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Vitellogenic responses of 17β-estradiol and bisphenol A in male Chinese loach (Misgurnus anguillicaudatus)
Environmental Toxicology and Pharmacology 24 (2007) 155–159
Vitellogenic responses of 17-estradiol and bisphenol A in male Chinese loach (Misgurnus anguillicaudatus) Xuefei Lv, Qunfang Zhou, Maoyong Song, Guibin Jiang ∗ , Jing Shao State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, P.O. Box 2871, 100085 Beijing, PR China Received 31 December 2006; received in revised form 18 April 2007; accepted 22 April 2007 Available online 27 April 2007
Abstract In this study, male Chinese loaches were exposed to 17-estradiol (E2), bisphenol A (BPA) and their mixtures, respectively, for 42 days using a semi-static exposure system. Plasma vitellogenin (Vtg) in male Chinese loaches was used as the determining endpoint. The results demonstrated that male Chinese loaches were sensitive to E2, and the vitellogenic responses showed time- and dose-dependent increase. Similarly, BPA induced the estrogenic effects in male Chinese loaches, and the vitellogenic response increased in a time- and dose-dependent manner. The synthesis of Vtg was initiated by the exposure to higher level of BPA (500 g/L) within 7 days, and a relative long-term exposure to lower concentrations of BPA (10–100 g/L) also led to the production of Vtg. The estrogenic effect of the binary mixture of E2 and BPA also showed a time- and dose-dependent response, which was more potent than that of individual compounds, and Vtg contents in the binary mixture group were higher than the summation of Vtg contents in the single-compound groups at the same concentration. © 2007 Elsevier B.V. All rights reserved. Keywords: Bisphenol A; Chinese loach; Chemical interaction; Endocrine disruption; Estradiol; Experimental animal; Misgurnus anguillicaudatus; Vitellogenin
1. Introduction Xenoestrogens have received considerable attention because of their adverse effect on the functions of endocrine system in wildlife and humans, and many of those compounds are industrial chemicals. Bisphenol A (BPA) has received particular attention because of its large-scale production and widespread use. BPA is primarily used in the production of polycarbonate plastic, epoxy resins, and other plastic products (Lindholst et al., 2000; Larsen et al., 2006). The estrogenic potency of BPA has been demonstrated in various in vitro (Kanno et al., 2004; Lutz et al., 2005; Oehlmann et al., 2006) and in vivo (Lindholst et al., 2000; Pait and Nelson, 2003; Yamaguchi et al., 2005) studies. E2, as the main natural estrogen, is present in the sewage treatment works (STWs) effluent, river water, and drinking water with the concentration at tens of nanograms per liter (Routledge et al., 1998). BPA is also detected in the STWs effluent, however, because of its relatively
∗
Corresponding author. Tel.: +86 10 62849334; fax: +86 10 62849179. E-mail address: [email protected] (G. Jiang).
1382-6689/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2007.04.007
low estrogenicity, the estrogenic effect of STWs effluent was mainly attributed to the presence of natural and synthetic estrogens (Thorpe et al., 2003; Brian et al., 2005). Current studies are usually focused on the assessment of the estrogenic potency of single chemicals, however, in the real-world situation, fish live in the environment containing many different kinds of endocrine disrupting chemicals (EDCs). Meanwhile, studies have demonstrated the estrogenic effects of mixtures of xenoestrogens were more potent than those of the individual chemicals at no-observed-effect concentrations (Payne et al., 2000; Rajapakse et al., 2002; Silva et al., 2002; Thorpe et al., 2003; Brian et al., 2005). So, it is much more important to assess the effects of the mixtures of estrogenic compounds than those of the isolated ones. Chinese loach (Misgurnus anguillicaudatus) was chosen as experimental animal in this study, as it was previously demonstrated to be sensitive to 17-estradiol (Lv et al., 2006). The estrogenicity of BPA in male Chinese loach remains to be investigated. The study of Rajapakse et al. (2001) demonstrated that BPA could contribute to the overall mixture effect at the presence of E2 using the yeast estrogen screen, which remains to be
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validated in in vivo studies. Until now, little is known about the estrogenic effect of the mixture of BPA and E2 in fish species including male Chinese loaches. In the current study, male Chinese loaches were exposed to various concentrations of E2, BPA, and binary mixtures of E2 and BPA at the fixed-ration of 1:100, respectively, to evaluate their estrogenic effect on this kind of fish. Plasma Vtg in male Chinese loaches was used as determining endpoint. 2. Materials and methods 2.1. Chemicals 17-Estradiol (E2, 98% purity) was purchased from Sigma; bisphenol A (BPA, 4,4 -isopropylidenediphenol, 97% purity) was obtained from Acros Organics (New Jersey). The stock solutions of E2 and BPA were prepared by dissolving suitable amounts of chemicals into methanol, sealed and stored at 4 ◦ C. The working solutions were made by diluting the stock solutions with ultrapure water (EASY pure LF).
2.2. Fish The fish used in this study were mature male Chinese loaches. Healthy Chinese loaches were obtained from the market of Beijing, China, and were acclimated in stainless steel tanks with dechlorinated tap water for at least 2 weeks prior to the exposure experiment. The mean weight and length of fish were 9.11 ± 1.55 g and 11.88 ± 0.65 cm, respectively. The day/night cycle was set as natural length. Throughout the study, water quality was monitored by detecting pH (6.9–7.9), oxygen concentration (5–7 mg/L), and temperature (22.5–25.5 ◦ C). At the beginning of the experiment, 15 fish were randomly allocated to 12 L glass tanks with a working volume of 5 L. Two aquaria were dosed at the same concentration. Water was changed every other day during the exposure. The fish were fed with Oligochaetes limnodirlus sp. twice a week.
2.3. Experimental design Male Chinese loaches were exposed either to nominal concentrations of E2 at 0.1, 0.5, 1, and 5 g/L, or to nominal concentrations of BPA at 10, 50, 100, and 500 g/L, or to fixed-ration binary mixtures of E2 and BPA (1:100) at nominal concentrations of 0.1 + 10, 0.5 + 50, and 1 + 100 g/L. The solventcontrol group (0.05‰ methanol) and the control group were also set up. Loaches were provided with dechlorinated tap water, and the whole exposure experiment lasted 42 days.
the coated plate, and then incubated with horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG at a final dilution of 1:2000 in PBST. Following this step, the enzyme substrate solution (0.05 M phosphate–citrate buffer, 0.4 mg/mL o-phenylenediamine, and 0.16% H2 O2 ) was added. Thirty minutes later, the reaction was stopped by 2 M HCl. The absorbance was measured at 490 nm using a microtiter plate reader (DNA Expert, TECAN, Austria).
2.6. Data expression and statistical analysis All the values were expressed as mean ± S.D. The ELISA data were processed by a four-parameter Boltzmann equation: y = a + b/[1 + exp(x − c)/d] where y represents the percentage binding of sample or standard relative to analyte free wells (Bi /B0 ) and x represents log dose. The effects of different treatments on vitellogenin concentrations were assessed using one-way analysis of variance (ANOVA). The level of significance was set as p < 0.05.
3. Results 3.1. Vtg induction in male Chinese loach exposed to 17β-estradiol Male Chinese loaches were exposed to 17-estradiol at nominal concentrations of 0.1, 0.5, 1, and 5 g/L for 42 days (Fig. 1). It was seen that Vtg concentrations in control and solvent-control groups were not detected within 28 days; however, on days 35 and 42, relative high levels Vtg can be detected, and the reasons remain to be unsolved. Vtg can be significantly enhanced at 1 and 5 g/L E2 within 7 days (p < 0.05) with average values of 18.12 ± 2.20 and 211.93 ± 27.06 g/mL, respectively, and the vitellogenic responses of the two exposed groups showed time- and dose-dependent increase during the whole experiment. 0.5 g/L E2 significantly induced the production of Vtg within 14 days (p < 0.05) with an average value of 43.04 ± 0.46 g/mL. However, when the concentration of E2 was 0.1 g/L, a 35 days exposure was needed to induce the synthesis of Vtg, though not significant, with an average value of 50.05 ± 19.11 g/mL.
2.4. Plasma collection Fish were randomly sampled on days 7, 14, 21, 28, 35, and 42 of the exposure (n = 4 for each dose). Fish were anesthetized with quinoline sulfate (40 mg/L), and after recording weight and length, blood samples were taken from the caudal vein using heparinized syringes and transferred to 1.5 mL centrifuge tubes in the presence of aprotinin (2.5 TIU, Roche, Germany). After centrifugation (3000 rpm, 4 ◦ C, 30 min), plasma samples were divided into aliquots and stored at −20 ◦ C.
2.5. Vitellogenin determination The plasma Vtg was purified with anion-exchange membrane (SartobindTM MA 15D, Germany) according to the methods described previously (Shi et al., 2003; Shao et al., 2005). The concentrations of Vtg in the plasma were determined with a competitive enzyme-linked immunosorbent assay (ELISA) described previously (Shao et al., 2005). Briefly, after coated with 750 ng/mL purified Vtg, the 96-well microtiter plate was blocked with 1% bovine serum albumin (BSA). Standards and samples, incubated with primary antibody solution at a final dilution of 1:16,000 in PBST (0.02 M phosphate buffer with 0.15 M NaCl and 0.05% Tween 20, pH 7.4) overnight at 4 ◦ C, were transferred to
Fig. 1. Vitellogenic responses of male Chinese loach exposed to 17-estradiol at the nominal concentrations of 0.1, 0.5, 1, and 5 g/L for 42 days. Values are means ± S.D. (n = 4). (*) Significant differences from the control groups at p < 0.05.
X. Lv et al. / Environmental Toxicology and Pharmacology 24 (2007) 155–159
Fig. 2. Vitellogenic responses of male Chinese loach exposed to BPA at the nominal concentrations of 10, 50, 100, and 500 g/L for 42 days. Values are means ± S.D. (n = 4). () The lack of samples for 50 g/L BAP on day 42. (*) Significant differences from the control groups at p < 0.05.
3.2. Vtg induction in male Chinese loach exposed to BPA Fig. 2 illustrates plasma Vtg levels in male Chinese loaches induced by various doses of BPA (10, 50, 100, and 500 g/L) during 42 days exposure. No Vtg was induced following exposure to 10, 50, and 100 g/L BPA for 28 days but Vtg were significantly enhanced after 35 days exposure (p < 0.05), and on day 42, Vtg levels in the groups of 10 and 100 g/L BPA were 360.08 ± 11.63 and 968.09 ± 92.69 g/mL, respectively. 31.98 ± 9.43 g/mL of Vtg were detected in the plasma of male Chinese loaches exposed to 500 g/L BPA within 7 days, which was significantly different from the control groups (p < 0.05), and from days 14 to 42, Vtg levels increased from 4.22 ± 0.02 to 47.61 ± 2.60 mg/mL. 3.3. Vtg induction in male Chinese loach exposed to the binary mixtures of E2 and BPA The vitellogenic responses of male Chinese loach exposed to the fixed-ration (1:100) binary mixtures of E2 and BPA at the nominal concentrations of 0.1 + 10, 0.5 + 50 , and 1 + 100 g/L for 42 days are shown in Fig. 3. Vtg concentrations in the binary mixture exposed group showed a timeand dose-increasing manner. 0.1 g/L E2 + 10 g/L BPA did not induce the synthesis of Vtg within 28 days, and compared with the control groups, Vtg levels were significantly induced by 0.1 g/L E2 + 10 g/L BPA after 35 days exposure (p < 0.05), and Vtg levels in this group increased to 1.31 ± 0.20 mg/mL on day 42. 0.5 g/L E2 + 50 g/L BPA significantly induced the synthesis of Vtg (6.81 ± 0.99 g/mL) within 7 days (p < 0.05), and after a 42 days exposure, Vtg levels increased to 1.38 ± 0.37 mg/mL. Exposure to 1 g/L E2 + 100 g/L BPA significantly induced the synthesis of Vtg (112.75 ± 3.21 g/mL) within 7 days compared with the control and solvent-control groups (p < 0.05). Moreover, Vtg levels in the co-exposed groups were all higher than the summation
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Fig. 3. Vitellogenic responses of male Chinese loach exposed to binary mixtures of E2 and BPA at the fixed-ration (1:100) (i.e. the nominal concentrations of 0.1 g/L E2 + 10 g/L BPA, 0.5 g/L E2 + 50 g/L BPA, and 1 g/L E2 + 100 g/L BPA) for 42 days. Values are means ± S.D. (n = 4). () The lack of sample for 1 g/L E2 plus 100 g/L BPA on day 14. (*) Significant differences from the control groups at p < 0.05.
of Vtg in the single-compound exposed groups, as shown in Fig. 4. 4. Discussion The attractive characteristics including moderate size, easy to culture and identify gender (by the shape of pectoral fin) make loach suitable as a candidate fish in field and laboratory studies (Shao et al., 2005). A previous study of our group has demonstrated that male Chinese loach was sensitive to 17-estradiol (Lv et al., 2006), and this was validated in the present study. The ability of E2 to induce the synthesis of Vtg in other species following waterborne exposure has also been documented, such as fathead minnow (Pimephales promelas) (Panter et al., 1998), rainbow trout (Oncorhynchus mykiss) (Routledge et al., 1998;
Fig. 4. Comparison between Vtg levels in the group of E2 + BPA treated fish (夽) and the summation of Vtg levels in the individual E2 and BPA groups () after exposure for 35 days.
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Lindholst et al., 2000), roch (Rutilus rutilus) (Routledge et al., 1998), zebrafish (Danio rerio) (Van den Belt et al., 2003), etc. Compared the results on the same species and those from other species, it can be found that different fish species, even the same species at different reproductive stages, might show different sensitivity; the applied Vtg quantification techniques and the exposure system (semi-static versus flow-through) might also have an impact on the differences in sensitivity (Van den Belt et al., 2003). Until now, no studies have been performed to address the estrogenicity of BPA in male Chinese loach by water exposure. Vitellogenin (Vtg) is normally produced in sexually maturing females. Under natural conditions, male fish cannot synthesize Vtg, However, they can be induced to synthesize Vtg when exposed to estrogens or estrogen mimics, so the abnormal level of Vtg in male fish has been used as a good biomarker to demonstrate estrogenic effects of xenoestrogens in the aquatic environment (Sumpter and Jobling, 1995; Marin and Matozzo, 2004). Therefore, the presence of Vtg in male Chinese loach was used to evaluate the estrogenicity of BPA using semi-static exposure system in the present study. The results (Fig. 2) demonstrated that BPA was estrogenic in male Chinese loach, although less potent than E2, and Vtg was induced in time- and dosedependent manner. A 28 days exposure to BPA at the nominal concentrations of 10, 50, and 100 g/L did not induce the production of Vtg, however 500 g/L BPA significantly induced the synthesis of Vtg within 7 days. Plasma Vtg synthesis in male Chinese loach can be compared with that in other fish species after similar exposure. Lindholst et al. (2000) found that BPA concentration in water lower than 70 g/L did not induce Vtg synthesis within 12 days in rainbow trout (O. mykiss), and 500 g/L BPA induced the production of Vtg with the value of 1168 g/mL within 6 days. Van den Belt et al. (2003) demonstrated that 1000 g/L BPA significantly increased plasma Vtg in male zebrafish and juvenile rainbow trout under semi-static conditions for 3 weeks, and both species exposed to lower BPA concentration (200 g/L) showed no response during the whole study. The results of Pait and Nelson (2003) indicated that 0.17 mg/mL plasma Vtg were induced in Fundulus heteroclitus (killifish) after intraperitoneal injection of 50 mg/kg BPA within 8 days. From the above-mentioned results, it can be found that the sensitivity of Chinese loaches to BPA was comparable with that in other fish species. Meanwhile, it also indicated that long-term exposure to low concentrations of BPA induced the synthesis of Vtg. Normally, the estrogenic effect of effluent was mainly attributed to the presence of natural and synthetic estrogens because of the low estrogenicity of alkylphenols and other estrogenic compounds. The results of our study demonstrated that the vitellogenic responses of male Chinese loaches to the binary mixtures of E2 and BPA were more potent than those produced in each compound tested alone, and Vtg levels induced in the binary mixture groups were even higher than the summation of Vtg levels produced in the single-compound exposed groups. The results indicated each estrogenic chemical contributed to the overall effects and any evaluation on the estrogenic effects of chemicals in the real-world situation should consider them as
a whole (Routledge et al., 1998). However, on the basis of the preliminary information obtained in the present study, it cannot be concluded that whether the effect between BPA and E2 was synergistic. In conclusion, the results of the present study indicated that male Chinese loaches were sensitive to E2; BPA was estrogenic in male Chinese loaches, although less potent than E2; the estrogenic effect of the binary mixture of E2 and BPA was more potent than that of individual compound, and Vtg levels induced in the binary mixture were higher than the summation of Vtg levels produced in each tested compound alone. In most cases, it was concluded that individual estrogenic compound posed no hazard to human and wildlife because of their very low concentrations. However, our study demonstrated that weak xenoestrogens are able to contribute to the overall mixture effect, even at low concentrations, and even in the presence of strong effects of endogenous steroidal estrogens. Therefore, during environmental hazard and risk assessment, we must take into account of the combination effects of mixtures of endocrine disrupting chemicals, which will not lead to the underestimation of potential hazards. Acknowledgements This work was jointly supported by the State High Tech Development Plan (2006AA06Z424), National Basic Research Program of China (2003CB415001), and Chinese Academy of Sciences (KJCX2-SW-H06). References Brian, J.V., Harris, C.A., Scholze, M., Backhaus, T., Booy, P., Lamoree, M., Pojana, G., Jonkers, N., Runnalls, T., Bonf`a, A., Marcomini, A., Sumpter, J.P., 2005. Accurate prediction of the response of freshwater fish to a mixture of estrogenic chemicals. Environ. Health Perspect. 113, 721–728. Kanno, S., Hirano, S., Kayama, F., 2004. Effects of phytoestrogens and environmental estrogens on osteoblastic differentiation in MC3T3-E1 cells. Toxicology 196, 137–145. Larsen, B.K., Bjørnstad, A., Sundt, R.C., Taban, I.C., Pampanin, D.M., Andersen, O.K., 2006. Comparison of protein expression in plasma from nonylphenol and bisphenol A-exposed Atlantic cod (Gadus morhua) and turbot (Scophthalmus maximus) by use of SELDI-TOF. Aquat. Toxicol. 78, S25–S33. Lindholst, C., Pedersen, K.L., Pedersen, S.N., 2000. Estrogenic response of bisphenol A in rainbow trout (Oncorhynchus mykiss). Aquat. Toxicol. 48, 87–94. Lutz, I., Bl¨odt, S., Kloas, W., 2005. Regulation of estrogen receptors in primary cultured hepatocytes of the amphibian Xenopus laevis as estrogenic biomarker and its application in environmental monitoring. Comp. Biochem. Physiol. 141C, 384–392. Lv, X.F., Shao, J., Song, M.Y., Zhou, Q.F., Jiang, G.B., 2006. Vitellogenic effects of 17-estradiol in male Chinese loach (Misgurnus anguillicaudatus). Comp. Biochem. Physiol. 143C, 127–133. Marin, M.G., Matozzo, V., 2004. Vitellogenin induction as a biomarker of exposure to estrogenic compounds in aquatic environments. Mar. Pollut. Bull. 48, 835–839. Oehlmann, J., Schulte-Oehlmann, U., Bachmann, J., Oetken, M., Lutz, I., Kloas, W., Ternes, T.A., 2006. Bisphenol A induced superfeminization in the ramshorn snail Marisa cornuarietis (Gastropoda; Prosobranchia) at environmentally relevant concentrations. Environ. Health Perspect. 114, 127– 133.
X. Lv et al. / Environmental Toxicology and Pharmacology 24 (2007) 155–159 Pait, A.S., Nelson, J.O., 2003. Vitellogenesis in male Fundulus heteroclitus (killifish) induced by selected estrogenic compounds. Aquat. Toxicol. 64, 331–342. Panter, G.H., Thompson, R.S., Sumpter, J.P., 1998. Adverse reproductive effects in male fathead minnow (Pimephales promelas) exposed to environmentally relevant concentrations of the natural oestrogens, oestradiol and oestrone. Aquat. Toxicol. 42, 243–253. Payne, J., Rajapakse, N., Wilkins, M., Kortenkamp, A., 2000. Predication and assessment of the effects of mixtures of four xenoestrogens. Environ. Health Perspect. 108, 983–987. Rajapakse, N., Ong, D., Kortenkamp, A., 2001. Defining the impact of weakly estrogenic chemicals on the action of steroidal estrogens. Toxicol. Sci. 60, 296–304. Rajapakse, N., Silva, E., Kortenkamp, A., 2002. Combing xenoestrogens at levels below individual no-observed-effect concentrations dramatically enhances steroid hormone action. Environ. Health Perspect. 110, 917–921. Routledge, E.J., Sheahan, D., Desbrow, C., Brighty, G.C., Waldock, M., Sumpter, J.P., 1998. Identification of estrogenic chemicals in STW effluent. 2. In vivo responses in trout and roach. Environ. Sci. Technol. 32, 1559–1565. Shao, J., Shi, G.Q., Jin, X.L., Song, M.Y., Shi, J.B., Jiang, G.B., 2005. Development and validation of an enzyme-linked immunosorbent assay for
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vitellogenin in Chinese loach (Misgurnus anguillicaudatus). Environ. Int. 31, 763–770. Shi, G.Q., Shao, J., Jiang, G.B., Wang, Q.X., Lu, Y.Q., Liu, J.F., Liu, J.M., 2003. Membrane chromatographic method for the rapid purification of vitellogenin from fish plasma. J. Chromatogr. B 785, 361–368. Silva, E., Rajapakse, N., Kortenkamp, A., 2002. Something from “nothing”eight weak estrogenic chemicals combined at concentrations below NOECs produce significant mixture effects. Environ. Sci. Technol. 36, 1751–1756. Sumpter, J.P., Jobling, S., 1995. Vitellogenesis as a biomarker for estrogenic contamination of the aquatic environment. Environ. Health Perspect. 103, 173–178. Thorpe, K.L., Cummings, R.I., Hutchinson, T.H., Scholze, M., Brighty, G., Sumpter, J.P., Tyler, C.R., 2003. Relative potencies and combination effects of steroids estrogens in fish. Environ. Sci. Technol. 37, 1142–1149. Van den Belt, K., Verheyen, R., Witters, H., 2003. Comparison of vitellogenin responses in zebrafish and rainbow trout following exposure to environmental estrogens. Ecotoxicol. Environ. Saf. 56, 271–281. Yamaguchi, A., Ishibashi, H., Kohra, S., Arizono, K., Tominaga, N., 2005. Short-term effects of endocrine-disrupting chemicals on the expression of estrogen-responsive genes in male medaka (Oryzias latipes). Aquat. Toxicol. 72, 239–249.
Vitellogenic responses of 17-estradiol and bisphenol A in male Chinese loach (Misgurnus anguillicaudatus) Xuefei Lv, Qunfang Zhou, Maoyong Song, Guibin Jiang ∗ , Jing Shao State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, P.O. Box 2871, 100085 Beijing, PR China Received 31 December 2006; received in revised form 18 April 2007; accepted 22 April 2007 Available online 27 April 2007
Abstract In this study, male Chinese loaches were exposed to 17-estradiol (E2), bisphenol A (BPA) and their mixtures, respectively, for 42 days using a semi-static exposure system. Plasma vitellogenin (Vtg) in male Chinese loaches was used as the determining endpoint. The results demonstrated that male Chinese loaches were sensitive to E2, and the vitellogenic responses showed time- and dose-dependent increase. Similarly, BPA induced the estrogenic effects in male Chinese loaches, and the vitellogenic response increased in a time- and dose-dependent manner. The synthesis of Vtg was initiated by the exposure to higher level of BPA (500 g/L) within 7 days, and a relative long-term exposure to lower concentrations of BPA (10–100 g/L) also led to the production of Vtg. The estrogenic effect of the binary mixture of E2 and BPA also showed a time- and dose-dependent response, which was more potent than that of individual compounds, and Vtg contents in the binary mixture group were higher than the summation of Vtg contents in the single-compound groups at the same concentration. © 2007 Elsevier B.V. All rights reserved. Keywords: Bisphenol A; Chinese loach; Chemical interaction; Endocrine disruption; Estradiol; Experimental animal; Misgurnus anguillicaudatus; Vitellogenin
1. Introduction Xenoestrogens have received considerable attention because of their adverse effect on the functions of endocrine system in wildlife and humans, and many of those compounds are industrial chemicals. Bisphenol A (BPA) has received particular attention because of its large-scale production and widespread use. BPA is primarily used in the production of polycarbonate plastic, epoxy resins, and other plastic products (Lindholst et al., 2000; Larsen et al., 2006). The estrogenic potency of BPA has been demonstrated in various in vitro (Kanno et al., 2004; Lutz et al., 2005; Oehlmann et al., 2006) and in vivo (Lindholst et al., 2000; Pait and Nelson, 2003; Yamaguchi et al., 2005) studies. E2, as the main natural estrogen, is present in the sewage treatment works (STWs) effluent, river water, and drinking water with the concentration at tens of nanograms per liter (Routledge et al., 1998). BPA is also detected in the STWs effluent, however, because of its relatively
∗
Corresponding author. Tel.: +86 10 62849334; fax: +86 10 62849179. E-mail address: [email protected] (G. Jiang).
1382-6689/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2007.04.007
low estrogenicity, the estrogenic effect of STWs effluent was mainly attributed to the presence of natural and synthetic estrogens (Thorpe et al., 2003; Brian et al., 2005). Current studies are usually focused on the assessment of the estrogenic potency of single chemicals, however, in the real-world situation, fish live in the environment containing many different kinds of endocrine disrupting chemicals (EDCs). Meanwhile, studies have demonstrated the estrogenic effects of mixtures of xenoestrogens were more potent than those of the individual chemicals at no-observed-effect concentrations (Payne et al., 2000; Rajapakse et al., 2002; Silva et al., 2002; Thorpe et al., 2003; Brian et al., 2005). So, it is much more important to assess the effects of the mixtures of estrogenic compounds than those of the isolated ones. Chinese loach (Misgurnus anguillicaudatus) was chosen as experimental animal in this study, as it was previously demonstrated to be sensitive to 17-estradiol (Lv et al., 2006). The estrogenicity of BPA in male Chinese loach remains to be investigated. The study of Rajapakse et al. (2001) demonstrated that BPA could contribute to the overall mixture effect at the presence of E2 using the yeast estrogen screen, which remains to be
156
X. Lv et al. / Environmental Toxicology and Pharmacology 24 (2007) 155–159
validated in in vivo studies. Until now, little is known about the estrogenic effect of the mixture of BPA and E2 in fish species including male Chinese loaches. In the current study, male Chinese loaches were exposed to various concentrations of E2, BPA, and binary mixtures of E2 and BPA at the fixed-ration of 1:100, respectively, to evaluate their estrogenic effect on this kind of fish. Plasma Vtg in male Chinese loaches was used as determining endpoint. 2. Materials and methods 2.1. Chemicals 17-Estradiol (E2, 98% purity) was purchased from Sigma; bisphenol A (BPA, 4,4 -isopropylidenediphenol, 97% purity) was obtained from Acros Organics (New Jersey). The stock solutions of E2 and BPA were prepared by dissolving suitable amounts of chemicals into methanol, sealed and stored at 4 ◦ C. The working solutions were made by diluting the stock solutions with ultrapure water (EASY pure LF).
2.2. Fish The fish used in this study were mature male Chinese loaches. Healthy Chinese loaches were obtained from the market of Beijing, China, and were acclimated in stainless steel tanks with dechlorinated tap water for at least 2 weeks prior to the exposure experiment. The mean weight and length of fish were 9.11 ± 1.55 g and 11.88 ± 0.65 cm, respectively. The day/night cycle was set as natural length. Throughout the study, water quality was monitored by detecting pH (6.9–7.9), oxygen concentration (5–7 mg/L), and temperature (22.5–25.5 ◦ C). At the beginning of the experiment, 15 fish were randomly allocated to 12 L glass tanks with a working volume of 5 L. Two aquaria were dosed at the same concentration. Water was changed every other day during the exposure. The fish were fed with Oligochaetes limnodirlus sp. twice a week.
2.3. Experimental design Male Chinese loaches were exposed either to nominal concentrations of E2 at 0.1, 0.5, 1, and 5 g/L, or to nominal concentrations of BPA at 10, 50, 100, and 500 g/L, or to fixed-ration binary mixtures of E2 and BPA (1:100) at nominal concentrations of 0.1 + 10, 0.5 + 50, and 1 + 100 g/L. The solventcontrol group (0.05‰ methanol) and the control group were also set up. Loaches were provided with dechlorinated tap water, and the whole exposure experiment lasted 42 days.
the coated plate, and then incubated with horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG at a final dilution of 1:2000 in PBST. Following this step, the enzyme substrate solution (0.05 M phosphate–citrate buffer, 0.4 mg/mL o-phenylenediamine, and 0.16% H2 O2 ) was added. Thirty minutes later, the reaction was stopped by 2 M HCl. The absorbance was measured at 490 nm using a microtiter plate reader (DNA Expert, TECAN, Austria).
2.6. Data expression and statistical analysis All the values were expressed as mean ± S.D. The ELISA data were processed by a four-parameter Boltzmann equation: y = a + b/[1 + exp(x − c)/d] where y represents the percentage binding of sample or standard relative to analyte free wells (Bi /B0 ) and x represents log dose. The effects of different treatments on vitellogenin concentrations were assessed using one-way analysis of variance (ANOVA). The level of significance was set as p < 0.05.
3. Results 3.1. Vtg induction in male Chinese loach exposed to 17β-estradiol Male Chinese loaches were exposed to 17-estradiol at nominal concentrations of 0.1, 0.5, 1, and 5 g/L for 42 days (Fig. 1). It was seen that Vtg concentrations in control and solvent-control groups were not detected within 28 days; however, on days 35 and 42, relative high levels Vtg can be detected, and the reasons remain to be unsolved. Vtg can be significantly enhanced at 1 and 5 g/L E2 within 7 days (p < 0.05) with average values of 18.12 ± 2.20 and 211.93 ± 27.06 g/mL, respectively, and the vitellogenic responses of the two exposed groups showed time- and dose-dependent increase during the whole experiment. 0.5 g/L E2 significantly induced the production of Vtg within 14 days (p < 0.05) with an average value of 43.04 ± 0.46 g/mL. However, when the concentration of E2 was 0.1 g/L, a 35 days exposure was needed to induce the synthesis of Vtg, though not significant, with an average value of 50.05 ± 19.11 g/mL.
2.4. Plasma collection Fish were randomly sampled on days 7, 14, 21, 28, 35, and 42 of the exposure (n = 4 for each dose). Fish were anesthetized with quinoline sulfate (40 mg/L), and after recording weight and length, blood samples were taken from the caudal vein using heparinized syringes and transferred to 1.5 mL centrifuge tubes in the presence of aprotinin (2.5 TIU, Roche, Germany). After centrifugation (3000 rpm, 4 ◦ C, 30 min), plasma samples were divided into aliquots and stored at −20 ◦ C.
2.5. Vitellogenin determination The plasma Vtg was purified with anion-exchange membrane (SartobindTM MA 15D, Germany) according to the methods described previously (Shi et al., 2003; Shao et al., 2005). The concentrations of Vtg in the plasma were determined with a competitive enzyme-linked immunosorbent assay (ELISA) described previously (Shao et al., 2005). Briefly, after coated with 750 ng/mL purified Vtg, the 96-well microtiter plate was blocked with 1% bovine serum albumin (BSA). Standards and samples, incubated with primary antibody solution at a final dilution of 1:16,000 in PBST (0.02 M phosphate buffer with 0.15 M NaCl and 0.05% Tween 20, pH 7.4) overnight at 4 ◦ C, were transferred to
Fig. 1. Vitellogenic responses of male Chinese loach exposed to 17-estradiol at the nominal concentrations of 0.1, 0.5, 1, and 5 g/L for 42 days. Values are means ± S.D. (n = 4). (*) Significant differences from the control groups at p < 0.05.
X. Lv et al. / Environmental Toxicology and Pharmacology 24 (2007) 155–159
Fig. 2. Vitellogenic responses of male Chinese loach exposed to BPA at the nominal concentrations of 10, 50, 100, and 500 g/L for 42 days. Values are means ± S.D. (n = 4). () The lack of samples for 50 g/L BAP on day 42. (*) Significant differences from the control groups at p < 0.05.
3.2. Vtg induction in male Chinese loach exposed to BPA Fig. 2 illustrates plasma Vtg levels in male Chinese loaches induced by various doses of BPA (10, 50, 100, and 500 g/L) during 42 days exposure. No Vtg was induced following exposure to 10, 50, and 100 g/L BPA for 28 days but Vtg were significantly enhanced after 35 days exposure (p < 0.05), and on day 42, Vtg levels in the groups of 10 and 100 g/L BPA were 360.08 ± 11.63 and 968.09 ± 92.69 g/mL, respectively. 31.98 ± 9.43 g/mL of Vtg were detected in the plasma of male Chinese loaches exposed to 500 g/L BPA within 7 days, which was significantly different from the control groups (p < 0.05), and from days 14 to 42, Vtg levels increased from 4.22 ± 0.02 to 47.61 ± 2.60 mg/mL. 3.3. Vtg induction in male Chinese loach exposed to the binary mixtures of E2 and BPA The vitellogenic responses of male Chinese loach exposed to the fixed-ration (1:100) binary mixtures of E2 and BPA at the nominal concentrations of 0.1 + 10, 0.5 + 50 , and 1 + 100 g/L for 42 days are shown in Fig. 3. Vtg concentrations in the binary mixture exposed group showed a timeand dose-increasing manner. 0.1 g/L E2 + 10 g/L BPA did not induce the synthesis of Vtg within 28 days, and compared with the control groups, Vtg levels were significantly induced by 0.1 g/L E2 + 10 g/L BPA after 35 days exposure (p < 0.05), and Vtg levels in this group increased to 1.31 ± 0.20 mg/mL on day 42. 0.5 g/L E2 + 50 g/L BPA significantly induced the synthesis of Vtg (6.81 ± 0.99 g/mL) within 7 days (p < 0.05), and after a 42 days exposure, Vtg levels increased to 1.38 ± 0.37 mg/mL. Exposure to 1 g/L E2 + 100 g/L BPA significantly induced the synthesis of Vtg (112.75 ± 3.21 g/mL) within 7 days compared with the control and solvent-control groups (p < 0.05). Moreover, Vtg levels in the co-exposed groups were all higher than the summation
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Fig. 3. Vitellogenic responses of male Chinese loach exposed to binary mixtures of E2 and BPA at the fixed-ration (1:100) (i.e. the nominal concentrations of 0.1 g/L E2 + 10 g/L BPA, 0.5 g/L E2 + 50 g/L BPA, and 1 g/L E2 + 100 g/L BPA) for 42 days. Values are means ± S.D. (n = 4). () The lack of sample for 1 g/L E2 plus 100 g/L BPA on day 14. (*) Significant differences from the control groups at p < 0.05.
of Vtg in the single-compound exposed groups, as shown in Fig. 4. 4. Discussion The attractive characteristics including moderate size, easy to culture and identify gender (by the shape of pectoral fin) make loach suitable as a candidate fish in field and laboratory studies (Shao et al., 2005). A previous study of our group has demonstrated that male Chinese loach was sensitive to 17-estradiol (Lv et al., 2006), and this was validated in the present study. The ability of E2 to induce the synthesis of Vtg in other species following waterborne exposure has also been documented, such as fathead minnow (Pimephales promelas) (Panter et al., 1998), rainbow trout (Oncorhynchus mykiss) (Routledge et al., 1998;
Fig. 4. Comparison between Vtg levels in the group of E2 + BPA treated fish (夽) and the summation of Vtg levels in the individual E2 and BPA groups () after exposure for 35 days.
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Lindholst et al., 2000), roch (Rutilus rutilus) (Routledge et al., 1998), zebrafish (Danio rerio) (Van den Belt et al., 2003), etc. Compared the results on the same species and those from other species, it can be found that different fish species, even the same species at different reproductive stages, might show different sensitivity; the applied Vtg quantification techniques and the exposure system (semi-static versus flow-through) might also have an impact on the differences in sensitivity (Van den Belt et al., 2003). Until now, no studies have been performed to address the estrogenicity of BPA in male Chinese loach by water exposure. Vitellogenin (Vtg) is normally produced in sexually maturing females. Under natural conditions, male fish cannot synthesize Vtg, However, they can be induced to synthesize Vtg when exposed to estrogens or estrogen mimics, so the abnormal level of Vtg in male fish has been used as a good biomarker to demonstrate estrogenic effects of xenoestrogens in the aquatic environment (Sumpter and Jobling, 1995; Marin and Matozzo, 2004). Therefore, the presence of Vtg in male Chinese loach was used to evaluate the estrogenicity of BPA using semi-static exposure system in the present study. The results (Fig. 2) demonstrated that BPA was estrogenic in male Chinese loach, although less potent than E2, and Vtg was induced in time- and dosedependent manner. A 28 days exposure to BPA at the nominal concentrations of 10, 50, and 100 g/L did not induce the production of Vtg, however 500 g/L BPA significantly induced the synthesis of Vtg within 7 days. Plasma Vtg synthesis in male Chinese loach can be compared with that in other fish species after similar exposure. Lindholst et al. (2000) found that BPA concentration in water lower than 70 g/L did not induce Vtg synthesis within 12 days in rainbow trout (O. mykiss), and 500 g/L BPA induced the production of Vtg with the value of 1168 g/mL within 6 days. Van den Belt et al. (2003) demonstrated that 1000 g/L BPA significantly increased plasma Vtg in male zebrafish and juvenile rainbow trout under semi-static conditions for 3 weeks, and both species exposed to lower BPA concentration (200 g/L) showed no response during the whole study. The results of Pait and Nelson (2003) indicated that 0.17 mg/mL plasma Vtg were induced in Fundulus heteroclitus (killifish) after intraperitoneal injection of 50 mg/kg BPA within 8 days. From the above-mentioned results, it can be found that the sensitivity of Chinese loaches to BPA was comparable with that in other fish species. Meanwhile, it also indicated that long-term exposure to low concentrations of BPA induced the synthesis of Vtg. Normally, the estrogenic effect of effluent was mainly attributed to the presence of natural and synthetic estrogens because of the low estrogenicity of alkylphenols and other estrogenic compounds. The results of our study demonstrated that the vitellogenic responses of male Chinese loaches to the binary mixtures of E2 and BPA were more potent than those produced in each compound tested alone, and Vtg levels induced in the binary mixture groups were even higher than the summation of Vtg levels produced in the single-compound exposed groups. The results indicated each estrogenic chemical contributed to the overall effects and any evaluation on the estrogenic effects of chemicals in the real-world situation should consider them as
a whole (Routledge et al., 1998). However, on the basis of the preliminary information obtained in the present study, it cannot be concluded that whether the effect between BPA and E2 was synergistic. In conclusion, the results of the present study indicated that male Chinese loaches were sensitive to E2; BPA was estrogenic in male Chinese loaches, although less potent than E2; the estrogenic effect of the binary mixture of E2 and BPA was more potent than that of individual compound, and Vtg levels induced in the binary mixture were higher than the summation of Vtg levels produced in each tested compound alone. In most cases, it was concluded that individual estrogenic compound posed no hazard to human and wildlife because of their very low concentrations. However, our study demonstrated that weak xenoestrogens are able to contribute to the overall mixture effect, even at low concentrations, and even in the presence of strong effects of endogenous steroidal estrogens. Therefore, during environmental hazard and risk assessment, we must take into account of the combination effects of mixtures of endocrine disrupting chemicals, which will not lead to the underestimation of potential hazards. Acknowledgements This work was jointly supported by the State High Tech Development Plan (2006AA06Z424), National Basic Research Program of China (2003CB415001), and Chinese Academy of Sciences (KJCX2-SW-H06). References Brian, J.V., Harris, C.A., Scholze, M., Backhaus, T., Booy, P., Lamoree, M., Pojana, G., Jonkers, N., Runnalls, T., Bonf`a, A., Marcomini, A., Sumpter, J.P., 2005. Accurate prediction of the response of freshwater fish to a mixture of estrogenic chemicals. Environ. Health Perspect. 113, 721–728. Kanno, S., Hirano, S., Kayama, F., 2004. Effects of phytoestrogens and environmental estrogens on osteoblastic differentiation in MC3T3-E1 cells. Toxicology 196, 137–145. Larsen, B.K., Bjørnstad, A., Sundt, R.C., Taban, I.C., Pampanin, D.M., Andersen, O.K., 2006. Comparison of protein expression in plasma from nonylphenol and bisphenol A-exposed Atlantic cod (Gadus morhua) and turbot (Scophthalmus maximus) by use of SELDI-TOF. Aquat. Toxicol. 78, S25–S33. Lindholst, C., Pedersen, K.L., Pedersen, S.N., 2000. Estrogenic response of bisphenol A in rainbow trout (Oncorhynchus mykiss). Aquat. Toxicol. 48, 87–94. Lutz, I., Bl¨odt, S., Kloas, W., 2005. Regulation of estrogen receptors in primary cultured hepatocytes of the amphibian Xenopus laevis as estrogenic biomarker and its application in environmental monitoring. Comp. Biochem. Physiol. 141C, 384–392. Lv, X.F., Shao, J., Song, M.Y., Zhou, Q.F., Jiang, G.B., 2006. Vitellogenic effects of 17-estradiol in male Chinese loach (Misgurnus anguillicaudatus). Comp. Biochem. Physiol. 143C, 127–133. Marin, M.G., Matozzo, V., 2004. Vitellogenin induction as a biomarker of exposure to estrogenic compounds in aquatic environments. Mar. Pollut. Bull. 48, 835–839. Oehlmann, J., Schulte-Oehlmann, U., Bachmann, J., Oetken, M., Lutz, I., Kloas, W., Ternes, T.A., 2006. Bisphenol A induced superfeminization in the ramshorn snail Marisa cornuarietis (Gastropoda; Prosobranchia) at environmentally relevant concentrations. Environ. Health Perspect. 114, 127– 133.
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