ABSTRACT
Objective:To investigate the effects of combined exposure of fluorine, arsenic, and fluorine-arsenic on the signaling pathway related protein expression of tumor necrosis factor receptor-related factor 6 (TRAF-6)/nuclear factor κB1(NF-κB1) in a co-culture system of mouse osteoblasts MC3T3-E1 and mouse monocyte macrophage RAW264.7.Methods:MC3T3-E1 cells were co-cultured with RAW264.7 cells after induction with osteogenic inducers. The cells were cultured for 7 days in vitro, and different doses of sodium fluoride (0.0, 0.1, 0.4, 1.6 mmol/L NaF, F), sodium arsenite (0.0, 0.5, 2.5, 12.5 μmol/L NaAsO 2, As) and different doses of fluorine and arsenic were added to the culture medium and cultured for 24 h using factorial design. The expression levels of nuclear factor κB receptor activating factor (RANK), TRAF-6, NF-κB1, T cell activating factor (NFATc1), and tartrate-resistant acid phosphatase (TRAP) protein were detected by Western blotting. Results:When fluorine was used alone, compared with the control group (F 0.0As 0.0, 1.00 ± 0.00), the expressions of RANK, NF-κB1 and TRAP proteins (1.11 ± 0.04, 1.29 ± 0.05, 1.38 ± 0.04, 1.24 ± 0.04, 1.13 ± 0.03, 1.34 ± 0.05, 1.12 ± 0.03, 1.24 ± 0.04, 1.61 ± 0.06) were increased ( P < 0.05); TRAF-6 protein expressions in F 0.1 and F 1.6 groups (1.23 ± 0.04, 1.35 ± 0.03) were increased ( P < 0.05). When arsenic was used alone, compared with the control group (F 0.0As 0.0), the expressions of RANK, TRAF-6, NF-κB1 proteins were increased in As 0.5 group ( P < 0.05), the expressions of RANK and NFATc1 proteins were reduced in As 12.5 group ( P < 0.05). When fluorine was combined with arsenic, at the same dose of fluorine, RANK protein expression in F 0.1As 0.5 group and TRAF-6 protein expression in F 0.1As 12.5, F 0.4As 0.5, F 0.4As 2.5 groups, NF-κB1 protein expression in F 0.1As 0.5 F 0.4As 2.5, F 0.4As 12.5 groups, NFATc1 protein expression in F 0.1As 0.5 and F 0.4As 0.5 groups, TRAP protein expression in F 0.1As 12.5 group were higher than the corresponding fluorine groups alone (F 0.1, F 0.4, P < 0.05), but lower than the sum of fluorine and arsenic alone. At the same dose of arsenic, RANK protein expression in F 0.1As 12.5 group, TRAF-6 protein expression in F 0.1As 12.5 and F 0.4As 2.5 groups, and NF-κB1 protein expression in F 0.1As 12.5, F 0.4As 2.5, F 0.4As 12.5, and F 1.6As 2.5 groups, TRAP protein expression in F 1.6As 2.5 and F 1.6As 12.5 groups were higher than the corresponding arsenic groups alone (As 2.5, As 12.5, P < 0.05), but lower than the sum of fluorine and arsenic alone. Fluorine had a major effect on the expressions of RANK, TRAF-6, NF-κB1, NFATc1, and TRAP proteins ( F=3.41, 341.73, 66.01, 56.49, 147.40, P < 0.05); arsenic also had a main effect on all protein indicators ( F=686.71, 174.96, 107.32, 235.80, 331.37, P < 0.05); the combined effect of fluorine and arsenic had an interaction effect on each protein indicator ( F=50.39, 234.94, 116.72, 67.77, 36.56, P < 0.05). Conclusions:In the co-culture system of MC3T3-E1 and RAW264.7 cells, fluorine can activate TRAF-6-mediated expression of NF-κB1 signaling pathway-related proteins, thereby promoting osteoclast differentiation; the effects of arsenic on the expression of related proteins are not completely consistent. The interaction of fluorine and arsenic exposure on TRAF-6-mediated expression of NF-κB1 signaling pathway-related proteins is mainly antagonistic.
ABSTRACT
Objective To investigate the effects of chronic fluoride and arsenic co-exposure on bone morphogenetic proteins 2 (BMP-2) and runt-related transcription factor 2 (Runx2) gene expressions of bone tissue in rats. Methods One hundred and sixty 8-week-old clean-grade Wistar rats weighting (200 ± 50) g were randomly divided into 16 groups by weight via the random number table method of 10 rats in each group by 2 × 4 factorial experimental design (half female and half male), and treated with different doses of fluoride, arsenite and fluoride plus arsenite in deionized water (untreated control group with 0.0 mg/kg fluoride and 0.0 mg/kg arsenite; low-, moderate- and high-fluoride groups were supplemented with 5.0, 10.0 and 20.0 mg/kg fluoride and 2.5, 5.0 and 10.0 mg/kg arsenite) for 6 months. Rats were divided into control (F0.0As0.0), low fluorine (F5.0As0.0), moderate fluorine (F10.0As0.0), high fluorine (F20.0As0.0), low arsenic (F0.0As2.5), moderate arsenic (F0.0As5.0), high arsenic (F0.0As10.0), low fluorine and low arsenic (F5.0As2.5), low fluorine and moderate arsenic (F5.0As5.0), low fluorine and high arsenic (F5.0As10.0), moderate fluorine and low arsenic (F10.0As2.5), moderate fluorine and moderate arsenic (F10.0As5.0), moderate fluorine and high arsenic (F10.0As10.0), high fluorine and low arsenic (F20.0As2.5), high fluorine and moderate arsenic (F20.0As5.0), high fluorine and high arsenic (F20.0As10.0) groups. The concentrations of urinary fluoride (UF) and urinary arsenic (UAs) were determined as exposure biomarkers via the fluoride ion selective electrode method and the flame atomic fluorescence method. The mRNA expressions of BMP-2 and Runx2 were measured with quantitive real-time PCR. Results There were no dental fluorosis found in F0.0As0.0, F0.0As2.5, F0.0As5.0 and F0.0As10.0 groups, and there was a dose-response relationship between the occurrence of dental fluorosis and fluoride doses. Under exposure of fluorine and arsenic combined with high dose of fluorine (20.0 mg/kg), with increasing of arsenic exposure doses, the degree of injury of dental fluorosis increased (χ2 = 9.124, P < 0.05). Compared with F0.0As0.0 (0.99 ± 0.08, 0.99 ± 0.07), the mRNA expressions of BMP-2 (1.01 ± 0.07, 1.06 ± 0.06, 1.21 ± 0.05) and Runx2 (1.03 ± 0.04, 1.24 ± 0.03, 1.33 ± 0.10) in F5.0As0.0, F10.0As0.0, F20.0As0.0 groups were increased with increasing of fluoride doses. Fluoride had a significant effect on mRNA expressions of BMP-2 and Runx2 (F=3.067, 2.927, P<0.05). There was a significant interaction between fluoride and arsenic combination and BMP-2 and Runx2 mRNA expression levels (F = 3.817, 4.802, P < 0.05). Conclusion When rat is co-exposed to fluorine and arsenic, fluorine plays a leading role on BMP-2 and Runx2 mRNA expressions, and arsenic is indirectly involved in fluoride-induced bone toxicity; fluorine and arsenic has a antagonistic effect on BMP-2 and Runx2 mRNA expressions.
ABSTRACT
Objective To analyze the role of osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) in bone toxicity in rats co-exposed to fluoride and arsenite.Methods One hundred and ninety-two 8-week-old clean-grade Wistar rats weighing (200 ± 50) g were divided into 16 groups by weight using random number table method of 12 rats in each group by 2 × 4 factorial experimental design (half female and half male),and treated with different doses of fluoride,arsenite and fluoride plus arsenite in deionized water (untreated control group containing 0.0 mg/kg fluoride and 0.0 mg/kg arsenite;low-,moderate-,and high-fluoride groups supplemented with 5.0,10.0 and 20.0 mg/kg fluoride and 2.5,5.0 and 10.0 mg/kg arsenite) for 6 months.Rats were divided into control (F0As0),low fluorine (F5.0As0),moderate fluoride (F10.0As0),high fluoride (F20.0As0),low arsenic (F0As2.5),moderate arsenic (F0As5.0),high arsenic (F0As10.0),low fluorine and low arsenic (F5.0As2.5),low fluorine and moderate arsenic (F5.0As5.0),low fluorine and high arsenic (F5.0As10.0),moderate fluorine and low arsenic (F10.0As2.5),moderate fluorine and moderate arsenic (F10.0As5.0),moderate fluorine and high arsenic (F10.0As10.0),high fluorine and low arsenic (F20.0As2.5),high fluorine and moderate arsenic (F20.0As5.0),high fluorine and high arsenic (F20.0As10.0) groups.The protein expressions of OPG and RANKL in bone were measured via the enzyme-linked immunosorbent assay method.The mRNA expressions of OPG and RANKL were measured with quantitative real-time PCR.Results Compared with F0As0 [(2.678 ± 0.136) ng/mg,(29.658 ± 0.662) pg/mg],the protein expressions of OPG [(2.857 ± 0.162),(2.983 ± 0.272),(3.117 ± 0.143) ng/mg],and RANKL [(32.533 ± 0.999),(32.698 ± 1.932),(33.331 ± 1.140) pg/mg] in F5.0As0,F10.0As0,F20.0As0 were increased with increasing of fluoride doses;increased first and then decreased was observed in levels of RANKL protein [(32.348 ± 2.838),(31.589 ±1.359),(28.843 ± 1.908) pg/mg] in F0As2.5,F0As5.0,F0As10.0 with increasing of arsenic doses (P＜0.05).Compared with F0As0 (0.83 ± 0.19,0.92 ± 0.23),the mRNA expressions of OPG (1.14 ± 0.27,1.33 ± 0.39,1.69 ± 0.77) and RANKL (1.02 ± 0.21,1.17 ± 0.15,1.25 ± 0.31) in F5.0As0,F10.0As0,F20.0As0 were increased with increasing of fluoride dose.Fluoride had a significant effect on protein and mRNA expressions of OPG and RANKL (F=11.530,21.765,6.320,3.543,P ＜ 0.05).There was interaction between fluoride and arsenite on the expressions of RANKL protein and mRNA,OPG protein (F =9.496,2.217,3.375,P ＜ 0.05).Conclusion When rat is co-exposed to fluorine and arsenic,fluorine plays a leading role in regulating RANKL and OPG,and arsenic is indirectly involved in the fluorine bone toxicity in rats,fluorine and arsenic has a antagonistic effect on OPG and RANKL expressions.