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Effect of Ginkgo biloba on acetaminophen-induced hepatotoxicity in male Long-Evans rats Cheung, Catherine Yuen Shan 2006

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EFFECT OF GINKGO BILOBA O N A C E T A M I N O P H E N - I N D U C E D IN M A L E L O N G - E V A N S RATS  HEPATOTOXICITY  by CATHERINE Y U E N S H A N C H E U N G B.Sc.(Hons.), The University of British Columbia, 1999 B.Sc.(Pharm.), The University of British Columbia, 2003  A THESIS SUBMITTED IN P A R T I A L F U L F I L L M E N T OF THE REQUIREMENTS FOR THE D E G R E E OF M A S T E R OF SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES  (Pharmaceutical Sciences)  THE UNIVERSITY OF BRITISH C O L U M B I A April 2006  © Catherine Yuen Shan Cheung, 2006  ABSTRACT Acetaminophen (APAP) is commonly used as an analgesic andan antipyretic. This drug is considered safe, but it may cause severe hepatotoxicity and even fatality in certain situations. Ginkgo biloba extract (GBE), which is a popular herbal medicine used mainly to improve memory, induces rat hepatic cytochrome P450 (CYP) enzymes, including CYP1A2, CYP2E1, and CYP3A. A P A P is bioactivated by these enzymes to the hepatotoxic metabolite, A'-acetyl-p-benzoquinoneimine. Hence, G B E may modulate APAP-induced hepatotoxicity. The current study investigated the influence of G B E (containing 6.2% w/w terpene trilactones and 21% w/w flavonol glycosides) on APAP-induced hepatotoxicity in male Long-Evans rats, as assessed by liver histological analysis and plasma alanine aminotransferase (ALT) levels. The validated plasma A L T assay was accurate, precise, and reproducible, although it had poor sensitivity and a narrow dynamic range. Control experiments indicated that 20% Tween 80 in 0.9% NaCl (the vehicle used to suspend APAP) did not increase plasma A L T levels, fasting was required to elicit APAP-induced hepatotoxicity, and maximal plasma A L T levels were obtained with A P A P at a dosage of 1000 mg/kg. G B E (500 mg/kg ip once daily for 8 days) did not increase plasma A L T levels, when compared to the 0.9% NaCl-treated control group, although histological analysis of hepatic tissues from GBE-treated rats showed that four of the five samples exhibited steatosis, necrosis, capsular inflammation, or sinusoidal dilatation. By comparison, G B E pretreatment (500 mg/kg ip once daily for 9 days) prevented the increase in plasma A L T levels in rats administered A P A P (1000 mg/kg ip). The plasma A L T levels were 41 ± 3 U / L (mean ± SEM), 114 ± 22 U / L , and 53 ± 7 U / L in the vehicle-treated control group, A P A P treated group, and G B E and APAP-treated group, respectively. In contrast, G B E was not  t  ii  able to attenuate the occurrence of steatosis, necrosis, capsular inflammation, or sinusoidal dilatation. These effects were accompanied by an increase (5-fold) in hepatic CYP3A23 mRNA expression, but no change in UDP-glucuronosyltransferase 1A6 mRNA expression. In summary, based on histological assessment, G B E exacerbated APAP-induced hepatotoxicity in male Long-Evans rats, and this might be related to increased bioactivation rather than reduced glucuronidation of A P A P by GBE.  iii  TABLE OF CONTENTS Abstract Table of Contents List of Tables List of Figures List of Abbreviations Acknowledgements  ii iv vii viii ix x  1. INTRODUCTION '. 1 1.1 Herbal Medicine 1 1.1.1 Definitions 1 1.1.2 Prevalence of Usage 1 1.1.3 Reasons for Use 2 1.2 Ginkgo biloba 2 1.2.1 Clinical Uses .2 1.2.2 Chemical Constituents 3 1.2.3 Pharmacokinetics of Some Terpene Trilactones and Flavonols 3 1.2.3.1 Absorption and Bioavailability 4 1.2.3.2 Distribution :...........5 1.2.3.3 Metabolism 5 1.2.3:4 Elimination : 8 1.2.3.5 Effect of Dosage Form on Pharmacokinetic Parameters 9 1.2.4 Adverse Effects .....9 1.2.5 Drug Interactions —..9 1.2.6 Inhibition of Drug-Metabolizing Enzymes 13 1.2.7 Induction of Drug-Metabolizing Enzymes 14 1.3 Acetaminophen : 15 .1.3.1 Clinical Uses .: : 15 1.3.2 Mechanism of Therapeutic Action 16 1.3.3 Pharmacokinetics 16 1.3.3.1 Absorption and Bioavailability 16 1.3.3.2 Distribution » 17 1.3.3.3 Metabolism . 17 1.3.3.4 Elimination ....19 .1.3.4 Adverse Effects ..; 19 1.3.5 Hepatotoxicity ..' 20 1.3.5.1 Mechanism of Hepatotoxic Action 20 1.3.5.2 Effect of Fasting 22 1.3.5.3 Strain Differences 23 1.3.5.4 Role of Nuclear Receptors in APAP-induced Hepatotoxicity .24 1.3.5.5 Other Modulators of APAP-induced Hepatotoxicity 26 1.4 Rationale 29 1.5 Research Hypothesis : • 30 1.6 Experimental Hypothesis 30 1.7 Specific Aims '. ......30 ;  iv  2. M A T E R I A L S A N D M E T H O D S 2.1 Chemicals and Reagents 2.2 Animals 2.3 Treatment 2.3.1 Positive Control Experiment 2.3.2 Effect of Fasting and A P A P 2.3.3 Effect of 20% Tween 80 in 0.9% NaCl (the vehicle for APAP) 2.3.4 Effect of the Dose of A P A P 2.3.5 Effect of G B E 2.3.6 Effect of the Combination of G B E and A P A P , 2.4 Termination of Animals, Blood Collection, and Preparation of Plasma 2.5 Alanine Aminotransferase (ALT) Assay 2.5.1 Principle of the Assay 2.5.2 Methodology 2.6 Validation of the A L T Assay 2.6.1 Lower Limit of Linearity 2.6.2 Limit of Quantitation 2.6.3 Upper Limit of Linearity 2.6.4 Dynamic Range 2.6.5 Accuracy and Precision 2.6.6 Intra-day and Inter-day Variabilities 2.6.7 Inter-laboratory Comparison of A L T Values 2.7 Histological Assessment of Liver Tissues 2.8 Data Analysis 3. RESULTS 3.1 Validation of the A L T Assay 3.1.1 Lower Limit of Linearity 3.1.2 Limit of Quantitation 3.1.3 Upper Limit of Linearity 3.1.4 A Representation Standard Curve 3.1.5 Accuracy and Precision 3.1.6 Intra-day and Inter-day Variabilities 3.1.7 Positive Control Experiment 3.1.8 Inter-laboratory Comparison of A L T Values 3.2 Control Experiments with A P A P 3.2.1 Effect of Fasting on Plasma A L T Levels in Rats Administered A P A P 3.2.2 Effect of the Vehicle on Plasma A L T Levels in Rats 3.2.3 Effect of the Dose of A P A P on Plasma A L T Levels in Rats 3.3 Experiments with G B E 3.3.1 Effect of G B E on Plasma A L T Levels in Rats 3.3.2 Effect of G B E on Liver Histology in Rats 3.4 Experiments with G B E and A P A P : 3.4.1 Effect of Pretreatment with G B E on Plasma A L T Levels in Rats Administered A P A P  v  32 32 33 33 33 34 34 34 35 35 36 36 36 38 39 39 39 39 40 40 40 41 41 41 43 43 43 44 46 47 48 49 52 54 ....57 57 59 61 63 63 65 66 66  3.4.2  Effect of Pretreatment with G B E on Liver Histology in Rats Administered A P A P  4. DISCUSSION 4.1 Validation of the A L T Assay 4.1.1 Accuracy 4.1.2 Precision 4.1.3 Reproducibility 4.1.4 Dynamic Range 4.1.5 Sensitivity 4.1.6 Summary 4.2 Control Experiments with A P A P 4.2.1 Effect of Fasting on Plasma A L T Levels in Rats Administered A P A P 4.2.2 Effect of the Vehicle on Plasma A L T Levels in Rats 4.2.3 Effect of the Dose of A P A P on Plasma A L T Levels in Rats 4.3 Effect of G B E on Liver Toxicity in Rats .: 4.3.1 Effect of G B E on Plasma A L T Levels in Rats 4.3.2 Effect of G B E on Liver Histology in Rats 4.4 Effect of G B E Pretreatment on Liver Toxicity in Rats treated with A P A P 4.4.1 Effect of G B E on Plasma A L T Levels in Rats treated with A P A P 4.4.2 Effect of other chemicals in G B E on Plasma A L T Levels in Mice treated with A P A P 4.4.3 Effect of G B E on Liver Histology in Rats treated with A P A P 4.4.4 Conflicting Results between A L T data and Histology data 4.4.5 Potential Mechanisms by which G B E Exacerbates APAP-induced Hepatotoxicity 4.4.5.1 Increased Bioactivation of A P A P by G B E 4.4.5.2 Decreased Detoxification of A P A P by G B E 4.4.5.3 Combination of the Effects 4.5 Comparison of Current Study to Published Studies 4.6 Limitations of the Study 4.7 Future Studies  68  74 74 74 ....75 75 77 77 77 78 78 81 82 84 84 85 85 85 85 86 87 88 88 89 90 91 92 92  5. S U M M A R Y A N D CONCLUSIONS  95  6. R E F E R E N C E S  96  7. APPENDICES 7.1 Appendix 1: The amount of ginkgolides, bilobalide, and flavonols in the G B E used in the study 7.2 Appendix 2: Effect of pretreatment with G B E on CYP3A23 mRNA expression in rats administered A P A P 7.3 Appendix 3: Effect of pretreatment with G B E on UGT1A6 mRNA expression in rats administered A P A P  vi  .107 107 109 ,...110  LIST OF T A B L E S Table 3.1. Accuracy and precision of the A L T assay  48  Table 3.2. Intra-day variability of the A L T assay  50  Table 3.3. Inter-day variability of the A L T assay  51  Table 3.4. Inter-laboratory comparison of A L T values  55  Table 3.5. Comparison of A L T levels obtained from the A L T assay performed  56  in our lab and those obtained from the Central Lab for Veterinarians Table 3.6. Effect of the vehicle on plasma A L T levels in rats  60  Table 3.7. Effect of G B E on liver histology scores in rats  65  Table 3.8. Effect of G B E on liver histology scores in rats administered A P A P Table A l . The amount of ginkgolides, bilobalide, and flavonols in the G B E used in the study  vii  ,  69 107  LIST O F FIGURES  Figure 1.1. A simplified scheme of the bioactivation and detoxification pathways of A P A P  ...19  Figure 1.2. Mechanisms of APAP-induced hepatotoxicity  22  Figure 1.3. Dose-response curve for hepatic necrosis in Long-Evans and SpragueDawley rats administered A P A P  24  Figure 2.1. Principle of the A L T assay (the first step in the reaction)  37  Figure 2.2. The second step in the A L T assay  37  Figure 3.1. Determination of the lower limit of linearity for the A L T assay  43  Figure 3.2. Determination of the limit of quantitation of the A L T assay  45  Figure 3.3. Determination of the upper limit of linearity for the A L T assay  46  Figure 3.4. A representative standard curve for the A L T assay  47  Figure 3.5. Effect of C C U on plasma A L T levels in rats  53  Figure 3.6. Effect of fasting on plasma A L T levels in rats treated with A P A P or the  58  vehicle Figure 3.7. Effect of the dose of A P A P on plasma A L T levels in rats  62  Figure 3.8. Effect of G B E on plasma A L T levels in rats  64  Figure 3.9. Effect of pretreatment with G B E on plasma A L T levels in rats  67  administered A P A P Figure 3.10. Summary of scores for steatosis in rat liver tissues  70  Figure 3.11. Summary of scores for necrosis in rat liver tissues  71  Figure 3.12. Summary of scores for capsular inflammation in rat liver tissues  72  Figure 3.13. Summary of scores for sinusoidal dilatation in rat liver tissues  73  Figure A l . Effect of pretreatment with G B E on CYP3A23 mRNA expression in rats administered A P A P Figure A2. Effect of pretreatment with G B E on UGT1A6 mRNA expression in rats administered A P A P viii  109 110  LIST O F ABBREVIATIONS ALT ANOVA APAP AST ATP AUC CAM CAR cDNA CV CYP dsDNA GBE ip LOQ mRNA NaCl NAPQI NSAIDS PCR PXR RNA SD SEM SULT UGT  alanine aminotransferase analysis of variance acetaminophen aspartate aminotransferase adenosine triphosphate area under curve complementary and alternative medicine constitutive androstane receptor complementary deoxyribonucleic acid coefficient of variation cytochrome P450 double-stranded deoxyribonucleic acid Ginkgo biloba extract intraperitoneal limit of quantitation messenger ribonucleic acid sodium chloride A-acetyl-/?-benzoquinoneimine non-steroidal anti-inflammatory drugs polymerase chain reaction pregnane X receptor ribonucleic acid standard deviation standard error of the mean sulfotransferase uridine diphosphoglucuronosyltransferase or UDP-glucuronosyltransferase  IX  ACKNOWLEDGEMENTS . I would like to sincerely thank my supervisor, Dr. Thomas Chang, for his continued patience and guidance throughout my undergraduate and graduate research experience. Thank you for not losing faith in me. Also, thanks to all my committee members, Dr. Stelvio Bandiera, Dr. Wayne Riggs, and Dr. Zhaoming Xu. Their support and helpful advice are greatly appreciated. Special thanks go to Dr. Ron Reid for chairing all my committee meetings, and Dr. Frank Abbott for being the external examiner. In terms of financial support, I want to thank Merck Frosst Canada Inc. for the James E. Frosst Postgraduate Pharmacy Fellowship, the Faculty of Pharmaceutical Sciences at the University of British Columbia for the Kam L i M a Scholarship in Pharmaceutical Sciences, Merck Research Laboratories (U.S.A.) for a partial M.Sc. traineeship, and the University of British Columbia for teaching assistantships. I would also like to acknowledge Canadian Institutes of Health Research (CIHR) for funding this research project. The Ginkgo biloba extract was generously provided by Indena S.A. (Tours, France). This study would not be made possible without the generous assistance from Dr. Ganesh Rajaraman, the postdoctoral fellow in our lab. Without his generous help in the animal experiments and continuous support, this project would not have run as smoothly. Thanks to our knowledgeable and hard-working technician, Jessie Chen, for her continued support and help in gathering all the mRNA expressions data.  1. I N T R O D U C T I O N 1.1  Herbal Medicine  1.1.1  Definitions Complementary and alternative medicine (CAM) is defined by the National Center  for Complementary and Alternative Medicine as "a group of diverse medical and health care systems, practices, and products that are not presently considered to be part of conventional medicine" (Barnes et al, 2004). "Complementary medicine is used together with conventional medicine", whereas "alternative medicine is used in place of conventional medicine" (Barnes et al, 2004). Herbal products belong to the "biologically based therapies" category, which is one of the five categories in C A M (Barnes et al, 2004). The World Health Organization has defined "traditional medicine (including herbal drugs) as comprising therapeutic practices that have been in existence, often for hundreds of years; before the development and spread of modern medicine and are still in use today. Traditional preparations comprise medicinal plants, minerals and organic matter etc. Herbal drugs constitute only those traditional medicines which primarily use medicinal plant preparations for therapy" (Pal and Shukla, 2003).  1.1.2  Prevalence of Usage Various factors such as place of residence, ethnicity, educational level, gender, age,  health and socio-economical status can affect the prevalence of usage of C A M among the public (Barnes et al, 2004). Kamboj (2000) estimated that about 75 to 80% of the people in the developing countries use herbal medicine as their primary health remedies. Numerous surveys were performed at a national level in the United States to more closely portray the use of C A M by the public in the United States, but the results of the surveys have been  1  inconsistent (Barnes et al, 2004). However, a more complete and comprehensive survey conducted by the National Center for Health Statistics in 2002 indicated that about 36% of respondents (N = 31044) used some form of C A M in the past 12 months (Barnes et al, 2004). Among these surveyed users of C A M throughout the United States, about 19% of them have used natural products in the past 12 months (Barnes et al, 2004). About 21% of these natural product users have used Ginkgo biloba in 2002, and this made Ginkgo biloba the third most popular natural product in the United States in that year according to this survey (Barnes et al, 2004).  1.1.3  Reasons for Use Different factors can affect why an individual would use herbal medicine (Ernst and  Pittler, 2002). Some potential reasons for trying herbal medicine include perceived safety, self-control over treatment, easier accessibility, affluence, rejection of established science and technology, desperation and dissatisfaction with conventional medicine due to ineffective conventional therapies, or experience of adverse effects from conventional drug treatments (Ernst and Pittler, 2002). Advertising may also be one of the reasons. It was also suggested that the general public tend to use herbal medicine for treating diseases and conditions that are relatively not severe and can heal by themselves (Barnes et al, 2003).  1.2  Ginkgo biloba  1.2.1  Clinical Uses Patients use Ginkgo biloba for many different reasons. Its purported uses include  anxiety, asthma, bronchitis, cardiovascular diseases, circulatory disorders, hearing loss, memory loss, Raynaud's disease, sexual dysfunction, stress, and tinnitus (SK Cancer Center,  2  2004). Other additional uses include age-related macular degeneration, vertigo, and altitude sickness (Ang-Lee et al, 2001). Ginkgo biloba is mainly used in the United States to help patients "enhance mental focus" in diseases such as dementia and Alzheimer's disease (Kudolo, 2001). In Europe, Ginkgo biloba is widely sold to treat "early-stage Alzheimer's disease, vascular dementia, peripheral claudication, and tinnitus of vascular origin" (Sierpina era/., 2003).  1.2.2  Chemical Constituents There are standardized Ginkgo biloba products available on the market. These  products are extracts from the leaves of Ginkgo biloba trees, and are standardized to the contents of flavonol glycosides (24.0% w/w on average, range of 22-27% w/w) and terpene trilactones (6.0% w/w on average, range of 5-7% w/w) (van Beek, 2002; Kressmann et al, 2002). Most of the flavonol glycosides in these standardized products are glycosides of quercetin, kaempferol, and isorhamnetin; and the terpene trilactones include ginkgolides A , B, C, and J and bilobalide (van Beek, 2002; Kressmann et al, 2002; Drago et al, 2002). Other chemical compounds such as proanthocyanidins, simple phenolic acids, glucose, rhamnose, and a minute amount of alkylphenols (ginkgolic acid) are also present in these Ginkgo biloba products (Ahlemeyer and Krieglstein, 2003). One of the commonly used standardized products of Ginkgo biloba is called EGb 761.  1.2.3  Pharmacokinetics of Some Terpene Trilactones and Flavonols Dietary quercetin and other flavonoids are absorbed in the intestine and undergo  intestinal metabolism by enterocytes before reaching the liver, entering the circulation, and ultimately excreted in the urine (Ross and Kasum, 2002; Murota and Terao, 2003). Most of  3  the terpene trilactones (ginkgolides A and B and bilobalide) are absorbed and excreted unchanged in the urine (Kleijnen and Knipschild, 1992).  1.2.3.1 Absorption and Bioavailability In a study of 12 volunteers (6 males, 6 females), each of them received an intravenous infusion of 100 mg of EGb 761 for 15 min, followed by an oral ingestion of 120 mg of EGb 761 solution. The bioavailability of ginkgolide A , ginkgolide B, and bilobalide were 80%, 88%o, and 79% respectively, whereas ginkgolide C was reported to be not bioavailable (Biber, 2003). A single oral dose of 120 mg of EGb 761 in capsule formulation was ingested by each of the 12 healthy male volunteers in a study by Kressmann et al. (2002). A U C values were reported to be 121.35, 59.88, and 217.24 ng h/mL for ginkgolide A , ginkgolide B, and bilobalide, respectively. C  m a x  values were reported to be 22.22, 8.27, and 54.42 ng/mL for  ginkgolide A , ginkgolide B, and bilobalide, respectively. Similar T  m a x  values (ranged from  1.17 to 1.54 h) were reported for all three chemicals (Kressmann et al., 2002). In a recent controlled, randomized, and crossover study of 12 males (age 18 to 35), the volunteers ingested a standardized G B E for 7 days (Drago et al., 2002). The regimen of 40 mg twice daily (vs. 80 mg once daily) led to a greater A U C , and longer half-life and mean residence time of ginkgolides A and B and bilobalide (Drago et al, 2002). However, the 80 mg once daily regimen led to a greater maximum peak concentration ( C  max  ) of these three  compounds. With both dosage regimens, time to maximum concentration ( T  max  ) of all three  compounds was reached after 2 to 3 h of oral administration (Drago et al, 2002). A study of ten adult volunteers in which each of them was given a single oral dose of 6 tablets of G B E reported absorption half-lives of quercetin and kaempferol to be 1.51 h and 1.56 h, respectively, whereas the T  m a x  of quercetin and kaempferol were reported as 2.30 h  4  and 2.68 h, respectively. In the same study, bioavailability of both quercetin and kaempferol was reported to be low (< 1%) in humans (Wang et al, 2003). Biber and Koch (1999) performed a pharmacokinetic study using male SpragueDawley rats by giving each of them an oral dose of the standardized G B E EGb 761 at one of the three doses (30, 55, and 100 mg/kg body weight). At the 30 mg/kg dose of EGb 761, the mean C x values were reported to be 68, 40, and 159 ng/mL for ginkgolide A , B and ma  bilobalide, respectively. The clearance values of ginkgolide A , B and bilobalide were very similar to each other, and were reported be 30.1, 29.0, and 29.2 mL/min/kg, respectively (Biber and Koch, 1999).  1.2.3.2 Distribution Kleijnen and Knipschild (1992) studied the pharmacokinetics of ginkgolide A , ginkgolide B, and bilobalide in humans administered GBE. In that study, the volume of distribution was reported to be 40 - 60 L for ginkgolide A and 60 - 100 L for ginkgolide B , whereas the volume of distribution for bilobalide was 170 L. According to the same study, the degree of protein binding was not known for the individual components of Ginkgo biloba (Kleijnen and Knipschild, 1992).  1.2.3.3 Metabolism In a study performed using female Wistar rats by Pietta et al (1995), rats were fed with an extract of Ginkgo biloba leaves at 4 g/kg body weight. Biological samples including urine, feces, and blood were collected from the rats for 5 days. Within 24 h, the flavonoids in the G B E were metabolized to seven different phenylalkyl acids: 3,4-dihydroxyphenyl-acetic acid, hippuric acid, 3-hydroxy-phenylacetic acid, homovanillic acid, benzoic acid, 3-(4-  5  hydrophenyl)propionic acid, and 3-(3-hydrophenyl)propionic acid. No glycosides or aglycones were detected in any of the biological samples (Pietta et al, 1995). Moon et al (2001) treated rats orally with quercetin aglycone and they were the first group to isolate a glucuronide metabolite of quercetin (quercetin 3-0-P-glucuronide) in rat plasma. In a separate study performed by Pietta et al. (1997), the flavonol metabolites identified after oral administration of an extract of Ginkgo biloba leaves to humans were different from those in rats. Substituted benzoic acids were detected in urine samples of volunteers - 4-hydroxybenzoic acid conjugate, 4-hydroxyhippuric acid, 3-methoxy-4hydroxyhippuric acid, 3,4-dihydroxybenzoic acid, 4-hydroxybenzoic acid and 3-methoxy-4hydroxybenzoic acid (vanillic acid). Also, no phenylacetic acid or phenylpropionic acid derivatives were found in the urine samples. Murota et al. (2000) have reported enhanced diffusion of quercetin aglycone, compared to its glycosides, across the Caco-2 cell monolayer. In addition, they suggested that quercetin glycosides would be converted to its aglycone forms by enterobacteria before conjugation occurred, producing sulfation and glucuronidation metabolites. For those glycosides that were not converted to aglycones but absorbed, it was suggested that they would mostly be conjugated to form metabolites before entering the circulation (Murota et al, 2000), except that in vivo studies in humans have reported the detection of low levels of flavonoids glycosides in plasma following ingestion of Ginkgo biloba tablets by four volunteers (Oliveira et al, 2002). In vitro studies using HepG2 cells yielded similar results as in studies using Caco-2 cells (Galijatovic et al, 1999). Wang et al. (2003) studied the disposition of quercetin and kaempferol in human subjects. Each adult human in the study was given an oral dose of six tablets of G B E (each  6  tablet containing 1.134 mg quercetin and 1.233 mg kaempferol). Both quercetin and kaempferol underwent sulfation and glucuronidation, and they were mainly excreted as glucuronides in the urine (Wang et al, 2003). This is in accordance with the studies performed in rats. Oliveira et al (2002) studied the metabolism of quercetin and kaempferol in rat hepatocytes. Four monoglucuronides of quercetin and two monoglucuronides of kaempferol (glucuronide metabolites not identified) were detected after 40 min incubation of quercetin and kaempferol with the rat hepatocytes. The glucuronide metabolites were reported to be the same ones formed after glucuronidation by UGT1A9 (Oliveira et al, 2002). It is generally believed that in both rats and human, quercetin and other flavonoids such as kaempferol and isorhamnetin undergo sulfate conjugation by phenol sulfotransferase, glucuronyl conjugation by UDP-glucuronosyltransferase, and methylation by catechol-0methyltransferase in the intestine before being absorbed into the circulation, with glucuronidation being the major metabolism pathway (Murota and Terao, 2003; Oliveira et al, 2002). A quercetin metabolite in rat plasma, quercetin 3-0-(3-D-glucuronide, was successfully isolated after oral administration of quercetin aglycone (Moon et al, 2001). In humans, a sulfate metabolite of quercetin in plasma was identified as quercetin 3'-0-sulfate (Daye* al, 2001). In addition to conjugation reactions, flavonoids are metabolized by the hepatic cytochrome P450 (CYP) enzymes. A n in vitro study performed by Breinholt et al. (2002) using human recombinant C Y P enzymes has shown 3'-hydroxylation of kaempferol to quercetin being mediated by CYP1A2. It has been reported that 70% of ginkgolide A , 50% of ginkgolide B, and 30% and bilobalide were excreted unchanged in the urine following oral  7  ingestion of G B E in humans, which indicated that some of the terpene trilactones in the G B E were not biotransformed after entering the human body (Kleijnen and Knipschild, 1992).  1.2.3.4 Elimination Biber (2003) reported the elimination half-lives for ginkgolide A , B , and bilobalide to be 4.5 h, 10.6 h, and 3.2 h, respectively, following an IV infusion of 100 mg of EGb 761 for 15 min in 12 volunteers. Kressmann et al. (2002) reported the terminal half-lives of elimination of ginkgolide A , ginkgolide B, and bilobalide to be 3.93 h, 6.04 h, and 3.19 h, respectively, in 12 healthy male volunteers. The results of these human studies are in contrast to the study performed in rats in which the elimination half-lives of ginkgolide A , ginkgolide B, and bilobalide were reported to be 1.7 h, 2.0 h, and 2.2 h, respectively (Biber and Koch, 1999). The elimination half-lives of these three chemicals in rats appeared to be shorter than those in human. Elimination half-lives of quercetin and kaempferol were reported to be 2.17 h and 2.76 h, respectively, after studying the urine of ten volunteers who ingested a single oral dose of 6 tablets of G B E (Wang et al, 2003). One of the Ginkgo biloba flavonol metabolite called benzoic acids were detected only in urine samples, but not in blood samples, of human volunteers after oral ingestion of G B E (Pietta et al, 1997). No phenylacetic acid or phenylpropionic acid derivatives were detected in human urine, whereas these metabolites were detected in rats. The metabolites detected in human urine contributed to only less than 30% of the flavonoids given which was similar as in rats (Pietta et al, 1997).  8  1.2.3.5 Effect of Dosage Form on Pharmacokinetic Parameters A study performed in 18 healthy volunteers compared three different dosage forms (capsules, drops, and tablets) of Ginkgo biloba. After administration of a single oral dose, the T  m a x  values of quercetin, kaempferol and isorhamnetin were reported to be prolonged in  the capsules formulation when compared with the drops and tablets formulation (Wojcicki et al, 1995). The use of phospholipid complex (Ginkgoselect Phytosome) form vs. the free (Ginkgoselect) form prolonged the T  m a x  values of ginkgolides A and B and bilobalide from  120 min to 180-240 min in a study of volunteers who were administered a single oral dose of G B E (Mauri et al, 2001).  1.2.4  Adverse Effects Patients considered Ginkgo biloba to be well tolerated in most clinical trials in that its  "adverse event profile was not different from that of the placebo" (Le Bars and Kastelan, 2000). Alkylphenols (ginkgolic acid) is always limited to less than 5 ppm of the products of Ginkgo biloba due to its potential toxicity in causing cell death and apoptosis (Ahlemeyer and Krieglstein, 2003). Occasionally, Ginkgo biloba may cause headache, nausea, gastric symptoms, diarrhea, or allergic skin reactions (Commission E Monograph). The Commission E Monograph listed "very seldom stomach or intestinal upsets, headaches, or allergic skin reaction" as the side effects of Ginkgo biloba (Commission E Monograph).  1.2.5  Drug Interactions In terms of pharmacodynamic drug interactions in human, there have been case  reports of patients taking recommended doses of Ginkgo biloba and experiencing spontaneous hyphema upon taking warfarin (Rosenblatt and Mindel, 1997) or intracerebral  9  haemorrhage upon taking aspirin (Matthews, 1998) as these drugs may intensify the platelet antagonist effect of Ginkgo biloba in humans. However, in a crossover randomized study performed in 12 healthy male subjects in which each ingested a single dose of 25 mg of warfarin after a pretreatment with 2 tablets of a standardized G B E (each tablet standardized to 9.6 mg of ginkgo flavonglycosides and 2.4 mg of ginkgolides and bilobalide) three times a day for 7 days, no significant changes in platelet aggregation and international normalized ratio (INR) of prothrombin time, compared to the parameters measured when they did not receive the pretreatment, were reported (Jiang et al, 2005). Also, Ginkgo biloba may increase blood pressure in humans when combined with a thiazide diuretic such as hydrochlorothiazide (Fugh-Berman, 2000). Y i n et al. (2004) investigated the pharmacokinetic drug interaction of Ginkgo biloba with omeprazole in 18 healthy Chinese volunteers. Ginkgo biloba was administered orally to each volunteer at a dose of 140 mg twice daily for 12 days. It was reported to induce omeprazole hydroxylation and reduce the A U C of omeprazole. According to the results of this study, Ginkgo biloba may reduce the effect of omeprazole to a significant level by increasing the clearance of omeprazole. In a crossover randomized study performed in 12 healthy male subjects in which each ingested a single dose of 25 mg of warfarin after a pretreatment with 2 tablets of a standardized G B E (each tablet standardized to 9.6 mg of ginkgo flavonglycosides and 2.4 mg of ginkgolides and bilobalide) three times a day for 7 days, no significant changes in warfarin enantiomer protein binding, warfarin enantiomer concentrations in plasma, as well as S-lhydroxywarfarin concentration in urine, compared to the parameters measured when they did not receive the pretreatment, were reported (Jiang et al, 2005).  10  Markowitz et al. (2003) investigated whether oral ingestion of 120 mg of the G B E called EGb 761 twice daily for 14 days in 12 humans would affect the pharmacokinetic parameters of dextromethorphan (a CYP2D6 substrate) and alprazolam (a CYP3A4 substrate). The mean A U C of alprazolam among all 12 subjects was found to have significantly decreased by 17% when compared to the baseline A U C before Ginkgo biloba treatment. The mean C  m a x  of alprazolam was not significantly decreased after Ginkgo biloba  treatment, although a significant decrease of 32% in C  m a x  was reported only in the six male  subjects. Mean half-life of elimination was not significantly decreased after Ginkgo biloba treatment, despite a significant decrease of 19% reported only in the six female subjects. No significant changes were reported in T  m a x  and apparent oral clearance in all subjects and in  either sex. Although the investigators concluded that the G B E was not likely to cause clinically significant drug interactions with medications metabolized by hepatic CYP2D6 and CYP3A4 based on the insignificant changes in apparent oral clearance, the results of this study suggested the potential for the G B E to significantly shorten half-life of alprazolam in female subjects and decrease A U C of alprazolam in male subjects (Markowitz et al, 2003). Considering the data from all the subjects, a reduction in A U C without shortening the elimination half-life of alprazolam suggested the effect of Ginkgo biloba on reduced oral absorption of alprazolam, either through induction of intestinal C Y P enzymes and/or inhibition of carrier-mediated transport, or through other mechanisms. Gurley et al (2002) investigated the effect of Ginkgo biloba on CYP2E1, CYP3A4, CYP2D6, and CYP1A2 drug metabolisms in 12 healthy volunteers. It was reported that Ginkgo biloba did not produce any significant effect on these four specific C Y P activities using chlorzoxazone, midazolam, dextromethorphan, and caffeine as the C Y P substrate for  11  CYP2E1, CYP3A4, CYP2D6, and CYP1A2, respectively. In terms of pharmacodynamic drug interaction studies in animals, Sugiyama et al. (2004b) have reported a potential drug interaction between G B E and tolbutamide in rats: Rats were either pretreated with a 0.1% w/w Ginkgo biloba diet for 5 days before they were orally administered tolbutamide at 40 mg/kg body weight, or they were given the tolbutamide dose simultaneously with 100 mg/kg body weight of Ginkgo biloba orally. Under both conditions, Ginkgo biloba was reported to affect the hypoglycemic action of tolbutamide, and this effect was more prominent in aged rats than in young rats (Sugiyama et al, 2004b). A study using a mouse model investigated the effect of Ginkgo biloba on barbitalinduced sleeping time (Brochet et al, 1999). It was reported that single injections of the G B E called EGb 761 (at 25 and 50 mg/kg body weight) decreased barbital-induced sleeping time when given 1 h before sodium barbital. Ginkgolide B and bilobalide were reported to be responsible for this interaction (Brochet et al, 1999). The results of this study were in accordance with a pharmacokinetic drug interaction study using a rat model. Kubota et al. (2004) fed rats a 0.1 % w/w, 0.5% w/w, or 1.0% w/w Ginkgo biloba diet for 2 weeks, and they reported a reduction in not only the Cm and A U C of phenobarbital, but also a reduction ax  in the phenobarbital-induced sleeping time in rats. In another study using rats to investigate the pharmacokinetic interaction between G B E and nicardipine (a calcium channel blocker), Kubota et al. (2003) reported that a 0.5% w/w Ginkgo biloba diet to rats for 14 days decreased the C  max  and A U C of nicardipine (30  mg/kg body weight administered orally to rats after 2 weeks). This was in contrast to the conclusion Yoshioka et al. (2004) reached after they co-administered G B E and nifedipine (another calcium channel blocker) in rats. Yoshioka et al. (2004) reported the oral co-  12  administration of nifedipine at 5 mg/kg body weight and G B E at 20 mg/kg body weight caused a significant increase in C  m a x  , A U C , and absolute bioavailability of nifedipine due to  the ability of Ginkgo biloba to inhibit CYP3A. In rats, a G B E called Ginkgolon-24 increased the bioavailability of diltiazem (Ohnishi et al., 2003).  1.2.6  Inhibition of Drug-Metabolizing Enzymes G B E was recently reported to competitively inhibit 7-ethoxyresorufin O-dealkylation  in hepatic microsomes prepared from rats whose hepatic C Y P 1 A had been induced by once daily intraperitoneal (ip) injections with /^naphthoflavone for 3 days (Kuo et al., 2004). However, in vitro inhibition of C Y P 1A activity in rats by G B E was not caused by the diglycosides of kaempferol, quercetin, or isorhamnetin, ginkgolides A , B , C, or J, or bilobalide. The aglycones of kaempferol, quercetin, and isorhamnetin were capable of inhibiting rat hepatic C Y P 1 A activity (Kuo et al., 2004). They were found to inhibit 7ethoxyresorufin O-dealkylation activity in human liver microsomes as well (Chang et al., in press). Kaempferol, quercetin, and isorhamnetin were reported to inhibit human recombinant CYP1 A l and CYP1A2 in vitro, as shown by their relatively low K i values (Chang et al., in press). One speculation was that the aglycones could be responsible for C Y P 1 A inhibition by the G B E in vivo, as flavonol glycosides can be biotransformed to their corresponding aglycones in the gastrointestinal tract (Walle, 2004). Gaudineau et al. (2004) were the first group to report CYP2C9 and CYP2E1 inhibition by G B E (EGb 761) using microsomes prepared from human B-lymphoblastoid cells expressing individual human C Y P . Their group demonstrated that the flavonoid fraction of EGb 761 was responsible for inhibition of human recombinant CYP2C9, CYP1A2, CYP2E1, and CYP3A4, and the terpenoid fraction of EGb761 was responsible for  13  inhibition of human recombinant CYP2C9 (Gaudineau et al, 2004). In vitro capability of quercetin to significantly inhibit tolbutamide 4-methylhydroxylation (IC50 of 35 uM) and testosterone 6/?-hydroxylation  ( I C 5 0  of 38 (j,M) has been reported in human liver microsomes  (He and Edeki, 2004). In the same study, ginkgolide A was reported to inhibit tolbutamide 4-methylhydroxylation with an IC50 of 200 u M .  1.2.7  Induction of Drug-Metabolizing Enzymes Rats that have been fed Ginkgo biloba were found to have their hepatic CYPs  induced. Sugiyama et al. (2004a) incorporated 0.5% w/w of a standardized G B E in the diet of male Wistar rats for 1 week, and they monitored the C Y P activities for another 1, 2, or 3 weeks. After 1 week of oral treatment of G B E , the liver weight, the total C Y P content in the liver microsomes, glutathione ^-transferases activity, and six hepatic C Y P marker enzyme activities (ethoxyresorufin O-deethylation, methoxyresorufin O-demethylation, pentoxyresorufin 0-dealkylation, iS-warfarin 7-hydroxylation,/>-nitrophenol hydroxylation, and testosterone 6/?-hydroxylation) were increased significantly. The total C Y P content and the six hepatic C Y P marker enzyme activities in the liver microsomes returned to basal level after discontinuing G B E for 1 week, whereas the glutathione 5-transferases activity returned to basal levels after discontinuing the extract for 3 weeks (Sugiyama et al, 2004a). In another study performed by Umegaki et al. (2002), rats were given G B E at 1, 10, 100, or 1000 mg/kg body weight daily by intragastric gavage for 5 days. The content of cytochrome P450 increased significantly compared to control starting at a dose of 10 mg/kg body weight of G B E . The activity of glutathione 5-transferases increased significantly starting at a dose of 1 mg/kg body weight of G B E . Activities of ethoxyresorufin Odeethylase, methoxyresorufin O-demethylase, pentoxyresorufin O-dealkylase, p-nitrophenol  14  hydroxylase, testosterone 6/?-hydroxylase, and S-warfarin 7-hydroxylase were all reported to increase significantly compared to control at 100 and 1000 mg/kg body weight of G B E treatment. The increase in pentoxyresorufin O-dealkylase activity was substantial (65-fold compared to control level). Hepatic mRNA expression of CYP2B1/2, C Y P 3 A 1 , and CYP3A2 (but not that of CYP1A1/2, CYP2E1, CYP2C11, or CYP4A1) were markedly increased in male rats on a diet containing 0.5% w/w G B E for 4 weeks (Shinozuka et al, 2002). In another study performed by the same group, rats were given G B E at 1000 mg/kg body weight intragastrically for 5 days. Western blot analysis using anti-rat CYP2B1/2B2 antibody showed increased expression of CYP2B1 and CYP2B2 proteins in liver microsomes prepared from these rats treated with Ginkgo biloba (Umegaki et al, 2002). Yang et al. (2003) investigated the effect of Ginkgo biloba on C Y P activities when it was administered to rats by oral gavage at 100 mg/kg or 200 mg/kg body weight for 10 days. At the Ginkgo biloba dose of 200 mg/kg body weight, methoxyresorufin O-demethylase activity, ethoxyresorufin O-deethylase activity, pentoxyresorufin O-dealkylase activity, aminopyrine A-demethylase activity, and aniline hydroxylase activity in rat liver microsomes all increased significantly compared to the control group. At the dose of 100 mg/kg body weight, all but the ethoxyresorufin O-deethylase and the aniline hydroxylase activities in rat liver microsomes increased significantly.  1.3 Acetaminophen 1.3.1  Clinical Uses A P A P is a commonly used analgesic and antipyretic that is available without a  prescription in North America. A P A P has little or no side effects if used as directed but it  15  may cause liver damage during an overdose. A survey conducted in 308 patients in the United States with acute liver failure by Ostapowicz et al. (2002) indicated that A P A P overdose accounted for 39% of all cases. Hepatotoxicity is more frequently observed in overdose of A P A P and in patients with alcoholism (Thummel et al, 2000).  1.3.2  Mechanism of Therapeutic Action A P A P has antipyretic and analgesic properties similar to non-steroidal anti-  inflammatory drugs (NSAIDs) such as aspirin and ibuprofen. However, in contrast to NSAIDs, A P A P does not have anti-inflammatory and anticoagulation activities as it is only a weak inhibitor of cyclo-oxygenase-1 and cyclo-oxygenase-2 enzymes (Warner et al, 1999). The mechanism of action of A P A P at the molecular level is not clear. Its pharmacological action is thought to be mediated through central (instead of peripheral in NSAIDs) inhibition of prostaglandin synthesis at the spinal cord and the hypothalamus (Muth-Selbach et al, 1999).  1.3.3  Pharmacokinetics  1.3.3.1 Absorption and Bioavailability A P A P is rapidly absorbed (absorption half-life of 30 min) upon oral administration and its elimination half-life is approximately 2 h (Rumack, 2004). Its volume of distribution is estimated to be 1 L/kg (Rumack, 2004) and its oral bioavailability is dose-dependent, ranging from 70% to 90% (Forrest et al. ,1982).  16  1.3.3.2 Distribution A P A P is not known to bind to any plasma proteins. A P A P was found to distribute to brain regions including the dorsal spinal cord and the cerebellum in rats (Courad et al, 2001). Volume of distribution in human was reported to be 66 L (Anderson et al, 2002).  1.3.3.3 Metabolism Data from human and animal studies have revealed that A P A P may undergo one of the three metabolic pathways upon oral administration. As shown in Figure 1.1, it may conjugate with sulfate, conjugate with glucuronide, or it may be metabolized by cytochrome P450 (CYP) enzymes (Bartlett, 2004). In humans, majority (96%) of A P A P undergoes glucuronidation or sulfation to form non-toxic conjugated metabolites, whereas CYP2E1 is the main C Y P enzyme that is responsible for the other 4% of the metabolism of A P A P to form the hepatotoxic metabolite - N-acetyl-p-benzoquinoneimine (NAPQI) (Hersh and Moore, 2004; Rumack, 2002; Manyike et al, 2000). Other C Y P enzymes such as CYP3A4, CYP1A2, CYP2A6 have also been suggested to be involved in the formation of the hepatotoxic metabolite (Bartlett, 2004). The hepatotoxic metabolite is then eliminated by binding to glutathione to form non-toxic metabolites in hepatocytes, a process that is catalyzed by glutathione 5-transferases (Kaplowitz, 2004). Overdose of A P A P may result in severe hepatic necrosis in humans and in various laboratory animals. Among laboratory animals, hamsters and mice are more sensitive, whereas rats, rabbits, and guinea pigs are less sensitive to APAP-induced hepatotoxicity (Bessems and Vermeulen, 2001). In adult male rats treated with A P A P alone, a 46% and a 41% inhibition of A P A P oxidation was observed when the liver microsomes were incubated with CYP2E1 and CYP1A2 antibodies, respectively, at an A P A P concentration of 1 m M ,  17  suggesting the important roles of these two C Y P enzymes in the metabolism of A P A P to form the hepatotoxic metabolite (Patten et al, 1993). This concentration of A P A P (1 mM) may not be relevant in in vivo rat studies because A P A P has been reported to reach a level of 1043 ng/mL (or 0.007 mM) in rat plasma 4 h after oral administration of A P A P at 5 mg/kg body weight (Abu-Qare and Abou-Donia, 2001). CYP3A1/2 antibodies showed a slight inhibition (10%), suggesting the involvement of CYP3A1/2 in the metabolism of A P A P , but to a lesser extent as compared to CYP2E1 and CYP1A2 (Patten et al, 1993). The 3hydroxylation of A P A P in rats was reported to exceed the formation of the hepatotoxic metabolite rendering the relative insensitivity of APAP-induced hepatotoxicity in rats (Bessems and Vermeulen, 2001). In terms of Phase II biotransformation reactions, A P A P is metabolized by sulfotransferase (SULT) enzymes and UDP-glucuronosyltransferase (UGT) enzymes to nontoxic metabolites for eliminiation. Kessler et al. (2002) suggested the UGT1A6 enzyme plays a key role in glucuronidation of A P A P as Gunn rats, which have nonfunctional UGT1A6, showed reduced rate of A P A P glucuronidation and increased AP AP-induced hepatotoxicity. The members of the SULT1 family of sulfotransferases were suggested to be responsible for the sulfation of A P A P (Duffel, 1997).  18  SULT1 non-toxic conjugated < === metabolites A  UGT1A6 APAP B C  non-toxic conjugated metabolites  CYP2E1, CYP1A2, C Y P 3 A  NAPQI D glutathione  Glutathione S-trarisferases  non-toxic metabolites Figure 1.1. A simplified scheme of the bioactivation and detoxification pathways of A P A P . A P A P can undergo sulfation in the presence of SULT1 (pathway A) or glucuronidation in the presence of UGT1A6 (pathway B) to form non-toxic conjugated metabolites. A P A P can also be metabolized by CYP2E1, CYP1A2, and C Y P 3 A to form the hepatotoxic metabolite, NAPQI (pathway C). In the presence of glutathione, NAPQI can be converted into non-toxic metabolites through the action of glutathione ^-transferases (pathway D).  1.3.3.4 Elimination The sulfate and glucuronide metabolites of A P A P are more water-soluble than the other metabolites and are eliminated mainly by urine. A small amount (glucuronide of APAP) is eliminated by bile. The hepatotoxic metabolite NAPQI, formed by the cytochrome P450 enzymes, can bind to and form a conjugate with glutathione. This conjugate can further be converted to mercapturic acid for elimination in the urine (Bessems and Vermeulen, 2001).  1.3.4  Adverse Effects In a study done in 66 human volunteers who each underwent a surgical dental  procedure, 1000 mg of A P A P was given to each volunteer after the procedure and about 17% of the subjects reported adverse effects (Olson et al, 2001). About 2% of the subjects  19  reported fever, 6% of them reported headache, 3% of them experienced nausea, and 4.5% of them reported somnolence as adverse drug reactions. However, no difference in adverse effects was reported between A P A P (1000 mg twice daily) and placebo in a randomized, single blind, placebo-controlled study of 72 healthy volunteers treated for 2.5 days (Jerussi et al, 1998). In another randomized, double blind, cross-over study of 24 healthy volunteers, there was no difference in occurrence of gastrointestinal lesions between A P A P (1000 mg four times a day) and placebo (Lanza et al, 1998). Nonetheless, when A P A P is ingested at a toxic dose of more than 150 mg/kg by a human adult, it can potentially cause acute liver toxicity (Wallace et al, 2002; Tenenbein, 2004). A -Acetylcysteine is a clinically useful antidote for APAP-induced poisoning, i f given 7  within a few hours after ingestion of toxic doses of A P A P (Thomas, 1993). Glutathione is a tripeptide that is poorly absorbed by cells. Af-acetylcysteine can act as a precursor for the synthesis of glutathione.  1.3.5  Hepatotoxicity  1.3.5.1 Mechanism of Hepatotoxic Action Many mechanisms leading to APAP-induced hepatotoxicity have been proposed. It is generally believed that the CYP-mediated formation of NAPQI from A P A P (pathway C in Figure 1.1) is a key event in causing the hepatotoxicity. At hepatotoxic doses of A P A P , the store of glutathione can be depleted, leading to increased accumulation of the hepatotoxic metabolite, NAPQI. This reactive metabolite of A P A P can potentially bind covalently to D N A and critical cellular protein(s), resulting in loss of function of the protein(s) and ultimately to cell death. These proteins have been postulated to be mitochondrial proteins and/or proteins responsible for cellular ion control (Nelson, 1990). Recently, it has been  20  hypothesized that A P A P toxicity occurs when there is increased oxygen and/or nitrogen stress in the system, and this increased oxidative/nitrosative stress can lead to mitochondrial permeability transition (Hinson et al, 2004). Mitochondrial permeability transition is "an abrupt increase in the permeability of the inner mitochondrial membrane to small molecular weight solutes" (Hinson et al, 2004). It has been hypothesized that this process can be lethal to the cells as it occurs with an increased release of superoxides. Superoxides are toxic to the cells because they can lead to peroxynitrite and tyrosine nitration (Kim et al, 2003). The intracellular signaling mechanisms leading to liver cell death during AP A P induced hepatotoxicity were summarized in detail in a recent review by Jaeschke and Bajt (2006). The hepatotoxic metabolite of A P A P (NAPQI) "first depletes cellular glutathione and subsequently covalently bind to cellular proteins. These initiating events lead to disturbances of the cellular C a  2+  homeostasis, with increase of the cytosolic C a  2+  levels, Bax  and Bid translocation to the mitochondria, and a mitochondrial oxidative stress and peroxynitrite formation. The Bcl-2 family members form pores in the outer mitochondrial membrane and release cytochrome c, Smac (the second mitochondria-derived activator of caspases), apoptosis-inducing factor, and endonuclease G from the mitochondrial intermembrane space. Reactive oxygen species and peroxynitrite induce the membrane permeability transition, which causes the collapse of the mitochondrial membrane potential, eliminates ATP synthesis, and causes further release of mitochondrial proteins." "Apoptosisinducing factor and endonuclease G translocate to the nucleus and induce D N A fragmentation." "The massive nuclear D N A damage and the rapid elimination of functional mitochondria, together with activation of intracellular proteases (calpains), lead to cell membrane failure and oncotic necrosis of the hepatocytes" (Jaeschke and Bajt, 2006).  21  Figure 1.2. Mechanisms of A P AP-induced hepatotoxicity. (Jaeschke and Bajt, 2006. Reproduced with written permission from Oxford University Press)  1.3.5.2 Effect of Fasting Price et al. (1987) have reported the effect of fasting on AP AP-induced hepatotoxicity in male Long-Evans rats. The rats experienced an overnight fast before receiving ip injections of A P A P . Fasting not only decreased the hepatic levels of glutathione, but also decreased clearance (due to reduced glucuronidation and sulfation) of A P A P . The depression in glucuronidation and sulfation hence increased the formation of the hepatotoxic metabolite NAPQI, rendering fasted-rats more susceptible to A P AP-induced hepatotoxicity (Price etai, 1987).  22  1.3.5.3 Strain Differences Strain differences in APAP-induced hepatotoxicity became evident in rats as early as 1986, when the susceptibility to APAP-induced hepatotoxicity was compared between Sprague-Dawley and Long-Evans rats (Price and Jollow, 1986). Both strains of rats were fasted for 24 h prior to ip injections of A P A P at 800 mg/kg. The Sprague-Dawley rats showed no evidence of liver injury, whereas 90% of the Long-Evans rats showed liver necrosis 48 h after the dose of A P A P . In the same study, both strains of rats were fasted and received ip injections of A P A P at 400, 600, 800, and 1200 mg/kg body weight, and were terminated 48 h after the A P A P dose. There was an obvious left shift in the dose-response curve of the Long-Evans rats compared to that of the Sprague-Dawley rats, indicating the Long-Evans strain of rats was more susceptible to APAP-induced hepatotoxicity (Figure 1.3).  23  400  600  800  1000  1200  Acetaminophen (mg/kg) Figure 1.3. Dose-response curve for hepatic necrosis in Long-Evans and Sprague-Dawley rats administered A P A P . The effect of various intraperitoneal doses of A P A P on liver necrosis in two strains of rats 48 h after administration of A P A P (original data from study done by Price and Jollow, 1986).  1.3.5.4 Role of Nuclear Receptors in APAP-induced Hepatotoxicity Recently, the pregnane X receptor (PXR) was suggested to play an important role in APAP-induced hepatotoxicity in mice (Wolfe;' al, 2005). P X R is a nuclear receptor that is abundant in the liver and the intestine. Upon ligand binding to PXR, a heterodimer will be formed between P X R and retinoid X receptor. This is followed by activation of "ER6 (everted repeat with a 6 nucleotide spacer) elements" upstream to the target C Y P genes, leading to increased transcription (Waxman, 1999). The PXR(-/-) mice were reported to be less susceptible, compared to wild-type mice, to hepatotoxicity induced by oral ingestion of  24  A P A P . Wild-type mice were also found to have greater hepatic CYP1A2 protein levels than PXR(-/-) mice. CYP1A2 was hence suggested to be critical in modulating A P A P hepatotoxicity in the wild-type mice, as caffeine (a CYP1A2 inhibitor) was able to protect these mice against the hepatotoxicity caused by A P A P . The same study also suggested the effect of P X R on intestinal APAP-transporting proteins, as wild-type mice absorbed A P A P from the intestine to a greater degree than PXR(-/-) mice. The absence of P X R in the PXR(/-) mice may lead to a decreased expression of these transporter proteins, leading to a decrease absorption of A P A P and hence a decreased susceptibility of APAP-induced hepatotoxicity in these PXR(-/-) mice. In contrast, Guo et al. (2004) reported enhanced hepatotoxicity, as shown by elevated serum A L T levels and worse hepatocyte necrosis, in PXR-null mice at 24 h after ip injection of A P A P . This enhanced APAP-induced hepatotoxicity in PXR-null mice was attributed to an increase in CYP3A11 mRNA expression and a decrease in glutathione 5-transferases -Pi mRNA expression (both by northern blot analysis) in PXR-null mice. Also, there was a lower mRNA expression of CYP1A2 in PXR-null mice, although the CYP2E1 mRNA expression were similar in both PXR-null and wild-type mice. However, enhanced A P A P induced hepatotoxicity was reported in wild-type mice upon treatment with a P X R activator, pregnenolone 16ct-carbonitrile. Enhanced APAP-induced hepatotoxicity was not seen in PXR-null mice, whose CYP3A11 expression and NAPQI formation were both lower, upon treatment with the same activator, suggesting the important role of P X R in APAP-induced hepatotoxicity. Both studies that investigated the effect of P X R in APAP-induced hepatotoxicity in mice found an increase in C Y P 3 A expression and a decrease in CYP1A2 expression in PXR-  25  null mice, when compared to wild-type mice. CYP2E1 expression in both PXR-null mice and wild-type mice were similar. However, AP AP-induced hepatotoxicity was enhanced in wild-type mice in the study by Wolf et al. (2005), whereas it was enhanced in PXR-null mice in the study by Guo et al. (2004). On the other hand, constitutive androstane receptor (CAR) was suggested to be another key regulator in AP AP-induced hepatotoxicity (Zhang et al, 2002). CAR(-/-) mice showed significantly lower A L T levels than wild-type mice at 5 h and 24 h after given 500 or 800 mg/kg dose of A P A P . Also, as early as 2 h after the dose of A P A P at 500 mg/kg, CYP1A2, CYP3A11, and glutathione S-transferases -Pi mRNA expression were all increased significantly in wild-type mice, but not in CAR-null mice, by 2.8-, 4.4-, and 3.9-fold, respectively. In the same study, both wild-type and CAR-null mice were pretreated with a C A R activator or agonist, either phenobarbital or l,4-bis[2-(3,5-dichloropyridyloxy)]benzene, before dosed with a non-hepatotoxic dose A P A P at 250 mg/kg body weight. Elevated A L T levels and liver necrosis were observed only in wild-type mice, but not in CAR-null mice, at 24 h after dosing with A P A P .  1.3.5.5 Other Modulators of APAP-Induced Hepatotoxicity Enhancement of AP AP-induced hepatotoxicity A few compounds have been reported to enhance A P AP-induced hepatotoxicity in laboratory models by modulation of Phase I or II drug-metabolizing enzymes. Drugs such as acarbose and troglitazone may potentiate the hepatotoxicity effect of A P A P by inducing the expression of CYP2E1 or C Y P 3 A in rats (Wang et al, 1999; L i et al, 2002). Acarbose, when included in a 3-week-diet of rats at 40 or 80 mg /100g body weight diet, was reported to increase the levels of A L T and the levels of aspartate aminotransferase (AST) by 1.5  26  (40 mg) and 2 (80 mg) times relative to A P A P control (APAP only) after ip injection of A P A P at 750 mg/kg body weight (Wang et al, 1999). In another study, rats were fed a troglitazone (0, 100, or 500 mg/kg body weight/day) diet for 3 weeks starting at an age of 10 weeks, followed by ip injection of A P A P at 0.75 g/kg body weight in a 0.9% saline solution (10 mL/kg) (Li et al, 2002). In the plasma, the levels of A L T , AST, and a-glutathione ^-transferases in the group pretreated with troglitazone at 500 mg/kg/day (700 mg drug per 100 g food) prior to A P A P vs. control (APAP without troglitazone) were increased by 1.6-fold, 1.3-fold, and 2.3-fold, respectively, suggesting the increased risk of APAP-induced toxicity when troglitazone was given in the diet (Li et al, 2002). At a troglitazone dose of 500 mg/kg/day (700 mg drug per 100 g food), the glutathione level was observed to be significantly less (83% of A P A P control level) compared to the A P A P control (Li et al, 2002). Ethanol can induce CYP2E1 leading to increased formation of the hepatotoxic metabolite, thereby increasing the risk of hepatotoxicity (Zand et al, 1993; Thummel et al, 2000). In the study by Thummel et al (2000), each of the ten healthy human volunteers received an intravenous infusion of ethanol, a CYP2E1 inducer, for 6 h. Each volunteer received an oral dose of A P A P at 500 mg 8 h after the end of the infusion. The formation of NAPQI was reported to be significantly increased by 22% in the group that received ethanol prior to A P A P , compared to the control group that received 5% dextrose in water infusion (Thummel et al, 2000). In cultured human hepatocytes, both phenobarbital (2 mM) and phenytoin (0.2 mM) have been reported to increase A P A P (5 mM) hepatotoxicity by inhibition of UGT1A6, UGT1A9, and UGT2B15 (Kostrubsky et al, 2005).  27  Protection against AP AP-induced hepatotoxicity Many compounds have been investigated for their protective potential against A P A P induced hepatotoxicity. For instance, chemicals such as stiripentol may have a protective effect on A P AP-induced hepatotoxicity in rats (Tran et al, 2001). Stiripentol (200 mg/kg body weight ip in corn oil), an in vitro inhibitor of hydroxylation of naphthalene by CYPs in cerebral tissues of rats (Mesnil et al, 1988), administered 30 min before an overdose of A P A P (two doses at 500 mg/kg body weight ip at 2.5 h apart) and 5 h after the first administration at 100 mg/kg body weight ip, lowered mortality rate of the rats from 63% (0.9% saline and corn oil) to 0% by inhibiting the formation of the hepatotoxic metabolite of A P A P by C Y P inhibition (Tran et al, 2001). Disulfiram pretreatment (500 mg orally at bedtime received by each volunteer 10 hour before a 500 mg oral dose of A P A P was administered) was reported to decrease formation of the hepatotoxic metabolite by 74% in humans (Manyike et al, 2000). Isoniazid is a CYP2E1 inhibitor upon a single dose (O'Shea et al, 1997). It was reported to inhibit the formation of A P A P thioether metabolites when given orally to ten volunteers as a single dose of 300 mg daily for 7 days (Zand et al, 1993). There was only one study in the literature that investigated the effect of G B E on A P AP-induced hepatotoxicity in rats (Shenoy et al, 2002). At an oral dose of A P A P at 2000 mg/kg once daily for 3 days and in the absence of any G B E treatment (Group II), the mean ± S E M plasma A L T level was 256 ± 25 IU/L. When the same A P A P treatment was followed by a vehicle treatment using 2% gum acacia for 7 days (Group III), plasma A L T level decreased slightly to 213 ± 19 IU/L. When G B E was given as an ip dose at 50 mg/kg simultaneously with an oral dose of A P A P at 2000 mg/kg once daily for 3 days (Group IV), plasma A L T level decreased significantly to 43 ± 7 IU/L (when compared to Group II). A  28  similar plasma A L T level (44 ± 6 IU/L) was obtained when G B E was given for 7 days after the same regimen (oral dose of A P A P at 2000 mg/kg once daily for 3 days) of A P A P (Group V). This was significantly less than that in Group III. Both the plasma A L T data and the liver histology data in this study by Shenoy et al. (2002) suggested a protective effect of G B E when it is given together with or after a hepatotoxic dose of A P A P in rats. However, Shenoy et al. (2002) failed to investigate the effect of a pretreatment with G B E to the rats before A P A P is given. Their study only addressed the effect of G B E on APAP-induced hepatotoxicity when G B E was given concurrently or after the treatment with A P A P .  1.4  Rationale A P A P is a widely used over-the-counter antipyretic and analgesic drug. Although  generally regarded as a well-tolerated medication, A P A P can cause liver toxicity. The majority of A P A P is converted to non-toxic sulfate and glucuronide conjugates of A P A P for excretion from the body (Thomas, 1993). APAP-induced hepatotoxicity can result when the conjugation pathways are saturated. During oxidative stress and in conditions where the level of hepatic glutathione is reduced (e.g. malnourishment), the risk of APAP-induced hepatotoxicity can also be increased (Vendemiale et al, 1996). The accumulation of the hepatotoxic metabolite NAPQI, potentially leading to oxidative stress and covalent binding to functional proteins rendering cellular damage, is directly associated with the hepatotoxicity caused by A P A P (Mitchell et al., 1973). This hepatotoxic metabolite is formed by a cytochrome P450 oxidation pathway mediated mainly by CYP2E1, CYP1A2, and C Y P 3 A (Patten et al, 1993; Thummel et al, 1993). The capacity of the glutathione 5-transferases to detoxify the hepatotoxic metabolite also plays a crucial role in alleviating the hepatotoxicity caused by A P A P .  29  G B E has been shown to significantly induce methoxyresorufin 0-demethylase, pnitrophenol hydroxylase, and testosterone 6y3-hydroxylase activities, which are thought to represent the activities of CYP1A2, CYP2E1, and CYP3A, respectively, in rats (Umegaki et al, 2002). Hence, it is plausible that G B E can exacerbate AP AP-induced hepatotoxicity by increasing formation of NAPQI as a result of CYP1A2, CYP2E1, and C Y P 3 A induction by G B E . G B E was shown to be hepatoprotective in rats pretreated or co-treated with A P A P (Shenoy et al, 2002). However, that study did not include a pretreatment regimen of G B E prior to A P A P . G B E is a supplement that people consume on a daily basis. It would be invaluable to investigate whether pretreatment of rats with G B E can modulate A P A P induced hepatotoxicity. Both A P A P and G B E are popular medications that are available over-the-counter without a prescription. The results obtained from the current study may have important clinical implications.  1.5  Research Hypothesis G B E will modify the degree of AP AP-induced hepatotoxicity.  1.6 Experimental Hypothesis G B E pretreatment will modulate AP AP-induced hepatotoxicity in male Long-Evans rats, as assessed by the plasma alanine aminotransferase (ALT) assay and histological analysis.  1.7 Specific Aims (1)  To validate the plasma A L T assay  (2)  To perform control experiments to verify some conditions (e.g. dose of A P A P , fasting) required to increase plasma A L T levels in male Long-Evans rats  30  To determine the effect of G B E pretreatment on plasma A L T levels and liver histology (steatosis, necrosis, capsular inflammation, and sinusoidal dilatation) male Long-Evans rats administered A P A P  31  2. M A T E R I A L S A N D M E T H O D S 2.1  Chemicals and Reagents Chemicals used in the study were purchased from the following:  B D H Inc. (Toronto, Ontario, Canada) Sodium chloride powder Best Foods Canada Inc. (Etobicoke, Ontario, Canada) 100% pure Mazola Corn oil Biotron Diagnostics Inc. (Hemet, California, USA) A L T Kit containing: A L T Reagent: 0.15 mg/L a-ketoglutaric acid, 8.9 mg/L dl-alanine in phosphate buffer and preservative Color Developer A : 0.02% w/v 2,4-dinitrophenyl-hydrazine, 8.6% hydrochloric acid, and preservative . Color Developer B: 1.6% w/v sodium hydroxide Abnormal Clinical Chemistry Control - lyophilized standard Fisher Scientific (Fair Lawn, New Jersey, USA) Tween 80 (Polysorbate 80) Indena S.A. (Milan, Italy) Ginkgo biloba extract (Lot no. 1306A) [the levels of terpene trilactone and flavonols are listed in Table A l in the appendix] M T C Pharmaceuticals (Cambridge, Ontario, Canada) Somnotol (Sodium pentobarbital for injection) Sigma-Aldrich Inc. (St. Louis, Missouri, USA) Acetaminophen SigmaUltra, minimum 99.0%  32  10% Formalin solution Carbon tetrachloride Heparin sodium salt  2.2  Animals Male and female Long-Evans rats were purchased from Charles River Canada Inc.  (Montreal, Quebec, Canada). The animals were housed in pairs in polycarbonate cages with corncob bedding for at least 5 days before any experiment was performed. Food (Lab Diet, Purina Mills, Inc.) and water were available ad libitum (except during fasting condition), and their room was under controlled temperature (20 °C) and lighting (lights on from 7 a.m. to 7 p.m.) during the course of each experiment. A l l animals weighed 100 - 150 g and were 30 37 days of age before the start of each experiment. Long-Evans rats were used because they are more susceptible to APAP-induced hepatotoxicity when compared to another strain of rats [Sprague-Dawley] (Price and Jollow, 1986). Mice are more susceptible to APAP-induced hepatotoxicity compared to rats (Bessems and Vermeulen, 2001) but their blood volume is too small to have sufficient amount of plasma isolated for repeated analyses of A L T levels in a single plasma sample.  2.3  Treatment  2.3.1  Positive Control Experiment Carbon tetrachloride (CCU) served as a positive control. It was diluted with corn oil  to a 1:1 v/v mixture for intraperitoneal (ip) injection at a dose of 2 ml/kg body weight. Animals were terminated 24 h after the injection. In the control group, animals were injected  33  with 4 ml/kg body weight of corn oil ip (this was used accidentally as the dose for corn oil instead of 2 ml/kg).  2.3.2  Effect of Fasting and A P A P Half of the animals were fasted overnight (from 6 pm to 8 am with food but not water  withheld), while the other half was provided free access to food. On the next day, each animal in the fasted group was given either an ip injection of A P A P at 1000 mg/kg body weight or an equivalent volume (injection volume of 3 ml/kg) of 20% Tween 80 in 0.9% NaCl, the vehicle used to suspend A P A P (Gardner et al, 1998). In the non-fasted group, each animal was given an ip injection of A P A P at 1000 mg/kg body weight or the vehicle (at the same injection volume of 3 ml/kg body weight). Food was returned to the fasted animals after the injections. Animals were terminated the next day at 24 h (Price et al, 1987) after injections of A P A P or the vehicle.  2.3.3  Effect of 20% Tween 80 in 0.9% NaCl (the vehicle for APAP) Animals were fasted overnight and injected ip with either 0.9% NaCl (at an injection  volume of 3 ml/kg body weight) or an equal volume (3 ml/kg body weight) of 20% Tween 80 in 0.9%) NaCl (the vehicle used to suspend APAP). Food was returned to the cages and the animals were terminated the next day at 24 h after the injections.  2.3.4  Effect of the Dose of A P A P In the dose response experiment, animals were fasted overnight and injected with an  ip dose of either 700, 1000, or 1300 mg/kg body weight of A P A P suspended in the 20% Tween 80 in 0.9% NaCl. In the control group, each animal was given an ip injection of the 20%) Tween 80 in 0.9% NaCl (the vehicle used to suspend APAP). Animals continued to be  34  fasted after the injections. Animals were sacrificed 8 h after the dose of A P A P or the vehicle. Animals in this experiment and subsequent experiments with A P A P were sacrificed at 8 h instead of 24 h. This was because the fold-increases in plasma A L T levels at 1000 mg/kg ip of A P A P (vs. respective control groups) at both 8 h and 24 h after dosing were similar, according to my preliminary A P A P experiments (data not shown).  2.3.5  Effect of G B E Animals were injected with either G B E at a dose of 500 mg/kg ip (dissolved in 0.9%  NaCl) or an equivalent volume of 0.9% NaCl (injection volume of 2 ml/kg) once daily for 8 consecutive days. They were fasted overnight and terminated on Day 9 at about 24 hours after the last injections.  2.3.6  Effect of the Combination of G B E and A P A P The animals were separated into three groups. One group received ip injections of  G B E at a dose of 500 mg/kg (dissolved in 0.9% NaCl) once daily for 8 consecutive days. The other two groups received ip injections of 0.9% NaCl (at the same injection volume of 2 ml/kg) once daily for 8 consecutive days. A l l animals were fasted overnight, and on the 9  th  day, each animal in the GBE-treated group received an additional dose of G B E at 500 mg/kg, while each animal in the other two groups received an additional dose of 0.9% NaCl. About 30 min after the last dose, each animal in the GBE-treated group and in one of the 0.9% NaCl-treated groups received an ip injection of A P A P at the dose of 1000 mg/kg (injection volume of 4 ml/kg). The animals in the other 0.9% NaCl-treated group each received an equivalent volume (4 ml/kg ip injection) of the vehicle for A P A P . Food was returned to the cages after the injections. Each animal was terminated about 8 hours after the last injection.  35  2.4  Termination of Animals, Blood Collection, and Preparation of Plasma Animals were anesthetized by ip injection of sodium pentobarbital at 65 mg/kg body  weight (Wawryko et al, 2004) at 8 or 24 h after the ip dose of A P A P (or carbon tetrachloride in the case of the positive control experiment) was given the previous day. The injection volume for sodium pentobarbital was 1 ml/kg body weight. Blood was then collected from the inferior vena cava using heparinized 3 ml syringe (pre-filled with 300 units of heparin , diluted with 0.9% NaCl). Each animal was terminated by decapitation after blood collection was completed. Our protocol used for termination of the animals was in accordance with the guidelines of the Canadian Council on Animal Care. Immediately after blood was collected from each rat into a syringe, the needle was inserted into a B D vacutainer (PLUS-with K E D T A 7.2 mg). After the blood was 2  transferred to the vacutainer, the tube was put on ice immediately. A l l tubes of whole blood were centrifuged at l,000xg for 10 min in a Beckman Model J-6B centrifuge with a JS 4.2 motor within 15 min of collection. Immediately after centrifugation, the supernatant (plasma) was transferred to a clean Eppendorf tube. A l l samples were put on ice before further analysis. Plasma samples were put into a -20 °C freezer when not used within the same day.  2.5 Alanine Aminotransferase (ALT) Assay 2.5.1  Principle of the Assay The A L T assay is a modified method by Reitman and Frankel (1957). The A L T  Reagent contains alanine and cx-ketoglutarate. A L T catalyzes the conversion of alanine to glutamate and a-ketoglutarate to pyruvate.  36  NH  3  .  H-C-COO" I  CH  3  0  0 C—COO" CH  2  CH  2  NH  C—COO"  ALT  CH  COO" a - Ketoglutarate  +  + 3  H —C—COO"  3  CH  2  CH  2  COO" Glutamate  Pyruvate  Figure 2.1.Alanine Principle of the A L T assay (the first step in the reaction). The diagram illustrates the conversion of alanine to pyruvate via A L T . This diagram was modified from the original obtained from: http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/partl/aacarbon.htm  Upon addition of 2,4-dinitrophenylhydrazine (Developer A), hydrazones of the pyruvate (2,4-dinitrophenylhydrazone of pyruvate) are formed, in the presence of NaOH (Developer B). These colored complexes formed from the reaction can be quantified by a spectrophotometer at the wavelength of 540 nm.  CH 3 \ "0^  „0 I  H C 3  HN  .NH  2  HN' NO.  -0  NO.  NaOH  N0  NO.  Pyruvate  COO  2,4-Dinitrophenylhydrazine  2  2,4-Dinitrophenylhydrazone of Pyruvate  Figure 2.2. The second step in the A L T assay. Pyruvate formed from the first step reacts with 2,4-dinitrophenylhydrazine (Developer A) to produce the colored complex (2,4dinitrophenylhydrazone of pyruvate) in the presence of NaOH. (Chemical structures shown here were obtained from: http://www.steve.gb.com/science/molecules.html)  37  2.5.2 Methodology A 0.8 ml aliquot of A L T Reagent was pipetted into each empty glass tube. Exactly 5 min after each tube was put into a 37 °C water bath, 100 ul of each sample (standard or plasma) was pipetted into each tube. In the blank tubes, 100 ui of distilled water was added in place of the standard or plasma. Each tube was then incubated for exactly 30 min before the addition a 0.5 ml aliquot of Developer A . After being left at room temperature for 20 min, a 2.0 ml aliquot of Developer B was added into each tube. Following the addition of each reagent, each tube was vortexed for at least 5 sec. Absorbance measurement did not start for at least 5 min after the addition of Developer B for color development at room temperature. Measurement was completed within 30 min of the addition of Developer B. Absorbance values were measured at 540 nm with a glass cuvette in a Beckman DU®64 Spectrophotometer. Incubation of samples, addition of reagents, and measurement of absorbance values were carried out under subdued light due to the light sensitive nature of the A L T reagents. Net absorbance for each sample was obtained by subtracting the absorbance of the blank from the absorbance of the sample. The A L T level for each sample was then calculated using the equation of the standard curve generated by the linear regression method according to each corresponding net absorbance. Plasma samples with A L T levels greater than the upper limit of linearity of the standard curve were diluted with 0.9% NaCl to be in the dynamic range of the standard curve and were re-assayed on the same day. A standard curve was performed on each day of the A L T assays. Each unknown sample was done in duplicate measurement.  38  2.6 Validation of the A L T Assay 2.6.1  Lower Limit of Linearity The standard (at an A L T level of 76 U/L) was diluted with distilled water to the  desired A L T levels (i.e. 15, 20, 25, and 30 U/L). Six determinations at each A L T level were carried out using the A L T assay. The net absorbance at 540 nm of each A L T level was plotted against its respective A L T level (15, 20, 25, and 30 U/L). The lower limit of linearity was determined to be the lowest A L T level that would still fall within the linear region of the standard curve.  2.6.2  Limit of Quantitation The same data set from the lower limit of linearity experiment was used for the  determination of the limit of quantitation. Mean, standard deviation, and coefficient of variation (CV) were determined for each A L T level (15, 20, 25, and 30 U/L). The LOQ was set as the lowest concentration where the precision of the assay (CV) was still acceptable (less than 15%) (Shah et al, 1992).  2.6.3  Upper Limit of Linearity The standard was diluted with distilled water to make 25, 30, 35, 40, 60 U/L. A n  appropriate volume of the standard was used to reach a final A L T level of 80,100, 120, 130, and 140 U / L in each sample during the A L T assay. Four trials of A L T assays were carried out at each of the A L T levels. The mean net absorbance at 540 nm for each A L T level was plotted against its respective A L T level. Linear regression analysis was performed. The upper limit of linearity was the greatest A L T level included in the linear regression analysis that would produce a coefficient of determination as close to 1 as possible.  39  2.6.4  Dynamic Range Dynamic range is the range between the lower and the upper limits of linearity in  which the assay is linear and able to quantify the amount of an analyte reliably. A standard curve that contains that dynamic range was performed prior to analysis of unknown plasma samples for the A L T levels. The standard was diluted using distilled water to 20, 40, and 60 U / L , and an appropriate volume of the standard was used to reach 80 U / L . A L T assays were carried out in duplicate (two determinations for the absorbance at 540 nm) at each of these A L T levels. There were three determinations for the absorbance of the blank (distilled water instead of standard in each assay) at 540 nm.  2.6.5  Accuracy and Precision To determine the accuracy (measured value reflects the true value in the sample) and  the precision (results are reproducible), A L T assays in quadruplicate were carried out using the standard (at an A L T level of 76 U/L) and the diluted standard (at an A L T level of 30 U/L). Accuracy is presented as a percentage difference [(measured value - expected value) / expected value x 100%] compared to the expected level. Precision is presented as C V at each A L T level. The two A L T levels were chosen to represent the lower and the higher end of the dynamic range of the A L T assay.  2.6.6  Intra-day and Inter-day Variabilities Three female Long-Evans rats (weighed 300 - 350 g) were terminated and plasma  was prepared as described above. Rat plasma was pooled into one sample and was divided into 5 aliquots and stored at -20 °C prior to A L T assays. For intra-day variability, 6 determinations of A L T level for the pooled plasma sample and the standard curve were done  40  on the same day. For inter-day variability, an A L T assay was done each day using the pooled plasma sample (6 replicates) and standards (4 levels in duplicate as described above for construction of the standard curve) for 5 consecutive days.  2.6.7  Inter-laboratory Comparison of A L T Values  A L T levels of the rat plasma samples in the positive control experiment were obtained using the plasma A L T assay in our lab. A n aliquot of each plasma sample was sent to the Central Lab for Veterinarians (Langley, B.C.) for A L T analysis. The A L T values obtained from our lab for each sample were then compared with those reported by the Central Lab for Veterinarians.  2.7 Histological Assessment of Liver Tissues Liver tissue sections were fixed in 10% v/v formalin solution and stained with hematoxylin-eosin (HE). Histological examination of the liver tissues was performed by Wax-it Histology Services (Vancouver, B.C., Canada). The pathologist who analyzed the liver tissues was blinded to the identity of the samples. Assessments of severity of liver damage were presented in scores. To grade steatosis, liver tissues were scored as follows: 0, negative; 1 - 6 , steatosis. To grade necrosis, liver tissues were scored as follows: 0, negative; 1, single cell necrosis, 2 - 5 , coagulative necrosis. In terms of capsular inflammation and sinusoidal inflammation, scoring was as follows: 0, negative; M I N I M U M , very scant amount and is less than mild; 1, mild; 2, moderate; 3, severe.  2.8 Data Analysis A l l data were presented as mean ± standard error of the mean (SEM). Data were analyzed using the one-tailed, unpaired Student's t-test (StudyResult 1.0), one-way A N O V A  41  with Student Newman-Keuls multiple comparison test, or two-way A N O V A (SigmaPlot). Results were considered to be statistically significant when the p-value (p) was less than 0.05.  42  3. RESULTS 3.1 3.1.1  Validation of the A L T Assay Lower Limit of Linearity The lower limit of linearity was the lowest A L T level at which the standard curve was  still linear. The standard was used to prepare the A L T levels to be tested (15, 20, 25, and 30 U/L). Figure 3.1 shows the net absorbance (at 540 nm) at each A L T level being tested. The standard curve was linear at an A L T level as low as 15 U / L . Therefore, the lower limit of linearity for the A L T assay was at least 15 U / L .  0.06  <  i  0.02 J  0.01 -  0.00 -I  1  T  ,  ,  r  1  0  5  10  15  20  25  30  ALT Level (U/L)  Figure 3.1. Determination of the lower limit of linearity for the A L T assay. The standard was used to prepare the A L T levels (15, 20, 25, and 30 U/L). The A L T assay was performed in six replicates at each A L T level. Mean net absorbance at 540 nm with S E M was plotted against each A L T level.  43  3.1.2  Limit of Quantitation The limit of quantitation (LOQ) was set to be at the lowest analyte concentration or  level where the coefficient of variation (CV), or precision of the assay, was still less than 15% (Shah et al, 1992). For the A L T assay, the LOQ was the lowest A L T level at which the assay still had acceptable accuracy and precision. Several A L T levels prepared using the standard were used to determine the LOQ. The coefficients of variation for the net absorbance at 540 nm for 15, 20, 25, and 30 U / L were 23%, 5%, 4%, and 5%, respectively. The LOQ was determined to be at least 20 U / L as the C V increased dramatically from 5% to 23% when the A L T level decreased from 20 U / L to 15 U / L . A coefficient of variation of more than 15% was considered to be unacceptable at the LOQ for an assay (Shah et al, 1992). Figure 3.2 shows the coefficient of variation at each A L T level.  44  25  20  A  c  o .2  15  CD > c CD  10  it CD O  o  5H  20  25  ALT Level (U/L)  Figure 3.2. Determination of the limit of quantitation of the A L T assay. The standard was diluted with distilled water to A L T levels of 15, 20, 25, and 30 U / L . A L T assay was done in six replicates at each A L T level. This was the same data set that was used in the determination of the lower limit of linearity. Net absorbance at 540 nm was calculated using the absorbance of the blank. Mean, standard deviation, and C V of the net absorbance at each level were calculated. C V was plotted for each A L T level.  45  3.1.3  Upper Limit of Linearity The upper limit of linearity was the highest A L T level at which the standard curve  deviates from linearity. For each A L T assay trial, the standard was used to prepare the A L T levels to be tested (25, 30, 35, 40, 60, 80, 100, 120, 130, and 140 U/L). Four trials were performed to determine the upper limit of linearity for the A L T assay. The net absorbance at 540 nm for each level was determined. The mean and S E M for each level were calculated and plotted in Figure 3.3. The curve started to deviate from linearity at 100 U/L. Therefore, 80 U / L was determined to be the upper limit of linearity for the A L T assay.  0.20  i  0.18 -  E c o LO  To  I  0.16 -  i  0.14 0.12 -  CD  o  C CD .Q O CO .Q  <  0.10 0.08 0.06 -  CD  z  0.04 0.02 0.00 -  — I —  20  i  40  60  80  100  120  140  ALT Level (U/L)  Figure 3.3. Determination of the upper limit of linearity for the A L T assay. The standard was used to prepare the following A L T levels: 25, 30, 35, 40, 60, 80, 100, 120, 130, and 140 U / L . Four trials were performed using these levels. Net absorbance at 540 nm was calculated, and the mean net absorbance with S E M was plotted for each level.  46  3.1.4  A Representative Standard Curve Based on the information from the limit of quantitation (LOQ), and the lower and the  upper limits of linearity for the assay, the dynamic range was determined to be from 20 to 80 U / L . Shown below is a representative standard curve for the A L T assay.  0.18 -i  0.02 0.00 -I 0  1  20  —i 40  1  60  —  1  80  ALT Level (U/L)  Figure 3.4. A representative standard curve for the A L T assay. The net absorbance was measured in duplicate at 540 nm for four A L T levels (20, 40, 60, and 80 U/L). Each point represents the mean net absorbance at 540 nm at that A L T level.  47  3.1.5  Accuracy and Precision The mean ± SD of plasma A L T level for the quality control sample (ALT = 76 U/L)  was 80 ± 3 U / L , with a C V of 5% (Table 3.1). The mean plasma A L T level was 5% greater than the expected A L T level of the quality control sample. The quality control sample was diluted to an A L T level of 30 U/L. The mean plasma A L T level was 29 + 6 U / L , with a C V of 20%. The mean plasma A L T level was 3% less than the expected level of 30 U/L. Table 3.1. Accuracy and precision of the A L T assay. The assay was performed in quadruplicate at A L T levels of 30 and 76 U / L . Accuracy was measured as the percentage difference between the measured and the expected A L T level. Precision is presented as C V . Expected A L T Level (U/L)  Measured A L T Level  30  (U/L) 23  Accuracy (% Difference)  Precision (CV in %)  ^3%  20%  +5%  5%  34 25 34 Mean  29  SD  6  76  80 78 76 84  Mean  80  SD  3  48  3.1.6  Intra-day and Inter-day Variabilities Three female Long-Evans rats were sacrificed and a pooled plasma sample was  collected for the intra-day and inter-day variability studies. The pooled plasma sample was divided into five aliquots. In the intra-day study, a standard curve was run together with six replicates of the plasma sample on the same day. The A L T level for each replicate was then calculated using the linear regression method. The mean ± SD for the A L T level of the pooled plasma sample on the same day was 68 + 2 U / L . The coefficient of variation was 3% (Table 3.2). In the inter-day study, a standard curve was run together with six replicates of the plasma sample once daily for five consecutive days. Table 3.3 shows the mean A L T level obtained on each day using the standard curve. The mean ± SD for the A L T level of the plasma sample was 67 ± 2 U / L . The coefficient of variation in the inter-day study was 2%.  49  Table 3.2. Intra-day variability of the A L T assay. The assay was performed in six replicates for a pooled plasma sample prepared from three female Long-Evans rats. This table shows the A L T level for each replicate, together with the mean, SD, and C V of the six replicates. Replicate No. 1  A L T level (U/L) 70  2  65  3  69  4  67  5  71  6  67  Mean  68  SD  2  CV^  3%  50  Table 3.3. Inter-day variability of the A L T assay. The assay was performed in six replicates using the pooled plasma sample for five consecutive days. This table shows the average A L T level obtained from each day, together with the mean, SD, and C V of the averages of the five days. Day 1  Average A L T level (U/L) 68  2  65  3  66  4•  66  5  69  Mean  67  SD  2  CV  2%  51  3.1.7  Positive Control Experiment A positive control experiment using carbon tetrachloride  (CCI4)  the A L T assay could detect increased A L T levels in the samples.  CCI4  was used to ensure was diluted with corn  oil to a 1:1 v/v mixture for ip injection to four male Long-Evans rats at a dose of 2 ml/kg body weight. In the. control group, 4 ml/kg of corn oil was injected to another four male Long-Evans rats. A l l eight animals were terminated 24 h after dosing. Figure 3.5 shows the mean A L T level of the plasma obtained from each treatment group. The mean ± S E M for the CCU-treated group was 311 + 127 U / L , with a C V of 81%. The mean ± S E M for the control group was 42 + 2 U / L , with a C V of 9%. The mean A L T value for the CCl -treated group 4  was significantly greater than that for the control group (p = 0.039, unpaired one-tailed Student's t-test). The mean A L T level of the plasma in the CCU-treated group was 7.5 times greater than that in the control group.  52  500  400  £  A  300  CD I— _ l  < CD  E  200  A  100  A  to _CD Q _  Corn Oil  CCL Treatment  Figure 3.5. Effect of C C U on plasma A L T levels in rats. Four rats were treated with 2 ml/kg C C I 4 ip and were terminated 24 h after dosing. The four rats in the control group were treated with 4 ml/kg of corn oil. Plasma from all rats was prepared and plasma A L T assay was performed using each plasma sample. This figure shows the mean and S E M of A L T level for each treatment group. * Significantly different from corn oil-treated group (p = 0.039)  53  3.1.8  Inter-laboratory Comparison of A L T Values A L T levels of the samples in the carbon tetrachloride experiment obtained through  the A L T assay were compared with those obtained from the Central Lab for Veterinarians. The mean ± S E M for the CCl -treated group was 313 ± 118 U / L with a C V of 75%, which 4  was not significantly different from the value obtained by us (311 + 127 U / L , p = 0.99, unpaired two-tailed Student's t-test). The mean ± S E M for the control group was 56 + 2 U / L , with a C V of 7%, which was significantly different from the value obtained by us (42 ± 2 U / L , p = 0.001, unpaired two-tailed Student's t-test). The mean A L T level in the CCUtreated group was significantly greater than that in the control group (p = 0.036, unpaired one-tailed Student's t-test). These A L T values obtained from the Central Lab for Veterinarians were compared with those obtained from the A L T assay performed in our lab. The A L T levels determined in each laboratory are listed in Table 3.4. The mean plasma A L T level in the CCL-treated group was 5.5 times greater than that in the control group, according to the independent laboratory assessment by the Central Lab for Veterinarians, whereas this fold-increase was 7.5 times according to the A L T assay performed in our lab. Table 3.5 shows the percentage differences in the A L T levels obtained using the A L T assay in our lab as compared to those obtained from the Central Lab for Veterinarians. On average, the percentage differences in the A L T levels in the CCU-treated group was -6%, whereas the percentage difference of the A L T levels in the control group was -25%.  54  Table 3.4. Inter-laboratory comparison of A L T values. Plasma samples were prepared from rats treated with GCI4 and corn oil. The plasma A L T values obtained from our lab were compared with those from the Central Lab for Veterinarians. The mean, S E M , and C V for each group are shown in this table. A L T Level (U/L) Central Lab for Our Lab Veterinarians  Treatment / Animal No.  CC1 : 4  1  76  93  2  565  557  3  111  131  4  492  473  Mean  311*  313*  127 .  118  81%  75%  5  43  59  6  38  51  7  46  57  8  40  59  SEM CV  Corn oil:  #  Mean  42  SEM  2  2  CV  9%  7%  # 5  6  *  Fold-Increase 5.5 7.5 in A L T Level with C C I 4 treatment Significantly different from the corn oil-treated group (p < 0.05) Significantly different from each other (p = 0.001)  55  Table 3.5. Comparison of A L T levels obtained from the A L T assay performed in our lab and those obtained from the Central Lab for Veterinarians. Percentage difference was calculated as (value obtained from our lab - value obtained from Central Lab for Veterinarians)/ value obtained from Central Lab for Veterinarians x 100%.  Treatment / Animal No. CC1 :  % Difference  1  -19%  2  +2%  3  -15%  4  +4%  Mean % difference  -6%  4  Corn oil: 5  -27%  6  -26%  7  -20%  8  -33%  Mean % difference  -25%  56  3.2  Control Experiments with A P A P  3.2.1  Effect of Fasting on Plasma A L T Levels in Rats Administered A P A P The purpose of this experiment was to determine the effect of fasting on plasma A L T  levels in rats injected with A P A P . Seventeen adult male rats were divided into four groups (four rats in the non-fasted/vehicle group, four rats in the non-fasted/APAP group, four rats in the fasted/vehicle group, and five rats in the fasted/APAP group). Rats were either fasted or not fasted overnight and received single injection of either 20% Tween 80 in 0.9% NaCl (the vehicle used to suspend APAP) or A P A P at 1000 mg/kg the next morning, followed by termination at 24 h after the last dose. The mean ± S E M of the plasma A L T level in the nonfasted/vehicle group was 38 ± 4 U / L , which was not significantly different from that in the non-fasted/APAP group (41 + 4 U/L) (Figure 3.6). In the fasted groups, however, the mean plasma A L T level in the fasted/APAP group was 245 ± 1 0 6 U / L , which was 4.5 times greater than that in the fasted/vehicle group (55 ± 1 U/L), in which the animals were also fasted but were injected with the vehicle instead of A P A P . The C V in the fasted group treated with A P A P was very large (97%) compared to the rest of the groups (ranged from 2% to 21%). There was not a statistical significant interaction between fasting and treatment (p = 0.167, two-way A N O V A ) .  57  400  300  A  > 200  < ro  E w ro  Q_  100  A  NF/Vehicle  NF/APAP  FA/ehicle  F/APAP  Treatment  Figure 3.6. Effect of fasting on plasma A L T levels in rats treated with A P A P or the vehicle. Animals were fasted overnight (F) or had free access to food (NF). They were then injected with either A P A P at 1000 mg/kg ip or 20% Tween 80 in 0.9% NaCl (vehicle) the next day. A l l animals were terminated 24 after the last injection. This figure shows mean and S E M of plasma A L T level for each group (N = 4 in the NF/Vehicle group; N = 4 in the N F / A P A P group; N = 4 in the F/Vehicle group; N = 5 in the F/APAP group).  58  3.2.2  Effect of the Vehicle on Plasma A L T Levels in Rats The purpose of this experiment was to investigate i f the vehicle used to suspend  A P A P (20% Tween 80 in 0.9% NaCl) would affect the plasma A L T levels in rats. Seven adult male rats were assigned into two groups (four rats in the 0.9% saline-treated group and three rats in the vehicle-treated group). Rats in both groups were fasted overnight before each was injected with either 0.9% NaCl (saline group) or 20% Tween 80 in 0.9% NaCl (vehicle group). They were terminated at 24 h after the last dose. The mean + S E M of the plasma A L T level in the rats treated with the vehicle was 63 ± 10 U / L (CV = 27%), which was not significantly different (p = 0.73, unpaired two-tailed Student's t-test) from the mean plasma A L T level in the rats treated with 0.9% NaCl (66 ± 3 U / L , with C V = 10%) (Table 3.6).  59  Table 3.6. Effect of the vehicle on plasma A L T levels in rats. This table shows individual plasma A L T level for each animal, together with the mean, S E M , and C V for each group.  Treatment / Animal No. 0.9% NaCl:  A L T Level (U/L)  1  75  2  66  3  63  4  61  Mean  66  SEM  3  CV  10%  20% Tween 80 in 0.9% NaCl (vehicle): 5  81  6  47  7  61  Mean  63  SEM  10  CV  27%  60  3.2.3  Effect of the Dose of A P A P on Plasma A L T Levels in Rats To study the effect of the dose of A P A P on the plasma A L T levels in rats, the  following experiment was performed. Twenty adult male rats were assigned into four groups (five rats in the control group, five rats in the 700 mg/kg group, four rats in the 1000 mg/kg group, and six rats in the 1300 mg/kg group). A l l rats were fasted overnight and rats in the different treatment groups were injected ip with various doses (700, 1000, and 1300 mg/kg body weight) of A P A P . In the control group, animals were injected with the vehicle. A l l rats were terminated at 8 h after the last injection. The mean ± S E M of the plasma A L T level was 30 ± 3 U / L (CV = 22%) in the control group. In the treatment groups, the mean plasma A L T level was 65 ± 11 U / L (CV - 37%), 137 ± 34 U / L (CV = 50%), and 136 ± 55 U / L (CV = 99%), in the 700 mg/kg, 1000 mg/kg, and 1300 mg/kg groups, respectively (Figure 3.7). The mean plasma A L T level in the 1300 mg/kg group was 4.5 times greater than the mean plasma A L T level in the control group (30 U/L). In the 700 mg/kg and the 1000 mg/kg groups, the mean plasma A L T level was twice and 4.6 times greater than the control group, respectively. The mean plasma A L T level of the 1000 mg/kg group was similar to that of the 1300 mg/kg group. However, the differences in the mean plasma A L T levels among the four groups were not statistically significant (p = 0.14, one-way A N O V A ) .  61  200  1000  700  1300  Acetaminophen (mg/kg)  Figure 3.7. Effect of the dose of A P A P on plasma A L T levels in rats. Animals were fasted overnight and injected ip with the vehicle (for the control group, N = 5) or different doses of A P A P (700 mg/kg for five rats, 1000 mg/kg for four rats, or 1300 mg/kg for six rats) the next day. Fasting continued and they were terminated 8 h after the last injection. The mean + S E M of the plasma A L T level for the treatment group is shown in this figure. The mean ± S E M of the plasma A L T value for the vehicle-treated control group was 30 ± 3 U / L .  62  3.3  Experiments with G B E  3.3.1  Effect of G B E on Plasma A L T Levels in Rats To determine whether G B E has any effect on the plasma A L T levels in rats, the  following study was performed. Five adult male rats were injected with 500 mg/kg of G B E ip (treatment group), whereas another five adult male rats were injected with 0.9% NaCl (control group) once daily for 8 consecutive days. Animals were sacrificed 24 h from the last injection after an overnight fast. The mean ± S E M of the plasma A L T level for the treatment group was 50 ± 3 U / L (CV = 14%), which was not significantly different from the mean plasma A L T level for the control group (54 ± 6 U / L , C V = 25%) (p = 0.59, unpaired twotailed Student's t-test) (Figure 3.8).  63  70  60  50 CD > CD  <  40  A  30  CO  E  CO  I  20 10  0.9% NaCl  Treatment  GBE  Figure 3.8. Effect of G B E on plasma A L T levels in rats. Animals were injected ip with either 0.9% NaCl (N = 5) or G B E (N = 5) at 500 mg/kg once daily for 8 days. Animals were terminated 24 h after the last dose. Plasma from each animal was prepared and the A L T level was determined for each plasma sample. The means and S E M for the plasma A L T levels of both groups were plotted in this figure.  64  3.3.2  Effect of G B E on Liver Histology in Rats In addition to plasma samples prepared from the rats as described in Section 3.3.1,  hepatic tissues were also excised from these rats and stored in 10% formalin immediately after termination. Histological analysis was performed on each piece of tissue and a score was assigned to each animal's liver in terms of steatosis, necrosis, capsular inflammation, and sinusoidal dilatation around the central vein (Table 3.7). G B E did not cause severe steatosis or necrosis to hepatic tissues as compared to the vehicle (with the exception of Animal D7, in which severe necrosis and moderate steatosis and capsule inflammation were reported). Severe capsular inflammation was reported for Animal D4.  Table 3.7. Effect of G B E on liver histology scores in rats. Rats were treated as described in the legend to Figure 3.8. Liver tissues from each animal were assessed and a score was assigned for each animal's liver histology in terms of steatosis, necrosis, capsular inflammation, and sinusoidal dilatation. Animal ID  Treatment  Steatosis  Necrosis  Capsular Inflammation  3 0 0-1 D4 1 0 0 D5 MINIMUM 1 0 GBE D6 2 1-2 3 D7 0 0 0 D8 0 0 0 Fl 1 0 o F2 0 0 0 0.9% NaCl F3 0 0 0 F4 0 0 0 F5 Steatosis: 0 = Negative, 1-6 = Steatosis Necrosis: 0 = Negative, 1 = Single cell necrosis, 2-5 = coagulative necrosis Capsular inflammation: 1 = Mild, 2 = Moderate, 3 = Severe Sinusoidal dilatation around central vein: 1 = Mild, 2 = Moderate, 3 = Severe M I N I M U M = very scant amount and is less than mild  65  Sinusoidal dilatation (Hepatocyte collapse) 0 0 MINIMUM MINIMUM 0 0 0-1 0 0 0  3.4  Experiments with G B E and A P A P  3.4.1  Effect of Pretreatment with G B E on Plasma A L T Levels in Rats Administered A P A P The purpose of this study was to investigate whether G B E pretreatment could  modulate A P AP-induced hepatotoxicity in rats. Twenty-two adult male rats were divided into three groups - five in the 0.9% NaCl/20% Tween 80 (vehicle-treated group) group, eight in the 0.9% NaCl/APAP (APAP-treated) group, and nine in the combination group (GBE and APAP). During the pretreatment period, rats in the vehicle-treated control group and the APAP-treated group were injected with 0.9% NaCl ip, whereas the rats in the combination group were injected with G B E at 500 mg/kg ip once daily for 9 consecutive days. Thirty minutes after the last dose, the rats in the vehicle-treated control group were injected with the vehicle ip, whereas those in the APAP-treated group and the combination group were injected with 1000 mg/kg A P A P ip. A l l rats were terminated 8 h after the last dose was given. As shown in Figure 3.9, the mean ± S E M of the plasma A L T level for the vehicletreated control group, the APAP-treated group, and the combination group, was 41 ± 3 U / L (CV = 16%), 114 ± 22 U / L (CV = 55%), and 53 ± 7 U / L (CV = 37%), respectively. There was a significant difference in the mean plasma A L T levels among the three groups (p = 0.008, one-way A N O V A ) . Moreover, the mean plasma A L T level for the APAP-treated group was shown to be significantly greater than both the combination group (p = 0.008, Student Newman-Keuls (SNK) multiple comparison test) and the vehicle-treated control group (p = 0.016, SNK multiple comparison test). The mean plasma A L T levels for the combination group and the vehicle-treated control group were not significantly different from each other (p = 0.624, SNK multiple comparison test).  66  160 -j 140 120 -  >  100  CD  80  < TO60 E  CO TO  Q_  40 20 H 0  ~i  1  0.9% NaCIA/ehicle  0.9% NaCI/APAP  GBE/APAP  Treatment  Figure 3.9. Effect of pretreatment with G B E on plasma A L T levels in rats administered A P A P . Animals received either G B E (500 mg/kg) or 0.9% NaCl ip once daily for 9 consecutive days (an overnight fast before the 9 dose). Thirty minutes after the last dose, they were injected ip with either A P A P (1000 mg/kg) or the vehicle. Animals were terminated 8 h thereafter. The mean and S E M of plasma A L T level for each group (vehicle, N = 5; A P A P , N = 8; G B E / A P A P , N = 8 because 1 rat died before plasma could be prepared) are shown in this figure. * Significantly different from both the vehicle and the G B E / A P A P groups (p < 0.05). th  67  3.4.2  Effect of Pretreatment with G B E on Liver Histology in Rats Administered A P A P In addition to plasma samples prepared from the twenty-two rats as described in  Section 3.4.1, hepatic tissues were also isolated from these rats and stored in 10% v/v formalin immediately after termination. Histological analysis was performed on each piece of tissue and a score was assigned to each animal's liver in terms of steatosis, necrosis, capsular inflammation, and sinusoidal dilatation around the central vein (Table 3.8). Hepatic tissues in all five animals in the vehicle-treated group did not show any steatosis, necrosis, capsular inflammation, or sinusoidal dilatation. The number of animals in each treatment group with a particular histology score for each category has been summarized in Figures 3.10, 3.11, 3.12, and 3.13. In terms of steatosis, two out of the eight rats in the APAP-treated group, while none of the rats in the combination group, scored 6 out of 6. However, three out of the nine rats in the combination group, while only one out of the eight rats in the A P A P treated group, scored 5 out of 6. In terms of hepatic necrosis, all eight rats in the A P A P treated group scored 2 (out of 5) or lower, whereas three out of the nine rats in the combination group scored 3 (out of 5) or higher. The slightly worse hepatic tissue necrosis reported from the combination group was in accordance with the higher capsular inflammation scores in the group. Two rats in the combination group showed severe (vs. none in the APAP-treated group) and five rats showed moderate (only one in the A P A P treated group) capsular inflammation. Also, more rats in the combination group (four out of the nine vs. two out of the eight in the APAP-treated group) showed moderate sinusoidal dilatation in the liver, although both groups had two rats that showed severe sinusoidal dilatation.  68  Table 3.8. Effect of G B E on liver histology scores in rats administered A P A P . Rats were treated as described in the legend to Figure 3.9. A l l rats were terminated 8 h after last dose. Scores were assessed for each animal's liver histology in terms of steatosis, necrosis, capsular inflammation, and sinusoidal dilatation.  Animal ID  CI C2 C3 C4 C5 Bl B2 B3 B4 B5 B6 B7 El Al A2 A3 A4 A6 A7 DI D2 D3  Treatment  0.9% NaCl/ Vehicle  0.9% NaCl/ APAP  GBE/ APAP  Steatosis  Necrosis  Capsular Inflammation  0 0 0 0 0 3 0 6 2 5 3 0 6 0 0 4 3 5 0 5 5 4  0 0 0 0 0 1 0 2 1 1 1 0 1 .0 5 1 4 3 2 1 1 1  0 0 0 0 0 1 0 2 1 1 1 0 0 0 3 2 2 2 3 2 MINIMUM 2  Steatosis: 0 = Negative, 1-6 = Steatosis Necrosis: 0 = Negative, 1 = Single cell necrosis, 2-5 = Coagulative necrosis Capsular inflammation: 1 = Mild, 2 = Moderate, 3 = Severe Sinusoidal dilatation around central vein: 1 = Mild, 2 = Moderate, 3 = Severe M I N I M U M = very scant amount and is less than mild  69  Sinusoidal dilatation (Hepatocyte collapse) 0 0 0 0 0 1 MINIMUM 3 MINIMUM 3 2 0 2 0 1 2 2 3 0 2 3 2  50  09 . % NaC/IAPAP F^l GBE/APAP  40 A CO CD  30  o  CD 20 H CD  -*—»  c CD O i_ CD  0_  1 0  A  0  Negative  1  2  3  4  Score (Steatosis)  Severe  Figure 3.10. Summary of scores for steatosis in rat liver tissues. Rats were treated as described in the legend to Figure 3.9. The percentage of rats in the two key treatment groups (0.9% NaCl/APAP and GBE/APAP) having a specific score is shown in this figure (0 = Negative, 1-6 = Steatosis).  70  09 . % NaC/IAPAP GBE/APAP  60 A CO  -4—' CD <4—  o  CD CD  40  ro -I—»  c CD O i_ CD  D_  20  o  Negative  LI 1  2  3  5  Severe  Score (Necrosis)  Figure 3.11. Summary of scores for necrosis in rat liver tissues. Rats were treated as described in the legend to Figure 3.9. The percentage of rats in each treatment group having a specific score is shown in this figure (0 = Negative, 1 = Single cell necrosis, 2-5 = Coagulative necrosis).  71  60  50  0.9% NaCI/APAP GBE/APAP  H  -4—• 40 CD  O  0 a  O) CD  30  -4—*  c  CD  o  £  20  10 4  I St;  Mi m  0  Negative  MIN S  _J 1  r  2  core (Capsular Inflammation)  3  Severe  Figure 3.12. Summary of scores for capsular inflammation in rat liver tissues. Rats were treated as described in the legend to Figure 3.9. The percentage of rats in each treatment group having a specific score is shown in this figure (1 = Mild, 2 = Moderate, 3 = Severe, M I N I M U M (MIN) = very scant amount and is less than mild).  72  50  ••I 09 . % NaC/IAPAP r—1 GBE/APAP  40 A co  -*—»  CD  4—  o CD O) CD  30  I 20  i_ CD Q_  10  Negative  MN I  score (Sinusoidal Dilatation)  3  Severe  Figure 3.13. Summary of scores for sinusoidal dilatation in rat liver tissues. Rats were treated as described in the legend to Figure 3.9. The percentage of rats in each treatment group having a specific score is shown in this figure (1 = Mild, 2 = Moderate, 3 = Severe, M I N I M U M (MIN) = very scant amount and is less than mild).  73  4. D I S C U S S I O N 4.1  Validation of the A L T Assay Assay validation is essential to ensure the accuracy and precision of an assay. A L T is  an enzyme that is produced in hepatocytes. The hepatotoxic metabolite of A P A P , NAPQI, can cause hepatocellular death and centrilobular liver necrosis, which can lead to "leaky membranes" of hepatocytes, resulting in leakage of A L T from cytosol of hepatocytes into the circulation, and hence leading to elevation of A L T level in the plasma. Another marker of liver damage is aspartate aminotransferase (AST). However, AST is a less specific marker compared to A L T because AST exists in tissues other than liver, such as in heart and skeletal muscles (Mason, 2004). In this study, only the plasma A L T assay was used because of problems in setting-up the AST assay.  4.1.1  Accuracy Accuracy is defined as the "closeness of agreement between the value that is accepted  either as a conventional true value or an accepted reference value and the value found" (Lundblad, 2001). A sample (in which nominal level was known) from the plasma A L T kit was used for determination of accuracy of the A L T assay. The percentage differences between the measured A L T level and the true A L T level in the quality control sample were - 3% and +5% at 30 U / L and 76 U/L, respectively (Table 3.1), which were within the acceptable level of 15% (Shah et al, 1992). Therefore, the assay was considered to be accurate.  74  4.1.2  Precision Precision is "the closeness of agreement (degree of scatter) between a series of  measurements obtained from multiple sampling of the same homogeneous sample under prescribed conditions" (Lundblad, 2001). At 76 U / L , the C V was less than 15% (Table 3.1), and hence was considered to be acceptable (Shah et al, 1992). However, the C V at 30 U / L was 20%) (more than 15%). The assay was precise at the greater A L T level of 76 U / L . At 30 U / L , the assay was not as precise. The greater variability at the lower A L T level could be explained by how the C V (SD/Mean x 100%) is calculated. A smaller mean value would result in a greater C V .  4.1.3  Reproducibility To know whether the assay would be reproducible on the same day and from a day-  to-day basis, intra-day and inter-day variabilities of the plasma A L T assay were investigated. The results of the study were reported in Section 3.1.6. As shown in Table 3.2 and Table 3.3, the relatively small intra-day and inter-day CVs (2% and 3%, respectively) were considered to be acceptable as they were both less than 15% (Shah et al, 1992). To investigate whether the plasma A L T assay could measure an elevated A L T level in rats with liver damage, a positive control experiment was performed using a known hepatotoxin, CC1 . The statistical significant elevation in plasma A L T level in the C C 1 4  4  treated group (as shown in Figure 3.5) revealed that the plasma A L T assay used in this study was able to quantify elevated plasma A L T levels as caused by liver damage. When compared to literature values, Janakat and Al-Merie (2002) also reported a statistical increase in plasma A L T levels in male albino Wistar rats at 24 h after CC1 administration (2 ml/kg ip 4  in olive oil). This was the same treatment regimen used in the present study. The mean ±  75  S E M of plasma A L T levels was 686 + 9 U / L at 24 h after the dose, which was a 7-fold increase compared to its control group (104 ± 4 U/L). Although the mean plasma A L T level in the CCU-treated group as quantified by our assay method (311 U/L) did not agree with the value (686 ± 9 U/L) reported from Janakat and Al-Merie (2002), the fold-increases compared to the control group were similar (7.5-fold increase from our assay vs. 7-fold increase from Janakat and Al-Merie, 2002). As part of validation, it was necessary to ensure the plasma A L T levels obtained from our plasma A L T assay were comparable to those obtained from an independent laboratory. In fact, inter-laboratory comparison of the plasma A L T values obtained from the carbon tetrachloride study showed that our plasma A L T assay was able to quantify similar plasma A L T levels as another laboratory (Table 3.4, Table 3.5). In both the CCU-treated and the control group, both our mean plasma A L T levels and the variabilities (CV) were similar to those from the other laboratory. The fold-increase in plasma A L T level in the CCU-treated group when compared to the control group was 7.5, according to our plasma A L T assay. This fold-increase was 5.5, as reported from another laboratory. This difference in foldincrease could be explained by the greater plasma A L T level in the control group as reported by another laboratory (56 ± 2 U / L was significantly greater than 42 ± 2 U / L , p = 0.001, unpaired two-tailed Student's t-test). Although the fold-increase obtained from our plasma A L T assay data was different from that reported from another laboratory, both laboratories were able to quantify a statistical significant increase in mean plasma A L T level in the C C U treated group when compared to the control group.  76  4.1.4  Dynamic Range A standard curve was necessary for quantification of A L T level in unknown plasma  samples. Considering the lower and upper limit of linearity, together with the LOQ, the standard curve was constructed using A L T levels between 20 U / L to 80 U / L (Figure 3.4). The plasma A L T assay used in the current study has a narrow dynamic range. Most of the samples with minimally elevated plasma A L T levels were beyond the upper limit of the dynamic range. This is a major limitation of the assay, as plasma samples suspected to have elevated A L T levels would have to be diluted. The dilution had to be done so that the diluted plasma sample would have an A L T level within the dynamic range.  4.1.5  Sensitivity Sensitivity is defined as "the slope of the calibration curve" and is commonly  misinterpreted as limit of detection (Karnes et al, 1991). A n assay is sensitive " i f small changes in concentration cause larger changes in analytical response" (Karnes et al, 1991). In the current study, the standard curve has a slope of 0.002 absorbance unit per unit of A L T per liter of plasma. Therefore, it is not a very sensitive assay for quantification of A L T levels, as it makes differentiation between samples with control levels very difficult.  4.1.6  Summary The plasma A L T assay used in this study was considered to be accurate, precise, and  reproducible, although it had a limited dynamic range and was not sensitive.  77  4.2  Control Experiments with A P A P  4.2.1  Effect of Fasting on Plasma A L T Levels in Rats Administered A P A P Fasting was necessary to achieve a greater plasma A L T level in the APAP-treated  group compared to the control group. As described in Section 3.2.1 and as illustrated in Figure 3.6, in the two non-fasted groups of rats, the mean plasma A L T levels in the group of rats given A P A P was not significantly different from those in rats that were given the vehicle used to suspend A P A P (20% Tween 80 in 0.9% NaCl). However, upon an overnight fast, the mean plasma A L T levels in rats dosed with A P A P became greater than those in non-fasted rats dosed with A P A P . Fasting per se did not cause an elevation in plasma A L T levels because the mean plasma A L T levels were not elevated in fasted rats dosed with the vehicle when compared to fasted rats dosed with 0.9% NaCl. Although the mean A L T plasma levels among the four groups in that study were not statistically different from each other, the results of this control experiment provided us with the insight that fasting the rats before dosing with A P A P was necessary to obtain an increase in plasma A L T levels. The lack of statistical significance in the results can be explained by the insufficient power of the study. The number of rats used in the study should be increased to counter the highly variable plasma A L T levels observed in the fasted rats treated with A P A P . However, the experiment was not repeated for ethnical reasons. This was only a control experiment to verify the need for fasting in subsequent experiments. The results of this experiment clearly suggested the need for fasting to have elevated plasma A L T levels in rats injected with A P A P . The finding of this experiment verified the results of many rodent studies that showed fasting was required to increase the susceptibility of the animals to AP AP-induced  78  hepatotoxicity. In some studies using Sprague-Dawley and albino Wistar rats, an overnight fast was required before ip injection (750, 800, or 1000 mg/kg) or oral administration (2000 mg/kg) of A P A P (Dehpour et al, 1999; Kukongviriyapan et al; 2003; Bauer et al, 2000; Brennan et al, 1994; Kucukardali et al, 2002). In one of the studies using male LongEvans rats, animals were fasted 20 h before and for 2 h after ip injection with A P A P at 650 mg/kg (Lim et al, 1995). Investigators fasted mice from 16 to 24 h prior to oral (300 or 800 mg/kg) or ip (300 or 500 mg/kg) injection of A P A P in some studies (Thomsen et al, 1995; Gamal el-din et al, 2003; Manautou et al, 1996; Hewawasam et al, 2003). There were a few studies that did not require fasting (or fasting was not mentioned) but the investigators were able to report a statistical significant elevation i n plasma A L T levels in rats after treatment of A P A P . For instance, Shenoy et al. (2002) reported an 8-fold increase compared to control in plasma A L T levels in adult male albino Wistar rats after receiving A P A P at 2000 mg/kg body weight orally once daily for 3 days. Gilani and Janbaz (1995) reported a 19-fold increase in plasma A L T levels in adult male albino Wistar rats at 24 h after a single oral dose of A P A P at 640 mg/kg body weight. Ahmed and Khater (2001) reported a 6-fold increase in plasma A L T levels in adult male Sprague-Dawley rats at 24 h after a single oral dose of A P A P at 640 mg/kg body weight. Porchezhian and Ansari (2005) reported a similar 6-fold increase at 48 h after an oral dose of A P A P at 3000 mg/kg body weight. The mechanism through which fasting potentiates APAP-induced hepatotoxicity were investigated by Price et al. (1987). A n overnight fast was initiated in Long-Evans Hooded rats 24 h prior to ip administration of various doses of A P A P from 150 mg/kg to 1000 mg/kg. As compared to fed rats, significantly greater plasma glutamic-oxaloacetic transaminase  79  (former name for AST) levels were reported in fasted rats 24 h after A P A P doses of 500 mg/kg or greater. Also, both the incidence and severity of liver necrosis (at 48 h after APAP) were increased in rats received A P A P doses of 300 mg/kg or greater. Hepatic glutathione and hepatic glycogen levels were significantly lower in fasted rats at 0, 2, 4, and 6 h after ip administration of A P A P at a dosage of 700 mg/kg. In addition, the elimination half-life of A P A P was reported to be significantly longer (1.98 h) in fasted rats than in fed rats (1.52 h). Hence, it was suggested that acute fasting increased susceptibility of rats to A P AP-induced hepatotoxicity by reducing hepatic glutathione levels and hepatic glycogen levels, leading to impairment of both the detoxification pathway of NAPQI and glucuronidation of A P A P elimination, respectively, in fasted rats (Price et al, 1987). Reduced levels of hepatic glutathione in fasted rats could potentially lead to accumulation of the hepatotoxic metabolite NAPQI as binding of glutathione to NAPQI is essential for its elimination. A reduced level of hepatic glycogen could lead to a lower supply of glucose for synthesis of UDP-glucuronic acid, which is a co-substrate for UGT. Hence, a reduced hepatic glycogen level upon fasting could potentially lead to a lower rate of glucuronidation, a lower rate of elimination of A P A P , and ultimately an accumulation of NAPQI (Price et al, 1987). The current study used a fasted animal model to conduct the drug-induced hepatotoxicity experiment. Hence, it would be important to address the clinical relevance of using a fasted model. Clinical data also suggested increased risk of AP AP-induced hepatotoxicity in patients who had been fasted (Whitcomb and Block, 1994). A n anorexic , patient had severe liver failure after ingestion of A P A P , although the effect could have been due to the concurrent administration of carbamazepine (Young and Mazure, 1998). Also, when a patient is sick (e.g. fever), appetite will likely decrease.  80  In summary, many studies revealed that overnight fasting prior to A P A P dosing was necessary to increase the susceptibility of rats and mice to A P AP-induced hepatotoxicity. This is in agreement with the results found in the current study.  4.2.2  Effect of the Vehicle on Plasma A L T Levels in Rats A P A P has low water solubility. In the process of identifying a suitable suspending  vehicle for A P A P , it was essential to ensure the vehicle itself would not cause liver toxicity. Hence, it was necessary to investigate the effect of the vehicle used in this study (20% Tween 80 in 0.9% NaCl, Gardener et al, 1998) on plasma A L T levels in rats. As discussed in Section 3.2.2, 24 h after the injection, the mean plasma A L T levels in rats injected with the vehicle was not significantly different from those in rats injected with 0.9% NaCl. Therefore, it was concluded that 20% Tween 80 in 0.9% NaCl was a suitable vehicle to suspend A P A P because it did not cause an increase in plasma A L T levels. The majority of studies published in the literature involved using a vehicle for A P A P for oral administration or ip injection. Many different kinds of vehicle such as 0.2% tragacanth gum (Jorgensen et al, 1988; Poulsen et al, 1985), 20% Tween 80 in 0.9% NaCl (Gardner et al, 1998; Kamanaka et al, 2003), 1% methylcellulose (Janbaz and Gilani, 2000; Ahmed and Khater, 2001), 50% propylene glycol (Devi et al, 2004), and dimethyl sulfoxide (Nakae et al, 1988), have been used in published studies involving A P A P . The vehicle used to suspend A P A P used in our study was 20% Tween 80 in 0.9% NaCl with reference to the studies performed by Gardner et al. (1998) and Kamanaka et al. (2003) who also used the same A P A P dose (1000 mg/kg ip) as in the current study. In the current study, 20% Tween 80 in 0.9% NaCl did not cause an elevation in plasma A L T levels.  81  4.2.3  Effect of the Dose of A P A P on Plasma A L T Levels in Rats The similar plasma A L T levels for both the 1000 mg/kg and the 1300 mg/kg groups  indicated that the dose-response curve (Figure 3.7) had reached a plateau at the dose of 1000 mg/kg. The variability (CV) of the mean plasma A L T levels in the current study increased with the dose of A P A P used. The C V in mean plasma A L T level gradually increased from 22% in the control group, to 37%, 50%, and 99%, in the 700 mg/kg, 1000 mg/kg, and 1300 mg/kg, respectively. There are no published data available that involve plasma A L T measurements in fasted male Long-Evans rats at 8 h after an A P A P dose of 1000 mg/kg ip. However, there was one study in which fasted female Sprague-Dawley rats were used (Bauer et al, 2000). The mean plasma A L T levels at 8 h after an A P A P dose of 1000 mg/kg ip was estimated (data were shown in a figure) to be at 165 U / L , which was about 5.5-fold greater than control level (estimated to be at 30 U/L). The fold-increase at this particular ip dose of A P A P and time-point was comparable to ours (4.6-fold, see Section 4.2.3), considering that a different strain of rats (Sprague-Dawley) and a different vehicle for A P A P (37 °C warm 0.9% NaCl with pH adjusted to 10 with 0.1 N NaOH) were used in the study by Bauer et al. (2000). In this study, the CVs ranged from 22% to 99% among the four groups. The large inter-animal variability in plasma A L T levels was not caused by technical problems in the A L T assay. Intra-day and inter-day variability data (CV) from the validation study ranged from 2% to 3% and hence provided direct evidence to support the reproducibility of the A L T assay. Also, C V was large (81%) for the CCU-treated group in the carbon tetrachloride study. The C V was similarly large (75%) for the CCU-treated group as determined by another laboratory in the inter-laboratory comparison of the data. The large variability  82  observed in the APAP-treated rats could be due to biological variability in the susceptibility of rats to A P AP-induced hepatotoxicity. Different rats have different basal glutathione levels and glutathione level has to reach below a certain threshold before toxicity is observed in the animals. Hence, the large inter-animal variability can be potentially explained by the differences in susceptibility of each rat to A P AP-induced hepatotoxicity. There are no published data on the variability of plasma A L T levels in Long-Evans rats at 8 h after being injected with an A P A P dose of 1000 mg/kg ip. However, plasma AST data are available from one study and it was reported that mean ± S E M of the plasma AST level in male Long-Evans rats at 24 h after A P A P dose at 1000 mg/kg was 3858 ± 590 U / L (Price et al, 1987). This represents a C V of 48%, which was comparable to both the C V for the 1000 mg/kg group in the dose-response study (50%, see Section 3.2.3) and that in the A P A P group in the combination study (55%, see Section 3.4.1), although that the plasma A L T levels were measured at 8 h after the A P A P dose. Although the differences in mean plasma A L T levels among the four groups were not statistically significant, the results of this study confirmed the use of 1000 mg/kg as the ip dose in elevating plasma A L T levels by A P A P . The lack of statistical significance in the study was caused by lack of power in the study. Increasing the number of subjects used in each group could potentially reduce the variability and hence increased the likelihood of a statistical significant result. Again, more animals were not used due to ethnical reasons and the fact that this was only one of the control experiments to verify the dose of A P A P being used in subsequent studies. Overall, results from our A P A P dose-response control experiment did agree with the results published by other groups (Price and Jollow, 1986; Price et al, 1987; Bauer et al,  83  2000). This ip dose of A P A P at 1000 mg/kg was used because it has been shown to cause hepatotoxicity in fasted Long-Evans rats up to 24 h after the A P A P dose (Price and Jollow, 1986; Price ef al, 1987).  4.3  Effect of G B E on Liver Toxicity in Rats  4.3.1  Effect of G B E on Plasma A L T Levels in Rats Treatment of rats with G B E at an ip dose of 500 mg/kg once daily for 8 consecutive  days did not increase plasma A L T levels. The mean plasma A L T level in rats treated with G B E did not differ significantly from the control group (treated with a once daily ip injection of 0.9% NaCl for 8 consecutive days) (Figure 3.8). Therefore, G B E (using the current treatment regimen) did not have any effect on the plasma A L T levels in rats. There were no other studies that used an ip dose of G B E at 500 mg/kg which made direction comparison of results difficult. Sener et al. (2005) injected male albino Wistar rats at 50 mg/kg/day ip once daily for 28 days. The average plasma A L T level in rats dosed with G B E was not significantly different from the control group. Shinozuka et al. (2002) fed a 0.5% w/w G B E diet to male Wistar rats for 4 weeks and no increase in A L T was observed compared to the control group. In the studies performed by Sener et al. (2005) and Shinozuka et al. (2002), the time between the last dose of G B E and the time of termination was not mentioned. Albino Wistar rats of both sexes were fasted 12 h prior to receiving two ip doses of G B E at 50 mg/kg/day (12 h apart) (Sakarcan et al, 2005). Average plasma A L T levels in rats injected with G B E were not significantly different from that in the control group at 24 h after termination (same time from G B E dose to termination as in our study).  84  4.3.2  Effect of G B E on Liver Histology in Rats In contrast to the plasma A L T data, the histology data indicated that G B E given at  500 mg/kg ip caused damage in the liver tissues. As shown in Table 3.7, saline treatment did not cause any effect in the liver tissues because all scores reported were "0" (normal) for all five animals in each of the four categories (steatosis, necrosis, capsular inflammation, and sinusoidal dilatation). However, mild to severe necrosis, capsular inflammation, and steatosis were observed in some of the liver tissues from the G B E group (Table 3.7), which indicated some effects of G B E on rats at an ip dose of 500 mg/kg once daily for 8 consecutive days.  4.4 4.4.1  Effect of G B E Pretreatment on Liver Toxicity in Rats treated with A P A P Effect of G B E on Plasma A L T Levels in Rats treated with A P A P In the combination study where pretreatment of rats with G B E for 9 days was  followed by a hepatotoxic dose of A P A P , the same G B E dose of 500 mg/kg was used. The results from the plasma A L T data suggested that G B E was blocking the A PAP-induced hepatotoxicity. The mean plasma A L T level in the combination group was not significantly different from the control group in which the rats were pretreated with 0.9% NaCl. Without the G B E pretreatment, the mean plasma A L T level was significantly elevated only in the APAP-treated group. There is no similar study published in the literature for comparison of the results involving G B E (at 500 mg/kg ip once daily for 9 days) and A P A P .  4.4.2  Effect of other chemicals in G B E on Plasma A L T Levels in Mice treated with A P A P Janbaz et al. (2002) reported a protective effect by a chemical (rutin) in G B E on  AP AP-induced hepatotoxicity in Swiss male mice using plasma A L T levels as a marker for  85  liver toxicity. Rutin is a diglycoside form of quercetin. This compound was quantified in the G B E used in the current study (Table A l ) . Assuming that all diglycosides present in the G B E used in the current study are rutin, a 500 mg/kg dose of G B E would be equivalent to 20 mg/kg of rutin. However, rutin was given orally, instead of intraperitoneally, to the rats in the study by Janbaz et al. (2002). The study by Janbaz et al. (2002) did show that pretreatment with four oral doses of rutin at 20 mg/kg (12 hours apart) was able to prevent an increase in plasma A L T levels caused by A P A P at an oral dose of 640 mg/kg. With the pretreatment of rutin, the mean ± S E M of the plasma A L T level was 61 ± 15 U / L , which was significantly lower than that for the APAP-only group (686 ± 2 1 9 U/L), although the time of termination (time between the A P A P dose and termination) was not mentioned. However, the plasma A L T levels were significantly less in the rats pretreated with rutin before dosing with A P A P when compared to those without the rutin pretreatment. This particular finding was consistent with the plasma A L T results our current study using G B E . Also, this study by Janbaz et al. (2002) only used plasma A L T level as a liver toxicity marker and no histological assessment was performed on the rat livers.  4.4.3  Effect of G B E on Liver Histology in Rats treated with A P A P In contrast to the plasma A L T data, the histology data (Figure 3.10 to Figure 3.13)  from the current study suggested that G B E at 500 mg/kg for 9 days seemed to have a negative impact on liver histology in the presence of A P A P , especially in terms of necrosis (Figure 3.11) and capsular inflammation (Figure 3.12). The relatively higher histology scores obtained from the liver tissues in G B E / A P A P group were unlikely caused by mishandling of tissues prior to the histology assessment because the liver tissues in the control group were completely normal.  86  4.4.4  Conflicting Results between A L T data and Histology data The effect of G B E on worsening of liver damage caused by A P A P when compared to  A P A P alone was not reflected in our A L T data. The histology data are more reliable. The histology data are direct evidence showing damage to liver tissues, whereas plasma A L T level is only an indirect marker of liver toxicity. Also, histological assessment was performed independently by another laboratory and the pathologist was blinded to the identity of the samples so any potential bias could be prevented. G B E contains many individual chemicals and one or more of these chemicals might have the potential to inhibit plasma A L T . The lower A L T level in the G B E / A P A P group, as compared to the A P A P group, could potentially be explained by A L T enzyme inhibition. If the plasma A L T enzyme was inhibited, it would fail to convert alanine to pyruvate (Figure 2.1), and hence the formation of 2,4-dinitrophenylhydrazone of pyruvate (Figure 2.2) would be inhibited as well. Inhibition of A L T by G B E is plausible, although there was 8.5 h between the last G B E dose and blood collection. Inhibition of A L T has been shown to occur. For example, Maekawa et al. (2002) reported the presence of an immunoglobulin G (IgG) inhibitor in a patient's serum, causing significantly reduced serum A L T activity. Further experiments will be required to investigate whether G B E inhibits A L T . Moreover, the plasma A L T level, although a commonly used liver toxicity marker, may not reflect the extent of hepatic necrosis (Zhao et al, 1998). A single ip injection of diallyl disulfide at 200 mg/kg to /5-naphthoflavone-pretreated mice 2 h before an ip injection of A P A P at 350 mg/kg was able to prevent the increase in the plasma A L T level by A P A P , but not able to improve the histological assessment or mortality. Without the treatment of diallyl disulfide, severe necrosis was observed in all the mice pretreated with /2-naphthoflavone at 4 h after A P A P .  87  4.4.5  Potential Mechanisms by which G B E Exacerbates APAP-induced Hepatotoxicity  4.4.5.1 Increased Bioactivation of A P A P by G B E G B E might have increased the bioactivation of A P A P . Increased bioactivation of A P A P could lead to increased accumulation of the hepatotoxic metabolite NAPQI, leading to liver toxicity. A mechanism by which this could occur is induction of CYP3A23 by G B E (pathway C in Figure 1.1), as shown by the CYP3A23 mRNA expression data from the current study (Figure A l ) . A pretreatment regimen of G B E at 500 mg/kg ip once daily for 9 days, followed by an ip dose of A P A P at 1000 mg/kg 30 min after the last G B E dose in rats, was able to cause a statistical significant 5-fold increase in hepatic CYP3A23 mRNA expression, when compared to the A P A P group (Figure A l ) . The inductive effect of G B E on CYP3A23 could potentially be mediated through P X R or C A R . P X R and C A R have been hypothesized as key regulators for C Y P 3 A and other C Y P gene expressions (Waxman, 1999). One or more of the chemicals present in G B E may activate P X R or C A R , leading to increased transcription of the CYP3 A23 gene. There is no published evidence that G B E can increase C Y P 3 A gene expression through activation of P X R or C A R . However, it has been reported that quercetin (a chemical present in GBE) increased CYP3A4 mRNA expression in human hepatocytes in culture (Raucy, 2003), but it was suggested that the mechanism by which quercetin induced CYP3 A might not involve activation of PXR. Inhibition of UGT enzymes by G B E might be another potential mechanism whereby G B E enhances APAP-induced hepatotoxicity. Currently, there are no published data on the effect of G B E on the catalytic activity of U G T enzymes. Kostrubsky et al. (2005) reported that some compounds such as phenobarbital and phenytoin enhanced APAP-induced  88  hepatotoxicity in both rat liver microsomes and cultured human hepatocytes by inhibiting U G T enzymes. Hepatotoxicity caused by A P A P can be worsened when the U G T enzymes are inhibited, potentially leading to decreased glucuronidation and hence increased bioactivation of A P A P to the hepatotoxic metabolite NAPQI (pathway B in Figure 1.1). Exacerbation of AP AP-induced hepatotoxicity by G B E through UGT inhibition is therefore possible but it will require further investigation. Another potential mechanism of how G B E can exacerbate A P AP-induced hepatotoxicity might be inhibition of SULT1. A n enzyme(s) in the SULT1 family plays a major role in transformation of A P A P to non-toxic metabolites (Duffel, 1997). If G B E can significantly inhibit the catalytic activity of SULT1 (pathway A in Figure 1.1), the extent of elimination of A P A P will decrease and hence more NAPQI will be produced from biotransformation of A P A P . The risk of AP AP-induced hepatotoxicity may increase when the amount of NAPQI increases and the body is not able to eliminate the excess NAPQI produced in time. However, there is currently no published information on whether G B E inhibits the catalytic activity of SULT enzymes.  4.4.5.2 Decreased Detoxification of A P A P by G B E G B E might have exacerbated A P AP-induced hepatotoxicity by suppressing the expression of UGT1A6 and/or SULT1 enzymes responsible for detoxifying A P A P . G B E might have decreased the rate of A P A P elimination by glucuronidation (pathway B in Figure 1.1) or sulfation (pathway A in Figure 1.1), leading to decreased elimination of A P A P and hence increased formation of the hepatotoxic metabolite (NAPQI) in the liver. This mechanism was not likely as the hepatic UGT1A6 mRNA expression was neither increased  89  nor decreased by G B E in the current study (Figure A2). There is no published information on whether G B E can increase or decrease the expression of SULT1. Glutathione ^-transferase is a conjugation enzyme that is directly responsible for the detoxification of the hepatotoxic metabolite of A P A P , NAPQI. Hence, decreased levels of glutathione S-transferase enzyme expression can potentially lead to reduced conjugation of glutathione with NAPQI, ultimately leading to increased risk of APAP-induced hepatotoxicity (pathway D in Figure 1.1). However, this mechanism is not likely, as several research groups have reported increased liver glutathione S-transferase activity in rats fed a G B E diet. Sugiyama et al. (2004b) reported a statistical significant 2-fold increase in liver glutathione 5-transferase activity in rats that were fed a 0.1% w/w G B E diet for just 5 days. Similarly, glutathione S-transferase activity in livers of male Wistar rats was significantly increased by 2.7 times compared to control group after being fed orally with a 0.5% w/w G B E diet for 1 week (Sugiyama et al, 2004a). For as long as a 4-week duration, rats were fed a 0.5% w/w G B E diet and their liver glutathione S-transferase activity was increased significant by 2.6 times as compared to control (Shinozuka et al, 2002).  4.4.5.3 Combination of the Effects Combination of any of the effects as discussed in Section 4.4.5.1 and 4.4.5.2 is possible. For instance, G B E is known to induce C Y P 3 A (in rats) and G B E may have an inhibitory effect on UGT1A6. Induction of C Y P 3 A per se can lead to increased formation of NAPQI from A P A P . UGT1A6 inhibition may lead to increased accumulation of A P A P and hence formation of NAPQI. Therefore, when both events are happening simultaneously the resultant detrimental effect on the liver can be worse than the toxic effect caused by either event alone.  90  4.5  Comparison of Current Study to Published Studies The study by Shenoy et al. (2002) used albino Wistar rats and the ip dose of G B E  used in that study was only at 50 mg/kg/day, a dose that is ten times lower than the dose used in the current study (500 mg/kg/day). Instead of a G B E pretreatment, the G B E was given to the rats either simultaneously with the A P A P or after the A P A P dose. The respective order of the G B E and the A P A P treatment may make a drastic difference in the results. Any , potential C Y P enzyme induction effect caused by G B E will take more than a few days to happen. Shenoy et al. suggested a protective effect of G B E when it is given together with or after a hepatotoxic dose of A P A P in rats, based on their assessment of plasma A L T and liver histology data. A problem with the study performed by Shenoy et al. (2002) was that there was no information regarding the amounts of terpene trilactones and flavonol glycosides in the G B E used in their study. Recent evidence revealed that ginkgolide A is a chemical that contributes to C Y P 3 A induction by G B E (Chang et al, 2006). It is possible that ginkgolide A was not present in that extract or was present at a level too low to result in CYP3 A induction. This could have explained the lack of enhanced hepatotoxicity in rats treated with G B E and A P A P in the study by Shenoy et al. (2002). Sener et al. (2006) suggested a protective effect of G B E against A P AP-induced toxicity in mice. A problem with this study is that only a low dosage of G B E (50 mg/kg ip) was used. This G B E dosage may not be sufficient to elicit any C Y P induction. C Y P induction is crucial for increased NAPQI formation from A P A P . Absence of C Y P induction might have prevented the investigators from finding an exacerbation of AP AP-induced hepatotoxicity by G B E . Also, G B E was given to the mice after A P A P injection. The effect  91  of pretreatment with G B E on A P AP-induced hepatotoxicity was not addressed in this study by Sener et al. (2006).  4.6  Limitations of the Study The degree of C Y P induction by G B E may vary from one species to the next. Hence,  the results of this study in rats may or may not represent the situation in humans. In the current study, the plasma A L T assay did not agree with the liver histology data. Hence, the data could only be explained in terms of either the plasma A L T levels or the liver histology, but not both. Liver histological analysis is more reliable because it is a direct way of assessing liver toxicity. Plasma A L T only served as a biochemical marker in assessing liver toxicity. It would be better if there were more than one way of assessing liver toxicity. Only one dosage of G B E was used in the current study. This relatively high dosage was used because it induces CYP3 A activity. However, smaller dosages of G B E could have been used in the study to determine the range of dosages that do not modulate AP AP-induced hepatotoxicity. G B E was given as a pretreatment to rats for 8 or 9 days. We did not investigate whether G B E given as a shorter pretreatment period would have similar effects on liver histology or plasma A L T levels.  4.7  Future Studies In the present study, there was enhanced AP AP-induced hepatotoxicity by G B E . It  will be important to investigate which chemical(s) in G B E is(are) responsible for this observed effect.  92  Some of the experiments can be repeated in the future with more animals in each group to increase the power of the highly variable data. This is necessary to confirm the effect of G B E pretreatment on APAP-induced hepatotoxicity using histology for assessment of liver damage in rats. It would be important to investigate whether the order of administering G B E and A P A P would affect APAP-induced hepatotoxicity. Published data suggested administering G B E together with or after A P A P prevented APAP-induced hepatotoxicity (Shenoy et al, 2002; Sener et al, 2006). Repeating the same published studies will allow us to know the effect of G B E co- and post-treatment with A P A P on APAP-induced hepatotoxicity. The lack of an increase in plasma A L T levels could be explained by a potential inhibition on the A L T enzyme by G B E . It will be interesting to investigate whether G B E inhibits plasma A L T enzyme and to identify the component(s) of G B E responsible for this inhibition. Smaller dosages of G B E should be used in future studies to investigate whether the increase in APAP-induced hepatotoxicity by GBE, as observed in the current study, will occur with smaller dosages. Different durations of pretreatment with GBE, either administered orally or intraperitoneally, can be studied to investigate whether it will play any role in modulating APAP-induced hepatotoxicity. The time between the last G B E dose and the time of termination can be another crucial factor. The effect of G B E pretreatment on expression and catalytic activities of certain drug metabolizing enzymes (e.g. CYP3A, CYP1A2, CYP2E1, SULT1, UGT1A6) in rats administered A P A P should be investigated in future studies to help elucidate the mechanism by which G B E modulates APAP-induced hepatotoxicity. In the current study, there was an  93  increase in CYP3A23 mRNA expression in rats treated with G B E and A P A P . It will be important to find out the individual chemical(s) in G B E that is responsible for any enzyme induction and inhibition effects. Also, quantifying the NAPQI metabolite may be helpful in explaining the modulation of APAP-induced hepatotoxicity by GBE.  94  5. SUMMARY AND CONCLUSIONS The plasma A L T assay used in this study was successfully validated. The assay had a dynamic range of 20 to 80 U / L , with a LOQ of 20 U / L . The inter-day and intra-day variabilities were acceptable based on published guidelines (Shah et al, 1992). The assay was accurate, precise, and reproducibility, although it had a narrow dynamic range and was not very sensitive. Fasting was required to elicit AP AP-induced hepatotoxicity. 20% Tween 80 in 0.9% NaCl (the vehicle used to suspend APAP) did not elevate the plasma A L T levels in rats. Maximal plasma A L T levels were obtained with A P A P at a dosage of 1000 mg/kg. G B E (500 mg/kg ip once daily for 8 days) did not increase plasma A L T levels, when compared to the 0.9% NaCl-treated control group, although histological analysis of hepatic tissues from GBE-treated rats showed that four of the five samples exhibited steatosis, necrosis, capsular inflammation, or sinusoidal dilatation. G B E at 500 mg/kg ip once daily for 9 days prevented the increase in plasma A L T levels in male Long-Evans rats, when assessed at 8 h after a single ip dose (1000 mg/kg) of A P A P . In contrast, pretreatment of male Long-Evans rats with G B E at 500 mg/kg ip once daily for 9 days did not attenuate the occurrence of hepatic steatosis, necrosis, capsular inflammation, or sinusoidal dilatation, when assessed at 8 h after a single ip dose (1000 mg/kg) of A P A P . Caution should be exercised when using plasma A L T levels as a marker to assess hepatotoxicity, especially in experiments involving G B E .  95  6. 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Zhao C, Sheryl D, Zhou Y X (1998) Effects of combined use of diallyl disulfide and A acetyl-cysteine on acetaminophen hepatotoxicity in beta-naphthoflavone pretreated mice. World J Gastroenterol 4:112-116.  106  7. APPENDICES 7.1  Appendix 1  Table A l . The amount of ginkgolides, bilobalide, and flavonols in the G B E used in the study.  Ginkgo biloba Extract  Amount (% w/w) Diterpene Ginkgolide A  1.1  Ginkgolide B  0.3  Ginkgolide C  1.4  Ginkgolide J  0.6  Total  3.4  Sesquiterpene Bilobalide  2.8  Total Terpene Trilactones  6.2  Flavonol and its Glycosides Kaempferol (aglycone)  none  Kaempferol (diglycosides)  1.9  Kaempferol (other glycosides)  4.4  Kaempferol (sum of aglycone and glycosides)  6.3  Quercetin (aglycone)  none  107  Quercetin (diglycosides)  4.0  Quercetin (other glycosides)  6.6  Qercetin (sum of aglycone and Glycosides)  10.6  Isorhamnetin (aglycone)  none  Isorhamnetin (3-O-rutinoside)  0.6  Isorhamnetin (other glycosides)  3.5  Isorhamnetin (sum of aglycone and glycosides)  4.1  Total Flavonol Glycosides  21  The amount of terpenes in Ginkgo biloba extract (lot no. 1306A) was quantified by gas chromatography (Indena S.A., Milan, Italy) and the amount of flavonols in the extract was quantified by liquid chromatography - mass spectrometry (ChromaDex, Inc., Santa Ana, CA).  108  7.2  <  Appendix 2  8000  to •D  CO  c CO CD  6000  Cl  O O  O  co co d)  4000  i—  CL X LU  £  E  2000  oo C N < O O  CL  5  0  Vehicle  APAP  GBE/APAP  Treatment Figure A l . Effect of pretreatment with G B E on CYP3A23 mRNA expression in rats administered A P A P . Rats were treated as described in the legend to Figure 3.9. CYP3A23 mRNA was quantified by real-time PCR and the mean and S E M of the CYP3A23 mRNA expression are shown here for each treatment group. CYP3 A23 mRNA expression was 6083 ±1021 copies per 1 ng of dsDNA for the group treated with both G B E and A P A P (combination group). This amount was significantly greater than the CYP3 A23 mRNA expression for both the APAP-treated group (1272 ± 275 copies per 1 ng of dsDNA) and the vehicle-treated group (359 ± 149 copies per 1 ng of dsDNA) (p < 0.001, one-way A N O V A and S N K multiple comparison test). The CYP3A23 mRNA expression of the APAP-treated group was not significantly different from the vehicle-treated group, according to the S N K multiple comparison test. The assay was performed by Jessie Chen. * Significantly different from the APAP-treated group and the vehicle-treated group (p < 0.001).  109  7.3  Appendix 3  140 Q to CO C  120  jg 100 "Q.  o o  ^  80 H  o 'co CO  2  60  <  40  CL X LU  Z  E  20  C D <  O  ZD  Vehicle  APAP  GBE/APAP  Treatment Figure A2. Effect of pretreatment with G B E on UGT1A6 mRNA expression in rats administered A P A P . Rats were treated as described in the legend to Figure 3.9. Mean and S E M of the UGT1A6 mRNA expression for each treatment group are presented in this figure. The pretreatment of G B E did not appear to affect hepatic UGT1A6 mRNA expression in rats administered A P A P . UGT1A6 mRNA expression was 82 ± 1 1 copies per 1 ng of dsDNA for the vehicle-treated group. The mean UGT1A6 mRNA expression for the APAP-treated group (107 ± 21 copies per 1 ng of dsDNA) and the combination group (96 ± 17 copies per 1 ng of dsDNA) were not significantly different from the vehicle-treated group (p = 0.69, one-way A N O V A and and S N K multiple comparison test). The assay was performed by Jessie Chen.  110  

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