Reproductive Toxicity of Endosulfan: Implication From Germ Cell Apoptosis Modulated by Mitochondrial Dysfunction and Genotoxic Response Genes in Caenorhabditis elegans.

Endosulfan, as an organochlorine pesticide (OCP), has been listed as a persistent organic pollutant (POP) by the Stockholm Convention and phased out termination of all manufactures and uses globally (UNEP-POP, 2011). However, the residues of endosulfan have been found ubiquitously in geographical regions ranging from temperate environments to the Arctic and various foods (Weber et al., 2010). These findings implied that a large number of ecosystem organisms and field workers may be potentially exposed to endosulfan via food chain and occupational routes. There have been growing concerns about the reproductive toxicity of endosulfan to various species in recent years (Rastogi et al., 2014). Exposure to endosulfan significantly reduced the brood size and caused embryo abnormalities in Daphnia magna (Palma et al., 2009). It could cause an 80% reduction in progeny production and the change of sex ratio in fighting fish Betta splendens (Balasubramani and Pandian, 2014). The exposure of endosulfan to pregnant rats not only increases fetal resorption and induces gross fetal anomalies but also decreases spermatogenesis in offspring (Milesi et al., 2012). It should be noted that the reproductive toxicity of endosulfan has been variable in different experiments and may be the result of the types of endosulfan used, different experimental animal species, age at exposure, doses, duration of exposure, or the nature of the biological end points. Thus, there is an urgent demand for a comprehensive study to illustrate the reproductive dysfunction of endosulfan in intact organisms. Apoptosis is a common programmed event in the development of reproductive system, which has critical functions in maintaining appropriate germ cell to Sertoli cell ratio, removing defective germ cells, and controlling the sperm production (Shukla et al., 2012). Endosulfan has been reported to induce apoptosis in Sertoli and Leyding cells of rabbit testis and cell death in Sertoli-germ cells in male rats (Rastogi et al., 2014). However, little is known about the underlying mechanism of germ cell apoptosis induced by endosulfan. Oxidative stress has been shown to play a pivotal role in endosulfan toxicity in multiple organs including the brain, liver, kidney, and testis (Wu et al., 2012). The antioxidant compounds, such as vitamins C and E, have shown to ameliorate the sperm and testicular toxicity of endosulfan in male rats (Takhshid et al., 2012). Moreover, endosulfan-induced mitochondrial membrane depolarization, resulting in the increase of reactive oxygen species (ROS) and apoptosis in rat testis (Aly and Khafagy, 2014; Sohn et al., 2004). These data suggest that ROS induced by endosulfan may further interact with the genetic material and cause germ cell DNA damage leading to abnormal transmission of genetic information. Among in vivo animal models, Caenorhabditis elegans is a powerful genetic model to explore environmental toxicology and human biological mechanisms (Leung et al., 2008). The transparent cuticle allows observing and distinguishing germ cells at organism level. The genome of C. elegans showed a high level of conservation with vertebrates (Cutter et al., 2009); approximately 60–80% of C. elegans’ genes are conserved with humans (Kaletta and Hengartner, 2006). The C. elegans germline apoptosis during oogenesis is a fundamentally important reproductive process and is evolutionarily conserved from worms to mammals, including humans. The validity of using C. elegans to study genes and pathways related to human health and disease has been well established by dissecting the pathway controlling apoptosis (Lettre and Hengartner, 2006). In addition, C. elegans is an ideal animal model for the study of ecotoxicology due to its abundance in ecosystems and its properties of short life cycle, small size, plenty of offspring, and ease of handling in laboratory (Riddle et al., 1997). Among the two types of isomers (α-: 64–67%; β-: 29–32%), α-endosulfan is supposed to be more poison and has higher soil permeability (Wan et al., 2005). In this study, the adverse effects of endosulfan on the reproductive system were assessed by the fecundity, the hatchability, germ cell apoptosis, and cell cycle arrest. Because oxidative stress has been implicated as a probable mechanism involved in the reproductive toxicity of endosulfan (Silva and Gammon, 2009), we speculated that the mitochondria which is the main source oxyradicals in vivo might mediate the germ cell apoptosis induced by endosulfan. To further explore the mechanism of endosulfan-induced toxicity, the highly conserved genotoxic stress response genes were determined by C. elegans mutants exposed to endosulfan. In the meantime, dichloro-diphenyl-trichloroethane (DDT) and lindane were included in the study for comparison with endosulfan as two other organochloride pesticides. Our results showed that endosulfan reduced the fecundity and hatchability in C. elegans. Moreover, the germ cell apoptosis induced by endosulfan was regulated by mitochondrial dysfunction and DNA damage response genes in a dose-dependent manner.