (all types of protein).

5.5 CONCLUSIONS

Hydrazine clearly affects protein synthesis much more strongly in vitro th an in vivo and it is plausible that nutritional factors are responsible in both systems. Hydrazine may induce a condition which mimics starvation, by decreasing both the production and utilisation of glucose, thus shifting the emphasis firom protein synthesis to protein degradation in order to supply the necessary amino acids for gluconeogenesis. In vivo there are homeostatic control mechanisms which intervene and attempt to return the system to normal, whereas there are no such influences in vitro. Thus in the latter system inhibition of protein synthesis may result from inhibition of protein degradation or a diversion of amino acids (supplied by protein degradation) to other metabolic pathways. However as the amount of radiolabelled amino acid incorporation into protein appeared to be related in some way to the cellular content of GSH, and also ATP in vitro, protein synthesis may be regulated by an amino acid independent mechanism. If GSSG is formed, even in minute quantity, protein synthesis could be affected at the level of initiation.

1 2 0 THE EFFECT OF AN ACUTE DOSE OF HYDRAZINE ON LIVER LIPIDS.

6.1 INTRODUCTION

There have been several studies undertaken to investigate the effect of hydrazine on liver lipids. The methods utilised involved extraction of liver lipids into chloroform-methanol and separation of neutral lipids from phospholipids by adsorption of the latter onto sihcic acid. Further identification of lipids was then achieved by titration, thin layer chromatography or other means (Lamb & Banks, 1979; Clark et al, 1970; Trout, 1966).

The use of proton NMR in the detection and quantitation of liver lipids has been compared with other techniques such as high performance liquid chromatography (HPLC) (Christie, 1985; Casu et al, 1991). Although the results obtained were very similar, more information on the structure and composition of lipids was obtained using NMR. For this reason proton NMR was utilised to analyse hepatic lipid content of rats previously exposed to hydrazine in the hope that novel information could be obtained. The following paragraphs will briefly outline the basic concepts of NMR.

The magnetic properties of atomic nuclei, such as the proton (hydrogen nucleus), form the basis of nuclear magnetic resonance spectroscopy. When exposed to an external magnetic field protons will preferentially exist in a low energy state. A nuclear magnetic moment is induced which attempts to align parallel to the applied field. Bombardment of the protons with radiowaves, causes energy absorption and a shift from the low to a high energy state. In this high energy state the nuclear magnetic moment deviates from its original position. The radiofrequency which exactly matches the difference in energy

1 2 1 between high and low energy states produces a resonance signal.

The radiofrequency at which a proton will resonate is influenced by several factors. Exposure of molecules to an external magnetic field causes the electrons surrounding the nuclei of hydrogen and neighbouring atoms to circulate. This causes an increase in electron density around the protons and as a result those protons are shielded from the applied magnetic field. Thus the local magnetic fields at the individual protons within the molecule differ according to chemical environment. The degree of magnetic shielding is proportional to the electron density and the resonance frequency (chemical shift) is inversely proportional to the degree of shielding.

Spin-spin coupling is another phenomenon which influences the shape of the signal and determines the multiplicity (splitting) of that signal. In this case there is magnetic interaction between individual protons which is transmitted by the bonding electrons which indirectly connect individual protons.

The frequency separation of resonance signals is represented as chemical shift, an arbitrary scale expressed in ppm. The intensity of the signal, which can be quantitated by calculation of the area under each signal peak relative to that of an internal standard, is directly proportional to the number of protons.

Another important factor to take into account whilst generating an NMR spectrum is relaxation time. This is the time taken for the absorbed energy to dissipate, returning the protons back to the ground state (Boltzman equilibrium). Allowance of total relaxation for all protons within a sample is vital for the acquisition of quantitatively accurate data. If protons are not allowed to relax the signal intensity is reduced and the peak width is too great.

1 2 2 6.2 METHODS

6.2.1 Treatment Regime

Male Sprague Dawley rats were weighed and randomly allocated to treatment groups. Food and water were given ad libitum prior to ip injection of 30mg/Kg body weight hydrazine (free base) after which food was withdrawn. Control animals received saline. Animals were sacrificed 24 hours post dose. Part of the liver was utilised to prepare microsomes, which were resuspended in 0.25M sucrose buffer containing 0.5mM dithiothreitol (pH was adjusted to 7.4 with IM potassium bicarbonate). Lipids were extracted from the rest of the liver as outlined below. Triglycerides and non-esterified free fatty acids (NEFA) were measured in serum.

6.2.2 Phosphatidate Phosphohydrolase Activity

There are two forms of this enzyme: the NEM-sensitive and Mg^^ requiring form which translocates to the endoplasmic reticulum from the cytoplasm in response to fatty acids (Cascales et al, 1984). This enzyme is responsible for diacylglycerol production to be utilised in triglyceride, phosphatidylcholine and phosphatidylethanolamine synthesis; the NEM-insensitive and Mg^^ independent form which is located primarily in the plasma membrane, and is possibly involved in signal transduction (Loffelholz, 1989). Similar activity can also be detected in microsomes (Jamal et uZ, 1991).

Enzyme activity was measured in microsomes using a modified version of the method of Jamal et al (1991). The reaction, catalysed by PAP, is as follows:

Phosphatidate ------> Diacylglycerol + P^

Enzyme activity was determined by detection of inorganic phosphate, which forms a blue coloured complex with molybdate reagent.

123 Prior to performing the assay one set of microsomes was incubated 1:1 with N- ethylmaleimide (8.4mM) for 10 minutes at 37°C and cooled on ice.

An aliquot (5pl) of untreated microsomes was preincubated in trisXmaleate buffer (185pl; O.IM containing dithiothreitol (ImM) and MgClg (3mM), pH6.5) for 5 minutes at 37°C. NEM-treated microsomes (lOpl) were also incubated in buffer (180pl) as above. A blank was prepared for each sample using boiled (denatured) microsomes.

The reaction was started by addition and mixing of the substrate phosphatidate (lOpl; 0.6mM, sonicated in trisXmaleate buffer containing 10% ethanol). The tubes were incubated for 15 minutes at 37°C after which the reaction was terminated with molybdate reagent (0.8ml; 1.25% ascorbic acid; 0.13% SDS; 0.32% ammonium molybdate; 0.375M HgSO^) and the samples cooled on ice. This reagent was also added to the standards (0-0.2mM sodium phosphate, 0.2ml final volume).

Colour was developed by incubation at 45°C for 20 minutes and the absorbance read at 820nm against a water blank.

6.2.3 Determination of Liver and Serum Lipids

Hepatic triglycerides were analysed using the method of Butler et al (1961) and serum triglycerides and NEFA were determined using automated assays as described in Chapter 2.

6.2.3.1 Extraction of Lipids for Proton NMR

An aliquot of crude liver bomogenate (see microsome preparation, Chapter 2) was added to twice the volume of cbloroformimetbanol (2:1). This was vortexed and subsequently centrifuged at 2,500rpm for 5 minutes. The lower chloroform layer was removed and retained. The resultant pellet was reextracted as described above.

124 The chloroform layers from each extraction were pooled and washed (twice) with an equal volume of potassium chloride (0.5M in 50% methanol), centrifuged and the aqueous supernatant discarded. The chloroform layer containing the lipids was decanted into a preweighed tube and the solvent evaporated to dryness under a stream of nitrogen. The tube was reweighed to assess the quantity of lipid extracted. This residue was then redissolved in chloroform (1ml; stock extract).

An aliquot (200ul) of the stock lipid extract was taken, the chloroform evaporated as previously described and the residue dissolved in deuterated chloroform (C^HClg):deuterated methanol (C^HgO^H) (0.6ml; 1:2) for NMR analysis.

6.2.S.2 Separation of Different Classes of Lipids

Another aliquot of the stock extract (400ul) was carefully pipetted directly onto an aminopropyl solid phase extraction column (Isolut; Jones Chromatography Ltd, Mid Glamorgan, UK) which had been previously washed with dry hexane. A series of different solvents were added onto the column in order to preferentially elute different classes of lipid. The columns were pulse centrifuged to 500g after addition of each solvent.

The solvents (8ml each) were as follows: Chloroform - for extraction of neutral lipids (those which do not have a polar head group) and cholesterol. Diethvlether with 2% acetic acid - for extraction of non- esterified (free) fatty acids. Methanol - for extraction of non-acidic phospholipids. 0.05M Ammonium acetate in chloroform:methanol (4:1 v/v) plus 2% (v/v) of 28% aqueous ammonium solution (w/v) - for extraction of acidic phospholipids.

Fractions eluted with the same solvent were pooled, the solvent removed under a stream of nitrogen. The residue was resuspended in the deuterated solvents

125 (0.6ml) as previously described and transferred to 5mm NMR tubes.

6.2.3.3 Proton NMR Spectroscopy

Spectra were recorded using a Bruker AM500 NMR spectrometer operating at 500.136 MHz. The data were processed using a Bruker Aspect 3000 data system. Proton chemical shifts were referenced to the internal standard trimethylsilane (TMS) (5 = Oppm). 4096 free induction decays were collected (over 6024 Hz), each consisting of 32768 data points, using 45° (3 microsecond) pulses with an acquisition time of 2.72 seconds. A further 4 second delay was added to ensure full relaxation. The integrals of individual lipid peaks were normalised to that of deuterated chloroform (Ô = 7.7ppm). NMR spectra and peak assignments are presented in Appendix X.

I would like to express my gratitude to Mire Zloh, of the chemistry department, for running my samples and to Professor Gibbons for his help with peak assignment and data interpretation.

6.3 RESULTS

It has been stated that translocation of phosphatidate phosphohydrolase (PAP) from the cytoplasm to endoplasmic reticulum is evidence of induction (Cascales et al, 1984). Indeed 24 hours after an acute dose of hydrazine there was a 2- fold increase in microsomal activity of PAP (Table 6.1) in hydrazine treated rats. Maximal induction of the enzyme parallels maximal accumulation of triglycerides (Haghighi & Honaijou, 1987) which were increased 3-fold at this time (Table 6.1). Serum triglycerides were also raised (2-fold) but free fatty acids were comparable to controls (Table 6.1).

Much information can be gained from the NMR spectrum derived from a stock chloroform extract of liver lipids. Nevertheless it is difficult to designate specific alterations to any particular class of lipids with any degree of certainty

126 as in several instances some lipid peaks are masked by others. For this reason the different classes of lipids were separated and analysed individually.

NMR analysis of lipids extracted from livers taken from rats treated with hydrazine 24 hours previously displayed some interesting changes (Table 6.2). There was a 3-fold increase in liver triglycerides in agreement with the value obtained using chemical analysis (Table 6.1). Cholesterol was similar to control at this time (Table 6.2). Non-acidic phospholipids, phosphatidylcholine and phosphatidylethanolamine were unaltered 24 hours after hydrazine treatment. Acidic phospholipid phosphatidylinositol, peaks for which were masked in the full spectrum, was found to be increased 3-5-fold (Table 6.2).

Total fatty acids, which constitute both free fatty acids and those bound to lipids, are represented by a single peak (chemical shift 0.85ppm), the area of which was elevated in lipid extracts taken from dosed animals. Further analysis indicated this increase was due to raised levels of free fatty acids and and those bound to neutral lipids. This was expected as the phospholipids remained relatively unchanged.

By comparing the integrals obtained for total unsaturated fatty acids (chemical shift 5.4ppm) with total fatty acids (chemical shift 0.85ppm) an estimate of the degree of saturation could be made (Table 6.2). The degree of unsaturation in acidic and non-acidic phospholipids was unchanged (control 69%, hydrazine 73%; and control 100%, hydrazine 100% respectively). Neutral lipids contained more unsaturated fatty acids (control 52%, hydrazine 81%) verified by analysis of specific peaks for linoleic, docosahexaenoic (hexaenoic) and arachidonic acids which were also increased in this lipid fraction (Table 6.2). On the other hand free fatty acids were predominantly saturated (80%) compared to controls (60%).

The NMR data presented in this chapter is only semiquantitative as there was the potential for peak overlap in the 1 dimensional NMR spectra.

127 Table 6.1

Alterations to Lipid Parameters in Liver and Serum 24 hours After an Acute Dose of 30mg/Kg Hydrazine.

Treatm ent Microsomal PAP Liver Triglyceride Serum Triglyceride Serum NEFA Group (nmol P / min/ mg (mg/g) (mmol/L) (mmol/L) protein)

Control 3.51±1.02 19.20±5.62 0.46±0.09 1.00±0.08 to 00 Hydrazine 7.oi±o.er** 69.70±32.04P=°°^^ 0.87±0.18** 0.98±0.16 30mg/Kg

PAP - phosphatidate phosphohydrolase Means ± S.D., n=4 Statistical significance: ** p<0.01, *** p<0.001 unpaired t-test. Table 6.2 Lipids Extracted From the Livers of Rats Dosed 24 hours Previously with 30mg/Kg Hydrazine. Peak Areas (Integrals) obtained by NMR Analysis of Chloroform Extracts.

Peak All Lipids N eutral Non-acidic Acidic NEFA % Total (Chemical shift ppm) Lipids Phospholipids Phospholipids

Triglyceride (4.35) 0.6±0.2 0.5±0.1 - - - 79

2.0±0.8" 1.6±0.7 - - - 80

Cholesterol (0.7) 0.6±0.1 0.5±0.2 -- - 89

0.5+G.l 0.4±0.1 - - - 86 to

(3.2) 3.8±0.3 - 3.5+0.3 - - 91

Phosphatidyl 0.5±0.1 - 0.4+0.1 -- 96

ethanolamine (3.1) 0.4±0.1 - 0.4±0.1 - - 100

Phosphatidylinositol - - - 1.5±1.1 -

(3.6) - - - 4.6±1.2" -

Hydrazine data are shown in bold. NEFA - non-esterified fatty acids. Integral values given are means ± S.D.; n=4; *p<0.05, **p<0.01 Unpaired T-test. Total % = the amount of each constituent in the original extract accounted for by summing the values obtained in all the other fractions. This should give an indication of how much material was lost during the separation procedures. Table 6.2 (continued)

Peak All Lipids N eutral Non-acidic Acidic NEFA % Total (Chemical shift ppm) Lipids Phospholipids Phospholipids

Arachidonic Acid 1.1±0.1 0.3 0.8±0.2 nm - 98

(2.1) 1.3±0.1' 0.6 0.7±0.1 n m - 100

Linoleic Acid (2.75) 1.3±0.1 0.7±0.1 0.3±0.1 0.2 - 86

2.8±0.9 1.9±0.7 0.3±0.1 0.2 - 87 p=0.054

00 Hexaenoic (2.4) 0.5±0.1 0.2 0.3±0.1 - - 94 o 0.7±0.1* 0.3±0.1 0.3±0.1 - - 87

Total Fatty Acids 9.9±0.2 5.5±0.4 3.9±0.9 0.6±0.3 0.5 100 (0.85) 15.5±3.4* 9.9±2.9 3.4±0.4 0.6±0.2 0.8 95

Unsaturated Fatty 9.8±0.6 2.9±0.4 4.8±1.2 0.4 0.2 85 Acids (5.4) 15.6±3.1* 8.0±2.8 4.0+0.6 0.4 0.1 81

Polyunsaturated 5.2±0.2 1.0±0.2 3.4±0.7 0.4±0.1 nm 91 Fatty Acids (2.8) 6.7±0.6" 2.5±0.6* 3.0±0.2 0.4±0.1 n m 87

nm = present at very low levels and thus not measured; where mean alone expressed n = 2. 6.4 DISCUSSION

The majority of reports to date concerning hydrazine-induced lipid changes have focused on the mechanisms of hepatic triglyceride accumulation and the temporal nature of events. The effect on phospholipids has received relatively little attention. Certain precursors, such as phosphatidate, are common to the synthetic pathways of both phospholipids and triglycerides (Bell & Coleman, 1980). In addition triglycerides can be utilised in phosphoHpid synthesis but in the presence of lipogenic precursors triglyceride catabolism is prevented (Borrowitz & Blum, 1974). Thus if one pathway is stimulated another may be compromised. NMR analysis of lipid extracts gives information on all classes of lipids in a single sample allowing relative determinations to be made. For this reason proton NMR was utilised to study hydrazine-induced lipid changes.

Analysis of triglycerides, using a colorimetric assay (Table 6.1) and NMR (Table 6.2), revealed a 3-fold increase in the livers of dosed animals. This was accompEinied by induction of PAP activity in microsomes (Table 6.1) as previously reported (Lamb & Banks, 1979; Haghighi, 1987). These data are consistent with de novo triglyceride synthesis. Examination of the spectrum obtained for neutral lipids also revealed that cholesterol was unchanged (Table 6.2).

An increase in hepatic fatty acids will promote triglyceride synthesis by inducing both PAP and DGAT activities (Haagsman & Van Golde, 1981; Butterwith et al, 1985). A small elevation of free fatty acids was detected in the livers of hydrazine treated rats 24 hours after dosing (Table 6.2). The source of these fatty acids is either adipose tissue, de novo synthesis in the liver, or both. Indeed hydrazine has been reported to induce both pathways. Mobilisation of fatty acids from adipose tissue is an early response to hydrazine administration (Trout, 1965) but one which can persist for up to 12 hours (Clark et al, 1970). In agreement with this serum free fatty acids were comparable to controls 24 hours post dose (Table 6.1).

Hydrazine also induces de novo synthesis of fatty acids in the liver but this

131 effect has only been studied at early time points (Marshall et al, 1983). The largest proportion of newly synthesised fatty acids are saturated (Duerden et al, 1990) and further analysis of the NMR data revealed that in the treated group 80% of total free fatty acids were saturated compared to 60% in controls (calculated from data in Table 6.2). This finding may be consistent with ongoing lipogenesis. This is plausible as hydrazine also induces the activity of glucose-6-phosphate dehydrogenase, maximal activity being achieved 24 hours post dose, thus potentially providing sufficient NADPH for fatty acid synthesis at this time (Haghighi & Ghanbari, 1991).

Decreased utilisation of fatty acids resulting from disruption of 8-oxidation within mitochondria would also encourage a rise in free fatty acids within the liver. However this is unlikely to be a contributory factor as this pathway has has been reported to be unaffected by hydrazine (Amenta, 1963), although recent preliminary data, in isolated rat hepatocytes in suspension, suggest B- oxidation may be stimulated (personal communication. Dr Cathy Waterfield). Whatever the reason for the initial increase in the concentration of hepatic fatty acids accumulation would ensue if the capacity for degradation and/or utilisation was exceeded.

Another cause of fatty liver is reduced triglyceride secretion and several authors have studied this pathway in relation to hydrazine with conflicting results (Trout, 1966; Amenta & Dominguez, 1965). Clark and coworkers (1970), after administering 40mg/Kg hydrazine to 48 hour starved rats, reported a 2-fold increase in serum triglycerides despite a 10-fold accumulation in the liver 24 hours post dose. They suggested that secretion may have been impaired due to reduced synthesis of specific phospholipids such as phosphatidylcholine, hepatic content of which was diminished (Clark et al, 1970).

In the present study, in which 30mg/Kg hydrazine was administered to fed rats, serum triglycerides were raised 2-fold whereas liver triglycerides were raised 3-fold (Table 6.1). An adequate supply of phosphatidylcholine is vital for the synthesis of VLDL (Yao & Vance, 1988) and as such the secretion of

132 triglycerides out of the liver. However the content of phospatidylcholine and phosphatidylethanolamine, and the degree of fatty add saturation in these lipids, were comparable to controls (Table 6.2) implying that synthesis of these lipids was not impaired.

The magnitude of response with respect to triglyceride accumulation in the present study is obviously much less than that reported by Clark (1970) and the discrepancy may be solely attributable to the nutritional state of the animals, as under conditions of starvation VLDL secretion is reduced (Gibbons & Burnham, 1991). However even if hydrazine does not effect VLDL secretion the secretory capadty of the liver would not be able to compensate for the strong increase in triglyceride production. Furthermore assembly of VLDL takes 30-40 minutes (Moir & Zammit, 1992) and only a proportion of newly synthesised triglyceride is secreted at any one time (Duerden et al, 1990). Under these conditions fatty liver would ensue.

Greater amounts of unsaturated fatty acids, such as linoleic, docosahexaenoic and arachidonic acids, were associated with neutral lipid (Table 6.2). However very few triglycerides contain docosahexaenoic and arachidonic acids as these are preferentially incorporated into phospholipids (Holub & Kuksis, 1971; Grove & Schimmel, 1982). Thus it is possible that a large proportion of these unsaturated fatty acids were esterified to diacylglycerols in readiness for phospholipid synthesis. Although diacylglycerol was difficult to detect as its resonances were masked by those of triglyceride (Casu et aly 1991), a 3-5 fold elevation of phosphatidylinositol was detected (Table 6.2). Indeed the most predominant species of phosphatidylinositol contain arachidonic or docosahexaenoic acid at the 2-position of glycerol (Holub & Kuksis, 1971). These data may indicate increased synthesis of this lipid.

The breakdown of phosphatidyl inositol in the plasma membrane generates inositides which are involved in cell signalling and thus increased synthesis of PI in the presence of hydrazine may indicate activation, either directly or indirectly, of this second messenger system. In addition prostaglandin synthesis may also be stimulated by the release of arachidonic acid during PI

133 degradation.

6.5 CONCLUSIONS

Alterations in the levels of specific hepatic lipids as well as some insight into their composition was gained using proton NMR. The greater degree of saturation of hepatic free fatty acids in dosed animals may be indicative of de novo synthesis. In turn this could result in accumulation of hepatic triglycerides, synthesis of which is stimulated in response to elevated hepatic free fatty acids. Elevated PAP activity in microsomes is consistent with this. Control levels of phosphatidylethanolamine and phosphatidylcholine 24 hours after hydrazine dosing seem to imply that their synthesis was undisturbed, although an earlier defect cannot be ruled out.

The importance of enhanced synthesis of phosphatidylinositol is as yet unknown, but hydrazine may stimulate the breakdown of this lipid, either directly or indirectly, to elicit changes in cellular function.

134 FINAL DISCUSSION

7.1 THE ROLE OF CYTOCHROMES P450 IN HYDRAZINE TOXICITY

7.1.1 Modulation of the Activities of Certain Cytochrome P450 Isoenzymes on Acute Hydrazine Toxicity In Vivo and In Vitro

To date it remains unclear as to whether hydrazine toxicity results from exposure to the parent compound or a metabolite, however as hydrazine causes toxicity in almost every tissue of the body this may indicate that the former is responsible. In order to clarify this, one of the aims of the research described in this thesis was to further elucidate the role of certain isoenzyme(s) of cytochrome P450 in hydrazine toxicity.

At the outset of this project it was known that hepatic metabolism of hydrazine was partly carried out by cytochrome P450 and more specifically cytochromes P4502B1/2 and P4501A1/2 were thought to catalyse detoxication pathways (Timbrell et al, 1982; Jenner & Timbrell, 1994a). Perhaps more interesting were data suggesting the enhancement of acute hydrazine hepatotoxicity by induction of cytochrome P4502E1 (Jenner & Timbrell, 1994a). This isoenzyme is present in human liver (Wrighton et al, 1986) and has been shown to be responsible for metabolism of substrates such as paracetamol and alcohol to reactive intermediates (Raucy et al, 1989; Albano et al, 1991; Morimoto et al, 1993). It was therefore deemed important to further assess the role of this enzyme in hydrazine metabolism.

The aim of the first study outlined in Chapter 3 was to assess the toxicity of hydrazine in isolated rat hepatocytes in suspension after pretreatment of the animals with agents which induced or inhibited cytochrome P4502E1. Although the toxicity was modulated by these pretreatments the results were

135 inconclusive as modulation of cytochrome P4502E1 activity was not as specific as hoped. Despite the fact that induction of this isoenzyme both increased and decreased hydrazine toxicity, there appeared to be some correlation between cytotoxicity, such that the greater the activity the less the toxicity. This was suggestive of a detoxication role.

As these results were contradictory to those reported in vivo (Jenner & Timbrell, 1994a) a further study was undertaken (Chapter 4) in an attempt to modify the acute toxicity of hydrazine in vivo after induction of cytochrome P4502E1 by pretreatment with hydrazine itself. Hydrazine pretreatment causes qualitatively similar changes in microsomal enzyme activities to isoniazid and as such has been assumed to induce cytochrome P4502E1 (Rice & Talcott, 1979; Ghatineh et al, 1990c; Jenner & Timbrell, 1994a/b). Unfortunately there was no modulation of hydrazine hepatotoxicity, the reasons for which are discussed elsewhere, casting some doubt as to whether hydrazine actually does induce cytochrome P4502E1 or another enzyme with p-nitrophenol hydroxylase activity.

The studies described above did, however, verify a role for cytochromes P4501A1/2 and P4502B1/2 in acute hydrazine toxicity, the activities of these isoenzymes correlating with the degree of toxicity both in vivo (depletion of ATP and GSH, and triglyceride accumulation) and in vitro (depletion of ATP and GSH, and loss of cell viability) such that the greater the activity the less the toxicity. As acute and chronic dosing inhibits the activities of EROD and, to a lesser extent, PROD, hydrazine may be limiting its own metabolism. If so, and the parent hydrazine is responsible for the toxicity, then this will influence the dose response.

7.1.2 Induction of Cytochrome P4502E1 by Repeated Exposure to Hydrazine

Some of the compounds which induce cytochrome P4502E1, such as ethanol and carbon tetrachloride, ultimately cause fibrotic and/or necrotic hepatic damage as a result of free radical generation during their metabolism (Glaser

136 & Mager, 1974; Johansen & Ingelman-Sundberg, 1985; Ray et al y 1989). In contrast, necrotic lesions are extremely rare with respect to hydrazine, in fact administration of 5mg/Kg/day throughout the entire lifespan of mice did not result in any detectable tissue damage (SteinhofF et al, 1990). Indeed there is only one case of human morbidity from occupational exposure to hydrazine and death resulted from lesions in the lung and kidney, although small areas of focal hepatic necrosis were observed (Sotaniemi et al, 1971). Although cytochrome P4502E1 is expressed in relatively small amounts in rat kidney and lung (Porter et al, 1989; Wu & Cederbaum, 1994), it is not expressed in these tissues in the human (De Waziers et al, 1990). This is inconsistent with metabolism of hydrazine by this isoenzyme being responsible for the toxicity in these organs.

Results obtained after subchronic exposure of rats to hydrazine in vivo (Chapter 4) support this concept. In fact the data are inconsistent with hydrazine induction of cytochrome P4502E1 as, despite a dose-dependent increase in p-nitrophenol hydroxylase, a marker enzyme activity, there was no apparent increase in the amount of cytochrome P4502E1 protein in the centrilobular region of the liver, illustrated by immunohistochemical staining of tissue slices. Nor was there any evident tissue damage. Nevertheless there was a rise in hepatic GSSG, indicative of oxidative stress, in the animals exhibiting the highest p-nitrophenol hydroxylase activity. Cytochrome P4502E1 exhibits a high rate of oxidase activity, even in the absence of substrate, leading to superoxide and hydrogen peroxide generation (Ekstrom & Ingelman-Sundberg, 1989). Reactive oxygen species can then oxidise GSH to GSSG. However as hydrazine disrupts many pathways of intermediary metabolism it is possible that lack of the necessary cofactors for the reduction of GSSG were responsible for the rise in GSSG.

Unfortunately more questions than answers have been raised with regard to the involvement of cytochrome P4502E1 in hydrazine toxicity. Further research is still necessary to unequivocally state whether or not cytochrome P4502E1 is induced by, and metabolises hydrazine. If future work discovers that hydrazine does induce cytochrome P4502E1, even if this is of no

137 consequence with regards to the toxicity of hydrazine itself, this would be of extreme importance when considering exposure to other chemicals/drugs which are bioactivated during their metabolism by this isoenzyme.

7.2 THE EFFECT OF HYDRAZINE ON LIVER LIPIDS

Fatty liver is a common response to many xenobiotics, including some of those that are metabolised by cytochrome P4502E1, such as ethanol (French et al, 1993). Indeed the only parameter of hydrazine hepatotoxicity to be affected during cytochrome P4502E1 induction by isoniazid was triglyceride accumulation (Jenner & Timbrell, 1994a), but this may or may not reflect the involvement of this enzyme in the pathogenesis of hydrazine-induced fatty liver.

If hydrazine-induced triglyceride accumulation was related to the induction of cytochrome P4502E1, one might expect to see accumulation of hepatic lipid in the centrilobular region, as this is where the greatest proportion of cytochrome P4502E1 is localised (Lindros et al, 1990; Dicker & Cederbaum, 1992). However subchronic exposure to hydrazine did not alter the content of hepatic lipid relative to control, despite apparently induced levels of cytochrome P4502E1. Furthermore acute exposure to hydrazine in hydrazine-pretreated rats resulted in periportal fatty degeneration of similar magnitude to that seen in control animals (Chapter 4). These data indicate that alterations to these parameters are not directly related, as has been suggested for ethanol (Forkert et al, 1991). Thus if hydrazine metabolism is not responsible for the steatosis, the intact molecule may directly or indirectly be the causative agent.

Periportal fat accumulation is usually associated with compounds that inhibit protein synthesis. Hydrazine may have caused an early, small decrease in protein synthesis in vivo (Chapter 5), which may have resulted in a transient fall in triglyceride secretion from the liver, as previously reported (Amenta & Dominguez, 1965). Thus it is possible that reduced secretion, as a result of protein synthesis inhibition may be partly responsible for the steatosis.

138 Analysis of liver lipids by NMR did not reveal any changes in phosphatidylcholine suggesting that synthesis of VLDL was normal (Chapter

6).

In agreement with previous reports the data suggest that increased synthesis may be the major cause for fat accumulation, based on stim ulated PAP activity, although enhanced de novo fatty acid synthesis may contribute. The fact that steatosis appears to be an acute phenomenon may again imply that hydrazine itself is the effector. As acute doses of hydrazine are much larger than those given repeatedly the metabolic capability of the liver to detoxify large doses may be saturated.

7.3 THE EFFECT OF HYDRAZINE ON PROTEIN SYNTHESIS

Discrepancies in the published literature concerning the effect of hydrazine on protein synthesis prompted us to investigate further. The results from the studies described in this thesis (Chapter 5) tend to suggest that in vivo protein synthesis may only be transiently inhibited by hydrazine whereas in vitro in isolated hepatocytes hydrazine strongly inhibited protein synthesis. The rapidity of the effect in the latter system may imply direct action of hydrazine on the synthetic machinery of the cell. It is possible that the effect in both systems was due to nutritional deprivation caused by hydrazine-induced hypoglycaemia, which can impede the maintenance of cellular thiols. The apparent relationship between protein synthesis and GSH levels in vitro supports this.

The mechanism of inhibition is at present unknown. A possible cause is the production of ammonia, which is capable of inhibiting protein synthesis indirectly by inhibiting protein degradation. This may be more relevant in hepatocytes in vitro for reasons previously discussed. A further possibility is the production of GSSG, which can inhibit protein synthesis initiation.

139 7.4 GENERAL CONCLUSIONS i Hydrazine may not necessarily induce cytochrome P4502E1. However metabolism of hydrazine by this isoenzyme may lead to detoxication. ii Cytochromes P4501A1/2 and P4502B1/2 catalyse detoxication pathways of hydrazine. iii Hydrazine-induced lipid accumulation appears to be independent of cytochrome P4502E1 activity. The mechanism of fatty liver appears to be predominantly due to stimulated triglyceride synthesis (via PAP induction) and possibly a transient decrease in triglyceride secretion. iv Hydrazine strongly inhibits protein synthesis in vitro and only very weakly in vivo. In the former system there is no recovery w hereas in the latter protein synthesis is stimulated 24 hours post dose. The mechanism of action is not currently known.

V The data imply that hydrazine itself, and not a metabolite, may be responsible for toxicity.

140 7.5 FUTURE STUDIES

7.5.1 Verification of Involvement of Cytochrome P4502E1 in Hydrazine Metabolism

The importance of cytochrome P4502E1 in the metabolism of numerous compounds with the potential for causing cancer in humans cannot be over emphasised. It is therefore of the utmost importance to verify, once and for all, whether hydrazine induces, and is metabolised by this isoenzyme.

The first line of research should attempt to positively identify induction of cytochrome P4502E1 by repeated exposure to hydrazine utilising electrophoretic analysis (Western immunoblots) of liver microsomes, accompanied by determination of catalytic activity using specific marker activities, such as chlorzoxazone 6-hydroxylation or iV-nitrosodimethylamine N- demethylation.

The second line of research should attempt to demonstrate the metabolism of hydrazine by cytochrome P4502E1 in vitro and in vivo. Toxicological endpoints can be used to assess hydrazine metabolism in isolated hepatocytes additionally exposed to specific inhibitors of cytochrome P4502E1, such as isoniazid and DEDC (Lindros et al, 1990; Albano et al, 1991; Guengerich et al, 1991; Lauriault & O’Brien, 1991). Hydrazine disappearance in control and cytochrome P4502E 1-induced microsomes could also be assessed in the presence of inhibitors, which could include specific anti-cytochrome P4502E1 antibodies.

When administered in vivo isoniazid competitively inhibits the metabolism of chlozoxazone, demonstrated by reduced urinary excretion of chlorzoxazone metabolites. Metabolism of the latter compound resumes after isoniazid withdrawal (Zand et al, 1993). A similar approach could be utilised to determine if hydrazine is a substrate for cytochrome P4502E1 or not.

141 7.5.2 Further Investigations into Hydrazine-induced Fatty Liver

The studies described in this thesis utilised the time of maximal triglyceride accumulation (24 hours post-dose; Amenta & Johnston, 1962) as the time at which to assess changes in other lipids. Approximately 70% of total liver triglyceride accumulated at 24 hours is present in the liver 4 hours after a dose of 60mg/Kg hydrazine (Amenta & Dominguez, 1965). With this in mind perhaps the study described in Chapter 6 should be repeated but at an earlier time.

There are many causes of fatty liver in response to xenobiotics and triglyceride synthesis and secretion have been studied previously with respect to hydrazine. Another cause of steatosis is inhibition of endogenous triglyceride hydrolysis by lysosomal acid lipases in the liver, one of the modes of action of carbon tetrachloride (Kato & Nakazawa, 1987). This aspect has not been studied in relation to hydrazine.

7.5.3 Further Investigations into the Effects of Hydrazine on Protein Synthesis

Protein synthesis is a very complicated process and there are numerous stages at which the process can be disrupted. In order to determine whether the mechanism of action of hydrazine is dependent on amino acids protein synthesis should be monitored in vitro in isolated rat hepatocytes in suspension incubated in amino acid supplemented medium. Simultaneous determination of intracellular amino acids should indicate any disruption to transport mechanisms. Amino acid imbalance has profound effects on protein synthesis (Nishihira et al, 1993) and this is thought to be due to altered tRNA charging (Ogilivieet al, 1979). Individual tRNA’s can be measured by deproteination, cold phenol extraction and ethanol precipitation of liver/hepatocyte homogenate followed by amino acid analysis (Airhart et al, 1974).

Other factors such as oxidative stress, ATP depletion and calcium release can inhibit protein synthesis at the stage of initiation (Lyon & Kisilevsky, 1990;

142 Kimball & Jefferson, 1990; Haussinger et a/, 1993). This may be the stage at which hydrazine inhibits protein synthesis as the response, in vitro at least, may have been related to cellular GSH and ATP content. Polysomal disaggregation with a rise in monomer population is an indication of inhibition of peptide chain initiation. Polysomes and ribosomal subunits can be isolated, separated on sucrose gradients and measured spectrophotometrically (Flaim et al, 1982b; Lyon & Kisilevsky, 1990). In addition, ternary complex formation, which involves many different factors, can be assessed by measuring the binding of (^®S)-methionine-tRNA to 408 ribosomal subunits, which together constitute the initiation complex (Flaim et al, 1982b).

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172 APPENDICES

APPENDIX I Determination of Protein

Reagents: Alkaline Copper Reagent consists of: A 2% NagCOg 20g NagCOg + 4g NaOH in IL B 1% CUSO4 5g CuS04.5Hg0 in 500ml C 2% NaK Tartrate lOg NaK Tartrate in 500ml Prior to use mix 49ml A : 0.5ml B : 0.5ml C.

Sample Preparation: Microsomes - 250-fold dilution with deionised water. Crude Liver Homogenate -1:1 dilution with IM NaOH and further 100-fold dilution with deionised water. Hepatocytes (IxlOVml) - 0.5ml suspension spun and supernatant removed. Cell pellet dissolved in 0.5ml IM NaOH and digest diluted 50-fold with deionised water.

APPENDIX II Determination of ATP using Bioluminescence

Equipment: The sample is placed in a light-tight compartment (held at -25°C to reduce background interference) and light emitted during the reaction is detected in a photomultiplier tube. Prior to any sample readings a dark count is carried out to assess how much stray light is entering the sample compartment. This figure is then automatically deducted from the sample counts.

Reagents: ATP Buffer consists of: 80mM MgS04.7 Hg0 9.86g/500ml

173 lOmM KH2PO4 0.68g/500ml lOOmM NagHAsO^.THgO 15.6g/500ml On the day of use 1:1:1 quantities of the above solutions were mixed and the pH adjusted to 7.4 if necessary.

Luciferase: 1 vial of firefly lantern extract resuspended with 5ml deionised water. Insoluble matter sedimented by centrifugation at lOOOrpm for 5 minutes. Luciferase is light and heat labile and so the supernatant was stored in a dark container and held on ice at all times.

Sample Preparation: Liver - at autopsy an amount of liver (approximately Ig) was homogenised as quickly as possible into preweighed tubes containing 4ml of ice-cold TCA (10% w/v). The tubes were re weighed and immediately frozen at -80°C. Prior to analysis the samples were thawed, centrifuged and the supernatant diluted (xlO-20) with 10% TCA so that the counts did not exceed that of the top standard.

Hepatocytes - 0.5ml of cell suspension (IxlOVml) at chosen time points was added directly to an equal volume of 20% TCA (5x10® cells in 1ml 10% TCA). The samples were centrifuged and the supernatant used for the assay.

APPENDIX III Determination of Hepatic Reduced and Oxidised Glutathione

Sample Preparation: At autopsy approximately Ig of liver was homogenised in a preweighed tube containing sulphosalicylic acid ( 2ml; 4% w/v). The tube was reweighed and immediately frozen at -80°C. Prior to assay the samples were thawed and centrifuged (3,000rpm, 5 minutes) to pellet precipitated protein.

For estimation of reduced glutathione (total non-protein sulphydryls) 0.5ml

174 supernatant was diluted 2/3-fold with acid. No dilution was necessary for the estimation of oxidised glutathione.

APPENDIX IV Analysis of Hepatic Triglycerides

Reagents: 0.4% Alcoholic potassium hydroxide (KOH) - 0.4g KOH added to 95% ethanol (100ml). Made fresh on the day of use. 0.2% Chromotropic acid - 2.24g sodium salt in 200ml water. Separately 600ml concentrated HgSO^ gradually added to 300ml of water. Both solutions were kept on ice at all times. When cooled the acid solutions were mixed together and stored in the dark. Triglyceride Standard - 0.5g commercial com oil dissolved in 100ml chloroform (5mg/ml stock). To obtain a working standard of O.lmg/ml chloroform this stock was diluted 50-fold.

Sample Preparation: An aliquot (0.5ml) of liver homogenate (retained from microsome preparation and thus approximately lg/5ml) was added to 4g activated zeolite (dehydrated at 60°C for 24hrs) previously moistened with 8ml chloroform. A further 12ml chloroform was added and the tubes vigorously shaken at intervals for 20 minutes to extract neutral lipids.

The extract was filtered through fat-fi'ee filter paper and depending on the expected triglyceride concentration 0.2-1ml was pipetted into 5 glass tubes/sample. The chloroform was evaporated overnight at 35°C. Working standard (0-lml of O.lmg/ml commercial com oil in chloroform) was also pipetted into tubes and the solvent evaporated as above.

175 APPENDIX V Isolation of Rat Hepatocytes

Reagents:

Krebs-Henseleit (K+H) Buffer (x2 concentrated stock): 100ml 16.09% NaCl 80.45g/500ml water 75ml 1.1% KCl 5.5g/500ml 12.5ml 0.22M KHfO^ 7.49g/500ml 25ml 2.74% Mg804.7Hg0 6.85g/250ml 50ml 0.12M CaCl2.2H20 8.82g/500ml 392.5ml UHQ water 500ml 0.97% NaHCOg 4.85g/500ml These solutions were mixed and gassed with carbogen 95% 02:5% CO2) for 5-10 minutes with the exception of NaHCOg which was added at the end to prevent precipitation of CaCOg.

Working K+H solutions made fresh on day of use: 150ml K+H stock 150ml UHQ water 0.9g HEPES (final concentration 12.6mM) The HEPES was dissolved and the solution gassed with carbogen for 2-3 m inutes.

K+H Alb - Ig albumin was dissolved in 100ml of working K+H

Hanks balanced salt solution (xlO concentrated stock): 80g NaCl 4gK C l 2g MgS0,.7H20 0.6g N a2HP04.2H20 0.6g KH2PO4 Made up to IL with ultra high quality (UHQ) water.

176 W orking Hank solution was prepared on the day of use: 50ml Hanks xlO stock 450ml UHQ water 1.5g HEPES (final concentration 12.6mM) 1.05g NaHCOg (final concentration 25mM) Mixed together and gassed with carbogen for 2-3 minutes.

This solution was then divided as follows: H ank I: 300ml working Hank; 68.4mg EGTA; 2g albumin. H ank II: 200ml working Hank; 2ml 5.88% CaClg. Collagenase (50mg) was weighed and kept at -20°C until added to 100ml Hank II immediately prior to use.

The above solutions were adjusted to pH7.4 with IM NaOH.

APPENDIX VI Determination of Lactate Dehydrogenase

Reagents: Pyruvate/Phosphate buffer - 3.75mg pyruvate was dissolved in 100ml of 0.05M phosphate buffer (6.8g KH^PO^ in IL; pH7.5).

Sample Preparation: For total LDH activity an aliquot of hepatocyte suspension (0.5ml; 5x10® cells) was taken after a 30 minute preincubation period (time zero) and sonicated to lyse the cells. The samples were spun to remove cell debris. Thereafter an aliqout of hepatocyte suspension was taken at each designated time point and spun to pellet the cells. The supernatant was retained for LDH estimation.

177 APPENDIX VII Determination of Reduced Glutathione in Isolated Hepatocytes

Reagent: Phosphate/EDTA Buffer: 6.8g KH2PO4 (O.IM) and 0.93g EDTA (5mM) made up to 450ml. The volume was made up to 500ml after adjusting the pH to 8 with IM NaOH. Prior to use 5.45ml of IM NaOH was added to 500ml of phosphate/EDTA buffer.

Sample Preparation: Hepatocyte suspension (0.5ml of 1x10® cells/ml) was centrifuged and the supernatant decanted and retained for LDH analysis (see Appendix VI). The pellet was resuspended in 6.5% TCA (0.5ml), centrifuged and the acid supernatant used for the determination of reduced glutathione.

APPENDIX VIII Determination of DNA - Diphenylamine Method

Working diphenylamine reagent: 1.5% (w/v) diphenylamine in glacial acetic acid with 1.5ml of concentrated H2SO4 added per 100ml. Make up a solution of aqueous acetaldehyde (16mg/ml) and add 0.1ml to every 20ml of the above reagent.

178 APPENDIX IX Immunohistochemical Staining of Cytochrome P4502E1 - Report from US Airforce Pathologist

Subject: Hydrazine Dosed Livers Date: 9 January, 1995

Rat liver tissues from study J3 were immunohistologically stained for PasniiEi and quantified using computer-based image analysis.

Method: Four rats were exposed to 2.5 mg/kg hydrazine (free base) in drinking water for 10 days and four additional rats served as pair-fed controls. The livers were fixed in 10.5% formalin for 24 hours and mounted in paraffin for histologic sectioning. A single sectioned tissue from each rat was processed using Biogenex reagents (San Ramon. CA)

for immunohistologic localization of P 45011E1 isoenzyme.

Tissue image analysis was performed using a Leica Quantimet 570C Image Analysis System (Deerfield, IL).. Positive labeled tissue area was directly quantified by color detection.. Total tissue area was calculated by thresholding the background area (that area without any tissue) and subtracting it from the total frame area examined. Positive labeled tissue fraction was calculated as positive labeled tissue area divided by total tissue area. Each liver section was analyzed at three random tissue areas at 100x magnification. The results were statistically analyzed to determine the mean, the standard deviation and the variance between individual measurements, between individual liver sections and between treatment groups.

Results: Immunohistologic localization of P^snuEi isoenzyme was strongest in the centrolobular and the mid-lobular regions and was generally absent in the periportal regions (see accompanying kodachromes). Statistical analysis failed to show a significant difference between the control group and the treated group.

John H. Grabau, DVM Pathologist Chief, Image Analysis Section

179 JHÎI--Ü7-95 WED 14:21 ARM LAB TOX DIV FAX NO, 513 255 1474 P. 01

s t /o-v*.

June 6 , 1995 /o i f - Dear Dr. Timbrell,

1 have completed the immunohistochemical staining of kidney and liver specimens from the second study with hydrazine, The specimens stained nicely after some preliminary adjuscments. Additionally, I did a routine histopathologic evaluation w hich failed to detect any apparentt r e a t m e n t - induced lesions in the liver or kidney. Subjectively, it was impo.^sible to say with certainty that there was a hydrazine-induced difference in expression of 2E1 detected by immunocytochemistiy. An attempt to quantify a significant difference in 2E1 detection is underway. I have suggested a strategy to Dr. Grabau (see page 4), our image analyst He will do his best (detecting relative staimng intensity is breaking new ground)!

I have recently written a paper (Fundamental and Applied Toxicology, in press) on the oncogcmc potential of inhaled hydrazine in the nose o f rats and hamsters, correlating the acute subchronic, and chronic lesions induced by hydrazine using a concentration x time saidy design. The manuscript should be published in the July issue. F II send you a copy if you like,

Sincerelv,

R L.A.TENDRESSE, DVM, PhD Diplomate, College of American Veterinary Pathologists

1 8 0 APPENDIX X NMR Spectra and Peak Assignments from Chloroform Extracts of Hepatic Lipids

Lipid Extract Without Separation

/L 1 J. . i'Jv, ^ __

Neutral Lipids and Cholesterol

4 J'\ Jh L ( liW Non-esterifled Fatty Acids

.Jj_.

Non-acidic Phospholipids

r Wv__

Acidic Phospholipids

7. no C. 00 s. 00 4. 00 00 00 I . 00

181 Peak Assignments of Lipids from NMR Spectra

Resonating protons in bold:

Unsaturated fatty acids 5.4ppm -CH=CH- (vinyl protons) Triglyceride 4.35ppm H-CH-OCOR

Phosphatidylinositol 3.6 ppm CHO- (CH0H)2-CH0H- (CH0H)2 Phosphatidylcholine 3.2ppm - 0P03-CH2CH2N^(CH3)3 Phosphatidylethanolamine 3.Ippm -OPO3-CH2CH2NH3+

Polyunsaturated fatty acids 2.8 ppm -CH3=CHCH2CH=CHCH2CH=CH

Linoleic acid 2.7 5ppm -CH=CHCH2CH=CHCH2 - (CH2)6C00' Docosahexaenoic acid 2.4ppm -CH=CHCH2CH2C00 ' Arachidonic acid 2.Ippm -CH=CHCH2CH2CH2C00’

Total fatty acids O.BSppm CH3- (CH2)n-C H 2-CH 2-COO'

Cholesterol 0.7ppm C38 methyl protons

182 PUBLICATIONS

Delaney, J., and Timbrell, J.A. Modulation of Hydrazine Toxicity In Vitro Using Various Inhibitors and Inducers of Cytochrome P4502E1. Hum. Exp. Toxicol. 13 (4): 292 (1994) Abstract

Delaney, J., and Timbrell, J.A. Repeated Exposure to Hydrazine In Vivo Protects Against Hydrazine Toxicity In Vitro. Hum. Exp. Toxicol. 13 (9): 646 (1994) Abstract

Delaney, J., and Timbrell, J.A. Effects of Repeated Exposure to Hydrazine: Biochemical Perturbations and Effects on Acute Hydrazine Toxicity. Hum. Exp. Toxicol. 14 (4): 755 (1995) Abstract

Delaney, J., Zloh, M., Gibbons, W.A., and Timbrell, J.A. The Effect of an Acute Dose of Hydrazine on Liver Lipids. Proceedings from lUTOX Meeting, Seattle (1995) Abstract

Waterfield, C.J., Delaney, J., and Timbrell, J.A. Evaluation of In Vitro Biochemical Markers of Hydrazine Toxicity in Hepatocytes: Comparison with In Vivo Data. Proceedings from lUTOX Meeting, Seattle (1995) Abstract

Delaney, J., and Timbrell, J.A. Role of Cytochrome P450 in Hydrazine Toxicity in Isolated Hepatocytes In Vitro. Xenohiotica 25 (12): 1399-1410 (1995)

Waterfield, C.J., Delaney, J., Ghatineh, S., Preece, N.E., Jenner, A.M., and Timbrell, J.A. A Comparison of the Biochemical Effects of Hydrazine In Vivo in Rats and In Vitro in Isolated Rat Hepatocytes. Hum. Exp. Toxicol. 15 (2): 149 (1996) Abstract

183 XENOBIOTICA, 1995, VOL. 25, NO. 12, 1399-1410

Role of cytochrome P450 in hydrazine toxicity in isolated hepatocytes in vitro

J. D EL A N EY and J. A. TIM BR ELL* Toxicology Department, School of Pharmacy, L'nh ersity of London, 29/39 Brunswick Square, London W CIN l.ÂX, UK

Receix ed 3 M a y 1995

1. Hepatocytes. isolated from the control, diethyldithiocarbamate (DEDC). acetone, isoniazid and hydrazine pretreated rat, were incubated with hydrazine (8-20mM/ for 3 h. Hydrazine caused a dose-dependent loss of \ iabilit), leakage of LDH, depletion of GSH and .ATP and an inhibition of the incorporation of 'H-leucint into protein, 2. Pretreatment with DEDC increased, whereas hydrazine and acetone pretreatments decreased the cytoxicity and biochemical effects of hydrazine. Pr etreatment with isoniazid slightly increased hydrazine cytotoxicity. .Acetone pretreatment reduced the inhibition of protein synthesis caused by hydrazine compared to the control.

3. 4-Nitrophenol hydroxylase activity (P4302E1 ) correlated with viability, LDH leakage. .ATP and GSH depletion in cells from the control, DEDC, acetone and hydrazine pretreated rats.

4. The activities of PROD (P4502B1 ) and EROD (P4501 .A 1, 1 .A21 also correlated with the above parameters for all treatments. The results suggest that three isoenzymes may be involved in the detoxication of hydrazine. Protein synthesis inhibition did not correlate with the activities of anv of the enzvmes measured.

Introduction Hydrazine is a highly reactive chemical that has a wide spectrum of uses including aerospace fuels, corrosion inhibitors, dyes and photographic chemicals. H\ drazine and its deriv atives have pharmaceutical use including hydrazine sulphate, used to treat cancer cachexia, isoniazid, an antitubercular agent and hydralazine, an antihypertensive agent. Exposure to hydrazine results from industrial use and as a metabolite of the drugs isoniazid and hydralazine. There are many toxic effects associated with exposure to hydrazine such as liver damage, particularly fatty infiltration (Amenta and Johnston 1962), hypoglycaemia (Fortney 1966) and central nervous system disorders such as convulsions (Witkin 1955) and interference with intermediary metabolism at various sites (Moloney and Prough 1983). There have been many studies investigating the metabolism of hydrazine and several metabolites have now been identified (figure 1). Unchanged h\ drazine (McKennis et al. 1955, Preece et al. 1992), mono- and diacetylhydrazine (Noda et al. 1985a), pyruvate and 2-oxoglutarate hydrazones (Moloney and Prough 1983) have all been detected in urine. Nelson and Gordon (1982) reported the formation of 1,4,5,6-tetrahydro-oxo-3-pyridazine carboxylic acid (THOPC), a cyclized derivative of oxoglutarate hydrazone, and Sanins et al. ( 1992) and Preece et al. (1991) also detected this in urine from the hydrazine-dosed rat using nmr. Nitrogen from hydrazine has been detected in expired air (Springer et al. 1981 ), Nelson and Gordon 1982) and has been shown to be incorporated into ammonia and urea (Preece et al.

* .Author for correspondence.

t|i)4V-S254, V5 SI (MX) t' 1943 Tiivlor & Francis Ltd. 1400 y. Delaney and J . A. Thnbrell

Cytochrome P450 ?

isozymes 2 E 1 , 231, 1 A 1 NH=NH

Acétylation NHj-NHj NH2-C-NH2

CH3 C-NHNH2 Pyruvate

0 a-Ketoglutarate COOK Acétylation

0 C=NHNH2

CH3 C-NHNH-CCH 3

COOH

Figure 1 . Esrablished and possible routes of metabolism for hydrazine.

1901). This suggests the N -N bond is clea\ ed in vivo but whether the process is enzymatic or chemical is presently unknown. A significant proportion (approxi mately 25"t)) of a dose of hydrazine is still of unknown fate (Springer et al. 1981 ). Hepatic P450 has been implicated in hydrazine oxidation (Noda et al. 1985a,b, 1988) but the isoenzymes responsible are unknown. Timbrell et al. ( 1982) and Jenner and Timbrell (1995) studied the disappearance of hydrazine from hepatic microsomal preparations and concluded that hydrazine was oxidised by the P450 enzyme system. The product was not identified. Several studies in vivo and in vitro have also been carried out to determine the effect of induction and inhibition of P450 isoenzymes on hydrazine toxicity. Noda et al. (1985a) pretreated rats with phenobarbitone and rifampicin and found decreased amounts of intact hydrazine excreted in the urine after a dose of h\ drazine sulphate. In hepatocytes isolated from rats pretreated with the enzyme inducers phenobarbitone and rifampicin there was increased disappearance of h\ drazine, whereas pretreatment with the enzyme inhibitors piperonyl butoxide and met\ rapone decreased the disappearance (Noda et al. 1987). Studies in vivo (Timbrell et al. 1982) showed that pretreatment of rats with phenobarbitone and piperonyl butoxide decreased and increased hydrazine induced lipid accumulation respectively. Induction of P4502E1 by acetone and isoniazid (Jenner and Timbrell 1994a) has been shown to increase the toxicity of hydrazine in vivo, increasing lipid accumulation compared with control animals. Induction of P4502E1 has also been shown to increase the toxicity of other chemicals such as carbon tetrachloride ( Ivindros et al. 1990). dimethylnitrosamine (Lauriaultet al. 1992) .V-nitrosomethylaniline (Quan et al. 1992) and acetaminophen (Koop et al. 1982). Repeated exposure to hydrazine itself in vivo has been shown to induce P4502E1, as indicated by aniline hydroxylase and 4-nitrophenol hydroxylase activity (Ghatineh et al. 1992, Jenner and Timbrell 1994b). Role of P450 071 hépatocytes 1401

The aim of this study was to ascertain the role, if any, of P450 isozymes in the toxicity and biochemical effects of hydrazine using isolated rat hepatocytes in vitro.

Materials and methods Chemicals Adenosine 5'-triphosphate (disodium salt), albumin (bo\ine fraction V), colla^enase (from Clostridium histolyticiim, type 1), dicumarol, dietbyldithiocarbamate (sodium salt). fireHy lantern extract, glutathione (reduced form), HEPES, hydrazine hydrate, isonicotinic acid hydrazide (isoniazid), 1.-leucine, N.ADH (yeast grade 111, disodium salt), NADPH (tetrasodium salt), 4-nitrocatechol (crystalline), 4-nitrophenol (spectrophotometer grade, crystalline), o-phthaldialdehyde, pyruvic acid (sodium salt, type II crystalline) were obtained from Sigma Chemical Co. (Poole, UK). Acetone was obtained from BDH (Poole, UK). l[3,4,5-' H(N)]-leucine (37MBq, 1 mCi/ml) was obtained from DuPont (NEN) (Stevenage, UK).

Animal pretreatment Male Sprague-Daw ley rats (200-3(.)0 g) w ere used and were pretreated prior to isolation of hepatocytes in the follow ing ways: w ith acetone (20",, \ /\- in distilled water, 5 ml/kg by gavage 24 h prior to isolation ); dietbyldithiocarbamate [DEDC] (75() mg/kg, i.p. in distilled water, 24 h prior to isolation); isoniazid ( 1 22 mg/kg in drinking water for 20 days prior to isolation); hydrazine (2 3 mg/kg in drinking water for 10 days prior to isolation). Control animals recei\ ed no pretreatment.

Hepatocyte preparation Hepatocytes were isolated by collagenase perfusion according to the method of Moldeus ei al. (197S). Cells w ere subsequently incubated in rotating siliconized flasks in Krebs-Henseleit buffer at 37°C. under an atmosphere of 95"„ 0:/5"« CO:, at a cell density of X1 10^'ml. Hydrazine was added to the incubation buffer to achieve a final concentration of 8-20m.M. Samples were taken after a 30-min preincubation period then at hourly intervals up to 3 h.

Biochemical determinations \ ’iability w as assessed by trypan blue dye exclusion and leakage of lactate dehydrogenase (LDH ) into the incubation medium. Acti\ ity of this cytosolic enzyme was determined in cell-free supernatant using the method of Bergermeyer et al. (1965). Total activity was measured in a separate sample, which had been sonicated to lyse the hepatocytes. Reduced glutathione (GSH) was measured in trichloroacetic acid (TCA) extracts of hepatocytes w ith o-phthaldialdehyde using the fluorimetric method of Hissin and Hi If (1976). ATP was measured by luciferase-linked bioluminescence (Stanley and Williams 1969) in TC.A extracts of hepatocytes using a firefly lantern extract, E ROD, an indicator of P4301 Al activity and PROD, an indicator of P4502B1 activity, were assessed by following the rate of production of resorufin at 37'C in hepatocyte homogenates using the methods of Lubet et al. ( 1985) and Burke ef al. (1985). 4-Nitrophenol hydroxylase (an indicator of P4502E1) activity was determined by following the production of 4-nitrocatechol from 4-nitrophenol at 37°C in the presence of NADPH, using the spectrophotometric method of Hammond and Fry ( 1990). The reaction was terminated with Oô-M perchloric acid, the samples centrifuged and the product measured at 5 10 nm in 1 ml supernatant after the addition of NaOH (IOm; 01 ml). Protein synthesis was determined by measuring the incorporation of '’H-leucine into cellular protein (Seglen 1978). ^H-leucine (1 ;/Ci in 5 mM unlabelled leucine/ml incubation buffer) was added to each flask after the 30-min preincubation period. Aliquots of cell suspension (0 5 ml), taken immediately after addition of radiolabel then at hourly intervals during the incubation w ith hydrazine, were added directly to TCA ( 10"„; w/v) to precipitate the protein. The protein pellet was washed (five times) with TCA (10%; w /\ ) and subsequently dissolved in NaOH (1 .M; 0-5 ml). An aliquot of protein digest (200;d) was analysed by scintillation counting to assess incorporation of radiolabel.

Statistical analysis Statistical analysis was assessed using the Student's and Dunnett's /-tests to compare pretreated w ith control values.

Results Hydrazine caused a concentration-dependent loss of viability over time (3 h) in cells isolated from untreated and pretreated rats (figure 2). The data at the three hour time point only is shown for this and all the other parameters measured. In all cases a concentration of 20-mM hydrazine was cytotoxic, However, loss of viability was significantly reduced by pretreatment with acetone and hydrazine compared with non-pretreated cells. In contrast, pretreatment with DEDC 1402 J. Delaney and J. A. Timbrell

100 -1

90 - 0)

E 80 -

0 ro - A ♦

0) 60 - to > 50 -

« 40 -

30 -

20 - CD < 0 > 0 0 5 10 15 20

concentration of hydrazine (mM)l

Figure 2. Effect of various pretreatments on loss of viability in isolated hepatoctyes after 3-h exposure to different concentrations of hydrazine. Pretreatments: # . control; ▲. acetone; ■. DEDC; ♦ , INH; hydrazine. Values are means ± SEM; = 4—5. “p < 0 05; ^p<0-01; significantly different from non-pretreated control at the same concentration by Dunnett’s test. *p < 0 05; •• p < 0 01 ; ••• p < 0-001, significantly different from non-exposed hepatocytes by paired /-test. significanrly increased the cytotoxicity of 8-mM hydrazine at 3 h (figure 2) and loss of viability was apparent at 2 h in cells exposed to 16 and 20-mM hydrazine (data not shown). The apparent increase in cytotoxicity in isoniazid-pretreated hepatocytes was not statistically significant (figure 2 ). Hydrazine caused a concentration-dependent leakage of LD H over time in hepatocytes from non-pretreated rats (figure 3). Leakage of L D H was significantly increased by pretreatment with D ED C compared with control cells exposed to the same concentration of hydrazine (12 mM hydrazine). The leakage of LD H from hepatocytes from the acetone pretreated rat exposed to 20-mM hydrazine was not significantly different from control hepatocytes not exposed to hydrazine. However, pretreatment with acetone significantly reduced the extent of LDH leakage compared with non-pretreated cells at an exposure level of 20-mM hydrazine (figure 3). As previously described (Preece et al. 1990) hydrazine causes a concentration- dependent depletion of ATP. This effect was, however, increased by DEDC pretreatment relative to non-pretreated cells exposed to the same concentration of hydrazine ( 8 and 12-mM hydrazine) and significantly decreased by acetone or hydrazine pretreatment (at 12-20-m.M hydrazine) (figure 4). Glutathione was significantly depleted by h\ drazine in a dose dependent manner (figure 5) as previously described (Ghatineh et al. 1992). This depletion was significantly decreased by pretreatment with acetone or hydrazine (8-20 m.M) (figure 5). In hepatocytes exposed to hydrazine there was a decrease in the incorporation of radiolabelled leucine relative to control cells indicative of inhibition of protein Role of P450 on hepatocytes 1403 O 500 Q) E

ro 400

Q) 350 ro > 300

0) 250 O) ro 200 ro CD 150

100 0 5 10 15 20

Concentration of hydrazine (mM)

Figure 3. Effect of various pretreatments on leakage of LDH by isolated hepatocytes after 3-h of exposure to different concentrations of hydrazine. Pretreatments: # . control; A, acetone; ■ . DEDC; ♦ , INH; hydrazine. \'alues are meansr SEM; n = +-5. < 0 05; significantly different from non-pretreated control at the same concentration by Dunnett's test. • p < 0 05; •• p < 0 01, *** p < 0 00]. significantly different from non-exposed hepatocytes by paired ?-test < 0 01 ; significantly different from non-pretreated control of same concentration by students /-test. synthesis. This inhibition was not clearly dose dependent over the concentation ranges studied nor was it maximal (figure 6 , inset). All of the pretreatments except hydrazine decreased the inhibition of protein synthesis although this was only statistically significant in the case of acetone pretreatment (figure 6 ). The effects of the various pretreatments on EROD, PROD and 4-nitrophenol hydroxylase activities are shown in table 1. D ED C pretreatment caused a significant decrease in all three enzyme activities. Acetone pretreatment significantly increased EROD, and both isoniazid and hydrazine pretreatment significantly increased 4-nitrophenol hydroxylase. However, isoniazid and hydrazine also significantly decreased PROD and EROD respectively. When the activities of the P450 isozymes were plotted against the biochemical parameters measured, significant correlations were observed (table 2, figures 7-9).

D iscussion The results clearly show that pretreatments of animals that alter the activities of P450s as indicated by EROD, PROD and 4-nitrophenol hydroxylase activities influence hydrazine toxicity and its biochemical effects in isolated hepatocytes in vitro (figures 2-5). Thus, thresholds for the cytotoxicity and the biochemical effects are altered. Furthermore, the data indicates that loss of viability, ATP and GSH depletion are all correlated. For example, A TP depletion is increased by DEDC pretreatment at a concentration of hydrazine ( 8 mM), which is not cytotoxic as 404 y. Delaney and J. A. Tinihrell

130 - _ 120 J '

(D 100 P i

ro Q)

ro i >

2 Û. H 2 < ïi

10 15 20

Concentration of hydrazine (mM)

'leure 4. EffecT of various pretrcatments on depletion o: Al'P in isolated hepatocytes after 3-h exposure to different concentrations of hydrazine. Pretreatments: # , control; À. acetone; ■. DEDC, ♦ . INH; %, hydrazine. \ ’alues are means % SEM. u = 4—5. ""p < 0 03; ^ p < 001; significantly different from non-pretreated control at the same concentration by Dunnett's test. “p< 0()5; ••p < 0-01; ♦’••p<0-()01. significantly different from non-exposcd hepatocytes by paired /-test

110-1 100 o

30 X 00 20 a-*-Te O 2 •*

0 5 10 15 20

Concentration of hydrazine (mM)

Figure 5. Effect of various pretreatments on depletion of GSH in isolated hepatocytes after 3-h exposure to different concentrations of hydrazine. Pretreatments; • . control; A, acetone; ■. DEDC; ♦ , INH; hydrazine. X'alues are means ± SEM; //= 4—5. ‘‘p<0-05; ^p < 0 01; significantly different from non-pretreated control at the same concentration by Dunnett's test. * p < 0 05; * * p < 0 01; * **p < 0 00l, significantly different from non-exposed hepatocytes by paired /-test. Role of P450 on hepatocytes 1405

115 1 I

c traiior o: o 90 J u ! 2» 75 z i 00 oo 60 -j • 00 C 30 -

Concentration of hydrazine (mM)

1'im.irt 6. Inhibitinn of prottin sxnthesis in isntnied heputocNtes after 3-h exposure to diîîerent concentrations of hydrazine t inset I. Effect of \ arious pretreatments on inhibition of protein synthesis (main histoeram). N'alues arc expressed as "n incorporation of'H-leucine into cells not exposed to h\ drazine. Pretreatments: .control: S . acetone: ^ . L4vDC. ■, I.\H; E . h\ drazine \'alues are means z SEi\l: n = -^3. ''/)< (l-i'S; sieniricantly different from non-pretreated control at the same concentration by Dunnett's test. * p < P (l3: siunihcantl\ different from non-pretreated hepatocv tes by Student's /-test.

I'able 1. Microsomal enzyme acti\ ities in isolated hepatocytes.

Eithoxy resorufin Pentoxy resorufin 4-Nitrophenol Number of .Animal o-deethylase w-depentylase hydroxylase replicates pretreatment (pmolmin lO'' cells) (pmolmin, 1 d" cells) (nmol'min 111" cells I

4 none 74 9 z : 4 61 7 z 4 7 (1 57 z 0 (12 4 DEDC 26-8 z 2-6‘ *-' 21-7 z 2 3**-' 0 (18 z UOl" 4 2E1 3 EROD,PROD acetone 1141 z 14 7* 67 3 z 14 8 0 81 z 017 3 isoniazid 65 3 z 11 5 31-4 z 31**" 2 73 z 0 24**" 4 h> drazine 65 6 z 2 2' 517 z 12 0 45 z 0-06"

Results are expressed as means r S.E.M. *p = 0-().s Dunnett's /-test. /) = b ill Dunnett's /-test. p = 0 (15 Students /-test. 1406 y. Delaney a n dj. A. Timbrell

Table 2. Correlation between P450 activities and biochemical parameters in isolated hepatoc> tes.

Indicators of P450 activity

PNPH Biochemical,toxicology parameter EROD PROD - INH - INH

\'iability r = 0-60 0-73 0-08 0-79 P < 0-01 <0-001 0 76 0-001 LDH leakage r = - 0-54 - 0-59 - 0-23 - 0 66 p < 0-05 0-01 0 34 <0-01 .ATP r = 0-66 0-65 0 15 0-74 (% IN IT) p<0-01 <0-01 0 54 <0-01 GSH r = 0-46 0 53 0 13 0-72 ("u I NIT) p = 0-06 <0-05 0-59 <0-01 Protein synthesis r = 0-23 - O-OS 0-19 - 0 20 ("o control) p = 0-36 0-76 0-43 0 46

EROD. ethyoxyresorufin o-deethylase; PROD, pentoxyresorufin o-depentylase; PNPH, 4-nitro- phcnol hydroxylase; INH, isoniazid. r. Correlation coefficient from linear regression. p. if < 0 05, statistically significant correlation.

3.4 -j

2.5 -, — Ü I 2.4 4 I l| %9 4 ■ c Ô 1.5 - II o 1.0 - - r iz ;> 0.5 ^ I: 0 -J 0 10 20 30 40 50 50 70 80 90

% Viability

Figure 7. Correlation between 4-nitrophenol hydroxylase acti\ ity (P4502E1) and "» \ lability (trypan blue dye uptake). Each point represents 4-nitrophenol h\ droxylase acti\ ity in cells prior to hydrazine exposure compared with loss of \ lability in cells after exposure to 1 2-m.\l hydrazine for 5 h (obtained from separate hepatocyte isolations). Data from untreated, DEDC acetone and hydrazine pretreatments: r = 0-795, p < 0-001. Correlation with data from all pretreatments, including isoniazid r = 0-0766, p = 0-755. Isoniazid data represented by stars.

indicated by LD H leakage. Thus the correlation does not seem to be due simply to increased cytotoxicity and consequential ATP and GSH depletion. The data also show that hydrazine causes a significant inhibition of the incorporation of ■'H-leucme, indicating inhibition of protein synthesis, an effect not previously shown in isolated hepatocytes in suspension. This inhibition occurs at a much lower concentration of hydrazine (Oôm.M) than the other biochemical effects, is not dose-dependent, or has a \ery steep dose-response relationship in the Od-O S-m.M range. Furthermore, except in the case of acetone pretreatment, the changes in Role of P450 on hepatocytes 1401

= 100 0) a

O 80 -

c Ê 60 -J o E Q. 40 -

u IV Oo cr 0 -J r CL 0 10 20 30 40 50 60 70 80 90

% Viability

Figure 8. Correlation between PROD actn ity ( P4502B1 ) and \ lability (trypan blue dye uptake). F.aeh point represents PROD activity in cells prior to hydrazine exposure compared with loss of viability in cells after exposure to 1 2-m\I hydrazine for 3 h (obtained from separate hepatocyte isolations i. Data from untreated, DEDC, acetone, hydrazine and isoniazid pretreatments are shown. < 0 0 0 1 , r = 0-727.

- 150 -, 140 4

c 120 -

E 100 - "Ô E 80 - CL 60 -

40 - «u Q 20 - O c LU 0 10 20 30 40 50 60 70 80 90

% Viability

Figure 9. Correlation between EROD activity (P4501 Al ) and viability (trypan blue dye uptake). Each point represents EROD activ ity in cells prior to hydrazine exposure compared with loss of viability in cells after exposure to 1 2-m.\I hydrazine for 3 h (obtained from separate hepatocyte isolations). Data from untreated, DEDC, acetone, hydrazine and isoniazid pretreatments are shown, r = 0-602, p < 0-01, 1408 y. Delaney and J. A. Timbrell inhibition of protein synthesis caused by the pretreatments did not correlate with changes in the other biochemical parameters. For example, increases in toxicity due to D ED C pretreatment were not accompanied by increased inhibition of protein synthesis. These data suggest that inhibition of protein synthesis is not a cause of the cytotoxicity and is not related to the other biochemical effects. The effects of the pretreatments also suggest that hydrazine may be detoxified by metabolism via P450s. This can also be seen from the correlations between viability and enzyme activity (table 2. figures 7-9). Thus hepatocyte preparations \\ ith the lowest activity for each of the three enzyme activities measured showed the greatest loss in viability and vice \ ersa. Similar correlations were seen between all the other parameters of toxicity except protein synthesis (table 2). Thus, D ED C pretreatment, which at the dose used inhibited all three of the isoenzymes (table 1 ), increased hydrazine toxicity. In vivo the P450 inhibitor piperonyl butoxide exacerbated hydrazine toxicity, manifested as accumulation of triglycerides and increased depletion of GSH and ATP in the liver (Jenner and Timbrell 1994a). Consistent with this is in vitro data from rat liver microsomes in which P450- mediated hydrazine metabolism was detected and inhibited by piperonyl butoxide (Jenner and Timbrell 1995). Conversely induction of P450 with phenobarbitone (PROD activity; 2B1) and with ^-naphthoflavone (EROD activity; 1A1/1A2) lowered hepatotoxicity in vivo. Acetone and isoniazid pretreatment, which induced 4-nitrophenol hydroxylase activity (2E1 ), increased the toxicity of hydrazinein vivo as indicated by triglyceride accumulation, but there was no significant increase in depletion of GSH and ATP (jenner and Timbrell 1994a). The results obtained in vitro with isoniazid pretreatment are consistent with this, showing increased cytotoxicity but no significant alteration in ATP and GSH depletion compared with controls. In this study acetone pretreatment, despite being the same as used in the in vivo study (Jenner and Timbrell 1994a), only significantly increased EROD (1A1/1A2) and protected against hydrazine toxicity in isolated hepatocytes. The reason for the discrepancy between the in vivo and in vitro observations may be due to the induction of EROD only and absence of any effect on 4-nitrophenol hydroxylase or to different mechanisms of toxicity in vitro and in vivo. Howe\ er. hydrazine pretreatment also reduced the cytotoxicity and biochemical effects of hydrazine exposure in hepatocytes yet induced 4-nitrophenol hydroxylase actu ity (2E1) approximately two-fold and reduced the activity of EROD (lA 10A 2i (significantly) and PROD (2B1) (table 1). Although isoniazid pretreatment also induced 4-nitrophenol hydroxylase activity (2E1) (five-fold) and also reduced the activities of EROD (1A1/1A2) and PROD (2B1) (significantly) (table 1) toxicity was slightly increased. Thus hydrazine and isoniazid pretreatments caused qualitatively similar effects on the three isoenzymes but dissimilar effects on the toxicity of hydrazine to hepatocytes. A correlation was found between 4-nitrophenol hydroxy lase activity and viability but only if the data for isoniazid was excluded (table 2, figure 7). The data points for hepatocytes from the isoniazid pretreated rat were clearly separated from the remainder (figure 7). This could be explained by inhibition of the isozyme as suggested by human in-vivo data with isoniazid and chlorzoxazone (Zand et al. 1993). Then if 2E1 was responsible for detoxication inhibition by isoniazid would block the further metabolism of hydrazine. .•Mternatively 2E1 may not be in\ olvcd with hydrazine metabolism and the changes are due to effects on other isozymes. In summarv this studv has shown that alteration of the activitv of P450 isozvmes Role of P450 on hepatocytes 1409 manifested as EROD, PROD and 4-nitrophenol hydroxylase acti\ ities influences the cytotoxicity and biochemical effects of hydrazine in isolated hepatocytes. Although the effects observed in vitro are not exactly comparable with those described in vivo, overall the data from both systems suggests that hydrazine may be metabolized and detoxified via several pathways catalysed by several P450 isozymes. Currently it is not possible to conclude definitively which isozymes are responsible for detoxication or if 2E1 is responsible for metabolic activation or detoxication. Taken together the in vivo and in vitro data suggests that metabolism of hydrazine by all three P450 isozymes leads to detoxification and therefore the cytotoxicity of hydrazine could be due to the parent compound. Consequently, altering the balance by induction and inhibition will increase or decrease the toxicity. Further studies in vitro with more specific inhibition of P450 isozymes or use of isolated P450 isozymes may help to clarify the data.

Acknowledgement This work was supported by a grant from EOARD, US Air Force.

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Modulation of Hydrazine Toxicity In Vitro Using Various Inhibitors and Inducers of Cytochrome P4502E1.

Jane Delaney* & John A.Timbrell, Toxicology Department, School of Pharmacy, 29 Brunswick Square, London WCIN 1AX.

Hydrazine is a hepatotoxic compound used extensively in industry and is a metatxilite of certain drugs, including isoniazid. Hepatotoxicity is manifested as steatosis accompanied by ATP and glutathione (GSH) depletion. Previous work has shown that induction of P4502E1 causes an increase in hydrazine toxicity in vivo (Jenner and Timbrell, 1991). The aim of this study was to assess the effect of induction and inhibition of P4502E1 on hydrazine toxicity in vitro Male Sprague Dawley rats (200-300g) were pretreated with acetone(2C% v/v,po,24h) or isoniazid (0.1% w/v,dhnking water,20 d), both inducers of P4502E1, the former also inducing P4502B1 (Ronis & Ingleman-Sundberg, 1989) or dietbyldithiocarbamate [DEDC](750mg/kg,ip,24h) an inhibitor of both P4502E1 and P4502B1 (Launault e t al. 1992). Hepatocytes were prepared from control and pretreated rats and incubated with 8,12,16 and 20 mM hydrazine hydrate for 3 hours. Samples were taken after a 30 min preincubation pehod then at hourly intervals and subsequently assayed for ATP, GSH and protein synthesis. Viability, assessed by dye exclusion and LDH leakage were also determined. p-Nitrophenol hydroxylase (P4502E1) activity was measureo in the same hepatocytes but without exposure to hydrazine.

Table 1. Biochemical effects of 12 mM hydrazine in hepatocytes after 3 h exposure.

PROTEIN SYNTHESIS pNP HYDROXYLASE N LDH (%TQTAL) VIABILITV(%INITIAL) ATP(%INITIAL) GSH|%INITIAL) r%CONTPQL)______(nm ol/m ig'1C ‘ cells)

CONTROL 4 41.00=4 14 76 00=4 64 33.75=5 56 25 25=2 75 33 00=158 0.228=0.007# ACETONE 5 39.20=4 09 94 40=4 41a 62 60=5 35’a 37 00=2 49a 69 60=13 00a 0 382=0 068 DEDC 4 93 25=17 72" 24 50=13 77"a 14 50=5 32a 16.75=8 88 41 75=7.75 0.084=0.020a ISONIAZID 4 53.25=7.00 61.75=11 16 31.25=5 32 26.25=4 2 7 59.20=19 80 0 902=0.123'a " p<0 05 Dunnetts I tes; a p<0 05 unpaired t-test. Means = SEM. N=numbers o1 determinations. # N.6

12 mM hydrazine was found to be the threshold concentration for cytotoxicity in vitro, manifested as LDH leakage, dye uptake, ATP and GSH depletion (dose and time dependent) and inhibition of protein synthesis. DEDC pretreatment rendered celts more susceptible to hydrazine toxicity at this concentration (see Table 1), causing an increase in LDH leakage and decrease in cell viability. Both GSH and ATP depletion were also Increased. Acetone pretreated cells remained viable throughout the experiment, ATP and GSH depletion being less than in controls. 16 mM hydrazine caused a depletion of GSH and ATP, to 12 and 15% of initial concentration respectively, in control cells after 3 h but in acetone pretreated cells depletion was 24 and 52% respectively. Isoniazid pretreatment did not significantly effect the cytotoxicity of hydrazine in isolated hepatocytes All pretreatments decreased inhibition of protein synthesis compared to controls although only after acetone was this statistically significant Pretreatments with isoniazid and DEDC significantly affected P4502E1 activity. In conclusion, inhibition of P4502E1 by DEDC increased the cytotoxicity of hydrazine whereas acetone pretreatment had a protective effect Isoniazid pretreatment had little effect on hydrazine toxiafy. These results indicate that P4502E1 may not be responsible for converting hydrazine to a toxic species and that other P450 isoenzymes, such as P4502B1, may be responsible for detoxication The effect of hydrazine on protein synthesis inhibition seem s to be independent of P4502E1 activity. Tne authors are gratetui for financial support from the EOARD, United States Air Force Jenner, A.M. & Timbrell, J.A. (1991) Hum. Exp. Toxicol. 10, 492-493. Lauriauft. V.V., Khan. S. & O Bnen, P.J. (1992) Chem-Biol. Interactions. 81, 271-289. Ronis, M.J.J. & Ingelman-Sundberg, M. (1989) Xenobiotica. 19(10), 1161-1165.

Repeated Exposure to Hydrazine in vivo Protects Against Hydrazine Toxicity in vitro

J Delanev & J.A. Timbrell, Toxicology Department, School of Pharmacy, 29 Brunswick Square, London WCiN 1 AX.

There are many toxic effects associated with exposure to hydrazine (Hz), which is a widely used industna! chemical and a metabolite of the dru isoniazid and hydralazine. As a result patients on long term therapy with these agents are potentially at risk from increased toxicity. It is importa therefore to elucidate which enzymes are involved in Hz metabolism and to assess their role in toxicity. Repeat doses of Hz in vivo indu P4502E1'. Pretreatment of rats with acetone^ and Isoniazid^ induces P4502E1 and causes increased Hz toxicity in vivo, manifested as steatos accompanied by ATP and glutathione (GSH) depletion. The purpose of this study was to investigate the influence of Hz pretreatment on the toxic of Hz in vitro and to determine the role, if any, of P4502E1 in Hz metabolism. Male Sprague Dawley rats (200-300g) were pretreated with (2.5mg/'kg, in the dnnking water for 10 days). Hepatocytes isolated from untreated and pretreated rats were incubated with 8,12,16 anc 20 ml Hz hydrate for 3 hours. Samples were taken at hourly intervals and subsequently assayed for viability (trypan blue dye exclusion), LDH leakag ATP, GSH and protein synthesis. p-Nitrophenol hydroxylase (P4502E1) activity was measured in the stock hepatocytes.

Table 1. Biochemical effects of 12 mM Hz in hepatocytes after 3 h exposure.

PROTEIN SYNTHESIS pNP HYDROXYLASE N LDH (-/.TOTAL) V1ABIL1TY(%1N1T1AL) ATP(%INIT1AL) GSH(%IN1TIAL) («/.CONTROL)______(nmol/miglQ' ceils)

(U) CONTROL 4 32.00=3.08 92.00=2.68 92.00=6 98 99.25=7 38 100.00 0.572=0 015 (U)*12mM 4 41.00=4.14 75.00=4 64-5 33.75=5.57-5 25 25=2 75-5 33 00=1.58-5 ...... (P) CONTROL 4 35.25=1.89 92.50=0 29 78.00=4 6 7 72.25=9 68 100 00 0 946=0 064" (P) T l2mM 4 33.00=1 29 95.25=1.1 Ta 53.75=5 36'a 46 75=4.3Ta 23.75=3.54 ......

U«untfeateo; P=preiraated, ' p<0,05 unpaired t-test. a) 12mM Hz U versus P. 5) (U) CONTROL versus (U) 12mM Hz. Means = s.e.m; N»num5ers ol determinations

The threshold concentration for cytotoxicity (expressed as loss of cell viability) in hepatocytes from untreated rats was 12 mM Hz. Lowt concentrations had little effect and higher concentrations caused significant ceil death. Concentrations of Hz below the threshold affected ATP, GS and protein synthesis but did not cause toss of viability. Hz pretreatment rendered cells less susceptible to Hz toxicity at all concentrations (Tab 1 for l2mM data). LDH leakage and loss of cell viability were reduced and both GSH and ATP depletion were decreased compared to cells fro untreated rats. Hydrazine-induced protein synthesis inhibition w as similar in cells from untreated and pretreated rats. P4502E1 activity was induce by Hz pretreatment (Table 1). EROD (P4501A1/2) and PROD (P4502B1/2) activities were also measured (results not shown) and were found t be reduced by Hz pretreatment. These results suggest that P4502E1 may alter the rate or route of Hz metabolism.

In conclusion, pretreatment of rats with Hz protects against Hz toxicity in vitro in isolated hepatocytes. This may be due to increased metabolisi and hence detoxication of Hz by P4502E1. There is clearly a discrepancy with in vivo studies employing other P4502E1 inducers. This is current unexplained. The effect of Hz on protein synthesis inhibition seem s to be independent of P4502E1, EROD and PROD activities. The authors are grateful for financial support from the EOARD, United States Air Force.

'Jenner. A.M. & Timbrell, J.A. (1992) Hum. Exp. Toxicol. 11, 411-412. ^Jenner, A.M. & Timbrell, J.A. (1991) Hum. Exp. Toxicol. 10, 492-493. Jenner, A.M. (1992) PhD Thesis. London. Effects of Repeated Exposure to Hydrazine; Biochemical Perturbations and Effects on Acute Hydrazine Toxicity

J. Delanev and J. A. Timbrell, Toxicology Department, School of Pharmacy. 29/39 Brunswick Square, London WC1N. 1AX.

We have previously shown that repeated exposure to hydrazine (Hz) increases enzyme activity attributable to cytochrome P450 2 E l\ Other inducers of this isozyme increase the hepatotoxicity of an acute dose of hydrazine whereas in vitro hydrazine toxicity to hepatocytes was decreased by induction of 2E1 by prior exposure to hydrazine itself. The objective of this study was to examine the biochemical effects of repeated hydrazine exposure and of induction of 2E1 on the acute toxicity of hydrazine.

Rats were exposed to hydrazine (2.5, 5 and 10 mg4tg/oay) for 10 days via the drinking water. Control animals were pair fed. At the end of the exposure period animals were either killed or half of those treated with 2.5 mg/kg/day were given a single acute dose of hydrazine (30 mg'kg) 24h prior to termination. Liver and kidneys were removed, weighed and taken for histology analysis. Blood and liver samples were taken for biochemical analysis. Liver was analysed for cytochrome P450 isozyme activities: ethoxyresorufin 0-dethylase (EROD), pentoxyresorufin O- depentylase (PROD) and p-nitrophenol hydroxylase (PNP). Liver was assayed for ATP, GSH, GSSG and triglycerides by standard assay techniques. The results are shown in Table 1. T a b l e 1 .

TREATMENT PNP nmol/min/mg EROD pmoLmia' PROD pmol/mia'' GSH pmoLg GSSG nmol/g protein mg protein mg protein liver liver

Control a 0.4 ± 0.1 22 7 : 58 45 ± 12 5.45 z 0.92 61 ± 16 a)2.5mg/Kg Hz 0.9 ± O.T 21 2 ± 46 50 ± 14 5.51 ± 0 .7 7 7 0 z 21 Control b 0.4 ± 0.2 204 ± 4 6 7 ± 44 5.28 ± 1.30 6 7 ± 44 b)5mg/Kg Hz 1.3 ± 0.4' 1 6 3 ± 43 61 z 5 4.36 ± 0.54 1 9 5 ± 105 Control c 0.4 ± 0.1 224 ± 37 72 ± 14 5.65 ±0.81 76 ±31 c)10mg/Kg Hz 1.6 ± 0 .2 ' 147 ± 7 48 ± 4 ’ 3.41 ± 0.73" 259 ± 95" Mean ± SD; ' p<0 05: ' p<0.01; p<0.001 Students t-Test

Repeated exposure to hydrazine significantly increased relative liver and kidney weights. The toxicity of an acute dose of hydrazine as indicated by liver and kidney weight, and liver levels of tnglycerides, GSH, GSSG and ATP was not increased by pnor treatment with hydrazine for 10 days. Thus we conclude that repeated exposure to hydrazine for a relatively short time does cause a number of biochemical and morphological effects. However these changes do not influence the toxicity of a subsequent acute dose of hydrazine.

The authors are grateful to U.S.A.F (EOARD) for funding.

’jenner, A. M. & Timbrell, J.A. (1994) Arch. Toxicol. 68, 240-245.

24-PF-8 THE EFFECT OF AN ACUTE DOSE OF HYDRAZINE ON LIVER LIPIDS. J Delaney, M Zloh\ W A Gibbons' and J A Timbrell. Toxicology and Chemistry Departments, School of Pharmacy, London, UK.

Acute exposure to hydrazine (Hz) causes fatty accumulation in the liver. This could arise from increased synthesis, decreased secretion or a combination of both. Male Sprague Dawley rats (200-250g) were given 30mg,TCg Hz ip and sacrificed 24H later. Serum and liver triglycerides were raised (x2 and x4 respectively) as was liver phosphatidate phosphohydrolase (PAP) activity (x2), a rate limiting enzyme in triglyceride synthesis. Analysis of liver extracts using proton NMR confirmed the increase in triglycerides but also showed a concomitant increase in fatty acids and little or no effect on phospholipids and cholesterol. The activity of PAP is thought to be induced by increased availability of precursors in the liver. The data showing increased fatty acids and triglycerides but unchanged phospholipids suggests de novo synthesis. The rise in serum triglycerides may be due to increased secretion from the liver or mobilization from fat stores. However Hz has also been shown to inhibit protein synthesis and thus apolipoprotein levels may be reduced impeding lipid secretion from the liver. Further research is necessary to clarify which mechanism(s) is involved in Hz induced fatty liver.

JD is grateful to U.S.A.F (EOARD) for funding. 73-P-5 EVALUATION OF IN VITRO BIOCHEMICAL MARKERS OF HYDRAZINE TOXICITY IN HEPATOCYTES: COMPARISON WITH IN VIVO DATA. C J Waterfield, J Delaney and J A Timbrell. Toxicology Department, School of Pharmacy, University' of London, London, U.K.

Hydrazine causes a variety of biochemical perturbations in vivo\ these were investigated to find out which were the most appropriate parameters to measure in in vitro toxicity studies. The following were evaluated in isolated hepatocytes: lactate dehydrogenase (LDH) leakage, taurine leakage and synthesis, ATP and GSH depletion and triglyceride, urea and protein synthesis. Apart from LDH leakage all have previously been reported to be influenced by hydrazine in vivo. Exposure of isolated rat hepatocytes to various concentrations of hydrazine (minimum effective concentration) resulted in significant depletion of ATP (8 mM) and GSH (4 mM), inhibition of urea (2 mM) and protein synthesis (0.5 mM) and leakage of taurine (20 mM) and LDH (16 mM). Despite increased triglyceride levels in vivo (2X, @3 h, 60 mg.kg'^), levels in vitro were slightly but not significantly greater than control. ATP and GSH are depleted in vitro but at higher concentrations than observed in vivo (0.2 mM). Overall, the most sensitive marker of hydrazine toxicity was inhibition of protein synthesis but urea synthesis was the most sensitive liver specific marker.

CJW is grateful for suppon from Glaxo Research and Develoomem and JD from USAF (E0.4RD).

A Comparison of the Biochemical Effects of Hydrazine In Vivo in Rats and In Vitro in Isolatec Rat Hepatoc^'tes

C. J. Waterfield, J. Delaney, S. Ghatineh, N. E. Preece, A. M. Jenner & J.A. Timbrell. Department of Toxicology. School of Pharm.acy, 29,29 Brunswick Sq., London WCIN 1AX.

We have compared the biochemical effects of the hepatotoxicant hydrazine in vivo with the effects in vitro. Rats (male, Sprague Dawley. 250-300 g) were given hydrazine (3 ■ 80 mg.kg ' free base p.o.) or saline (controls). Hydrazine was measured in plasma and liver taken at post mortem 10, 30, 90 or 270 min after dosing’. Hepatocytes were isolated by collagenase perfusion and either used in suspension or cultured for 24 h then exposed to hydrazine (0.1 ■ 20 mM). Several biochemical parameters were measured after 3 h exposure.

Hydrazine caused a dose dependent accumulation of triglyceridesin v iv d and depletion of ATP and total non-protein sulphydryls/glutathione within 3 h of dosing in vivo and in hepatocytes in suspension in vitro. Although hydrazine did not cause liver necrosis in vivo (serum levels of LDH, AST and ALT w ere not raised), in vitro cytotoxicity was indicated by LDH leakage and uptake of Trypan blue. Urea synthesis was investigated as a more liver specific marker. This was found to be significantly inhibited by hydrazinein vitro but not in liver or serum in vivo 3 h after dosing. The final parameter measured was protein synthesis as incorporation of (’Hj-leucine into proteins in vitro and total protein/g liver in vivo. Protein synthesis was significantly inhibited in vitro and at 3 h in vivo there was significant reduction in liver protein but not in [^H)-leucine incorporation.

Table. EC.^ values fmM) for hydrazine toxicity both in vivo and in vitro.

ATP TNPSH/GSH LDH TB TRIG UREA Protein In vivo 0.1 0.05 NE 0.3 - 0.26 NE 0.3 0.05 (total) Cells in suspension 10 4 16 16 >8 2 0.5 (pH)-leucine) Cells in culture' >20 >20 20 ND ND ND 0.5 (pH]-leucine)

Values are mM hydrazine in media or maximal plasma levels. In vivo data mean of 4-15 rats and in vitro data from 5 or more experiments; TNPSH (total non-protein sulphydryls); GSH (Glutathione): TB (Trypan blue); TRIG (triglycerides); ND (no data); NE (no effect at this plasma concentration).

Comparison of the concentration of hydrazine required to cause the biochemical effects observed in vivo with those used in vitro revealed a 10-100 fold difference. Thus the EC50 concentration of hydrazine in plasma in vivo after a toxic dose was 0.05 - 0.26 mM depending on the parameter measured but in vitro it was 0.5 - >20 mM. In conclusion, although two of the biochemical effects of hydrazine observed in vivo (ATP and TNPSH/GSH depletion) were observed in vitro the concentrations required in vitro are much higher. The other effects observed in vivo (triglyceride accumulation) and those observed in vitro (cytotoxicity, inhibition of urea synthesis and ('H)-leucine incorporation into proteins) did not correlate.

The authors thank Glaxo Research and Development and EOARD. Air Force and SERC for financial support

’Preece N.E., Ghatineh S. & Timbrell J.A. (1992) Hum. Expt, Toxicol. 11. 121-127 ’Jenner A.M. & Timbrell J.A. (1994) Arch. Toxicol. 68. 349-357. ’Ghatineh S. & Timbrell J.A. (1994) Toxic in Vitro. 8. 393-399.