Combined Toxicity Of Co-exposure To Formaldehyde And Acrolein On Human Lung Cells

Aldehydes are common air pollutants,which are closely related to the occurrence and development of respiratory diseases.They often coexist in cigarette smoke,cooking oil smoke,automobile exhaust and so on.However,combined toxic effects and mechanisms of aldehyde mixtures have not been completely understood so far.For understanding the possible joint toxicity of aldehydes,formaldehyde and acrolein were chosen as the typical representatives of saturated and unsaturated aldehydes,respectively,to obtain a simple model of aldehyde mixtures.Based on human bronchial epithelial BEAS-2B cells and human lung adenocarcinoma A549 cells,the combined effects of aldehyde mixtures were evaluated in terms of cytotoxicity and genotoxicity,then mechanisms of joint toxicity of aldehyde mixtures were further explored.This study can provide references in fields of revealing the potential risks associated with respiratory diseases and the prevention research about co-exposure to aldehyde mixtures.The primarily results are as follows:1.Combined cytotoxicity:Cell counting kit-8(CCK-8)assay,colony formation assay and apoptosis assay were used to evaluate the cytotoxicity after 1-24 h of(individual or combined)treatment with formaldehyde and acrolein at concentrations varying from no observed effect concentrations(NOECs)to sub-cytotoxic concentrations(?IC20)based on uniform design method.The combined action was analyzed with two way ANOVA and Interaction Factor(IF).The results showed that individual and combined exposure of formaldehyde and acrolein could increase the cytotoxicity and cell death in the concentration/time-dependent manner.Formaldehyde and acrolein primarily showed synergism on inducing cytocxixcity.Based on the apoptosis,the mechanism of synergistic cell death induced by formaldehyde and acrolein mixtures was further discussed in terms of oxidative stress-related apoptosis pathway and DNA damage-related apoptosis pathway with using flow cytometry,immunofluorescence and western blotting.The results illustrated that(1)formaldehyde could potentiate acrolein-induced apoptosis through TRAIL death receptor and mitochondrial pathway;(2)aldehyde mixture could synergistically induce reactive oxygen species(ROS),lipid peroxidation,elevation of intracellular Ca2+ level,mitochondrial membrane potential change and nuclear factor-E2-related factor 2(Nrf2)pathway,which resulted in imbalance of redox homeostasis;(3)formaldehyde potentiated acrolein-induced deoxyribonucleic acid(DNA)damage,and inhibition of p53 and AKT pathway;(4)oxidative stress inhibitor inhibited ROS and DNA damage,as well as blocked death receptor and mitochondrial pathway,but could not rescue p53 and AKT pathway.Therefore,formaldehyde and acrolein mixtures could elevate apoptosis through ROS-mediated death receptor and mitochondrial pathway,and inactivation of p53 and AKT pathway.2.Combined genotoxicityWith the same exposure style,concentration,time,concentration combination and combined action evaluating,?H2AX assay,single cell gel electrophoresis test,micronucleus test and hypoxanthine-guanine phosphoribosyl transferase(HPRT)gene mutation assay were used to evaluate the individual and combined genotoxicity of formaldehyde and acrolein in terms of DNA single/double strand break,chromosome damage(mononuclear micronucleus(MMN)and cytoplasmic block micronucleus(CBMN)assay)and gene level.The results suggested that single aldehydes and aldehyde mixtures induced DNA single/double strand break and formation of MMN in a sigmoid or linear concentration/time-effect relationship,but an inverted-U concentration/time-effect relationship was found for inducing CBMN and HPRT mutation.Additionally,formaldehyde and acrolein had synergistic effects on DNA single/double strand break and formation of MMN,but had antagonistic effects on HPRT gene mutation and formation of CBMN.Based on combined effects of formaldehyde and acrolein mixtures on genotxicity,the mechanism of combined genotoxicity was further discussed in terms of indirect mechanism(oxidative stress),direct mechanism(DNA-protein crosslink(DPC)),cell sycle and DNA damage-repair pathway with using flow cytometry,PCR array assay and western blotting.The results implied that(1)formaldehyde-induced DNA damage primarily resulted from DNA-protein crosslink(DPC),while acrolein-induced DNA damage was mainly caused through oxidative stress;(2)although aldehyde mixture-induced DNA damage included the formaldehyde-induced DNA damage by DPC,the total DNA damage was mainly caused by acrolein-induced oxidative DNA damage;(3)formaldehyde potentiated acrolein-induced cell stress,cell cycle regulation,up-regulation of DNA damage-related apoptosis genes and down-regulation of DNA repair-related genes/proteins,which enhanced aldehyde mixture-induced DNA strand breaks and MMN formation.Meanwhile,aldehyde mixture-induced antagonistic effects on CBMN formation and HPRT gene mutation resulted from inactivation of DNA repair and toxicity of cell division induced by formaldehyde and acrolein mixtures.Briefly,formaldehyde promoted acrolein-induced oxidative stress,which resulted in that co-exposure of formaldehyde and acrolein induced synergistic effects on DNA strand breaks and formation of MMN.Meanwhile,because of inactivation of aldehyde mixture-induced DNA repair,the synergistic DNA and chromosome damage was further enhanced,which not only further resulted in the synergistic cytotoxicity and cell death(which could be mediated through death receptor and mitochondrial apoptotic pathway together),but also led to antagonistic HPRT gene mutation and formation of CBMN.