| Plasticizer is a kind of polymer additives. It is widely used in plastic products toimprove the flexibility of plastic material. Because of its high mobility in theenvironment, it has become one of the most widespread pollutions in the world. Theoutbreak of "plasticizer storm" in Taiwan in2011has attracted more and moreattentions on the toxicity of plasticizer all over the world. Due to the fact that theaquatic organisms usually fertilized and developed in vitro, the reproductive anddevelopmental toxicity of plasticizer may interrupt the future of aquatic organisms.Up to date, studies on the toxicity of plasticizers to aquatic organisms are scarce, andsystematic and effective methods specific for the evaluation of the toxicity ofplasticizer have not yet been established, thus limiting the prevention andmanagement of plasticizers.Firstly, we examined the acute toxic effects of the three kinds of plasticizers ofdiethylhexyl phthalate (DEHP), dibutyl phthalate (DBP) and acetyl tributyl citrate(TBAC) to the in vitro cultured FG cells, derived from the gill tissues of the marineflatfish flounder. In the cytotoxicity test, the toxic effects of the above-mentionedthree plasticizers on the cellular activity, inhibition of cell growth, damage of cellularmembrane and induction of apoptosis in the exposed FG cells were analyzed by MTTassay, neutral red (NR) absorption assay, lactate dehydrogenase (LDH) release assay,acridine orange and ethidium bromide (AO/EB) double fluorescent dye staining andDNA ladder assay, respectively.Observations from the cell morphology of the exposed FG cells to all the threeplasticizers demonstrated cytotoxic effetcs. The cells started to shrink and becamerounded in shape. The number of rounded cells increased in a dose-and time-dependent manner for all the three tested plasticizers. The lowest observed effectconcentration (LOEC) for DEHP, DBP and TBAC were150μg/mL,50μg/mL and150μg/mL, respectively.In the MTT assay, it was found that, all the three kinds of plasticizers testedinhibited the dehydrogenase activity of the exposed FG cells. At the lowconcentrations (0.005~50μg/mL), a similar and low cytotoxicity was obtained in theexposed cells for all the three plasticizers tested. The inhibition rates for cellularactivity after24h and48h exposure were about96%and94%, respectively. At theconcentration of150μg/mL，significant cytotoxicity was observed in the exposedcells for all the three plasticizers tested with the toxicity order of DBP>TBAC>DEHP.The inhibition rates after24h exposure to DEHP, DBP and TBAC were96±0.19%,80.8±3.7%and91.9±3.9%, respectively. And the inhibition rates after48h exposureto DEHP, DBP and TBAC were94.8±1.5%,62.8±2.5%and73.8±0.58%, respectively.The results obtained showed that the cytotoxicity of plasticizers DEHP, DBP andTBAC was time-dependent, i.e., the cytotoxicity of plasticizers to FG cells increasedwith the exposure time.In the NR absorption assay, all the three plasticizers tested produced obviousgrowth inhibition on the exposed FG cells in dose-and time-dependent manner, thetoxicity order for them is DBP>TBAC>DEHP. At the concentration of0.005μg/mL,the inhibition rates after24h exposure to DEHP, DBP and TBAC were96.7±0.79%,95.8±0.65%and95.9±1.1%, respectively. Likewise, the inhibition rates after48hexposure to DEHP, DBP and TBAC were95.9±1.1%,93.3±0.8%and94.1±1.4%,respectively.The24h-IC50values for DEHP, DBP and TBAC were290.4(242.0-348.6),144.8(99.23-211.2) and217(176.3-267.2) μg/mL (95%confidence interval), respectively; the48h-IC50values for DEHP, DBP and TBACwere213.8(166.6-274.3),108.3(71.05-165.0) and135.8(97.54-189.1) μg/mL (95%confidence interval), respectively. Thus, the NR absorption assay seems moresuitable for detecting the toxic effects of the three above-mentioned plasticizers to FGcells. In the LDH release assay, the LDH activity released into the supernatant mediumof the exposed FG cells increased in a dose-dependent manner for all the threeplasticizers tested, and the toxicity order for them is also DBP>TBAC>DEHP. At theconcentration of0.005μg/mL, low relative LDH activities after24h exposure toDEHP, DBP and TBAC were obtained, they were21.1±5.8%,33.4±0.8%and30.2±0.8%, respectively; at the concentration of150μg/mL, higher relative LDHactivities after24h exposure to DEHP, DBP and TBAC were obtained, and they were48.4±0.5%,72.6±1.6%and52.2±5.7%, respectively. The results obtained indicatedthat the cellular membrane had been damaged by the plasticizers tested and thus theLDH enzymes had been leaked from the exposed FG cells into the supernatant of themedium.The results of AO/EB double fluorescent dye staining showed that, percentage ofapoptotic cells induced by the three plasticizers tested is very low, and even at thehigher concentration of150μg/mL, the apoptosis percentage in the FG cells exposedto DEHP, DBP and TBAC for24h were only2.4%,28%and6%, respectively. Thiscan account for the failure to detect the DNA ladder in the exposed FG cells.Secondly, we compared the embryo toxicity of the above-mentioned threeplasticizers using zebrafish embryos. In the embryo toxicity test, the terotogenicity bythe three plasticizers in the early stage of development of zebrafish were examined,and the lethality at24h post fertilization (hpf) and the hatching rates at72h postfertilization in the exposed zebrafish embryos were calculated. The results obtained inthe embryo toxicity test showed that,(1) all the three plasticizers affected thedevelopment of the exposed zebrafish embryos, resulting in autopepsia at4hpf, slowepiboly at8hpf, failure in the formation of somites at12hpf, failure in the formationof head at24hpf, reduction or lack of pigment at48hpf, decrease in hatching rate andappearance of pericardial cyst at72hpf, spinal curvature at96hpf;(2) results for thelethality test at24hpf showed that the embryonic death rate increased in adose-dependent manner. At the concentration of0.005μg/mL, the death rates ofzebrafish embryos exposed to DEHP, DBP and TBAC were16.7±5.8%,76.7±7.6% and18.3±2.9%, respectively. The toxicity of DBP to zebrafish embryos was thehighest, and all embryos died at the concentration of50and150μg/mL. However,only65±5%and75±5%of the zebrafish embryos exposed to150μg/mL of DEHPand TBAC died, respectively;(3) with the increase of the dose of the plasticizers, thehatching rates at72hpf decreased rapidly in a dose-dependent manner. At theconcentration of0.005μg/mL, the hatching rate of zebrafish embryos exposed toDBP was as low as16.7±8.2%. But at the concentration of50and150μg/mL, thehatching rate of zebrafish embryos exposed to DBP decreased to0, i.e., no embryossuccessfully hatched. However, at the concentration of150μg/ml, the hatching ratesof zebrafish embryos exposed to DEHP and TBAC were21.7±12.6%and10±13.2%,respectively.Finally, the possible toxic mechanisms by these three different plasticizers werediscussed. TBAC has toxic effetcts on both FG cells and zebrafish embryos and itstoxicity is higher than that of DEHP. However, the result is not consistent with thepreviously reported toxic effects of TBAC.In conclusion, the NR absorption assay is more suitable for detecting the toxiceffects of the three kinds of plasticizers to FG cells than MTT assay, and the result isconsistent with what showed in the LDH release assay. The three plasticizers of DEHP,DBP and TBAC may not induce the death of FG cells by apoptosis. There exits threatto the zebrafish embryos by the plasticizers of DEHP, DBP and TBAC, evidenced bythe presence of teratogenicity, embryonic lethality and reduced hatching rate. All thetoxic data obtained in both FG cells and zebrafish embryos showed the same toxicityorder of DBP>TBAC>DEHP. |
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