BPDE and B[a]P induce mitochondrial compromise by ROS-mediated suppression of the SIRT1/TERT/PGC-1α pathway in spermatogenic cells both in vitro and in vivo

Highlights

• Mitochondria are the target of male reproductive toxicity induced by BPDE/B[a]P.

• BPDE and B[a]P induce mitochondrial compromise in spermatogenic cells.

• SIRT1/TERT/PGC-1α pathway accounts for BPDE/B[a]P-induced mitochondrial damage.

• BPDE/B[a]P-induced ROS causes the suppression of SIRT1/TERT/PGC-1α pathway.

Abstract

There is increasing evidence that indicates benzo[a]pyrene (B[a]P) and its active metabolite benzo[a]pyrene-7, 8-dihydrodiol-9, 10-epoxide (BPDE) are endocrine disruptors that can cause reproductive toxicity. Nevertheless, the underlying mechanisms are still obscure. The present study investigates the impacts of B[a]P and BPDE on mitochondria, a sensitive target affected by multiple chemicals, in spermatogenic cells. It showed that BPDE treatment induced mitochondrial dysfunction and the inhibition of mitochondrial biogenesis in mouse spermatocyte-derived cells (GC-2). These effects were efficiently mitigated by pretreatment with ZLN005, an activator of PGC-1α, in GC-2 cells. TERT knockdown and re-expression cell models were established to demonstrate that TERT regulated the BPDE-induced mitochondrial damage via PGC-1α signaling in GC-2 cells. Moreover, upregulating or knockdown SIRT1 expression attenuated or aggravated BPDE-induced mitochondrial compromise by activating or inhibiting, respectively, the TERT and PGC-1α molecules in GC-2 cells. Finally, we observed that BPDE markedly elevated oxidative stress in GC-2 cells. Resveratrol and N-acetylcysteine, as reactive oxygen species (ROS) scavengers, attenuated BPDE-mediated mitochondrial damage by increasing SIRT1 activity and expression in GC-2 cells. The in vitro results were corroborated by in vivo experiments in rats treated with B[a]P for 4 weeks. B[a]P administration caused mitochondrial damage and mitochondria-dependent apoptosis in spermatogenic cells, as well as the decreased expression of SIRT1, TERT, and PGC-1α. In summary, the results of the present study demonstrate that B[a]P and BPDE induce mitochondrial damage through ROS production that suppresses SIRT1/TERT/PGC-1a signaling and mediate B[a]P- and BPDE-mediated reproductive toxicity.

Introduction

Benzo[a]pyrene (B[a]P), as a ubiquitous and extensively studied polycyclic aromatic hydrocarbon (PAH), is mainly generated by the incomplete combustion of organic material (Yin et al., 2017). Besides well-documented carcinogenicity and mutagenicity, growing evidence indicates that B[a]P is a representative endocrine disruptor that may exert notable male reproductive toxicity (Banerjee et al., 2016; el SA et al., 2010). Previous epidemiological research efforts have confirmed the production of B[a]P-DNA adducts in human sperm and suggested that PAHs exposure in humans is associated with reduction in semen quality that might result in a higher risk of infertility (Oliveri Conti et al., 2017; Han et al., 2011). B[a]P following oral administration in rats can be metabolized in reproductive tissues, such as testis, and get transformed into various metabolites by cytochrome P450 enzymes, including the most-studied metabolic product benzo[a]pyrene-7, 8-dihydrodiol-9, 10-epoxide (BPDE) which has been identified as an active metabolite that can cause reproductive damage (Ramesh et al., 2001; Sipinen et al., 2010). Our previous study has implied that sub-chronic exposure to B[a]P results in the atrophy of seminiferous epithelium and decreases sperm count in male Sprague Dawley rats (Chen et al., 2011). Other animal studies have also revealed that exposure to B[a]P causes a significant decline in sperm count and motility (el SA et al., 2010; Meier et al., 2017). These particular evidences demonstrate that B[a]P can induce appreciable spermatogenic disorder, which may then exert further adverse effects on male reproductive health.

Mitochondria are eukaryotic organelles that produce energy through the Krebs cycle and the electron transport chain that then can result in the synthesis of adenosine triphosphate (ATP). Previous in vivo and in vitro research efforts have revealed that exposure to B[a]P as well as BPDE can induce obvious mitochondrial compromise in somatic cells (Zha et al., 2017; Zhang et al., 2015). Moreover, mitochondria in spermatogenic cells play importance roles in spermatogenesis and male fertility (Rajender et al., 2010). A previous cross-sectional study conducted by our laboratory found a significant relationship between urinary concentrations of PAH-metabolites and decreased mitochondrial DNA copy number (mtDNAcn) in sperm, which implied that mitochondria might be the target organelle involved in the B[a]P-induced spermatogenic disorder (Ling et al., 2017). However, the precise cellular and biochemical mechanisms by which B[a]P causes mitochondrial damage in spermatogenic cells and triggers spermatogenic disorder have not yet been identified.

The peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC-1α), as a transcriptional coactivator, fundamentally regulates mitochondrial biogenesis as well as the mitochondrial function. Inactivated PGC-1α remains in its acetylated form in cells and is deacetylated by silent information regulator type-1 (SIRT1), which generates the active form of PGC-1α. Deacetylated PGC-1α cooperates with the nuclear respiratory factors (NRFs), such as NRF1, to trigger expression of the mitochondrial transcriptional factor A (Tfam) and various nuclear-encoded genes (Dorn 2nd et al., 2015). Besides SIRT1-mediated regulation, PGC-1α can also be regulated by telomerase reverse transcriptase (TERT), indeed a critical component of telomerases (Sahin et al., 2011). Telomerase, including the TERT and the telomerase RNA, is an RNA-containing reverse transcriptase that adds repeats of telomeric DNA to the ends of the chromosomes, which then cap and protect the integrity of the chromosomal ends.

Previous studies indicate that TERT deficiency causes telomeres dysfunction, which inhibits PGC-1α-mediated mitochondrial biogenesis and elevates oxidative stress through the activation of p53 (Sahin et al., 2011). Our study also previously reported that B[a]P exposure decreased the expression and activity of TERT, which caused senescence and apoptosis via the DNA damage response in the spermatogenic cells in vitro and in vivo (Ling et al., 2018). However, whether suppression of TERT is involved in B[a]P-induced reproductive damage via regulation of PGC-1α remains still largely unknown.

Mitochondria are the primary source of cellular ROS production (Yue et al., 2016). Although reactive oxygen species (ROS) is necessary for several vital signaling pathways to maintain physiologic homeostasis (Dostert et al., 2008; Tschopp and Schroder, 2010), the excessive production of ROS is closely related to the inhibition of mitochondrial biogenesis and mitochondrial dysfunction (Murphy, 2013; Soeur et al., 2015). Several studies have reported that exposure to B[a]P induces incremental ROS production both in vivo and in vitro (Banerjee et al., 2016; Omidian et al., 2017; Yang et al., 2017), and oxidative stress can decrease the expression of SIRT1 and agonism of SIRT1 through resveratrol or SIRT1 reconstitution can mitigate ROS-induced damages (Lin et al., 2018; Kerksick and Willoughby, 2005). Thus, further investigations are needed to elucidate whether excessive ROS production can cause mitochondrial compromise through a SIRT1 signal in B[a]P-induced male reproductive damage.

We hypothesized that mitochondrion is the sensitive targeted organelle that is involved in B[a]P-mediated spermatogenic cell damage. Meanwhile, B[a]P-induced mitochondrial compromise may be closely related to the alterations of SIRT1, TERT, and PGC-1α that can be regulated by elevated oxidative stress. To test this hypothesis, we examined mitochondrial damage in mouse spermatocyte-derived GC-2 spd (ts) cells (GC-2 cells) and Sprague-Dawley rats treated with BPDE and B[a]P, respectively. We also modified the ROS level as well as the molecules mentioned above to further ascertain they are involved in B[a]P-induced mitochondrial compromise and male reproductive toxicity.