Effect of PFOA on DNA Methylation and Alternative Splicing in Mouse Liver

Highlights

• DNA methylation alterations and alternative RNA splicing in hepatocellular steatosis were noted.

• PFOA exposure’s effects on Dnmts and Tets in liver tissues were evaluated.

• PFOA exposure alters gene expression in mTOR pathway and decreases Pten expression.

• Tissue specific changes in RNA binding proteins were noted.

• Changes in alternative splicing factors were identified upon PFOA exposure.

Abstract

Perfluorooctanoic acid (PFOA) is a persistent organic pollutant prevalent in the environment and implicated in damage to the liver leading to a fatty liver phenotype called hepatocellular steatosis. Our goal is to provide a basis for PFOA-induced hepatocellular steatosis in relation to epigenetic alterations and mRNA splicing. Young adult female mice exposed to different concentrations of PFOA showed an increase in liver weight with decreased global DNA methylation (5-mC). At higher concentrations, the expression of DNA methyltransferase 3A (Dnmt3a) was significantly reduced and the expression of tet methycytosine dioxygenase 1 (Tet1) was significantly increased. There was no significant change in the other Dnmts and Tets. PFOA exposure significantly increased the expression of cell cycle regulators and anti-apoptotic genes. The expression of multiple genes involved in mTOR (mammalian target of rapamycin) signaling pathway were altered significantly with reduction in Pten (phosphatase and tensin homolog, primary inhibitor of mTOR pathway) expression. Multiple splicing factors whose protein but not mRNA levels affected by PFOA exposure were identified. The changes in protein abundance of the splicing factors was also reflected in altered splicing pattern of their target genes, which provided new insights on the previously unexplored mechanisms of PFOA-mediated hepatotoxicity and pathogenesis.

Introduction

Perfluorooctanoic acid (PFOA) is a synthetic compound that belongs to a group of per- and polyfluoroalkyl substances (PFAS). PFOA consists a long hydrophobic chain with eight carbons, saturated with fluorine atoms and a hydrophilic polar functional group (Banks et al., 1994; Kissa, 2001; Taylor, 1999). PFOA is one of the most frequently used perfluoroalkyl compounds since the 1940s; and is abundant in the environment, with deleterious consequences due to exposure through food and drinking water (US EPA, 2001; Martin et al., 2003; Ericson et al., 2008; Vestergren et al. 2008). PFOA has been detected in human cord serum and breast milk (Apelberg et al., 2007; Liu et al., 2010; Mondal et al., 2012). In the human system, PFOA is known to be hard to metabolize due to its long half-life (mean estimated half-life is 2.7 years) as noted in past works (Li et al., 2018; Burris et al., 2002; Olsen et al., 2007). Experiments in animal model showed that PFOA predominantly accumulated in the liver, kidney and serum (Ylinen et al., 1990; Vanden Heuvel et al., 1991; Cui et al., 2009), resulting in hepatotoxicity, developmental toxicity, immunotoxicity, and neurotoxicity (DeWitt et al., 2008; Johansson et al., 2008a, 2008b; Gallo et al., 2012; Christopher and Martin, 1977; Metrick and Marias, 1977; Yang et al., 2000; Yang et al., 2001; Sibinski et al., 1987; Johansson et al., 2008a, 2008b).

Past in vitro and in vivo studies show a lack of genotoxicity associated with PFOA (Fernández Freire et al., 2008; Eriksen et al., 2010; Florentin et al., 2011; Stefani et al., 2014). Thus, one plausible way by which PFOA can exert its effect is through epigenetic programming. DNA methylation is one of the most important and well-established epigenetic indicators that especially plays a role in embryonic development and cellular differentiation (Jähner et al., 1982; Razin et al., 1984). Alteration at regions where cytosine-guanine dinucleotide (CpG sites) are frequent (CpG islands) could typically lead to the repression of gene expression (Hackett and Surani, 2013). CpG methylation is regulated by a group of enzymes known as DNA methyltransferases (Dnmts). Three isoforms of Dnmts, namely DNA methyltransferase 1 (Dnmt1), DNA methyltransferase 3 alpha (Dnmt3a), and DNA methyltransferase 3 beta (Dnmt3b), all have unique functions to perform (Lyko et al., 1999; Tucker et al., 1996). Apart from Dnmts, ten-eleven translocation methylcytosines (Tets), including Tet1, Tet2, and Tet3, also play major roles in DNA methylation status maintenance. These Tet enzymes catalyze the oxidation of 5-methylcytosine (5-mC) to generate 5-hydroxymethylcytosine (5-hmC), causing demethylation in CpG islands (Tahiliani et al., 2009; Ito et al., 2011); thus activating gene transcription. They can also subsequently catalyze the oxidation of 5-mC to 5-formylcytosine (5-fC) and 5-fC to 5-carboxylcytosine (5-caC) (Ito et al. 2011).

The effects of PFOA exposure on methylation have been noted in some in vitro and in vivo studies (Wen et al., 2020; Wan et al., 2010; Rashid et al., 2020; Tian et al., 2012). Prenatal PFOA exposure has been associated with reduced Insulin Line Growth Factor 2 (IGF2) methylation in cord blood (Kobayashi et al., 2017), lowered global DNA cytosine methylation in neonates (Guerrero-Preston et al., 2010), and increased methylation of Long Interspersed Nuclear Element-1 (LINE-1) (Watkins et al., 2014). Other studies have reported changed expression of genes in cholesterol metabolism (Fletcher et al., 2013) and lipid metabolism (Wen et al., 2020). However, none have explored genome-wide methylation alterations and its impact in liver where PFOA accumulation can reach high levels.

PFOA-induced liver enlargement is an evident consequence as noted in past studies (Christopher and Martin, 1977; Metrick and Marias, 1977). Hepatocellular hypertrophy/ cytomegaly and increased liver mass were observed in male rats exposed to PFOA (Goldenthal, 1978; Perkins et al., 2004a, 2004b), which are notable features of non-alcoholic fatty liver disease (NAFLD) (Liang et al., 2014a, 2014b). Several animal studies have connected PFOA exposure to fatty liver disease, also known as hepatic steatosis (Sibinski et al. 1987). In chronic studies with Sprague-Dawley rats, PFOA exposure was known to induce hepatocellular adenomas, with increasing liver weight and hepatic β-oxidation activity (Biegel et al., 2001). Study has also reported on epigenetic alterations induced by PFOA in relation to lipid metabolism genes in liver cell model (Wen et al., 2020).

Besides epigenetic changes, gene or protein expression could also be influenced by changes in mRNA splicing and translational factors upon exposure to PFOA. Transcriptome-wide profiling studies have been previously carried out on human livers with NAFLD (Lake et al., 2011; Moylan et al., 2014); but as with most studies they only examined changes in overall mRNA abundance and did not attempt to monitor changes in splice isoforms (Shackel et al., 2006). Nonetheless, when mRNA level changes were profiled in the liver samples from insulin-resistant obese population, the key pathways downregulated in obese liver samples were related to alternative mRNA processing and splicing (Pihlajamäki et al., 2011). Alternative splicing decisions are determined by splice site strength, cis-acting regulatory elements within pre-mRNAs that promote or inhibit exon recognition, and expression/activity of trans-acting factors that bind to these cis elements and regulate the accessibility of the spliceosome to splice sites (Chen and Manley, 2009; Kalsotra and Cooper, 2011; Lee and Rio, 2015). Notably, several new studies have shown that proper expression of alternative splicing factors is important for hepatocyte differentiation and function, implicating its major role in maintaining normal liver physiology (Pihlajamäki et al., 2011; Sen et al., 2013; Elizalde et al., 2014; Sen et al., 2015; Bhate et al., 2015, Cheng et al., 2016; Bangru et al., 2018a, 2018b; Kumar et al., 2019).

Although numerous biochemical studies have been performed on PFOA exposed liver samples, none of the studies have investigated the mechanisms of epigenetic alterations or alternative splicing variations in PFOA exposed liver. Specifically, our study tested the hypothesis that PFOA induces hepatic hypertrophy and steatosis through specific alterations of DNA methylation patterns and changes in alternative splicing factors.