The Molecular Toxic Mechanism Of The Three Phthalate Acid Esters Separate To Eiseniafetida

Phthalic acid esters (PAEs) have formed serious threat to the ecological system. Earthworm is one of the important components and largest groups which played the important role in the soil ecosystem. Earthworm has become a bioindicator organism to reflect soil pollution, which provides the scientific evidence for the rational assessment ecological risk to soils. From cells and macromolecular level, the toxic effects of the three phthalate esters (DMP, DEP, DBP) were studied individually using Eisenia foetide. Besides, human breast cancer estrogen receptor positive MCF-7cells was used to investigate estrogenic activity of DMP, DEP, DBP by in-vitro assays to make up the inadequate datas on endocrine disrupting effects of PAEs and offer scientific evidences of the toxic effects of PAEs. The results were showed as follow:(1) The acute toxic effects the single pollutions of DMP and DEP on Earthworms(Eisenia were studied by filter paper contact method. The results showed that LD50(DMP) and LD50(DEP) were129.603μg/cm2and145.336μg/cm2, respectively, when the earthworms were exposed to DMP and DEP for48 h, and earthworms showed the similar symptom after poisoned. When earthworms were exposed to DMP and DEP for14d by natural soil method, LD50(DMP) and LD50(DEP) were1560.120mg/kg and1516.186mg/kg, respectively.(2) The natural soil test method was used to study the effects of DMP, DEP and DBP on growth and the activity of earthworm enzemys. The activities of superoxide dismutase (SOD), catalase (CAT) and acetylcholinesterase (AChE) were measured, including Neutral Red Retention Time (NRRT) measurement. The results showed that the growth of earthworms decreased with the increase of the concentration of DMP, DEP and DBP and the exposure time as well. CAT activity was significantly affected by DMP, DEP and DBP concentration and the duration of exposure; SOD activity was significantly affected by DMP, DEP and DBP concentration but not the duration of exposure. The effects of application of different concentrations of DEP and DBP in soil on AChE activity in earthworms are similar:first AChE activity was inhibited, then induced, and finally recovered to the control level at28d or exhibit tendency to recovery. Neutral Red Retention Time (NRRT) for earthworms decreased significantly with the increase of the concentration of DMP, DEP and DBP and the exposure time as well.(3) The effects of17β-estradio and DMP, DEP, DBP on human breast cancer estrogen receptor positive MCF-7cells viabiility were determined by MTT assay. The effects on estrogen receptor genes expression in MCF-7cells were detected with expression of GAPDH as the reference. The results showed that10-12～10-6mol/L17β-E2and10-7～10-3mol/L DMP、DEP、DBP induced cell viability increased significantly, and the activity of cell reached the maximum at10-9moL/L and10"4moL/L,10-4mol/L,10-5mol/L, respectively. DMP, DEP and DBP could lower the expressing level of ERa mRNA、ER β mRNA and PGRMC1mRNA, and increase the level of pS2mRNA in the cell. DMP, DEP, DBP demonstrated an estrogenic activity, and estrogenic potency with increasing carbon chain length, that is DBP>DEP>DMP.