Understanding environmental contaminants’ direct effects on non-alcoholic fatty liver disease progression

Abstract

Purpose of review:

Environmental contaminants are considered one of major factors in the development and progression of NAFLD, the most common liver disease in the U.S.

Recent findings:

The evolving knowledge of mechanisms of hepatic steatosis and steatohepatitis has recently been reviewed and characterized as ALD, NAFLD, and TAFLD. The most recent mechanistic studies on PFAS and PCBs have revealed a greater role for toxicants in the initiation of not only TAFLD, but also NAFLD and the more progressive inflammatory stage of NAFLD, non-alcoholic steatohepatitis. In addition to insecticides, recent studies support a significant contribution of fungicides and herbicides to NAFLD.

Summary:

The mechanisms of PFAS, PCBs and fungicides in contributing to the increased prevalence of NAFLD remains unclear. Addressing whether chronic, low-dose exposures could result in liver pathology and whether real-world exposure to mixtures of environmental contaminants pose a significant risk factor for NAFLD is paramount to understand the impact of NAFLD on populations today.

Introduction

The liver is a vital organ in the maintenance of metabolic homeostasis. It is considered the first organ of exposure prior to entering systemic circulation, following oral absorption, and is responsible for the metabolism, distribution, and excretion of exogenous chemicals. Therefore, liver cells are exposed to significant concentrations of chemicals and chemical- or drug-induced liver injury (hepatotoxicity) has become a growing concern. Hepatoxicity is a common endpoint in risk assessment of many environmental contaminants, including persistent organic pollutants (POPs) and pesticides. There are various pathological conditions associated with hepatotoxicity, but it is recently suggested that fatty liver, or steatosis, is the most prevalent pathology associated with toxicant exposure (1). Steatosis is defined by a significant increase in hepatic lipid content (>5%), which can remain a benign pathology or progress to steatohepatitis. Steatohepatitis, is a pro-inflammatory state associated with significant liver injury that can progress even further to conditions of liver fibrosis, cirrhosis, or hepatocellular carcinoma. As previously reviewed, three of the most common and characterized factors contributing to the development of steatosis and steatohepatitis are alcohol, obesity or metabolic syndrome, and environmental toxicants; also known as, alcoholic liver disease (ALD), non-alcoholic liver disease (NAFLD), and toxicant-associated liver disease (TAFLD), respectively (2).

NAFLD is considered the most common cause of liver disease in children and adults in the United States. It affects 83.1 million Americans and progresses to the more severe condition of non-alcoholic steatohepatitis (NASH) in 20% of patients diagnosed with NAFLD or an estimated 3–5% of the population (3). NASH-related liver cirrhosis is currently the first leading cause of liver transplantation, which is estimated to increase by 62% by 2030 due to NAFLD (4–6). NAFLD is characterized as a progressive disease based on contributions of parallel conditions and factors that act synergistically, known as the “multiple parallel hits hypothesis” (7). The now accepted multi-hit hypothesis was previously reviewed, but briefly, insulin resistance, obesity, adipose tissue dysregulation, gut dysbiosis, and genetic factors are identified as the major mechanisms that contribute to the pathogenesis of NAFLD (8). As previously addressed, alcohol, fat deposition and other toxicants are known to cause the same pathology, but the mechanism in developing NAFLD and TAFLD may share more mechanistic similarities (2). A myriad of environmental chemicals have been identified to be potentially associated with the development of TAFLD, a term coined by Cave and colleagues (9), but exposure levels may not demonstrate a direct human relevance (1). The rise in steatosis and steatohepatitis is alarming and warrants continued research. Moreover, environmental contaminants must be considered as factors in disease progression of NAFLD, not rather than direct hepatotoxicants. The most current definition, prevalence, and risk factors associated with NAFLD has been reviewed (10). This review will focus on the mechanisms by which environmental chemicals contribute to the development and progression of NAFLD, emphasizing the major developments in mechanistic research regarding perfluoroalkylated substances (PFAS) contribution to steatosis, polychlorinated biphenyl (PCBs) association with steatohepatitis, and the emerging role of fungicides/herbicides in NAFLD within the last 5 years.

POPs Contribute to the Progression of NAFLD

The role of environmental contaminants contribution to the development and progression of the multi-factorial disease of NAFLD has become a growing area of research. The most recent mechanistic research on the role of PFAS and PCBs in the development of steatosis and steatohepatitis, respectively, will be reviewed to demonstrate the growing concern of low-dose chronic exposure to environmental contaminants contribution to disease.

PFAS and Development of Steatosis/NAFLD

Polyfluorinated chemicals (PFCs) are synthetic surfactants used in industrial and consumer products. PFAS have been identified as POPs and endocrine disruptors, with bioaccumulation demonstrated in humans. PFCs predominantly accumulate in blood, liver, and kidneys due to their lipophobic and hydrophobic properties; therefore, dietary exposure is limited while indoor air contains 25–100 times more PFAS due to the presence of the contaminate in indoor dust (11). In the most recent analysis of the C8 Health Study population exposed to perfluorooctanoic acid (PFOA) in contaminated drinking water (12), the serum biomarker CK18 M30 was positively correlated to PFAS levels; in addition, inflammatory cytokine levels, specifically tumor-necrosis factor (TNFα), were decreased (13). These data support the role of hepatocyte apoptosis in liver toxicity induced by PFAS and favor the development of fatty liver compared to the development of toxicant-associated steatohepatitis (TASH). The most recent analysis of NHANES 2011–2014 data showed that previously described associations of liver function tests in previous NHANES populations (14; 15) persist at lower level exposures of PFAS (16). Lin et al. (14) also suggested that obese participants may be a more susceptible population, in which PFOA, perfluorohexanesulfonic acid (PFHxS) and perfluorononanoic acid (PFNA) levels resulted in a significant positive association with serum liver function biomarkers in both non-obese and obese participants. This association was then demonstrated to be markedly more significant in obese participants (16). These epidemiological studies demonstrate a role of PFAS exposure in the development of steatosis, and suggest a role of PFAS in the progression of NAFLD.

PFAS are shown to induce steatosis and hepatic triglyceride levels in rodent models via induction of the genes involved in fatty acid and triglyceride synthesis, including peroxisome proliferator-activated receptor (Ppar) α and fatty acid transporters fatty acid translocase (Cd36) and Very low-density lipoprotein receptor (Vldlr), in mice treated with PFOA, PFNA, and PFHxS (17). These effects were demonstrated for PFAS exposure in Pparα-null mice, confirming PPARα-independent effects predominantly for PFNA and PFHxS; in addition, the PPARα activator, WY-14643, did not cause this disruption of fatty acid uptake, synthesis, and oxidation (17). In Balb/c mice exposed to PFOA for 28 days, PPARα activation was shown via up-regulation of target genes (Acyl-CoA Oxidase 1, Cyp4a10, Fatty acid binding protein 1, and Vldlr), and increased sterol regulatory element binding protein 1 (Srebp1) mRNA levels corresponded to induced gene expression levels (Fatty acid synthetase, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, Low density lipoprotein receptor, Lipoprotein lipase (Lpl), and Stearoyl-CoA desaturase-1 (18). It was mechanistically determined that increased lipid synthesis occurred due to SREBP activation by PFOA, shown by a dose-dependent increase in cleavage fragments and decreased Srebp1 and Srebp2 precursors upon exposure; therefore, it is proposed that SREBP maturation and regulation of lipid metabolism have a role in PFOA steatotic effects in vivo (18). In addition to SREBP-dependent effects, an acute 48-hr exposure of PFOA in Kunming mice resulted in induced lipid deposition in the liver, altered blood cholesterol levels, and most significantly induced expression of Cd36 (19). Cd36, also known as fatty acid translocase (FAT), is a key membrane protein responsible for fatty acid uptake in hepatocytes. Cd36 is a target gene of Pparγ, which is in concordance with noted induction of Pparγ with PFOA in rodent models (18; 20). Most recent publications have elucidated a direct role of Cd36 in NAFLD disease progression by suppression or knock-out of Cd36 resulting in the attenuation of steatosis and NAFLD (21,22).

At concentrations seen in workers exposed to PFCs the PFAS, PFOA and perfluorooctanesulfonic acid (PFOS) favored carcinogenesis through the decrease in protein levels of hepatocyte nuclear factor 4-alpha (HNF4α), therefore altering the regulation of key genes, Claudin 1/early growth response protein 1 (CLDN1/Egr1) and Aldo-keto reductase family 1 member (AKR1B10/Akr1b7), in primary human hepatocytes and murine livers (23). In addition to HNF4α regulation of genes involved hepatocellular carcinoma and metastasis, HNF4α is known to contribute to lipid homeostasis via regulation of apolipoprotein B (ApoB) and other genes involved in very low-density lipoprotein (VLDL) secretion. VLDL secretion was mechanistically demonstrated to be down-regulated favoring the development of steatosis via suppressed ApoB mRNA and ApoB-100 levels due to retention of HNF4α in the cytoplasm of C57BL/6 mice fed a high-fat diet (HFD) for 12-weeks (24). It is suggested though that altered lipid metabolism genes through PPARα and PPARγ induction pathways via RNA-sequencing of human hepatocytes exposed to PFOS and PFOA persist in PPARα-null mice due to HNF4α regulation of PPARα itself (23). Further investigation is warranted as HNF4α mRNA levels were reduced without changes in target gene expression following PFOA treatment (18). PFOS and PFOA may promote steatosis in a PPARα-independent manner through the decrease HNF4α protein levels or inhibition of endogenous ligand binding demonstrated in human hepatocytes and murine livers.

PFAS are also suggested to act through the nuclear receptor constitutive androstane receptor (CAR). PFOA treatment increased CAR target genes Cyp2b10 and Aldehyde dehydrogenase 1 (Aldh1) to a greater extent than the CAR activator TCPOBOP in mouse livers via increased CAR nuclear localization (25). Mechanistically, it was shown that CAR is indirectly activated by PFCs in mouse and human hepatocytes; but most notably, PFCs are suggested to be dual agonists of CAR and PPARα suggesting a crosstalk between these nuclear receptors due to overlapping regulation of hepatocarcinogenesis and energy metabolism (25). These key nuclear receptors, SREBP, HNF4α, CAR, PPARγ, and PPARα, are responsible for the maintenance of liver homeostasis, and their role in rodent models of PFAS-induced liver toxicity emphasizes the dysregulation of lipid metabolism and development of steatosis.

PFOA overall has been demonstrated to act on key nuclear receptors, induce lipid accumulation, alter inflammatory pathways, and induce cell proliferation in rodent livers. The most recent publication to address the role of PFOA in NAFLD surprisingly found that PPARα activation was enhanced in PFOA and HFD feeding, favoring fatty acid oxidation and clearance, and thus reducing the severity of macrovesicular steatosis and secondary fibrosis (20). These findings demonstrate a time-dependent effect that supports long-term exposure to PFOA may be due to tissue crosstalk and fatty acid availability from white adipose tissue (20). Of note, this was a model of pre-existing NAFLD and no dose-dependent effects were studied. A co-administration of PFOA and HFD resulted in the same paradoxical effect of protection against HFD-induced NAFLD (26). The most significant mechanistic finding was the attenuation of HFD-induced Cd36 levels by PFOS, favoring the attenuation of HFD-induced steatosis as mentioned previously (26). These mechanistic studies have elucidated conflicting hypotheses in regard to PFOA and PFOS exposures (Table 1). Therefore, continued studies are needed to identify whether PFAS overall contribute to steatosis and steatohepatitis in humans via direct toxicant-induced effects, which differs from all current hypotheses supporting that PFAS would exacerbate NAFLD.

Table 1: Perfluoroalkylated substances (PFAS).

Key physiological and molecular endpoints from studies conducted between 2015–2019 are summarized by publication, rodent in vivo and/or in vitro model, PFAS, and exposure.

Citation	Species	PFAS	Exposure	Physiological and Molecular Endpoints
Yan et al. 2015	Balb/c	PFOA (water)	Oral gavage 0.08, 0.31, 1.25, 5, 20 mg/kg/day (0.26 to >120 µg/g in liver); 28 days	Dose-dependent effects: ↓Total liver cholesterol ↑PParγ mRNA ↑Pparα response genes ↑Srebp1 and Srebp2 cleavage products (active) and response genes
Beggs et al. 2016	Primary human hepatocytes	PFOA PFOS (DMSO)	10 nM to 10µM; 48–96 hrs.	PFOA and PFOS effects: No cellular cytotoxicity (↔ALT release) ↓HNF4α protein at 10 µM; ↔HNF4α mRNA ↓CLDN1, CYP7A1, TAT, ADH1B mRNA (10 µM) ↑CCND1, AKR1B10, PLIN2 mRNA (10 µM) ↑NANOG protein (10 µM) RNA-Seq Ingenuity Pathway Analysis: PFOA-gene expression associated with hepatic steatosis/lipid metabolism PFOS-gene expression associated with liver necrosis and carcinogenesis
	CD-1	PFOA PFOS (0.5% Tween-20)	Oral gavage 3 mg/kg day PFOA 10 mg/kg/day PFOS; 7 days	PFOA and PFOS effects: ↑Liver:body weight ratio ↓HNF4α protein ↑Akrlb7, Egr1, Ccnd1 mRNA ↑PCNA liver staining
Wu et al. 2017	Kunming	PFOA	Intragastrically 5 mg/kg single dose; 48 hrs.	Trends for: ↑Serum ALT, AST, total Bilirubin ↑Pancreatic insulin ↓Pancreatic glucagon ↑Serum LDL-cholesterol ↓ Serum HDL-cholesterol ↑CD36 positive cells; protein; mRNA
Das et al. 2017	SV129 (Wild-type) Pparα-null	PFOA, PFNA, PFHxS	Oral gavage 10 mg/kg/day; 7 days	Pparα-independent effects (Both WT and Null mice): ↑Liver weight and liver:body weight ratio ↑ Cell area ↓ DNA content ↑Liver lipid content Differential effects in null mice: Trend ↑ body weight ↔ PFOA liver lipid content ↑Liver triglycerides in WT and only in null mice exposed to PFNA
Abe et al. 2017	C57BL/6	PFOA	Intraperitoneally 20 mg/kg single dose; 4 or 24 hrs.	↑CAR protein ↑Cyp4a10, Acox1, Aldh1 mRNA (4 and 24 hrs.) ↑Cyp2b10 mRNA (24 hrs.) ↓Pck1 mRNA (4 hrs.)
	C57BL/6 Car-null	PFOA	Intraperitoneally 20 mg/kg/day; 3 days	↑Liver/body weight % ↑Cyp4a10 and Acox1 mRNA ↑Cyp2b10 mRNA in WT only ↑Acat1 mRNA in Car-null only
	C57BL/6	PFOA PFNA	Intraperitoneally 20 mg/kg/day; 3 days	↑Liver/body weight % ↑CAR protein ↑Cyp4a10 and Cyp2b10 mRNA ↔CAR reporter activity ↑Pparα reporter activity
Huck et al. 2018	C57BL/6J	PFOS	Supplemented Diet 1 mg/kg (0.0001%); 6 weeks Control or HFD-fed	PFOS effects on CD-fed Mice: ↑Body weight, WAT weight ↑Liver weight:body weight ratio ↑Hepatic lipids and triglycerides ↑Hepatic Cd36 mRNA PFOS effects on HFD-fed Mice: ↔ Body weight, WAT weight, food intake ↓ Hepatic lipids and triglycerides ↓Hepatic Cd36 mRNA ↑Hepatic Srebf1 mRNA
Li et al. 2019	C57BL/6J 16 weeks Control or HFD-fed	PFOA (water)	Oral gavage 1 mg/kg/day; 2, 8, or 16 weeks	PFOA effects on HFD-fed Mice: ↓Body weight at all time points ↔Liver weight or liver:body weight ratio ↓Serum ALT activities ↓Steatosis and hepatic triglycerides ↓Hepatic collagen ↓Col1a1 and Timp1 mRNA ↑BrdU staining ↑Ccnd1 and Cmyc mRNA

Polychlorinated Biphenyls (PCBs) and Development of Steatohepatitis/NASH

PCBs were commercially produced and used in the United States until 1977 when they were banned due to toxicity. PCB mixtures were used in industrial applications in electrical capacitors and transformers, hydraulic fluid, lubricants, plasticizers, and heat-transfer liquids. These chemicals are still present in pre-1979 products, and persist in the environment due to their resistance to biodegradation. PCBs were detectable in 100% of participants in the analysis of NHANES data from 2003–2004, and 20 PCBs were individually associated with the 10.6% of the U.S. population that had unexplained elevations of the activities of alanine aminotransferase (ALT) (27). PCB exposure has been correlated to liver dysfunctions associated with NAFLD, and are well known to exert their toxic effects through the aryl hydrocarbon receptor (AhR) and the generation of reactive oxygen species (ROS). Ancolor 1260, a mixture of PCBs relevant to human exposure, and dioxin-like PCBs, not only activate AhR, but act on the nuclear receptors, pregnane X receptor (PXR) and CAR, and induce downstream genes CYP1A1 (AhR target), CYP3A4 (PXR target), and CYP2B6 (CAR target) in HepG2 cells (28).

The PCB mixture of Aroclor 1260 demonstrated greater effects on inflammation and the progression of steatohepatitis in models of obesity and NAFLD. C57BL/6 mice co-exposed to Aroclor 1260 and HFD showed a significant development of steatohepatitis compared to steatosis in HFD feeding alone with elevated serum ALT activities and hepatic Tnfα expression (29). Aroclor 1260 altered hepatic fat metabolism upon HFD feeding via induction of Srebp1c mRNA levels in a dose-dependent manner compared to HFD alone (29). Most notably, Cyp2b10 and Cyp3a11 were induced with Ancolor 1260 as expected (29), and a more recent study was conducted to determine the role of CAR and PXR. Aroclor 1260 induced CAR and Cyp2b10 in WT mice, and a significant induction was noted in PXR^−/− mice even though CAR itself is upregulated by PXR deficiency (30). The presence of both PXR and CAR are required for Aroclor 1260 to induce inflammation, as only wild-type mice had increased hepatic and serum Tnfα levels (30). It is suggested that CAR and PXR are crucial in maintaining energy homeostasis, as major alterations between knock-out models and wild-type mice were noted. These nuclear receptors may modulate the transition to steatohepatitis, but they are not solely responsible for Ancolor 1260 induced hepatotoxicity (30). Therefore, PCBs are considered to be involved in the progression of NAFLD and are suggested to be metabolic disruptors.

In considering individual PCB exposure, PCB 153, a non-dioxin like PCB, has previously been studied for its role in steatosis and obesogenic effects with focus on adipose regulation (31–33), but PCB 126 has most recently been studied for its role in the progression of steatohepatitis and accounts for the majority of human levels of dioxin-like PCBs (27). PCB 126 was previously demonstrated to induce lipid deposition in human hepatocytes and rats exposed to PCB 126 via increased Srebp1c expression and plasma triacylglycerols (34). In an acute study in rats (9 hrs to 12 days), PCB 126 showed a time dependent increase in lipid accumulation in the liver that correlated to diminished PPARα in a time- and dose-dependent manner measured by the downregulation of mRNA for target genes acyl-CoA oxidase 1 and 3-Hydroxy-3-Methylglutaryl-CoA Synthase 2 (Hmgcs2) (35). There was an AhR-dependent Phosphoenolpyruvate carboxykinase downregulation that resulted in reduced gluconeogenesis, that precedes hepatic steatosis development (35). PCB 126 would be suggested to contribute to hepatic steatosis, but recent mechanistic studies suggest a direct role in the progression of NASH via metabolomic and transcriptomic analyses.

Similar effects were observed in human models. A human hepatocyte cell line (HepaRG) exposed to PCB 126 resulted in 30 differentially measured metabolites and 264 differentially expressed transcripts compared to control (36). PCB 126 caused a decrease in polyunsaturated fatty acids (PUFAs) and an increase in sphingolipids that were associated with a decrease in triglyceride metabolism genes, Apolipoprotein A2, HMGCS2, and LPL, demonstrating induced oxidative stress and decreased triglyceride breakdown (36). Decreased PUFA content has been previously observed in patients with NAFLD, supporting a mechanistic role of PCB 126 in NAFLD development.

NAFLD is associated with increased production of ROS and induced oxidative stress, in which oxidative stress-mediated liver injury contributing to NASH has been reviewed (37). PCB 126 induced ROS and exacerbated the production of ROS in a liver injury model (methionine-choline deficient diet), suggesting a role in the progression to NASH/steatohepatitis (38). These inflammatory effects can contribute to cardiovascular effects associated with NAFLD, which is in concordance with increased inflammatory cytokines and early markers of vascular inflammation in the same mouse model and the subsequent development of atherosclerosis (39,40). Specifically, PCB 126 is shown to decrease glutathione levels and decrease antioxidant defense genes (Glutathione peroxidase 1, Superoxide dismutase 1, Heme Oxygenase 1, Glutaredoxin 1 and 2) irrespective of diet, but redox metabolites are elevated by PCB 126 in the liver injury model contributing to the exacerbation of oxidative stress on an MCD-diet (40). Overall, PCBs are well-established for their contribution to hepatotoxicity and have been implicated in the development of steatosis, but the more recent mechanistic studies demonstrate a role in the progression from steatosis to steatohepatitis (Table 2).

Table 2: Polychlorinated Biphenyls (PCBs).

Key physiological and molecular endpoints from studies conducted between 2015–2019 are summarized by publication, rodent in vivo and/or in vitro model, PCBs, and exposure.

Citation	Species	PCB	Exposure	Physiological and Molecular Endpoints
Boucher et al. 2015	Human hepatocytes	PCB 126 (DMSO)	2.5 µM;72 hrs	↑Oil red O staining and quantification
	Sprague- Dawley rats	PCB 126 (corn oil)	Intraperitoneally, 1.05 µmol/kg single dose; 7 days	↑Hepatic and plasma triglycerides ↓MTP protein levels ↑Srebp1c and Dgat2 protein levels
Gadupudi et al. 2016	Sprague Dawley rats	PCB 126 (tocophe rol-stripped soy oil)	Intraperitoneally, 5 µmol/kg bw single injection; time-course (12, 6, 3, 1.5 days and 18 and 9 hrs post-injection)	↑ liver:body weight ratio at 6 and 12 days ↑Cyp1a1 mRNA levels at all time points ↑Hepatic lipid in a time-dependent manner (osmium tetroxide stain) ↓Serum glucose levels at all time points ↑Serum triglycerides levels initially and ↓levels by 12 days ↓Pepck and Glut2 mRNA levels 6- and 12-days post injection ↔Pparα and Hmgcs2 mRNA levels over time; ↓Acox1 mRNA levels at only 12 days
		PCB 126 (tocophe rol-stripped soy oil)	Intraperitoneally, 1 µmol/kg bw or 5 µmol/kg bw single injection; 2 weeks	↓Pepck, Glut2, Pparα, Acox1, Hmgcs2 mRNA levels at both doses (trend of dose-dependent)
	Rat hepatocytes (H4IIE cells)	PCB 126 (DMSO) CH2231 91(AhR inhibitor)	3 pM – 300 nM; 24 hrs.	↑Cyp1a1 mRNA level 0.3 nM – 300 nM ↑Cyp1a1 mRNA levels (3 nM); inhibited by CH223191 co-treatment ↓Pepck-c mRNA levels (3 nM); inhibited by CH223191 co-treatment
Deng et al. 2019	C57BL/6 4-week diet – control diet or MCD	PCB 126 (corn oil)	Intragastric gavage 1.53 µmol/kg 14 weeks on diet	↑Uric acid levels further in MCD-PCB 126 mice compared to PCB-126 alone. ↑HNE-GSH and oxylipids in MCD-PCB 126 mice only. ↓CoA, Cysteine, Glutathione, Cysteinylglycine levels with PCB 126 independent of diet. ↓GSH/GSSG ratio with PCB 126 treatment independent of diet. ↓Gsta1 mRNA were further reduced in MCD-PCB 126 mice. ↓Gpx1, Grx1, Grx2, Sod1, Hmox1 mRNA with PCB 126 treatment alone.
Wahlang et al. 2014	C57BL/6J	Aroclor 1260 (corn oil) + Diet (control or HFD)	Oral gavage, 20 mg/kg single dose (week 1 of feeding) or 200 mg/kg (50 mg/kg weeks 1, 3, 5, and 7); 12 weeks of feeding	Effects of Arcolor 1260 compared to HFD control: ↓ % BW increase (200 mg/kg only) ↓ % Fat composition / adipose weight ↔ Adipocyte size ↑ Serum ALT, Il-6, tPAI-1 (20 mg/kg only); ↓Resistin (200 mg/kg only) ↓HOMA-IR (200 mg/kg only); ↔ GTT and fasting blood glucose ↓Fas ↑Srebp1c mRNA dose-dependent ↑Cyp2b10 mRNA dose-dependent ↑Cyp1a2 mRNA (200 mg/kg only) ↑Serum LDL levels (200 mg/kg only) Effects of Arcolor 1260 in control diet: ↔ BW, % Fat, adipose weight or size ↔Serum ALT, AST, Il-6, tPAI-1, Leptin, and Resistin ↔HOMA-IR or GTT(AUC), but ↓ in fasting blood glucose dose-dependent ↑Cd36 and Il-6 mRNA (20 mg/kg only) ↑Cyp2b10, Cyp3a11 mRNA ↑Cyp1a2 mRNA (200 mg/kg only)
Wahlang et al. 2016	C57BL/6J (WT) Car-null PXR-null	Aroclor 1260 (corn oil) + HFD	Oral gavage, 20 mg/kg single dose (week 1 of feeding); 12 weeks of HFD feeding	Effects of Aroclor 1260 compared to control within genotype: ↑Car mRNA in WT mice only ↑Cyp2b10 mRNA in WT, and greater extent in Pxr −/− mice. ↑Pxr mRNA in Car −/− mice only ↔Cyp3a11 mRNA in all genotypes ↑Ugt1a1 mRNA in WT and Car −/− ↑Liver injury (H&E/CAE staining) ↔Serum ALT and AST levels ↑Tnfα mRNA and serum levels in WT mice only ↓Plasma insulin, HOMA-IR, QUICKI in WT mice only ↑Pparα and Cd36 mRNA in WT and CAR −/− mice ↓Fas mRNA in WT mice only ↑Cpt1a in all genotypes

Research on the mechanism by which PFASs and PCBs contribute to hepatotoxicity, specifically in relation to steatosis and steatohepatitis, respectively, provide essential data to support the increasing role of environmental contaminants in the development and progression of the most common cause of liver disease in the United States.

Emerging Role of Fungicides/Herbicides in NAFLD

Insecticides have been recently reviewed for their role in the development of NAFLD (41), but it has been determined that fungicides and herbicides make up a greater number of the 10% of pesticides that are associated with toxicant-induced steatosis (1). Five fungicides (cyproconazole, dazomet, fluazinam, hexaconazole, and pyrasulfotole meatoblite) had lowest effect levels at less than 10 mg/kg/day in at least one animal study, demonstrating a correlation of fungicide exposure to fatty liver endpoints (1). Two identified fungicides belong to the same class, the conazoles, which are established hepatotoxicants via CAR-mediated hepatocellular hypertrophy and hepatomegaly in rodent models (42–44). In the most recent studies the non-carcinogenic conazole, myclobutanil, has been studied in an in vitro model of steatosis for its contribution to the development of NAFLD. Myclobutanil was shown to promote a significant increase in fatty acid-induced steatosis in HepG2 cells, and contributed to hepatoxicity (increased LDH levels) along with diminished anti-apoptotic markers (Bcl-xL/Bak and Mcl-1/Bak) (45). Mancozeb, another fungicide, was also demonstrated to contribute to fatty acid-induced steatosis and cellular damage in HepG2 cells (46). These observations suggest that exposure to the fungicides may worsen NAFLD, but a mechanism remains elusive and in vivo studies are warranted. Specifically, myclobutanil is only known to cause hepatomegaly and its effects on lipid deposition in the liver have not been characterized. Preliminary unpublished data, within our laboratory, have suggested that exposure to pesticides via oral route of exposure may alter gut homeostasis. Therefore, some hepatotoxic effects of pesticides in vivo may be due to gut dysbiosis, another key factor in the development of NAFLD.

Glyphosate, the active ingredient in Roundup® (commercially available herbicide), has demonstrated pathological findings in the liver and digestive tract due to long-term exposure (47). Metabolomics data from rats exposed to low-dose Roundup formulation showed increased plasma triglyceride levels, which was supported by changes in triglyceride metabolism and lipotoxic conditions in the metabolome (48). Glutathione depletion was not significant, but markers of oxidative stress contributing to glutathione depletion were markedly disrupted (48). The most significant initial findings from a transcriptome profile of liver and kidney observed in a 2-year rat study demonstrated that the altered transcripts were significantly different from transcript levels of liver necrosis provoked by classical hepatotoxicants (49). A proteome analysis of C57BL/6 mice exposed to the active ingredient glyphosate for 7 days demonstrated a potential mechanism via the induced levels of the reactive metabolite glyoxylate, contributing to inhibition of fatty acid oxidation enzymes and altered lipid profiles and fat storage in the liver (50). The overall metabolome, transcriptome and proteome findings associated with glyphosate do not provide a direct in vivo mechanism, but demonstrate the importance of determining if chronic exposure to fungicides/herbicides via food contaminants contributes to or worsens NAFLD and NASH etiologies.

Conclusion

The liver can be considered a major organ of toxicity to environmental toxicants and hepatotoxic effects may not always result in overt toxicities, but can contribute to prevalent liver disease etiologies of steatosis and steatohepatitis. With the growing prevalence of NAFLD and NASH in the U.S. among both adults and children, we must consider all risk factors and mechanisms of the multifactorial disease. POPs have begun to be elucidated for these effects and the contribution to novel mechanisms of disease development, as demonstrated by PFAS and PCB most current mechanistic research in the field. Mechanisms of PFAS liver toxicity have demonstrated an association with the development of hepatic steatosis and worsening of NAFLD, whereas recent PCB research have elucidated mechanisms by which the POP promotes NASH. Pesticides have been a prevalent concern in society, but the recent findings that fungicides and herbicides may contribute more to hepatotoxic effects warrants further investigation into their role in NAFLD development, especially since primary chronic exposure is through our food supply. A recent study also addresses the importance that we are continually exposed to mixtures and not individual chemicals, and demonstrated that environmentally relevant mixtures of PCBs and the well-studied class of organochlorine pesticides contributed to steatosis via dysregulated lipid metabolism in a mouse model of obesity (51). Emphasis should be placed on determining the mechanisms by which these environmental exposures can induce toxic effects, and then be applied to models of relevant disease in order to determine the significance of low-dose chronic exposure to the initiation and/or progression of disease etiologies. Lastly, emphasis must also be placed on the importance of organ cross-talk in the progression of NAFLD and NASH as mechanisms of liver steatosis and steatohepatitis may not always develop from direct liver effects, but also from gut dysbiosis and adipose tissue dysregulation. Continued research on environmental contaminates’ mechanistic toxicities are essential in fully understanding and being able to address the burden of NAFLD.

Abbreviations

AhR

Aryl hydrocarbon receptor

ALD

Alcoholic liver disease

ALT

Alanine aminotransferase

ApoB

Apolipoprotein B

CAR

Constitutive androstane receptor

Cd36 / FAT

Fatty acid translocase

CYP

Cytochrome P450

HFD

High fat diet

Hmgcs2

3-Hydroxy-3-Methylglutaryl-CoA Synthase 2

HNF4

Hepatocyte nuclear factor 4

Lpl

Lipoprotein lipase

MCD

methionine-choline deficient

NAFLD

Non-alcoholic fatty liver disease

NASH

Non-alcoholic steatohepatitis

PFAS

Perfluoroalkylated substances

PFCs

Polyfluroinated chemicals

PFHxS

Perfluorohexanesulfonic acid

PFNA

Perfluorononanoic acid

PFOA

Perfluorooctanoic acid

PFOS

Perfluorooctanesulfonic acid

POPs

Persistent organic pollutants

PPAR

Peroxisome proliferator-activated receptor

PUFA

polyunsaturated fatty acids

PXR

Pregnane X receptor

ROS

Reactive Oxygen Species

SREBP

Sterol regulatory element-binding protein

TAFLD

Toxicant-associated fatty liver disease

TASH

Toxicant-associated steatohepatitis

Tnf

Tumor-necrosis factor

VLDL

Very low-density lipoproteins

Vldlr

Very low-density lipoprotein receptor