Polychlorinated biphenyls alter hepatic m6A mRNA methylation in a mouse model of environmental liver disease
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.
Section snippets
Animal studies
The experimental design is modeled in Supplementary Fig. 1. The mouse protocol for PCB exposure and HFD was ratified by the University of Louisville Institutional Animal Care and Use Committee (Jin et al., 2020). Adult male C57BL/6 mice (8 wks old) were purchased from Jackson Laboratory and randomized (n = 10) into four equal groups. All mice were fed ad libitum a high-fat diet (HFD, 15.2, 42.7, and 42.0% of total calories from protein, carbohydrate, and fat, respectively; TekLad TD88137)
Examination of m6A levels in mRNA isolated from livers of HFD-mice +/- PCB exposure
To identify hepatic m6A epitranscriptome changes mediated by the combination of HFD and PCB exposure, male C57Bl/J6 mice were exposed to 12 wks of HFD and a single oral dose of Ar1260, PCB126, or Ar1260 + PCB126 in combination (the experimental design is in Supplementary Fig. 1). As assayed by a colorimetric assay against a standard curve, PCB126 exposure reduced m6A modification in RNA isolated from the livers of the HFD-fed mice compared to PCB vehicle control or Ar1260-exposed mice (Fig. 1).
m6A readers
Discussion
We previously reported that the exposure of mice to HFD and PCBs impairs liver function and results in TASH (Hardesty et al., 2019a; Jin et al., 2020, 2021; Shi et al., 2019; Wahlang et al., 2014b). HFD-fed mice exposed to the NDL PCB mixture Ar1260 show more hepatic inflammation compared to mice exposed to the DL PCB126 that show more hepatic metabolism disruption and the co-exposure to Ar1260 + PCB126 shows more steatosis with less inflammation compared to Ar1260 (Jin et al., 2020; Wahlang et
Funding sources
This work was funded by National Institutes of Health (R21ES031510-01, ES031510-01S1, R35ES028373, P30ES030283, R01ES032189, T32ES011564, P42ES023716 and P20GM113226; P50AA024337, the Kentucky Council on Postsecondary Education (PON2 415 1900002934); and the Wendell Cherry Endowed Chair. This work was supported in part by a grant from the Jewish Heritage Fund for Excellence Research Enhancement Grant Program at the University of Louisville, School of Medicine. Part of this work was performed
Author contributions
Belinda J. Petri: investigation, formal analysis, editing; Kellianne M. Piell: investigation, formal analysis, and editing; Banrida Wahlang: investigation and editing; Kimberly Z. Head, investigation; Kalina Andreeva, formal analysis; Eric C. Rouchka: formal analysis and editing; Matthew C. Cave: conceptualization, resources, supervision and editing; Carolyn M. Klinge: conceptualization, supervision, formal analysis, writing, and editing.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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