| BackgroundCarbon disulfide (CS2), one kind of volatile organic solvents as well as raw material, is widely used in various industrial processes, such as thiolation of viscose fiber, fumigating grain, oil extraction, and manufacturing viscose rayon fibers. CS2 is existent in the workshop in the productive process due to its volatility. In the production of viscose rayon fibers, a large number of female employees are occupationally exposed to CS2.CS2 is one kind of multi-system poisons, and has effects on nervous system, cardiovascular system, reproduction, liver and kidney. The studies on the reproductive toxicity of CS2 indicated that CS2 exposure could induce sexual disorder, atrophy of testis, dyszoospermia, quality of semen decreased and rate of teratospermia increased significantly in male employees. CS2 exposure also induced menstrual disorder, dysmenorrheal, menostaxis, pleonemia and abnormal labor, such as spontaneous abortion and premature birth among female employees. Our previous work showed that, the time-to-pregnancy was longer for women who intended to be pregnant in the exposed group than the unexposed women. And among the women who intended to be pregnant, the incidence of the embryo loss in clinically unrecognized pregnancy loss was 48.7% in the women who exposed to CS2, whereas it was only 26.3% in unexposed group by detected the level of human chorionic gonadotrophin in the urine of them. But the mechanism of toxic action of CS2 was yet unclear. Many researches showed that the maternal immune tolerance to fetus was the key in maintaining normal pregnancy, in which lymphocytes and their cytokine production profiles played an important role. But there were few studies on the damage of maternal-fetal immune tolerance induced by CS2 exposure.ObjectiveTo observe the effects of CS2 exposure on the maternal immune tolerance and to analyze the relationship between the status of immune tolerance and embryo implantation after mice exposed to CS2 in the phase of implantation in order to explore the mechanism of embryo implantation failure of mice exposed to CS2 and to provide scientific basis for the protection of the health of the occupational employees and future generation.Contents1. Mice were exposed CS2 at different dosages of CS2 or at different time-points during the phase of implantation in order to observe the maternal toxicity and embryotoxicity induced by CS2.2. Mice were exposed CS2 at different dosages of CS2 or at different time-points during the phase of implantation in order to detect the DNA damage of lymphocytes in spleens by single cell gel electrophoresis (SCGE).3. Mice were exposed to CS2 at different time-points during the phase of implantation, and the levels of Thl-type cytokines interleukin-2 (IL-2) and interference-γ(IFN-γ) and Th2-type cytokines IL-4 and IL-10 in uterus tissue were detected by enzyme-linked immuno sorbent assay (ELISA).Methods1. Laboratory animals and grouping Sexually matured virgin female Kun-Ming mice (aged 8-10 weeks, weighted between 28-32g) were mated with mature male mice (weighted between 36-40g), and the pregnant mice were divided into groups which consist of exposed groups and control groups according to their body weight.2. Ovulation inductionsEach female mouse was administered intraperitoneally with lOu PMSG firstly and with 10u HCG 48h later.3. Exposure to CS23.1 Exposed to different dosages of CS2:0.1 LD50 (157.8mg/kg),0.2 LD50 (315.7mg/kg) and 0.4 LD50(631mg/kg).3.2 CS2 exposure occurred on the third day (GD3, in abbreviated form) or on GD4, GD5, GD6, and the experiments were terminated at GD4, GD5, GD6, GD7, GD9, respectively.3.3 Exposure was only one time.3.4 Exposure treatment:Administration route was intraperitoneal injection. The volume for injection was O.lml/lOg body weight. Mice in the control group were injected with the olive oil which was the solvent of CS2.4. Experiment methods4.1 The experiment of embryotoxicity:Mice were treated by designed exposure dosages and time-points and were sacrificed by cervical dislocation on GD9. The livers, spleens, kidneys, uteri, ovaries and conceptuses were harvested and weighed, and the number of implanted conceptus was counted.4.2 The experiment of DNA damage of lymphocytes:Mice were sacrificed by cervical dislocation 24h after exposure, the splenic lymphocytes were extracted by lymphocytes separation medium, cell viability was detected, and DNA damage of lymphocytes was detected by SCGE.4.3 The experiment of expression of Thl/Th2 cytokines:Mice were sacrificed by cervical dislocation on designed end time-points. Uterus tissues were homogenized manually and the content of total protein was assayed by BCA (bicinchoninic acid) method. Then, the levels of Thl-type cytokines IL-2 and IFN-y and Th2-type cytokines IL-4 and IL-10 were detected by ELISA.5. Statistical analysesResults were analyzed by a computerized statistical program (SPSS 16.0). Values of the results are presented as means±standard deviation. The variances between groups were analyzed by Homogeneity-of-variance test first. If the variances were equal, the data was analyzed by analysis of variance (ANOVA), and then the difference between exposure and control groups were determined by Dunnett t test; if not, the data was analyzed by nonparametric Kruskal-Wallis test, and the difference between groups were determined by Mann-Whitney U test. Differences were considered statistically significant if P<0.05.Results1. Embryotoxicity of mice induced by CS2 exposure during the phases of implantationEmbryotoxicity:①After the exposure to CS2 at different dosages during the phase of implantation, the number of embryo, total weight of uteri and embryo and total weight of embryo in 0.4LD50 exposure group were decreased obviously by 79.03%, 75.53% and 90.67% respectively (P<0.05), but there was no significant difference on the mean weight of conceptus after adjusting the number of embryo (P>0.05).②After the exposure to CS2 at different time-points during the phase of implantation, the number of embryo and total weights of embryo in GD4 and GD5 exposure groups were decreased obviously (P<0.05), but there was no significant difference on the mean weight of conceptus after adjusting the number of embryo (P>0.05).Maternal toxicity:After the exposure to CS2 at different dosages or different time-points during the phase of implantation, there were no statistically significant differences on the bodyweight gain and indicators of livers and spleens in mice.2. DNA damage of lymphocytes in mice spleen induced by CS2 exposure during the phases of implantation①After the exposure to CS2 at different dosages during the phase of implantation, the indicators of single cell gel electrophoresis were statistically significant different among 0.2LD50 and 0.4LD50 exposure groups on HDNA%, TDNA%, Tail Length, Comet Length, Tail Moment and Olive Tail Moment, compared with those in the control and 0.1LD50 exposure groups (P<0.05).②fter the exposure to CS2 at different time-points during the phase of implantation, the indicators of single cell gel electrophoresis were statistically significant different among GD4, GD5 and GD6 exposure groups on HDNA%, TDNA%, TL, CL, TM and OTM, compared with those in the control group (P<0.01).3. Abnormal expressions of Thl/Th2 cytokines in mice uterus tissue induced by CS2 exposure during the phases of implantation①Expression of Thl-type cytokines IL-2 and IFN-γ:Compared with the control group, the levels of IL-2 at GD5 end point in GD3 exposure group and at GD5 and GD9 end points in GD4 exposure group were increased; the levels of IFN-y at GD7 and GD9 end points in GD4 exposure group were increased (P<0.05).②Expression of Th2-type cytokines IL-4 and IL-10:Compared with the control group, the level of IL-4 at GD4 end point in GD3 exposure group was decreased; the level of IL-10 at GD6 end point in GD5 exposure group was decreased; the levels of IL-4 and IL-10 at GD9 end point in all exposure groups were decreased (P<0.05).③The ratio of Thl/Th2 cytokines:Most of the ratios of Thl/Th2 cytokines at each end point in all exposure groups were increased (P<0.05).Conclusions1. Exposure to CS2 during the phase of implantation could induce obvious embryotoxicity. The numbers of embryo in 0.4LD50 exposure group and in GD4 and GD5 exposure groups were decreased, which indicated that 0.4LD50 (631mg/kg) was the sensitive dosage and GD4 and GD5 were the sensitive time-points for CS2 toxic activity.2. Exposure to CS2 during the phase of implantation could induce DNA damage of lymphocytes in mice spleen, and the degree of damage was aggravated with the increase of CS2 concentration. These indicated that CS2 exposure at the phase of implantation could interfere with the maternal immune tolerance to embryo by damaging the DNA of lymphocytes.3. Exposure to CS2 during the phase of implantation could interfere with the maternal immune tolerance to embryo by affecting the expression of Thl-type and Th2-type cytokines, which was attribute to the DNA damage of lymphocytes, and this might be one of important mechanism for embryo implantation failure. |
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