Combined effects of fluoride and arsenic exposure on proliferation, differentiation and beta-catenin expression in rat osteoblasts

Abstract

Objective To investigate the combined effects of fluoride (NaF) and arsenate (NaAsO2) exposure on proliferation, differentiation and bata-catenin expression in SD rat osteoblasts. Methods Osteoblasts were isolated from calvarias of twelve SD rats born in 1-3 days and cultured. The method was divided into 9 groups [F0.0As0.0 (control group), F0.5As0.0, F4.0As0.0, F0.0As0.1, F0.0As10.0, F0.5As0.1, F0.5As10.0, F4.0As0.1, F4.0As10.0] by factorial experiment design (3 factors and 2 levels). Osteoblasts were exposed to NaF (F-: 0.0, 0.5, 4.0 mmol/L, F0.0, F0.5, F4.0), NaAsO2 (As3+: 0.0, 0.1, 10.0 μmol/L, As0.0, As0.1, As10.0) and cultured for 72 hours. The proliferation and alkaline phosphatase (ALP) was determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl (MTT) and Enzyme-linked immunosorbent assay (ELISA). The expression of beta-catenin was analyzed by quantitative real-time PCR (qPCR) and Western blotting after 72 hours of experiment. Results ① There was significant difference in cell proliferation and the activity of ALP among groups after 72 hours (F= 14.022, 14.425, all P < 0.05). Compared with control group, the proliferation and the activity of ALP were significantly induced in F0.5 treated osteoblasts groups (0.313±0.023 vs 0.455±0.152, 4.46 ± 0.72 vs 6.09±0.68, all P < 0.05), and the proliferation was significantly suppressed in F4.0As0.0 group (0.029 ± 0.014, P < 0.05), the activity of ALP was significantly induced in F0.0 As10.0 group(0.156 ± 0.010, 6.29 ± 0.67) and the proliferation was significantly suppressed in F0.0As0.1 group (0.370 ± 0.029, 3.68 ± 0.45, all P < 0.05). ② There was statistical difference in beta-catenin mRNA and protein expressions among groups with F- (F= 7.782, 559.455, all P < 0.05) at As0.0 condition. There was significant difference in the expression of beta-catenin mRNA and beta-catenin protein in F0.5As0.0 group compared with control group (1.00 ± 0.32 vs 1.99 ± 0.14, 3.56 ± 0.15 vs 5.11 ± 0.26, all P < 0.05), the beta-catenin protein was significantly suppressed in F4.0As0.0 group (1.10 ± 0.02, P < 0.05). There was significant difference in the expression of beta-catenin protein of all groups with As3+ (F= 154.736, P < 0.05) at F0.0 condition. ③ Factorial analysis showed that fluoride or arsenic alone could affect the proliferation and the expression level of beta-catenin mRNA and protein (F= 82.081, 11.991, 514.741; 19.302, 8.753, 523.698, all P < 0.05), the effect of arsenic on ALP activity of osteoblasts was also the main effect (F= 17.444, P < 0.05); and there was an interaction between fluoride or arsenic to cell proliferation and the activity of ALP and the expression of beta-catenin mRNA and protein (F= 13.085, 18.157, 4.936, 426.036, all P < 0.05). Conclusions A biphasic pattern of fluoride or arsenic on proliferation and differentiation has been induced in SD rat osteoblasts. Fluoride or arsenic can affect bone metabolic by beta-catenin. Key words: Fluoride; Arsenate; Combined effect; Osteoblasts; Beta-catenin