Russell A. Prough

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.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants associated with metabolic disruption and nonalcoholic 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 and 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 nonadditive 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.

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. 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.

1. Aryl hydrocarbon receptor (AhR) ligands, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and polychlorinated biphenyls (PCBs), are endocrine disrupting chemicals associated with nonalcoholic fatty liver disease. This study documents the species-specific differences between mouse (high affinity mAhR) and human AhR (hAhR) activation by PCB congeners and Aroclor mixtures. 2. AhR activation by TCDD or PCBs 77, 81, 114, 114, 126, and 169 was measured using luciferase reporter constructs transfected into either Hepa1c1c7 mouse or HepG2 human liver cell lines. The EC₅₀ values were lower in Hepa1c1c7 cells than HepG2 cells for all compounds tested except PCB 81. The results for TCDD and PCB 126 were validated in primary human and mouse hepatocytes by measuring CYP1A1 gene transcript levels. 3. Because humans are exposed to PCB mixtures, several mixtures (Aroclors 1254; 1260; and 1260 + 0.1% PCB126 each at 10 µg/ml) were then tested. Neither Aroclor 1254 nor Aroclor 1260 increased luciferase activity by the transfected AhR reporter construct. The Aroclor 1260 + 0.1% PCB 126 mixture induced mAhR-mediated transactivation, but not hAhR activation in cell lines. 4. In summary, significant concentration-dependent differences exist between human and mouse AhR activation by PCBs. Relative effect potencies differed, in some cases, from published toxic equivalency factors.

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 were to identify and validate the pathways, transcription factors, and mechanisms responsible for the steatohepatitis associated with PCB and HFD coexposures. 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. Liver proteomics detected 1103 unique proteins. Aroclor 1260 upregulated 154 and downregulated 93 of these. Aroclor 1260 + HFD coexposures 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. 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.