| Background and objectivesBenzene is an environmental pollutant that can cause diseases of the blood system.Due to the widespread use of benzene in the petrochemical industry and manufacturing,long-term exposure can lead to chronic benzene poisoning,causing hematopoietic dysfunction,whose main symptoms are hypoplastic myelodysplastic and peripheral blood cytopenia.Studies have shown that hypoxia-inducible factor-1α(HIF-1α)is involved in benzene-induced hematopoietic toxicity.In this study,we first established the benzene poisoning model with high expression of HIF-1α(HIF-1α~+)mouse to study the effects of benzene exposure on hematopoietic toxicity and oxidative stress in mice,and to explore the regulatory role of HIF-1αin oxidative damage of hematopoietic cells in benzene-exposed mice and its effect on downstream genes.In the previous study,CHIP-Seq technology has been used to screen out the HIF-1αdownstream target genes related to benzene toxicity.Here,target genes related with oxidative damage were selected using the GO gene function and verficated with ChIP-qPCR,namely RBM45 and ASB15.Furthermore,RBM45 and ASB15 high expression cell lines were constructed to study the cytotoxicity of 1,4-benzoquinone.Meanwhile,whether the over-expression of RBM45 and ASB15 play a protective role on the cytotoxicity of1,4-benzoquinone by inhibiting oxidative stress was also investigated.Finally,the mRNA expression of RBM45,ASB15 in peripheral blood between occupational benzene exposed population and control population was compared,and the feasibility of these two genes as biomarkers of benzene hematopoiesis toxicity was explored.Chapter1:Role of HIF-1αin oxidative damage of hematopoietic cells in benzene-exposed mice and its effect on downstream genesSpecific-Pathogen-Free(SPF)grade C57BL/6 and HIF-1αstable high expression(HIF-1α~+)male mice were divided into two groups respectively:control group(0 mg/kg,n=8)and benzene exposure group(150 mg/kg,n=8).The mice were continuously exposed to benzene by subcutaneous injection for 15 days,and the body weight was recorded during the exposure.After poisoning,the mice were sacrificed to separate the extremities bones and main organs,collect samples,calculate the organ coefficient,analyze the changes of blood routine indexes and the proportion of LSK cells,etc.The results showed that compared with the control group,benzene exposure significantly decreased the coefficient of thymus,lung and spleen,peripheral blood leukocytes,red blood cells and hemoglobin in the two kinds of mice.The results of flow cytometry indicated that the percentage of LSK cells decreased significantly and the apoptosis rate increased significantly after benzene exposure,while the percentage of LSK cells in HIF-1α~+mice was higher than that in C57BL/6 mice and the apoptosis rate was lower.The results of oxidative damage test showed that benzene exposure increased the level of MDA,tail length,tail DNA%and Olive tail distance in bone marrow cells of mice,and the oxidative damage indexes in HIF-1α~+mice were significantly lower than those in C57BL/6 mice.Meanwhile,the RBM45,ASB15 protein expression level decreased with benzene exposure,which was consistent with reduced HIF-1αprotein level as previously reported by our research group.ChIP-qPCR was used to examine the binding level of HIF-1αwith RBM45,ASB15 in bone marrow samples of C57BL/6 mice in benzene group and control group.It was found that the specific binding level of HIF-1αantibody with RBM45,ASB15in benzene group was 0.045,0.206 times as those in control group.The comprehensive results pointed that benzene exposure can inhibit hematopoiesis,cause oxidative damage in bone marrow cells,and decrease the level of HIF-1αresponse gene RBM45,ASB15 in mice.High expression of HIF-1αcan reduce benzene-induced hemopoietic damage to a certain extent by inhibiting radical oxidation.Chapter 2:The role of HIF-1αdownstream target genes RBM45 and ASB15 in benzene toxicity and related mechanismsIn this chapter,the lentivirus was used to infect K562 cells to obtain novel cell lines with high expression of target genes(RBM45~+,ASB15~+)and their corresponding control cell lines(NC-RBM45~+,NC-ASB15~+).Cell transfection efficiency was verified by RT-PCR and WB methods.Cells were treated with 1,4-benzoquinone at different concentrations(0、10、20μmol/L).Then MTT assay,comet assay and flow cytometry were performed to detect cell proliferation,DNA damage,cell apoptosis rate and cell cycle.Moreover,downstream pathway protein expression was also tested by WB.The relative mRNA expression of RBM45 in transfected cells was 5 times higher than that of control cells by RT-PCR,and ASB15 mRNA expression was even as high as 7620 times in ASB15~+cells.The difference was statistically significant,indicating that the RBM45 and ASB15 high expression cell lines were successfully constructed.MTT results demonstrated that the cell relative proliferation rate decreased significantly after treated with 10,20μmol/L 1,4-benzoquinone for 24h and 48h.Apoptosis results showed that there was a dose-response relationship between the apoptosis rate and the concentration of 1,4-benzoquinone.Compared with control cells exposed to1,4-benzoquinone at the same dose,the apoptosis rate of RBM45~+cells and ASB15~+cells were significantly lower.In the presence of 1,4-benzoquinone,the cell cycle of RBM45~+cells and ASB15~+cells and their control groups were mainly arrested in G2 phase.And the tail length,tail DNA%and Olive tail moment were significantly increased.Compared with the NC-RBM45~+and NC-ASB15~+cells treated with the same dose of 1,4-benzoquinone,the tail length,tail DNA%and Olive tail distance of RBM45~+and ASB15~+cells were significantly decreased.The WB results revealed the expression of RBM45 protein and its downstream pathway protein NRF2,KEAP1,along with ASB15 protein and its downstream pathway protein PI3K,AKT were decreased after1,4-benzoquinone treatment.In summary 1,4-benzoquinone could inhibit cell proliferation,increase cell apoptosis,change the cell cycle,cause oxidative damage,and inhibit NRF2/KEAP1 and PI3K/AKT pathway.The up-regulation of RBM45/ASB15 genes play an anti-oxidative role by inhibiting oxidative stress,which can promote cell proliferation,decrease apoptosis rate and reduce DNA damage.Chapter3:RBM45,ASB15 expression decreased and were related to blood indicators in occupational benzene-exposed workersWorkers exposed to benzene from four enterprises in Yangzhou were collected as exposure group,and healthy occupational workers without benzene exposure in Nanjing were used as control group.One hundred fifty cases of benzene exposed population and one hundred fifty cases of healthy control population were matched by gender and age.Human peripheral blood leukocytes were isolated,RNA was extracted and real-time quantitative PCR was performed to detect RBM45and ASB15 expression.Peripheral blood indexes showed that white blood cell,neutrophil count,red blood cell and platelet count in benzene exposed population were lower than those of control group.The results of RT-qPCR displayed that the mRNA relative expression of RBM45 and ASB15 in peripheral blood leukocytes of benzene-exposure group decreased by 51%and 62%respectively compared with the control group.In addition,the RBM45 expression had significantly positive correlations with the white blood cell(P<0.05),neutrophil count(P<0.05)and red blood cell(P<0.05)levels.And the ASB15 expression was positively correlated with white blood cell(P<0.05),neutrophil count(P<0.05)and platelet count(P<0.05).The above results suggest that decreased expression of RBM45 and ASB15 may be related to the hematotoxicity of benzene.It is indicated that RBM45 and ASB15 may be used as a potential candidate biomarker for benzene exposure.ConclusionsIn conclusion,we found that the high expression of HIF-1αcould reduce oxidative stress by regulating its downstream target genes RBM45 and ASB15,and reduce the damage of hematopoietic system induced by benzene to a certain extent.The study of K562 cell line with over-expression of RBM45,ASB15 showed that RBM45,ASB15 high expression could reduce the benzene-induced damage,including proliferation inhibition,pro-apoptosis and DNA damage.The possible mechanism may be related with reducing the level of oxidative damage and regulating the NRF2/KEAP1 or PI3K/AKT pathways,respectively.It was also found that the expression of RBM45,ASB15 in peripheral blood leukocytes of benzene-exposed population was significantly lower and positively correlated with basic blood indicators,indicating that RBM45 and ASB15 may be the early potential biomarkers for oxidative damage and hematopoiesis toxicity of benzene exposure. |
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