Polychlorinated biphenyl disrupts bone metabolism in goldfish Koji Yachiguchi, 1 * Noriko Matsumoto, 1 Yuki Haga, 2 Motoharu Suzuki, 2 Chisato Matsumura, 2 Masahiro Tsurukawa, 2 Toshihiro Okuno, 2 Takeshi Nakano, 2 Kimi Kawabe, 3 Kei-ichiro Kitamura, 4 Akira Toriba, 3 Kazuichi Hayakawa, 3 Atsuhiko Hattori, 5 and Nobuo Suzuki, 1 1 Noto Marine Laboratory, Institute of Nature and Environmental Technology, Kanazawa University, Housu-gun, Ishikawa , Japan; 2 Hyogo Prefectural Institute of Environmental Sciences, Kobe, Hyogo , Japan : 3 School of Pharmaceutical Sciences, College of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kakuma, Ishikawa , Japan; 4 School of Health Sciences, College of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kodatsuno, Ishikawa , Japan; 5 Department of Biology, College of Liberal Arts and Sciences, Tokyo Medical and Dental University, Ichikawa, Chiba , Japan P Experiment 1:Effect of the PCB on scale osteoclasts and osteoblasts and plasma calcium in goldfish (in vivo experiment) Immature goldfish (Carassius auratus), in which the endogenous effects of sex steroids are negligible, were used. PCB-118 (Figure 3) was solubilized in dimethyl sulfoxide (DMSO) at a concentration of 10 ppm. Then, the PCB solution (10µl/g body weight) was injected intraperitoneally. At 1 and 2 days after injection, scales of the goldfish were removed under anesthesia. Thereafter, blood samples were collected from the gill. The goldfish in the control group were injected with DMSO in the same manner as the experimental goldfish. The tartrate-resistant acid phosphatase (TRAP) in scale osteoclasts and alkaline phosphatase (ALP) in scale osteoblasts and plasma calcium level were measured. The measurement of TRAP and ALP activities according to method by Suzuki and Hattori (Life Sci., 2003). 1)Plasma calcium levels increased significantly at 2 days after PCB administration. 2)On the experiment in vivo, scale TRAP activity in PCB-injected goldfish increased significantly, while ALP activity did not change significantly. 3)To evaluate the effect of PCB on osteoclasts and osteoblasts in vitro, scales were incubated in the media supplemented with PCB. Scale TRAP activity increased with low concentration PCB than ALP activity of osteoblasts. 4) PCB-118 was proved to disrupt the bone metabolism of the goldfish. RESULTS Fig.3 Chemical structure of 2,3’,4,4’5- pentachlorobiphenyl(PCB-118) **:P<0.01 Fig.5 Effect of PCB on the plasma calcium level in goldfish (Experiment 1). Plasma calcium levels increased significantly at 2 days after PCB administration as did in osteoclastic activity. The data were analyzed by t-test. **:P<0.01 Experiment 2: Effect of the PCB on osteoclasts and osteoblasts of goldfish scales (in vitro experiment) Scales were collected from mature female goldfish (Carassius auratus) (n=12) and incubated in the media (L 15-medium, Gibco) supplemented with PCB-118 of 0.025, 0.25 and 2.5ppm for 6 and 18hrs. After incubation, TRAP in scale osteoclasts and ALP in osteoblasts activities were measured for analyzing the effect of PCB on bone metabolism. Fig. 4 Effect of PCB on the osteoclastic and osteoblastic activities of goldfish scales (Experiment 1). At 1 day after injection, scale osteoclasic and osteoblastic activities in PCB-treated goldfish tended to increase. At 2 days, scale osteoclastic activity in PCB-injected goldfish increased significantly. The data were analyzed by t-test. **:P<0.01 Fig.6 Effect of PCB on osteoclastic and osteoblastic activities in the scales of goldfish at 6 hrs of incubation (Experiment 2). After incubation in the PCB at 0.25 ppm for 6 hrs, TRAP activity significantly increased in the compared with control. On the other hand, ALP activity increased at 2.5ppm PCB. The data were analyzed by paired t-test. *:P<0.05 Fig.7 Effect of PCB on osteoclastic and osteoblastic activities in the scales of goldfish at 18 hrs of incubation (Experiment 2). Experiment 2 (in vitro) After the scales had been incubated for 18 hrs, TRAP activities significantly increased in the PCB at ppm to 2.5 ppm compared with control. ALP activity increased only at 2.5 ppm PCB. The data were analyzed by paired t-test. *:P<0.05; ***: P<0.001 Fig.1 Scheme of fish epidermis and scale. It has been reported that polychlorinated biphenyl (PCB) congeners acting as endocrine-disrupting compounds have the potential to mimic sex steroids and inhibit or increase their secretion. However, the detailed effects of PCBs on osteoclasts and osteoblasts have not yet been elucidated. The teleost scale which contains osteoblasts, osteoclasts, and bone matrix is very similar to mammalian membrane bone (Figure 1). Therefore, we have developed original assay system using goldfish scales. To analyze the effect of PCBs on bone metabolism, we examined osteoclastic and osteoblastic activities, as well as plasma calcium levels, in the scales of PCB-injected goldfish. In addition, we detected activities of osteoclasts and osteoblasts in scales by i n vitro assay. INTRODUCTION MESTHODS SUMMARY Fig.2 Goldfish (Carassius auratus) and its scale. Fish scale