Formaldehyde-induced Hematotoxicity In Mice With Or Without Benzene Exposoure

Formaldehyde(FA)is a ubiquitous environmental pollutant,which is classified as a human leukemogen by both the International Agency for Research on Cancer and the U.S.National Toxicology Program based on epidemiological studies.However,the biological plausibility of FA-induced leukemia is controversial.Animal studies generally reported no leukemia and related diseases associated with FA exposure.Existed animal studies of FA-induced hematotoxicity only stayed at the level of blood or bone marrow(BM),and not went deep into the level of hematopoietic stem cells(HSCs)or progenitor cells(HPCs).The present study determined the hematotoxicity of FA from three levels of blood,hematopoietic organs,and HPCs in mice,which gave a comprehensive evaluation of FA-induced hematotoxicity.As only one single factor involved in animal studies while many other factors may also play a role in epidemiological studies at the same time,inconsistency between these two different types of studies is reasonable.We hypothesized that FA may be a causative cofactor involved in hematotoxicity or leukemia.Therefore,benzene,a known leukemogen,was used to establish the mouse hematotoxicity model.Then FA-induced hematotoxicity was studied with or without BZ exposure.FA(3.0 mg/m~3,8 hr/day,5 days/week,for 2 weeks)alone could not significantly alter peripheral complete blood count(CBC)in male BALB/c mice after two weeks of exposure compared to the control group;however,all types of leukocyte and hemoglobin dramatically decreased when BZ existed(p<0.01).Moreover,compared with the BZ group(150 mg/kg diluted in com oil,once daily,5 days/week,for 2 weeks),BZ-combined with FA(BZ+FA)decreased the counts of white blood cells,lymphocytes,monocytes,and granulocytes to 59.0%(p<0.01),55.5%(p<0.01),59.7%,and 66.0%(p<0.05),respectively.The number of nucleated BM cells in femurs of mice in all three exposure groups were reduced drastically compared with the control group(p<0.01),especially the BZ+FA group.Reactive oxygen species(ROS)in the nucleated BM cells of each exposure group,malondialdehyde(MDA)and DNA-protein crosslink(DPC)content in the nucleated BM cells of the BZ group,and ROS in the spleen nucleated cells of FA group were all higher than the control group(p<0.05,p<0.01),but MDA content in nucleated spleen cells only rose in the BZ+FA group(p<0.05).Both ROS and DPC levels were higher in the BZ+FA group than in the BZ or FA group(p<0.01).BZ and BZ+FA caused obvious apoptosis in nucleated BM cells(p<0.05,compared with the control group),but FA alone did not Cleaved caspase-3 expression level was upregulated in the nucleated spleen cellk in all three exposure groups.To farther clarify the reasons why peripheral CBC and nucleated BM cells decreased in the BZ and BZ+FA groups,colony forming unit of myeloid progenitors were assayed in growth factor-containing semisolid methylcellulose culture medium.Results showed that the quantity of colony-forming unit granulocyte-macrophage(CFU-GM)and burst-forming unit erythroid(BFU-E)in all three exposure groups,especially the BZ+FA group,was drastically reduced(p<0.01)in relation to the control group.Compared with the BZ and FA groups,colonies of CFU-GM in BZ+FA group reduced by 18.3%(p<0.05)and 22.1%(p<0.01),respectively;colonies of BFU-E in BZ+FA group decreased by 33.3%(p>0.05)and 49.2%(p<0.01),respectively.ROS and apoptosis levels in CFU-GM cells were higher in each exposure group compared with the control group(p<0.05,p<0.01).DPC content only increased in BZ+FA group(p<0.01).ROS and DPC levels in BFU-E cells of BZ group were higher than the control group(p<0.05,p<0.01),but little change observed in other exposure groups.IL-3 and GM-CSF levels in the cuture supernatant of nucleated spleen cells in the BZ and BZ+FA groups were decreased obviously in relation to the control group(p<0.05,p<0.01),and the two colony stimulating factor levels in the BZ+FA group decreased more significantly(p<0.01,compared with BZ group),but no pronounced change in the FA group.EPO mRNA in kidney and IL-3Ra in myeloid progenitors of BZ and BZ+FA groups were upregulated compared to the control group(p<0.05,p<0.01),while GM-CSFRa and EPOR expression in myeloid progenitors severely downregulated(p<0.01).To further evaluate the risk of FA-induced leukemia,the recovery of hematopoietic function in mice of each treated group was assessed one week after the final exposure.BALB/c mice showed strong recovery ability.Except for monocytes and mean corpuscular volume in peripheral blood of BZ+FA group,there was no significant difference in the number of other cell types in peripheral blood of three exposure groups compared with the control group.ROS,DPC,MDA and apoptosis levels in nucleated BM and spleen cells of mice in all exposure groups were almost comparable to the control group.H&E staining showed that BM recovered well,but there were still pathological changes to a certain degree in spleen.Proliferation ability of myeloid progenitors regained after 1-week recovery period.Colonies of CFU-GM and BFU-E were no longer reduced in BZ,FA,and BZ+FA groups;on the contrary,colonies of CFU-GM increased by 12.3%(p>0.05),10.7%(p>0.05)and 21.7%(p<0.01)compared to the control group,respectively.Levels of ROS,DPC,apoptosis,IL-3Ra and GM-CSFRa expression in CFU-GM,and the generation of IL-3 in each exposure group were all amost the same as the control group.DPC and apoptosis levels in BFU-E of all treated groups were also comparable to the control group.Nevertheless,the generation of GM-CSF was lower in FA and BZ+FA groups than in the control group(p<0.05).ROS remained at a high level in BFU-E of each exposure group,especially the BZ+FA group.Moreover,expression level of EPOR in BFU-E was still downregulated(p<0.05,p<0.01).In summary,FA-induced hematotoxicity is obvious.It can cause oxidative stress and pathological changes in BM and spleen.It also downregulated the expression levels of GM-CSFR?and EPOR in myeloid progenitors,which leaded to a decrease in the number of nucleated BM cells,and colonies of CFU-GM and BFU-E.Although FA alone did not result in a significant reduction in peripheral CBC,it can promote hematotoxicity in the presence of BZ.BZ+FA not only caused serious pathological changes,oxidative damage,DNA damage and apoptosis in BM and spleen,but also drastically reduced the generation of IL-3 and GM-CSF,and downregulated expression levels of GM-CSFR? and EPOR in myeloid progenitors,which leaded to a significant decrease in CFU-GM and BFU-E colonies as well as nucleated BM cells.In addition,all kinds of leukocyte count and hemoglobin levels in peripheral blood were significantly reduced.The above indicates that there is a synergistic effect in hematotoxicity induced by FA and BZ.FA can promote and aggravate hematotoxicity in the presence of BZ.Although hematopoietic function in mice largely recovered one week after exposure,FA still seems to show some risks of hematotoxicity.It significantly reduced the generation of GM-CSF and expression of EPOR in mice regardless of whether BZ existed;however,the colony-forming units of myeloid progenitors were not reduced.In particular,colonies of CFU-GM increased significantly in BZ+FA group.It suggested that myeloid progenitors were able to respond to trace amounts of colony stimulating factors and proliferate rapidly,which caused a possible risk of transforming normal HSCs or HPCs to leukemia stem cells or progenitor cells.Therefore,the potential of hematotoxicity induced by FA can not be ignored.