Research Of Toxic Effects Of Formaldehyde And Related Mechanism In BM-MSCs

Backgroud and objective: Formaldehyde (FA) is a known human carcinogen(group A1) that causes leukemia, however, the mechanisms remain unclear. Severalstudies have confirmed that can reach and exert a toxic effect on hematopoietic stemcells. However, to date, the ability of FA exposure to disrupt BM-MSCs has not beenreported. As one component of bone marrow, bone marrow mesenchymal stem cells(BM-MSCs) are notable for their multi-differentiation potential. Also, BM-MSCs canhelp hematopoietic stem cells maintain function and primary properties. Thus, ifformaldehyde induces cytotoxic and genotoxicity in BM-MSCs, BM-MSCs will losetheir normal control over hematopoietic stem cells, leading to myeloid leukemia. Thepresent study was performed to detect the cytotoxic, genotoxic, cell cycle andapoptosis effect of formaldehyde on mouse BM-MSCs, and try to elucidate themolecular mechanism, thus provides the biological evidence for the formaldehyde tolead to leukemia.Methods: Proliferation activity was measured by MTT assay; DNA damage wasdetected by comet assay, KCl-SDS precipitation assay, sister chromatid exchange(SCE) test and micronucleus (MN) test; RT2profiler PCR array was used toinvestigate the changes in the expression of important genes in DNA damagesignaling pathways; Cell cycle and apoptosis of BM-MSCs were detected by usingflow cytometry and phalloidine/hoechst33258double dyed cells, respectively. Andwestern blot method was used to detect the expression of proteins related cell cycleand apoptosis.Results:(1)75~200μmol/L formaldehyde can inhibit the BM-MSCsproliferation (P<0.01) in a dose-dependent manner and without a time-dependentmanner.(2) The standard alkaline comet assay showed that formaldehyde-induced DNAstrand breakage increased gradually at increasing concentrations below125μmol/L. However, when formaldehyde concentration was over125μmol/L, DNA strandbreakage decreased. There are significant differences (P<0.01) between75~150μmol/L formaldehyde group and the control group. On the other hand, the proteinaseK-modified comet assay showed that formaldehyde-induced DNA strand breakagewas increased in a dose-dependent manner from75to200μmol/L. OTM values ofproteinase K modified comet assay were significiant higher than that of standardcomet assay in125μmol/L and above groups (P<0.01).(3) Induction of DNA-protein crosslinks, SCE in≥125μmol/L and MN in≥150μmol/L formaldehyde groups were significant higher than in control group (P<0.01).(4) Xpa and Xpc of the nucleotide excision repair (NER) pathway and Brca2,Rad51and Xrcc2of the homologous recombination (HR) pathway were allup-regulated in both75and125μmol/L formaldehyde. However, same genes weredown-regulated with175μmol/L formaldehyde. The expressions of Chek1and Hus1,which are involved in cell cycle regulation, were altered in the same manner with75,125and175μmol/L formaldehyde.(5) G2cell percentages in≥125μmol/L formaldehyde groups were significanthigher than in control group (P<0.01).(6) BM-MSCs exposed to≥125μmol/L formaldehyde were shrinking,cytoskeleton arrange turbulence, nuclear enrichment, hyperchromatic and split and theformation of apoptotic bodies, the apoptosis rates were significantly higher thancontrol (P<0.01).(7) The expression of P53and ERK1/2protein in75,125and175μmol/Lformaldehyde groups were no significant difference compared with control (P>0.05);The expression of p-P53, Chk1and Bax protein at75,125and175μmol/Lformaldehyde were significantly higher than control (P<0.01), and p-ERK1/2weresignificantly lower than control (P<0.01); The expression of Bcl-2protein in125,175μmol/L formaldehyde were significantly lowerthan control (P<0.01).Conclusions:(1) Formaldehyde has cytotoxic and genotoxic effects onBM-MSCs, thus will provides the biological evidence for the formaldehyde to lead toleukemia.(2) Formaldehyde could induce DNA strand breakage in a dose-dependentmanner, but also induce the formation of DPCs, SCE and MN.(3) Formaldehyde makes BM-MSCs cell cycle arrest in G2phase, and causeBM-MSCs apoptosis at≥125μmol/L. (4) Xpa, Xpc, Brca2, Rad51, Xrcc2, Chek1and Hus1were essential for DNArepair of BM-MSCs after treatment with formaldehyde.(5) P53, Chk1, ERK1/2, Bax and Bcl-2were participate in the formaldehydeinduced BM-MSCs G2cycle arrest and apoptosis.(6) The comet assay modified with proteinase K may be a suitable method forthe detection of DNA strand breakage induced by formaldehyde.