Study On Female Reproductive Toxicity Of Titanium Dioxide Nanoparticle In Vitro

As new types of photo-catalyst, anti-ultraviolet light agents, and photoelectric effect agents, titanium dioxide (TiO₂) nanoparticles are widely used commercially in the fields of paints, cosmetics, waste water treatment, sterilization, ceramic materials, etc,it is one of the most highly manufactured nanomaterials worldwide. TiO₂ is believed to be highly inert and safe,using TiO₂ as the color additive for food had achieved the US Food and Drug Administration (FDA) approval. TiO₂ nanoparticles have a bigger surface area compared with the materials of conventional size. Surface atoms, surface energy and surface tension of TiO₂ nanoparticles increase rapidly with the decreasing diameter of particles. Their physicochemical characteristics are different from those of conventional size TiO₂ because of the small-size effects, surface effect, quantum-size effect, and macroscopic quantum tunneling effect of TiO₂. According to the existent research, the toxicity of materials had close relation with their physicochemical characteristics. However, the effects of TiO₂ to environment and human healthy were not very clear yet, so it is necessary to study the toxicity of TiO₂ to evaluate its safety and risk.In the study of male reproductive toxicity TiO₂ nanoparticles were taken up by leydig cells, and affected the viability, proliferation and gene expression. The study of toxicity of nanoparticles and bulk TiO₂ discovered that they both can affect the reproductive ability of nematode Caenorhabditis elegans and the reproductive toxicity of TiO₂ nanoparticles showed more toxicity than the bulk TiO₂. TiO₂ nanoparticles could inhibit the proliferation of several kinds of cell. Reports of TiO₂ being genotoxic are inconsistent:some of researches showed that ultrafine TiO₂ induces DNA and chromosome damage and gene mutation, while some of them showed negative results.We apply in-vitro rat preantral follicle culture system to study the effects of TiO₂ nanoparticle on the rat follicle development and oocyte maturation. Under the foundation of this we study the effect of TiO₂ nanoparticle on the proliferation of granulosa cell and if it can cause the chromosomal aberration of oocyte. The aim of this study was to study whether nanoparticle could cause female reproductive toxicity, and provide evidences of TiO₂ on its safety evaluation.1. The effects of titanium dioxide nanoparticle on the development and maturation of in-vitro rat preantral follicleOBJECTIVE: Applying in-vitro rat preantral follicle culture system to study the effects of TiO₂ nanoparticles on the rat follicle development and oocyte maturation. METHODS: Mechanically dissected ovaries to gain the preantral follicles and allocated the preantral follicles to 96-well plates. Five groups were setup randomly: three groups of 25nmTiO₂ (12.5μg/ml,25μg/ml and 50μg/ml); one negative group used medium, another control group used conventional TiO₂. After subsequently cultured individually for 10 days, the follicles were induced ovulation, observed the condition of follicle development and oocyte maturation.RESULTS:Along with the increase of dosage,the rates of survival, antral follicles and COCs releasing had a tendency to reduce(P<0.05).Comparing with the negative group ,there was no abnormal effects of conventional TiO₂ group on the follicle development and oocyte maturation; Comparing the two control groups,≥25μg/mL 25nmTiO₂ caused significant different(P<0.05) survival rate , rate of antral follicles, and caused abnormal follicle morphological changes. 50μg/mL 25nmTiO₂ caused significant reduced rate of oocyte maturation.CONCLUSION:≥25μg/mL 25nmTiO₂ could inhibit the in-vitro rat follicle development and 50μg/mL 25nmTiO₂ could reduce the rate of oocyte maturation, while the conventional TiO₂ group did not show any obviously effect on follicle development and oocyte maturation.2. The effect of titanium dioxide nanoparticle on the proliferation of rat ovarian granulosa cellOBJECTIVE: To study the effect of TiO₂ nanoparticle on the proliferation of rat ovarian granulose cell.METHODS: In this research 21²3 days SD rats were used with subcutaneous injection of PMSG, after 48h ovarian granulosa cells were collected and cultured in the DMEM-F12 medium. Cells were exposed to different doses of 25μg/ml, 50μg/ml and 100μg/ml TiO₂ nanoparticle for 24h or 48h respectively, the proliferation rate of cells was analyzed by CCK-8 assay. RESULTS:After exposed to TiO₂ nanoparticle,the morphous of granulosa cells changed, a part of cell could not adherence. 25nm TiO₂ nanoparticle depressed the proliferation of granulosa cells, the dose raised accompanying the inhibition ratio increased too. The inhibition ratio of the group of high dose after 24h and 48h exposed was 70.87% and 65.73%. Following the increase of the dosage , the survival rates of granulosa cells reduced gradually, the tendency of curves of dose-effects for 24h and 48h was consistent.CONCLUSION:≥25μg/mL 25nmTiO₂ could inhibit the proliferation of rat ovarian granulose cell in vitro.3. The effect of titanium dioxide nanoparticle on rat oocyte chromosomeOBJECTIVE: To study if TiO₂ nanoparticle and conventional TiO₂ could cause oocyte chromosome aberration. METHODS: This study based on the part one, harvested oocytes of negative control group, another control group used conventional TiO₂ and 50μg/ml 25nm TiO₂. After added to 1% sodium citrate for 30min, used stationary liquid ( glacial acetic acid and methanol) to oocytes. Giemsa staining solution dyed slide 30min. Tap water wash slide, observed them after drying. One hundred of oocytes were observed for each group, and computed the chromosome aberration rate.RESULTS:Each chromosome aberration rate was less than 5%, comparing to the negative control, the difference has no statistical significance (p<0.05). This study showed that 50μg/ml 25nm TiO₂ has not obvious effects on chromosome aberration of oocyte, it may has not genetoxic.CONCLUSION: TiO₂ nanoparticle has no obvious effects on chromosome aberration of oocyte under the dosage which could cause remarkable follicle toxicity.Summary1. TiO₂ nanoparticle could inhibit the in-vitro rat follicle development and oocyte maturation, while the conventional TiO₂ did not show any obviously effect on follicle development and oocyte maturation.2.≥25μg/mL 25nmTiO₂ could inhibit the proliferation of rat ovarian granulose cell in vitro.3. TiO₂ nanoparticle has no obvious effects on chromosome aberration of oocyte under the dosage which could cause remarkable follicle toxicity.