Pesticides have adverse effects on cells. A study systematically tested the cytotoxic potential of 13 commonly used agricultural pesticides on three human HepG2, Hek293, HeLa cells, and three insect Tn5B1-4, Sf-21, and Drosophila S2 cells and found that some pesticides promoted cell proliferation, while some pesticides have a significant inhibitory effect on cell viability (Yun, Huang et al. 2017). They showed that even 24h exposure to methomyl at 20 μg/ml produced little cytotoxicity to the tested human and insect cells (Yun, Huang et al. 2017). However, human umbilical vein endothelial cells exposed to methomyl for 24 h exhibited significant proliferation inhibition at higher concentrations (500 and 1000 μM) (Saquib, Siddiqui et al. 2021). It is suggested that different cells have different susceptibility to the same insecticide. Folic acid, a member of the vitamin B9 family, is essential for the biosynthesis of nucleotides, amino acids, and S-adenosyl-l-methionine (Li, Ma et al. 2019, Koohpeyma, Goudarzi et al. 2020, Wusigale, Hu et al. 2020). Animal studies have demonstrated that methomyl reduces testosterone levels and impairs sperm quality in male rats, while folic acid improves methomyl's impairment of reproductive performance (Shalaby, El Zorba et al. 2010, Sakr, Hassanien et al. 2018). In this study, we found that 500 and 1000 μM methomyl significantly increased cytotoxicity after 24h in vitro treatment of GC-1 spermatogonia, TM4 Sertoli cells, and TM3 Leydig cells, while folic acid attenuated the testis cytotoxicity of methomyl in a dose-dependent manner.
The testis is composed of seminiferous epithelium and mesenchyme (Hai, Hou et al. 2014). The former is a unique site of spermatogenesis, mainly composed of spermatogonia and Sertoli cells (Wen, Tang et al. 2016), while the latter is the place where mesenchymal cells synthesize and secrete androgens (Zhou, Wu et al. 2019). It is known that spermatogenesis begins with the proliferation of spermatogonia. Spermatogonia continue to self-renew and proliferate throughout adult life to maintain stem cell reserves while providing cells for the spermatogenesis cycle (Manku and Culty 2015). Therefore, the proliferation and apoptosis of spermatogonia are crucial for spermatogenesis. In the testis, the blood-testis barrier composed of a variety of structural proteins (such as TJP1, Cx43, N-cadherin, Occludin, E-cadherin, etc.) between adjacent Sertoli cells, can maintain normal spermatogenesis by participating in the construction of the spermatogenic microenvironment, providing an immune barrier, and conferring cell polarity in the seminiferous epithelium (Cheng and Mruk 2012). Meanwhile, steroidogenesis in Leydig cells involves the conversion of cholesterol to testosterone through several intermediate steroids, a process regulated and catalyzed by P450scc, StAR, Hsd3b1, CYP17a1, and HSD17b1 (Hong, Chen et al. 2016, Zhao, Xiao et al. 2021). We found that in spermatogonia, methomyl at 1000 μM significantly inhibited the expression of proliferation genes Ki67 and PCNA, and increased the expression of apoptosis genes Caspase3 and Bax at various doses, while folic acid could reverse the changes in related gene expression caused by methomyl; In Sertoli cells, methomyl inhibited the expression of blood-testis barrier function genes TJP1, Cx43, N-cadherin in a dose-dependent manner, but did not affect the expression of Occludin and E-cadherin, while folic acid significantly up-regulated the expression of TJP1, Cx43, and Occludin; in Leydig cells, methomyl dose-dependently inhibited the expression of steroid synthase P450scc, StAR, Hsd3b1 and down-regulated the level of testosterone, but did not affect Cyp17a1 and Hsd17b1, while folic acid could significantly up-regulate the expression of P450scc, StAR, Hsd3b, Cyp17a1, and Hsd17b1, and reverse testosterone level downtrend. All in all, Methomyl can damage the cell function of testicular spermatogonia, Sertoli cells, and Leydig cells, while folic acid can reduce this damage.
Epigenetic information encoded by 5-methylcytosine (5mC) plays a key role in mammalian development and human disease. De novo production and maintenance of 5mC is regulated by DNA methyltransferases, namely DNMT1, DNMT3A, and DNMT3B. DNA methylation in mammals is thought to be critical for essential functions, including gene silencing leading to genomic imprinting, X chromosome inactivation, and repression of transposable elements (Liu, Liu et al. 2020). Folic acid is known to be critical in the process of supplying methyl groups for methylation in one-carbon metabolism, and low folate status is associated with an increased risk of cardiovascular disease, various cancers, and neural tube defects (Crider, Yang et al. 2012). Studies have found that folic acid can restore hepatic triglyceride accumulation induced by a high-fat sucrose diet by altering the methylation pattern of fatty acid synthase promoter (Cordero, Gomez-Uriz et al. 2013), inhibiting amyloid β-peptide production by modulating the activity of DNMTs in N2a-APP cells (Li, Jiang et al. 2015), and also alleviate oxidative stress-induced apoptosis in vivo and in vitro by increasing DNMTs activity and VPO1 promoter methylation level (Luo, Zhang et al. 2013). Thus, by altering DNA methylation, the use of folic acid can be used as a treatment modality for many diseases. In this paper, we found that methomyl did not affect the expression of Dnmt1, Dnmt3A, Dnmt3B genes in testicular cells, while folic acid dose-dependently up-regulated the expression of Dnmt1, Dnmt3A and Dnmt3B in spermatogonia, Dnmt1, and Dnmt3B in Sertoli cells, and Dnmt1 and Dnmt3A in Leydig cell. It is suggested that folic acid attenuates the damage of methomyl to testicular cells by changing the DNA methylation environment.