Research PaperPrenatal exposure to environmentally relevant levels of PBDE-99 leads to testicular dysgenesis with steroidogenesis disorders
Graphical Abstract
Introduction
Polybrominated diphenyl ethers (PBDEs) are a class of brominated flame retardants (BFRs). PBDEs are widely used in plastics, textiles, electronic appliances, and other products owing to their excellent flame-retarding effect (Schmitt et al., 2021). However, due to the unstable chemical combination between PBDEs and various products, PBDEs are readily released into the surrounding environment by volatilization, exudation, and other means. They easily bioaccumulate in the environment and last for years because of their high lipophilicity and long half-lives (Shin et al., 2017, Thuresson et al., 2006). PBDEs have been detected in air, dust, soil, and water resources (Deng et al., 2021, Feng et al., 2021, Labunska et al., 2014). Moreover, many studies have detected the presence of PBDEs in human blood, milk, and adipose tissue in recent years (Drobná et al., 2019, Guo et al., 2020b, Kaihan et al., 2020, Toms et al., 2009). The concentration of PBDEs in biota has been continuously increasing, and the impact of PBDEs on human health has attracted worldwide attention.
A large number of studies have confirmed that the accumulation of PBDEs in organisms can cause developmental and reproductive toxicity (Sarkar et al., 2019, Sarkar and Singh, 2021). PBDE exposure during pregnancy can result in PBDEs entering the fetus through the placental barrier, which has an impact on the growth and development of the fetus (Chen et al., 2018, Gómara et al., 2007). Population cohort studies and animal experiments show that PBDE exposure is closely related to male reproductive system abnormalities, such as the decrease of male reproductive organ weight, anogenital distance (AGD), and semen quality (García-Villarino et al., 2018, Kuriyama et al., 2005, Tseng et al., 2013, Zhang et al., 2020).
PBDEs have 209 different congeners according to the number and position of the bromine atoms of the PBDE molecule (Sjödin et al., 2003). PBDE-99 is one of the most abundant homologues detected in humans, with a biological half-life of more than 40 days in vivo (Geyer et al., 2004). Labunska et al. (2014) have estimated that the median PBDE exposure levels around e-waste recycling sites were 130.9 and 614.1 ng/kg bw/day for adults and children, respectively. In particular, PBDE 99 exposure is about 1.5 times higher than the U.S. Environmental Protection Agency reference dose (Labunska et al., 2014). The highest reported PBDE-99 concentration in human adipose tissue was up to 1.38 mg/kg (Johnson-Restrepo et al., 2005). It has been reported in an animal experiment that prenatal exposure to PBDE-99 at a level of 10 mg/kg body weight (bw) can reduce AGD in male offspring (Lilienthal et al., 2006). Additionally, a population-based study has found that the anogenital index (AGI), which is AGD divided by weight, is inversely associated with PBDE-99 concentrations in boys at 18 months (García-Villarino et al., 2018). A recent case–control study has examined the association between PBDE concentrations in maternal hair and the risk of cryptorchidism in male infants, and found that maternal exposure to PBDE-99 may be associated with abnormal migration of testes in the male offspring (Goodyer et al., 2017). Another case–control study has found that mothers of children with hypospadias were exposed during pregnancy to significantly higher levels of PBDE-99 and seven more PBDEs (Poon et al., 2018).
AGD or AGI is widely used as a measure of in utero exposure to hormonally active agents (Kızılay et al., 2020, Swan et al., 2005, van den Driesche et al., 2017), and both cryptorchidism and hypospadias are considered as clinical manifestations of testicular dysgenesis (Skakkebaek et al., 2001, Skakkebaek et al., 2016, Welsh et al., 2008). The above findings highlight the anti-androgenic properties of PBDE-99, suggesting that PBDE-99 exposure plays an important role in the pathogenesis of testicular dysgenesis.
Testicular dysgenesis seriously affects male fertility and poses a great threat to male reproductive health. It has been reported that the incidence of testicular dysgenesis-related diseases in men is more than 10% (Jørgensen et al., 2016, van den Driesche et al., 2017). It is considered to be the source of male reproductive dysfunction in the immature stage (Kilcoyne and Mitchell, 2019). Recent studies have found that the occurrence of testicular dysgenesis is closely related to genetic and environmental factors, in which environmental factors play a major role, but the relevant molecular mechanism has not been fully elucidated (Dalgaard et al., 2012, Ma et al., 2020, Macdonald et al., 2018). Therefore, it is of great practical significance to study the effect of PBDE-99, a kind of endocrine-active compound, on testicular dysgenesis and its underlying mechanism.
Past toxicology research on hazardous material exposure was mainly based on hypotheses. The hypothesis-driven research mode is an attempt to verify hypotheses, and verification is performed according to the existing pathophysiological molecular mechanisms. Researchers may be constantly frustrated by the fact that the hypothesis does not agree with the real bioprocess, particularly in the study of low-dose exposure effects of environmentally relevant concentrations. Currently, with the development of high-throughput sequencing technology, the data-driven mode provides a credible research direction for evaluating biological changes and the involved mechanism occurring after the exposure to environmentally relevant levels of a chemical. Omics techniques with in-depth bioinformatics exploration are emerging as powerful data-driven research tools (Fan et al., 2019, Guo et al., 2020a, Pedersen et al., 2020, Wang et al., 2021).
In the present work, environmentally relevant levels of PBDE-99 were applied to both the in vivo and in vitro studies, which are similar to the human exposure doses. We demonstrated the adverse effects of prenatal exposure to PBDE-99 on testicular development in male offspring, and clarified the potential mechanism through integrated transcriptomic analysis and subsequent experimental verification. We aimed to explain the molecular mechanism of testicular dysgenesis induced by prenatal exposure to PBDE-99 based on data-driven research.
Section snippets
Animal care and maintenance
Research ethics approval for the animal study was obtained from the Institutional Animal Care and Use Committee of Guangzhou Huateng Biomedical Technology Co., Ltd., and the certificate of approval is available upon request. Adult ICR mice were obtained from Beijing Huafukang Bioscience Co., Inc. (Certificate No. SCXK 2019-0008, Grade SPF), and were raised in a 12-h dark/light cycle at a temperature of 25 ± 2 °C and relative humidity of 50 ± 5%. All mice were given free access to standardized
Prenatal exposure to PBDE-99 induces testicular dysgenesis
To evaluate the effect of prenatal exposure to PBDE-99 on the development of the reproductive system in male offspring, AGI, incidence of cryptorchidism, testicular organ coefficient, and testicular histological morphology were analyzed and are shown in Fig. 1.
AGI was counted based on AGD and body weight, which are presented in Fig. S1. On days 1, 7, 21, and 35 after birth, the AGI values of male offspring in all three PBDE-99 exposure groups were significantly lower than that in the control
Discussion
Despite increasing regulation, exposure to PBDEs remains a serious public health concern due to their accumulation in the environment and ability to biomagnify up the food chain (Lin et al., 2020, Zhao et al., 2020c). PBDEs are associated with endocrine-disrupting effects including adverse reproductive outcomes that could disturb the normal development of the male reproductive system (Kahn et al., 2020, Luan et al., 2019). PBDE-99 is one of the most common PBDE congeners accumulated in the
Conclusions
Prenatal exposure to environmentally relevant levels of PBDE-99 leads to testicular dysgenesis in male offspring, which is characterized by decreased AGI and testicular organ coefficient, increased incidence of cryptorchidism, and disturbed testicular histology. PBDE-99 exposure induced Leydig cell injury is involved in the testicular dysgenesis with steroidogenesis disorders. This could be mediated by a dual mechanism. First, PBDE-99 exposure increases the ROS levels and activates the ERK1/2
Funding
This study was supported by the Guangdong Basic and Applied Basic Research Foundation [Grant number 2020A1515110796]; the National Natural Science Foundation of China [Grant number 81803320]; the Key-Area Research and Development Program of Guangdong Province [Grant number 2019B020227001]; the Guangdong Provincial Key Laboratory of Research in Structural Birth Defect Disease [Grant number 2019B030301004]; the Guangzhou Institute of Pediatrics [Grant number YIP-2019-036]; and the Guangzhou
CRediT authorship contribution statement
Tianxin Zhao: Conceptualization, Funding acquisition, Investigation, Visualization, Writing – original draft. Xiangliang Tang: Investigation, Data curation, Visualization, Software. Dian Li: Methodology, Software. Jinglu Zhao: Methodology, Visualization. Rui Zhou: Investigation, Validation, Methodology. Fangpeng Shu: Investigation, Data curation. Wei Jia: Methodology, Validation. Wen Fu: Investigation, Validation. Huimin Xia: Conceptualization, Investigation. Guochang Liu: Conceptualization,
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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