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PCB126 Modifies the Murine Hepatic Transcriptome and Metallome to Promote Alcohol-associated Liver Disease Pathogenesis

ABSTRACT: Background: Environmental pollutants, including polychlorinated biphenyls (PCBs) have been shown to alter and promote the development of alcohol-associated liver disease (ALD). Our group recently demonstrated that PCB126 promoted steatosis, hepatomegaly, and modulated intermediary metabolism in an ALD rodent model. Objectives: To better understand how PCB126 promoted ALD, the current study adopts transcriptomic and metallomic approaches to identify mechanistic pathways involved in this promotion. Methods: Briefly, male C57BL/6J mice were exposed to 0.2 mg/kg PCB126 or corn oil vehicle prior to ethanol feeding in the chronic-binge model. Liver tissues were collected for RNA sequencing and ICP-MS metals quantification. Results: PCB126 uniquely modified the transcriptome in EtOH-fed mice in terms of variance. EtOH feeding alone resulting in >4000 differentially expressed genes (DEGs) and PCB126 exposure resulted in more DEGs in the EtOH-fed group over the pair-fed group. Top gene ontology (GO) biological processes indicated ‘peptidyl tyrosine modifications’ and GO molecular function processes showed a loss of ‘metal, and ion, and zinc binding’. Western blot analysis depicted that the JAK2-STAT5 signaling axis was disrupted by the enhanced loss of phosphorylated JAK2 in EtOH+PCB126 mice. Quantified liver essential metal levels were overall depleted by EtOH feeding, and potassium, magnesium, cobalt, and zinc were further decreased by PCB126. Discussion: The results suggests that phosphorylation and metal binding are disrupted in EtOH+PCB126 mice, implying that evolutionarily conserved homeostatic signaling mechanisms are modified by pollutant exposure in EtOH-fed mice. The loss of phosphorylation and essential metals are two suggestive modes of action that may explain the promotion of disease by PCB126 in ALD.

ORGANISM(S): Mus musculus

PROVIDER: GSE244388 | GEO | 2024/05/01

REPOSITORIES: GEO

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Project description:Environmental pollutants, including polychlorinated biphenyls (PCBs) have been implicated in the pathogenesis of liver disease. To better understand how PCB126 promoted alcohol-associated liver disease (ALD) in our previous model, the current study utilized a phosphoproteomic approach for hypothesis generation regarding mechanisms of action. Briefly, male C57B/6J mice were exposed to 0.2 mg/kg polychlorinated biphenyl PCB126 or corn oil vehicle prior to ethanol (EtOH) or control diet feeding in the chronic-binge alcohol feeding model. Liver tissues were harvested during euthanasia and tissue collection, snap frozen in liquid nitrogen, and then were archived in a -80C freezer. For phosphoproteome analysis, six random liver tissues from four groups (N=24) were homogenized in 2% sodium dodecyl sulfate (SDS) radioimmunoprecipitation (RIPA) buffer with 1X HALT protease and phosphatase inhibitors. Lysates were trypsinized at a 1:20 (enzyme:sample) ratio and prepared following ProtiFi S-Trap mini spin column digestion protocol. Phospho-peptides were enriched with MagReSyn titanium ion - immobilized metal affinity chromatography (Ti-IMAC) microparticles following ReSyn Biosciences MagReSyn Ti-IMAC protocol. Samples were then purified using C18 PROTO 300 A Ultra MicroSpin columns, dried under a SpeedVac, and stored at -80C prior to peptide concentration assessment. Digests were fractionated by UPLC nanoflow liquid chromatography using PepMap RSLC C18 EASY-spray separating column at 40C with a 90-minute linear gradient. Eluates were introduced into an Exploris480 using an EASY-spray source (320C and 1.8kV). Full MS-ddMS2 method with a 3 second cycle time was generated in Xcalibur (v4.5.445.18) operating in positive polarity. RAW data files were separately searched in PeaksXpro (Bioinformatics Solutions Inc.; Waterloo, ON, Canada) using Denovo, PeaksDB, and PeaksPTM algorithms against the UniprotKB Mus musculus canonical protein sequences (proteomics ID: UP000000589) downloaded March 17, 2023. Label Free Quantification algorithm was then used with PeaksPTM results utilizing a mass error tolerance of 10 ppm and retention time shift tolerance of 6-minutes. Peptides were accepted at a 1% FDR threshold for consideration by the Label Free Quantification algorithm.

Project description:Background: The high number of heavy metal resistance genes in the soil bacterium Cupriavidus metallidurans CH34 makes it an interesting model organism to study microbial responses to heavy metals. Results: In this study the transcriptional response of this bacterium was measured after challenging it to a wide range of sub-lethal concentrations of various essential or toxic metals. Considering the global transcriptional responses for each challenge as well as by identifying the overlap in upregulated genes between different metal responses, the sixteen metals could be clustered in three different groups. Additionally, next to the assessment of the transcriptional response of already known metal resistance genes, new metal response gene clusters were identified. The majority of the metal response loci showed similar expression profiles when cells were exposed to different metals, suggesting complex cross-talk at transcriptional level between the different metal responses. The highly redundant nature of these metal resistant regions – illustrated by the large number of paralogous genes – combined with the phylogenetic distribution of these metal response regions within evolutionary related and other metal resistant bacteria, provides important insights on the recent evolution of this naturally soil dwelling bacterium towards a highly metal-resistant strain found in harsh and anthropogenic environments. Conclusions: The metal-resistant soil bacterium Cupriavidus metallidurans CH34 displays myriads of gene expression patterns when exposed to a wide range of heavy metals at non-lethal concentrations. The interplay between the different gene expression clusters points towards a complex cross-regulated regulatory network governing heavy metal resistance in C. metallidurans CH34. Keywords: Cupriavidus metallidurans CH34, transcriptional regulation, heavy metal resistance Two-condition experiments. Comparing samples after induction with heavy metals versus non-induced samples. Biological duplicate or triplicate. Each array contains 3 or 4 technical replicates.

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PCB126 Modifies the Murine Hepatic Transcriptome and Metallome to Promote Alcohol-associated Liver Disease Pathogenesis

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