- Email: [email protected]
Induction of tributyltin-binding protein type 2 in Japanese flounder, Paralichthys olivaceus, by exposure to tributyltin-d27
Marine Pollution Bulletin 62 (2011) 412–414
Contents lists available at ScienceDirect
Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul
Baseline
Induction of tributyltin-binding protein type 2 in Japanese flounder, Paralichthys olivaceus, by exposure to tributyltin-d27 Mohamed Nassef a, Takahiko Tawaratsumita a, Yumi Oba a, Hina Satone a, Kei Nakayama b, Yohei Shimasaki a, Tsuneo Honjo a, Yuji Oshima a,⇑ a b
Laboratory of Marine Environmental Science, Faculty of Agriculture, Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka 812-8581, Japan Center for Marine Environmental Studies, Ehime University, 2-5 Bunkyo-cho, Matsuyama 790-8577, Japan
a r t i c l e
i n f o
Keywords: Tributyltin-binding proteins Protein induction
a b s t r a c t In this study, individual Japanese flounder were intraperitoneally injected with 2 lg tributyltin-d27 (TBTd27) fish1. Blood samples were collected on day 7 after injection. TBT-binding protein types 1 and 2 (TBT-bp1, -bp2) in the blood serum were quantified by western blotting analysis. As a result, the concentration of TBT-bp2 in TBT-d27 treated group increased to 220% of that in the solvent control, whereas the TBT-bp1 concentration decreased to 65% of that in the solvent control. Additionally, a positive relationship between the concentrations of TBT-bp2 and TBT was observed in blood sera of wild and cultured flounder. We suggest that TBT-bp2 is produced in response to TBT exposure and may play an important role in fish physiology. Ó 2010 Elsevier Ltd. All rights reserved.
Tributyltin (TBT) has strong toxicity and endocrine-disrupting actions in marine organisms. TBT can cause masculinization in Japanese flounder, Paralichthys olivaceus (Shimasaki et al., 2003) and deformity in medaka embryos, Oryzias latipes (Hano et al., 2007). TBT is detected at significant levels in tissues of fish living in Japanese coastal areas (Inoue et al., 2006; Murai et al., 2008; Ueno et al., 2004). High levels of TBT accumulation in the blood serum of Japanese flounder were accompanied by the appearance of TBT-binding proteins (TBT-bps) (Shimasaki et al., 2002). TBT bound by TBT-bp type 1 (TBT-bp1) in Japanese flounder is excreted outside via skin mucus, suggesting that TBT-bp1 has detoxification functions (Satone et al., 2008). Based on their genome structure, TBT-bp1 and TBTbp type 2 (TBT-bp2) genes are similar to the lipocalin group of proteins, including retinol-binding protein, a1-acid glycoprotein (AGP), and pufferfish saxitoxin- and tetrodotoxin-binding protein (PSTBP), which have roles in detoxification and elimination of xenobiototic compounds (Satone et al., 2008; Oba et al., 2007). Furthermore, TBT-bp1 in medaka is up-regulated by exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Volz et al., 2005) and down-regulated in the self-fertilizing fish, Kryptolebias marmoratus, exposed to bisphenol A (Lee et al., 2007). Thus, TBT-bps may respond to chemical exposure. This study aimed to elucidate the response of TBT-bps in flounder administered TBT-d27 by intraperitoneal injection.
⇑ Corresponding author. Tel.: +81 92 642 2905; fax: +81 92 642 2908. E-mail address: [email protected] (Y. Oshima). 0025-326X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2010.12.005
For the TBT-d27 exposure study, cultured flounder were purchased from Nisshin Marinetech Co., Ltd. (Kanagawa, Japan) in October 2005. All flounder were maintained in 80-L flow-through aquaria (10 fish per aquarium) under natural daylight and at natural water temperature, without being fed. For a treatment solution, 100 lg TBT-d27 (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) was dissolved in 1 mL nonane (Wako Pure Chemical Industries Ltd., Osaka, Japan) and added to 9 mL of corn oil (Wako). Thirty flounder were each intraperitoneally injected with 200 ll of TBTd27 solution (2 lg fish1) and 10 flounder were intraperitoneally injected with 200 ll of corn oil containing 10% nonane as the solvent control. Ten untreated flounder were also established as the control. On day 7 following injection, blood was collected from the caudal vein of each fish in all groups and stored at 4 °C overnight. The blood was then centrifuged at 17,000g for 5 min to separate the serum. Serum samples were diluted to 50 or 10 times their original volume with phosphate-buffered saline (PBS; 137 mM NaCl, 8.1 mM Na2HPO412H2O, 2.68 mM KCl, 1.47 mM KCl2) and stored at 80 °C until western blotting analysis. For analyzing the correlations between TBT and TBT-bps concentrations in serum of Japanese flounder, fish were purchased from a fish market and wild Japanese flounder were collected from the coastal area around Fukuoka, Japan in 2003. Blood was collected from the caudal vein of each fish, centrifuged at 17,000g for 5 min, and stored at 4 °C overnight. The serum obtained was diluted to 50 times its original volume with PBS and stored at 80 °C for later western blotting analysis. TBT-bp1 and TBT-bp2 in the blood serum were detected by western blotting analysis as described by Satone et al. (2008).
413
M. Nassef et al. / Marine Pollution Bulletin 62 (2011) 412–414
Relative concentration of TBT-bps in flounder serum
4 Control
*
Solvent control 3
2
TBT-exposed
*
1
0 TBT-bp1
TBT-bp2
Fig. 1. Changes in concentrations of TBT-bp1 and TBT-bp2 in the blood serum of Japanese flounder injected with TBT-d27 at 2 lg TBT-d27 fish1. Blood was collected and serum separated 1 week after injection. Concentrations of TBT-bps were determined by western blotting analysis. Data represent means ± standard deviation. ⁄P < 0.01.
1.6
A Relative amount of TBT-bp1
1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 0
50
100
150
200
250
300
350
400
450
500
550
Concentration of TBT in the serum (ng/g)
2.5
B Relative amount of TBT-bp2
Concentrations of TBT-bp1 and TBT-bp2 in the blood serum were semi-quantified by western blotting analysis. A captured image of each gel was analyzed and the areas of the targets were quantified. The relative concentrations of TBT-bp1 and TBT-bp2 were determined from the areas of bands normalized to those of standard sample #1 (randomly numbered). In case of TBT-bp2, two bands were detected around 46.5 kDa and they were combined into a single value because these bands might be a variant of TBT-b2. The TBT-d27 concentration in P. olivaceus blood serum was analyzed by using a gas chromatograph equipped with a mass spectrometer (GC–MS; model 6890 gas chromatograph, model 5973 mass spectrometer, Hewlett–Packard, Avondale, PA, USA), as described by Inoue et al. (2004). The accuracy of the abovementioned analytic method was checked using certified reference biological materials NIES No. 11 (National Institute for Environmental Studies, Tsukuba, Japan). Differences between TBT-bps concentrations of solvent control and TBT-d27-treated fish were analyzed by the Mann–Whitney U-test. Correlations between TBT concentrations and relative concentrations of TBT-bps in the serum of wild and cultured Japanese flounders were calculated by Spearman’s rank test using the Statistical Package for the Social Sciences Analysis software (SPSS, Chicago, IL, USA). Fig. 1 shows the concentrations of TBT-bps in the blood serum of flounder exposed to TBT-d27. The concentration of TBT-bp1 decreased to 65% of that in the solvent control (P < 0.01). However, the concentration of TBT-bp2 increased to 220% of that in the solvent control (P < 0.01). Fig. 2 shows the relationships between concentrations of TBT and TBT-bps in the blood serum of wild and cultured Japanese flounders. There was a weak correlation (rs = 0.210) between concentrations of TBT and TBT-bp1 (Fig. 2A). In contrast, there was a significant positive correlation (rs=0.547; P < 0.05) between concentrations of TBT-bp2 and TBT (Fig. 2B). In the TBT exposure tests, average TBT-bp1 concentration in TBT-treated fish was lower than in the solvent control. This result agrees with that of Nassef et al. (2011) showing down-regulation of the TBT-bp1 gene in medaka exposed to TBT. Down-regulation of the TBT-bp1 gene has also been reported in K. marmoratus exposed to bisphenol A (Lee et al., 2007). However, up-regulation has been reported in medaka exposed to TCDD (Volz et al., 2005). Response of TBT-bp1 gene may depend on exposed chemical.
2.0
1.5
1.0
0.5
0.0 0
50
100
150
200
250
300
350
400
450
500
550
Concentration of TBT in the serum (ng/g)
Fig. 2. Relationships between TBT concentration and relative amounts of TBT-bp1 (A) and TBT-bp2 (B) in serum of wild and cultured Japanese flounders, as analyzed by western blotting.
In the present study, the concentration of TBT-bp2 increased to 220% of that in controls after exposure to TBT. Also, the concentration of TBT-bp2 in flounder collected from a fish market was positively correlated to that of TBT in blood serum. Structural analysis of the TBT-bp2 gene has revealed a sequence similar to that of the glucocorticoid-response element (GRE) in the transcriptional regulation region of the TBT-bp2 gene (Oba et al., 2007). Expression of the TBT-bp2 gene may be regulated by a glucocorticoid, because the gene bears a putative GRE-like element in its 50 flanking region. Atanasov et al. (2005) suggested that TBT exposure may cause excessive glucocorticoid levels by inhibition of glucocorticoid metabolism. However, the mechanism related to the decreasing level of TBT-bp1 by exposure to TBT is unknown. TBT is also well known to have high cytotoxicity and to trigger apoptosis (Aw et al., 1990; Reader et al., 1999). Thus, toxic stress due to TBT administration may induce expression of the gene encoding TBT-bp2, AGP, to which TBT-bp2 may be related, is known to have an anti-inflammatory function (Fournier et al., 2000; Hochepied et al., 2003). Our results suggest that TBT-bp2 is produced in response to TBT exposure and could play an important role in the toxic response of fish. In the available literature we have very few studies on TBT-bps in fish. Satone et al. (2011) demonstrated that recombinant TBT-bp1 can restore osteoblastic activity inhibited by TBT in goldfish scales. TBT-bps might function to protect the fish against TBT toxicity. In summary, TBT-bp2 production was induced by injection of TBT-d27. A positive correlation between TBT-bp2 and TBT concentrations in the blood serum was observed, suggesting that TBT-bp2 is produced in response to TBT exposure and could play an important role in fish physiology. Further studies are required to clarify the functions of TBT-bps.
414
M. Nassef et al. / Marine Pollution Bulletin 62 (2011) 412–414
References Aw, T.Y., Nicotera, P., Manzo, L., Orrenius, S., 1990. Tributyltin stimulates apoptosis in rat thymocytes. Arch. Biochem. Biophys. 283, 46–50. Atanasov, A.G., Nashev, L.G., Tam, S., Baker, M.E., Odermatt, A., 2005. Organotins disrupt the 11beta-hydroxysteroid dehydrogenase type 2-dependent local inactivation of glucocorticoids. Environ. Health Perspect. 113, 1600–1606. Fournier, T., Medjoubi, N.N., Porquet, D., 2000. Alpha-1-acid glycoprotein. Biochim. Biophys. Acta 1482, 157–171. Hano, T., Oshima, Y., Kim, S.G., Satone, H., Oba, Y., Kitano, T., Inoue, S., Shimasaki, Y., Honjo, T., 2007. Tributyltin causes abnormal development in embryos of medaka Oryzias latipes. Chemosphere 69, 927–933. Hochepied, T., Berger, F.G., Baumann, H., Libert, C., 2003. Á1-Acid glycoprotein, an acute phase protein with inflammatory and immunomodulating properties. Cytokine Growth Factor Rev. 14, 25–34. Inoue, S., Oshima, Y., Nagai, K., Yamamoto, T., Go, J., Kai, N., Honjo, T., 2004. Effect of maternal exposure to tributyltin on reproduction of the pearl oyster (Pinctada fucata martensii). Environ. Toxicol. Chem. 23, 1276–1281. Inoue, S., Abe, S., Oshima, Y., Kai, N., Honjo, T., 2006. Tributyltin contamination of bivalves in coastal areas around northern Kyushu. Jpn. Environ. Toxicol. 21, 244–249. Lee, M.Y., Rhee, J.S., Hwang, D.S., Kim, I.C., Raisuddin, S., Lee, J.S., 2007. Mining of biomarker genes from expressed sequence tags and differential display reverse transcriptase-polymerase chain reaction in the self-fertilizing fish, Kryptolebias marmoratus and their expression patterns in response to exposure to an endocrine-disrupting alkylphenol, bisphenol A. Mol. Cells 23, 287–303. Murai, R., Sugimoto, A., Tanabe, S., Takeuchi, I., 2008. Biomagnification profiles of tributyltin (TBT) and triphenyltin (TPT) in Japanese coastal food webs elucidated by stable nitrogen isotope ratios. Chemosphere 73, 1749–1756. Nassef, M., Kato-Unoki, Y., Furuta, T., Nakayama, K., Satone, H., Shimasaki, Y., Honjo, T., Oshima, Y., 2011. Molecular cloning, sequencing, and gene expression
analysis of tributyltin-binding protein type1 in Japanese medaka fish, Oryzias latipes. Jpn. Zool. Sci., in press. Oba, Y., Shimasaki, Y., Oshima, Y., Satone, H., Kitano, T., Nakao, M., Kawabata, S., Honjo, T., 2007. Purification and characterization of tributyltin-binding protein type 2 from plasma of Japanese flounder, Paralichthys olivaceus. J. Biochem. 142, 229–238. Reader, S., Moutardier, V., Denizeau, F., 1999. Tributyltin triggers apoptosis in trout hepatocytes: the role 55 of Ca2+, protein kinase C and proteases. Biochim. Biophys. Acta 1448, 473–485. Satone, H., Oshima, Y., Shimasaki, Y., Tawaratsumida, T., Oba, Y., Takahashi, E., Kitano, T., Kawabata, S., Kakuta, Y., Honjo, T., 2008. Tributyltin-binding protein type 1 has a distinctive lipocalin-like structure and is involved in the excretion of tributyltin in Japanese flounder, Paralichthys olivaceus. Aquat. Toxicol. 90, 292–299. Satone, H., Lee, J.M., Oba, Y., Kusakabe, T., Akahoshi, E., Miki, S., Suzuki, N., Sasayama, Y., Nassef, M., Shimasaki, Y., Kawabata, S., Honjo, T., Oshima, Y., 2011. Tributyltin-binding protein type1, a lipocalin, prevents inhibition of osteoblastic activity by tributyltin in fish scales. submitted for publication. Shimasaki, Y., Oshima, Y., Yokota, Y., Kitano, T., Nakao, M., Kawabata, S., Imada, N., Honjo, T., 2002. Purification and identification of a tributyltin-binding protein from serum of Japanese flounder, Paralichthys olivaceus. Environ. Toxicol. Chem. 21, 1229–1235. Shimasaki, Y., Kitano, T., Oshima, Y., Inoue, S., Imada, N., Honjo, T., 2003. Tributyltin causes masculinization in fish. Environ. Toxicol. Chem. 22, 141–144. Ueno, D., Inoue, S., Takahashi, S., Ikeda, K., Tanaka, H., Subaramanian, A.N., Fillmann, G., Zheng, J., Muchtar, M., Prudente, M., Chung, K., Tanabe, S., 2004. Global pollution monitoring of butyltin compounds using skipjack tuna as a bioindicator. Environ. Pollut. 127, 1–12. Volz, D.C., Bencic, D.C., Hinton, D.E., Law, J.M., Kullman, S.W., 2005. 2,3,7,8Tetrachlorodibenzo-p-dioxin (TCDD) induces organ-specific differential gene expression in Japanese medaka (Oryzias latipes). Toxicol. Sci. 85, 572–584.
Contents lists available at ScienceDirect
Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul
Baseline
Induction of tributyltin-binding protein type 2 in Japanese flounder, Paralichthys olivaceus, by exposure to tributyltin-d27 Mohamed Nassef a, Takahiko Tawaratsumita a, Yumi Oba a, Hina Satone a, Kei Nakayama b, Yohei Shimasaki a, Tsuneo Honjo a, Yuji Oshima a,⇑ a b
Laboratory of Marine Environmental Science, Faculty of Agriculture, Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka 812-8581, Japan Center for Marine Environmental Studies, Ehime University, 2-5 Bunkyo-cho, Matsuyama 790-8577, Japan
a r t i c l e
i n f o
Keywords: Tributyltin-binding proteins Protein induction
a b s t r a c t In this study, individual Japanese flounder were intraperitoneally injected with 2 lg tributyltin-d27 (TBTd27) fish1. Blood samples were collected on day 7 after injection. TBT-binding protein types 1 and 2 (TBT-bp1, -bp2) in the blood serum were quantified by western blotting analysis. As a result, the concentration of TBT-bp2 in TBT-d27 treated group increased to 220% of that in the solvent control, whereas the TBT-bp1 concentration decreased to 65% of that in the solvent control. Additionally, a positive relationship between the concentrations of TBT-bp2 and TBT was observed in blood sera of wild and cultured flounder. We suggest that TBT-bp2 is produced in response to TBT exposure and may play an important role in fish physiology. Ó 2010 Elsevier Ltd. All rights reserved.
Tributyltin (TBT) has strong toxicity and endocrine-disrupting actions in marine organisms. TBT can cause masculinization in Japanese flounder, Paralichthys olivaceus (Shimasaki et al., 2003) and deformity in medaka embryos, Oryzias latipes (Hano et al., 2007). TBT is detected at significant levels in tissues of fish living in Japanese coastal areas (Inoue et al., 2006; Murai et al., 2008; Ueno et al., 2004). High levels of TBT accumulation in the blood serum of Japanese flounder were accompanied by the appearance of TBT-binding proteins (TBT-bps) (Shimasaki et al., 2002). TBT bound by TBT-bp type 1 (TBT-bp1) in Japanese flounder is excreted outside via skin mucus, suggesting that TBT-bp1 has detoxification functions (Satone et al., 2008). Based on their genome structure, TBT-bp1 and TBTbp type 2 (TBT-bp2) genes are similar to the lipocalin group of proteins, including retinol-binding protein, a1-acid glycoprotein (AGP), and pufferfish saxitoxin- and tetrodotoxin-binding protein (PSTBP), which have roles in detoxification and elimination of xenobiototic compounds (Satone et al., 2008; Oba et al., 2007). Furthermore, TBT-bp1 in medaka is up-regulated by exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Volz et al., 2005) and down-regulated in the self-fertilizing fish, Kryptolebias marmoratus, exposed to bisphenol A (Lee et al., 2007). Thus, TBT-bps may respond to chemical exposure. This study aimed to elucidate the response of TBT-bps in flounder administered TBT-d27 by intraperitoneal injection.
⇑ Corresponding author. Tel.: +81 92 642 2905; fax: +81 92 642 2908. E-mail address: [email protected] (Y. Oshima). 0025-326X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2010.12.005
For the TBT-d27 exposure study, cultured flounder were purchased from Nisshin Marinetech Co., Ltd. (Kanagawa, Japan) in October 2005. All flounder were maintained in 80-L flow-through aquaria (10 fish per aquarium) under natural daylight and at natural water temperature, without being fed. For a treatment solution, 100 lg TBT-d27 (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) was dissolved in 1 mL nonane (Wako Pure Chemical Industries Ltd., Osaka, Japan) and added to 9 mL of corn oil (Wako). Thirty flounder were each intraperitoneally injected with 200 ll of TBTd27 solution (2 lg fish1) and 10 flounder were intraperitoneally injected with 200 ll of corn oil containing 10% nonane as the solvent control. Ten untreated flounder were also established as the control. On day 7 following injection, blood was collected from the caudal vein of each fish in all groups and stored at 4 °C overnight. The blood was then centrifuged at 17,000g for 5 min to separate the serum. Serum samples were diluted to 50 or 10 times their original volume with phosphate-buffered saline (PBS; 137 mM NaCl, 8.1 mM Na2HPO412H2O, 2.68 mM KCl, 1.47 mM KCl2) and stored at 80 °C until western blotting analysis. For analyzing the correlations between TBT and TBT-bps concentrations in serum of Japanese flounder, fish were purchased from a fish market and wild Japanese flounder were collected from the coastal area around Fukuoka, Japan in 2003. Blood was collected from the caudal vein of each fish, centrifuged at 17,000g for 5 min, and stored at 4 °C overnight. The serum obtained was diluted to 50 times its original volume with PBS and stored at 80 °C for later western blotting analysis. TBT-bp1 and TBT-bp2 in the blood serum were detected by western blotting analysis as described by Satone et al. (2008).
413
M. Nassef et al. / Marine Pollution Bulletin 62 (2011) 412–414
Relative concentration of TBT-bps in flounder serum
4 Control
*
Solvent control 3
2
TBT-exposed
*
1
0 TBT-bp1
TBT-bp2
Fig. 1. Changes in concentrations of TBT-bp1 and TBT-bp2 in the blood serum of Japanese flounder injected with TBT-d27 at 2 lg TBT-d27 fish1. Blood was collected and serum separated 1 week after injection. Concentrations of TBT-bps were determined by western blotting analysis. Data represent means ± standard deviation. ⁄P < 0.01.
1.6
A Relative amount of TBT-bp1
1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 0
50
100
150
200
250
300
350
400
450
500
550
Concentration of TBT in the serum (ng/g)
2.5
B Relative amount of TBT-bp2
Concentrations of TBT-bp1 and TBT-bp2 in the blood serum were semi-quantified by western blotting analysis. A captured image of each gel was analyzed and the areas of the targets were quantified. The relative concentrations of TBT-bp1 and TBT-bp2 were determined from the areas of bands normalized to those of standard sample #1 (randomly numbered). In case of TBT-bp2, two bands were detected around 46.5 kDa and they were combined into a single value because these bands might be a variant of TBT-b2. The TBT-d27 concentration in P. olivaceus blood serum was analyzed by using a gas chromatograph equipped with a mass spectrometer (GC–MS; model 6890 gas chromatograph, model 5973 mass spectrometer, Hewlett–Packard, Avondale, PA, USA), as described by Inoue et al. (2004). The accuracy of the abovementioned analytic method was checked using certified reference biological materials NIES No. 11 (National Institute for Environmental Studies, Tsukuba, Japan). Differences between TBT-bps concentrations of solvent control and TBT-d27-treated fish were analyzed by the Mann–Whitney U-test. Correlations between TBT concentrations and relative concentrations of TBT-bps in the serum of wild and cultured Japanese flounders were calculated by Spearman’s rank test using the Statistical Package for the Social Sciences Analysis software (SPSS, Chicago, IL, USA). Fig. 1 shows the concentrations of TBT-bps in the blood serum of flounder exposed to TBT-d27. The concentration of TBT-bp1 decreased to 65% of that in the solvent control (P < 0.01). However, the concentration of TBT-bp2 increased to 220% of that in the solvent control (P < 0.01). Fig. 2 shows the relationships between concentrations of TBT and TBT-bps in the blood serum of wild and cultured Japanese flounders. There was a weak correlation (rs = 0.210) between concentrations of TBT and TBT-bp1 (Fig. 2A). In contrast, there was a significant positive correlation (rs=0.547; P < 0.05) between concentrations of TBT-bp2 and TBT (Fig. 2B). In the TBT exposure tests, average TBT-bp1 concentration in TBT-treated fish was lower than in the solvent control. This result agrees with that of Nassef et al. (2011) showing down-regulation of the TBT-bp1 gene in medaka exposed to TBT. Down-regulation of the TBT-bp1 gene has also been reported in K. marmoratus exposed to bisphenol A (Lee et al., 2007). However, up-regulation has been reported in medaka exposed to TCDD (Volz et al., 2005). Response of TBT-bp1 gene may depend on exposed chemical.
2.0
1.5
1.0
0.5
0.0 0
50
100
150
200
250
300
350
400
450
500
550
Concentration of TBT in the serum (ng/g)
Fig. 2. Relationships between TBT concentration and relative amounts of TBT-bp1 (A) and TBT-bp2 (B) in serum of wild and cultured Japanese flounders, as analyzed by western blotting.
In the present study, the concentration of TBT-bp2 increased to 220% of that in controls after exposure to TBT. Also, the concentration of TBT-bp2 in flounder collected from a fish market was positively correlated to that of TBT in blood serum. Structural analysis of the TBT-bp2 gene has revealed a sequence similar to that of the glucocorticoid-response element (GRE) in the transcriptional regulation region of the TBT-bp2 gene (Oba et al., 2007). Expression of the TBT-bp2 gene may be regulated by a glucocorticoid, because the gene bears a putative GRE-like element in its 50 flanking region. Atanasov et al. (2005) suggested that TBT exposure may cause excessive glucocorticoid levels by inhibition of glucocorticoid metabolism. However, the mechanism related to the decreasing level of TBT-bp1 by exposure to TBT is unknown. TBT is also well known to have high cytotoxicity and to trigger apoptosis (Aw et al., 1990; Reader et al., 1999). Thus, toxic stress due to TBT administration may induce expression of the gene encoding TBT-bp2, AGP, to which TBT-bp2 may be related, is known to have an anti-inflammatory function (Fournier et al., 2000; Hochepied et al., 2003). Our results suggest that TBT-bp2 is produced in response to TBT exposure and could play an important role in the toxic response of fish. In the available literature we have very few studies on TBT-bps in fish. Satone et al. (2011) demonstrated that recombinant TBT-bp1 can restore osteoblastic activity inhibited by TBT in goldfish scales. TBT-bps might function to protect the fish against TBT toxicity. In summary, TBT-bp2 production was induced by injection of TBT-d27. A positive correlation between TBT-bp2 and TBT concentrations in the blood serum was observed, suggesting that TBT-bp2 is produced in response to TBT exposure and could play an important role in fish physiology. Further studies are required to clarify the functions of TBT-bps.
414
M. Nassef et al. / Marine Pollution Bulletin 62 (2011) 412–414
References Aw, T.Y., Nicotera, P., Manzo, L., Orrenius, S., 1990. Tributyltin stimulates apoptosis in rat thymocytes. Arch. Biochem. Biophys. 283, 46–50. Atanasov, A.G., Nashev, L.G., Tam, S., Baker, M.E., Odermatt, A., 2005. Organotins disrupt the 11beta-hydroxysteroid dehydrogenase type 2-dependent local inactivation of glucocorticoids. Environ. Health Perspect. 113, 1600–1606. Fournier, T., Medjoubi, N.N., Porquet, D., 2000. Alpha-1-acid glycoprotein. Biochim. Biophys. Acta 1482, 157–171. Hano, T., Oshima, Y., Kim, S.G., Satone, H., Oba, Y., Kitano, T., Inoue, S., Shimasaki, Y., Honjo, T., 2007. Tributyltin causes abnormal development in embryos of medaka Oryzias latipes. Chemosphere 69, 927–933. Hochepied, T., Berger, F.G., Baumann, H., Libert, C., 2003. Á1-Acid glycoprotein, an acute phase protein with inflammatory and immunomodulating properties. Cytokine Growth Factor Rev. 14, 25–34. Inoue, S., Oshima, Y., Nagai, K., Yamamoto, T., Go, J., Kai, N., Honjo, T., 2004. Effect of maternal exposure to tributyltin on reproduction of the pearl oyster (Pinctada fucata martensii). Environ. Toxicol. Chem. 23, 1276–1281. Inoue, S., Abe, S., Oshima, Y., Kai, N., Honjo, T., 2006. Tributyltin contamination of bivalves in coastal areas around northern Kyushu. Jpn. Environ. Toxicol. 21, 244–249. Lee, M.Y., Rhee, J.S., Hwang, D.S., Kim, I.C., Raisuddin, S., Lee, J.S., 2007. Mining of biomarker genes from expressed sequence tags and differential display reverse transcriptase-polymerase chain reaction in the self-fertilizing fish, Kryptolebias marmoratus and their expression patterns in response to exposure to an endocrine-disrupting alkylphenol, bisphenol A. Mol. Cells 23, 287–303. Murai, R., Sugimoto, A., Tanabe, S., Takeuchi, I., 2008. Biomagnification profiles of tributyltin (TBT) and triphenyltin (TPT) in Japanese coastal food webs elucidated by stable nitrogen isotope ratios. Chemosphere 73, 1749–1756. Nassef, M., Kato-Unoki, Y., Furuta, T., Nakayama, K., Satone, H., Shimasaki, Y., Honjo, T., Oshima, Y., 2011. Molecular cloning, sequencing, and gene expression
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