Toxicity Of Bisphenol S Exposure To Mouse Leydig Cells And Its Mechanisms Of Action

Objective:On account of restrictions regarding the use of Bisphenol A(BPA),Bisphenol S(BPS),as its main substitute,is employed in numerous industrial applications and consumer products.BPS is ubiquitously present in a variety of environmental matrices,and can enter human body through various routes such as dermal contact,oral intake or respiratory inhalation.Recently,studies have shown that BPS exposure is associated with testosterone deficiency and male reproductive dysfunction.High levels of testosterone in the testes are indispensable for the maintenance of spermatogenesis and male fertility.Endogenous testosterone is primarily produced by Leydig cells in the testis.Considering the important role of Leydig cells in testosterone synthesis and spermatogenesis,this study aimed to explore the toxicity of BPS to mouse Leydig cells and its underlying mechanism,and provided a research basis for further revealing the effects of BPS exposure on mammals,especially human health.Methods:The TM3 cells were cultured to logarithmic growth phase in DMEM/Ham’s F12 medium supplement with 2.5% fetal bovine serum and 5% horse serum.The TM3 cells cultured in vitro were randomly divided into 4 groups,and separately challenged with BPS at doses of 0,100,200 and 400 μM for 48 h.Then the viability of cells was examined by CCK8 method,and the changes of cell morphology were observed via a microscope;The level of reactive oxygen species(ROS)in TM3 cells was detected with DCFH-DA fluorescent probe dyeing;The content of lipid peroxide malonaldehyde(MDA)and the activities of catalase(CAT)and superoxide dismutase(SOD)in cells were tested by visible light spectrophotometry;The openness of mitochondrial permeability transition pore(m PTP)were determined by Calcein AM fluorescent probe;The alterations of mitochondrial membrane potential(ΔΨm)in TM3 cells were detected by JC-1 fluorescent probe;The content of ATP was determined by chemiluminescence;Targeted metabolomics was performed to analyze cellular energy metabolite levels;The expression of autophagy marker protein LC3 B,Beclin1and P62,as well as apoptosis-related proteins BAX,BCL-2 was detected by Western blot;The activity of intracellular Caspase-3 was detected by colorimetric method;The generation of intracellular autophagosome was observed using transmission electron microscope(TEM).Results:(1)The CCK8 detection results showed that BPS concentrations below 100 μM had no significant effect on the viability of TM3 cells.However,exposed to relatively high concentrations of BPS(100,200,400,800 μM)for 48 h,the cell viability decreased in a dose-dependent manner(P<0.05).In the microscopic examination,BPS exposure caused a marked decline in cell density with altered cell morphology.(2)BPS exposure could induce oxidative damage in TM3 cells.BPS treatment significantly increased the production of ROS and MDA in TM3 cells compared with the control group(P<0.05).At the same time,the activities of antioxidant enzymes CAT and SOD were significantly decreased in 200 μM and 400 μM BPS-treated cells(P<0.05).(3)BPS treatment induced mitochondrial dysfunction in TM3 cells.The results of JC-1 fluorescent staining showed that with the increase of BPS concentration,the ratio of red fluorescence to green fluorescence gradually decreased(P<0.05),indicating that BPS exposure caused the collapse of ΔΨm.Meanwhile,BPS treatment elicited excessive opening of m PTP,which was evidenced by a significant decrease in Calcein fluorescence intensity.In addition,BPS incubation prominently suppressed ATP production in a dose-dependent pattern(P<0.05).(4)Targeted metabolomics demonstrated that exposure to an intermediate dose of BPS(200 μM)for 48 h led to a significant alternation of energy metabolism.L-malic acid(LMA)and adenosine monophosphate(AMP)were up-regulated significantly,while thiamine pyrophosphate(TPP),adenosine 5′-triphosphate(ATP),beta-D-fructose 6-phosphate(β-D-F6-P),cis-aconitate,phosphoenolpyruvate(PEP),cyclic AMP(c AMP),D-Glucose 6-phosphate(D-G6-P)and dihydroxyacetone phosphate(DHAP)were down-regulated.These results suggested that BPS might affect the process of glucose metabolism in TM3 cells.(5)BPS exposure induced apoptosis in TM3 cells.Western blot’s results revealed that with the increase of BPS concentration,the expression of anti-apoptotic protein BCL-2 significantly reduced,the expression of pro-apoptotic protein BAX augmented,with a conspicuous decline in BCL-2/BAX protein expression ratio.Additionally,the activity of Caspase-3 was notably elevated in the BPS treatment group with medium dose(200 μM)and high dose(400 μM).(6)Western blot results found that treatment with intermediate and high doses of BPS could up-regulate the expression levels of autophagy marker proteins LC3 B,Beclin1 and P62,as well as the ratio of LC3B-II/LC3B-I.Moreover,TEM examination exhibited that medium and high doses of BPS induced a large amount of undegraded autophagosome accumulation and mitochondrial swelling,suggesting that BPS exposure could lead to autophagy disorder in TM3 cells.Conclusion:BPS exposure in vitro impaired the viability of TM3 cells,induced ROS production,disrupted mitochondrial function,and further lead to apoptosis,autophagy disorder and energy metabolism alternation.As a consequence,TM3 cells may be one of the targets of BPS-induced testicular toxicity,and mitochondrial oxidative stress may be the key mechanism of BPS-induced reproductive toxicity injury.Our findings further highlight the latent hazard of BPS as a substitute to BPA.