Oxidative Stress in NAFLD: Role of Nutrients and Food Contaminants

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.


General Overview
Non-alcoholic fatty liver disease (NAFLD) frequently co-exists with metabolic syndrome and thus often is defined as the liver expression of dyslipidemia, insulin resistance, and obesity. However, it can also occur in patients with low body mass index (<25 kg/m 2 ), a condition known as lean NAFLD [1][2][3]. NAFLD is characterized by fat accumulation in hepatocytes and comprises a spectrum of disease. This spectrum ranges from simple steatosis (non-alcoholic fatty liver or NAFL) to non-alcoholic steatohepatitis (NASH) with inflammation, hepatocyte ballooning, and various levels of fibrosis, associated with a significant risk for progression to cirrhosis and liver cancer (hepatocellular carcinoma (HCC)). In fact, only 20% of NAFLD patients develop NASH, and 4% of patients with NAFL and The strong association between NAFLD and obesity further reinforces the role of dietary factors in the pathogenesis of NAFLD. Hence, in the absence of an approved drug treatment, as noted, a lifestyle intervention that targets weight loss is the primary therapy for the management of NAFLD. The effectiveness of weight reduction was recently confirmed in a meta-analysis of 22 randomized clinical trials that included more than 2500 patients with NAFLD. The results showed that weight loss intervention always improved their serum and histologic parameters, although no changes in liver fibrosis were observed [90].
In general, patients with NAFLD have a higher daily energy intake compared with healthy controls [91], and calorie restriction leading to weight loss has beneficial effects in these patients [92]. Both acute and chronic calorie-restricted diets with either a low-fat or low-carbohydrate content are associated with reduced liver fat in patients with obesity [93,94]. Hypocaloric diets are also effective in improving NAFLD in patients with diabetes and children with obesity [95,96]. In experimental models, calorie restriction extends lifespan and reduces the hepatic steatosis associated with obesity [92,97,98].

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Gut microbiota Some nutrients known to promote NAFLD, such as fructose, affect the gut microbiota and cause dysbiosis (an altered gut microbiota composition). These changes impair the permeability of the gut barrier and trigger the development of metabolic syndrome and its associated perturbations. In contrast, intake of dietary fiber, probiotics, and prebiotics associated with physical training is effective in improving NAFLD [99][100][101].
Of note, the gut microbiota is also required for the hepatic clock daily oscillations that regulate metabolic gene expression for optimal liver function [102][103][104]. In fact, the liver and gut are in close anatomical and functional relation via the portal vein. Diet, gut microbiota, and the liver constitute an axis that can promote either liver health or NAFLD progression. Analyses of the variations in gut microbiota composition in NAFLD have identified specific bacterial species that directly impact NAFLD progression [105]. In support of a beneficial effect, the administration of butyrate, a short chain FA produced by the gut microbiota, improves hepatic inflammation and fat accumulation in NAFLD mouse models [106]. However, despite many animal studies demonstrating a relationship between dysbiosis and NAFLD, only a limited number of cross-sectional human studies have investigated the role of the gut microbiota in NAFLD or NASH, with variable results [107]. Intestinal microbiota dysbiosis also has been associated with other liver diseases in animals and humans, including alcohol-related liver disease, cirrhosis, and HCC [107].
• Food contaminants: the example of pesticides Pesticides are considered as risk factors for human health. Various epidemiological studies reported a correlation between occupational exposure to these products and the development of pathologies, such as cancers, neurodegenerative diseases, and metabolic disorders [108][109][110][111]. Moreover, recent population studies (Nutrinetcohort in France) showed that the consumption of organic food correlated with a significant decreased risk of metabolic disease and cancer [112][113][114]. In addition, pesticides are bioactive compounds that exert an impact on various pathways underlying diseases [115]. Environmental contaminants are considered to play major roles in the etiology and progression of NAFLD. Endocrine-disrupting compounds were first described as inducing reproductive disorders but can be involved in the etiology of diabetes and other metabolic disorders independently of their hormone signaling effects [116][117][118][119][120][121][122]. In addition to contaminants such as polychlorinated biphenyls (PCBs), other compounds such as dioxins [123], bisphenol A [122], and pesticides are suspected to contribute to the increased prevalence of NAFLD.
Perinatal exposure to some pesticides has been correlated with metabolic changes [162], increased insulin levels in newborns [163], and overweight in childhood [164,165]. Experimental studies evaluating the consequences of pesticide exposure during the critical window of pre-and postnatal development (pregnancy and lactation) confirm that early life exposure to pesticides may have a role in metabolic disruption later in life (Table 1). A metabolic impact of perinatal exposure to pesticides in animal models has been reported for insecticides, such as chlorpyrifos [140,166] and imidacloprid [142]; herbicides, such as glyphosate [150] and 2-4 dichlorophenoxyacetic acid (2,4-D) [155]; and pesticide mixtures [167,168]. The metabolic consequences of early life exposure to organophosphate have been reviewed previously [61,74,169]. Early life exposure to fungicides has been tied to metabolic disturbances, reduced liver weight, and histopathological changes [170].

Type of Pesticide
The fact that some pesticides have antimicrobial activity implies that they affect the composition and metabolic functions of the gut microbiota and may be metabolized by the microbiota [212,213]. For example, the organochloride dichloro-diphenyl-trichloroethane (DDT) is converted to DDD by the rat and human microbiota (Eubacterium limosum) [214]. When administered by oral gavage in mice, DEE, a metabolite of DDT, impacts bile acid metabolism and reduces the relative abundance of Bacteroidetes, Verrucomicrobia, and Actinobacteria, whereas that of Firmicutes and Proteobacteria is increased. Furthermore, genes involved in bile acid synthesis are upregulated in the liver [215]. Many studies mainly using rats have analyzed the impact of other insecticides on the microbiota, and important changes in its composition have been reported (reviewed in [213]). Relatively little is known about how glyphosate, the most widely used herbicide worldwide, affects NAFL in terms of gut microbiota, but glyphosate has a profound effect on both gut microbiota composition [216] and metabolism [217] in rodents. Roundup, whose active compound is glyphosate, is suspected to lead to greater alterations in the gut microbiota composition than glyphosate alone, alterations that are similar to those reported in NAFLD, obesity, and systemic inflammation [213,218,219]. Finally, fungicides also cause an important dysbiosis and, one of them, carbendazim, has been associated with hepatic oxidative stress [220]. Overall, pesticides in general are associated with important dysbiosis but the link among pesticides, dysbiosis, and hepatic oxidative stress in NAFLD is not well established, and further studies are warranted. Table 2. Metabolic impact of pesticide exposure in high-fat diet-fed animal models. Acceptable Daily Intake (AD)I values were from https://ephy.anses.fr/ and https://ec.europa.eu/food/plant/pesticides/eu-pesticides-database/. ADI value is not reported for some compounds due to insufficient data (organochlorine compounds, prinomectin, moxidectin, GW4064, PCB). ALT, alanine aminotransferase; BW, body weight; FFA, free fatty acid; CPT, carnitine palmitoyl-transferase; FXR, farnesoid X receptor; GD, gestational day; GIP, glucose-dependent insulinotropic peptide; GLP-1, glucagon-like peptide-1; HFD, high-fat diet; HOMA-IR, homeostasis model assessment of insulin resistance. NEFA, non-esterified fatty acid; NOAEL, no observable adverse effect level; ND, normal diet; PCB polychlorinated biphenyl; PP, pancreatic polypeptide; PUFA, polyunsaturated fatty acid; PND, postnatal day; SFA, saturated fatty acid; TG, triglyceride; UCP, uncoupling protein; VLDL, Very low density lipoprotein; WT, wild type; ADI, Acceptable Daily Intake. Increased antioxidant enzyme activities more in ND-than in HFD-fed animals • Increased UCP2 mRNA levels in ND-and HFD-fed animals [203] • ND-and HFD-fed male rats exposed to 100 µkg/BW/day DDE in drinking water for 12 weeks • Increased accumulation in fatty acid (SFA and PUFA) content in both ND-and HFD-fed animals [204] Insecticide  • ND-and HFD-fed male mice • Co-exposure by daily gavage with 5 mg/kg for 12 weeks • Increased BW in both ND-and HFD-fed mice • Impaired glucose metabolism and insulin resistance in both HFD-and ND-fed mice [209] • ND-and HFD-fed male rats • Co-exposure by daily oral gavage to 0.3 and 3 mg/kg BW for 9 weeks    [202] Other environmental factors can affect NAFLD development and progression. Although the effect of active smoking on NAFLD development remained controversial [222] a recent systematic review and meta-analysis from 20 observational studies revealed a slight but significant association between active and passive smoking and NAFLD [223]. The link between a modest alcohol consumption with NAFLD development is described in a very recent review [224].

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Aim of the review There is currently no effective pharmacological therapy for the treatment of NAFLD. Weight loss, achieved through lifestyle changes including diet modifications and exercise, remains the most effective strategy for NAFLD management. However, these lifestyle interventions are difficult to maintain for most patients. Vitamin E and the anti-diabetic agent Pioglitazone are the two classic therapies currently used to treat NAFLD patients but they can both induce adverse effects and their benefit on liver fibrosis is either absent or still not clear. Most of the other molecules that are under investigation target the metabolic comorbidities of NAFLD such as obesity, insulin resistance, and dyslipidemia [225]. A better understanding of the development and progression of this complex pathology will allow the development of novel therapeutic strategies for the treatment of NAFLD.
As is well known, alterations in lipid metabolism are key to the development of NAFLD and its progression. They can affect different reactive oxygen species (ROS) generators, such as mitochondria, endoplasmic reticulum (ER), and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) [226]. Although many aspects of the contributions of these disturbances to NAFLD remain unexplored, much progress has been accomplished in recent years. Knowledge is accumulating on how increased ROS generation triggers changes in insulin sensitivity and the activity of key enzymes of lipid metabolism, innate immune system signaling, and the level of inflammatory responses. Our review addresses the mechanisms and consequences of excessive ROS production in the liver that drives NAFLD progression, and the roles of nutrients and food contaminants in these processes ( Figure 1).
Our discussion on the roles of oxidative stress on NAFLD development covers knowledge gained from both animal models, mainly rodent models, and humans. It is important to keep in mind, while reading the review, that rodents and humans NAFLD are not equivalent, although the FDA encourages the use of appropriate animal models for developing new treatments against pre-cirrhotic NASH [227]. The current animal models mimicking the progression of the human NAFLD phenotype rely on genetic alterations, chemically induced liver damage or diets deficient in essential nutrients. Therefore, they do not fully match, mechanistically and biochemically, the lifestyle-associated NAFLD in humans [228]. There are several models used currently, each presenting advantages and limitations [229,230]). A useful animal model, from a clinician's view point, should reflect the important metabolic deteriorations seen in human NAFLD patients including hyperglycemia and insulin resistance, hyperlipidemia, and obesity with liver fat accumulation. Fatty liver should show progression from simple steatosis to the inflamed liver stage with ballooning and fibrosis monitored by histopathological examination and quantified by recognized scoring systems. The progression should be relatively fast allowing the evaluation of candidate drugs and nutritional interventions. Despite relentless efforts and the advances made in recent years, the ideal rodent model comprising all desirable characteristics is not yet available. Therefore, studies in different models are necessary to perform all the analyses required for a better understanding of human NAFLD progression. progression. They can affect different reactive oxygen species (ROS) generators, such as mitochondria, endoplasmic reticulum (ER), and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) [226]. Although many aspects of the contributions of these disturbances to NAFLD remain unexplored, much progress has been accomplished in recent years. Knowledge is accumulating on how increased ROS generation triggers changes in insulin sensitivity and the activity of key enzymes of lipid metabolism, innate immune system signaling, and the level of inflammatory responses. Our review addresses the mechanisms and consequences of excessive ROS production in the liver that drives NAFLD progression, and the roles of nutrients and food contaminants in these processes ( Figure 1).  . 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.

Methods
We performed a PubMed search of the literature. Studies that were not published in English or published in the grey literature were excluded. We tried to focus on articles published during the last 10 years. We established a list of keys words (below) and combined them according to each author's dedicated chapter in order to get a representative overview of the literature. Each author reviewed the chapter of the other co-authors. Furthermore the corresponding authors, worked on the homogeneity and uniformity of the different chapters. The following lists of keywords were used: (i) "Oxidative stress" OR "oxidative phosphorylation" OR "oxidation-reduction potential" OR "reactive oxygen species" OR "ROS" OR "reactive nitrogen species " OR "RNS" OR "redox stress" OR "peroxidation" OR "free radicals" OR "lipotoxicity" OR mitochondria OR endoplasmic reticulum OR glutathione OR antioxidant pathways OR Nrf2 OR SIRT OR SOD OR GPX OR Catalase (ii) "metabolic hepatitis" OR "metabolic liver" OR "Non Alcoholic Fatty Liver Disease" OR "Nonalcoholic Fatty Liver Disease" OR "Fatty Liver" OR "Nonalcoholic Fatty Livers" OR "Nonalcoholic Liver" OR "Nonalcoholic Fatty" OR "Nonalcoholic Fatty Liver" OR "Nonalcoholic Steatohepatitis" OR "Nonalcoholic Steatohepatitides" OR "Steatohepatitides" OR "Nonalcoholic Steatohepatitis" OR "cirrhosis" OR "carcinogenesis" OR "hepatocarcinoma" OR "hepatocellular carcinoma" OR "carcinoma" OR "hepatoma" OR malignant hepatoma" (iii) "mice" OR "mouse" OR "mus musculus" OR "clinical studies" OR "therapeutic" (iv) "pesticides" OR "herbicides" OR "insecticides" OR "fungicides" OR "plant protection product" (v) "oxidant" OR "antioxidant" OR "diet" OR "cholesterol" OR "fructose" OR "nutrients" OR "micronutrients", "macronutrients" OR "fat" OR "western diet" OR "sugars" OR "fibers" OR "vitamins" OR "fatty acid".

Role of Oxidative Stress in NAFLD Pathogenesis
Oxidative stress is defined as an imbalance between the cellular levels of antioxidants and that of pro-oxidants, including ROS and reactive nitrogen species (Figure 2), which causes cellular damage and, in most cases, cell death. In healthy physiological conditions, cells maintain a basal level of ROS to promote the balanced redox signaling required for various processes such as cell metabolism, cell differentiation and survival, immune defense, and modulation of transcription factor activity and epigenetic state [226]. Upon oxidative stress, the antioxidant enzyme superoxide dismutase (SOD) generates hydrogen peroxide (H 2 O 2 ) from the superoxide radical (O 2 .− ), which is then processed to oxygen (O 2 ) and water (H 2 O) through glutathione peroxidase (Gpx) or catalase enzyme activities ( Figure 2) [231]. Within cells, ROS are mainly produced in the mitochondria, the peroxisomes, and the ER, but cytoplasmic production of ROS also occurs. High levels of ROS alter these organelles, further enhancing oxidative stress and creating a vicious circle.

Role of Oxidative Stress in NAFLD Pathogenesis
Oxidative stress is defined as an imbalance between the cellular levels of antioxidants and that of pro-oxidants, including ROS and reactive nitrogen species (Figure 2), which causes cellular damage and, in most cases, cell death. In healthy physiological conditions, cells maintain a basal level of ROS to promote the balanced redox signaling required for various processes such as cell metabolism, cell differentiation and survival, immune defense, and modulation of transcription factor activity and epigenetic state [226]. Upon oxidative stress, the antioxidant enzyme superoxide dismutase (SOD) generates hydrogen peroxide (H2O2) from the superoxide radical (O2 .− ), which is then processed to oxygen (O2) and water (H2O) through glutathione peroxidase (Gpx) or catalase enzyme activities ( Figure 2) [231]. Within cells, ROS are mainly produced in the mitochondria, the peroxisomes, and the ER, but cytoplasmic production of ROS also occurs. High levels of ROS alter these organelles, further enhancing oxidative stress and creating a vicious circle. 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 Ca 2+ 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) Ca 2+ 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; 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 Ca 2+ 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) Ca 2+ 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 Ca 2+ -ATPase; JNK, c-Jun N-terminal kinase; NF-κB, nuclear factor-kappa B.

Evidence for a Role of Oxidative Stress in NAFLD
Oxidative stress promotes activation of enzymatic or non-enzymatic-mediated antioxidant mechanisms that counteract ROS production. Both clinical and experimental studies show that these antioxidant pathways are modulated during NAFLD progression. In fact, activity of the antioxidant enzymes SOD and Gpx increases in patients with NAFLD (Figures 2-5) [232]. In vitro, hepatic stellate cells deficient for the glutathione peroxidase 7 (Gpx7) isoform present increased expression of profibrotic and pro-inflammatory genes in response to FFA exposure. Consistent with these results, overexpression of GPX7 in these cells decreases ROS generation and expression of profibrotic and pro-inflammatory genes. In vivo, Gpx7 deficiency exacerbates choline-deficient, L-amino-defined, high-fat diet-induced NASH fibrosis [233]. The expression of glutaminase 1 (GLS1) is increased in both NASH preclinical mouse models and clinical NASH liver biopsies. GLS1 promotes glutamine fueling of anaplerotic mitochondrial metabolism resulting in increased ROS production. In methionine choline-deficient (MCD) diet-fed mice, GLS1 inhibition decreases hepatic TG accumulation by restoring VLDL TG export and diminishes oxidative stress by lowering ROS production. GLS1 deficiency in this model is also associated with decreased lipid peroxidation [234]. Paraoxonase-1 is a liver antioxidant enzyme that hydrolyses peroxide and lactones associated with lipoproteins. In a cohort of 81 patients with NAFLD, the serum Paraoxonase-1 concentration was decreased, which could reflect higher oxidative stress in these patients [235].

Evidence for a Role of Oxidative Stress in NAFLD
Oxidative stress promotes activation of enzymatic or non-enzymatic-mediated antioxidant mechanisms that counteract ROS production. Both clinical and experimental studies show that these antioxidant pathways are modulated during NAFLD progression. In fact, activity of the antioxidant enzymes SOD and Gpx increases in patients with NAFLD (Figures 2-5) [232]. In vitro, hepatic stellate cells deficient for the glutathione peroxidase 7 (Gpx7) isoform present increased expression of profibrotic and pro-inflammatory genes in response to FFA exposure. Consistent with these results, overexpression of GPX7 in these cells decreases ROS generation and expression of profibrotic and pro-inflammatory genes. In vivo, Gpx7 deficiency exacerbates choline-deficient, L-amino-defined, high-fat diet-induced NASH fibrosis [233]. The expression of glutaminase 1 (GLS1) is increased in both NASH preclinical mouse models and clinical NASH liver biopsies. GLS1 promotes glutamine fueling of anaplerotic mitochondrial metabolism resulting in increased ROS production. In methionine choline-deficient (MCD) diet-fed mice, GLS1 inhibition decreases hepatic TG accumulation by restoring VLDL TG export and diminishes oxidative stress by lowering ROS production. GLS1 deficiency in this model is also associated with decreased lipid peroxidation [234]. Paraoxonase-1 is a liver antioxidant enzyme that hydrolyses peroxide and lactones associated with lipoproteins. In a cohort of 81 patients with NAFLD, the serum Paraoxonase-1 concentration was decreased, which could reflect higher oxidative stress in these patients [235].
The peroxisomal antioxidant enzyme catalase plays a key role in protecting cells from oxidative damage by reducing H2O2 concentration ( Figure 3). In HFD-fed mice deficient for catalase, lipid accumulation and oxidative stress are exacerbated [236]. These mice develop an imbalanced redox status in peroxisomes because of increased H2O2 levels, which in turn induces ER stress in the liver. Catalase inhibition in HepG2 cells increases ROS production by peroxisomes, causing ER stress and FA accumulation [237]. In line with this observation, human liver cells with impaired peroxisome biogenesis show decreased ER stress, oxidative stress, and apoptosis [238].     Mitochondrial ROS production results from dysfunction in complexes I and II of the mitochondrial respiratory chain. Various mechanisms leading to this dysfunction and ROS production have been described in NAFLD. In ob/ob mice, the quantity of mitochondrial respiratory chain complex I is decreased. In this model, mitochondrial oxidative stress causes mitochondrial DNA (mtDNA) damage, resulting in the reduction of mtDNA-encoded subunits of respiratory chain complex I. mtDNA is in the mitochondrial matrix near the mitochondrial respiratory chain where most ROS are generated. Thus, it is particularly prone to oxidative damage compared with nuclear DNA, which is more protected inside the cell nucleus. Furthermore, mtDNA lacks protective histones, which together with the absence of an mtDNA repair system, makes it more susceptible to damage [246]. Mitochondrial complex I dysfunction can also arise from mitochondrial protein 3-tyrosine nitration by peroxynitrite anion. Peroxynitrite originates from a combination of nitric oxide (NO . ) with superoxide (O2 .− ), which are both elevated in ob/ob mice and in patients with NAFLD. In vitro, incubation of mitochondrial proteins from wild-type (WT) mice with peroxynitrite induces their 3- . 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 2H 2 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 H 2  The peroxisomal antioxidant enzyme catalase plays a key role in protecting cells from oxidative damage by reducing H 2 O 2 concentration (Figure 3). In HFD-fed mice deficient for catalase, lipid accumulation and oxidative stress are exacerbated [236]. These mice develop an imbalanced redox status in peroxisomes because of increased H 2 O 2 levels, which in turn induces ER stress in the liver. Catalase inhibition in HepG2 cells increases ROS production by peroxisomes, causing ER stress and FA accumulation [237]. In line with this observation, human liver cells with impaired peroxisome biogenesis show decreased ER stress, oxidative stress, and apoptosis [238].
Expression of genes encoding antioxidant enzymes and regulators of the glutathione pathway (glutamate-cysteine ligase catalytic subunit, glutamate-cysteine ligase modifier subunit, GPX2) is regulated by the transcription factor erythroid2-like 2 (Nrf2) [239]. Upon oxidative stress, Nrf2, which is anchored in the cytoplasm via binding to Keap1, dissociates from it and translocates into the nucleus, where it interacts with specific DNA sequences called antioxidant response elements in the promoter of its target antioxidant enzyme genes [240]. Nrf2 expression is increased in the first stage of NAFLD in preclinical models [241], and pharmacological activation of Nrf2 in mice fed a high-fat and high fructose diet decreases NASH parameters (insulin resistance, weight gain, TG, ALT) via the transcriptional regulation of genes involved in inflammation, apoptosis, fibrosis, ER stress, and oxidative stress [239]. Consistently, Nrf2 deficiency in mice fed an MCD or an HFD promotes progression of steatosis to NASH by increasing oxidative stress, inflammation, and hepatic FA accumulation [242,243]. In addition, Nrf2-deficient mice fed an HFD develop a more severe NASH phenotype than their WT counterparts [241]. In in vivo and in vitro models of NAFLD, the reduction of Nrf2 expression by microRNA is associated with a decreased expression of its target genes, heme oxygenase, Sod2, and NAD(P)H dehydrogenase quinone 1 and an increase in ROS production [244]. Nrf2 also directly affects lipid metabolism by activation of genes involved in FA oxidation (acyl-CoA Oxidase 2, carnitine palmitoyltransferase 1), TG export (apolipoprotein B), and the lipogenic transcription factor sterol regulatory element binding transcription factor 1 (Srebp-1) [239]. Nfr2 deficiency in HFD-fed mice diminishes phosphorylation of acetyl-CoA carboxylate (ACC), a rate-limiting enzyme of hepatic FA synthesis, and thus increases its activity [241]. Collectively, these data indicate that alterations in antioxidant pathways are associated with NAFLD, suggesting a role of oxidative stress in disease progression.
Biomolecules 2020, 10, x FOR PEER REVIEW 28 of 69 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 Ca 2+ -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; Ca 2+ , calcium.
To summarize, ER stress induced by lipid accumulation during NAFLD promotes ER ROS production and calcium homeostasis disruption that both act to increase oxidative stress during disease progression ( Figure 5). ER stress also modulates expression of proteins involved in lipid and glucose metabolism, thus contributing directly to hepatic lipid accumulation [262]. Finally, in addition to mitochondria and ER, the peroxisomes, which are important in H2O2 production by peroxisomal FA oxidation, contribute to oxidative stress in NAFLD (Figure 3) [226,237,238,263].

Cytoplasmic Production of ROS
Patients with NAFLD present with altered expression of cytochrome P (CYP)450 enzymes, which are involved in detoxification, FA oxidation, inflammation, and oxidative stress ( Figure 6) [264]. In these patients, for example, increased serum lipid peroxidation correlates with a higher level of CYP4A11. In contrast, in the hepatocyte cell line HepG2 exposed to FFA, CYP4A11 inhibition led to reduced ROS production associated with decreased lipid accumulation and reduced proinflammatory cytokine levels, such as tumor necrosis factor α (TNF-α), interleukin (IL)-6, and IL-1β [265]. In HFD-fed mice, CYP4A deficiency reduces hepatic ER stress, apoptosis, insulin resistance, and steatosis, suggesting a role for CYP4A in NAFLD pathogenesis. CYP2E1, which is expressed in 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 Ca 2+ -ATPase (SERCA) activity, leading to calcium leakage and decreased mitochondrial electron transport chain (ETC) activity, resulting in ROS production. Other abbreviations: O 2 , oxygen; FFA, free fatty acid; ROS, reactive oxygen species; Ca 2+ , calcium.

Mitochondria-Mediated Oxidative Stress
Mitochondria are intracellular sites of oxygen (O 2 ) consumption. In case of energy imbalance, such as lipid accumulation, mitochondria ROS production increases considerably because of mitochondrial respiratory chain alteration and a reduction in electron capture through various mechanisms discussed below (Figure 4). Generation of mitochondrial ROS results in impairment of the mitochondrial membrane potential generated by proton pumps and activation of the JNK (c-Jun N-terminal kinase) and AMPK (5' AMP-activated protein kinase) pathways. All of these mechanisms enhance oxidative stress and lipid accumulation and promote inflammation, thereby contributing to the development of obesity and metabolic diseases, including NAFLD [245].
Mitochondrial ROS production results from dysfunction in complexes I and II of the mitochondrial respiratory chain. Various mechanisms leading to this dysfunction and ROS production have been described in NAFLD. In ob/ob mice, the quantity of mitochondrial respiratory chain complex I is decreased. In this model, mitochondrial oxidative stress causes mitochondrial DNA (mtDNA) damage, resulting in the reduction of mtDNA-encoded subunits of respiratory chain complex I. mtDNA is in the mitochondrial matrix near the mitochondrial respiratory chain where most ROS are generated. Thus, it is particularly prone to oxidative damage compared with nuclear DNA, which is more protected inside the cell nucleus. Furthermore, mtDNA lacks protective histones, which together with the absence of an mtDNA repair system, makes it more susceptible to damage [246]. Mitochondrial complex I dysfunction can also arise from mitochondrial protein 3-tyrosine nitration by peroxynitrite anion. Peroxynitrite originates from a combination of nitric oxide (NO . ) with superoxide (O 2 .− ), which are both elevated in ob/ob mice and in patients with NAFLD. In vitro, incubation of mitochondrial proteins from wild-type (WT) mice with peroxynitrite induces their 3-tyrosine nitration, which results in decreased complex I activity and mitochondrial oxygen consumption. This decrease is amplified by reduced expression of prohibitin, a protein that protects mitochondrial complexes against degradation [246]. Complex I or II activity is also regulated by the mitochondrial histone deacetylase sirtuin (SIRT) 3.
Mice fed an HFD have decreased SIRT3 expression, which results in a higher level of membrane transport chain complex I acetylation, causing mitochondrial dysfunction. MCD-fed mice deficient in SIRT3 develop more severe liver lesions, inflammation, and fibrosis compared with WT mice [247]. The proper functioning of the mitochondrial respiratory chain requires ATP, and in turn, impaired ATP production leads to transport chain complex dysfunction. In NAFLD, oxidative phosphorylation (OXPHOS) and the TCA cycle are disturbed, which causes impaired ATP production leading to an alteration in mitochondrial respiration and ROS production. In patients with NASH, antioxidant capacity and ATP production decreases, which enhances hepatocellular damage and insulin resistance, promoting NAFLD progression [248]. Mitochondria are dynamic organelles, and fission and fusion are crucial for maintaining their function under environmental stress. Both processes are regulated by specific proteins, Fis-1 and Drp-1 for fission and Mfn-2 for fusion, which show decreased expression in WD-fed mice, suggesting that mitochondrial dynamism is affected in NAFLD [249]. ROS accumulation in mitochondria leads to modification of several mechanisms associated with oxidative stress and NAFLD development. Phosphorylated JNK under lipotoxicity or ER stress conditions phosphorylates SH3-domain binding protein 5 at the outer mitochondrial membrane, leading to the inactivation of mitochondrial c-Src, which regulates the phosphorylation of respiratory chain components. This Src inactivation alters electron transport, which promotes increased ROS release [250]. Phosphorylated JNK is also associated with insulin resistance, which further suggests its involvement in NAFLD development [251]. Oxidative stress induced in HFD-fed mice causes liver damage through mitochondrial membrane potential alteration [245]. ROS accumulation in mouse models of NAFLD affects mitochondrial depolarization potential through the formation of aldehydes (malondialdehyde [MDA] and 4-hydroxynonenal [4-HNE]) by lipid peroxidation or an increase in mitochondrial sensitivity to Ca 2+ .
Mitochondrial depolarization is a dysfunction that appears early in NAFLD development and contributes to mitochondrial homeostasis deregulation. In fact, WD-fed mice show mitochondrial depolarization at an early stage of NASH, with an increased protein level of PINK1 (PTEN-induced kinase 1), a mediator of mitochondrial autophagy (mitophagy), which is associated with mitochondrial dysfunction. In these situations of mitochondrial depolarization, the mitophagic burden increases, mitochondrial biogenesis declines, and mitochondrial depletion occurs after 2 to 6 months.
These alterations are thought to be important in promoting steatosis, inflammation, and progression to fibrosis [249].
The ROS H 2 O 2 , produced by mitochondria, activates AMPK through modification of the ATP/ADP ratio [252], which further regulates antioxidant enzyme gene expression through Nrf2 activation [253]. The AMPK pathway may be activated by an antioxidant component Peroxiredoxin 5 (Prx5). Indeed, in HepG2 cells exposed to FFAs, mitochondrial Prx5 activates the AMPK pathway to regulate the activity of lipogenic enzymes (ACC, SREBP-1, FA synthase) [254]. AMPK stimulates glucose and FA oxidation by induction of CPT-1 and acyl-CoA dehydrogenase expression via PPAR gamma coactivator 1-alpha and PPARα [253]. Thus, although the AMPK pathway represents a protective response against oxidative stress and lipid accumulation, prolonged oxidative stress alters this pathway, leading to lipid accumulation and further enhanced oxidative stress. A recent study showed that the AMP kinase pathway is inhibited in NASH, leading to the activation of caspase 6-associated cell death [255]. Similarly, other antioxidant responses may have deleterious effects. Early upregulation of uncoupling protein-2 (UCP-2) during steatosis protects hepatocytes against ROS production through ATP production, whereas a further increase in UCP-2 during NASH leads to chronic ATP depletion [256].
Overall, in NAFLD, mitochondria are an important source of ROS because of alterations in complex I and II activity that initially lead to the activation of antioxidant mechanisms. However, during prolonged oxidative stress, these mechanisms participate in oxidative damage and enhance mitochondrial oxidative stress, thus contributing to the development of NAFLD (Figure 4).

ER-Mediated Oxidative Stress
The ER is an organelle connected to the nuclear membrane in eukaryotic cells and is abundant in hepatocytes because of their high metabolic activity. It has multiple functions including protein synthesis, folding, modification, and trafficking, and synthesis of lipids and steroid hormones. Alteration of ER homeostasis, i.e., ER stress, can induce oxidative stress ( Figure 5). The ER stress response is defined by activation of the unfolded protein response (UPR) pathway. Prolonged ER stress increases the PKR-like ER protein kinase (PERK) and activated transcription factor (ATF) 6 pathways that both lead to ROS generation through the proapoptotic C/EBP homologous protein (CHOP), a specific protein of the ER stress response ( Figure 5). CHOP is involved in oxidative stress induction in mouse models of type 2 diabetes and MCD-induced steatohepatitis [226,257]. Recently, ER stress was shown to be involved in lipogenesis and in the transition of NAFLD to NASH through caspase 2 induction [258]. ER also contributes to the regulation of calcium (Ca 2+ ) homeostasis and particularly Ca 2+ storage in the ER lumen. In animal models of diabetes and obesity, the decreased activity of the ER Ca 2+ pump sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) in hepatocytes leads to ER stress and induces apoptosis [257]. Moreover, impaired Ca 2+ homeostasis in the ER induces mitochondria dysfunction [226].
Other studies have provided evidence for the involvement of the Ca 2+ flux in oxidative stress and ER stress induction. In Buffalo rat liver (BRL-3A) cells exposed to a high FFA concentration to mimic NAFLD, induction of oxidative stress stimulates transcription and translation of the protein calcium release-activated calcium channel protein 1 (Orai1), a plasma membrane protein involved in the Ca 2+ cytosolic flux. The resulting increase in Ca 2+ entry into the cell activates nuclear factor-kappa B (NF-κB) signaling, which further exacerbates the Ca 2+ flux, mitochondrial impairment, and ROS generation, enhancing ER stress. Other mechanisms activated by lipid accumulation and Ca 2+ homeostasis impairment in the liver also enhance ER stress in NAFLD. For instance, in a mouse model of MCD-induced steatohepatitis, increased expression and activation of liver protein kinase Cδ (PKCδ) are associated with ER stress activation. Similar results have been obtained in vitro in mouse hepatic cells cultivated in a palmitic acid-enriched MCD medium. In addition, PKCδ deficiency reduces MCD-and palmitic acid-induced ER stress activation, hepatic TG accumulation, and cell death [259]. These effects may involve CHOP, because in a human hepatic cell line (L02) exposed to palmitic acid, PKCδ deficiency reduces ER stress through decreased CHOP expression. In this model, PKCδ silencing also increases SERCA activity, which improves Ca 2+ homeostasis [260]. In HFD-fed rats, increased plasma FFA is associated with increased expression of markers of oxidative stress and ER stress, as well as Orai1 and NF-κB [261]. Mitochondria and peroxisome functional alterations also can induce ER stress. For example, an imbalance in redox signaling in peroxisomes upon catalase inhibition induces FA accumulation, which leads to ROS production and ER stress [237] (Figure 5).
To summarize, ER stress induced by lipid accumulation during NAFLD promotes ER ROS production and calcium homeostasis disruption that both act to increase oxidative stress during disease progression ( Figure 5). ER stress also modulates expression of proteins involved in lipid and glucose metabolism, thus contributing directly to hepatic lipid accumulation [262]. Finally, in addition to mitochondria and ER, the peroxisomes, which are important in H 2 O 2 production by peroxisomal FA oxidation, contribute to oxidative stress in NAFLD (Figure 3) [226,237,238,263].

Cytoplasmic Production of ROS
Patients with NAFLD present with altered expression of cytochrome P (CYP)450 enzymes, which are involved in detoxification, FA oxidation, inflammation, and oxidative stress ( Figure 6) [264]. In these patients, for example, increased serum lipid peroxidation correlates with a higher level of CYP4A11. In contrast, in the hepatocyte cell line HepG2 exposed to FFA, CYP4A11 inhibition led to reduced ROS production associated with decreased lipid accumulation and reduced pro-inflammatory cytokine levels, such as tumor necrosis factor α (TNF-α), interleukin (IL)-6, and IL-1β [265]. In HFD-fed mice, CYP4A deficiency reduces hepatic ER stress, apoptosis, insulin resistance, and steatosis, suggesting a role for CYP4A in NAFLD pathogenesis. CYP2E1, which is expressed in ER, mitochondria, and cytosol, is increased in experimental models of NAFLD and in patients with NASH, resulting in ROS generation and inactivation of SOD and catalase [236,266]. CYP2E1 has a high NOX activity that promotes lipid peroxidation [265,266]. Furthermore, overexpression of CYP2E1 in mice is associated with severe steatohepatitis and upregulation of antioxidant enzymes (SOD2, catalase, and GPX) [267]. CYP1A1 induces ROS production by the reduction of O 2 to H 2 O 2 and O 2 .− . It also has a role in ω-hydroxylation of PUFAs. The expression of CYP1A1 is enhanced in oleic acid-stimulated HepG2 cells. In these same cells, CYP1A1 small interfering RNA inhibits lipid peroxidation, whereas overexpression of CYP1A1 stimulates lipid peroxidation and reduces SOD expression. Collectively, these observations unveil a regulatory role for CYP1A1 in hepatic lipid peroxidation [264]. CYP450 epoxygenase-derived epoxyeicosatrienoic acids (EETs) are a class of lipid mediators that are abundant in liver and that mediate cytoprotective and anti-inflammatory properties. A CYP450-induced EET increase in HFD-fed mice protects against NAFLD progression. Moreover, overexpression of the CYP2J2 leads to higher EET levels in serum that are associated with decreased hepatic TG levels, inhibition of the NF-κB/JNK signaling pathway, and increased antioxidant enzyme levels. Treatment of HepG2 cells with EET protects against palmitic acid-induced lipotoxicity, oxidative stress, and inflammation [268].
In cytosol, ROS are also produced by many enzymes including xanthine oxidase (XO), cyclo-oxygenase lipoxygenase, and NOX ( Figure 6) [226,269,270]. NOX-mediated H 2 O 2 production causes liver damage [271] and induces a pro-inflammatory response [269]. Patients with NAFLD have a higher NOX2 activity and increased NOX2-derived peptide, a marker of systemic NOX activation [271,272]. In one cohort of children with NAFLD, NOX2 activity increased in parallel with disease severity [271]. Furthermore, HFD-fed mice deficient in NOX2 develop steatosis but not NASH because of reduced OXPHOS dysfunction [273]. In agreement with this finding, NOX2-induced cellular oxidative stress causes inhibition of OXPHOS activity induced by FFA treatment [270]. Modulation of NOX expression also has been reported in experimental models. HFD-fed mice fed have increased NOX expression and activity [273], and NOX1 expression is increased in a mouse model of obesity [274], leading to ROS generation associated with nitrotyrosine protein expression that counteracted oxidative damage [275]. NOX1 also induces ROS production in a mouse model of steatosis [274].
(SOD2, catalase, and GPX) [267]. CYP1A1 induces ROS production by the reduction of O2 to H2O2 and O2 .− . It also has a role in ω-hydroxylation of PUFAs. The expression of CYP1A1 is enhanced in oleic acid-stimulated HepG2 cells. In these same cells, CYP1A1 small interfering RNA inhibits lipid peroxidation, whereas overexpression of CYP1A1 stimulates lipid peroxidation and reduces SOD expression. Collectively, these observations unveil a regulatory role for CYP1A1 in hepatic lipid peroxidation [264]. CYP450 epoxygenase-derived epoxyeicosatrienoic acids (EETs) are a class of lipid mediators that are abundant in liver and that mediate cytoprotective and anti-inflammatory properties. A CYP450induced EET increase in HFD-fed mice protects against NAFLD progression. Moreover, overexpression of the CYP2J2 leads to higher EET levels in serum that are associated with decreased hepatic TG levels, inhibition of the NF-κB/JNK signaling pathway, and increased antioxidant enzyme levels. Treatment of HepG2 cells with EET protects against palmitic acid-induced lipotoxicity, oxidative stress, and inflammation [268].
In cytosol, ROS are also produced by many enzymes including xanthine oxidase (XO), cyclooxygenase lipoxygenase, and NOX ( Figure 6) [226,269,270]. NOX-mediated H2O2 production causes liver damage [271] and induces a pro-inflammatory response [269]. Patients with NAFLD have a higher NOX2 activity and increased NOX2-derived peptide, a marker of systemic NOX activation [271,272]. In one cohort of children with NAFLD, NOX2 activity increased in parallel with disease In humans, NOX4 mRNA expression is upregulated in the liver of patients with NASH [275,276]. The NOX4 single nucleotide polymorphism rs3017887 is associated with increased ALT levels in liver biopsies from patients with NAFLD, indicating hepatocyte damage [269]. In a mouse model of diet-induced NAFLD, hepatocyte-specific deletion of NOX4 or its pharmacological inhibition reduces oxidative stress and fibrosis. Cell culture findings show that NOX4 promotes oxidative stress by ER stress activation [276]. In summary, increased NOX isoform expression and activity are associated with NAFLD and lead to oxidative stress induction via ROS generation concomitant with activation of ER and mitochondrial stress (Figures 4 and 5).
Oxidized phospholipids (OxPLs) play a role in oxidative stress induction through generation of cellular ROS, which promote fibrosis and inflammation that result in NASH progression. OxPLs are produced upon overnutrition or in patients with NAFLD. Treatment of primary hepatocytes from Ldlr −/− hyperlipidemic mice with an OxPL mixture promotes ROS accumulation in the cytoplasm and all organelles, resulting in mitochondrial dysfunction [277]. Treatment of the immortalized human hepatic stellate cell line with OxPLs stimulates fibrogenic gene expression [278]. Furthermore, in a mouse model of NASH, the neutralization of OxPL decreases inflammatory mechanisms [277]. Toll-like receptor (TLR)-4 and TLR-2 are expressed in Kupffer cells and stellate cells, which are associated with fibrosis. Mice fed an HFD supplemented with lecinoxoids (synthetic OxPLs), which inhibit these TLRs, show reduced liver fibrosis and inflammation (reduced IL-1B and IL-6) [279]. Furthermore, the neutralization of OxPL in a mouse model of NAFLD inhibits progression to HCC [277].
Iron metabolism, which is modified in patients with NAFLD [75,[280][281][282], leads to ROS generation because of the ability of the ferrous iron to catalyze the production of OH − from H 2 O 2 , known as the Fenton reaction [257] (Figures 2-4). This point is further developed below in Section 5.

Gut Microbiota and Liver Oxidative Stress
Mice lacking the gut microbiota (germ-free mice) display an altered hepatic pool of glutathione [283]. The gut microbes also alter levels of amino acids and N-acetylated amino acids circulating in the mouse portal vein, affecting host amino acid and glutathione metabolism in the intestine and the liver [284]. A recent study showed that the gut microbiome induces the Nrf2 antioxidant response pathway in the liver [285] and that exogenous administration of Lactobacilli could amplify this response and protect against oxidative liver injury. Of great interest, these authors identified a Lactobacilli-derived metabolite (namely 5-methoxyindoleacetic acid) that could activate hepatic Nrf2 and in part mediate these beneficial effects. The relevance of this gut-liver mechanism in humans remains to be studied.
Unhealthy eating modifies the gut microbiota, as others have reviewed [286,287]. This dysbiosis participates in the development of metabolic diseases and plays a major role in NAFLD pathogenesis [288,289]. In fact, the development of NAFLD correlates with (i) dysbiosis; (ii) a leaky intestinal barrier; (iii) impaired mucosal immunity; (iv) bacteria and bacterial components reaching the liver through the portal vein, as well the bacterial metabolites such as lipopolysaccharides, trimethylamine-N-oxide, N,N,N-trimethyl-5-aminovaleric acid, and endogenous ethanol; and (v) impaired bile acid homeostasis [286,287]. These alterations participate in increased hepatic inflammation and oxidative stress and promote NAFLD development and progression to NASH.
Altogether, whereas the role of oxidative stress in NAFLD pathogenesis has been demonstrated in both clinical and animal studies, the underlying mechanisms are complex and not completely understood. Alterations in mitochondrial respiratory complex activity because of lipid accumulation in NAFLD make the mitochondria the main source of ROS in this condition. ER stress, cytochromes, NOX, and OxPLs are also key components in ROS generation. The consequences of ROS production in NAFLD are enhanced oxidative stress and altered lipid metabolism, which further promote lipid accumulation. In addition, increased ROS production may modulate insulin signaling and inflammatory processes, thus promoting insulin resistance and inflammation, which are important features of NAFLD progression [226] (Figure 2); however, the specific mechanisms underlying oxidative stress-promoted NAFLD need further study. Furthermore, several environmental factors, such as the nutrients and contaminants discussed below, affect oxidative stress in NAFLD.

Oxidative Stress in NAFLD: Role of Nutrients
An unhealthy lifestyle is strongly associated with NAFLD. Of note, dietary constituents are key factors in NAFLD pathogenesis. Such factors include a high intake of calories and an excessive consumption of saturated fats, refined carbohydrates, and animal proteins. Several nutrients are well known to affect the metabolic pathways leading to hepatic fat accumulation in the first steps of NAFLD development. Accumulating data also support the idea that diet-mediated alterations include increased ROS production and oxidative stress, which are key factors in the progression of NAFLD to NASH, as noted above (Figure 7). Given the essential role of nutrition in the etiology of NAFLD, several epidemiological studies have evaluated its association with dietary habits.

Dietary Oxidants in NAFLD
A correlation between consumption of different nutrients and oxidative stress has been previously shown in patients with NASH. Intake of saturated fat is associated with a lower ratio of reduced to oxidized glutathione (GSH/GSSG), suggesting a pro-oxidant effect. In contrast, consumption of fiber, MUFAs, PUFAs, vitamin E, selenium, and folate protect against oxidative stress [290].

Dietary Oxidants in NAFLD
A correlation between consumption of different nutrients and oxidative stress has been previously shown in patients with NASH. Intake of saturated fat is associated with a lower ratio of reduced to oxidized glutathione (GSH/GSSG), suggesting a pro-oxidant effect. In contrast, consumption of fiber, MUFAs, PUFAs, vitamin E, selenium, and folate protect against oxidative stress [290]. 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; 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.
HFDs induce obesity, insulin resistance, and hepatic steatosis. As noted above, an excess of lipids in hepatocytes is associated with lipotoxicity and mitochondrial overload, resulting in impaired FA catabolism and ROS production. Increased exogenous FA delivery by liver perfusion or by HFD increases mitochondrial oxidative metabolism, resulting in hepatic oxidative stress during NAFLD in mice [248]. Of interest, FA storage as TG, which avoids accumulation of hepatic FFAs, protects against hepatic lipotoxicity and oxidative stress [291]. In line with this observation, the level of TG and free cholesterol increases in the liver in patients with NAFLD, whereas the level of FFAs is unaltered [292].
A limited number of studies have assessed the relationship between dietary saturated FA intake and oxidative stress. Medium-chain saturated FAs have shown antioxidant effects in rats through reduced lipid peroxidation and increased activity of antioxidant enzymes [293,294]. In contrast, saturated FAs in the steatotic rat liver induce hepatocyte ER stress and apoptosis [295]. In elderly Japanese patients, a correlation has been reported between high consumption of saturated FAs, particularly short-chain saturated FAs and medium-chain saturated FAs, and decreased urinary 8-hydroxy-2'-deoxyguanosine (an oxidative stress marker) and ameliorated hypertension [296]. Overall, the effect of saturated FAs on oxidative stress in NAFLD remains unclear and needs further investigation. Differences in FA chain lengths may contribute to differences in their effects on oxidative stress: short-chain saturated FAs and medium-chain saturated FAs may be antioxidant, whereas long-chain saturated FAs increase oxidative stress.
In addition, a high ratio of omega-6 to omega-3 FAs reduces mitochondrial respiratory functions and increases ROS levels in human hepatoma cells. Decreasing this ratio prevents diet-induced NASH and hepatic oxidative stress in rats [297,298].
FAs are not the only compounds that accumulate in the mitochondria. Cholesterol is consistently elevated in human and mouse fibrotic NASH, and its mechanistic link to NASH development has been explored recently [59]. In hepatocytes from a NAFLD rat model, mitochondrial cholesterol accumulation decreases mitochondrial glutathione, which is crucial for scavenging ROS produced during mitochondrial metabolism, and sensitizes mitochondria to inflammatory cytokines [299].
Mice fed a high-cholesterol diet show a modified transcriptome, including modulation of several pathways involved in cell death and oxidative stress. Cholesterol accumulation increases ROS generation, leads to expression of antioxidant genes such as SOD2, and modifies expression of genes regulating the glutathione pathway (decreased GPx-1, increased GPx-4) [300]. In addition, oxidation of free cholesterol generates oxysterols, which are elevated in NAFLD and may participate in lipid synthesis and inflammation [301]. In patients with NASH, cholesterol accumulation decreases mitochondrial membrane permeability and induces defective mitochondrial GSH transport from cytosol to the mitochondria [256].
Interactions among different lipid classes may play a role in oxidative stress induction. For example, dietary fat and cholesterol act in synergy to promote NAFLD progression through impairment of mitochondrial function and biogenesis. Mice fed a high-fat, high-cholesterol diet develop more severe liver lesions, including inflammation and fibrosis, and show mitochondrial dysfunction such as altered respiration, increased H 2 O 2 production, and impaired ATP homeostasis, compared to each diet (high fat or high cholesterol) fed separately [302,303]. The combination of dietary cholesterol with PUFAs leads to high levels of oxidized peroxiredoxins and protein carbonyls, indicating severe hepatic oxidative stress [304]. Of interest, a ketogenic diet increases rat liver antioxidant capacity [305].
In the liver, fructose is mainly metabolized by fructokinase, which uses ATP to phosphorylate fructose into fructose-1-phosphate. Thus, fructose metabolism decreases ATP levels, resulting in oxidative stress induction and uric acid generation, which also has pro-oxidative effects [50,306]. Fructose drinking in rodents also induces gut microbiota changes and CYP2E1-dependent intestinal oxidative stress, which both lead to endotoxemia and increased severity of steatohepatitis with fibrosis [307]. Rats fed a diet rich in fructose for 3 weeks show decreased catalase activity and GSH levels in the liver, whereas the level of NOX and the content of protein carbonyl groups, a marker of oxidative stress, are increased [308,309]. These effects are associated with changes in carbohydrate metabolism and increased hepatic TGs, suggesting a switch towards lipid synthesis rather than mitochondrial oxidation, which may protect against ROS production [309]. A fructose-rich diet also causes oxidative damage to hepatic lipids, proteins, and mitochondrial DNA, as shown by increased lipid peroxidation and nitrotyrosine levels in the liver and higher levels of plasma 8-hydroxydeoxyguanosine generated by oxidative damage to DNA. In addition, administration of the antioxidant lipoic acid or an inhibitor of NOX protects against fructose-induced hepatic insulin resistance, oxidative stress, and inflammation, reinforcing the relationship between hepatic oxidative stress and high fructose intake [308,310]. Fructose intake also enhances hepatic oxidative stress of maternal HFD offspring because of decline in the antioxidant defense system [311].
In patients with NASH and hyperferritinemia, iron reduction by a combination of iron-restricted diet and phlebotomy reduces serum iron and ferritin levels, liver enzymes, and hepatic oxidative damage to DNA [312]. In preclinical models, iron excess leads to hepatic TG accumulation, but the effect on oxidative stress is controversial. For example, dietary iron supplementation enhances HFD-induced hepatic steatosis and inflammation, whereas no change in hepatic oxidative stress is observed [313]. In contrast, iron supplementation in genetically obese mice causes hepatocellular ballooning and hepatic oxidative stress by increasing lipid peroxidation and decreasing expression of antioxidant genes [314]. A recent study indicated that the hepatocyte-specific deletion of the iron chaperone, poly r(C) binding protein 1 (PCBP1), results in liver iron depletion, hepatic steatosis and inflammation, increased lipid peroxidation, and activation of the Nrf2 and glutathione pathways. Both an iron-restricted diet and treatment with vitamin E can prevent steatosis and hepatocyte damage associated with PCBP1 deficiency, suggesting that iron mediates its toxic effects in part through the production of ROS [315]. Liver biopsies of 222 patients (men and women) with NAFLD showed that serum ferritin levels, which are biomarkers of iron metabolism, are positively associated with a higher NAFLD activity score [316]. The relation between ferritin level and NAFLD is mostly significant in men and associated with hepatic iron deposition, fibrosis, and NASH [77,317]. A correlation between ferritin level and NAFLD development also has been observed in a cohort of postmenopausal women [317].
Collectively, observations from dietary interventions show that cholesterol and fructose promote oxidative stress in NAFLD pathogenesis. Meat processing of meat also causes protein oxidation, which may be associated with oxidative stress [318]. In addition, the interactions among different types of lipids may play a role in the induction of oxidative stress during disease development. The pro-oxidant effects of saturated lipids, animal-based proteins, and iron are not well understood and need further investigation in experimental and large-scale clinical studies. Although excess of certain nutrients is clearly associated with oxidative stress induction in NAFLD, other nutrients have antioxidant properties. Below, we discuss the antioxidant role of these nutrients in preventing NAFLD development.

Dietary Antioxidants in NAFLD
Several meta-analyses have demonstrated the effectiveness of omega-3 PUFA supplementation in the dietary management of NAFLD, especially for liver fat content, although a detailed mechanism of its effect on liver inflammation and fibrosis is still unclear [319][320][321][322][323]. Omega-3 PUFAs reduce liver TG accumulation mainly through regulation of hepatic lipid metabolism and inflammation [324]. In experimental studies, supplementation with omega-3 PUFAs, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), reduces HFD-induced steatosis in part through induction of antioxidant responses [325]. Furthermore, omega-3 PUFA supplementation reduces hepatic oxidative stress in HFD-fed mice, as shown by a decreased GSSG/GSH ratio and protein carbonylation content in the liver [326]. Omega-3 PUFA supplementation also reduces the WD-induced NOX pathway [327] and can decrease oxidative stress through indirect mechanisms such as Nrf2 upregulation because the oxidation products of DHA and EPA are potent Nrf2 activators [328]. Dietary DHA is more efficient than EPA in reducing WD-induced NASH and hepatic oxidative stress [327,329]. In humans, the effects of PUFAs on oxidative stress in NAFLD are less clear. According to a recent systematic review of clinical trials, the effect of omega-3 PUFAs on oxidative stress in human NAFLD is inconclusive and varies from study to study depending on the oxidative stress marker measured [323].
MUFAs are the main FAs found in extra-virgin olive oil, which is associated with the prevention of diet-induced liver oxidative stress [330]. However, a specific mechanism is unknown, and antioxidant compounds such as phenols, which are present in high quantity in extra-virgin olive oil, are more likely responsible for these beneficial effects [331].
A few dietary amino acids affect NAFLD pathogenesis in several ways, including gut epithelium integrity, inflammation, fibrosis, and glucose homeostasis. We will only briefly address amino acids here because a recent review has described their effects in some detail [332]. Branched-chain amino acids repress inflammation, apoptosis, and fibrosis by downregulation of the transforming growth factor-β and Wnt/β-catenin pathway, and steatosis and mitochondrial dysfunction by downregulating FA synthase and upregulating mTOR. Glutamine attenuates inflammation by upregulating PPARγ. The α-amino acids citrulline and arginine downregulate TLR-4, TNFα, IL-6, and endotoxins, which ameliorates inflammation and apoptosis, and serine increases AMPK activity to repress steatosis and mitochondrial dysfunction. Serine is also a GSH precursor, and its supplementation has been reported to decrease hepatic steatosis in a proof-of-concept human study [333]. Of interest, glycine supplementation increases thermogenic activity in hepatic mitochondria, mitigates liver steatosis, improves insulin sensitivity, and reduces serum lipid levels by repressing p38, JNK, and TLR-4, and increasing glucagon-like peptide-1/glucagon [332]. Methionine also has an important hepatic impact because compromised levels in methyl-group donors result in alterations promoting the development of NAFLD in animal models and humans. In mice, both dietary methionine deficiency and high methionine supplementation induce anomalies associated with NAFLD progression, including an effect on lipid metabolism and one-carbon metabolic pathways disturbances. Oxidative and ER stress is also increased [334].
Choline, which is not an amino acid, is also an essential methyl donor, and its deficiency results in impaired VLDL secretion and hepatic fat accumulation and liver damage, including impaired mitochondrial function. The result is reduced FA oxidation and stimulated production of ROS with downstream deleterious effects. Of interest, the MCD diet has become a classical diet for triggering NASH. The absence of two main ingredients, methionine and choline, alters mitochondrial β-oxidation and VLDL synthesis [335], causing relatively rapid fatty liver development, together with oxidative stress and modifications in NASH-promoting adipokine and cytokine profiles, contributing to liver injury [336]. MCD-fed rodents undergo significant weight loss and present with decreased serum TG, cholesterol, glucose, insulin, and leptin [337]. These effects are a limitation of this diet model because the metabolic profile it induces is very different from that of human NASH, which must be considered when using the MCD in studies [338].
Taurine is a sulfur-containing β-amino acid that is not used for protein synthesis. The main sources of taurine are fish, meat, and dairy. It has several health benefits, and people also take it as a supplement to improve physical performance. In a mouse NAFLD model, taurine treatment alleviated HFD-induced reduction of catalase and SOD activity, and, in HepG2 cells, it suppresses FA-induced lipid accumulation and reduces ROS production in concert with the FA-induced alteration in mitochondrial membrane potential. These findings supporting an antioxidative effect of taurine show that it can attenuate NAFLD progression in animals [339].
Micronutrients are required in microgram or milligram quantities for physiological functions, such as cellular metabolism and tissue function [340]. These compounds, including vitamins, minerals, and phytochemicals, are also important in NAFLD pathogenesis [72,341], mainly through modulation of anti-inflammatory and antioxidant pathways. Liver plays an essential role in micronutrient metabolism by contributing to their uptake, transport, and storage, and by producing binding, transport, and regulatory proteins required for their action at the cellular level [72]. Micronutrients with antioxidant capacity include vitamin D, vitamin E, and phytochemicals such as carotenoids and polyphenols.
Vitamin D is often deficient in liver diseases and is associated with NAFLD and NASH in epidemiological studies. In fact, the anti-inflammatory, antifibrotic, and antiproliferative effects of vitamin D on NAFLD and NASH have been documented in both human cohorts and animal models [342][343][344][345]. Preclinical models have provided critical information for designing possible related therapeutic strategies for the treatment of NAFLD/NASH [342].
Vitamin E supplementation in adults with NAFLD does not improve fibrosis but ameliorates hepatic steatosis and inflammation [346]. Animal studies show that vitamin E improves NAFLD/NASH by repressing oxidative stress and inflammation [347]. We have recently reviewed the impact of vitamin E on NAFLD/NASH more extensively [348].
Natural polyphenols found in vegetables, fruits, coffee, and wine are a class of phytochemicals that include several different compounds sharing a common phenolic structure. They are classified as flavonoids and non-flavonoids. Their beneficial effects mainly trace to their antioxidant properties, and they also influence glucose and lipid metabolism and exert anti-inflammatory, antifibrogenic, and antitumoral effects [349]. In addition, they reduce de novo lipogenesis by regulating the activity of SREBP-1c and stimulate FA β-oxidation possibly via activation of AMPK [350]. Furthermore, several compounds of the polyphenol family, including green tea catechins, curcumin, resveratrol, and quercetin, appear to reduce liver enzymes, lipid peroxidation, and inflammation markers [342]. Carotenoids also have beneficial effects on the liver, mainly through their antioxidant capacity and by regulating expression of genes involved in inflammation and lipid metabolism [351]. Furthermore, many other natural products present in the diet confer hepatoprotective effects by mechanisms related to improved gut dysbiosis and ameliorated intestinal barrier permeability, and inhibiting steatosis by mechanisms acting on inflammation, oxidative stress, fibrosis, and apoptosis [286].

Gut Microbiota and Dietary Antioxidants in NAFLD
Natural dietary supplements can support microbiota homeostasis or improve it when it is disturbed [286,287]. The use of probiotics (Lactobacillus and Bifidobacterium) can ameliorate NAFLD [352][353][354][355]. Similarly, liver health can be supported by well-planned consumption of several classes of food-derived compounds that are possibly involved in microbiota maintenance. Among these are functional oligosaccharides (fructo-oligosaccharides, galacto-oligosaccharides, and chitosan oligosaccharides) [275,356,357]. Dietary fiber is another, catabolized through fermentation by the lower gastrointestinal tract microbiota to short-chain FAs comprising butyrate, propionate, and acetate that ameliorate NAFLD pathogenesis [358,359]. The microbiota can convert functional amino acids, such as L-tryptophan, to indoles and its derivatives, which improve gut and liver health. In preclinical studies, oral administration of another amino acid, L-glutamine, has been proved protective against diet-induced NASH progression [287,360]. Carotenoids improve the gut barrier and immune homeostasis and, as noted, are known for their anti-inflammatory and antioxidant properties [361]. Polyphenols repress insulin resistance, oxidative stress, and inflammation and stimulate FA β-oxidation, which collectively confer a protective effect against NAFLD. The ω-3 PUFAs have a broad range of beneficial effects in preclinical and clinical studies in preventing or treating NAFLD. They improve intestinal barrier integrity and thus reduce bacterial translocation, ameliorate bile acid homeostasis, and repress liver inflammation via activation of the nuclear farnesoid X receptor [287].
These effects of various compounds in interaction with the gut microbiota indicate that nutritional interventions offer promise in NAFLD/NASH prevention and treatment and that their implementation will evolve toward personalized dietary therapies with consideration of each patient's genomic, metabolic, and microbiotic profile. The doses of individual micronutrients appear to be important, with some doses offering benefits and others resulting in side effects [72]. In addition, oxidative stress is an important factor in cancer initiation but may also offer some beneficial toxicity against cancer cells. Thus, antioxidants could have harmful effects in patients with HCC, for instance. More studies investigating the long-term effects and outcomes in patients with advanced NASH and liver cancer are needed [362].
ROS can be produced from several sources during metabolism, as a result of biotransformation reactions of pesticides (herbicides, insecticides, fungicides, miticides) by CYP450 enzymes, which catalyze the oxidation reactions [192,[385][386][387]. For example, oxidative stress generated by exposure to the insecticide permethrin is linked to the detoxifying activities of CYP450 enzymes, and other enzymes involved in oxidation and hydrolysis processes [364]. Organochlorine compounds such as DDT lead to an increased ROS production through activation of CYP450 gene expression involved in its detoxifying pathway in exposed rat liver [211]. The biotransformation reactions of organophosphorus compounds, during which an excess of free radicals is generated, also produces ROS [187]. Table 3. Impact of pesticide exposure on oxidative stats in in vivo models. Acceptable Daily Intake (ADI) values were from https://ephy.anses.fr/ and https: //ec.europa.eu/food/plant/pesticides/eu-pesticides-database/. ALT, alanine aminotransferase; AST, aspartate aminotransferase; BW, body weight; CYP, cytochrome P450; EROD, ethoxyresorufin-O-deethylase; MDA, malondialdehyde; GPX, glutathione peroxidase; GSH, reduced glutathione; GSSG, oxidized glutathione; GST, glutathione-S-transferase; IL-1 IL-6, Interleukin 1 and 6; ROS, reactive oxygen species; SIRT, sirtuin; SOD, superoxide dismutase; TNFα, Tumor necrosis factor α.    Moreover, all mitochondrial complexes, except complex IV, are targets of at least one pesticide family (Table 4). In chloroplasts, herbicides are photosynthesis inhibitors and mediate their actions by inhibiting the electron transport chain. Some functional similarity between plant chloroplasts and mammalian mitochondria may explain the mitochondrial toxicity of the herbicide triazine, which in plants inhibits the photosynthetic process that it targets [147]. Indeed, the triazine herbicide blocks the activity of the oxidative phosphorylation complex I, the NADH dehydrogenase complex, in an animal model (Table 4) [148]. Atrazine inhibits complexes III and V [148,389]. Other herbicides such as Diuron and glyphosate inhibit complex III [390,391]. Organochlorine insecticides affect complexes II and III, which contain the enzyme succinate dehydrogenase, and causes inhibition of succinate translocation, likely explaining insecticide suppression of mitochondrial respiration [392]. Organophosphate insecticides inhibit mitochondrial complex I in birds [393], and effects on complex IV and/or V have been reported in mammalian models [394,395]. Complex V is also inhibited by imidacloprid, abamectin, and DDE [392,396,397].
The strobilurin family of fungicides, known as succinate dehydrogenase inhibitors (SDHIs), inhibits fungus mitochondrial respiration via blockade of electron transfer between cytochrome b and cytochrome c1, which results in oxidative stress [378]. In addition to the inhibitory effect on succinate dehydrogenase, other SDHI fungicides inhibit complex III (ubiquinol cytochrome c reductase) with varying efficiency in human and animal cells [184,381,398,399]. The herbicide glyphosate targets the mitochondrial electron transport chain in duckweed, in particular complex III, resulting in ROS production; however, its impact on mammalian mitochondria is not yet documented [400]. Some pesticides may affect more than one mitochondrial complex [148,395] (Table 4). For example, SDHI fungicides act on mitochondrial complexes I, II, and III (Table 4). In brief, various chemical families of insecticides, except DDE, alter complex V, whereas rotenone and malathion act on complexes I and II and I, IV, and V, respectively. Fungicides affect complexes II and III of the mitochondrial respiratory chain, and herbicides target complexes I, III, and V (Table 4). It is noteworthy that some fungicides (fludioxonil and Maneb) and the herbicide paraquat induce oxidative stress by acting on both mitochondria complexes and cytosolic NADPH oxidase [401,402].
Pesticide exposure affects antioxidant enzyme expression and/or activity and/or levels of antioxidant compounds such as GSH (Table 3). Pesticides can influence antioxidant pathways by modifying the expression of Nrf2. In HepG2 cells, DDE exposure decreases Nrf2 protein level and subsequently glutamyl-cysteine synthase activity [403]. Changes in Nrf2 promoter methylation have been identified in germ cells from adult mice exposed to the herbicide methyl-parathion for 5 days [404]. The exposure of rats to the pyrethroid insecticide bifenthrin for 60 days at a high dose led to an induction in gene expression, including of Nrf2, in the hippocampus [405], and the fungicide prochloraz induced Nrf2 activity in a gene reporter assay [406]. Exposure to pesticides such as atrazine, chlorpyrifos, zinc dimethyldithiocarbamate, vinclozolin, paraquat, and rotenone alters Sirt expression (mainly Sirt1, 3, and 6)-all members of a NAD + -dependent type III deacetylase enzyme family considered to operate as stress adaptors under oxidative, genotoxic, and metabolic stresses [407][408][409][410][411][412][413]. The mitochondrial scavenger of ROS, UCP-2, is also induced by the organochlorine pesticide DDE [203]. Pesticides induce ER stress in association with the development of some pathologies but without metabolic disturbance [414]. Additionally, the antiparasitic niclosamide induces ROS generation that enhances ATF3 and CHOP expression and ER stress in hepatoma HepG2 cells [415]. However, the pyrethroid insecticide permethrin induces ROS production without inducing ER stress in HepG2 cells [366]. Altogether, these findings indicate that pesticides affect several processes that result in oxidative stress: the CYP450 system, mitochondrial respiration, antioxidant pathways, the stress adaptor system, and scavenger ROS activity. Insecticide Rotenone x Isolated mitochondria from exposed HL-60 cells [405] DDT/DDE x x x Rat liver or heavy beef heart isolated mitochondria or sonicated submitochondria particles [392] Monocrotophos x Rat muscle mitochondria isolated from exposed animals [394] Imidacloprid x Rat liver isolated mitochondria [397] Malathion x x x Rat muscle mitochondria isolated from exposed animals [395] Herbicide Atrazine x Rat liver, isolated mitochondria [389] x x Rat muscle mitochondria isolated from exposed animals [148] Diuron Antiparasitic Abamectin x Rat liver, isolated mitochondria [396] Besides oxidative stress, pesticide exposure may lead to NAFLD through their impact on lipid metabolism by (i) modifying fatty acid uptake and efflux [171] and modulating FFA transport as described for insecticides and herbicides, (ii) increasing lipogenesis ( [173,406] (Table 1), (iii) altering oxidation pathways [175]; insecticides, herbicides, and fungicides affect both β-oxidation and lipogenesis (Tables 1 and 2), (iv) interacting with nuclear receptors [416][417][418][419][420][421] involved in the control of metabolism as demonstrated for pyrethroid and neonicotinoid insecticides [176][177][178], and imidazole or triazole fungicides [158,382] or a pesticide mixture [193], but implication in NAFLD is not proven.
Some pesticides may ameliorate lipid metabolism. For example, environmental doses of Boscalid, a widely used fungicide, induce gene expression that promotes β-oxidation and inhibits lipogenesis in the zebrafish liver [179]. Similarly, oral exposure to the carbamate fungicide propamocarb in mice during a 4-week period leads to a down regulation of genes involved in glycolysis, TG, and fatty acid synthesis in the liver [180]. Pesticide alter glucose metabolism through activation of glucose uptake, glycogenolysis, gluconeogenesis (as reviewed for insecticides in [136]) or through inhibition of the mitochondrial respiratory complex (Table 4), and modulation of Chrebp gene expression levels [185], change in the expression of PPARβ/δ and genes involved in glucose metabolism (FoxO1 and CREB) [186]. Pesticides also induce insulin resistance by acting on insulin signaling pathways (for a review see [192]). Liver inflammation was also reported upon exposure to organophosphorus [188], OC [189], neonicotinoid [142,143] pyrethroid insecticides [144], or to triazole or imidazole fungicides [190] ( Table 1).
In summary this section on the role of pesticides in oxidative stress in NAFLD shows that pesticides are pro-oxidant compounds that can induce ROS production through various mechanisms: (i) detoxification via P450-dependent processes, (ii) effects on mitochondrial respiratory complexes, and (iii) action on Sirt-and or Nrf2-dependent antioxidant responses (Figure 8). It is noteworthy that the data do not consistently come from studies performed in the liver and/or related to a metabolic impact of pesticides. Although pesticide exposure and NAFLD are not clearly correlated, the prooxidative properties of pesticides as well as their impact on various pathways involved in the regulation of metabolic homeostasis support their putative role in the development or in the progression of hepatopathy. Moreover, various experimental studies showed that pesticides could affect various pathways involved in the regulation of metabolic and hepatic homeostasis strongly suggesting their role in NAFLD. Pesticides affect oxidative stress in NAFLD, but in many studies (reported in Tables 1  and 2), animals were exposed to high doses of pesticide and durations of exposure that are not relevant for human exposure, making any human translation of results difficult. In fact, to better decipher the role of pesticide-induced oxidative stress in the development of NAFLD, more studies are needed that link different pesticide cellular targets and/or mechanisms of action underlying ROS production with hepatic processes involved in the control of energy metabolism, such as lipid metabolism, inflammation, and gene regulation by nuclear receptors. Interestingly, nuclear receptors, such as PPARs, progesterone receptor (PR), mineralocorticoid receptor (MR), glucocorticoid receptor (GR), aryl hydrocarbon receptor (AhR), retinoic acid receptors (RARs), farnesoid X receptor (FXR), and liver X receptors (LXRs) are established or suspected targets of endocrine disrupting chemicals including diverse agrochemicals, but further analyses are needed to determine whether this can lead to the development of NAFLD/NASH [410,411].

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.

Conclusions
Many challenges remain in understanding the etiology of NAFLD because the precise staging of its progression in the absence of liver biopsy is not yet possible. Furthermore, no therapy has been approved by the U.S. Food and Drug Administration, and lifestyle modifications (diet, exercise) are difficult to implement and adhere to, which has contributed to the failure to reduce NAFLD prevalence. In terms of implemented therapeutic interventions, vitamin E and the PPARγ ligand pioglitazone have beneficial effects on steatosis and inflammation, but they do not improve fibrosis, the strongest indicator of mortality in NAFLD/NASH. However, sustained research has led to a better understanding of the pathogenesis and progression of this condition and to the development of promising compounds to block and possibly reverse fibrosis, which are presently in clinical trials [348,412]. It is well accepted that disturbances in lipid metabolism are key to the development of NAFLD and its progression. They can affect different ROS generators, such as mitochondria, ER, and NOX [226]. Although many aspects of the contributions of ROS generators to NAFLD remain unexplored, much progress has been made in understanding how increased ROS levels trigger changes in insulin sensitivity, the activity of key enzymes of lipid metabolism, innate immune system signaling, and the level of inflammatory responses. Both macro-and micronutrients impact oxidative stress in NAFLD pathogenesis. An excess of certain macronutrients, such as cholesterol and fructose, exerts pro-oxidant effects, whereas several micronutrients have the potential to decrease NAFLD through their antioxidant properties. However, human diets are complex, and it is challenging to mimic them in experimental models. The contribution of specific dietary nutrients to oxidative stress in NAFLD is not fully elucidated and needs further research. It is noteworthy that epidemiological studies did not report a correlation between pesticide exposure and NAFLD in human. However, pesticides exposure was correlated in occupationally exposed population with metabolic disorders. Moreover, various experimental studies showed that pesticides can affect various pathways involved 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.

Conclusions
Many challenges remain in understanding the etiology of NAFLD because the precise staging of its progression in the absence of liver biopsy is not yet possible. Furthermore, no therapy has been approved by the U.S. Food and Drug Administration, and lifestyle modifications (diet, exercise) are difficult to implement and adhere to, which has contributed to the failure to reduce NAFLD prevalence. In terms of implemented therapeutic interventions, vitamin E and the PPARγ ligand pioglitazone have beneficial effects on steatosis and inflammation, but they do not improve fibrosis, the strongest indicator of mortality in NAFLD/NASH. However, sustained research has led to a better understanding of the pathogenesis and progression of this condition and to the development of promising compounds to block and possibly reverse fibrosis, which are presently in clinical trials [348,412]. It is well accepted that disturbances in lipid metabolism are key to the development of NAFLD and its progression. They can affect different ROS generators, such as mitochondria, ER, and NOX [226]. Although many aspects of the contributions of ROS generators to NAFLD remain unexplored, much progress has been made in understanding how increased ROS levels trigger changes in insulin sensitivity, the activity of key enzymes of lipid metabolism, innate immune system signaling, and the level of inflammatory responses. Both macro-and micronutrients impact oxidative stress in NAFLD pathogenesis. An excess of certain macronutrients, such as cholesterol and fructose, exerts pro-oxidant effects, whereas several micronutrients have the potential to decrease NAFLD through their antioxidant properties. However, human diets are complex, and it is challenging to mimic them in experimental models. The contribution of specific dietary nutrients to oxidative stress in NAFLD is not fully elucidated and needs further research. It is noteworthy that epidemiological studies did not report a correlation between pesticide exposure and NAFLD in human. However, pesticides exposure was correlated in occupationally exposed population with metabolic disorders. Moreover, various experimental studies showed that pesticides can affect various pathways involved in the regulation of metabolic and hepatic homeostasis strongly suggesting a role in NAFLD. Pesticides affect oxidative stress in NAFLD, but in many studies (reported in Tables 1 and 2), animals were exposed to high doses of pesticide and durations of exposure that are not relevant for human exposure, making any human translation of results difficult. In rodents, perinatal exposure to organophosphorus insecticides worsens metabolic perturbations induced by HFD [197] or by overfeeding [198,206]. The lifelong effects of perinatal exposure with respect to NAFLD/NASH development, and health in general, remain an open and crucial area of research.

Conflicts of Interest:
The authors declare no conflict of interest.