The role of STAT3/p53 and PI3K-Akt-mTOR signaling pathway on DEHP-induced reproductive toxicity in pubertal male rat
Introduction
Phthalate esters (PAEs) are a class of organic chemicals that are widely used as plasticizers in commercial and industrial application. More than 8 million tons of PAEs were manufactured in 2011 (Net et al., 2015). Currently, there are more than 20 kinds of PAEs (Abdel daiem et al., 2012), among which DEHP has the largest production and consumption, accounting for more than 50% of the total PAEs (Lu et al., 2014). DEHP, as one of the most common PAEs, is extensively added in food packaging materials, building decoration materials, children's toys and medical equipment, as well as other products (Garcia Ibarra et al., 2018). Because it is noncovalently bound to plastic substrates, DEHP can constantly be released from plastic products to pollute the surface water, groundwater, atmosphere, soil (Gao et al., 2019b; Zhu et al., 2019). House dust is a major source of environmental exposure of DEHP for human. It was reported that the mean concentration of DEHP in house dust is 1596.9 μg/g and with a maximum detected of 80,116.4 μg/g in summer of China (Fan et al., 2018). And the mean concentration is 304 with a maximum detected of 9650 μg/g in the USA. DEHP in dust can be absorbed via inhalation, ingestion, and dermal contact. It was estimated that the DEHP ingestion from house dust is 2.6–45 μg/kg/day based on US studies (Ginsberg et al., 2016). In addition, the European Chemicals Bureau (ECB) reported that the occupational exposure level is up to 1060 μg/kg/day by inhalation and 928 μg/kg/day by dermal exposure (ECB, 2008). DEHP is a known environmental endocrine disruptor (Ahuactzin-Perez et al., 2018) with antiandrogenic effect and is considered of great concern for its wide spectrums of toxic effects on human and animal health, such as developmental toxicity (Lyche et al., 2009), carcinogenicity (Ito and Nakajima, 2008), hepatotoxicity (Ha et al., 2016), reproductive toxicity (Rowdhwal and Chen, 2018).
The impact of DEHP exposure on male reproductive function has been identified for many years. Human studies showed that DEHP exposure leaded to a decrease of semen volume, sperm motility and serum testosterone. Moreover, DEHP could disrupt sperm DNA integrity and induce spermatozoa malformation and apoptosis (Wang et al., 2016a; Wang et al., 2016b; Wang et al., 2019b). In vivo exposure of DEHP caused a decrease in male fertilization ability with dysfunction of testicular development and decreased spermatozoa in mice (Jones et al., 2015).
Oxidative stress that is a common pathological process acts as a bridge between endocrine disruptors and reproductive dysfunction (Kim et al., 2014; Rahman et al., 2019). The specific composition of sperm with abundant polyunsaturated fatty acids also increases the risk of oxidative damage (Saez and Drevet, 2019). So oxidative stress-mediated spermatogenic disorder could be a potential etiology for male infertility (Aitken and Baker, 2006). Apoptosis is a common process in spermatogenesis to sustain the genetic integrity and prevent abnormal spermatogenesis (Kaushik et al., 2018). But excessive apoptosis induced by oxidative stress leads to alteration of testicular histopathology and disruption of spermatogenesis resulting in male infertility (Beigi Harchegani et al., 2018). Accumulating evidence has shown that DEHP administration destroys the balance of ROS and antioxidants in germ cells and Leydig cells and thereby promotes the production of oxidative stress in testis (Zhang et al., 2014; Ha et al., 2016).
Autophagy is an evolutionarily conserved process in all eukaryotic organisms (Levine and Klionsky, 2004). Moderate autophagy contributes to cell survive by degrading intracellular misfolded, aggregated or damaged proteins and organelles and recycling nutrients. But excessive autophagy destroys cellular composition and ultimately results in autophagic cell death (Codogno and Meijer, 2005). ROS as a direct trigger of oxidative stress can provoke autophagy as a cytoprotective mechanism (Lin and Baehrecke, 2015) and can also promote programmed cell death in stress conditions by regulating AMPK/Akt-mTOR, JNK pathways (Duan et al., 2016). Studies showed that autophagy is involved in DEHP-induced testicular cytotoxicity (Sun et al., 2018; Gan et al., 2020).
Puberty is a period characterized by testicular development, secretion of gonadotropin-releasing hormone, sperm formation and the acquisition of reproductive capacity. And this developmental stage is one of the windows of susceptibility to environmental exposure of harmful substrates (Abreu and Kaiser, 2016; Ho et al., 2017). Therefore, we hypothesize that oxidative stress-related autophagy plays a role in DEHP-induced testicular apoptosis, spermatogenesis disorder and reproductive toxicity in puberty male rats. The present study aims to investigate adverse reproductive effects of DEHP on male puberty rats and to explore the possible role of STAT3/p53 and PI3K-Akt-mTOR signaling pathway in testis damage in puberty rats.
Section snippets
Chemicals and reagents
Di-(2-ethylhexyl) phthalate (CAS: 117–81-7) with 99% analytical standard was obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). The testosterone assay kit, superoxide dismutase (SOD), glutathione peroxidase (GSH-PX) and malondialdehyde (MDA) kit were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). BCA Protein Quantification Kit and High-Sensitive ECL chemiluminescence Detection Kit were purchased from Vazyme Biotech Co., Ltd. (Nanjing, China).
Effect of DEHP on rat body weight, organ coefficients of rat testes and sperm count
As shown in Fig. 1A, after 28-day exposure to DEHP, a significant increase in body weight was only observed in the 1000 mg/kg/day DEHP group (p < .05). Although there was no difference in the testis weight among these four groups (Fig. 1B), the organ coefficient of testis in the 500 and 1000 mg/kg/day DEHP groups was significantly reduced as compared with the control group (p < .05) (Fig. 1C). In addition, the sperm count decreased significantly in the 500 and 1000 mg/kg/day DEHP groups (p
Discussion
The current study demonstrated that DEHP caused distinct toxicity effect on the testes of pubertal rats, leading to decreased testis coefficient and injury microstructure and abnormal ultrastructure of testes. DEHP also decreased serum testosterone levels and sperm counts. These pathological changes induced by DEHP may be explained by increased oxidative stress levels and testicular cell apoptosis. Moreover, activation of autophagy was accompanied with elevated autophagosomes and
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.
Acknowledgments
This work was financially supported by Natural Science Foundation of Hubei Province (CN) (2018CFB638), Hubei Province Key Laboratory of Occupational Hazard Identification and Control (OHIC2019G03) and Health Commission of Hubei Province Scientific Research Project (WJ2019H255), Excellent Youth Foundation of Hubei Scientific Committee (2018CFA040), Excellent Young and Middle-aged Scientific Research and Innovation Team Fund, Wuhan University of Science and Technology (2018TDZ03), Innovation and
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These authors contributed equally to this work.