The Liver: Oxidative Stress and Dietary Antioxidants

The Liver

Oxidative Stress and Dietary Antioxidants

Edited by

Vinood B. Patel

Reader, University Westminster, United Kingdom

Rajkumar Rajendram

Department of Anaesthesia and Intensive Care, Stoke Mandeville Hospital,

Aylesbury, Buckinghamshire, United Kingdom; Department of Internal and

Perioperative Medicine, King Abdulaziz Medical City, Ministry of National

Guard Hospital Affairs, Riyadh Saudi Arabia; King’s College London,

Faculty of Life Sciences and Medicine, London, United Kingdom

Victor R. Preedy

Professor, King’s College London, School of Biomedical and Health Sciences,

London, United Kingdom

Table of Contents

Cover

Title page

Copyright

List of Contributors

Chapter 1: Oxidative Stress in Hepatitis C Infection

Abstract

Abbreviations

Introduction

Reactive oxygen species—generation and scavenging

Hepatitis C virus

Oxidative stress in HCV-infected patients

Mechanisms of HCV-induced ROS generation

Mechanisms of HCV-induced ROS scavenging

Role of ROS in the HCV life cycle

Role of ROS and antioxidants in the pathology of chronic hepatitis C

Conclusions

Acknowledgments

Chapter 2: Role of Paraoxonase 1 as an Antioxidant in Nonalcoholic Steatohepatitis

Abstract

Abbreviations

NAFLD and NASH

Structure

PON1: role as an antioxidant

PON1 in liver

PON 1 in NASH

Chapter 3: Alterations in Nucleocytoplasmic Localization of the Methionine Cycle Induced by Oxidative Stress During Liver Disease

Abstract

Abbreviations

Key facts

Introduction

S-adenosylmethionine synthesis

S-adenosylmethionine utilization

S-adenosylhomocysteine elimination and adenosine recycling

Methionine resynthesis and homocysteine catabolism

Regulation of the methionine cycle by oxidative stress

Alterations in nucleocytoplasmic distribution and their link to pathology

Concluding remarks and future perspectives

Acknowledgments

Chapter 4: Oxidative Stress in Iron Toxicity of the Liver

Abstract

Abbreviations

Key facts

Introduction

Iron homeostasis

Fenton and Haber–Weiss Chemistry in biological systems

Inherent protection against oxidative stress

Causes of iron excess

Free iron and ROS in action

Oxidative pathophysiology in iron toxicity

Liver is affected by iron-induced oxidative stress

Overview of therapeutics

Chapter 5: Cardiac Glycosides and Oxidative Stress in Liver Cancer

Abstract

Abbreviations

Liver cancer

Oxidative stress in liver cancer

Cardiac glycosides

The anticancer effect of cardiac glycosides in liver cancer

Chapter 6: Acetaldehyde Effects on Cellular Redox State

Abstract

Key facts

Introduction

Acetaldehyde: generation, reactivity, and toxicity

Acetaldehyde and redox homeostasis

Acetaldehyde induces mitochondria dysfunction

Mitochondrial GSH

The cholesterol-endoplasmic reticulum paradigm

Acetaldehyde targets antioxidant enzymes and GSH

Acetaldehyde induces prooxidant cytokines

Conclusions

Acknowledgments

Chapter 7: The Role of Glyoxalase-I in Oxidant Stress of Liver Damage

Abstract

Abbreviations

Key facts

Introduction and main text

Chapter 8: Reactive Oxygen and Nitrogen Species and Liver Ischemia-Reperfusion Injury: An Overview

Abstract

Abbreviations

Introduction

Hepatic ischemia/reperfusion injury

Conclusions

Chapter 9: Reactive Oxygen and Nitrogen Species and Liver Ischemia- Reperfusion Injury: Role of Glutamine

Abstract

Abbreviations

Introduction

Glutamine and its functions

Conclusions

Chapter 10: Reactive Oxygen and Nitrogen Species and Liver Ischemia-Reperfusion Injury: Role of Lipoic Acid

Abstract

Abbreviations

Key facts

Introduction

Conclusions

Chapter 11: Oxidative Stress as a Crucial Factor in Liver Associated Disorders: Potential Therapeutic Effect of Antioxidants

Abstract

Introduction

Chapter 12: l-NAME as a Synthetic Antioxidant in Liver Injuries

Abstract

Abbreviations

Introduction

Reactive nitrogen species (RNS)

Nω-nitro-l-arginine methyl ester (l-NAME)

Conclusions

Chapter 13: Chemoprotective Role of Vitamin C in Liver Diseases

Abstract

Abbreviations

Introduction

Vitamin C metabolism

Transport of ascorbic acid and dehydroascorbic acid

Role of vitamin C in liver disease

Dietary vitamin C and liver health

Conclusions

Acknowledgments

Chapter 14: Protective Role of Lycopene Against Oxidative Stress in Liver

Abstract

Key facts

Introduction

Oxidative stress and liver diseases

Lycopene

Absorption of lycopene

Antioxidant and anticancer mechanisms of lycopene

Lycopene: a key protective carotenoid against the liver diseases

Conclusions and future prospects

Acknowledgment

Chapter 15: Vitamin E Protection Against Hyperthyroidism-Induced Liver Oxidative Stress

Abstract

Key facts

Introduction

Liver oxidative stress due to hyperthyroidism

RNS and ROS production in hyperthyroid state

Antioxidant defense system in hyperthyroid liver

Sensibility to oxidants of the hyperthyroid liver

Vitamin E management of thyroid hormone induced oxidative stress

Antioxidant action of vitamin E

Vitamin E effect on hyperthyroidism-induced liver injury

Effects of vitamin E administration on hyperthyroid liver

Conclusions

Chapter 16: Citric Acid an Antioxidant in Liver

Abstract

Abbreviations

Key facts

Introduction

Sources of free radicals

Antioxidant mechanisms

Oxidative stress in liver disease

Citric acid

Sources of citric acid

Citric acid is protective in the liver

Lipopolysaccharide-mediated liver injury

Organophosphate-induced liver injury

Mechanism (s) underlying hepatic protection by citric acid

Antiinflammatory effects of citric acid

Metabolic effects of citric acid

Chapter 17: Resveratrol and Protection in Hepatic Steatosis: Antioxidant Effects

Abstract

Abbreviations

Key facts

Physiopathology of nonalcoholic fatty liver disease

Resveratrol: a bioactive stilbene

Effects of resveratrol on different models of liver steatosis

Effects of resveratrol on liver steatosis in humans

Concluding remarks

Acknowledgments

Chapter 18: Astaxanthin, a Marine Carotenoid Against Hepatic Oxidative Stress: a Systematic Review

Abstract

Abbreviations

Key facts

Introduction

Absorption

Bioavailability

Safety

Structure and antioxidant property

Oxidative stress in liver disorders

Multiple hepatoprotective effects of astaxanthin

In vitro studies

Parenchymal cells (hepatocytes)

Nonparenchymal cells (hepatic stellate cells)

Hepatoma cell lines

In vivo studies

Alcohol induced liver injury

Radiation-induced liver damage

Ischemia/reperfusion (I/R) injury

Autoimmune hepatitis

Liver damage by chemical agents

Insulin resistance and type 2 diabetes

Obesity and fatty liver

Liver fibrosis

Liver malignancies

Clinical studies

Conclusions

Chapter 19: Effect of Melatonin as an Antioxidant in the Liver

Abstract

Key facts

Introduction

Brief on melatonin

Oxidative stress and antioxidant

Melatonin as antioxidant

Effects of melatonin on hepatic apoptotic pathways

Liver transplantation and melatonin

Limitations of melatonin administration

Conclusions

Chapter 20: The Liver: Oxidative Stress and Dietary Antioxidants

Abstract

Introduction

Resource of ROS production

Effect of oxidative stress in liver

Alcohol-induced liver injury

Drug-induced liver injury

Antioxidant for the treatment of oxidative stress

Dietary antioxidants

Phenolics

Flavonoids

Conclusions

Chapter 21: Endogenous Mitochondrial Aldehyde Dehydrogenase-2 as an Antioxidant in Liver

Abstract

Abbreviations

Key facts

Oxidative stress and aldehyde toxicity

Introduction of mitochondria aldehyde dehydrogenase-2

The role of oxidative stress and aldehydes in the development of liver diseases

ALDH2 genetic polymorphisms

ALDH2 acts as an antioxidant in liver

Conclusions

Chapter 22: Comparing Antioxidants in Liver Disease: l-Carnitine, N-Acetylcysteine, and Genistein

Abstract

Abbreviations

Introduction

Molecular pathogenesis in liver diseases

l-Carnitine

N-Acetylcysteine

Genistein

A comparison of l-Carnitine, N-Acetylcysteine, and Genistein

Chapter 23: The Hepatoprotective Effects of Coenzyme Q10 Against Oxidative Stress

Abstract

Abbreviations

Key facts

Introduction

CoQ10 functions

CoQ10 tissue distribution

Absorption, transportation, metabolism, and excretion of CoQ10

Dietary sources of CoQ10

CoQ10 and inflammation

Conclusions

Chapter 24: Carnosine as a Putative Antioxidant in Usage Against Liver Disease

Abstract

Abbreviations

Key facts

Introduction

Carnosine against oxidative liver damage

Chapter 25: Dioscin as a Natural Saponin and Protection in Oxidative Stress in Hepatic Fibrosis

Abstract

Abbreviations

Introduction

Dioscin exerts protection in oxidative stress in hepatic fibrosis

Conclusions

Chapter 26: Gallic Acid as a Putative Antioxidant in Usage Against Liver Disease

Abstract

Gallic acid

Steatosis and insulin resistance

Liver fibrosis

Hepatocarcinoma

Hepatitis C virus infection

Conclusions

Chapter 27: Oleuropein as an Antioxidant and Liver Protect

Abstract

Key facts

Introduction

Chemical structure, biosynthesis, and metabolism

Bioavailability of oleuropein

Role of oxidative stress on liver diseases

Role of oleuropein in liver protection

Conclusions

Chapter 28: Garlic and Liver Diseases

Abstract

Key facts

Introduction

Oxidative stress and liver disease

Garlic as a health promoting food/drug

Application of garlic in liver disease

Perspectives

Index

Copyright

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List of Contributors

Omar M.E. Abdel-Salam,     National Research Centre, Cairo, Egypt

Leixuri Aguirre,     University of the Basque Country and CIBERobn Physiopathology of Obesity and Nutrition, Carlos III Health Institute (ISCIII), Vitoria-Gasteiz, Spain

Pezhman Alavi Nezhad,     Research Institute for Infectious Diseases of the Digestive System, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran

Fabienne T.E. Alban,     Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

Carani V. Anuradha,     Annamalai University, Chidambaram, Tamil Nadu, India

Sıla Appak,     Germany Cancer Research Centre, Heidelberg, Germany

Juan J. Aragón,     Instituto de Investigaciones Biomédicas Alberto Sols (CSIC-UAM), Madrid, Spain

Juan Ascacio-Valdés,     Autonomous University of Coahuila, Saltillo, Coahuila, Mexico

Rengul Cetin Atalay,     Middle East Technical University (ODTU), Ankara, Turkey

Adnan Ayhancı,     Eskişehir Osmangazi University, Eskişehir, Turkey

Robert D. Baker,     Digestive Diseases and Nutrition Center, Women and Children’s Hospital of Buffalo, State University of New York at Buffalo, Buffalo, NY, United States

Susan S. Baker,     Digestive Diseases and Nutrition Center, Women and Children’s Hospital of Buffalo, State University of New York at Buffalo, Buffalo, NY, United States

Daniela Barone,     University of Naples Federico II, Naples, Italy

Birke Bartosch,     Cancer Research Center Lyon, INSERM U1052, and CNRS 5286, Lyon, France

Ruth Belmares-Cerda,     Autonomous University of Coahuila, Saltillo, Coahuila, Mexico

Vijay K. Bharti,     Defence Institute of High Altitude Research (DIHAR), Defence Research and Development Organisation, Leh, Jammu and Kashmir, India

Birdal Bilir,     Winship Cancer Institute, Emory University, Atlanta, GA, United States

Mustafa Cengiz,     Siirt University, Siirt, Turkey

Denise Clavijo-Cornejo,     National Institute of Rehabilitation, Mexico City, Mexico

Arul Ananth D.,     Bharathidasan University, Tiruchirappalli, Tamil Nadu, India

Kaan Demiroren,     University of Medical Sciences, Yuksek Ihtisas Teaching Hospital, Bursa, Turkey

Semra Doğru-Abbasoğlu,     Istanbul University, Istanbul, Turkey

Irem Durmaz,     Middle East Technical University (ODTU), Ankara, Turkey

Farnaz Farsi,     Colorectal Research Center, Iran University of Medical Sciences and Health Services, Tehran, Iran

Alfredo Fernández-Quintela,     University of the Basque Country and CIBERobn Physiopathology of Obesity and Nutrition, Carlos III Health Institute (ISCIII), Vitoria-Gasteiz, Spain

Smilin Bell Aseervatham G.,     Bharathidasan University, Tiruchirappalli, Tamil Nadu, India

Napolitano Gaetana,     University of Naples Federico II, Naples, Italy

Hao Gao,     Jinan University, Guangzhou, China

Hasan Gencoglu,     Firat University, Elaziğ, Turkey

Arup Giri,     Defence Institute of High Altitude Research (DIHAR), Defence Research and Development Organisation, Leh, Jammu and Kashmir, India

Luis E. Gomez-Quiroz,     Autonomous Metropolitan University Iztapalapa, Mexico City, Mexico

Marcela González,     National University of Litoral, National Council of Scientific and Technical Research (CONICET), Santa Fe, Argentina

Mayela Govea-Salas,     Autonomous University of Coahuila, Saltillo, Coahuila, Mexico

María C. Gutiérrez-Ruiz,     Autonomous Metropolitan University Iztapalapa, Mexico City, Mexico

Daniel Gyamfi,     Kwame Nkrumah University of Science and Technology, Kumasi, Ghana

Hanaa A. Hassan

Faculty of Science, Taibah University, Al-Ula, Kingdom of Saudi Arabia

Faculty of Science, Mansoura University, Mansoura, Egypt

Weiyang He,     Institute of Hepatobiliary Diseases of Wuhan University, Wuhan, China

Michal Heger,     Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

Marcus Hollenbach,     Martin-Luther University Halle-Wittenberg, Halle, Germany

Alexander V. Ivanov,     Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia

Olga A. Khomich,     Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia

Necla Koçak-Toker,     Istanbul University, Istanbul, Turkey

Omer Kucuk,     Winship Cancer Institute, Emory University, Atlanta, GA, United States

Asier Léniz,     University of the Basque Country and CIBERobn Physiopathology of Obesity and Nutrition, Carlos III Health Institute (ISCIII), Vitoria-Gasteiz, Spain

Rocio I.R. Macias,     Experimental Hepatology and Drug Targeting (HEVEFARM); National Institute for the Study of Liver and Gastrointestinal Diseases (CIBERehd); Institute for Biomedical Investigation of Salamanca (IBSAL); University of Salamanca, Salamanca, Spain

Jose J.G. Marin,     Experimental Hepatology and Drug Targeting (HEVEFARM); National Institute for the Study of Liver and Gastrointestinal Diseases (CIBERehd); Institute for Biomedical Investigation of Salamanca (IBSAL); University of Salamanca, Salamanca, Spain

Óscar H. Martínez-Costa,     Instituto de Investigaciones Biomédicas Alberto Sols (CSIC-UAM), Madrid, Spain

Kosha Mehta,     Institute of Hepatology, London, United Kingdom

Iñaki Milton-Laskibar,     University of the Basque Country and CIBERobn Physiopathology of Obesity and Nutrition, Carlos III Health Institute (ISCIII), Vitoria-Gasteiz, Spain

Jesus Morlett-Chávez,     Autonomous University of Coahuila, Saltillo, Coahuila, Mexico

Nava Morshedzadeh,     Shahid Beheshti University of Medical Sciences, Tehran, Iran

Enayat A. Omara,     National Research Centre, Cairo, Egypt

Dolores Pérez-Sala,     Centro de Investigaciones Biológicas (CSIC), Madrid, Spain

María A. Pajares,     Instituto de Investigaciones Biomédicas Alberto Sols (CSIC-UAM), Madrid, Spain

Venditti Paola,     University of Naples Federico II, Naples, Italy

Jinyong Peng,     Dalian Medical University, Dalian, China

Maria J. Perez,     Experimental Hepatology and Drug Targeting (HEVEFARM); National Institute for the Study of Liver and Gastrointestinal Diseases (CIBERehd); Institute for Biomedical Investigation of Salamanca (IBSAL); University of Salamanca, Salamanca, Spain

Maria P. Portillo,     University of the Basque Country and CIBERobn Physiopathology of Obesity and Nutrition, Carlos III Health Institute (ISCIII), Vitoria-Gasteiz, Spain

Ana M. Rivas-Estilla,     Autonomous University of Nuevo Leon, Monterrey, Nuevo Leon, Mexico

Raul Rodríguez-Herrera,     Autonomous University of Coahuila, Saltillo, Coahuila, Mexico

Kazim Sahin,     Firat University, Elaziğ, Turkey

Maria A. Serrano,     Experimental Hepatology and Drug Targeting (HEVEFARM); National Institute for the Study of Liver and Gastrointestinal Diseases (CIBERehd); Institute for Biomedical Investigation of Salamanca (IBSAL); University of Salamanca, Salamanca, Spain

Nermeen M. Shaffie,     National Research Centre, Cairo, Egypt

Arturo Simoni-Nieves,     Autonomous Metropolitan University Iztapalapa, Mexico City, Mexico

Sarita Singhal,     Digestive Diseases and Nutrition Center, Women and Children’s Hospital of Buffalo, State University of New York at Buffalo, Buffalo, NY, United States

Rajendra S. Srivastava,     Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, India

Sivasudha T.,     Bharathidasan University, Tiruchirappalli, Tamil Nadu, India

Xufeng Tao,     Dalian Medical University, Dalian, China

George L. Tipoe,     University of Hong Kong, Hong Kong, China

Müjdat Uysal,     Istanbul University, Istanbul, Turkey

Rowan F. van Golen,     Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

Yanfeng Wang,     Institute of Hepatobiliary Diseases of Wuhan University, Wuhan, China

Jia Xiao,     Jinan University, Guangzhou, China

Noha N. Yassen,     National Research Centre, Cairo, Egypt

Seung K. Yoon,     The Catholic University Liver Research Center, WHO Collaborating Center of Viral Hepatitis, Seoul St. Mary’s Hospital, Catholic University of Korea, Seoul, South Korea

Rui Zhang,     Jinan University, Guangzhou, China

Lixin Zhu,     Digestive Diseases and Nutrition Center, Women and Children’s Hospital of Buffalo, State University of New York at Buffalo, Buffalo, NY, United States

Alexander Zipprich,     Martin-Luther University Halle-Wittenberg, Halle, Germany

Liangliang Zou,     Shunde Polytechnic, Foshan, China

Alejandro Zugasti-Cruz,     Autonomous University of Coahuila, Saltillo, Coahuila, Mexico

Chapter 1

Oxidative Stress in Hepatitis C Infection

Alexander V. Ivanov*

Olga A. Khomich*

Birke Bartosch**

*    Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia

**    Cancer Research Center Lyon, INSERM U1052, and CNRS 5286, Lyon, France

Abstract

Chronic infection with hepatitis C virus (HCV) is characterized by liver inflammation, fibrosis, and hepatocellular carcinoma (HCC). Due to the long delay with which HCC occurs, malignant transformation is thought to be driven by indirect mechanisms. Indeed, HCV is known to create an oxidative microenvironment in the liver, which in turn stimulates repair and regeneration processes. Proliferative repair processes that occur in the context of strong oxidative stress are thought to facilitate fixation and accumulation of genetic mutations and to drive neoplastic transformation. In this review, we summarize knowledge on oxidative stress and oxidative stress responses induced by HCV. We describe the molecular mechanisms by which HCV modulates cellular systems that generate or eliminate oxidative stress and control cellular redox homeostasis. The impact of an altered cellular redox homeostasis on HCV replication, as well as the on the course and outcome of liver fibrosis and hepatocarcinogenesis are discussed.

Keywords

hepatitis C virus

reactive oxygen species

oxidative stress

fibrosis

hepatocellular carcinoma

inflammation

Summary Points

• This chapter focuses on the role of oxidative stress in chronic hepatitis C infection.

• Hepatitis C virus augments the production of reactive oxygen species in infected cells by several different molecular mechanisms.

• The reactive oxygen species produced by HCV are amplified by inflammation and cause hepatic fibrogenesis.

• Hepatitis C virus replication is inhibited by oxidative stress and in particular by lipid peroxides.

• Hepatitis C virus directly and indirectly activates enzymes that scavenge reactive oxygen species, but in the long term, levels of oxidative stress increase in vitro and in HCV-infected patients.

• The accumulation of oxidative stress markers correlates with fibrosis progression and levels of inflammation in patients.

• Hepatitis C virus induced oxidative stress plays a primordial role in hepatocarcinogenesis.

Abbreviations

ALT alanine aminotransferase

ARE antioxidant response elements

AST aspartate aminotransferase

CAT catalase

CHC chronic hepatitis C

CK2 protein (casein) kinase 2

COX2 cyclooxygenase 2

CTGF connective tissue growth factor

CYP2E1 cytochrome P450 2E1

DHCR24 24-dehydrocholesterol reductase

eIF2α eukaryotic initiation factor 2α

ER endoplasmic reticulum

ERAD ER-associated protein degradation

Ero1α ER oxidoreductin 1α

GCL glutamate-cysteine ligase

GPx glutathione peroxidases

GR glutathione reductase

GS glutathione synthase

gt genotypes

HCC hepatocellular carcinoma

HCV hepatitis C virus

HIF1α hypoxia-inducible factor 1α

HNE 4-hydroxynonenal

HO-1 heme oxygenase 1

IP3R inositol 1,4,5-triphosphate receptor

MAM mitochondria-associated membrane

MCU mitochondrial calcium uniporter

MDA malondialdehyde

Nqo1 NAD(P)H:quinoneoxidoreductase 1

Nrf2 NF-E2-related factor 2

PBMCs peripheral blood mononuclear cells

PDGF platelet-derived growth factor

PI3K phosphoinositide-3-kinase

Prdx peroxiredoxins

PPARα peroxisome proliferator-activated protein α

ROS reactive oxygen species

SOD superoxide dismutases

TGFβ transforming growth factor β

UPR unfolded protein response

VDAC voltage-dependent anion channel

VEGF vascular endothelial growth factor

8-oxoG 7,8-dihydro-8-oxoguanine

Introduction

According to the Centers for Disease Control and Prevention, the American Cancer Society, the National Cancer Institute, and the North American Association of Central Cancer Registries, the incidence of liver cancer is rapidly increasing at a rate of 2.3% per year (from 2003 to 2012). Hepatocellular carcinoma (HCC) is the most common histologic type of liver cancer and the fastest increasing cause of cancer-related deaths in industrialized countries.¹ Liver cancer is the second most common cause of cancer death for men and women combined.² Chronic infection with hepatitis B and C viruses are the most important risk factors for HCC and at least 30% of HCC cases are attributed to chronic infection with hepatitis C virus in particular.¹,³

In the acute phase of infection, an estimated 20% of patients manage to eliminate hepatitis viruses, in ca. 80% of cases, infection becomes chronic. Both hepatitis B and C have evolved complex strategies to escape the host’s immune responses and to establish permanent infection. In order to survive in the host and to ensure a cellular environment optimal for viral replication, hepatitis viruses are known to alter hepatic physiology and metabolism. While many of these changes ensure optimal viral fitness or persistence, the downside is cellular stress signaling, oxidative stress, and chronic inflammatory processes. These features are particularly evident in chronic hepatitis C infection.

Chronic inflammation is characterized by an oxidative microenvironment in the liver and stimulates regenerative liver fibrosis and ultimately cirrhosis. At advanced stages of fibrosis (cirrhosis), the risk of HCC incidence in chronic hepatitis C increases considerably. Besides elevated risk of HCC, cirrhotic patients also display the most elevated level of treatment failure, and this is particularly in the case of genotype 3 (gt3) infected cirrhotics. With the arrival of direct acting antivirals for curing hepatitis C, it is becoming clear that fibrosis and even cirrhosis are reversible. However, this is not the case in all patients, and in particular, patients with advanced stage fibrosis remain at risk of HCC upon successful antiviral treatment, in some cases to the same extent as before the treatment.

HCV driven hepatocarcinogenesis is multifactorial. But one key factor underlying the oncogenic effects of HCV is the viruss’ capacity to induce oxidative stress.⁴,⁵ Liver regeneration/fibrosis in the context of an oxidative and inflammatory microenvironment is likely to be a driving force and the long-term accumulation of nonreversible DNA, protein, and lipid oxidation and damage may explain why at late stages of disease fibrosis regresses, but the risk of HCC does not decline back to baseline. Here, we comprehensively review the molecular mechanisms by which hepatitis C virus induces oxidative stress and triggers reactive oxygen species-sensitive signaling cascades and inflammatory processes.

Reactive oxygen species—generation and scavenging

, hydroxyl radical (HO·), singlet oxygen (¹O2), and hydrogen peroxide (H2O2).and HO·, is relatively stable with a half-life of ∼1 ms.⁸ Due to its selective reactivity and long half-life, it is cell permeable and an important trigger of cell-signaling cascades. Noteworthy, most ROS types are interconvertible.

Figure 1.1   ROS producing and scavenging enzymes.

is converted by superoxide dismutases (SOD) into H2O2. H2O2 is neutralized by catalase (CAT), peroxiredoxins (Prdx), and glutathione peroxidases (GPx).

Eukaryotic cells dispose two ROS scavenging systems: one based on low molecular weight antioxidants, such as glutathione, α-tocopherol, and vitamin C which can directly neutralize ROS, another based on phase II ROS-scavenging enzymes (is converted by the family of superoxide dismutases (SOD) into H2O2. H2O2 in turn is neutralized by catalase (CAT), peroxiredoxins (Prdx) 1–6 and glutathione peroxidases (GPx) 1–8. Most ROS scavenging enzymes exist as several isoforms with different cellular localizations. Of note, GPx4 is unique in its ability to scavenge lipid peroxides.

Dysregulation of ROS-producing and ROS-scavenging mechanisms is implicated in carcinogenesis. Chronically elevated ROS favor inflammation, genomic instability, and increased mutation rates, as well as metabolic adaptations to glycolysis, required to sustain high proliferation rates. Chronic activation of ROS scavenging systems in transforming cells can favor resistance to cellular and oxidative stress, as well as anticancer drugs.

Hepatitis C virus

HCV affects 130–210 million people worldwide with a global prevalence of ca. 2.8%¹⁰ and was originally identified as the major causative agent of non-A non-B hepatitis in the 1980s. Albeit the molecular cloning of HCV in 1989, it was not possible to culture HCV in vitro until 2005. Since then tremendous advances have been made in the analysis of the viral life cycle, the molecular mechanisms by which the virus induces fibrosis and hepatocarcinogenesis, as well as the development of direct acting antivirals, which have recently entered the clinic.

HCV is a member of the Flaviviridae family.¹¹ Its genome is constituted by a (+)-strand RNA of approximately 9.6 kb, with a single open reading frame flanked by 5′- and 3′-untranslated regions. Translation of the genome and subsequent cleavage of the resulting polypeptide chain by cellular and viral proteases produces 10 viral proteins: 3 structural proteins including the viral capsid (core) and the 2 glycoproteins E1 and E2, which form a heterodimer on the viral surface. The other proteins are not part of the virion structure but have functions required to ensure viral propagation. They include the ion channel protein p7, proteases NS2 and NS3, the protease cofactor NS4A, the ER localized transmembrane protein NS4B, the phosphoprotein NS5A, and finally the RNA-dependent RNA polymerase NS5B.

Upon interaction with a set of cellular attachment and coreceptors, HCV is internalized by clathrin-mediated endocytosis.¹² The entry process of HCV is strongly dependent on lipids. Indeed, the serum of patients, the virus is frequently found associated with very low-density lipoproteins. This association is thought to increase the specific infectivity of virions and to shield the virus from recognition by neutralizing antibodies. Among the cell entry receptors and cofactors of HCV are a number of specific and unspecific lipoprotein receptors, such as syndecans, scavenger receptor BI, very low-density lipoprotein receptor, Niemann-Pick C1-like 1 cholesterol absorption receptor, and others.¹² How the lipid association modifies the entry and uptake process of HCV is unknown. Intracellular replication and assembly are also strongly dependent on the hepatic lipid and cholesterol mechanism. Replication of the virus occurs then in membranous web structures that are part of the outer ER membrane and in tight interactions with lipid droplets, where core protein is localized.¹¹ Replication and assembly are highly dependent on lipid and cholesterol biosynthesis pathways. 3-hydroxy-3-methylglutaryl-coenzyme A reductase and diglyceride acyltransferase, for example, are proteins known to be required for productive HCV assembly.¹³

While many steps of the HCV life cycle show a strong dependence on the hepatic lipid and cholesterol metabolism, the virus induces in turn changes to the hepatic glucose and lipid metabolism. The virus is known to induce insulin resistance/type 2 diabetes, steatosis, as well as iron overload. Clinically, these metabolic dysfunctions are associated with accelerated hepatic fibrosis progression, the development of HCC and some cardiovascular events, such as stroke.¹⁴ These disorders are intrinsically linked to and driven by oxidative stress, underlining the importance of hepatic redox balance in disease progression of chronic hepatitis C (CHC). In particular, the replication efficiency and infectivity of virions is strongly sensitive to peroxidation of lipids, which alters membrane fluidity and permeability, again stressing the importance of lipids in the viral life cycle.¹⁵,¹⁶

Oxidative stress in HCV-infected patients

Few patients can clear the virus upon infection. In an estimated 80% of cases, the virus establishes a persistent infection. Independent of the stage of infection, all forms of CHC, acute, chronic, and occult are associated with increased oxidative stress.¹⁷,¹⁸ However, ROS levels, as well as content of electron acceptor molecules, such as reduced glutathione are generally higher in the acute compared to the chronic stage of infection.¹⁸ In occult hepatitis C, the changes to the hepatic redox balance are less manifest and generally milder compared to classical CHC.¹⁷

Several publications have reported appearance of markers of strong oxidative stress in CHC patients at all stages of disease: in liver biopsies a 2–5 log elevation of the levels of ROS¹⁹ and elevated levels of DNA, lipid, and protein oxidation products, such as 7,8-dihydro-8-oxoguanine (8-oxoG), malondialdehyde (MDA), and 4-hydroxynonenal (HNE), as well as protein adducts were observed.²⁰,²¹ Similarly, levels of MDA,²⁰–²⁵ lipid peroxides,²⁵ oxysterols,²⁶–²⁸ and protein carbonyl content²² were increased in sera, plasma, and peripheral blood mononuclear cells (PBMCs)²⁹–³¹ and 8-isoprostane levels were found increased in urine samples.³² Besides oxidative stress markers, levels of clastogenic factors were increased in sera of CHC patients,²⁰,²³ and this correlated with up to 20 times more apurine/apyrimidine sites compared to DNA from uninfected control patients.¹⁷ Consistent with this finding, HCV has been shown to deregulate damage responses to single- and double-stranded DNA lesions.³³ Another characteristic of CHC patients are decreased levels of total and reduced glutathione in liver³⁴ and blood,³¹ as well as reduced levels of vitamins C and E,³² pointing to a reduced antioxidant capacity. In conclusion, in most, but not all studies, levels of oxidative stress markers in serum or blood seemed to correspond and reflect intrahepatic levels of oxidative stress.

Mechanisms of HCV-induced ROS generation

HCV triggers ROS production by a variety of mechanisms (Fig. 1.2). Different HCV genotypes (gt) differ in their ability to promote ROS production: oxidative stress is strongest in gt1 infection and decreases as follows:1a/4 > 2a/c2b > 3a.²⁹ To date, six HCV proteins have been shown to stimulate ROS production: core,³⁰,³⁵–⁴⁰ E1,³⁵ E2,³¹,³⁵ NS3,⁴¹ NS4B,³⁵,⁴² and NS5A,³⁵,³⁹,⁴³,⁴⁴ with core and NS5A considered to be the major triggers. HCV core is considered to be the strongest and NS5A the earliest inducer of ROS.³⁵,³⁹ However, we failed to see noticeable differences in the timing of ROS induction by the two proteins using similar settings.³⁵

Figure 1.2   HCV-induced sources of ROS.

The mechanisms by which HCV or particular HCV proteins trigger ROS production include mitochondrial dysfunctions, altered metabolic activity in mitochondria, altered Ca²+ fluxes, induction of NADPH oxidases 1, 2, and 4, cytochrome P450 2E1 (CYP2E1), ER oxidoreductin 1α (Ero1α), as well as induction of ER stress and concomitant unfolded protein response (UPR). For detailed explanations see in the text.

The mechanisms by which HCV triggers ROS production include mitochondrial dysfunctions, altered Ca²+ fluxes, induction of NADPH oxidases 1, 2, and 4, cytochrome P450 2E1 (CYP2E1), ER oxidoreductin 1α (Ero1α), as well as induction of ER stress and concomitant unfolded protein response (UPR). Several lines of evidence show furthermore links and dependencies between these mechanisms. The role of mitochondria in HCV-induced oxidative stress has been studied mostly in cells expressing core protein.³⁰,³⁶,⁴⁰ HCV core localizes to multiple organelles in the cell: the ER, lipid droplets, the nucleus, and the outer membrane of mitochondria.⁴⁵ Core induces mitochondrial calcium uniporter (MCU)-mediated redistribution of Ca²+ between ER and mitochondria,³⁰,³⁷,⁴⁰ ER stress with concomitant Ca²+ depletion from the ER⁴⁶ and altered Ca²+ flux from the ER to mitochondria due to altered mitochondria associated membrane (MAM) structure.³⁷ MAM represent sites of contact between ER and mitochondria and unpublished data from our laboratory demonstrate the presence of not only core, but also other viral proteins at MAMs. MAMs physically connect calcium transporters of the ER and mitochondria: ER inositol 1,4,5-triphosphate receptor (IP3R) and voltage-dependent anion channel (VDAC) located at the ER and on the outer membrane of mitochondria, respectively.³⁰ Li and coworkers revealed that the core protein affects calcium flux to mitochondria through IP3R and not the general efflux from the ER as in case of the unfolded protein response.³⁷ However, direct flux of calcium ions from ER to mitochondria is controlled by the IP3R binding partner Ero1α, which is part of the protein folding machinery in ER lumen. Our laboratory has shown that expression of HCV core leads to accumulation of Ero1α, while Ero1α knock down partially prevents core-mediated superoxide anion production.⁴⁰ Thus, MAMs are implied in the accumulation of calcium ions in mitochondria in core-expressing cells.

For NS5A, the first data by Gong and coworkers reported that ROS production is a result of an efflux of Ca²+ from ER into mitochondria, since the process was sensitive to the cell-permeable calcium chelator BAPTA-AM.⁴³ However, in our hands BAPTA-AM had only a minor effect on NS5A-induced oxidative stress, and an MCU inhibitor did not affect ROS production at all.⁴⁴ Neither could we detect induction of Ero1α by NS5A. In line with these findings, Dionisio and coworkers pointed out that altered Ca²+ fluxes may be the consequence and not cause of the enhanced ROS production in NS5A-expressing cells.⁴⁷

Accumulation of calcium ions in mitochondria can result in altered respiratory chain functions, one major source of ROS in mitochondria. Decreased activity of respiratory complex I and its restoration by inhibitors of Ca²+ transporters has been demonstrated in cells overexpressing core.³⁰,³⁶ However, in our hands, productively replicating virus seems to directly interfere with and augment the activity of respiratory complex I and associated ROS production, suggesting that changes to Ca²+ levels are not sufficient to impact respiration (data not shown). Also, additional ROS-producing systems in mitochondria, such as alpha-ketoglutarate dehydrogenase or pyruvate dehydrogenases may contribute to augmentation of mitochondrial ROS in infected cells.

via phagocyte NADPH oxidase (NOX2).⁴¹ However, the physiological relevance of this finding is questionable, as NS3 is not normally excreted. In addition, induction of NOX1 and NOX4 was demonstrated not only in vitro , these data need validation.

HCV core and NS5A-mediated induction of NOX1 and NOX4 expression is thought to be mediated on the transcriptional level by transforming growth factor β1 (TGFβ1).⁴⁴,⁴⁸ We showed that anti-TGFβ1 antibodies block NS5A-mediated induction of NOX1 and NOX4 via a cascade involving TGFβ1→NOX1→COX2→NOX4, where COX2, or cyclooxygenase 2 synthesizes proinflammatory prostaglandin E2.⁴⁴ Consistent findings were reported in productively infected cells.⁴⁹ In core-expressing cells, NOX1 and NOX4 were induced by TGFβ1 independently, without an involvement of COX2.³⁵

A third factor that increases ROS production in HCV infection is ER stress and concomitant UPR, triggered in turn by accumulation of misfolded proteins.⁵¹ To resolve ER stress a cell induces expression of (1) chaperones and other proteins of the folding enzymes, (2) ER-associated protein degradation (ERAD) machinery to digest the accumulated unfolded polypeptides, and (3) the cell blocks cap-dependent translation to alleviate pressure on the ER.⁵¹ This is achieved through the ATF6, Ire1/Xbp1, and PERK/GADD153 pathways, respectively. Of the HCV proteins, core⁴⁶, E1/E2 glycoproteins,⁵² and NS4B protein⁴²,⁵³ are all known to trigger ER stress and UPR. The ER is a cellular store for Ca²+, and ER stress can lead to ER overload response which is characterized by Ca²+ release into the cytoplasm leading to their subsequent accumulation in mitochondria. The latter has been shown by Li and coworkers in NS4B-expressing cells.⁴² Furthermore, the PERK/GADD153 branch of the UPR controls expression of Ero1α and IP3R activity⁵⁴ thus regulating direct flux of Ca²+ to mitochondria through MAMs. Noteworthy, Ero1α produces H2O2 as a by-product, and in our hands downregulation of Ero1α inhibits production of ROS in cells expressing HCV core.⁴⁰ Third, the UPR can be induced by ER-residing NOX4, and as mentioned earlier NOX4 transcription is enhanced by HCV proteins.⁴⁴ Although the interplay between the UPR and NOX4 has never been studied in HCV infection, Chusri and coworkers recently reported that inhibition of NADPH oxidases with DPI efficiently prevented UPR in HCV-infected cells.⁵⁵ Finally, activation of the UPR can result in production of TGFβ1⁵⁵ and amplify NOX1 and NOX4 expression.

Another source of reactive oxygen species in HCV-infected cells is CYP2E1, an enzyme found in the ER and mitochondria that is involved in catabolism of a wide array of endogenous and exogenous compounds including ethanol. We observed induction of CYP2E1 and concomitant increase in ROS production in cells expressing core and NS5A proteins.⁴⁰,⁴⁴ The Weinman laboratory reported augmented oxidative stress upon coexpression of HCV core and CYP2E1.³⁸ Moreover, increased CYP2E1 levels were reported in CHC patients with mild fibrosis.⁵⁶ Thus, CYP2E1 is regarded as a prominent source of ROS in HCV infection, which may explain why alcohol consumption aggravates disease progression.³⁸

Mechanisms of HCV-induced ROS scavenging

HCV-induced oxidative stress is accompanied by changes in expression of the enzymes that are involved in neutralization of ROS. Major discrepancies regarding the induction of the antioxidant defense system by HCV exist between in vitro and in vivo data, as well as between reports from different groups. It is generally accepted that HCV infection reduces the total antioxidant status in blood serum, increases levels of oxidized glutathione and decreases total glutathione content.²⁹,⁵⁷,⁵⁸ The impact of HCV infection on SOD or catalase is less clear. Transgenic mice expressing HCV core in the liver or Huh7 cells harboring subgenomic HCV replicons display elevated levels of catalase, but CAT levels remain unaffected in the HCVcc system, as well as Huh7 cells overexpressing core protein.¹⁶,⁵⁷,⁵⁸ Similar discrepancies exists for SOD: SOD2 induction was observed in HCV-infected and core-expressing cells, whereas overexpression of nonstructural HCV proteins diminished levels of SOD1 and SOD2.¹⁶,⁵⁷ Levels of these enzymes in liver specimens from CHC patients were also determined by several groups. Abdalla and coworkers did not detect changes in CAT or SOD expression,⁵⁹ whereas Jacobs and coworkers observed reduction of CAT and induction of SOD1.⁶⁰ The same group also revealed elevated levels of SOD2 in liver biopsies from the patients with chronic hepatitis C and mild fibrosis.⁶¹ Different results might be attributed to heterogeneity of expression between patients and influence of stage of liver disease.⁶¹

Peroxiredoxins (Prdx) and glutathione peroxidases (GPx) represent additional groups of peroxide scavenging enzymes. Proteomic analysis by Diamond and coworkers suggested that expression of Prdx1, 2, and 5 is elevated in livers of a majority of CHC patients with mild fibrosis.⁶¹ We observed induction of GPx1 and GPx4 in HCVcc-infected cells and in CHC liver biopsies.¹⁶ NS3/4A protease of HCV is also known to cleave the C-terminus of ER-residing GPx8.⁶² Interestingly, GPx8 is thought to scavenge H2O2 produced by Ero1α in the ER in order to prevent leakage into the cytoplasm.⁶³ HCV was also shown to enhance expression of thioredoxin and glutaredoxin,⁶⁰ proteins involved in the regeneration of oxidized peroxiredoxins.

Several independent groups have shown that HCV⁶⁴,⁶⁵ as well as core, E1/E2, NS4B, and NS5A³⁵ activate the Nrf2/ARE pathway. Several signaling cascades are implied in this activation—dependent or independent of ROS: mitogen-activated protein kinases,⁶⁴ protein kinase C, phosphoinositide-3-kinase (PI3K), and protein (casein) kinase 2 (CK2).³⁵ However, the Nrf2/ARE pathway has also been shown to be blocked by HCV in infected Huh7.5 cells and primary human hepatocytes, as well as in the liver biopsies from CHC patient.⁶⁶ Mechanistically, this inhibition was shown to be due to retention of sMAFs by NS3 in the presence of HCV core. The reasons for these observed discrepancies are unknown as all studies were based on the same HCVcc system and used at similar time points. Quantification of Nrf2-target genes, such as NAD(P)H:quinoneoxidoreductase 1 (Nqo1), HO-1, or a variety of glutathione-metabolizing enzymes including glutamate-cysteine ligase (GCL), glutathion synthase (GS), and glutathione reductase (GR) did not resolve the issue either. Several groups described increased expression of HO-1 in CHC liver biopsies and in vitro infections,⁶⁷ but other groups reported suppressed expression in vivo and in various other models (e.g., see Ref. [59]). We have observed elevated levels of glutathione synthase in livers from HCV carriers and in infected cell cultures.¹⁶ Further transcriptomic and proteomic studies also showed suppression of classical Nrf2-target genes and other genes involved in protection against oxidative stress (Nqo1, CAT, glutathione-S-transferases, etc.).⁶⁰,⁶⁸ The reasons for these discrepant results may be that prolonged HCV-induced ROS production exhausts the antioxidant defense system. Alternatively, TGFβ1 has recently been shown to suppress the Nrf2/ARE pathway.⁶⁵ Fibrosis in CHC is frequently accompanied by the elevation of TGFβ1 levels,⁶⁹ whereas treatment of patients with anti-HCV agents reduces the levels of this cytokine.⁷⁰ Therefore, taking into account the duration of the disease and levels of TGFβ1 and other cytokines in future may allow to resolve this discrepancy.

Role of ROS in the HCV life cycle

Several steps of the HCV life cycle are known to be controlled by ROS and the cellular redox balance and in particular a sensitivity to lipid peroxidation has been described.¹⁵,¹⁶ HCV is known to replicate and assemble intracellularly in close association with intracellular membranes, which the virus is known to remodel into membranous web structures. These structures are enriched in cholesterol, sphingolipids, and phosphatidylinositol-4-phosphate.¹⁵ Accumulation of lipidperoxides and their degradation products have been shown to be detrimental for HCV replicase activity, possibly by interfering with the interactions between HCV NS proteins within these complexes. Interestingly, patients with a null genotype of glutathione-S-transferases, namely gstt1 and gstm1, enzymes that detoxify endogenous compounds including lipid peroxides or break down xenobiotics, are characterized by decreased rates of the spontaneous resolution of acute HCV infection.⁷¹

Exogenous H2O2 has also been shown to inhibit HCV replication,⁷² but this effect is mediated by Ca²+.⁷³ However, knock-down of GPx1 and GPx8, as well as of SOD1 or SOD2, in infected cells had no significant impact on HCV replication.¹⁶,⁶² ROS may also affect the translation of the HCV genome,⁷⁴ an effect that is mediated through the induction of UPR, and in particularl activation of the PERK branch.⁷⁴ PERK phosphorylates eukaryotic initiation factor 2α (eIF2α), leading to the suppression of cap-dependent translation and often switching ribosomes to cap-independent translation, which might account for the observed effects.⁵¹ However, contrary observations have also been reported.⁷⁵

Another important consequence of ROS induction is the stimulation of autophagy, a catabolic process that protects cellular homeostasis from metabolic and oxidative stress by degrading cytoplasmic constituents. HCV replication has indeed been shown to depend on the induction of several autophagic factors, however, the exact interrelationship between autophagy and HCV replication remains unknown.

HCV cell entry has also been shown to be sensitive to oxidative stress. While association of virus with lipoproteins or production of viro-lipo particles has been shown to optimize viral infectivity, presence of oxidized LDL particles is known to compete with viral infection and spread.¹² Another indirect effect of ROS on HCV persistence is their contribution to the increase in HCV genome heterogeneity, which ensures viral escape from the immune system.⁷⁶

While amplification and spread of HCV are intrinsically associated with generation of ROS, the virus has also evolved mechanisms to counteract ROS induction in order to ensure viral viability. The virus has been shown to induce the lipid peroxide scavenger GPx4 in vitro and in vivo. Downregulation of GPx4¹⁶ and consequential accumulation of lipid peroxides induces a moderate suppression of HCV replication and strong suppression of virion infectivity due to decreased fusogenic activity.⁷⁷ These effects could be rescued by tocopherol. Similarly, the downregulation of GPx8 by RNA interference impairs virus particle assembly.⁶² Moreover, the SEC14L2 protein, also known as tocopherol-binding protein 1, was recently identified as the cellular factor crucial for pan-genotype HCV replication in hepatocytes.⁷⁸ Its effect was attributed to enhanced uptake and activity of vitamin E,⁷⁸ one of two bona fide antioxidants⁷⁹ that, like GPx4, protect from lipid peroxidation. Saeed and coworkers showed that SEC14L2 ensured protection of HCV replication from lipid peroxidation, since the effect of the protein was observed even in cells harboring subgenomic replicons of the virus.⁷⁸ Overall, lipid peroxidation can inhibit several steps of the viral life cycle: replication, assembly, virion infectivity, and fusion. Finally, ROS may impact HCV replication via the Nrf2/ARE pathway. Yu and coworkers reported that induction of the Nrf2-target gene heme-oxygenase 1 inhibited HCV replication by two different modes: through induction of interferon α and through bilirubin induction, which in turn inhibited NS3 protease activity.⁸⁰

Role of ROS and antioxidants in the pathology of chronic hepatitis C

Oxidative stress in CHC is intrinsically linked to the metabolic alterations induced by the virus. The ROS produced by the virus causes direct damage to hepatocytes and drive inflammatory processes within infected hepatocytes by activating inflammatory signaling cascades, such as the ROS-sensitive NF-κB pathway, as well as induction of ROS-inducible cytokines TNF-α and IL-8. ROS can furthermore diffuse and activate neighboring stellate cells directly to produce inflammatory cytokines and fibrogenic molecules. Furthermore, ROS favor neoplastic transformation due to their direct oxidizing activity. Indeed, levels of oxidative stress markers, such as MDA or 8-isoprostane in serum or urine, respectively, correlate with levels of inflammation, the stage of fibrosis²⁰,²³,³² as well as markers of liver damage, such as alanine or aspartate aminotransferase (ALT, AST)⁸¹ and correlate inversely with the antioxidant capacity.⁶⁵ Indeed, levels of the antioxidants in the liver are higher in patients with mild compared to moderate-to-severe inflammation and fibrosis.²¹

Activation of hepatic stellate cells into activated myofibroblasts that start to secrete extracellular matrix components is a major driver of inflammation and fibrosis in CHC (Fig. 1.3). The processes of HSCs activation and proliferation are triggered by a panel of different molecules that are produced by hepatocytes and stellate cells. Platelet-derived growth factor (PDGF), connective tissue growth factor (CTGF), osteopontin, or TGF-β1 are all profibrogenic.⁸² TGF-β1 expression/secretion can be triggered by HCV directly in the infected hepatocyte. This effect is mediated by core, NS3/4A, NS4B, and NS5A proteins through ROS- and calcium-dependent mechanisms.⁸³ Furthermore, proteolytic processing of TGF-β1 into its active form depends on mitochondrial Ca²+ uptake and is thus ROS-dependent.⁸³ Levels of TGF-β1 are elevated in serum and liver of CHC patients and correlate with fibrosis scores.⁸⁴,⁸⁵

Figure 1.3   Activation of hepatic stellate cells is a major trigger of fibrosis.

HCV triggers ROS and release of cytokines and profibrogenic mediators, such as osteopontin and TGF-β1 from infected hepatocytes. These molecules, as well as hepatocyte apoptosis activate hepatic stellate cells in the immediate vicinity and convert them into activated myofibroblasts. In addition, the virus activates Kupffer cells and circulating leukocytes, which also contribute to proinflammatory cytokine secretion and thus amplify activation of hepatic stellate cells. Activated hepatic stellate cells are not the only but thought to be the predominant cell type responsible for extracellular matrix alterations that are characteristic of fibrosis, such as secretion of collagens and other extracellular matrix constituents.

Levels of oxidative stress markers in CHC patients correlate with the incidence of HCC and HCC recurrence upon liver transplantation.⁸⁶ As already mentioned, ROS induce DNA damage, accumulation of mutations and on the long term genetic instability.⁸⁷ And this is underlined by the fact that levels of DNA oxidation markers, such as 8-oxoG or thioredoxin increase with fibrosis progression and are most elevated in liver cancer.⁸⁸,⁸⁹ Furthermore, chronic oxidative stress, instead of inducing apoptosis, can induce prosurvival programs and in particular the Nrf2/ARE pathway, thus ensuring survival of cells exposed to a strongly oxidative microenvironment. Apoptosis is suppressed by ROS via upregulation of 24-dehydrocholesterol reductase (DHCR24), or suppression of p14, both of which disrupt the p53-Mdm2 pathway.⁹⁰ In addition, HCV also counteracts apoptosis through the ROS-dependent activation of peroxisome proliferator-activated protein α (PPARα)⁹¹ and NS5A-mediated suppression of potassium ion channel Kv2.1.⁹²

The cell cycle checkpoint inhibitor p21/Cip1/WAF1, induced by ROS, can activate Nrf2 signaling directly via a p21-Nrf2 interaction under low stress conditions. This is thought to be important in order to reduce ROS while the cell is in cell cycle arrest and busy to repair DNA damage. The direct interaction of p21 with Nrf2 blocks the degradation of the latter protein. However, at high levels of oxidative stress, p21 normally mediates apoptosis. HCV interferes with the p21-mediated regulation of apoptosis. Both, core and NS5A have been implied in this process and thus render Nrf2-depedent stress response, less sensitive tothe p21-regulation. Thus, HCV may favor continuous cell proliferation under moderate, as well as severe stress.⁹³ This mechanism has been confirmed in mice, where depletion of p21 leads to a continuous proliferation of severely injured hepatocytes. While survival of these animals augments tumor incidence and development are significantly increased. In humans, p21 expression levels in the cancerous tissues of liver biopsies appear to be significantly reduced compared to those in the surrounding noncancerous tissues,⁹⁴ and histological negativity for p21 is a negative prognostic factor for survival after HCC resection.⁹⁵ However, this latter finding has been contradicted.⁹⁶

Enhanced intracellular ROS production in response to a hypoxic microenvironment plays an important role in the initiation of the metabolic adaptations a cancer cell requires to sustain proliferation. Increased ROS levels lead to the stabilization of hypoxia-inducible factor 1α (HIF1α). HIF1α is transcription factor that induces the Warburg effect, consisting of induction of glycolysis and the metabolic adaptations necessary for a cell to produce ATP and the metabolic intermediates required for cell survival and proliferation in an oxygen deprived environment. HIF1α has been shown to be induced in HCV infection.⁹⁷ ROS also regulate metabolic adaptations via Nrf2 induction. Nrf2 in turn activates transcription of many metabolic enzymes that regulate carbohydrate metabolism and NADPH production, glutaminolysis, lipid oxidation, and others.⁹⁸ Besides triggering a pro-proliferative metabolism, ROS also induce angiogenesis via induction of COX2 and vascular endothelial growth factor (VEGF).⁹⁹

Given the primordial role of ROS in HCV-induced fibrogenesis, inflammation, and hepatocarcinogenesis, a number of clinical studies have investigated antioxidants for their antifibrotic and antiviral effects. Vitamin E, for example, has been shown to reduce ALT levels in CHC patients. Zinc has been shown to reduce hepatic fibrosis and possibly also HCV replication, while the antioxidant N-acetylcysteine has not shown any convincing antiviral or antifibrotic activity. The free radical scavenger glycyrrhizin also diminished transaminase enzyme levels, and decreased cellular damage in patients with chronic HCV infections. Silibinin and mitoquinone A showed both antifibrotic activities, but their antiviral effect is not clear. Combination antioxidant therapies have been tested as well in clinical trials and were again shown to decrease HCV-induced liver damage; however, their antiviral effects were not clear cut. These data are discussed in detail in reviews.¹⁰⁰ The fact that antioxidants emerge to not have an evident and strong antiviral activity is clearly in line with the idea that viral replication is sensitive to oxidative stress and in particular lipid peroxidation. Indeed, HCV induces a strong scavenging program in order to control the cellular redox homeostasis, even if on the long term ROS accumulate in infected cells.

Conclusions

It is well established that HCV infection induces strong cellular oxidative stress in infected hepatocytes, which is amplified by inflammatory processes and activates hepatic stellate cells in the surrounding microenvironment. Therefore HCV-induced oxidative stress is intrinsically linked to metabolic disorders, fibrosis, and hepatocarcinogenesis. While HCV may depend on certain metabolic alterations associated with ROS production, such as HIF1α-mediated glycolysis or altered TCA cycle rates, there is also clear evidence that HCV replication is sensitive to the induction of oxidative stress. Oxidation of lipids in particular seems to be detrimental to viral replication. Thus ROS are a double edged sword for HCV. Indeed, the virus activates a series of ROS scavenging mechanisms by a variety of direct and indirect mechanisms, but cannot prevent on the long term accumulation of ROS and oxidative damage. This knowledge is important for our understanding of the pathology underlying CHC and needs to be taken into account for the design of antifibrotic and antiinflammatory treatment.

Acknowledgments

A.I. acknowledges support from the Russian Ministry of Education and Science (Agreement 14.616.21.0043, the unique identification number RFMEFI61615X0043). B.B. acknowledges support from the French National Agency for AIDS and Viral Hepatitis Research, Comité de Saône-et-Loire de la Ligue contre le cancer, Agence Nationale de Recherche, DevWeCan French Laboratories of Excellence Network (Labex, Grant ANR-10-LABX-61), the OpeRa IHU program (GRANT ANR-10-IBHU-004), and the PHC Kolmogorov program.

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