DNA Damage And Apoptosis Of Mice Endometrial Cell In Implantation Phase Induced By Carbon Disulfide Exposure

BACKGROUNDCarbon disulfide (CS2), a volatile organic solvent, is widely applied in various industrial processes, such as thiolation of viscose fiber, fumigating grain, oil extraction, and manufacturing viscose rayon fibers. It is worth noting that many workers in the developing countries still are exposed to CS2. In China, a large number of female employees are exposed to CS2 in the industry of the production of synthetic fibers.We all know that CS2 can mainly influence the central and peripheral nervous systems, and exposure to CS2 can damage blood vessels. Furthermore, it is noteworthy that the wide use of CS2 is associated with various types of reproductive disturbance. Many studies have focused on the relationship between exposure of CS2 and male reproductive health. The evidence for the adverse effects of occupational and environmental exposures to CS2 on male reproduction was strongly supported in well-designed epidemiological studies. CS2 could induce atrophy of testis, dyszoospermia, sexual disorder and decline of sex hormone, and quality of semen decreased significantly in workers exposed to CS2. Previous researches reported that CS2 induced menstrual disorders and abnormal labor, such as spontaneous abortion and premature birth among female employees. Some studies showed that female workers exposed to CS2 had a higher risk of early pregnancy loss. Our previous work showed that, among female employees who intended to be pregnant, the incidence of clinically unrecognized pregnancy loss was observable increase. And the time-to-pregnancy was longer for the women under exposure. It was indicated that the process for implantation of embryo might be influenced after women's exposure to CS2. The embryotoxicity of CS2 and its sensitive points were found through the animal experiments. Embryo implantation is sensitive time-point for embryotoxicity.OBJECTIVEEstablish the animal models of implantation embryo development and embryo implantation to detect the damage of endometrium cells induced by CS2, to analyze the embryotoxicity of CS2 and the relationship between the extent of DNA damage and apoptosis and the intensity of CS2 exposure, in order to provide scientific basis for the protection of the health of the occupational employees and future generation.METHODSLaboratory animals and grouping:Sexually-matured Kunming mice in sterile grade,8-12 weeks of age, approximate 27g to 30g for females and 30g to 35g for males, were fed on free diet and under natural light. The males were caged separately and the females were divided randomly into 4 groups based on their weights.Ovulation induction:the female mice were injected through intraperitoneal route with 10u PMSG, and 10u hCG 48h later. The males and females were placed 1:1 overnight to impregnate. Mating was evidenced by the appearance of a vaginal plug on the following morning. Plugged females designated day 1 of pregnancy (recorded as D1). In vitro:Single cell suspension was treated together with a series of concentrations from 0 to 2500μmol/L of CS2 for 1h. In vivo:Based on the obtained from the female mice through intraperitoneal injection in our previous research, the dosage was designed to be 0.4LD50(631mg/kg),0.2LD50,0.1LD50. The injection volume was 0.1ml/lOg. Administration route was intraperitoneal injection. CS2 was specially prepared prior to the administration on the basis of the weights of the mice to be treated, with the olive oil being the solvent. Mice in the control group were injected with olive oil with body weight-dependent volumes.The DNA damage was determined by SCGE. And the apoptosis rate was determined by flow cytometry stained with Annexin V-FITC/PI and TUNEL.Statistical treatment:SPSS16.0 statistical software was used for statistical treatment. Variances were evaluated by Homogeneity-of-variance test first. If variances were equal, statistical treatment were performed with factorial analysis of variance (ANOVA), followed by LSD's post hoc tests. If variances were unequal, statistical differences were evaluated by Brown-Forsythe test. Statistical significance was determined at level of a=0.05.RESULTS1. CS2 exposure induced embryotoxicity in phase of implantationEmbryotoxicity:in 0.1LD50, 0.2LD50 and 0.4LD50 exposure groups, the number of implantation was decreased by 32.3%,65.1% and 67.4%; Total weight of uteri and embryos was decreased by 0.3%,57.9% and 71.9%; Total weight of embryos was decreased by 54.7%,68.2% and 85.4% respectively (P<0.05). But compared with control group, the mean weight of embryos was not obvious decreased (P>0.05).Maternal toxicity: The CS2 exposure could lead to the statistically significant differences on the bodyweight gain and the weight of liver in all of pregnant mice group and pregnant mice without embryo implantation group (P<0.05). The results mentioned a little of maternal toxicity.2. DNA damage of mice endometrial cell in phase of implantation induced by CS2In vitro:The index of single cell gel electrophoresis was statistically significant different among different dosages of CS2 on Head DNA%, Tail DNA%, Tail Length, Comet Length, Tail Moment and Olive Tail Moment, compared to the control respectively. The result of regression analysis showed that regression coefficient between Head DNA%, Tail DNA%, TL, TM, OTM and the doses were-13.78,13.78, 0.05,4.38 and 3.23, respectively (P<0.001).In vivo:DNA damage of implantation mice endometrial cells:The index of single cell gel electrophoresis was statistically significant different among different dosages of CS2 on Head DNA%, Tail DNA%, Tail Length, Comet Length and Tail Moment, compared to the control respectively. OTM in 0.2LD50 and 0.4LD50 dosages groups increased 5.88 and 9.21, respectively (P<0.001). Regression analysis showed that regression coefficient between Head DNA%, Tail DNA%, TL, TM, OTM and the doses were-0.021,0.021,0.014,0.043 and 0.062 (P<0.001).3. Apoptosis of mice endometrial cell in phase of implantation induced by CS2Apoptosis of implantation mice endometrial cells:The apoptosis rate was determined by flow cytometry stained with Annexin V-FITC/PI, and in 0.2LD50 and 0.4LD50 dosages groups, the apoptosis rate increased 89.26% and 274.50%, respectively (P<0.05). The apoptosis rate was determined by TUNEL, and the apoptosis rate in 0.2LD50 and 0.4LD50 dosages groups increased 89.9% and 121.8%, respectively (P<0.05). But the apoptosis rate in 0.1LD50 was no statistical significance between the two detection methods.CONCLUSIONS1. CS2 exposure could induce significant embryotoxicity, such as reduce the number of embryo implantation, decrease the total weight embryo, and result in pregnancy loss.2. CS2 exposure could induce DNA damage and apoptosis of implantation mice endometrial cells, the damage of endometrial cells was aggravated, with the increase of CS2 concentration.