Oxidative Stress in NAFLD: Role of Nutrients and Food Contaminants

Abstract

Non-alcoholic fatty liver disease (NAFLD) is often the hepatic expression of metabolic syndrome and its comorbidities that comprise, among others, obesity and insulin-resistance. NAFLD involves a large spectrum of clinical conditions. These range from steatosis, a benign liver disorder characterized by the accumulation of fat in hepatocytes, to non-alcoholic steatohepatitis (NASH), which is characterized by inflammation, hepatocyte damage, and liver fibrosis. NASH can further progress to cirrhosis and hepatocellular carcinoma. The etiology of NAFLD involves both genetic and environmental factors, including an unhealthy lifestyle. Of note, unhealthy eating is clearly associated with NAFLD development and progression to NASH. Both macronutrients (sugars, lipids, proteins) and micronutrients (vitamins, phytoingredients, antioxidants) affect NAFLD pathogenesis. Furthermore, some evidence indicates disruption of metabolic homeostasis by food contaminants, some of which are risk factor candidates in NAFLD. At the molecular level, several models have been proposed for the pathogenesis of NAFLD. Most importantly, oxidative stress and mitochondrial damage have been reported to be causative in NAFLD initiation and progression. The aim of this review is to provide an overview of the contribution of nutrients and food contaminants, especially pesticides, to oxidative stress and how they may influence NAFLD pathogenesis.

Keywords: food contaminant; macronutrients; micronutrients; mitochondria; non-alcoholic fatty liver disease (NAFLD); non-alcoholic steatohepatitis (NASH); oxidative stress; reactive oxygen species (ROS); steatosis.

Figure 1

Factors involved in non-alcoholic fatty liver disease (NAFLD) pathogenesis. Multiple causes, including metabolic factors, gut microbiota, and environmental factors, operate in the context of the specific genetic background of individuals for the development of non-alcoholic fatty liver disease (NAFLD). Lipid overload induces lipotoxicity that affects different reactive oxygen species (ROS) generators, such as mitochondria, peroxisomes, and endoplasmic reticulum (ER). As environmental factors, solid and liquid foods and their contaminants contribute to ROS production. ROS generation triggers changes in insulin sensitivity, the activity of key enzymes of lipid metabolism, innate immune system signaling, inflammatory responses, and liver apoptosis. High levels of ROS enhance oxidative stress, thus creating a vicious circle. Collectively, these conditions contribute to NAFLD development and progression. Other abbreviations: NAFL, non-alcoholic fatty liver; NASH, non-alcoholic steatohepatitis.

Figure 2

Role of oxidative stress in NAFLD. Lipid and cholesterol accumulation in hepatocytes causes lipotoxicity, resulting in activation of different pathways involved in oxidative stress. Lipid entry and alteration of mitochondrial Ca²⁺ homeostasis cause electron transport chain (ETC) dysfunction that leads to mitochondrial reactive oxygen species (ROS) production, which in turn induces Sirtuin (SIRT)3 expression, protein 3-tyrosine nitration, mitochondrial DNA damage, and membrane depolarization. SIRT3 and protein nitration also cause ETC dysfunction and exacerbate mitochondrial ROS production. Mitochondrial biogenesis dysfunction additionally induces ROS production. Lipotoxicity activates endoplasmic reticulum (ER) stress, leading to ROS production by (i) unfolded protein response (UPR) activation, which stimulates C/EBP homologous protein (CHOP) expression and (ii) Ca²⁺ homeostasis dysfunction. ROS generation in cytoplasm is induced by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) enzyme activation, oxidized phospholipids (OxPL), several cytochrome p450 enzymes (CYP450), and lipid peroxidation. ROS accumulation in hepatocytes leads to impairment of several pathways involved in NAFLD development, such as lipid metabolism, insulin signaling, inflammation, and apoptosis. Other abbreviations: mtDNA, mitochondrial DNA; TCA, tricarboxylic acid cycle; MDA, malondialdehyde; 4-HNE, 4-hydroxynonenal; SERCA, ER calcium pump sarco/endoplasmatic reticulum Ca²⁺-ATPase; JNK, c-Jun N-terminal kinase; NF-κB, nuclear factor-kappa B.

Figure 3

ROS production in peroxisomes. In peroxisomes, fatty acid (FA) oxidation leads to the formation of H₂O₂, which is decomposed by the antioxidant enzymes GPX1, CAT, or peroxiredoxin (PRX) 1 or 5, leading to the formation of O₂ and H₂O. O₂ can react with XO to produce O₂^.−. Nitric oxide synthase (NOS) interaction with L-arginine (L-Arg) leads to the formation of NO^., which interacts with O₂^.− to form ONOO⁻. ONOO⁻ is converted by Prx5 to a peroxynitrite radical ONO^.. SOD2 dismutates O₂^.− to H₂O₂. The Fenton reaction also occurs in peroxisomes and leads to membrane damage. The H₂O₂ is then exported to the cytoplasm, where it acts as a signaling molecule that exacerbates oxidative stress. Other abbreviations: XO, xanthine oxidase; CAT, catalase; GPX1, glutathione peroxidase 1; SOD, superoxide dismutase; Fe²⁺, ferrous iron; O₂, oxygen; H₂O, water; NO^., nitric oxide; ONOO⁻, peroxynitrite; HO^., hydroxyl radical; ONO^., peroxynitrite radical; O₂^.−, superoxide; H₂O₂, hydrogen peroxide.

Figure 4

ROS production in mitochondria. Acyl-CoA imported into the mitochondria is converted by β-oxidation into acetyl-CoA, which enters the tricarboxylic acid cycle (TCA), resulting in the production of nicotinamide adenine dinucleotide (NADH) and succinate. Mitochondria have two respiratory chains composed of different complexes: (i) complexes I (NADH-ubiquinone oxidoreductase), III (cytochrome b-c1), and IV (cytochrome c oxidase). Complex I reduces NADH to NAD⁺ and H⁺ after electron (e−) release, and (ii) complexes II (succinate-quinone oxidoreductase), III, and IV. Complex II catalyzes the dehydrogenation and oxidation of succinic acid into furamate and 2H₂ after e−release. Released electrons pass through complexes and other molecules in the mitochondria inner membrane ubiquinone (Q) and cytochrome c (cyt c). Complex V (F1F0-ATP synthase) uses the chemiosmotic proton gradient to power the synthesis of ATP from adenosine-diphosphate (APD) and Pi. In red: impairment of the electron transport chain results in the leakage of e− that react directly with oxygen to form the superoxide anion radical transformed in H₂O₂ through SOD2 activity. H₂O₂ can react with Fe²⁺ to form OH^. (Fenton reaction). H₂O₂ is processed into H₂O and O₂ by the antioxidant enzymes GPX1 and catalase. Mitochondrial ROS production causes oxidative mtDNA, lipid, and protein damage. Other abbreviations: CAT, catalase; GPX1, glutathione peroxidase 1; SOD2, superoxide dismutase; Fe²⁺, ferrous iron; O₂, oxygen; H₂O, water; HO^., hydroxyl radical; O₂^.−, superoxide; H₂O₂, hydrogen peroxide; ATP, adenosine-triphosphate; Pi, inorganic phosphate; NAD⁺, nicotinamide adenine dinucleotide; H⁺, hydrogen; mtDNA, mitochondrial DNA; GSH, glutathione; GSSG, oxidized glutathione; ROS, reactive oxygen species.

Figure 5

ROS production in endoplasmic reticulum. Lipid overload causes accumulation of unfolded proteins in the endoplasmic reticulum (ER) by the reduction of reduced glutathione (GSH) to oxidized glutathione (GSSH), resulting in the breakage of protein disulfide bonds (SH). The ER resident proteins, protein disulfide isomerase (PDI) and ER oxidoreductin 1 (ERO1), are involved in disulfide bond formation and superoxide (O₂^.−) and hydrogen peroxide (H₂O₂) release through electron transfer. ROS production and ER stress activate the unfolded protein response (UPR), which leads to a decrease in GSH. UPR is regulated by three transmembrane proteins: protein kinase RNA-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring signaling protein 1 (IRE1), which promote the transcription of CCAAT/enhancer-binding protein homologous protein (CHOP) that further induces ROS generation. ER stress is associated with reduced sarco/endoplasmatic reticulum Ca²⁺-ATPase (SERCA) activity, leading to calcium leakage and decreased mitochondrial electron transport chain (ETC) activity, resulting in ROS production. Other abbreviations: O₂, oxygen; FFA, free fatty acid; ROS, reactive oxygen species; Ca²⁺, calcium.

Figure 6

ROS production in the cytoplasm. In the cytoplasm, interaction of NADPH oxidase (NOX) and xanthine oxidase (XO) with oxygen (O₂) leads to the formation of superoxide radical (O₂^.−). O₂^.− dismutation by the antioxidant enzyme superoxide dismutase (SOD) forms hydrogen peroxide (H₂O₂), which is decomposed to O₂ and water (H₂O) by the enzyme catalase (CAT) or the enzyme glutathione peroxidase 1 (GPX1), leading to the oxidation of reduced GSH into glutathione disulfide (GSSG). Reactive nitrogen species (RNS) are derived from nitric oxide (^.NO) and superoxide (O₂^.−) produced via specific enzymes such as NADPH oxidase; the reaction of ^.NO with O₂^.− produces peroxynitrite (ONOO⁻). ONOO⁻ can react with other molecules to form additional types of RNS including nitrogen dioxide (NO₂^.) as well as other types of chemically reactive free radicals (^.OH). H₂O₂ forms a hydroxyl radical (HO^.), which causes lipid peroxidation leading to malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), or interacts with DNA causing DNA damage. H₂O₂ can also react with ferrous iron (Fe²⁺) to form hydroxyl radical (OH^.) and OH⁻, called the Fenton reaction. Other abbreviations: NADH, nicotinamide adenine dinucleotide; H₂O₂, hydrogen peroxide; DNA, Deoxyribonucleic acid; XO, xanthine oxidase; ECT, electron transport chain; H₂O, water.

Figure 7

Role of nutrients in hepatic ROS production. High intake of diets rich in fructose or cholesterol promotes hepatic oxidative stress. Fructose metabolism leads to adenosine triphosphate (ATP) depletion and uric acid production, which both trigger reactive oxygen species (ROS) generation in the liver. High fructose consumption can also cause gut dysbiosis, which influences the liver oxidative status. Dietary cholesterol induces mitochondrial dysfunction and further mitochondrial ROS production. In contrast, several micronutrients such as vitamins, polyphenols, carotenoids, some amino acids (glycine, serine), and others have antioxidant properties and may be beneficial in NAFLD. Other abbreviations: ADP, adenosine diphosphate; F1P, fructose-1-phosphate; Nrf2, nuclear factor erythroid-2-related factor 2; GSH, reduced glutathione; FAs, fatty acids; PUFAs, polyunsaturated fatty acids.

Figure 8

Mechanisms of pesticide-induced oxidative stress. Reactive oxygen species (ROS) can be produced from several sources during metabolism, as a result of biotransformation of pesticides by cytochrome P450 enzymes, which catalyze the oxidation reactions. Moreover, the mitochondrial complexes, except complex IV, are targets of at least one pesticide family, leading to a disruption in the electron transport chain (ETC) and ROS production. Pesticides can affect antioxidant pathways by (i) modifying the expression of erythroid2-like 2 (Nrf2), (ii) changing Nrf2 promoter methylation, and (iii) altering the expression of sirtuins (Sirts; mainly Sirt1, 3, and 6), which are members of a NAD⁺-dependent type III deacetylase enzyme family. They are considered to be stress adaptors to oxidative, genotoxic, and metabolic stress. ARE, antioxidant response element; SOD, superoxide dismutase; GPX glutathione peroxidase.