The Toxic Effects And Mechanisms Of Benzene Exposure On The Formation Of Hematopoietic Stem Progenitor Cells During Embryonic Development

BackgroundBenzene is the simplest and most common aromatic hydrocarbon,and is common air pollutants present in both occupational and non-occupational environments.Non-occupational exposure of benzene is mainly caused by exposure or use of products containing benzene by the population.Benzene is a component of gasoline,automobile exhaust,industrial emissions and tobacco smoke,and air exposure is the common exposure mode.Previous evidence suggested that long-term exposure to benzene can cause chronic benzene poisoning,leading to a decrease in white blood cells(RBCs)and platelets(PLTs)in peripheral blood,as well as the occurrence of anemia.Further development can lead to aplastic anemia and leukemia.Although benzene has been listed as a known carcinogen,the US Environmental Protection Agency believed that the reproductive and developmental toxicity of benzene was insufficient.Researches have found that there was a correlation between benzene exposure during pregnancy and the occurrence of childhood leukemia,but existing evidence on the damage and mechanisms of offspring caused by benzene exposure during pregnancy was limited.In recent years,the development of stem cell toxicology has promoted the study on reproductive and developmental toxicity.The process of differentiation of pluripotent stem cells(PSCs)into hematopoietic stem progenitor cells(HSPCs)in vitro simulated the development of embryonic hematopoiesis in vivo.Based on differentiation experiments in vitro and animal experiments of benzene exposure during pregnancy in vivo,the toxicity and related mechanisms of benzene metabolites on hematopoietic development were investigated.This study provided more evidence for the toxic damage of benzene exposure during pregnancy to the hematopoietic development of offspring.Methods1.Establishment of hematopoietic differentiation model based on human induced pluripotent stem cells and study on the toxic effects of benzene metabolitesFirstly,the pluripotency of human induced pluripotent stem cells(hiPSCs)was comprehensively identified,including morphology,expression of pluripotent factors,karyotype,and ability to differentiate into three germ layers.On this basis,a self-renewal model of hiPSCs,a spontaneous differentiation three germ layer model based on embryoid bodies(EBs),and a hematopoietic stem progenitor cell differentiation model based on EBs were constructed.Three cell models were used to evaluate the effects of different stages of hematopoietic differentiation in early life.Two main metabolites of benzene,hydroquinone(HQ)and 1,4-benzoquinone(1,4-BQ),were used to investigate totoxic effects using three models.The CCK8 method was used to determine the doses of HQ and 1,4-BQ,and the treated doses of 0,0.1,0.5,and 1μM of HQ and 1,4-BQ were determined.After treated experiments,the marker genes in three stages,apoptosis in hiPSCs and trilineage cells(TLCs),and the proportion of HSPCs were detected.2.The effects of benzene metabolites on genetic variation in different stages of hematopoietic differentiationhiPSCs,TLCs,and HSPCs were collected from the control group and the highest dose group after treated with HQ and 1,4-BQ.DNA was extracted and performed whole exome sequencing(WES).After filtering and comparing the offline data,identification and annotation of single nucleotide variation(SNV),insertion-deletion(In Del),structural variation(SV),and fusion genes were performed.Further analysis was conducted to investigate the SNVs caused by HQ and 1,4-BQ in cells at different stages of hematopoietic differentiation,including the number and chromosomal distribution of SNVs,the mutation spectrum of SNVs,harmful mutations based on SIFT score and Poly Phen2 score,and the genetic disease spectrum.The number and distribution of In Dels on three types of cells,the number of In Dels of different lengths,and the mutation types of In Dels were investigated.The number and distribution of SVs and fusion genes on three types of cells were also investigated.3.The effects of PRIM2 mutation on telomere length in different stages of hematopoietic differentiationThe same SNV of PRIM2 was found in three cell models both exposed to HQ and 1,4-BQ after further analysis of WES data.It was found that PRIM2 can affect the POLA1/TERT pathway,thereby affecting telomere length by protein interaction analysis.The DNA of three cell models treated with HQ and 1,4-BQ was extracted,and the relative telomere length(RTL)of cells was measured by quantitative real-time polymerase chain reaction(q RT-PCR).The RNA of three cell models treated with HQ and 1,4-BQ was extracted,and the relative expression levels of PRIM2,POLA1,TERT,and telomerase related genes DKC1 and TERC were measured by q RT-PCR.The proteins of three cell models treated with HQ and 1,4-BQ were extracted,and the relative expression levels of PRIM2,POLA1 and TERT were measured by western blotting(WB).4.Study on the effects of benzene exposure during pregnancy on hematopoietic toxicity in pregnant mice and their offspringFinally,animal experiments were conducted to investigate the effects of maternal exposure to benzene during pregnancy on hematopoietic toxicity in both the mother and offspring mice,while verifying the effects of the PRIM2/POLA1/TERT pathway and telomere length.Female and male C57BL/6 mice were matched in 1:1,and vaginal plugs were checked on the second day.If there were vaginal plugs,the female mice were in a pregnant state,and that day was recorded as 0.5 days of pregnancy.Pregnant mice were divided into four groups,with 12 mice in each group.Corn oil was used as a solvent,and mice were subcutaneously injected with benzene at concentrations of 0,2,10,and 50 mg/(kg·d)until the pregnant mice gave birth.After the female mice give birth,6 female mice in each group are randomly selected and killed to test their relevant indicators.The remaining 6 female mice continued to breastfeed their offspring until 3 weeks after weaning.At this time,both the mother and offspring mice were euthanized to detect relevant indicators.The body weight and organ weight of mother mice and offspring weaned mice were measured and organ coefficients were calculated.Peripheral blood cells were analyzed by automated hematology analyzer.The proportion of hematopoietic stem cells(Lin~-Sca-1~+c-Kit~+,LSK)in bone marrow was detected by flow cytometry.The DNA RTL of bone marrow cells was measured by q RT-PCR on weaned mouse.The relative expression levels of PRIM2,POLA,TERT,DKC1 and TERC were measured by q RT-PCR.The relative proteins expression levels of PRIM2,POLA1,and TERT were measured by WB.Results1.Establishment of hematopoietic differentiation model based on human induced pluripotent stem cells and study on the toxic effects of benzene metabolitesThis study first identified the morphology of stably cultured hiPSCs,and it was observed that they grew in a clone like manner with tight intercellular adhesion,high nucleocytoplasmic ratio,and distinct nucleoli.Immunofluorescence identification revealed that stable cultured hiPSCs highly expressed specific markers of pluripotent stem cells,including nuclear markers OCT4,SOX2,and NANOG,and cell surface markers including SSEA4 and TRA-1-60.Karyotype identification revealed that the copy number of the autosomes was around 2,while the copy number of the X chromosome was 1.The results of the study on the differentiation of the three germ layers showed that all cells in the three germ layers expressed the related germ layer marker genes.EBs with consistent size and normal morphology were constructed from hiPSCs by using EBs formation medium.A 7-day spontaneous differentiation three germ layer model and a 12-day hematopoietic stem progenitor cell differentiation model were successfully constructed based on EBs.After HQ and 1,4-BQ treated,the m RNA expression of pluripotency related marker genes OCT4 and NANOG in hiPSCs were significantly reduced.The m RNA expression of ectodermal related genes(PAX6,OTX2,NES and DLK1),the mesoderm related genes(NCAM1,SAVA,HAND1 and MIXL1)and the endodermal related genes(CXCR4,GATA6,SOX17 and FOX2)in TLCs were significantly reduced.The m RNA expression of hematopoietic differentiation related marker genes RUNX1 and SCL in HSPCs were significantly reduced.With the increase of HQ and 1,4-BQ concentrations,the apoptosis of hiPSCs and TLCs significantly increased,and the proportion of CD34~+cells significantly decreased.2.The effects of benzene metabolites on genetic variation in different stages of hematopoietic differentiationThrough WES analysis of cells in the control,HQ treated and 1,4-BQ treated groups,it was found that common SNVs caused by HQ and 1,4-BQ were 1275,1135 and 944 in hiPSCs,TLCs,and HSPCs respectively.Common SNVs were mainly distributed in the intron,exon,and inter gene regions.The mutation spectrum of common SNVs was mainly characterized by A>G/T>C and G>A/C>T mutations,while the SNVs in the exon regions were mainly non synonymous mutations.The numbers of non synonymous mutations on hiPSCs,TLCs,and HSPCs were 102,117,and 112,respectively.The numbers of harmful mutations on hiPSCs,TLCs,and HSPCs were 6,11,and 12 based on the SIFT score and Poly Phen2 score.The harmful mutation genes on hiPSCs were HLA-DRB5,MUC6,ESRRA,NPIPB6,and COLEC12.The harmful mutation genes on TLCs were OR2T8,ZNF717,FAM185A,PRSS1,BRI3BP,and SLC25A5.The harmful mutation genes on HSPCs were ZNF717,SLC9B1,HLA-DRB5,PRSS1,MUC6,ESRRA,and MAP2K3.OMIM analysis found that the diseases related to hematopoietic development in hiPSCs mainly included schneckenbecken dysplasia,WHIM syndrome,thrombophilia,type 2 anemia,platelet PLCB2 deficiency,and chronic myeloid leukemia.In TLCs,the diseases related to hematopoietic development mainly included schneckenbecken dysplasia,WHIM syndrome,osteogenesis imperfecta,hyperphenylalaninemia,and bleeding disorders.In HSPCs,the diseases related to hematopoietic development mainly included type 2 hyperproliinemia,schneckenbecken dysplasia and fanconi anemia.The numbers of common In Dels caused by HQ and 1,4-BQ on hiPSCs,TLCs,and HSPCs were 605,515,and 580,respectively.Common In Dels mutated mainly within 3 bases.The mutation regions were mainly in the intron region,exon region,and inter gene region.The common In Dels in the exon region were mainly non frameshift mutations.The numbers of common SVs caused by HQ and 1,4-BQ on hiPSCs,TLCs,and HSPCs were 40,17,and 22,respectively.The mutation regions were mainly in the intron and exon regions.The number of common fusion genes caused by HQ and 1,4-BQ on hiPSCs,TLCs,and HSPCs is 50,11,and 10,respectively.The main genes involved in gene fusion are TDG,KIR2DL3,and KIR2DL1.3.The effects of PRIM2 mutation on telomere length in different stages of hematopoietic differentiationThrough further analysis of SNVs data,six genes with mutation site changes were screened out for three cell models after treated with HQ and 1,4-BQ,including SLC35D1,PRIM2,ANKRD20A4-ANKRD20A20A20P,WASH8P,GOLGA6L4,and SLC35G4.The mutation site of SLC35D1 was located in the intron region.The mutation sites of PRIM2 and SLC35G4 were located in the exon region.The mutation sites of WASH8P and GOLGA6L4were is located in the intergenic region.The mutation site of ANKRD20A4-ANKRD20A20P was located in non-coding RNA region.The PRIM2 gene was selected for further research.Protein-protein interaction analysis found that related proteins included PRIM1,POLA1,POLA2,WRAP53,and TERT.The interaction relationships included direct interaction,co-expression,co-localization,and prediction of the existence of interactions.The main functions of these proteins include telomere maintenance,telomere assembly,DNA replication,and DNA biological processes.Ultimately,it was determined that PRIM2 can affect the POLA1/TERT pathway,and then affected telomere length.The RTL results showed that compared with the control group,RTL was significantly reduced in all three cell models of the high-dose HQ and 1,4-BQ treatment groups.The q RT-PCR results showed that the relative expression level of the PRIM2/POLA1/TERT pathway genes decreased with increasing exposure concentration,while the relative expression levels of telomerase related genes TERC and DKC1 genes also significantly decreased.The WB results also found that the relative protein expression of the PRIM2/POLA1/TERT pathway decreased with increasing exposure concentration.4.Study on the effects of benzene exposure during pregnancy on hematopoietic toxicity in pregnant mice and their offspringThe results of animal experiments in vivo showed that the body weight of pregnant mice in each group increased significantly with the increase of pregnancy days,and there was no significant difference in weight between groups when giving birth.There were no statistically significant differences in the organ coefficients of the liver,kidney,and spleen among the pregnant mice of each group.Peripheral blood tests showed no significant differences in the red blood cell count(RBCs),levels of hemoglobin(Hb),and PLTs count between the exposed group and the control group pregnant mice.The WBCs count in the medium and high dose group pregnant mice was significantly lower than the control group mice.The proportion of LSK cells in the bone marrow of the pregnant mice in the medium and high dose group were significantly lower than those in the control group.The offspring mice exposed to the medium and high dose groups during the fetal period showed delayed development,and the body weights of the offspring weaned mice in the medium and high dose groups were significantly lower than the control group.There were no statistically significant differences in the organ coefficients of the liver,kidney,and spleen of offspring weaned mice from each group.There were no significant differences in the RBCs count,levels of Hb,and PLTs count of weaned mice among different groups.The WBCs count of offspring weaned mice in the high-dose group was significantly lower than the control group.The proportion of LSK cells in the bone marrow of offspring weaned mice in the medium to high dose group were significantly lower than those in the control group.The RTL measurement results of bone marrow cells in offspring weaned mice showed that the RTL of bone marrow cells in the high-dose group was significantly lower than the control group.The q RT-PCR results showed a significant decrease in the relative expression levels of the PRIM2/POLA1/TERT pathway and telomerase related genes TERC and DKC1 in bone marrow cells.The WB results also found that the relative protein expression of the PRIM2/POLA1/TERT pathway decreased with increasing exposure concentration.ConclusionThis study successfully constructed a self-renewal model of hiPSCs,a spontaneous differentiation three germ layer model based on EBs,and a hematopoietic stem progenitor cell differentiation model based on EBs in vitro.Animal toxicity experiments during pregnancy in vivo were also performed.The study firstly found that pluripotency,three-germ layer and hematopoietic differentiation related marker genes were significantly reduced after HQ and1,4-BQ treated.The WES results confirmed the occurrence of genetic variations related to hematopoietic development after exposed to HQ and 1,4-BQ.At all stages,the mutation of PRIM2 gene was found and further affect the POLA1/TERT pathway and RTL.This study provided more theoretical basis for the potential hematopoietic toxicity of maternal exposure to benzene in the embryonic stage on offspring.