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Alterations in physiological processes in pancreas have been associated with various metabolic dysfunctions and can result from environmental exposures, such as chemicals and diet. It was reported that environmental vinyl chloride (VC) exposure, a common industrial organochlorine and environmental pollutant, significantly exacerbated metabolic-related phenotypes in mice fed concurrently with high-fat diet (HFD) but not low-fat diet (LFD). However, little is known about the role of the pancreas in this interplay, especially at a proteomic level. The present study was undertaken to examine the protein responses to VC exposure in pancreas tissues of C57BL/6J mice fed LFD or HFD, with focus on the investigation of protein expression and/or phosphorylation levels of key protein biomarkers of carbohydrate, lipid, and energy metabolism, oxidative stress and detoxification, insulin secretion and regulation, cell growth, development, and communication, immunological responses and inflammation, and biomarkers of pancreatic diseases and cancers. We found that the protein alterations may indicate diet-mediated susceptibility in mouse pancreas induced by HFD to concurrent exposure of low levels of inhaled VC. These proteome biomarkers may lead to a better understanding of pancreas-mediated adaptive or adverse response and susceptibility to metabolic disease.

The pathogenesis of non-alcoholic fatty liver disease (NAFLD), the most prevalent chronic liver disease, is associated with zinc deficiency. Previous studies show zinc supplementation improves steatosis and glucose metabolism, but its therapeutic effects in patients with established NAFLD remain unclear. We developed an in vivo model to characterize the effects of zinc supplementation on high-fat diet (HFD) induced NAFLD and hypothesized that the established NAFLD would be attenuated by zinc supplementation. Male C57BL/6J mice were fed a control diet or HFD for 12 weeks. Mice were then further grouped into normal and zinc-supplemented diets for 8 additional weeks. Body composition and glucose tolerance were determined before and after zinc supplementation. At euthanasia, plasma and liver tissue were collected for characterization and downstream analysis. As expected, 12 weeks of HFD resulted in reduced glucose clearance and altered body composition. Eight weeks of subsequent zinc supplementation did not alter glucose handling, plasma transaminases, steatosis, or hepatic gene expression. Results from our model suggest 8-week zinc supplementation cannot reverse established NAFLD. The HFD may have caused NAFLD disease progression beyond rescue by an 8-week period of zinc supplementation. Future studies will address these limitations and provide insights into zinc as a therapeutic agent for established NAFLD.

Dioxin-like molecules have been associated with endocrine disruption and liver disease. To better understand aryl hydrocarbon receptor (AHR) biology, metabolic phenotyping and liver proteomics were performed in mice following ligand-activation or whole-body genetic ablation of this receptor. Male wild type (WT) and Ahr–/– mice (Taconic) were fed a control diet and exposed to 3,3′,4,4′,5-pentachlorobiphenyl (PCB126) (61 nmol/kg by gavage) or vehicle for two weeks. PCB126 increased expression of canonical AHR targets (Cyp1a1 and Cyp1a2) in WT but not Ahr–/–. Knockouts had increased adiposity with decreased glucose tolerance; smaller livers with increased steatosis and perilipin-2; and paradoxically decreased blood lipids. PCB126 was associated with increased hepatic triglycerides in Ahr–/–. The liver proteome was impacted more so by Ahr–/– genotype than ligand-activation, but top gene ontology (GO) processes were similar. The PCB126-associated liver proteome was Ahr-dependent. Ahr principally regulated liver metabolism (e.g., lipids, xenobiotics, organic acids) and bioenergetics, but it also impacted liver endocrine response (e.g., the insulin receptor) and function, including the production of steroids, hepatokines, and pheromone binding proteins. These effects could have been indirectly mediated by interacting transcription factors or microRNAs. The biologic roles of the AHR and its ligands warrant more research in liver metabolic health and disease., Graphical abstract The aryl hydrocarbon receptor regulated liver metabolism and endocrine function impacting systemic energy homeostasis and body composition. This regulation appeared to involve both direct and indirect mechanisms.Image 1

The prevalence of alcohol-associated liver disease (ALD), a subtype of fatty liver disease (FLD), continues to rise. ALD is a major cause of preventable death. Polychlorinated biphenyl (PCB) 126 is an environmentally relevant, dioxin-like pollutant whose negative metabolic effects have been well documented. In human and animal studies, PCB has been associated with the severity of nonalcoholic fatty liver disease (NAFLD). However, few studies have investigated whether exposures to environmental toxicants can worsen ALD. Thus, the objective of the current study was to develop an alcohol-plus-toxicant model to study how an environmental pollutant, PCB 126, impacts rodent ALD pathology.Briefly, male C57BL/6J mice were exposed to 0.2 mg/kg PCB 126 or corn oil vehicle four days prior to ethanol feeding using the chronic-binge (10-plus-one) model.Concentrations of macromolecules, including hepatic lipids, carbohydrates, and protein (albumin) were impacted. Exposure to PCB 126 exacerbated hepatic steatosis and hepatomegaly in mice exposed to the chemical and fed an ethanol diet. Gene expression and the analysis of blood chemistry showed a potential net increase and retention of hepatic lipids and reductions in lipid oxidation and clearance capabilities. Depletion of glycogen and glucose was evident, which contributes to disease progression by generating systemic malnutrition. Granulocytic immune infiltrates were present but driven solely by ethanol feeding. Hepatic albumin gene expression and plasma levels were decreased by ~50% indicating a potential compromise of liver function. Finally, gene expression analyses indicated that the aryl hydrocarbon receptor and constitutive androstane receptor were activated by PCB 126 and ethanol, respectively.Various environmental toxicants are known to modify or enhance FLD in high-fat diet models. Findings from the present study suggest that they interact with other lifestyle factors such as alcohol consumption to reprogram intermediary metabolism resulting in exacerbated ethanol-associated systemic malnutrition in ALD.

Graphical abstract, Highlights • Aroclor1260 worsened diet-induced steatohepatitis in CAR and PXR knockout mice. • CAR and PXR ablation increased gut microbiome diversity and modified composition. • Improved bacterial diversity did not protect mice from PCB-mediated toxicity. • Toxicant-nuclear receptor interactions modulate the gut-liver axis., 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.

BACKGROUND AND AIMS: Per- and polyfluoroalkyl substances (PFAS) are widespread and persistent pollutants that have been shown to have hepatotoxic effects in animal models. However, human evidence is scarce. We evaluated how prenatal exposure to PFAS associates with established serum biomarkers of liver injury and alterations in serum metabolome in children. APPROACH AND RESULTS: We used data from 1,105 mothers and their children (median age, 8.2 years; interquartile range, 6.6–9.1) from the European Human Early-Life Exposome cohort (consisting of six existing population-based birth cohorts in France, Greece, Lithuania, Norway, Spain, and the United Kingdom). We measured concentrations of perfluorooctane sulfonate, perfluorooctanoate, perfluorononanoate, perfluorohexane sulfonate, and perfluoroundecanoate in maternal blood. We assessed concentrations of alanine aminotransferase, aspartate aminotransferase, and gamma-glutamyltransferase in child serum. Using Bayesian kernel machine regression, we found that higher exposure to PFAS during pregnancy was associated with higher liver enzyme levels in children. We also measured child serum metabolomics through a targeted assay and found significant perturbations in amino acid and glycerophospholipid metabolism associated with prenatal PFAS. A latent variable analysis identified a profile of children at high risk of liver injury (odds ratio, 1.56; 95% confidence interval, 1.21–1.92) that was characterized by high prenatal exposure to PFAS and increased serum levels of branched-chain amino acids (valine, leucine, and isoleucine), aromatic amino acids (tryptophan and phenylalanine), and glycerophospholipids (phosphatidylcholine [PC] aa C36:1 and Lyso-PC a C18:1). CONCLUSIONS: Developmental exposure to PFAS can contribute to pediatric liver injury.

Background & Aims Acute alcoholic hepatitis (AAH) is a major cause of liver-related morbidity and mortality; there are no good blood biomarkers for diagnosis or determining magnitude of cell death. Keratin 18 (KRT18, also called K18), found in epithelial cells, is released from hepatocytes upon death. We investigated whether level of K18 is a better marker of hepatocyte death than standard biomarkers and might be used to identify patients with AAH at risk for death within 90 days. Methods We analyzed data from 173 participants in a large trial performed at 4 medical centers. Participants with AAH were classified as severe (n = 57, model for end-stage liver disease [MELD] scores above 20) or moderate (n = 27, MELD scores from 12 to 19); 38 participants had alcohol use disorder with mild (n = 28) or no liver injury (n = 10); 34 participants had nonalcoholic steatohepatitis; and 17 participants were healthy (controls). We quantified serum levels of K18 using ELISAs and APOPTOSENSE kits. Results Serum level of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and the ratio of AST:ALT did not correlate with MELD scores. Patients with alcohol use disorder had higher serum levels of ALT than patients with severe AAH. Levels of K18M65 and K18M30 had statistically significant increases as liver disease worsened, as did the degree of necrosis (ratio of K18 M65:M30). The ratio of K18M65:ALT was increased in serum from patients with AAH compared with controls. Serum levels of K18 identified patients who died within 90 days with greater accuracy than commonly used static biomarkers. Conclusions There is a stronger association between serum level of keratin 18 and amount of hepatocyte death and liver disease severity than for other biomarkers (AST, ALT, and the AST:ALT ratio). The ratio of K18M65:M30 might be used as marker of mechanism of hepatocyte death, and the ratio of K18M65:ALT might be used to distinguish patients with AAH from patients with nonalcoholic steatohepatitis. Serum levels of K18 might be used to identify patients with severe AAH at risk for death. ClinicalTrials.gov identifier # NCT01922895 and NCT01809132 .

Polychlorinated biphenyls (PCBs) are persistent organic pollutants associated with metabolic disruption and non-alcoholic fatty liver disease (NAFLD). Based on their ability to activate the aryl hydrocarbon receptor (AhR), PCBs are subdivided into two classes: dioxin-like (DL) and non-dioxin-like (NDL) PCBs. Previously, we demonstrated that NDL PCBs compromised the liver to promote more severe diet-induced NAFLD. Here, the hepatic effects and potential mechanisms (by untargeted liver proteomics) of DL PCBs, NDL PCBs or co-exposure to both in diet-induced NAFLD are investigated. Male C57Bl/6 mice were fed a 42% fat diet and exposed to vehicle control; Aroclor1260 (20 mg/kg, NDL PCB mixture); PCB126 (20 μg/kg, DL PCB congener); or a mixture of Aroclor1260 (20 mg/kg)+PCB126 (20 μg/kg) for 12 weeks. Each exposure was associated with a distinct hepatic proteome. Phenotypic and proteomic analyses revealed increased hepatic inflammation and phosphoprotein signaling disruption by Aroclor1260. PCB126 decreased hepatic inflammation and fibrosis at the molecular level; while altering cytoskeletal remodeling, metal homeostasis, and intermediary/xenobiotic metabolism. PCB126 attenuated Aroclor1260-induced hepatic inflammation but increased hepatic free fatty acids in the co-exposure group. Aroclor1260+PCB126 exposure was strongly associated with multiple epigenetic processes, and these could potentially explain the observed non-additive effects of the exposures on the hepatic proteome. Taken together, the results demonstrated that PCB exposures differentially regulated the hepatic proteome and the histologic severity of diet-induced NAFLD. Future research is warranted to determine the AhR-dependence of the observed effects including metal homeostasis and the epigenetic regulation of gene expression.

Background & AimsVinyl chloride (VC) monomer is a volatile organic compound commonly used in industry to produce polyvinyl chloride (PVC). At high exposure levels, VC causes liver cancer and toxicant-associated steatohepatitis. However, lower exposure levels (i.e., < regulatory exposure limits) that do not directly damage the liver, enhance injury caused by Western diet (WD). Although these lower exposure levels are considered ‘safe,’ it is unknown if the long-term impact of transient low-concentration VC enhances the risk of liver cancer development. Low-concentration VC is especially a concern given that fatty liver disease is in and of itself a risk factor for the development of liver cancer.MethodsC57Bl/6J 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 (ResultsFeeding WD for 1 year caused significant hepatic injury, including steatohepatitis and moderate fibrosis, 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.ConclusionsThese data indicate that VC sensitizes the liver to other stressors (e.g., WD), resulting in enhanced tumorigenesis. These data raise concerns about a potential interaction between VC exposure and WD. Furthermore, it also emphasizes that current safety restrictions may be insufficient to account for other factors that can influence hepatotoxicity.

Background Residents of Anniston Alabama were highly exposed to polychlorinated biphenyls (PCBs) due to longstanding manufacturing in the area. The Anniston Community Health Surveys (ACHS I-2005–2007 and II, 2014) have linked these exposures with a variety of deletereous health outcomes. In addition to PCBs, these individuals were likely simultaneously exposed to other persistent organic pollutants including per and polyfluoroalkyl substances (PFAS), which are an emerging class of ubiquitous industrial chemicals that are measurable in the blood of most individuals and have themselves been linked increased risk of some non communicable diseases. Methods To characterize PFAS exposures in ACHS I and ACHS II, we measured eight environmentally significant PFAS in serum by UPLC coupled electrospray ionization tandem mass spectrometry. Perfluorooctane sulfonate (PFOS), Perfluorooctanoic acid (PFOA), Perfluorononanoate (PFNA), Perfluorohexane sulfonate (PFHxS), Perfluoroheptanoic acid (PFHpA), Perfluorobutanesulfonic acid (PFBS), Hexafluoropropylene oxide dimer acid (HFPO-DA), and 4:2 Fluorotelomer sulfonic acid (4.2 FTS) were extracted from matched serum samples of individuals who participated in the original ACHS I (2005–2007; n = 297) and the follow up ACHS II (2014; n = 336). Data were collected in negative multiple reaction monitoring (MRM) mode with monitoring of quantitation and qualifier ions for all target PFAS analytes, surrogates and internal standards. VARCLUS procedure was used to create hierarchical clusters between PFAS and other legacy persistent organic pollutants which may share similar exposure routes. Results Overall, circulating PFAS levels decreased approximately 50% from ACHS I (2005–2007) to ACHS II (2014), but these changes varied by compound. Mean levels of PFOS were >3 times higher in ACHS I subjects than in conpemporaneous NHANES subjects (2005–2006; ACHS I mean: 71.1 ng/ml; NHANES mean: 20.2 ng/mL), and this relationship persisted in ACHS II subjects (2014: ACHS II mean: 34.7 ng/ml; NHANES mean: 5.92 ng/mL). PFNA was also higher in both ACHS I and ACHS II subjects in comparision to NHANES whereas levels of PFOA and PFHxS were lower than in NHANES. Finally, cluster analysis revealed that in ACHS II, most PFAS tracked with polybrominated diphenyl ethers, except PFNA and PFHpA which clustered with industrial PCBs. In ACHS I, PFAS analytes correlated more closely with industrial PCBs and chlorinated pesticides. Conclusions Participants in the Anniston Community Health Surveys have higher levels of PFOS and PFNA than the general population with average PFOS levels >3 times contemporaneous NHANES levels. Since PFAS were not known to be manufactured in the area, more work needs to be completed to determine if population demographics, proximity to a military base, or regional manufacturing can explain the elevated levels.

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

Exposure to polychlorinated biphenyls (PCBs) has been associated with liver injury in human cohorts and with nonalcoholic steatohepatitis (NASH) in mice fed a high fat diet (HFD). N (6)-methyladenosine (m6A) modification of mRNA regulates transcript fate, but the contribution of m6A modification on the regulation of transcripts in PCB-induced steatosis and fibrosis is unknown. This study tested the hypothesis that PCB and HFD exposure alters the levels of m6A modification in transcripts that play a role in NASH in vivo. Male C57Bl6/J mice were fed a HFD (12 wks) and administered a single oral dose of Aroclor1260, PCB126, or Aroclor1260 + PCB126. Genome-wide identification of m6A peaks was accomplished by m6A mRNA immunoprecipitation sequencing (m6A-RIP) and the mRNA transcriptome identified by RNA-seq. Exposure of HFD-fed mice to Aroclor1260 decreased the number of m6A peaks and m6A-containing genes relative to PCB vehicle control whereas PCB126 or the combination of Aroclor1260 + PCB126 increased m6A modification frequency. ~41% of genes had one m6A peak and ~49% had 2–4 m6A peaks. 117 m6A peaks were common in the four experimental groups. The Aroclor1260 + PCB126 exposure group showed the highest number (52) of m6A-peaks. qRT-PCR confirmed enrichment of m6A-containing fragments of the Apob transcript with PCB exposure. A1cf transcript abundance, m6A peak count, and protein abundance was increased with Aroclor1260 + PCB126 co-exposure. Irrespective of the PCB type, all PCB groups exhibited enriched pathways related to lipid/lipoprotein metabolism and inflammation through the m6A modification. Integrated analysis of m6A-RIP-seq and mRNA-seq identified 242 differentially expressed genes (DEGs) with increased or reduced number of m6A peaks. These data show that PCB exposure in HFD-fed mice alters the m6A landscape offering an additional layer of regulation of gene expression affecting a subset of gene responses in NASH.

International audience; Obesity is a multifactorial disease with both genetic and environmental components. The prevailing view is that obesity results from an imbalance between energy intake and expenditure caused by overeating and insufficient exercise. We describe another environmental element that can alter the balance between energy intake and energy expenditure: obesogens. Obesogens are a subset of environmental chemicals that act as endocrine disruptors affecting metabolic endpoints. The obesogen hypothesis posits that exposure to endocrine disruptors and other chemicals can alter the development and function of the adipose tissue, liver, pancreas, gastrointestinal tract, and brain, thus changing the set point for control of metabolism. Obesogens can determine how much food is needed to maintain homeostasis and thereby increase the susceptibility to obesity. The most sensitive time for obesogen action is in utero and early childhood, in part via epigenetic programming that can be transmitted to future generations. This review explores the evidence supporting the obesogen hypothesis and highlights knowledge gaps that have prevented widespread acceptance as a contributor to the obesity pandemic. Critically, the obesogen hypothesis changes the narrative from curing obesity to preventing obesity.

Single-cell RNA-sequencing (scRNA-seq) is a recent high-throughput sequencing technique for studying gene expressions at the cell level. Differential Expression (DE) analysis is a major downstream analysis of scRNA-seq data. DE analysis the in presence of noises from different sources remains a key challenge in scRNA-seq. Earlier practices for addressing this involved borrowing methods from bulk RNA-seq, which are based on non-zero differences in average expressions of genes across cell populations. Later, several methods specifically designed for scRNA-seq were developed. To provide guidance on choosing an appropriate tool or developing a new one, it is necessary to comprehensively study the performance of DE analysis methods. Here, we provide a review and classification of different DE approaches adapted from bulk RNA-seq practice as well as those specifically designed for scRNA-seq. We also evaluate the performance of 19 widely used methods in terms of 13 performance metrics on 11 real scRNA-seq datasets. Our findings suggest that some bulk RNA-seq methods are quite competitive with the single-cell methods and their performance depends on the underlying models, DE test statistic(s), and data characteristics. Further, it is difficult to obtain the method which will be best-performing globally through individual performance criterion. However, the multi-criteria and combined-data analysis indicates that DECENT and EBSeq are the best options for DE analysis. The results also reveal the similarities among the tested methods in terms of detecting common DE genes. Our evaluation provides proper guidelines for selecting the proper tool which performs best under particular experimental settings in the context of the scRNA-seq.

Nonalcoholic fatty liver disease (NAFLD) is a major global public health concern affecting more than 25% of the world's population. Although obesity and diabetes are major risk factors for NAFLD, they cannot account for all cases, indicating the importance of other factors such as environmental exposures. Cadmium (Cd) exposure is implicated in the development of NAFLD; however, the influence of early life, in utero Cd exposure on the development of diet-induced NAFLD is poorly understood. Therefore, we developed an in vivo, multiple-hit model to study the effect of whole-life, low dose Cd exposure on high fat diet (HFD)-induced NAFLD. Adult male and female C57BL/6 J mice fed normal diets (ND) were exposed to 0, 0.5 or 5 ppm Cd-containing drinking water for 14 weeks before breeding. At weaning, offspring were fed ND or HFD and continued on the same drinking water regimen as their parents for 24 weeks. Cd exposure at different concentrations differentially altered HFD-associated adverse health effects, including liver injury. HFD-induced increased body weight, decreased glucose tolerance. Liver injury and lipid deposition were exacerbated by 5 ppm Cd exposure but attenuated by 0.5 ppm Cd exposure. Further, HFD blunted the response of metallothionein, a major Cd detoxification protein, in mice exposed to 5 ppm Cd but enhanced the response in mice exposed to 0.5 ppm Cd, suggesting a possible mechanism for Cd alteration of HFD-induced NAFLD. These results confirm the multi-hit nature of NAFLD and show whole life, low dose Cd exposure alters HFD-induced NAFLD with outcomes dependent on Cd concentration.

Polychlorinated biphenyls (PCBs) have been associated with abnormal liver enzymes and suspected nonalcoholic fatty liver disease (NAFLD) in cohort studies. NAFLD affects greater than 25% of the global population and may result in liver-related mortality. Both dioxin-like and non-dioxin-like PCBs have been associated with NAFLD, but their effects and mechanisms differ. Dioxin-like PCBs altered the gut:liver axis and microbiome and caused hepatic steatosis by disrupting hepatic lipid metabolism. In contrast, NDL PCBs reduced the liver's protective responses to promote diet-induced NAFLD. Mechanisms included the disruption of phosphoprotein signaling resulting in altered nuclear receptor function.

Vinyl chloride is an organochlorine mainly used to manufacture its polymer polyvinyl chloride, which is extensively used in the manufacturing of consumer products. Recent studies suggest that chronic low dose vinyl chloride exposure affects glucose homeostasis in high fat diet-fed mice. Our data suggest that even in the absence of high fat diet, exposure to vinyl chloride (0.8 ppm, 6h/day, 5day/week, for 12 weeks) induces glucose intolerance (1.0 g/kg, i.p) in male C57BL/6 mice. This was accompanied with the depletion of hepatic glutathione and a modest increase in lung interstitial macrophages. Vinyl chloride exposure did not affect the levels of circulating immune cells, endothelial progenitor cells, platelet-immune cell aggregates, and cytokines and chemokines. The acute challenge of vinyl chloride-exposed mice with LPS did not affect lung immune cell composition or plasma IL-6. To examine the effect of vinyl chloride exposure on vascular inflammation and atherosclerosis, LDL receptor-KO mice on C57BL/6 background maintained on western diet were exposed to vinyl chloride for 12 weeks (0.8 ppm, 6h/day, 5day/week). Unlike the WT C57BL/6 mice, vinyl chloride exposure did not affect glucose tolerance in the LDL receptor-KO mice. Plasma cytokines, lesion area in the aortic valve, and markers of lesional inflammation in vinyl chloride-exposed LDL receptor-KO mice were comparable with the air-exposed controls. Collectively, despite impaired glucose tolerance and modest pulmonary inflammation, chronic low dose vinyl chloride exposure does not affect surrogate markers of cardiovascular injury, LPS-induced acute inflammation in C57BL/6 mice, and chronic inflammation and atherosclerosis in the LDL receptor-KO mice.

Non-alcoholic fatty liver disease (NAFLD), the most common form of liver disease, affects over 30% of the US population. Our group and others have previously demonstrated that low-level environmental pollutant exposures were associated with increased odds ratios for unexplained alanine aminotransferase (ALT) elevation, a surrogate biomarker for NAFLD, in the adult National Health and Nutrition Examination Survey (NHANES). However, recently, more sensitive and lower ALT cut-offs have been proposed. The objective of this observational study is to utilize these ALT cut-offs to determine new associations between environmental chemicals and the surrogate NAFLD biomarker. Adult NHANES 2003–2004 participants without viral hepatitis, hemochromatosis, or alcoholic liver disease were analyzed in this cross-sectional study. ALT elevation was defined as >30 IU/L in men and >19 IU/L in women. Odds ratios adjusted for potential confounders for ALT elevation were determined across exposure quartiles for 17 pollutant subclasses comprised of 111 individual pollutants. The overall prevalence of ALT elevation was 37.6%. Heavy metal and organochlorine insecticide subclasses were associated with dose-dependent increased adjusted odds ratios for ALT elevation of 1.6 (95% CI 1.2–2.3) and 3.5 (95% CI 2.3–5.5) respectively, for the highest vs. lowest exposure quartiles (p(trend)

Diversity analysis and taxonomic profiles can be generated from marker-gene sequence data with the help of many available computational tools. The Quantitative Insights into Microbial Ecology Version 2 (QIIME2) has been widely used for 16S rRNA data analysis. While many articles have demonstrated the use of QIIME2 with suitable datasets, the application to pre-clinical data has rarely been talked about. The issues involved in the pre-clinical data include the low-quality score and small sample size that should be addressed properly during analysis. In addition, there are few articles that discuss the detailed statistical methods behind those alpha and beta diversity significance tests that researchers are eager to find. Running the program without knowing the logic behind it is extremely risky. In this article, we first provide a guideline for analyzing 16S rRNA data using QIIME2. Then we will talk about issues in pre-clinical data, and how they could impact the outcome. Finally, we provide brief explanations of statistical methods such as group significance tests and sample size calculation.

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.

Exposures to persistent environmental pollutants like polychlorinated biphenyls (PCBs) has been associated with liver diseases such as toxicant-associated steatohepatitis (TASH). However, previously published PCB hepatotoxicity studies evaluated mostly male animal models. Moreover, epidemiologic studies on PCB-exposed cohorts evaluating sex differences are scarce. Therefore, the objective of this study was to examine hepato-toxicological responses of PCB exposures in the context of sex-dependent outcomes. Male and female C57Bl/6 mice were exposed to Aroclor 1260 (20 mg/kg), and PCB126 (20 μg/kg), by gavage for two weeks. Female mice appeared to be more sensitive to PCB-induced hepatotoxic effects as manifested by increased liver injury markers, namely, hepatic Serpine1 expression. Additionally, compared to their male counterparts, PCB-exposed females exhibited dysregulated hepatic gene expression favoring lipid accumulation rather than lipid breakdown; accompanied by dyslipidemia. Sex differences were also observed in the expression and activation of PCB targets such as the epidermal growth factor receptor (EGFR) while PCB-induced pancreatic toxicity was similar in both sexes. Importantly, PCB exposure appeared to cause pro-androgenic, anti-estrogenic along with sex-dependent thyroid hormone effects. The overall findings demonstrated that the observed PCB-mediated hepatotoxicity was sex-dependent; confirming the existence of sex differences in environmental exposure-induced markers of TASH and warrants further investigation.

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: (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. SUMMARY: 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.

BACKGROUND: Environmental pollution contributes to fatty liver disease pathogenesis. Polychlorinated biphenyl (PCB) exposures have been associated with liver enzyme elevation and suspected steatohepatitis in cohort studies. Male mice treated with the commercial PCB mixture, Aroclor 1260 (20 mg/kg), and fed high fat diet (HFD) for 12 weeks developed steatohepatitis. Receptor-based modes of action including inhibition of the epidermal growth factor (EGF) receptor were previously proposed, but other mechanisms likely exist. OBJECTIVES: To identify and validate the pathways, transcription factors, and mechanisms responsible for the steatohepatitis associated with PCB and HFD co-exposures. METHODS: Comparative proteomics analysis was performed in archived mouse liver samples from the aforementioned chronic exposure study. Pathway and transcription factor analysis (TFA) was performed, and selected results were validated. RESULTS: Liver proteomics detected 1103 unique proteins. Aroclor 1260 upregulated 154 and downregulated 93 of these. Aroclor 1260+HFD co-exposures affected 55 pathways including glutathione metabolism, intermediary metabolism, and cytoskeletal remodeling. TFA of Aroclor 1260 treatment demonstrated alterations in the function of 42 transcription factors including downregulation of NRF2 and key nuclear receptors previously demonstrated to protect against steatohepatitis (e.g., HNF4α, FXR, PPARα/δ/γ, etc.). Validation studies demonstrated that Aroclor 1260 significantly reduced HNF4α protein levels; while Aroclor 1260+HFD reduced expression of the HNF4α target gene, albumin, in vivo. Aroclor 1260 attenuated EGF-dependent HNF4α phosphorylation and target gene activation in vitro. Aroclor 1260 reduced levels of NRF2, its target genes, and glutathione in vivo. Aroclor 1260 attenuated EGF-dependent NRF2 upregulation, in vitro. Aroclor 1260 indirectly activated hepatic stellate cells in vitro via induction of hepatocyte-derived TGFβ. CONCLUSION: PCB exposures adversely impacted transcription factors regulating liver protection, function, and fibrosis. PCBs, thus, compromised the liver by reducing its protective responses against nutritional stress to promote diet-induced steatohepatitis. The identified mechanisms by which environmental pollutants influence fatty liver disease pathogenesis require confirmation in humans.

Exposure to high fat diet (HFD) and persistent organic pollutants including polychlorinated biphenyls (PCBs) is associated with liver injury in human populations and non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) in animal models. Previously, exposure of HFD-fed male mice to the non-dioxin-like (NDL) PCB mixture Aroclor1260, dioxin-like (DL) PCB126, or Aroclor1260 + PCB126 co-exposure caused toxicant-associated steatohepatitis (TASH) and differentially altered the liver proteome. Here unbiased mRNA and miRNA sequencing (mRNA- and miRNA- seq) was used to identify biological pathways altered in these liver samples. Fewer transcripts and miRs were up- or down- regulated by PCB126 or Aroclor1260 compared to the combination, suggesting that crosstalk between the receptors activated by these PCBs amplifies changes in the transcriptome. Pathway enrichment analysis identified "positive regulation of Wnt/β-catenin signaling" and "role of miRNAs in cell migration, survival, and angiogenesis" for differentially expressed mRNAs and miRNAs, respectively. We evaluated the five miRNAs increased in human plasma with PCB exposure and suspected TASH and found that miR-192-5p was increased with PCB exposure in mouse liver. Although we observed little overlap between differentially expressed mRNA transcripts and proteins, biological pathway-relevant PCB-induced miRNA-mRNA and miRNA-protein inverse relationships were identified that may explain protein changes. These results provide novel insights into miRNA and mRNA transcriptome changes playing direct and indirect roles in the functional protein pathways in PCB-related hepatic lipid accumulation, inflammation, and fibrosis in a mouse model of TASH and its relevance to human liver disease in exposed populations.

Background: High-level occupational vinyl chloride (VC) exposures have been associated with hepatic hemangiosarcoma, which typically develops following a long latency period. Although VC is genotoxic, a more comprehensive mode of action has not been determined and diagnostic biomarkers have not been established. The purpose of this study is to address these knowledge gaps through plasma metabolomics. Methods: Plasma samples from polyvinyl chloride polymerization workers who developed hemangiosarcoma (cases, n = 15) and VC exposure-matched controls (n = 17) underwent metabolomic analysis. Random forest and bioinformatic analyses were performed. Results: Cases and controls had similar demographics and routine liver biochemistries. Mass spectroscopy identified 606 known metabolites. Random forest analysis had an 82% predictive accuracy for group classification. 60 metabolites were significantly increased and 44 were decreased vs. controls. Taurocholate, bradykinin and fibrin degradation product 2 were up-regulated by greater than 80-fold. The naturally occurring anti-angiogenic phenol, 4-hydroxybenzyl alcohol, was down-regulated 5-fold. Top affected ontologies involved: (i) metabolism of bile acids, taurine, cholesterol, fatty acids and amino acids, (ii) inflammation and oxidative stress, and (iii) nicotinic cholinergic signaling. Conclusions: The plasma metabolome was differentially regulated in polyvinyl chloride workers who developed hepatic hemangiosarcoma. Ontologies potentially involved in hemangiosarcoma pathogenesis and candidate biomarkers were identified.

Background: Polychlorinated biphenyls (PCBs) are signaling disrupting chemicals that exacerbate nonalcoholic steatohepatitis (NASH) in mice. They are epidermal growth factor receptor (EGFR) inhibitors that enhance hepatic inflammation and fibrosis in mice. Objectives: This study tested the hypothesis that epidermal growth factor (EGF) administration can attenuate PCB-related NASH by increasing hepatic EGFR signaling in a mouse model. Methods: C57BL/6 male mice were fed a 42% milk fat diet and exposed to Aroclor 1260 (20mg/kg) or vehicle for 12 wk. EGF (0.2μg/g) or vehicle were administered daily for 10 d starting at study week 10. Liver and metabolic phenotyping were performed. The EGF dose was selected based on results of an acute dose–finding study (30 min treatment of EGF at 0.2, 0.02, 0.002μg/g of via intraperitoneal injection). Hepatic phosphoproteomic analysis was performed using liver tissue from this acute study to understand EGFR’s role in liver physiology. Results: Markers of EGFR signaling were higher in EGF-treated mice. EGF+PCB–exposed mice had lower hepatic free fatty acids, inflammation, and fibrosis relative to PCB-only exposed mice. EGF-treated mice had higher plasma lipids, with no improvement in hepatic steatosis, and an association with higher LXR target gene expression and de novo lipogenesis. EGF-treated mice showed more severe hyperglycemia associated with lower adiponectin levels and insulin sensitivity. EGF-treated mice had higher hepatic HNF4α, NRF2, and AhR target gene expression but lower constitutive androstane receptor and farnesoid X receptor target gene expression. The hepatic EGF-sensitive phosphoproteome demonstrated a role for EGFR signaling in liver homeostasis. Discussion: These results validated EGFR inhibition as a causal mode of action for PCB-related hepatic inflammation and fibrosis in a mouse model of NASH. However, observed adverse effects may limit the clinical translation of EGF therapy. More data are required to better understand EGFR’s underinvestigated roles in liver and environmental health. https://doi.org/10.1289/EHP8222

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.

International audience; Obesity is a chronic, relapsing condition characterized by excess body fat. Its prevalence has increased globally since the 1970s, and the number of obese and overweight people is now greater than those underweight. Obesity is a multifactorial condition, and as such, many components contribute to its development and pathogenesis. This is the first of three companion reviews that consider obesity. This review focuses on the genetics, viruses, insulin resistance, inflammation, gut microbiome, and circadian rhythms that promote obesity, along with hormones, growth factors, and organs and tissues that control its development. It shows that the regulation of energy balance (intake vs. expenditure) relies on the interplay of a variety of hormones from adipose tissue, gastrointestinal tract, pancreas, liver, and brain. It details how integrating central neurotransmitters and peripheral metabolic signals (e.g., leptin, insulin, ghrelin, peptide YY(3-36)) is essential for controlling energy homeostasis and feeding behavior. It describes the distinct types of adipocytes and how fat cell development is controlled by hormones and growth factors acting via a variety of receptors, including peroxisome proliferator-activated receptor-gamma, retinoid X, insulin, estrogen, androgen, glucocorticoid, thyroid hormone, liver X, constitutive androstane, pregnane X, farnesoid, and aryl hydrocarbon receptors. Finally, it demonstrates that obesity likely has origins in utero. Understanding these biochemical drivers of adiposity and metabolic dysfunction throughout the life cycle lends plausibility and credence to the "obesogen hypothesis" (i.e., the importance of environmental chemicals that disrupt these receptors to promote adiposity or alter metabolism), elucidated more fully in the two companion reviews.

The endocrine disrupting chemicals, polychlorinated biphenyls (PCBs), have been associated with nonalcoholic steatohepatitis (NASH) and diabetes. However, an integrative analysis of the effects of PCBs on the liver and pancreas has never been performed for the two major PCB subtypes, dioxin-like (DL) and nondioxin-like (NDL), and a mixture of NDL/DL PCBs. Therefore, male C57BL/6 J mice fed a control synthetic diet were treated with either a NDL PCB mixture, Aroclor 1260 (20 mg/kg); a single DL PCB congener, PCB 126 (20 μg/kg); a NDL/DL mixture, Aroclor 1260 plus PCB 126; or vehicle control for 2 weeks. PCB126 had the greatest impact on hepatic lipid metabolism. It caused steatosis due to increased hepatic lipid import with associated hypolipidemia. However, all PCB exposures impacted expression of hepatic lipid metabolism genes in different manners. The ‘NASH gene’, Pnpla3, was elevated by Aroclor 1260, but decreased by all other exposures. The expression of hepatokines implicated in metabolic syndrome (Fgf21, Igf1, and betatrophin) were differentially regulated. The NDL/DL PCB mixture had the greatest effects on pancreatic histology, including acinar cell atrophy, mild steatosis, and fibrosis without ductal changes or immune cell infiltration. It decreased expression of insulin and altered the expression of genes regulating islet identity. None of these exposures was associated with altered HOMA-IR or HOMA-B. In summary, PCB exposures differentially regulated liver and pancreas structure and function. Novel mechanisms for PCB-induced endocrine/metabolic disruption included altered hepatokines and Pnpla3 as well as ‘PCB pancreatopathy’ that was associated with altered expression of pancreatic islet identity factors. More research is required to understand fully these findings in the context of human NASH and diabetes.

INTRODUCTION: Exposures to volatile organic compounds and metals have previously been associated with liver diseases including steatohepatitis, although more data are needed. Benzene, toluene, ethylbenzene, xylenes, styrene (BTEXS) and metals were measured in blood samples collected between May 2012-July 2013 from volunteers participating in home visits for the Gulf Long-term Follow-up (GuLF) Study. This cross-sectional analysis evaluates associations of exposure biomarkers with serum liver injury and adipocytokine biomarkers in a sample of 214 men. METHODS: Adult nonsmoking men without a history of liver disease or heavy alcohol consumption were included. The serologic disease biomarkers evaluated were the hepatocellular injury biomarker, cytokeratin 18 [whole (CK18 M65) and caspase-cleaved fragment (CK18 M30)]; and adipocytokines. Confounder-adjusted beta coefficients were determined using linear regression models for the overall sample (primary endpoints) and for obesity-classified sub-groups (secondary endpoints). A product interaction term between the exposure of interest and a dichotomized indicator of obesity was included to determine the disease modifying effects of obesity on the biomarker associations. RESULTS: The study sample was 57% white and 51% obese. In the overall sample, lead was positively associated with CK18 M30 (β=21.7±6.0 (SE), p=0.0004); IL-1β (β=32.8±5.2, p

Zinc deficiency is a frequent complication of alcohol abuse for multiple reasons including poor intake, increased excretion, internal redistribution and altered transporters. Zinc deficiency has been postulated to play a role in the development/progression of alcoholic liver disease (ALD). This study aimed to relate serum zinc levels with alcohol intake, serum albumin concentration and markers of inflammation and liver injury. One hundred and eight male and female very heavy drinking (≥10 drinks/day) individuals without clinical evidence of ALD were grouped by serum zinc concentration: normal-zinc group (zinc level≥71 μg/dl) included 67 patients, and low-zinc group (zinc level71 μg/dl) included 41 patients. Data were collected on demographics, drinking history in last 90 days (heavy drinking days, HDD90 and total drinks, TD90), lifetime drinking history (LTDH) and clinical/ laboratory assessments. Our data show that in a very well-characterized, chronically heavy-drinking population without clinical evidence of liver disease, about 40% of subjects had low serum zinc levels. Frequency of heavy drinking days (HDD90) was significantly higher in the low-zinc group. Total drinks in past 90 days, LTDH and HDD90 showed significant associations with low zinc levels. The group with the low serum zinc had a higher aspartate aminotransferase/alanine aminotransferase ratio (good marker of alcoholic liver disease). Those in the low-zinc group had the lower albumin levels, a marker of hepatic synthetic function, and the highest C-reactive protein level, a biomarker of inflammation.

Purpose: Referral access to subspecialty care for patients with gastrointestinal (GI) diseases is not well defined, but has significant importance to patients. We hypothesized that patients experience barriers to care in two common gastroenterology subspecialties, Hepatology and Motility, in a university medical center. Methods: Two hundred thirteen clinic patients (mean age 46.5 years; 66.5% female; 85.6% Caucasians) completed a formatted questionnaire on access to care. Hepatology patients were older (49.7 years, p=0.008); motility patients predominantly female (76.8%, p