Sodium fluoride induces hypercalcemia resulting from the upregulation of both osteoblastic and osteoclastic activities in goldfish, Carassius auratus

Abstract

The influence of sodium fluoride (NaF) on calcium metabolism was examined in goldfish (fresh water teleost). At 2 days after administration of NaF (500 ng/g body weight; 5µg/g body weight)(around 10 -5 to 10 -4 M in goldfish), we indicated that plasma calcium levels upregulated in both doses of NaF-treated goldfish. To examine the mechanism of hypercalcemia by NaF treatments, therefore, direct effects of NaF on osteoblasts and osteoclasts in goldfish were investigated by an original assay system using teleost scale which has osteoblasts, osteoclasts and bone matrix. Alkaline phosphatase activity in the scales increased with the treatment of NaF (10 -6 and 10 -5 M) during 6 hours of incubation. Also, tartrate-resistant acid phosphatase activity increased after exposure to NaF (10 -5 M) at the 6 hours of incubation. To investigate the osteoclastic activation, the mRNA expression of osteoclastogenesis related factors were examined. The receptor activator of the nuclear factor-κB ligand (RANKL) which is known as a factor for osteoclastogenesis, increased in the NaF-treated scales after 6 hours of incubation.The ratio of RANKL/osteoprotegerin (osteoclastogenesis inhibitory factor) significantly increased after 6 hours of incubation. Resulting from the increase of RANKL mRNA level, the expression of transcription-regulating factors was significantly increased. Furthermore, the expression of functional genes, cathepsin K and matrix metalloproteinase-9 mRNA, was significantly increased. In our knowledge, this is the first report concerning the effects of NaF on osteoblasts and osteoclasts in teleosts. We concluded that NaF influences calcium metabolism via osteoclastic activation in goldfish.3 Keywords: sodium fluoride; osteoblasts; osteoclasts; scales; calcium metabolism; RANKL;OPG; goldfish Abbreviations: ALP, alkaline phosphatase; CTSK, cathepsin K; MMP-9, matrix metalloproteinase-9; NFATc1, nuclear factor of activated T-cells and cytoplasmic 1; TRAF6, TNF receptor-associated factor 6; TRAP, tartrate-resistant acid phosphatase; OPG, osteoprotegerin; RANK, receptor activator of the nuclear factor-κB ; RANKL, receptor activator of the nuclear factor-κB ligand 4

Sodium fluoride influences calcium metabolism resulting from the suppression of osteoclasts in the scales of nibbler fish Girella punctata

“…We previously reported that NaF induces hypercalcemia in goldfish (freshwater teleosts) [17]. We believe that the response to NaF in marine teleosts is different from that in freshwater teleosts.…”

“…Furthermore, in rat osteoblasts, fluoride activated Wnt/β-catenin signaling [27]. In goldfish, we recently reported that OPG mRNA expression did not change significantly between NaF-treated and control scales, although NaF significantly increased RANKL mRNA expression [17]. In goldfish, therefore, the ratio of RANKL/OPG in NaF-treated scales was significantly higher than that in control scales, and then NaF treatment induced the activation of osteoclasts [17].…”

“…After blood sampling, NaF (purity: 99.0%, Wako Pure Chemical Industries, Ltd.) (5 µg/g of body weight) was injected intraperitoneally. The dose of NaF was determined in accordance with our previous report [17].…”

“…These experimental periods were adopted because hormonal and toxicological effects appeared in goldfish in 2 days [16][17][18][19][20][21]. In addition, our preliminary experiment with nibbler fish indicated that the changes in plasma calcium levels were highest 2 days after injection.…”

“…This fish scale assay has been utilized to analyze hormonal functions [7,11,15,16]. Using the scale assay system, we recently indicated that in goldfish Carassius auratus (freshwater teleosts), sodium fluoride (NaF) affects both osteoblasts and osteoclasts [17]. In marine teleosts, however, the direct effect of fluoride on osteoblasts and osteoclasts has not yet been reported.…”