Mechanisms Of Indoor Dust Induced Toxicity In Normal Human Corneal Epithelial Cells

Indoor pollution is relevant to health because people spend most of their time indoors.In the indoor environment,the need for the reliable assessment of human exposure to indoor pollutants is still attracting increasing attention.Indoor dust is a complex heterogeneous mixture of particles of different sources,which contains both harmful microbes and various contaminants.Epidemiological studies have linked human health issues,including respiratory,allergenic,and ocular surface diseases with exposure to indoor dust.The contaminants in indoor dust are probably responsible for the adverse health effects.Human cornea is highly susceptible to damage by dust.Continued daily exposure to indoor dust has been associated with increasing risks of corneal injury,however,the underlying mechanism has not been elucidated.Human cancer cell lines and immortalized cell lines are widely used in dust toxicological studies,however,cancer cells and immortalized cell lines are demonstrated to loss many physiological features after long term passages in vitro.Thus,toxicologists suggest that primary human cells,with many in vivo physiological features are an ideal candidate for toxicology.In this context,this Ph.D.program mainly aimed to investigate and compare the pollution characteristic of indoor dust from household and office and to figure out the toxic effects and the relevant molecular mechanisms of human normal corneal epithelial cells after exposure to indoor dust or dust extracts.In addition,the molecular mechanisms of TDCPP induced human normal corneal epithelial cells apoptosis was emphatically investigated.The specific work in this program is followed as:(1)Dust samples were collected from air conditioner filter of both residential house(n ?16)and commercial offices(n = 15)in Nanjing,China using a vacuum cleaner with a paper bag.All dust samples were then mixed into one composite sample as house and office dust and sieved through nylon sieve(<100 ?m).The particle size and total organic carbon(TOC)contents and dust particle diameter in dust were characterized.Water and organic extracts were prepared from both dust samples.The concentrations of heavy metals in the dust and dust extracts were determined by inductively coupled plasma mass spectrometry(ICP-MS),with office dust having greater concentrations of heavy metals than house dust.In addition,polycyclic aromatic hydrocarbons(PAHs),organochlorine pesticides(OCPs),phthalic acid esters(PAEs),and organophosphorus flame retardants(OPFRs)in dust extracts were detected by GC-MS.The toxicity equivalent concentrations of individual PAHswere calculated based on the toxic equivalency factor(TEF)with respect to benzo(a)pyrene values.The total concentration of 16 PAHs in office dust was 25.3 mg/kg,which was N3 folds of that in house dust(7.94 mg/kg).The concentration of high molecular weight(HMW,4-to 6-rings)PAHs in office dust at 23.8 mg/kg was greater than that in house dust at 6.36 mg/kg.The toxic equivalent quantity for office dust was 5.6 folds as high as house dust.Among the five PAEs,DEHP was the most abundant PAEs in both house and office dust.For the ?5sPFRs,the house dust(51.52 mg/kg)was significantly high than office dust(5.94 mg/kg).Taken together,the concentrations of heavy metals and PAHs in office dust were higher than that in house dust,whilewith house dust having greater concentrations of PAEs,PFRs,and OCPs.(2)In this study,a composite housedust sample was tested for its cytotoxicity on normal human corneal epithelial(HCE)cells,which were exposed to dust at 5-320 ?g/100 ?L for 24 h.HCE cell viability showed a concentration-dependent toxic effect,attributing to elevated intracellular ROS.Moreover,when exposed at>20-80 ?g/100 ?L,dust-induced oxidative damage was evidenced by increased malondialdehyde and 8-hydroxy-2-deoxyguanosine(1.3-2.3 folds)and decreased antioxidative capacity(1.6-3.5 folds).Alteration of mRNA expression of antioxidant enzymes(SOD1,CAT,HO-1,TRXR1,GSTM1,GSTP1,and GPX1)and pro-inflammatory mediators(IL-1?,IL-6,IL-8,TNF-a,and MCP-1)were also observed.Furthermore,the mitochondrial transmembrane potential was dissipated from 9.2 to 82%.Our results suggested that dust-induced oxidative stress probably played a vital role in the cytotoxicity in HCE cells,which may have contributed to dust-induced impairment of human cornea.(3)In this study,indoor dust from residential houses and commercial offices in Nanjing,China was collected and the effects of organic and water soluble fraction of dust on normal HCE cells were examined.Based on LC50,organic extract was more toxic than water extract,and office dust was more toxic than house dust.Accordingly,the organic extracts induced more ROS,malondialdehyde,and 8-Hydroxydeoxyguanosine and higher expression of inflammatory mediators(IL-1?,IL-6,and IL-8),and AhR inducible genes(CYP1A1,and CYP1B1)than water extracts(p<0.05).Extracts of office dust presented greater suppression of superoxide dismutase and catalase activity than those of house dust.In addition,exposure to dust extracts activated NF-?B signal pathway except water extract of house dust.The results suggested that both water and organic soluble fractions of dust caused cytotoxicity,oxidative damage,inflammatory response,and activation of AhR inducible genes,with organic extracts having higher potential to induce adverse effects on normal HCE cells.The results based on normal HCE cells demonstrated the importance of reducing contaminants in indoor dust to reduce their adverse impacts on human eyes.(4)In this study,we investigated the cell apoptosis in normal human corneal epithelial cells(HCECs)after TDCPP exposure and elucidated the underlying molecular mechanisms.Our data indicated a dose-dependent decrease of cell viability after TDCPP exposure with LC50 at 202?g/mL.A concentration-dependent apoptotic sign was observed in HCECs after exposing to ? 2?g/mL TDCPP.Endoplasmic reticulum stress induction was evidenced by up-regulation of its biomarker genes(ATF-4,CHOP,BiP,and XBP1).Furthermore,alternation of Bcl-2/Bax expression,mitochondrial membrane potential loss,cellular ATP content decrease,and caspase-3 and-9 activity increase were observed after exposing to 2 or 20 ?g/mL TDCPP.Taken together,the data implicated the involvement of endoplasmic reticulum stress in TDCPP-induced HCEC apoptosis,probably mediated by mitochondrial apoptotic pathway.Our findings showed TDCPP exposure induced toxicity to human cornea.Due to TDCPP's presence at high levels in indoor dust,further study is warranted to evaluate its health risk on human corneas.