1,2-dichloroethane Induces Reproductive Toxicity Mediated By The CREM/CREB Signaling Pathway In Male NIH Swiss Mice

Objective1,2-Dichloroethane（1,2-DCE）is an organic solvent.1,2-DCE is currently widely used in industry because of its good performance and low price.It can easily enter the human body through a variety of ways and causes occupational poisoning.The current research mainly focuses on the toxic effects of 1,2-DCE on the nervous system,liver and kidney system and respiratory system.At present,there was little studies on male reproductive toxicity caused by 1,2-DCE exposure,and the mechanism of poisoning is unknown.In order to reveal the male reproductive toxicity induced by 1,2-DCE and to elucidate its potential toxicity mechanism,this study aimed to simulate 1,2-DCE occupational exposure through animal models of inhalation exposure and to explore the male reproductive toxicity caused by 1,2-DCE and its pathogenic mechanism.Methods1.1,2-DCE exposureExposure to 1,2-DCE was performed using a systemic inhalation method.Two hundred specific pathogen free healthy 7-weeks-old male NIH Swiss mice were randomly divided into control and low-dose,medium-dose,high-dose group.The mice were exposed to 1,2-DCE at dosage of 0,100,350,700 mg/m³ for 6 hours per day for continuous exposure to 7 days a week by dynamic inhalation.The experiment is divided into three parts.1)80 mice were used for 1,2-DCE kinetic experiments which were collected for blood and urine concentrations detected;2)60 mice for 1-week exposure experiments;3)60 mice for4-weeks exposure experiments.During the experiment,the temperature,humidity,oxygen,carbon dioxide,ammonia and aerosol particle sizes in the cabinet were monitored daily.2.Detection of 1,2-DCE subacute male reproductive toxicityAfter exposure,the weight of male NIH mice in each group were counted,and the testis organ coefficients were weighed and calculated.Pathological examination of testis and epididymis tissue.Routine parameters,abnormal rate and sperm cell damage were detected in epididymal spermatozoa.The levels of testosterone-related hormones in the plasma and testicular tissue homogenate of mice were analyzed by ELISA kits.3.Molecular mechanisms of 1,2-DCE-induced male reproductive toxicityQuantitative real-time PCR and western blot were used to detect the expression levels of testosterone synthesis pathway,CREM/CREB signaling pathway and testicular apoptosis signaling pathway related genes and proteins,respectively.TUNEL fluorescence assay was used to quantify the level of testicular germ cell apoptosis,and TUNEL colorimetric assay with DAB reaction was used to qualitatively analyze the testicular apoptosis cells and stage specificity.Results1.1,2-DCE exposureThe concentrations of 1,2-DCE in the control group and the low,middle and high-dose groups were 0.30±0.10,102.70±7.05,356.04±20.45 and 707.01±39.33 mg/m³ respectively during the exposure period.There was no significant difference in experimental quality control data（P>0.05）.2.Male reproductive toxicity induced by 1,2-DCE subacute exposureAt 4^th week,the high-dose mice showed a significant decrease in body weight with increased testis/body weight ratio,vacuolar degeneration of germ cells in the seminiferous tubules of testes,and the sperm in the epididymis decreased after exposed to 1,2-DCE.The sperm concentration of high-dose group decreased in the 1^st week and the 4^thh week（P<0.05）.Moreover,the sperm abnormal rate of the low-,middle-,high-dose groups were increased（P<0.05）.The levels of testosterone and luteinizing hormone in plasma and testicular tissue in the medium-and high-dose groups increased in the fourth week of exposure（P<0.05）.Gonadotropin-releasing hormone and cyclic adenosine monophosphate in testis increased at the 4^th week of exposure（P<0.05）.3.Molecular mechanisms under 1,2-DCE-induced male reproductive toxicityThe expression of luteinizing hormone receptors mRNA and proteins in the low-,middle-and high-dose groups increased at the 4^th week after exposure（P<0.05）.1,2-DCE exposure inhibits CREM/CREB signaling pathway in mice testis.CREB and CREM were both significantly inhibited by 1,2-DCE.This is consistent with the declines in the TORC1and ACT,which results in the decrease in LDH-C and TESK1 in the testes（P<0.05）.Moreover,the activation of p53 and Bax with the inhibition of Bcl-2 might be the reason for the upregulation of caspase 3in the apoptosis,as detected by TUNEL assay in the testes induced by1,2-DCE.Conclusions1.1,2-DCE subacute exposure can cause reproductive toxicity in male NIH mice.2.1,2-DCE inhibits CREM/CREB signaling cascade and subsequently induces apoptosis associated with p53 activation and mitochondrial dysfunction.This also results in induced malformation of spermatozoa,reduced sperm concentration and pathological impairment of the testes.