Oxidative stress and DNA damage in utero and embryo implantation of mice exposed to carbon disulfide at peri-implantation

Abstract

Carbon disulfide (CS₂) has reproductive toxicity but the mechanism remains unclear. The aim of the present study was to investigate the effect of oxidative stress and DNA damage on embryo implantation of mice exposed to CS₂ at peri-implantation. CS₂ exposure was on gestational day 3 (GD3), GD4, GD5 and GD6, separately, and the number of embryonic day 9 (E9) mouse embryos was obtained. DNA damage of endometrial cells, oxidative stress and 8-hydroxy-2′-deoxyguanosine (8-OH-dG) level in uterus tissues were tested with time series at different end points after exposure. The number of E9 mouse embryos significantly decreased in all CS₂ exposure groups, especially on GD4 exposure. The rates of embryo implantation decreased by 43.05%, 63.74%, 60.45% and 47.26% for CS₂ exposure on GD3, GD4, GD5 and GD6, respectively. Oxidative stress significantly increased within 24 h and reached the top level at 18 h after exposure. The same time-dependent trend was observed no matter when the exposure happened at peri-implantation. 8-OH-dG significantly increased at 18 h and 24 h after exposure by 893.8% and 647.4%, respectively, when compared with the control. The indexes of DNA damage significantly increased at 6 h after exposure, which appeared earlier than the changes of oxidative stress and 8-OH-dG. Besides, both oxidative stress and DNA damage showed a strong negative correlation with the number of E9 mouse embryos. The present study illustrated that CS₂ directly induced DNA damage in endometrial cells and enhanced the action through oxidative stress, both of which were responsible for CS₂-induced embryo loss.

Keywords

Carbon disulfide oxidative stress DNA damage embryo implantation

Introduction

Carbon 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.¹ CS₂ has multisystem toxicity and long-term exposure to low concentrations of CS₂ can cause neurological, cardiovascular, psychological and hormonal effects.² Furthermore, it is noteworthy that wide use of CS₂ is associated with various types of reproductive disturbance. CS₂ could induce atrophy of testis, dyszoospermia, sexual disorder, decline of sex hormone and decrease in semen quality.³ Many studies have shown that CS₂ could induce menstrual disorders and abnormal labor, such as spontaneous abortion and premature birth among female employees.⁴ 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 CS₂.⁵ We also found that the number of implanted embryos significantly decreased when mice were exposed to CS₂ at the phase of embryo implantation.⁶ These results suggested that CS₂ could disturb embryo implantation and induce embryo loss, but the mechanism was still unclear.

Oxidative stress induces lipid peroxidation, structurally and functionally alters protein and DNA and promotes apoptosis.⁷ It was reported that reduced plasma levels of antioxidant increased risk of spontaneous abortion.^8,9 Chronic exposure to CS₂ could cause coronary heart disease by oxidative stress.¹⁰ Our previous study revealed that CS₂ exposure could induce DNA damage in the endometrial cells of mice at the phase of implantation.¹¹ Therefore, we hypothesized that oxidative stress and DNA damage were a single or joint participation in disturbing embryo implantation after CS₂ exposure. In the present study, the mice were exposed to CS₂ at peri-implantation. Oxidative stress, DNA damage and the oxidative DNA damage product 8-hydroxy-2′-deoxyguanosine (8-OH-dG)¹² were observed and evaluated with time series to explore the mechanism of CS₂-induced embryo loss.

Materials and methods

Animals

Adult Kunming mice, female (7–9 weeks old, 23–25 g) and male mice (8–10 weeks, 30–35 g), were provided by Vital River Laboratories (Beijing, China). All the animals were kept under controlled environmental conditions at room temperature (22 ± 2°C) with 50% ± 10% humidity and an automatically controlled cycle of 12 h light and 12 h dark. Standard laboratory animal feed (purchased from commercial supplier) and water were provided ad libitum. Animals were acclimatized to the experimental conditions for a period of one week prior to the commencement of the experiment. Then the female mice were mated with the male mice. The day on which sperm plug was found after the mating procedure was designated as gestational day 1 (labeled GD1) and the mice were randomly grouped. All the animal experiment protocols were approved by the Medical Ethics Committee of Shandong University.

Experimental design

The experiment was divided into two parts. Part 1 was designed to establish the animal model of CS₂ exposure 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. Part 2 was to evaluate the levels of DNA damage and 8-OH-dG with time series after exposure at the sensitive exposure time obtained from part 1.

Animal treatment

In part 1, mice on GD1 were randomly divided into four groups and each group contained two types of exposure (CS₂ exposure and solvent exposure). Mice in group 1 were exposed on GD3, group 2 on GD4, group 3 on GD5 and group 4 on GD6, respectively. There were a total of 26 end points in four groups. The end points were introduced in detail as follows. Eight end points in group 1 were designed at 6 h, 12 h, 18 h, 24 h, 48 h, 72 h, 96 h and 144 h (GD9) after exposure. Seven end points in group 2 were at 6 h, 12 h, 18 h, 24 h, 48 h, 72 h and 120 h (GD9). Six end points in group 3 were at 6 h, 12 h, 18 h, 24 h, 48 h and 96 h (GD9). Five end points in group 4 were at 6 h, 12 h, 18 h, 24 h and 72 h (GD9) after exposure. They are illustrated in Figure 1. The aim of the last end point (GD9) was to observe the maternal toxicity and embryo implantation, and the aim of other end points was to measure the level of oxidative stress in the uterine tissue.

Figure 1.

A schematic diagram of the arrangements of exposure points and end points in part 1. The exposure points of mice in group 1 were on GD3, group 2 on GD4, group 3 on GD5 and group 4 on GD6, respectively. Eight end points in group 1 were at 6 h, 12 h, 18 h, 24 h, 48 h, 72 h, 96 h and 144 h after exposure. Seven end points in group 2 were at 6 h, 12 h, 18 h, 24 h, 48 h, 72 h and 120 h. Six end points in group 3 were at 6 h, 12 h, 18 h, 24 h, 48 h and 96 h. Five end points in group 4 were at 6 h, 12 h, 18 h, 24 h and 72 h. GD: gestational day.

In part 2, CS₂ exposure was arranged on GD4 according to the results of part 1. Four end points were designed at 6 h, 12 h, 18 h and 24 h after CS₂ or solvent exposure, respectively. There were six gestational mice in each end point in part 1 and part 2.

Each animal received a single dose intraperitoneal injection of CS₂ (Guangcheng chemical reagent corporation, Tianjin, China) or olive oil (Shanghai Kerry Food Industries Co., Ltd, Shanghai, China) at the designed exposure time. CS₂ dissolved in olive oil was 631.4 mg/kg body weight, 0.4 median lethal dose (LD₅₀), based on the data from the acute toxicity test for female Kunming mice in our previous research.¹¹ The injection volume was 0.1 mL/10 g body weight.

Sample collection

The mice for the last end point (GD9) in all groups in part 1 were weighed and killed on GD9. The utero weight on GD9 was harvested and weighed, and the number of embryonic day 9 (E9) mouse embryos for each litter was counted. Uterus, ovary, liver, spleen and kidney were isolated and weighed and then frozen at −80°C in a refrigerator for further analysis.

The mice in other end points, except for the last one in all groups in part 1, were killed at the designed end points. Half of each uterine tissue was separately frozen at −80°C for quantitating the protein content of the homogenate and testing oxidative stress and 8-OH-dG levels. 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.

Measurement of malondialdehyde, superoxide dismutase, glutathione peroxidase and catalase in uterine tissue

Malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-PX) and catalase (CAT) in the uterine tissue homogenate were measured with commercial chemical assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) by an UV-2450 spectrophotometer (Shimadzu Co., Kyoto, Japan). The following wavelengths were used: 532 nm for MDA, 550 nm for SOD, 412 nm for GSH-PX and 405 nm for CAT. The results were expressed as nanomoles of MDA per milligram of protein (nmol/mgprot) for MDA, units per milligram protein (U/mgprot) for SOD, GSH-PX and CAT. The experimental procedures were in strict accordance with the kit instructions. Results were determined according to the manufacturer’s guide.

Determination of the total protein content

The total protein content was determined with Coomassie blue method assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) by an UV-2450 spectrophotometer. Wavelength used was 595 nm, and the result was expressed as gram per liter (g/L). Results were determined according to the manufacturer’s guide. Determination of the total protein was to standardize the indexes of oxidative stress.

Endometrial cell comet assay

The comet assay was carried out with slight modifications of the standard protocol as described by Singh et al.¹³ Two solutions, one containing 0.8% normal melting point agarose (NMA; Amresco, Solon, Ohio, USA) and the other containing 0.8% low melting point agarose (LMA; Amresco) dissolved in Ca²⁺, Mg²⁺-free Phosphate Buffered Saline (PBS), were prepared. Endometrial cell suspension of 25 µL and 75 µL LMA were distributed onto microscope slides precoated with 100 µL of 0.8% NMA. After solidifying the agarose, slides were immersed in ice-cold freshly prepared lyses solution (2.5 M sodium chloride, 100 mM diaminetetraacetic acid (Na₂EDTA) (Amresco), 10 mM Tris (Sigma, St. Louis, Missouri, USA), pH 10.0 containing 1% Triton X-100 (Amresco) and 10% dimethyl sulfoxide (Amresco)) for 1 h to lyse the cells and to allow DNA unfolding. Then the slides were placed on a horizontal electrophoresis chamber and DNA was allowed to unwind for 20 min in an alkaline solution containing (300 mM sodium hydroxide and 1 mM Na₂EDTA, pH 13). Electrophoresis was conducted at 20 V, 150 mA for 20 min. After electrophoresis, the slides were gently washed three times for 5 min with neutralization buffer (0.4 M Tris-base, pH 7.4). The slides were drained and SYBR-Green I (30 μL/slide; Sigma) was added. To prevent additional DNA damage, all the steps described above were run under red light. Analyses were performed immediately under a fluorescence microscope (Nikon, Tokyo, Japan) and the photographs were individually measured at 200× magnification using a calibration scale. A total of 50 individual cells were screened per slide to determine the following parameters for the DNA damage analysis: tail length (TL, in μm), tail moment (TM), tail moment olive (OTM) and tail DNA% (TDNA%) with image analysis software (CASP, Poland¹⁴). TL is the maximum distance that the damaged DNA migrates from the center of the cell nucleus. TDNA% is the total DNA that migrates from the nucleus into the comet tail. TM is the product of the TL and TDNA%, which gives a more integrated measurement of overall DNA damage in the cell. OTM is the product of TDNA% and median migration distance. All slides were coded and scored blindly, and each experiment was reproduced independently at least three times.

Measurement of 8-OH-dG in uterine tissue

The mouse 8-OH-dG ELISA kit (Shanghai BlueGene Biotech Co., Ltd, Shanghai, China) was used to measure the 8-OH-dG in uterine tissue homogenate. The whole experiment process was according to the manufacturer’s guide. Reading the optical density (OD) at 450 nm within 5 min after the termination of the reaction by Thermo Scientific Multiskan MK3 (Thermo Fisher, Boston, Massachusetts, USA). The result was expressed as nanograms per milliliter 8-OH-dG.

Statistical analysis

Data were shown as mean ± SD for each group. Statistical analysis was performed using the SPSS 20.0 statistical 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 < 0.05 was considered to be statistically significant. Linear correlation analysis was performed in order to observe the correlation between the number of E9 mouse embryos and different other parameters.

Results

Maternal toxicity and embryo implantation

CS₂ exposure on GD3, GD4, GD5 and GD6 all failed to induce a statistically significant difference in maternal net body weight on GD9 (Figure 2(a)). Furthermore, no statistically significant changes were observed in the weight of the uterus, ovary, liver, spleen and kidney between CS₂ exposure and solvent control in four groups (Figure 2(b)). Yet, a statistically significant reduction was observed in the number of E9 mouse embryos when compared with the control (Figure 2(c)). The rates of embryo implantation were 56.95%, 36.26%, 39.55% and 52.74% for CS₂ exposure on GD3, GD4, GD5 and GD6, respectively, when compared with each corresponding control. No statistically significant change was found in the mean weight of conceptus in each CS₂ exposure when compared with the control (Figure 2(d)).

Figure 2.

Maternal toxicity and blastocyst implantation after exposing mice to CS₂ at different time in peri-implantation. (a) Maternal net body weight gain in each group; (b) the weight of maternal uterus, ovary, liver, spleen and kidney in each group; (c) the number of E9 mouse embryos in each group; (d) mean weight of conceptus in each group. All the values are expressed as mean ± SD. **p < 0.01 compared with control. The control is the merged control of four groups. CS₂: carbon disulfide; E9: embryonic day 9.

Oxidative stress after mice exposed to CS₂ at peri-implantation

The level of MDA, SOD, GSH-PX and CAT in uterine tissue

MDA content as an indicator of lipid peroxidation had a significant increase at 18 h after CS₂ exposure on GD3, GD4, GD5 and GD6 when compared with the control (Figure 3(a)). At 18 h after CS₂ exposure, the MDA level increased by 131.4% in GD4, which is higher than 120.8%, 121.6% and 104.9% in GD3, GD5 and GD6 CS₂ exposure 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 at 18 h, GSH-PX at 12 h and 18 h and CAT at 12 h when compared with each control (Figure 3(b) to (d)).

Figure 3.

Oxidative stress levels in uterine tissue after exposing gestational mice to CS₂ at different time in peri-implantation. (a) MDA level in each group; (b) SOD level in each group; (c) GSH-PX level in each group; (d) CAT level in each group. All the values are expressed as mean ± SD. **p < 0.01 compared with control. CS₂: carbon disulfide; MDA: malondialdehyde; SOD: superoxide dismutase; GSH-PX: glutathione peroxidase; CAT: catalase.

The trends with time for oxidative stress

MDA levels, in all CS₂ exposure groups, significantly increased within 24 h after CS₂ exposure and then returned to normal after 24 h. No statistically significant changes were observed at 24 h and after 24 h between each CS₂ exposure and their control. The variation trend of MDA was the same no matter when CS₂ exposure happened on GD3, GD4, GD5 or GD6. SOD, GSH-PX and CAT levels all decreased in 24 h and returned to normal after 24 h (Figure 4(a) to (d)).

Figure 4.

Trends with time for oxidative stress levels in uterine tissue after exposing mice to CS₂ at different time in peri-implantation. (a) The trends with time for MDA in each group; (b) the trends with time for SOD in each group; (c) the trends with time for GSH-PX in each group; (d) the trends with time for CAT in each group. The values are expressed as mean. CS₂: carbon disulfide; MDA: malondialdehyde; SOD: superoxide dismutase; GSH-PX: glutathione peroxidase; CAT: catalase.

The relationship between oxidative stress and embryo implantation

The correlation analysis showed a strong negative correlation between MDA levels at 18 h after exposure and the number of E9 mouse embryos in each group (r = −0.783, p < 0.01; Figure 5).

Figure 5.

The correlation analysis between MDA levels at 18 h after exposure and the number of E9 mouse embryos in each group. CS₂: carbon disulfide; MDA: malondialdehyde; E9: embryonic day 9.

DNA damage of endometrial cells in mice after CS₂ exposure at peri-implantation

After CS₂ exposure 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 part 2 (Table 1). Comet parameters appeared to significantly increase at 6 h after CS₂ exposure and reached the top level at 18 h, and then these comet parameters decreased gradually (Figure 6(a) to (e)). The correlation analysis showed a strong negative correlation between the number of E9 mouse embryos and TM, OTM, TL and TDNA% after CS₂ exposure at 18 h (r = −0.804, −0.847, −0.934 and −0.863, respectively, p < 0.01).

Figure 6.

DNA damage of endometrial cell after exposing mice to CS₂ on GD4. Control (a), 6 h (b), 12 h (c), 18 h (d) and 24 h (e) of the comet assay in the mice endometrial cell at ×200, respectively.

Table 1.

DNA damage of endometrial cells for mice exposed to CS₂ on GD4.

End point (h)	The number of animals	The number of cells	TL (μm)	TM (μm)	OTM (μm)	TDNA% (%)
^aControl	6 × 4	50 × 6 × 4	4.90 ± 2.15	0.08 ± 0.05	0.39 ± 0.29	1.92 ± 1.53
6	6	50 × 6	22.00 ± 6.50^b	1.55 ± 1.08^b	2.71 ± 1.31^b	6.25 ± 2.84^b
12	6	50 × 6	20.96 ± 7.20^b	1.18 ± 0.91^b	2.26 ± 1.21^b	4.69 ± 2.16^b
18	6	50 × 6	35.20 ± 5.50^b	5.92 ± 2.45^b	7.93 ± 2.14^b	16.17 ± 4.75^b
24	6	50 × 6	28.53 ± 5.57^b	2.96 ± 1.47^b	4.28 ± 1.37^b	9.83 ± 3.21^b

CS₂: carbon disulfide; GD4: gestational day 4; 8-OH-dG: 8-hydroxy-2′-deoxyguanosine; TL: tail length; TM: tail moment; OTM: tail moment olive; TDNA%: tail DNA%.

^a The control is the merged control of four groups.

^b p < 0.01 compared with control.

The level of 8-OH-dG in uterine tissue after CS₂ exposure at peri-implantation

Statistically significant increases in the 8-OH-dG were observed at 18 h and 24 h after exposure when compared with the control in uterine tissue (Figure 7). The increased rate of the 8-OH-dG level was 893.8% at 18 h and 647.4% at 24 h after exposure when compared with the control.

Figure 7.

Oxidative DNA damage induced by CS₂ in uterine tissue after exposing mice to CS₂ on GD4. The 8-OH-dG in uterine tissue of pregnant mice killed at the end point of 6 h, 12 h, 18 h and 24 h, respectively, and their controls. **p < 0.01 compared with control. CS₂: carbon disulfide; GD4: gestational day 4; 8-OH-dG: 8-hydroxy-2′-deoxyguanosine.

The relationship between oxidative stress, DNA damage and 8-OH-dG level

TDNA%, TL, TM and OTM showed statistically significant difference at 6 h after exposure, whereas MDA showed a statistical increase at 18 h after exposure. MDA change that indicated oxidative stress occurred after DNA damage. Then TDNA%, TL, TM, OTM and MDA all showed a decrease at 24 h after exposure. The correlation analysis showed a strong positive correlation between the 8-OH-dG level and TDNA%, TL, TM and OTM (r = 0.766, 0.688, 0.738 and 0.771, respectively, p < 0.01). Figure 8 shows the relationship between oxidative stress, DNA damage and 8-OH-dG after exposing mice to CS₂ on GD4.

Figure 8.

The relationship between oxidative stress, DNA damage and 8-OH-dG after exposing mice to CS₂ on GD4. Point 0 on the X-axis is the merged control for each index. CS₂: carbon disulfide; GD4: gestational day 4; 8-OH-dG: 8-hydroxy-2′-deoxyguanosine.

Discussion

Embryo implantation in rats and mice occurs between days 4 and 5 after mating,¹⁵ which is called the implantation window.¹⁶ In the present study, CS₂ exposure was arranged on GD3, GD4, GD5 and GD6, respectively, to find out the effect of CS₂ exposure on embryo implantation at different exposure time. Therefore, the mice only received a single exposure, which could not induce maternal toxicity, but had a significant effect on embryo implantation. Based on our previous study,¹¹ 0.4 LD₅₀ (631.4 mg/kg) was the appropriate dose. The results showed a statistically significant reduction in the number of E9 mouse embryos at single dose exposure, especially on GD4. Meanwhile, no significant maternal toxicity and the changes in mean weight of conceptus were observed. These results illustrated that CS₂ exposure at different time of peri-implantation disturbed embryo implantation, and the sensitive exposure time was GD4. According to the results of embryology studies, successful embryo implantation depends on endometrial receptivity in the implantation window period,¹⁷ and many signals and molecular pathways that induce or regulate the complex series of interactions required for implantation.¹⁸ If the condition of maternal uterus was disturbed, the loss of embryos would happen.¹⁹ In our previous studies, CS₂ exposure at peri-implantation disturbed expression of MMP(metalloproteinase)-2, MMP-9,²⁰ leukemia inhibitory factor,²¹ adhesion molecule fibronectin and its ligand, and integrin β1 in the uterus of gestational mice,²² all these are related to implantation failure. Therefore, we speculated that CS₂ exposure might affect the environment of the uterus and disturb the development of the implantation window to disturb the process of embryo implantation.

Luo et al.²³ reported that CS₂ exposure could induce oxidative stress and reduce the levels of antioxidative enzymes in the blood. Song et al.²⁴ showed that CS₂ exposure could induce oxidative–antioxidative system disorders and significant changes in oxidative–antioxidative function in rat nerve tissues. Determination of free radicals that could indicate oxidative stress is difficult because of their relatively short half-lives. Instead, they can be evaluated indirectly by measurement of the levels of some antioxidant enzymes such as SOD, CAT and GSH-PX, and by measurement of products of lipid peroxidation such as MDA.^25,26 It has been reported that the increase in SOD in human endometrial stromal cells with decidualization is an important component of implantation,^27,28 and both ROS and SOD may act as second messengers to regulate endometrial function.²⁹ In the present study, oxidative stress was assessed using MDA and antioxidant enzymes. Within 24 h after CS₂ exposure, MDA increased significantly in uterine tissue; the antioxidant enzymes were initially induced and then significantly inhibited and GSH-PX decreased before SOD and CAT. The reasons for the above evidence may be that ROS increased rapidly, which led to tissue cell damage and produced MDA by lipid peroxidation after CS₂ exposure.²⁴ The increased ROS induced compensation function and resulted in the increase of enzyme activity. However, the increased ROS not only consumed a lot of enzymes but also changed the structure and function of protein and DNA,³⁰ which significantly reduced the activities of the antioxidant enzyme. Furthermore, the GSH-PX may be more sensitive to CS₂ exposure. The result showed a strong negative correlation between the number of E9 mouse embryos and the level of MDA. Moreover, positive correlations between the number of E9 mouse embryos and the level of SOD, CAT and GSH-PX were observed. The above results showed that oxidative stress was one of the mechanisms responsible for CS₂-induced embryo loss. In addition, no matter when the exposure happened on GD3, GD4, GD5 or GD6, the trend of change for oxidative stress parameters above in uterine tissue of gestational mice was the same along with the reduced number in each exposed group. Coincidentally, oxidative stress was more severe in uterine tissue of mice on GD4 exposure that was corresponding to the least number of E9 mouse embryos on GD4 exposure. It can be speculated that oxidative stress destroyed the endometrial receptivity, and GD4 was the most sensitive period for CS₂ exposure.

Reactive oxygen species is known to produce membrane peroxidation and MDA formation and attack on DNA.³¹ Studies reported that exposure to lower concentration of CS₂ could cause certain damage to human buccal cell DNA³² and mice sperm cell DNA.³³ Based on the evidence and the results in part 1, GD4 exposure was chosen to further assess the relationship between oxidative stress, DNA damage and embryo implantation. Endometrial cell comet assay revealed that CS₂-induced DNA damage began at 6 h after exposure and reached the top level at 18 h. To ensure the role of the increased oxidative stress in the induction of oxidative DNA damage, quantification of 8-OH-dG was performed. It has been reported that the levels of the oxidative DNA damage marker 8-OH-dG were significantly elevated in infertile male patients.³⁴ In the present study, a significant increase in 8-OH-dG was observed at 18 h and 24 h after exposure and has been well correlated with the parameters of endometrial cell DNA damage. Furthermore, the linear correlation analysis showed a strong negative correlation between the number of E9 mouse embryos and DNA damage. Thus, it can be inferred that CS₂ destroys the function of endometrium to accept embryo implantation by DNA damage and oxidative stress. Interestingly, DNA damage was found earlier than oxidative stress, and the 8-OH-dG and DNA damage increased significantly after oxidative stress returned to basal levels. This result was inconsistent with the traditional theory that DNA damage was caused by oxidative stress. Hence, we conjecture that CS₂ exposure can directly cause DNA damage, which was strengthened by the oxidative stress action. Additionally, further research are required to explain why oxidative DNA still had statistically significant changes after oxidative stress return to basal levels.

The part 1 of this study can provide the optimum exposure point for further confirmation and mechanistic evaluation, and the part 2 can provide better and more informative results. Based on the results of the present study, if the women at peri-implantation stage cannot avoid contacting with CS₂, we recommend taking interventions on oxidative stress to reduce the damage caused by CS₂. Embryo implantation is a complex process, it is essential to characterize the embryotoxicity of CS₂ using other surrogate end points in order to reduce the potential risk in women who are exposed to CS₂ in workshop, especially those of reproductive age.

Footnotes

Conflict of interest

The authors declared no conflicts of interest.

Funding

This work was supported by the projects of National Natural Science Foundation of China [grant numbers 30671745 and 81072275].

References

Tsai

Chang

Huang

Liu

. In vivo exposure to carbon disulfide increases the contraction frequency of pregnant rat uteri through an indirect pathway. Life Sci 1999; 66: 201–208.

Gelbke

Göen

Mäurer

Sulsky

. A review of health effects of carbon disulfide in viscose industry and a proposal for an occupational exposure limit. Crit Rev Toxicol 2009; 39: 1–126.

Qing

The effects of carbon disulfide on male sexual function and semen quality. Toxicol Ind Health. 2010; 26: 375–382.

Pieleszek

. The effect of carbon disulphide on menopause, concentration of monoamines, gonadotropins, estrogens and androgens in women. Ann Acad Med Stetin 1997; 43: 255–267.

Wang

Hou

. Very early pregnancy loss of women occupationally exposed to carbon disulphide. Chin J Ind Hyg Occup Dis 1999; 17: 204–207.

Wang

Xie

. Embryotoxicity of carbon disulfide on the phase of follicle developing and implantation of blastocysis in mice. J Toxicol 2005; 19: 184–186.

Ames

Shigenaga

Hagen

. Oxidants, antioxidants, and the degenerative diseases of aging. Proc Natl Acad Sci USA 1993; 90: 7915–7922.

Barrington

Lindsay

James

Smith

Roberts

. Selenium deficiency and miscarriage: a possible link? Br J Obstet Gynaecol 1996; 103: 130–132.

Vural

Akgül

Yildirim

Canbaz

. Antioxidant defence in recurrent abortion. Clin Chim Acta 2000; 295: 169–177.

10.

Wronska-Nofer

Nofer

Stetkiewicz

Evidence for oxidative stress at elevated plasma thiol levels in chronic exposure to carbon disulfide (CS₂) and coronary heart disease. Nutr Metab Cardiovasc Dis 2007; 17: 546–553.

11.

Shen

Wang

Sun

. DNA damage of mice endometrial cells at implantation phase exposed to carbon disulfide. Chin J Ind Hyg Occup Dis 2010; 28: 268–271.

12.

Leinonen

Lehtimäki

Toyokuni

New biomarker evidence of oxidative DNA damage in patients with non-insulin-dependent diabetes mellitus. FEBS Lett 1997; 417: 150–152.

13.

Singh

McCoy

Tice

Schneider

. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 1988; 175: 184–191.

14.

Konca

Lankoff

Banasik

A cross-platform public domain PC image-analysis program for the comet assay. Mutat Res 2003; 534: 15–20.

15.

Psychoyos

. Uterine receptivity for nidationa. Ann NY Acad Sci 1986; 476: 36–42.

16.

Lessey

. Endometrial receptivity and the window of implantation. Best Pract Res Clin Obstet Gynaecol 2000; 14: 775–788.

17.

Nikas

Psychoyos

. Uterine pinopodes in peri implantation human endometrium clinical relevance. Ann N Y Acad Sci 1997; 816: 129–142.

18.

Tabibzadeh

Babaknia

. The signals and molecular pathways involved in implantation, a symbiotic interaction between blastocyst and endometrium involving adhesion and tissue invasion. Mol Hum Reprod 1995; 1: 179–202.

19.

Chen

Peng

Zhang

Lei

Cao

Duan

. Embryo implantation: a time for recalling and forwarding. Chin Sci Bull 2009; 54: 4083–4093.

20.

Sun

Wang

Xie

. Effect of carbon disulfide on expression of matrix metalloproteinase-2 and metalloproteinase-9 in embryo and uterus of pregnant mice. Chin J Ind Hyg Occup Dis 2010; 28: 881–884.

21.

Zhang

Sun

Wang

. Effect of carbon disulfide exposure during peri-implantation on expression of leukaemia inhibitory factor in the uterus of mice. J Shandong Univ (Health Sci) 2011; 26: 215–217.

22.

Sun

Wang

Xie

. Effects of carbon disulfide on adhesion molecule of mice in different reproductive phases. Chin J Public Health 2011; 27: 200–202.

23.

Luo

JCJ

Shih

Chang

Huang

. Blood oxidative stress in Taiwan workers exposed to carbon disulfide. Am J Ind Med 2011; 54: 637–645.

24.

Song

Pan

Zeng

Xie

. Effect of carbon disulfide on oxidation–antioxidation function of rat nerve tissues. Chin J Ind Hyg Occup Dis 2007; 25: 641–644.

25.

Gutteridge

. Lipid peroxidation and antioxidants as biomarkers of tissue damage. Clin Chem 1995; 41: 1819–1828.

26.

Leff

. Autoimmune and inflammatory diseases. In: Armstrong

(ed). Free radicals in diagnostic medicine. New York, NY: Plenum Press, 1994, pp. 199–213.

27.

Sugino

Shimamura

Takiguchi

Changes in activity of superoxide dismutase in the human endometrium throughout the menstrual cycle and in early pregnancy. Hum Reprod 1996; 11: 1073–1078.

28.

Sugino

Kashida

Takiguchi

Nakamura

Kato

. Induction of superoxide dismutase by decidualization in human endometrial stromal cells. Mol Hum Reprod 2000; 6: 178–184.

29.

Sugino

. The role of oxygen radical-mediated signaling pathways in endometrial function. Placenta 2007; 28: 133–136.

30.

Ruder

Hartman

Blumberg

Goldman

. Oxidative stress and antioxidants: exposure and impact on female fertility. Hum Reprod Update 2008; 14: 345–357.

31.

Marnett

. Oxyradicals and DNA damage. Carcinogenesis 2000; 21: 361–370.

32.

Chen

Tan

. Studies on DNA damage in workers with long-term exposure to lower concentration of carbon disulfide. Chin J Prev Med 2004; 38: 36–38.

33.

Tang

Xuan

. Detection of DNA damage induced by carbon disulfide in mice sperm with single-cell gel electrophoresis assay. Chin J Ind Hyg Occup Dis 2003; 21: 440–443.

34.

Kasai

. Analysis of a form of oxidative DNA damage, 8-hydroxy-2′-deoxyguanosine, as a marker of cellular oxidative stress during carcinogenesis. Mutat Res 1997; 387: 147–163.

Oxidative stress and DNA damage in utero and embryo implantation of mice exposed to carbon disulfide at peri-implantation

Abstract

Keywords

Introduction

Materials and methods

Animals

Experimental design

Animal treatment

Sample collection

Measurement of malondialdehyde, superoxide dismutase, glutathione peroxidase and catalase in uterine tissue

Determination of the total protein content

Endometrial cell comet assay

Measurement of 8-OH-dG in uterine tissue

Statistical analysis

Results

Maternal toxicity and embryo implantation

Oxidative stress after mice exposed to CS2 at peri-implantation

The level of MDA, SOD, GSH-PX and CAT in uterine tissue

The trends with time for oxidative stress

The relationship between oxidative stress and embryo implantation

DNA damage of endometrial cells in mice after CS2 exposure at peri-implantation

The level of 8-OH-dG in uterine tissue after CS2 exposure at peri-implantation

The relationship between oxidative stress, DNA damage and 8-OH-dG level

Discussion

Footnotes

Conflict of interest

Funding

References

Oxidative stress after mice exposed to CS₂ at peri-implantation

DNA damage of endometrial cells in mice after CS₂ exposure at peri-implantation

The level of 8-OH-dG in uterine tissue after CS₂ exposure at peri-implantation