Diepoxybutane Induces Oxidative Stress-mediated DNA Damage And Cell Cycle Arrest In Mouse Germ Cells

Objective 1,3-Butadiene(BD) is an important industrial chemical used in the synthesis of nylon, plastics and rubber production. Exposed to EB, health damage was observed in occupational groups. It is also commonly found in automobile exhaust and cigarette smoke and identified as one of the nine priority air pollutants in residental environment. The International Agency for Research on Cancer(IARC, 2008) has classified BD as Group 1 carcinogen. 1,2,3,4-diepoxybutane(DEB), a major carcinogenic metabolite of BD, has been shown its potent genotoxicity including gene mutation and chromosome-level damage. However, it is not clear that relationship of DEB-induced DNA damage and cell cycle arrest in germ cells and the related molecular mechanism also remains to be elucidated.Methods In the present study, GC-2 cell line which was established from mouse spermatocyte was used as a research model to evaluate the male reproductive damage induced by DEB. After exposure to different doses of DEB(100, 300, 500 mM), the cell proliferation was detected by CCK-8 and Ed U incorpation assay, respectively. The effect of DEB on cell cycle, apoptosis and reactive oxygen species was analyzed by flow cytometry. Cell mitotic index was detected by Giemsa staining in GC-2 cells. Meanwhile, the genotoxic effects of DEB in GC-2 cells were investigated using the alkaline comet assay, γ-H2 AX expression detection and cytokinesis-block micronucleus(CBMN) assay. The signaling pathway in response to cell cycle was detected by western blot. The level of Chk1/cdc25c/cdc2/cyclin B1 protein expression and the change of phosphorylation were observed. The distribution of Cyclin B1 in GC-2 cell nucleus was detected by immunofluorescence. On the basis of the above research, pretreatment with N-acetyl-l-cysteine(NAC) for 1h following exposed to DEB, we measured the effect of NAC on ROS generation, the g-H2 AX protein expression, G2/M cell cycle distribution and the key molecular of cell cycle. We used UCN-01 for 4 h in advance, a potent inhibitor of Chk1 kinase, to detect the change of cell cycle arrest in GC-2 cells and the expression changes of downstream effectors of phospho-Chk1, phospho-Cdc25 c and phospho-Cdc2 after DEB treatment. To further confirm the importance of Chk1, we conducted RNA interference to detect the expression changes of downstream effectors of phospho-Chk1. In addition to in vitro cell experiment, we confirmed the potential effect of DEB on reproductive cell cycle in male mice. After one week adaptive feed, 24 Kun-Ming mice(10-12 weeks old) were randomly assigned to four group. The mice received a single intraperitoneal injection of different dose of DEB(0, 17, 34.5, 85mg/kg) on day 1, 3 and 5 injection intervals. The animals were sacrificed 7 days after the last treatment. The change of testis morphology in male mice was observed by hematoxylin and eosin(H&E) stain. The effect of DEB on mice sperm parameters was dected by CASA. Typical DNA content distribution was analyzed by flow cytometry. The expression of cell cycle-related protein in mouse testis was detected by immunohistochemistry. The level of GSH-Px, MDA and SOD was detected by Nan Jing kit.Results After exposure to different doses of DEB, we found the cell viability was decreased and cell proliferation was inhibited dose-dependently, respectively. Furthermore, flow cytometry analysis showed that DEB could induce obvious cell cycle arrest at G2/M phase but not apoptosis. Compared with the control group, DEB treatment group markedly increased the mean DCF fluorescence(p < 0.05), indicating that DEB could cause a significant increase in cellular ROS levels. The results of comet assay showed that the tail length, tail moment and the DNA content in tail of cells treated with DEB were markedly increased compared with the control cells(p <0.05). Western blot analysis indicated the level of γ-H2 AX protein expression was up-regulated in a dose-dependent manner with DEB treatment. The CBMN assay found that the frequencies of micronucleus(MN), nuclear bud(NBUD) and nucleoplasmic bridge(NPB) in DEB-treated cells were significantly higher than the control(p < 0.05). These results suggested DEB could induce DNA oxidative damage in spermatocyte GC-2 cells. Giemsa staining showed that treatment with DEB significantly increased mitosis index, immunofluoresence showed that the distribution of Cyclin B1 gradually reduced in GC-2 cell nucleus. Meanwhile, western blot analysis found that the level of phospho-ATM(Ser1981), phospho-chk1(Ser 345), Phospho-cdc25C(Ser 216), Phospho-cdc2(Tyr15) was significantly up-regulated and the level p-H3, a M phase marker protein, was significantly down-regulated with DEB treatment, indicating that DEB could induce G2 pahse arrest in GC-2 cells. NAC was applied to determine whether oxidative stress is responsible for DNA damage and G2 arrest induced by DEB, the results showed NAC pretreatment significantly decreased the production of ROS in DEB-treated GC-2 cells, the level of γ-H2 AX protein expression was down-regulated, the G2 phase proportion in NAC plus DEB group was significantly lower than DEB treatment alone(p < 0.05), also, the increased phosphorylation level of Chk1 and Cdc25 c were relatively reversed with NAC pretreatment. The results indicated that NAC could partly attenuated G2-phase arrest induced by DEB. To detect the importance of Chk1 in G2 phase, after treatment with UCN-01, a potent inhibitor of chk1 kinase, we found that the proportion of cells in G2 phase in UCN-01 plus DEB group was significantly lower than DEB treatment alone(p < 0.05). Also, the expression of phospho-Chk1(Ser 345), Phospho-cdc25C(Ser 216) and Phospho-cdc2(Tyr15) protein was downregulated in UCN-01+DEB group compared to that of DEB treatment alone. Meanwhile, transfection of Chk1 si RNA also abrogated partly the changes of phospho-Chk1, phospho-Cdc25 c and phospho-cdc2 in DEB treatment group, indicating that Chk1 played an important role in G2 phase. In vitro cell experiment of intraperitoneal injection of DEB in male mice, the rate of sperm deformation was significantly increased and sperm motility was significantly decreased. H&E staining results suggest that compared with the control, the decrease in the number of spermatogenic cells and mature sperm, disorder of spermatogenic cells, vacuolation and pycnotic nuclei were observed in DEB treatment group. Flow cytometry analysis showed that the proportion of tetraploid in DEB treatment group was increased markedly compared to control(p < 0.05). Immunohistochemical results showed that the mean density of p-Chk1 protein expression in DEB treatment group was increased significantly compared to control and the mean density of p-H3 protein expression was gradually decreased. In mice liver tissue, the oxidation level of MDA was increased significantly and the antioxidant level of GSH-Px and SOD was significantly induced.Conclusion DEB could induce reproductive cell damage and arrests cell cycle at G2 phase by oxidative stress. This study also presents a mechanistic basis for the adverse effects of DEB on male reproductive system.