Oxidative Stress and Redox Signaling in the Pathophysiology of Liver Diseases

Abstract

The increased production of derivatives of molecular oxygen and nitrogen in the form of reactive oxygen species (ROS) and reactive nitrogen species (RNS) lead to molecular damage called oxidative stress. Under normal physiological conditions, the ROS generation is tightly regulated in different cells and cellular compartments. Any disturbance in the balance between the cellular generation of ROS and antioxidant balance leads to oxidative stress. In this article, we discuss the sources of ROS (endogenous and exogenous) and antioxidant mechanisms. We also focus on the pathophysiological significance of oxidative stress in various cell types of the liver. Oxidative stress is implicated in the development and progression of various liver diseases. We narrate the master regulators of ROS-mediated signaling and their contribution to liver diseases. Non-alcoholic fatty liver diseases (NAFLD) are influenced by a “multiple parallel-hit model” in which oxidative stress plays a central role. We highlight the recent findings on the role of oxidative stress in the spectrum of NAFLD, including fibrosis and liver cancer. Finally, we provide a brief overview of oxidative stress biomarkers and their therapeutic applications in various liver-related disorders. Overall, the article sheds light on the significance of oxidative stress in the pathophysiology of the liver.

The Concept and Definition

In 1936, Hans Selye introduced the “stress concept” in understanding the response of animals to acute nonspecific nocuous agents such as cold, surgical injury, and drugs (349). He defined stress as a nonspecific response of the body to any demand (348). In the later 1970s, Paniker et al. showed that exposure of red blood cells to hydrogen peroxide (H₂O₂) induces “oxidative stress,” which is associated with glutathione reductase activity (299). In the 1980s, the term oxidative stress was introduced in redox and medicine biology in an introductory chapter (366). The review by Helmut Sies “Biochemistry of Oxidative Stress” described the biology of oxidative stress in biological systems, including the pro-oxidants, antioxidants, cause and effect, defense and repair mechanisms, and also control of oxidative stress. Oxidative stress is defined as the “state of condition wherein the cellular pro-oxidant and antioxidant balance is altered in favor of a pro-oxidant state.” In other words, oxidative stress occurs in response to increased production of reactive oxygen species and decreased production of antioxidants (135, 364, 365).

The concept of oxidative stress is associated with free radicals in biology and medicine (317). Free radicals are the molecules or atoms characterized by highly reactive unpaired electrons or atoms (39, 313, 412). All the free radicals are not equally toxic. The degree of reactivity or chemical nature of free radicals and their reactants depends on the extent of damage to the biological system. The reactivity of free radicals estimated by one-electron reduction potentially reflects the molecule’s affinity compared with hydrogen. Thus, most free radical products are assumed to be oxygen-based hydroxyl radicals and nitrogen-based peroxynitrite anion (241, 317).

Oxidative Stress-classification

In modern biology and medicine, several reactive species were identified. Among them, the most well-studied reactive species in the mammalian system include reactive oxygen species (ROS), reactive nitrogen species (RNS), reactive carbonyl species (RCS), and reactive sulfur species (RSS). Other biologically critical reactive species include selenium, chlorine, and bromine species.

Reactive oxygen species (ROS)

ROS comprise radical and nonradical oxygen species formed by the partial reduction of oxygen. ROS are majorly represented by superoxide anions (O₂^•−), hydroxyl radicals (HO^•), and hydrogen peroxide (H₂O₂). Superoxide anions and hydroxyl radicals exist in a free-radical form characterized by highly unstable unpaired electrons (e.g., O₂^•− and HO^•). While H₂O₂ is a nonradical, chemically stable, freely diffusible, and long-lived molecule (282, 365). ROS is formed through endogenous mechanisms such as mitochondrial electron transport chain (ETC), flavin-dependent oxidation, and microsomal oxidation (188, 280). ROS generation also occurs in response to exogenous pathways such as xenobiotic metabolism (e.g., antimycin and adriamycin) (285, 367). Furthermore, ROS could also be generated through exposure to nutrients, pollutants, and physical factors such as ultraviolet light, ultrasound, and X-rays (367). Auto-oxidation products such as flavins and hemoglobin also could lead to the formation of ROS (326, 347).

In the biological system, H₂O₂ is considered as the major ROS that plays a critical role in redox regulation (129, 324, 379). The production of H₂O₂ takes place in response to metabolic cues, cytokine, and chemokines (343). Under physiological levels, the redox signaling of H₂O₂ is mediated through oxidation of sulfur proteins, reversible methionine oxidation, selenoprotein, oxidation of metal centers, and lipids (408). However, the generation of higher amounts of H₂O₂ results in unspecific oxidation of proteins leading to cell growth arrest and death (128). The major endogenous enzymatic sources for H₂O₂ are NADPH oxidases and the mitochondrial ETC. Excess H₂O₂ is removed by peroxiredoxins and glutathione peroxidases, apart from catalases (141, 249). Besides, H₂O₂ levels are maintained by the exchange between organelle systems such as ER, mitochondria, and peroxisomes (453).

Reactive nitrogen species (RNS)

RNS is a family of nitrogen-associated molecules produced when nitrogen interacts with oxidants and reductants like superoxide and hydrogen peroxide, either endogenously or exogenously. The most common RNS includes nitric oxide (NO^•) and peroxynitrite (ONOO⁻). Like ROS, RNS also exists in a free radical form (nitric oxide, nitrogen dioxide, and nitrite) or nonradical form (nitrous acid, peroxynitrite, and nitrosyl anions). The half-life, solubility, and biological reactivity of RNS depend on their precursors (1, 82, 297, 320).

Nitric oxide is the foremost and critical RNS known to play a significant role in redox biology. Nitric oxide is an easily diffusible free radical with a short half-life and is central to the formation of other RNS (320, 328). The generation of RNS begins with the synthesis of nitric oxide by the enzyme nitric oxide synthase (NOS), which exists in three forms; includes neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). The eNOS and nNOS are constitutively expressed by the endothelial cells and neuronal cells, respectively, while iNOS expression is regulated at the transcriptional level in various cells (104, 105). In addition to nitric oxide, peroxynitrite is considered a major cellular nitrating agent, which derives from the reaction of NO with superoxide anion. Peroxynitrite is generated by the plasma membrane NADPH oxidases and mitochondrial respiratory chain. Although peroxynitrite is short-lived, it is a potent inducer of cell death (319, 320, 388).

Other species

In addition to ROS and RNS, several other highly reactive species regulate redox signaling. Among them, reactive sulfur species (RSS) comprises sulfur-containing reactive biomolecules that range from small molecules to proteins. The common RSS includes hydrogen sulfide (H₂S), protein thiols, low-molecular-mass compounds such as glutathione, trypanothione, sulfenic acids (RSOH), and nitrosothiols (RSNO) (76, 122). Most of the RSS formed as a by-product of major thiols or oxidation of sulfite or sulfate molecules. Importantly, the per/polysulfides encompassing cysteine persulfide and polysulfide are the most abundant RSS identified in the mammalian and other biological systems (182, 203). Besides, several studies have shown that H₂S is the most common short-lived RSS that has prolonged biological effects in the mammalian system (329). Recent studies have demonstrated a role for RSS in various diseases, including atherosclerosis, fatty liver, inflammation, and viral hepatitis (65, 189, 217). Further, a recent work by Zhang et al. shows that cellular polysulfides inhibit lipopolysaccharide-induced proinflammatory responses in the macrophages through toll-like receptor 4 (TLR4) signaling (462).

RCS are the biological compounds with one or more carbonyl groups that include metabolically generated aldehydes and electronically excited carbonyl molecules. Some of the RCS include acrolein, crotonaldehyde, glyoxal, acetone, and formaldehyde (350). RCSs are majorly generated through nonenzymatic processes such as lipid peroxidation, amino acid oxidation, and glycation (253, 369). Although RCSs exert beneficial effects, their overproduction, known as carbonyl stress, is the major contributing factor for aging, metabolic diseases, and other neurodegenerative diseases (95, 144). Mechanistically, RCS induce biological damage by forming α- and β-unsaturated aldehydes, dialdehydes, and keto-aldehydes, which are more reactive than saturated forms (5, 254). In aging, RCS accumulates in various tissues and peripheral blood in the form of advanced glycation end products (AGEs) (304, 400).

Physiological Significance of ROS

The cells generate ROS during oxidative metabolism by various chemical, environmental, and dietary cues, as described above (12). Maintaining a delicate balance between the oxidant-antioxidant mechanisms is modulated by its production, location, and inactivation. At physiological levels, ROS, considered as “redox biology,” plays a significant function in regulating signal transduction, gene expression, and cell proliferation (268, 292, 458). Recent studies have emphasized their role in blood pressure control (410, 418) and embryonic development (222, 437). Thus, ROS monitors cell fate indicating the existence of a “ROS rheostat” in the cells.

Under physiological conditions, mitochondrial reactive oxygen species (mROS) regulates biological functions such as autophagy, immunity, differentiation, longevity, and adaption to hypoxia (79, 351). As a defense mechanism, mitochondria are equipped with antioxidant enzyme machinery to minimize the risk of aberrant increases in ROS. For example, superoxide dismutase (SOD) families of enzymes such as SOD1, 2, and 3, peroxiredoxins, glutathione peroxidase (GPX), and catalases are localized in mitochondrial intermembrane space or matrix (172, 175, 421). Peroxiredoxins turn ROS signaling off (303), while GPXs buffers the excess ROS and bring them to normal levels (250).

The physiological significance of ROS is well-studied in mitophagy, a quality control process wherein damaged mitochondria are continuously removed from the cells (185). Impairment of mitophagy results in the accumulation of damaged mitochondria leading to a further increase in mROS/total ROS. Several studies have shown that mROS is required to induce mitophagy (92, 208). For instance, under starvation mROS drives the formation of autophagosome through the activation of PI3K pathway (342). Besides, mitophagy preserves mitochondrial bioenergetics, attenuates cell injury and progression of liver diseases by reducing oxidative stress (33, 243, 432). The other important physiological significance of mROS was demonstrated under hypoxic conditions. This concept has arrived from the data that the cells depleted of mitochondrial DNA do not stabilize HIFs under hypoxia (55). Thus, when the cells encounter low oxygen levels, they undergo an adaptive mechanism triggered by enhanced mROS generation. Similarly, several studies have shown that mROS regulate the immune system (106, 325), aging (211, 266, 332), and stem cell differentiation (161, 295). Thus, the level of ROS serves as an alarm to report the changing environment in the cell.

Liver and Oxidative Stress

The liver is the second-largest and key metabolic organ in the mammalian system (271). The liver consists of parenchymal (hepatocytes) and nonparenchymal cells. Hepatocytes are the major structural and functional units of the liver. The nonparenchymal cells include Kupffer cells, sinusoidal endothelial cells, stellate cells, periportal fibroblasts, and hepatic dendritic cells (404). The liver is the main site for synthesis, secretion, degradation, and coupled inter-conversion and biotransformation of amino acids, carbohydrates, and lipids. It is also involved in the storage and transport of micronutrients such as vitamins and minerals. In addition, the liver is the major site of detoxification of drugs, alcohol, and hormone metabolism and to some extent, filtration of blood. Collectively, it performs five essential functions, namely, (i) metabolism, (ii) storage, (iii) excretion and secretion, (iv) detoxification, and (v) blood filtration. These activities account for approximately 25% of the total metabolic rate. The liver is considered as the metabolic hub as it connects various organs in coordinating whole-body homeostasis of bio-molecules, hormones, macro-, and micronutrients (201, 314, 404).

Given its role in xenobiotic metabolism, the liver generates several oxygen (ROS) and nitrogen (RNS)-based free radical species. However, their persistent production results in oxidative stress leading to the dysregulation of liver homeostasis (69, 256). The initiation of ROS in the liver takes place due to increased mitochondrial respiration. mROS generated in the hepatocytes is responsible for the oxidative damage of lipids, proteins, and DNA (265, 280). Recent studies have reported that endoplasmic reticulum and peroxisomes also contribute to hepatic ROS levels (38, 207). Besides mitochondrial respiration, hepatic xanthine oxidase, which converts hypoxanthine to xanthine and xanthine to uric acid, generates superoxide anion and hydrogen peroxide (26).

Hepatocytes

Hepatocytes, which occupy nearly 80% of the liver volume, are the first cells that respond to the dietary contents after absorption and are prone to injuries from ingested toxins, alcohol, and other drugs. Hepatocytes prevent liver damage by storing free fatty acids in the form of lipid droplets. (14). Lipotoxicity impairs mitochondrial function and changes the redox state. Over time, dysfunctional mitochondria reduce ATP production resulting in increased ROS production and hepatocyte death (246). Thus, ROS released from the injured hepatocytes acts as a major stimulus for the progression of various liver diseases via activation of immune and hepatic stellate cells.

ROS generated in the hepatocytes also influences the function of the neighboring cells through various mechanisms. For example, ROS activates TGF-β and fibromodulin in the hepatocytes, which induces the migration and proliferation of HSCs leading to liver fibrosis (37). Apoptosis or necrosis of the hepatocytes also increases mROS releases and contributes to fibrogenesis (48, 416). Furthermore, increased hepatocyte ROS destroys the critical function of cellular macromolecules such as DNA, proteins, and lipids. For example, ROS oxidizes the protein kinase and phosphatases that regulate major signaling pathways such as mitogen-activated protein kinases (MAPKs) (153, 370). Hepatocytes also promote inflammation and oxidative stress by releasing hepatokines such as fetuin A, fetuin B, and IL-18. Further, hepatocytes store a large amount of iron and Fe-S containing mitochondria proteins, which are highly reactive toward O₂⁻ (389, 414). Thus, the excessive production of ROS generated from the hepatocytes plays a significant role in the development and progression of liver diseases (Figure 1).

Figure 1.

Mechanisms of generation of ROS-mediated oxidative stress in the liver. Increased lipid accumulation in hepatocytes via fatty acid uptake and de novo lipogenesis using several lipogenic enzymes augments mitochondrial β-oxidation leading to incessant generation of ROS. Hepatic nonparenchymal cells are also involved in ROS generation by activating the toll-like receptors (TLRs) on the Kupffer cells and HSCs. In Kupffer cells, TLR4 in response to SFA and TLR1/2/6 for FFA activation triggers NOX-2-mediated ROS generation, resulting in oxidative stress. In HSCs, accumulation of cholesterol and acetyl-CoA generates huge amount of ROS through glucose metabolism.

Kupffer cells

In the liver, Kupffer cells are the most abundant resident macrophages that eliminate invading microbes and their products. Kupffer cells reside in the hepatic sinusoidal cells, and their numbers are tightly regulated by various factors (91, 163). Although it seems that resident macrophages are essential to fight invading microbes, their activation plays a significant role in the initiation and progression of acute liver injuries and fatty liver disease (269). Recent evidence demonstrates that the recruitment and activation of Kupffer cells also occur in response to dietary and environmental factors (102, 391). The recruited macrophages initiate fatty liver and fibrosis in NAFLD (152, 406).

Under pathological conditions, Kupffer cells are activated by various inflammatory cells, chemokines, and other growth-modulating factors (93, 215). Also, the accumulation of ROS activates Kupffer cells (110). Further, activated Kupffer cells could also contribute to ROS generation under various acute and chronic liver injuries. In support of this notion, the treatment of rats and mice with carbon tetrachloride (CCl₄) results in the activation of Kupffer cells, thereby increasing the reactive oxygen intermediates (9, 176, 327). In addition to resident macrophages, recruited macrophages also produce ROS through various mechanisms, including mitochondrial damage, ER stress, and increased NADPH oxidases (NOXs) (83, 115, 132). The iNOS expressed by the Kupffer cells generate RNS and its redox derivatives. NOX-derived ROS involves the production of proinflammatory cells and other chemokines in response to LPS and other fattyacids (Figure1) (142, 181, 228, 311). Recent studies show that the phenotypic switch in the resident macrophages from classically activated inflammatory macrophages (M1) to alternatively activated anti-inflammatory macrophages (M2) is strongly associated with increased generation of ROS in the liver (169, 290).

Hepatic stellate cells (HSCs)

HSCs located in the space of Disse occupy nearly 8% to 12% of the total liver cell population. HSCs play a significant role in maintaining hepatic architecture and blood flow by regulating the synthesis/degradation of the extracellular matrix. HSCs also exhibit immune function by secreting various cytokines, chemokines, and growth factors. HSCs store retinoids including vitamin A and its isoforms. Because of these characteristics, HSCs regulate the functions of hepatocytes and other liver cells through a paracrine and juxtracrine mechanism (Figure 1) (340, 353, 354). ROS generated by the Kupffer cells and leukocytes could activate HSCs. A positive correlation between ROS and HSCs activation was demonstrated using various models such Fas the CCl₄-induced and diet-induced liver injury in rodent animals. Although CCL₄ does not affect the HSCs directly, substances released from the injured hepatocytes, including ROS activates HSCs (27, 272). Activation of HSCs leads to excessive synthesis and deposition of extracellular matrix proteins resulting in liver fibrosis (457). Although the exact mechanisms of HSC activation are unclear, several studies demonstrated that inflammatory cytokines and chemokines drive the activation and proliferation of HSCs (6, 216).

Endothelial cells

The liver sinusoidal endothelial cells (LSEC) are highly specialized and distinctive micro-vascular cell types that play a key role in maintaining the liver microenvironment. LSECs are considered as the first defense barrier and contribute to metabolite transport, inflammation, and angiogenesis by interacting with various neighboring cell types in the liver (42). LSECs also govern the regenerative process in response to liver injury (84, 309, 417). Thus, the dysfunction of LSECs leads to the initiation and progression of various liver-related diseases, including liver fibrosis and cirrhosis (137, 257). Free fatty acids, triglycerides, ethanol, and HCV core protein could trigger LSEC dysfunction (18, 140, 263). Inflammatory cells and activated HSCs also drive liver injury by altering LSEC response to injury (174, 360). It has been shown that ROS selectively targets LSEC during prolonged liver injury. Importantly, LSECs are highly sensitive to ROS compared to other liver cells due to higher expression of NADPH oxidase (NOX) 2 and 4, the major source of ROS via NADPH oxidases, and their reduced capacity to enzymatically detoxify H₂O₂ (259, 267, 358). Moreover, impairment in autophagy in LSEC results in an improper response to oxidative damage and leading to increased ROS generation. This further helps LSEC recruit and activate macrophages and modulating the expression of proinflammatory cytokines and, thereby disease progression (136, 330).

Immune cells

The liver is considered as “the organ of the immune system.” The liver immune cells are distributed and localized strategically in various compartments and circulate in the sinusoids. The liver immune cells majorly include dendritic cells, neutrophils, lymphocytes (B & T), and natural killer cells (74, 284). The hepatic immune cell population is established as early as early embryonic life and creates a niche for blood surveillance. Although liver immune cells are prerequisites for proper host immune responses, excessive accumulation or recruitment of immune cells could lead to several liver-related diseases. For example, neutrophils infiltrated at the site of injury recruit blood monocytes and other immune cells, which further activates dendritic cells and macrophages (160, 431, 447).

One of the hallmarks of inflammation is the generation of large quantities of superoxide radicals via NADPH oxidase complex in the liver immune cells. In addition, most of the oxidants, except superoxide and hydrogen peroxide, are produced by the enzyme myeloperoxidase (MPO) in the immune cells, especially neutrophils (197). ROS generated in the immune cells induces tissue damage and disease progression. Indeed, studies have shown that neutrophils were the primary cell that displays NADPH oxidase activity and generates a whole spectrum of both radicals and nonradical relevant to ROS (382, 447). T cells also generate ROS via NADPH oxidase and act as the primary source for mROS (352). This concept is tested in several studies wherein pharmacologic treatment of primary T cells with antioxidants attenuated the proliferation of T cells (168, 277). Similarly, continuous generation of ROS in primary B cells in response to B cell antigen receptor stimulation results in proliferation and activation of B cells (430). Likewise, oxidative stress in the liver also enhances the dendritic cell response leading to T cell activation (289, 296).

Cellular Organelles and ROS

In the cells, ROS is generated in various cellular compartments, including the cytoplasm, mitochondria, ER, lysosomes, and peroxisomes.

Mitochondria

Mitochondria play an essential role in the generation of ATP through the oxidation of metabolic intermediates through the ETC. Mitochondria account for nearly 90% of oxygen consumption and therefore acts as the most redox-active compartment in the mammalian cells (46, 464). However, the accumulation of excess fatty acids creates an imbalance between the delivery and outflow of electrons to the respiratory chain in the mitochondria leading to the production of ROS (287, 377). In general, the mitochondrial ROS (mROS) in ETC occurs in the form of superoxide anion radical and its dismutation product H₂O₂ (40, 210). Although mitochondria are occupied with several complexes in generating energy, complex I and III are the major sites of electron transfer to O₂ to generate O₂^•− (62, 195). Studies have shown that ROS could also be generated from pyruvate dehydrogenase (44), α-ketoglutarate dehydrogenase (378), glycerol-3-phosphate dehydrogenase (293), and monoamine oxidase (375). Under pathological conditions, several molecular mechanisms drive the overproduction of mROS. For example, mitochondria in the apoptotic cells produce superoxide in response to the release of cytochrome c (130, 166). Thus, mitochondria are considered the main source of ROS.

Given its role in ROS production, mitochondria are well equipped with antioxidant and scavenging mechanisms such as manganese superoxide dismutase (Mn-SOD), copper zinc-superoxide dismutase (CuZn-SOD), mitochondrial glutathione (mGSH), glutathione peroxidase, and catalase. In addition, the mitochondrial matrix is equipped with two antioxidant enzymatic systems: GSH-dependent glutathione peroxidase and NADPH-dependent thioredoxin-2 systems (338, 351). These enzymatic reactions help to maintain the balance between the pro and antioxidants. However, under pathological conditions, mitochondria generate huge amounts of ROS due to dysregulation in one or several of the aforementioned oxidant and antioxidant mechanisms. The well-studied liver pathologies that involve increased mitochondrial ROS include alcoholic-fatty liver disease (AFLD), nonalcoholic fatty liver disease (NAFLD), cirrhosis, viral hepatitis, and hepatocellular carcinoma (HCC). For example, chronic alcohol consumption leads to enhanced ROS production in the mitochondria partly due to the accumulation of cholesterol in the inner mitochondrial membrane and further disturbing the mGSH import from the cytosol. The significance of the mitochondrial import of GSH was demonstrated by restoring the membrane fluidity by administering the antioxidant N-acetylcysteine. Moreover, alcohol-induced ROS causes oxidative damage to the mitochondrial DNA, thereby increasing the risk of double-strand breaks and somatic mutations (32, 53, 118, 138). Thus, several studies have demonstrated targeting oxidative stress as a potential therapeutic avenue for liver-related diseases such as AFLD and NAFLD.

Endoplasmic reticulum

The endoplasmic reticulum (ER) is the major site for protein synthesis, folding, modification, and trafficking (41). ER is also involved in the biosynthesis of steroids, lipids, and carbohydrates (154). Oxidative protein folding in the ER generates ROS, and in fact, the overall estimate is that nearly 25% of ROS generated in the cell is contributed by the ER. An oxidizing environment needs to be maintained in the ER lumen to introduce the disulfide bonds during protein folding and trafficking (346). Interestingly, ER is impermeable to GSSH; therefore, it relies on its capacity to generate GSSH. The electron transport required to generate GSSH in the ER membrane is regulated by the protein disulfide isomerase (PDI) and ER oxidoreductin 1(ERO1) (36, 312). Under pathological conditions, overactivation of unfolded protein response results in the generation of ROS and is strongly associated with the progression of NASH and cirrhosis (207, 374). Furthermore, ER stress results in the accumulation of calcium in the mitochondria, which promotes exacerbated mitochondrial ROS production leading to disease progression (126, 134). ER is also the home for the enzyme cytochrome 2E1 (CYP2E1), the enzyme responsible for ethanol catabolism. Thus, the ER plays a central role in ethanol-induced ROS production in the hepatocytes (265). Moreover, in HCV infections, viral replication and their gene products induce ROS production from ER (133, 307).

Peroxisomes

Peroxisomes are the oxidation sites of very long-chain and branched-chain fatty acids that cannot directly enter into the mitochondria. The dynamic nature of peroxisomes to enlarge, elongate, and proliferate confers its ability to involve in oxidative and detoxification reactions (205). In other words, peroxisomes majorly serve two functions to protect against liver diseases: (i) by the degradation of very long-chain fatty acids and (ii) disposing of excess ROS. Peroxisomes produce large amounts of H₂O₂ from the continuous oxidation of fatty acids (107, 165, 288). As a protective mechanism, peroxisomes are equipped with high detoxifying enzymes such as catalase, GPx, Mn-SOD, and CuZn-SOD (288, 398). Thus, structural and functional disturbances in peroxisomes of hepatocytes are sufficient to induce spontaneous hepatic steatosis through excessive generation and release of ROS (158).

Lysosomes

Lysosomes are the major nutrient-sensitive organelle involved in autophagy, including the removal of damaged mitochondria through a process called mitophagy (204). Recent studies have demonstrated a reciprocal relationship between ROS and autophagy (219). For example, Atg4, a cysteine protease, was identified as a direct target of ROS (342). Further, starvation concomitantly increases autophagy and mitochondrial ROS production through the inhibition of mTOR pathway (359). This concept was supported by a study wherein nutrient deprivation in hepatocytes results in the accumulation of defective mitochondria and increased oxidative stress (180). Moreover, ROS-mediated mitophagy is known to play a critical role in the first hit (lipid accumulation) and second hit (oxidative stress and inflammation) of NASH pathogenesis (212). The other fascinating role of lysosomes in oxidative stress arises from their ability to accumulate iron, wherein it catalyzes the Fenton reaction with H₂O₂ in various liver disease models (227, 399). Thus, lysosomal disorders associated with several chronic diseases are mainly related to increased generation of mitochondrial ROS.

External Mediators of Oxidative Stress

In addition to intracellular sources, several external factors trigger ROS generation. They include alcohol, drugs, nutrients, toxicants, pollutants, and physical stressors (UV, X-ray, and ionizing radiation). All these factors promote the initiation and progression of various liver-related diseases by altering the redox signaling.

Alcohol

Alcohol metabolism occurs in the liver with the help of enzymes alcohol dehydrogenase and aldehyde dehydrogenase resulting in the generation of one molecule of NADH. During this process, the respiratory activity is increased, resulting in increased oxygen consumption and the generation of greater amounts of ROS. When alcohol consumption is excessive, other enzymes such as NADH-dependent cytochrome C reductase, aldehyde oxidase, and xanthine oxidase drive ROS generation (52, 455). Alcohol also increases ROS generation by noncanonical mechanisms. For example, alcohol induces oxidative stress by increasing intestinal iron absorption leading to iron overload in the liver (157). Several studies have shown that the accumulation of iron in the liver is strongly associated with oxidative stress and the development of several liver-associated disorders (58, 99, 405, 411). Further, alcohol increases cytochrome P2E1 (CYP2E1) activity, an important player in the metabolism of alcohol. The induction of CYP2E1 strongly correlates with the generation of hydroxyethyl radicals and other lipid radicals (109, 276). For example, the exposure of HepG2 cells expressing human CYP2E1 to alcohol results in ROS generation, mitochondrial damage, and cell toxicity (435). Studies have also established that oxidative stress is the primary cause of alcohol-induced hepatocyte toxicity (54, 202). In support of this notion, alcohol-induced hepatocyte injury and cell death are mitigated by the administration of antioxidants (60, 231, 424).

Lipids

In the mammalian system, lipids act not only as the source of energy but also provide the structural components of the cell membranes. Lipids can also act as signaling molecules by forming a permeability barrier of cells and lipid bilayer in subcellular organelles. The fluidity of the membrane is highly dependent on the composition of fatty acids (i.e., degree of saturation: unsaturation fatty acids) in the lipid bilayer (384, 438). Thus, it becomes essential to maintain not only the lipid content but also the ratio of saturated to unsaturated fatty acids. Lipids are more prone to damage from several exogenous stimuli. Most importantly, excessive accumulation of ROS in subcellular organelle could directly damage the lipids (449). The most common forms of the ROS that affect the lipids are hydroxyl radical and hydroperoxyl. ROS damages the lipids through the process of lipid peroxidation, wherein they attack the lipids containing carbon-carbon double bond(s), especially the polyunsaturated fatty acids (PUFAs), which consists of two or more double bonds belonging to omega-3 (n−3) and omega-6 (n−6) fatty acids (315, 448). In addition, a wealth of literature has shown that cholesterol, membrane phospholipids, and glycolipids also target lipid peroxidation (51, 310). Lipid peroxidation generates a variety of lipid hydroperoxides such as MDA, 4-HNE, propanal, and hexanal. Among them, MDA is considered as the most mutagenic product of lipid peroxidation, whereas 4-HNE is the most toxic form. Further, MDA and 4-HNE are used as bioactive markers of lipid peroxidation, which could signal and regulate various transcriptional factors (16, 23).

Diet/Nutrition

The diet has a profound effect on redox biology by regulating the balance between the pro- and antioxidant mechanisms. The major dietary micro- and macronutrients that play a critical role in the production of ROS include proteins, lipids, carbohydrates, vitamins, and minerals (127, 390). For example, excessive consumption of carbohydrates or high-fat diet increases mitochondrial respiration, subsequently producing high levels of superoxides and free radicals. In particular, the consumption of high fructose diet results in lipid peroxidation, cytokine secretion, and ROS production. These diets induce hepatic mitochondrial dysfunction by supplying electrons continuously to the ETC by upregulating the TCA cycle and thereby impair mitochondrial complex IV activity. Thus, a higher release of ROS favors the development and progression of fructose-induced NAFLD (50, 397). Further, Mohanty et al. have shown that increased lipid and protein intake is strongly associated with the ROS generation in polymorphonuclear leukocytes and mononuclear cells (270).

Vitamins and minerals essentially prevent the generation of excess ROS by upregulating the antioxidant enzyme activities. Among them, vitamins E (α-tocopherol), C, and B₁₂ are widely studied for their antioxidant properties (49, 334). For example, vitamin E deficiency reduces antioxidant enzymes such as liver GSH peroxidase, glutathione reductase, and catalase resulting in increased lipid peroxidation (403). Further, vitamin E and C have been shown to improve the clinical symptoms of NAFLD, enhance glucose metabolism, and lower liver injury (94, 291). Similarly, minerals such as selenium, magnesium, manganese, and copper prevent mitochondrial dysfunction and free radical-induced liver damage (429). For example, selenium acts as a cofactor for several enzymes, including glutathione peroxidase and selenoprotein P (402). Several studies have linked mineral deficiency to oxidative stress and increased susceptibility to lipid peroxidation (220, 441, 465).

Aging

Aging is a natural process that involves the loss of tissue and organ function over time. During aging, macromolecules (lipids, DNA, and proteins) undergo various structural and functional changes due to the accumulation of ROS and RNS from endogenous and exogenous sources (229). Although the exact mechanism(s) of ROS-induced aging is unclear, several lines of studies have elucidated a role for ROS in cellular senescence. Oxidative stress induces cellular senescence through multiple mechanisms. For example, the accumulation of ROS and RNS increases the proinflammatory cytokines, chemokines, and other growth factors that are central to the progression of liver diseases (20, 103). ROS also increases the expression of several MMPs such as MMP-1, 2, 7, and 9, which affects the mechanical properties of the extracellular matrix leading to the development of senescence (80, 108, 387). Further, ROS and RNS decrease the expression of forehead box protein (FOXO), sirtuins, and sarco-endoplasmic reticulum Ca²⁺-ATPase activities, which are involved in various pathways associated with age-related diseases (100, 186, 279, 368).

It is also well-established that mitochondrial integrity and function decline with age due to mitochondrial DNA damage. The increased sensitivity of mitochondrial DNA to oxidative stress led to the concept of a “vicious cycle” wherein ROS-induced impairment of mitochondrial function leads to a further increase in oxidant production (308, 362). It is also accepted that old mitochondria show impaired mitochondrial morphology and function due to significant impairment in electron transport( 260). Furthermore, studies with genetically modified animals such as SOD1, SOD2, and p66shc-deficient animals show that mitochondrial dysfunction could trigger premature aging (294, 413, 426). The epigenetic and DNA methylation modifications induced by ROS also play a role in the aging mechanisms (117, 131, 322).

Pathophysiological Sources of ROS

Inflammation

Oxidative stress and inflammation are tightly interrelated and often present simultaneously, making it difficult to discern the causal role of oxidative stress in inflammatory diseases such as NASH. Oxidative stress plays a dual role in tissue damage by triggering innate immune response and infiltration of inflammatory cells (neutrophils, monocytes, and lymphocytes). For example, oxidative stress due to impaired mitophagy triggers an innate immune response by activating the NOD-like receptor protein 3 (NLRP3), a pattern recognition receptor (461). NLRP3 regulates genes involved in cytoprotective and antioxidant protection. A recent study found that the NLRP3 inflammasome accentuated oxidative stress by suppressing the nuclear factor (erythroid-derived 2; Nrf2), a basic leucine zipper (bZIP) transcription factor (149). On the other hand, sustained NRF2 activation protects mice from NASH progression by inhibiting oxidative stress and inflammation (440).

Further, cytokines, chemokines, and nitric oxide released by the activated inflammatory cells induce tissue damage, thereby augment oxidative stress. Thus, a vicious feed-forward regulation of inflammation by oxidative stress and vice versa plays a critical role in the initiation and progression of liver diseases. Therefore, antioxidant and anti-inflammatory therapy is beneficial in the management of liver diseases. Consistently, enhancing the scavenging of ROS by SIRT1 deacetylases (stem cell therapy) decreases oxidative stress and inflammation in NASH (221).

In NASH patients, ROS induces necroptosis of hepatocytes by upregulating IL-1β, IL-8, and TNF-α. It is also possible that the hypomethylated CpG islands and formyl peptides released from the damaged mitochondria stimulate innate immune response (281). For example, glutathionylated peroxiredoxin 2 and thioredoxin released from the activated macrophages induce oxidative stress in a TNFα-dependent manner (335). Sometimes, the release of mitochondrial DNA into the cytosol induces cGAS-Stimulator of Interferon (STING) pathway, further aggravating liver injury (61, 415). In a mouse model of NAFLD, activation of STING in macrophages aggravates hepatic inflammation and fibrosis (420). Exposure of formyl peptides also stimulates the innate immune system in a CXC chemokine receptor 2 (CXCR2)-dependent recruitment of neutrophils in acute liver injury models (218).

Insulin resistance

Insulin regulates glucose homeostasis by promoting peripheral glucose uptake and utilization in various tissues. When insulin fails to promote glucose metabolism, a condition known as insulin resistance (IR), blood glucose is chronically elevated. Insulin secretion from pancreatic islets is increased as compensation to IR (173). Thus, chronic hyperinsulinemia is the hallmark of IR. However, sustained hyperglycemia eventually leads to failure of the pancreatic islets leading to type 2 diabetes. The polygenic nature of IR makes it challenging to elucidate its origin.

Nonetheless, over three decades of research indicate ROS as an integral part of insulin signaling. High levels of ROS are associated with IR. For example, the thiol-dependent enzymes, namely protein tyrosine phosphatase (PTP) regulated by the cellular redox state, play a crucial role in inhibiting insulin signaling (409). Moreover, insulin increases H₂O₂ generation by inducing NOX4, and low levels of H₂O₂ are essential for insulin-mediated translocation of glucose transporter 4 (Glut4) via PI3K/PLC (72). While physiological levels of ROS promote insulin sensitivity by promoting Glut4 translocation, an overload of ROS suppresses Glut1 translocation to the plasma membrane by downregulating AKT phosphorylation (101).

Oxidative stress also reduces insulin packaging and impairs glucose-stimulated insulin secretion (101, 113). Furthermore, oxidative stress inhibits β-cell differentiation via its effect on transcription factors such as pancreatic and duodenal homeobox 1 (Pdx-1), homeobox protein Nkx6.1, neurogenin-3 (Ngn.3), FOXO, and MafA (121). Higher concentrations of free radicals inhibit insulin gene expression at the transcriptional level by repressing Pdx-1 (insulin promoter factor 1) and MafA (a transcription factor) (143). However, a recent study in adipocytes demonstrated that ROS-mediated transcriptional response is insufficient to cause IR. Nonetheless, overexpression of PRDX3 and MnSOD decreased blood glucose levels and improved insulin sensitivity in HFD-induced obesity (59). The contribution of mitochondrial ROS to IR is further evidenced by a rapid reversal of IR by inhibitors of mitochondrial respiratory complexes and uncouplers of oxidative phosphorylation (7). Patients with NASH present with high levels of nonesterified fatty acids sufficient to induce oxidative stress and IR through activation of JNK/p38MAPK pathway (116). These results suggest that approaches that prevent ROS generation provide excellent protection against IR.

Oxidative Stress and Cellular Dysfunction

Mitochondrial dysfunction

Mitochondrial dysfunction could arise from impaired mitochondrial biogenesis and clearance of damaged mitochondria (273, 274). Several transcription factors are essential for mitochondrial biogenesis and function, including PGC-1α and TFAM that are decreased in fatty liver disease (3, 283). Moreover, the mitochondria appear swollen with disrupted architecture in NASH patients (337). In addition to aberrant mitochondrial structures, the expression and activity of mitochondrial respiration enzymes are decreased in both alcoholic and nonalcoholic patients. In rat models, alcohol-containing diets severely impair the expression and activity of mitochondrial respiratory complexes (almost all I, III, IV, and V) (71, 118). Although the efficacy is affected by the route of administration (intragastric ingestion vs. oral feeding), the ultimate effect of alcohol on mitochondrial dysfunction does not vary significantly (138). Similarly, high-fat diet-fed mice display reduced cytochrome b-complex, ATP complex subunits, and citrate synthase enzyme expression and activity (119). Moreover, the mitochondrial defects attribute to the decreased half-life of mitochondrial respiratory proteins. Due to reduced mitochondrial ATP production, these animals exhibit impaired mitophagy, enhancing ROS generation.

The mitochondrial respiratory chain produces a substantial amount of ROS as it consumes molecular oxygen during oxidative phosphorylation. Efficient scavenging of ROS in the NASH models improves mitochondrial integrity and ameliorates NASH. A recent study demonstrated that sirtuin (SIRT) 3 improves mitochondrial integrity and protects hepatocytes from oxidative injury by enhancing ROS scavenging (232). Moreover, overexpression of SIRT1 or SIRT3 increased antioxidants in chronic hepatocyte injury models (Figure 2) (232).

Figure 2.

Putative sources of ROS and their contribution to pathologies of liver diseases. Under physiological conditions, organelle systems generate steady-state ROS levels that promote redox signaling. On the other hand, under pathological conditions, excess ROS produced various danger signals such as DAMPS and ATP from the mitochondria and unfolded/misfolded proteins from the ER and oncogenes that promote mitochondrial dysfunction, ER stress, and DNA damage. All these factors lead to the development of liver pathologies such as inflammation, fibrosis, and even cancer.

Endoplasmic reticulum (ER) stress

ER is the site of protein synthesis, folding, and maturation. Misfolded or unfolded proteins undergo either refolding or degradation. Aberrations in protein folding or clearance of misfolded proteins induce an unfolded protein response (UPR) mediated by protein kinase RNA-like ER kinase (PERK), activating transcription factors (ATFs), and inositol-requiring signaling protein 1 (IRE1). PERK temporarily halts the global protein translation by phosphorylating the eukaryotic translation initiation factor 2a (eIF2a). Activated eIF2a phosphorylates nuclear factor erythroid 2-related factor 2 (Nrf2), leading to the dissociation of the Nrf2 from Kelch-like ECH-associated protein 1 (Keap1) complex, which eventually induces the expression of antioxidant enzymes, including heme oxygenase-1 (HO-1) (41, 209). ATF4 induces CCAAT/enhancer-binding protein homologous protein (CHOP) to activate an antioxidant response. IRE1 and ATF6 co-operate to upregulate several UPR target genes, heat-shock protein 70 (Hsp70), and other chaperones to restore the ER redox state. ATF6 also induces X-box binding protein-1 (XBP1), which stimulates an inflammatory response through NFκB and C-Jun N-terminal kinase (JNK) signaling pathways. Thus, UPR helps the cells to recover from ER stress and restore ER equilibrium. If UPR persists, IRE1 increases tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2)-induced JNK activation, which then triggers cell death by inducing the expression of proapoptotic BH3-only proteins including p53 upregulated modulator of apoptosis (PUMA), Bcl-2 like protein (BIM), and BH3-interacting-domain death agonist (BID).

Similar to mitochondria, the redox state is critical for the metabolic homeostasis of ER. In NASH patients and model organisms, ER stress occurs due to oxidative damage (445). Chronic ER stress increases ROS production by upregulating CHOP. HO-1 induced by Nrf2 helps to mitigate oxidative stress. Figure 2 with genetic deletion of Nrf2 mice are highly susceptible to oxidative stress and show aggravated NASH phenotype (264).

Overexpression of HO-1 protects against oxidative stress due to increased expression of antioxidant chaperones, enzymes, and anti-inflammatory cytokines such as IL-22 (57). Paradoxically, PERK inactivates Nrf2 signaling during ER stress; however, its physiological significance is unclear (373). In alcohol-induced liver injury, ER stress is strongly associated with oxidative stress. Alcohol feeding increases the expression of ER stress-associated proteins such as Hsp70, binding immunoglobulin protein (BiP), and Grp 94, CHOP as well as caspase 12 as early as 2-weeks (139). Exaggerated hepatocyte apoptosis with no difference in hepatic steatosis in alcohol-fed CHOP null mice suggests that ER stress plays a significant role in injury-mediated apoptosis (162). Further, hepatic overexpression of the ER chaperones, such as ORP150/HYOU1 and GRP78/BiP improved insulin sensitivity and hepatic steatosis (451). This suggests that ER stress response plays a vital role in maintaining hepatic homeostasis during metabolic insults. Interestingly, hepatocyte-specific IRE1 knockout mice exhibit steatosis when exposed to tunicamycin, while deletion of IRE1 in the hepatic stellate cells attenuates HCC progression (70, 460). Thus, a considerable difference in the effect of ER stress and UPR response between various cell types in the liver poses a severe limitation in therapeutic targeting ER stress and UPR pathways for the treatment of NASH.

DNA damage

Several endogenous and exogenous genotoxic insults induce spontaneous mutations in the DNA. Therefore, precise DNA repair mechanisms are critical in maintaining genome integrity. Genomic instabilities are the characteristic feature of HCC due to erroneous DNA repair mechanisms. ROS, UV light, radiation, and environmental mutagen are the major DNA damage inducers. Oxidative stress and ROS co-operate with the mutagens to drive the progression of NASH to HCC. DNA damage activates p53-mediated cell apoptosis (81, 376). A recent review has elaborated on how dysregulation of p53 function leads to metabolic disorders, including NASH and HCC (196). However, the mechanisms involved in p53-mediated metabolic dysregulation are not fully understood. Studies show that p53-binding protein 1 (p53BP1), a DNA damage response protein, forms nuclear foci in response to double-strand DNA breaks. The number of p53BP1 foci increases in NASH patients implying significant DNA double-strand breaks (4). ROS and p53 have a versatile partnership. ROS can act upstream and downstream to p53 to regulate cellular processes. ROS modifies DNA by forming stable covalent bonds leading to base modifications, including thymidine glycol, 5-hydroxylmethyluracil, and 8-OHdG. Thus, hepatitis viral infections that generate 8-OHdG often lead to liver cancer. An elevated level of 8-OHdG is used as a novel prognostic marker in HCC and is often associated with poor patient survival (225). 8-OHdG also serves as a marker in hemochromatosis, Wilson’s disease, chronic hepatitis, hepatoblastoma, and primary biliary cirrhosis (73). ROS also induces double-stranded DNA breaks, thereby increasing mutations or chromosomal aberrations resulting in tumorigenesis (Figure 2) (22).

Advanced glycation end products (AGEs)

In obesity and NASH, AGEs levels are significantly elevated (63). AGEs that include methylglyoxal, glyoxal, and HNE, reduce sarcoplasmic (ER) reticulum Ca2+ ATPase (SERCA) activity (428). AGE products also possess highly reactive moieties and interact with a wide range of biomolecules, including proteins. AGE could also signal through several receptors called receptors for AGE (RAGE), whose identity is revealed in recent investigations. For example, galectin 3a and Oligosaccharyltransferase-48 (OST48) are characterized as AGE binding proteins. OST48 is an integral part of the ER membrane that acts as a clearance receptor for AGE. Overexpression of OST48 is associated with a high risk of liver fibrosis and liver failure (468). RAGE/TLR4 signaling also induces ER stress-mediated liver fibrosis. For example, a recent study shows that high mobility group protein (HMGB1) released from the injured hepatocyte induces ER stress in hepatic stellate cells via RAGE/TLR4 signaling leading to liver fibrosis (146). The significance of these mechanisms in NASH or ASH patient needs further investigation. Uncoupling protein (UCP) 2, an important regulator of ER stress in a wide range of tissues, intersect with AGE/RAGE signaling. For instance, UCP2 deficiency decreases hepatic glyoxalase-1 resulting in a reciprocal increase in methylglyoxal associated with high mortality in young mice (194).

Molecular Master Regulators of Oxidative Stress

The oxidative stress is regulated by numerous transcription factors, which helps to minimize tissue damage. These transcription factors either promote or suppress oxidative stress depending on their expression levels.

Nuclear factor erythroid 2-related factor 2 (Nrf2)

The transcription factor, Nrf2 regulates numerous antioxidant genes by binding to the antioxidant response elements (ARE) on the target gene promoters. The transcriptional activity of Nrf2 is regulated by p62 and Keap1. Oxidative stress increases the binding of NRF2 to p62, thereby limiting the interaction with Keap1, a cullin-3-type ubiquitin ligase that degrades Nrf2 (190). Tank binding kinase 1 (TBK1) further acts as an upstream regulator of Nrf2 by stabilizing p62(67). Recent studies demonstrate that noncoding RNAs and micro-RNAs are also involved in stabilizing Nrf2 during oxidative stress (425).

Nrf2 expression is a cardinal feature of acute or chronic oxidative stress. Targets of Nrf2 include HO-1, SOD1, catalase, and glutathione S-transferase (GST). Therefore, Nrf2 could potentially control the oxidative stress response as it targets an array of genes that possess stress-responsive elements similar to GST. Mice with hepatocyte-specific Keap1 deletion, display enhanced Nrf2 levels leading to elevated expression of enzymes involved in glutathione synthesis, including GST-peroxidase and glutamate-cysteine ligase (233). Initially, Nrf2 expression is thought to be protective against almost all liver injury models ranging from acute hepatitis to cholangitis and NASH to liver cancer. However, a growing body of evidence suggests that its expression is vital in the progression of liver diseases (183, 392). Although the hepatocyte-specific Keap1 knockout mice enhanced Nrf2 expression and showed a protective phenotype from cadmium-induced acute liver injury (233), Nrf2 knockout mice are highly susceptible to ethanol-induced liver fibrosis and steatosis (199). Wang et al. found that genetic deletion of Nrf2 improved liver phenotype by suppressing the expression of very-low-density lipoprotein receptors in alcohol-fed mice(422). The latter observation was further supported by clinical studies showing that Nrf2 activation increases with the incidence of HCC (156). Moreover, Nrf2 knockout mice display severe NASH symptoms when fed on a methionine choline-deficient diet (199). Nevertheless, these observations indicate that NRF2 could be a sensitive player to define the state of the liver phenotype in various experimental settings of liver diseases (Figure 3).

Figure 3.

Master regulators of oxidative stress response leading to inflammation and insulin resistance: Oxidative stress causes severe DNA damage sensed by p53 accumulation. Oxidative stress exerts its effects through the ER and mitochondrial stress (complete events mentioned in the review). NRF2 is the cumulative stress response marker induced by the ER and mitochondrial stress. ER stress-induced inflammation is mediated by the recently discovered HMGB1 transcription factor, which further intersects with RAGE signaling. C-GAS/STING pathway is the intracellular DNA-sensing pathway activated by the mitochondrial DNA leaked into the cytosol. NLRP3 is either directly activated by TRPM2 or could be upstream of C-GAS/STING pathway. Thus, NLRP3 co-operates with C-GAS/STING pathway to promote inflammation and insulin resistance.

Nuclear factor-kappa B (NF-κB)

Canonical and noncanonical pathways regulate the activation of NF-κB. In the canonical mechanism, phosphorylation of inhibitory kappa Bα (IκBα) by IκB kinase (IKK) induces degradation of the IκBα from NF-κB:IκBα complex, resulting in the nuclear translocation of NF-κB. In contrast, the noncanonical mechanism involves NF-κB-inducing kinase (NIK)-mediated processing of NF-κB2 p100 into NF-κB2 p52, which complex with RelB to induce the expression of NF-κB target genes (177, 363).

NF-κB plays a dual role in liver injury, where its deletion in hepatocytes is deleterious but protective against liver injury in other cell types. A moderate activation of NF-κB signaling in hepatocytes is protective in states of low-grade lipogenesis in the absence of inflammation (240, 380). Therefore, it appears that NF-κB-induced by oxidative stress helps to prevent hepatocyte death and promote a compensatory proliferation of hepatocytes. In contrast, the deletion of IKK2α in unstressed hepatocytes does not affect oxidative stress markers (237). Thus, NF-κB serves as the master regulator of inflammation and cell death. The role of NF-κB in inducing cell death appears to be dependent on its ability to activate its downstream target JNK/p38 MAPK (discussed below).

NF-κB activation in acute or chronic liver disease is partly mediated by the inflammatory response (442). NF-κB transcriptionally regulates several antioxidants, and pro-oxidant gene expression in a spatial-temporal manner, and their details are reviewed elsewhere (275). ROS stimulates NF-κB activation in the cytoplasm but inhibits NF-κB DNA binding activity in the nucleus (164). ROS could also regulate DNA binding activity modifying specific residues in NF-κB (184).

Forkhead box protein O (FOXO)

Forkhead box protein O (FOXO) transcription factors are highly conserved in higher organisms with an essential role in hepatic glucose and lipid metabolism. There are four FOXO proteins expressed in mammals, among which FOXO1, 3, and 4 express ubiquitously, while FOXO6 expression is specific to neuronal cells. In mouse models, the deletion of FOXO1 and 3 induced spontaneous hepatic steatosis and decreased glucose metabolism (459). Mouse with triple knockouts for FOXO1, 3, and 4 also show similar phenotypes in hepatic glucose and lipid metabolism (393). FOXO-dependent suppression of fatty acid synthase and nicotinamide phosphoribosyl transferase (NMPT) regulates hepatic steatosis. Emerging evidence shows a crucial role for FOXO transcription factors in oxidative stress. For example, FOXO can control oxidative stress via NAD-dependent deacetylase SIRT1, as NMPT is essential for the synthesis of NAD (393). A recent study showed that FOXO1 expression is associated with fatty liver, whereas pharmacological inhibition of FOXO1 improved hepatic steatosis (89). This contradicting data is due to high levels of ER stress and hepatocyte necroptosis in these models (89). An independent study using triple knockout mice (FOXO1, 3, and 4) further established the significance of FOXO signaling in suppressing oxidative stress (10). Thus, the controversial data suggest a complex yet significant role of FOXOs in regulating redox biology in the liver.

JNK

c-JUN N-terminal kinases (JNK) and p38 are the principal members of MAPK family, which phosphorylate and activate several transcription factors, including c-Jun, ATF-2, and TCF/Elk-1 (87). Vertebrates express three isoforms of JNKs, where JNK1 and 2 are ubiquitously expressed, and JNK3 is expressed only in the heart, brain, and testicles. Although JNK1/2 are important mediators of liver injury, there appears to be a specific role for each JNK isoform in liver disease depending on their binding partners and downstream signaling mediators (167, 187, 371). For example, JNK promotes TNF-α-induced cell death when NF-κB is not active in hepatocytes (333). JNK then phosphorylates and activates its downstream targets such as c-JUN, JUN-B, and JUN-D. The targets of JNK pathway involve apoptosis-related genes such as BCL-2 and Bax (433). A recent study shows that JNK activation induces Bim in hepatocytes, enhancing oxidative stress leading to IR and steatosis (230). Thus, Bim knockout mice exhibit improved mitochondrial function, reduced oxidative stress, and are protected against diet-induced obesity and IR (230).

MAP3K, MLK3, and ASK1 are implicated in the regulation of hepatic JNK signaling in NASH models. Mice with the knockout of MLK3 alone or double knockout for MLK3 and MLK2 are protected from diet-induced obesity due to attenuated JNK activation (112, 170). ASK1 inhibitors (GS-4997, GS-444217) are currently on phase 2 clinical trials for liver fibrosis in humans (235). Whether ASK1 inhibitors ameliorate hepatic fibrosis through inhibition of JNK-oxidant signaling needs further investigation (235).

Sirtuins

Sirtuins are a class of NAD+-dependent deacetylases, which confers protection against a wide range of metabolic disorders, including obesity and age-associated diseases. Accumulating evidence suggests that sirtuins regulate autophagy in response to physiological and environmental stresses (8, 90). Humans express seven sirtuins that include SIRT1 to SIRT7. Spatiotemporal expression of sirtuins mediates their specific role from gene expression to cell cycle regulation. Among the sirtuins, SIRT3, SIRT4, and SIRT5 are exclusively localized to the mitochondria and involve in posttranslational modifications of mitochondrial proteins. In response to oxidative stress, mitochondrial sirtuins deacetylate a network of proteins involved in the TCA cycle and oxidative phosphorylation complexes (56).

SIRT1 expression protects against liver injury by maintaining mitochondria health. Insults such as hypoxia, ischemia, and pro-oxidants that are highly prevalent in fatty liver deplete SIRT1 levels and impair autophagy (407). Thus, the ectopic expression of SIRT1 restores autophagy dramatically. Surprisingly, SIRT1 interacts with mitofusin-2, a mitochondrial fusion protein, suggesting that SIRT1 is a linker between the nucleus and mitochondria under stress conditions (34). Moreover, SIRT1 overexpression restores autophagy in MFN2 deficient cells indicating that SIRT1 plays a role in alleviating mitochondrial dysfunction, thereby decreasing oxidative stress. Small molecules for SIRT1 such as SRT2104 and SRT1720 show a profound effect on lipid peroxidation and carbonylation of proteins in the liver (43). SIRT1 also regulates p53 activity by acetylation and stabilization of p53 in damaged or stress-induced cells (155). SIRT1 is also an essential regulator of insulin signaling and is downregulated in conditions such as obesity and T2DM. Inhibition of SIRT1 recapitulates IR phenotype, while overexpression of SIRT1 improved insulin sensitivity, mainly via repressing PTP1B (381). These studies indicate that SIRT1 has an essential role in depleting oxidative stress developed during disease progression. However, studies with liver and hepatocyte-specific knockdown of SIRT3 show a negative role for SIRT3 on the regulation of autophagy, lipotoxicity, and susceptibility to chronic alcohol consumption (223, 244).

NOD-like receptor protein 3 (NLRP3)

The inflammasome nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) is often investigated in liver diseases. NLRP3 belongs to the innate immune response complex consisting of NLRP3, pathogen/danger associated recognition receptors, apoptosis-associated speck-like protein (ASC), and pro-caspase-1. NLRP3 activation converts pro-caspase-1 into active caspase-1 leading to the induction of mediators of local inflammation and pyroptosis such as IL-1β, IL-17, and IL-18 (198, 238). Studies with NLRP-3 knockout mice show that alcohol-induced liver inflammation and fibrosis were attenuated in the absence of NLRP3 (86, 278). Global and myeloid cell-specific Nlrp3 overexpression mice display hyperactive NLRP3, which induces inflammation, hepatocyte pyroptosis, and fibrosis in the liver (434). It has been demonstrated that mROS is required for the activation of NLRP3 and TRPM2, a calcium channel required for this pathway (466). However, it warrants future studies to investigate the role of mROS in NLRP3 signaling and its implications in NASH. Hepatitis-B virus toxin induces NLPR3-mediated inflammation and hepatocyte pyroptosis in ROS (H₂O₂)-dependent manner (439). In diabetic conditions, increased NLRP3 activation from elevated oxidative stress induces hepatocyte pyroptosis during ischemic reperfusion injury (361). Studies using NLRP3, ASC, and caspase 1 deletions also suggest the role of NLRP3 inflammasome in the pathogenesis of NAFLD in obesity (148).

Oxidative Stress and Pathophysiology of Liver Disease

Oxidative stress and NAFLD/NASH

The presence of cytoplasmic triglyceride lipid droplets in more than 5% of hepatocytes or triglyceride content accounting for more than 5% of the liver weight is defined as hepatic steatosis/fatty liver. Hepatic steatosis is classified as microvesicular (smaller lipid droplets) and macrovesicular (large lipid droplets). Macrovesicular steatosis results from an imbalance in triglyceride synthesis, whereas microvesicular steatosis develops due to mitochondrial dysfunction (defective fatty acid oxidation) (206, 372). Simple hepatic steatosis is often self-limiting; however, it can progress to nonalcoholic steatohepatitis (NASH) characterized by hepatocyte injury (hepatocyte ballooning and cell death) and infiltration of immune cells. NASH is followed by induction of liver fibrosis leading to cirrhosis culminating in hepatocellular carcinoma (HCC) (11, 258). The prevalence of NAFLD is estimated to be around 20% to 30% in Western countries and 5% to 18% in Asia (31, 341).

The pathogenesis of NAFLD is explained by “multiple parallel-hit hypothesis” wherein the “first hit” involves the accumulation of free fatty acids (FFAs) leading to lipotoxicity and oxidative stress denoted as “second hit.” The accumulation of FFAs in the liver alters mitochondrial function and increases ROS generation (28, 47). Therefore, the development and progression of NAFLD are strongly associated with the continuous generation of ROS/RNS and oxidative damage to the organelles. This is further accentuated by dysfunction in the counteractive antioxidant mechanisms. The mitochondrial abnormalities range from lipid-related changes to mitochondrial DNA damage to sirtuin imbalance (214, 301, 383). For example, mitochondrial DNA damage and alterations in genes encoding mitochondrial proteins result in a rapid increase in oxidative stress and, thereby, trigger the progression of simple steatosis to fibrosis (251). The mitochondrial dysfunction mostly alters fatty acid oxidation leading to the accumulation of fat in the hepatocytes, exacerbating steatosis (88). Oxidative stress also affects lipid metabolism in hepatocytes. For example, Seo et al. demonstrated that treatment of Huh7 and AML12 cell with H₂O₂ increased mRNA expression levels of genes involved in lipid (lipin) and cholesterol metabolism (SREBP-2) and further, the lipid overload in the hepatocytes increases inner mitochondrial membrane permeability, loss of membrane potential, and ATP synthesis capacity (355). Further, hepatocyte-derived ROS releases highly unstable reactive aldehydic derivatives that impair mitochondrial respiration and drive disease progression (78).

NAFLD is strongly associated with an increase in nonesterified fatty acids (NEFA) and free cholesterol (FC). Further, the NEFA and FC undergo oxidation with the help of lipoxygenases, cyclogeneses and cytochromes P450 family and, thereby, produce lipid peroxides such as malondialdehyde (MDA) and 4-hydroxynonenal (HNE) (30). Several studies have a positive correlation between lipid peroxidation and NAFLD. For example, plasma levels 8-isoprostane, a product of lipid peroxidation was significantly increased in patients of NAFLD (191). Recent studies show that lipid peroxidation is increased in pediatric NAFLD; however, no change with liver CYP 2E1 expression was observed (29). In addition, lipid peroxides such as MDA and 4-HNE generated in NAFLD act as biomarkers of NAFLD and NASH (450). Further, 4-HNE activates and increases ROS production in HSCs (456). Thus, alteration of lipid metabolism during NAFLD leads to accumulation of toxic lipids and oxidative stress and helps to the progression to NASH (Figure 4).

Figure 4.

The mechanisms of ROS-induced oxidative stress in the pathogenesis of NAFLD. ROS can oxidize stored lipids through the process of lipid peroxidation, releasing lipid peroxidation reactive aldehydes, which result in lipotoxicity. Lipotoxicity involves in the production of several hepatic inflammatory mediators. ROS also increases the production of danger signals and mtDNA stimulating the innate immune system and inflammatory cytokines to promote liver inflammation. ROS-associated lipid peroxidation and cytokines contribute to the inflammatory cell infiltrate. On the other hand, ROS-mediated oxidative stress is a feature of liver fibrosis that activates HSCs by releasing several profibrotic stimuli and growth factors such as TGF-β, leptin, AGEs, and PDGF. Further, ROS induces DNA damage, resulting in cancer cell transformation.

Oxidative stress and liver inflammation

The accumulation of toxic lipids and lipid peroxides in the liver is one of the common manifestations of inflammation in both ALD and NAFLD/NASH. Inflammation is the physiological responses to tissue injury and infection represented by various inflammatory cells, including cytokines and other lipid mediators such as eicosanoids. Numerous studies have shown that the persistence of inflammatory cells and inflammatory mediators in the liver cells play a significant role in the pathogenies of ALD and NAFLD (345, 394). It is believed that liver inflammatory cells rapidly recruit and activate liver macrophages, neutrophils, and other immune cells that promote hepatic injury and thereby NASH progression (114). Further, liver inflammation is triggered by extrahepatic tissues such as the adipose tissue and the gut by releasing various lipid mediators and innate immune response mediators (68).

Most importantly, the common causative factor for the sustained activation of inflammatory cells during the NAFLD/NASH is oxidative stress. The lipid peroxide-induced oxidative stress plays a critical role in the NASH progression through inflammatory signaling. For instance, inflammation and oxidative stress are inter-linked with several liver diseases (69, 323). Under physiological levels, ROS helps to kill the pathogens and modulate signaling events through redox regulation (298). However, during hepatocyte injury, ROS from leukocytes triggers the release of damage-associated molecular patterns (DAMPs), including heat shock proteins, DNA, and RNA (19, 193). Studies have shown that DAMPs and their associated complexes activate the liver resident Kupffer cells and neutrophils via toll-like receptors (TLRs) (Figure 4) (261, 306). For example, TLR4 activates the superoxide-producing enzymes such as NADPH oxidase and MPO in the neutrophils and thereby promotes inflammation (300). Further, MPO is also shown to be involved in the production of hypochlorous acid and contributes to the formation of chlorotyrosine protein and oxidative stress protein adducts (35). Thus, all these changes result in increased production of ROS in various liver cells and help in the NASH progression.

The oxidative stress or reactive species in NAFLD/NASH also involves in the activation of various inflammatory mediators such as TNF-α, IL-1, IL-6, IL-18, and MCP-1 and some of the inflammatory pathways and among them, the most common is NF-κB activation. The ROS is known to activate the NF-κB through several inflammatory cytokines and oxidized lipids and also LPS (124). For example, H₂O₂ is shown to activate the NF-κB even at micromolar concentrations in human neutrophils and is further rescued by treatment with antioxidant N-acetylcysteine (344). The ROS-induced NF-κB activation takes place in various liver cells in NASH or injury models influencing the survival of hepatocytes, Kupffer cells, and HSCs (171). Thus, given its broad roles, NF-κB acts as the central player in various chronic liver diseases.

Oxidative stress and liver fibrosis/cirrhosis

Liver fibrosis is a complex phenomenon wherein continuous production and accumulation of extracellular matrix (ECM) in the liver occurs due to repeated injury and inflammation. Hepatic steatosis, inflammation, alcohol consumption, viral hepatitis, cholestasis, and iron overload are some of the major causative factors of liver fibrosis (25, 192, 396). The generation of ROS in the liver plays a critical role in the initiation and progression of fibrogenesis through its effect on hepatocytes, Kupffer cells, and HSCs (179, 239). The oxidative products and lipid peroxidation augment the production and release of other pro-fibrogenic factors such as inflammatory cytokines (17, 245). Mechanistically, ROS-driven hepatocyte injury increases the secretion of several pro-fibrotic mediators such as TNF-α and transforming growth factor (TGF-β), which further aggravate the inflammatory and fibrotic responses (Figure 4) (234). Oxidative stress in hepatocytes could also increase fibrogenesis through indirect mechanisms via suppression of antioxidant enzymes (GSH) in a NF-κB-dependent manner (236). In addition to the hepatocytes, Kupffer cells and resident macrophages produce a significant amount of ROS upon activation by TGF-β (150, 151). A study has shown that the treatment of macrophages with 4-HNE results in TGF-β-mediated myofibroblast activation and, thereby, fibrosis (213). Further, activation of Kupffer cells with profibrogenic toxins such as iron, copper, and dichlorobenzene generates abundant oxidative stress in the liver (213, 336). Hydrogen peroxide produced by the Kupffer cells increases collagen type 1 from HSCs (120). Similarly, HSCs co-cultured with stimulated neutrophils show a significant increase in the procollagen α1 expression due to the activation of HSCs from ROS released from the neutrophils (467). HSCs themselves express Nox1 and 2 involved in liver fibrosis in association with TLRs (200, 228). Thus, oxidative stress drives liver fibrosis through a complex mechanism that involves the activation of HSCs in the liver.

Oxidative stress and liver cancer

Cirrhosis often progresses to hepatocellular carcinoma (HCC), the leading cause of cancer-related deaths worldwide. Hepatitis B virus, hepatitis C virus, and metabolic diseases are some of the risk factors associated with the development of HCC (21, 395, 452). Oxidative stress plays a pivotal role in every stage of HCC (302, 357, 423). For example, in NASH, inflammation induced-ROS and RNS cause DNA damage and significantly impair the DNA repair mechanisms resulting in mutations leading to HCC (Figure 4) (81, 446). Recent studies have shown that oxidative stress directly regulates cancer cell proliferation, survival, invasion, and/or metastasis of HCC (2, 145, 427).

The role of oxidative stress in HCC was demonstrated with the decreased levels of antioxidant enzymes, including the SODs, glutathione reductase, and glutathione peroxidase. For example, under chronic HCV-induced oxidative stress, the glutathione levels were significantly reduced, and the ratio between the oxidized and reduced forms of glutathione was increased (64, 385). A recent study has shown that thioredoxin reductase and glutathione reductase-null mice are more susceptible to chemical-induced liver cancer through elevated DNA damage (262). Oxidative stress also represses the Nrf2-Keap1 complex and increases the expression of thioredoxin reductase and glutathione reductase above basal levels (386). Further, oxidative stress also enhances Nrf2 and 8-OHdG levels in HCC cell lines, and these effects could be rescued through antioxidant mechanisms (255). Oxidative stress also dysregulates autophagy mechanisms and thereby, activates Nrf2 leading to the proliferation and survival of HCC (24, 190). Although the Nrf2 is considered as an antioxidant, persistent activation of oxidative stress-induced Nrf2 is one of the critical mechanisms that control the development of HCC.

HCV infections also increase the expression of the NADPH oxidase family, thereby causing DNA damage and tissue remodeling (75, 96, 436). The interaction of core proteins of HCV with the inner mitochondrial enzymes results in ROS generation through the oxidation of glutathione and reduction of NADPH content in the liver (226, 419). The HBV-mediated ROS also alters DNA methylation and suppresses SOCS3 expression, which results in the proliferation of the cancer cells (454). The other important environmental factor associated with increased ROS production in the HCC patients is iron-related Fenton reactions. Several studies have demonstrated a strong link between iron toxicity and HCC via ROS generation (15, 125, 252).

Oxidative Stress-biomarkers

Estimation of serum alanine transaminase (ALT) and aspartate transaminase (AST) levels is the gold standard to evaluate liver injury. However, AST levels increase in conditions such as muscle injury, celiac disease, and even pregnancy. Moreover, 20% of patients with cirrhosis may have normal ALT levels. Therefore, several studies aimed at identifying a reliable biomarker for liver diseases. The estimation of oxidative stress and antioxidants have a significant impact on the clinical management of liver injuries. Direct measurement of ROS is highly variable depending on their species, origin, and hyperreactivity. Therefore, instead of considering actual free radical species, considering their reactive products such as lipids, DNA, amino acids, and glycated proteins could yield a better and reliable estimation of ROS in redox state (286). The by-products of ROS reaction with lipids are MDA, 4-HNE, and 8-isoprostane. Nucleic acid-derived oxidative products are 8-hydroxy-2^′-deoxyguanosine (8-OH-2dG) and 8-hydroxyguanine (8-OH-G). Proteins or amino acid-derived oxidative products are hydroxyproline, 3-nitrotyrosine, and 2-oxohistidine. AGE products also serve as a valuable biomarker of oxidative stress. Different methodologies estimate these markers depending on the disease context. For example, thiobarbituric acid reactive substances (TBARS), MDA, and 4-HNE are commonly used to assess lipid peroxidation in the serum of NASH patients. Among these, MDA is the most indicative of oxidative stress (111). Oxidative stress markers can also be identified in the liver or serum by simple ELISA and fluorescence-based detection methods. Mass spectrometry could be the most powerful methodology to identify the species of ROS (45). The determination of ROS levels combined with the standard liver function tests will be highly beneficial in predicting the clinical outcome of liver diseases (13).

Oxidative Stress as a Therapeutic Approach

The generation or maintenance of a minimal amount of ROS is essential for proper liver function. It is clear from the literature that antioxidants play a significant role in balancing ROS levels. Antioxidants have a high affinity for reducing the reactivity of ROS-generated free radicals. The most commonly used antioxidants include curcumin, resveratrol, coffee, flavonoids, and silymarin. These antioxidants reduce the risk of liver injury by activating various signaling cascades (49, 247, 248, 334). For example, plant-derived flavonoids protect the liver from ROS-mediated damages such as inflammation, liver fibrosis, and cancer (224, 463). One of the most studied flavonoids as hepatoprotectant is silymarin, a complex of seven flavonolignans present in the milk of thistle extracts (98, 123). Similarly, the antioxidant potential of flavonolignans silybin is tested in a spectrum of NAFLD, including liver inflammation, fibrosis, and cirrhosis (97). Besides, silybin also improves mitochondrial function and thereby attenuates the progression of NAFLD to end-stage liver disease (356). In addition, the polyphenolic compound curcumin ameliorates oxidative stress in the liver by increasing the expression of Nrf2 and HO-1 and also various antioxidants such as glutamate-cysteine ligase, activating transcription factor (ATF), peroxiredoxin 3, SOD, and catalase. (77, 331, 443). Resveratrol, also a polyphenolic compound that has been shown to improve mitochondrial function by increasing AMPK and SIRT1 activity in the liver (147, 401). In comparison, quercetin reduces the risk of progression of liver fibrosis by attenuating the expression of TGF-β, CTGF, and collagen-1α (321, 444). Most importantly, vitamins, including vitamin E and C and superoxide scavengers such as N-acetylcysteine (NAC) and tempol are well known for their antioxidant properties. For example, vitamin E reduces the incidence of NAFLD and its progressive forms by limiting lipid peroxidation, inflammation, and hepatic fibrosis (159, 305, 316, 339). Recently, a study has shown that superoxide dismutase mimetics, NAC, and tempol attenuated the development of liver pathologies such as cell necrosis, fibrosis, and ER stress through suppressing mTORC1 signaling (66). Currently, vitamin E, D, C, and NAC are very effective in treating chronic liver cancer patients who do not respond well with interferon and chemotherapy-based treatments (85, 178, 242, 318). Thus, targeting oxidative stress in NAFLD patients provides immediate and effective therapy.

Conclusion

In modern redox biology and medicine, ROS signaling is one of the comprehensive and unified central homeostatic mechanisms at molecular, organellar, cellular, and tissue level. Several studies have emphasized its significance in health and diseases. Since the liver acts as the hub of various metabolic and detoxification processes, all most all the liver cells are involved in redox homeostasis. Any perturbation in hepatic redox homeostasis dysregulates the fundamental cellular processes such as insulin signaling, mitophagy, cell proliferation, and hypoxic signaling leading to the initiation and progression of liver diseases. Especially in NASH, oxidative stress plays a critical role in the pathogenesis, independent of heterogeneous cellular damage. Furthermore, oxidative stress is the hallmark of liver cirrhosis/cancer. However, targeting oxidative stress in liver-related diseases is partially successful because of its diverse role in regulating physiological processes such as mitophagy and hypoxic signaling. Thus, by considering all these traits, there has been continued interest in the therapeutic targeting of redox medicine/biology to treat liver-related diseases.

Didactic Synopsis.

Major teaching points

• In biology and medicine, oxidative stress is defined as a “state of condition wherein the cellular pro-oxidant and antioxidant balance is altered in favor of a pro-oxidant state.”

• The most common free radicals are (a) reactive oxygen species (ROS), such as superoxide anions (O₂^•−), hydroxyl radicals (HO^•), and hydrogen peroxide (H₂O₂); and (b) reactive nitrogen species (RNS), such as nitric oxide (NO^•) and peroxynitrite (ONOO⁻). In addition, several other reactive species, such as reactive sulfur species (RSS) and reactive carbonyl species (RCS), have been characterized.

• ROS generation is driven by endogenous (mitochondrial electron transport chain and transmembrane NADPH oxidases) and exogenous mechanisms (nutrients, drugs, toxicants, and physical stressors).

• The liver acts as the metabolic hub and the detoxication center in our body and, therefore, generates a significant amount of ROS.

• All the cell types in the liver are equipped with a battery of enzymatic (superoxide anion dismutase, catalase, glutathione peroxidases) and nonenzymatic antioxidants (vitamins A, C, GSH) to defend against oxidative stress.

• At physiological levels, ROS plays a significant role in diverse cellular mechanisms, including signal transduction, gene expression, mitophagy, cell proliferation, and immune regulation.

• The key molecular mediators/regulators of Redox signaling are nuclear factor erythroid 2-related factor 2 (Nrf2), NF-κB, sirtuins, and forkhead box protein O (FOXO).

• Imbalance in the liver Redox biology is one of the critical components of “multiple parallel-hit hypotheses” responsible for the initiation and progression of liver diseases, including nonacholic fatty liver diseases (NAFLD), liver cirrhosis, and HCC.

• Studies have identified several biomarkers of oxidative stress such as protein carbonyls, AGEs, 4-hydroxynonenal, and malondialdehyde (MDA), which could be detected by ELISA, fluorescence-based methods, and mass spectrophotometry.

• Redox-modulation by natural or pharmacological antioxidants appears to be an attractive and viable therapeutic option in several liver-related diseases.