The Study On The Toxicological Mechanism Of Decabromodiphenylethane And Polystyrene Microspheres On A549 Cells And Caco-2 Cells

Decabromodiphenylethane(DBDPE)is a new type of brominated flame retardant,and has been detected in various environmental media and even human bodies.The diameter of the micro plastic is less than 5 mm.It is widely found in water,soil,atmosphere and even in organisms.Due to the mass production and wildly used of polystyrene(PS)and DBDPE,the release amount of both is also increasing but the degradation is difficult.This situation will cause an increased possibility for both of them coexistence in the environment.Therefore,it is of great significance to explore the toxicological mechanism of the single and combined effects in vitro.OBJECTIVE: To study the adverse reactions and related molecular mechanisms of single and combined DBDPE and PS microspheres(5 ?m)on two human cell lines(A549 cells and Caco-2 cells).This study will help complement the toxicological data of DBDPE and PS microspheres and provide a theoretical basis for assessing their potential risks to human health.METHODS: In the DBDPE single exposure experiment,cell viability was measured by the MTT method.Then,three concentration exposure groups were set according to cell viability(cell viability ?80%).After exposure of these three concentrations of DBDPE for 24 h,cell morphology was observed.And then,we determined oxidative stress indicators,apoptosis,apoptosis-related genes and protein expression levels.PS microsphere single exposure experiments showed that the viability of two kinds of was changed in after exposure to different concentrations of PS microspheres for 24 h.The oxidative stress indicators of the cells were detected,transcriptome sequencing and differentialgenes obtained by transcriptome sequencing were verified,and Analysis of related protein expression levels.According to the three single exposure experimental concentrations of DBDPE and PS,cell morphology was observed after 24 h in the combined exposure experiment,and then,cell viability,oxidative stress index and related gene expression levels were determined.After DBDPE and PS single and combined exposure,the changes of various indicators in the two cells are as follows:(1)The morphology and viability of A549 cells and Caco-2 cells changed after DBDPE exposure for 24 h.Cell viability decreased with exposure concentration increased.The detection of cellular oxidative stress index indicated that DBDPE can increase both levels of MDA and ROS,but decreased the SOD activity and GSH content.This change broken the balance of oxidation/reduction within cell.The expression of apoptosis-related genes showed that after exposure to 100 ?g/L DBDPE,the expression of Fas and Caspase-8 in A549 cells increased,which would activate the death receptor pathway.In Caco-2 cells,the expression of p53,Bax/Bcl-2 and Caspase-9 genes increased,which activated the mitochondrial pathway.Finally,the two cells induced apoptosis by activating Caspase-3.(2)The morphology and viability of A549 cells and Caco-2 cells did not significantly change after PS microspheres exposure for both 24 h.By detecting oxidative stress indicators,we found that high concentrations of PS microspheres(50mg/L)can reduce CAT activity in Caco-2 cells,while SOD activity,GSH content and MDA content in both cells were no significant difference,indicating that PS microspheres had less effect on cellular oxidative damage.Compared with the control,PS microspheres significantly down-regulated the expression of proliferation-related genes(Ras,ERK,MEK,CDK4,and Cyclin D1)and phosphorylation level of ERK1/2protein of two kinds of cells,and inhibited cell proliferation by activating the Ras-MAPK pathway.In addition,for Caco-2 cells,the expression of inflammation-related genes TRPV1,iNOS,IL-? and IL-8 were significantly increased after exposure to PS microspheres,indicating that it can induce Caco-2 cells produces an inflammatory response.(3)After the combined exposure of DBDPE and PS microspheres,the viability of A549 cells and Caco-2 cells was significantly reduced,and the expression of apoptosis-related gene Caspase-3 was significantly increased.However,these changes were not significant compared to DBDPE single exposure.This indicates that combined exposure leads to decreased cell viability and apoptosis may be caused byDBDPE alone.After 24 hours of combined exposure,the expression of proliferation-related genes(CDK4 and Cyclin D1)in the two cells decreased significantly compared with the control(P value < 0.05),indicating that cell proliferation was inhibited.The decrease in the CDK4 and Cyclin D1 relative to the control in all the combined exposure groups of two kinds of cells was greater than or equal to the decrease in the single exposure groups(except for 0.05 mg/L DBDPE +50 mg/L PS and 0.1 mg/L DBDPE + 12.5 mg/L PS combined exposure group in A549cells).These results suggest that DBDPE and PS microspheres may increase the inhibitory effect of two kinds of cells through addition or synergy.In summary,single exposure of DBDPE can cause oxidative damage in cells,inducing apoptosis of A549 cells and Caco-2 cells through death receptor pathway and mitochondrial pathway.PS microsphere exposure can inhibit cell proliferation and induce cells inflammation.Combined with the results of single exposure,combined exposure also induces apoptosis,but this may be related to the effect of DBDPE.However,compared with single exposure,combined exposure has more obvious inhibition of cell proliferation.Therefore,when evaluating the toxicity of pollutants,we should not only focus on the toxic effects of single pollutants,but also investigate the toxicological mechanism of pollutants.This will provide more effective guidance for environmental risk assessment.