The Mechanistic Study Of MLL Gene Translocations And Differential Disease Gene Expressions Induced By Chlorpyrifos In Human Fetal Liver Hematopoietic Stem Cells

Objective: Chlorpyrifos(O,O-diethyl O-3,5,6-trichloro-2-pyridyl phosphorothioate,CPF)is a widely used insecticide.With long-term exposure of low level CPF had shown adverse effects on the human health,especially prenatal exposure.Epidemiological studies showed that in utero exposure to CPF during pregnancy can increase the risk of MLL gene recombinations and the incidence of acute leukemia in children,which is the main pathological feature of infant acute leukemia.In the early development of the fetus,the liver is a hematopoietic organ,which is capable of generating a large number of CD34+ hematopoietic stem cells(CD34+HSC).Xenobiotic exposure leads to DNA damage has become the basic pathogenesis of some blood diseases in utero,and mixed lineage leukemia(MLL)usually involves 11q23 gene recombinant.MLL gene rearrangements are the main pathological features of infant acute leukemia,through either Topoisomerase II(Topo II)inhibition or oxidative damage and apoptosis.This study aims to:(1)To evaluate CPF of CD34+HSC in the Topo II produced the effect;(2)To further investigate the possible mechanism underlying CPF-induced MLL gene rearrangements in CD34+HSC;(3)To identifiy the differential global gene expressions in CD34+HSC induced by CPF.Methods:(1)The establishment of human embryonic liver CD34+HSC CPF exposure system;(2)The application of Cell Counting Kit-8(CCK-8)detection of CPF after exposure to HSC activity,Lactate dehydrogenase(LDH)on HSC the release of cytotoxic reaction for detection of CPF;(3)Using neutral comet assay for DNA CD34+HSC fragmentation after CPF exposure;(4)The trapped in agarose DNA immunostaining(TARDIS)assay after exposure to CPF Topo II-DNA cleavable complexes;(5)Using fluorescence in situ hybridization(FISH)by recombinant DNA dual color probes for detecting CD34+HSC MLL gene the occurrence;(6)Using Gene Chip Human Genome U133 Plus 2 Array gene expression microarray for detection of HSC in different treatment groups of genes;(7)Using real-time fluorescence quantitative PCR technology to verify the difference Gene expression.Results:(1)Human embryo liver CD34+HSC is from the fetal liver about 18 to 24 weeks of pregnancy.On early CD34+HSC separation and purification,to include CD34-FITC labeled with BD FACS flow cytometry is extracted after verification by the human CD34 multisort kit,extraction efficiency is close to 100% and extracted purity is more than 95%;(2)Through the release of LDH is detected and exposed on 1,5,10,25,50 and 100 μM CPF concentration,cell viability of CD34+HSC is respectively 84.48%,58.62%,35.63%,35.63%,12.07% and 8.62%,exposure in positive control of the 1 μM VP-16 CD34+HSC cell viability is 58.62%,indicating that the CPF 1 μM of CD34+HSC have a significant impact.The rate of cell proliferation was detected by CCK-8 in 72 h,and the results showed that the cell proliferation inhibition rate showed a dose-dependent increase with the CPF concentration;(3)Neutral comet assay 1,10,and 50 μM CPF or 10 μM VP-16 exposure increased CD34+HSC DNA double strand breaks,and dose response relationship,specific control non genotoxic compounds in the test range(i.e.cytotoxic levels)can induce DNA double strand breaks;(4)FISH assay showed that the recombinant CD34+HSC gene can induce MLL in 1 and 10 μM CPF exposure group,respectively 0.5% and 3%,a dose-dependent increase.HSC exposed to 1 μM VP-16,FISH assay showed that the recombinant MLL gene;(5)TARDIS experiments showed that 0,10 and 50 μM CPF or 10,50 μM VP-16 in CD34+HSC under the Topo IIα cleavage complexes in vivo formation of a Topo IIβ VP-16 cleavage complexes CPF drug dose dependent.0,10 μM and 10 and 50 μM formed in the cells in a dose-dependent manner;(6)CPF 1,10 μM and positive control VP-16 1 μM for HSC differential gene expression was detected by whole genome microarray.The results showed that 1 μM CPF caused 1 genes is up-regulated and 15 genes are down regulated and 10 μM CPF has 17 up-regulated genes and 62 genes were down regulated and down regulated genes covered by the 1 μM CPF group with 13 down regulated genes,(CCL2,G0S2,TNIP3,CCL8,PHLDA2,IL7 R,TFPI2,IL6,IL1 B,CCL7,IL1 A and CXCL5),and in a dose response relationship,and 1 μM VP-16 induced 21 genes up-regulated and 14 down-regulated genes,and with CPF gene without any overlap;(7)Fluorescence quantitative PCR on the part of the expression differences of genes were verified,including three CPF low and high dose group are down regulated genes(G0S2,TNIP3 and PHLDA2),CPF 10 μM group 2 up-regulated genes(CEACAM6,CDKN1C),1 μM of VP-16 group 2 up-regulated genes(CDKN1A,MDM2)and three down regulated genes(E2F8,HELLS,CCNE2).The G0S2,TNIP3 and PHLDA2 expression trend and the microarray results are consistent,extent proved the trustworthiness of gene chip data.Other genes currently still in the validation,although after adjusting primer design,PCR condition control,but many aspects need further improve.Conclusion:(1)CPF in CD34+HSC can form DNA-Topo IIα-CPF and DNA-Topo IIβ-CPF cleavage complexes,and therefore can inhibit the activity of Topo II in HSC,which leads to DNA double strand breaks and MLL gene recombination;(2)Although CPF and VP-16 both can cause cytotoxicity,DNA injuries,Topo II inhibition and MLL gene recombinations in HSC,the underlying mechanisms and pathways can be different for the two compounds.