Toxic Effects And Mechanisms Of Typicl Organophosphate Flame Retardants In Protozoan Tetrahymena Thermophila

Tris?2-chloroethyl?phosphate?TCEP?,triphenyl phosphate?TPHP?and tris?clorisopropyl?phosphate?TCPP?were the three typical organophosphate flame retardants,which were frequently detected in various environmental media.Previous studies reported the toxic effects of these chemicals on vertebrates,but toxic effects of which on lower trophic aquatic organisms were rarely reported.Therefore,in this study,the lower trophic aquatic organism,Tetrahymena thermophila was selected as a model to evaluate the acute toxicity of TCEP,TPHP and TCPP.In addition,the toxic effects and molecular mechanisms of TCEP and TPHP in Tetrahymena thermophila after subchronic exposure were evaluated through phenotypic observations,transcriptome sequencing analysis and real-time quantitative PCR detection.The main results were as follows:1. Acute exposure?12 h and 18 h?to great concentrations of TCEP,TPHP or TCPP in Tetrahymena thermophila showed dose-and time-dependent effects on population density and population activity.When the population density was used as a monitoring index,the median effect concentration(EC₅₀)at 12h of TCEP,TPHP and TCPP were 19.26,23.46 and42.17 mg/L,respectively,and the 18h-EC₅₀ were 11.85,17.47 and 32.27 mg/L,respectively.When the population activity was used as a monitoring index,the 12h-EC₅₀ of TCEP,TPHP and TCPP were 13.09,18.04 and 43.27 mg/L,respectively,and the 18h-EC₅₀ were 9.86,9.97 and 39.09 mg/L,respectively.2. After exposure to 0.05,0.5,or 5 mg/L TCEP for 5 days,the theoretical population,cell viability,number of cilia,and cell size of Tetrahymena thermophila decreased in a time-and dose-dependent manner.Meanwhile,RNA-Seq analysis indicated that there were significant changes in the expression levels of 2932 genes?up-regulation:1228;down-regulation:1704?in Tetrahymena thermophila in the 5 mg/L TCEP exposure group compared with the control.Further KEGG enrichment analysis revealed that 9,10,and 17genes were down-regulated in SNARE interactions in vesicular transport,proteasome,and endocytosis pathways,respectively.Therefore,exposure to great concentrations?mg/L?of TCEP might affect the development and reproduction of Tetrahymena thermophila by down-regulating the gene transcription levels of proteins related to vesicle trafficking,proteasome and endocytosis.3. After expourse to 0.05,0.5,or 5 mg/L TPHP for 5 days,the cell viability and number of cilia of Tetrahymena thermophila were significantly reduced.In addition,exposure to0.5 or 5 mg/L TPHP significantly reduced the theoretical population and cell size.At the same time,RNA-Seq identified 4105 genes up-regulated and 4487 genes down-regulated after exposure to 5 mg/L TPHP.Further analysis by KEGG enrichment revealed that these differentially expressed genes were significantly enriched in ribosome,spliceosome,phagosome,proteasome,and protein processing in endoplasmic reticulum pathways.Therefore,great concentrations?mg/L?of TPHP might affect the development and reproduction of Tetrahymena thermophila by affecting pathways related to cell cycle regulation,protein processing and synthesis utilization,biomolecule synthesis and metabolism.