The Toxic Effects And Mechanism Of Man-made Nanoparticles Of ZnO To Flounder Cells FG And Zebrafish Embryos

Nanosized particles of zinc oxide （nZnO） have been widely used in the textiles,cosmetics, toothpaste and antibacterial agents, and so on, in our daily life since theirunique features like the light-catalysed activity, ultraviolet absorption ability andantiseptic effects. Thus more attentions have been paid to the concurrent toxic effectsof nZnO on exposed organisms.The main purposes of the present study were to examine the toxic effects of threesized particles of ZnO （30nm,80-150nm and2μm） to the in vitro cultured FG cells,derived from the gill tissues of the marine flatfish flounder, and to the embryos ofzebrafish, in order to understand the correlation of the toxic effects of ZnO particlewith its size. In the cytoxicity test, the toxic effects of three sized ZnO particles onthe growth inhibition, cellular membrane injury and apoptosis induction of theexposed FG cells were detected by the MTT assay, lactate dehydrogenase （LDH）assay, Hoechst33342/PI fluorescent dyes and DNA ladder, respectively. In theembryotoxicity test, the teratogenic potency of the three sized ZnO particles on theexposed zebrafish embryos was examined, and the inhibitive effects on the hatchingrate of exposed zebrafish embryos were calculated. In order to explore thecontribution of the released zinc ion (Zn²⁺)（from the ZnO particles） to thecorresponding toxicity of the ZnO particles, we also examined the cytoxicity ofdifferent concentration of ZnCl₂to the FG cells, as well as the amount of releasedZn²⁺into the PBS buffer by the different concentrations of the three sized ZnOparticles, respectively. In order to evaluate the correlation of the degree ofagglomeration of the three sized ZnO particles with the corresponding toxicity, wealso compared the toxic effects of ultrasonicated and un-ultrasonicated ZnO particleson FG cells, and the effects of different concentration of serum on the degree ofagglomeration of the tested ZnO particles. The results obtained in this paper will promote the understanding of the toxic effects and mechanisms of action of theman-made nanosized ZnO particles and lay a good foundation for the establishment ofbioassay systems for the detection of the toxicity man-made nanosized ZnO particleson environmental organisms.In the MTT assay, all the three sized ZnO particles showed obvious growthinhibition on the exposed FG cells in a dose-and time-dependent manner. The24h-IC₅₀values for30nm,80-150nm and2μm sized ZnO particles were5.136（3.9-6.8）,5.826（4.1-8.3）and6.473（4.3-9.7）μg/ml （95%confidence interval）,respectively. Thus the toxicity order for them is30nm＞80-150nm＞2μm,indicating an obvious correlation of the size with toxicity of the tested ZnO particles.That is, with the decrease of the size, the toxicity of the tested ZnO particles increased,and this phenomenon was more obvious when the concentration of ZnO particles isrelatively low.In the LDH assay, the LDH activity in the medium of exposed FG cells increasedwith the increase of the amount of the tested ZnO particles in a dose-dependentmanner, indicating the occurrence of cellular membrane damage by the tested ZnOparticles and the leakage of LDH enzyme from exposed FG cells into the medium.The relative LDH activity obtained ranged from25%to50%.Hoechst33342/PI fluorescent staining results showed that, all the three sized ZnOparticles could induce apoptosis and/or necrosis to the treated FG cells in adose-dependent manner. In another word, the higher the concentration of ZnOparticles, the more apoptotic and/or necrosis cells will be induced. And also, theapoptotic cells predominated the necrotic cells after exposure of FG cells to the threesized ZnO particles, even at higher concentration （100and50μ g/ml） of ZnOparticles with increased number of dead cells. However, no fragmentation of genomicDNA was detected in the FG cells exposed to the highest dose of the three sized ZnOparticles.The results obtained in the test of the released zinc ions from the ZnO particlesshowed that, all the three sized ZnO particles had released certain amount of Zn²⁺into the PBS buffer, and the amount of released Zn2+increased with the increase of theconcentration of tested ZnO particles. Like ZnO particles, ZnCl₂also impose obviousgrowth inhibition on FG cells in a dose-dependent manner. The24h-IC50values for30nm,80-150nm and2μm sized ZnO particles and ZnCl₂were0.08,0.072,0.063and0.075umol/ml, respectively. And the amount of the released Zn2+from30nm,80-150nm and2μm sized ZnO particles at the top concentration of100μg/ml were1.12±0.054,1.86±0.08and1.54±0.065ug/ml （i.e.0.023±0.001,0.029±0.0012and0.017±0.0008μmol/ml） in PBS buffer. In contrast to the corresponding IC50value,the released Zn²⁺level （no more than0.03umol/ml） was too low to account for theentire toxicity of the three sized ZnO particles on exposed FG cells. Thus it can beconcluded that the released Zn²⁺contributed partially to the cytotoxicity of the testedZnO particles.It was also found that, for all the three sized ZnO particles, the ultrasonicatedZnO particles imposed higher cytotoxicity on the exposed FG cells than theun-ultrasonicated ZnO particles, and this became more significant at the higherconcentration of50-100μg/ml. This indicates that ultrasonication treatment help todisperse the ZnO particles and reduce their degree of agglomeration, thus more ZnOparticles can go through the cellular membrane and enter into the cells. Otherwise, theun-ultrasonicated ZnO particles will reunite into larger masses, which can hinder thepenetration of the ZnO particles into the cellss, thus reduce the toxicity. We also foundthat the presence of serum helped to reduce the degree of agglomeration of ZnOparticles, thus decrease the corresponding toxicity. In short, the degree ofagglomeration of ZnO particles is another key factor in relation to their toxicity.The resuts obtained in the embryotoxicity test showed that, all the three sizedZnO particles effected the development of the exposed zebrafish embryos, resulting instagnation at9h post fertilization, and most of the stagnated embryos usually died at24h post fertilization; at72h post fertilization, for all the three sized ZnO particles,the hatching rates of the exposed zebrafish embryos decreased markedly than thecontrol；and the teratogenicity and lethality of all the three sized ZnO particles on the exposed zenrafish embryos showed no dose-dependent effects, and in particular, foreach of the tested ZnO particles, the teratogenicity percentage at9h was almost thesame as the mortality percentage at24h. But obvious dose-dependent effects on thehatching rates of the exposed zebrafish embryos were observed in each of the threesized ZnO particles. At the higher concentration of50¹00μg/ml, none of theexposed zebrafish embryos hatched. At the concentration of10μg/ml, the hatchingrates of zebrafish embryos exposed to the30nm,80-150nm and2μm sized ZnOparticles were0,11.54%±2.2%and36.7%±7.3%, respectively. The above resultsobtained indicated that the smaller the size of ZnO particles, the higher toxicity on thehatching rate of the exposed zebrafish embryo.In conclusion, all the above cytotoxic and embryotoxic results of the threesized ZnO particles indicated that, the particle size, the amount of released Zn²⁺andthe degree of agglomeration are all involved in the biological toxicity of ZnO particles.The smaller the size of ZnO particles, the higher the toxicity. The embryotoxicity ofthe three sized ZnO particles mainly appears as stagnation, mortality and reducedhatching rate.