Endocrine disrupting chemicals: A promoter of non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is the most prevalent liver disorder. With the improvement in human living standards, the prevalence of NAFLD has been increasing in recent years. Endocrine-disrupting chemicals (EDCs) are a class of exogenous chemicals that simulate the effects of hormones in the body. There has been growing evidence regarding the potential effects of EDCs on liver health, especially in NAFLD. This paper aims to summarize the major EDCs that contribute to the growing burden of NAFLD and to raise public awareness regarding the hazards posed by EDCs with the objective of reducing the incidence of NAFLD.


Introduction
Non-alcoholic fatty liver disease (NAFLD) is an important public health issue that affects a large portion of the global population. Estimates indicate that the worldwide prevalence of NAFLD ranges from 13% in Africa to 42% in Southeast Asia (1). NAFLD encompasses a spectrum of liver conditions, including simple steatosis or non-alcoholic fatty liver (NAFL), which has a milder course, and non-alcoholic steatohepatitis (NASH), with potential progression to fibrosis or cirrhosis and hepatocellular carcinoma (HCC) (2,3). The hallmark of NAFLD is the accumulation and deposition of excessive fat in liver cells, which may be related to genetic, dietary, and environmental factors (4). These factors promote the onset of insulin resistance (IR) in adipose tissue, leading to adipocyte dysfunction and increased influx of free fatty acids (FFAs) into the liver (5). These FFAs and their consequent lipotoxic intermediates have been shown to have adverse effects such as abnormal lipids metabolism, oxidative stress, and chronic liver inflammation, all of which contribute to the progression of NAFLD (6,7).
Environmental endocrine disrupting chemicals (EDCs) are a class of exogenous chemicals that mimic the effects of hormones in the body, causing hormonal dysregulation and mediating various metabolic disorders. The liver, an organ crucial to metabolism and detoxification (8), has been shown to be impacted by EDCs, with studies suggesting exposure to these chemicals can lead to metabolic changes and liver disease (9)(10)(11)(12). Because of the difficulty in getting biopsy-confirmed NAFLD histological specimens, the liver injury is typically assessed using serum biomarkers of hepatoxicity of NAFLD. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and γ-glutamyl transferase (GGT) are considered specific biomarkers of liver injury, they are widely used to evaluate the progression of NAFLD. Given the role of EDCs in the worldwide deterioration of metabolic health, it is imperative that the scientific community continues to study and understand their effects.
We aimed to provide an overview of EDCs and summarize the effects of EDC exposure on NAFLD.

Overview of EDCs
Environmental endocrine disruptors (EDCs) are a heterogenous group of chemicals that are widely distributed and easily enriched, present in many forms. In daily life, an increasing number of substances have been identified as EDCs; they enter the body through the digestive tract, respiratory tract, or skin, and produce adverse effects. EDCs can be classified as natural or synthetic, based on their origin. Natural EDC include phytoestrogens and mycotoxins, while synthetic EDCs include chemicals used as industrial solvents and their byproducts (polychlorinated biphenyls, polybrominated biphenyls, and dioxins), plastics (bisphenol A), plasticizers (phthalates), fungicides (vinclozolin), pesticides (methoxychlor and chlorpyrifos), heavy metals (mercury and lead), and pharmaceutical agents present in human and animal foods (11,(13)(14)(15)(16).
Studies have shown that EDCs can interfere with various aspects of hormone regulation in the body, including production, release, transport, metabolism, binding, action, and elimination, leading to hormonal dysregulation and contributing to various metabolic disorders (17, 18). It's also been found that EDCs play a potential role in the regulation of genomic expression, promoting epigenetic modifications that result in the development of pathologies by mediating carcinogenic, neurotoxic, hepatotoxic, and immunotoxic effects (19-21). The challenge in understanding the impact of EDCs is compounded by the fact that humans are exposed, not to a single environmental pollution compound, but to a cocktail of EDCs, making it even more difficult to predict the net effect and evincing the association between a specific EDC and disease (13,22). Moreover, exposure to persistent EDCs may initiate and promote the pathogenesis of NAFLD (23). EDCs affect the progression of NAFLD through the interaction of nuclear receptors. Activating transcription factors, triggering the imbalances between lipid flow/outflow in the liver, promoting mitochondrial dysfunction, and mediating the hepatic inflammatory are the possible mechanism of NAFLD (17).

Methods
In this study, we focused on several key EDCs that are closely related to human health, including per−/polyfluorinated substances, bisphenol A, polychlorinated biphenyls, and phthalates (as outlined in Table 1). Systematic search of PubMed and Embase databases was conducted from January 1, 2010, to December 28, 2022, to identify human studies investigating the relationship between non-alcoholic liver disease and these EDCs. Detailed search strategies are presented in Table 2. After screening the retrieved studies based on their titles and abstracts, we excluded studies without human data, case reports, non-original reports, studies without NAFLD outcomes, and pharmacological or ecological studies.
The liver is one of the main target organs of PFAS toxicity (40). However, the exact mechanism of PFAS hepatotoxicity remains unclear. PFAS are thought to act as ligands for peroxisome proliferatoractivated receptors (PPARs), which promote liver inflammation and

Substance
Abbreviation Source Characteristic Frontiers in Public Health 03 frontiersin.org triglyceride accumulation through the activation of PPARα and lead to liver injury or NAFLD (41-43). The complementary mechanism also includes activation of the constitutive androstane receptor (CAR) (42,44), downregulation of nuclear factor erythroid 2-related factor 2 (NRF2) (45,46), and upregulation of nuclear factor kappa-lightchain-enhancer of activated B cells nuclear factor-kappa B (NF-ĸ B) (47). Animal and epidemiological studies have shown that PFAS can cause an obviously increase in liver lipid volume, induce mitochondrial dysfunction and oxidative stress, and promote inflammatory responses in NAFLD progression (41,48,49). A large number of nuclear receptors (NRs) were expressed in liver, making it a critical target for PFAS. In this review, we noticed that a few studies have evaluated the hepatic enzyme abnormalities associated with PFAS exposure. PFOA and PFNA exposure is usually positively associated with higher ALT and GGT levels, suggesting that changes in serum biomarkers are often accompanied by histopathological changes or liver disease (34,39,(50)(51)(52)(53). However, for AST, studies have reported different outcomes in different crowds. Khalil (37) found that there was no relationship between serum PFAS and AST levels in obese children, while PFOA and PFOS were positively correlated with AST in Japanese children (52). In addition, Attanasio et al. reported sex-specific histological effects of PFAS exposure and found positive associations between PFAS and ALT in female adolescents, but conversely in male adolescents, which could be mediated by sex hormones (54). Evidence for sex-specific differences was also found in rats, with ALT increasing more frequently in male rats (55)(56)(57). The authors suggest that PFAS exposure may play an important role in the development of NAFLD and carcinoma ( Table 3). The PFAS was positively associated with lobular inflammation in adults undergoing bariatric surgery in Northern Europe, however, the reason for this association is unclear. It may be related to changes in lipid and bile acid metabolism (9,59). In animal models, exposure to subchronic PFOS has been found to enhance hepatic stellate cell (HSC) activation and exacerbate carbon tetrachloride (CCl 4 )-induced liver fibrosis (60), consistent with the outcomes of Sen's study, which showed that PFOS was positively associated with hepatic fibrosis in adults (9). In contrast, a study including adults from the C8 Health Project in the USA showed that cumulative PFOA exposure had no effect on all liver diseases, enlarged liver, or cirrhosis (58). Additionally, a study included 1,105 motherchild pairs from the European Human Early-Life Exposome (HELIX) cohort showed that higher exposure to PFAS during pregnancy was

Substances
Database Search terms NAFLD PubMed NAFLD OR NASH OR "nonalcoholic fatty liver disease" OR "nonalcoholic steatohepatitis" OR "nonalcoholic fatty liver" OR "fatty liver" OR steatosis OR "liver enzymes" OR"liver damage" OR "liver injury" OR "liver fibrosis"  Frontiers in Public Health 05 frontiersin.org associated with higher liver enzyme levels in children (38). PFAS can cross the placenta barrier efficiently and deposit in fetal tissues (61), they altered some amino acid (e.g., valine, leucine, phenylalanine) and lipid (e.g., glycerophospholipid) metabolism that related to NAFLD pathogenesis, which exerted adverse effects in liver (38).

Polychlorinated biphenyls
Polychlorinated biphenyls (PCBs) are also a type of POP that are manufactured and used commercially as dielectric fluids in transformers (62). They are thermodynamically stable polyhalogenated aromatic hydrocarbons consisting of up to ten chlorine substituents attached to a biphenyl ring (63). Based on their structures, PCB congeners have been subclassified into dioxin-like (DL) and nondioxin-like (NDL). DL PCBs have a coplanar structure, whereas NDL PCBs have a noncoplanar structure, which can be attributed to receptor-based modes of action. PCBs persist in the environment and accumulate in soil, aquatic sediments, and in species that consume these sources (fish, cows, dairy) (64). Despite a ban on their production and emission in 1979, human exposure to PCBs usually occurs through contaminated air, water, or food. As POPs, PCBs accumulate in adipose tissue and are gradually released into the bloodstream (65,66).
The liver appears to be both a target and an effector organ for PCB-induced endocrine disruption. The occurrence of NAFLD is due to an imbalance between lipid production and elimination, which promotes excessive accumulation of hepatic lipids (17). PCBs have been documented to be related to this phenomenon, as they can induce pathological fat aggregation, both in the DL and NDL groups (67). DLPCBs activate the aryl hydrocarbon receptor (AhR) and peroxisome proliferator-activated receptors alpha and gamma (PPARα/γ) (63,68), exerting a multimodal effect on lipid accumulation and causing steatosis by disrupting liver lipid metabolism. NDL PCBs, on the other hand, activate the constitutive androstane receptor (CAR) and pregnane X receptor (PXR) (69), which could reduce the protective response of the liver to promote diet induced NAFLD (67).
Studies have shown that PBC exposure can cause NAFLD related metabolic disorders, including insulin resistance, obesity, and lipid metabolic dysfunction. PCBs differentially regulate hepatic lipid metabolism and several related genes. PCB126 exposure increases hepatic lipids and causes toxicant-associated steatosis (mild smalldroplet macro vesicular steatosis) (63). Mono-exposure to either PCB126 or Aroclor1260 increased hepatic lipid uptake while decreasing lipid biosynthesis, but these effects were abrogated by exposure to the NDL/DL PCB mixture (63). Pnpla3, a lipase implicated in NAFLD (70), was significantly upregulated by Aroclor1260 exposure, but was suppressed by either PCB126 or Aroclor1260/PCB126 exposure, potentially due to activation of different receptors among NDL or DL PCBs. PCBs have also been associated with the promotion of toxicant-associated steatohepatitis (TASH), which can ultimately lead to secondary liver necrosis, potentially due to a loss of protein phosphorylation and downregulation of the hepatic kinome (71). Positive associations were also observed between PCB concentrations and NAFLD-related biomarkers (Table 4), with most PCB congeners positively correlated with elevated alanine aminotransferase (ALT) levels. A study of 4,582 adults conducted by Cave et al. (76) showed that 10.6% of participants had unexplained ALT elevation, with older age significantly associated with total PCB levels in the highest quartile. Of those participants aged 70 years or older, 71.7% had high PCB levels compared to only 2.2% of those aged under 30 years. A study of 1,108 mother-child pairs from six countries by Midya (72) discovered that prenatal exposure to PCBs is a potential risk factor for pediatric NAFLD, and were further associated with increased CK-18 levels (a novel marker of hepatocyte apoptosis and NAFLD). Notably, researchers have identified potential therapeutic targets for improving PCB-induced NAFLD, including the anti-fibrotic compound recombinant FGF21, which reduced the overexpression of hepatic lipocalin-2 (LCN2), a group of transporters of small lipophilic molecules that are upregulated in several liver diseases, and attenuated NAFLD (62, 77).

Bisphenol A
Bisphenol A (BPA), which consists of two phenol rings attached by a methyl bridge with two methyl groups (78), is a plasticizer mainly used for polycarbonate plastics and epoxy resins in many consumer products (79). BPA exposure can occur via various sources such as plastic containers, toys, water bottles, food packaging materials, office supplies, and dental sealants (80,81). BPA has low lipophilicity and degrades rapidly with a half-life of 4-5 h (82). Due to its broad application, BPA is detected in more than 90% of people, and the median urine BPA concentration in adults is 2.24-6.17 ng/mL (83, 84).
The liver is the main organ that metabolizes and transforms BPA into glucuronidation; therefore, it is more susceptible to BPA than other organs (85). BPA increases the risk of NAFLD owing to fat accumulation, obesity, and oxidative stress. Upregulation of lipogenic enzymes and transcription factors, such as sterol regulatory element binding protein-1c (srebp-1c), the carbohydrate responsive element binding protein (ChREBP), and liver X receptor (LXR) (86), promotes de novo lipogenesis (DNL) (87), increasing the risk of lipid accumulation and obesity. In addition, exposure to high doses of BPA decreased the activities of antioxidant indicators, such as superoxide dismutase (SOD) and glutathione (GSH), causing excessive accumulation of free radicals, such as reactive oxygen species (ROS), promoting liver damage and hepatotoxicity (88).
Aminotransferases are the most widely used biomarkers in experiments, and are released into the bloodstream following liver injury (89). Epidemiological studies have shown that BPA exposure could have negative effects on NAFLD-related biomarkers (  (98). In addition to liver biomarkers, urinary BPA levels were positively associated with the prevalence of NAFLD in adults and adolescents. In the Korean National Environmental Health Survey (83), which included 3,476 participants with a mean age of 52.96 ± 0.25 years old, the geometric mean concentration of BPA in the NAFLD group was significantly higher than in the non-NAFLD group (2.56 ug/L vs. 2.24 ug/L, p = 0.001). A study analyzing adolescents (12-19 years old) from NHANES in the USA also showed that the risk of suspected NAFLD (ALT≥30 U/L) was increased in participants in higher quartiles of BPA exposure (93). In terms of pathological findings, mice and rats treated with BPA showed liver tissue dilatation of sinusoids,  GC-MS, gas chromatograph-mass spectrometry; GC-MS/MS, gas chromatography coupled to tandem mass spectrometry; HRGC/HRMS, high-resolution gas chromatography/isotope dilution high-resolution mass spectrometry; HRGC-MS, high-resolution gas chromatograph coupled to mass spectrometry.  Frontiers in Public Health 08 frontiersin.org congestion, inflammation, and necrosis in a dose-dependent manner (99). Although BPA can be excreted quickly from the body, people are constantly exposed to it throughout their lives and BPA exposure is associated with metabolic health in offspring. Prenatal BPA exposure has been shown to alter gene expression profiles and result in peripheral insulin resistance and liver lipotoxicity (100,101). Gestational BPA exposure can promote the development of NAFLD in rodent models through the perturbation of the nuclear transcription factor activity (102). Another study indicated that exposure to BPA may diminish the immune response following hepatitis B vaccination (79).

Di-(2-ethylhexyl) phthalate
Phthalates are a large group of ubiquitous industrial chemicals that are commonly used in a variety of products such as plasticizers in food wrapping and packaging, coatings, cosmetics, adhesives, medical tubes, and toys (103,104). Phthalates may enter the human body through the skin, respiratory tract, digestive tract, or even intravenous injection, as they are prone to leaching and transfer to air, soil, or food (105, 106). These chemicals are usually rapidly metabolized and excreted within 24-48 h. Diester phthalates could hydrolyze into monoester phthalates, then excreted as glucuronide conjugates, in the urine (107).
The development of NAFLD may be related to the adverse effects of DEHP on lipid metabolism and oxidative stress. DEHP and its active metabolite mono-(2-ethylhexyl) phthalate (MEPH) can affect hepatic accumulation of TGs and exacerbate NAFLD in rodents (108). MEHP also can affect the lipid accumulation in BRL-3A hepatocytes through the inhibition of the Janus kinase 2/Signal transducer and activator of transcription 5 (JAK2/STAT5) pathway, suggesting that the regulation of STAT5 by MEPH plays a critical role in the activation of enzymes involved in fatty acid metabolism (109). Furthermore, DEHP also mediates the deterioration of antioxidant machinery and induces oxidative stress. Higher levels of ROS were observed in MEHP-treated cells, indicating the effect of ROS on pro-inflammatory cytokine production and apoptosis of hepatocytes by inducing NF-κB (110, 111). Other experimental studies in animals have shown that the toxicity of phthalates drives liver fibrosis by oxidative stress pathways (112,113).
Phthalate exposure is strongly associated with the NAFLD prevalence (114) ( Table 6). A study involving 5,800 Korean adults demonstrated that the prevalence of NAFLD defined by the hepatic steatosis index (HSI) was associated with high urinary levels of many types of phthalates, and higher quartiles of MEHHP revealed a significantly higher risk (OR 1.39, 95% CI:1.00-1.92) of NAFLD (119). In addition, NAFLD measured using vibration-controlled transient elastography (VCTE) was also found to be positively associated with MECPP and MEHHP exposure (116). Unlike in adults, DEHP exposure also affects the prevalence of NAFLD in adolescents. Berman et al. (115) studied 387 mother-child pairs in Australia and found that mid-level prenatal exposure to MnBP was associated with a greater incidence of NAFLD at 17 years old. Table 6 also lists epidemiologic studies on the relationship between NAFLD biomarkers. A study involving 102 males aimed to examine the influence of MEP and MEHP on liver function and found that phthalate exposure may be associated with a statistically significant increase in ALT and AST serum levels, while urinary phthalate levels may be correlated with increased serum TG and decreased HDL cholesterol levels (104). A transversal study also demonstrated that serum MEHP levels were correlated with GGT (122). Thus, DEHP may interfere with thyroid function and induce NAFLD. Yang et al. divided 2,308 adults with subclinical hypothyroidism (SCH) into NAFLD and non-NAFLD groups according to the HSI score and found that the levels of phthalate metabolites in urine are positively associated with NAFLD with SCH (118). DEHP possesses a thyroid receptor antagonistic function, while thyroid hormones can activate TH-Receptor β (a potential target in NAFLD therapy) and decrease hepatic steatosis, which may further induce NAFLD (123)(124)(125). However, not all studies have discovered an association between phthalates and thyroid hormones, and further studies ought to be conducted to investigate this association.

Sex differences of association between EDCs and NAFLD
Liver expresses androgen and estrogen receptors (126), thus research on sex-specific differences in EDCs has been a hot topic, but no consistent conclusion has been made so far. In the analyses of NHANES 2013-2016, it was observed that an opposite direction of the statistically significant association between PFAS and liver enzyme by sex, elevated AST is associated with increased PFOA in female adolescents, whereas there is an inverse association with increased PFOA, PFNA and PFHxS in males (54). However, Borghese et al. using the data from Canadian Health Measures Survey found that the association between PFOA and AST was twice as strong among men vs. women, this could be because menstruation, pregnancy, and breastfeeding are all prominent excretion pathways for PFAS in women (25). The sex difference was also reported in PCB, Li et al. (127) found that increased mortality from hepatic disease in PCB-exposed, it may be explained by sex-specific effects of estrogenic PCB congeners (73,128). Relationships between BPA exposure and liver function at puberty were observed, serum AST levels were positively associated with BPA in boys, and the effect sizes were larger for all indicator in boys (94). Due to the differences in sex hormone associated BPA metabolism, women expressed higher levels of the UGT2B1 to catalyze BPA glucuronidation and accelerate the clearance of BPA (129,130). Trasande et al. (131) identified a near-significant interaction of DEHP metabolites with sex, suggesting a possible role of reduced androgen activity. EDC's sex difference is complicated, further research is needed to be done.

Conclusion
Exposure to environmental chemicals is ubiquitous and poses a threat to human metabolic health. EDCs affect NAFLD by interacting with nuclear receptors (NRs) and activating transcriptional factors, which promote hepatic lipid accumulation, oxidative stress, and liver dysfunction. Data from epidemiological studies prove an interrelationship between EDCs exposure and NAFLD. However, several challenges remain. For example, EDC mixtures are a complicated issue, and it is difficult to predict the net effect of EDC mixtures at the individual level in humans because it is difficult to perform laboratory detection for individual isomer and every human has a unique exposome (22,90). In addition, the interpretation of the results on the effects of EDCs has been complicated by using different      (132). In the future, research with larger samples, longer follow-up periods, and a multidisciplinary approach to explore the effect of EDCs in the human body is required. Moreover, the scientific community should help draw public attention to the hazards of EDCs and promote more regulation of industrial pollution.

Author contributions
YC: design, information retrieval, draft writing, and article review and editing. YW: information retrieval, draft writing, and article review and editing. ZC: information retrieval, draft writing, and information visualization. WL: methodology, review, and information visualization. BL: methodology and review. QZ: article review and editing. XZ: article review and editing. JD: methodology, and article review and editing. JC: conceptualization, design, funding acquisition, and article review and editing. All authors contributed to the article and approved the submitted version.