PHASE IIa CHEMPREVENTIN TRIAL WITH GREEN TEA PLYPHENLS IN HIGH-RISK PPULATIN F LIVER CANCER by HAITA LU, B.Med., M.Med. A DISSERTATIN IN ENVIRNMENTAL TXICLGY Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for the Degree of DCTR F PHILSPHY Approved Jia-Sheng Wang Chairperson of the Committee Todd Anderson Barbara Pence Leslie Shen Christopher Theodorakis Accepted John Borrelli Dean of the Graduate School AUGUST, 2005
ACKNWLEDGEMENTS I would like to express deep gratitude to my advisor, Dr. Jia-Sheng Wang, who has been helping me everywhere in my life and in my Ph.D. study, and guiding me through the whole process to reach completion of this dissertation. It has been a wonderful chance to work with Dr. Wang on this green tea intervention trial, which is very meaningful and has attracted significant attention from media and academia in two AACR meetings. I learned from my advisor not only how to work hard, but also how to work efficiently, and the experiences in many comprehensive instruments and techniques practiced in Dr. Wang s lab have become an invaluable treasure in my academic career. I greatly appreciate my other Advisory Committee members, Professors Todd Anderson, Barbara Pence, Leslie Shen, and Christopher Theodorakis. It has been a tedious work for them to guide my readings, to review my writings, to provide comments and suggestions, and to attend my meetings after meetings. During this Ph.D. program, even I myself could get bored and frustrated sometime, but what they presented was always a smile and encouragement that made me keep moving ahead. A lot of required classes that first appeared irrelevant and boring to me turned out to be so intimate in my study. I feel grateful to all those professors who have been so important in building my knowledge structure. Besides Drs. Todd Anderson, Christopher Theodorakis, and Jia-Sheng Wang who also served in my Advisory Committee, I clearly remember Drs. Michael Hooper, George Cobb, Ernest Smith, Stephen Cox, and Richard Dickerson for their knowledge and their presentations. I like their classes and have ii
benefited a lot from the knowledge they impart, especially in the writing of my own dissertation. During the analyses of more than one thousand samples, I was not alone. All my colleagues and classmates have been helping me. Dr. Hengqian Liu taught me the HPLC- ECD instrument step by step, and Dr. Lili Tang and Xia He taught me the RIA technique. Hongmei Wu and Madhavi Billam helped me analyze GTP, Qingsong Cai and Meng Tang helped me analyze urinary AFB 1 metabolites, Yuntian Tang helped me analyze serum AFB 1 metabolites, and Hongxia Guan helped me in the HPLC system. It has been a painful process analyzing so many samples, but it was also filled with laughter because of their help and accompaniment. I appreciate everyone s hard work that made this dissertation possible. At last, my wife, Linxia Dong, supported my academic pursuit by not only cooking, but also patience and understanding, even at the cost of our honeymoon. As so much time was spent in the lab, I feel sorry for my family, but also feel lucky for having such a wonderful family that is always supportive. If this dissertation would ever be a success, it s not my achievement, it s theirs, it s all those people around me, who guided me, and helped me. My sincere appreciation goes to everyone who has contributed to this dissertation, to my degree, and to humanity s knowledge about health and disease. iii
TABLE F CNTENTS ACKNWLEDGEMENTS.................................................ii ABSTRACT............................................................vi LIST F TABLES......................................................viii LIST F FIGURES...................................................... ix LIST F ABBREVIATINS............................................. xiii CHAPTER I. INTRDUCTIN...................................................1 II. LITERATURE REVIEW............................................. 7 2.1 verview of Hepatocellular Carcinoma.............................. 7 2.2 Major Etiologic Factors of HCC.................................... 9 2.2.1 Aflatoxins................................................ 9 2.2.2 Hepatitis B Virus..........................................21 2.2.3 ther Etiological Factors................................... 24 2.3 xidative DNA Damage and Biomarker.............................25 2.4 Chemoprevention and Surrogate Endpoints.......................... 30 2.5 Green Tea Polyphenols.......................................... 33 2.6 Significance................................................... 44 III. MATERIALS AND METHDS...................................... 46 3.1 Materials and Instruments........................................ 46 3.2 verall Design and Procedure of Chemoprevention Trial............... 49 3.3 Analysis of Urinary Excretion of Green Tea Polyphenols............... 54 iv
3.4 Analysis of Plasma Green Tea Polyphenols.......................... 60 3.5 Analysis of Urinary Excretion of AFM 1 and AFB 1 -NAC............... 62 3.6 Analysis of Serum AFB 1 -Albumin Adducts..........................66 3.7 Analysis of Urinary Excretion of 8-HdG........................... 73 3.8 Data Management and Statistical Analysis........................... 77 IV. RESULTS........................................................81 4.1 Conduct of Chemoprevention Trial.............................. 81 4.2 Validation of Urinary Biomarkers for GTP Consumption............... 85 4.3 Validation of Plasma Biomarkers for GTP Consumption................96 4.4 Modulation of Urinary Excretion of AFB 1 Metabolites................ 115 4.5 Modulation of Serum Levels of AFB 1 -Albumin Adducts.............. 121 4.6 Modulation of Urinary Excretion of 8-HdG........................128 V. DISCUSSIN....................................................137 5.1 verall Features...............................................137 5.2 Safety of GTP Intervention and Quality of Chemoprevention Trial.......139 5.3 Validation of GTP Biomarkers and Modulation of GTP Phase II Metabolism..................................... 142 5.4 Modulation of AFB 1 Phase I and Phase II Metabolites.................147 5.5 Modulation of xidative DNA Damage............................ 153 5.6 Conclusion...................................................156 VI. CNCLUSIN...................................................162 REFERENCES........................................................ 164 v
ABSTRACT Hepatocellular carcinoma (HCC) is the third leading cause of cancer deaths in the developing world, and major risk factors have been identified as chronic infection with hepatitis B virus (HBV) and dietary exposure to aflatoxin B 1 (AFB 1 ). ne of the current challenges in this field is how to manage high-risk individuals who have been exposed to these risk factors for many years. Chemoprevention was proposed as a good tool to target these individuals. Among various chemopreventive agents, green tea polyphenols (GTP) have been shown to be safe and effective in most in vitro and in vivo studies. To further investigate GTP s safety and efficacy in humans, a randomized, double-blind, and placebo-controlled phase IIa chemoprevention trial with GTP was carried out in a highrisk population of HCC in Fusui, Guangxi, China. After screening 1200 sera samples from local residents, a total of 124 healthy adults who were sero-positive for HBV and AFB 1 was voluntarily recruited, randomized into 3 groups, and received either placebo, 500-mg GTP, or 1,000-mg GTP daily for 3 months. Urine and blood samples at baseline, 1 month, and 3 months of the intervention were collected from these participants and were analyzed for various GTP biomarkers and carcinogen specific biomarkers to assess the efficacy of the trial. An overall compliance of 99% with mild and insignificant sideeffects was observed. Baseline data for all biomarkers analyzed showed homogeneity among the 3 study groups. The placebo group had slight fluctuations in biomarker levels during the course of intervention, whereas urinary excretion of (-)-epigallocatechin (EGC) and (-)-epicatechin (EC) and plasma concentration of (-)-epigallocatechin gallate (EGCG) vi
and (-)-epicatechin gallate (ECG) were dose-dependently elevated in the GTP-treated groups. Urinary excretion of AFB 1 -mercapturic acid, a detoxified metabolite of AFB 1, was significantly increased, while the serum levels of AFB 1 -albumin adduct, a macromolecule damaged by AFB 1, was significantly diminished after GTP intervention. The oxidative DNA damage biomarker, urinary 8-hydroxy-2 -deoxyguanosine, was also significantly decreased by GTP intervention. This study validated GTP biomarkers and proved the relative safety of GTP in humans. GTP intervention significantly inhibited carcinogen biomarkers and enhanced activities of detoxifying enzymes. vii
LIST F TABLES 3.1. Gradient of Mobile Phase in HPLC for GTP Analysis........................57 3.2. Gradient of Mobile Phase in HPLC for AFM 1 and AFB 1 -NAC Analysis....................................... 65 3.3. Gradient of Mobile Phase in HPLC for 8-HdG Analysis............................................... 76 4.1. Demographic Distribution of Study Participants in Chemoprevention Trial............................................82 4.2. Compliance in Chemoprevention Trial....................................82 4.3. Adverse Effects in Chemoprevention Trial................................ 84 4.4. Urinary GTP Recovery Test............................................89 4.5. Parameter Estimation in Multi-level Model for Change for Urinary GTP Levels......................................89 4.6. Plasma GTP Recovery Test........................................... 101 4.7. Parameter Estimation in Multi-level Model for Change for Plasma GTP Levels..................................... 104 4.8. Urinary 8-HdG Recovery Test........................................132 4.9. Parameter Estimation in Multi-level Model for Change for Urinary 8-HdG Levels..................................133 viii
LIST F FIGURES 2.1. Structures of Frequently Encountered Aflatoxins............................10 2.2. Structures of AFB 1-8,9-epoxide Stereoisomers..............................12 2.3. Pathway of AFB 1 -DNA Adduct Formation............................... 14 2.4. Mercapturic Acid Pathway for AFB 1-8,9-exo-epoxide Metabolism....................................16 2.5. Pathway of AFB 1 -lysine Adduct Formation............................... 18 2.6. Pathway of 8-H-Gua Adduct Formation................................. 27 2.7. Structures of Major Green Tea Polyphenols................................34 2.8. Glucuronidation of EGCG............................................. 37 2.9. Sulfation of EGCG................................................... 38 2.10. Methylation of EGCG................................................39 3.1. verall Design of Phase IIa Chemoprevention Trial......................... 50 3.2. Procedure for Urinary GTP Extraction....................................55 3.3. Procedure for Urinary Creatinine Analysis.................................59 3.4. Procedure for Plasma GTP Extraction....................................61 3.5. Procedure for Urinary AFM 1 and AFB 1 -NAC Extraction.....................63 3.6. Procedure for Serum Aflatoxin Extraction.................................67 3.7. Procedure for Radioimmunoassay of Free Aflatoxin Levels...................69 3.8. A Standard Series for AFB 1 Quantification by RIA......................... 71 3.9. Procedure for Urinary 8-HdG Extraction................................ 74 ix
4.1. Chromatograph of Standard GTP Components by HPLC-ECD Analysis............................................. 86 4.2. Calibration Curves for Individual GTP Components.........................88 4.3. Chromatograph Showing Separation and Detection of All 4 GTP Components in ne Urine Sample............................ 88 4.4. Urinary EGC Levels in Three Groups at Each Collection..................... 90 4.5. Urinary EC Levels in Three Groups at Each Collection.......................91 4.6. Prototypical Trajectories for Urinary GTP Levels...........................94 4.7. ne Urine Sample Treated with No Enzyme, with Sulfatase, and with Beta-glucuronidase for HPLC-ECD Analysis....................... 95 4.8. Urinary EGC Levels Analyzed for Conjugation Percentage................... 97 4.9. Urinary EC Levels Analyzed for Conjugation Percentage.....................98 4.10. Chromatograph of A Plasma Sample as Compared with the Same Sample Spiked......................................... 99 4.11. Plasma EGC Levels in Three Groups at Each Collection................... 102 4.12. Plasma EC Levels in Three Groups at Each Collection.....................103 4.13. Plasma EGCG Levels in Three Groups at Each Collection..................106 4.14. Plasma ECG Levels in Three Groups at Each Collection................... 107 4.15. Prototypical Trajectories for Plasma GTP Levels.........................109 4.16. Chromatograph Showing EGC and EC from A Plasma Sample Treated with No Enzyme, with Sulfatase, and with Beta-glucuronidase for HPLC-ECD Analysis....................... 110 4.17. Chromatograph Showing EGCG and ECG from A Plasma Sample Treated with No Enzyme, with Sulfatase, and with Beta-glucuronidase for HPLC-ECD Analysis...............................................110 x
4.18. Plasma EGC Levels Analyzed for Conjugation Percentage..................112 4.19. Plasma EC Levels Analyzed for Conjugation Percentage...................113 4.20. Plasma EGCG Levels Analyzed for Conjugation Percentage................ 114 4.21. Plasma ECG Levels Analyzed for Conjugation Percentage..................116 4.22. Chromatograph of Standard AFB 1 -NAC and AFM 1 by HPLC-fluorescence Analysis......................................117 4.23. Standard Curves for AFB 1 -NAC and AFM 1............................. 119 4.24. Chromatograph Showing Separation and Detection of Both AFB 1 -NAC and AFM 1 by HPLC-fluorescence Analysis...................119 4.25. The Ratio of Urinary Levels of AFB 1 -NAC/ AFM 1 at 1-month and 3-month s Collections................................. 120 4.26. Urinary Levels of AFB 1 -NAC at Both 1-month and 3-month Collections............................................... 122 4.27. Urinary Levels of AFM 1 at Both 1-month and 3-month Collections............................................... 123 4.28. Inhibition Curve for RIA............................................ 125 4.29. Calibration Curve for RIA........................................... 125 4.30. Estimated Marginal Means of Serum AFB 1 -Albumin Adduct Levels over Time within Each Group............................126 4.31. The Levels of Serum AFB 1 -Albumin Adducts at Three Collections in Each Group....................................127 4.32. The Levels of Serum AFB 1 -Albumin Adducts in Three Groups at Each Collection....................................129 4.33. Chromatograph Showing Retention Time and Response Pattern of Standard 8-HdG by HPLC-ECD Analysis.................... 131 4.34. Calibration Curve for Urinary 8-HdG................................. 131 xi
4.35. Chromatograph Showing Separation and Detection of 8-HdG in Urine Samples Versus a Standard......................... 132 4.36. Urinary 8-HdG Levels in Three Groups at Each Collection................ 135 4.37. Prototypical Trajectories for Urinary 8-HdG Levels......................136 5.1. The utlined Metabolism Pathways for AFB 1, AFB 1-8,9-exo-epoxide, and Three Metabolites...........................149 5.2. The Comprehensive Pathways of AFB 1, HBV and RS as Modulated by GTP...............................................160 xii
LIST F ABBREVIATINS 8-HdG AF 8-hydroxy-2 -deoxy-guanosine aflatoxin AFB 1 aflatoxin B 1 AFB 1 -FAPY AFB 1 -formamidopyrimidine AFB 1 -N7-Guanine trans-8,9-dihydro-8-(n7-guanyl)-9-hydroxy- AFB 1 AFB 1 -NAC AFB AFB 1 -mercapturic acid AFB 1-8,9-exo-epoxide AFM 1 aflatoxin M 1 ALT AP AP-1 ARE AST BCG BER BUN CMT CV CX CYP/P450 alanine aminotransferase alkaline phosphatase activator protein-1 antioxidant response element aspartate aminotransferase bromcresol green base-excision repair blood urea nitrogen catechol--methyltransferase coefficient of variation cyclooxygenase cytochrome P450-dependent polysubstrate monooxygenase enzyme xiii
DMS EC ECG EGC EGCG FAP FDA gamma-gt GCG GLM GPX GSH GST GST GTP HBsAg HBV HCC HCV hgg1 IARC ins dimethylsulfoxide (-)-epicatechin (-)-epicatechin-3-gallate (-)-epigallocatechin (-)-epigallocatechin-3-gallate adenomatous polyposis Food and Drug Administration gamma-glutamyltranspeptidase gallocatechin gallate general linear model glutathione peroxidase reduced glutathione GSH-S-transferase glutathione S-transferase green tea polyphenols hepatitis B surface antigen hepatitis B virus hepatocellular carcinoma hepatitis C virus Human GG1 International Agency for Research on Cancer inducible nitric oxide synthase xiv
MeH ML NER NF-kB NTP DC GG1 PAPS PBS RIA RS SAM SD SPE SULT TEAF TNF TPA UDP-GA UGT methanol maximum likelihood nucleotide excision repair nuclear factor-kb National Toxicology Program ornithine decarboxylase 8-H-Gua glycosylase 3 -phosphoadenosine-5 -phosphosulfate phosphate buffering solution radioimmunoassay reactive oxygen species S-adenosylmethionine superoxide dismutase solid phase extraction phenolsulfotransferases triethylammonium formate tumor necrosis factor 12--tetradecanoylphorbol-13-acetate uridine-5 -diphospho-alpha-d-glucuronic acid UDP-glucuronosyltransferases xv
CHAPTER I INTRDUCTIN Ever since the role of pathogens was realized in infectious diseases, human heath has been dramatically improved as more and more infectious pathogens are characterized and controlled. The idea is straightforward: an infectious disease is caused by a specific pathogen and removal of this pathogen will ultimately prevent or cure such a disease. This progress in human health has been demonstrated by the eradication of several infectious diseases on regional levels and smallpox on a worldwide scale. 1 The next problem in human health is more challenging. With the emergence of public health concerns about chronic diseases such as cancer, cardiovascular diseases, and neurodegenerative diseases, the situation becomes more complicated and the former straightforward strategy does not apply. These chronic diseases are not directly caused by one or more pathogens (even though they might well be a risk factor) and removal of these pathogens can not guarantee human s health from these diseases. A new era was revealed where diseases are caused by multiple factors through a multi-stage process. With the statistical concepts becoming more and more integral to almost every field of science and society, and because of the uncertainty about the disease outcome caused by various factors, it is now widely accepted that chronic-disease outcomes can not be predicted definitively, but at best be predicted in terms of risk. In this new, uncertain era of public health, the goal is not to eliminate such a chronic disease from human beings, but to reduce the risk and incidence of these diseases in human populations. 1
With successful control of most, if not all, infectious diseases, chronic diseases become the major obstacle in the way toward a better human health situation. Cancer, along with cardiovascular diseases and neuro-degenerative diseases, has become the number one cause of death in many Asian countries 2 and the number two cause of death in the western world, 3 and is expected to outrank cardiovascular diseases in the near future. 4 Liver cancer is one of the most important cancers worldwide, ranked number three in cancer mortality due to a high prevalence and poor prognosis, 5 and the risk is especially high in certain areas in Southeast Asia and Sub-Saharan Africa. 6 The longterm goal of this dissertation study is to reduce the risk of liver cancer in high-risk populations in the developing world by chemoprevention with green tea polyphenols (GTP). The primary form of liver cancer is hepatocellular carcinoma (HCC), comprising 90% of all liver cancers in the world. 7 Several etiological risk factors for HCC have been characterized. These include aflatoxin B 1 (AFB 1 ) dietary exposure and hepatitis B virus (HBV) infection in the developing world, and hepatitis C virus (HCV) infection and alcohol abuse in the developed world. As there is no established cure for HCC currently, prevention becomes an extremely important alternative to reduce the risk of HCC. While several of the etiological factors can be eliminated through primary prevention for younger generations, there is a large fraction of the contemporary generation that has been exposed to these etiological factors for decades and therefore can not benefit much from primary prevention. These populations are at severely high risk for HCC, and become the first priority in research to reduce HCC risk in humans. Chemoprevention 2
focuses on these populations and is expected to provide a solution to decrease the risk for HCC in these high-risk populations. Among various chemopreventive agents, green tea and its polyphenolic compounds have attracted much attention in the last two decades with respect to their anti-carcinogenic activities. Tea (Camellia sinensis) has been consumed by humans for thousands of years, and is still the most widely consumed beverage in the world, second only to water. GTP are the major biologically-active components in green tea, and have been shown to be effective in reducing the risk and severity of HCC development in several animal studies, presumably due to their well-known anti-oxidant properties among others. As tea is popularly consumed as a beverage and effective in disease-risk reduction in many studies, it is reasonable to extend the concept of tea (and GTP) consumption as a normal drink into a chemopreventive agent in HCC high-risk populations. In spite of numerous lab research evidence supporting GTP protective effects against carcinogenesis, epidemiological studies have given inconsistent results as to tea s effect as protective, neutral, or aggravating. The potential role of tea consumption in human carcinogenesis remains controversial. To bridge animal data to humans, the most direct and convincing evidence would have to come from human studies. In classic epidemiological studies, the gross estimation of tea consumption and the lack of biomarkers as surrogate endpoints have probably masked a negative correlation between tea consumption and cancer risk, as suggested repeatedly in animal studies. Well designed prospective studies, especially GTP intervention trials incorporating biomarker 3
measurements as surrogate endpoints, are expected to be the most time- and costeffective means to elucidate tea s role in human health with a better accuracy and persuasion. Such a GTP intervention trial was carried out in this study. The general hypothesis of this study is that GTP has a protective effect against hepatocellular carcinogenesis in humans through the mechanism of modulating hepato-carcinogen metabolism and reducing oxidative DNA damage. In this study, a randomized, doubleblind, and placebo-controlled phase IIa chemoprevention trial with GTP was conducted in one of the HCC high-risk populations in China for three months. Participants were monitored for adverse effects by GTP intervention, and blood and urine samples were collected for analysis of various parameters to validate GTP biomarkers as well as to evaluate GTP safety and efficacy in human populations at high risk for HCC. The specific aims of this study include: 1. Conduct a phase IIa chemoprevention trial with GTP in one of the HCC highrisk populations and monitor any possible adverse effects by consumption of GTP during the period of this trial. 2. Validate GTP biomarkers in humans and examine GTP metabolism ratio in this population by analysis of GTP levels, free versus conjugated, in body fluids (blood and urine) of participants. 4
3. Test the hypothesis of GTP effect on modulating AFB 1 metabolism toward detoxification by analysis of different AFB 1 metabolites in serum and urine samples of participants. 4. Test the hypothesis of GTP effect on reducing oxidative DNA damage in humans by analysis of 8-hydroxy-2 -deoxyguanosine (8-HdG) levels in urine samples of participants. This study was exclusively dedicated to elucidate the safety and efficacy of GTP in humans, exploring a possibility of applying GTP as a chemopreventive agent in populations at high risk of HCC. Though tea and tea products have been consumed for centuries implicating safety, purified GTP products intended for use in chemoprevention have not been formally evaluated for safety and tolerable dosages in human populations. Even though numerous researchers have studied GTP efficacy in a variety of circumstances, none have been able to administer precise dose of GTP for extended periods of time in human populations for the purpose of efficacy evaluation. A three month intervention appears insignificant as compared to the decades necessary for HCC development, and no decrease in HCC incidence would be expected from a short-term intervention and observation. However, participants were all high-risk individuals who were assumed to be compromised and therefore potentially more responsive to a chemopreventive agent. In addition, various biomarkers were applied as surrogate endpoints that are more sensitive and indicative of GTP efficacy. There is no data extrapolation necessary from the results of this study, as these subjects represent the 5
ultimate target population. By its prospective nature, this chemoprevention study also better minimized bias and ensured more convincing results in elucidation of the effects of GTP in human health. 6
CHAPTER II LITERATURE REVIEW 2.1 verview of Hepatocellular Carcinoma Hepatocellular Carcinoma (HCC) is the predominant histological subtype of primary liver cancer, comprising 90% of all liver cancer cases.7 n a global scale, HCC is one of the leading causes of cancer morbidity and mortality, although its incidence varies widely by geographical area. With an estimated 530,000 new cases per year worldwide, 8 HCC is listed the fifth most common malignancy in men, the eighth most common malignancy in women, 9 and the sixth when pooled, in the world. 10 Men are at least two to three times more likely to develop HCC than women, 11 and the risk increases with age, reaching a peak between 70-75 years. 12 However, younger HCC cases are not uncommon, especially in certain high-incidence areas. Because of its poor prognosis, HCC has a mortality approaching its incidence and is ranked the third largest cause of cancer mortality in the world. 13 The burden of HCC is even more significant in certain areas of the world because of the extremely wide geographical variation in HCC incidence. Eighty percent of the world s HCC cases arise in the developing world in Southeast Asia and Sub-Saharan Africa,6 leaving the developed countries in America and Europe rare with this cancer. For example, the annual HCC incidence is 35-40/100,000 in China and Japan, as compared 14, 15 with that of only 2/100,000 in the United States. Significant variation in HCC incidence within a country has also been noticed, resulting in several HCC endemic 7
regions. In China, Fusui County in Guangxi Zhuang Autonomous Region and Qidong City in Jiangsu Province are two typical endemic regions with an annual average HCC incidence of more than 50/100,000.13 Developed countries in North America and Europe, although rarely suffered from HCC in the past, are experiencing an increase in HCC incidence probably attributed to hepatitis C virus (HCV) infection and excessive alcohol consumption. The incidence of HCC in the United States has doubled over the past 25 years, affecting all ethnic groups, both sexes, younger and elder age groups, and is expected to double again over the next 20 years. Caucasians, although the least affected, constitute the majority of HCC cases in the United States because they are the largest ethnic group. 16 More severely affected groups include Asian-, Hispanic-, and Africa- Americans. 17 In Europe, UK and France have also experienced a 2-4 fold increase in HCC incidence over the past 25 years. 18,19 HCC is unique among many other cancers in that most of its etiological factors have been characterized by human epidemiological studies and/or animal studies. The major factors contributing to HCC risk include aflatoxins especially aflatoxin B1 (AFB 1 ) ingestion, hepatitis B virus (HBV) infection, hepatitis C virus (HCV) infection, and alcohol abuse. 20 The alcohol abuse and HCV infection are more closely related to HCC cases in the developed world while in developing countries, AFB 1 ingestion and HBV infection are the major concerns.6 Considering that 80% of HCC cases arise in the developing world,6 intense efforts have been focused on HBV and AFB 1 research. 8
2.2 Major Etiologic Factors of HCC 2.2.1 Aflatoxins Aflatoxins are the best known and most extensively characterized mycotoxins, which were first identified as a contributing agent in Turkey X-disease in England in the early 1960s. 21 Aflatoxins are secondary metabolites produced from acetate and malonyl precursors in fungi. They play no role in the growth of the producing organisms, but are presumably involved in their ecology. 22 Four species of Aspergillus are known to yield aflatoxins: A. flavus, A. parasiticus, A. nomius, and A. tamari. nly the former two are important agronomically, with A. flavus being the predominate species on the majority of commodities contaminated by aflatoxins. Aspergillus fungi contamination can occur before or after harvest, but the major concern is pre-harvest fungi contamination and post-harvest aflatoxin production. 23 Aflatoxins consist of nearly 20 similar fungal metabolites, with aflatoxins B 1, B 2, G 1, G 2, and M 1 most frequently encountered. 24 The B, G, and M represent blue, green, and milk respectively, as aflatoxin B has a blue-violet fluorescence and aflatoxin G has a green fluorescence, while aflatoxin M was originally isolated from milk, though it also has a blue-violet fluorescence. 25 The structures of these most frequently found aflatoxins are shown in Figure 2.1.25 bviously, AFB 2, AFG 2, and AFM 2 are 8,9- dihydro-analogues of corresponding aflatoxins where the double bond between C8 and C9 becomes saturated, thus less likely to form the toxic 8,9-epoxides. In humans, CYP 1A2 catalyzes the 9a-hydroxylation of AFB 1 to produce AFM 1, which is excreted through milk and urine. 26 AFM 1 is less carcinogenic than AFB 1, approximately 30% as AFB 1 in 9
CH 3 CH 3 AFB1 AFB2 CH 3 CH 3 AFG1 AFG2 H H CH 3 CH 3 AFM1 AFM2 Figure 2.1. Structures of Frequently Encountered Aflatoxins. 10
trout 27 and 10% as AFB 1 in rats, 28 and is considered one of the primary detoxification products of AFB 1. 29 Aflatoxin B 1 (AFB 1 ) is the most toxic and most thoroughly studied aflatoxin, and it also ranks as the most potent naturally occurring mycotoxin known. 30 f the toxic effects associated with AFB 1 in humans, the most serious and threatening is its carcinogenicity. AFB 1 is, however, not carcinogenic by itself, and a bioactivation is necessary for its carcinogenicity. 31 The initial metabolism of AFB 1 involves 4 types of reactions: -dealkylation, hydroxylation, epoxidation, and ketoreduction. Except for the reductions, most other reactions are primarily catalyzed by the cytochrome P450- dependent polysubstrate monooxygenase enzyme superfamily (P450, CYP). AFB 1 can be epoxidated by P450 into its reactive intermediate, AFB 1-8,9-epoxide, which is believed responsible for most of mutagenicity and carcinogenicity of AFB 1.31 As shown in Figure 2.2, AFB 1-8,9-epoxide has two stereoisomers. 32 The endo-stereoisomer has the epoxide ring positioned above the furan plane and trans to the 5a and 9a protons, whereas the exostereoisomer has the epoxide ring extended below the furan plane and cis to the 5a and 9a protons. 33 Both stereoisomers are produced by human liver microsomes, but the exoepoxide predominates and is also the one that binds covalently to DNA and forms adducts.32 Multiple forms of human P450 are capable of activating AFB 1 to its 8,9-epoxide, including CYP1A2, CYP2A6, and CYP3A4. Among these, CYP1A2 is the high-affinity human P450 capable of activating AFB 1 at low substrate concentrations encountered with human dietary exposure, 34 and CYP3A4 is the one constitutively 11
CH 3 AFB1 CYP 1A2 CYP 3A4 H H H H CH 3 AFB1-8,9-endo-epoxide H H H CH 3 H AFB1-8,9-exo-epoxide Figure 2.2. Structures of AFB 1-8,9-epoxide Stereoisomers. 12
expressed at a higher level in human liver. 35 The major DNA adduct formed in vivo and in vitro is trans-8,9-dihydro-8-(n7- guanyl)-9-hydroxy-afb 1 (AFB 1 -N7-Guanine). 36,37 The AFB 1-8,9-exo-epoxide (AFB) intercalates between base pairs in the DNA helix, where it attacks the nitrogen atom at the 7 position of guanine by the epoxide ring to form a trans adduct at the carbon 8 position of AFB 1. AFB 1-8,9-endo-epoxide also intercalates into the double stranded DNA, but its spatial orientation precludes such an adduction.32 AFB 1 -N7-Guanine DNA adduct is unstable because the positively charged imidazole ring will either promote a depurination to form an apurinic site and an excised AFB 1 -N7-guanine to excrete into urine, or the imidazole ring will open to form a chemically and biologically stable derivative, AFB 1 - formamidopyrimidine (AFB 1 -FAPY). 38 Production of AFB 1 -FAPY in vivo is significant, and the accumulation of this derivative is time dependent, non-enzymatic, and possibly of more potential significance due to its apparent persistence in DNA. 39 It is speculated that the initial unstable AFB 1 -N7-Gua DNA adduct, and the subsequently formed AFB 1 - FAPY or the apurinic site, individually or collectively represent the chemical precursors to the genotoxic effects of AFB 1.38 The structures of AFB 1 -N7-Gua, AFB 1 -FAPY, and apurinic site are shown in Figure 2.3. As DNA is being constantly repaired, the AFB 1 - N7-Guanine DNA adduct usually has a biological half-life of only 8 hours, and urinary excretion of AFB 1 -N7-Guanine has been established as a biomarker to reflect very recent exposure to aflatoxins. 40 13
H H H CH 3 H AFB1-8,9-exo-epoxide DNA H HN N CH 3 H 2 N N N AFB1-N7-Gua DNA Adduct - 3 P P - 3 H HN N CH 3 H 2 N N N H + H AFB1-N7-Guanine AP Site H 2 N HN N H N CH 3 CH NH AFB1-FAPY DNA Adduct - 3 P P - 3-3 P P - 3 Figure 2.3. Pathway of AFB 1 -DNA Adduct Formation 14
In addition to binding to DNA to form aflatoxin-dna adducts and presumably contributing to carcinogenesis, the AFB also goes through conjugation to form AFB 1 - mercapturic acid, or hydrolysis to form AFB 1 -albumin adducts, both of which are established biomarkers for AFB 1 exposure. 41 Mercapturic acid is formed via reaction of AFB 1 with Reduced glutathione (GSH). GSH is a physiological tripeptide consisting of cysteine, glycine, and gamma-glutamine. It is the most abundant sulfhydryl compound mainly present in the cytosol in almost all living cells, and its conjugation with reactive xenobioticd is one of the most notable reactions in phase II metabolism. 42 This conjugation reaction is catalyzed by several glutathione-s-transferase (GST) isoenzymes, with the metabolic end-product being mercapturic acid. 43 AFB can be detoxified by conjugation with GSH to form AFB 1 -GSH, where the epoxide ring opens and C8 of AFB 1 bonds to the sulfhydryl of the cysteine moiety in the middle of GSH. The AFB 1 - GSH is further catalyzed by glutamyltranspeptidase to cleave the gamma-glutamyl moiety of GSH on one side, by cysteinylglycinase to cleave the glycine moiety of GSH on the other side, and by cysteine conjugate N-acetyltransferase to add an acetyl to the nitrogen atom of the cysteine moiety in the middle of GSH. The N-acetylcysteineattached AFB 1 (AFB 1 -NAC), also called AFB 1 -mercapturic acid, is excreted into urine as a detoxified end-product and becomes a valid biomarker for AFB exposure.43,44 1 The mercapturic acid pathway for AFB metabolism and structure of metabolites are shown in Figure 2.4. Both AFB 1-8,9-exo-epoxide and AFB 1-8,9-endo-epoxide can undergo rapid nonenzymatic hydrolysis to form AFB 1-8,9-dihydrodiol, which further undergoes a slow, 15
H H H CH 3 H AFB1-8,9-exo-epoxide Glutathione-S-transferase GSH CH H CH2.CH 2.CH 2.C.NH NH 2 CH.CH 2.S CH 3 C.NH.CH 2. CH AFB1-GSH γ Glu Glutamyltranspeptidase H NH 2 CH.CH 2.S CH 3 Cysteinylglycinase C.NH.CH 2. CH AFB1-Cys.Gly. Gly H NH 2 CH.CH 2.S CH 3 CH AFB1-Cys Cysteine conjugate N-acetyltransferase Acetyl CoA CH 3 C H CoA NH CH.CH 2.S CH 3 CH AFB1-Nac.Cys Figure 2.4. Mercapturic Acid Pathway for AFB 1-8,9-exo-epoxide Metabolism 16
base-catalyzed ring opening and oxidation to form dialdehyde phenolate ion. 45 AFB 1 - dialdehyde does not bind to DNA, but can form Schiff bases with the primary amine group of lysine to form protein adducts, 46 and albumin is the only protein found in serum that can bind AFB 1 to any significant extent in monkeys and rats. 47 These protein adducts are not involved in the mechanism of carcinogenesis, neither are they repaired. 48 But they form a stable repository of accumulated AFB 1 exposure over the lifetime of corresponding proteins. As the AFB 1 -albumin adduct stays with albumin for the same half-life of 2-3 weeks, 49 it has been established as a valid biomarker to reflect a long-term exposure to aflatoxins.26 The pathway and structure of these metabolites are shown in Figure 2.5. ther biotransformed metabolites, including AFM 1, AFQ 1, AFP 1, and AFB 2 B, are hydroxylated products and generally less than 4% as potent as AFB 1 in terms of mutagenicity. Although toxic and mutagenic somehow, these metabolites are usually considered detoxification metabolites. 50 AFB 1 is a potent hepatocarcinogen that induces liver tumors in many species of animals, including fish, bird, rodents, and nonhuman primates.49 In humans, numerous retrospective and prospective epidemiological studies have revealed a positive association of AFB 1 contamination with HCC risk,20 although many of them involved exposures to a mixture of aflatoxins rather than pure AFB 1 for the sake of natural cocontaminations. A case-control study conducted in Philippines showed a 4.5 times higher aflatoxin exposure in HCC patients. 51 In Nigeria, a study of only 22 case-control pairs found significantly higher percentage in HCC patients with elevated levels of blood 17
H H H H H CH 3 H AFB1-8,9-exo-epoxide H CH 3 H AFB1-8,9-endo-epoxide H H AFB1-dihydrodiol CH 3 H CH 3 AFB1-dialdehyde N H CH 3 CH 2 CH 2 CH 2 CH 2 H C CH NH 2 AFB1-lysine adduct Figure 2.5. Pathway of AFB 1 -lysine Adduct Formation. 18
aflatoxins. 52 More convincing evidence came from prospective studies, wherein the cohort in Guangxi, China uncovered a 10 times higher HCC incidence in aflatoxin heavily exposed populations after only 5 to 8 years of follow-up. 53 Within the cohort of 18,000 male Shanghai residents in China, a nested case-control study incorporating molecular biomarker measurements revealed a significantly higher risk for aflatoxin exposure. 54 Another case-control study nested within a 6,000 person cohort in Taiwan also indicated a significantly elevated risk for AFB 1 exposure, when measurements of the serum biomarkers were combined. 55 A comprehensive list of AFB 1 -HCC epidemiological studies is beyond the scope of this section, but two general features characterize most of these studies: one is the utilization of nested case-control studies within much larger cohorts due to the difficulties of performing a real cohort analysis, especially when molecular biomarkers were included and many years are followed up; the other is the greater impact of HBV infection in analysis, for almost all of these studies found AFB 1 s contribution second to HBV in an increased HCC risk, and frequently, a synergistic effect between HBV and AFB 1 was demonstrated. For example, in the nested case-control study within Shanghai cohort, there is an increased HCC risk of 7.3 for HBV infection, 3.4 for aflatoxins exposure, and an astonishing 59 for the two factors combined.54 Among various mechanisms by which AFB 1 induces HCC, p53 mutation is the most common feature. The p53 gene is a tumor suppressor gene that plays an important role in regulating cell cycle, DNA repair, apoptosis, and others. Mutation at p53 not only leads to the loss of its house-keeping function, but also possibly converts it into an oncogene. The role of p53 in carcinogenesis can be supported by the finding that more 19
than 50% of all human tumors have p53 mutations. 56 In an in vitro system, the p53 gene was mostly attacked by AFB 1 at the site of codon 249 to form the AFB1-N7-guanine adducts, 57 and in human HCC, occurrence of G T transversion at base pair 3 of codon 249 in the p53 gene was frequently found in areas with high exposure to AFB 1. In HCCs from Qidong, China and from Mozambique, two areas with high AFB 1 contamination, this characteristic p53 mutation was found at frequencies of 30-50%, while in HCCs from North America, Europe and Japan, such mutations were rarely observed. 58 This specific mutation spectrum in p53 gene has been widely accepted as closely related with AFB 1 exposure. 59 Albeit AFB 1 s causative role in HCC risk was at first confounded by concomitant HBV infections and not widely accepted, more intricate epidemiological studies have successfully analyzed the effect of AFB 1 isolated from that of many other factors, especially HBV. With accumulating evidence from epidemiological studies and lab research that supports the etiological role of AFB 1 in HCC risk, the International Agency for Research on Cancer (IARC) classified AFB 1 as a Group I Human carcinogen in 1993, 60 and reaffirmed this classification in 2002 with more recent research data. 61 The National Toxicology Program (NTP) classified AFB1 as known to be human carcinogens in 1980, and reaffirmed this classification in the 11 th edition of Report on Carcinogens in 2004. 62 20
2.2.2 Hepatitis B Virus Hepatitis B Virus (HBV) is the prototype member of the Hepadnaviridae (hepatotropic DNA virus) family. It has a strong preference for infecting liver cells. 63 The entire virion, also known as the Dane particle, is 40 to 42 nm in diameter. 64 This double-shelled particle consists of an outer lipoprotein envelope (surface antigens) and an inner polypeptide nucleocapsid (core). The genome enclosed in the nucleocapsid is a relaxed-circular, partially duplex DNA of 3.2 kb, constituting only four long open reading frames: Presurface-surface region (PreS-S), Precore-core region (PreC-C), P coding region, and the X region. By differential initiation of translation, the PreS-S region encodes the S protein (surface antigen, HBsAg), L protein (pres1), and M protein (pres2), which collectively assemble the outer envelope of the virion. Likewise, the PreC-C region encodes the C protein (core antigen, HBcAg) constituting the inner nucleocapsid, and the E protein (e antigen, HBeAg) with unknown function in viral assembly thus far. The P coding region encodes the viral polymerase involved in DNA synthesis and RNA encapsidation, and the X region encodes the X protein (HBx) which modulates host-cell signal transduction and is necessary for virus replication and spread.63 HBV is not directly cytotoxic to liver cells, 65 which, in the case of hepatitis B, are usually injured by the host immune responses, especially T-cell responses, to viral antigens displayed on the infected cells, and by the cytotoxic by-products of the secondary antigen-nonspecific inflammatory responses, such as tumor necrosis factor (TNF), free radicals, and proteases.63 21
HBV is transmitted through body fluids especially through the blood, and can remain alive in dried blood for longer than one week. 66 While the primary infections in healthy adults, whether symptomatic or not, are usually self-limited and followed by virus clearance and immunity development, 67 the impact to susceptible (non-immune) infants and children are much more of concern. When infected, 90% of infants and 30% of children under year 5 will progress into chronic infection because of their immature immune systems. 68 Chronic HBV infection is defined as HBsAg positivity for at least six months, symptomatic or not. 69 People with abnormal serum aminotransferase levels and liver biopsy findings are classified as having chronic hepatitis B, and those with subclinical persistent infection but normal liver function and histological features are termed asymptomatic chronic HBV carriers.63 Chronic HBV carriers have a risk of developing into HCC 100 fold higher than that of non-carriers. 70 There are an estimated 350 million chronic HBV carriers worldwide, 71 with a marked geographic variation in the prevalence. In North and South America, Western Europe, and Australia, less than 2% of the population has HBsAg positivity. 72 n the other hand, 10% to 20% of the populations of Southeast Asia and Sub-Saharan Africa are HBsAg carriers, with the majority infected perinatally or in infancy.71 The geographic variation in HBV infection prevalence corresponds well with HCC incidence. High levels of HBV endemicity are concentrated in the developing world in Southeast Asia and Sub- Saharan Africa, which is also the area of highest HCC incidence.72 About 80% of HCC cases are associated with HBV infection.20 Since the early 1970s, more than 100 epidemiological studies have shown compelling evidence on 22
HBV s role in the etiology of HCC.15 A prospective study of 22,000 men in Taiwan during the 1970s showed a 223 times higher risk of developing HCC in HBV carriers compared to non-carriers, with a HCC death rate 351/100,000 carriers. 73 A similar figure (214/100,000) was also found in 1,069 HBV carriers followed in Toronto, implying a similar etiologic effect from HBV even in western populations. 74 The mechanism of HBV s effect in HCC remains incompletely understood, although numerous lab research studies have proposed several hypotheses with supporting evidence. In HBV related HCC tissues, more than 90% have clonally integrated HBV-DNA sequences. 75 However, no known proto-oncogene was found in HBV s genome, and no specific region in the host s genome was noticed for the integration of HBV DNA. HBV X protein has been found to bind and inactivate tumor suppressor p53 protein in HCC cells, and the pres2/s proteins were found to transactivate cellular oncogenes in vitro. 76 Because of persistent infection, repeated damage to liver cells permanently initiates regeneration, giving rise to the risk of mutation and thus serves as a tumor promoter. The possible initiation and promotion actions by HBV make it a complete carcinogen. 77 Due to the intensive, compelling evidence from accumulated epidemiological studies and lab research, the IARC has classified HBV as Category I known human carcinogen,15 and the NTP has classified HBV as known to be a human carcinogen in the 11 th Report on Carcinogens.62 23
Albeit 2.2.3 ther Etiological Risk Factors Besides AFB 1 and HBV, the most important etiological factor of HCC is hepatitis C virus (HCV) infection, which accounts for 24% of all liver cancers in the world10 and is mainly responsible for most HCC cases in Europe, North America,71 Japan, 78 and other economically developed regions. HCV is a positive-sense, single-stranded RNA virus with no reverse transcriptase and does not integrate into the host genome. 79 The major route of HCV transmission seems to be the parenteral exposure to contaminated blood, such as via transfusion, needle-sharing drug abuses and other accidental medical injections. 80 An estimated 85% of HCV infections will develop into persistence with the majority asymptomatic, 81 and no vaccine is currently available for active prevention.71 HCV chronic infection alone will increase HCC risk by 24-fold, 82 with 20-40% patients progressing into liver cirrhosis which is the very risk state for HCC. 83 Being a nonintegrating virus, HCV is less likely to act as an initiator in HCC. 72 the mechanisms have not been elucidated, a prolonged period of hepatocellular damage progressing from chronic hepatitis to cirrhosis to HCC is usually required for HCV-related malignancy, 84 suggesting a promoting role of HCV. Due to convincing data correlating HCV chronic infection and HCC development, IARC Working Group categorized HCV as being carcinogenic to humans,15 and NTP classified HCV as known to be a human carcinogen.62 Excessive alcohol intake has long been suggested to be an important contributor to HCC risk by earlier clinical studies in North America, and was confirmed by following epidemiological data worldwide.11 The alcohol-induced cirrhosis accounts for 12% HCC 24
cases in Milan, Italy, 85 15% in the United States, 86 and 6% in Japan. 87 The mechanism seems to be repeated unspecific damage to liver tissues which induce cirrhosis that is usually the common precursor of HCC. 88 Cigarette smoke has multiple chemical components that are hepatic carcinogens in animals, 89 and epidemiological studies have suggested cigarette smoking as an independent risk factor for HCC. 90 Exogenous sex hormones also have been suggested to increase HCC risk. 91 Estrogens are strong promoters of hepato-carcinogenesis in animals, 92 and oral contraceptive use has been associated with increased HCC risk by a number of epidemiological studies. 93,94 A series of case reports also documented the occurrence of HCC in relatively young men who were androgenic steroids users, 95, 96, 97 consistent with experimental findings that male animals are more susceptible to chemically induced HCC than female.11 These risk factors for HCC, albeit suggested as causative by epidemiological studies, generally play a minor role on the global scale as compared to that of HBV, HCV, and AFB. However, additive 98 and synergistic71 effects do exist for these minor factors which may exaggerate the situation for HCC risk if co-act with major and/or other minor risk factors. 2.3 xidative DNA Damage and Biomarker There is a background oxidative DNA damage in living organisms physiologically, due to the endogenous reactive oxygen species (RS) generated intentionally by inflammation responses, and inadvertently by the imperfect electron transport chains in aerobic respiration. 99 The background oxidative burden and DNA 25