An investigation into the inflammatory properties of tenofovir in HepG2 human liver cells by SONGEZO VAZI Submitted in fulfilment of the requirements for the degree of Master of Medical Science (Physiology) in the Department of Basic Medical Sciences School of Biomedical Sciences Faculty of Health Sciences University of the Free State 2023 Supervisor: Dr C Tiloke Co-supervisor: Dr S Van Zyl Co-supervisor: Dr Vorster-de Wet i DECLARATION I hereby declare that this work, submitted for the degree Master of Medical Science with specialisation in Physiology at the University of the Free State, is my original work and has not previously been submitted to any other institution of higher learning for degree purposes or otherwise. I further declare that all sources cited or quoted are indicated and acknowledged through a comprehensive list of references. Copyright hereby cedes to the University of the Free State. -------------------------- ----------------------- Name and Surname Date 24 July 2023 ii DEDICATION I dedicate this thesis to my family Nokwandisa Vazi, Zimkhatha Vazi, Kwakhanya Vazi and Liyema Vazi I appreciate your continuous motivation, words of encouragement, and prayers. iii ACKNOWLEDGEMENTS SUPERVISORY TEAM Dr Charlette Tiloke, Dr Sanet van Zyl and Dr Roné Vorster-de Wet, I appreciate your guidance and advice throughout the study. The study would not have been possible without your valuable suggestions and constructive feedback. I am also grateful for the skills that you have given me to develop into a better researcher. Your valuable time spent with us in the laboratory after hours, during the weekends and holidays is much appreciated. I am blessed to be supervised by you. Thank you for your endless inspiration, motivation and dedication in sharing your knowledge. Thank you for all your efforts in developing me. Thank you for your assistance in the manuscript preparations. I thank you for your presence throughout my master’s degree journey. MY FRIENDS Mr Bopape Abel Matlola, Miss Saki Mbasakazi and Miss Moremane Malebogo, thank you for your unwavering support, assistance and teamwork. I am grateful to have classmates like you. Thank you for your friendship. BASIC MEDICAL SCIENCES DEPARTMENT STAFF Dr Claudia Matlakala Ntsapi and Dr Abrahams Beynon, and the rest of the staff, thank you for your support and assistance throughout this thesis. I am so grateful to have met such kind, motivating, hardworking individuals. MY FAMILY Thank you for your love, support and encouragement. To my parents, this project would not have been possible without you. Thank you for your financial support. It’s greatly appreciated. I thank you so much for all the sacrifices you have made to help me reach this goal. FUNDING I thank the University of the Free State tuition bursary for the master’s partial funding opportunity. The Avacare bursary from Farmovs also supported the study. Thank you for recognising the importance of research. iv PUBLICATIONS 1) Vazi, S., van Zyl, S., Vorster-de Wet., R, Tiloke, C. 2023. A review into the inflammatory properties of tenofovir in HepG2 human liver cells. Health Sciences Review 8(2023): 100114. v PRESENTATIONS The study titled: An investigation into the inflammatory properties of tenofovir in HepG2 human liver cells by Vazi, S., van Zyl, S., De Wet., P.C., Tiloke, C was presented to the Evaluation Committee (Oral): 1. Department of Basic Medical Sciences 10 August, University of the Free State, Bloemfontein, South Africa, 2021 The study titled: An investigation into the inflammatory properties of tenofovir in HepG2 human liver cells by Vazi, S., van Zyl, S., De Wet., P.C., Tiloke, C was presented at the Departmental Journal Club (Oral): 1. Department of Basic Medical Sciences 21 September, University of the Free State, Bloemfontein, South Africa, 2022 The study titled: An investigation into the inflammatory properties of tenofovir in HepG2 human liver cells by Vazi, S., van Zyl, S., De Wet., P.C., Tiloke, C was presented at the Health Sciences Faculty meetings (Oral): 1. Department of Haematology 14 March, University of the Free State, Bloemfontein, South Africa, 2023 The study titled: An investigation into the inflammatory properties of tenofovir in HepG2 human liver cells by Vazi, S., van Zyl, S., De Wet., P.C., Tiloke, C will be presented at a local conference (Oral): 1. Faculty of Health Sciences Research Forum 22-25 August, University of the Free State, Bloemfontein, South Africa, 2023 vi PREFACE ARRANGEMENT OF THE THESIS The following outline provides the reader with an overview of the arrangement of the thesis: Chapter 1: This chapter briefly explored the global and local burden of HIV, introducing the background and problem statement of the study, followed by the research rationale, aims, research questions and objectives of the study. The chapter concluded with the significance, the value of the study and the arrangement of the thesis. Chapter 2: This chapter provides an overview of existing literature on HIV/AIDS, antiretroviral drugs, inflammation and tenofovir’s properties. Chapter 3: Outlines the material and methods used in the research study, followed by a detailed description of the study design, techniques and procedures used during the research project. Chapter 4: This chapter presents the core findings of this study derived from methods. Chapter 5: The discussion provides the interpretation and description of the significance of the research findings considering what is already known about tenofovir and its mechanism in liver inflammation. Chapter 6: Finally, chapter 6 presents the study conclusions and recommendations for future research areas. This chapter also includes limitations for the study. Lastly, references and appendices are incorporated at the end of the thesis. vii TABLE OF CONTENTS DECLARATION ............................................................................................................................. i DEDICATION ................................................................................................................................ ii ACKNOWLEDGEMENTS ........................................................................................................... iii PUBLICATIONS .......................................................................................................................... iv PRESENTATIONS ......................................................................................................................... v PREFACE ...................................................................................................................................... vi TABLE OF CONTENTS .............................................................................................................. vii LIST OF FIGURES ..................................................................................................................... xiii LIST OF TABLES ........................................................................................................................ xv LIST OF APPENDICES .............................................................................................................. xvi LIST OF ABBREVIATIONS ..................................................................................................... xvii ABSTRACT .................................................................................................................................. xx CHAPTER 1: ORIENTATION OF THE STUDY ......................................................................... 1 1.1 INTRODUCTION .................................................................................................................. 1 1.2 PROBLEM STATEMENT ..................................................................................................... 4 1.3 RESEARCH RATIONALE .................................................................................................... 5 1.4 AIM OF THE STUDY ............................................................................................................ 6 1.5 RESEARCH QUESTIONS..................................................................................................... 6 1.6 OBJECTIVES OF THE STUDY ............................................................................................ 6 1.7. METHODOLOGY.................................................................................................................. 7 1.8. SIGNIFICANCE AND VALUE OF THE STUDY ............................................................... 7 1.8.1 Significance ....................................................................................................................... 7 1.8.2 Value ................................................................................................................................. 7 1.9 ARRANGEMENT OF THE THESIS.................................................................................... 8 1.10. CONCLUSION ...................................................................................................................... 9 CHAPTER 2: LITERATURE REVIEW ...................................................................................... 10 2.1 INTRODUCTION ............................................................................................................... 10 2.2 HUMAN IMMUNODEFICIENCY VIRUS / ACQUIRED viii IMMUNODEFICIENCY SYNDROME ............................................................................ 10 2.2.1 BACKGROUND ................................................................................................................. 10 2.2.2 Classification of human immunodeficiency virus ............................................................... 11 2.2.3 Transmission of HIV/AIDS ................................................................................................. 11 2.2.4 The HIV replication cycle and the immune response .......................................................... 11 2.2.5 Management techniques of HIV/AIDS ................................................................................ 14 2.3 ANTIRETROVIRAL DRUGS ........................................................................................... 15 2.3.1 Classes of antiretroviral drug therapy .................................................................................. 15 2.3.2 Tenofovir–lamivudine–dolutegravir, a fixed-dose combination therapy ............................ 18 2.3.2.1 Lamivudine ................................................................................................................ 19 2.3.2.2 Dolutegravir ............................................................................................................... 20 2.3.2.3 Tenofovir .................................................................................................................... 21 2.3.3 The effect of tenofovir on inflammation ............................................................................ 22 2.3.4 Effect of antiretroviral drugs .............................................................................................. 23 2.4. ANATOMY AND PHYSIOLOGY OF THE LIVER ....................................................... 24 2.4.1 Metabolism of tenofovir..................................................................................................... 25 2.4.2. Antiretroviral drugs effect on the liver .............................................................................. 26 2.4.3 ART-induced mitochondrial toxicity ................................................................................. 26 2.5 INFLAMMATION ............................................................................................................ 27 2.5.1 Liver inflammation linked to tenofovir .............................................................................. 27 2.5.2 Regulation of the inflammatory response .......................................................................... 28 2.5.3 NF-κB signalling pathway ................................................................................................. 28 2.5.4 Inflammatory cytokines ..................................................................................................... 30 2.6 Antiretroviral drug investigations in HepG2 liver cell model ............................................ 31 2.7 CONCLUSSION ................................................................................................................ 32 CHAPTER 3: MATERIALS AND METHODS ........................................................................ 33 3.1 INTRODUCTION .................................................................................................................. 33 3.2 RESEARCH DESIGN ............................................................................................................ 33 ix 3.3 MATERIALS .......................................................................................................................... 35 3.4 RESEARCH METHODS ....................................................................................................... 35 3.4.1 CELL CULTURE ................................................................................................................ 35 3.4.1.1 Introduction ....................................................................................................................... 35 3.4.1.2 Protocol ............................................................................................................................. 36 3.4.2 EXPERIMENTAL TREATMENT ...................................................................................... 36 3.5 DATA COLLECTION ........................................................................................................... 37 3.5.1 ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA). .......................................... 37 3.5.1.1 Introduction ....................................................................................................................... 37 3.5.1.2 Properties of ELISA .......................................................................................................... 37 3.5.1.3 Preparation of reagents...................................................................................................... 37 3.5.1.4 Sample Preparation ........................................................................................................... 38 3.5.1.5 Preparation of Standards ................................................................................................... 38 3.5.1.6 Protocol ............................................................................................................................. 38 3.5.2 DETERMINATION OF PROTEIN EXPRESSION (WESTERN BLOT) ......................... 39 3.5.2.1 PROTEIN ISOLATION ................................................................................................... 39 3.5.2.1.1 Introduction .................................................................................................................... 39 3.5.2.1.2 Protocol .......................................................................................................................... 39 3.5.3 QUANTIFICATION AND STANDARDISATION OF PROTEINS ................................. 40 3.5.3.1 Introduction ....................................................................................................................... 40 3.5.3.2 Protocol ............................................................................................................................. 40 3.5.4. WESTERN BLOTTING ..................................................................................................... 41 3.5.4.1 Introduction ....................................................................................................................... 41 3.5.4.2 Preparation of buffers........................................................................................................ 42 3.5.4.3 Sample preparation ........................................................................................................... 43 3.5.5 SDS-PAGE........................................................................................................................... 43 3.5.5.1 Introduction ....................................................................................................................... 43 3.5.5.2 Protocol ............................................................................................................................. 43 x 3.5.6. PROTEIN TRANSFER (ELECTRO-BLOTTING) ........................................................... 44 3.5.6.1 Introduction ....................................................................................................................... 44 3.5.6.2 Protocol ............................................................................................................................. 44 3.5.7 BLOCKING ......................................................................................................................... 45 3.5.7.1 Introduction ....................................................................................................................... 45 3.5.7.2 Protocol ............................................................................................................................. 45 3.5.8. ANTIBODY INCUBATION .............................................................................................. 46 3.5.8.1 Introduction ....................................................................................................................... 46 3.5.8.2 Primary antibody incubation ............................................................................................. 46 3.5.8.3 Secondary antibodies ........................................................................................................ 47 3.5.9. IMAGING ........................................................................................................................... 48 3.5.10 RE-PROBING .................................................................................................................... 48 3.5.10.1 Protocol ........................................................................................................................... 48 3.5.11 NORMALISATION .......................................................................................................... 48 3.5.11.1 Introduction ..................................................................................................................... 48 3.5.11.2 Protocol ........................................................................................................................... 49 3.5.12 QUANTITATIVE POLYMERASE CHAIN REACTION (qPCR) .................................. 49 3.5.12.1 Introduction ..................................................................................................................... 49 3.5.12.2 RNA ISOLATION .......................................................................................................... 50 3.5.12.3 RNA PURIFICATION ................................................................................................... 50 3.5.12.3.1 Preparation of buffers................................................................................................... 50 3.5.12.4 RNA QUANTIFICATION ............................................................................................. 51 3.5.12.5 cDNA SYNTHESIS ........................................................................................................ 51 3.5.12.6 Quantitative polymerase chain reaction (qPCR) ............................................................. 52 3.6 DATA ANALYSIS ................................................................................................................. 54 3.7 VALIDITY AND RELIABILITY .......................................................................................... 55 3.7.1 Validity................................................................................................................................. 55 3.7.2 Reliability ............................................................................................................................. 55 xi 3.8 ETHICAL CONSIDERATIONS ............................................................................................ 55 3.8.1 Approval............................................................................................................................... 55 3.9 CONCLUSION ....................................................................................................................... 56 CHAPTER 4: RESULTS OF THE QUANTITATIVE DATA .................................................... 57 4.1 INTRODUCTION .................................................................................................................. 57 4.2 CELL CULTURE ................................................................................................................... 59 4.2.1 HepG2 cells ........................................................................................................................... 59 4.3 ENZYME LINKED-IMMUNOSORBENT ASSAY (ELISA) .............................................. 59 4.3.1 Quantification of IL-6 levels ................................................................................................ 60 4.3.2 Quantification of IL-1β levels .............................................................................................. 61 4.3.3 Quantification of TNF-α levels ............................................................................................ 62 4.3.4 Quantification of IL-10 levels .............................................................................................. 63 4.4 QUANTITATIVE POLYMERASE CHAIN (qPCR) ............................................................ 65 4.4.1 Introduction .......................................................................................................................... 65 4.4.2 Determination of gene expression at different time intervals. ............................................. 66 4.4.2.1 Determination of NF-κBp65 expression at different time intervals .................................. 66 4.4.2.2 Determination of IκBα expression .................................................................................... 67 4.5 WESTERN BLOT .................................................................................................................. 68 4.5.1 Introduction .......................................................................................................................... 68 4.5.2 Determination of protein expression of the NF-κB signaling pathway. .............................. 68 4.5.2.1 Determination of NF-κBp65 protein at different time intervals. ...................................... 69 4.5.2.2 Determination of p-NF-κBp65 protein at different time intervals .................................... 70 4.5.2.3 Determination of IκBα protein at different time intervals. ............................................... 71 4.5.2.4 Determination of p-IκBα protein at different time intervals ............................................. 71 4.6 SUMMARY OF THE RESULTS ........................................................................................... 72 4.7 CONCLUSION ....................................................................................................................... 73 CHAPTER 5: DISCUSSION OF THE FINDINGS ..................................................................... 74 5.1 INTRODUCTION .................................................................................................................. 74 xii 5.2 DISCUSSION ......................................................................................................................... 76 5.3 TENOFOVIR’S EFFECT ON NF-ΚB SIGNALING PATHWAY AT ACUTE EXPOSURE .............................................................................................................................................. …….76 5.4 TENOFOVIR’S EFFECT ON NF-ΚB SIGNALLING PATHWAY AT CHRONIC EXPOSURE .................................................................................................................................. 78 CHAPTER 6: CONCLUSION, RECOMMENDATIONS AND LIMITATIONS OF THE STUDY. ........................................................................................................................................ 81 6.1 INTRODUCTION .................................................................................................................. 81 6.2 CONCLUSION ...................................................................................................................... 81 6.3 RECOMMENDATIONS ........................................................................................................ 82 6.4 LIMITATIONS OF THE STUDY .......................................................................................... 82 REFERENCES.............................................................................................................................. 83 APPENDICES ............................................................................................................................ 104 xiii LIST OF FIGURES Chapter 1 ......................................................................................................................................... 1 Figure 1.1: Line graph showing new HIV infections and related deaths in the South African population........................................................................................................................................ 2 Chapter 2 ....................................................................................................................................... 10 Figure 2.1: Structure of the Human Immunodeficiency Virus (HIV)........................................... 12 Figure 2.2: The HIV replication cycle .......................................................................................... 14 Figure 2.3: Chemical structure of lamivudine .............................................................................. 19 Figure 2.4: Chemical structure of dolutegravir ............................................................................. 20 Figure 2.5: Chemical structure of tenofovir .................................................................................. 21 Figure 2.6: Tenofovir’s effects on inflammatory markers at different time exposures ................ 23 Figure 2.7: Anatomy of the human liver ....................................................................................... 25 Figure 2.8: NF-κB signalling and inflammation ........................................................................... 29 Chapter 3 ....................................................................................................................................... 33 Figure 3.1: Schematic representation of the research design of the study .................................... 34 Figure 3.2: Serial dilutions ............................................................................................................ 38 Figure 3.3: Overview of Western blot procedure ......................................................................... 42 Figure 3.4: Gel electrophoresis ..................................................................................................... 44 Figure 3.5: Electro-blotting ........................................................................................................... 45 Chapter 4 ....................................................................................................................................... 57 Figure 4.1: Schematic overview of quantitative experimental assays used in the study .............. 58 Figure 4.2: Quantitative ELISA analysis of IL-6 levels in HepG2 cells following exposure to tenofovir at 24h (A) and 120h (B) ................................................................................................ 61 Figure 4.3: Quantitative ELISA analysis of IL-1β levels in HepG2 cells following exposure to tenofovir at 24h (A) and 120h (B) ................................................................................................ 62 Figure 4.4: Quantitative ELISA analysis of TNF-α levels in HepG2 cells following exposure to tenofovir at 24h (A) and 120h (B) ................................................................................................ 63 xiv Figure 4.5: Quantitative ELISA analysis of IL-10 levels in HepG2 cells following exposure to tenofovir at 24h (A) and 120h (B) ................................................................................................ 64 Figure 4.6: NF-κBp65 expression in HepG2 cells after exposure to tenofovir at 24h (A) and 120h (B) ..................................................................................................................................... ...67 Figure 4.7: IκBα expression in HepG2 cells after exposure to tenofovir at 24h and 120h ........... 68 Figure 4.8: NF-κBp65 protein expression in HepG2 cells after exposure to tenofovir at 24h (A) and 120h (B).................................................................................................................................. 70 Figure 4.9: p-NF-κBp65 protein expression in HepG2 cells after exposure to tenofovir at 24h (A) and 120h (B) ........................................................................................................................... 71 Figure 4.10: IκBα protein expression in HepG2 cells after exposure to tenofovir for 24h (A) and 120h (B) ........................................................................................................................................ 72 Figure 4.11: p-IκBα protein expression in HepG2 cells after exposure to tenofovir for 24h (A) and 120h (B).................................................................................................................................. 73 Chapter 5 ....................................................................................................................................... 75 Figure 5.1: Tenofovir’s pro- and anti-inflammatory properties in HepG2 human liver cells at different time exposures ................................................................................................................ 76 xv LIST OF TABLES Chapter 2 ....................................................................................................................................... 10 Table 2.1: HIV treatment regimens............................................................................................... 16 Table 2.2: Examples of pro- and anti-inflammatory cytokines .................................................... 30 Chapter 3 ....................................................................................................................................... 33 Table 3.1: Primary antibody and dilutions used during the Western blot procedure.................... 46 Table 3.2: Secondary antibodies ................................................................................................... 47 Table 3.3: Reaction volume and components of the High-Capacity RNA-to-cDNA kit ............. 52 Table 3.4: Volumes of TE buffer used to prepare gene primer main stock .................................. 53 Table 3.5: qPCR reaction mixture................................................................................................. 53 Table 3.6: Primer sequences used in qPCR assay ......................................................................... 54 Chapter 4 ....................................................................................................................................... 57 Table 4.1: Concentrations of IL-6 following 24h and 120h exposure to tenofovir ...................... 60 Table 4.2: Concentrations of IL-1β following 24h and 120h exposure to tenofovir .................... 61 Table 4.3: Concentrations of TNF-α following 24h and 120h exposure to tenofovir .................. 62 Table 4.4: Concentrations of IL-10 following 24h and 120h exposure to tenofovir .................... 64 Table 4.5: Cytokine concentrations in tenofovir-treated cells following 24h............................... 65 Table 4.6: Cytokine concentrations in tenofovir-treated cells following 120h............................. 66 xvi LIST OF APPENDICES APPENDIX A: LITERATURE REVIEW PUBLICATION ........................................................ 98 APPENDIX B: IL-6 standard curve.............................................................................................. 99 APPENDIX C: IL-1β standard curve ........................................................................................ 101 APPENDIX D: TNF-α standard curve. ...................................................................................... 103 APPENDIX E: IL-10 standard curve .......................................................................................... 105 APPENDIX F: BCA standard curve ........................................................................................... 107 APPENDIX G: qPCR raw data and calculations ........................................................................ 108 APPENDIX H: Western blot raw data .......................................................................................... 76 APPENDIX I: HepG2 cell images ................................................................................................. 78 APPENDIX J: HepG2 cell images ................................................................................................ 79 APPENDIX K: Hoechst stain 33342 ............................................................................................ 80 APPENDIX L: Health Sciences Research Ethics Committee (HSREC) Initial approval letter. .. 81 APPENDIX M: Health Sciences Research Ethics Committee (HSREC) Sub approval .............. 83 APPENDIX N: Language editing declaration .............................................................................. 85 xvii LIST OF ABBREVIATIONS 3TC Lamivudine 3TC-TP Lamivudine triphosphate AIDS Acquired Immunodeficiency Syndrome ART Antiretroviral therapy ARV Antiretroviral APC Antigen-presenting cell APP Acute phase proteins AZT Zidovudine CHB Chronic hepatitis B CRP C-reactive proteins CYP450 Cytochrome p450 dATP Deoxyadenosine-5’-triphosphate DC Dendritic cells DM Diabetes mellitus DMSO Dimethyl sulfoxide DTG Dolutegravir ER Endoplasmic reticulum FDA Food and Drug Administration FOHS Faculty of Health Sciences GAPDH Glyceraldehyde 3-phosphate dehydrogenase GM-CSF Granulocyte-macrophage colony-stimulating factor GP120 Glycoprotein 120 HAART Highly active antiretroviral therapy HBV Hepatitis B viruses HCV Hepatitis C viruses xviii HepG2 Human hepatoma cells HIV Human immunodeficiency virus HR1 Heptad repeat 1 HR2 Heptad repeat 2 HRP Horse-radish peroxidase HSREC Health Sciences Research Ethics Committee IKK IĸB kinase IL Interleukin Kg Kilograms mtDNA Mitochondrial DNA MtROS Mitochondrial reactive oxygen species NASH Nonalcoholic steatohepatitis NIAID National Institute of Allergy and Infectious Diseases NK Natural killer cells NF-ΚB Nuclear factor-kappa b NNRTIs Non-nucleoside reverse transcriptase inhibitors NRTIS Nucleoside reverse transcriptase inhibitors O2 Oxygen OD Optical density PAMP Pathogen-associated molecular pattern PBMCS Peripheral blood mononuclear cells PIs Protease inhibitors PRR Pattern recognition receptors Pol-γ Polymerase gamma qPCR Quantitative polymerase chain reaction ROS Reactive oxygen species xix RT Reverse transcriptase SA South Africa SAHPRA South African Health Products Regulatory Authority TCR T-cell receptor TDF Tenofovir disoproxil fumarate TLD Tenofovir, lamivudine, and dolutegravir TLE Tenofovir-lamivudine-efavirenz TLRS Toll-like receptors TLR4 Toll-like receptor 4 TNF-α Tumour necrosis factor-alpha UFS University of the Free State UNAIDS United Nations Programme on HIV/AIDS USA United States of America WHO World Health Organization xx ABSTRACT INTRODUCTION: Since the introduction of antiretroviral (ARV) drugs in 1996, the life expectancy of HIV-infected individuals has been nearly comparable to that of HIV-uninfected individuals. However, increasing evidence shows that antiretroviral therapy (ART) is associated with increased metabolic disorders, systemic inflammation, and hepatotoxicity. Tenofovir induces oxidative stress via mitochondrial DNA polymerase inhibition in HepG2 cells at chronic exposure. Although in vitro and in vivo studies have been performed to determine the effect of tenofovir on the inflammatory response, the inflammatory effect of this antiretroviral drug in liver cells still needs elucidation. AIM: This study aimed to investigate tenofovir's potential pro- and anti-inflammatory properties in HepG2 human liver cells at different time frames. METHODOLOGY: HepG2 cells were treated with tenofovir (1.2 µM) over 24h and 120h; pro- and anti-inflammatory cytokines levels were assessed using a SimpleStep human ELISA kit specific to each analyte (IL-6, IL-1β, TNF-α, IL-10). Protein expression of p-NF-ĸBp65, NF-ĸBp65, p-IĸBα, and IĸBα was determined with Western blotting. A quantitative polymerase chain reaction assessed the mRNA expression of NF-κBp65 and IkBα. RESULTS: Tenofovir significantly increased IL-6 and 10 levels, NF-κBp65 mRNA expression and NF-κBp65, p-NF-κBp65 and p-IκBα protein expression. Additionally, a significant decrease in IL- 1β levels and IκBα mRNA expression at 24h were observed. After 120h, tenofovir-treated cells showed increased p-NF-κBp65 and IκBα protein expression. Furthermore, a significant decrease in IL-6 and IL-10 levels, NF-κBp65 and IκBα mRNA expression and NF-κBp65 and p-IκBα protein expression were observed. CONCLUSION: The study demonstrated that tenofovir elevated the anti-inflammatory cytokines at acute exposure. Tenofovir increased pro-inflammatory cytokines and downregulated anti- inflammatory cytokines at chronic exposure of tenofovir in HepG2 human liver cells. The knowledge xxi obtained from tenofovir-induced inflammatory changes can provide valuable information regarding tenofovir’s clinical use. KEYWORDS: Tenofovir, HepG2 cell line, ART, HIV/AIDS, inflammatory properties 1 CHAPTER 1: ORIENTATION OF THE STUDY 1.1 INTRODUCTION Globally, the Human Immunodeficiency Virus (HIV) had a sustained negative impact on health since the report of the first cases in 1981 (Pant and Singh, 2018; Bosh et al.,2021). HIV can lead to Acquired Immunodeficiency Syndrome (AIDS), occurring at a later stage of infection (Stevenson et al., 2020). The immune system is affected by viral infections, increasing the risk of other diseases and infections, such as renal dysfunction, liver disease, and cardiovascular disease (Mahy et al., 2019). In 2020, an estimated 37.7 million individuals lived with HIV worldwide. This includes children, with 1.5 million newly infected people and a mortality rate of 680 000 from AIDS-related causes worldwide (WHO, 2021). The global HIV prevalence in adults is 0.8%, with approximately 7.1 million individuals unaware of their HIV-positive status (UNAIDS, 2020). South Africa (SA) is largely affected by HIV/AIDS (UNAIDS, 2020). In 2020, approximately 7.7 million people were HIV-positive, with a prevalence rate of up to 20.4% (UNAIDS, 2020). This prevalence increases among gay men, transgender women, sex workers, and drug addicts (Stevenson et al., 2020). Figure 1.1 illustrates the number of new HIV infections and deaths in SA (Marcus and MacDonell, 2020). The graph illustrates a declining trend of HIV infections since 1996. The decrease was followed by a sharp decline in HIV death observed from 2005. Subsequently, the number of HIV deaths is lower than HIV new infections, meaning more people living with HIV. This resulted from introducing antiretroviral therapy in 1996 and its wide use starting in 2005 (Trickey et al., 2017). 2 Figure 1.1: Line graph showing new HIV infections and related deaths in the South African population (Marcus and MacDonell, 2020) The introduction of antiretroviral (ARV) drugs is one of the most noticeable signs of progress in HIV/AIDS control (Schwetz and Fauci, 2019). In the 1980s, an AIDS diagnosis typically resulted in a 1-year life expectancy (NIAID, 2020). Today, with the combination of antiretroviral drug treatments, HIV-positive individuals can have a normal life expectancy (Smiley et al., 2021). However, long-term usage of antiretroviral drugs is associated with many adverse effects, such as mitochondrial toxicity, hepatotoxicity, and lactic acidosis (Calza et al., 2017). According to UNAIDS (2021), the affordability of a high generic combination of drugs such as tenofovir/lamivudine/dolutegravir (TLD) can help achieve the 95-95-95 strategy. This strategy was introduced in 2014 to diagnose 95% of HIV-positive people and aimed to suppress viral load, with 95% of patients receiving antiretroviral drugs by 2030. Today, 84% of HIV-positive people have been diagnosed, and only 90% are virally suppressed (UNAIDS, 2021). Among HIV-infected patients, liver disease is the most common non-AIDS-related cause of death (Morrison et al., 2019). The liver is the primary site of antiretroviral drug metabolism through the cytochrome P450 system (McMillan et al., 2018). Chronic use of antiretroviral drugs is associated with mitochondrial toxicity causing liver damage (Margolis et al., 2014; van Welzen et al., 2019). 3 Nucleotide reverse transcriptase inhibitors (NRTIs) cause mitochondrial toxicity by inhibiting mitochondrial DNA polymerase gamma (Pol-γ) (Smith et al., 2017). This inhibition reduces oxidative phosphorylation in the electron transport chain resulting in oxidative stress (Ganta and Chaubey, 2019). Tenofovir is a NRTI drug used with lamivudine and dolutegravir (Venter et al., 2019). A study by Nagiah and colleagues in 2015 has shown that tenofovir induces oxidative stress in human hepatoma (HepG2) liver cells, compared to other regimens (Nagiah et al., 2015). Lingappan (2018) found that oxidative stress affects NF-κB-related activities. NF-κB is a transcription factor activated in response to external stimuli, regulating pro-inflammatory genes (Liu et al., 2017). Antiretroviral therapy causes liver toxicity as one of its most serious side effects (Bordes et al., 2020). Several studies have been performed to determine tenofovir's cytotoxic effect; however, inflammatory response in liver cells still needs to be fully elucidated (Nagiah et al., 2015; Zhang et al.,15; Vidal et al., 2006). Therefore, in the present study, tenofovir’s inflammatory properties were investigated at different time exposures by assessing pro- and anti-inflammatory markers in HepG2 human liver cells. This study also focused on the role of oxidative stress on inflammatory mediators of the NF- κB signalling pathway. 4 1.2 PROBLEM STATEMENT A major problem linked to the successful application of antiretroviral therapy in HIV-positive people is liver disease (Ganesan et al., 2018). Tenofovir is one of the ARV drugs used as a first-line regimen known to suppress HIV viral load successfully. However, its clinical application is limited by a lack of understanding of its inflammatory response in human liver cells. Liver toxicity has been linked to long-term use of highly active antiretroviral therapy (HAART) (Baynes et al., 2017); thus, the need for antiretroviral agents that are safer and more effective. South Africa has 84.6% of HIV-positive people, with 70.7% on ART (Marinda et al., 2020). Chronic antiretroviral therapy is linked to increased metabolic disorders such as metabolic syndrome, dyslipidemia, and systemic inflammation (Calza et al., 2017). Furthermore, a study by Bakasis and Androutsakos (2021). identified liver dysfunction, which is usually related to the inflammatory mechanism and pharmacological impact of ART (Bakasis and Androutsakos., 2021). While these complications are common in the general population, the sub-Saharan Africa population has a prevalence rate of deaths related to liver disease of 2.5 (Spearman, 2023). Tenofovir is the preferred drug in the NRTI class. It is deemed a safer alternative to treat HIV/AIDS and hepatitis B. On the other hand, tenofovir has been shown to cause mitochondrial structural changes and dysfunction while retaining mtDNA levels in the kidneys, resulting in nephropathy (Zanger and Schwab, 2013) and, in the liver, elevating mitochondrial reactive oxygen species (MtROS) induction resulting in hepatotoxicity (Abraham et al., 2013). 5 1.3 RESEARCH RATIONALE The Southern African HIV Clinicians Society guidelines recommend the combination of tenofovir with dolutegravir and lamivudine as a first-line regimen (SAHCS, 2012). Tenofovir is regarded as a potent drug for HIV treatment; however, recently, it has been associated with many adverse effects, such as lactic acidosis, hepatic steatosis, and liver cirrhosis (Wassner et al., 2020). According to Nagiah et al. (2015), tenofovir induces oxidative stress via mitochondrial DNA polymerase inhibition in HepG2 cells. Enhanced generation of reactive oxygen species can activate the NF-κB signalling pathway through the IĸB kinase (IKK) complex system (Adebayo et al., 2020). NF-κB is an important pro-inflammatory transcription factor that plays a significant role in oxidative stress-induced inflammation. Following its activation, it can increase the transcription of various genes and subsequently regulate inflammation. Based on the above information, tenofovir exhibits its cytotoxic effect via induced mitochondrial dysfunction; however, its effect on liver inflammation is yet to be determined. Therefore, the overall goal of this study was to broaden the understanding of the inflammatory properties of tenofovir by investigating its effect on HepG2 cells. Investigating the inflammatory properties of tenofovir in acute and chronic exposure can provide physiologically relevant insight, supplement available information on tenofovir and inform targeted therapeutic intervention. 6 1.4. AIM OF THE STUDY The study aimed to determine tenofovir's potential pro- and anti-inflammatory properties in HepG2 human liver cells at different time exposures. 1.5 RESEARCH QUESTIONS This study aimed to answer the following research questions: 1.5.1 How does tenofovir affect pro- and anti-inflammatory cytokines in HepG2 cells? 1.5.2 Which inflammatory proteins will be expressed after exposure to tenofovir in HepG2 cells? 1.5.3 Will the NF-κB signalling pathway be induced after exposure to tenofovir in HepG2 cells? 1.6. OBJECTIVES OF THE STUDY From the research questions, the following objectives were identified: 1.6.1 To quantify pro- and anti-inflammatory cytokine levels (IL-6, IL-1β, TNF-α, IL-10) after exposure to tenofovir in HepG2 human liver cells. 1.6.2 To measure inflammatory mRNA (NF-ĸBp65 and IĸBα) following exposure to tenofovir in HepG2 human liver cells. 1.6.3 To determine the protein expression of the NF-κB signalling pathway (p-NF-ĸBp65, NF-ĸB- p65, p-IĸBα, and IĸBα) after exposure to tenofovir in HepG2 human liver cells. 7 1.7. METHODOLOGY This study followed an experimental research design. An in vitro assessment of pro- and anti- inflammatory markers was done after exposure of HepG2 cells to tenofovir. Treatment with tenofovir and untreated controls was conducted over two time periods, acute (24h) and chronic (120h) exposure. Pro- and anti-inflammatory cytokines were assessed using a SimpleStep human ELISA kit specific to each analyte (IL-6, IL-1β, TNF-α, IL-10). Protein expression of the NF-κB signalling pathway was determined with Western Blot. A quantitative polymerase chain reaction assessed the mRNA expression of NF-κBp65 and IκBα. The knowledge obtained from tenofovir-induced inflammatory changes can provide valuable information regarding tenofovir’s clinical use. 1.8. SIGNIFICANCE AND VALUE OF THE STUDY 1.8.1 Significance This study contributes to scientific knowledge by elucidating tenofovir’s effect on the human liver cell inflammatory response. Understanding tenofovir’s inflammatory properties might lead to advanced tools to gain insight into pro- and anti-inflammatory cytokines profiles. Comprehension of tenofovir’s possible effect on inflammatory cytokines and transcription factors elucidates this drug’s acute and chronic response mechanism. 1.8.2 Value The value of this study is to provide a scientific basis for treatment management, reducing the chances of developing liver inflammation. The lack of information contributing to understanding the effects of tenofovir on inflammation justifies the initiation of the present study. This study aims to broaden the understanding of the inflammatory properties of tenofovir in HepG2 cells. Therefore, allowing proactive measures to be taken in the prevention of liver inflammation. 8 1.10 CONCLUSION Chapter 1 provided an orientation to this study entitled: An investigation into the inflammatory properties of tenofovir in HepG2 human liver cells. It briefly introduced the problem statement, research rationale and aims, research questions and objectives of the study. It also provided a brief overview of the significance and value of the study. 9 CHAPTER 2: LITERATURE REVIEW 2.1 INTRODUCTION Chapter 2 provides an overview of existing literature on HIV/AIDS, antiretroviral drugs, tenofovir’s properties, its association with inflammation and its effect on the liver. Furthermore, available literature regarding antiretroviral effects on HepG2 human liver cell models will be discussed. In this study, literature searches were conducted using several search engines such as Google Scholar, PubMed, Science direct, UFS electronic journals, EBSCO web and Google Chrome. 2.2 HUMAN IMMUNODEFICIENCY VIRUS / ACQUIRED IMMUNODEFICIENCY SYNDROME 2.2.1 BACKGROUND Acquired Immunodeficiency Syndrome (AIDS) was first described in 1981 (CDC 1982; Greene 2007). After several years, the causative lentivirus that emerged as the Human Immunodeficiency Virus-1 (HIV-1) was identified. However, HIV-1 and HIV-2 originated from the Simian Immunodeficiency Viruses (SIVs) of primates (Sharp and Hahn, 2011). Thus, HIV-1 and HIV-2 had a zoonotic derivation but are currently spread from human to human (Sharp and Hahn, 2011). The transmission of the SIVs to humans remains a mystery. Still, it is suspected that it may have occurred during the hunting of the primates by indigenous people from Central and Western Africa (Schneider, 2021). Medical specialists apprehend that HIV has become a worldwide pathogen capable of manifesting in almost every organ, causing severe illnesses, especially in the advanced stage of the disease (Alonzo and Reynolds, 1995). 10 2.2.2 Classification of Human Immunodeficiency Virus The two main types of HIV viruses, HIV-1 and HIV-2, belong to the family of Retroviruses in the genus of Lentiviruses (Sharp and Hahn, 2011). However, the disease appearances are similar (Castro- Nallar et al., 2012). Retroviruses are associated with autoimmune diseases, malignancies, and immunodeficiency syndromes (Blattner, 1989). HIV-1 has been classified into four subtypes: major (M), new (N), outlier (O), and putative (P); each subtype represents an independent transmission of SIV into humans (Sharp et al., 2001). However, the major (M) group is the predominant subtype of HIV, with more than 90% of HIV/AIDS cases resultant from HIV-1 (Spira et al., 2003). HIV-2 shows molecular heterogeneity with five subtypes, A to E, with Subtype A and B viruses considered epidemic (Gao et al., 1994). The HIV-1 strain is observed globally and has high virulence, while the HIV-2 strain is confined to areas of West Africa and has inadequate virulence (Serra et al., 2021). 2.2.3 Transmission of HIV/AIDS HIV can be transmitted in two ways, namely, horizontal and vertical transmission (Taqaddas, 2020). In horizontal transmission, the virus is transmitted from one individual to another via unprotected sex, contaminated blood, and sharing of needles (Shamsi, 2019). Vertical transmission refers to mother-to-child transmission through childbirth and breastfeeding (Edwards et al., 2006). 2.2.4 The HIV replication cycle and the immune response The HIV genome comprises two identical single-stranded RNA molecules encased within the inner core of the virus (Figure 2.1). The outer phospholipid bilayer consists of constituents that are significant for the virus’s infection and disease development. The viral envelope glycoprotein 120 (gp120) is visible on the surface of HIV. This envelope, gp120, interacts with the host cell receptors on normal healthy cells such as CD4+ lymphocytes, macrophages, and monocytes (Paoletti et al., 11 2019). Glycoprotein 120 is equivalent to gp41, the envelope transmembrane viral protein necessary for viral-cell membrane fusion (Finzi et al., 2010). Glycoprotein 120 interact with the CD4+ receptors through chemokine receptor CCR5 (macrophage- trophic) and CXCR4 (T-cell-trophic); these co-receptors allow cell binding and entry of the virus (Shearer, 1998). HIV viral infection impairs cellular functions, characterised by a decline in CD4+ cell count. This decline increases susceptibility to opportunistic, viral, bacterial, protozoa and fungal infections (Sadiq et al., 2018). This immune deficiency is known as AIDS (Gallo and Montagnier, 2003). Figure 2.1 below illustrates the structure of HIV and its essential parts for viral replication. Figure 2.1: Structure of the Human Immunodeficiency Virus (HIV) (Dawany, 2010) The HIV replication cycle can be divided into six stages, summarised in Figure 2.2 and discussed below: 1. Binding and Fusion: The initial stage of the replication cycle begins with virus particles adhering to a CD4+ receptor and with co-receptors on the surface of a CD4+ T-lymphocyte. The virus merges with the host cells releasing its RNA into the host cell cytoplasm (Sperber, 2021). 12 2. Reverse Transcription: The enzyme reverse transcriptase is crucial for converting the single- stranded RNA to double-stranded DNA. This conversion of RNA to DNA allows HIV to combine with the cell's genetic material (Shcherbatova et al., 2020). 3. Integration: The integrase enzyme integrates newly formed viral DNA into the host cell's nucleus, forming a provirus that can be activated to produce viral proteins (Anderson and Maldarelli, 2018). 4. Transcription: The process of copying information from a DNA strand into a new shorter strand of RNA called messenger RNA (mRNA). The mRNA is utilised as a blueprint to synthesise long chains of HIV proteins (Sperber, 2021). 5. Assembly: The newly produced HIV proteins and RNA translocates into the cell’s surface and assemble into immature HIV (Shcherbatova et al., 2020). 6. Budding: The newly assembled immature HIV pushes itself out of the host CD4+ cell. The protease enzyme breaks up long protein chains in the immature virus creating the mature infectious virus. The new copies of HIV can now infect other CD4+ cells (Shcherbatova et al., 2020). 13 Figure 2.2: The HIV replication cycle (NIAID, 2018) 2.2.5 Management techniques for HIV/AIDS Medical scientists and clinical practitioners continue searching for an HIV vaccine, treatment, and cure (Pitman et al., 2018). Treatment with antiretroviral drugs can control HIV, but there is no cure for it. Developing new medications and treatment strategies has greatly improved HIV infection management over the past decade (Tseng et al., 2015). The use of combination antiretroviral therapy (cART) generally resulted in effective control of HIV viremia. It maintained the increase in CD4+ T- cell numbers. Both HIV-1 and HIV-2 lead to AIDS in affected individuals; therefore, disease monitoring includes CD4+ cell count, while treatment includes antiretroviral drugs (Volberding et al., 2004). 14 According to Chibawara et al. (2019): “HIV/AIDS has struck regions, countries, and populations in unusual ways. With the introduction of antiretroviral drugs, people living with HIV (PLHIV) have a much better prognosis.” In the 1980s, HIV/AIDS mortality rose steadily and peaked in 1995 (Taqaddas, 2020). This disease weakens the body’s immune system making it vulnerable to other infections. However, after investigating the HIV mechanisms of action and its replication cycle, antiretroviral therapy was introduced in 1996, giving HIV-positive individuals a better prognosis. 2.3 ANTIRETROVIRAL DRUGS Antiretroviral (ARV) drugs are used to treat HIV (WHO, 2014). Antiretroviral therapy (ART) has reduced HIV-associated morbidity and mortality (Calza et al., 2017). The primary aim of ARV drugs is to provide a better quality of life for HIV-positive individuals by restoring immunologic functions and viral suppression. These ARV drugs inhibit different phases of the HIV replication cycle; thus, they are classified into six classes. 2.3.1 Classes of antiretroviral drug therapy Over 30 treatment regimens (Table 2.1) are classified into six different classes according to their molecular mechanisms and resistance profiles (Lu et al., 2018). The mechanism of action of each drug type differs (Table 2.1). Generally, drugs from two or sometimes three classes are combined to ensure the necessary efficacy (Calza et al., 2017). However, the antiretroviral drug classes share a common goal: to prevent the virus from replicating and allow the immune system to produce more CD4+ T cells (Calza et al., 2017). Table 2.1 below summarises classes of HIV treatment regimens. 15 Table 2.1: HIV treatment regimens (compiled by the researcher, S Vazi) Class of Antiretroviral drugs Antiretroviral drugs Mechanism of action References Nucleoside reverse transcriptase inhibitors (NRTIs) - Tenofovir - Lamivudine - Stavudine Reverse transcriptase Inhibition Gulick, 2003 Non-nucleoside reverse transcriptase inhibitors (NNRTIs) - Efavirenz - Nevirapine - Etravirine Reverse transcriptase Inhibition De Clercq, 1995 Protease inhibitors (PIs) - Saquinavir - Tipranavir Protease inhibition De Clercq, 1995 Fusion inhibitors (FIs) - Aplaviroc - Ibalizumab Fusion inhibition of HIV to CD4+ T cells Gulick, 2003 Co-receptor inhibitors (CRIs) - Maraviroc - Vicriviroc Block the virus from binding to the co-receptor De Clercq, 1995 Integrase inhibitors (INIs) - Dolutegravir - Raltegravir Inhibit viral DNA strand transfer Lataillade and Kozal, 2006 HIV treatment regimens, as mentioned in Table 2.1, have different mechanisms of action. After the drug has been introduced into viral DNA, nucleoside reverse transcriptase inhibitors (ARVs that induce viral DNA termination are classified as class 1) block reverse transcription by inducing chain termination (Table 2.1) (Sahin, 2020). When reverse transcriptase joins viral DNA, the NRTIs that lack the 3'-OH group function as chain terminators (Edagwa et al., 2017). NRTIs are activated intracellularly by phosphotransferases and nucleoside kinases into an active form (Holec et al., 2017). Drugs commonly used in this class include tenofovir, which is used in combination with lamivudine (3TC) and dolutegravir (DTG) to suppress HIV effectively (Pau and George, 2014). The shape of the catalytic site of reverse transcriptase is altered by non-nucleoside reverse transcriptase inhibitors (ARVs that directly target enzyme reverse transcriptase classified as Class 2 16 drugs) through direct inhibition (Table 2.1) (Edagwa et al., 2017). Two subunits (p66 and p51) form a heterodimer called HIV reverse transcriptase (Sahin, 2020). NNRTIs bind the p66 subunit in a hydrophobic pocket away from the enzyme’s active site (Fletcher et al., 2020). Due to the non- competitive binding, the enzyme undergoes a conformational change, altering the active site and restricting its activity (Edagwa et al., 2017). The Class 3 drugs (ARVs targeting protease enzyme), Protease inhibitors (PIs), are the most effective anti-HIV drugs but are associated with many adverse effects such as gastrointestinal (diarrhoea and vomiting) and metabolic complications (dyslipidemia and insulin resistance) (Nagiah et al., 2015). PIs are intended to suppress viral proteases at the later stage of viral replication and maturation. During viral maturation, protease separates Gag and Gag-Pol polypeptide precursors (Pau and George, 2014). The inhibition of the enzyme prohibits a mature infectious virus from developing (Pau and George, 2014). For Class 4 drugs (ARVs that target glycoprotein 41), the fusion process of the viral life cycle is dependent on the communication of two areas of the gp41 transmembrane protein heptad repeat 1 and 2 (HR1 and HR2) (Mzoughi et al., 2019). Combining these two motifs results in a hairpin structure that pushes the cell membrane toward the viral membrane (Mzoughi et al., 2019). Fusion inhibitors imitate one of these domains and stop intramolecular interaction from occurring. Fortunately, these drugs also make the virus more susceptible to anti-gp41 antibodies by prolonging the virus’s exposure during fusion (Nagiah et al., 2015). Co-receptor inhibitors (ARVs that target glycoprotein 120 are classified as Class 5 drugs) act as allosteric viral entry inhibitors (Shamsabadi, 2014). A co-receptor, CCR5, is activated by gp120 during viral replication to initiate fusion (Nagiah et al., 2015). CCR5 antagonists are small molecules that bind to the hydrophobic pockets between the receptors (Shamsabadi, 2014). These small molecules alter the CCR5 receptor’s conformation, rendering it unrecognisable to the virus (Hashemi, 2019). 17 Class 6 drugs (ARVs that target an enzyme integrase), Integrase inhibitors (IN) process the 3' end of viral DNA and aid in the viral DNA strand joining to host DNA (Nagiah et al., 2015). Integrase inhibitors are the most recent advancement in ARV therapy (Cames et al., 2018). Integrase inhibitors bind to the complex formed by viral RNA and Integrase to prevent viral DNA strand transfer (Nagiah et al., 2015). Integrase inhibitors have a metal-binding pharmacophore, allowing them to interact with magnesium ion cofactors, which is essential for Integrase function (Sahin, 2020). A few regimens with appropriate antiretroviral strength are currently available, consisting of three or four antiretroviral drugs (Eggleton and Nagalli, 2020). In 2018, the combination of tenofovir- lamivudine-efavirenz (TLE) was introduced as the first-line regimen for HIV-1 treatment (Kouanfack et al., 2019). Nevertheless, TLE has a low genetic barrier to drug resistance (Raffi et al., 2014). Therefore, the South African National Department of Health replaced TLE with tenofovir– lamivudine–dolutegravir (TLD), a fixed-dose combination (Mendelsohn and Ritchwood, 2020). 2.3.2 Tenofovir–lamivudine–dolutegravir, a fixed-dose combination therapy Tenofovir–lamivudine–dolutegravir (TLD) is a fixed-dose combination of antiretroviral medication used to treat HIV/AIDS (Mendelsohn and Ritchwood, 2020). It is a combination of two NRTIs (tenofovir and lamivudine) and one PI (dolutegravir) (Eggleton and Nagalli, 2020). In October 2019, the South African National Department of Health introduced TLD (Mendelsohn and Ritchwood, 2020). The TLD treatment is proven to provide effective viral suppression and a high genetic barrier to resistance compared to other combination therapy (Umar et al., 2020). Detailed information on each drug is given below. 18 2.3.2.1 Lamivudine Lamivudine belongs to NRTIs class, inhibiting viral DNA synthesis through DNA chain termination (Max and Sherer, 2000). Inactive 3TC is phosphorylated by nucleoside kinase into active lamivudine triphosphate (3TC-TP) (Taylor et al., 2020). The 3TC-TP competes for the viral binding site with endogenous triphosphate. Lamivudine is well-tolerated in combination with other antiretroviral drugs in HIV-infected individuals (Dumitrescu et al., 2020). Figure 2.3 below illustrates the chemical structure of lamivudine. Figure 2.3: Chemical structure of lamivudine (Matta et al., 2012) Lamivudine is a monothioacetal that consists of cytosine having a (2R,5S)-2-(hydroxymethyl)-1,3- oxathiolan-5-yl moiety in the first carbon (Sohrabi and Zarkesh, 2014; Reis et al., 2020). The 3TC- TP inhibit HIV-1 and HIV-2 reverse transcriptase enzyme activity; therefore, it is essential for HIV/AIDS and hepatitis B treatment (Taylor et al., 2020). 19 2.3.2.2 Dolutegravir Dolutegravir (DTG) is an orally bioavailable integrase strand transfer inhibitor (Mohan et al., 2021). DTG hinders the activity of the integrase enzyme by binding to its active site. The integrase enzyme catalyses the integration of viral DNA into chromosomal DNA, resulting in viral replication (Kandel and Walmsley, 2015). DTG is metabolised in the liver by uridine 5'-diphospho- glucuronosyltransferase and cytochrome P450. DTG is a well-tolerated ARV drug which has fewer side effects when compared to Efavirenz and other ARV drugs (Fantauzzi and Mezzaroma, 2014). Figure 2.4 depicts the chemical structure of dolutegravir. Figure 2.4: Chemical structure of dolutegravir (Han et al., 2020) Dolutegravir is a monocarboxylic acid amide and an organic heterocyclic compound with a sodium moiety (Zamora et al., 2019). DTG inhibits the active site of the integrase enzyme, which catalyses the integration of viral DNA into chromosomal DNA, inducing viral replication (Kandel and Walmsley, 2015). 20 2.3.2.3 Tenofovir Tenofovir is an adenosine acyclic nucleotide analogue used along with other HIV therapeutic agents (Hoofnagle, 2013). Tenofovir blocks reverse transcriptase, an enzyme needed for viral replication (Holec et al., 2017). The reduced viral replication impacts the HIV viral load. In 2001, tenofovir was approved in the United States of America (USA) (Ustianowski and Arends, 2015), followed by SA in 2004 (Williams et al., 2011). Tenofovir must be phosphorylated to become pharmacologically active in two steps (Fletcher et al., 2020). First, tenofovir is phosphorylated into tenofovir monophosphate by an enzyme adenylate kinase and then phosphorylated to active tenofovir-diphosphate (Hamlin et al., 2019). In competition with the natural substrate 5′‐triphosphate, tenofovir diphosphate inhibits the action of HIV-1 reverse transcriptase and terminates the DNA chain after its incorporation into the DNA strand (Holec et al., 2017). Although tenofovir has been demonstrated to be effective in treating HIV, there is still more to be understood regarding its inflammatory properties. Figure 2.5 illustrates the chemical structure of tenofovir. Figure 2.5: Chemical structure of tenofovir (Grigsby et al., 2010) As illustrated in Figure 2.5, tenofovir is a methyl phosphonic acid having the methyl hydrogen substituted by a [(2R)-1-(6-amino-9H-purin-9-yl) propan-2-yl] oxy group (Grigsby et al., 2010). Tenofovir inhibit HIV-1 and HIV-2 reverse transcriptase enzyme activity; therefore, it is important 21 for HIV/AIDS treatment (Holec et al., 2017). Several studies assessed tenofovir’s toxicity in HepG2 cells and found mitochondrial DNA (mtDNA) depletion, which causes mitochondrial toxicity and possible inflammation (Nagiah et al., 2015). 2.3.3. The effect of tenofovir on inflammation Hoofnagle recommended antiretroviral treatment with nucleoside analogue tenofovir to patients with hepatitis to reduce liver disease progression (Hoofnagle, 2013). Tenofovir suppresses the synthesis of interleukin (IL)-8, an inflammatory cytokine, during cellular stress or damage in HIV patients with liver disease (Deng et al., 2018). Interleukin (IL)-8 is a potent chemoattractant for neutrophils and contributes to acute hepatitis (Kaspar and Sterling, 2017). In addition, the drug raises the levels of IL- 12, thus responding to other infectious pathogens, and maintaining low levels of IL-10, preventing the body from inhibiting the immune response (Dayakar et al., 2019). Different studies have been carried out in different models, such as peripheral blood mononuclear cells (PBMCs) and mouse cells, investigating the inflammatory properties of tenofovir (Figure 2.6) (Olojede et al., 2022; Castillo-Mancilla et al., 2015; Biswas et al., 2014: Melchjorsen et al., 2011). These studies formulated the same conclusion that tenofovir upregulates pro-inflammatory cytokines and downregulate anti-inflammatory cytokines in acute and chronic exposure. However, tenofovir’s mechanism of action is not fully understood in human liver cells. Figure 2.6 indicates tenofovir’s effects on inflammatory properties. 22 Figure 2.6: Tenofovir’s effects on inflammatory markers at different time exposures (compiled by the researcher, S Vazi) 2.3.4 Effect of antiretroviral drugs The prevalence of opportunistic infections due to immune suppression has declined in HIV-positive patients on HAART, remarkably reducing mortality. However, the chronic nature of the anti-HIV treatment has seen the emergence of long-term side effects ranging from low intolerance to life- threatening effects (Edagwa et al., 2017), with NRTI-associated mitochondrial dysfunction resulting in lactic acidosis, pancreatitis, peripheral neuropathies, and liver toxicity (Holec et al., 2017). A study by Macías and colleagues found that long-term usage of HAART is associated with hepatic steatosis (Macías et al., 2017). Nassir et al. (2015) further explained hepatic steatosis as a disorder in which the liver forms triacylglycerol-containing vacuoles due to excessive lipid retention and disrupted lipid metabolism (Nassir et al., 2015). Consistently, several researchers found that NRTIs induce hepatic steatosis through mitochondrial toxicity, disrupting lipid metabolism and promoting inflammation (Macías et al., 2017; Nagiah et al., 2015; Nassir et al., 2015). 23 Overall, effective antiretroviral therapy is the most significant intervention in averting opportunistic infections in an HIV-positive patient. However, antiretroviral drugs are associated with several side effects arising during their metabolism (Thet and Siritientong, 2020). The liver is a key organ required for the normal homeostasis and metabolism of drugs (Zanger and Schwab, 2013). 2.4 ANATOMY AND PHYSIOLOGY OF THE LIVER The liver weighs approximately 1.36 to 1.59 kilograms, which makes it one of the largest internal solid organs (Zhang et al., 2020). The human liver is positioned in the right upper quadrant of the abdomen, receiving oxygenated blood from the heart via the hepatic artery (Yang, 2021). Furthermore, the liver receives deoxygenated blood from the hepatic portal vein containing newly absorbed nutrients, drugs, and toxins from the gastrointestinal tract (Yang, 2021). The most significant role of the liver is the detoxification of harmful substances, chemicals, and metabolic waste products (Hall and AC, 2015). Additionally, the liver functions as an exocrine and endocrine organ. In exocrine function, the liver secretes bile, which contains waste products, cholesterol, and bile acids essential for intestinal absorption and digestion. The endocrine function entails large amounts of secreted serum factors, including albumin and protein components of lipoproteins (Zhang et al., 2020). Approximately 80% of the liver comprises hepatocytes, which perform the main roles of the liver. Hepatocytes are organised into plates and are parted by sinusoids (Kuntz and Kuntz, 2006). The sinusoids are connected by endothelial cells and macrophages called Kupffer cells and separated from hepatocytes by the space of Disse responsible for vitamin A storage (Abdulrasool and Briggs, 2018). The thin layer between sinusoids and the basal surface of the hepatocyte aids in the exchange of substances between the blood and the liver cells (Szafranska et al., 2021). The hepatic portal vein and hepatic artery supply the liver with blood, while the hepatic vein drains the liver. Through this arrangement, the liver can control blood flow from the gastrointestinal tract and pancreas to the rest of the body (Abdulrasool and Briggs, 2018). 24 The liver is an essential organ with many functions, including the metabolism of key nutrients such as carbohydrates, proteins, and lipids (Zhang et al., 2020). Additionally, it is responsible for the detoxification of harmful substances such as drugs and toxins (Costanzo, 2018). Figure 2.7 illustrates both the anterior and posterior view of the human liver. Figure 2.7: Anatomy of the human liver (Mahadevan, 2020) 2.4.1 Metabolism of tenofovir In the liver, drugs are metabolised primarily (Almazroo et al., 2017), which occurs via the cytochrome P450 (CYP450) enzyme located in the endoplasmic reticulum (ER) (Zanger and Schwab, 2013). However, tenofovir is not metabolised through the CYP450 system (Chittick et al., 2006). Tenofovir is commercially accessible as a pro-drug, tenofovir disoproxil fumarate (TDF) (Fletcher et al., 2020). After oral intake, TDF is quickly converted to tenofovir in the intestinal walls entering the cell through its transporters (Cressey et al., 2020). Intracellularly, tenofovir is phosphorylated by adenylate kinases and subsequently phosphorylated by nucleoside diphosphate kinases into its active form, tenofovir-diphosphate (Hamlin et al., 2019). Tenofovir-diphosphate is an analogue of deoxyadenosine-5’-triphosphate (dATP), a regular substrate for DNA polymerase (Holec et al., 2017). Tenofovir diphosphate terminates the viral DNA chain 25 elongation by competing with dATP to be incorporated into viral DNA (Fernandez-Fernandez et al., 2011). Tenofovir is excreted by the kidneys via glomerular filtration and tubular secretion (James et al., 2012). 2.4.2 Antiretroviral drugs effect on the liver In treating HIV-positive patients, NRTI’s long-term usage is associated with hepatic toxicity (Fletcher et al., 2020). Findings from preclinical and clinical-based studies have also linked ART with hepatotoxicity, which is influenced by oxidative stress (Elias et al., 2013). Hepatotoxicity is function impairment triggered by exposure to drugs, alcohol and environmental toxicants (Paniagua and Amariles, 2017). The tenofovir drug has been associated with severe lactic acidosis and hepatic steatosis (Wassner et al., 2020). The possible mechanism behind tenofovir causing the latter complications is the inhibition of mitochondrial DNA (mtDNA) polymerase gamma (γ) (Chhatwani et al., 2016). 2.4.3 ART-induced mitochondrial toxicity Nucleoside reverse transcriptase inhibitors are frequently used antiretroviral drugs as a backbone for first-line treatment regimens (Edagwa et al., 2017). A study by Sahin in 2020 suggests that NRTIs’ interaction with DNA Polymerase gamma (Pol-γ) results in evident mitochondrial dysfunction. Most NRTIs can act as substrates for Pol-γ disturbing mtDNA synthesis, diminishing mtDNA content, oxidative phosphorylation, and ROS overproduction (Ahmed et al., 2018). These modifications result in changes in nucleotide phosphorylation and mitochondrial toxic effects on mitochondrial respiration (Blas‐García et al., 2010). Tenofovir has been proven to inhibit the mitochondrial adenylate kinase and adenosine nucleotide translocator in isolated mitochondria. This inhibition results in mitochondrial dysfunction through ROS overproduction and inhibition of the electron transport chain (Ahmed et al., 2018; Feeney et al., 2012). 26 In 2019, Zhang and colleagues determined that mitochondrial dysfunction results in damage and depletion of mtDNA and the release of mtDNA (Zhang et al., 2019). The mtDNA molecule encodes 13 polypeptides of the oxidative phosphorylation system (Mustafa et al., 2020). MtDNA lesions could aggravate mitochondrial oxidative stress, damaging hepatocytes (Zhang et al., 2019). Due to the damage of hepatocytes, mtDNA leaves the confines of mitochondria to the cytoplasm. The circulating mtDNA act as damage-associated molecular patterns (DAMPs) to stimulate the Toll-like receptor 9 (TLR9) and inflammasomes, promoting inflammation (Xuan et al., 2020). Antiretroviral drugs are generally metabolised through cytochrome P450 enzyme activity except for tenofovir. The tenofovir is metabolised by intracellular enzymes adenylate kinases and nucleoside diphosphate kinases into active tenofovir diphosphate. However, tenofovir is associated with hepatotoxicity by inhibiting mtDNA polymerase gamma (γ), resulting in mtROS. The mtROS damages the hepatocyte, releasing mtDNA, which promotes inflammation. 2.5. INFLAMMATION According to Lonardo et al. (2021), liver inflammation substantially negatively impacts HIV-positive patients receiving ART. Chen et al. (2018) defined liver inflammation as a reaction that occurs when a foreign substance attacks the liver cell. Liver inflammation results from hepatotoxicity through exposure to substances such as dietary supplements, toxic chemicals, and drugs (Singh et al., 2016). ART-induced hepatotoxicity aggravates liver disease by activating an inflammatory response (Ganesan et al., 2018). 2.5.1 Liver inflammation linked to tenofovir The liver highly depends on mitochondria to produce Adenosine 5′-triphosphate (ATP), which is crucial for biosynthetic pathways (Spinelli and Haigis, 2018). Various studies that assessed tenofovir’s mitochondrial toxicity found that tenofovir induces mtROS resulting in hepatocyte damage through mtDNA depletion (Nagiah et al., 2015; Abraham et al., 2013). The damaged 27 hepatocytes release mtDNA into the extracellular environment and circulation (Zhang et al., 2019). As mentioned in the previous section, cytosolic mtDNA acts as DAMP, activating TLR9 and inflammasomes resulting in liver inflammation (Xuan et al., 2020). Due to variations in the duration of inflammatory processes, inflammation has two categories (Deng et al., 2018). Inflammation is acute or chronic (Deng et al., 2018), and acute inflammation is the first rapid response to a dangerous stimulus (Kany et al., 2019). Chronic inflammation refers to long-term tissue damage and repair, often associated with fibrosis. Cell regeneration is achieved by releasing inflammatory cytokines such as interleukin-1β and tumour necrosis factor-alpha (TNF-α) (Kany et al., 2019). 2.5.2. Regulation of the inflammatory response The inflammation process involves a series of reactions which protect the host from infections and tissue damage (Zhang and Sun, 2015). These reactions include recruiting immune cells and plasma proteins to the inflammation site (Liu et al., 2017). Generally, inflammation is beneficial to the host and can be resolved promptly; however, deregulated inflammation is linked with chronic tissue damage, resulting in acute or chronic inflammatory diseases (Kany et al., 2019). The NF-κB pathway induces pro-inflammatory gene expression (Liu et al., 2017). 2.5.3. NF-κB signalling pathway NF-κB is a significant transcription factor regulating immune and inflammatory responses (Liu et al., 2017). The NF-κB family comprises five members: p50, p52, p65 (RelA), and c-Rel and RelB proteins, which facilitate the transcription of specific genes (Nagel et al., 2014). The p65 is a REL- associated protein involved in NF-κB heterodimer formation and nuclear translocation and is typically activated during the inflammatory response (Ghosh and Hayden, 2012). Mitochondrial ROS generation occurs at the electron transport chain during oxidative phosphorylation (Zhao et al., 2019). The generated ROS can activate the NF-κB signalling pathway (Figure 2.8). NF-κB is a transcription factor that regulates the function of the innate and adaptive 28 immune system, which mediates the inflammatory response by expression of proinflammatory genes (Giuliani et al., 2018; Liu et al., 2017). In addition, NF-κB expresses inflammatory cytokines that are key components in immune response regulations (Liu et al., 2017). Figure 2.8: NF-κB signalling and inflammation (adapted from Morgan and Liu, 2011) As illustrated in Figure 2.8, increased generation of ROS acts as an inflammatory stimulus. The increase in ROS leads to the phosphorylation of the IκB kinase (IKK) complex. This phosphorylation results in the dissociation of IκB from NF-κB. The NF-κB translocates into the nucleus, activating specific genes for pro-inflammatory cytokines. Elevated pro-inflammatory cytokines are necessary for acute inflammation and the retention of chronic inflammatory responses. These pro-inflammatory cytokines also mediate inflammation as a response to infection and injury. Furthermore, pro- inflammatory cytokines IL-1β and TNF-α trigger NF-κB and form a forward feed loop in response to the activation of NF-κB. The transcription factor, NF-κB, regulates the expression of several pro- inflammatory genes and helps to coordinate an inflammatory response (Dorrington and Fraser, 2019). 29 2.5.4. Inflammatory cytokines Cytokines are proteins that are important in controlling other immune system cells and blood cells (Zhang and Bansal, 2020). Inflammatory cytokines play a role in introducing the inflammatory response and controlling the host defence against pathogens mediating the innate immune response (Chen et al., 2018). Two categories of inflammatory cytokines (Table 2.2) exist: pro-inflammatory cytokines and anti-inflammatory cytokines. Pro-inflammatory cytokines are involved in initiating the inflammatory response. In contrast, anti-inflammatory cytokines regulate the pro-inflammatory cytokine response. Table 2.2 below categorises inflammatory cytokines that are quantified in this study. Table 2.2: Examples of pro- and anti-inflammatory cytokines (compiled by the researcher, S Vazi) Pro-inflammatory cytokines Anti-inflammatory cytokines Interleukin 1-β Interleukin 10 Tumour necrosis factor-alpha Interleukin 6 Inflammatory cytokines regulate the host’s defence against pathogens mediating the innate immune response (Gulati et al., 2016). As a result, the levels of inflammatory cytokines naturally produced by CD4+ T cells during HIV infection are expected to decrease. Inflammatory mechanisms are present in acute and chronic liver diseases, with increased expression of various pro- and anti-inflammatory cytokines in the liver (Hernaez et al., 2017). Kupffer cells are essential to the immune response by releasing inflammatory cytokines (Zhang and Bansal, 2020). Inflammatory cytokines play a significant role in triggering the inflammatory response and controlling host defence against pathogens through the innate immune system (Chen et al., 2018). 30 In antiretroviral-treated HIV infection, cytokines play a role in chemical signalling pathways that control cell growth, tissue repair, immune response, and inflammation (Kany et al., 2019). 2.6. Antiretroviral drug investigations in HepG2 liver cell model HepG2 cells, a human hepatoma, is commonly used in liver metabolism studies and toxicity of drugs (Xuan et al., 2016). Scientists have conducted numerous studies on antiretroviral drugs using HepG2 cells and found that tenofovir, lamivudine, and dolutegravir fixed-dose combination exhibited the lowest cytotoxic effect compared to other NRTIs (WHO, 2018). In 2017, Paemanee and colleagues found that mitochondrial dysfunction is linked to the induction of apoptosis of HepG2 cells treated with Nevirapine (Paemanee et al., 2017). Tunicamycin and thapsigargin, both oxidative stress inducers, caused a significant increase in Fibroblast growth factor 21 (FGF21) protein release in HepG2 cells treated with ART (Moure et al., 2018). In human hepatic cell lines, NNRTIs and PI groups have been identified for the first time as causing disturbances in the FGF21/KLB system (Moure et al., 2018). Nagiah et al. (2015) found that tenofovir, stavudine, and zidovudine exhibited mitochondrial toxicity and oxidative stress, especially at chronic (5 days) exposure in HepG2 cells. Tenofovir caused mitochondrial dysfunction without lowering the levels of mitochondrial DNA. In conclusion, tenofovir is scientifically proven to inhibit HIV-1 and HIV-2 reverse transcriptase enzyme activity. However, it is associated with many adverse effects, such as lactic acidosis, hepatic steatosis and mitochondrial toxicity (Wassner et al., 2020). Tenofovir is found to exhibit mitochondrial toxicity and induce oxidative stress. Oxidative stress indirectly induces inflammation through the activation of the NF-κB signalling pathway. Currently, there are limited reports on tenofovir’s inflammatory activity in human HepG2 human liver cells. 31 2.7. CONCLUSION This chapter aimed to summarise the literature and recent finding on antiretroviral therapy, specifically tenofovir, the drug of interest and its possible influence on liver inflammation. The review explored the research gap in tenofovir’s properties. The existing literature stipulated that tenofovir induces ROS, which activates the NF-κB signalling pathway. This study also investigated tenofovir’s influence on pro- and anti-inflammatory markers. 32 CHAPTER 3: MATERIALS AND METHODS 3.1 INTRODUCTION This chapter presents the research design, materials, and methods used in the study. A quantitative research design (Figure 3.1) was followed to address the research questions mentioned in Chapter 1. Furthermore, this chapter provides an in-depth description and justification of the methods used. 3.2 RESEARCH DESIGN This study followed an in vitro experimental research design. The researcher created an experimental and a control group. This study treated the experimental group with tenofovir and the control group with regular complete culture media (CCM) for 24h and 120h. After that, pro- and anti-inflammatory markers were quantified. The Enzyme-linked Immunosorbent Assay (ELISA), quantitative polymerase chain reaction (qPCR) and Western blot techniques were employed. The experimental techniques were carried out in the Department of Basic Medical Sciences and Human Molecular Biology Unit laboratories. Figure 3.1 represents a schematic overview of the research design and methodology that was followed. 34 Figure 3.1: Schematic representation of the research design of the study (compiled by the researcher, S Vazi 35 3.3 MATERIALS HepG2 cells were obtained from the University of KwaZulu Natal (UKZN) (Durban, SA). Cell culture reagents were purchased from ThermoFisher Scientific (Johannesburg, SA). Tenofovir (SML1795- SMG) was purchased from Sigma–Aldrich (St. Louis, MO). The Enzyme-linked immunosorbent assay (ELISA) kits were purchased from Sigma–Aldrich (Johannesburg, SA). Antibodies used for the Western blot were obtained from Cell Signaling Technology, Inc (Beverly, MA). The Western blot reagents and buffers were purchased from Bio-Rad, SA. The iScriptTM cDNA synthesis kit and IQTM SYBR® green Supermix were purchased from Bio-Rad, SA. All other reagents and consumables were purchased from Merck (Darmstadt, Germany). 3.4 RESEARCH METHODS 3.4.1 CELL CULTURE 3.4.1.1 Introduction Cell culture is the process of growing cells under a controlled environment (Haycock, 2011). These cells are used in biochemical, cytogenetic and molecular laboratories for diagnostic and research studies (Langdon, 2010). Therefore, cell culture provides a platform to investigate cell biology, biochemistry, physiology and metabolism. Cells cultured in the laboratory can be classified into three categories: Primary, transformed, and self-renewing. Primary cells are isolated directly from living tissue (Gordon and Amini, 2021). Transformed cells are transferred genetic material from one cell to another or genetically altered in order to change a recipient cell's genome (Panganiban et al., 2013). Self-renewing cells are stem cells that divide, maintaining an undifferentiated state. For this study, the HepG2 cell line was used. The HepG2 cell line is derived from the liver tissue of a 15-year-old Caucasian male with differentiated hepatocellular carcinoma (Donato et al., 2015). HepG2 cells are commonly used for drug metabolism and hepatotoxicity studies (Negoro et al., 2021). 36 3.4.1.2 Protocol The HepG2 cell line was obtained from UKZN and stored at -80°C. The frozen vial was thawed at 37°C using a bead bath (Dri-blockTM, DB200/3). The thawed cell line was re-suspended inside a sterile hood (Labtech) into a tissue culture flask 75cm2 (T75) parent flask, followed by the addition of 10 ml of pre-warmed complete culture media (CCM) to the culture flask. The CCM was prepared by supplementing Minimum Essential Medium (MEM) (gibco, 2534308) with 10% foetal bovine serum (10500-064), 1% L-glutamine (25030-024) and 1% penicillin-streptomycin (15140-122). Thereafter, the cells were placed in an incubator (Heal Force CO2 incubator, HFZ40) with conditions for optimal cell growth achieved with 5% CO2 and 95% relative humidity at 37°C. Following 24h of incubation, cells were viewed under the light-inverted microscope (Bio-Smart Scientific, 10109371) for health, attachment and confluency. The CCM was changed as required. Before adding a new CCM, 5 ml of Phosphate buffered saline (PBS) (gibco, 2235064) was used to wash the cells three times. Thereafter, 10 ml of fresh pre-warmed CCM was added, placed in the incubator (Heal Force CO2 incubator, HFZ40), and monitored for growth. Once the HepG2 cells were 80% confluent, the T75 parent flask was trypsinised with 2 ml trypsin (cat no. 25300-054). After that, cells were counted using trypan blue (MKBW2465) in a TC 20TM Automated Cell Counter (Bio-Rad). Following cell count, cells were seeded at the appropriate density into four T25 flasks and subjected to treatment once the cells were 80% confluent. 3.4.2 EXPERIMENTAL TREATMENT A 1.436 mg of tenofovir powder was dissolved in 5 ml of dimethyl sulfoxide (DMSO) to make a 1 mM stock solution. A volume of 6 µL was required to make 1.2 µM of tenofovir plasma concentration (Nagiah et al., 2015; Venhoff et al., 2007). Acute one-day (24h) and chronic five-day (120h) time duration treatments and untreated controls were conducted. A fresh cell culture medium containing tenofovir was replenished for the chronic five-day (120h) treatment after 48h (Nagiah et al., 2015). 3.5 DATA COLLECTION 37 3.5.1 ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) 3.5.1.1 Introduction ELISA technique is used to quantify the levels of a target protein in a sample (Ma et al., 2011). The samples used in ELISAs include plasma, serum, cell lysate, saliva, urine and cell culture supernatants (Shah and Maghsoudlou 2016). The ELISA principle is based on an antigen and antibody interaction. The first step involved antibody coating on a polystyrene plate. The second step was adding samples to the wells to be captured by the antibodies. The third step required detecting an antibody-antigen reaction that was visualised using enzymes linked to antibodies such as horseradish peroxidase (HRP) (Sakamoto et al., 2018). 3.5.1.2 Properties of ELISA This technique utilises an antibody to determine and quantify the presence of the target antigen in biological samples. The target antigen is captured directly (labelled primary antibody) or indirectly (labelled secondary antibody). There are four types of ELISAs: direct, indirect, competitive and sandwich ELISA. For this study, sandwich ELISA was used. This assay measures the antigen using a capture antibody and detects the antibody-antigen complexes with a detection antibody linked to an enzyme. Detection involves assessing conjugated enzyme activity via incubation with the substrate to generate measurable luminescence. The produced colour is measured, and the colour intensity is directly proportional to sample concentration. The data is analysed to determine the concentration of the target molecule in the samples. This study measured levels of released extracellular pro- and anti- inflammatory cytokines at different time exposures. 3.5.1.3 Preparation of reagents All the reagents were equilibrated to room temperature before use. The following reagents were prepared as needed on the day of the experiment: The 1X Wash buffer PT was prepared by combining 5 ml of Wash Buffer 10X with 45 ml of deionised water; The 10X Capture antibody was prepared by 38 re-suspending the lyophilised capture antibody with indicated volume of the Sample Diluent NS in the vial; The antibody cocktail was prepared by combining 300 µL of Capture antibody and 300 µL of Detector antibody with 2.4 ml antibody diluent 5BI, followed by gentle mixing 3.5.1.4 Sample preparation Following the treatment period, the supernatant was collected from the treatment flasks. After that, the supernatant was centrifuged at 2000 x g for 10 minutes using a centrifuge (Hermle, Z 32 HK) and stored at -20℃ until experimentation. 3.5.1.5 Preparation of standards The standard stock solution was prepared by reconstituting the targeted protein using the volume of sample diluent indicated on the vial. After that, eight eppendorfs were labelled from 1 to 8. Following the manufacturer’s guidelines, an appropriate volume was added to each eppendorf. The prepared stock standard was used to complete a serial dilution of 8 concentrations. Eppendorf 8 had no protein and was used as a blank control. Figure 3.2 illustrates the serial dilutions series method. Figure 3.2: Serial dilutions (adapted from the manufacturer’s guidelines (ab185986)) 3.5.1.6 Protocol The pro- and anti-inflammatory cytokine levels (IL-6, IL-1β, TNF-α, IL-10) were quantified using SimpleStep human ELISA Kits (ab178013), (ab214025), (ab181421), and (ab185986), respectively. 39 A volume of 50 µL of sample and standard was added to appropriate wells, followed by 50 µL of antibody cocktail. The plate was sealed and incubated for one hour at room temperature on a plate shaker (Bio–Smart Scientific, DIAB, SK-0110-B) set at 400 rpm. Following incubation, the wells were washed thrice with 350 µL of 1X Wash Buffer PT. The plate was blotted against a paper towel to remove excess liquid during the final wash. Then a volume of 100 μL of Tetramethylbenzidine (TMB) Substrate was added to each well. A plate shaker (Bio–Smart Scientific, DIAB, SK-0110-B) was used to incubate the plate for 10 minutes set at 400 rpm. Following incubation, 100 μL of stop solution was added to each well and mixed on a shaker (Bio–Smart Scientific, DIAB, SK-0110-B) for 1 minute. The absorbance was read within 15 minutes at 450 nm on an ELISA plate reader GloMaxTM Discovery (Promega,