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Investigation of valproic acid-associated oxidative stress and hepatotoxicity Tong, Vincent 2005

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INVESTIGATION OF VALPROIC ACID-ASSOCIATED OXIDATIVE STRESS AND HEPATOTOXICITY b y VINCENT TONG B.Sc, Simon Fraser University, 1997 M.Sc, The University of British Columbia, 2000 A thesis submitted in partial fulfilment of the requirements for the degree of DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES PHARMACEUTICAL SCIENCES THE UNIVERSITY OF BRITISH COLUMBIA February, 2005 © Vincent Tong, 2005 A B S T R A C T A serious adverse reaction of valproic acid (VPA), a widely used anti-epileptic, is a rare but potentially fatal hepatotoxicity affecting mostly children under 2 years of age with developmental delay and who are on V P A polytherapy. The mechanism of the hepatotoxicity remains unknown. This thesis describes in vivo and in vitro studies to examine the hypothesis that V P A increases oxidative stress in rats that in turn is associated with liver toxicity. 15-F 2 t -Isoprostaglandin (15-F 2 t- lsoP), a free-radical catalyzed, lipid peroxidation breakdown product of arachidonic acid, was measured as an indicator of oxidative stress. Our findings showed a dose-dependent elevation in plasma and liver 15-F 2 t - lsoP with plasma concentration-time profiles similar to that of the drug in rats given a single high dose of V P A . This increase in 15-F 2 t - lsoP did not involve cytochrome P450-mediated biotransformation even though rats treated with both phenobarbital (PB) and V P A resulted in greater levels of 15-F 2 t - lsoP compared to rats treated with only V P A . These results prompted further mechanistic studies in rats to establish an association between V P A glucuronidation and 15-F 2 t - lsoP levels. V P A glucuronidation, the major V P A biotransformation pathway, correlated with 15-F 2 t - lsoP formation. Levels of both 15-F 2 t - lsoP and VPA-1-O-acyl glucuronide (VPA-G) were elevated by P B and reduced by inhibitors of VPA-glucuronidation in rats after a single dose of V P A . The fluorinated analogue of V P A (a-fluoro-VPA), which was a poor substrate for glucuronidation, did not elevate 15-F 2 t - lsoP levels. To determine whether there was a temporal relationship between VPA-associated oxidative stress and hepatotoxicity, rats were given high daily doses of V P A for 14 days. V P A elevated levels of 15-F 2 ,- lsoP prior to the onset of hepatic necrosis and steatosis. An in vitro model using primary cultured rat hepatocytes also demonstrated that V P A induces oxidative stress as measured by elevated levels of 15-F 2 t - lsoP and 2',7'-dichlorofluorescein (DCF). In hepatocytes with reduced levels of glutathione, these oxidative stress biomarkers were further elevated, and ii were accompanied by mitochondrial dysfunction and hepatocyte toxicity. The work presented is significant in that it supports the hypothesis that VPA-associated oxidative stress occurs prior to hepatotoxicity and the link of acyl glucuronidation of V P A to the production of reactive oxygen species is unique. T A B L E O F C O N T E N T S ABSTRACT ii TABLE OF C O N T E N T S iv LIST OF F IGURES viii ABBREVIATIONS AND S Y M B O L S xii A C K N O W L E D G M E N T S xv DEDICATION xvi 1 I N T R O D U C T I O N 1 1.1 VALPROIC ACID 1 1.2 THERAPEUTIC USE AND MECHANISM OF ACTION 2 1.3 PHARMACOKINETICS 2 1.4 METABOLISM 3 1.5 VPA HEPATOTOXICITY 6 1.5.1 VPA-induced metabolic and biochemical disturbances 7 1.5.2 Coenzyme A depletion hypothesis 8 1.5.3 Carnitine deficiency hypothesis 9 15.4 Reactive metabolite hypothesis 10 1.6 OXIDATIVE STRESS HYPOTHESIS 16 1.6.1 Oxidative stress 16 1.6.2 Lipid peroxidation and biomarkers 16 1.6.3 Antioxidant defense 20 1.6.4 Valproic acid and oxidative stress 21 1.7 RESEARCH RATIONALE & HYPOTHESIS 23 1.8 RESEARCH OBJECTIVES 24 1.9 REFERENCES 25 2 T H E E F F E C T O F V P A O N 1 5 - F 2 T - I S O P R O S T A N E L E V E L S IN R A T S 36 2.1 INTRODUCTION 36 2.2 MATERIALS & METHODS 37 2.2.1 Reagents 3 7 2.2.2 Animals 3 8 iv 2.2.3 Treatment of animals and sample collection 38 2.2.4 15-F2t-lsoPenzyme immunoassay (EIA) 39 2.2.5 Validation of the 15-F2rlsoP EIA 40 2.2.6 Thiobarbituric acid reactive substances (TBARS) assay 41 2.2.7 Lipid hydroperoxide (LPO) assay 42 2.2.8 VPA and metabolite assay 43 2.2.9 . Gas chromatography-mass spectrometry (GC/MS) instrumentation 43 2.2.10 Statistical analysis 46 2.3 RESULTS 47 2.3.7 15-F2t-lsoP EIA 47 2.3.2 Dose-dependent effect of VPA on plasma levels of free 15-F2t-lsoP 47 2.3.3 Time-dependent effect of VPA on plasma levels of free 15-F2t-lsoP 51 2.3.4 Effect of PB on plasma and liver levels of free 15-F2rlsoP, TBARS, LPO, and VPA metabolites in rats treated with VPA 52 2.3.5 Lack of an effect by SKF-525A on VPA-induced increase in plasma 15-F2t-lsoP levels 56 2.3.6 Effect ofABT on plasma levels of free 15-F2rlsoP in VPA-treated rats 57 2.3.7 Effect of SKF-525A on the enhancement of plasma levels of free 15-F2rlsoP by PB in rats treated with VPA 58 2.3.8 Lack of an effect on plasma 15-F2t-lsoP levels by octanoic acid 61 2.4 DISCUSSION .62 2.5 REFERENCES 67 3 V A L P R O I C A C I D G L U C U R O N I D A T I O N IS A S S O C I A T E D WITH I N C R E A S E S IN 1 5 - F 2 T - I S O P R O S T A N E L E V E L S IN R A T S 72 3.1 INTRODUCTION 72 3.2 MATERIALS & METHODS 73 3.2.7 Materials 73 3.2.2 Instrumentation and analytical methods 74 3.2.3 Animals 75 3.2.4 Isolation of VPA-1-O-acyl glucuronide (VPA-G) and fH6]-VPA-1-0-acyl glucuronide (fH6]-VPA-G) 75 3.2.5 Treatment of animals and sample collection 76 3.2.6 Sample preparation for VPA-G assay by LC/MS 77 3.2.7 Validation of the LC/MS VPA-G assay 77 v 3.2.8 15-F2t-lsoP Assay 78 3.2.9 Statistical analysis 78 3.3 RESULTS 79 3.3.1 Biosynthesis and identification of VPA-glucuronide 79 3.3.2 Validation of LC/MS VPA-G assay 83 3.3.3 Dose-dependent increases in plasma and hepatic 15-F2t-lsoP and VPA-G levels in VPA-treated rats 84 3.3.4 Effect of (-)-borneol on VPA-glucuronide and 15-F2t-lsoP levels in rats treated with VPA 87 3.3.5 Effect of salicylamide on VPA-glucuronide and 15-F2t-lsoP levels in rats treated with VPA 87 3.3.6 Effect of PB on VPA-glucuronide and 15-F2t-lsoP levels in rats treated with VPA 88 3.3.7 Effect of (-)-borneol on PB-induced increases in VPA-glucuronide and 15-F2t-lsoP levels in rats treated with VPA 88 3.3.8 Effect of a-F-VPA on a-F-VPA-G and 15-F2t-lsoP levels 93 3.3.9 Effect ofPB on a-F-VPA-G and 15-F2,-lsoP levels in rats treated with a-F-VPA 93 3.4 DISCUSSION 95 3.5 REFERENCES 101 4 TIME-COURSE OF OXIDATIVE STRESS BIOMARKERS AND LIVER TOXICITY IN RATS TREATED WITH VALPROIC ACID 106 4.1 INTRODUCTION 106 4.2 METHODS & MATERIALS 107 4 . 2 . 7 Chemicals 107 4.2.2 Animal experiments 107 4.2.3 Determination of oxidative stress biomarkers 708 4.2.4 Determination of serum alpha glutathione S-transferase (a-GST) as a liver toxicity marker 108 4.2.5 Histopathology 109 4.2.6 Determination of VPA metabolites 109 4.2.7 Instrumentation and analytical methods 7 7 0 4.2.8 Statistical Analysis 110 4.3 RESULTS 111 4.3.1 Time course for 15-F2t-lsoP during VPA treatment 7 7 7 4.3.2 TBARS and LPO levels during VPA treatment 7 7 7 vi 4.3.3 Serum a-GSTlevels and histology during VPA treatment 116 4.3.4 Levels of VPA metabolites during VPA treatment 120 4.4 DISCUSSION 123 4.5 REFERENCES 129 5 INVESTIGATION O F V P A - A S S O C I A T E D O X I D A T I V E S T R E S S IN P R I M A R Y C U L T U R E D R A T H E P A T O C Y T E S 132 5.1 INTRODUCTION 132 5.2 METHODS & MATERIALS 134 5.2.1 Chemicals 134 5.2.2 Animals 134 5.2.3 Rat hepatocyte isolation and culture 135 5.2.4 15-F2t-lsoP Assay 136 5.2.5 DCF-DA assay 137 5.2.6 Measurement of mitochondrial membrane potential (A 138 5.2.7 Cell viability 138 5.2.8 Glutathione (GSH) assay 139 5.2.9 Statistical analysis 140 5.3 RESULTS 141 5.3.1 VPA-induced oxidative stress in primary culture rat hepatocytes 141 5.3.2 VPA-induced oxidative stress in GSH-depleted rat hepatocytes 143 5.3.3 Effect of VPA on mitochondrial membrane potential (ATJ in primary culture rat hepatocytes 147 5.3.4 Effect of VPA on cell viability 147 5.4 DISCUSSION 150 5.5 REFERENCES 155 6 G L O B A L S U M M A R Y , F U T U R E S T U D I E S , C O N C L U S I O N S 159 6.1 SUMMARY OF FINDINGS AND FUTURE STUDIES 159 6.2 CONCLUSION 164 6.3 REFERENCES 165 vii L I S T O F F I G U R E S Figure 1-1: Structure of V P A . 1 Figure 1-2: S u m m a r y of the metabol ic pathways (P450 , (3-oxidation, and glucuronidation) for major V P A metabol i tes in human. 5 Figure 1-3: Metabol ic pathways of V P A leading to reactive metabol i tes and their G S H conjugates. 12 Figure 1-4: S c h e m a t i c descr ib ing the concept of oxidat ive s t ress a s a ba lance between R O S production and R O S removal . 17 Figure 1-5: S c h e m a t i c descr ib ing the formation of lipid peroxidat ion b iomarkers : lipid hydroperox ides, malond ia ldehyde, and 15-F 2 t - i sopros tane. 18 Figure 1-6: Format ion of F 2 - i sopros tane reg io- isomers dur ing non-enzymat ic peroxidation of a rach idon ic ac id . 19 Figure 1-7: A schemat i c descr ib ing the glutathione perox idase ( G S H - P x ) - glutathione reductase (GSH- reduc tase ) antioxidant sys tem. 21 Figure 2-1: Structure of M D A - T B A R S adduct . 41 Figure 2-2: Reduct ion/ox idat ion react ion s c h e m e involved in the L P O assay . 42 Figure 2-3: Influence of s ingle d o s e V P A on free p lasma 1 5 - F 2 t - l s o P levels. 49 Figure 2-4: The effect of C C I 4 treatment on levels of 1 5 - F 2 t - l s o P . 50 Figure 2-5: T ime-cou rse of p l asma V P A and 1 5 - F 2 t - l s o P . 51 Figure 2-6: Effect of P B pretreatment on levels of 1 5 - F 2 r i s o P measu red as the free form in the p lasma and liver of V P A treated rats. 53 Figure 2-7: S K F - 5 2 5 A pretreatment on 1 5 - F 2 t - l s o P levels in V P A - t r e a t e d rats. 56 Figure 2-8: Effect of A B T pretreatment on free p l asma 1 5 - F 2 t - l s o P levels in V P A -treated rats. 57 Figure 2-9: PB and SKF-525A pretreatment on plasma 15-F 2 t - lsoP levels in VPA-exposed rats. 59 Figure 2-10: Comparison between V P A and octanoic acid on the effect of plasma 15-F 2 t - lsoP levels in rats. 61 Figure 3-1: Collision-induced dissociation mass spectra of (A) VPA-1-O-acyl glucuronide (VPA-G) and (B) [ 2H 6 ]-VPA-1-0-acyl glucuronide ([ 2H 6 ]-VPA-G) purified from rat bile obtained by L C / M S / M S with negative ion electropspray ionization. 80 Figure 3-2: 1 H - N M R spectra (D 2 0) of biosynthesized (A) 2-propyl-pentanoyl-1-0-acyl glucuronide (VPA-G) and (B) 2-(propyl-3,3,3-d3) pentanoyl-5,5,5-d 3-1-0-acyl glucuronide ([ 2H 6 ]-VPA-G) purified from rat bile. 81 Figure 3-3: Representative L C / M S chromatograms of V P A - G (m/z 319) (A) as biosynthesized standard spiked in blank liver homogenate, and from liver homogenates from rats treated ip with (B) 0.9% saline, (C) V P A (500 mg/kg), or (D) (-)-borneol (320 mg/kg) followed by V P A (500 mg/kg) 0.5 hr later. 82 Figure 3-4: Dose-dependent effect of V P A on (A) plasma, (B) free and (D) total liver levels of 15-F 2 t - lsoP, and (C) liver V P A - G . 86 Figure 3-5: Effect of (-)-borneol pretreatment on levels of (A) plasma 15-F 2 t - lsoP, (B) free liver 15-F 2 t - lsoP, and (C) total liver 15-F 2 t - lsoP in VPA-treated rats. 90 Figure 3-6: Effect of salicylamide (SAL) pretreatment on levels of (A) plasma 15-F 2 t -isoP, (B) free liver 15-F 2 t - isoP, and (C) total liver 15-F 2 t- isoP in VPA-treated rats. 91 Figure 3-7: Effect of (-)-borneol on the PB-induced levels of (A) plasma, (B) free and (C) total liver 15-F 2 t - lsoP in VPA-treated rats. 92 Figure 3-8: Comparison of V P A and a -F -VPA in producing 15-F 2 t - lsoP and V P A - G or a -F -VPA-G, respectively, in control rats and in PB-pretreated rats. 94 Figure 4-1: Levels of (A) plasma, (B) free liver and (C) total liver (esterified and non-esterified) 15-F 2 t - lsoP in rats treated with V P A (500 mg/kg once daily, ip) for 2, 4, 7, 10, or 14 consecutive days. 113 ix Figure 4-2: Leve ls of l iver L P O in rats treated with V P A (500 mg/kg o n c e dai ly, ip) for 2, 4, 7, 10, or 14 consecut ive days . 114 Figure 4-3 : Leve ls of (A) p l asma and (B) liver T B A R S in rats treated with (500 mg/kg once daily, ip) for 2, 4 , 7, 10, or 14 consecut ive days . 115 Figure 4-4: Leve ls of se rum a - G S T in rats treated with V P A (500 mg/kg once daily, ip) for 2, 4, 7, 10, or 14 consecut ive days . 116 Figure 4-5: Pho tomic rographs of liver sect ions from rats admin is tered ip with V P A at 500 mg/kg once daily for up to 14 days showing progress ive inc idence of liver d a m a g e . 119 Figure 4-6: S u m m a r y of results indicating the t ime-course of hepatotoxici ty and oxidative s t ress events . 123 Figure 5-1: S c h e m a t i c descr ib ing the mechan i sm of the D C F - D A a s s a y . 137 Figure 5-2: G S H recycl ing in the p resence of glutathione reduc tase and E l lman 's reagent ( D T N B , 5,5'-dithiob/ 's-2-nitrobenzoic acid). 140 Figure 5-3: T ime-cou rse and dose - response relat ionship for the product ion of (A) 15-F 2 t - l s o P and (B) D C F in f reshly isolated rat hepatocytes treated with V P A . 142 Figure 5-4: Total G S H levels determined in freshly isolated rat hepatocytes pretreated with a combinat ion of DL-buthionine-[S,R]-sul foximine ( B S O ) and diethylmaleate (DEM) . 144 Figure 5-5: Dose -dependen t changes in 1 5 - F 2 t - l s o P by V P A in f reshly isolated rat hepatocytes. 145 Figure 5-6: C o m p a r i s o n of the t ime- and concentrat ion-dependent effect of V P A on the production of D C F in control , and B S O and D E M pretreated rat hepatocytes . 146 Figure 5-7: T h e t ime-course and concentrat ion-dependent effects of V P A on mitochondrial membrane potential (A^J a s s e s s e d by the J C - 1 f luorescent probe. 148 Figure 5-8: The effect of V P A on cell viability as measured by W S T - 1 assay . 149 x L I S T O F T A B L E S Table 2-1: D iagnost ic ions and retention t imes of P F B / T B D M S der ivat ives of V P A , V P A metabol i tes and deuterated ana logues from standard reference s a m p l e s in rat p lasma. 45 Tab le 2-2: Inter-assay variat ion of the 1 5 - F 2 t - l s o P e n z y m e immunoassay . 48 Tab le 2-3: T B A R S and lipid hydroperoxide levels detected in p l asma and liver of V P A -treated rats and rats pretreated with P B . 54 Tab le 2-4: Effect of P B pretreatment on p lasma and liver levels of V P A metabol i tes in rats treated with V P A . 55 Tab le 2-5: Effect of 1-aminobenzotr iazole on p lasma levels of V P A metabol i tes. 58 Tab le 2-6: Effect of S K F - 5 2 5 A on p lasma levels of V P A metabol i tes in rats treated with both P B and V P A . 60 Tab le 3-1: Inter- and in t ra-assay variation of the V P A - G a s s a y by L C / M S . 85 Tab le 3-2: T h e effects of pre-treatment with P B , (-)-borneol, and sa l icy lamide on V P A -G levels measu red in liver homogenate . 89 Tab le 4 -1 . V P A - a s s o c i a t e d necros is and steatosis in rats treated with V P A for 14 consecut ive days . 118 Tab le 4-2 : Liver V P A metabol i te levels (u.g/g t issue) in rats treated with V P A for 2, 4, 7, 10, or 14 days . 121 Tab le 4 -3 : P l a s m a V P A metabol i te levels (ug/ml) in rats treated with V P A for 2, 4, 7, 10, or 14 days . 122 xi A B B R E V I A T I O N S A N D S Y M B O L S ° c (E)-2,4-diene V P A (£)-2-ene V P A (E,E) -2 ,3 ' -d iene V P A (E,Z)-2,3 ' -d iene V P A a - F - 4 - e n e A / P A a- f luoro-VPA (a-F-VPA) ul 15 -F 2 t - l soP 2,3-DHBA 2 - P G A 2 - P S A 3- e n e - V P A 3-keto-VPA 3- O H - V P A 4- e n e A / P A 4-keto-VPA 4- O H - V P A 5- O H - V P A A B T a m u A N O V A B S O CAT C C I 4 Cmax C o A C V D C F D C F - D A Mitochondr ia l membrane potential Deg rees Celsius (E)-2-propyl-2,4-pentadienoic acid (E)-2-propyl-2-pentenoic ac id (E,E)-2-propyl -2,3 ' -pentadienoic ac id (E,Z)-2-propyl-2,3 ' -pentadienoic ac id 2-f luoro-2-propyl-4-pentenoic acid 2-f luoro-2-propylpentanoic acid Microl i ter 15 -F 2 t - l sopros tane 2,3-d ihydroxybenzoic ac id 2-propylglutaric ac id 2-propylsuccin ic acid 2-propyl-3-pentenoic acid 2-propyl -3-oxopentanoic ac id 2-propyl-3-hydroxypentanoic ac id 2-propyl-4-pentenoic acid 2-propyl -4-oxopentanoic ac id 2-propyl -4-hydroxypentanoic ac id 2-propyl-5-hydroxypentanoic ac id 1-Aminobenzotr iazole A tomic m a s s unit Ana lys i s of var iance Buthionine sul foximine Ca ta l ase C a r b o n tetrachloride Max ima l p lasma concentrat ion C o e n z y m e - A Coeff ic ient of variation 2 ' ,7 ' -Dichlorof luorescein 2 ' ,7 ' -Dichlorof luorescin d iacetate DCFH 2',7'-Dichlorofluorescin DEM Diethylmaleate DPPD N,N'-diphenyl-p-phenylenediamine EDTA Ethylenediaminetetraacetic acid EIA Enzyme linked immunoassay ESP+ Positive electrospray F C C P carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone g Gram G A B A y-aminobutyric acid G C Gas chromatography G C / M S Gas chromatography-mass spectrometry G S H Glutathione, reduced GSH-reductase Glutathione reductase G S S G Glutathione, oxidized G S T Glutathione S-transferases GST-Px Glutathione peroxidase H&E Hematoxylin-eosin H 2 0 2 Hydrogen peroxide H B S S Hank's Balanced Salt Solution HCI Hydrochloric acid HO Hydroxyl radical H P L C High pressure liquid chromatography I.D. Internal diameter ip Intraperitoneal kg Kilogram kV Kilovolts LC/MS Liquid chromatography-mass spectrometry LC /MS/MS Liquid chromatography/tandem mass spectrometry LOQ Limit of quantitation LPO Lipid hydroperoxides m C L C C P carbonyl cyanide m-chlorophenylhydrazone MDA Malondialdehyde mg Milligram xiii min Minutes ml Milliliter mm Millimeter mM Millimolar MTBSTFA N-(te/t.-butyldimethylsilyl)-N-methyltrifluoroacetamide NADPH Nicotine adenine dinucleotide NaOH Sodium hydroxide ND Not detected ng Nanogram NCI Negative ion chemical ionization nmol Nanomole NMR Nuclear magnetic resonance P450 Cytochrome P-450 PA 4-pentenoic acid PB Phenobarbital, sodium P B S Phosphate-buffered saline PFBBr Pentafluorylbenzyl bromide pg Picogram PSI Pounds per square inch Q C Quality control r2 Coefficient of determination R O S Reactive oxygen species S E M Standard error of means SIM Single ion monitoring SOD Superoxide dismutase T B A R S Thiobarbituric acid reactive substances TBDMS Te/t-butyl-dimethylsilyl chloride TMA Trimethylacetic acid tmax Time of occurrence of maximal plasma concentration U D P G A UDP-Glucuronic acid UGT UDP-Glucuronosyltransferase V Volts V P A Valproic acid V P A - G Valproyl-1-O-acyl glucuronide xiv A C K N O W L E D G M E N T S Firstly, I would like to extend my sincere thanks to my supervisors, Drs. Frank Abbott and Tom Chang for their guidance and excellent training with regards to their knowledge, advice and the entire spectrum of experiences not only related to my project, but to research in general. Although valproic acid is a superb learning tool, my supervisors have made my program most enjoyable and rewarding. Next, I would like to thank the members of my committee, Drs. Gail Bellward (Chair), Wayne Riggs, and Kevin Farrell, all of whom I have maintained a terrific relationship with over the years. I enjoyed the interactions and the learning environment that each of the above committee members have provided. Last but not least, I would like to acknowledge and thank all the fellow lab inmates with whom I had the pleasure of working within the "bowels" of the Faculty of Pharmaceutical Sciences at UBC. Dr. Xiaowei Teng has been an excellent addition to the Abbott lab and has helped me considerably with the completion of my research project. I believe we both have learned a lot from each other and I would like to wish her good luck in solving the mysteries of valproic acid. Mr. Roland Burton for everything he taught me about mass spectrometry and more. I want to thank Dr. Stoyan Karagiozov for his kind help and advice with life, chemistry and the synthesis of metabolites. Thanks to Dr. Aspasia Michoulas from B C Children and Women's Hospital who obtained ethics approval and organized clinical samples for the clinical component of the grant. Thanks also to Jessie Chen and Tony Kiang, for their reliable help and involvement with the project whenever it was needed. I thank the funding agencies, Canadian Institutes for Health Research for the Doctoral Scholarship Award (years 2002-2005) and the PMAC-Rx&D Health Research Foundation for the Doctoral Scholarship Award (years 2000-2002). xv D E D I C A T I O N This thesis is dedicated to my family: Mom, Dad, Vivian, Gloria, Chuck, and Baba. Chapter 1: Introduction 1 Introduction 1.1 V A L P R O I C A C I D Valpro ic ac id (2-n-propyl-pentanoic ac id , V P A , Figure 1-1) is an 8 ca rbon , short -chain, branched fatty acid that w a s serendip i tously d iscovered to demonst ra te ant iconvulsant propert ies when it was used as a drug veh ic le for other ant iconvulsant test c o m p o u n d s (Meunier et al. 1963). Subsequent ly , va lpro ic ac id ( V P A ) w a s introduced into cl inical s tud ies in F rance in 1967 and w a s approved by the F o o d and Drug Administrat ion in 1978 (Loscher 1999a). V P A is avai lable for use as the free ac id , its sod ium salt, and as a therapeut ic combinat ion of the parent compound and its sod ium salt in a 2:1 molar ratio (divalproex sod ium) (Davis et al. 1994). C O O H Figure 1-1: Structure of VPA. 1 Chapter 1: Introduction 1.2 T H E R A P E U T I C U S E A N D M E C H A N I S M O F A C T I O N Valproic acid is a broad-spectrum antiepileptic drug requiring relatively high-doses (25-60 mg/kg/day) for the treatment of several epileptic seizure types including absence, myoclonic, and partial and generalized tonic-clonic seizures (Loscher 1999b; Sarisjulius and Dulac 1999; Willmore 1999). More recently, the use of V P A has been therapeutically extended for the management of bipolar disorder (Lennkh and Simhandl 2000; Post et al. 1996) and migraine (Silberstein and Wilmore 1996). The mechanism of the antiepileptic action of V P A is not well understood; however, it is most likely related to its ability to potentiate the effects of the inhibitory neurotransmitter, y-aminobutyric acid (GABA), by either increasing its production or inhibiting its degradation. V P A has been implicated in the activation of glutamic acid decarboxylase, an enzyme involved in the activation of glutamate which is required for G A B A synthesis (Loscher 1989; Loscher and Schmidt 1981). In addition, it appears to inhibit G A B A -transaminase and succinic semialdehyde dehydrogenase, two enzymes involved in the successive degradation of G A B A (Loscher et al. 1985; van der Laan et al. 1979; Zeise et al. 1991). In another proposed mechanism, V P A appears to have a non-specific membrane stabilizing effect by reducing high frequency repetitive firing of neurons through action at sodium and/or potassium channels (McLean and Macdonald 1986; Slater and Johnston 1978). None of the mechanisms alone can adequately explain VPA 's broad-spectrum anticonvulsant effects; thus, it is likely that multiple mechanisms may be involved. 1.3 P H A R M A C O K I N E T I C S The gastrointestinal absorption of V P A is rapid and complete in humans with > 90% bioavailability from all available formulations (Shen 1999). The high bioavailability stems from the fact that it readily crosses the intestinal mucosa with no site specificity (Levy and Shen 1995). Plasma peak concentrations are dependent on formulation and are achieved within 1-3 2 C h a p t e r 1: Introduction h for immediate release preparations and 3-8 h for sustained-release formulations (Davis et al. 1994). V P A is characterized by a relatively low volume of distribution that ranges from (0.1-0.2 L/kg in adults, and 0.15-0.4 L/kg in neonates, infants, and children (Cloyd et al. 1993; Gugler and von Unruh 1980; Hall et al. 1983; Herngren et al. 1991; Herngren and Nergardh 1988; Irvine-Meek et al. 1982). The low volume of distribution is attributed to the compound's extensive and saturable (nonlinear) plasma protein binding (-90% at therapeutic concentrations) and its high degree of ionization at physiological pH (pKa 4.8), which act to confine V P A largely to the vascular space and extracellular fluids (Levy and Shen 1995). V P A is not subject to significant first pass effect and is a low clearance compound (unbound plasma clearance ranging from 1-3 ml/min/kg) with elimination half-lives ranging from 9-18 h (Davis et al. 1994; Rettenmeier et al. 1987; Zaccara et al. 1988). The clearance is dose-dependent with nonlinearities resulting from saturable protein binding and/or saturable metabolism (Bowdle et al. 1980). Elimination of V P A is primarily via hepatic metabolism with only 1 to 3% of the dose excreted as the parent compound in urine (Bowdle et al. 1980; Dickinson etal. 1989; Gugler and von Unruh 1980). 1.4 METABOLISM Although the structure of V P A is relatively simple, its metabolic fate is extremely complex with approximately 50 metabolites identified, of which 16 are consistently observed in humans (Figure 1-2) (Abbott and Anari 1999; Baillie and Sheffels 1995). V P A is primarily conjugated to glucuronic acid with approximately 10-70% of the dose recovered in human urine (Dickinson et al. 1989; Levy era/ . 1990) as the 1-O-acyl-B-D-ester linked glucuronide (Dickinson era/ . 1979). V P A also competes with endogenous fatty acids for mitochondrial and peroxisomal p-oxidation resulting in the formation of the metabolites (E)-2-propyl-2-pentenoic acid ((E)-2-ene-VPA), 2-3 Chapter 1: Introduction propyl-3-pentenoic acid (3-ene-VPA), 2-propyl-3-oxopentanoic acid (3-keto-VPA) (Abbott and Anari 1999; Baillie and Sheffels 1995; Bjorge and Baillie 1991; Granneman et al. 1984b; Ponchaut et al. 1992). To a lesser extent, V P A undergoes P450-dependent co and co-1 oxidation to form 2-propyl-5-hydroxypentanoic acid (5-OH-VPA) and 2-propyl-4-hydroxypentanoic acid (4-OHA/PA), respectively (Rettenmeier et al. 1987). The formation of 3-OH-VPA is also formed via direct cytochrome P-450-mediated microsomal hydroxylation of the parent compound (Rettenmeier et al. 1987). Further oxidation of 5 -OH-VPA results in the dicarboxylic acid, 2-propylglutaric acid (2-PGA). Oxidation of 4 -OHA/PA leads to the formation of 2-propyl-4-oxopentanoic acid (4-keto-VPA) and 2-propylsuccinic acid (2-PSA) (Granneman et al. 1984b). The mono-unsaturated metabolite 2-propyl-4-pentenoic acid (4-ene-VPA) is catalyzed by P450 enzymes from a distinct carbon-centre radical after hydrogen abstraction (Rettie et al. 1987). Subsequent (3-oxidation of 4-ene-VPA results in the di-unsaturated (E)-2,4-diene-VPA, both which have been implicated in the pathogenesis of hepatotoxicity by V P A (Gerber et al. 1979; Kesterson et al. 1984; Lewis et al. 1982; Zimmerman and Ishak 1982). 4 Chapter 11; Introduction Figure 1-2: Summary of the metabolic pathways (P450, p-oxidation, and glucuronidation) for frequently observed VPA metabolites in human (Abbott and Anari 1999). 5 C h a p t e r 1: Introduct ion 1.5 VPA HEPATOTOXICITY The hepatotoxicity induced by V P A is considered to fall into two categories (Dreifuss et al. 1989; Dreifuss et al. 1987). One is the reversible hepatic dysfunction associated with V P A therapy (Itoh et al. 1982; Mathis et al. 1979; Powell-Jackson et al. 1984; Thygesen and Boesen 1982; Ware and Millward-Sadler 1980; Willmore et al. 1978) that is characterized by low plasma fibrinogen concentrations and dose-related elevation in liver enzymes in blood (Sussman and McLain 1979). In most cases, the above abnormalities are reversed upon dosage reduction or discontinuation of V P A therapy. The most serious V P A side effect reported in man is irreversible and potentially fatal hepatic damage that is described as being unpredictable, rare, and dose-independent. The first cases of fatal hepatotoxicity were reported in 1979 (Gerber et al. 1979; Mathis et al. 1979; Suchy et al. 1979); since then, over 100 cases have been reported (Bryant and Dreifuss 1996; Dreifuss et al. 1989; Dreifuss et al. 1987; Itoh et al. 1982; Scheffner et al. 1988; Zimmerman and Ishak 1982). The primary risk factors of fatal hepatotoxicity associated with V P A therapy were found to be co-administration of other anti-epileptics such as phenytoin or phenobarbital, as well as young age (children less than 2 years old) (Dreifuss et al. 1987). Between the years 1978-1984, the hepatic fatality rate of patients on V P A monotherapy under 2 years of age was 1/7000 (Dreifuss et al. 1987). This rate decreased to 1/45000 in patients that were older than 2 years of age (Dreifuss et al. 1987). With polytherapy, the fatality rate increased to 1/500 for patients less than 2 years old and 1/12000 for those older than 2 years (Dreifuss et al. 1987). Patients in the highest-risk group typically had severe epilepsy and other medical conditions including mental retardation, developmental delay, congenital anomalies, and other neurological diseases (Appleton et al. 1990; Dreifuss et al. 1987). The prescribing patterns for V P A have changed since the high risk population was identified and a follow-up retrospective study between the years 1985-1986 reported a decreased incidence (« 6 Chapter 1: Introduction 5- fold) of hepatic fatalities despite a significant increase in the overall use of V P A (Dreifuss et al. 1989). The common clinical symptoms associated with V P A hepatotoxicity are nausea, vomiting, anorexia, lethargy, edema, fever, hypoglycemia, hemorrhage, jaundice, and finally hyperammonemic coma and death (Cotariu and Zaidman 1988). Jeavons reviewed 67 cases of VPA-associated hepatotoxicity and reported the occurrence of hepatotoxicity within 3 months of the onset of V P A treatment in 75% of the cases (Jeavons 1984). Liver biopsy revealed microvesicular steatosis, a condition characterized by the accumulation of numerous small lipid vesicles in the hepatocyte (Fromenty er al. 1997), and often accompanied by centrizonal necrosis (Jeavons 1984; Zimmerman and Ishak 1982). In the 67 cases reviewed, microvesicular steatosis accompanied by necrosis was found in 22 cases, necrosis alone in 20 cases, and steatosis alone in 8 cases (Jeavons 1984). Electron-microscopy of liver biopsies revealed a pattern of primary parenchymal-organelle injury characterized by the following ultrastuctural changes: enlarged and densely packed mitochondria (megamitochondria), large mitochondrial granules in the matrix, altered cristae with crystalline inclusions, and disorganized rough endoplasmic reticulum (Itoh et al. 1982; Mathis et al. 1979). In rats, high doses of V P A were shown to cause hepatic steatosis (Kesterson et al. 1984; Lewis et al. 1982; Sobaniec-Lotowska et al. 1993) and to result in swollen and ruptured mitochondria (Jezequel et al. 1984). 1.5.1 VPA-induced metabolic and biochemical disturbances The pathogenesis of severe V P A hepatotoxicity is not clear, but the observation of microvesicular steatosis is consistent with a disturbance in mitochondrial function and/ or fatty acid metabolism (Fromenty et al. 1997; Fromenty and Pessayre 1997; Powell-Jackson et al. 1984). Investigations have centred on mitochondrial dysfunction as a likely explanation for valproate-induced toxicity. The histological data appear to suggest that the fatal liver failure due 7 Chapter 1: Introduction to V P A resembled Reye's syndrome, a condition for which a mitochondrial origin had been established (Mitchell et al. 1980; Partin ef al. 1971), and is associated with inhibition of the mitochondrial p-oxidation system (Jezequel ef al. 1984; Mortensen 1980). This was evident in early metabolic studies of V P A in humans in which the urinary excretion of C6-C10 dicarboxylic acids, indicative of impaired hepatic fatty acid p-oxidation, was significantly increased after a dose of V P A (Mortensen ef al. 1980). The lipid content of liver increased within 2-4 h after V P A administration to fasted rats, mainly in the periportal lobular regions (Jezequel ef al. 1984; Olson ef al. 1986). V P A also inhibited ketogenesis in the same regions of the lobule in the perfused rat liver (Olson ef al. 1986) and in isolated rat hepatocytes (Coude ef al. 1983). As a further indication of impaired fatty acid metabolism, the p-oxidation of palmitic acid (a long chain fatty acid) in isolated rat hepatocytes (Coude ef al. 1983) and of decanoic acid (a medium chain fatty acid) in rat liver homogenate (Bjorge and Baillie 1985) were inhibited by VPA . Several major hypotheses have developed to rationalize the observed inhibition of mitochondrial p-oxidation by V P A and the resultant hepatotoxicity. Some of these include: I) drug - associated coenzyme-A deficiency; II) carnitine deficiency; III) reactive toxic metabolites of V P A , and IV) a combination of increased oxidative stress and reduced free radical scavenger activity. 1.5.2 Coenzyme A depletion hypothesis A major hypothesis developed to explain the inhibition of mitochondrial p-oxidation and the associated hepatotoxicity was the sequestering of coenzyme A (CoA) by V P A as valproyl-CoA (Kesterson ef al. 1984; Thurston ef al. 1983). The transport and subsequent p-oxidation of fatty acids require prior activation to CoA thioesters, which is catalyzed by the ATP-dependent acyl CoA synthetase located on the outer mitochondrial membrane. Thus, at the high dose of V P A employed, depletion of the intracellular free CoA pool due to the conversion of V P A to its acyl-CoA thioester may impair mitochondrial p-oxidation (Brass 1994). In rat mitochondrial 8 Chapter 1: Introduction incubations, V P A was converted to the corresponding CoA thioester, and in addition, the following CoA esters of the p-oxidation metabolites were identified: 2-ene-VPA, 3-OHA/PA, and 3-keto-VPA (Li et al. 1991). Valproyl-CoA was poorly hydrolyzed in liver preparations (Moore et al. 1988) and 3-keto-VPA-CoA appeared to resist cleavage by 3-ketoacyl-CoA thiolase (Li et al. 1991), resulting in the sequestering of CoA. The resulting depletion of the free CoA pool in the mitochondrial matrix was considered a major contributing factor for V P A hepatotoxicity (Harris et al. 1991). In support of this hypothesis, incubation of V P A with isolated rat hepatocytes resulted in dose-dependent decreases in free concentrations of CoA, and this was accompanied by.an inhibition of the oxidation of long- and medium-chain fatty acids (Becker and Harris 1983; Turnbull et al. 1983). However, it may be argued that the depletion of CoA should be V P A dose-dependent, and yet the hepatotoxicity appears to be idiosyncratic in nature (dose-independent and rare). 1.5.3 Carnitine deficiency hypothesis Carnitine deficiency has been associated with Reye's syndrome (Glasgow et al. 1980) and was postulated to be involved in VPA-associated impairment of fatty acid metabolism (Coulter 1984). L-carnitine (L-p-hydroxy-gamma-nitro-aminobutyric acid) is an essential cofactor in the p-oxidation pathway by facilitating transport of fatty acyl-CoA from the mitochondrial intermembrane space across the inner mitochondrial membrane to the matrix (Coulter 1991). Prior to transport across the inner mitochondrial membrane, fatty acyl-CoA is esterified into fatty acyl-carnitine via carnitine palmitoyltransferase I. The transported fatty acyl-carnitine is subsequently converted back to the fatty acyl-CoA by carnitine palmitoyltransferase II (located on the matrix side of the inner mitochondrial membrane) for subsequent p-oxidation reactions in the matrix. Treatment of V P A has been associated with decreased levels of serum carnitine (Bohles et al. 1982; Laub et al. 1986; Murphy et al. 1985; Ohtani et al. 1982). Another study 9 C h a p t e r 1: Introduct ion revealed an increased acylcarnitine/ free carnitine ratio that was accompanied by detectable levels of acetylcarnitine and valproylcarnitine in the urine of patients on V P A therapy (Millington et al. 1985). Thus, patients on V P A therapy with inborn errors of carnitine metabolism would theoretically be predisposed to idiosyncratic VPA-hepatotoxicity. Although carnitine supplementation has been recommended in the pediatric population on V P A therapy (Raskind and El-Chaar 2000), the overall significance of VPA-induced carnitine deficiency in causing impairment of p-oxidation of fatty acids is not clear. 1.5.4 Reactive metabolite hypothesis It has been postulated that V P A hepatotoxicity is mediated by one or more of its metabolites (Kesterson ef al. 1984; Lewis et al. 1982; Zimmerman and Ishak 1982). The metabolic pathways of V P A leading to reactive metabolites and their glutathione conjugates are outlined in Figure 1-3. The similarity of VPA-induced hepatotoxicity to Reye's syndrome and Jamaican Vomiting Sickness (microvesicular steatosis due to inhibition of p-oxidation, hypoglycemia due to impaired gluconeogenesis, hyperammonemia due to interference with the urea cycle) led researchers to postulate that 4-ene-VPA was responsible for the liver injury because the metabolite was structurally similar to two known hepatotoxicants, i.e. methylene-cyclopropylacetic acid (a metabolite of hypoglycin) and 4-pentenoic acid (Gerber et al. 1979; Zimmerman and Ishak 1982). V P A undergoes desaturation to 4-ene-VPA in rat liver microsomes catalyzed by P450 (Rettie et al. 1988; Rettie et al. 1987), and more specifically by the recombinant human P450's CYP2A6, C Y P 2 C 9 (Ho ef al. 2003; Sadeque et al. 1997) and CYP2B6 (Anari et al. 2000). Overall, the pathway is relatively minor, but it has been shown that the metabolic flux through the 4-ene-VPA pathway is elevated by co-administration of P450 inducers (phenytoin, P B , or carbamazepine), a known risk factor for VPA-induced hepatotoxicity (Cotariu and Zaidman 1988; Dreifuss et al. 1987; Levy ef al. 1990; Lewis et al. 1982; 10 Chapter 1: Introduction Zimmerman and Ishak 1982). Further investigations demonstrated that microvesicular steatosis could be reproduced by chronic administration of 4-ene-VPA and (£)-2,4-diene-VPA, an intermediate in the p-oxidation pathway of 4-ene-VPA, to rats (100 mg/kg for 5 days) (Granneman ef al. 1984a; Kesterson et al. 1984; Lewis et al. 1982). When tested in vitro, 4-ene-VPA demonstrated the greatest toxicity to rat hepatocyte cultures compared to 3-OH-VPA, 4-OH-VPA, 5-OH-VPA, and 2 -PGA (Kingsley ef al. 1983; Kingsley et al. 1980) and was an effective inhibitor of mitochondrial p-oxidation in rat liver preparations (Bjorge and Baillie 1985; Ponchaut et al. 1992). Radiolabeled 4-ene-VPA was seen to bind covalently to rat liver proteins (Porubek et al. 1989) and P450 enzymes (Prickett and Baillie 1986). A proposed mechanism of 4-ene-VPA hepatotoxicity suggests that the mitochondrial biotransformation of 4-ene-VPA to (E)-2,4-diene-VPA will lead to the formation of 3-keto-4-ene-VPA (Baillie 1988). Based on the evidence that the p-oxidation of 4-pentenoic acid produces the highly reactive electrophile, 3-keto-4-pentenoic acid, that irreversibly inhibits 3-ketoacyl-CoA thiolase (Schulz 1983), 3-keto-4-ene-VPA was suspected to alkylate and thereby irreversibly inhibit p-oxidative enzyme(s) in a similar manner (Baillie 1988). Although 3-keto-4-ene-VPA was not directly identified, there exist several lines of indirect evidence for the existence of this putative metabolite. Consistent with this mechanism, the activity of acetoacetyl-CoA thiolase was observed to be irreversibly inhibited in rat hepatocyte cultures treated with 4-ene-VPA (Porubek et al. 1991). Two precursors of 3-keto-4-ene-VPA, i.e. (E)-2,4-diene-VPA and 3-OH-4-ene-VPA were identified using perfused rat livers (Rettenmeier et al. 1985) and 3-keto-4-ene-VPA was indirectly identified in very low amounts as its G S H conjugate, 5-GS-3-keto-VPA (Kassahun et al. 1994). 11 C h a p t e r 1: Introduction CYTOPLASM P450 ^ ^ J ° ° ^ ^ (4,5-epoxy VPA) (4-ene VPA) Cytosolic GSH (5-GS-40H VPA lactone) + SG COOH (4-GS-5-OH VPA) COOH C O O H Cytosolic GSH ((E)-2,4-diene VPA) MITOCHONDRIA -ox idat ion c o s e OA ((E)-2-ene VPA CoA ester) R-oxidation Mitochondrial GSH ((E)-2,4-diene VPA CoA ester) SG (5-GS-3-ene VPA) + COOH COS Co A (3-keto-4-ene VPA CoA ester) SG (5-GS-2-ene VPA) Mitochondrial GSH (5-GS-3-keto VPA) Figure 1-3: Metabolic pathways of V P A leading to reactive metabolites and their G S H conjugates (Tang et al. 1995). Subsequently, the GSH conjugates undergo further mercapturic acid metabolism to produce the respective N-acetylcysteine (NAC) conjugates. The N A C conjugates of 3-heptanone (the suspected decarboxylated product of 5-GS-3-keto-VPA), 5-GS-4-OH-VPA y-lactone and 4-GS-5-OH-VPA have been identified in rat urine of 4-ene-VPA treated rats only, and not in 4-ene-VPA treated guinea pigs nor in patients on V P A therapy (Gopaul et al. 2000b). 12 Chapter 1: Introduction Further evidence of chemically reactive intermediates of 4-ene-VPA was provided by the detection of G S H and N-acetylcysteine conjugates of (£)-2,4-diene-VPA in the bile and urine, respectively, of rats dosed with 4-ene-VPA or (£)-2,4-diene-VPA (Kassahun et al. 1991). The major metabolite of 4-ene-VPA was determined to be (E)-2,4-diene-VPA and the G S H conjugation of this reactive metabolite was believed to occur in the mitochondria (Kassahun et al. 1991; Kassahun et al. 1994). A number of other G S H conjugates were identified in the bile of rats treated with 4-ene-VPA: 5-GS-3-keto-VPA from the conjugation of G S H with 3-keto-4-ene-VPA; 5-GS-3-ene-VPA from the conjugation of G S H with (E)-2,4-diene (Kassahun et al. 1991; Kassahun et al. 1994); and 5-GS-4-OH-VPA lactone from the conjugation of G S H with 4,5-dihydroxy-VPA-y-lactone (Prickett and Baillie 1986). The latter is produced from the spontaneous hydrolysis of 4,5-epoxy-VPA, a P450-catalyzed reactive metabolite identified in the bile of 4-ene-VPA treated rats (Kassahun etal. 1994). The V P A metabolite, (E)-2,4-diene-VPA, which is produced from either microsomal P450-catalyzed dehydrogenation of (E)-2-ene-VPA and/or the mitochondrial p-oxidation of 4-ene-VPA, was shown to be hepatotoxic when administered to rats (Kesterson et al. 1984). Two novel G S H conjugates, CoA-activated and glucuronide-activated G S H conjugates of (E)-2,4-diene-VPA, have been identified in the mitochondria and cytoplasm of rat liver homogenate, respectively, in (E)-2,4-diene-VPA treated rats (Tang and Abbott 1996). These findings suggest that glucuronide formation activates (E)-2,4-diene-VPA to further conjugate with G S H via a Michael addition reaction. Based on the principle that the role of G S H may serve as a trap for reactive species, the conjugation reactions may produce a localized depletion of the finite pool of mitochondrial G S H that would result in oxidative stress, covalent binding and subsequent inactivation of enzymes with accompanying hepatocellular damage (Kassahun etal. 1991; Kassahun etal. 1994). To reiterate, VPA-mediated hepatotoxicity is presumed to result from mitochondrial p-oxidation of the P450-dependent V P A metabolite, 4-ene-VPA, to (E)-2, 4-diene-VPA, which in the CoA 13 C h a p t e r 1: Introduct ion thioester form either depletes G S H and/or produces a putative inhibitor of p-oxidation enzymes. Acyl-CoA dehydrogenase enzymes catalyze the initial step in mitochondrial fatty acid p-oxidation by converting fatty acyl-CoA thioesters to their corresponding trans-2,3-enoyl-CoA derivatives (Schulz 1991). The enzymes are believed to function by a mechanism involving a -proton abstraction by an active site base followed by transfer of the p-hydride (Thorpe and Kim 1995). To further investigate VPA-induced hepatotoxicity, a mechanistic study was conducted in which the a-fluorinated 4-ene-VPA analogue (a -F-4-ene-VPA) was synthesized (Tang et al. 1995) with the rationale that the substitution of a fluorine atom at the a-position to the carboxylic acid group provides a derivative that contains a chemically and enzymatically inert carbon centre. This analogue is expected to be inert to p-oxidation to its reactive (£)-2,4-diene-VPA metabolite via acyl-CoA dehydrogenases, and thus reduce/prevent hepatotoxicity. Administration of a -F -4 -ene-VPA (113 mg/kg x 5 days, ip) to rats resulted in the absence of hepatic microvesicular steatosis, while 4-ene-VPA (100 mg/kg x 5 days, ip) induced severe hepatic microvesicular steatosis and mitochondrial alterations (Tang et al. 1995). Consistent with these findings, the major metabolites of 4-ene-VPA, (£)-2,4-diene-VPA and its N A C conjugate, were not detected in rats administered a -F -4 -ene-VPA (Tang et al. 1995). Mitochondrial G S H levels were also reduced to 68% of control in the rats administered 4-ene-VPA. This observed decrease in hepatotoxicity was further investigated and results indicated that a -F-4-ene-VPA did not form the corresponding acyl-CoA derivative in liver extracts of a - F -4-ene-VPA-treated rats (Grillo ef al. 2001). The data suggest that VPA-associated hepatotoxicity involves a sequence of events: (1) P450-dependent formation of 4-ene-VPA, (2) 4-ene-VPA activated to its 4-ene-VPA-CoA thioester, and (3) its subsequent p-oxidation to the electrophilic metabolite, (£)-2,4-diene-VPA-CoA that potentially depletes mitochondrial G S H . This localized depletion of G S H would make the cell more susceptible to oxidative stress, reactive metabolite covalent binding, and ultimately to mitochondrial dysfunction. 14 Chapter 1: Introduction In support of a reactive metabolite mechanism, the N-acetylcysteine conjugate of (E)-2,4-diene-VPA, was identified in the urine of patients receiving V P A therapy, with the levels being 3-4 times higher in those who had developed VPA-related hepatotoxicity (Kassahun et al. 1991). Furthermore, treatment with oral N-acetylcysteine was associated with clinical and biochemical recovery and survival of a small number of patients diagnosed with severe hepatotoxicity (i.e. microvesicular steatosis) while on V P A therapy (Farrell and Abbott 1991; Farrell et al. 1989a). It appeared that serum concentrations of 4-ene-VPA were higher in pediatric patients than in older patient groups (Abbott et al. 1986; Tatsuhara et al. 1987), the former suggested to be at most risk to V P A hepatotoxicity (Dreifuss ef al. 1987). Furthermore, patients who had taken V P A co-administered with the P450 enzyme inducers, phenytoin, PB , or carbamazepine, were more susceptible to VPA-induced liver injury than those under V P A monotherapy. On the other hand, there is evidence suggesting that the VPA-mediated hepatotoxicity does not correlate with plasma levels of 4-ene-VPA or its derivatives. V P A metabolite profiles of patients were determined and the hepatotoxicity appeared to be independent of 4-ene-VPA formation (Siemes et al. 1993). A similar conclusion was also reached following comparative studies of V P A and 2-ene-VPA in rats where the incidence of liver microvesicular steatosis was observed to be independent of plasma levels of 4-ene-VPA and (E)-2,4-diene-VPA. It was therefore suggested that these metabolites were not the decisive factors in VPA-induced hepatotoxicity (Loscher 1993). However, it remains questionable whether or not levels of 4-ene-VPA or (E)-2,4-diene-V P A are delineating factors for VPA-mediated hepatotoxicity. Recent studies in our laboratory demonstrated the applicability of measuring N-acetylcysteine conjugates arising from the reactive (£)-2,4-diene-VPA in patient urines as an indicator of reactive metabolite exposure (Gopaul et al. 2000a, b; Tang et al. 1995) Furthermore it was demonstrated that higher doses of V P A associated with younger patients and those on V P A polytherapy resulted in a greater exposure of these patients to reactive metabolites of V P A (Gopaul ef al. 2003). 15 C h a p t e r 1: Introduct ion 1.6 OXIDATIVE STRESS HYPOTHESIS 1.6.1 Oxidative stress Cellular generation of oxygen radicals and peroxides (reactive oxygen species, ROS) is a continuous and physiological event upon oxygen consumption (Chance et al. 1979; Sies and Moss 1978). Free radical intermediates (e.g. superoxide anion, 02*~) are formed through the univalent reduction of molecular oxygen, and are highly toxic to cellular components. The superoxide radical undergoes dismutation to hydrogen peroxide (H 2 0 2 ) and molecular oxygen (0 2 ) enzymatically via superoxide dismutase (SOD). Hydrogen peroxide can undergo an iron-catalyzed conversion to hydroxyl radical (OH*) (Reed 1995). The hydroxyl radical is one of the most chemically reactive species known and it can react at the site of formation with neighbouring tissue macromolecules. Oxidative stress is a term used to describe a condition of excessive production of R O S and/or decrease in antioxidant levels (see section 1.6.3) (Sies 1991). This pro-oxidant and anti-oxidant imbalance towards the former (Figure 1-4) can result in damage to tissue macromolecules such as lipids, proteins, and DNA. 1.6.2 Lipid peroxidation and biomarkers Lipid peroxidation can be defined as the oxidative deterioration of polyunsaturated fatty acids (PUFA), which are abundantly located in phospholipid cell membranes (Halliwell and Gutteridge 1999). Lipid peroxidation is initiated by hydrogen abstraction on the phospholipid side chain (i.e. arachidonate, Figure 1-5) by free radical species (e.g. the hydroxyl radical). The resultant reactive carbon-centre radical reacts with 0 2 to form peroxyl radicals (ROO*), which can propagate the lipid peroxidation chain reaction by removing another hydrogen atom from a neighbouring P U F A side chain generating more lipid hydroperoxides (Halliwell and Gutteridge 1999). Malondialdehyde (MDA, Figure 1-5) is a low molecular weight, reactive aldehyde, formed via the decomposition of certain primary and secondary lipid peroxidation products (e.g. 16 C h a p t e r 1: Introduction cyclic endoperoxides) (Esterbauer et al. 1991). MDA is commonly detected by the thiobarbituric acid reactive substances (TBARS) assay by forming a 1:2 adduct (MDA:thiobarbituric acid) which can be measured by fluorometry (Janero 1990). The isoprostanes are prostaglandin analogs formed during the free radical-catalyzed lipid peroxidation of arachidonic acid (Figure 1-5 and Figure 1-6) (Morrow et al. 1990b). F 2-lsoprostranes are also used as biomarkers of lipid peroxidation, and in particular, 15-F 2 t-isoprostane (15-F 2 t- lsoP, also known as 8-isoprostane) has been shown to be elevated in animal models of oxidative stress and also in humans (Gopaul et al. 1995; Morrow et al. 1995; Morrow et al. 1990a; Morrow et al. 1990b; Morrow et al. 1994). Recently, 1 5 - F 2 r l s o P has become a commonly used marker of oxidative stress that can be determined either by G C / M S or an enzyme-linked immunoassay (EIA) methods (Proudfoot et al. 1999). A ROS ROS production removal Figure 1-4: Schematic describing the concept of oxidative stress as a balance between ROS production and ROS removal. (A) A balance exists between ROS production through normal cellular respiration and ROS removal by antioxidants. (B) Oxidative stress is a term that describes an imbalance between ROS production and ROS removal. 17 C h a p t e r 1: Introduct ion C H 2 — O - F a t t y Acid C H 2 - 0 - A r a c h i d o n a t e Phospholipid in tissue I C H 2 — O - P O 3 - R + Initiation ROS (-o2« R* ) C H 2 — O - F a t t y Acid CH 2 —O-Arach idonate* C H 2 — O - P O 3 — R CH 2 —0—Fat ty Acid i CH 2 —O—Peroxyl radical C H 2 — O - P O 3 — R RH V £ Propagation H 6 6 Lipid hydroperoxides COOH Rearrangement C H 2 — O - F a t t y Acid CH 2 —O—Endoperoxide C H 2 — O - P O 3 — R H H + other aldehydes malondialdehyde Reduction C H 2 — O - F a t t y Acid CH 2 —O—Isoprostanes C H , — O - P O - , — R Phospholipase A 2 C O O H 15-F 2 t - isoprostane Figure 1-5: Schematic describing the formation of lipid peroxidation biomarkers: lipid hydroperoxides, malondialdehyde, and 15-F 2 t-isoprostane (modified from Cayman Chemical Co., 15-F 2 t-lsoprostane assay kit booklet). 18 Chapter 1: Introduction Phospholipid-bound arachidonic acid H » 1 — O - P - O . " o ^ ^ N + ( C H 3 ) n 1 Non-enzymatic lipid peroxidation Esterified F2-lsoprostanes 1 PHOSPHOLIPASE J Free F2-lsoprostane regio-isomers HO COOH HO OH C O O H IV Figure 1-6: Formation of F2-isoprostane regio-isomers during non-enzymatic peroxidation of arachidonic acid. Regio-isomer IV represents 15-F2t-lsoP (Gopaul ef al. 1995). 19 Chapter 1: Introduction 1.6.3 Antioxidant defense To regulate the free radical reactions, a defense system exists which includes enzymes (SOD, catalase (CAT), glutathione peroxidase (GSH-Px) and small molecules (Vitamins C and E, uric acid, G S H , albumin or bilirubin), and repair systems that prevent the accumulation of oxidatively damaged molecules (Pincemail 1995). Superoxide dismutase (SOD), a cytosolic and mitochondrial enzyme, serves as the first line of defense against the accumulation of toxic superoxide free radicals by catalyzing their dismutation to form hydrogen peroxide. Catalase, a cytoplasmic heme-enzyme found in high concentrations in peroxisomes, catalyzes the divalent reduction of hydrogen peroxide to water. Glutathione (L-y-glutamyl-L-cysteinyl-glycine, GSH) is a ubiquitous tripeptide thiol synthesized in the cytosol by the consecutive actions of y-glutamylcysteine synthetase and glutathione synthetase reactions (Meister 1995a) and is transported into the mitochondria (Griffith and Meister 1985; Meister 1995b). G S H serves not only as an important biomolecule for the conjugation of reactive metabolites, but also as an antioxidant to protect cells against R O S (Reed 1990). G S H is directly involved in the enzymatic reduction of R O S via the actions of the glutathione peroxidase - glutathione reductase system (Figure 1-7). GSH-peroxidase biotransforms hydrogen peroxide to water through the oxidation of G S H . The process represents detoxification when levels of electrophile are low, but toxic consequences may occur if cellular G S H is depleted (Reed 1990). Based on the principle that G S H may serve as a trap for reactive V P A metabolites and/or reactive oxygen species (i.e. lipid hydroperoxides) formed in the mitochondria, its conjugation reaction may result in a localized depletion of mitochondrial G S H that would result in oxidative stress, mitochondrial dysfunction, and ultimately lead to hepatocellular damage. 20 C h a p t e r 1: Introduction GSH - reductase 2 GSH GSSG Lipid • hydroperoxides 2H 2 Q Polyunsaturated fatty acid alcohol Figure 1-7: A schematic describing the glutathione peroxidase (GSH-Px) - glutathione reductase (GSH-reductase) antioxidant system. 1.6.4 Valproic acid and oxidative stress There is also a growing body of evidence suggesting that idiosyncratic hepatic toxicity may be mediated, at least in part, by the excessive generation of free radical intermediates as a consequence of xenobiotic bioactivation. Adverse effects during V P A therapy in patients has been associated with alterations in antioxidant enzymes characterized with reduced G S H -peroxidase and elevated G S H reductase activities (Graf et al. 1998). In addition, V P A therapy produced significant depletion of zinc and selenium in plasma of rats and humans, trace elements required for antioxidant enzyme activity (Graf ef al. 1998; Hurd et al. 1984). Both total hepatic and hepatic mitochondrial G S H levels have been depleted with V P A (Cotariu et al. 1990; Seckin ef al. 1999) and 4-ene-VPA (Tang ef al. 1995) exposure in rats. GSH-reductase and GSH-peroxidase activities were reported to be reduced, parallel to the depletion of G S H and NADPH after a single dose of V P A in rats (Cotariu ef al. 1990). The cytotoxicity of 4-ene-V P A in GSH-depleted rat hepatocytes was attenuated with vitamin C and E, while a-F-4-ene-V P A did not elicit toxicity under similar conditions (Jurima-Romet ef al. 1996). These findings 21 Chapter 1: Introduction are consistent with increased levels of R O S after V P A exposure. V P A treatment of human lymphocytes from patients who had developed hepatotoxicity while on V P A therapy demonstrated marked increases in cell death compared to lymphocytes from VPA-treated patients that did not show liver toxicity (Farrell and Abbott 1991; Farrell et al. 1989a). This concentration-dependent toxicity could be inhibited by inactivation of the microsomal metabolizing system in the incubation (Farrell ef al. 1988, 1989b). A further study demonstrated that the lymphocyte toxicity induced by V P A could also be inhibited by the addition of G S H or N-acetylcysteine. This result prompted the use of N-acetylcysteine in the successful treatment of three children who developed severe hepatotoxicity while receiving V P A . A rapid in vitro assay was developed for the detection of metabolism-dependent cytotoxicity on isolated human lymphocytes and provided a method to study the mechanisms underlying lymphocyte toxicity exhibited by V P A (Tabatabaei ef al. 1997). Mechanistic studies were carried out to evaluate the contribution of reactive metabolite and reactive oxygen species in the mechanism of the metabolism-dependent VPA-induced lymphocyte toxicity (Tabatabaei ef al. 1999). The addition of catalase or 10-phenanthroline (a cell permeable iron chelator) to the incubation mixture resulted in significant reduction in cytotoxicity without altering V P A metabolite levels. However, the cell impermeable iron chelator, desferroximine, did not reduce cytotoxicity. The data support the hypothesis that the observed cytotoxicity in the lymphocyte model was the result of the microsomal metabolism-dependent generation of hydrogen peroxide in the medium that can readily cross cell membranes and subsequently interact with intracellular iron to produce the highly reactive hydroxyl free radicals (Tabatabaei ef al. 1999). Further evidence implicating the involvement of oxidative stress with V P A treatment was demonstrated with the use of salicylate (2-hydroxybenzoic acid) as a specific hydroxyl radical (OH*) trap. This produces the stable hydroxylation product, 2,3-dihydroxybenzoic acid (2,3-DHBA) that can be specifically identified and quantified by L C / M S (Tabatabaei and Abbott 1999). V P A (100 mg/kg) treatment in rats resulted in increased formation of 2,3-DHBA with a mean maximal plasma level 2.5-fold greater 22 C h a p t e r 1: Introduct ion than the control group. In a separate study, vitamin E (a-tocopherol) and N,N'-diphenyl-p-phenylenediamine (DPPD) protected rat hepatocyte cultures against VPA-induced toxicity (Buchi et al. 1984). In a latter study, a protective effect by vitamin C and E against 4-ene-VPA-induced cytotoxicity in hepatocytes was reported (Jurima-Romet et al. 1996). Based on the available evidence, the induction of oxidative stress by V P A might be responsible or contribute to the mechanism of hepatotoxicity, and the idiosyncratic nature of this toxicity may occur in those individuals who have compromised anti-oxidant defenses. 1.7 R E S E A R C H R A T I O N A L E & H Y P O T H E S I S V P A is a widely used antiepileptic compound having desirable broad-spectrum activity. Although V P A is generally considered safe, it is limited by a rare but potentially fatal hepatotoxicity that was associated with the deaths of over one hundred children in the early to mid 1980's. The mechanism of fatal hepatotoxicity is unknown, and this has stimulated extensive research to investigate possible mechanisms. Traditionally, our laboratory has taken the approach to investigate the reactive metabolite theory by identifying reactive metabolites and examining patient risk factors that would help explain the idiosyncratic nature of the toxicity. Recently, we have found evidence for the generation of reactive oxygen species with V P A treatment in an in vitro lymphocyte model and an in vivo tat model as a possible mechanism of VPA-associated hepatotoxicity. Based on the available evidence, the hypothesis to be tested in this thesis is that V P A is associated with oxidative stress through the formation of reactive oxygen species and the depletion of G S H as a consequence of V P A biotransformation, and that these are key factors contributing to the hepatotoxicity. Therefore, evidence for oxidative stress associated with V P A treatment is expected to occur prior to hepatotoxicity. 23 C h a p t e r 1: Introduct ion 1.8 RESEARCH OBJECTIVES The objectives of the proposed research are as follows: 1. To determine if V P A is associated with oxidative stress in rats as determined by in vivo biomarkers of lipid peroxidation. 2. To investigate the effect of modulating the biotransformation pathways of V P A (P450, glucuronidation, and p-oxidation) on oxidative stress in rats. 3. To examine the effects of multiple V P A dosing on the temporal relationship between oxidative stress and hepatotoxicity. 4. To develop an in vitro model to investigate the relationship between oxidative stress, mitochondrial dysfunction, and toxicity by V P A in primary cultured rat hepatocytes. 24 C h a p t e r 1: Introduct ion 1.9 REFERENCES Abbott, F. S., and Anari, M. R. (1999). Chemistry and biotransformation. In Valproate: Milestones in Drug Therapy (W. 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VPA Increases Levels of 15-F2t-lsoprostane 2 The Effect of VPA on 15-F2rlsoprostane Levels in Rats 2.1 INTRODUCTION Recently, an in vitro assay consisting of an incubation system with PB-induced rabbit microsomes and human lymphocytes was developed as a model to investigate the involvement of reactive oxygen species and the role of biotransformation in VPA-induced cytotoxicity (Tabatabaei et al. 1997). The removal of NADPH from the system or the addition of catalase or 1,10-phenantholine (a cell permeable iron chelator) to the incubation mixture resulted in significant reduction in lymphocyte cytotoxicity (Tabatabaei et al. 1999). These results support the hypothesis that the observed cytotoxicity was the result of the generation of hydrogen peroxide by a microsomal-dependent pathway, and that the produced hydrogen peroxide can readily cross cell membranes and subsequently react with intracellular iron to produce highly reactive hydroxyl free radicals. Another method for the measurement of oxidative stress was demonstrated using salicylate as a specific hydroxyl radical (HO*) trap that produces stable hydroxylation products (Halliwell ef al. 1991). Our laboratory has developed an LC/MS assay to quantify 2, 3-dihydroxybenzoic acid (2, 3-DHBA) as a specific, non-enzymatic hydroxylation product of salicylate (Tabatabaei and Abbott 1999). In vivo studies with rats demonstrated that plasma levels of 2,3-DHBA were elevated 2.5- and 6.5-fold following exposure to V P A (100 mg/kg) and 1,1,1-trichloroethane (700 mg/kg), respectively, compared to saline control (Tabatabaei 1998). The HO* trap approach is not without limitations because it involves the administration of salicylate, which 36 C h a p t e r 2. V P A Inc reases L eve l s of 15 -F 2 i - l sop ros tane may influence the biotransformation (i.e. P450, p-oxidation, glutathione conjugation) of the test compound that may possibly confound the study. In this study, we have focused on the measurement of a 15-series F 2-isoprostane (15-F 2 t-lsoP), formerly known as 8-iso-prostaglandin F 2 a (8- iso-PGF 2 a ) (Cracowski et al. 2002) as a direct, in vivo index of oxidative stress. The endogenous formation of 15-F 2 t - lsoP is by a non-cyclooxygenase-dependent mechanism involving the free radical-catalyzed breakdown product of arachidonic acid (Morrow et al. 1990). This marker is useful because of its potential application in pediatric patients on V P A therapy. The objectives of the present study were to investigate the effect of V P A in rats on the levels of 15-F 2 t - lsoP and to determine if modulation of the P450-dependent V P A biotransformation pathways influences the plasma and liver levels of 15-F 2 t - lsoP. For comparison, we also measured thiobarbituric acid reactive substances (TBARS) and lipid hydroperoxide levels, even though they are generally considered to be less sensitive and less specific than 15-F 2 t - lsoP as markers for oxidative stress. 2.2 MATERIALS & METHODS 2.2.1 Reagents V P A (sodium salt), SKF-525A (proadifen), 1-aminobenzotriazole (ABT), octanoic acid, and carbon tetrachloride (CCI 4) were purchased from Sigma-Aldrich (Oakville, O N , Canada). Sodium phenobarbital (PB) and anhydrous sodium sulphate were purchased from BDH Chemicals Inc., (Toronto, ON , Canada). The 8-iso-prostaglandin F 2 a (8- iso-PGF 2 a ) EIA kit was purchased from Cayman Chemical Co. (Ann Arbor, Ml). Dimethylformamide and the gas chromatography derivatizing reagents pentafluorobenzyl bromide (PFBBr) and N-(tert-37 Chapter 2. VPA Increases Levels of 15-F2t-lsoprostane butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) were purchased from Pierce Chemical Co. (Rockford, IL). N,N-diisopropylethylamine and fe/t-butyldimethylsilyl chloride were obtained from Aldrich (Milwaukee, WI). HPLC grade ethyl acetate, acetonitrile, methanol, chloroform, and n-hexanes (GC/MS resolved) were purchased from Fisher Scientific (Vancouver, BC, Canada). 2.2.2 Animals Adult male Sprague-Dawley rats (225-250 g) were obtained from the University of British Columbia Animal Care Facility. They were fed with rat diet (Labdiet 5001 rodent diet, PMI Feeds Inc., Richmond, IN) and water ad libitum and maintained in a room on a 12 h light/12 h dark cycle at constant temperature (22°C) and humidity. All animal experiments were approved by the University of British Columbia Animal Care Committee and conducted in accordance with the guidelines of the Canadian Council on Animal Care. 2.2.3 Treatment of animals and sample collection Rats were administered with V P A , octanoic acid, or saline at the doses and duration indicated in each figure legend. To modulate P450 enzyme activity, rats were injected ip with SKF-525A (80 mg/kg, single dose), ABT (100 mg/kg, single dose), or P B (80 mg/kg once daily for 4 consecutive days) prior to V P A administration (500 mg/kg, ip). At a predetermined time after V P A treatment, as indicated in each figure legend, rats were sacrificed by decapitation, trunk blood was collected, and plasma was prepared. An aliquot of the plasma was used immediately for the determination of 15-F 2 t - lsoP levels and the remainder was stored at -80°C for the subsequent analysis of V P A , its metabolites, T B A R S , and lipid hydroperoxide levels. Livers were weighed and homogenized in 20 ml of 50 mM phosphate buffer (pH 7.4) under cold conditions. The homogenate was processed immediately for the analysis of R O S biomarkers 38 Chapter 2. VPA Increases Levels of 15-F2t-lsoprostane (described below). Aliquots were stored at -70°C for the later determination of V P A metabolite levels. As a positive control, carbon tetrachloride (CCI 4 , 2 ml/kg, ip) or corn oil (vehicle control) was given to Sprague-Dawley rats and animals were sacrificed 4 h later, the time point when plasma 15-F 2 t - lsoP levels were found to be maximal at the given C C I 4 dose (Morrow ef al. 1992). 2.2.4 15-F2rlsoPenzyme immunoassay (EIA) Plasma and liver levels of 15-F 2 t - lsoP were determined by an EIA assay according to the manufacturer's protocol. The assay is based on competition between unknown amounts of free 15-F 2 t - lsoP present in the samples and fixed amounts of 15-F 2 t-lsoP-acetylcholinesterase conjugate (15-F 2 t-lsoP-tracer) for 15-F 2 t-lsoP-specific rabbit antiserum. The 1 5 - F 2 r l s o P and 15-F 2 t - lsoP tracer complex with rabbit antiserum and bind to the mouse monoclonal anti-rabbit IgG antibody coated on a 96-well plate. After an incubation period of 20 h, the plates were washed and treated with Ellman's reagent (Cayman Chemical Co., Ann Arbor, Ml), which contains a mixture of acetylthiocholine iodide (5%) and 5,5'-Dithiobis-(2-nitrobenzoic acid) (10%), to produce an enzymatic product that was determined spectrophotometrically at 405 nm on a Labsystems Multiscan Ascent multi-well plate reader (Thermo Electron Corp., Burlington, O N , Canada). The intensity of color is proportional to the amount of 15-F 2 t - lsoP tracer bound to the well, which is inversely proportional to the amount of free 15-F 2 t - lsoP present in the sample. Free plasma 15-F 2 t - lsoP was measured directly by adding 50 ul of fresh rat plasma to 96-well plates coated with mouse monoclonal anti-rabbit IgG. For the determination of total 15-F 2 t - lsoP in plasma (100 u.1) and liver (500 ul), samples were subject to alkaline hydrolysis by incubation with an equal volume of 15% K O H for 1 h at 40°C followed by protein precipitation with 12 M HCI (final sample pH 1-2). For the determination of free liver 15-F 2 , - lsoP, liver homogenate was subjected only to protein precipitation. The samples were centrifuged (1600 x g for 10 min) to 39 Chapter 2. VPA Increases Levels of 15-F2t-lsoprostane pellet the protein, and processed by liquid-liquid extraction with ethyl acetate (5 ml) by gentle rotation for 30 min. The samples were centrifuged again (1600 x g for 10 min) to aid in the separation of the layers. The organic layer was dried under a stream of N 2 (30°C), reconstituted in acidified water (2 ml, pH 3), and processed by a solid phase extraction procedure adapted from Nourooz-Zadeh et al. (1995). Samples were extracted on a 24-channel Vac-Elut® Vacuum Manifold (Varian Inc., Lexington, MA) using Waters Oasis® C18 cartridges (Waters Ltd., Mississauga, O N , Canada). The cartridges were pre-conditioned with 2 ml methanol and 2 ml water (pH 3). Samples were washed with 3 ml water (pH 3) followed by 3 ml acetonitrile/water (15/85, v/v) and eluted with 6 ml hexane/ethyl acetate/propan-2-ol (30/65/5, v/v). The eluant was evaporated under N 2 and the residue reconstituted in 1 ml of EIA phosphate buffer (Cayman Chemical Co. , Ann Arbor, Ml) for 15-F 2 t - lsoP determination. Solid phase extraction recovery was performed by comparing extracted 15-F 2 t - lsoP quality control (QC) samples spiked with blank plasma and liver matrices to non-extracted Q C samples. 2.2.5 Validation of the 15-F2rlsoP EIA The 15-F 2 t - lsoP EIA was validated over a concentration range of 4-150 pg/ml in EIA buffer, plasma and liver matrices. Method validation was performed by determining inter-assay accuracy (% bias) and precision (% CV) using quality control samples at low (10 pg/ml), mid (50 pg/ml), and high (125 pg/ml) concentrations. This was accomplished by analyzing calibration curves and Q C samples on separate days in EIA buffer (n = 6), plasma (n = 6), and liver (n = 3) matrices. Quantitation of Q C samples was performed by analyzing the calibration curve standards and back calculating the concentration of each Q C sample from the obtained slope and intercept. A bias and precision of < 20% at the low Q C and < 15 % for all other Q C samples was considered to be acceptable. 40 C h a p t e r 2. V P A In c r eases Leve l s of 15 -F 2 t - l s op ros tane 2.2.6 Thiobarbituric acid reactive substances (TBARS) assay The concentration of T B A R S was calculated as malondialdehyde (MDA) equivalents (Figure 2-1) using a commercial kit (Oxi-Tek®, Zeptometrix Corporation, Buffalo, NY). Samples (100 ul) of plasma or supernatant from 10,000 x g, 15 min centrifugation of liver homogenate were mixed with an equal volume of sodium dodecyl sulfate solution and 2.5 ml of 5% thiobarbituric acid/ acetic acid reagent. Samples were incubated for 60 min at 95°C. After centrifugation at 1600 x g, samples were transferred into a 96-well plate and fluorescence was monitored on a Cytofluor® Series 4000 multi-well plate reader (Applied Biosystems, Bedford, MA) with excitation set at 508 nm (20 nm band width) and emission at 560 nm (20 nm bandwidth). This assay used an MDA standard to construct a standard curve (0.5 - 5 nmol/ml MDA). Figure 2-1: Structure of MDA-TBARS adduct. MDA forms a 1:2 adduct with thiobarbituric acid and is detected by fluorometry (ZeptoMetrix Co., T B A R S assay kit booklet). 41 Chapter 2. VPA Increases Levels of 15-F2t-lsoprostane 2.2.7 Lipid hydroperoxide (LPO) assay Hydroperoxide levels were determined using a commercial kit (Cayman Chemical Co., Ann Arbor, Ml). Plasma and liver homogenate samples (500 u,l) were de-proteinated and extracted under acidic conditions with 1 ml of ice-cold de-oxygenated chloroform. The chloroform extract was removed following centrifugation (1600 x g for 5 min at 0°C) for hydroperoxide determination. The L P O assay is based on redox reactions with hydroperoxides and ferrous ions to produce ferric ions and the resulting ferric ions are detected using thiocyanate ion as the chromagen (Figure 2-2). Chloroform extracts (500 u.l) were mixed with 50 ul chromagen reagent (2.3 mM ferrous sulfate in 0.2 M HCI and 1.5% methanolic solution of ammonium thiocyanate), and 300 u.l samples were transferred to a glass 96-well plate and absorbance was determined at 500 nm. This assay used 13-hydroperoxyoctadecadienoic acid as a lipid hydroperoxide standard to construct standard curves (0.5 - 5 nmol lipid hydroperoxide) against which unknown samples were plotted. ROOH + Fe2+ • RO* + Fe3+ RO* + Fe2+ + H+ — • ROH + Fe3+ Fe3+ + 5SCN • Fe(SCN), 2-Figure 2-2: Reduction/oxidation reaction scheme involved in the L P O assay (Cayman Chemical Co. , Lip id hydroperoxide assay booklet). 42 Chapter 2. VPA Increases Levels of 15-F2t-lsoprostane 2.2.8 VPA and metabolite assay The assay for V P A and its metabolites was from a modified method (Anari et al. 2000). Rat plasma (10-100 ul) or liver (50 ul) samples, and 50 ul of an internal standard mixture containing 2 ug/ml of each deuterated metabolite standard were added to borosilicate glass screw top culture tubes. The mixture was acidified to pH 3.5 and made up to 1 ml with 1 M K H 2 P 0 4 buffer (pH 3.5) and extracted with 6 ml of ethyl acetate by gentle rotation of the phases for 30 min. The organic extract was transferred to another borosilicate glass screw top test tube containing anhydrous sodium sulfate, vortex mixed, and centrifuged at 1600 x g for 10 min. The ethyl acetate was dried to approximately 50-100 ul under a gentle stream of N 2 (0.5 P S I at 30°C) using a Zymark Turbo-Vap® LV Evaporator (Zymark Co., Hopkinton, MA). N,N-diisopropylethylamine (30 ul) and pentafluorobenzyl bromide (10 were added to the mixture and heated at 40°C for 1 h for the pentafluorobenzyl ester derivatization of the carboxylic acid groups. The mixture was cooled at room temperature and treated with 20 (J of dimethylformamide and 30 pi of the mixture of 2% ferf.-butyldimethylsilyl chloride in MTBSTFA, and heated at 60°C for 2 h for the second step ferf.-butyldimethylsilylation of hydroxyl groups. The derivatized sample was cleaned up to remove non-volatiles by drying it down to a residue under a stream of N 2 and reconstituting it in 150 ul of hexane by vortex mixing. The mixture was centrifuged (1600 x g, 10 min) and the hexane layer was transferred to an autosample vial for injection onto the gas chromatograph (GC). 2.2.9 Gas chromatography-mass spectrometry (GC/MS) instrumentation G C / M S analysis of V P A and its metabolites was carried out using a HP 6890 G C interfaced to a HP5973 mass selective detector (Hewlett-Packard, Avondale, PA). The G C was equipped with a capillary splitless injector and a HP7683 autosampler. The mass spectrometric data acquisition and handling software, HP Enhanced Chemstation Software G1701BA (V B.01.00), 43 Chapter 2. VPA Increases Levels of 15-F2t-lsoprostane were used to control the operation of all instruments. Metabolite separation was carried out on a fused-silica narrow bore G C column (60 m x 0.25 mm I.D., 0.25 um film thickness) coated with the non-polar stationary phase SolGel 1-ms (SGE Inc., Austin, TX) in combination with a fused-silica 5-meter G C Z-guard column (Phenomenex, Torrance, CA). The carrier gas was helium with a constant column flow rate of 0.5 ml/min, a column head pressure of 18.5 PSI, and a septum purge flow rate of 23 ml/min. Samples (1 ul) were injected in the splitless mode (injector temperature, 250°C) and cold-trapped at 40°C. The column oven temperature was raised rapidly to 140°C and programmed linearly as follows: 1°C/min to 160°C, then 10°C/min to 270°C and held at 270°C for 5 min. Solvent delay was set at 17 min and the total run time was 37.8 min. The mass spectrometer was operated in negative chemical ionization (NCI) mode with single ion monitoring and a fixed filament emission current and electron energy of 50 uA and 150 eV, respectively. The ion source temperature and quadrupole temperatures were set at 200°C and 106°C, respectively. The G C interface was held at 270°C. Methane was used as a reagent gas for NCI operation with the reagent gas pressure set to 0.18 mTorr. Synthesized standards and deuterated internal standards were monitored at the specified m/z and retention time as shown in Table 2-1. The assay was based on negative ion selected ion monitoring of the (M-181) - anions of the double derivatized (PFB and TBDMS) analytes that corresponded to the loss of the P F B moiety at the carboxylate group (Anari et al. 2000). A standard curve was constructed for each metabolite over the concentration range of 2 to 500 ng/ml. 44 Chapter 2. VPA Increases Levels of 15-F2t-lsoprostane Table 2-1: Diagnostic ions and retention times of PFB/TBDMS derivatives of VPA, V P A metabolites and deuterated analogues from standard reference samples in rat plasma. Compound Ion monitored Retention time (min) [2H7] 4-ene VPA 148 18.67 4-ene VPA 141 18.81 [2H7] VPA 150 18.88 VPA 143 19.12 3-ene VPA 141 19.32 (E)-2-ene-[2H7] VPA 148 22.38 (E)-2-ene VPA 141 22.59 (E,Z)-2,3'-diene VPA 139 23.20 (E)-2,4-diene VPA 139 23.45 (£f£)-2,3'-diene VPA 139 24.52 4-keto-[2H7] VPA 164 25.57 4-keto VPA 157 25.70 4-OH-[2H7] VPA 280 31.17, 31.45 4-OH VPA 273 31.22, 31.50 3-OH-[2H7] VPA 280 31.28 3-OH VPA 273 31.34 3-keto-[2H7] VPA 278 32.44 3-keto VPA 271 32.49 5-OH-[2H7] VPA 280 32.54 5-OH VPA 273 32.59 2-PSA 339 34.02 2-PGA 353 35.08 45 C h a p t e r 2. V P A Inc reases Leve l s of 15 -F 2 t - l s op ro s t ane 2.2.10 Statistical analysis Statistical significance of the difference between the means of multiple groups was analyzed by one-way analysis of variance and, where appropriate, was followed by the Student Newman-Keul's multiple range test. The level of significance was set a priori at p < 0.05. 46 Chapter 2. VPA Increases Levels of 15-F2t-lsoprostane 2.3 RESULTS 2.3.1 15-FzrlsoPEIA Calibration curves of 15-F 2 t - lsoP demonstrated linearity over the range 4 - 1 5 0 pg/ml with linear regression coefficients > 0.99 (r2). The inter-assay variability (% CV) and precision (% bias) based on Q C samples in buffer, plasma, and liver matrices were < 15% for the high and mid concentrations, and < 20% at the low concentration (Table 2-2). The mean analytical recovery of 15-F 2 t - lsoP from the liquid-liquid/solid phase extraction procedure was performed by comparing extracted Q C samples from non extracted Q C samples. All Q C samples spiked with liver homogenate demonstrated approximately 90% recovery, while Q C samples spiked with plasma demonstrated approximately 90% recovery at the high and mid concentration Q C samples and 80% recovery at low concentration QC samples. 2.3.2 Dose-dependent effect of VPA on plasma levels of free 15-F2rlsoP To determine the effect of V P A on the plasma levels of 15-F 2 t - lsoP, adult male rats were injected with a single ip dose (50, 100, 250, or 500 mg/kg body weight) of V P A or an equivalent volume of saline (vehicle control). As shown in Figure 2-3, V P A at doses of 250 and 500 mg/kg increased plasma levels of 15-F 2 t - lsoP approximately 2.2-fold, when compared to the control group. In contrast, the lower doses had no effect. The positive control CCI 4 (2 ml/kg, ip), resulted in a 7-fold increase in plasma 15-F 2 t- lsoP levels compared to the corn oil vehicle control group with values of 221 ± 20 and 32 ± 6 pg/ml, respectively (n=6, mean ± S E M , Figure 2-4). 47 C h a p t e r 2. V P A In c r eases Leve l s of 15 -F 2 t - l s op ro s t ane Table 2-2: Inter-assay variation of the 15-F 2 t-lsoP enzyme immunoassay. 3 Matrix Parameter QC low" QC midc QC high" EIA Buffer % C V 5 13 4 (n=6) % Bias -4 8 2 P l a s m a % C V 5 12 4 (n=6) % Bias -4 8 3 Liver % C V 7 3 7 (n=3) % B ias -3 -4 6 aMethod validation was performed by evaluating inter-assay accuracy (% bias) and precision (coefficient of variation, % CV) of the low, mid, and high Q C concentrations in EIA buffer, blank rat plasma, and blank rat liver on separate days. Quantitation of Q C samples was performed by analyzing the calibration curve standards and back calculating the concentration of each Q C sample from the obtained slope and intercept. b Q C low (10 pg/ml). C Q C mid (50 pg/ml). ' 'QC high (125 pg/ml). 48 Chapter 2. VPA Increases Levels of 15-F2t-lsoprostane VPA Dose (mg/kg) Figure 2-3: Influence of single dose V P A on free plasma 15-F 2 t-lsoP levels. Adult male Sprague-Dawley rats (n = 4/group) were injected with a single ip dose of V P A (50, 100, 250 or 500 mg/kg). Control rats received an equivalent volume (1 ml/kg) of the vehicle (0.9% NaCl). At 0.5 h after V P A treatment rats were sacrificed and blood was collected. Values are means ± SEM. *Significantly different from control, p < 0.05. 49 Chapter 2. VPA Increases Levels of 15-F2t-lsoprostane Figure 2-4: The effect of CCI 4 treatment on levels of 15-F 2 t-lsoP. Male Sprague-Dawley rats were treated with a single ip dose of corn oil (vehicle control) or CCI 4 (2 ml/kg). At 4 h after treatment, rats were sacrificed and blood was collected. *Significantly different from control, p < 0.05. 50 C h a p t e r 2. V P A Inc reases L eve l s o f 15 -F 2 t - l s op ro s t ane 2.3.3 Time-dependent effect of VPA on plasma levels of free 15-F2rlsoP To characterize the time-course of the elevation of plasma levels of 15-F 2 t - lsoP by VPA, rats were administered a single ip dose of V P A (500 mg/kg) and blood samples were collected at 0, 0.5, 1, 1.5, 2, 4 and 8 h thereafter. As shown in Figure 2-5, the plasma concentration of 15-F 2 t -IsoP was the greatest at 0.5 h after V P A administration and returned to basal levels by 4 h post-dosing. Interestingly, the temporal profile of plasma 15-F 2 t - lsoP (tm a x = 0.5 h and C m a x = 46 + 9 pg/ml, mean + S E M , n = 4 rats per group) was similar to that of plasma V P A (tm a x = 1 h and C m a x = 578 ± 49 pg/ml). Overall, based on the dose-response and time-course experiments, the greatest increase in plasma 15-F 2,- lsoP levels after a single dose of V P A was 2.3-fold, when compared with the saline-treated control group. Time (hr) Figure 2-5: Time-course of plasma V P A and 15-F 2 t-lsoP. Adult male Sprague-Dawley rats (250-300g) were injected with a single ip dose of V P A (500 mg/kg). Control rats received an equivalent volume (1 ml/kg) of the vehicle (0.9% NaCl). At various times after V P A treatment (0.5, 1, 1.5, 2, 4, and 8 h), rats were sacrificed and blood was collected. Values are means ± SEM (n = 4/time point). 51 Chapter 2. VPA Increases Levels of 15-F2t-lsoprostane 2.3.4 Effect ofPB on plasma and liver levels of free 15-F2rlsoP, TBARS, LPO, and VPA metabolites in rats treated with VPA To determine the effect of inducing P450-mediated biotransformation of V P A on the elevation of plasma 15-F 2 t - lsoP levels by this drug, rats were pretreated with PB (80 mg/kg, ip) or an equal volume of saline (vehicle control) once daily for four consecutive days. Animals were treated with saline vehicle or a single dose (500 mg/kg) of V P A on the following day and sacrificed 0.5 h after V P A administration. PB pretreatment and V P A exposure (500 mg/kg) resulted in a 2-fold increase of free plasma 15-F 2 t - lsoP compared to V P A treatment alone, and a 5-fold increase compared to PB pretreatment and saline vehicle controls (Figure 2-6A). A similar trend in the elevation of free liver 15-F 2 t - lsoP levels was determined in liver samples of the treatment groups (Figure 2-6B). Plasma and liver levels of T B A R S and hydroperoxides did not differ among the treatment groups, including the CCI 4-treated rats (Table 2-3). 52 C h a p t e r 2. V P A Inc reases L eve l s of 15 -F 2 t - l s op ros tane Figure 2-6: Effect of PB pretreatment on levels of 15-F 2 t-isoP measured as the free form in the plasma and liver of VPA-treated rats. Adult male Sprague-Dawley rats were pretreated with sodium phenobarbital (PB, 80 mg/kg, ip, once daily for 4 days) or saline (vehicle control). Animals were treated with a single dose of V P A (500 mg/kg, ip) or saline on the following day. At 0.5 h after treatment, rats were sacrificed, and blood and liver were collected. Values are means ± SEM (n = 6/group). *Significantly different from control, p<0.05; **Significantly different from the VPA-treated group, p < 0.05. 53 Chapter 2. VPA Increases Levels of 15-F2t-lsoprostane Table 2-3: T B A R S and lipid hydroperoxide levels detected in plasma and liver of V P A treated rats and pretreated with PB . a Treatment (n=6/group) Plasma (nmol/ml) Liver (nmol/g tissue) TBARS Saline 1.0 ± 0.1 11.9 ±0.2 (MDA equivalents) P B a 1.1 ± 0.1 12.8 ± 0.7 V P A " 1.2 ± 0.1 12.2 ± 0.6 PB+VPA 0 1.1 ±0.1 13.8 ±0.6 Treatment (n=6/group) Plasma (nmol/ml) Liver (nmol/ g tissue) Lipid hydroperoxides Saline ND 72 ± 7 P B a ND 64 ± 5 V P A " ND 61 ± 5 P B + V P A c ND 62 + 6 "Values represent the mean ± S E M (n=6). One-way analysis of variance using Newman-Keuls test was performed on the data. b Rats were pretreated with saline vehicle followed by V P A (500 mg/kg, ip). °Rats were pretreated with PB (80 mg/kg/day for 4 days) and by V P A (500 mg/kg, ip). ND, not detected. 54 C h a p t e r 2. V P A In c r eases Leve l s of 15 -F 2 t - l s op ro s t ane PB pretreatment and V P A exposure was associated with increased levels of 4-ene VPA , (£)-2, 4-diene V P A , 4 -OH-VPA, 4-keto-VPA, 3-OH-VPA and 5-OH-VPA compared to V P A treated rats (Table 2-4). Liver V P A metabolite levels showed a similar pattern with the exception that 5-OH-V P A was not different. The p-oxidation metabolites (2-ene-VPA, 3-keto-VPA, 3-ene-VPA, (E,£)-2,3'-diene-VPA) and the dicarboxylic acids (2-PSA and 2-PGA) were not different between the two groups (data not shown). Table 2-4: Effect of PB pretreatment on plasma and liver levels of V P A metabolites in rats treated with VPA . a Plasma (x 10 2 ng/ml) Liver (x 10 2 ng/g tissue) Saline" P B C Saline b PB c 4-ene V P A 1.3 ± 0.17 6.5 ± 1.3* 0.96 ± 0.05 3.9 ± 0.10 # (E)-2,4-diene V P A < LOQ 0.73 ± 0.08 * < L O Q 0 . 1 5 ± 0 # 3-OH V P A 3.9 ± 0.32 19 + 3.7* 3.7 + 0.08 12 ± 0.40 # 4-OH V P A 18 + 2.6 121 ± 19* 8.8 ± 0.42 6 9 ± 2 . 4 # 5-OH V P A 30 ±2 .6 52 ± 6 . 4 * 40 ± 1.4 36 ± 1.2 4-keto V P A 4.0 ± 0.44 45 ± 5 . 5 * 1.3 ±0.07 17 ± 0.48 # 'Values represent the mean ± S E M (n=6). One-way analysis of variance using Newman-Keuls test was performed on the data. 6 Rats were pretreated with saline vehicle followed by V P A (500 mg/kg, ip). c Rats were pretreated with P B (80 mg/kg/day for 4 days, ip), followed by V P A (500 mg/kg, ip) on the next day, and sacrificed 0.5 h later. "Indicates a statistically significant difference in the plasma compared to the saline pretreatment group, ind icates a statistically significant difference in the liver compared to the saline pretreatment group. < LOQ, below the limit of quantitation (2 ng/ml) for the G C / M S assay of V P A metabolites. 55 Chapter 2. VPA Increases Levels of 15-F2t-lsoprostane 2.3.5 Lack of an effect by SKF-525A on VPA-induced increase in plasma 15-F2r IsoP levels. To determine the effect of inhibition of P450-mediated biotransformation of V P A on the elevation of plasma 15-F 2 t - lsoP levels by this drug, rats were pretreated with SKF-525A (80 mg/kg, ip) or saline (vehicle control) 30 min prior to the administration of V P A . SKF-525A is known to inhibit multiple rat hepatic P450 enzymes (Buening and Franklin 1976; Murray 1988). As shown in Figure 2-7, SKF-525A exposure resulted in similar levels of 15-F 2 t - lsoP compared to the saline control, and SKF-525A pretreatment did not influence the elevated levels of 15-F 2 t - lsoP observed in V P A exposed rats. Control SKF-525A VPA SKF-525A +VPA Figure 2-7: SKF-525A pretreatment on 15-F 2 t-lsoP levels in VPA-treated rats. Adult male Sprague-Dawley rats were pretreated with a single ip dose of SKF-525A (80 mg/kg) or saline (vehicle control). A single ip dose of V P A (500 mg/kg) or saline (vehicle control) was administered 0.5 h after the pretreatment. At 0.5 h after V P A treatment rats were sacrificed and blood was collected. *Significantly different from control, p < 0.05. 56 Chapter 2. VPA Increases Levels of 15-Fa-lsoprostane 2.3.6 Effect ofABT on plasma levels of free 15-F2rlsoP in VPA-treated rats ABT is a mechanism-based inactivator of multiple constitutive P450 enzymes .(Ortiz de Montellano and Mathews 1981). To determine the effect of inhibition of P450-mediated biotransformation of V P A on the elevation of plasma 15-F 2 t - lsoP levels by this drug, ABT (100 mg/kg, ip) was administered to rats 2 h prior to V P A treatment (250 mg/kg). This regimen of ABT treatment is known to produce maximal inhibition (Mugford ef al. 1992). ABT had no effect on the elevation of plasma 15-F 2 t - lsoP levels by V P A (Figure 2-8), even though treatment with this compound resulted in undetectable levels of plasma 4-ene-, 3-OH-, 4-OH-, 5-OH-, and 4-keto-VPA concentrations (Table 2-5). saline ABT VPA ABT + VPA Figure 2-8: Effect of A B T pretreatment on free plasma 15-F 2 t-lsoP levels in VPA-treated rats. Adult male Sprague-Dawley rats were pretreated with a single ip dose of 1-aminobenzotriazole (ABT, 100 mg/kg) or saline. A single ip dose of V P A (250 mg/kg) or saline (vehicle control) was administered 2 h after the pretreatment. At 0.5 h after V P A treatment rats were sacrificed and blood was collected. Values are means ± SEM (n = 6/group). *Significantly different from control, p < 0.05. 57 Chapter 2. VPA Increases Levels of 15-F2Hsoprostane Table 2-5: Effect of 1-aminobenzotriazole on plasma levels of V P A metabolites. 3 S a l i n e b (x 10 2 ng/ml) A B T c (x 10 2 ng/ml) 4-ene V P A 1.2 ± 0 . 0 7 < L O Q (E)-2,4-diene V P A < L O Q < L O Q 3-OH V P A 2.6 ± 0 . 3 7 < L O Q 4 - O H V P A 11 ± 0 .67 < L O Q 5-OH V P A 27 ± 1.6 < L O Q 4-keto V P A 4.2 ± 0.22 < L O Q 'Values represent the mean ± S E M (n=4). "Rats were pretreated with saline vehicle (ip), followed by V P A (250 mg/kg) 2 h after. °Rats were pretreated with ABT (100 mg/kg, ip) followed by V P A (250 mg/kg) 2 h after. ND, not detected. 2.3.7 Effect of SKF-525A on the enhancement of plasma levels of free 15-F2rlsoP by PB in rats treated with VPA SKF-525A is an effective inhibitor of PB-inducible P450 enzymes in rat liver (Buening and Franklin 1976; Murray 1988). To determine the effect of inhibiting V P A biotransformation by PB-inducible P450 enzymes on plasma levels of 15-F 2 t - lsoP, PB-pretreated (80 mg/kg/day, ip, for 4 days) rats were administered SKF-525A (80 mg/kg, ip) or saline at 0.5 h prior to V P A administration (500 mg/kg, ip). SKF-525A pretreatment did not alter the elevated levels of 15-F 2 t - lsoP by PB in the VPA-treated rats (Figure 2-9). According to G C / M S analysis, SKF-525A administration reduced the levels of 4-ene-VPA, (E)-2,4-diene-VPA, 4-OH-VPA, 4-keto-VPA, 3-OH-VPA, and 5-OH-VPA in rats treated with the combination of PB and V P A (Table 2-6). 58 C h a p t e r 2. V P A Inc reases L eve l s of 15 -F 2 t - l s op ro s t ane 160 n V P A S K F - 5 2 5 A S K F - 5 2 5 A + V P A Figure 2-9: PB and SKF-525A pretreatment on plasma 15-F 2 t-lsoP levels in VPA-exposed rats. Adult male Sprague-Dawley rats were pretreated with PB (80 mg/kg/day, ip) for 4 consecutive days. A single dose of SKF-525A (80 mg/kg, ip) or saline (vehicle control) was given on the following day. At 0.5 h following SKF-525A administration, rats were treated with V P A (500 mg/kg) or saline. At 0.5 h after V P A treatment rats were sacrificed and blood was collected. Values are means ± SEM (n = 6/group). *Significantly different from control, p < 0.05. 59 Chapter 2. VPA Increases Levels of 15-F2t-lsoprostane Table 2-6: Effect of SKF-525A on plasma levels of V P A metabolites in rats treated with both PB and V P A . 3 Saline b (x 10 2 ng/ml) S K F - 5 2 5 A c (x 10 2 ng/ml) 4-ene V P A 11+1 .5 1.8 ± 0 . 2 8 * (£)-2,4-diene V P A 0.60 ±0 .08 < L O Q 3-OH V P A 17 + 2.8 7.3 ± 1.1 * 4 -OH V P A 260 ± 4 . 0 49 ± 5 . 8 * 5-OH V P A 82 ± 1.2 46 ± 3 . 5 * 4-keto V P A 120 ± 2 . 5 15 ± 3 . 0 * 3 Values represent the mean ± S E M (n=6). One-way analysis of variance using Newman-Keuls test was performed on the data. b Rats were pretreated with PB (80 mg/kg/day for 4 days), followed by saline on the next day and V P A (500 mg/kg, ip) 0.5 h later. c Rats were pretreated with P B (80 mg/kg/day for 4 days), followed by SKF-525A (80 mg/kg, ip) on the next day and V P A (500 mg/kg, ip) 0.5 h later. Rats were sacrificed 0.5 h after V P A treatment, indicates a statistically significant difference from the saline control group. < LOQ, below the limit of quantitation (2 ng/ml) for the G C / M S assay of V P A metabolites. 60 Chapter 2. VPA Increases Levels of 15-F2t-lsoprostane 2.3.8 Lack of an effect on plasma 15-F2rlsoP levels by octanoic acid. Octanoic acid (Figure 2-10), an 8-carbon straight chain fatty acid, was compared with V P A to determine if the elevation of plasma 15-F 2 t - lsoP levels by V P A occurs with other fatty acids of similar structure. Rats were administered ip with octanoic acid at an equivalent dose (500 mg/kg) of V P A that resulted in maximal elevation of plasma 15-F 2 t - lsoP levels or 0.9% saline (vehicle control). There was no significant difference between the plasma levels of 15-F 2 t - lsoP in the octanoic acid-treated group (24 ± 5 pg/ml) and the control group (22 ± 3 pg/ml). Saline VPA Octanoic acid Figure 2-10: Comparison between VPA and octanoic acid on the effect of plasma 15-F 2 t-IsoP levels in rats. Adult male Sprague-Dawley rats were treated with a single dose of VPA or octanoic acid (each at 500 mg/kg, ip). The control group was treated with saline vehicle. At 0.5 h after treatment, rats were sacrificed, and blood was collected for the immediate preparation of plasma. Values are means ± S E M (n = 4/group). *Significantly different from the saline-treated control group, p < 0.05. 61 Chapter 2, VPA Increases Levels of 15-F2t-lsoprostane 2.4 DISCUSSION It has been hypothesized that the responsible mediator of VPA-induced hepatotoxicity is produced by the P450-dependent formation of reactive metabolites, such as 4-ene-VPA and its subsequent mitochondrial p-oxidation metabolite, (£)-2,4-diene-VPA (Baillie 1992; Granneman et al. 1984; Kassahun ef al. 1991; Kassahun ef al. 1994; Kesterson ef al. 1984; Porubek et al. 1989; Tang and Abbott 1996; Tang et al. 1995). This hypothesis was based on earlier observations that 4-ene-VPA was structurally similar to the known hepatotoxins 4-pentenoic acid and methylenecyclopropylacetic acid (Gerber et al. 1979), and that patients on V P A antiepileptic polytherapy with other antiepileptic compounds were at increased risk of V P A -induced idiosyncratic hepatotoxicity (Bryant and Dreifuss 1996; Dreifuss et al. 1989; Dreifuss et al. 1987). Another hypothesis involves the generation of R O S upon V P A exposure (Pippenger et al. 1991; Wilder and Hurd 1991), which may or may not be dependent on V P A biotransformation. In the present study in rats, experiments were performed to determine the effect of V P A on plasma and hepatic levels of 15-F 2 t - lsoP, a sensitive marker of R O S . The application of 15-F 2 t - lsoP as a stable, in vivo marker for oxidative stress was demonstrated in animal models of free radical injury and lipid peroxidation (Morrow et al. 1990), and is increasingly being used as an in vivo index of lipid peroxidation. The formation of F 2 -isoprostanes has been shown to increase significantly in rats exposed to CCI 4 and diquat (Morrow et al. 1992; Morrow ef al. 1990) and in heavy smokers (Morrow et al. 1995). Quantitation of F 2-isoprostanes has been traditionally carried out by G C / M S (Morrow and Roberts 2002) and more recently by EIA methods. However, studies comparing both methods have demonstrated a lack of correlation due to inadequate separation and antibody cross-reactivity of closely related isoprostanes using G C / M S and immunoassay, respectively (Bessard et al. 2001; Mori et al. 1999; Proudfoot et al. 1999). The current study uses a commercially 62 Chapter 2. VPA Increases Levels of 15-F2t-lsoprostane available immunoassay that was validated in our laboratory and was shown to detect low concentrations (pg/ml levels) of 15-F 2 t - lsoP in a small volume of sample (50 ul of plasma). In our study, saline-treated rats (control) had basal free plasma levels of 15-F 2 t - lsoP in the range of 10-35 pg/ml. These levels of 15-F 2 t - lsoP increased in a dose-dependent manner following single exposures of V P A , and the temporal profile of 15-F 2 t - lsoP concentration in plasma followed that of V P A . These findings indicate that V P A treatment results in an increase in lipid peroxidation, and that the liver may be the source of the lipid peroxidation products. Conflicting data exist in the literature with respect to the effect of V P A on lipid peroxidation. In a 2-year prospective study, epileptic children on V P A therapy demonstrated an elevation in the extent of lipid peroxidation as measured by the T B A R S assay as compared to healthy controls, with a concomitant reduction in glutathione peroxidase levels and an increase in superoxide dismutase levels (Yuksel ef al. 2001). Also, increased lipid peroxidation was observed in mice chronically treated with VPA , as demonstrated by increased T B A R S (malondialdehyde equivalents) in the liver (Raza et al. 1997). However, other studies investigating the effects of V P A did not show significant differences in lipid peroxidation using the same biomarker for oxidative stress. For instance, rats treated with single doses of 500 or 1000 mg/kg V P A did not result in changes in T B A R S levels in both liver homogenates and hepatic mitochondrial fractions (Seckin ef al. 1999). A similar lack of increase in T B A R S level by V P A treatment (1 mM) was also demonstrated in a rat hepatocyte model (Klee et al. 2000). However, in another study, the free radical scavengers vitamin E and A/,A/'-diphenyl-p-phenylenediamine protected against V P A toxicity in a rat hepatocyte model, suggesting that V P A hepatotoxicity is associated with lipid peroxidation (Buchi et al. 1984). In our results, although levels of T B A R S and lipid hydroperoxides in plasma and liver were not affected by single doses of V P A , 15-F 2 t - lsoP was found to be elevated suggesting that this is a more sensitive biomarker of oxidative stress. To further verify this difference in response of biomarkers, a 7-fold increase in plasma 15-F 2 t - lsoP 63 Chapter 2. VPA Increases Levels of 15-F2t-lsoprostane was observed with carbon tetrachloride (data not shown) with no elevation in plasma or liver T B A R S . This result was consistent with other studies that show that an elevation of 15-F 2 t - lsoP in CCI 4-treated rats is not accompanied by an increase in T B A R S levels (Basu 1999; Sodergren etal. 2001). In the present study, rats pretreated with PB prior to V P A administration generated marked increases in levels of free 15-F 2 t- lsoP in plasma and liver compared to the saline control and VPA-treated groups. Levels of total 15-F 2 t - lsoP (sum of free and esterified) were also measured in plasma and liver. A single large dose of V P A , regardless of P B pretreatment, did not result in changes in total 15-F 2 t - lsoP even though free 15-F 2 t - lsoP levels in plasma and liver were elevated (data not shown). A possible explanation of this result is the combination of PB pretreatment followed by V P A treatment enhances the transition of the esterified form of 15-F 2 t -IsoP to the free form. Thus the basal ratio of total/free 15-F 2 t - lsoP in plasma and liver decreases from 3.3 and 4.6, respectively, after V P A to approximately 1.5 after P B / V P A treatment. This transition from esterified to free 15-F 2 t - lsoP may also be inferred from a study that investigated the effect of vitamin E on liver levels of 1 5 - F 2 r l s o P , where free 15-F 2 t - lsoP was attenuated while total 15-F 2 t - lsoP remained unchanged (Sodergren et al. 2001). However, in our study, this phenomenon was not observed in animals pretreated with P B alone, suggesting that PB does not simply enhance the release of the esterified form of 15-F 2 t - lsoP. Although levels of total 15-F 2 t - lsoP were not altered after a single dose of V P A , regardless of P B pretreatment, total levels of liver 15-F 2 t - lsoP were elevated after chronic treatment of V P A for 2 weeks, suggesting that multiple doses of V P A are required to observe an overall increase in total lipid peroxidation in the liver. The elevation of free 1 5 - F 2 r l s o P in P B / V P A treated rats corresponded with increased plasma levels of 4-ene-VPA and (E)-2, 4-diene-VPA among other P450-dependent V P A metabolites. 64 Chapter 2. VPA Increases Levels of 15-F2t-lsoprostane These findings are consistent with the reactive metabolite hypothesis that suggests that an increase in the metabolic flux through the PB-inducible P450 pathway leads to higher levels of 4-ene-VPA and (£)-2,4-diene-VPA, which may be responsible for the marked elevation in plasma 15-F 2 t - lsoP levels. However, SKF-525A, which attenuated P450-derived V P A metabolite levels, did not influence the PB-induced elevation in plasma levels of 15-F 2 t - lsoP. This finding suggests that the mechanism by which PB increases the elevation of 15-F 2 t - lsoP in VPA-treated rats does not involve V P A metabolites formed by pathways inhibited by SKF-525A. P450 systems have been demonstrated to contribute to the in vitro formation of R O S as monitored by the formation of 2',7'-dichlorofluorescin (DCF) by an NADPH-dependent process, which can be blocked by SKF-525A (Bondy and Naderi 1994). In vitro induction of P450 by dexamethasone and PB resulted in marked production of 2, 3-DHBA as an index of hydroxyl radical production, which was attenuated by SKF-525A (Strolin-Benedetti et al. 1999). Therefore, if the pathway leading to the P450-dependent formation of 4-ene-VPA, and thus (£)-2,4-diene-VPA, contributes directly or indirectly to the production of R O S , it should then be possible to influence 15-F 2 t - lsoP production with the use of P450 inhibitors. Our study, however, demonstrated the lack of effect of P450 inhibitors (ABT and SKF-525A) on 15-F 2 t - lsoP plasma levels. Based on the results obtained with P450 inhibitors, there appears to be a discrepancy between the in vitro studies using human lymphocytes (Tabatabaei et al. 1999) and our in vivo studies regarding the importance of P450-dependent V P A biotransformation on R O S generation. The discontinuity may reflect differences in biotransformation between models (microsomes vs. whole animal), the effector cells (lymphocytes vs. whole animal), and the R O S biomarker measured ( H 2 0 2 vs. 15-F 2 t- lsoP). To help clarify the in vivo and in vitro discrepancy, research is currently underway to evaluate in vitro data seen with V P A using primary cultures of rat hepatocytes. 65 C h a p t e r 2, V P A In c r eases Leve l s of 15 -F 2 t - l s op ro s t ane A possible explanation for the influence of PB on the VPA-dependent generation of R O S may involve the transactivation of the constitutive androstane receptor (CAR) by PB . This nuclear receptor has been shown to mediate the expression of multiple enzymes, including C Y P 2 B (Honkakoski et al. 1998), glutathione S-transferase (GST) Pi (Zhang et al. 2002), UDP-glucuronosyltransferase (UGT) 1A1 (Sugatani ef al. 2001), and a peroxisomal p-oxidation enzyme 3-hydroxyacyl-CoA dehydrogenase (Kassam et al. 2000). In a recent study, known C A R activators (e.g. PB and 1,4-bis[2-(3,5-dichloropyridyl-oxy)]benzene) in combination with acetaminophen treatment resulted in hepatotoxicity in wild-type but not in CAR-null mice (Zhang et al. 2002). Conceivably, C A R may also play a role in the observed enhancement of R O S levels by PB in rats administered VPA. Studies are now in progress to elucidate the cellular and molecular mechanisms by which V P A produces oxidative stress, how PB is able to modulate this effect, and the impact of VPA-dependent R O S generation on mitochondrial dysfunction and hepatotoxicity. The in vivo studies described in the manuscript do raise issues regarding the importance of oxidative stress and/or the possibility of covalent binding, the latter not being ruled out, in the proposed mechanism of VPA-associated hepatotoxicity. In summary, our data demonstrate that V P A results in a dose- and time-dependent increase in free plasma levels of 15-F 2 t - lsoP that follow the plasma profile of V P A in rats. PB-pretreatment significantly enhanced free plasma and liver levels of 15-F 2 t - lsoP by a mechanism that remains unclear; however, this was not due to enhanced production of P450-dependent metabolites such as 4-ene-VPA. 66 Chapter 2. VPA Increases Levels of 15-F2t-lsoprostane 2.5 REFERENCES Anari, M. R., Burton, R. W., Gopaul, S., and Abbott, F. S. (2000). Metabolic profiling of valproic acid by cDNA-expressed human cytochrome P450 enzymes using negative-ion chemical ionization gas chromatography-mass spectrometry. J Chromatogr B Biomed Sci Appl 742, 217-27. 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Science 298, 422-4. \ 71 C h a p t e r 3: Va l p r oa t e -g l u cu ron i de and 15-F 2 t - i sop ros tane 3 Valproic Acid Glucuronidation is Associated with Increases in 15-F2rlsoprostane Levels in Rats 3.1 INTRODUCTION F 2-isoprostanes, a series of prostaglandin F 2 a- isomers, are produced by a free-radical-catalyzed (cyclooxygenase-independent) lipid peroxidation of arachidonic acid. Among the F 2 -isoprostanes, 15-F 2 t-isoprostane (15-F 2 t- lsoP) is used as a marker for lipid peroxidation (Morrow et al. 1992; Morrow ef al. 1990a; Morrow ef al. 1990b; Morrow ef al. 1994). Recently, we reported that V P A was associated with an increase in oxidative stress as measured by serum and liver levels of 15-F 2 t - lsoP in rats (Tong et al. 2003). The increase in 15-F 2 t - lsoP was dose-dependent and its plasma concentration-time profile was similar to that of V P A . Interestingly, this increase in 15-F 2 t - lsoP by V P A was further enhanced by pretreatment with PB. However, the elevation in 15-F 2 t - lsoP was not associated with P450-dependent VPA-biotransformation because pretreatment with P450 inhibitors did not influence the levels of 15-F 2 t - lsoP, even though there was a dramatic decrease in the levels of V P A oxidative and desaturated metabolites (Tong ef al. 2003). The effect of PB on the VPA-associated increase in 15-F 2 t - lsoP was an interesting observation and prompted us to investigate the role of V P A glucuronidation, a major V P A biotransformation pathway, on 15-F 2 t - lsoP levels. V P A forms the 1-O-acyl glucuronide conjugate (VPA-G) and this is catalyzed by UDP-glucuronosyltransferase (UGT) enzymes, which transfer the glucuronide moiety from UDP-glucuronic acid (UDPGA) to the carboxylate group of VPA. PB induces specific UGT enzymes and elevates levels of hepatic U D P G A (Granneman ef al. 1984; 72 C h a p t e r 3: Va l p roa te -g l u cu ron i de and 15-F2t-isoprostane Heinemeyer ef al. 1985; Watkins and Klaassen 1982). Specifically, rat UGT2B1 and human UGT2B7 (both are PB-inducible), UGT1A3, UGT1A6, and UGT1A9 have all been reported to catalyze V P A glucuronidation (Ethell et al. 2003; Ritter 2000; Sakaguchi ef al. 2004). V P A - G undergoes extensive enterohepatic recycling in the rat (Dickinson et al. 1979; Pollack and Brouwer 1991) and it was demonstrated that greater than 90% of the V P A in rat bile was in the form of the glucuronide conjugate (Watkins and Klaassen 1981). Glucuronidation is dependent upon intracellular UGT activity as well as U D P G A concentration (Boelsterli 2002). The present study in rats investigated the influence of modulating V P A glucuronidation on oxidative stress, as measured by plasma and hepatic levels of 15-F 2 t - lsoP. For comparison, we also determined plasma and hepatic levels of 15-F 2 t - lsoP in rats treated with a-fluoro-VPA (a-F-VPA) , which is an analogue of V P A demonstrated to be a poor substrate for glucuronidation (Tang et al. 1997). A novel and intriguing finding in our study is that V P A - G formation is associated with oxidative stress. This report also describes a novel assay for the direct measurement of V P A - G by an LC/MS method. 3.2 MATERIALS & METHODS 3.2.1 Materials 2-Propyl-pentanoic acid (sodium salt, VPA) , (-)-borneol, salicylamide, D-saccharic acid 1,4-lactone (D-saccharolactone), and 1-chlorobutane were purchased from Sigma Chemicals Co. (Oakville, ON , Canada). 2-(Propyl-3,3,3-d3) pentanoic-5,5,5-d 3 acid ([ 2H 6]-VPA, 99.5 atom % D) was obtained from C D N Isotopes (Pointe-Claire, Q C , Canada). Sodium phenobarbital (PB) was purchased from BDH Chemicals Inc. (Toronto, ON , Canada). The 15-F 2 t-isoprostane (8-isoprostane) EIA kit was purchased from Cayman Chemical Co. (Ann Arbor, Ml). Ethyl acetate, acetonitrile (both H P L C grade), N-hexanes (GC/MS resolved), and diethyl ether were purchased 73 Chapter 3: Valproate-glucuronide and 15-F2t-isoprostane from Fisher Scientific (Vancouver, BC, Canada). a -F-VPA was synthesized in our laboratory by a previously described method (Tang etal. 1997). 3.2.2 Instrumentation and analytical methods 1 H-NMR spectra were obtained on a Bruker (Silberstreifen, Germany) Avance 300 Spectrometer in the Department of Chemistry at the University of British Columbia. Chemical shifts are expressed relative to tetramethylsilane. LC/MS analysis of VPA-1-O-acyl glucuronide was performed using a Fisons V G Quattro tandem mass spectrometer (Micromass, Montreal, Canada) interfaced with a Hewlett Packard (Avondale, PA, USA) 1090 II Liquid chromatograph. Instrument operation, data acquisition and integration were controlled by MassLynx® (v3.1, Micromass, Montreal, Canada) software. Negative electrospray was used as the means of ionization. The collision energy was 15 eV, source temperature 140°C, capillary voltage 3 kV, and cone voltage 15 V with skimmer offset by 5 V. The multipliers 1 and 2 were set at 650 V. The low mass and high mass resolutions were set at 5.0 for MS1. To record the full daughter ion spectra of V P A - G , mass resolutions were set at 12.5 (MS1) and 5.0 (MS2), MS/MS dwell times were adjusted to provide a scan rate of 1 sec/100 amu, and collision-induced dissociation used argon gas at a pressure of 3.0 x 10"4 mbar. The H P L C was fitted with a Phenomenex Columbus C18 column (150 x 2 mm, 5 um, Torrance, CA). The H P L C autoinjector syringe and sample loop volumes were 25 ul and 250 ul, respectively. The mobile phase consisted of a mixture of acetonitrile/water with 10 mM ammonium acetate and delivered at 0.2 ml/min at room temperature. Linear gradient conditions were as follows: 20% to 80% acetonitrile from 0 to 5 min and a return to 20% acetonitrile at 5.5 min and held at 20% acetonitrile for 3.5 min for equilibration. Total run time was 9 min. 74 C h a p t e r 3: Va l p roa te -g l u cu ron i de and 15-F 2 t - i sop ros tane 3.2.3 Animals Adult male Sprague-Dawley rats (250-300 g) were obtained from the University of British Columbia Animal Care Facility. They were fed with rat diet (Labdiet 5001 rodent diet, PMI Feeds Inc., Richmond, IN) and water ad libitum and maintained in a room on a 12 hr light/12 hr dark cycle at constant temperature (22°C) and humidity. All animal experiments were approved by the University of British Columbia Animal Care Committee. 3.2.4 Isolation of VPA-1-O-acyl glucuronide (VPA-G) and [2H6]-VPA-1-0-acyl glucuronide ([2H6]-VPA-G) Rats were anesthetized with urethane (1.2 g/kg ip) in an aqueous solution (0.4 g/ml) and their bile ducts cannulated with PE-10 tubing. Control bile was collected for 20 min. Rats (3 individual rats per group) were administered either V P A (100 mg/ml) in saline) or [ 2H 6]-VPA (100 mg/ml) prepared with an equimolar amount of 2 mmol NaOH and 0.9% saline) at 100 mg/kg every 2 h (4 doses in total) by ip injection, and bile was collected on ice for a total of 8 hours. The purification of V P A - G and [ 2H 6 ] -VPA-G was performed using the method by Cannell et al. (Cannell et al. 2002) Briefly, aliquots of rat bile (4 ml) were acidified with 5 M phosphoric acid to pH 2 and centrifuged (3000 x g, 10 min, room temperature). The supernatant (4 ml) was extracted with 1-chlorobutane ( 3 x 1 2 ml) to remove free V P A or [ 2H 6 ]-VPA, and the aqueous fraction was extracted with diethyl ether (3x12 ml). The diethyl ether extracts were evaporated in vacuo to dryness to give a dark-green residue. The residue was reconstituted in mobile phase (15% acetonitrile/85% water in 10 mM ammonium acetate) and subjected to further purification by H P L C . Bile extracts were injected on a Zorbax 300SB-C8 semi-preparative column (9.4 mm I.D. x 250 mm) and eluted at a flow rate of 3 ml/min with a linear gradient of 15% to 33% acetonitrile in water containing 10 mM ammonium acetate over a run time of 12 min. HPLC-purified fractions were collected, pooled, and subjected to lyophilization. 75 Chapter 3: Valproate-glucuronide and 15-F2t-isoprostane LC/MS/MS mass spectrum of 2-propyl-pentanoyl-1-0-acyl glucuronide (VPA-G, Figure 3-1 A), m/z(%): 319 ([M-H]", 30), 175 (G -2H, 50), 143 (VPA-H, 50), 113 (G - H O C H 2 C H 2 O H , 100), 103 (11), 59 (8). 1 H NMR (D 20) (Figure 3-2A): S0.75 (t, 6H, JHH = 7.2 Hz, 5,5'), 1.21-1.14 (m, 4H, 4,4'), 1.51-1.38 (m, 4H, 3,3'), 2.44 (m, 1H, 2), 3.42 (m, 3H, G, 2",3",4"), 3.75 (d, 1H, JHH = 9 Hz, G, 5"), 5.42 (d, 1H, J H H = 7.8 Hz, G, 1"). The 1 H NMR spectrum for V P A - G was in agreement with the spectrum obtained by Azaroual ef al. (2000). LC/MS/MS mass spectrum of 2-(propyl-3,3,3-d3) pentanoyl-5,5,5-d 3-1-0-acyl glucuronide ([2H6]-V P A - G , Figure 3-1B), m/z(%): 325 ([M-HT, 35), 258 (9), 199 (5), 175 (G -2H, 50), 149 ( d 6 - V P A -H, 45), 113 (G - H O C H 2 C H 2 O H , 100), 103 (12), 59 (12). 1 H NMR (D 20) (Figure 3-2B): £1.16 (t, 4H, J H H = 7.1 Hz, 4, 4'), 1.45 (m, 4H, 3, 3'), 2.44 (m, 1H, 2'), 3.43 (m, 3H, G, 2",3",4"), 3.79 (d, 1H, J H H = 7.9 Hz, G, 5'), 5.43 (d, 1H, J H H = 7.8 Hz, G, 1"). 3.2.5 Treatment of animals and sample collection Rats were administered with V P A , a-F-VPA, or 0.9% saline (vehicle control) at the doses and duration indicated in each figure legend. To modulate V P A glucuronidation, rats were injected ip with PB (80 mg/kg/day for 4 consecutive days in 0.9% saline), (-)-borneol (320 mg/kg in corn oil), salicylamide (1 mmol/kg in 50% glycerol), or combination of P B for 4 days and (-)-borneol treatment prior to V P A dosing (500 mg/kg, ip) at a time point indicated in each figure legend. At 0.5 h after V P A administration, rats were sacrificed by decapitation, trunk blood was collected, and plasma was prepared. An aliquot of the plasma was used immediately for the determination of 1 5 - F 2 r l s o P levels. Livers were weighed and homogenized in 30 ml of 50 mM phosphate buffer (pH 7.4) with 5 mM D-saccharolactone under ice-cold conditions. The homogenate was processed immediately for the analysis of 15-F 2 t - lsoP levels. Aliquots of liver homogenate were stored at -80°C for subsequent determination of V P A - G . 76 C h a p t e r 3: Va l p roa te -g l u cu ron i de and 15-F2t-isoprostane 3.2.6 Sample preparation for VPA-G assay by LC/MS Working stock solutions of 1 mg/ml V P A - G and 0.1 mg/ml [ 2 H 6 ] -VPA-G were prepared in distilled water, aliquots stored at -80°C, and were used for all calibration curve standards. Calibration standards (at concentrations of 0.5, 1, 5, 10, 25 and 50 ug/ml) were prepared in triplicate by adding appropriate volumes of the working stock solution in water to a final volume of 1 ml. Rat liver homogenate samples (0.5 ml) were diluted with water (0.5 ml). Samples were acidified by the addition of 1 ml of 1 M K H 2 P 0 4 (pH 2.5). [ 2 H 6 ] -VPA-G (50 pi of a 0.1 mg/ml solution) served as the internal standard and was added to each sample. Liquid-liquid extraction was performed twice with methyl ferf-butyl ether (5 ml for each extraction) and the extracts were pooled and evaporated to dryness under nitrogen. The residue was reconstituted in water and filtered using Acrodisk® (0.2 micron, Gelman Sciences) syringe filters directly into autosampler vials. An aliquot (10 pi) was injected on the HPLC. In a-F-VPA-treated rats, liver samples were processed as described above and the a -F -VPA-G levels were determined indirectly by L C / M S using the same response factor as the V P A - G standard since no synthetic standard for a -F -VPA-G was available. 3.2.7 Validation of the LC/MS VPA-G assay Assay validation was performed by evaluating inter-assay and intra-assay accuracy (% bias) and precision (coefficient of variation, %CV) of the low (0.5 pg/ml), mid (8 pg/ml), and high (40 pg/ml) concentration quality control (QC) samples. The Q C samples were prepared in blank liver homogenate containing 5 mM D-saccharolactone, flash-frozen in liquid nitrogen, stored at -80°C, and aliquots thawed for daily use. This was accomplished by analyzing 5 sets of calibration curves (triplicate readings) and Q C samples (6 replicates) on 5 separate days (inter-assay) and on the same day (intra-assay). Quantification of Q C samples was performed by 7 7 Chapter 3: Valproate-glucuronide and 15-F2t-isoprostane analyzing the calibration curve standards and back calculating the concentration of each Q C sample from the obtained slope, intercept, and the peak area ratios. The accuracy of the assay was assessed as the % bias of the nominal concentration observed for the spiked Q C s and a bias of < 15% at each concentration was considered to be acceptable accuracy. The precision of the assay (% CV) was determined from the variance observed for the mean of replicate Q C s of low, mid and high concentrations. Precision values of < 15% C V at the mid and high Q C concentrations and < 20% at the low Q C concentration were considered to meet the validation acceptance criteria (Karnes ef al. 1991). The mean percent recovery of V P A - G spiked in blank rat liver homogenate was determined at concentrations (0.5, 1, 5, 10, 25, and 50 ug/ml) representing the entire range of the calibration curve. 3.2.8 15-F2rlsoP Assay Plasma and hepatic levels of 15-F 2 t - lsoP were determined using an EIA assay kit (Cayman Chemical Co., Ann Arbor, Ml) as described previously in section 2.2.4. 3.2.9 Statistical analysis Statistical significance of the difference between the means of multiple groups was analyzed by one-way analysis of variance and was followed by the Student-Newman-Keuls multiple range post hoc test. Comparison between pretreated and treated groups ( 2 x 2 format) was analyzed by two-way analysis of variance and was followed by the Student Newman-Keuls multiple range post hoc test. The level of significance was set a priori at p < 0.05. 78 Chapter 3: Valproate-glucuronide and 15-F2t-isoprostane 3.3 RESULTS 3.3.1 Biosynthesis and identification of VPA-glucuronide V P A - G and its deuterated internal standard, [ 2 H 6 ] -VPA-G, were isolated from rat bile for the purpose of developing and validating an LC/MS assay. The isolation and purification of the glucuronides from the bile of rats treated with V P A or [ 2 H 6 ] -VPA were achieved by extraction with 1-chlorobutane and diethyl ether followed by purification by H P L C . The glucuronide conjugates were identified by LC /MS (Figure 3-1) and by 1 H - N M R (Figure 3-2). Under LC /MS using multiple reaction monitoring mode, V P A - G and [ 2 H 6 ] -VPA-G resulted in glucuronide fragments: transitions m/z 319 ->175 and m/z 325 ->175, respectively, and in diagnostic fragments: transitions m/z 319 ->143 and 325 ->149, corresponding to the respective V P A and [ 2H 6]-VPA moieties (Figure 3-1). The analysis of V P A - G from liver homogenates of rats treated with V P A was performed by L C / M S using reverse phase H P L C with gradient elution and single ion monitoring (m/z 319 for V P A - G and m/z 325 for [ 2H 6 ]-VPA-G). Results show that both V P A -G and its internal standard, [ 2 H 6 ] -VPA-G were detected in whole liver homogenate having retention times of 4.9 min, with the internal standard eluted approximately 0.04 min earlier (Figure 3-3A). The V P A - G standard isolated from the bile of rats treated with V P A was shown to co-elute with the analyte detected in the liver homogenates of VPA-treated rats (Figure 3-3C). No peak-elution for V P A - G was detected in liver homogenates of control rats treated with 0.9% saline (Figure 3-3B). 79 C h a p t e r 3: Va l p roa te -g l u cu ron i de and 15 - F 2 t- isoprostane 100i fi 0> a> %1 P3 Gluc-62 ( - H O C H 2 C H 2 O H ) 11 59 103 75 Ul H O 143 175 117 / 157 1 143 (VPA,-H) C O O 175-...J (Glue, -2H) [M-H]-50 100 150 200 250 300 350 m/z B 100] fl 0> » % -5 113 59 103 ,iil,.,L HO 149 175 115 150 coo -1 4 9 ( [ 2 H 6 ]-VPA, -H) C D 3 C D 3 175...J O (Glue, -2H) 325 [M-H]-199 258 326 50 100 150 200 250 300 350 m/z Figure 3-1: Collision-induced dissociation mass spectra of (A) VPA-1-O-acyl glucuronide (VPA-G) and (B) [ 2H 6]-VPA-1-0-acyl glucuronide ([ 2H 6]-VPA-G) purified from rat bile obtained by LC/MS/MS with negative ion electropspray ionization. Glue = glucuronide. 80 C h a p t e r 3: Va l p roa te -g l u cu ron i de and 15-F 2 t - i sop ros tane Figure 3-2: 1 H-NMR spectra (D 2 0) of biosynthesized (A) 2-propyl-pentanoyl-1-0-acyl glucuronide (VPA-G) and (B) 2-(propyl-3,3,3-d3) pentanoyl-5,5,5-d 3-1-0-acyl glucuronide ([ 2H 6]-VPA-G) purified from rat bile. 81 C h a p t e r 3: Va l p roa te -g l u cu ron i de a n d 15-F 2 t - i s op ros tane A 100i o c a < 0 t 100i [ 2 H 6 ] -VPA-G (m/z 325) V P A - G (m/z 319) 4.92 min I32890 4.96 min 72699 100i % 0 100] 1.00 2.00 3.00 4 .00 5.00 6.00 7.00 8.00 [ 2 H 6 ] -VPA-G (m/z 325) 4-90 min 21329 4.94 min 3 V P A - G 245415 (m/z 319) • J — i — i — i — i — , — , — , — . — . — I 1.00 2.00 3 .00 4 .00 5.00 6.00 7.00 8.00 B O c •a a 3 < 100] V P A - G (m/z 319) 4.93 min 0] [ 2 H 6 ] -VPA-G 38629 / o (m/z 325) 0 l = . , r - . . , . - ' I 1.00 2.00 3 .00 4 .00 5.00 6.00 7.00 8.00 Time (min) D 100i 0' 100] [ 2 H 6 ] -VPA-G (m/z 325) 4.91 min 23212 V P A - G (m/z 319) 4.92 min 1.00 2.00 3.00 4 .00 5.00 6.00 7.00 8.00 Time (min) Figure 3-3: Representative LC/MS chromatograms of VPA-G (m/z 319) (A) as biosynthesized standard spiked in blank liver homogenate, and from liver homogenates from rats treated ip with (B) 0.9% saline, (C) VPA (500 mg/kg), or (D) (-)-borneol (320 mg/kg) followed by V P A (500 mg/kg) 0.5 hr later. [ 2H 6]-VPA-G (m/z 325), the internal standard, was spiked into each sample. 82 C h a p t e r 3: Va l p r oa t e -g l u cu ron i de and 15-F 2 t - i sopros tane 3.3.2 Validation ofLC/MS VPA-G assay A quantitative assay for V P A - G in liver homogenate was developed using liquid-liquid extraction with methyl tert-butyl ether as the extraction solvent after samples were acidified with 1M K H 2 P 0 4 (1 ml, pH 2.5). The mean percent recovery of V P A - G spiked in blank rat liver homogenate was determined at concentrations (0.5, 1,5, 10, 25, and 50 ug/ml) representing the entire range of the calibration curve and was found to be approximately 78%. Calibration curves were prepared in triplicate in water and demonstrated linearity over the range 0.5-50 ug/ml (r2 > 0.99) with mean slope and intercept values of 0.277 ± 0.004 and -0.008 ± 0.007, respectively (n = 5 validation days). The inter-assay and intra-assay variability and precision based on quality control (QC) samples of V P A - G at high (40 ug/ml), mid (8 ug/ml) and low (0.5 ug/ml, representing the LOQ) concentrations in blank liver homogenate were all less than 15% CV and less than ± 15% bias (Table 3-1). The LOQ for the V P A - G assay was estimated to be 0.5 ug/ml, based on the validation results in accordance of having a C V of < 20% (Karnes et al. 1991) and a signal to noise ratio of greater than 5 to 1. Although no matrix interferences were observed in the liver homogenate samples, we observed a trend of negative bias in both inter-and intra-assay determination which increased at the high Q C sample, suggesting sample degradation. However, the sample degradation observed was minimal as evidenced by the bias values shown in Table 3-1, which in all cases fell within the acceptance criteria. When D-saccharolactone was not included in the homogenization buffer, marked sample degradation in the liver homogenate was noted with extraction recoveries of < 20% (data not shown). To minimize the potential degradation of V P A - G during processing, samples were thawed quickly, immediately placed on ice, and acidified with cold phosphate buffer prior to extraction. Samples were stable at -80°C as demonstrated by a lack of increasing negative assay bias in the Q C samples with time over the 5 validation days and in subsequent analysis runs post-validation. 83 Chapter 3: Valproate-glucuronide and 15-F2t-isoprostane 3.3.3 Dose-dependent increases in plasma and hepatic 15-F2rlsoP and VPA-G levels in VPA-treated rats A dose-dependent increase in plasma 15-F 2 t - lsoP levels was observed following V P A treatment with marked elevation at 250 mg/kg (73 ± 9 pg/ml) and 500 mg/kg doses (82 ± 5 pg/ml) (Figure 3-4A) compared to the saline-treated control group (31 ± 4 pg/ml). However, liver (free and total) 15-F 2 t - lsoP levels were only significantly elevated after 500 mg/kg doses (Figure 3-4C and D, respectively). Hepatic V P A - G levels increased over the dose range and reached a maximum at doses of 250 and 500 mg/kg (Figure 3-4B). 84 Chapter 3: Valproate-glucuronide and 15-F2t-isoprostane Table 3-1: Inter- and intra-assay variation of the VPA-G assay by LC/MS. a Inter-assay validation Day QC low QC mid QC high 1 0.52 8.37 37.7 2 0.57 8.38 37.6 3 0.51 7.43 36.8 4 0.43 7.72 35.8 5 0.42 7.71 36.2 Nominal cone, (mg/ml) 0.50 8.00 40.0 Mean cone, (mg/ml) 0.49 7.92 36.8 SD 0.06 0.43 0.85 C V . (%) 13.1 5.4 2.3 BIAS (%) -2.21 -0.96 -7.91 Intra-assay validation Replicate # QC low QC mid QC high 1 0.61 7.21 36.7 2 0.45 7.52 37.1 3 0.44 7.52 35.8 4 0.54 7.47 36.8 5 0.51 7.53 36.9 6 0.49 7.33 37,5 Nominal cone, (mg/ml) 0.50 8.00 40.0 Mean cone, (mg/ml) 0.51 7.43 36.8 SD 0.06 0.13 0.57 C V . (%) 12.5 1.8 1.5 BIAS (%) 1.10 -7.10 -7.94 a Method validation was performed by evaluating inter-assay accuracy (% bias) and precision (coefficient of variation, % CV) of the low, mid, and high Q C concentrations in blank rat liver homogenate on separate days. Quantitation of Q C samples was performed by analyzing the calibration curve standards and back calculating the concentration of each Q C sample from the obtained slope and intercept. 85 C h a p t e r 3: Va l p roa te -g l u cu ron i de and 15-F 2 i - i sop ros tane VPA Dose (mg/kg) VPA Dose (mg/kg) Figure 3-4: Dose-dependent effect of VPA on (A) plasma, (B) free and (D) total liver levels of 15-F 2 t-lsoP, and (C) liver VPA-G. Rats were injected with a single ip dose of V P A (50, 100, 250 or 500 mg/kg). Control rats received an equivalent volume (1 ml/kg) of saline vehicle. Results are shown as mean ± SEM for 4 individual rats per treatment group. Significantly different from control, p < 0.05; 6 Not significantly different from the 250 mg/kg group, p > 0.05. 86 Chapter 3: Valproate-glucuronide and 15-F2t-isoprostane 3.3.4 Effect of (-)-borneol on VPA-glucuronide and 15-F2rlsoP levels in rats treated with VPA To determine the effect of U D P G A depletion on VPA-glucuronidation and on the plasma and liver levels of 15-F 2 t - lsoP, rats (n = 8 individual rats per group) were pretreated with a single dose of (-)-borneol (320 mg/kg, ip) or an equal volume of corn oil (vehicle control). At 0.5 h after (-)-borneol pretreatment, rats were treated with saline vehicle or a single dose of V P A (500 mg/kg, ip). This regimen of (-)-borneol treatment is a modification of described procedures (Watkins and Klaassen 1982) and demonstrated maximal inhibition of the glucuronidation of 2-phenylpropionic acid, a carboxylic acid similar in structure to V P A (Li ef al. 2003). (-)-Borneol inhibited V P A - G formation by approximately 97% compared to animals treated with V P A alone (Table 3-2 and Figure 3-3D). The concentration of V P A - G detected in VPA-treated rats pretreated with (-)-borneol was 9 ± 3 u.g/g tissue, which was significantly lower than that determined in livers of rats treated with only V P A (315 ± 26 u.g/g tissue). Concomitant with this decrease in V P A - G , the apparent level of 15-F 2 t - lsoP detected in plasma of rats treated with (-)-borneol and V P A (60 ± 4 pg/ml) was significantly lower than the levels seen in VPA-treated rats (93 ± 4 pg/ml), but still greater than levels seen in control rats (35 + 4 pg/ml) (Figure 3-5A). A similar trend was also seen in liver; however, the levels of free and total 15-F 2 t - lsoP were decreased in the (-)-borneol/VPA group to levels seen in the vehicle control group (Figure 3-5B and Figure 3-5C). (-)-Borneol alone did not have an effect on plasma or liver 15-F 2 t - lsoP levels compared to vehicle control rats. 3.3.5 Effect of salicylamide on VPA-glucuronide and 15-F2rlsoP levels in rats treated with VPA Salicylamide pretreatment, at a dose (2 mmol/kg) and a time point (0.5 h prior to VPA) shown to maximally deplete U D P G A (Howell et al. 1986; Kamisako et al. 1990), produced similar results 87 Chapter 3: Valproate-glucuronide and 15-F2t-isoprostane as observed with (-)-borneol with respect to V P A - G and 15-F 2 t - lsoP levels. Liver V P A - G levels significantly decreased from 247 + 54 ug/g tissue in VPA-treated animals to 60 ± 19 ug/g in salicylamide/VPA-treated animals. Furthermore, in the salicylamide-pretreated rats, 15-F 2 t - lsoP levels in plasma and liver were significantly decreased after V P A dosing compared to V P A -treated animals without pretreatment with salicylamide (Figure 3-6A-C). 3.3.6 Effect of PB on VPA-glucuronide and 15-F2rlsoP levels In rats treated with VPA When rats were pretreated with PB prior to V P A dosing, there was a further 2.2-fold increase in plasma 15-F 2 t - lsoP levels (149 + 18 pg/ml) compared to the VPA-treated group (68 + 5 pg/ml) (Figure 3-7A). Free and total liver 15-F 2 t - lsoP levels were also elevated in PB-pretreated animals (369 ± 37 and 3194 ± 325 pg/g tissue, respectively) compared to rats treated with V P A only (237 ± 29 and 2664 ± 182 pg/g tissue, respectively) (Figure 3-7C and D). Associated with the increase in 15-F 2 t - lsoP levels in P B / V P A treated rats, was a marked elevation in V P A - G levels (356 + 11 ug/g tissue) compared to the V P A group (266 ± 17 ug/g tissue) (Table 3-2). 3.3.7 Effect of (-)-borneol on PB-induced increases in VPA-glucuronide and 15-F2rlsoP levels in rats treated with VPA When PB-pretreated rats were treated with a single dose of (-)-borneol prior to V P A treatment, the levels of plasma 15-F 2 t - lsoP were reduced from 149 ± 18 (PB/VPA group) to 109 ± 12 pg/ml (Figure 3-7A-C). In the same treatment group, the free liver and total liver 15-F 2 t - lsoP were reduced to vehicle control levels. Associated with the observed decrease in 15-F 2 t - lsoP in the PB/borneol/VPA group was a significant decrease in V P A - G levels (129 + 12 ug/g tissue) compared to both P B / V P A (356 ± 11 ug/g tissue) and V P A groups (266 ± 17 ug/g tissue) (Table 3-2). 88 Chapter 3: Valproate-glucuronide and 15-F2t-isoprostane Table 3-2: The effects of pre-treatment with PB, (-)-borneol, and salicylamide on VPA-G levels measured in liver homogenate. 3 Pretreatment Treatment Sample size VPA-G (pg/g tissue) Corn o i l 6 V P A n = 8 259 ± 20 (-)-Borneol V P A n = 8 12 + 4* G lycero l 0 V P A n = 4 247 ± 54 Salicylamide V P A n = 4 60 ± 19* Saline (4d)d/ corn oil V P A n = 8 266 ± 17 PB (4d)d/ corn oil V P A n = 8 356 ± 1 1 * PB (4d)7 (-)-Borneol V P A n = 8 129 ± 12** 'Values represent the mean ± S E M . Rats were pretreated with "corn oil vehicle or (-)-borneol (320 mg/kg, ip), g lycerol vehicle (50%) or salicylamide (1 mmol/kg, ip), "saline vehicle or P B (80 mg/kg/day, 4 consecutive days, ip) followed by VPA-treatment (500 mg/kg, ip) 0.5 h later. e Rats were pretreated with P B (80 mg/kg/day, 4 consecutive days, ip), followed by (-)-borneol on day 5 at 0.5 h prior to V P A treatment (500 mg/kg, ip). *Significantly different from the vehicle pretreated + V P A group (p < 0.05). "Significantly different from all other groups (p< 0.05). 89 C h a p t e r 3: Va l p roa te -g l u cu ron i de and 15-F 2t- isoprostane % 150 tn 100 T— O) « a (0 50 control borneol VPA borneol + VPA control borneol VPA borneol + VPA o 3000 w 4- = 2000 g> o) 1000 _j a « 0 •g w control borneol VPA borneol I- + VPA Figure 3-5: Effect of (-)-borneol pretreatment on levels of (A) plasma 15-F 2 t-lsoP, (B) free liver 15-F 2 t-lsoP, and (C) total liver 15-F 2 t-lsoP in VPA-treated rats. Sprague-Dawley rats were pretreated ip with (-)-borneol (320 mg/kg) or corn oil (vehicle). At 0.5 h later, the rats were treated ip with a single dose of VPA (500 mg/kg) or 0.9% saline (vehicle). At 0.5 h after V P A treatment, rats were sacrificed, and blood and liver were collected. Results are shown as mean ± S E M for 8 individual rats per treatment group. "Significantly different from the control group, p<0.05; Signif icantly different from the V P A group, p<0.05. 90 C h a p t e r 3: Va l p roa te -g l u cu ron i de and 15-F 2 t - i sop ros tane ft. O (A • E E (ft re control SAL* VPA SAL + VPA (ft (ft 600 uTS> • & 400 2 o) 200 • B cu a) n 6000 uT§ 4500 w ~ 3000 CD ^ g 1500 control SAL VPA SAL + VPA control SAL VPA S A L + VPA Figure 3-6: Effect of salicylamide (SAL) pretreatment on levels of (A) plasma 15-F 2 t-isoP, (B) free liver 15-F 2t-isoP, and (C) total liver 15-F 2 t-isoP in VPA-treated rats. Adult male Sprague-Dawley rats were pretreated ip with salicylamide (1 mmol/kg) or 50% glycerol (vehicle). At 0.5 h later, the rats were treated ip with a single dose of V P A (500 mg/kg) or 0.9% saline (vehicle). At 0.5 h after VPA treatment, rats were sacrificed, and blood and liver were collected. Results are shown as mean + S E M for 4 individual rats per treatment group. Signif icantly different from all other groups, p < 0.05. 91 Chapter 3: Valproate-glucuronide and 15-F2t-isoprostane CL O re B E </) w C L C L O W L L 3 .A W > D) _ i a. CD I? w "(5 o c o n t r o l V P A P B PB+ PB+ PB+ borneol V P A borneol+ V P A 600 i B 400 200 0 a,b • l l . l l c o n t r o l V P A P B P B + P B + P B + borneol V P A borneol+ o 4000 V P A 3000 2000 1000 a,b •lull c o n t r o l V P A P B PB + PB + PB + Borneol V P A Borneol + V P A Figure 3-7: Effect of (-)-borneol on the PB-induced levels of (A) plasma, (B) free and (C) total liver 15-F 2 t-lsoP in VPA-treated rats. Rats were pretreated ip with PB (80 mg/kg) or 0.9% saline (vehicle) once daily for 4 days. On day 5, rats were treated ip with (-)-borneol (320 mg/kg) or corn oil (vehicle) at 0.5 h prior to VPA administration (500 mg/kg in 0.9% saline vehicle). At 0.5 h after VPA treatment, rats were sacrificed, and blood and liver were collected. Results are shown as mean ± S E M for 8 individual rats per treatment group. "Significantly different from the control group, p < 0.05; "Significantly different from the VPA group, p < 0.05; Significantly different from the V P A and PB/borneol/VPA groups, p < 0.05. 92 Chapter 3: Valproate-glucuronide and 15-F2 t-isoprostane 3.3.8 Effect of a-F-VPA on a-F-VPA-G and 15-F2rlsoP levels a-F-VPA was selected for comparison with V P A for the effect on 15-F 2 t - lsoP because a-F-VPA was demonstrated to form the glucuronide conjugate in only minor amounts (Tang et al. 1997). Rats were treated with a single dose of 3.47 mmol/kg V P A (equivalent to 500 mg/kg) or a-F-V P A (3.47 mmol/kg) and sacrificed at 0.5 h later. Plasma levels of 15-F 2 t - lsoP following a-F-V P A treatment (44 ± 2 pg/ml) were similar to those of saline-treated vehicle control rats (36 ± 2 pg/ml), but significantly less than that in the VPA-treated group (83 ± 3 pg/ml) (Figure 3-8A-C). 3.3.9 Effect of PB on a-F-VPA-G and 15-F2rlsoP levels in rats treated with a-F-VPA a-F-VPA was further investigated in rats pretreated with PB to see if PB could elevate the levels of 15-F 2 t - lsoP as observed in rats treated with VPA . However, when a -F -VPA was administered to PB-pretreated rats, levels of plasma (66 ± 17 pg/ml) and free liver (110 ± 17 pg/g tissue) 15-F 2 ,- lsoP were not elevated and were similar to those of the saline control group. By comparison, PB-pretreated rats demonstrated significant elevation in plasma (169 ± 20 pg/ml) and free liver (197 ± 28 pg/g tissue) 15-F 2 t - lsoP in the VPA-treated group compared to the saline-treated group (68 ± 6 pg/ml and 118 ± 6 pg/g tissue) (Figure 3-8E-G). Following a single dose of a -F-VPA (3.47 mmol/kg), a -F -VPA-G levels were 44 + 6 pg/g tissue and 36 ± 7 ug/g tissue in control rats (Figure 3-8D) and PB-pretreated rats (Figure 3-8H), respectively. These values are approximately 19% and 11% of the amount of V P A - G seen following an equimolar dose of V P A to control rats (Figure 3-8D) and PB-pretreated rats (Figure 3-8H), respectively. 93 Chapter 3: Valproate-glucuronide and 15-F2t-isoprostane O ) re S E to Li. 3 saline V P A a-F-VPA PB + PB + PB+ saline VPA a-F-VPA 400 -I B 400 300 a 300 S cn 200 j • 200 1 i I a CO — IOO-I ata H _ L - ioo saline V P A a-F-VPA PB + PB + PB + saline V P A a-F-VPA 4000 4- a 3000 VPA-G a-F-VPA-G " VPA-G a-F-VPA-G Control rats PB-pretreated rats Figure 3-8: Comparison of V P A and a-F-VPA in producing 15-F 2 t-lsoP and VPA-G or a-F-VPA-G, respectively, in control rats and in PB-pretreated rats. Rats were treated with a single dose of 0.9% saline (vehicle), VPA, or a-F-VPA, both at 3.47 mmol/kg, and sacrificed at 0.5 h later (A-D). In some groups, rats were pretreated ip with PB at 80 mg/kg once daily for 4 days (E-H). Results are shown as mean ± S E M for 4 individual rats per treatment group. a Significantly different from all other groups, p < 0.05. 94 Chapter 3: Valproate-glucuronide and 15-F2Hsoprostane 3.4 DISCUSSION Our recent study indicated that the administration of V P A (250 mg/kg and 500 mg/kg) increased plasma and hepatic levels of 15-F 2t- lsoP, a marker of oxidative stress (Tong et al. 2003). This finding was confirmed in the present study. A novel and intriguing finding from the present study is the association between the levels of 15-F 2 t - lsoP and V P A - G in rats treated with VPA , as shown in a series of in vivo studies that modulated the acyl-glucuronidation of VPA. Another novel aspect of this study is our new method for the direct LC /MS quantification of V P A - G in rat liver homogenate. The method is advantageous because levels of V P A - G are determined directly, as opposed to indirect determination that requires chemical hydrolysis with sodium hydroxide and/or enzymatic hydrolysis with (3-glucuronidase. Furthermore, the assay is relatively rapid and provides highly specific detection. In the present study, V P A at the maximal dose of 500 mg/kg consistently elevated 15-F 2 t - lsoP levels and PB pre-treatment had an enhanced effect with V P A resulting in an even greater elevation of 15-F 2 t - lsoP. These results were consistent with previous findings that also demonstrated the effect of P B on VPA-induced 15-F 2 ,- lsoP (Tong ef al. 2003). This increase in plasma and liver 15-F 2 t - lsoP by PB-pretreatment coincided with a 40% increase in liver V P A - G compared to saline-pretreated rats administered VPA. This 40% increase in liver V P A - G levels was similar to the magnitude of increase of V P A - G observed in PB-pretreated animals seen in other studies (Booth et al. 1996; Watkins and Klaassen 1982). Studies investigating the effects of P B on VPA-associated hepatotoxicity showed that lower doses of V P A (300 mg/kg) are required to produce the same hepatotoxic events in PB-pretreated rats as compared to rats given a single large dose of V P A (700 mg/kg) (Kesterson ef al. 1984; Sugimoto et al. 1987). This enhancement in V P A hepatotoxicity by PB has been associated with elevated levels of potentially reactive 4-ene-VPA and (E)-2, 4-diene-VPA via induction of P-450 enzymes (Cotariu 95 Chapter 3: Valproate-glucuronide and 15-F»-isoprostane and Zaidman 1988; Dreifuss et al. 1987; Levy et al. 1990; Zimmerman and Ishak 1982). However, our recent study indicated that the PB-induced increase in hepatic and plasma levels of 15-F 2 t - lsoP in VPA-treated rats was not associated with P450-mediated biotransformation (Tong ef al. 2003). PB also induces specific UGT enzymes that catalyze the glucuronidation of VPA, such as UGT2B1 in rat and UGT2B7 in humans (Ritter 2000), and increases the levels of hepatic U D P G A by 1.3-fold (Watkins and Klaassen 1982). As shown in the present study, (-)-borneol and salicylamide lowered liver V P A - G to approximately 10% and 25%, respectively, of the levels seen in control rats (Table 2). (-)-Borneol and salicylamide are compounds that deplete U D P G A (Howell ef al. 1986; Kamisako ef al. 1990; Watkins and Klaassen 1982) and were previously used to inhibit the acyl glucuronidation of V P A (Watkins and Klaassen 1982) and 2-phenylpropionic acid (Li ef al. 2003). The inhibition of V P A - G by both (-)-borneol and salicylamide decreased plasma and liver 15-F 2 t - lsoP levels and our results suggest a positive correlation between V P A - G and 15-F 2 t - lsoP levels. In a subsequent experiment, the combination of (-)-borneol and V P A in PB-pretreated rats was used to examine if the elevation in 15-F 2 t - lsoP due to PB-pretreatment was associated with the increased V P A - G formation. The results demonstrated that the VPA-associated increase in 15-F 2 t - lsoP levels was manipulated by chemically modulating the VPA-glucuronidation pathway in either direction. The liver V P A - G levels observed in the P B + (-)-borneol + V P A (500 mg/kg) group were attenuated to equivalent levels seen following a single low dose of V P A (50-100 mg/kg) that was not associated with significant increases in 15-F 2 t - lsoP levels (Fig. 4). Our study also used a - F - V P A as a mechanistic probe to gain insight into the relationship between VPA-acy l glucuronidation and 15-F 2 t - lsoP levels. Previous studies that used fluorinated analogues of V P A to investigate the reactive metabolite hypothesis of V P A -96 Chapter 3: Valproate-glucuronide and 15-F2t-isoprostane associated hepatotoxicity showed that 4-ene-VPA, but not the a-fluorinated analogue (a - F - 4 -ene-VPA), produced microvesicular steatosis and G S H (total and mitochondrial) depletion in rats (Tang et al. 1995). In vitro models of hepatotoxicity also demonstrated greater cytotoxicity and G S H depletion with V P A and 4-ene-VPA when compared to their respective a-fluorinated analogues (Jurima-Romet et al. 1996; Neuman et al. 2001). In contrast to V P A , a - F - V P A undergoes acyl-glucuronidation to only a minor extent (< 5% of the dose) and is not a substrate for fatty acid p-oxidation in rats (Grillo ef al. 2001; Tang et al. 1997). Based on these observations, a - F - V P A was examined in rats for its ability to affect the levels of 15-F 2 t - lsoP. As shown in this study, the rats treated with a - F - V P A produced significantly less plasma and liver 15-F 2 t - lsoP compared to the V P A group at equimolar doses. Consistent with our hypothesis that V P A acyl-glucuronidation is associated with oxidative stress, there was significantly less a -F-VPA-G than V P A - G observed in the liver. Furthermore, in PB-pretreated rats, levels of a - F -V P A - G and 15-F 2 t - lsoP were similar to the levels seen in the saline control group, whereas both V P A - G and 15-F 2 t - lsoP were further elevated after an equivalent dose of VPA . In an earlier pharmacokinetic study using mice, both V P A and a - F - V P A (0.83 mmol/kg) exhibited similar serum tm a x of 15-30 min; however, a - F - V P A was characterized with higher peak serum concentrations (4.39 umol/ml vs. 2.88 u.mol/ml) and a lower elimination rate constant (1.50 x 10" 3/min vs. 2.02 x 10"2/min) than those of VPA, respectively (Tang ef al. 1997). The relatively lower clearance of a - F - V P A compared to V P A was ascribed to the absence of p-oxidation and the markedly reduced glucuronidation of a - F - V P A . The fluorine substituent increases the acidity of the carboxylate group (pKa = 3.55) compared to V P A (pKa = 4.80) (Tang ef al. 1997), which may influence its binding to UGT and subsequent conjugation to the glucuronide moiety. Increasing the degree of substitution at the a-carbon position with the fluorine group may also decrease the reactivity of the glucuronide conjugate since there appears to be an inverse relationship between the degree of substitution at the alpha-carbon to the carboxylic acid and its 97 Chapter 3: Valproate-glucuronide and 15-F2t-isoprostane covalent binding to protein (Benet ef al. 1993). It would be reasonable to assume that the distinction in the ability of V P A and a -F -VPA to produce 15-F 2 t - lsoP may be due to the inherent differences in their metabolism to their respective glucuronide conjugates rather than their peak serum concentrations and their rate of elimination in serum. Acyl glucuronide conjugates are electrophilic intermediates with the intrinsic potential to be reactive. The reactivity of acyl-glucuronides manifests themselves via potential pathways such as hydrolysis (resulting in the regeneration of parent compound), rearrangement (isomerization via intramolecular acyl migration), and covalent adduct formation with neighbouring macromolecules (Bailey and Dickinson 2003; Benet ef al. 1993; Boelsterli 2002). Although V P A - G undergoes non-specific enzymatic hydrolysis in the plasma and liver, and p-glucuronidase-dependent hydrolysis in the intestine, it is generally considered to be an unreactive metabolite based on in vitro incubation studies (Williams ef al. 1992). However, V P A - G has been demonstrated to undergo non-enzymatic, intramolecular rearrangement into various p-glucuronidase-resistant isomers in slightly alkaline environments, such as bile (Dickinson ef al. 1984). The effect of PB-pretreatment is non-specific and capable of inducing not only UGTs, but other drug-metabolizing enzymes such as P450 enzymes involved in V P A biotransformation. In PB-pretreated animals, significant elevations in levels of P450-dependent oxidative and desaturated V P A metabolites were observed (Tong ef al. 2003), all of which could be subsequently glucuronide conjugated. One possibility is that the acyl-glucuronide of P450-dependent V P A metabolites (i.e. 3-OH-, 4-OH-, 5-OH-, 4-ene-VPA) may be directly involved in the production of 15-F 2 t - lsoP. The reactive nature of these secondary acyl glucuronide metabolites and their ability to undergo rearrangement via intramolecular acyl migration to yield potentially reactive, p-glucuronidase-resistant, positional isomers has not been investigated. 98 C h a p t e r 3: Va l p roa te -g l u cu ron i de and 15-F 2 t - i sop ros tane A possible explanation for the association between oxidative stress and the formation of V P A - G may involve the enterohepatic cycling of VPA. V P A - G , which is extensively hydrolyzed in the liver, intestine, and to a minor extent in whole blood, is subject to recirculation and theoretically should generate glucuronic acid in the process. Given the large V P A doses employed, this systemic cycling of V P A to V P A - G and back to V P A may lead to a substantial release of free glucuronic acid. In a recent study, glucuronic acid was shown to induce oxidative stress in vitro, as measured by the fluorescence marker 2',7'-dichloro-dihydrofluorescein diacetate (Kim et al. 2004). The effects of hepatic and systemic glucuronic acid exposure on oxidative stress, as well as the determination of glucuronic acid levels during VPA-treatment will need to be examined to test the possibility that glucuronic acid plays a role in the elevation of 15-F 2 t - lsoP during VPA-treatment. The importance of hepato-biliary recirculation as a mechanism of methapyrilene hepatotoxicity was illustrated when cannulation and exteriorization of the bile duct to interrupt enterohepatic recirculation resulted in a reduction in necrosis (Ratra ef al. 2000). Thus, similar bile duct exteriorization to divert bile during V P A administration may be an informative study to determine whether or not enterohepatic recirculation plays a role in V P A -associated 15-F 2 t- lsoP elevation. An explanation for the elevation in oxidative stress in VPA-treated rats following PB pretreatment is the effect of P B on the biliary excretion of V P A . V P A has a choleretic effect (Dickinson et al. 1979; Watkins and Klaassen 1981), which was ascribed to the osmotic effect driven by the excretion of V P A conjugates across the canalicular membrane into bile. P B pretreatment prior to V P A dosing decreases bile flow and subsequent biliary excretion of V P A - G (Booth ef al. 1996; Watkins ef al. 1982). The canalicular egress of glucuronide conjugates has been shown to involve the saturable ATP-dependent MRP-2 organic anion transport protein (Wright and Dickinson 2004). Since PB-pretreatment decreases bile flow, elevates liver V P A - G formation, and because biliary excretion is saturable at the high doses of V P A used (Watkins 99 C h a p t e r 3: Va l p r oa t e -g l u cu ron i de and 15-F 2 t - i sop ros tane and Klaassen 1982), there may be a shift to higher V P A - G levels in the liver and increased egress of V P A - G from the hepatocyte into blood. In summary, our novel data demonstrate that V P A - G formation is associated with increases in hepatic and plasma levels of 15-F 2 t - lsoP in rats. The mechanism by which 15-F 2 t - lsoP is elevated by VPA-glucuronidation remains unclear. 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N., and Klaassen, C. D. (1982). Induction studies on the functional heterogeneity of rat liver UDP- glucuronosyltransferases. Toxicol Appl Pharmacol 64, 439-46. Watkins, J . B., and Klaassen, C. D. (1981). Choleretic effect of valproic acid in the rat. Hepatology 1, 341-7. Watkins, J . B., and Klaassen, C. D. (1982). Effect of inducers and inhibitors of glucuronidation on the biliary excretion and choleretic action of valproic acid in the rat. J Pharmacol Exp Ther. 220, 305-10. Williams, A. M., Worrall, S., de Jersey, J . , and Dickinson, R. G. (1992). Studies on the reactivity of acyl g!ucuronides--lll. Glucuronide- derived adducts of valproic acid and plasma protein and anti-adduct antibodies in humans. Biochem Pharmacol 43, 745-55. 104 Chapter 3: Valproate-glucuronide and 15-F2t-isoprostane Wright, A. W., and Dickinson, R. G. (2004). Abolition of valproate-derived choleresis in the mrp2 transporter-deficient rat. J Pharmacol Exp Ther. 310, 584-8. Zimmerman, H. J . , and Ishak, K. G. (1982). Valproate-induced hepatic injury: analyses of 23 fatal cases. Hepatology 2, 591-597. 105 Chapter 4: Time-course of VPA-associated oxidative stress and hepatotoxicity 4 Time-course of oxidative stress biomarkers and liver toxicity in rats treated with valproic acid 4.1 INTRODUCTION A recent study demonstrated that the administration of V P A led to a dose-dependent elevation of plasma and liver levels of the lipid peroxidation marker, 15-F 2t-isoprostaglandin (15-F 2 t- lsoP, also called 8-isoprostane or 8-iso-prostaglandin F2 c c) (Tong ef al. 2003). While the effects of V P A on 15-F 2 t - lsoP levels were consistent with an induction of oxidative stress, two other independent measures of oxidative stress, thiobarbituric acid reactive substances (TBARS) and lipid hydroperoxides (LPO), were unchanged by a single dose of V P A . Based on this finding, the question remains whether the elevation in 15-F 2 t - lsoP precedes liver toxicity. The objective of the present study was to determine the temporal relationship between oxidative stress and hepatotoxicity in rats treated with V P A , and in doing so, to provide new insight into the potential role of oxidative stress in VPA-mediated hepatotoxicity in rats. If the hepatotoxicity were induced through the formation of oxidative stress, an expected increase or accumulation of oxidative stress markers would occur prior to hepatotoxicity. 15-F 2 t - lsoP, T B A R S , and LPO were used as markers of oxidative stress, and histopathological findings (necrosis and steatosis), as well as serum a-glutathione-S-transferase (a -GST) levels, were used as markers of hepatotoxicity. V P A metabolites, in particular 4-ene-VPA and (£)-2,4-diene-VPA, were also monitored to investigate whether the observed oxidative stress and hepatotoxic events were associated with increases in these putative reactive metabolites. This novel study establishes the time-course for hepatotoxicity and oxidative stress in VPA-treated rats. 106 Chapter 4: Time-course of VPA-associated oxidative stress and hepatotoxicity 4.2 METHODS & MATERIALS 4.2.1 Chemicals 2-Propyl-pentanoic acid (sodium valproate, VPA) , D-saccharic 1,4-lactone monohydrate (D-saccharolactone), and 10% phosphate-buffered formalin were purchased from Sigma-Aldrich Canada Ltd. (Oakville, O N , Canada). The 15-F 2 t-isoprostane EIA kit and lipid hydroperoxide assay kit were purchased from Cayman Chemical Co. (Ann Arbor, Ml). The Oxi-Tek T B A R S assay kit was purchased from Zepto-Metrix Co. (Buffalo, NY). Dimethylformamide and the G C derivatizing reagents pentafluorobenzyl bromide and N-(terf.-butyldimethylsilyl)-N-methyltrifluoroacetamide were purchased from Pierce Chemical Co. (Rockford, IL). N,N-diisopropylethylamine and te/t-butyldimethylsilyl chloride were obtained from Aldrich (Milwaukee, WI). Ethyl acetate (HPLC grade) and n-hexanes (GC/MS resolved) were purchased from Fisher Scientific (Vancouver, BC, Canada). 4.2.2 Animal experiments Adult male Sprague-Dawley rats (250 - 300 g) were from the University of British Columbia Animal Care Facility. They were fed with rat diet (Labdiet 5001 rodent diet, PMI Feeds Inc., Richmond, IN) and water ad libitum and maintained in a room on a 12 h light/12 h dark cycle at constant temperature (22°C) and humidity. The University of British Columbia Animal Care Committee approved all animal experimentation. Rats were treated with an aqueous solution of V P A (dissolved in 0.9% saline) and injected ip at a dose of 500 mg/kg once a day for 2, 4, 7, 10, or 14 consecutive days. Our previous study demonstrated that the dose of 500 mg/kg V P A produced maximum elevation in plasma and liver levels of 15-F 2 t- lsoP (see Figure 2-3). The control group was treated with 0.9% saline solution (vehicle control, 1 ml/kg, ip) for 14 consecutive days. Based on the tm a x (30 min, see Figure 2-5) of plasma 15-F 2 t - lsoP following a single 500 mg/kg V P A dose, rats were sacrificed 30 min following the last injection by 107 Chapter 4: Time-course of VPA-associated oxidative stress and hepatotoxicity decapitation and trunk blood collected in Vacutainer® blood collection tubes. Serum and plasma were immediately prepared and snap-frozen in liquid nitrogen for the analysis of V P A metabolites, oxidative stress and toxicity biomarkers. The livers were weighed, rinsed with ice-cold phosphate buffered saline (pH 7.4), and homogenized in 50 mM phosphate buffer (pH 7.4) with 5 mM D-saccharolactone and 0.005% butylated hydroxytoluene on ice. The homogenate was snap-frozen in liquid nitrogen for the determination of V P A metabolites and oxidative stress biomarkers. All biological samples were stored a t -80°C. 4.2.3 Determination of oxidative stress biomarkers Plasma and liver levels of 15-F 2 t- lsoP were determined by an EIA assay as described in 2.2.4. The concentration of T B A R S in plasma and liver were calculated as malondialdehyde (MDA) equivalents using a fluorescent assay as described in 2.2.6. Lipid hydroperoxide levels were determined spectrophotometrically using a commercial kit as described in 2.2.7. 4.2.4 Determination of serum alpha glutathione S-transferase (cc-GST) as a liver toxicity marker Fresh serum was prepared immediately after the rats were sacrificed and a -GST was measured in rat serum as a marker for hepatic damage by a commercially available enzyme immunoassay method (Biotrin Rat Alpha-GST EIA, Biotrin, Dublin, Ireland). a -GST, a cytosolic enzyme located predominantly in liver parenchyma, was demonstrated to be a sensitive and specific biomarker of hepatocyte injury (Clarke ef al. 1997; van Wagensveld ef al. 1997). The quantitative immunoassay is based on the sequential addition of serum sample (1:50 dilution) and rabbit anti-rat a -GST IgG conjugated to streptavidin-peroxidase complex to micro-assay wells coated with anti-rat a -GST IgG with washing in-between each step. After the peroxidase substrate was added to the sandwich ELISA, the resultant color intensity was determined on a 108 Chapter 4: Time-course of VPA-associated oxidative stress and hepatotoxicity multi-well plate reader (absorbance at 450 nm) and is proportional to the amount of a-GST present in the sample. The assay was linear from 1.56-25 u.g/ml based on a-GST standard solution. 4.2.5 Histopathology Livers from all treated groups were rinsed with ice-cold saline and a small cross-section of the liver was obtained and fixed in 10% formalin-phosphate buffered saline when the rats were sacrificed. Hematoxylin and eosin stain was used for light microscopy. The severity of liver pathology was assessed as follows: necrosis (the percentage of the cross section containing necrotic foci) was scored as 1+ with < 25% of the area containing necrotic cells; 2+, with 26 to 50% of the area containing necrotic cells; 3+, with 51 to 75% of the area containing necrotic cells; and 4+, with > 75% of the area containing necrotic cells. Steatosis (expressed as the percentage of liver cells containing fat) was scored in a similar way as described for the determination of necrosis. At least 2 different sections were examined per liver sample and the pathologist was blind to the treatment groups when assessing the histology. 4.2.6 Determination of VPA metabolites Oxidative and de-saturated V P A metabolites were determined by G C / M S using negative ion chemical ionization and single ion monitoring. The sample preparation and assay procedures are described in a previous study (Tong ef al. 2003). VPA-1-O-acyl glucuronide (VPA-G) levels were determined in liver homogenate by an LC/MS assay using negative electrospray ionization and single ion monitoring. The assay utilizes purified V P A - G as a standard and [ 2 H 6 ] -VPA-G as its internal standard to measure V P A - G levels directly by a validated method described elsewhere (section 3.2.6). 109 Chapter 4: Time-course of VPA-associated oxidative stress and hepatotoxicity 4.2.7 Instrumentation and analytical methods G C / M S analysis of V P A and its metabolites was carried out using an HP 6890 gas chromatograph interfaced to an HP5973 mass selective detector (Hewlett-Packard, Avondale, PA). The gas chromatograph was equipped with a capillary splitless injector and an HP7683 autosampler. The mass spectrometric data acquisition and handling software, HP Enhanced Chemstation Software G1701BA (V B.01.00) was used to control the operation of all instruments. LC/MS analysis of VPA-1-O-acyl glucuronide was performed using a Fisons V G Quattro tandem mass spectrometer (Micromass, Montreal, Canada) interfaced with a Hewlett Packard (Avondale, PA, USA) 1090 II Liquid chromatograph. Instrument operation and data acquisition were controlled by MassLynx® (v3.1, Micromass) software. Fluorescent analysis for the T B A R S assay was performed on a Cytofluor® Series 4000 (Applied Biosystems, Bedford, MA) multi-well fluorescent plate reader. Spectrophotometric analyses for the 15-F 2 t - lsoP, LPO, and a - G S T assays were performed on a Labsystems Multiscan Ascent® multi-well plate reader (Thermo Electron Corp., Burlington, O N , Canada). 4.2.8 Statistical Analysis Statistical significance of the difference between the means of multiple groups was analysed by one-way analysis of variance and, where appropriate, followed by Bonferonni's multiple comparison post hoc test. The level of significance was set a priori at p < 0.05. 110 Chapter 4: Time-course of VPA-associated oxidative stress and hepatotoxicity 4.3 RESULTS 4.3.1 Time-course for 15-F2rlsoP during VPA treatment To characterize the time-course for changes in plasma and liver 15-F 2 t - lsoP levels following V P A treatment, rats were administered V P A (500 mg/kg, ip) once a day for 2, 4, 7, 10, or 14 consecutive days. At 0.5 h after the last dose of VPA , rats were terminated and plasma and liver 1 5 - F 2 r l s o P levels were determined (Figure 4-1A-C). Plasma 15-F 2 t - lsoP levels were found to be maximal on day 2 with a « 3-fold increase (102 ± 14 pg/ml) compared to the saline (vehicle)-treated control group (30 ± 3 pg/ml) and these elevated levels were similar after 4, 7, 10 and 14 days (Figure 4-1 A). Free and total liver 15-F 2 t - lsoP levels were also maximally elevated after day 2 (370 ± 1 6 0 and 1763 ± 149 pg/g tissue, respectively) compared to the control group (134 ± 8 and 877 ± 81 pg/g tissue, respectively), and these levels were similar to those seen after 4, 7, 10, and 14 days (Figure 4-1B and 1C). 4.3.2 TBARS and LPO levels during VPA treatment Liver LPO (Figure 4-2) and plasma and liver T B A R S (Figure 4-3A and B) were measured as other independent indicators of oxidative stress following 2, 4, 7, 10 and 14 consecutive days of V P A treatment (500 mg/kg/day, ip). Liver LPO levels were significantly elevated in the V P A -treated groups after 7 days (105 ± 5 nmol/g tissue) compared to the saline-treated control group (72 ± 6 nmol/g tissue) (Figure 4-2). The elevated levels of L P O were maximal after day 7 and were similar to levels observed on days 10 and 14. LPO levels in plasma were below the limit of detection of the assay in all groups (data not shown). 111 Chapter 4: Time-course of VPA-associated oxidative stress and hepatotoxicity Plasma and liver T B A R S were significantly elevated after 14 days in the VPA-treated group (2.4 ± 0.1 nmol MDA/ml plasma and 34 ± 1.7 nmol MDA/g tissue, respectively) compared to the saline-treated control group (1.6 ± 0.1 nmol MDA/ml plasma and 16.3 ± 2.1 nmol MDA/g tissue, respectively) (Figure 4-3A and B). 112 Chapter 4: Time-course of VPA-associated oxidative stress and hepatotoxicity 0 2 4 7 10 14 Days Figure 4-1: Levels of (A) plasma, (B) free liver and (C) total liver (esterified and non-esterified) 15-F 2 t-lsoP in rats treated with VPA (500 mg/kg once daily, ip) for 2, 4, 7, 10, or 14 consecutive days. Control rats were treated with 0.9% saline vehicle for 14 days. Plasma and liver levels of 15-F 2 t-lsoP were measured by an enzyme immunoassay method as described under Materials and Methods. Results are expressed as mean ± SEM, n = 5 individual rats per group (day 2, 4, 7, and 10 groups) and n = 16 rats per group (day 14 and vehicle control groups). Significantly different compared to the saline vehicle control group (p<0.05). 113 Chapter 4: Time-course of VPA-associated oxidative stress and hepatotoxicity Days Figure 4-2: Levels of liver LPO in rats treated with V P A (500 mg/kg once daily, ip) for 2, 4, 7, 10, or 14 consecutive days. The standard curve (0-5 nmol lipid hydroperoxide) was generated using 13-hydroperoxyoctadecadienoic acid as a lipid hydroperoxide standard. Control rats were treated with 0.9% saline (vehicle) for 14 days. Liver LPO was measured by a colormetric assay as described under Materials and Methods. Results are expressed as mean ± SEM, n = 5 individual rats per group (day 2, 4, 7, and 10) and n = 16 individual rats per group (day 14 and vehicle control). *Significantly different compared to the saline vehicle control group (p<0.05). 114 Chapter 4: Time-course of VPA-associated oxidative stress and hepatotoxicity Days Figure 4-3: Levels of (A) plasma and (B) liver T B A R S in rats treated with (500 mg/kg once daily, ip) for 2, 4, 7, 10, or 14 consecutive days. The standard curve for the T B A R S assay was generated using malondialdehyde (MDA) and the results are expressed as MDA equivalents. Control rats were treated with 0.9% saline (vehicle) for 14 days. Plasma and liver TBARS were measured by a fluorometric assay as described under Materials and Methods. Results are expressed as mean + SEM, n = 5 individual rats per group (day 2, 4, 7, and 10) and n = 16 individual rats per group (day 14 and vehicle control). *Significantly different compared to the saline-treated vehicle control group (p < 0.05). 115 Chapter 4: Time-course of VPA-associated oxidative stress and hepatotoxicity 4.3.3 Serum a-GST levels and histology during VPA treatment. Serum levels of a -GST were significantly elevated after 4 days of V P A treatment (251 ± 17 pg/L) compared to basal levels determined in the control group treated with saline for 14 days (57 ± 7 pg/L) (Figure 4-4). The a-GST levels remained elevated to the same extent on days 7, 10 and 14. Days Figure 4-4: Levels of serum a-GST in rats treated with V P A (500 mg/kg once daily, ip) for 2, 4, 7, 10, or 14 consecutive days. Control rats were treated with 0.9% saline vehicle for 14 days. a-GST was measured by an enzyme immunoassay method as described under Materials and Methods. Serum a-GST levels (pg/L) are expressed as mean + SEM, n = 5 individual rats per group (day 2, 4, 7, and 10) and n = 16 individual rats per group (day 14 and vehicle control). *Significantly different compared to the saline-treated vehicle control group (p < 0.05). 116 Chapter 4: Time-course of VPA-associated oxidative stress and hepatotoxicity Mortality was observed during the 14-day V P A treatment with an incidence of 1 out of 5 rats in the 4- and 7-day groups, and 3 out of 16 rats in the 14-day group (Table 4-1). In all treated groups, the most common feature was scarring of the liver capsule surface (liver "capsulitis") and this abnormality was attended by a mild inflammatory reaction localized only to the surface and usually consisted of lymphocytes and infrequent monocytes (Figure 4-5C). The frequency of "capsulitis" increased with duration of treatment from 1 out of 5 animals affected on day 2 to almost 100% frequency from day 4 to 14, while none were seen in the saline-treated control group. Liver necrosis was always associated with "capsulitis" but not vice versa, and without any signs of inflammation such as cellular infiltration into the parenchyma. In general, massive necrosis was observed without a consistent zonal pattern and involved irregular areas of hepatic tissue. All necrotic livers were subjectively scored (+4, +3, +2, or +1) to describe the severity of the cross-sectional area affected (>75%, 50-75%, 25-49%, or <25%, respectively). The 14-day treatment group (Figure 4-5F) had the highest incidence of massive necrosis with scores of +3 (3 rats), +2 (2 rats), and +1 (2 rats). In other treatment groups, 2 rats in the 4-day group and 1 rat in the 7-day group, widespread liver "capsulitis" was observed and this was accompanied with hepatocellular degeneration (necrosis score of +1) that extended locally into the parenchyma. Liver steatosis was observed in 7 rats (Table 4-1) and the incidence of lipid accumulation appeared to increase with the duration of V P A treatment: 1 rat in the 4-day group, and 2 rats in the 7-day group. Four out of five animals in the 10-day group exhibited steatosis (Figure 4-5E). The observed steatosis was of zonal distribution conforming to the centrilobular zones (periacinar zone 3). In all cases, steatosis was never extensive in area, and considered mild to 117 Chapter 4: Time-course of VPA-associated oxidative stress and hepatotoxicity moderate affecting approximately 10-25% of the liver cross-section, with the exception of one animal on day 10. Table 4-1. VPA-associated necrosis and steatosis in rats treated with V P A for 14 consecutive days. 3 Treatment Total Number of Incidence of Incidence of necrosis Incidence of Duration (days) number deaths "capsulitis" +1 +2 +3 +4 steatosis 0 16 0 0% 0 0 0 0 0 2 5 0 2 0 % 0 0 0 0 0 4 5 1 100 % 50% 0 0 0 25% 7 5 1 100% 25% 0 0 0 50% 10 5 0 100% 0 0 0 0 80% 14 16 3 9 2 % 15% 15% 0 23% 0 3 Male Sprague Dawley rats (250-300 g) treated with VPA (500 mg/kg, ip) once a day for 14 consecutive days. The 0-day treatment group received saline vehicle (1 ml/kg, ip) for 14 days. The rats were sacrificed at 0.5 h following the last dose. Liver necrosis and steatosis were determined by light microscope examination of Hematoxylin-eosin stained liver samples. b"Capsulit is" is described as extensive, widespread scarring of the liver capsule. 0 Necrosis was subjectively scored on the area (%) of liver cross-section affected and are described as follows: +1 (< 25%), +2 (26-50%), +3 (51-75%) and +4 (> 75%). 118 Chapter 4: Time-course of VPA-associated oxidative stress and hepatotoxicity i.Jm.. >' V ".' - . • ' 7 * Figure 4-5: Photomicrographs of liver sections from rats administered ip with V P A at 500 mg/kg once daily for up to 14 days showing progressive incidence of liver damage. The 0-day treatment group received saline for 14 days. Livers were fixed in 10% phosphate buffered formalin and cross-sections (10 pm) were stained with hematoxylin and eosin. Original magnification, 40X. 119 Chapter 4: Time-course of VPA-associated oxidative stress and hepatotoxicity 4.3.4 Levels of VPA metabolites during VPA treatment Levels of oxidative and mono- and di-desaturated V P A metabolites were monitored in liver (Table 4-2) and plasma (Table 4-3). There was a trend of decreasing V P A metabolite levels in liver with increasing duration of V P A treatment. Significant decreases were observed with some of the liver V P A metabolites after 14 days of treatment, in particular 3-OH-VPA, 2-ene-VPA, 3-ene-VPA and (£,£)- and (£,Z)-2,3'-diene VPA. The putative reactive V P A metabolites 4-ene-V P A and (£)-2,4-diene-VPA were not elevated throughout the duration of the study. Plasma V P A metabolites (Table 4-3) and hepatic V P A - G (Table 4-2) were similar in all groups treated with V P A over the duration of 14 days, with the exception of plasma (£)-2,4-diene-VPA, which was below the LOQ (2 ng/ml) by day 14. 120 Chapter 4: Time-course of VPA-associated oxidative stress and hepatotoxicity Table 4-2: Liver V P A metabolite levels (pg/g tissue) in rats treated with V P A for 2, 4, 7, 10, or 14 days. 3 Liver metabolites 2 days 4 days 7 days 10 days 14 days 4-ene 0.23 ± 0.04 0.15 ±0.03 0.18 ±0.03 0.14 ±0.03 0.11 ± 0 . 0 1 * 4-OH 1.13 ± 0.15 0.79 + 0.14 0.86 ±0.31 1.18±0.28 1.09 ±0.1.8 3-OH 0.59 + 0.07 0.42 ± 0.04 0.46 ±0.10 0.41 ±0.09 0.31 ±0.03* 5-0 H 0.99 + 0.11 0.68 ±0.05 0.75 ± 0.17 0.91 ±0.16 0.75 ±0.08 2-ene 2.79 ±0.17 2.23 ±0.14 2.43 ±0.33 2.03 ±0.30* 1.50 ±0.06* 3-keto 3.45 ± 0.35 2.57 ± 0.36 2.82 ±0.32 3.05 ±0.29 2.78 ± 0.23 4-keto 0.22 ± 0.04 0.14 ±0.02 0.15 ±0.04 0.19 ±0.04 0.18 ±0.02 3-ene 3.57 ±0.29 2.66 ±0.21 2.90 ±0.39 2.43 ±0.35 1.89 ±0.24* (E,E)-2,3'-diene 0.58 + 0.05 0.46 ± 0.04 0.51 ±0.09 0.53 ±0.08 0.30 ± 0.03* (£,Z)-2,3'-diene 0.12 ±0.02 0.07 + 0.01 0.07 ± 0.02 0.12 ±0.02 0.04 ± 0 . 0 1 * (E)-2,4-diene < LOQ < LOQ < LOQ < LOQ < LOQ V P A - G 249 ± 45 224 ± 34 271 ± 38 275 ± 36 273 ± 21 a Male Sprague-Dawley rats (250-300 g) treated ip with V P A (500 mg/kg) or 0.9% saline (vehicle; 1 ml/kg) once a day for 14 consecutive days. The rats were sacrificed at 0.5 h following the last dose. V P A metabolites were determined by a G C / M S assay. *Significantly different compared to the 2-days V P A treated group by one-way A N O V A with Bonferonni's multiple comparison post hoc test (p < 0.05). 121 Chapter 4: Time-course of VPA-associated oxidative stress and hepatotoxicity Table 4-3: Plasma V P A metabolite levels (ug/ml) in rats treated with V P A for 2, 4, 7,10, 14 days. 3 Plasma metabolites 2 days 4 days 7 days 10 days 14 days 4-ene 0.14 ±0.02 0.15 ±0.03 0.16 ±0.03 0.15 ±0.04 0.16 ±0.04 4-OH 2.07 ± 0.34 2.40 + 0.39 2.44 ± 0.47 2.40 ± 0.65 2.03 ±0.35 3-OH 0.93 ±0.15 0.80 ±0.08 0.70 ±0.15 0.59 ±0.14 0.49 ± 0.06 5-OH 2.32 ±0.17 2.31 ±0.19 2.09 ±0.19 1.97 ±0.29 1.71 ±0.14 2-ene 1.75 ±0.12 1.38 ±0.02 1.71 ±0.23 1.52 ±0.21 1.39 ±0.09 3-keto 3.36 ± 0.24 3.57 ± 0.45 3.09 ± 0.55 2.86 ±0.31 3.30 ±0.10 4-keto 0.59 ±0.08 0.39 ±0.07 0.43 ±0.10 0.32 ±0.07 0.30 ± 0.04 3-ene 0.52 ± 0.10 0.60 ± 0.05 0.62 ± 0.07 0.52 ± 0.06 0.60 ± 0.02 (E,E)-2,y-diene 0.75 ±0.09 0.85 + 0.06 1.04 ± 0.16 0.92 ± 0.09 0.70 + 0.05 (E,Z)-2,3'-diene 0.14 ±0.02 0.09 ±0.01 •0.15 ±0.02 0.14 ±0.01 0.08 + 0.01 2,4-diene 0.06 ±0.01 0.03 ±0.01 0.03 ±0.01 0.03 ±0.01 < LOQ 3 Male Sprague-Dawley rats (250-300 g) treated ip with V P A (500 mg/kg) or 0.9% saline (vehicle, 1 ml/kg) once a day for 14 consecutive days. The rats were sacrificed at 0.5 h following the last dose. V P A metabolites were determined by a G C / M S assay. *Significantly different compared to the 2-days V P A treated group by one-way A N O V A with Bonferonni's multiple comparison post hoc test (p < 0.05). 122 Chapter 4: Time-course of VPA-associated oxidative stress and hepatotoxicity 4.4 DISCUSSION Our previous study in rats showed increased levels of 15-F 2 t - lsoP after a single dose of VPA , but this did not involve P450-mediated VPA-biotransformation, as indicated in subsequent mechanistic experiments (Tong er al. 2003). The current study investigated the levels of oxidative stress biomarkers in relation to the occurrence of hepatotoxicity over a 2-week treatment period with V P A . The results from this study demonstrated that high doses of V P A given over a time-course of 14 days in rats resulted in an elevation in plasma and liver 15-F 2 t -IsoP that preceded the occurrence of hepatotoxicity (Figure 4-6). Hepatotoxicity: - Serum a-GST - necrosis 15-F2t-lsoP LPO i steatosis severe necrosis TBARs I Day 0 10 14 Figure 4-6: Summary of results indicating the time-course of hepatotoxicity and oxidative stress events. Rats were administered 500 mg/kg V P A ip once daily for up to 14 days. The 0-day treatment group received saline for 14 days. 123 Chapter 4: Time-course of VPA-associated oxidative stress and hepatotoxicity In this study, hepatotoxicity was observed from 4 to 14 days of V P A treatment and the liver histology was characterized predominantly by inflammation and extensive scarring of the liver capsule. The first incidence of focal necrosis was also observed after day 4 and increased in severity to massive necrosis that appeared to have no consistent zonal pattern and involved large irregular areas of hepatic tissue. The presence of necrosis from days 4 to 14 coincided with increased serum levels of ct-GST, which is a sensitive and specific biomarker of hepatocyte injury (Trull ef al. 1994). a -GST, which is found in high concentrations throughout the liver parenchyma, was demonstrated to be an earlier and more sensitive marker of hepatocyte injury than the release of conventionally used liver enzymes, such as the transaminases and lactate dehydrogenase (Clarke ef al. 1997; van Wagensveld ef al. 1997). In this study, alanine aminotransferase and aspartate amino transferase were also measured in the serum by colormetric assays (procedure not described). However, these results were highly variable among animals due to the variable hemolysis observed during the sample collection (data not shown). Fatty liver was not a predominant lesion observed in the current study. Other studies showed microvesicular steatosis as a common feature in rats at a near-lethal dose of 750 mg/kg over 48 h, but not with a lower dose of 350 mg/kg (Lewis ef al. 1982). Kesterson et al. (Kesterson ef al. 1984) demonstrated fatty liver in rats treated with 700 mg/kg/day for 4 days, but with a lower occurrence (2 out of 7 rats) at 600 mg/kg/day for 5 days. In another study, light microscopy did not reveal steatosis following the 200 or 600 mg/kg doses in 24 h; however, an increase in liver lipid and triglyceride levels as observed by 3 h at the higher dose (Jezequel ef al. 1984). In the above studies, electron microscopy revealed numerous lipid vacuoles and ultrastructural changes to the mitochondria believed to be a result of mitochondrial dysfunction that is consistent with inhibition of p-oxidation. In the present study, the first incidence of fatty liver, although never extensive in area, occurred on day 4 of treatment (1 out of 4 rats) with 124 Chapter 4: Time-course of VPA-associated oxidative stress and hepatotoxicity increasing occurrence observed after 10 days of treatment (4 out of 5 rats) at the dose of 500 mg/kg/day. The lack of steatosis on day 14 was peculiar and it was speculated to be related to the observation that necrosis was more severe at this time. In other studies that used the same 500 mg/kg dose of V P A , microvesicular steatosis was observed after a single dose in fasted rats by osmium tetroxide staining and biochemical analysis (Olson et al. 1986) and with repeated dosing also at 500 mg/kg for 7 consecutive days by electron microscopy in rats provided with food ad libitum (Sugimoto et al. 1987). Although steatosis was first observed on day 4 under light microscopy, the extent of steatosis may be underestimated in our study since electron microscopy and staining with oil-red O or osmium tetroxide was not performed. The reported studies suggest that V P A induces steatosis in a dose- and time-dependent manner in rats. The elevation in plasma and liver 15-F 2 t - lsoP preceded the occurrence of hepatotoxicity, as determined by histological assessment and by levels of serum a -GST. After the second day of dosing, plasma and serum 15-F 2 t - lsoP increased compared to the saline-treated group, and these levels remained elevated to the same extent throughout the 14-day study period. According to our data, the formation of 15 -F 2 r l soP did not increase over time with repeated dosing. The question of whether or not the observed increase in 15-F 2 t - lsoP following V P A administration is reflective of oxidative stress still remains to be determined. 15-F 2 t - lsoP is a member of the F 2-isoprostanes, a series of prostaglandin F 2 a- isomers that is produced by a free radical-catalyzed (non-enzymatic) lipid peroxidation of arachidonic acid (Roberts and Morrow 2000). It has been generally accepted that the cascade from arachidonic acid to 15-F 2 t - lsoP is independent of cyclooxygenase (COX). However, in extrahepatic tissues, evidence exists for a COX-dependent formation of 15-F 2 t - lsoP involving contribution from constitutive COX-1 and/or inducible C O X - 2 isoforms in a kidney model for ischemia-reperfusion injury (Favreau et al. 2004), an isolated rat kidney glomeruli model (Klein et al. 2001) and in thrombin or arachidonic 125 Chapter 4: Time-course of VPA-associated oxidative stress and hepatotoxicity acid activated platelets (Klein ef al. 1997; Pratico ef al. 1995). In these reported studies, the non-selective C O X inhibitors indomethacin and diclofenac were able to suppress the formation of 15-F 2 t - lsoP. This suggests that 15 -F 2 r l soP may have a dual origin such that both free radicals and C O X enzymes may contribute to their formation. However, the effects of V P A on 15-F 2 t - lsoP are unlikely to be due to C O X activity. V P A treatment has been associated with reduced arachidonic acid turnover in rats (Chang ef al. 2001) and attenuation of the arachidonic acid cascade as evidenced by reduced synthesis of COX-dependent products (Kis ef al. 1999; Szupera ef al. 2000). A recent study reported that chronic administration of V P A to rats reduced protein levels of COX-1 and COX-2 , total C O X activity, and the metabolites of arachidonic acid produced via C O X (Bosetti ef al. 2003). While taking into consideration of the reported lack of an effect of V P A on C O X expression, our interpretation is that 1 5 - F 2 r l s o P levels are indeed reflective of oxidative stress and precede the onset of hepatotoxicity in VPA-treated rats (Figure 4-6). My subsequent experiments with cultured rat hepatocytes indicate that oxidative stress, as measured by the 15-F 2 t - lsoP and DCF-DA assays, can occur in the absence of hepatocyte toxicity (See Chapter 5, Figure 5-3 and Figure 5-8). Other biomarkers of oxidative stress, LPO and T B A R S , were also examined in our study in relation to the onset of hepatotoxicity. We found that levels of liver L P O were elevated after the onset of hepatotoxicity (day 7) and remained elevated with an increasing trend to day 14. Liver and serum T B A R S were not elevated until day 14, which corresponded to the time point when necrosis was most prevalent. These results are consistent with previous findings that a single dose of V P A did not alter liver and plasma T B A R S and liver LPO levels (Tong etal. 2003). The increase in LPO and T B A R S detected at time points after the first onset of hepatic necrosis may imply that necrosis occurred first, giving rise to the elevated biomarkers of lipid peroxidation. However, it may also be argued that LPO and T B A R S are less sensitive markers of lipid peroxidation and the colormetric assays to be less specific and indirect as compared to the 126 Chapter 4: Time-course of VPA-associated oxidative stress and hepatotoxicity measurement of 15-F 2 t - lsoP by EIA. On the other hand, 15-F 2 t - lsoP may represent an earlier lipid peroxidation event, while MDA, a degradation product of polyunsaturated fatty acid hydroperoxides detected using the T B A R S assay, may simply represent later lipid peroxidation events as a consequence of the necrosis. A lack of temporal correlation among R O S biomarkers was also reported in another study that examined pulmonary artery endothelial cells treated with H 2 0 2 (Hart et al. 1998). Specifically, the levels of T B A R S and LPO were not significantly elevated in cells treated with H 2 0 2 (50-100 uM) at a time point that produced a maximal increase in 15-F 2 t - lsoP; however, only at the highest concentration of H 2 0 2 (250 uM) were increases in levels of T B A R S and LPO observed. These results were ascribed to differences in biomarker sensitivity. Our study is the first to simultaneously characterize V P A metabolite profiles, hepatotoxicity, and oxidative stress events within the same time-frame of V P A treatment in the whole animal. With respect to the reactive metabolite hypothesis, there was no elevation in hepatic and plasma levels of the putative V P A reactive metabolites 4-ene-VPA and (E)-2,4-diene-VPA, which suggests that these metabolites are not responsible for the oxidative stress or hepatotoxicity observed in the present study. These findings are consistent with reported studies that examined V P A metabolite profiles and hepatotoxicity in patients and showed a lack of correlation between hepatotoxicity and serum levels of 4-ene-VPA (Siemes ef al. 1993). A similar conclusion was also reached following comparative studies of V P A and 2-ene-VPA in rats where the incidence of liver microvesicular steatosis was observed to be independent of plasma levels of 4-ene-VPA and (£)-2,4-diene-VPA (Loscher ef al. 1993). It was therefore suggested that these metabolites were not the decisive factors in VPA-induced hepatotoxicity, whereas more recent studies indicated that urinary N-acetylcysteine conjugates of (E)-2,4-diene-VPA were a better indicator of reactive metabolite exposure (Gopaul et al. 2000a, b). Furthermore, the B-oxidation-related metabolites 2-ene-VPA, 3-ene-VPA, 3-OH-VPA, and (E,E)-127 Chapter 4: Time-course of VPA-associated oxidative stress and hepatotoxicity and (£,Z)-2,3'-diene-VPA were decreased in the liver by days 10-14, suggesting that inhibition of p-oxidation did not occur until after the onset of oxidative stress and hepatotoxicity. An interesting observation is that V P A - G levels in the liver were unchanged throughout the study period. Our previous results (see Chapter 3) linked V P A - G and 15-F 2 t - lsoP levels in the liver following a single large dose of V P A to rats. Since the liver V P A - G levels remained similar and did not decrease throughout the study, this finding is consistent with the observation that V P A - G levels correlated with the elevated levels of 15-F 2 t - lsoP seen from day 2. 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Effects of valproate and E-2-ene-valproate on functional and morphological parameters of rat liver. II. Influence of phenobarbital co-medication. Epilepsy Res 15, 113-31. Olson, M. J . , Handler, J . A., and Thurman, R. G. (1986). Mechanism of zone-specific hepatic steatosis caused by valproate: inhibition of ketogenesis in periportal regions of the liver lobule. Mol Pharmacol 30, 520-5. Pratico, D., Lawson, J . A., and FitzGerald, G. A. (1995). Cyclooxygenase-dependent formation of the isoprostane, 8-epi prostaglandin F2 alpha. J Biol Chem 270, 9800-8. 130 Chapter 4: Time-course of VPA-associated oxidative stress and hepatotoxicity Roberts, L. J . , and Morrow, J . D. (2000). Measurement of F(2)-isoprostanes as an index of oxidative stress in vivo. Free Radio Biol Med 28, 505-513. Siemes, H., Nau, H., Schultze, K., Wittfoht, W., Drews, E., Penzien, J . , and Seidel, U. (1993). Valproate (VPA) metabolites in various clinical conditions of probable VPA-associated hepatotoxicity. 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The effects of valproate on intermediary metabolism in isolated rat hepatocytes and intact rats. Biochem Pharmacol 32, 1887-92. van Wagensveld, B. A., Scheepers, J . J . , van Gulik, T. M., Frederiks, W. M., Bleeker, W. K., Obertop, H., and Gouma, D. J . (1997). Alpha glutathione S-transferase as novel parameter for hepatocellular damage in the isolated perfused rat liver. Transplant Proc 29, 3449-51. 131 Chapter 5: VPA and oxidative stress in rat hepatocytes 5 Investigation of VPA-Associated Oxidative Stress in Primary Cultured Rat Hepatocytes 5.1 INTRODUCTION Primary cultured rat hepatocytes have been well utilized to study VPA-associated hepatotoxicity. Early studies demonstrated that V P A caused dose-dependent toxicity to rat hepatocytes (Kingsley ef al. 1983). It was also demonstrated that V P A was associated with biochemical disturbances such as inhibition of fatty acid oxidation, gluconeogenesis, ketogenesis, urea synthesis, and reduced levels of acetyl-CoA in rat hepatocyte cultures (Becker and Harris 1983; Coude ef al. 1983; Turnbull ef al. 1983). In support of the oxidative stress hypothesis of V P A hepatotoxicity, it was first suggested that lipid peroxidation was involved in V P A hepatotoxicity when the antioxidants, vitamin E, a-tocopherol, and N, N'-diphenyl-p-phenylenediamine, conferred protection against V P A toxicity in rat hepatocyte cultures (Buchi et al. 1984; Jurima-Romet ef al. 1996). Our previous work demonstrated that a single dose of V P A administered in rats led to a dose-dependent elevation in plasma and liver levels of the endogenous lipid peroxidation marker, 15-F 2 t-isoprostaglandin (15-F 2 t- lsoP) (Tong ef al. 2003). High daily doses of V P A to rats for 14 consecutive days produced an elevation in 15-F 2 t - lsoP that preceded the onset of liver necrosis and steatosis (Tong et al. 2005, manuscript submitted). In the same study, two other independent measures of oxidative stress, thiobarbituric acid reactive substances and lipid hydroperoxides, were elevated at later time points. These findings are consistent with the hypothesis that V P A is associated with oxidative stress; however, the question remains whether the elevation in 15-F 2 t - lsoP levels truly reflects oxidative stress and whether this results in mitochondrial dysfunction and hepatocyte toxicity. 132 Chapter 5: V PA and oxidative stress in rat hepatocytes There is evidence that G S H homeostasis may be altered, either as a consequence of reactive metabolites and/or reactive oxygen species produced during V P A treatment (Cotariu ef al. 1990; Graf ef al. 1998; Olson et al. 1986; Raza et al. 1997; Yuksel ef al. 2000). Reduced G S H is an important cell protecting biomolecule against chemical-induced cytotoxicity by direct or enzymatic (glutathione-S-transferase) conjugation with electrophilic compounds (Reed 1990). G S H is also an important cellular antioxidant that is capable of direct or enzymatic (glutathione peroxidase) conjugation with R O S such as lipid hydroperoxides and hydrogen peroxide (Meister 1983). A useful approach to understanding the role of G S H in chemical toxicity is to determine the consequences of reducing or depleting cellular G S H . This was demonstrated when G S H -depleted rat hepatocytes exhibited greater toxicity to 4-ene-VPA than normal control hepatocytes, and the toxicity was attenuated with the addition of antioxidants, vitamin C and vitamin E (Jurima-Romet et al. 1996). The successful treatment of severe V P A hepatotoxicity with N-acetylcysteine in a small number of pediatric epileptic patients provided further evidence for a protective role of G S H (Farrell and Abbott 1991). The objective of the present study using freshly isolated rat hepatocytes was to determine the acute effects of V P A on: (1) oxidative stress, as measured by the 5-(and-6)-carboxy-2',7'-dichlorofluorescin diacetate (DCF-DA) and 15-F 2 t - lsoP assays; (2) mitochondrial membrane potential (AT™), as determined by the JC-1 assay; and (3) hepatocyte toxicity, as determined by the WST-1 assay. Furthermore, the role of G S H was investigated by comparing the effects of V P A on the above endpoints in GSH-reduced hepatocytes and in normal control hepatocytes. Findings from the present study support the hypothesis that V P A produces oxidative stress prior to hepatotoxicity, which is consistent with our previous in vivo findings (Tong ef al. 2005, manuscript submitted), and that GSH-depletion exacerbates oxidative stress, mitochondrial membrane depolarization, and cytotoxicity in rat hepatocytes treated with high concentrations of VPA. 133 Chapter 5: VPA and oxidative stress in rat hepatocytes 5.2 METHODS & MATERIALS 5.2.7 Chemicals 2-Propyl pentanoic acid (sodium valproate, VPA) , DL-buthionine-[S,R]- sulfoximine (BSO), diethylmaleate (DEM), butylated hydroxytoluene, trypsin inhibitor (Type ll-soybean) and collagenase (Type IV) were purchased from Sigma-Aldrich Canada Ltd. (Oakville, ON, Canada). Matrigel basement membrane matrix and Hepato-Stim medium were purchased from BD Biosciences (Mississauga, O N , Canada). The 15-F 2t-isoprostane EIA kit and the glutathione assay kit were purchased from Cayman Chemical Co. (Ann Arbor, Ml). WST-1 was obtained from Hoffman-La Roche (Mississauga, ON, Canada). JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide) and DCF-DA were purchased from Molecular Probes (Eugene, OR). Liver Perfusion® medium, Hepatocyte Wash® medium, Hepatozyme, Hank's Balanced Salt Solution (HBSS) and phosphate buffered saline (PBS) were purchased from Invitrogen (Burlington, ON , Canada). Percoll was purchased from Amersham Biosciences Inc. (Baie d'Urfe, Q C , Canada). 5.2.2 Animals Adult male Sprague-Dawley rats (250-300 g) were obtained from the University of British Columbia Animal Care Facility. They were fed with rat diet (Labdiet 5001 rodent diet, PMI Feeds Inc., Richmond, IN) and water ad libitum and maintained in a room on a 12 h light/12 h dark cycle at constant temperature (22°C) and humidity. All animal experiments were approved by the University of British Columbia Animal Care Committee and conducted in accordance with the guidelines of the Canadian Council on Animal Care. 134 Chapter 5: VPA and oxidative stress in rat hepatocytes 5.2.3 Rat hepatocyte isolation and culture Rats were anesthetized with sodium pentobarbital (60 mg/kg, ip) and the abdomen was opened by a midline incision. The liver, inferior vena cava and the hepatic portal vein were exposed. Silk sutures were tied loosely around the inferior vena cava, the superior vena cava, and the hepatic portal vein. The hepatic portal vein was cannulated with a 21 gauge Teflon catheter, the needle removed, and the catheter secured. A two-step, collagenase perfusion technique first involved perfusing the liver with Ca 2 + - f ree Liver Perfusion medium at a rate of 25 ml/min for 6 to 8 min using a peristaltic pump. The inferior vena cava was severed immediately to allow for the efflux of the perfusate. The perfusion solution was changed to a H B S S digest media (1.40 g/L CaCI 2 , 2.38 g/L H E P E S , 0.35 g/L N a H C 0 3 , 0.05 g/L trypsin inhibitor (Type ll-soybean), 0.5 g/L collagenase (Type IV, pH 7.4). Perfusion with digest media was subjectively determined to be complete (approximately 6-8 min). All perfusion media were warmed to 37°C. Upon digestion, the liver was excised and placed in a sterile petri-dish containing Hepatocyte Wash medium. Cells were mechanically dispersed using a blunt glass rod. The cell suspension was filtered through sterile 60 um Nytex® mesh cloth into 50 ml Falcon® centrifuge tubes on ice. The suspension was centrifuged (50 x g) for 3 min to pellet the hepatocytes and the pellet re-suspended in fresh wash medium. A Percoll solution (26.1 ml sterile Percoll and 3.9 ml of 10 x HBSS) was mixed with 20 ml of the cell suspension by inversion of the tube. The mixture was centrifuged at 4°C at 50 x g for 5 min and the supernatant containing the dead cells was decanted. The pellet was resuspended and washed once with Hepato-Stim medium and the viability and cell concentration was determined by trypan blue exclusion. The exclusion criterion for hepatocyte viability was > 90%. Cells were diluted to 4 x 10 5 cells/ml in Hepato-Stim medium and seeded as a monolayer culture on Matrigel matrix coated 24- or 96-well sterile tissue culture plates at a density of 2 x 10 5 or 4 x 10 4 cells/well, respectively. Cells were 135 Chapter 5: VPA and oxidative stress in rat hepatocytes allowed to attach for 2 h in a 37°C, 5% C 0 2 incubator prior to experimental treatment. Attachment efficiency was determined to be approximately 95%. In experiments involving hepatocytes depleted of G S H , hepatocytes were pretreated for 2 h prior to V P A treatment with a combination of DL-buthionine-[S,R]- sulfoximine (BSO) and diethylmaleate (DEM). B S O is a potent and specific transition state inhibitor of y -glutamylcysteine synthetase that depletes cellular G S H pools by blocking its synthesis (Griffith 1981). Diethylmaleate (DEM), an electrophilic compound, was also used to deplete cellular G S H directly by undergoing extensive conjugation with G S H in a reaction catalyzed by glutathione-S-transferases (Maellaro et al. 1990; Meredith and Reed 1982; Miccadei et al. 1988; Plummeref al. 1981). 5.2.4 15-F2rlsoP Assay 15-F 2 t - lsoP was determined in rat hepatocyte cultures (2 x 10 5 cells/well in 24-well plates) as an index of oxidative stress. After 2-h cell attachment, the hepatocytes were treated with V P A (0-1000 ug/ml dissolved in Hepatozyme medium). At specific time points, 2% butylated hydroxytoluene (10 u.l) was added to each culture to prevent sample auto-oxidation during storage and processing and the culture medium (1 ml) including the cell monolayer was transferred to polypropylene Eppendorf™ tubes and immediately snap-frozen in liquid nitrogen. All samples were stored at -80°C and processed the next day for the determination of 15-F 2 t -IsoP as previously described (Tong et al. 2003). The results were normalized per 10 6 cells and expressed as the mean ± S E M . 136 Chapter 5: VPA and oxidative stress in rat hepatocytes 5.2.5 DCF-DA assay Production of intracellular R O S was also monitored by the fluorescence emission of 2',7'-dichlorofluorescein (DCF). After 2-h cell attachment, the hepatocytes (4 x 10 4 cells/well in a 96-well plate) were preloaded with 5 uM DCF-DA (dissolved in Hepatozyme medium) for 20 min (37°C, 5% C 0 2 ) . The diacetate form of 2',7'-dichlorofluorescin (DCFH) diffuses across the cell membrane and is hydrolyzed by intracellular esterases to yield the non-fluorescent D C F H . D C F H , upon reacting with low molecular weight hydroperoxides (i.e. H 2 0 2 ) , is oxidized to its highly fluorescent, 2-electron oxidation product, 2',7'-dichlorofluorescein (DCF) (Figure 5-1). Following 20 min of DCF-DA preloading, cells were washed once with Hepatozyme medium and treated with V P A (0-1000 ug/mL, dissolved in Hepatozyme medium). Fluorescence was determined immediately after addition of V P A or its vehicle, and at 5, 15, 30, 45, 60, 90, and 120 min on a Cytofluor® Series 4000 (Applied Biosystems, Bedford, MA) multi-well fluorescent plate reader (excitation: 485 nm with slit width of 25 nm; emission: 530 nm with a slit width of 20 nm). The concentration of DCF was determined from calibration curves prepared from DCF (Polysciences Inc., Warrington, PA) standard and the results were reported as pmol DCF/10 6 cells. Figure 5-1: Schematic describing the mechanism of the DCF-DA assay. DCF-DA is taken up into cells, where intracellular esterases cleave the diacetate groups to produce the non-fluorescent DCFH. DCFH serves as a substrate for the iron-catalyzed oxidation by H 2 0 2 to yield the fluorescent DCF as a indirect measure of oxidative stress. esterases D C F H - D A hepa t o cy t e H 2 ° 2 D C F F e 2 + , peroxidase ^©x: 4 8 5 n m Xem: 530 nm 137 Chapter 5: VPA and oxidative stress in rat hepatocytes 5.2.6 Measurement of mitochondrial membrane potential (A Wm) AY™ was measured using the mitochondrial specific dual-fluorescence probe, JC -1 , based on modified methods (Reers et al. 1995; Reers ef al. 1991). JC-1 is a ratiometric dye that is internalized as a monomer dye (green fluorescence, emission wavelength: 530 nm) and is concentrated by respiring mitochondria with negative inner membrane potential into J-aggregate dye (red fluorescence, emission wavelength: 590 nm) (Smiley ef al. 1991). Consequently, mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio. Cells in culture (4 x 10 4 cells/well in a 96-well plate) were treated with various compounds up to 2 h. Positive controls to which V P A (0 - 1000 pg/ml, dissolved in Hepatozyme medium) were compared included valinomycin (50 pM), a potassium ionophore, and carbonyl cyanide m-chlorophenylhydrazone ( m C L C C P , 50 pM) and carbonyl cyanide 4-(trifluoromethoxy)-phenylhydrazone ( F C C P , 50 pM), both uncouplers of oxidative phosphorylation. After the 2-h treatment, the medium was removed and replaced with 100 pi of 10 pg/ml JC-1 in Hepatozyme medium. Cells were incubated with the dye for 10 min (37°C, 5% C 0 2 / 9 5 % air) followed by a wash with P B S , and cells were allowed to equilibrate at room temperature in the dark for 10 min. A CytoFiuor® Series 4000 plate reader (Applied Biosystems, Bedford, MA) was set to an excitation wavelength of 485 nm (slit width: 20 nm) to monitor the fluorescence intensities for the monomer and the aggregated JC-1 molecules (emission wavelengths: 530 nm with a slit width of 25 nm, and 590 nm with a slit width of 5 nm, respectively). 5.2.7 Cell viability Cell viability was measured by the WST-1 method in 96-well plates. This method is based on the cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenases in viable cells to a water-soluble formazan dye. After the 2-h attachment, cells (4 x 10 4 cells/well in 96-well plates) were treated with V P A (using Hepatozyme medium as a vehicle) for 8 h (37°C, 5% C 0 2 ) 138 Chapter 5: VPA and oxidative stress in rat hepatocytes followed by the addition of 10 pi WST-1 stock solution per 100 ul of medium in each well. After 30 min of incubation (37°C, 5% C 0 2 ) , absorbance was determined at 450 nm on a Labsystems Multiskan Ascent® multi-well plate reader (Thermo Electron Corp., Burlington, O N , Canada). A decrease in cell viability was indicated by a decrease in the amount of formazan dye (decrease in absorbance). The positive control, 4-pentenoic acid, was used to compare the cytotoxicity of VPA. 5.2.8 Glutathione (GSH) assay G S H levels in hepatocytes were determined using a commercially available kit (Cayman Chemical Co. , Ann Arbor, Ml) that is based on an enzymatic recycling method, using glutathione reductase, for the quantitation of total G S H levels (Figure 5-2). The rat hepatocytes were collected after pretreatment with B S O and DEM for 2 h and homogenized in 0.5 ml of cold 50 mM phosphate buffer (pH 7.4) containing 1 mM EDTA. The homogenate was centrifuged at 10,000 x g (15 min at 4°C) and the supernatant was collected and stored on ice. For the deproteination procedure, the samples were acidified with an equal volume of meta-phosphoric acid (10% solution in water), mixed on a vortex mixer, and allowed to stand at room temperature for 5 min. Samples were centrifuged (2000 x g at room temperature) to pellet the protein, and the supernatant (0.5 ml) collected. The pH of the sample was increased to approximately 8 with the addition of 25 u.l of a 4 M solution of triethanolamine, prior to G S H determination. Total G S H levels were determined with a standard curve over a concentration range of 0 - 16 uM G S H (reduced form). 139 Chapter 5: VPA and oxidative stress in rat hepatocytes Glutathione Reductase G S S G • 2 GSH DTNB TNB Figure 5-2: GSH recycling in the presence of glutathione reductase and Ellman's Reagent (DTNB, 5,5'-dithioo/s-2-nitrobenzoic acid). TNB (5-thio-2-nitrobenzoic acid) is monitored spectrophotmetrically (A,MAX = 405 nm) as an indirect measure of G S H levels. (Cayman Chemical Co., G S H assay kit booklet). 5.2.9 Statistical analysis The results are reported as means ± S E M . Statistical significance of the difference between the means of groups were analyzed by one-way A N O V A and followed by the Student-Newman-Keul's multiple pairwise comparison post hoc test. Groups receiving different treatment concentrations of V P A (Figure 5-3) or B S O and D E M (Figure 5-4) were compared to normal hepatocytes at the same time point. In addition, either normal hepatocytes were compared to B S O and DEM-pretreated hepatocytes with different V P A concentrations at the same time point, or different time points at the same V P A treatment (Figure 5-5, Figure 5-6, and Figure 5-7). The level of significance was set a priori at p < 0.05. 140 Chapter 5: VPA and oxidative stress in rat hepatocytes 5.3 RESULTS 5.3.1 VPA-induced oxidative stress in primary culture rat hepatocytes V P A produced a time- and concentration-dependent increase in the lipid peroxidation biomarker, 15-F 2 t - lsoP, in rat hepatocyte cultures (Figure 5-3A). Treatment with 1000 ug/ml V P A for 30 min increased the level of 15-F 2 t- lsoP compared to the control group (199 ± 18 and 104 + 22 pg/ml/10 6 cells, respectively). At the 120 min time point, treatment with V P A at all concentrations (100 - 1000 ug/ml) resulted in significantly elevated 15-F 2 t - lsoP levels by up to 1.9-fold compared to the 0 ug/ml control group. To verify the increase in oxidative stress by VPA , an independent indicator of intracellular oxidative stress was measured with DCF fluorescence intensity in VPA-treated hepatocytes. Oxidation of D C F H to D C F was elevated in a time- and concentration-dependent manner, with significant increases in D C F detected in the 10, 250 and 1000 ug/ml VPA-treated cells at time points from 30 to 120 min of incubation (Figure 5-3B). 141 Chapter 5: VPA and oxidative stress in rat hepatocytes A 0 30 60 90 120 150 180 Incubation Time (min) B Incubation Time (min) Figure 5-3: Time-course and dose-response relationship for the production of (A) 15-F 2 t-IsoP and (B) DCF in freshly isolated rat hepatocytes treated with VPA. (A) Freshly isolated rat hepatocytes were incubated with 0, 100, 500, and 1000 pg/ml VPA (4 x 10 s cells/well in a 24-well plate) from 0 - 1 2 0 min, at 30 min intervals. The medium was removed at each time point for the determination of 15-F 2 t-lsoP. Data are reported as the mean ± SEM (n = 5 experiments). Significant differences between 0 pg/ml vs. *1000 pg/ml, **1000 and 500 pg/ml, ***1000, 500, and 100 pg/ml groups at each time point (p < 0.05). (B) Hepatocytes (4 x 10 4 cells/well in 96-well plates) were preloaded with 5 uM DCF-DA for 20 min and treated with V P A (0 - 1000 pg/ml). Intracellular oxidative stress was measured with DCF fluorescence intensity (Ex: 485, Em: 530 nm). Data are reported as the mean ± SEM (n = 8 experiments). "Significantly different between the 10, 250, and 1000 pg/ml V P A groups compared to the 0 pg/ml V P A (control) group (p<0.05). 142 Chapter 5: VPA and oxidative stress in rat hepatocytes 5.3.2 VPA-induced oxidative stress in GSH-depleted rat hepatocytes To verify the effects of B S O and D E M pretreatment, total cellular G S H levels were measured in hepatocytes treated with varying concentrations of these two chemicals, either individually or in combination. Total intracellular G S H levels were decreased to a maximum of 50% with 8 mM B S O and 37% with 2 mM D E M compared to control levels (data not shown). To enhance G S H depletion, the combination of 2 mM B S O and 0.5 mM D E M resulted in maximal reduction of G S H (21% of control) (Figure 5-4). In all subsequent experiments, hepatocytes were pretreated with the combination of 2 mM B S O and 0.5 mM D E M . Reduction of G S H in hepatocytes resulted in greater increases of 15-F 2 t - lsoP when compared to the non-GSH reduced control group at the concentrations of V P A tested: 1000 ug/ml (1.7-fold), 500 ug/ml (1.4-fold increase), and 100 ug/ml (1.4-fold increase) (Figure 5-5). The time-dependent and dose-dependent formation of DCF was compared between control and BSO + DEM-pretreated hepatocytes treated with VPA. D C F levels were significantly elevated in B S O + DEM-pretreated hepatocytes compared to control hepatocytes treated with 1000 ug/ml V P A over the time-course from 45 to 120 min (Figure 5-6A). Furthermore, BSO + DEM-pretreated hepatocytes produced a 1.3 to 1.5-fold increase in D C F levels compared to control hepatocytes treated with 250, 500 and 1000 ug/ml V P A at the fixed incubation time of 120 min (Figure 5-6B). Strictly within the GSH-reduced hepatocytes, V P A (1000 ug/ml) produced a maximum 4.9-fold increase in D C F levels compared to the control group (0 pg/ml VPA) at a fixed incubation time of 2 h (Figure 5-6B). 143 Chapter 5: VPA and oxidative stress in rat hepatocytes 80 n BSO(mM): 0 0.5 1 2 4 8 DEM (mM): Q 0.13 0.25 0.5 1 2 Figure 5-4: Total G S H levels determined in freshly isolated rat hepatocytes pretreated with a combination of DL-buthionine-[S,R]-sulfoximine (BSO) and diethylmaleate (DEM). Rat hepatocytes were pretreated with serial dilutions of a mixture of B S O (8 mM) and DEM (2 mM) for 2 h. Results are normalized per 10 6 cells and expressed as mean ± S E M (n = 4 experiments). "Significantly different compared with the control group (p < 0.05). 144 Chapter 5: VPA and oxidative stress in rat hepatocytes 0 -I 1 1 1 1 , 0 200 400 600 800 1000 VPA (jLig/ml) Figure 5-5: Dose-dependent changes in 15-F 2 t-lsoP by V P A in freshly isolated rat hepatocytes. Hepatocytes were isolated from male Sprague-Dawley rats and were pretreated with a combination of BSO (2 mM) and DEM (0.5 mM) for 2 h. Hepatocytes were treated with V P A (0 - 1000 ug/mL) for an additional 2 h. Data are reported as the mean + SEM (n = 5 experiments) *Significant differences between control hepatocytes vs. BSO and DEM pretreated hepatocytes (p < 0.05). 145 Chapter 5: VPA and oxidative stress in rat hepatocytes A Incubation Time (min) B 20 -i —•— BSO+DEM pre-treated hepatocytes 0 -I—•• 1 1 1 1 1 0 200 400 600 800 1000 VPA (ug/ml) Figure 5-6: Comparison of the time- and concentration-dependent effect of V P A on the production of DCF in control, and BSO and DEM-pretreated rat hepatocytes. (A) Hepatocytes were treated with 1000 pg/ml VPA and DCF levels measured over time. (B) Hepatocytes were treated with VPA (0 - 1000 pg/ml) and DCF levels were measured at a fixed incubation time of 2 h. Data are reported as the mean ± S E M (n = 8 experiments). 'Significantly different between BSO + DEM pretreated hepatocytes vs. control hepatocytes at each time point (p < 0.05). 146 Chapter 5: VPA and oxidative stress in rat hepatocytes 5.3.3 Effect of VPA on mitochondrial membrane potential (A^m) in primary culture rat hepatocytes To examine the effect of V P A on A ^ , the hepatocytes were treated with J C - 1 , a cationic, lipophilic dual fluorescence dye that exhibits potential-dependent accumulation in mitochondria (Reers ef al. 1995; Reers ef al. 1991). The accumulation of dye aggregates is indicated by a fluorescence shift from green (emission: 530 nm) to red (emission: 590 m). A loss in A*¥m is indicated by a decrease in red/green fluorescence intensity ratio. V P A did not affect the A*Fm in control rat hepatocytes with respect to both time (Figure 5-7A) and concentration (Figure 5-7B). A loss in A ^ m was observed in GSH-depleted hepatocytes treated with 500 and 1000 ug/mL V P A (Figure 5-7B). The positive controls, mitochondrial un-couplers F C C P , m C L C C P and the potassium ionophore, valinomycin, resulted in A T m depolarization as indicated by a 50 - 60% decrease in the fluorescence ratio intensities by 15 min of treatment compared to their respective 0 min controls. 5.3.4 Effect of VPA on cell viability As measured by the conversion of the tetrazolium salt WST-1 to a water-soluble formazan dye (absorbance at 450 nm) in rat hepatocytes, cell viability was not affected by V P A treatment (0 -1000 ug/ml) for 8 h of incubation in normal control hepatocytes (Figure 5-8). A significant loss in cell viability (25% of control) was detected in GSH-depleted hepatocytes at 1000 ug/ml V P A . The hepatotoxin, 4-pentenoic acid (4-PA, 0 - 1000 pg/ml, n = 2 experiments) which was used as a positive control, resulted in a dose-dependent decrease in cell viability. 147 Chapter 5: VPA and oxidative stress in rat hepatocytes 0 B 1000 ng/ml VPA — 500 ng/ml VPA 250 ng/ml VPA —a— 125 ng/ml VPA — 0 ng/ml VPA - © — 50 nM Valinomycin — • — 50 nM FCCP 50 nM mCLCCP 20 40 60 Time (min) 80 Control hepatocytes BSO and DEM-pretreated hepatocytes 0 200 400 600 800 1000 VPA (ng/ml) Figure 5-7: The time-course and concentration-dependent effects of VPA on mitochondrial membrane potential ( A ^ m ) assessed by the JC-1 fluorescent probe. (A) Rat hepatocytes (4 x 10 4 cells/well in 96-well plates) were treated with VPA (0 - 1000 pg/ml, n = 3 experiments) or positive control compounds valinomycin, FCCP, m C L C C P (50 pM each, n=2 experiments) for up to 60 min. (B) Control and BSO+DEM-pretreated rat hepatocytes were treated with V P A (0-1000 pg/ml) for 2 h. Values are expressed as mean ± SEM (n = 5 experiments). *Significantly different between the control vs. BSO+DEM pretreated hepatocytes (p < 0.05). 148 Chapter 5: VPA and oxidative stress in rat hepatocytes E c o ^ «-* o ra c CD O (0 o o ^ (0 n < V P A (control hepatocytes) V P A (BSO+DEM hepatocytes) 4 - P A (normal hepatocytes) 0 2 0 0 4 0 0 6 0 0 8 0 0 VPA or 4-PA (^ig/ml) 1 0 0 0 Figure 5-8: The effect of V P A on cell viability as measured by WST-1 assay. Control and BSO + DEM-pretreated rat hepatocytes (4 x 10 4 cells/well in 96-well plates) were treated with VPA (0-1000 ug/ml_) for 8 h. Absorbance values are expressed as percentage of control (0 pg/ml VPA) and represent mean + SEM (n = 5 experiments). The positive control was 4-pentenoic acid (4-PA, n = 2 experiments). "Significantly different between the control vs. B S O + DEM pretreated hepatocyte (p<0.05). 149 Chapter 5: VPA and oxidative stress in rat hepatocytes 5.4 DISCUSSION Our previous study in rats showed increased levels of 15-F 2 t - lsoP after a single dose of V P A , but this did not involve P450-mediated VPA-biotransformation, as indicated in previous mechanistic experiments (Tong et al. 2003). In a subsequent study, daily doses of V P A for 14 days in rats resulted in an elevation in plasma and liver 15-F 2 t - lsoP that preceded the occurrence of necrosis and steatosis (Tong et al. 2005, manuscript submitted). In the same study, other biomarkers of oxidative stress, thiobarbituric acid reactive substances and lipid hydroperoxides, were measured and were found to be elevated after the initial occurrence of hepatotoxicity. Emanating from these findings, the current study is an in vitro approach to address: (1) if the rise in 15-F 2 t - lsoP by V P A is reflective of oxidative stress, (2) whether or not the oxidative stress precedes hepatotoxicity, and (3) the involvement of G S H in these effects by VPA. The overall objective of this study was to investigate the relationship between oxidative stress, mitochondrial membrane potential, and toxicity in freshly isolated rat hepatocytes treated with V P A . The experiments were designed to test the hypothesis that V P A treatment is associated with oxidative stress and mitochondrial dysfunction and that depletion of cellular G S H augments these responses by V P A . The first part of the study was to determine if oxidative stress could be detected following acute exposure to V P A in normal hepatocytes. The results indicated that cultured rat hepatocytes treated with V P A showed early increases in 15-F 2 t - lsoP at the 30 min incubation time point (Figure 5-3A), corresponding to approximately the plasma tm a x of 15-F 2 t -IsoP observed in a previous study following a single dose of V P A (500 mg/kg, ip) to rats (Tong ef al. 2003). In addition, intracellular oxidative stress was confirmed by monitoring the oxidation of DCFH to the fluorescent DCF as an independent indicator of oxidative stress. Significant elevation in D C F was first observed in hepatocyte cultures also at the 30 min time point 150 Chapter 5: VPA and oxidative stress in rat hepatocytes following V P A treatment (Figure 5-3B), coinciding with the rise in 15-F 2 t - lsoP. At this time, this study is the first to correlate chemically-induced oxidative stress using the biomarkers DCF and 15-F 2 t - lsoP in the same study. Another important finding was that the elevation in oxidative stress was not accompanied with hepatocyte toxicity during acute exposure to VPA . This in vitro observation is in agreement with previous in vivo findings that V P A elevated levels of 15-F 2 t - lsoP occurred prior to signs of liver necrosis and steatosis (Tong et al. 2005, manuscript submitted). Another objective of this study was to investigate the role of G S H in VPA-mediated oxidative stress in the hepatocyte model. The hypothesis to be tested is that cells with reduced levels of G S H have compromised antioxidant capabilities, and as a consequence, are more susceptible to VPA-induced oxidative stress. Rat hepatocytes pretreated with the combination of B S O and DEM had significantly reduced total G S H levels (to « 20% of control levels), and as a consequence, resulted in significantly elevated oxidative stress compared to control hepatocytes over the same range of V P A concentrations. This suggests that G S H does play a protective role as an antioxidant against VPA-mediated oxidative stress. V P A treatment in rats and patients has also been linked to a decrease in G S H levels and alterations in glutathione peroxidase, a key intracellular antioxidant enzyme (Cengiz et al. 2000; Cotariu ef al. 1990; Graf ef al. 1998; Raza ef al. 1997; Yuksel ef al. 2000). Individuals deficient in G S H , either due to inborn errors (Bruggemann ef al. 2004), malnutrition (Bray and Taylor 1993), or disease states such as those associated with hepatitis, hepatic cirrhosis, or HIV (White ef al. 1994) may be theoretically more susceptible to xenobiotic-induced oxidative stress. Based on the available evidence, V P A may mediate its toxicity by a mechanism that involves the production of R O S in combination with decreased antioxidant capabilities (decreased levels of GSH) that ultimately leads to oxidative stress. 151 Chapter 5: VPA and oxidative stress in rat hepatocytes The hypothesis of a reactive metabolite of V P A contributing to the observed A*Fm and cytotoxicity upon reduction of cellular G S H by B S O and D E M can not be ruled out. Of the many metabolites, only 4-ene-VPA and (£)-2,4-diene-VPA have been shown to cause hepatic steatosis in the rat (Kesterson ef al. 1984). Furthermore, indicators of reactive metabolite exposure, G S H and N-acetylcysteine conjugates of (E)-2,4-diene-VPA, have been identified in rats and patients (Gopaul ef al. 2000b; Kassahun ef al. 1991). The formation of (E)-2,4-diene-V P A and the subsequent depletion of G S H in the mitochondria provided a rationale for a reactive metabolite mechanism for mitochondrial dysfunction and microvesicular steatosis (Tang ef al. 1995). However, in a recent study that involved high daily doses of V P A for 14 days to rats (Tong ef al. 2005, manuscript submitted), there was no detectable increase in the measured V P A metabolites that accompanied the observed oxidative stress and hepatotoxicity. It may be argued that metabolite levels, in particular 4-ene-VPA and (E)-2,4-diene-VPA, may not be the delineating factor for VPA-mediated hepatotoxicity (Loscher ef al. 1993; Siemes ef al. 1993). More recent studies indicated that G S H or N-acetylcysteine conjugates of (£)-2,4-diene-V P A would be a better indicator of reactive metabolite exposure (Gopaul ef al. 2000a, b; Tang ef al. 1996). To establish a link between reactive metabolite exposure and oxidative stress, it would be useful in future studies to measure the levels of G S H conjugates of V P A metabolites in isolated hepatocytes treated with VPA. The pathogenesis of severe V P A hepatotoxicity is not clear, but the observation of microvesicular steatosis is consistent with a disturbance in mitochondrial function and/ or fatty acid metabolism (Fromenty and Pessayre 1995; Powell-Jackson ef al. 1984). The present study also investigates the effect of V P A on the ATm of rat hepatocytes as an indicator of mitochondrial dysfunction as determined by the fluorescent molecular probe, JC -1 . Our findings indicate that V P A alone does not decrease the in control hepatocytes; however, in hepatocytes with reduced G S H levels, the A T m was significantly decreased at the highest V P A 152 Chapter 5: VPA and oxidative stress in rat hepatocytes concentration tested (1000 ug/ml) (Figure 5-7). The decrease in A ^ was also associated with significant cytotoxicity in GSH-reduced hepatocytes. These findings suggest that the added stress of G S H removal during V P A treatment is associated with increased oxidative stress levels, mitochondrial dysfunction, and cytotoxicity. Recently, similar studies with acetaminophen (Masubuchi et al. 2005; Reid et al. 2005), salicylate (Trost and Lemasters 1997), and clofibrate (Qu ef al. 2001) established an association between elevated oxidative stress and the loss in AWm by induction of the mitochondrial membrane transition, both occurring before the onset of cell toxicity. Other factors known to decrease A ^ m include K + and C a 2 + ionophores, cyanide compounds known to un-couple oxidative phosphorylation such as F C C P , pH-dependent ischemia-reperfusion, and oxidative stress (Lemasters ef al. 1998). ferf-Butylhydroperoxide, a short chain lipid hydroperoxide analog, was demonstrated to generate R O S within the mitochondria that induced the mitochondrial membrane transition, and resulted in the loss in A T ™ and A T P depletion prior to hepatocyte toxicity (Imberti ef al. 1993; Nieminen ef al. 1997). In many models of oxidative stress, there is a general agreement that G S H must be depleted below a certain critical threshold, before the extent of cytotoxicity correlates with the magnitude of G S H depletion (Kaplowitz and Tsukamoto 1996). Our study is consistent with a mechanism of VPA-hepatotoxicity that involves oxidative stress through the combination of R O S production and G S H depletion, resulting in the subsequent loss in A * F m , and ultimately leading to cytotoxicity. Future studies would be required to establish a causal link between V P A -induced oxidative stress, mitochondrial depolarization, and cytotoxicity. One approach would be to block the mitochondrial membrane transition with cyclosporine A or trifluoperazine (Lemasters ef al. 1998) to see if VPA-induced cytotoxicity can be prevented in GSH-depleted hepatocytes. In summary, we demonstrated that V P A is capable of producing oxidative stress in freshly isolated rat hepatocytes as measured by elevated levels of 15-F 2 t - lsoP and DCF. However, the 153 Chapter 5: VPA and oxidative stress in rat hepatocytes oxidative stress did not result in mitochondrial dysfunction or hepatocyte toxicity. By comparison, in hepatocytes pretreated with BSO and DEM to reduce total G S H , the levels of the oxidative stress biomarkers were further elevated, and this was accompanied by mitochondrial dysfunction (as detected by a decrease in A T m ) and cytotoxicity in hepatocytes treated with high concentrations of V P A . These results are significant because they support the hypothesis that VPA-associated oxidative stress occurs prior to hepatotoxicity, and indicate that G S H serves a protective role to mitigate the deleterious effects of high concentrations on V P A on mitochondrial function and cell viability. 154 Chapter 5: VPA and oxidative stress in rat hepatocytes 5.5 REFERENCES Becker, C. M., and Harris, R. A. (1983). Influence of valproic acid on hepatic carbohydrate and lipid metabolism. Arch Biochem Biophys 223, 381-92. Bray, T. M., and Taylor, C. G. (1993). Tissue glutathione, nutrition, and oxidative stress. Can J Physiol Pharmacol 71, 746-51. Bruggemann, L. W., Groenendaal, F., Ristoff, E., Larsson, A., Duran, M., van Lier, J . A., Dorland, L , Berger, R., and de Koning, T. J . (2004). Glutathione synthetase deficiency associated with antenatal cerebral bleeding. J Inherit Metab Dis 27, 275-6. Buchi, K. N., Gray, P. D., Rollins, D. E., and Tolman, K. G. (1984). Protection against sodium valproate injury in isolated hepatocytes by alpha-tocopherol and N,N'-diphenyl-p-phenylenediamine. J Clin Pharmacol 24, 148-54. Cengiz, M., Yuksel, A., and Seven, M. (2000). The effects of carbamazepine and valproic acid on the erythrocyte glutathione, glutathione peroxidase, superoxide dismutase and serum lipid peroxidation in epileptic children. Pharmacol Res 41, 423-5. Cotariu, D., Evans, S., Zaidman, J . L , and Marcus, O. (1990). Early changes in hepatic redox homeostasis following treatment with a single dose of valproic acid. Biochem Pharmacol 40, 589-93. Coude, F. X. , Grimber, G. , Parvy, P., Rabier, D., and Petit, F. (1983). Inhibition of ureagenesis by valproate in rat hepatocytes. Role of N- acetylglutamate and acetyl-CoA. Biochem J 216, 233-6. Farrell, K., and Abbott, F. S. (1991). Role of N-acetylcysteine in the treatment of valproate hepatotoxicity. In Idiosyncratic Reactions to Valproate: Clinical Risk Patterns and Mechanisms of Toxicity. (R. H. Levy and J . K. Penry, eds.), pp. 149-153. Raven Press, New York. Fromenty, B., and Pessayre, D. (1995). Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity. Pharmacol Ther 67, 101-54. Gopaul, S. V., Farrell, K., and Abbott, F. S. (2000a). Gas chromatography/negative ion chemical ionization mass spectrometry and liquid chromatography/electrospray ionization tandem mass spectrometry quantitative profiling of N-acetylcysteine conjugates of valproic acid in urine: application in drug metabolism studies in humans. J Mass Spectrom 35, 698-704. 155 Chapter 5: VPA and oxidative stress in rat hepatocytes Gopaul, S. V., Farrell, K., and Abbott, F. S. (2000b). Identification and characterization of N-acetylcysteine conjugates of valproic acid in humans and animals. Drug Metab Dispos 28, 823-32. Graf, W. D., Oleinik, O. E., Glauser, T. A., Maertens, P., Eder, D. N., and Pippenger, C. E. (1998). Altered antioxidant enzyme activities in children with a serious adverse experience related to valproic acid therapy. Neuropediatrics 29, 195-201. Griffith, O. W. (1981). Depletion of glutathione by inhibition of biosynthesis. Methods Enzymol 77, 59-64. Imberti, R., Nieminen, A. L , Herman, B., and Lemasters, J . J . (1993). Mitochondrial and glycolytic dysfunction in lethal injury to hepatocytes by t-butylhydroperoxide: protection by fructose, cyclosporin A and trifluoperazine. J Pharmacol Exp Ther 265, 392-400. Jurima-Romet, M., Abbott, F. S., Tang, W., Huang, H. S. , and Whitehouse, L. W. (1996). Cytotoxicity of unsaturated metabolites of valproic acid and protection by vitamins C and E in glutathione-depleted rat hepatocytes. Toxicology 112, 69-85. Kaplowitz, N., and Tsukamoto, H. (1996). Oxidative stress and liver disease. Prog Liver Dis 14, 131-59. Kassahun, K., Farrell, K., and Abbott, F. S. (1991). Identification and characterization of the glutathione and N-acetylcysteine conjugates of (E)-2-propyl-2,4-pentadienoic acid, a toxic metabolite of valproic acid, in rats and humans. Drug Metab Dispos 19, 525-535. Kesterson, J . W., Granneman, G. R., and Machinist, J . M. (1984). The hepatotoxicity of valproic acid and its metabolites in rats. I. Toxicologic, biochemical and histopathologic studies. Hepatology A, 1143-52. Kingsley, E., Gray, P., Tolman, K. G , and Tweedale, R. (1983). The toxicity of metabolites of sodium valproate in cultured hepatocytes. J Clin Pharmacol 23, 178-85. Lemasters, J . J . , Nieminen, A. L., Qian, T., Trost, L. C , Elmore, S. P., Nishimura, Y., Crowe, R. A., Cascio, W. E., Bradham, C. A., Brenner, D. A., and Herman, B. (1998). The mitochondrial permeability transition in cell death: a common mechanism in necrosis, apoptosis and autophagy. Biochim Biophys Acta 1366, 177-96. Loscher, W., Rundfeldt, C , and Honack, D. (1993). Pharmacological characterization of phenytoin-resistant amygdala- kindled rats, a new model of drug-resistant partial epilepsy. Epilepsy Res 15, 207-19. Maellaro, E., Casini, A. F., Del Bello, B., and Comporti, M. (1990). Lipid peroxidation and antioxidant systems in the liver injury produced by glutathione depleting agents. Biochem Pharmacol 39, 1513-21. 156 Chapter 5: VPA and oxidative stress in rat hepatocytes Masubuchi, Y., Suda, C , and Horie, T. (2005). Involvement of mitochondrial permeability transition in acetaminophen-induced liver injury in mice. J Hepatol 42, 110-6. Meister, A. (1983). Selective modification of glutathione metabolism. Science 220, 472-7. Meredith, M. J . , and Reed, D. J . (1982). Status of the mitochondrial pool of glutathione in the isoalted hepatocyte. J Pediatr 257, 3747-3753. Miccadei, S., Kyle, M. E., Gilfor, D., and Farber, J . L. (1988). Toxic consequence of the abrupt depletion of glutathione in cultured rat hepatocytes. Arch Biochem Biophys 265, 311-20. Nieminen, A. L , Byrne, A. M., Herman, B., and Lemasters, J . J . (1997). Mitochondrial permeability transition in hepatocytes induced by t -BuOOH: NAD(P)H and reactive oxygen species. Am J Physiol 272, C1286-94. Olson, M. J . , Handler, J . A. , and Thurman, R. G. (1986). Mechanism of zone-specific hepatic steatosis caused by valproate: inhibition of ketogenesis in periportal regions of the liver lobule. Mol Pharmacol 30, 520-5. Plummer, J . L , Smith, B. R., Sies, H., and Bend, J . R. (1981). Chemical depletion of glutathione in vivo. Methods Enzymol 77, 50-9. Powell-Jackson, P. R., Tredger, J . M., and Williams, R. (1984). Hepatotoxicity to sodium valproate: a review. Gut 25, 673-81. Qu, B., Li, Q. T., Wong, K. P., Tan, T. M., and Halliwell, B. (2001). Mechanism of clofibrate hepatotoxicity: mitochondrial damage and oxidative stress in hepatocytes. Free Radic Biol Med 31, 659-69. Raza, M., Al-Bekairi, A. M., Ageel, A. M., and Qureshi, S. (1997). Biochemical basis of sodium valproate hepatotoxicity and renal tubular disorder: time dependence of peroxidative injury. Pharmacol Res 35, 153-7. Reed, D. J . (1990). Glutathione: toxicological implications. Annu Rev Pharmacol Toxicol 30, 603-31. Reers, M., Smiley, S. T., Mottola-Hartshorn, O , Chen, A., Lin, M., and Chen, L. B. (1995). Mitochondrial membrane potential monitored by JC-1 dye. Methods Enzymol 260, 406-17. Reers, M., Smith, T. W., and Chen, L. B. (1991). J-aggregate formation of a carbocyanine as a quantitative fluorescent indicator of membrane potential. Biochemistry 30, 4480-6. Reid, A. B., Kurten, R. O , McCullough, S. S., Brock, R. W., and Hinson, J . A. (2005). Mechanisms of acetaminophen-induced hepatotoxicity: role of oxidative stress and mitochondrial permeability transition in freshly isolated mouse hepatocytes. J Pharmacol Exp Ther 312, 509-16. 157 Chapter 5: VPA and oxidative stress in rat hepatocytes Siemes, H., Nau, H., Schultze, K., Wittfoht, W., Drews, E., Penzien, J . , and Seidel, U. (1993). Valproate (VPA) metabolites in various clinical conditions of probable VPA-associated hepatotoxicity. Epilepsia 3 4 , 332-46. Smiley, S. T., Reers, M., Mottola-Hartshorn, C , Lin, M., Chen, A., Smith, T. W., Steele, G. D., Jr., and Chen, L. B. (1991). Intracellular heterogeneity in mitochondrial membrane potentials revealed by a J-aggregate-forming lipophilic cation J C - 1 . Proc Natl Acad Sci U S A 88, 3671-5. Tang, W., Borel, A. G. , and Abbott, F. S. (1996). Conjugation of glutathione with a toxic metabolite of valproic acid, (E)-2-propyl-2,4-pentadienoic acid, catalyzed by rat hepatic glutathione-S-transferases. Drug Metab Dispos 2 4 , 436-46. Tang, W., Borel, A. G , Fujimiya, T., and Abbott, F. S. (1995). Fluorinated analogues as mechanistic probes in valproic acid hepatotoxicity: hepatic microvesicular steatosis and glutathione status. Chem Res Toxicol 8, 671-82. Tong, V., Chang, T. K., Chen, J . , and Abbott, F. S. (2003). The effect of valproic acid on hepatic and plasma levels of 15-F(2t)-isoprostane in rats. Free Radio Biol Med 3 4 , 1435-46. Trost, L. C , and Lemasters, J . J . (1997). Role of the mitochondrial permeability transition in salicylate toxicity to cultured rat hepatocytes: implications for the pathogenesis of Reye's syndrome. Toxicol Appl Pharmacol 1 4 7 , 431-41. Turnbull, D. M., Bone, A. J . , Bartlett, K., Koundakjian, P. P., and Sherratt, H. S. (1983). The effects of valproate on intermediary metabolism in isolated rat hepatocytes and intact rats. Biochem Pharmacol 3 2 , 1887-92. White, A. C , Thannickal, V. J . , and Fanburg, B. L. (1994). Glutathione deficiency in human disease. J Nutr Biochem 5, 218-226. Yuksel, A., Cengiz, M., Seven, M., and Ulutin, T. (2000). Erythrocyte glutathione, glutathione peroxidase, superoxide dismutase and serum lipid peroxidation in epileptic children with valproate and carbamazepine monotherapy. J Basic Clin Physiol Pharmacol 1 1 , 73-81. 158 Chapter 6: Summary, future studies, conclusion 6 Global Summary, Future Studies, Conclusions 6.1 SUMMARY OF FINDINGS AND FUTURE STUDIES In vivo and in vitro studies were conducted with VPA , a broad-spectrum anticonvulsant, in order to investigate whether oxidative stress is associated with VPA-hepatotoxicity. As there is limited and conflicting evidence for the involvement of oxidative stress during V P A treatment, investigational toxicity studies with emphasis on probing the metabolic pathways of V P A while measuring oxidative stress biomarkers will provide a better understanding of the role of V P A biotransformation (i.e. cytochrome P450, UDPGT) and its association with oxidative stress. Our initial investigation measured levels of 15-F 2 t - lsoP as a biomarker for lipid peroxidation after a single high dose of V P A (500 mg/kg, ip) to rats (Tong ef al. 2003). Dose-dependent elevations in plasma and liver 15-F 2 t - lsoP levels were observed with a plasma-concentration time profile similar to that of V P A (tm a x of approximately 30 min). Other independent measures of oxidative stress, thiobarbituric reactive acid substances and lipid hydroperoxides, were not elevated and raised the question if the increased levels of 15-F 2 t -IsoP were truly reflective of oxidative stress. The formation of 1 5 - F 2 r l s o P is assumed to involve a free-radical, non-cyclooxygenase-dependent mechanism; however, literature evidence exists that cyclooxygenase (COX-1 and COX-2) isoforms may contribute to its production (Pratico ef al. 1995). Future studies in our laboratory would be to examine the effects of indomethacin, a non-selective'COX inhibitor, on the VPA-induced 15-F 2 t - lsoP levels observed in rats. However, in our favour, V P A was demonstrated to down-regulate both COX-1 and COX-2 protein levels 159 Chapter 6: Summary, future studies, conclusion and total C O X activity (Szupera et al. 2000), making it unlikely that the increase in 15-F 2,- lsoP by V P A was COX-dependent. In support of our in vivo findings, V P A elevated levels of 15-F 2 t - lsoP and DCF in rat hepatocyte cultures. With the available evidence, the overall interpretation at the present time is that the increases in 15-F 2 t- lsoP do reflect oxidative stress. Further mechanistic experiments examined the effects of chemical inducers (PB) and inhibitors (ABT and SKF-525A) of cytochrome-P450 on 15-F 2 t - lsoP levels (Tong et al. 2003). Our results demonstrated a lack of involvement of cytochrome-P450 biotransformation on the elevated 15-F 2 t - lsoP levels. However, an interesting finding was that rats treated with both P B and V P A showed levels of 15-F 2 t - lsoP greater than those seen with rats treated with V P A alone, whereas PB itself had no effect on 15-F 2 t - lsoP levels. The results with the P B - and VPA-treated animals prompted further studies examining the role of VPA-glucuronidation, the major V P A biotransformation pathway that is also inducible by PB , on oxidative stress (Tong ef al. 2004, manuscript submitted). Mechanistic experiments used chemical inducers (PB) and inhibitors ((-)-borneol and salicylamide) to modulate levels of liver V P A - G . An intriguing finding was the association between the levels of V P A - G and 15-F 2 t - lsoP in rats treated with a single dose of VPA. This is the first study to associate V P A -glucuronidation, which is normally considered a detoxification pathway, with oxidative stress. This novel finding prompts further studies to address the mechanism of how V P A -glucuronidation can give rise to oxidative stress. One possible mechanism may be a consequence of the extensive hydrolysis of V P A - G in the liver compartment at the site of formation and in the intestinal compartment as a result extensive enterohepatic recirculation. As a result, the systemic cycling of V P A to V P A - G and back to V P A may lead not only to 160 Chapter 6: Summary, future studies, conclusion potentially reactive p-glucuronidase-resistant isomers of V P A - G , but to theoretically high concentrations of free glucuronic acid due to the large doses of V P A employed. Glucuronic acid has recently been associated with oxidative stress as measured by DCF formation (Kim et al. 2004). Future studies are required to investigate the possibility that the enterohepatic recirculation of V P A - G and the glucuronic acid generated in the process are involved in the formation of 15-F 2 t -IsoP, and several approaches can be taken to investigate this hypothesis. The enterohepatic recirculation of V P A - G would be abolished in bile duct-cannulated rats dosed with VPA , and because the bile is exteriorized, lower levels of 15-F 2 t - lsoP would be expected. The multi-drug resistance protein-2 (MRP-2) has been identified to transport V P A - G across the canalicular membrane into bile; therefore, MRP-2 knockout rats dosed with V P A would be expected to have decreased levels of V P A - G excreted in the bile, decreased enterohepatic recirculation, and less V P A - G hydrolysed, that would ultimately lead to lower levels of free glucuronic acid, and theoretically lower levels of 15-F 2 t - lsoP. Another approach would be to inhibit p-glucuronidase activity, the enzyme predominantly responsible for the hydrolysis of V P A - G , and this should result in elevated systemic V P A - G levels and sequestering of glucuronic acid. Direct measurement of plasma and liver levels of free glucuronic acid would be required for the correlation with oxidative stress. The question remains whether the enzymatic formation of V P A - G via UDP-glucuronosyl transferases or the product itself (VPA-G) is responsible for the rise in 15-F 2 t - lsoP. Another question, one perhaps of clinical importance, emanating from these findings is whether or not other compounds (i.e. diclofenac, acetaminophen, lamotrigine) that are subject to glucuronidation can also generate oxidative stress as measured by 15-F 2 t - lsoP. In support of the hypothesis that VPA-glucuronidation is associated with oxidative stress, a comparative study with V P A and its a-fluorinated analog, a-f luoro-VPA, was investigated in rats 161 Chapter 6: Summary, future studies, conclusion with regard to their inherent ability to generate 15-F 2 t - lsoP (Tong ef al. 2004, manuscript submitted). a-Fluoro-VPA, a poor substrate for glucuronidation, did not elevate levels of 15-F 2 t -IsoP. However, the possibility that VPA-induced oxidative stress may have involved mitochondrial fatty acid (3-oxidation, the second largest metabolic pathway for VPA , cannot be ruled out. a-Fluoro-VPA is also inert to p-oxidation, and based on the lack of induction on 15-F 2 t - lsoP levels, the results also support the hypothesis that the source of oxidative stress may originate from the mitochondria and involve fatty acid oxidation of V P A . Octanoic acid, an 8-carbon linear chain analog of V P A that undergoes extensive p-oxidation, was examined to see whether another fatty acid of similar structure is capable of elevating 15-F 2 t - lsoP levels. Our findings showed that octanoic acid did not elevate 15-F 2 t - lsoP, which suggests that the increase in 15-F 2 t - lsoP was specific to VPA . As a future study to further investigate the role of p-oxidation on VPA-associated oxidative stress, the use of trimethylacetic acid (TMA) will be used as a chemical inhibitor of p-oxidation by depleting acetyl coenzyme-A, a cofactor required for fatty acid transport. In a preliminary study in rats pre-treated with a single dose of TMA (500 mg/kg) prior to V P A dosing, the results indicated that the 1 5 - F 2 r l s o P levels remained elevated compared to VPA-treated rats (Tong ef al., unpublished data). However, TMA pre-treatment was only capable of reducing p-oxidation metabolites of V P A by « 30-40%, and future studies are required to optimize the TMA dose to maximally inhibit p-oxidation and to measure the effects on the associated oxidative stress. More detailed in vivo toxicity studies investigated whether the VPA-induced oxidative stress could be associated with hepatotoxicity in rats (Tong ef al. 2005a, manuscript submitted). Our findings indicated that high daily doses of V P A given for 14 days elevated 15-F 2 t - lsoP levels prior to hepatotoxicity. Liver histology revealed that inflammation of the liver capsule was the 162 Chapter 6: Summary, future studies, conclusion most common feature, and this was accompanied by increasing severity and frequency of necrosis by day 14. Hepatic steatosis increased in frequency from days 4-10 and was never considered severe. Levels of T B A R S and LPO, which were not affected after a single dose of VPA , were found to be elevated after multiple doses of V P A and after the initial onset of hepatotoxicity. The question remains whether there is a causal relationship between oxidative stress and hepatotoxicity, and this may be addressed with studies that involve supplementing antioxidants (i.e. vitamin C, vitamin E) and/or N-acetylcysteine to rats given V P A for the same 2-week period. The levels of V P A metabolites, in particular the putative reactive metabolites 4-ene-VPA and (£)-2,4-diene-VPA, were not elevated throughout the study and may also be inadequate indicators of reactive metabolite exposure. G S H and N-acetylcysteine conjugates of V P A metabolites may be more informative indicators of reactive metabolite exposure (Gopaul et al. 2000a, b; Tang ef al. 1996) and these conjugates should be determined in the 14-day toxicity study in rats. The p-oxidation metabolites of V P A in the liver decreased in the late stages of the toxicity study (between days 10 to 14) which suggests that inhibition of p-oxidation in the liver occurred after the onset of hepatotoxicity. To strengthen the evidence that oxidative stress occurs prior to VPA-hepatotoxicity, a second indicator of oxidative stress should be monitored in VPA-treated rats. The ratio of oxidized to reduced glutathione ( G S S G / G S H ) levels would be another independent indicator to monitor changes in oxidant status during V P A treatment. Our laboratory is currently developing an assay for the direct and simultaneous determination of G S H and G S S G by L C / M S / M S . In vitro studies using primary cultured rat hepatocytes provide more evidence that oxidative stress occurs prior to VPA-hepatotoxicity (Tong ef al. 2005b, manuscript submitted). Levels of 15-F 2 t - lsoP and D C F were both elevated following V P A treatment at a time point similar to the tm a x seen in vivo, without evidence of hepatocyte toxicity. G S H plays a protective role in V P A toxicity since GSH-depleted hepatocytes exhibited greater levels of oxidative stress at high 163 Chapter 6: Summary, future studies, conclusion levels of V P A and this was accompanied by the loss of mitochondrial membrane potential and cytotoxicity. This in vitro model provides a platform that allows a similar investigation for structure-activity relationships using a variety of V P A analogs (i.e. a -F-VPA) with the ultimate goal of developing a new antiepileptic compound similar in efficacy to VPA , but devoid of undesirable properties such as oxidative stress, mitochondrial dysfunction, and cytotoxicity. 6.2 CONCLUSION This data presented in this thesis provide strong evidence that V P A is associated with oxidative stress that precedes hepatotoxicity. The finding that V P A exacerbates oxidative stress, mitochondrial dysfunction, and toxicity in GSH-reduced hepatocytes indicates that the mechanism of hepatotoxicity may be multi-factorial, such that the combination of high doses of VPA, and oxidative stress in the form of insufficient levels of antioxidants (e.g. GSH) and elevation in lipid peroxidation may make an individual more susceptible to hepatotoxicity. This thesis also makes a significant overall contribution to the field of V P A research, as it is the first in vivo study to examine VPA-associated oxidative stress and hepatotoxicity within the same study period. In addition, this study describes a novel quantitative assay for V P A - G by LC/MS and is the first report implicating V P A - G to be associated with oxidative stress. 164 Chapter 6: Summary, future studies, conclusion 6.3 REFERENCES Gopaul, S. V., Farrell, K., and Abbott, F. S . (2000a). Gas chromatography/negative ion chemical ionization mass spectrometry and liquid chromatography/electrospray ionization tandem mass spectrometry quantitative profiling of N-acetylcysteine conjugates of valproic acid in urine: application in drug metabolism studies in humans. J Mass Spectrom 35, 698-704. Gopaul, S. V., Farrell, K., and Abbott, F. S. (2000b). Identification and characterization of N-acetylcysteine conjugates of valproic acid in humans and animals. Drug Metab Dispos 28, 823-32. Kim, Y. M., Kim, H. J . , Song, E . J . , and Lee, K. J . (2004). Glucuronic acid is a novel inducer of heat shock response. Mol Cell Biochem 259, 23-33. Pratico, D., Lawson, J . A., and FitzGerald, G. A. (1995). Cyclooxygenase-dependent formation of the isoprostane, 8-epi prostaglandin F2 alpha. J Biol Chem 270, 9800-8. Szupera, Z., Mezei, Z., Kis, B., Geese, A., Vecsei, L., and Telegdy, G. (2000). The effects of valproate on the arachidonic acid metabolism of rat brain microvessels and of platelets. Eur J Pharmacol 387, 205-10. Tang, W., Borel, A. G. , and Abbott, F. S. (1996). Conjugation of glutathione with a toxic metabolite of valproic acid, (E)-2-propyl-2,4-pentadienoic acid, catalyzed by rat hepatic glutathione-S-transferases. Drug Metab Dispos 24, 436-46. Tong, V., Chang, T. K., Chen, J . , and Abbott, F. S. (2003). The effect of valproic acid on hepatic and plasma levels of 15-F(2t)-isoprostane in rats. Free Radic Biol Med 34, 1435-46. Tong, V., Teng, X. , Chang, T.K., and Abbott, F.S. (2004). Valproic acid glucuronidation is associated with increases in 15-F 2 t-isoprostane in rats. Free Radic. Biol. Med., manuscript in press. Tong, V., Teng, X., Chang, T.K., and Abbott, F.S. (2005a). Valproic acid I: time-course of lipid peroxidation biomarkers, liver toxicity, and valproic acid metabolite levels in rats. Toxicol Sci, manuscript submitted. Tong, V., Teng, X. , Chang, T.K., and Abbott, F.S. (2005b). Valproic acid II: effects on oxidative stress, mitochondrial membrane potential, and cytotoxicity in glutathione-depleted rat hepatocytes. Toxicol Sci, manuscript submitted. 165 

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