Biochemical Effects Of Chronic Cyanide Exposure In The Chicken And Their Relevance To The Mechanism By Which Cyanide Alleviates Selenium Toxicity. by JANE SARAH CRASTQN A thesis submitted for the degree of Doctor of Philosophy. U n iv ersity o f London Wye College February 1991 ABSTRACT Chronic cyanide exposure is more widespread than acute, but less well characterised biochemically. Selenosis is becoming increasingly important. Cyanide ameliorates selenium toxicity, but the mechanism of this interaction has not yet been adequately explained. Selenium and cyanide metabolism and toxicity have been reviewed. The biochemical effects of chronic cyanide exposure in chickens and their relevance to the mechanism by which cyanide alleviates selenosis have been investigated. Sodium nitroprusside proved a suitable experimental source of dietary cyanide. Hepatic glycogen concentration was reduced in chronic cyanide exposure and dietary supplementation with gluconeogenic precursors, L-alanine, L-lactate and L-serine, exacerbated cyanide toxicity, indicating the importance of anaerobic glucose catabolism. Cyanide caused little alteration in the redox state of hepatic pyridine nucleotides, but decreased total glutathione and GSH, increased GSSG and reduced the GSH:GSSG ratio. Selenium produced changes consistent with an increased demand for GSH and NADPH. Liver NADH and NADPH concentrations were decreased and NADPf increased resu ltin g in elevated NAD+ :NADH and NADP4" : NADPH ratios. Hepatic total glutathione and GSH were increased. Cyanide reversed these effects. Dimethylselenide exhalation by chicks consuming high selenium diets was decreased by cyanide. Cyanide reduced liver total selenium, decreased the proportion present as selenite and increased the percentage of selenoamino acids. An adequate supply of methionine or cysteine was required for the latter two effects and for alleviation of selenosis. - 2 - Cyanide exerted greater influence in the cytoplasm than in whole liver or the mitochondrion. Thiocyanate ions were not responsible for the amelioration of selenosis by cyanide. The mechanism of the alleviation of selenosis appears to involve cyanide-mediated alteration of the form of selenium in tissues. No evidence has been obtained to show that cyanide achieves its effects through changes in cellular redox state. - 3 - TABLE OF CONTENTS PAGE T itle ^ Abstract 2 Table of Contents 4 List of Figures H List of Tables 12 Abbreviations 15 Dedication 17 Acknowledgements 18 Chapter 1 Introduction 19 Chapter 2 Literature Review 22 2.1 Sources of cyanide 22 2.2 Cyanide toxicology 22 2.2.1 Chronic exposure 22 2.2.2 Acute exposure 23 2.3 Metabolic effects of acute cyanidetoxicity 23 2.3.1 Enzyme in h ib itio n 23 2.3.2 Carbohydrate metabolism 25 2.4 Metabolic effects of chronic cyanide toxicity 28 2.5 Cyanide metabolism 29 - 4 - Cyanide metabolism and sulphur supply 33 Sodium nitroprusside as a source of cyanide in experimental diets 34 Natural occurrence of selenium 37 Essentiality of selenium 37 Functions of selenium 38 Absorption of selenium 39 Plasma transport of selenium 40 Tissue distribution and forms of selenium 40 Selenium metabolism 41 Selenium excretion and homeostasis 48 Selenium toxicity 50 2.16.1 Biochemical lesions 50 2.16.2 Factors affecting selenium toxicity 52 2.16.3 The interaction between cyanide and selenium 54 Glutathione metabolism 57 Materials and Methods 61 M aterials 61 3.1.1 Animals and diets 61 3.1.2 Chemicals and biochemicals 61 3.1.3 Spectrophotometry and fluorimetry 61 3.1.4 Homogenisation and centrifugation 62 - 5 - Animal husbandry 64 Specimen collection 64 3.3.1 Livers for storage as whole tissue 64 3.3.2 Blood or plasma samples 64 Preparation of reagents 64 3.4.1 Acid mixture 64 3.4.2 Alcohol dehydrogenase 65 3.4.3 Alcoholic potassium hydroxide 65 3.4.4 Amyloglucos idase 65 3.4.5 Chloramine-T-phosphate 65 3.4.6 Copper sulphate reagent 65 3.4.7 Cysteine solution 66 3.4.8 2-3 Diaminonaphthalene (DAN) solution 66 3.4.9 5-5'-Dithio-bis (2-nitrobenzoic acid) (DTNB) solution 66 3.4.10 Diphosphate buffer 66 3.4.11 Folin-Ciocalteu phenol reagent 66 3.4.12 Glucose-6-phosphate dehydrogenase 67 3.4.13 Glutamate dehydrogenase 67 3.4.14 Glutathione reductase 67 3.4.15 Lactate dehydrogenase 67 3.4.16 NADPH/buffer solution 67 3.4.17 Phosphate/EDTA buffer 68 3.4.18 Pyrazalone-pyridine reagent 68 3.4.19 Substrate mixture 68 3.4.20 Triethanolamine (TEA)/ phosphate mixture 68 3.4.21 Tris chloride buffer 68 Methods 69 3.5.1 Estimation of cyanide in biological fluids 69 3.5.2 Estimation of cyanide in chick gut contents 70 3.5.3 Analysis of plasma glucose concentration 70 3.5.4 Glutathione and glutathione disulphide assays 70 - 6 - 3.5.5 Measurement of glutathione reductase activity in liver 72 3.5.6 Determination of liver glycogen concentration 73 3.5.7 Measurement of liver L-lactate and L-pyruvate concentrations 74 3.5.8 Estimation of total protein concentration in biological samples 75 3.5.9 Analysis of pyridine nucleotides by HPLC 75 3.5.10 Fluorometric analysis of pyridine nucleotides 75 3.5.11 Selenium estimation: glassware decontamination 80 3.5.12 Collection of selenium in expired air 81 3.5.13 Determination of the oxidation state of selenium in liver: sample preparation 82 3.5.14 Selenium estimation: assay 83 3.5.15 Measurement of sodium nitroprusside concentration in blood plasma 84 Statistical methods 86 Calculation of ratios 86 Results and Discussion 87 Sodium nitroprusside as a source of cyanide in metabolic studies 87 4.1.1 Intestinal cyanide content in chicks consuming SNP (experiment 1) 87 4.1.2 Cyanide content of blood and alimentary tract in chicks fed SNP (experiment 2) 89 4.1.3 SNP concentration in the plasma of chicks given diets containing this compound (experiment 3) 91 4.1.4 Discussion of experiments 1-3 92 The effect of dietary potassium thiocyanate on the chronic toxicity of SNP (experiment 4) 92 4.2.1 Discussion of experiment 4 93 - 7 - The effect of chronic exposure to dietary SNP and/or selenium on carbohydrate metabolism in the chicken 95 4.3.1 The effect of short-term dietary exposure to sublethal doses of SNP and/or selenium on liver glycogen and plasma glucose concentrations (experiment 5) 95 4.3.2 The effect of long-term dietary exposure to sublethal doses of SNP and/or selenium on liver glycogen concentration (experiment 6) 97 4.3.3 The effect of dietary exposure to sublethal doses of SNP and/or selenium on liver lactate and pyruvate levels in chicks (experiment 7) 105 4.3.4 Discussion of experiments 5-7 107 The effect of pyruvate donors on the chronic toxicity of cyanide 111 4.4.1 The effect of dietary cystine, alanine and lactate on the chronic toxicity of SNP (experiment 8) 111 4.4.2 The effect of dietary alanine and serine on the chronic toxicity of SNP (experiment 9) 112 4.4.3 Discussion of experiments 8 and 9 116 The effect of dietary supplementation with L-cystine on the chronic toxicity of SNP (experiment 10) 117 4.5.1 Discussion of experiment 10 118 The effect of dietary selenium and/or SNP on the redox state of pyridine nucleotides in chicken liver (experiment 11) 119 4.6.1 Discussion of experiment 11 124 - 8 - The effect of dietary selenium and/or SNP on the redox state of the glutathione system (experiments 12-14) 126 4.7.1 The effect of dietary selenium and/or SNP on the concentration and redox state of hepatic glutathione (experiment 12) 126 4.7.2 The effect of dietary selenium and/or SNP on the concentration and redox state of blood glutathione (experiment 13) 129 4.7.3 The effect of dietary L-cystine and/or SNP on the concentration and redox state of hepatic glutathione (experiment 14) 133 4.7.4 Discussion of experiments 12-14 134 The effect of dietary supplementation with DL-methionine on the chronic toxicity of selenium and/or SNP, and on the interaction between these toxins (experiments 15 and 16) 136 4.8.1 The effect of dietary supplementation with DL-methionine on the selenium-cyanide interaction (experiment 15) 137 4.8.2 The effect of dietary DL-methionine on the growth performance of chicks fed selenium and/or SNP (experiment 16) 138 4.8.3 Discussion of experiments 15 and 16 142 The effect of dietary SNP on the exhalation of volatile selenium compounds (experiment 17) 144 4.9.1 Discussion of experiment 17 147 The effects of dietary DL-methionine, L-cystine and SNP on the toxicity of selenium and on the subcellular distribution and redox state of this element in the liver (experiment 18) 148 Discussion of experiment 18 156 - 9 - Chapter 5 Final Discussion 165 5.1 The effects of chronic cyanide exposure on glucose catabolism and cellular redox state 165 5.2 The effects of chronic selenium exposure on glucose catabolism and cellular redox state 168 5.3 The importance of effects on glucose catabolism and cellular redox state in the mechanism by which cyanide alleviates selenosis 169 5.4 The effects of dietary supplementation with sulphur amino acids on the growth performance of chicks consuming selenium and/or SNP 169 5.5 The effects of cyanide on DmSe exhalation and tissue selenium levels 171 5.6 The effect of cyanide on the form in which selenium is present in the tissues 174 5.7 SNP as a source of dietary cyanide in nutritional and biochemical studies 175 5.8 Suggestions for further study 176 5.9 Concluding remarks 177 References 179 -10- LIST OF FIGURES An alternative pathway for carbohydrate metabolism in the presence of cyanide.