Calcium Phosphate And Titanium Dioxide (tiO₂ ) Nanoparticles Affected Hormone Production And Cell Cycle In `granulosa Cells

Objectives: Many studies had shown that nanoparticles affects the reproductive health of wildlife and possibly of humans. In this study, we had investigated in vivo the effects of titanium dioxide (TiO₂) nanoparticles on the main organs of female mice, especially on female reproductive gland., and the toxicity of calcium phosphate and TiO₂ nanoparticles in human luteinized granulosa cells were evaluate.Methods:Female mice, aged 5 weeks, were intraperitoneally injected with 200 or 500 mg/kg of titanium dioxide (TiO₂) nanoparticles, respectively, every other day for 5 times. Control group was injected equal volume of saline. After one week of treatment, the coefficients of organs were measured ; serum biochemical parameters and gonadal hormone (E2 and P) were tested. Histology of main organs of mice were observed.Human's luteinized granulosa cells were exposed to different concentrations of calcium phosphate nanoparticles or TiO₂ nanoparticles for 72h. Calcium phosphate's or TiO₂ nanoparticles's uptake was evaluated by transmission electron microscopy. The cell cycle was assessed by flow cytometry with propidium iodide-stained (distribution of cells in G0/G1, S, and G2/M phases). The patterns of cell death (necrosis and apoptosis) were analyzed with annexin V-FITC/PI staining. P450scc mRNA, P450arom mRNA and StAR mRNA expression was determined by RT-PCR , and progesterone and estradiol levels were examined by radioimmunoassay kits.Results:Compared with control group, the coefficients of main organs were not significantly changed in both doses of 200 mg/kg group and 500 mg/kg of TiO₂ . The changes of serum ALT/AST ratio in the two treated groups (P<0.05), suggesting the small hepatic injury after treatment with TiO₂ nanoparticles. UA level in 500 mg/kg group raised significantly (P<0.05), showing that TiO₂ nanoparticles had small nephrotoxicity if treated with larger dosage. The significant change of serum alpha-HBDH in 500 mg/kg groups (P<0.05) suggested the limited myocardial damage compared with the control group. However, there were no significant histological changes in main organs, including ovary and uterus. Levels of E2 and P were not significantly changed in two doses of TiO₂ nanoparticles . TEM results confirmed that calcium phosphate nanoparticles were able to enter into human luteinized granulose cells, and distribute in the cytoplasm and intracellular vesicles under electronic microscop. An increased percentage of S phase cells cultured with nanoparticles indicated that there was an arrest at the checkpoint from phase S-to-G2/M (from 6.28±1.55% to 11.18±1.73%,P< 0.05), while a increase in the S/(G2/M) ratio implied inhibition of DNA synthesis or impairment in the transition or the S progression stage. The apoptosis study showed that the mean apoptotic population of human luteinized granulosa cells was 11.18±1.73%, however, the apoptotic population increased to 16.53±5.56%(P<0.05)in the cells treated with 100μmol/L calcium phosphate nanoparticles for 48 h . The results showed that the apoptotic population of cells increased from 5.69±3.64% to 15.76±7.02%(P<0.05)in the cells exposed to 100uM calcium phosphate nanoparticles for 72h . The apoptotic population increased from 14.51±7.10% to 27.78±15.48%(P<0.05)in the cells treated with 200μg/ml TiO₂ nanoparticles for 48 h ; while from 10.51±0 .66% to 19.21±7.54%(P<0.05)for 72h。Treatment of human luteinized granulosa cells with calcium phosphate nanoparticles at concentrations of 10μmol/L to 100μmol/L did not caused any changes in progesterone and estradiol levels, neither to P450scc mRNA, P450arom mRNA and StAR mRNA expression after a 48h or 72h incubation period. The granulosa cells treated with TiO₂ nanoparticles had the same results.Conclusions:There were slight damage in the function of liver , kidneys and heart of mice that treated with large dose of TiO₂ nanoparticles. However, there are no significant effect on the levels of E2 and P and histology of mice ovary, when treated with two doses of TiO₂ nanoparticles. In conclusion, TiO₂ nanoparticles under 500 mg/kg dose might be safe for female reproductive system. These results in vitro suggest that calcium phosphate and TiO₂ nanoparticles may slightly interrupt ovarian granulosa cells by interfering cell cycle and increasing cells apoptosis.