To evaluate the effects of inorganic mercury (InHg) and methylmercury (MeHg) on bone metabolism in a marine teleost, the activity of tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) as indicators of such activity in osteoclasts and osteoblasts, respectively, were examined in scales of nibbler fish (Girella punctata). We found several lines of scales with nearly the same TRAP and ALP activity levels. Using these scales, we evaluated the influence of InHg and MeHg. TRAP activity in the scales treated with InHg (10 -5 and 10 -4 M) and MeHg (10 -6 to 10 -4 M) during 6 hrs of incubation decreased significantly. In contrast, ALP activity decreased after exposure to InHg (10 -5 and 10 -4 M) and MeHg (10 -6 to 10 -4 M) for 18 and 36 hrs, although its activity did not change after 6 hrs of incubation. As in enzyme activity 6 hrs after incubation, mRNA expression of TRAP (osteoclastic marker) decreased significantly with InHg and MeHg treatment, while that of collagen (osteoblastic marker) did not change significantly. At 6 hrs after incubation, the mRNA expression of metallothionein, which is a metal-binding protein in osteoblasts, was significantly increased following treatment with InHg or MeHg, suggesting that it may be involved in the protection of osteoblasts against mercury exposure up to 6 hrs after incubation. To our knowledge, this is the first report of the effects of mercury on osteoclasts and osteoblasts using marine teleost scale as a model system of bone.

To analyze the effect of polychlorinated biphenyl (PCB) 118 on fish bone metabolism, we examined osteoclastic and osteoblastic activities, as well as plasma calcium levels, in the scales of PCB (118)-injected goldfish. In addition, effect of PCB (118) on osteoclasts and osteoblasts was investigated in vitro. Immature goldfish, in which the endogenous effects of sex steroids are negligible, were used. PCB (118) was solubilized in dimethyl sulfoxide at a concentration of 10 ppm. At 1 and 2 days after PCB (118) injection (100 ng/g body weight), both osteoclastic and osteoblastic activities, and plasma calcium levels were measured. In an in vitro study, then, both osteoclastic and osteoblastic activities as well as each marker mRNA expression were examined. At 2 days, scale osteoclastic activity in PCB (118)-injected goldfish increased significantly, while osteoblastic activity did not change significantly. Corresponding to osteoclastic activity, plasma calcium levels increased significantly at 2 days after PCB (118) administration. Osteoclastic activation also occurred in the marker enzyme activities and mRNA expressions in vitro. Thus, we conclude that PCB (118) disrupts bone metabolism in goldfish both in vivo and in vitro experiments.

Using our original in vitro assay system with goldfish scales, we examined the direct effect of prostaglandin E₂ (PGE₂) on osteoclasts and osteoblasts in teleosts. In this assay system, we measured the activity of alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) as respective indicators of each activity in osteoblasts and osteoclasts. ALP activity in scales significantly increased following treatment at high concentration of PGE₂(10⁻⁷ and 10⁻⁶ M) over 6 hrs of incubation. At 18 hrs of incubation, ALP activity also significantly increased in the PGE₂ (10⁻⁹ to 10⁻⁶ M)-treated scale. In the case of osteoclasts, TRAP activity tended to increase at 6 hrs of incubation, and then significantly increased at 18 hrs of incubation by PGE₂ (10(-7) to 10⁻⁶ M) treatment. At 18 hrs of incubation, the mRNA expression of osteoclastic markers (TRAP and cathepsin K) and receptor activator of the NF-κB ligand (RANKL), an activating factor of osteoclasts expressed in osteoblasts, increased in PGE₂ treated-scales. Thus, PGE₂ acts on osteoblasts, and then increases the osteoclastic activity in the scales of goldfish as it does in the bone of mammals. In an in vivo experiment, plasma calcium levels and scale TRAP and ALP activities in the PGE₂-injencted goldfish increased significantly. We conclude that, in teleosts, PGE₂ activates both osteoblasts and osteoclasts and participates in calcium metabolism.

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The influence of sodium fluoride (NaF) on calcium metabolism was examined in nibbler fish (marine teleosts). Two days after the administration of NaF (5 µg/g of body weight)(around 10-4 M in fish), we showed that plasma calcium levels significantly decreased in NaF-treated nibbler fish. In addition, we detected fluoride in the treated scales by scanning electron microscope with an energy-dispersive X-ray microanalysis, indicating that NaF directly affects their scales. Therefore, the influence of NaF on osteoblasts and osteoclasts in the scales was examined. In the scales of NaF-injected nibbler fish, tartrate-resistant acid phosphatase (TRAP) (osteoclastic marker enzyme) decreased, although alkaline phosphatase (osteoblastic marker enzyme) was activated. To confirm the effect of NaF on osteoclasts, furthermore, the mRNA expressions of osteoclastic markers (matrix metalloproteinase-9 and TRAP) were decreased significantly 2 days after incubation. In barred knifejaws, plasma calcium levels decreased as they did in nibbler fish. Therefore, NaF functions in both osteoblasts and osteoclasts and then influences calcium metabolism in marine fish. In the marine environment, high levels of fluoride (1.2 to 1.5 mg F-/l) (around 10-5 to 10-4 M) are present in seawater. It is probable that teleosts living in seawater efficiently use fluoride to regulate their blood calcium levels. Keywords Sodium fluoride ・ Osteoblasts ・ Osteoclasts ・ Scales ・ Calcium metabolism ・ Element analysis・Nibbler fish

We have previously reported that microgravity promotes the activation of osteoclasts in cultured regenerating scales. This osteoclastic activation was induced by increased levels of receptor activator of the nuclear factor-κB ligand (RANKL). Therefore, we determined that RANKL is an important factor in evaluating osteoclastogenesis in bone tissue. However, the role of RANKL in fish scales is poorly understood. In the present study, we prepared antiserum against goldfish RANKL in rabbits and detected RANKL-producing cells in regenerating goldfish scales. Furthermore, we studied osteoclastic activation by the addition of RANKL to examine exogenous RANKL on osteoclastogenesis in regenerating goldfish scales. As a result, RANKL immunepositive cells were detected in grooves of regenerating scales. In addition, treating the regenerating scales with mammalian RANKL for 3 h significantly increased the expression of the nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), which is essential for osteoclast differentiation. After 6 h of incubation with RANKL, the expression of cathepsin K, a functional osteoclastic gene, significantly increased. Furthermore, the molecules for osteoclast multinucleation and differentiation significantly increased following treatment with mammalian RANKL. Therefore, in fish scales as well as mammalian bone, we concluded that RANKL plays an important role in osteoclastogenesis. ©2020 Jpn. Soc. Biol. Sci.

We have developed an original in vitro bioassay using teleost scale that has osteoclasts, osteoblasts, and bone matrix as each marker: alkaline phosphatase (ALP) for osteoblasts and tartrate-resistant acid phosphatase (TRAP) for osteoclasts. Using this scale in vitro bioassay, we examined the effects of seawater polluted with highly concentrated polycyclic aromatic hydrocarbons (PAHs) and nitro-polycyclic aromatic hydrocarbons (NPAHs) on osteoblastic and osteoclastic activities in the present study. Polluted seawater was collected from 2 sites (the Alexandria site on the Mediterranean Sea and the Suez Canal site on the Red Sea). Total levels of PAHs in the seawater from the Alexandria and Suez Canal sites were 1364.59 and 992.56 ng/l, respectively. We can detect NPAHs in both seawater samples. Total levels of NPAHs were detected in the seawater of the Alexandria site (12.749 ng/l) and the Suez Canal site (3.914 ng/l). Each sample of polluted seawater was added into culture medium at dilution rates of 50, 100, and 500 and incubated with the goldfish scales for 6 hrs. Thereafter, ALP and TRAP activities were measured. As a result, ALP activity was significantly suppressed by both polluted seawater samples diluted at least 500 times, although TRAP activity did not change. In addition, mRNA expressions of osteoblastic markers (ALP, osteocalcin, and the receptor activator of the NF-B ligand) decreased significantly, as did the ALP enzyme activity. Actually, ALP activity decreased with selected PAHs and NPAHs treatments. We conclude that seawater polluted with highly concentrated PAHs and NPAHs influenced bone metabolism in teleosts.

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