Pendimethalin induces apoptosis in testicular cells via hampering ER-mitochondrial function and autophagy☆
Graphical abstract
Possible mechanisms of action of pendimethalin (PDM) in mouse Leydig and Sertoli cells.
Introduction
Recently, various pesticide and industrial chemical residues were detected in remote parts of the globe, such as glaciers in the Arctic region, throwing light on the extent and severity of pesticide contamination (Hermanson et al., 2020). Pendimethalin (PDM) is a nitroaniline herbicide employed for cereal grain crops and leguminous plants. Although the carcinogenic potential of PDM in pancreatic cancers has been reported (Andreotti et al., 2009), it has garnered approval and has been widely used in almost all countries in Europe, America, and Asia for decades, according to the Pesticide Properties Database (PPDB) (Lewis et al., 2016). Depending on the environmental conditions, the half-life of PDM can persist for 12–90 days (Chopra et al., 2015; Vighi et al., 2017). Although the residual quantities of this pesticide may not pose severe toxicity concerns, its levels still require monitoring (Chopra et al., 2015; Jursik et al., 2017; Vighi et al., 2017). Even low concentrations of pesticides that are not gravely hazardous to human health could impinge on early embryo development, depending on various exposure routes (Greenlee et al., 2004). In addition, PDM exerts genotoxic effects on human primary lymphocytes and the liver and kidney tissues of rats (Ahmad et al., 2018; Ansari et al., 2018). PDM also induces significant DNA fragmentation in Chinese hamster ovary (CHO) cells at a concentration as low as 10 μM (Demir et al., 2017). Furthermore, pesticide exposures are now considered infertility-inducing factors in humans, based on results from epidemiological studies (Clementi et al., 2008; Masata et al., 2005; Sengupta and Banerjee, 2014). In South Korea, the use of pesticides in agriculture has decreased since 2001, but there is still a need to perform research regarding the various toxic effects of pesticides (Cha et al., 2014; Choi, 2014). Similarly, there are numerous studies detailing the cytotoxic potential of PDM; however, research on the mode of action of PDM in exerting detrimental effects on testis cells, especially in mammals, remains insufficient.
Male fertility is directly linked to the number of healthy sperms generated from germ cells in the testis (Jones and Lopez, 2013). In general, Sertoli cells offer an optimal environment for the proliferation and maturation of germ cells; a single Sertoli cell can support approximately 30–50 germ cells (Jones and Lopez, 2013). In addition, the Leydig cell population is dependent on Sertoli cell numbers (Rebourcet et al., 2017). Immature Sertoli cells possess active proliferative capacity to secure an adequate-sized pool, since they are rendered non-proliferative in the mature state. Therefore, the factors crucial for fertility include being in a state of good health and possessing a sizeable population of Leydig and Sertoli cells before attaining adulthood (Sharpe et al., 2003). In the present study, we examined the effects of PDM on mouse Leydig (TM3) and Sertoli (TM4) cell lines with regard to: 1) cellular viability and proliferation; 2) calcium regulation and mitochondrial dysfunction; 3) activation of endoplasmic reticulum (ER) stress and autophagy signaling pathway with/without 2-aminoethoxydiphenyl borate (2-APB); and 4) cell cycle arrest and apoptosis. These experiments were employed to determine whether PDM affects the survival of mouse testis cells and the mechanisms through which PDM leads to testicular cell death, focusing on cellular organelles, in particular, the endoplasmic reticulum and mitochondria.
Section snippets
Chemicals
Pendimethalin (Cat No. 36191, PDM) was purchased from Sigma-Aldrich and diluted with dimethyl sulfoxide (DMSO) at a concentration of 20 mM; 2-APB (Cat No. D9754) was purchased from Sigma-Aldrich. Antibodies against Bip (Cat No. sc-13968), Chop (Cat No. sc-7351), and Tuba (Cat No. sc-32293) were purchased from Santa Cruz Biotechnology for Western blot analysis. Ire1α (Cat No. 3294), phosphor-Ulk1 (Cat No. 5869), total-Ulk1 (Cat No. 8054), phosphor-p62 (Cat No. 16177), total-p62 (Cat No. 88588),
Pendimethalin suppressed mouse Leydig (TM3) and Sertoli (TM4) cell proliferation in 2D and 3D cultures
First, we confirmed whether PDM affects the viability and proliferation of mouse testis cell lines. Upon PDM treatment in a dose-dependent manner (0, 5, 10, and 20 μM), the viabilities of TM3 (Leydig) and TM4 (Sertoli) cells were decreased to 67% and 58%, respectively, compared to the vehicle-treated cells (94%–96%) (Fig. 1A and B). Proliferation of TM3 cells was reduced by 50% after 10 and 20-μM PDM treatment (Fig. 1C). Additionally, proliferation of TM4 cells declined to 54% and 38% in
Discussion
TM3 (Leydig) and TM4 (Sertoli) cells are different cell types with different functions, namely hormone production (steroidogenesis) or protecting germ cells (spermatogenesis), respectively. In addition, they display mutually exclusive hormone receptor expression; the luteinizing hormone (LH) receptor in Leydig cells and the follicle-stimulating hormone (FSH) receptor in Sertoli cells (Mather, 1980). Although they have distinct features and functions, these two cell types have a common purpose
Conclusion
Collectively, we demonstrated the adverse effects of PDM on the mammalian testes using mouse Leydig and Sertoli cell lines (Fig. 7). We investigated whether PDM affects the proliferation and apoptosis of TM3 and TM4 cells, focusing on the ER and mitochondria. We further verified that PDM-induced activation of ER and autophagy was mediated by calcium release via IP3R. Although Ire1a and LC3B were not significantly suppressed by 2-APB (a finding that will need to be explained through further
Author statement
Jiyeon Ham: Investigation, Data curation, Writing – original draft, Writing – review & editing. Whasun Lim: Conceptualization, Data curation, Validation, Writing – original draft, Writing – review & editing. Gwonhwa Song: Conceptualization, Methodology, Software, Data curation, Writing – original draft, Writing – review & editing
Declaration of competing interest
The authors have declared no conflict of interest.
Acknowledgement
This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Ministry of Science and ICT (MSIT) (grant number: 2018R1C1B6009048) and suppported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Minisry of Education (grant number: 2020R1A6A3A13074214).
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This paper has been recommended for acceptance by Da Chen.