Polychlorinated biphenyls alter hepatic m6A mRNA methylation in a mouse model of environmental liver disease

Highlights

• PCB exposures altered the m6A landscape in liver genes in HFD-fed mice.

• Common and PCB exposure-specific m6A peaks in genes were identified.

• Pathway analysis of DEGs with changes in m6A peaks include lipoprotein metabolism.

• qRT-PCR confirmed m6A peak enrichment in the Apob transcript with PCB exposure.

Abstract

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.

Introduction

Polychlorinated biphenyls (PCBs) are lipophilic, persistent organic pollutants that are “legacy contaminants” (Taylor et al., 2019). PCBs act as endocrine- and metabolism-disrupting chemicals (EDCs, MDCs) and increase susceptibility to obesity-associated diseases including insulin-resistance, type 2 diabetes, and nonalcoholic fatty liver disease (NAFLD) (Heindel et al., 2017a; Simhadri et al., 2020). Population studies have linked community PCB exposure in Anniston, Alabama, the site of a PCB manufacturing plant, to NAFLD (Cave et al., 2022; Clair et al., 2018; Wahlang et al., 2019a). NAFLD may progress from steatosis to steatohepatitis (NASH), featuring increased liver injury and inflammation with or without fibrosis, to cirrhosis and to hepatocellular carcinoma (HCC) (Cave et al., 2016). Despite improvements in treatment and diagnosis, the prevalence of chronic liver disease has increased significantly since 2000 and, in the absence of FDA-approved therapeutics to prevent disease progression, liver transplantation cannot meet demand (Bram et al., 2021). While volatilization and inhalation is one route of human PCB exposure (Christensen et al., 2021), the major route of current PCB exposure is dietary, due to contamination of the food supply, e.g., meat, dairy, fish, and eggs (Lebelo et al., 2021; Ng and von Goetz, 2017; Papadopoulou et al., 2019; Schecter et al., 2010). Due to their lipophilic nature, PCBs accumulate in adipose tissue; hence, circulating PCB levels increase after weight reduction surgery in obese individuals (Heindel et al., 2017b; Sargis et al., 2019). There are 209 theoretical PCB congeners which are classified as ‘dioxin-like (DL)’ or ‘non-dioxin-like (NDL)’ based on their activation of the aryl hydrocarbon receptor (AHR). Both DL and NDL PCB congeners have been associated with fatty liver disease (Heindel et al., 2017b; Wahlang et al., 2019b).

Aroclor1260 (Ar1260) is an environmentally relevant NDL PCB mixture (Wahlang et al., 2014a) that causes toxicant-associated steatohepatitis (TASH) in male C57Bl/6J mice fed a high-fat diet (HFD) (Wahlang et al., 2014b). Ar1260 exposure resulted in more severe HFD-induced hepatic necrosis, inflammation, and fibrosis compared to vehicle control-treated HFD-fed mice (Hardesty et al., 2019b; Wahlang et al., 2014b). Although PCBs act via nuclear receptors, e.g., pregnane X receptor (PXR, NR1I2), constitutive androstane receptor (CAR, NR1I3), and androgen receptor (AR) activation and inhibition of farnesoid X receptor (FXR), estrogen receptor α (ERα), peroxisome proliferator-activated receptor α (PPARα), hepatocyte nuclear factor 4α (HNF4α), NRF2 (NFE2L2) (reviewed in (Cave et al., 2016)) and epidermal growth factor receptor (EGFR) (Hardesty et al., 2017, 2018; Hardesty Josiah et al., 2021), these receptor-mediated activities only partially explain PCB-induced transcriptional reprogramming promoting steatohepatitis (Hardesty Josiah et al., 2021). Evidence of the contribution of the human gut microbiome in liver disease and the pathogenesis of NAFLD as well as evaluating disease severity has been reported (Canfora et al., 2019; Liu et al., 2022). Microbiome diversity was associated with greater hepatic and intestinal inflammation, dysregulated lipid metabolism, and TASH in HFD + Ar1260 exposed Car-/- and Pxr-/- mice compared to C57Bl/6J wildtype (WT) mice, implicating CAR and PXR have a protective effect by affecting the gut microbiome which regulates the gut-liver axis (Wahlang et al., 2021).

PCB126 is a dioxin-like (DL) PCB that binds and activates AHR (Safe, 1994) and increases the expression of hepatic AHR targets in mouse liver (Jin et al., 2021). PCB126 also inhibits hepatic EGFR signaling (Hardesty et al., 2018) and PCB126 exposure has been associated with liver metabolism disruption and variably with hepatic steatosis with at least some degree of AHR dependence (Jin et al., 2021; Wahlang et al., 2019a). In combination with HFD, PCB126 increases steatosis, lipid uptake, and fatty acid (FA) biosynthesis, but when combined with Ar1260, PCB126 protects against the inflammatory liver injury associated with Ar1260 exposure in male mice (Jin et al., 2020).

PCB exposures have been associated with epigenetic changes in DNA methylation (Pittman et al., 2020), changes in circulating miRNAs (Cave et al., 2022) (Klinge et al., 2021), and changes in the global RNA modifications. Epitranscriptomic mRNA modifications affect transcript processing, stability, splicing, transport, and translation (Chen et al., 2021; Edupuganti et al., 2017). N (6)-methyladenosine (m6A) is the most common dynamic modification of the transcriptome and regulates the transcription, processing, stability and the cellular location of eRNAs, mRNAs, long non-coding RNAs (lncRNAs), and primary microRNAs (pri-miRNAs) (Lee et al., 2021; Zaccara et al., 2019). The m6A modification occurs within the conserved RRACH (DRACH) motif (R = A, G; H = A, C, U) (Jenjaroenpun et al., 2020) which is recognized by the METTL3 complex that is the ‘writer’ of the m6A mark (Knuckles and Bühler, 2018; Licht and Jantsch, 2016). Protein ‘readers’ of m6A regulate transcript fate by altering splicing, subcellular location, stability, degradation, and translation (Cayir et al., 2020; Licht and Jantsch, 2016; Zaccara et al., 2019). Demethylases FTO and ALKBH5 are the ‘erasers’ of m6A methylation (Zhao et al., 2020b), although FTO is nuclear and may only demethylate m6Am in the 5′ N-terminal cap (Mauer et al., 2019). The state of knowledge of m6A epitranscriptomics to hepatic function and liver disease has been reviewed (Zhao et al., 2020b).

Only a few studies have examined the impact of environmental chemicals and toxicants, e.g., B [a]P, aflatoxin B1, bisphenol A (BPA), pesticides, and arsenic, on the m6A readers, writers, erasers, and global m6A epitranscriptome in a limited number of cell types or tissues (reviewed in (Cayir et al., 2020; Malovic et al., 2021)). More studies have examined the expression levels of the m6A readers, writers, and erasers in NAFLD and how their silencing impacts global m6A levels in HeLa and HEK-293T cells (Malovic et al., 2021). m6A RNA immunoprecipitation (RIP) followed by RNA sequencing (m6A RIP-seq or Me-RIP-seq) is used to identify gene transcripts with the m6A mark. There is only one study examining the impact of PCBs on m6A levels by m6A-RIP-seq (Aluru and Karchner, 2021). That study exposed zebrafish embryos (72 h post-fertilization) to 10 nM PCB126 for 6 h and identified 15 m6A marked transcripts that were differentially methylated (Aluru and Karchner, 2021). We reported that the global abundance of m6A in total RNA was reduced in HFD-fed male mice exposed to Ar1260 or PCB126, but increased in mice exposed to Ar1260 + PCB126 in combination (Klinge et al., 2021). No one has examined m6A modification of mRNA in mouse liver after PCB exposure, reflecting innovation of our study.

Animal models demonstrate that PCB exposures reprogram hepatic gene expression thereby promoting more severe HFD-induced fatty liver disease (Wahlang et al., 2019a). The mechanisms by which reprogramming occurs remain to be fully elucidated. Here we examined the hypothesis that exposure of HFD-fed male C57Bl/6J mice to a single oral dose of PCB126, Ar1260, or the combination of Ar1260+ PCB126 will alter m6A RNA modification levels in specific liver transcripts. Indeed, m6A-RIP-seq analysis revealed changes in the distribution of m6A peaks in hepatic transcripts with PCB exposure compared to PCB vehicle control. PCB126 exposure reduced m6A levels in total RNA. However, PCB126 or Ar1260 + PCB126 exposure increased the number of m6A peaks/gene and per chromosome. Integrated analysis of m6A-RIP-seq and the differentially expressed genes (DEGs) with PCB exposure from mRNA seq data (Petri et al., 2022) identified DEGs with increased or reduced number of m6A peaks. These data identify changes in m6A as a new molecular basis for hepatic responses to environmental chemicals in a HFD-fed mouse model of NAFLD.