DNA Damage And The Intactness Of Leukemia Inhibitory Factor Gene In Endometrial Cells Of Pregnancy Mice Exposed To CS₂at Peri-implantation Period

BackgroundCarbon disulfide (CS2) is one kind of volatile organic solvents as well as raw materials and widely used in various industrial processes, such as the manufacture of viscose rayon fibers. CS2exists in the workshop in the process of production due to its volatility, which has multi-system toxicity. Furthermore, in the production process of viscose rayon fibers, a large number of female employees are occupationally exposed to CS2.The previous study in our project team showed that exposed to different doses of CS2could induce the significant reduction of implanted embryos. Meanwhile, it resulted in DNA damage of endometrial cells, which showed a dose-response relationship. In addition, the decrease of implanted embryos was related with the level of DNA damage. In vivo, there exists the repair of DNA damage, which plays a crucial role in life survival and hereditary stability. Until now, the time effect of CS2exposure during the period of embryo implantation on DNA damage of endometrial cells is unclear. Hence, we detect the time variation in level of DNA damage in endometrial cells of mice exposed to CS2, to observe the time effect of CS2exposure during the period of embryo implantation on DNA damage of endometrial cells and reveal the genetic mechanism of embryotoxicity induced by CS2.In addition, the previous results indicated that the decrease of implanted embryos induced by CS2exposure during the period of embryo implantation was related with the decreased expression of protein and mRNA of adhesion molecules and cytokines, especially for Leukemia inhibitory factor (LIF), which was the maker of endometrial receptivity. However, the mechanism for the reduction of protein and mRNA of LIF is unclear.Comet-FISH, the combination of Comet assay and fluorescence in situ hybridization, is the method of detecting the damage and repair of special gene in the single cell level. By far, it is mainly used for detecting the effect of toxicant on tumor suppressor gene TP53. This study employs Comet-FISH to detect the effect of CS2exposure during the period of embryo implantation on the intactness of LIF gene, in order to find the reason for the decreased expression of protein and mRNA for LIF gene and reveal the genetic mechanism of embryotoxicity induced by CS2.ObjectiveTo design the animal model in which mice are exposed to CS2at different time points of the embryo implantation period and observe the effect of CS2exposure on the pregnancy mice and embryos. To detect the time variation of DNA damage and the intactness of LIF gene in endometrial cells at different endpoints of observation, in order to reveal the genetic mechanism of implantation failure induced by CS2.Methods1. Establishment of animal modelSexually-mature8-12-week-old Kunming mice were obtained from the Experimental Animals Production Center, Shandong University (Jinan. China. Batch No. SCXK (LU)20090001). The animals weighed approximately27-30g for females and30-35g for males. They were raised in a specific-pathogen-free (SPF) animal room and maintained under controlled conditions of temperature (20±2℃) and humidity (50%～60%) under a12-h light/dark cycle. Standard laboratory animal feed (purchased from a commercial supplier) and water were provided ad lihitum. All animals had at least1week of acclimatization prior to treatment. After mating procedure, the day of sperm plug detection was designated gestational day0(GDl). All the animal experiment protocols were approved by the institution animal ethics committee and with their prior approval for using the animals. The mice at GD1were randomly assigned into8groups, that is,4exposure groups and4control groups. The exposure time was designed at GD3, GD4, GD5and GD6. and each group was consisted of12mice. The exposure dose was0.4LD50(631.4mg/kg) based on the obtained data from the acute toxicity test for female Kunming mice in our previous research (LD50=1578.5mg/kg), and the injection volume was0.1ml/lOg body weight. CS2was specially prepared prior to the administration. Mice in the control group were injected with olive oil with body weight-dependent volumes. Mice of each group were sacrificed at GD9to weigh the livers, spleens, kidneys, uteri and ovaries and count the number of implanted embryos.We chose the group, in which the number of implanted embryos was significantly decreased, as the target group. Mice were randomly assigned into14groups, that is,7exposure groups and7control groups. The time points of observation were6h,12h,18h,24h,2d,3d and5d after exposure. Mice were sacrificed at each time point of observation, and endometrial cells were collected for use.2. Collection of endometrial cellsUteri were excised and gently washed three times with ice-cold Ca2+, Mg2+-free PBS in5ml hollow Petri dishes. The uteri were cut open longitudinally and added into1ml ice-cold Ca2+, Mg2+-free PBS. The endometrium was manually scraped with flat bamboo stick in sterile gently and was isolated into single cell by repeated mechanical percussion with pipet which was confirmed by the microscope view. Cell viability was detected by trypan blue staining and the number of cells was counted by automatic cell counter (Invitrogen, USA). Cell density was adjusted into2*105～4*105cells/ml by ice-cold Ca2+, Mg2+-free PBS for comet assay and flow cytometry.3. Detection of DNA damage in endometrial cellsThe comet assay was carried out with slight modifications of the standard protocol as described by Singh et al. We established the experimental condition of Comet assay to detect the DNA damage of endometrial cells in mice.4. Detection of LIF gene breakage in endometrial cellsWe prepared the Fluorescently-labeled probe combined with LIF gene, and employed Comet-FISH to detect the breakage of LIF gene in endometrial cells of mice.5. Statistical analysesData were analyzed by SPSS16.0statistical software. Data were expressed as mean±standard deviation. For each quantitative measure, dose was first treated as a categorical condition. Variances were evaluated by homogeneity of variance test first. If variances were equal, statistical analysis was performed with one-way analysis of variance (ANOVA), followed by Dunnett-t tests. If variances were unequal, statistical differences were evaluated by Brown-Forsythe tests. P values<0.05were considered to be significant.Results1. The effect of CS2exposure during the period of embryo implantation on pregnancy mice and embryosMaternal toxicity:After the exposure to CS2at different time points of embryo implantation, no significant difference was found in GD1, GD9body weights and the gain of body weights, and the weights of uteri, ovaries, livers, spleens, kidneys as well. However, the coefficient of uterus at GD4and the coefficient of ovary at GD5were significantly decreased compared with the control. The results indicated that CS2exposure during the period of embryo implantation could induce litter maternal toxicity.Embryotoxicity:Compared with the control, the numbers of implanted embryos at GD4and GD5were significantly decreased.2. The effect of CS2exposure in the period of embryo implantation on DNA damage of endometrial cellsMice were exposed to CS2at GD4. The indicators of DNA damage were significantly higher at6h,12h,18h,24h and2d after CS2exposure, compared with the control. At3d and5d after CS2exposure, no significant difference was found.3. The effect of CS2exposure in the period of embryo implantation on the breakage of LIF gene in endometrial cellsMice were exposed to CS2at GD4. Only at18h after CS2exposure, the breakage of LIF gene in endometrial cells was detected by Comet-FISH.Conclusion1. CS2exposure during the period of embryo implantation could induce obvious embryotoxicity, which showed that the number of implanted embryos was significantly decreased, and GD4was the sensitive time point for embryotoxicity induced by CS2.2. Mice were exposed to CS2at GD4, and the DNA damage of endometrial cells showed a definite time variation. During the18h after CS2exposure, the level of DNA damage was gradually accelerated, and at18h, the level of DNA damage reached the top. The results indicated that18h after CS2exposure was the sensitive time for DNA damage in endometrial cells induced by CS2.3. Mice were exposed to CS2at GD4. At18h after CS2exposure, the breakage of LIF gene in endometrial cells was detected by Comet-FISH. The results indicated that the LIF gene in endometrial cells was split at18h after CS2exposure, which may be the reason for the decreased expression of protein and mRNA for LIF gene.