DNA methylation changes induced by BDE-209 are related to DNA damage response and germ cell development in GC-2spd

Highlights

• BDE-209 led to genome wide DNA methylation changes in GC-2 cells.

• The differentially methylated genes were functionally related to gene transcription regulation, cell growth and development, signal transduction pathways.

• BDE-209 might affect cell growth and sperm development by the related gene methylation changes.

• BDE-209 increased gene expressions in p53-dependent DNA damage response.

Abstract

Decabrominated diphenyl ether (BDE-209) is generally utilized in multiple polymer materials as common brominated flame retardant. BDE-209 has been listed as persistent organic pollutants (POPs), which was considered to be reproductive toxin in the environment. But it still remains unclear about the effects of BDE-209 on DNA methylation and the induced-male reproductive toxicity. Due to the extensive epigenetic regulation in germ line development, we hypothesize that BDE-209 exposure impacts the statue of DNA methylation in spermatocytes in vitro. Therefore, the mouse GC-2spd (GC-2) cells were used for the genome wide DNA methylation analysis after treated with 32 μg/mL BDE-209 for 24 hr. The results showed that BDE-209 caused genomic methylation changes with 32,083 differentially methylated CpGs in GC-2 cells, including 16,164 (50.38%) hypermethylated and 15,919 (49.62%) hypomethylated sites. With integrated analysis of DNA methylation data and functional enrichment, we found that BDE-209 might affect the functional transcription in cell growth and sperm development by differential gene methylation. qRT-PCR validation demonstrated the involvement of p53-dependent DNA damage response in the GC-2 cells after BDE-209 exposure. In general, our findings indicated that BDE-209-induced genome wide methylation changes could be interrelated with reproductive dysfunction. This study might provide new insights into the mechanisms of male reproductive toxicity under the environmental exposure to BDE-209.

Introduction

Persistent organic pollutants (POPs) are born with the rapid development of industrialization, which have been ubiquitous in the surrounding environment (Chen et al., 2019a). Due to the persistence and bioaccumulation, POPs have brought global problems of environmental pollution (Wong et al., 2005). In the past few decades, POPs have caused adverse effects on wild life and human health (Li et al., 2006). Polybrominated diphenyl ethers (PBDEs) are a group of environmental persistent chemicals, which have been widely used as brominated flame retardants (BFRs) on household consumer products since 1970s. Decabrominated diphenyl ethers (BDE-209), a common homologue of PBDEs, which has been listed in the Stockholm Convention on POPs (UNEP, 2017). Exposure to BDE-209 could have multiple harmful effects on the environment and organisms (Chen et al., 2018b; Hou et al., 2019; Wang et al., 2018). Both animal and epidemiologic studies have proved that PBDEs induces biological toxicity in male reproduction and birth outcomes (Chen et al., 2018a; Zhang et al., 2020a).

Epigenetics plays a vital role in normal embryonic development and human disease etiology by participating in the regulation of cellular gene expression (Illum et al., 2018). DNA methylation is one of the most common mechanisms of epigenetic regulation, which has been considered to be an essential process in normal development of mammal. The addition of a methyl group to the 5′ position of cytosine is the important modifier of DNA methylation for genome organization (Zemach and Zilberman, 2010). DNA methylation has extensive impacts on the transcriptional regulation (Bird, 2002), and plays a major role in fetal growth and sperm maturation (Bernstein et al., 2007; Lambrot et al., 2013).

The abnormal gene methylation in germ lines may lead to the failure of spermiogenesis or embryonic development. A meta-analytic study found that male infertility is associated with altered sperm methylation (Santi et al., 2017). PBDEs exposure could increase aberrant DNA methylation in epididymal sperms (Suvorov et al., 2018). Besides, we previously reported that BDE-209 caused decreased sperm quality and quantity with DNA double-strand breaks (DSBs) and DNA damage response (DDR) (Li et al., 2019). Tseng et al. (2013) has also suggested the involvement of sperm DNA damage in BDE-209-induced male reproductive effects. However, it remains unclear whether DDR is associated with BDE-209-induced DNA methylation in male reproduction. There are different cell types and dynamically expressed DNA methyltransferases during spermatogenesis due to the active cell differentiation and meiosis in mammals (Uysal et al., 2016). Considering the essential differentiation of spermatocyte into mature sperm, we chose the stable GC-2spd cell line of mouse spermatocytes to accurately investigate the DNA methylation status after BDE-209 exposure.

In this study, we analyzed the global DNA methylation changes in mouse GC-2spd (GC-2) cells after BDE-209 treatment for 24 hr by using microarray technology. Remarkably, p53-related DNA damage response was determined on the basis of Gene Ontology (GO) enrichment. Gene verification related to cell growth and spermatogenesis was also conducted by qRT-PCR assay. This study aimed to clarify the underlying impacts of BDE-209 on the epigenetic mechanism of spermatocyte, focused on the gene transcription changes in germ cell development. Our findings revealed a preliminary epigenetic perspective to address the biological mechanism of BDE-209-induced spermatogenesis disorder, which might provide evidence for elucidating the male reproductive toxicity of BDE-209.