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ROS in Liver Cancer Promotion

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Le Thi Thanh Thuy, Hoang Hai, Pham Tuan Anh, Nguyen Bui Tam Chi, Pham Minh Duc, Michelle L. Hermiston

Submitted: 31 October 2025 Reviewed: 05 November 2025 Published: 12 March 2026

DOI: 10.5772/intechopen.1013837

Reactive Oxygen Species - An Overview IntechOpen
Reactive Oxygen Species - An Overview Edited by Rizwan Ahmad

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Reactive Oxygen Species - An Overview [Working Title]

Rizwan Ahmad

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Abstract

Reactive oxygen species (ROS) play a pivotal role in the pathogenesis and progression of liver cancer, particularly hepatocellular carcinoma (HCC). While physiological levels of ROS are essential for cellular signaling, excessive ROS generation leads to oxidative stress, DNA damage, lipid peroxidation, and chronic inflammation – all of which contribute to malignant transformation and tumor promotion. This chapter explores the dual nature of ROS in liver physiology and tumorigenesis, highlighting their involvement in hepatocyte injury, fibrosis, and activation of pro-oncogenic pathways such as NF-κB, STAT3, and PI3K/AKT. We also discuss how antioxidant agents, including cytoglobin and neuroglobin, regulate ROS levels to protect the liver from fibrosis and cancer progression. Understanding the mechanisms by which ROS promote liver cancer can inform novel therapeutic strategies targeting redox homeostasis and oxidative stress responses in HCC.

Keywords

  • ROS
  • antioxidant
  • liver cancer
  • cytoglobin
  • neuroglobin
  • tumor promotion

1. Introduction

1.1 Background on hepatocellular carcinoma (HCC)

1.1.1 Epidemiology and global burden of HCC

Hepatocellular carcinoma represents the majority of primary liver cancers, accounting for approximately 75–90% of cases worldwide [1]. The global incidence of HCC remains highly heterogeneous: Age‑standardized incidence rates vary across regions, with the highest burden observed in East Asia and sub‑Saharan Africa, and lower rates seen in many Western countries [2]. Contemporary estimates indicate that in 2020, roughly 900,000 people were diagnosed with liver cancer and ~ 830,000 died from the disease, underscoring its high case-fatality rate [3]. The rough equivalence of mortality rates to incidence globally reflects, at least in part, diagnosis at advanced stages and a lack of effective curative treatments [4]. Beyond incidence and mortality, the burden of HCC in terms of disability‑adjusted life years has also increased globally, driven by population growth and aging – even where age‑standardized rates have plateaued or declined [1]. Sex and age disparities are also evident: Men are consistently affected at higher rates (2–4 times) compared to women, and the median age of diagnosis varies by region [5]. Forward-looking analyses suggest that, without intensified prevention, annual new liver cancer cases could rise from ~ 0.87 million (2022) to ~ 1.52 million by 2050, keeping HCC among the leading causes of cancer death worldwide [6]. Given this backdrop, HCC remains one of the most formidable oncologic challenges worldwide, demanding ongoing efforts in prevention, early detection, and mechanistic research.

1.1.2 Major risk factors

The pathogenesis of HCC is multifactorial. Chronic HBV and HCV remain dominant drivers globally, with regional variation in their relative contributions [1, 5]. Despite antiviral progress, both viruses exert direct oncogenic effects and promote oxidative and inflammatory milieus that favor carcinogenesis [7]. Metabolic liver disease has risen rapidly. In 2023, an international, multisociety Delphi consensus updated terminology: Nonalcoholic fatty liver disease (NAFLD) was replaced by metabolic dysfunction-associated steatotic liver disease (MASLD), and nonalcoholic steatohepatitis (NASH) was replaced by metabolic dysfunction-associated steatohepatitis (MASH), under the umbrella of steatotic liver disease (SLD) – a change now reflected in major society communications and being incorporated into clinical practice. This shift matters because MASLD- /MASH-related HCC is increasing in many high-income countries [1]. Additionally, excess alcohol intake accelerates steato-inflammation, fibrosis, and malignant transformation – partly via CYP2E1-driven ROS generation and lipid peroxidation [8]. Among the many human carcinogens that are environmental factors, only two agents, aflatoxin and vinyl chloride, have been definitively linked to liver cancer. Aflatoxin increases the risk of HCC, while vinyl chloride causes liver angiosarcoma [9].

Together, these risk factors converge on the common pathways of chronic liver injury, inflammation, regeneration, and fibrosis/cirrhosis, ultimately setting the stage for malignant transformation.

1.2 Importance of oxidative stress in liver cancer biology

1.2.1 ROS as a physiological signaling molecule vs. pathological agent

ROS encompass a variety of chemically reactive molecules derived from oxygen (e.g., superoxide anion, hydrogen peroxide, and hydroxyl radical). Under normal physiological conditions, low-to-moderate levels of ROS play essential roles as signaling messengers in processes such as proliferation, differentiation, immune responses, and adaptive stress responses [10]. However, when ROS production exceeds the capacity of antioxidant defense systems, oxidative stress ensues, resulting in damage to lipids, proteins, and nucleic acids, and triggering cellular dysfunction, senescence, or death [11]. In the context of cancer, ROS thus play a dual role. On one hand, moderate ROS levels may promote oncogenic signaling, proliferation, and survival; on the other hand, excessive ROS can induce cell death or senescence, acting as a barrier to tumorigenesis [12]. Recent reviews reaffirm this “double-edged sword” paradigm and highlight context-specific thresholds and localization of ROS as key determinants of outcome [13]. In the liver, the balance between physiological ROS signaling and pathological oxidative stress is especially critical, given the organ’s high metabolic and detoxification activity.

1.2.2 Overview of redox homeostasis in liver physiology

The liver is a key metabolic organ, responsible for detoxification, synthesis, and storage of metabolites, as well as the handling of xenobiotics. As such, it is continuously exposed to reactive intermediates and oxidative challenges [11]. Under healthy conditions, hepatocytes maintain redox homeostasis through a network of enzymatic antioxidants (e.g., superoxide dismutases (SODs), catalase, glutathione peroxidase) and non-enzymatic systems (e.g., glutathione, thioredoxin, vitamins C/E). These ensure that ROS are generated and scavenged in a controlled fashion, allowing ROS to fulfill physiological signaling roles without causing damage [14].

When liver injury, inflammation, viral infection, steatosis, or toxin exposure occurs, ROS production increases (via mitochondria, NADPH oxidases, cytochrome P450 metabolism, peroxisomal oxidation) and antioxidant defenses may be overwhelmed, tipping the scale toward oxidative stress [11]. This redox imbalance contributes to hepatocellular damage, activation of stellate cells (fibrosis), genomic instability, and eventually carcinogenesis.

2. Role of ROS in liver diseases

2.1 Physiological roles of ROS in the liver

Under normal conditions, ROS are not merely unwanted metabolic by-products, but rather play critical roles as signaling molecules that maintain hepatic homeostasis.

First, mitochondria are a major endogenous source of ROS. During oxidative phosphorylation, a small fraction of electrons leaks from Complexes I and III of the electron-transport chain, reducing molecular oxygen to superoxide (O₂•⁻). Superoxide is rapidly converted to hydrogen peroxide (H₂O₂) by SODs. This transient H₂O₂ signal is required for normal liver regeneration; for example, in mouse models of partial hepatectomy, early increases in mitochondrial H₂O₂ are observed, and experimental scavenging of H₂O₂ with catalase or antioxidants markedly impairs the regenerative response [15]. Second, ROS participate directly in metabolic regulation. For instance, insulin-stimulated hepatocytes display a short-lived ROS burst (mainly H₂O₂), which transiently inhibits protein tyrosine phosphatases, thereby enhancing insulin-receptor autophosphorylation and downstream signaling. In this sense, moderate ROS act as second messengers that fine-tune insulin sensitivity and hepatic metabolic responses [16]. Third, the hepatic microsomal cytochrome P450 system, especially CYP2E1, generates ROS during the metabolism of xenobiotics, fatty acids, and ethanol. At physiological levels, this ROS production contributes to detoxification and adaptive responses [17]. Meanwhile, resident liver macrophages (Kupffer cells) deploy the NADPH–oxidase (NOX2) complex to generate superoxide during the respiratory burst, which is crucial for pathogen killing and clearance of apoptotic cells. Likewise, recruited neutrophils during acute inflammation release ROS to neutralize microbes [18].

2.2 Sources of ROS in the liver

The liver is continuously challenged by ROS generation, both from endogenous metabolism and exogenous stimuli [11]. Endogenous ROS sources (Figure 1) include the following:

  • Mitochondrial oxidative phosphorylation: Electron leak leads to superoxide and H2O2 formation.

  • Peroxisomal fatty-acid β-oxidation: Peroxisomes generate H2O2 during fatty-acid chain shortening, especially in steatotic states.

  • Microsomal cytochrome P450 enzymes (notably CYP2E1 in alcohol metabolism): These generate ROS as part of detoxification reactions and sometimes uncoupled electron-leak events.

  • NADPH oxidases in inflammatory or non-parenchymal liver cells (e.g., NOX family in Kupffer cells, stellate cells): These are “designed” ROS sources for signaling or host defense.

Figure 1.

Endogenous sources of ROS in the liver. ROS in the liver arise from endogenous sources, including mitochondria (electron-transport chain leakage), the endoplasmic reticulum (ER) (CYP2E1 system), peroxisomal β-oxidation, and NADPH oxidases (NOX family). These NOX genes are expressed in hepatocytes, Kupffer cells, hepatic stellate cells (HSCs), and endothelial cells. (Created in biorender.com).

Beyond these, several special conditions amplify the ROS burden:

  • Iron overload: Excess hepatic iron (as in hereditary hemochromatosis or iron overload due to excessive red blood cell transfusions) catalyzes the Fenton reaction (Fe2⁺ + H2O2 → Fe3⁺ + •OH + OH⁻), generating highly reactive hydroxyl radicals. This drives lipid-peroxidation and hepatocyte injury [19].

  • Gut-liver axis: Disruption of the intestinal barrier (e.g., due to dysbiosis or a high-fat diet) allows lipopolysaccharide (LPS) and other microbial products into the portal circulation. These activate Kupffer cells via TLR4 and trigger NADPH-oxidase-dependent ROS production in the liver [20]. In metabolic liver disease, elevated portal LPS correlates with hepatic inflammation and ROS generation.

  • Exogenous stimuli: Chronic ethanol consumption and acetaminophen (APAP) overdose are classic examples of ROS over-production. Ethanol induces CYP2E1, driving superoxide/H₂O₂ generation, mitochondrial dysfunction, and inflammation. APAP toxicity depletes glutathione, accumulates the reactive metabolite NAPQI, triggers a mitochondrial ROS burst, and JNK-mediated hepatocyte necrosis/apoptosis. Both illustrate how metabolic or toxic stress overwhelms antioxidant defenses and drives oxidative liver injury [17].

2.3 Pathological accumulation of ROS

In pathological settings, ROS accumulation can exceed the capacity of antioxidant defenses, triggering a cascade of molecular and cellular damage that perpetuates liver injury. In the context of drug-induced liver injury, hepatotoxic drugs and xenobiotics generate ROS as part of their metabolism. A prime example is acetaminophen (APAP) overdose: The reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI) depletes intracellular glutathione (GSH), tipping the redox balance toward uncontrolled oxidative stress. Once GSH is exhausted, ROS levels rise, initiating lipid-peroxidation, protein nitration, mitochondrial permeability transition, and hepatocyte necrosis or apoptosis [21].

In ischemia–reperfusion (IR) injury, as in liver transplantation or shock, the restoration of blood flow to an ischemic liver induces a sudden ROS burst. During ischemia, the xanthine oxidase and mitochondria accumulate reduced intermediates; upon reperfusion, abundant oxygen fuels rapid superoxide/H₂O₂ generation [22]. These ROS injure sinusoidal endothelial cells (SECs) and hepatocytes directly, while activating NF-κB and other pro-inflammatory pathways. Neutrophil infiltration during reperfusion adds a secondary ROS wave and proteases, creating a feed-forward loop of oxidative damage. IR injury is, therefore, a prototypical example where targeting ROS (ischemic pre-conditioning, NOX inhibition, antioxidants) can attenuate hepatocellular damage [23].

Beyond acute injury, pathological ROS accumulation is central to chronic liver diseases – viral hepatitis, alcoholic liver disease (ALD), and MASLD. In these settings, sustained ROS overproduction drives HSC activation, fibrogenesis, genomic instability, and ultimately hepatocarcinogenesis. Recent reviews highlight oxidative stress as a hallmark of MASLD progression to fibrosis and HCC [24]. Oxidative damage underpins both the parenchymal injury and the pro-tumorigenic microenvironment [25]. Similarly, experimental mouse models have demonstrated that high-fat diets or chemically induced liver injuries or carcinogenesis promote hepatic damage through ROS- and RNS-mediated mechanisms [26–29] (Figure 2).

Figure 2.

Distinct sources of ROS/RNS (reactive oxygen species/reactive nitrogen species) derived from multiple animal models of liver injuries induced by high-fat diet-induced steatohepatitis and liver fibrosis (CDAA), chemically induced liver fibrosis including TAA (thioacetamide), DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine), CCl4 (carbon tetrachloride), carcinogenesis (DEN, diethylnitrosamine), or acute liver damage (APAP, acetaminophen). These triggers caused hepatic inflammation and HSC activation, leading to fibrosis, while CYGB (cytoglobin) acts as a key antioxidative factor that attenuates oxidative stress and suppresses fibrogenesis. (Created in biorender.com).

3. Mechanisms of ROS-induced liver fibrosis and cancer

3.1 Molecular pathways activated by ROS

ROS exert profound effects on cellular macromolecules – DNA, lipids, and proteins – initiating molecular damage and redox signaling cascades that promote fibrogenesis and malignant transformation in the liver. The major ROS-driven mechanisms include DNA damage and mutagenesis, lipid peroxidation and toxic aldehyde formation, and protein oxidation leading to enzyme dysfunction.

3.1.1 DNA damage and mutagenesis

Among the four DNA bases, guanine (Gua) has the lowest redox potential, rendering it particularly susceptible to oxidative attack. The oxidation of guanine through a one-electron loss produces 7,8-dihydro-8-oxo-2′-guanine (8-oxoGua), while a one-electron gain forms 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua). Both are recognized as biomarkers of oxidative stress [30]. During DNA replication, 8-oxoGua can adopt a syn-conformation and mispair with adenine (A), which, after two replication cycles, yields G:C to T:A transversions, a mutational signature frequently detected in HCC [31]. These oxidative stress-induced lesions contribute to genomic instability and favor the accumulation of driver mutations.

A key molecular target of ROS-induced DNA damage is p53, a tumor suppressor mutated in over half of human cancers [32]. p53 orchestrates cellular responses to genotoxic stress by activating DNA repair, cell-cycle arrest, or apoptosis [33]. Structurally, its DNA-binding domain contains multiple redox-sensitive cysteine residues, rendering p53 directly susceptible to oxidative modification [34].

At basal or moderate ROS levels, p53 induces antioxidant genes – including SOD2 (MnSOD), GPx1, SESN1/2, GLS2, and TIGAR – enhancing NADPH production and maintaining redox balance [35]. However, under excessive oxidative stress, p53 can repress antioxidant responses by downregulating SOD2 [36] and the master antioxidant regulator Nrf2 [37]. This bidirectional regulation establishes p53 as a redox-sensitive switch: promoting survival and repair at low ROS but driving apoptosis or senescence when oxidative burden surpasses adaptive capacity [38].

In chronic liver injury, persistent 8-oxoG accumulation and p53 pathway disruption synergize with inflammation and fibrogenesis, creating a mutagenic environment that fosters malignant transformation.

3.1.2 Lipid peroxidation and toxic aldehyde adduct formation

Lipid membranes are inherently vulnerable to oxidative damage due to their high content of polyunsaturated fatty acids (PUFAs), which readily undergo peroxidation. Once initiated, lipid radicals propagate chain reactions that amplify ROS-induced injury [39, 40]. The resulting reactive aldehydes, such as 4-hydroxy-2-nonenal (4-HNE) and malondialdehyde (MDA), not only form adducts with DNA and proteins but also act as secondary messengers that modulate signaling and cell death [41, 42].

Two major pathways mediate lipid peroxidation, including: (1) non-enzymatic (autoxidation) – an iron-dependent radical chain reaction initiated by hydroxyl radicals (·OH), proceeding through initiation, propagation, and termination stages. Reactive intermediates – 4-HNE, MDA, isoprostanes – emerge as cytotoxic and signaling molecules [43]; 2) enzymatic – catalyzed by lipoxygenases (LOX), such as ALOX5 and ALOX12, which oxygenate arachidonic and linoleic acids, generating lipid hydroperoxides that influence redox signaling [44].

In consequence, lipid peroxidation products regulate multiple signaling cascades implicated in apoptosis and inflammation, such as the NF-κB pathway, in which reactive aldehydes activate NF-κB by promoting IκB degradation and phosphorylation-mediated inactivation of anti-apoptotic Bcl-2, thereby enhancing pro-inflammatory signaling [45]. Lipid peroxidation-derived electrophiles modulate the mitogen-activated protein kinase (MAPK) pathway via JNK and ERK signaling, linking oxidative stress to proliferation and survival [46]. Several PKC isoforms, including PKCδ, are activated by lipid peroxidation intermediates or DAG/IP₃ cascades. PKCδ cleavage by caspase-3 produces a constitutively active fragment that amplifies apoptosis [47].

In mitochondria, cardiolipin peroxidation destabilizes the inner membrane and promotes cytochrome c release – an initiating event of intrinsic apoptosis [48]. Collectively, lipid peroxidation products not only inflict structural damage but also act as bioactive mediators, integrating ROS signaling into pathways governing apoptosis, inflammation, and fibrogenesis [49].

3.1.3 Protein oxidation and enzyme dysfunction

Proteins are continuous targets of oxidative modification, leading to carbonylation, S-glutathionylation, glycoxidation, and cross-linking [50]. These post-translational changes disrupt enzymatic function, alter protein–protein interactions, and compromise proteostasis.

The proteasome system serves as the principal machinery for the degradation of oxidized or misfolded proteins. The 20S proteasome can degrade oxidatively modified substrates in an ATP-independent manner [51]. Upon oxidative challenge, proteasomal activity may increase transiently; yet, prolonged ROS exposure promotes 26S proteasome disassembly into 20S and 19S subunits [52], reducing ubiquitin-dependent degradation capacity [53].

While the 20S complex shows higher oxidative resistance, persistent exposure to ROS ultimately leads to proteasome carbonylation and functional impairment [50, 54]. The immunoproteasome, particularly in association with the 11S regulator, can compensate by degrading oxidized proteins more efficiently [55].

Chronic ROS exposure thus creates a vicious cycle: Protein oxidation impairs proteasomal degradation, resulting in further accumulation of damaged proteins that exacerbate oxidative stress [56]. This proteotoxic stress contributes to hepatocyte dysfunction, ER stress, and inflammatory activation – key drivers of fibrosis and hepatocarcinogenesis.

Taken together, ROS act through multilayered molecular mechanisms to promote liver injury and carcinogenesis (Table 1). DNA oxidation triggers mutagenesis and p53 dysregulation; lipid peroxidation yields bioactive aldehydes that modulate apoptosis and inflammatory signaling; and protein oxidation disrupts proteostasis, perpetuating cellular stress. Together, these interlinked processes create a pro-fibrotic, pro-mutagenic microenvironment that underlies the transition from chronic liver injury to cirrhosis and HCC.

ROS target Molecular event Key biomarkers/products Signaling pathways affected Pathophysiological consequences References
DNA Oxidation of guanine → 8-oxoG, FapyG, replication mispairing. 8-oxoGua, p53 oxidation. p53, DNA repair, cell-cycle checkpoints. Mutagenesis, genomic instability, malignant transformation. [24–34]
Lipids PUFA peroxidation (enzymatic + non-enzymatic) 4-HNE, MDA, isoprostanes. NF-κB, MAPK, PKC. Apoptosis, inflammation, stellate-cell activation, fibrosis. [35–58]
Protein Carbonylation, S-glutathionylation, glycoxidation, cross-linking. 26S proteasome → 20S and 19S subunits. Enzymatic function, protein–protein interactions, proteostasis. Hepatocyte dysfunction, inflammatory activation, ER stress, fibrosis, hepatocarcinogenesis. [59–69]

Table 1.

Molecular pathways activated by ROS.

3.2 ROS and liver fibrosis

ROS play a central role in the pathogenesis of liver fibrosis by regulating hepatocellular injury, inflammatory signaling, and activation of HSCs. Among various mediators, transforming growth factor-β (TGF-β) and MAPK pathways are key effectors that integrate redox signaling with fibrogenic gene expression.

3.2.1 Activation of HSCs via TGF-β/Smad and MAPK signaling

TGF-β is the prototypical pro-fibrogenic cytokine upregulated in nearly all chronic fibrotic disorders. Canonical Smad2/3 signaling is a well-established mechanism of TGF-β-driven extracellular-matrix (ECM) synthesis; however, accumulating evidence demonstrates that ROS act both upstream and downstream of TGF-β, forming a self-amplifying redox loop.

TGF-β1 stimulates ROS generation from mitochondria and NADPH oxidases, while ROS, in turn, activate latent TGF-β1 and enhance its transcription – thereby reinforcing fibrogenic signaling [57]. In parallel, TGF-β suppresses antioxidant defenses, further shifting the intracellular redox state toward oxidation. Collectively, this feed-forward TGF-β–ROS circuit sustains myofibroblast differentiation and fibrotic matrix deposition.

3.2.2 TGF-β-induced mitochondrial ROS production

Mitochondria are the primary intracellular source of ROS, and TGF-β1 markedly enhances their production across multiple cell types, contributing to apoptosis, senescence, epithelial–mesenchymal transition (EMT), and fibrotic gene expression associated with myofibroblast differentiation [58].

In hepatocytes, studies confirm that TGF-β1 drives EMT by inducing mitochondrial dysfunction, characterized by elevated mtROS and reduced membrane potential. This occurs because TGF-β disrupts mitochondrial quality control: it inhibits critical mitophagy pathways via Aldose Reductase and suppresses antioxidant enzymes (e.g., the SIRT3/SOD2 pathway). Restoring mitochondrial function – by either activating mitophagy or boosting SIRT3 – abolishes the excess mtROS and reverses the TGF-β-induced EMT. This confirms that mitochondrial ROS directly mediate TGF-β-dependent transcriptional control in hepatocytes [59, 60].

Mechanistically, several mitochondrial complexes are involved. Yoon et al. found that TGF-β inhibits complex IV activity in mink lung epithelial (Mv1Lu) cells [61], whereas Byun et al. identified GSK3α/β phosphorylation as a mediator of complex IV inhibition and ROS accumulation [62]. Conversely, Jain et al. reported that TGF-β suppresses complex III in human lung fibroblasts, leading to sustained ROS generation and profibrotic gene induction; scavenging mitochondrial ROS or inhibiting complex III attenuated these effects without affecting Smad signaling, confirming a Smad-independent mitochondrial mechanism [63].

3.2.3 TGF-β-mediated induction of NADPH oxidases (NOXs)

Beyond mitochondria, NADPH oxidases (NOXs) represent specialized enzymatic sources of ROS. The NOX family comprises seven isoforms (NOX1–5, DUOX1, DUOX2). TGF-β upregulates several NOX isoforms – particularly NOX1, NOX2, and NOX4 – in multiple cell types. Among these, NOX4 is most consistently implicated in fibrosis [64].

Unlike other NOX isoforms that require activation by cytosolic subunits, NOX4 is constitutively active and widely expressed in epithelial cells, fibroblasts, endothelial cells, and macrophages. TGF-β induces NOX4 through both Smad-dependent and Smad-independent routes, including PI3K, MAPK, and RhoA/ROCK pathways. The resulting ROS promotes HSC activation, upregulates collagen I and α-SMA, and perpetuates ECM accumulation [65].

3.2.4 Suppression of the antioxidant defense system by TGF-β

TGF-β also exacerbates oxidative stress by inhibiting cellular antioxidant capacity. The tripeptide glutathione (GSH) – the most abundant intracellular thiol – is central to detoxifying electrophiles and maintaining thiol homeostasis. Decreased GSH levels are consistently reported in chronic liver and other fibrotic diseases [66].

Mechanistically, TGF-β reduces GSH synthesis by repressing glutamate-cysteine ligase (GCL), the rate-limiting enzyme for GSH biosynthesis. Furthermore, TGF-β downregulates or inhibits the activities of SOD, catalase, and glutaredoxin (Grx). Together, these effects disable the cellular antioxidant network, maintaining a pro-oxidant microenvironment that favors fibrogenesis [66].

3.2.5 ROS-dependent activation of MAPK signaling

ROS also influences liver fibrogenesis through redox-sensitive MAPK pathways, including ERK, JNK, and p38. Many stimuli that generate ROS concurrently activate MAPKs [67]. One mechanism involves the direct oxidation of key cysteine residues in kinases or phosphatases [68].

A paradigmatic example is apoptosis signal-regulating kinase 1 (ASK1), a MAP3K upstream of JNK/p38. Under basal conditions, ASK1 binds to reduced thioredoxin (Trx); upon oxidative stress, Trx oxidation releases ASK1, permitting its oligomerization and activation [69]. In Ask1-knockout mice, JNK/p38 activation is markedly diminished after oxidative challenge [70]. A paradigmatic example is ASK1, a MAP3K upstream of JNK/p38. Under basal conditions, ASK1 binds to reduced thioredoxin (Trx); upon oxidative stress, Trx oxidation releases ASK1, permitting its oligomerization and activation [69]. In Ask1-knockout mice, JNK/p38 activation is markedly diminished after oxidative challenge [70].

ROS also stabilizes MAPK activation indirectly by inactivating MAPK phosphatases (MKPs): Oxidation of catalytic cysteines prevents their dephosphorylation activity [71], and oxidative stress can induce MKP degradation [72]. These mechanisms extend MAPK signaling duration, reinforcing profibrotic transcription programs in HSCs and hepatocytes.

3.2.6 Redox crosstalk among HSCs, Kupffer cells, and hepatocytes

Fibrosis is a multicellular process coordinated through oxidative intercellular communication. Hepatocyte injury – triggered by ER stress, mitochondrial dysfunction, or xenobiotics – produces ROS, mitochondrial DNA, and apoptotic bodies that activate Kupffer cells (KCs) and HSCs (HSCs). These immune and stromal interactions further amplify redox signaling (Figure 3).

Figure 3.

Redox crosstalk among HSCs, Kupffer cells, and hepatocytes. TAA-induced hepatocyte injury leads to excessive ROS generation and depletion of antioxidant defenses, releasing DAMPs and apoptotic bodies that activate Kupffer cells. The resulting ROS and cytokines (TNF-α, IL-6) from Kupffer cells further stimulate HSC activation and ECM deposition through TGF-β signaling, thereby establishing a self-amplifying redox loop that drives liver fibrosis. (Created in biorender.com).

Cytokines and growth factors such as angiotensin II, leptin, platelet-derived growth factor (PDGF), and TGF-β stimulate NOX enzymes in both macrophages and HSCs, driving excessive ECM deposition. SECs also participate, releasing ROS and vasoactive mediators that modulate HSC contraction and migration.

In hepatocytes, excessive ROS – especially when antioxidant defenses or unfolded-protein response mechanisms fail – activate pro-apoptotic signaling [73–75]. Conversely, in myofibroblasts, ROS enhance proliferation and survival [76].

Within the liver, the principal NOX isoforms are NOX1, NOX2, NOX4, and DUOXs [77]. NOX1/NOX2 primarily generate superoxide, while NOX4 produces hydrogen peroxide. Activation of NOX1/NOX2 requires the assembly of cytosolic subunits, whereas NOX4 is constitutively active and associates with p22^phox in HSCs, hepatocytes, and SECs.

NOX enzymes are activated by diverse mediators, including PDGF [78], angiotensin II [79], leptin [80, 81], alcohol [82], and phagocytosis of apoptotic hepatocyte bodies [83]. Functional studies confirm their non-redundant fibrogenic roles:

  • Deletion of Nox1 or Nox2 reduces fibrosis in CCl₄ and bile-duct-ligation models [83–85].

  • Hepatocyte-specific Nox4 deletion protects against ER-stress-induced fibrosis in NASH [86].

  • Nox4-deficient HSCs exhibit impaired activation and collagen I synthesis.

These findings establish NOX-derived ROS as critical mediators of fibrogenic signaling and identify selective NOX inhibition – especially targeting non-phagocytic NOXs – as a promising anti-fibrotic strategy [87, 88].

3.3 ROS in tumor promotion

3.3.1 ROS promotes the proliferation of HCC

Accumulating evidence demonstrates that moderate elevation of ROS stimulates HCC cell proliferation through redox-sensitive signaling cascades, whereas excessive ROS can trigger cell death.

In in vitro studies, several HCC cell lines, including HepG2, Huh7, HLE, HLF, and SNU, show enhanced proliferation in response to ROS induction. Moderate oxidative stress induced by H₂O₂, mitochondrial leakage, or NADPH oxidase activation promotes phosphorylation of ERK1/2, AKT, and STAT3, thereby accelerating DNA synthesis and cell-cycle progression [89]. For instance, ROS exposure in HLE and SNU cells upregulates cyclin D1 and activates the PI3K/AKT pathway, effects that are completely abolished by N-acetylcysteine or catalase, confirming their ROS dependency[89]. In agreement, Trepiana et al. (2017) reported that long-term culture of HepG2 and Huh7 cells under physiological hypoxia (8% O₂) increased intracellular ROS and p66^Shc, correlating with greater migration, metabolic flexibility, and oxidative-stress resistance, hallmarks of an aggressive HCC phenotype [90].

Beyond cell culture systems, the tumor-promoting effects of ROS are validated in in vivo models. In SOD1⁻/⁻ mice, chronic superoxide accumulation leads to spontaneous hepatic tumors accompanied by elevated proliferation markers and mitochondrial DNA oxidation [89]. Similarly, HBV-, HCV-, and CYP2E1-transgenic models exhibit persistent ROS production and lipid peroxidation, activating NF-κB and MAPK signaling that enhance hepatocyte proliferation and tumor growth. Treatment with antioxidants such as vitamin E or NADPH-oxidase inhibitors markedly reduces both ROS burden and tumor incidence, confirming a causal link between oxidative stress and hepatocellular proliferation [89].

Mechanistically, ROS exert their pro-tumorigenic effects mainly through activation of ERK/MAPK, PI3K/AKT, and STAT3 pathways – representing a paradigm shift from viewing ROS solely as cytotoxic molecules to recognizing them as key signaling intermediates in HCC development.

3.3.2 ROS promotes HCC metastasis

ROS not only drive HCC proliferation but also promote migration and metastasis through the activation of redox-sensitive signaling pathways such as NF-κB, ERK, AKT, and c-Jun [91].

In in vitro models, moderate ROS levels in HepG2 and Huh7 cells activate ERK and AKT, leading to the transcriptional induction of mesenchymal markers (Snail, vimentin) and matrix metalloproteinases (MMP-2/9), while concurrently repressing epithelial E-cadherin [91]. This molecular reprogramming facilitates EMT and enhances cell motility. Supporting this EMT-promoting role, HGF-induced ROS oxidize the chaperone proteins HSP60 and PDI, thereby activating ERK and enhancing wound-healing and transwell migration; these effects are abolished by antioxidants [92]. In parallel, mitochondrial Ca2+ influx through MCU elevates ROS, activates the JNK/c-Jun cascade, and upregulates MMP-2/9, culminating in enhanced invasion in vitro and spontaneous lung metastasis in vivo [93].

Collectively, these findings establish ROS as critical enablers of HCC metastasis, orchestrating EMT, proteolytic matrix degradation, and distant organ colonization through coordinated activation of ERK/AKT, JNK, and NF-κB signaling [91], thereby providing a mechanistic basis for the link between oxidative stress and poor clinical outcomes in HCC.

3.3.3 ROS promotes angiogenesis in HCC

ROS and the resulting oxidative stress (OS) are key drivers of tumor angiogenesis in HCC. High OS correlates with increased micro vessel density (MVD) and high VEGF expression in HCC tissues [94]. Mechanistically, ROS stabilizes HIF-1 and upregulates VEGF by activating several pathways.

  • PI3K/AKT pathway: ROS mediates hypoxia-induced VEGF expression by activating the PI3K/AKT/HIF-1 signaling pathway in HCC cells (HepG2) and in HCC tissues. The resulting activation of Akt enhances VEGF expression. Inhibiting ROS production significantly blocks this activation cascade and subsequent VEGF expression [94, 95]. ROS-dependent activation of PI3K/AKT/HIF-1 upregulates the Mxi1-0 protein in HepG2. Intriguingly, this Mxi1-0 then reciprocally promotes further ROS generation, forming a self-sustaining positive feedback loop that drives prolonged VEGF expression [95].

  • The ROS/NF-κB/HIF-1 pathway: It is activated in HCC cells (such as HepG2 and Huh7) under acute hypoxic conditions, initiating the pro-angiogenic response. Specifically, acute hypoxia causes an increase in ROS, which mediates the activation and phosphorylation of NF-κB. This NF-κB activation subsequently enhances the expression of HIF-1 and its critical downstream target, VEGF, thereby promoting tumor vascularization [96].

  • Impaired degradation: ROS stabilizes HIF-1 protein by impairing the activity of prolyl hydroxylase (PHD) in HepG2. This prevents the hydroxylation of HIF-1, thereby blocking its recognition and subsequent degradation by the VHL-mediated proteasomal system [96, 97].

This robust upregulation and stabilization of HIF-1α ultimately drives the transcription of VEGF, dramatically stimulating endothelial cell proliferation and tumor angiogenesis.

4. Antioxidant agents in protection against liver fibrosis and cancer

In Part 3, we outlined how ROS drives fibrosis and tumor promotion through DNA damage, lipid peroxidation, proteostasis failure, and pro-oncogenic signaling (NF-κB/STAT3, PI3K/AKT/mTOR, HIF-1α/VEGF, EMT). These same redox pressures also define therapeutic opportunities: selectively restoring redox balance can blunt fibrogenesis, constrain inflammatory loops, and weaken malignant adaptations – provided physiological ROS signaling is not indiscriminately suppressed.

4.1 Endogenous antioxidants

Antioxidants are molecules or enzymes that limit oxidation by scavenging reactive species or catalyzing their reduction. The body deploys endogenous systems (enzymatic and small-molecule) and relies on exogenous inputs from diet/supplements. Together, they maintain hepatic redox homeostasis against continuous oxidative challenges from metabolism, xenobiotics, and inflammation (Figure 4) [98].

Figure 4.

Antioxidant agents. (Created in biorender.com).

4.1.1 Enzymatic systems

Superoxide dismutases are the first line of defense, dismutating superoxide (O2•⁻) into H2O2 and O2. Three isoforms operate in complementary compartments: SOD1 (Cu/Zn-SOD) in the cytosol/intermembrane space, SOD2 (Mn-SOD) in the mitochondrial matrix, and SOD3 extracellularly [99–102]. In liver disease, SOD activity can initially rise as an adaptive response (e.g., pediatric cholestasis) but often declines with progression to HCC; SOD2 downregulation in tumors correlates with size, recurrence, and poor survival, potentially via loss of gene copy number [103–105]. Given that mitochondria are a dominant ROS source in hepatocytes, the loss of SOD2 is particularly deleterious.

Catalase (CAT) is a high-turnover, peroxisomal heme enzyme that decomposes H2O2 generated during fatty-acid oxidation and downstream of SOD [102, 106]. CAT expression/activity frequently declines during malignant transformation, including HCC, and lower CAT levels are linked to worse outcomes; conversely, higher tumor-to-normal CAT ratios are associated with longer survival [107–109]. Reduced CAT allows H2O2 to accumulate at signaling-competent levels, which can promote proliferation and angiogenesis.

Glutathione peroxidases (GPx) are selenium-containing enzymes that reduce H2O2 and lipid hydroperoxides using GSH, generating GSSG [102]. Roles are isoform-specific: GPx1 detoxifies H2O2 broadly, whereas GPx4 uniquely reduces phospholipid hydroperoxides within membranes, preventing the propagation of lipid peroxidation [102]. GPx responses can be compensatory in acute injury [110], yet chronic disease/HCC often shows impairment. Genetic variants (e.g., GPx1 Pro198Leu) are associated with advanced fibrosis/HCC risk [78]. GPx4 behaves as a tumor suppressor in liver models: overexpression limits oxidative stress, tumor growth, and angiogenesis; downregulation is linked to poor prognosis, underscoring the importance of restraining lipid peroxidation [111].

4.1.2 Non-enzymatic systems

The liver’s small-molecule network coordinates with enzymes to keep redox tone within a physiological window [112, 113]. Key components of this network include the following:

Glutathione (GSH): GSH is the most abundant low-molecular-weight antioxidant, synthesized primarily in the liver by GCL and GSH synthetase [114, 115]. Functions include direct ROS/RNS scavenging, control of cellular redox potential, and Phase II detoxification via GST-mediated conjugation (e.g., of NAPQI) to facilitate biliary/urinary excretion [114, 115]. Therapeutically, repleting hepatic GSH is rational; small clinical studies report improved liver enzymes with IV/oral GSH in NAFLD/ALD, and derivatives such as S-allyl-glutathione improve fibrosis in animals [116, 117]. However, clinical evidence remains limited; bioavailability and study design warrant more rigorous trials [117].

Globin family (CYGB, MB, NGB): Beyond oxygen transport, several globins buffer ROS/RNS in a compartmentalized manner, with CYGB and NGB being of particular relevance to hepatic fibrosis (expanded in Sections 4.2–4.3 below) [112].

Vitamins C and E: Vitamin E (α-tocopherol) is a lipophilic chain-breaking antioxidant that halts lipid peroxidation, while vitamin C (ascorbate) scavenges aqueous ROS and recycles vitamin E [98]. Evidence is mixed: some animal NAFLD models favor vitamin C, whereas human NASH trials (e.g., PIVENS) showed histologic improvement with high-dose vitamin E, albeit with limited impact on fibrosis; combinations of high-dose C + E improved fibrosis in one trial [118, 119]. However, other observational studies have shown evidence of harm (e.g., supplemental vitamin C and liver cancer risk in predisposed populations), thus leading to caution against non-specific, high-dose antioxidant use [120].

Uric acid: Uric acid is a paradoxical molecule: a major plasma antioxidant and RNS scavenger, yet pro-oxidant/inflammatory intracellularly. In hepatocytes/adipocytes, urate can stimulate NOX-dependent ROS; as a DAMP, extracellular urate activates innate pathways and directly promotes HSC activation[121–123]. Hyperuricemia associates with NAFLD severity and fibrosis and links to hepatobiliary cancer risk and HCC recurrence after resection. Moreover, Mendelian randomization analyses suggest a causal contribution of elevated urate to fibrosis and HCC [121, 124, 125].

Thus, endogenous antioxidants form a hierarchical, compartmentalized network. In chronic liver disease, this network is progressively subverted – especially at mitochondria and membranes – tilting ROS from signaling to pathology.

4.2 Cytoglobin (CYGB)

ROS amplify TGF-β/NOX signaling in HSCs (Section 3.2) and sustain tumor-promoting circuits (Section 3.3); an HSC-enriched antioxidant could interrupt fibrosis at its source and, secondarily, lower HCC risk.

Our group has identified CYGB, which is a 21-kDa, hexacoordinated globin (∼25% identity to Mb/Hb; ∼16% to NGB) with two cysteines that influence O2 binding via disulfide formation; its structure and ligand reactivity are redox-sensitive [126]. In the liver, CYGB is expressed almost exclusively in HSCs, making it a selective HSC marker in both human and rodent liver [126]. Genetic knock out of Cygb promotes hepatic fibrosis and the development of HCC, accompanied by increased markers of oxidative stress under the administration of diethylnitrosamine [28] or a choline-deficient amino acid-defined diet [29]. Both primary HSCs isolated from Cygb-deficient mice and those isolated from wild-type mice, in which Cygb mRNA was silenced, exhibited increased ROS generation and upregulated collagen alpha1(I), TIMP-1, IL-6, and TNFα expression [29]. In humans, decreased expression of Cygb was also found in patients with HCV-induced liver fibrosis [127], NASH, and HCC [29].

In multiple preclinical models, recombinant human CYGB (His-CYGB), delivered intravenously to mice with established cirrhosis (e.g., TAA, DDC), suppressed inflammation, reduced oxidative damage, and regressed fibrosis. Biodistribution favored HSC uptake, aligning the mechanism with the effect; humanized-liver safety studies detected no adverse signals [27]. These outcomes position CYGB as a targeted anti-fibrotic/antioxidant biologic.

Taken together, the overall effect of CYGB appears protective by curbing oxidative stress, restraining HSC activation, reducing matrix stiffening and hypoxia, and ultimately diminishing tumor-promoting conditions.

4.3 Neuroglobin (NGB)

Discovered in neurons, NGB is a stress-inducible globin with strong antioxidant and anti-apoptotic actions; although not a native hepatic protein, it displays translatable protective effects in liver models [112]. Recombinant NGB is endocytosed by human HSCs and localizes to membranes/cytoplasm. It scavenges intracellular ROS, deactivates HSCs, suppresses COL1A1 transcription, and increases MMP-1, promoting collagen degradation [112]. Mechanistically, NGB also binds released cytochrome-c, blocking apoptosome formation (caspase-9 activation) and preventing intrinsic apoptosis under oxidative/hypoxic stress – principles well-described in neurons and relevant to hepatic injury [128]. In vivo, systemic NGB, myoglobin (MB), and CYGB administration in CCl₄-induced fibrosis dampened inflammation and slowed fibrogenesis without toxicity in treated mice, indicating a feasible safety window and a globin-based therapeutic class concept for fibrotic liver disease [112] (Figure 5).

Figure 5.

Recombinant human myoglobin (MB), neuroglobin (NGB), and cytoglobin (CYGB) administration prevented the aggravation of CCl4-induced liver fibrosis and reduced levels of lipid peroxidation, 4-hydroxynonenal (4-HNE).

4.4 Other emerging antioxidant molecules

4.4.1 Heme oxygenase-1 (HO-1), peroxiredoxins (PRXs), thioredoxins (TRXs)

HO-1, the inducible, cytoprotective heme-degrading enzyme, generates biliverdin/bilirubin (antioxidants), CO (signaling), and Fe2+ (sequestered) [129]. In rodent liver disease, HO-1 induction decreases injury and inflammatory cytokines and slows fibrosis [130]. Yet, as with NRF2, tumor contexts complicate interpretation: HO-1 is overexpressed in some cancers, including HCC, where it actively supports tumor progression by enhancing survival via mechanisms such as increasing cancer cell proliferation, conferring resistance to chemo- and radiotherapy, and promoting angiogenesis, as indicated by its strong correlation with VEGF expression and microvascular invasion. This highlights the importance of further study to understand its potential role in cancer [129].

Peroxiredoxins (Prx) are a family of proteins that reduce peroxides and shape redox signaling. Prx6 supports mitochondrial function and guards against NAFLD, while Prx3 detoxifies mitochondrial ROS in alcoholic fatty liver disease. Some members (e.g., Prx1) can be upregulated in injury and, in certain contexts, exacerbate damage, illustrating isoform- and context-specific roles [131–134].

Thioredoxin (Trx) is a central reductase with anti-oxidant/anti-inflammatory actions, dysregulated across ALD, NAFLD, and HCC, and often correlating with disease severity [135–137]. Trx couples redox buffering with signaling (e.g., ASK1 regulation), positioning it at the interface of stress responses elaborated in Part 3.

4.4.2 Phytochemicals and nutraceuticals with ROS-modulating capacity

Many plant-derived compounds modulate endogenous defenses (e.g., NRF2 activation, NF-κB suppression) rather than merely scavenging radicals.

  • Silymarin: It activates NRF2 and inhibits NF-κB; it exhibits anti-fibrotic effects by restraining HSC activation/proliferation [138].

  • Curcumin: NRF2 activator/NF-κB inhibitor; it improves insulin resistance and reduces steatosis, inflammation, and oxidative stress in NAFLD models [139].

  • Resveratrol: It activates NRF2 and SIRT1, improving mitochondrial function and attenuating steatosis/oxidative stress [140].

  • Green-tea catechins (EGCG): antioxidant/anti-inflammatory; meta-analyses in NAFLD show improved ALT/AST and metabolic parameters [138].

  • Quercetin: It broadly reduces oxidative stress and inflammation, with reported improvements in liver enzymes and histology across preclinical models [141].

An important caveat of these agents is that benefits may be context-dependent and influenced by dosing, formulation, and bioavailability REF. Consistent with the above-mentioned dual-role paradigm, non-specific high-dose antioxidant supplementation can be neutral or paradoxically harmful; targeting specific ROS nodes or restoring endogenous tone is preferable.

5. Future directions

5.1 Redox-modulating and combination therapies

As discussed earlier, ROS form an intricate network that links chronic inflammation, fibrosis, and carcinogenesis. The next stage of translational research should focus on selective redox modulation – not simple radical scavenging. Several classes of redox-modulating drugs, such as mitochondria-targeted antioxidants, NOX inhibitors, and NRF2 activators, are progressing through preclinical and early clinical testing [142]. These agents will likely show the greatest benefit when used in combination therapy rather than as stand-alone antioxidants.

Combining ROS modulators with immune checkpoint inhibitors (ICIs) or molecularly targeted therapies offers particular promise. Oxidative stress profoundly shapes the tumor immune microenvironment by inducing T-cell exhaustion and promoting immune exclusion. Reducing pathological ROS can restore effector T-cell function and increase ICI responsiveness [143]. Likewise, pairing antioxidants with kinase inhibitors, such as sorafenib or lenvatinib, may alleviate oxidative injury and delay drug resistance mediated by ferroptosis or mitochondrial dysfunction [144]. Preclinical findings suggest that supplementation with CYGB or NGB proteins can further normalize the fibrotic, hypoxic niche, enhancing immune infiltration and treatment sensitivity [27, 145].

5.2 Precision medicine approaches

The success of redox-based therapy will depend on precision stratification. Traditional antioxidant trials have failed largely because they treated redox imbalance as a uniform process. New tools – single-cell and spatial transcriptomics – now reveal cell-type-specific redox programs, identifying NOX4-positive myofibroblasts, TREM2⁺ macrophages, and oxidative-metabolic hepatocytes as major ROS-regulated populations [145, 146]. Integrating these datasets with circulating or tissue biomarkers – such as 8-hydroxy-2′-deoxyguanosine, lipid peroxidation products, or CYGB levels – could guide patient selection and dosing for antioxidant therapy [147].

Artificial-intelligence-driven multiomics models may soon predict individual “redox signatures,” enabling clinicians to choose the right redox-modulating agent for each pathological stage of liver disease. Such biomarker-guided therapy exemplifies the shift toward personalized hepatic oncology.

5.3 Gene and protein-replacement strategies

Among emerging biologics, CYGB and NGB stand out as endogenous protectors with defined cellular targets. Viral or non-viral overexpression systems restoring CYGB in HSCs have markedly reduced fibrosis and hepatocarcinogenesis in mice [27, 112]. Recombinant His-CYGB protein shows efficient HSC uptake and regression of established cirrhosis without adverse effects, positioning it as a prototype antifibrotic biologic. Future work may adapt this strategy into mRNA-based therapeutics or pegylated protein formulations with improved stability and targeted delivery.

5.4 Challenges and perspectives

The central challenge in ROS-directed therapy remains balancing elimination with physiological necessity. Moderate ROS levels are indispensable for regeneration, xenobiotic metabolism, and immune surveillance; excessive suppression risks compromising cytotoxic lymphocyte function or favoring tumor survival. Overactivation of antioxidant pathways, such as NRF2 or HO-1, can likewise promote chemoresistance [129]. Hence, future strategies must emphasize spatiotemporal control – administering antioxidants at defined disease stages, in specific cell compartments, and at titrated doses.

Integrating omics-defined redox phenotypes, real-time ROS imaging, and computational modeling will be essential to achieve this precision. The long-term goal is to transform redox modulation from an empirical adjunct into a rational, biomarker-guided therapeutic axis for liver fibrosis and HCC.

6. Conclusion

Reactive oxygen species play a dual role in the liver – serving as indispensable messengers in normal physiology while acting as pathological drivers when chronically elevated. The continuum from oxidative stress to fibrosis and ultimately HCC underscores the necessity of maintaining redox homeostasis rather than indiscriminately suppressing ROS.

Recent insights highlight that antioxidant strategies, especially those centered on globin-based molecules such as cytoglobin (CYGB) and neuroglobin (NGB), can actively reverse fibrotic remodeling and weaken the pro-tumor microenvironment. Combined with advances in single-cell biology, nanomedicine, and gene therapy, these approaches promise a new generation of precision redox therapeutics.

Ultimately, preventing and treating liver cancer will require an integrated translational framework linking molecular redox biology with clinical intervention. By bridging mechanistic understanding and targeted therapy, the next decade may witness redox medicine evolve from a conceptual model into a cornerstone of hepatology and oncology.

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Written By

Le Thi Thanh Thuy, Hoang Hai, Pham Tuan Anh, Nguyen Bui Tam Chi, Pham Minh Duc, Michelle L. Hermiston

Submitted: 31 October 2025 Reviewed: 05 November 2025 Published: 12 March 2026

© 2026 The Author(s). Licensee IntechOpen. This content is distributed under the terms of the Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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