Investigating the effects of long-term Aroclor 1260 exposure on fatty liver disease in a diet-induced obesity mouse model.

Introduction: Polychlorinated biphenyls (PCBs) are persistent environmental toxicants that have been implicated in numerous health disorders including liver diseases such as non-alcoholic fatty liver disease (NAFLD). Toxicant-associated NAFLD, also known as toxicant-associated fatty liver disease (TAFLD), consists of a spectrum of disorders ranging from steatosis and steatohepatitis to fibrosis and hepatocellular carcinoma. Previously, our group demonstrated that 12-week exposure to the PCB mixture, Aroclor 1260, exacerbated steatohepatitis in high-fat diet (HFD)-fed mice; however, the longer-term effects of PCBs on TAFLD remain to be elucidated. This study aims to examine the longer-term effects of Aroclor 1260 (>30 weeks) in a diet-induced obesity model to better understand how duration of exposure can impact TAFLD. Methods: Male C57BL/6 mice were exposed to Aroclor 1260 (20 mg/kg) or vehicle control by oral gavage at the beginning of the study period and fed either a low-fat diet (LFD) or HFD throughout the study period. Results: Aroclor 1260 exposure (>30 weeks) led to steatohepatitis only in LFD-fed mice. Several Aroclor 1260 exposed LFD-fed mice also developed hepatocellular carcinoma (25%), which was absent in HFD-fed mice. The LFD+Aroclor1260 group also exhibited decreased hepatic Cyp7a1 expression and increased pro-fibrotic Acta2 expression. In contrast, longer term Aroclor 1260 exposure in conjunction with HFD did not exacerbate steatosis or inflammatory responses beyond those observed with HFD alone. Further, hepatic xenobiotic receptor activation by Aroclor 1260 was absent at 31 weeks post exposure, suggesting PCB redistribution to the adipose and other extra-hepatic tissues with time. Discussion: Overall, the results demonstrated that longer-term PCB exposure worsened TAFLD outcomes independent of HFD feeding and suggests altered energy metabolism as a potential mechanism fueling PCB mediated toxicity without dietary insult. Additional research exploring mechanisms for these longer-term PCB mediated toxicity in TAFLD is warranted.

RISING STARS: Sex differences in toxicant-associated fatty liver disease.

Based on biological sex, the consequential health outcomes from exposures to environmental chemicals or toxicants can differ in disease pathophysiology, progression, and severity. Due to basal differences in cellular and molecular processes resulting from sexual dimorphism of organs including the liver and additional factors influencing 'gene-environment' interactions, males and females can exhibit different responses to toxicant exposures. Associations between environmental/occupational chemical exposures and fatty liver disease (FLD) have been well-acknowledged in human epidemiologic studies and their causal relationships demonstrated in experimental models. However, studies related to sex differences in liver toxicology are still limited to draw any inferences on sex-dependent chemical toxicity. The purpose of this review is to highlight the present state of knowledge on the existence of sex differences in toxicant-associated FLD (TAFLD), discuss potential underlying mechanisms driving these differences, implications of said differences on disease susceptibility, and emerging concepts. Chemicals of interest include various categories of pollutants that have been investigated in TAFLD, namely persistent organic pollutants, volatile organic compounds, and metals. Insight into research areas requiring further development is also discussed, with the objective of narrowing the knowledge gap on sex differences in environmental liver diseases. Major conclusions from this review exercise are that biological sex influences TAFLD risks, in part due to (i) toxicant disruption of growth hormone and estrogen receptor signaling, (ii) basal sex differences in energy mobilization and storage, and (iii) differences in chemical metabolism and subsequent body burden. Finally, further sex-dependent toxicological assessments are warranted for the development of sex-specific intervention strategies.

Western diet unmasks transient low-level vinyl chloride-induced tumorigenesis; potential role of the (epi-)transcriptome.

BACKGROUND & AIMS: Vinyl chloride (VC) monomer is a volatile organic compound commonly used in industry. At high exposure levels, VC causes liver cancer and toxicant-associated steatohepatitis. However, lower exposure levels (i.e., sub-regulatory exposure limits) that do not directly damage the liver, enhance injury caused by Western diet (WD). It is still unknown if the long-term impact of transient low-concentration VC enhances the risk of liver cancer development. This is especially a concern given that fatty liver disease is in and of itself a risk factor for the development of liver cancer. METHODS: C57Bl/6 J mice were fed WD or control diet (CD) for 1 year. During the first 12 weeks of feeding only, mice were also exposed to VC via inhalation at sub-regulatory limit concentrations (<1 ppm) or air for 6 h/day, 5 days/week. RESULTS: Feeding WD for 1 year caused significant hepatic injury, which was exacerbated by VC. Additionally, VC increased the number of tumors which ranged from moderately to poorly differentiated hepatocellular carcinoma (HCC). Transcriptomic analysis demonstrated VC-induced changes in metabolic but also ribosomal processes. Epitranscriptomic analysis showed a VC-induced shift of the modification pattern that has been associated with metabolic disease, mitochondrial dysfunction, and cancer. CONCLUSIONS: These data indicate that VC sensitizes the liver to other stressors (e.g., WD), resulting in enhanced tumorigenesis. These data raise concerns about potential interactions between VC exposure and WD. It also emphasizes that current safety restrictions may be insufficient to account for other factors that can influence hepatotoxicity.

Associations between residential volatile organic compound exposures and liver injury markers: The role of biological sex and race.

While occupational exposures to volatile organic compounds (VOCs) have been linked to steatohepatitis and liver cancer in industrial workers, recent findings have also positively correlated low-dose, residential VOC exposures with liver injury markers. VOC sources are numerous; factors including biological make up (sex), socio-cultural constructs (gender, race) and lifestyle (smoking) can influence both VOC exposure levels and disease outcomes. Therefore, the current study's objective is to investigate how sex and race influence associations between residential VOC exposures and liver injury markers particularly in smokers vs. nonsmokers. Subjects (n = 663) were recruited from residential neighborhoods; informed consent was obtained. Exposure biomarkers included 16 urinary VOC metabolites. Serological disease biomarkers included liver enzymes, direct bilirubin, and hepatocyte death markers (cytokeratin K18). Pearson correlations and generalized linear models were conducted. Models were adjusted for common liver-related confounders and interaction terms. The study population constituted approximately 60% females (n = 401) and 40% males (n = 262), and a higher percent of males were smokers and/or frequent drinkers. Both sexes had a higher percent of White (75% females, 82% males) vs. Black individuals. Positive associations were identified for metabolites of acrolein, acrylamide, acrylonitrile, butadiene, crotonaldehyde, and styrene with alkaline phosphatase (ALP), a biomarker for cholestatic injury; and for the benzene metabolite with bilirubin; only in females. These associations were retained in female smokers. Similar associations were also observed between these metabolites and ALP only in White individuals (n = 514). In Black individuals (n = 114), the styrene metabolite was positively associated with aspartate transaminase. Interaction models indicated that positive associations for acrylamide/crotonaldehyde metabolites with ALP in females were dose-dependent. Most VOC associations with K18 markers were negative in this residential population. Overall, the findings demonstrated that biological sex, race, and smoking status influence VOC effects on liver injury and underscored the role of biological-social-lifestyle factor(s) interactions when addressing air pollution-related health disparities.

Circulating MicroRNAs, Polychlorinated Biphenyls, and Environmental Liver Disease in the Anniston Community Health Survey.

BACKGROUND: Polychlorinated biphenyl (PCB) exposures have been associated with liver injury in human cohorts, and steatohepatitis with liver necrosis in model systems. MicroRNAs (miRs) maintain cellular homeostasis and may regulate the response to environmental stress. OBJECTIVES: We tested the hypothesis that specific miRs are associated with liver disease and PCB exposures in a residential cohort. METHODS: Sixty-eight targeted hepatotoxicity miRs were measured in archived serum from 734 PCB-exposed participants in the cross-sectional Anniston Community Health Survey. Necrotic and other liver disease categories were defined by serum keratin 18 (K18) biomarkers. Associations were determined between exposure biomarkers (35 ortho-substituted PCB congeners) and disease biomarkers (highly expressed miRs or previously measured cytokines), and Ingenuity Pathway Analysis was performed. RESULTS: The necrotic liver disease category was associated with four up-regulated miRs (miR-99a-5p, miR-122-5p, miR-192-5p, and miR-320a) and five down-regulated miRs (let-7d-5p, miR-17-5p, miR-24-3p, miR-197-3p, and miR-221-3p). Twenty-two miRs were associated with the other liver disease category or with K18 measurements. Eleven miRs were associated with 24 PCBs, most commonly congeners with anti-estrogenic activities. Most of the exposure-associated miRs were associated with at least one serum hepatocyte death, pro-inflammatory cytokine or insulin resistance bioarker, or with both. Within each biomarker category, associations were strongest for the liver-specific miR-122-5p. Pathways of liver toxicity that were identified included inflammation/hepatitis, hyperplasia/hyperproliferation, cirrhosis, and hepatocellular carcinoma. Tumor protein p53 and tumor necrosis factor α were well integrated within the top identified networks. DISCUSSION: These results support the human hepatotoxicity of environmental PCB exposures while elucidating potential modes of PCB action. The MiR-derived liquid liver biopsy represents a promising new technique for environmental hepatology cohort studies. https://doi.org/10.1289/EHP9467.

Associations Between Residential Exposure to Volatile Organic Compounds and Liver Injury Markers.

Occupational exposures to volatile organic compounds (VOCs) have been associated with numerous health complications including steatohepatitis and liver cancer. However, the potential impact of environmental/residential VOC exposures on liver health and function is largely unknown. To address this knowledge gap, the objective of this cross-sectional study is to investigate associations between VOCs and liver injury biomarkers in community residents. Subjects were recruited from six Louisville neighborhoods, and informed consent was obtained. Exposure biomarkers included 16 creatinine-adjusted urinary metabolites corresponding to 12 parent VOCs. Serological disease biomarkers measured included cytokertain-18 (K18 M65 and M30), liver enzymes, and direct bilirubin. Associations between exposure and disease biomarkers were assessed using generalized linear models. Smoking status was confirmed through urinary cotinine levels. The population comprised of approximately 60% females and 40% males; White persons accounted 78% of the population; with more nonsmokers (n = 413) than smokers (n = 250). When compared with nonsmokers, males (45%) and Black persons (26%) were more likely to be smokers. In the overall population, metabolites of acrolein, acrylonitrile, acrylamide, 1,3-butadiene, crotonaldehyde, styrene, and xylene were positively associated with alkaline phosphatase. These associations persisted in smokers, with the exception of crotonaldehyde, and addition of N,N-dimethylformamide and propylene oxide metabolites. Although no positive associations were observed for K18 M30, the benzene metabolite was positively associated with bilirubin, irrespective of smoking status. Taken together, the results demonstrated that selected VOCs were positively associated with liver injury biomarkers. These findings will enable better risk assessment and identification of populations vulnerable to liver disease.

Polychlorinated biphenyls altered gut microbiome in CAR and PXR knockout mice exhibiting toxicant-associated steatohepatitis.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants associated with non-alcoholic fatty liver disease (NAFLD). Previously, we demonstrated that the PCB mixture, Aroclor1260, exacerbated NAFLD, reflective of toxicant-associated steatohepatitis, in diet-induced obese mice, in part through pregnane-xenobiotic receptor (PXR) and constitutive androstane receptor (CAR) activation. Recent studies have also reported PCB-induced changes in the gut microbiome that consequently impact NAFLD. Therefore, the objective of this study is to examine PCB effects on the gut-liver axis and characterize the role of CAR and PXR in microbiome alterations. C57Bl/6 (wildtype, WT), CAR and PXR knockout mice were fed a high fat diet and exposed to Aroclor1260 (20 mg/kg, oral gavage, 12 weeks). Metagenomics analysis of cecal samples revealed that CAR and/or PXR ablation increased bacterial alpha diversity regardless of exposure status. CAR and PXR ablation also increased bacterial composition (beta diversity) versus WT; Aroclor1260 altered beta diversity only in WT and CAR knockouts. Distinct changes in bacterial abundance at different taxonomic levels were observed between WT and knockout groups; however Aroclor1260 had modest effects on bacterial abundance within each genotype. Notably, both knockout groups displayed increased Actinobacteria and Verrucomicrobia abundance. In spite of improved bacterial diversity, the knockout groups however failed to show protection from PCB-induced hepato- and intestinal- toxicity including decreased mRNA levels of ileal permeability markers (occludin, claudin3). In summary, CAR and PXR ablation significantly altered gut microbiome in diet-induced obesity while Aroclor1260 compromised intestinal integrity in knockout mice, implicating interactions between PCBs and CAR, PXR on the gut-liver axis.

Hepatic Injury Caused by the Environmental Toxicant Vinyl Chloride is Sex-Dependent in Mice.

Vinyl chloride (VC), a common industrial chemical, has been associated with hemangiosarcoma and toxicant-associated steatohepatitis (TASH) in men working at rubber-production plants. Our group previously demonstrated that chronic VC inhalation at environmentally relevant levels (< 1 ppm) in male mice exacerbated hepatic injury caused by high-fat diet (HFD) feeding. Because VC studies on TASH have only been performed in male models, the objective of this study is to examine VC inhalation in female mice in the context of TASH mechanisms. Male and female C57Bl/6 mice were fed either a low-fat diet or HFD and exposed to VC or room air using an inhalation chamber, for 12 weeks (6 h, 5 days/week); and plasma and liver samples were collected after euthanasia. Compared with males, females were less susceptible to HFD+VC-induced obesogenic effects demonstrated by lower body weight and fat composition. Histological analysis revealed that whereas VC exacerbated HFD-induced steatosis in males, this effect was absent in females. In addition, females were more resistant to VC-induced hepatic inflammation whereas males had increased liver weights and higher hepatic Tnfα mRNA levels. Systemic markers of hepatic injury, namely alanine aminotransaminase and thrombin/antithrombin levels were increased by HFD+VC co-exposures only in males. In addition, females did not show significant cell death as previously reported in males. Taken together, the results suggested that VC inhalation led to sex-dependent liver and metabolic toxicity. This study implicated the importance of assessing sex differences in environmental basic science and epidemiologic studies to better identify at-risk populations in both men and women.

Phosphodiesterase 4 Inhibition as a Therapeutic Target for Alcoholic Liver Disease: From Bedside to Bench.

Alcoholic liver disease (ALD) is a major cause of liver-related mortality. There is still no US Food and Drug Administration-approved therapy for ALD, and therefore, identifying therapeutic targets is needed. Our previous work demonstrated that ethanol exposure leads to up-regulation of cAMP-degrading phosphodiesterase 4 (PDE4) expression, which compromises normal cAMP signaling in monocytes/macrophages and hepatocytes. This effect of ethanol on cAMP signaling contributes to dysregulated inflammatory response and altered lipid metabolism. It is unknown whether chronic alcohol consumption in humans alters hepatic PDE4 expression and cAMP signaling and whether inadequate cAMP signaling plays a pathogenic role in alcohol-induced liver injury. Our present work shows that expression of the PDE4 subfamily of enzymes is significantly up-regulated and cAMP levels are markedly decreased in hepatic tissues of patients with severe ALD. We also demonstrate the anti-inflammatory efficacy of roflumilast, a clinically available PDE4 inhibitor, on endotoxin-inducible proinflammatory cytokine production ex vivo in whole blood of patients with alcoholic hepatitis. Moreover, we demonstrate that ethanol-mediated changes in hepatic PDE4 and cAMP levels play a causal role in liver injury in in vivo and in vitro models of ALD. This study employs a drug delivery system that specifically delivers the PDE4 inhibitor rolipram to the liver to avoid central nervous system side effects associated with this drug. Our results show that PDE4 inhibition significantly attenuates ethanol-induced hepatic steatosis and injury through multiple mechanisms, including reduced oxidative and endoplasmic reticulum stress both in vivo and in vitro. Conclusion: Increased PDE4 plays a pathogenic role in the development of ALD; hence, directed interventions aimed at inhibiting PDE4 might be an effective treatment for ALD.

Mechanisms of Environmental Contributions to Fatty Liver Disease.

PURPOSE: Fatty liver disease (FLD) affects over 25% of the global population and may lead to liver-related mortality due to cirrhosis and liver cancer. FLD caused by occupational and environmental chemical exposures is termed "toxicant-associated steatohepatitis" (TASH). The current review addresses the scientific progress made in the mechanistic understanding of TASH since its initial description in 2010. RECENT FINDINGS: Recently discovered modes of actions for volatile organic compounds and persistent organic pollutants include the following: (i) the endocrine-, metabolism-, and signaling-disrupting chemical hypotheses; (ii) chemical-nutrient interactions and the "two-hit" hypothesis. These key hypotheses were then reviewed in the context of the steatosis adverse outcome pathway (AOP) proposed by the US Environmental Protection Agency. The conceptual understanding of the contribution of environmental exposures to FLD has progressed significantly. However, because this is a new research area, more studies including mechanistic human data are required to address current knowledge gaps.

Environmental perfluoroalkyl acid exposures are associated with liver disease characterized by apoptosis and altered serum adipocytokines.

Exposures to perfluoroalkyl substances (PFAS) including perfluoroalkyl acids (PFAAs) are associated with increased liver enzymes in cohort studies including the C8 Health Study. In animal models, PFAAs disrupt hepatic lipid metabolism and induce apoptosis to cause nonalcoholic fatty liver disease (NAFLD). PFAAs are immunotoxic and inhibit pro-inflammatory cytokine release from stimulated leukocytes in vitro. This cross-sectional study tests the hypothesis that environmental PFAAs are associated with increased hepatocyte apoptosis and decreased pro-inflammatory cytokines in serum. Biomarkers previously associated with PFAS exposures and/or NAFLD were evaluated as secondary endpoints. Two hundred adult C8 Health Study participants were included. Measured serum biomarkers included: perfluorohexane sulfonate (PFHxS); perfluorooctanoic acid (PFOA); perfluorooctane sulfonate (PFOS); perfluorononanoic acid (PFNA); cytokeratin 18 M30 (CK18 M30, hepatocyte apoptosis); adipocytokines; insulin; and cleaved complement 3 (C3a). Confounder-adjusted linear regression models determined associations between PFAS and disease biomarkers with cut-offs determined by classification and regression tree analysis. CK18 M30 was positively associated with PFHxS (ß = 0.889, p = 0.042); PFOA (ß = 2.1, p = 0.005); and PFNA (ß = 0.567, p = 0.03). Tumor necrosis factor α (TNFα) was inversely associated with PFHxS (ß = -0.799, p = 0.001); PFOA (ß = - 1.242, p = 0.001); and PFOS (ß = -0.704, p < 0.001). Interleukin 8 was inversely associated with PFOS and PFNA. PFAAs were also associated with sexually dimorphic adipocytokine and C3a responses. Overall, PFAA exposures were associated with the novel combination of increased biomarkers of hepatocyte apoptosis and decreased serum TNFα. These data support previous findings from cohorts and experimental systems that PFAAs may cause liver injury while downregulated some aspects of the immune response. Further studies of PFAAs in NAFLD are warranted and should evaluate sex differences.

Exposure to persistent organic pollutants: impact on women's health.

This literature review focuses on the causal relationship between persistent organic pollutants (POPs) exposure and women's health disorders, particularly cancer, cardio-metabolic events and reproductive health. Progressive industrialization has resulted in the production of a multitude of chemicals that are released into the environment on a daily basis. Environmental chemicals or pollutants are not only hazardous to our ecosystem but also lead to various health problems that affect the human population worldwide irrespective of gender, race or age. However, most environmental health studies that have been conducted, until recently, were exclusively biased with regard to sex and gender, beginning with exposure studies that were reported mostly in male, occupational workers and animal studies being carried out mostly in male rodent models. Health-related issues pertaining to women of all age groups have not been studied thoroughly and rather disregarded in most aspects of basic health science research and it is therefore pertinent that we address these limitations in environmental health. The review also addresses studies looking at the associations between health outcomes and exposures to POPs, particularly, polychlorinated biphenyls (PCBs), dioxins and pesticides, reported in cohort studies while accounting for gender differences. Considering that current levels of POPs in women can also impact future generations, informative guidelines related to dietary patterns and exposure history are needed for women of reproductive age. Additionally, occupational cohorts of highly exposed women worldwide, such as women working in manufacturing plants and female pesticide applicators are required to gather more information on population susceptibility and disease pathology.

Role of cAMP and phosphodiesterase signaling in liver health and disease.

Liver disease is a significant health problem worldwide with mortality reaching around 2 million deaths a year. Non-alcoholic fatty liver disease (NAFLD) and alcoholic liver disease (ALD) are the major causes of chronic liver disease. Pathologically, NAFLD and ALD share similar patterns of hepatic disorders ranging from simple steatosis to steatohepatitis, fibrosis and cirrhosis. It is becoming increasingly important to identify new pharmacological targets, given that there is no FDA-approved therapy yet for either NAFLD or ALD. Since the evolution of liver diseases is a multifactorial process, several mechanisms involving parenchymal and non-parenchymal hepatic cells contribute to the initiation and progression of liver pathologies. Moreover, certain protective molecular pathways become repressed during liver injury including signaling pathways such as the cyclic adenosine monophosphate (cAMP) pathway. cAMP, a key second messenger molecule, regulates various cellular functions including lipid metabolism, inflammation, cell differentiation and injury by affecting gene/protein expression and function. This review addresses the current understanding of the role of cAMP metabolism and consequent cAMP signaling pathway(s) in the context of liver health and disease. The cAMP pathway is extremely sophisticated and complex with specific cellular functions dictated by numerous factors such abundance, localization and degradation by phosphodiesterases (PDEs). Furthermore, because of the distinct yet divergent roles of both of its effector molecules, the cAMP pathway is extensively targeted in liver injury to modify its role from physiological to therapeutic, depending on the hepatic condition. This review also examines the behavior of the cAMP-dependent pathway in NAFLD, ALD and in other liver diseases and focuses on PDE inhibition as an excellent therapeutic target in these conditions.

Epidermal Growth Factor Receptor Signaling Disruption by Endocrine and Metabolic Disrupting Chemicals.

The purpose of this study is to identify an environmentally relevant shared receptor target for endocrine and metabolism disrupting chemical pollutants. A feature of the tested chemicals was that they induced Cyp2b10 in vivo implicating activation of the constitutive androstane receptor (CAR). Recent studies suggest that these compounds could be indirect CAR activators via epidermal growth factor receptor (EGFR) inhibition. Assays included a CAR activity reporter assay, EGF endocytosis assay, and EGFR phosphorylation assay. Docking simulations were used to identify putative binding sites for environmental chemicals on the EGFR. Whole-weight and lipid-adjusted serum mean pollutant exposures were determined using data from the National Health and Examination Survey (NHANES) and compared with the IC50 values determined in vitro. Chlordane, trans-nonachlor, PCB-126, PCB-153, and atrazine were the most potent EGFR inhibitors tested. PCB-126, PCB-153, and trans-nonachlor appeared to be competitive EGFR antagonists as they displaced bound EGF from EGFR. However, atrazine acted through a different mechanism and could be an EGFR tyrosine kinase inhibitor. EGFR inhibition relative effect potencies were determined for these compounds. In NHANES, serum concentrations of trans-nonachlor, PCB-126, and PCB-153 greatly exceeded their calculated IC50 values. A common mechanism of action through EGFR inhibition for three diverse classes of metabolic disrupting chemicals was characterized by measuring inhibition of EGFR phosphorylation and EGF-EGFR endocytosis. Based on NHANES data, EGFR inhibition may be an environmentally relevant mode of action for some PCBs, pesticides, and herbicides.

The environmental pollutant, polychlorinated biphenyls, and cardiovascular disease: a potential target for antioxidant nanotherapeutics.

Despite production having stopped in the 1970s, polychlorinated biphenyls (PCBs) represent persistent organic pollutants that continue to pose a serious human health risk. Exposure to PCBs has been linked to chronic inflammatory diseases, such as cardiovascular disease, type 2 diabetes, obesity, as well as hepatic disorders, endocrine dysfunction, neurological deficits, and many others. This is further complicated by the PCB's strong hydrophobicity, resulting in their ability to accumulate up the food chain and to be stored in fat deposits. This means that completely avoiding exposure is not possible, thus requiring the need to develop intervention strategies that can mitigate disease risks associated with exposure to PCBs. Currently, there is excitement in the use of nutritional compounds as a way of inhibiting the inflammation associated with PCBs, yet the suboptimal delivery and pharmacology of these compounds may not be sufficient in more acute exposures. In this review, we discuss the current state of knowledge of PCB toxicity and some of the antioxidant and anti-inflammatory nanocarrier systems that may be useful as an enhanced treatment modality for reducing PCB toxicity.

Polychlorinated biphenyls disrupt hepatic epidermal growth factor receptor signaling.

1. Polychlorinated biphenyls (PCBs) are persistent environmental pollutants that disrupt hepatic xenobiotic and intermediary metabolism, leading to metabolic syndrome and nonalcoholic steatohepatitis (NASH). 2. Since phenobarbital indirectly activates Constitutive Androstane Receptor (CAR) by antagonizing growth factor binding to the epidermal growth factor receptor (EGFR), we hypothesized that PCBs may also diminish EGFR signaling. 3. The effects of the PCB mixture Aroclor 1260 on the protein phosphorylation cascade triggered by EGFR activation were determined in murine (in vitro and in vivo) and human models (in vitro). EGFR tyrosine residue phosphorylation was decreased by PCBs in all models tested. 4. The IC50 values for Aroclor 1260 concentrations that decreased Y1173 phosphorylation of EGFR were similar in murine AML-12 and human HepG2 cells (∼2-4 µg/mL). Both dioxin and non-dioxin-like PCB congeners decreased EGFR phosphorylation in cell culture. 5. PCB treatment reduced phosphorylation of downstream EGFR effectors including Akt and mTOR, as well as other phosphoprotein targets including STAT3 and c-RAF in vivo. 6. PCBs diminish EGFR signaling in human and murine hepatocyte models and may dysregulate critical phosphoprotein regulators of energy metabolism and nutrition, providing a new mechanism of action in environmental diseases.

Human receptor activation by aroclor 1260, a polychlorinated biphenyl mixture.

Polychlorinated biphenyls (PCBs) are persistent environmental toxicants, present in 100% of U.S. adults and dose-dependently associated with obesity and non-alcoholic fatty liver disease (NAFLD). PCBs are predicted to interact with receptors previously implicated in xenobiotic/energy metabolism and NAFLD. These receptors include the aryl hydrocarbon receptor (AhR), pregnane xenobiotic receptor (PXR), constitutive androstane receptor (CAR), peroxisome proliferator-activated receptors (PPARs), liver-X-receptor (LXRα), and farnesoid-X-receptor (FXR). This study evaluates Aroclor 1260, a PCB mixture with congener composition mimicking that of human adipose tissue, and selected congeners, as potential ligands for these receptors utilizing human hepatoma-derived (HepG2) and primate-derived (COS-1) cell lines, and primary human hepatocytes. Aroclor 1260 (20 µg/ml) activated AhR, and PCB 126, a minor component, was a potent inducer. Aroclor 1260 activated PXR in a simple concentration-dependent manner at concentrations ≥10 µg/ml. Among the congeners tested, PCBs 138, 149, 151, 174, 183, 187, and 196 activated PXR. Aroclor 1260 activated CAR2 and CAR3 variants at lower concentrations and antagonize CAR2 activation by the CAR agonist, CITCO, at higher concentrations (≥20 µg/ml). Additionally, Aroclor 1260 induced CYP2B6 in primary hepatocytes. At subtoxic doses, Aroclor 1260 did not activate LXR or FXR and had no effect on LXR- or FXR-dependent induction by the agonists T0901317 or GW4064, respectively. Aroclor 1260 (20 µg/ml) suppressed PPARα activation by the agonist nafenopin, although none of the congeners tested demonstrated significant inhibition. The results suggest that Aroclor 1260 is a human AhR, PXR and CAR3 agonist, a mixed agonist/antagonist for CAR2, and an antagonist for human PPARα.

Polychlorinated biphenyl 153 is a diet-dependent obesogen that worsens nonalcoholic fatty liver disease in male C57BL6/J mice.

BACKGROUND: Polychlorinated biphenyls (PCBs) are persistent environmental pollutants that are detectable in the serum of all American adults. Amongst PCB congeners, PCB 153 has the highest serum level. PCBs have been dose-dependently associated with obesity, metabolic syndrome and nonalcoholic fatty liver disease (NAFLD) in epidemiological studies. OBJECTIVE: The purpose of this study is to determine mechanisms by which PCB 153 worsens diet-induced obesity and NAFLD in male mice fed a high-fat diet (HFD). METHODS: Male C57BL6/J mice were fed either control or 42% milk fat diet for 12 weeks with or without PCB 153 coexposure (50 mg/kg ip ×4). Glucose tolerance test was performed, and plasma and tissues were obtained at necropsy for measurements of adipocytokine levels, histology and gene expression. RESULTS: In control diet-fed mice, addition of PCB 153 had minimal effects on any of the measured parameters. However, PCB 153 treatment in high-fat-fed mice was associated with increased visceral adiposity, hepatic steatosis and plasma adipokines including adiponectin, leptin, resistin and plasminogen activator inhibitor-1 levels. Likewise, coexposure reduced expression of hepatic genes implicated in ß-oxidation while increasing the expression of genes associated with lipid biosynthesis. Regardless of diet, PCB 153 had no effect on insulin resistance or tumor necrosis factor alpha levels. CONCLUSION: PCB 153 is an obesogen that exacerbates hepatic steatosis, alters adipocytokines and disrupts normal hepatic lipid metabolism when administered with HFD but not control diet. Because all US adults have been exposed to PCB 153, this particular nutrient-toxicant interaction potentially impacts human obesity/NAFLD.

Toxicant-associated steatohepatitis.

Hepatotoxicity is the most common organ injury due to occupational and environmental exposures to industrial chemicals. A wide range of liver pathologies ranging from necrosis to cancer have been observed following chemical exposures both in humans and in animal models. Toxicant-associated fatty liver disease (TAFLD) is a recently named form of liver injury pathologically similar to alcoholic liver disease (ALD) and nonalcoholic fatty liver disease (NAFLD). Toxicant-associated steatohepatitis (TASH) is a more severe form of TAFLD characterized by hepatic steatosis, inflammatory infiltrate, and in some cases, fibrosis. While subjects with TASH have exposures to industrial chemicals, such as vinyl chloride, they do not have traditional risk factors for fatty liver such as significant alcohol consumption or obesity. Conventional biomarkers of hepatotoxicity including serum alanine aminotransferase activity may be normal in TASH, making screening problematic. This article examines selected chemical exposures associated with TAFLD in human subjects or animal models and concisely reviews the closely related NAFLD and ALD.

Mechanistic insights into PEPT1-mediated transport of a novel antiepileptic, NP-647.

The present study, in general, is aimed to uncover the properties of the transport mechanism or mechanisms responsible for the uptake of NP-647 into Caco-2 cells and, in particular, to understand whether it is a substrate for the intestinal oligopeptide transporter, PEPT1 (SLC15A1). NP-647 showed a carrier-mediated, saturable transport with Michaelis-Menten parameters K(m) = 1.2 mM and V(max) = 2.2 µM/min. The effect of pH, sodium ion (Na(+)), glycylsarcosine and amoxicillin (substrates of PEPT1), and sodium azide (Na(+)/K(+)-ATPase inhibitor) on the flux rate of NP-647 was determined. Molecular docking and molecular dynamics simulation studies were carried out to investigate molecular interactions of NP-647 with transporter using homology model of human PEPT1. The permeability coefficient (P(appCaco-2)) of NP-647 (32.5 × 10(-6) cm/s) was found to be four times higher than that of TRH. Results indicate that NP-647 is transported into Caco-2 cells by means of a carrier-mediated, proton-dependent mechanism that is inhibited by Gly-Sar and amoxicillin. In turn, NP-647 also inhibits the uptake of Gly-Sar into Caco-2 cells and, together, this evidence suggests that PEPT1 is involved in the process. Docking and molecular dynamics simulation studies indicate high affinity of NP-647 toward PEPT1 binding site as compared to TRH. High permeability of NP-647 over TRH is attributed to its increased hydrophobicity which increases its affinity toward PEPT1 by interacting with the hydrophobic pocket of the transporter through hydrophobic forces.