Oxidative Stress, DNA Damage And The Expression Of DNA Methyltransferase In Utero After Mice Exposed To Carbon Disulfide At Peri-implantation

BackgroundCarbon disulfide (CS2), a volatile organic solvent, is often used in various industrial processes, such as vulcanizing rubber, fumigating grain, oil extraction and manufacturing viscose rayon fibers. CS2has multisystem toxicity and long-term exposure to low concentrations of CS2can cause neurological, cardiovascular, psychological and hormonal effects. Furthermore, it is noteworthy that wide use of CS2is associated with various types of reproductive disturbance. CS2could induce atrophy of testis, dyszoospermia, sexual disorder, decline of sex hormone and decrease in semen quality. Many studies have shown that CS2could induce menstrual disorders and abnormal labor, such as spontaneous abortion and premature birth among female employees. In a prospective cohort study, we found that the incidence of clinically unrecognized pregnancy loss increased markedly, and the time-to-pregnancy was extended in the women employees exposed to CS2. We also found that the number of implanted embryos significantly decreased when mice were exposed to CS2at the phase of embryo implantation. These results suggested that CS2could disturb embryo implantation and induce embryo loss, but the mechanism was still unclear.Oxidative stress induces lipid peroxidation, DNA damage and the structural and functional change of protein. It was reported that the reduced levels of antioxidant increased the risk of spontaneous abortion. Exposure to CS2could cause oxidative stress. Our previous study revealed that CS2exposure could induce DNA damage in the endometrial cells of mice at the phase of implantation. DNA methylation involved in the formation of the embryo before implantation, growth and development and DNA methyltransferase participated in this process. Oxidative stress could change the status of DNA methylation. Therefore, we hypothesized that oxidative stress, DNA damage and DNA methylation were a single or joint participation in disturbing embryo implantation after CS2exposure. In the present study, oxidative stress, DNA damage, the oxidative DNA damage product8-hydroxy-2’-deoxyguanosine (8-OH-dG) and DNA methyltransferase (DNMT) were observed and evaluated with time series to explore the mechanism of CS2-induced embryo loss.ObjectiveCS2exposure was on gestational day3(GD3), GD4, GD5and GD6, separately, and the number of embryonic day9(E9) mouse embryos was obtained. DNA damage of endometrial cells, oxidative stress,8-OH-dG and DNA methyltransferase level in uterus tissues were tested with time series at different end points after exposure. The aim of the present study was to investigate the effect of oxidative stress, DNA damage and DNA methylation on embryo implantation after mice exposed to CS2at peri-implantation and to provide scientific basis for the protection of the health of the occupational employees.MethodsStudy design:The experiment was divided into three parts. Part1was designed to establish the animal model of CS2exposure at different time of peri-implantation in order to find the sensitive exposure time, and to determine the association between embryo implantation and oxidative stress. Part2and Part3were to evaluate the levels of DNA damage,8-OH-dG and DNMT with time series at the sensitive exposure time obtained from part1.Animal treatment:Pregnant mice received a single dose intraperitoneal injection of CS2or olive oil at the designed exposure time (GD3, GD4, GD5and GD6). CS2dissolved in olive oil was631.4mg/kg body weight,0.4median lethal dose (LD50), based on the data from the acute toxicity test for female Kunming mice in our previous research. The injection volume was0.1ml/10g body weight. Mice in group1were exposed on GD3, group2on GD4, group3on GD5and group4on GD6, respectively. There were a total of26end points in four groups. The end points were introduced in detail as follows. Eight end points in group1were designed at6h,12h,18h,24h,48 h,72h,96h and144h (GD9) after exposure. Seven end points in group2were at6h,12h,18h,24h,48h,72h and120h (GD9). Six end points in group3were at6h,12h,18h,24h,48h and96h (GD9). Five end points in group4were at6h,12h,18h,24h and72h (GD9) after exposure.Sample collection:The mice for the last end point (GD9) in all groups in part1were weighed and killed on GD9. The utero weight on GD9was harvested and weighed, and the number of embryonic day9(E9) mouse embryos for each litter was counted. Uterus, ovary, liver, spleen and kidney were isolated and weighed and then frozen at-80℃in a refrigerator for further analysis. The mice in other end points, except for the last one in all groups in part1, were killed at the designed end points. Half of each uterine tissue was separately frozen at-80℃for quantitating the protein content of the homogenate and testing oxidative stress,8-OH-dG and DNMT1levels. The endometrium of the other half uterine was manually scraped with a flat bamboo stick gently and isolated into a single cell for assessing DNA damage.Detection of oxidative stress, DNA damage,8-OH-dG and DNMT1: Malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-PX) and catalase (CAT) in the uterine tissue homogenate were measured with commercial chemical assay kits. The total protein content was determined with Coomassie blue method assay kit. DNA damage was determined by comet assay. The mouse8-OH-dG ELISA kit was used to measure the8-OH-dG in uterine tissue homogenate. The levels of DNMT1were detected by western blotting.Statistical analysis:Data were shown as mean±SD for each group. Statistical analysis was performed using the SPSS20.0statistical software. The data were first analyzed by the homogeneity test for variance. If variances were equal, one-way analysis of variance was performed followed by Dunnett’s t test if the results were significant. If variances were unequal, statistical differences were evaluated by the Brown-Forsythe test. P<0.05was considered to be statistically significant. Linear correlation analysis was performed in order to observe the correlation between the number of E9mouse embryos and different other parameters.Results1Maternal toxicity and embryo implantationCS2exposure on GD3, GD4, GD5and GD6all failed to induce a statistically significant difference in maternal net body weight on GD9. Furthermore, no statistically significant changes were observed in the weight of the uterus, ovary, liver, spleen and kidney between CS2exposure and solvent control in four groups. Yet, a statistically significant reduction was observed in the number of E9mouse embryos when compared with the control. The rates of embryo implantation were56.95%,36.26%,39.55%and52.74%for CS2exposure on GD3, GD4, GD5and GD6, respectively, when compared with each corresponding control. No statistically significant change was found in the mean weight of conceptus in each CS2exposure when compared with the control.2Oxidative stress after mice exposed to CS2at peri-implantationMDA content as an indicator of lipid peroxidation had a significant increase at18h after CS2exposure on GD3, GD4, GD5and GD6when compared with the control. At18h after CS2exposure, the MDA level increased by131.4%in GD4, which is higher than120.8%,121.6%and104.9%in GD3, GD5and GD6CS2exposure groups when compared with the control. In addition, SOD, GSH-PX and CAT initially resulted in no significant increase, whereas subsequent statistically significant decreases at different time points were observed:SOD at18h, GSH-PX at12h and18h and CAT at12h when compared with each control. The correlation analysis showed a strong negative correlation between MDA levels at18h after exposure and the number of E9mouse embryos in each group (r=-0.783, P<0.01).3DNA damage in mice after CS2exposure at peri-implantationAfter CS2exposure on GD4, the parameters of comet assay such as TL, TM, OTM and TDNA%all showed statistically significant increases when compared with the control in part2. Comet parameters appeared to significantly increase at6h after CS2exposure and reached the top level at18h, and then these comet parameters decreased gradually. The correlation analysis showed a strong negative correlation between the number of E9mouse embryos and TM, OTM, TL and TDNA%after CS2exposure at18h (r=-0.804,-0.847,-0.934and-0.863, respectively, P<0.01).4The level of8-OH-dG in uterine tissue after CS2exposure at peri-implantationStatistically significant increases in the8-OH-dG were observed at18h and24h after exposure when compared with the control in uterine tissue. The increased rate of the8-OH-dG level was893.8%at18h and647.4%at24h after exposure when compared with the control. 5The expression of DNMTl in uterine tissue after mice exposed to CS2at peri-implantationAfter CS2exposure on GD4, the expressions of DNMT1showed significant difference between the exposed group and the control group at6h and12h (P<0.01). The correlation analysis showed a positive correlation between the expression of DNMT1at6h after exposure and the number of embryo implantation (r=0.379, P<0.05); a negative correlation between the expression of DNMT1at12h after exposure and the number of embryo implantation (r=-0.433, P<0.01).6The relationship between oxidative stress, DNA damage and8-OH-dG levelTDNA%, TL, TM and OTM showed statistically significant difference at6h after exposure, whereas MDA showed a statistical increase at18h after exposure. MDA change that indicated oxidative stress occurred after DNA damage. Then TDNA%, TL, TM, OTM and MDA all showed a decrease at24h after exposure. The correlation analysis showed a strong positive correlation between the8-OH-dG level and TDNA%, TL, TM and OTM (r=0.766,0.688,0.738and0.771, respectively, P<0.01).Conclusions1Exposed to CS2at peri-implantation induced obvious and transient oxidative stress, and oxidative stress showed a definite time variation. The same time-dependent trend was observed whenever the exposure happened at peri-implantation.2Exposed to CS2at peri-implantation directly induced DNA damage, and the DNA damage of endometnal cells showed a definite time variation. In addition, DNA damage of endometrial cells was enhanced by oxidative DNA damage.3The level of oxidative stress in uterine tissue and DNA damage of endometrial cells was consistent with the degree of embryo loss, both of which might be responsible for CS2-induced embryo loss.4Exposed to CS2at peri-implantation disrupted the expression of DNMT1induced by oxidative stress and DNA damage, which was associated with the regulatory mechanism of the endometrial receptivity.