Toxic Effects And Mechanisms Of Tris(1,3-dichloro-2-propyl) Phosphate On Protozoan Tetrahymena Thermophila

Tris(1,3-dichloro-2-propyl)phosphate(TDCIPP)is a typical organophosphate ester flame retardant.Due to its excellent flame-retardant properties,as well as the phase-out of brominated flame retardants(e.g.,polybrominated diphenyl ethers),the production and usage of TDCIPP has increased in recent years.Because the physical addition of TDCIPP is applied to the associated products,it is easily released into the environment.Environmental monitoring has shown that TDCIPP has a high detection rate in air,natural water,soil,and wildlife organizations,threatening the survival and reproduction of wildlife and humans.Toxicological studies have shown that TDCIPP can cause a variety of toxic effects in vertebrates,but the effects on lower organisms and multi-generation toxic effects remains unknown.In the present study,the free-living ciliate protozoa Tetrahymena thermophila,was used as a model.Tetrahymena thermophila were exposed to TDCIPP at high concentrations(0.01,0.1 and 1 ?mol/L)for sub-chronic(5 d),and at environmentally-relevant concentrations of TDCIPP(0,300 or 3000 ng/L)for 60 d(e.g.,theoretically 372 generations)treatment followed by a period of recovery,during which T.thermophila were not exposed to TDCIPP,and phenotypic observation,RNA-Seq,RT-q PCR and transmission electron microscopy imaging technologies were used to evaluate the toxic effects of TDCIPP and explore the molecualr mechanisms of its action.The main results were as follows:(1)Acute exposure(8 h)at high concentrations(1,10,100,1000,and 10000 ?mol/L)was used to study the dose-dependent inhibition of TCDCPP on T.thermophila biomass,and the speculated EC50 was found to be 615.1 ?mol/L.Subchronic exposure to 0.01,0.1 or 1 ?mol/L of TDCIPP caused a significantly decreased the relative biomass by reducing number of cells,size of cells and quantity of cilia in a dose-dependent manner,indicating that inhibition of individual growth and proliferation was the major toxicity of TDCIPP.RNA-Seq analysis found that expression of twenty-one ribosome protein genes of ribosome large and small subunits was down-regulated and these genes were enriched in “ribosome” term in KEGG pathway analysis.Furthermore,down-regulation of these genes was accompanied by decreased ribosome quantity in rough endoplasmic reticulum and cytoplasm and enlarged ribosome size.The results of this experiment indicated that TDCIPP might affect the growth and reproduction of T.thermophila by targeting the ribosome.(2)In a multi-generation toxicity effect evaluation experiment,T.thermophila were exposed environmentally-relevant concentrations of TDCIPP(0,300 or 3000 ng/L)for about 372 generations(60-day),followed by about 372 generations of recovery.The results showed that TDCIPP caused a significantly decreased numbers of populations,sizes of individuals,number of cilia,and depth and diameter of basal bodies of cilia,and up-regulated expressions of genes(ift family of 3 genes: ift52,ift81 and ift172,and tcp-1 family of 7 genes: tcp-1-3700.m00089,tcp-1-3706.m00106,tcp-1-3715.m00106,tcp-1-3731.m00045,tcp-1-3698.m00091,tcp-1-2.m02183 and tcp-1-16.m00478)related to assembly and maintenance of cilia.In the 60-day recovery experiment,the population growth returned to the normal levels,however,the effects on individual growth,cilia number,basal bodies of cilia structure and related genes were partially restored,and most of the effects were still significant.Therefore,this study for the first time indicated that multi-generation of environmentally relevant doses of TDCIPP exposure could cause developmental and reproductive toxicity of T.thermophila,and some of the effects were not recoverable within the experimental time.(3)In order to further elucidate the molecular mechanisms of TDCIPP-causing multigenerational effects,exposure of TDCIPP to 300 and 3000 ng/L in T.thermophila for about 372 generations,and subsequent recovery of about 248 generations were carried out.The inhibitory effects of TDCIPP on the phenotype of T.thermophila were consistent with previous multigenerational exposure results.In addition,this study also found that TDCIPP caused cisternae of the rough endoplasmic reticulum were extended and resulted in loosely interconnected curvilinear profiles,and the intracellular ultrastructural changes in the recovery phase still existed.By RNA-Seq and KEGG enrichment analysis,it was found that the down-regulated differently expressed genes(DGEs)were significantly enriched in “ribosome” term.During exposure and recovery,ribosome protein genes of large and small subunits induced by TDCIPP were generally down-regulated in a dose-dependent manner,indicating that TDCIPP inhibited ribosome function.Additionally,the up-regulated DGEs were significantly enriched in “ribosome biogenesis” term,during the recovery period.It was speculated that this might be a positive recovery response of T.thermophila to the TDCIPP inhibited ribosome function.