- Email: [email protected]
Distribution of vitellogenin in Japanese flounder (Paralichthys olivaceus) for biomarker analysis of marine environmental estrogens
Aquatic Toxicology 216 (2019) 105321
Contents lists available at ScienceDirect
Aquatic Toxicology journal homepage: www.elsevier.com/locate/aqtox
Distribution of vitellogenin in Japanese flounder (Paralichthys olivaceus) for biomarker analysis of marine environmental estrogens
T
⁎
Zhenzhong Zhanga, Jun Wanga, , Zongbao Panb, Yabin Zhanga, Xiaona Zhanga, Hua Tiana, ⁎ Wei Wanga, Shaoguo Rua, a b
College of Marine Life Sciences, Ocean University of China, Qingdao, 266003, China Zhejiang Institute of Hydraulics & Estuary, Hangzhou, 310020, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Biomarker Environmental estrogens Direct immunofluorescence Lipovitellin Paralichthys olivaceus
Estrogen pollution in marine environments has become a research hotspot due to its adverse effects on the reproduction of wild organisms. To early detection of estrogen pollution, this study developed two methods for detecting Japanese flounder vitellogenin (Vtg), a sensitive biomarker for environmental estrogens. Firstly, monoclonal antibodies (mAb) specific to Vtg were prepared using purified lipovitellin (Lv), a main Vtg-derived yolk protein. Anti-Lv mAb (C1F1) had the highest titer (1:256,000) and was labeled with fluorescein isothiocyanate to establish a direct immunofluorescence (DIF) method for histological detection of Vtg in tissues. Additionally, using the purified Lv and mAb, an enzyme-linked immunosorbent assay (ELISA) was developed and this assay had a detection limit of 0.75 ng/mL and a working range of 1.95–250 ng/mL. Furthermore, Vtg induction in the plasma of Japanese flounder exposed to 17β-estradiol (E2), 17α-ethinylestradiol (EE2), and bisphenol A (BPA) were quantified by ELISA, and Vtg induction in the liver of EE2-exposed Japanese flounder were measured by DIF. Finally, the distribution of Vtg in Japanese flounder was detected using these two methods. The results revealed that Vtg mainly appeared in the terminal tail fin, liver, kidney, intestine, and spleen. Considering the high concentration of Vtg and easy sample collection, the terminal tail fin could be a new alternative to plasma for Vtg quantification, while kidney and liver are suitable for histological detection of Vtg.
1. Introduction During the past three decades, environmental estrogens have aroused great concern for their adverse effects on gonadal development and reproduction in aquatic organisms (Segner et al., 2013; Xu et al., 2014). These pollutants enter the ocean through surface runoff and have been frequently detected in offshore seawater. For example, 17αethinylestradiol (EE2), a synthetic estrogen compound, had concentrations of 4.7 and 24 ng/L in surface waters of the Acushnet River Estuary (USA) and Jiaozhou Bay (China) (Zuo et al., 2006; Zhou et al., 2011). Environmental estrogens could disturb the growth and reproduction of marine organisms at a low ng/L level. For example, 1 ng/L EE2 induced feminization in male gulf pipefish (Syngnathus scovelli) and reduced their mating opportunities (Partridge et al., 2010). Also, Hu et al. (2017) reported that 0.05 ng/L 17β-estradiol (E2) could cause malformation in embryos of clearhead icefish (protosalanx hyalocranius). Given the potential ecological risk of estrogenic pollution to marine organisms, there is an urgent need to establish reliable methods for detecting these emerging pollutants. ⁎
To date, several biomarkers have been developed for monitoring environmental estrogens, of which vitellogenin (Vtg) is the most widely used (Sumpter and Jobling, 1995; Tyler et al., 1999). Vtg is a femalespecific protein and is generally synthesized by hepatocytes of sexually mature females in response to 17β-estradiol, released into the blood circulation, and deposited in developing oocytes, where it is cleaved into lipovitellin (Lv), phosvitin (Pv), and other yolk proteins (Hara et al., 2016; Verderame and Scudiero 2017). Although juvenile and male fish do not have detectable Vtg, estrogenic mimics can easily induce them to synthesize Vtg. Thus, the abnormal production of Vtg in juvenile and male fish can sensitively indicate estrogenic pollution in aquatic environments (Tollefsen et al., 2003; Ihara et al., 2015). Recently, most studies evaluated the estrogenic activities of chemicals by measuring Vtg concentrations in the plasma of male fish (Madsen et al., 2013; Flick et al., 2014). Compared to plasma samples, the liver was considered to be the main tissue that synthesizes Vtg, and Vtg induction in the liver may be an early indicator for estrogenic pollution (Hiramatsu et al., 2005; Sawaguchi et al., 2005). In addition, many extrahepatic tissues also have the potential to synthesize or store Vtg. In
Corresponding authors at: College of Marine Life Sciences, Ocean University of China, 5 Yushan Road, Qingdao, 266003, Shandong province, China. E-mail addresses: [email protected] (J. Wang), [email protected] (S. Ru).
https://doi.org/10.1016/j.aquatox.2019.105321 Received 28 July 2019; Received in revised form 22 September 2019; Accepted 26 September 2019 Available online 27 September 2019 0166-445X/ © 2019 Elsevier B.V. All rights reserved.
Aquatic Toxicology 216 (2019) 105321
Z. Zhang, et al.
labeled anti-Lv mAb was obtained by semi-saturated ammonium sulfate precipitation and redissolved in PBS (containing 0.01% NaN3 and 0.5% BSA).
zebrafish (Danio rerio), Vtg was detected in the heart (Yin et al., 2009), intestine, muscles (Wang et al., 2005), skin (Jin et al., 2008), and fin (Zhong et al., 2014). As for marine fish, there is no report on tissue distribution of Vtg, and it is unclear whether some tissues other than blood are more suitable for detecting Vtg as a biomonitor of marine estrogenic pollution. Japanese flounder (Paralichthys olivaceus), a marine benthic fish widely distributed in Northeast Asia, was recommended as a good sentinel species for detecting environmental estrogens for its large size, easy blood collection, strong survival ability and weak exercise capacity (Cao et al., 2010; Huang et al., 2010; Liu et al., 2017). In this study, an enzyme-linked immunosorbent assay (ELISA) for quantifying Japanese flounder Vtg and a direct immunofluorescence (DIF) method for histological detection of Vtg in tissues were established using anti-Lv monoclonal antibody (mAb). Moreover, their reliability for detection of exogenous estrogens were validated by measuring Vtg induction in juvenile Japanese flounder exposed to three common environmental estrogens. Finally, the distribution of Vtg in different tissues of Japanese flounder were investigated using the above-mentioned methods to identify new alternative tissues for easy and sensitive detection of the Vtg biomarker.
2.4. DIF The liver tissue of Japanese flounder was fixed in 4% paraformaldehyde for the preparation of 5-μm paraffin sections according to the standard procedure (Aljandal et al., 2017). The paraffin sections were deparaffinized in xylene for 15 min and hydrated in a series of graded ethanol. After hydration, the sections were immersed in citrate buffer (0.01 M, pH 6) for antigen retrieval and then blocked in 5% BSA for 1 h at 37 °C to reduce non-specific binding. Finally, tissue sections were incubated with diluted FITC-labeled anti-Lv mAb for 1 h at room temperature and observed under a fluorescence microscope (Nikon Eclipse 50i, Florida). 2.5. Sandwich ELISA Sandwich ELISA was established according to previously reported methods (Li et al., 2018). The plates were firstly coated with anti-Lv mAb (100 μL/well) and then blocked by 1% BSA. After washing, 100 μL of samples or standards (0.95–1000 ng/mL purified Lv) were added into wells and incubated at 37 °C for 1 h. Afterwards, wells were incubated with different dilutions of HRP-labeled anti-Lv IgG solution, and color was developed using TMB substrate (Solarbio, China). The absorbance at 450 nm was measured using a plate reader (Multiskan MK2, Thermo USA). In addition, the precision, sensitivity, and specificity of the assay were analyzed according to methods of Wang et al. (2017).
2. Materials and methods 2.1. Purification and identification of Lv Ovulated eggs were collected from female Japanese flounder and homogenized to prepare an egg homogenate according to the methods of Wang et al. (2015b), and the purification of Lv was conducted using water precipitation method established in our previous study (Zhang et al., 2019). The purity of the obtained protein was analyzed using native polyacrylamide gel electrophoresis (Native-PAGE). Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF/TOF MS) analysis was used to determine the amino-acid sequence of the purified protein (Prasatkaew et al., 2019). Briefly, the protein band was cut from Native-PAGE and digested overnight at 37 °C with 0.02 μg/μL trypsin. Peptide was measured in a MALDI-TOF/TOF MS mass spectrometer (Applied Biosystems, Foster City, USA) in a positive MS reflector mode. The peptide mass fingerprint was analyzed using MASCOT V2.3 search engine (Matrix Science Ltd., London, U.K.), and peptide sequences were identified from the databases at NCBI-bony vertebrates.
2.6. Vtg induction in juvenile Japanese flounder exposed to environmental estrogens Juvenile Japanese flounder (n = 6 per concentration, 18.36 ± 2.94 g, 8.27 ± 1.03 cm) were exposed to nominal concentrations of 0, 2, 10, and 50 ng/L EE2 (Sigma-Aldrich) in 100-L aquaria. Moreover, sexually mature male Japanese flounder (n = 5 per concentration, 800 ± 100 g, 35 ± 5 cm) were exposed to 10 μg/L E2, 10 μg/L EE2, and 100 μg/L BPA. The stock solution of E2, EE2, and BPA was prepared in dimethyl sulfoxide (DMSO) and kept at 4 °C. The DMSO concentration in the control and exposure groups was below 0.01%. To maintain constant exposure concentrations, half of the volume seawater was renewed daily in all tanks. After 7 days, blood was collected from sexually mature Japanese flounder, and after 21 days blood was also collected from the caudal vein of juvenile Japanese flounder. The plasma was obtained by centrifugation, and plasma Vtg was detected by Western blot and sandwich ELISA. The actual concentrations of EE2 in the seawater of 2, 10, and 50 ng/L exposure groups were 0, 7.69 ± 0.72 ng/L, and 45.72 ± 0.99 ng/L, respectively, after 24 h of exposure (Zhang et al., 2019).
2.2. Production of anti-Lv mAb The anti-Lv mAb was prepared according to the method of Li et al. (2005). Briefly, Balb/c mice were immunized with 100 μg of purified Lv for three times in two-week intervals. Then the spleen was removed from the immunized mice, and the splenocytes were fused with the SP2/0 myeloma cells using polyethylene glycol 4000. After three rounds of screening and subcloning, the positive hybridoma cells were injected intraperitoneally into Balb/c mice, and anti-Lv mAb was purified from the ascitic fluid by affinity chromatography on a HiTrap Protein G column (GE Healthcare). The titer, molecular weight, and specificity were determined using conventional procedures (Ling et al., 2015).
2.7. Tissue distribution of Vtg after EE2 exposure After 21 days of exposure to 50 ng/L EE2, the liver, kidney, intestine, spleen, muscle, surface mucus, and terminal tail fin were collected from each fish. Each sample was divided into three parts: (i) for Vtg quantification, the tissues were homogenized in five volumes of ice-cold PBS containing protease inhibitor cocktails (Sigma-Aldrich); (ii) for quantification of Vtg1 and Vtg2 genes, the tissues were used for RNA extraction, reverse transcription and quantitative real-time PCR (qPCR), according to the methods of Yue et al. (2017), details were described in supplementary material; (iii) for histological detection of Vtg, tissues were fixed in 4% paraformaldehyde to prepare paraffin sections. Vtg in the homogenates were measured by Western blot and sandwich ELISA, Vtg1 and Vtg2 from samples were quantified by qPCR, and Vtg in tissue sections were visualized by DIF.
2.3. Preparation of FITC-labeled antibodies Anti-Lv mAb was covalently labeled with fluorescein isothiocyanate (FITC, Sigma-Aldrich) according to the methods of Zhang et al. (2014a) with minor modifications. Briefly, anti-Lv mAb was dialyzed three times in crosslinking agent (7.56 g/L NaHCO3, 1.06 g/L Na2CO3, and 7.36 g/L NaCl, in ddH2O, pH 9.0) at 4 °C. FITC was dissolved in the crosslinking agent (1 mg/mL) and added into the dialyzed mAb solution at a ratio of 1:6, followed by an agitation at 4 °C for 8 h in dark. FITC2
Aquatic Toxicology 216 (2019) 105321
Z. Zhang, et al.
50 ng/L EE2 exposure groups significantly increased to 66.1 ± 5.3 μg/ mL and 433.2 ± 75.9 μg/mL (P < 0.05, Fig. 3B).
2.8. Statistics Data were presented as mean ± standard deviation (SD), and the differences between the control and exposed groups were assessed by one-way analysis of variance followed by a Tukey’s post hoc test. Prior to parametric analysis, data were log-transformed to achieve variance homogeneity. All statistical tests were conducted using SPSS 18.0 software (SPSS Inc., USA), and values were determined as significant when P < 0.05.
3.4. Establishment of DIF for visible detection of Vtg
3. Results
When FITC-labeled anti-Lv mAb was diluted at 1:400, DIF showed the best result (Fig. S8). Vtg was undetected in the liver of Japanese flounder in the control and 2 ng/L EE2 exposure groups, but 10 ng/L EE2 induced clear green fluorescence near sinusoid vessels in the liver (Fig. 4C). In the 50 ng/L EE2 group, strong green fluorescence was observed near the central vein of liver (Fig. 4D).
3.1. Specificity of anti-Lv mAb to Japanese flounder Vtg
3.5. Distribution of Vtg in Japanese flounder
The protein purified from the egg homogenate showed a single band in Native-PAGE (Fig. S1) and matched well with amino acid sequences of Japanese flounder Vtg (XP_019936246.1, Fig. S2, S3). In Western blot analysis, five mAbs were obtained which could all recognize the purified Vtg and Lv, but not the plasma from the control male fish. Specifically, mAb C1F1 detected Vtg subunits with molecular weights of ˜120, ˜100, and ˜92 kDa, while other mAbs (C3F4, F4C4, F4F4 and D3E2) detected Vtg subunits with lower molecular weights (˜64, ˜56, and ˜46 kDa, Fig. 1). In addition, mAb C1F1 and C3F4 had the highest absorption and their titers were 1:256 000 and1:32 000, respectively (Fig. S4, S5).
After exposure to 50 ng/L EE2, positive bands were observed in the tail fin, liver, kidney, intestine, spleen, muscle, and surface mucus by Western blot analysis using anti-Lv mAb as the primary antibody (Fig. 5A). ELISA results showed that the highest Vtg level was detected in the tail fin (174.37 ± 9.31 μg/mg), followed by the liver (93.69 ± 1 4.06 μg/mg), kidney (93.69 ± 14.06 μg/mg), intestine (78.25 ± 15.18 μg/mg), spleen (70.20 ± 43.87 μg/mg), and muscle (44.10 ± 12.00 μg/mg) (Fig. 5B). Surface mucus had the lowest Vtg level (7.80 ± 0.59 μg/mg). Meanwhile, the results of qPCR showed that Vtg1 and Vtg2 mRNA were expressed at different levels in the six tissues. The highest expression levels of Vtg1 and Vtg2 were found in the liver, followed by the spleen and kidney. In contrast, relatively low transcription levels of Vtg1 and Vtg2 were found in the tail fin, intestine, and muscle (Fig. 6). The results of DIF showed that no fluorescent signal was detected in any of the juvenile Japanese flounder tissues from the control group. In the 50 ng/L EE2 groups, strong green fluorescent signals were observed in the kidney and liver, and relatively intense fluorescence signals were found in the spleen and intestine (Fig. 7). Conversely, the muscle showed little fluorescence signal.
3.2. Sandwich ELISA When HRP-labeled anti-Lv pAb was diluted at 1:10000, the sandwich ELISA had a wide working range of 1.95–250 ng/mL (y = 1.3649x-0.7707, R² = 0.98) and a limit of detection (LOD) of 0.75 ng/mL (Fig. 2A, B). Moreover, dilution curves of plasma from E2exposed males were parallel to the Lv standards (Fig. 2C). The intraand inter-assay coefficients of variation (CVs) of sandwich ELISA were 1.32–8.21% and 1.52–8.01%, respectively (Table S2).
4. Discussion
3.3. Vtg induction in plasma of Japanese flounder exposed to E2, EE2 and BPA
This study not only established an ELISA method to quantify Vtg concentration, but also developed a DIF method to locate Vtg in tissues based on anti-Lv mAb. Moreover, these two methods were combined to investigate the distribution of Vtg in marine fish. Fish Vtg is easily degraded during the purification process, and the breakdown products have more immunogenic than Vtg itself, which usually leads to the deviation of quantification of Vtg concentration (Magalhães et al., 2004; Brodeur et al., 2006). Lv, a highly stable Vtg-derived yolk protein, has shown the same immunogenicity as Vtg and was recommended as a better antigen to develop robust immunoassays for Vtg (Wang et al., 2015a). In our current study, Japanese flounder Lv was successfully purified by water precipitation and showed a single band in Native-PAGE, indicative of high purity. The result of mass spectrometry further confirmed that the purified protein was Japanese flounder Lv. Subsequently, using this purified Lv as an antigen, five mAbs were prepared which had good positive reaction with purified Vtg, while no band was observed in the plasma of the control male fish, demonstrating that the prepared mAbs had good specificity to Vtg (Luo et al., 2011; Garnayak et al., 2013). Moreover, mAb C1F1 had a very high titer, which was approximately eight times higher than pAb against zebrafish Lv (1:32,000) (Wang et al., 2017). Thus, our prepared anti-Lv mAbs were highly specific and sensitive for the development of Vtg immunoassays. To date, anti-Vtg pAb-based ELISAs were the most commonly used methods for quantifying plasma Vtg (Selcer and Verbanic, 2014; Gong et al., 2016), and their working ranges were usually 30–1000 ng/mL (Hennies et al., 2003; Roy et al., 2004; Scott et al., 2006). Our study developed a sandwich ELISA using anti-Lv mAb, and this assay had a LOD of 0.75 ng/mL, which was at least nine times lower than the LODs
Vtg concentrations in plasma of sexually mature male Japanese flounder exposed to BPA, E2 and EE2 were 207.26 ± 25.54 ng/mL, 359.12 ± 46.45 ng/mL, and 3501.99 ± 187.65 ng/mL, respectively, which were significantly higher than that in the control group (20.80 ± 0.03 ng/mL, Fig. S7). For juvenile Japanese flounder, no band was observed in either control or 2 ng/L EE2 exposure group, while plasma from 10 and 50 ng/L EE2 exposure groups showed several well-defined bands (Fig. 3A). Compared to the control plasma (0.2 ± 0.026 μg/mL Vtg), concentrations of Vtg in plasma of 10 and
Fig. 1. Western blot analysis of plasma from the control male (lane 1), purified Vtg (lane 2), and purified Lv (lane 3) using anti-Lv mAbs as primary antibodies. 3
Aquatic Toxicology 216 (2019) 105321
Z. Zhang, et al.
Fig. 2. Determination of optimal dilution of antibodies (A) for the sandwich ELISA using Japanese flounder Lv as the standard (B), and the detection of plasma dilution curves of the control male and E2-exposed male fish (C).
of ELISAs for Spotted wolffish (Anarhichas minor, 6.7 ng/mL, Maltais et al., 2014) and sea bass (Lates calcarifer) Vtg (40 ng/mL, Prasatkaew et al., 2019). The working range of the Vtg ELISA established in this study was 1.95–250 ng/mL, which was comparable with values of mAbbased sandwich ELISAs for Japanese medaka (Oryzias latipes) and Crucian carp (Carassius carassius) Vtgs (Nishi et al., 2002; An et al., 2007). Moreover, the robustness and specificity of this assay for quantifying Vtg were confirmed by low intra- and inter-assay CVs and parallelism between the Lv standard curve and plasma dilutions of E2exposed male fish (Roy et al., 2004; Wang et al., 2015a). The practicability and reliability of the method was verified by quantifying the plasma Vtg concentrations in the E2, EE2, and BPA exposure groups. The results showed that the order of estrogenic potency based on the Vtg induction was EE2 > E2 > BPA, which coincides with the results of a previous study (Petersen et al., 2013). These results demonstrated that the developed ELISA for Japanese flounder Vtg could be used to test estrogenic properties of chemicals. In addition, a DIF method was developed to visually detect Vtg induction in tissues using fluoresceinlabeled anti-Lv mAb. The DIF method could detect obvious fluorescence
Fig. 3. Vtg in plasma of juvenile Japanese flounder exposed to 0 (Lane 1), 2 (Lane 2), 10 (Lane 3), and 50 ng/L of 17α-ethinylestradiol (Lane 4) were detected by Western blot (A) and sandwich ELISA (B).
Fig. 4. Direct immunofluorescence analysis of Vtg in the liver of juvenile Japanese flounder exposed to 0 (A), 2 (B), 10 (C), and 50 ng/L 17α-ethinylestradiol (D) for 21 days. Arrows indicate sinusoidal veins. V indicate vein vessels. 4
Aquatic Toxicology 216 (2019) 105321
Z. Zhang, et al.
stronger fluorescence signal and weaker background. Thus, DIF is faster and more sensitive for histological detection of Vtg in tissues. Distribution of Vtg in Japanese flounder were investigated using these developed immunological methods and qPCR. The results of Western blot and sandwich ELISA revealed that Vtg was present not only in the liver, but also in various extrahepatic tissues, including muscle, spleen, intestine, kidney, and tail fin. Surface mucus was found to contain high concentrations of Vtg in goldfish (Carassius auratus) intraperitoneally injected with 3 mg/kg of E2 (Wang et al., 2015b). In our study, Japanese flounder mucus had the lowest Vtg concentration after 50 ng/L EE2 exposure. It is suspected that low induction of Vtg would be easily eliminated through the kidney, but excessive Vtg has to be excreted through the skin. It is interesting to note that tail fin contains the highest concentration of Vtg, but yet relatively low mRNA levels, which might be associated with the abundant blood vessels in this tissue. Due to the ease of sample collection and less damage to fish, the terminal tail fin could be a suitable alternative to plasma and liver for Vtg biomarker analysis. The distribution of Vtg in these tissues of Japanese flounder exposed to 50 ng/L EE2 were also detected by DIF. The results showed that strong fluorescence signals mainly appeared in liver, kidney, intestine, and spleen, which was consist with the results of Western blot and ELISA analysis in our study. Moreover, other studies also found the similar results in Liver (Licata et al., 2018), kidney (Del Giudice et al., 2012), intestine and spleen (Zhang et al., 2014b). In juvenile and male fish exposed to exogenous estrogens, Vtg was considered to be firstly produced in the RER/Golgi region of hepatocytes (Van der Ven et al., 2003; Mortensen and Arukwe, 2007), released into blood circulation by gathering in vein vessels, and then delivered into kidney for Vtg elimination (Del Giudice et al., 2012; Verderame and Scudiero, 2017). Normally, the molecular mass of proteins in glomerular filtrate does not exceed that of albumin (66 kDa, Koger et al., 1999). Since Japanese flounder Vtg has a molecular mass of 520 kDa (Zhang et al., 2019), it could not be resorbed and remained within kidney tubule cells, and thus caused obvious fluorescence signals in kidney tubule. Vtg in extrahepatic tissues, especially the spleen and intestines, were mainly originating from the circulating blood which contained the Vtg produced in the liver. So, strong fluorescent signal was also found in the spleen sinusoidal veins and intestinal epithelial cells (Zhang et al., 2014b). Nevertheless, the kidney showed similar fluorescence intensity to the liver, indicating that both tissues would be suitable for histological detection of Vtg. In conclusion, this study developed a sandwich ELISA and DIF for detection of Vtg in Japanese flounder, and the two methods could effectively detect estrogenic activity of environmentally relevant concentrations of EE2. Furthermore, this study demonstrated that Vtg was not only detected in the liver, but also in extrahepatic tissues. Considering the high concentration of Vtg, the terminal tail fin is suggested as an alternative tissue for Vtg quantification, whereas both kidney and liver are recommended for histological detection of Vtg. These methodological improvements could be used to early detect estrogen pollution in marine environments.
Fig. 5. Detection of Vtg in different tissues of juvenile Japanese flounder expose to 50 ng/L 17α-ethinylestradiol by Western blot (A) and sandwich ELISA (B). Lanes: Mc, Surface mucus; Ms, Muscle; S, Spleen; I, Intestine; K, Kidney; L, liver; F, Tail fin.
Fig. 6. Real-time quantitative PCR analysis of the expression levels of Vtg1 and Vtg2 mRNA in muscle, intestine, fin, kidney, spleen, and liver of juvenile Japanese flounder exposed to 50 ng/L EE2. Values are means ± SD (n = 5), and were normalized against β-actin as a housekeeping gene.
signals in the liver of juvenile Japanese flounder exposed to 10 and 50 ng/L EE2, and this was consistent with the results of plasma Vtg induction detected by Western blot and ELISA. Moreover, the results of DIF showed that Vtg firstly appeared in the sinusoidal veins and then gathered in vein vessels, which was similar to the results of Arukwe and Røe (2007). The above results demonstrated that the established DIF could be used for detection of Vtg induction. To date, indirect IF is normally used for in situ detection of specific protein in tissues (Aoki et al., 2010). For piscine Vtg, indirect IF has been developed for only two species, medaka and bluegill (Lepomis macrochirus) (Kobayashi et al., 2005; Allner et al., 2016). Compared to indirect IF, the DIF method developed in our study detected Vtg antigens in a single step, and did not need additional incubations. Moreover, DIF had a much
Declaration of Competing Interest We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.
Acknowledgements This research was supported by the Fundamental Research Funds for the Central Universities (201964025) and National Natural Science Foundation of China (21607144).
5
Aquatic Toxicology 216 (2019) 105321
Z. Zhang, et al.
Fig. 7. Direct immunofluorescence of Vtg in muscle, spleen, intestine, kidney, and liver of juvenile Japanese flounder exposed to 0 and 50 ng/L 17α-ethinylestradiol for 21 days. V indicate vein vessels. R indicate renal tubules. E indicate epithelial cells of the intestine. S indicate sinusoidal veins.
Appendix A. Supplementary data
development of an ELISA f`or the detection of estrogenic effects. Sci. Total Environ. 309, 93–103. https://doi.org/10.1016/S0048-9697(03)00005-6.lss. Hiramatsu, N., Cheek, A.O., Sullivan, C.V., Matsubara, T., Hara, A., 2005. Chapter 16 vitellogenesis and endocrine disruption. Biochem. Mol. Biol. Fishes. 6, 431–471. https://doi.org/10.1016/S1873–0140(05)80019–0. Hu, S., Zhang, H., Shen, G., Yuan, Z., Xu, T., Ji, R., 2017. Effects of 17β-estradiol and 17αethinylestradiol on the embryonic development of the clearhead icefish (Protosalanx hyalocranius). Chemosphere 176, 18–24. https://doi.org/10.1016/j.chemosphere. 2017.02.094. Huang, W., Cao, L., Ye, Z., Yin, X., Dou, S., 2010. Antioxidative responses and bioaccumulation in Japanese flounder larvae and juveniles under chronic mercury exposure. Comp. Biochem. Physiol. 152C, 99–106. https://doi.org/10.1016/j.cbpc.2010.03. 005. Ihara, M., Kitamura, T., Kumar, V., Park, C.B., Ihara, M.O., Lee, S.J., et al., 2015. Evaluation of estrogenic activity of wastewater: comparison among in vitro ERα reporter gene assay, in vivo vitellogenin induction, and chemical analysis. Environ. Sci. Technol. 49, 6319–6326. https://doi.org/10.1021/acs.est.5b01027. Jin, Y., Wang, W., Sheng, G.D., Liu, W., Fu, Z., 2008. Hepatic and extrahepatic expression of estrogen-responsive genes in male adult zebrafish (Danio rerio) as biomarkers of short-term exposure to 17β-estradiol. Environ. Monit. Assess. 146, 105–111. https:// doi.org/10.1007/s10661-007-0063-y. Kobayashi, K., Tamotsu, S., Yasuda, K., Oishi, T., 2005. Vitellogenin-immunohistochemistry in the liver and the testis of the medaka, Oryzias latipes, exposed to 17beta-estradiol and p-nonylphenol. Zool. Sci. 22, 455–461. https://doi.org/10. 2108/zsj.22.453. Koger, C.S., The, S.J., Hinton, D.E., 1999. Variations of light and temperature regimes and resulting effects on reproductive parameters in medaka (Oryzias latipes). Biol. Reprod. 61, 1287–1293. https://doi.org/10.1095/biolreprod61.5.1287. Li, C.R., Kim, K.T., Kang, Y.J., Kim, A., Kang, S.W., Park, J.S., 2005. A novel enzymelinked immunosorbent assay system for the quantitative analysis of Carassius auratus vitellogenin. Chemosphere 59, 997–1003. https://doi.org/10.1016/j.chemosphere. 2004.11.063. Li, Y., Wang, J., Zheng, M., Zhang, Y., Ru, S., 2018. Development of ELISAs for the detection of vitellogenin in three marine fish from coastal areas of China. Mar. Pollut. Bull. 133, 415–422. https://doi.org/10.1016/j.marpolbul.2018.06.001. Licata, P., Tardugno, R., Pergolizzi, S., Capillo, G., Aragona, M., Colombo, A., Gervasi, T., Pellizzeri, V., Cicero, N., Calò, M., 2018. In vivo effects of PCB-126 and genistein on vitellogenin expression in zebrafish. Nat. Prod. Res. 2, 1–8. https://doi.org/10.1080/ 14786419.2018.1455048. Ling, S., Wang, R., Gu, X., Wen, C., Chen, L., Chen, Z., Chen, Q.A., Xiao, S., Yang, Y., Zhuang, Z., Wang, S., 2015. Rapid detection of fumonisin B1 using a colloidal gold immunoassay strip test in corn samples. Toxicon 108, 210–215. https://doi.org/10. 1016/j.toxicon.2015.10.014. Liu, J., Zhou, N., Fu, R., Cao, D., Si, Y., Li, A., Zhao, H., Zhang, Q., Yu, H., 2017. The polymorphism of chicken-type lysozyme gene in Japanese flounder (Paralichthys olivaceus) and its association with resistance/susceptibility to Listonella anguillarum. Fish Shellfish Immunol. 66, 43–49. https://doi.org/10.1016/j.fsi.2017.05.010. Luo, W., Zhou, Q., Jiang, G., 2011. Development of enzyme-linked immunosorbent assays for plasma vitellogenin in Chinese rare minnow (Gobiocypris rarus). Chemosphere 84, 681–688. https://doi.org/10.1016/j.chemosphere.2011.03.025. Madsen, L.L., Korsgaard, B., Pedersen, K.L., Aagaard, T., Bjerregaard, P., 2013. Vitellogenin as biomarker for estrogenicity in flounder Platichthys flesus in the field and exposed to 17α-ethinylestradiol via food and water in the laboratory. Mar. Environ. Res. 92, 79–86. https://doi.org/10.1016/j.marenvres.2013.09.002.
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.aquatox.2019.105321. References Aljandal, N., Saeed, T., Azad, I., Alsubiai, S., Alzekri, W., Hussain, S., Alhasan, E., 2017. Impact of endocrine disrupting compounds in sewage impacted coastal area on seabream. Ecotoxicol. Environ. Saf. 150, 280–288. https://doi.org/10.1016/j.ecoenv. 2017.12.035. Allner, B., Hennies, M., Lerche, C.F., Schmidt, T., Schneider, K., Willner, M., Stahlschmidt-Allner, P., 2016. Kinetic determination of vitellogenin induction in the epidermis of cyprinid and perciform fishes: evaluation of sensitive enzyme-linked immunosorbent assays. Environ. Toxicol. Chem. 35, 2916–2930. https://doi.org/10. 1002/etc.3475. An, L., Hu, J., Zhu, X., Deng, B., Zhang, Z., Yang, M., 2007. Crucian carp (Carassius carassius) VTG monoclonal antibody: development and application. Ecotoxicol. Environ. Saf. 66, 148–153. https://doi.org/10.1016/j.ecoenv.2005.11.005. Aoki, V., Sousa, J.X.J., Fukumori, L.M., Périgo, A.M., Freitas, E.L., Oliveira, Z.N., 2010. Direct and indirect immunofluorescence. An. Bras. Dermatol. 85, 490–500. https:// doi.org/10.1590/S0365-05962010000400010. Arukwe, A., Røe, K., 2007. Molecular and cellular detection of expression of vitellogenin and zona radiata protein in liver and skin of juvenile salmon (Salmo salar) exposed to nonylphenol. Cell Tissue Res. 331, 701–712. https://doi.org/10.1007/s00441-0070543-y. Brodeur, J., Woodburn, K., Zhang, F., Bartels, M.J., Klecka, G.M., 2006. Plasma sampling and freezing procedures influence vitellogenin measurements by enzyme-linked immunoassay in the fathead minnow (Pimephales promelas). Environ. Toxicol. Chem. 25, 337–348. https://doi.org/10.1897/05-368R.1. Cao, L., Huang, W., Liu, J.H., Yin, X.B., Dou, S.Z., 2010. Accumulation and oxidative stress biomarkers in Japanese flounder larvae and juveniles under chronic cadmium exposure. Comp. Biochem. Physiol. 151C, 386–392. https://doi.org/10.1016/j.cbpc. 2010.01.004. Del Giudice, G., Prisco, M., Agnese, M., Verderame, M., Rosati, L., Limatola, E., Andreuccetti, P., 2012. Effects of nonylphenol on vitellogenin synthesis in adult males of the spotted ray Torpedo marmorata. J. Fish Biol. 80, 2112–2121. https:// doi.org/10.1111/j.1095-8649.2011.03172.X. Flick, R.W., Bencic, D.C., See, M.J., Biales, A.D., 2014. Sensitivity of the vitellogenin assay to diagnose exposure of fathead minnows to 17α-ethynylestradiol. Aquat. Toxicol. 152, 353–360. https://doi.org/10.1016/j.aquatox.2014.04.026. Garnayak, S.K., Mohanty, J., Rao, T.V., Sahoo, S.K., Sahoo, P.K., 2013. Vitellogenin in Asian catfish, Clarias batrachus: Purification, partial characterization and quantification during the reproductive cycle by ELISA. Aquaculture 392–395, 148–155. https://doi.org/10.1016/j.aquaculture.2013.02.020. Gong, B., Murray, K.D., Trimmer, J.S., 2016. Developing high-quality mouse monoclonal antibodies for neuroscience research approaches, perspectives and opportunities. N. Biotechnol. 33, 551–564. https://doi.org/10.1016/j.nbt.2015.11.007. Hara, A., Hiramatsu, N., Fujita, T., 2016. Vitellogenesis and choriogenesis in fishes. Fish. Sci. 82, 187–202. https://10.1007/s12562-015-0957-5. Hennies, M., Wiesmann, M., Allner, B., Sauerwein, H., 2003. Vitellogenin in carp (Cyprinus carpio) and perch (Perca fluviatilis): purification, characterization and
6
Aquatic Toxicology 216 (2019) 105321
Z. Zhang, et al.
vitellogenin. Environ. Toxicol. Chem. 18, 337–347. https://doi.org/10.1002/etc. 5620180234. Van der Ven, L.T., Holbech, H., Fenske, M., Van den Brandhof, E.J., Gielis-Proper, F.K., Wester, P.W., 2003. Vitellogenin expression in zebrafish Danio rerio: evaluation by histochemistry, immunohistochemistry, and in situ mRNA hybridization. Aquat. Toxicol. 65, 1–11. https://doi.org/10.1016/S0166-445X(03)00103-6. Verderame, M., Scudiero, R., 2017. Estrogen-dependent, extrahepatic synthesis of vitellogenin in male vertebrates: a mini-review. C. R. Biol. 340, 139–144. https://doi. org/10.1016/j.crvi.2017.01.005. Wang, H., Tan, J.T., Emelyanov, A., Korzh, V., Gong, Z., 2005. Hepatic and extrahepatic expression of vitellogenin genes in the zebrafish. Danio rerio. Gene. 356, 91–100. https://doi.org/10.1016/j.gene.2005.03.041. Wang, J., Ma, S., Zhang, Z., Zheng, M., Dong, Y., Ru, S., 2017. Vitellogenin induction in caudal fin of guppy (Poecilia reticulata) as a less invasive and sensitive biomarker for environmental estrogens. Sci. Rep. 7, 7647. https://doi.org/10.1038/s41598-01706670-6. Wang, J., Shan, R., Zhang, X., Tian, H., Wang, W., Ru, S., 2015a. Development of a lipovitellin-based sandwich ELISA for quantification of vitellogenin in surface mucus and plasma of goldfish (Carassius auratus). Ecotoxicol. Environ. Saf. 120, 80–87. https://doi.org/10.1016/j.ecoenv.2015.05.010. Wang, J., Wang, W., Zhang, X., Tian, H., Ru, S., 2015b. Development of a lipovitellin–based goldfish (Carassius auratus) vitellogenin ELISA for detection of environmental estrogens. Chemosphere 132, 166–171. https://doi.org/10.1016/j. chemosphere.2015.03.038. Xu, E.G.B., Liu, S., Ying, G.G., Zheng, G.J.S., Lee, J.H.W., Leung, K.M.Y., 2014. The occurrence and ecological risks of endocrine disrupting chemicals in sewage effluents from three different sewage treatment plants, and in natural seawater from a marine reserve of Hong Kong. Mar. Pollut. Bull. 85, 352–362. https://doi.org/10.1016/j. marpolbul.2014.02.029. Yin, N., Jin, X., He, J., Yin, Z., 2009. Effects of adrenergic agents on the expression of zebrafish (Danio rerio) vitellogenin Ao1. Toxicol. Appl. Pharmacol. 238, 20–26. https://doi.org/10.1016/j.taap.2009.04.004. Yue, Z., Yu, M., Zhang, X., Dong, Y., Tian, H., Wang, W., Ru, S., 2017. Semicarbazideinduced thyroid disruption in Japanese flounder (Paralichthys olivaceus) and its potential mechanisms. Ecotoxicol. Environ. Saf. 140, 131–140. https://doi.org/10. 1016/j.ecoenv.2017.02.043. Zhang, C., Zheng, L., Nurnberg, J., Vacari, B.M., Zhou, J., Wang, Y., 2014a. Cleavage of pro-tumor necrosis factor alpha by ADAM metallopeptidase domain 17: a fluorescence-based protease assay cleaves its natural protein substrate. Anal. Biochem. 445, 14–19. https://doi.org/10.1016/j.ab.2013.09.022. Zhang, X., Xiong, L., Liu, Y., Deng, C., Mao, S., 2014b. Histopathological and estrogen effect of pentachlorophenol on the rare minnow (Gobiocypris rarus). Fish Physiol. Biochem. 40, 805–816. https://doi.org/10.1007/s10695–013–9887–2. Zhang, Z., Wang, J., Gao, M., Li, X., Cheng, Y., Zhang, X., Tian, H., Wang, W., Ru, S., 2019. New methods for purification of Paralichthys olivaceus lipovitellin and immunoassay-based detection of vitellogenin. Ecotoxicol. Environ. Saf. 180, 624–631. https://doi.org/10.1016/j.ecoenv.2019.04.087. Zhong, L., Yuan, L., Rao, Y., Li, Z., Zhang, X., Liao, T., Xu, Y., Dai, H., 2014. Distribution of vitellogenin in zebrafish (Danio rerio) tissues for biomarker analysis. Aquat. Toxicol. 149, 1–7. https://doi.org/10.1016/j.aquatox.2014.01.022. Zhou, X., Lian, Z., Wang, J., Tan, L., Zhao, Z., 2011. Distribution of estrogens along Licun River in Qingdao, China. Procedia. Environ. Sci. 10, 1876–1880. https://doi.org/10. 1016/j.proenv.2011.09.293. Zuo, Y., Zhang, K., Deng, Y., 2006. Occurrence and photochemical degradation of 17αethinylestradiol in Acushnet River Estuary. Chemosphere 63, 1583–1590. https:// doi.org/10.1016/j.chemosphere.2005.08.063.
Magalhães, I., Ledrich, M.L., Pihan, J.C., Falla, J., 2004. One-step, non-denaturing purification method of carp (Cyprinus carpio) vitellogenin. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 799, 87–93. https://doi.org/10.1016/j.jchromb.2003.10. 026. Maltais, D., Dupont-Cyr, B.A., Roy, R.L., Le François, N.R., 2014. Purification and partial characterization of vitellogenin from spotted wolffish (Anarhichas minor) and development of an enzyme-linked immunosorbent assay for the determination of gender and sexual maturity. Fish Physiol. Biochem. 40, 279–294. https://doi.org/10.1007/ s10695-013-9843-1. Mortensen, A.S., Arukwe, A., 2007. Effects of 17α-ethynylestradiol on hormonal responses and xenobiotic biotransformation system of Atlantic salmon (Salmo salar). Aquat. Toxicol. 85, 113–123. https://doi.org/10.1016/j.aquatox.2007.08.004. Nishi, K., Chikae, M., Hatano, Y., Mizukami, H., Yamashita, M., Sakakibara, R., Tamiya, E., 2002. Development and application of a monoclonal antibody-based sandwich ELISA for quantification of Japanese medaka (Oryzias latipes) vitellogenin. Comp. Biochem. Physiol. 132C, 161–169. https://doi.org/10.1016/S1532-0456(02) 00058-3. Partridge, C., Boettcher, A., Jones, A.G., 2010. Short-term exposure to a synthetic estrogen disrupts mating dynamics in a pipefish. Horm. Behav. 58, 800–807. https:// doi.org/10.1016/j.yhbeh.2010.08.002. Petersen, K., Fetter, E., Kah, O., Brion, F., Scholz, S., Tollefsen, K.E., 2013. Transgenic (cyp19a1b-GFP) zebrafish embryos as a tool for assessing combined effects of oestrogenic chemicals. Aquat. Toxicol. 138–139, 88–97. https://doi.org/10.1016/j. aquatox.2013.05.001. Prasatkaew, W., Nanthanawat, P., Khongchareonporn, N., Kingtong, S., 2019. A monoclonal antibody against Lates calcarifer vitellogenin and a competitive ELISA to evaluate vitellogenin induction after exposure to xenoestrogen. J. Environ. Sci (China) 75, 325–333. https://doi.org/10.1016/j.jes.2018.05.001. Roy, R.L., Morin, Y., Courtenay, S.C., Robichaud, P., 2004. Purification of vitellogenin from smooth flounder (Pleuronectes putnami) and measurement in plasma by homologous ELISA. Comp. Biochem. Physiol. 139B, 235–244. https://doi.org/10.1016/j. cbpc.2004.07.006. Sawaguchi, S., Koya, Y., Yoshizaki, N., Ohkubo, N., Andoh, T., Hiramatsu, N., Sullivan, C.V., Hara, A., Matsubara, T., 2005. Multiple vitellogenins (Vgs) in mosquitofish (Gambusia affinis): identification and characterization of three functional Vg genes and their circulating and yolk protein products. Biol. Reprod. 72, 1045–1060. https://doi.org/10.1095/biolreprod.104.037895. Scott, A.P., Katsiadaki, I., Witthames, P.R., Hylland, K., Davies, I.M., McIntosh, A.D., Thain, J., 2006. Vitellogenin in the blood plasma of male cod (Gadus morhua): a sign of oestrogenic endocrine disruption in the open sea? Mar. Environ. Res. 61, 149–170. https://doi.org/10.1016/j.marenvres.2005.08.003. Selcer, K.W., Verbanic, J.D., 2014. Vitellogenin of the northern leopard frog (Rana pipiens): development of an ELISA assay and evaluation of induction after immersion in xenobiotic estrogens. Chemosphere 112, 348–354. https://doi.org/10.1016/j. chemosphere.2014.04.073. Segner, H., Casanova-Nakayama, A., Kase, R., Tyler, C.R., 2013. Impact of environmental estrogens on Yfish considering the diversity of estrogen signaling. Gen. Comp. Endocrinol. 119, 191–201. https://doi.org/10.1016/j.ygcen.2013.05.015. Sumpter, J.P., Jobling, S., 1995. Vitellogenesis as a biomarker for estrogenic contamination of the aquatic environment. Environ. Health Perspect. 103, 173–178. https://doi.org/10.1289/ehp.95103s7173. Tollefsen, K.E., Mathisen, R., Stenersen, J., 2003. Induction of vitellogenin synthesis in an Atlantic salmon (Salmo salar) hepatocyte culture: a sensitive in vitro bioassay for the oestrogenic and anti-oestrogenic activity of chemicals. Biomarkers 8, 394–407. https://doi.org/10.1080/13547500310001607827. Tyler, C.R., Aerle, R.V., Hutchinson, T.H., Maddix, S., Trip, H., 1999. An in vivo testing system for endocrine disruptors in fish early life stages using induction of
7
Contents lists available at ScienceDirect
Aquatic Toxicology journal homepage: www.elsevier.com/locate/aqtox
Distribution of vitellogenin in Japanese flounder (Paralichthys olivaceus) for biomarker analysis of marine environmental estrogens
T
⁎
Zhenzhong Zhanga, Jun Wanga, , Zongbao Panb, Yabin Zhanga, Xiaona Zhanga, Hua Tiana, ⁎ Wei Wanga, Shaoguo Rua, a b
College of Marine Life Sciences, Ocean University of China, Qingdao, 266003, China Zhejiang Institute of Hydraulics & Estuary, Hangzhou, 310020, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Biomarker Environmental estrogens Direct immunofluorescence Lipovitellin Paralichthys olivaceus
Estrogen pollution in marine environments has become a research hotspot due to its adverse effects on the reproduction of wild organisms. To early detection of estrogen pollution, this study developed two methods for detecting Japanese flounder vitellogenin (Vtg), a sensitive biomarker for environmental estrogens. Firstly, monoclonal antibodies (mAb) specific to Vtg were prepared using purified lipovitellin (Lv), a main Vtg-derived yolk protein. Anti-Lv mAb (C1F1) had the highest titer (1:256,000) and was labeled with fluorescein isothiocyanate to establish a direct immunofluorescence (DIF) method for histological detection of Vtg in tissues. Additionally, using the purified Lv and mAb, an enzyme-linked immunosorbent assay (ELISA) was developed and this assay had a detection limit of 0.75 ng/mL and a working range of 1.95–250 ng/mL. Furthermore, Vtg induction in the plasma of Japanese flounder exposed to 17β-estradiol (E2), 17α-ethinylestradiol (EE2), and bisphenol A (BPA) were quantified by ELISA, and Vtg induction in the liver of EE2-exposed Japanese flounder were measured by DIF. Finally, the distribution of Vtg in Japanese flounder was detected using these two methods. The results revealed that Vtg mainly appeared in the terminal tail fin, liver, kidney, intestine, and spleen. Considering the high concentration of Vtg and easy sample collection, the terminal tail fin could be a new alternative to plasma for Vtg quantification, while kidney and liver are suitable for histological detection of Vtg.
1. Introduction During the past three decades, environmental estrogens have aroused great concern for their adverse effects on gonadal development and reproduction in aquatic organisms (Segner et al., 2013; Xu et al., 2014). These pollutants enter the ocean through surface runoff and have been frequently detected in offshore seawater. For example, 17αethinylestradiol (EE2), a synthetic estrogen compound, had concentrations of 4.7 and 24 ng/L in surface waters of the Acushnet River Estuary (USA) and Jiaozhou Bay (China) (Zuo et al., 2006; Zhou et al., 2011). Environmental estrogens could disturb the growth and reproduction of marine organisms at a low ng/L level. For example, 1 ng/L EE2 induced feminization in male gulf pipefish (Syngnathus scovelli) and reduced their mating opportunities (Partridge et al., 2010). Also, Hu et al. (2017) reported that 0.05 ng/L 17β-estradiol (E2) could cause malformation in embryos of clearhead icefish (protosalanx hyalocranius). Given the potential ecological risk of estrogenic pollution to marine organisms, there is an urgent need to establish reliable methods for detecting these emerging pollutants. ⁎
To date, several biomarkers have been developed for monitoring environmental estrogens, of which vitellogenin (Vtg) is the most widely used (Sumpter and Jobling, 1995; Tyler et al., 1999). Vtg is a femalespecific protein and is generally synthesized by hepatocytes of sexually mature females in response to 17β-estradiol, released into the blood circulation, and deposited in developing oocytes, where it is cleaved into lipovitellin (Lv), phosvitin (Pv), and other yolk proteins (Hara et al., 2016; Verderame and Scudiero 2017). Although juvenile and male fish do not have detectable Vtg, estrogenic mimics can easily induce them to synthesize Vtg. Thus, the abnormal production of Vtg in juvenile and male fish can sensitively indicate estrogenic pollution in aquatic environments (Tollefsen et al., 2003; Ihara et al., 2015). Recently, most studies evaluated the estrogenic activities of chemicals by measuring Vtg concentrations in the plasma of male fish (Madsen et al., 2013; Flick et al., 2014). Compared to plasma samples, the liver was considered to be the main tissue that synthesizes Vtg, and Vtg induction in the liver may be an early indicator for estrogenic pollution (Hiramatsu et al., 2005; Sawaguchi et al., 2005). In addition, many extrahepatic tissues also have the potential to synthesize or store Vtg. In
Corresponding authors at: College of Marine Life Sciences, Ocean University of China, 5 Yushan Road, Qingdao, 266003, Shandong province, China. E-mail addresses: [email protected] (J. Wang), [email protected] (S. Ru).
https://doi.org/10.1016/j.aquatox.2019.105321 Received 28 July 2019; Received in revised form 22 September 2019; Accepted 26 September 2019 Available online 27 September 2019 0166-445X/ © 2019 Elsevier B.V. All rights reserved.
Aquatic Toxicology 216 (2019) 105321
Z. Zhang, et al.
labeled anti-Lv mAb was obtained by semi-saturated ammonium sulfate precipitation and redissolved in PBS (containing 0.01% NaN3 and 0.5% BSA).
zebrafish (Danio rerio), Vtg was detected in the heart (Yin et al., 2009), intestine, muscles (Wang et al., 2005), skin (Jin et al., 2008), and fin (Zhong et al., 2014). As for marine fish, there is no report on tissue distribution of Vtg, and it is unclear whether some tissues other than blood are more suitable for detecting Vtg as a biomonitor of marine estrogenic pollution. Japanese flounder (Paralichthys olivaceus), a marine benthic fish widely distributed in Northeast Asia, was recommended as a good sentinel species for detecting environmental estrogens for its large size, easy blood collection, strong survival ability and weak exercise capacity (Cao et al., 2010; Huang et al., 2010; Liu et al., 2017). In this study, an enzyme-linked immunosorbent assay (ELISA) for quantifying Japanese flounder Vtg and a direct immunofluorescence (DIF) method for histological detection of Vtg in tissues were established using anti-Lv monoclonal antibody (mAb). Moreover, their reliability for detection of exogenous estrogens were validated by measuring Vtg induction in juvenile Japanese flounder exposed to three common environmental estrogens. Finally, the distribution of Vtg in different tissues of Japanese flounder were investigated using the above-mentioned methods to identify new alternative tissues for easy and sensitive detection of the Vtg biomarker.
2.4. DIF The liver tissue of Japanese flounder was fixed in 4% paraformaldehyde for the preparation of 5-μm paraffin sections according to the standard procedure (Aljandal et al., 2017). The paraffin sections were deparaffinized in xylene for 15 min and hydrated in a series of graded ethanol. After hydration, the sections were immersed in citrate buffer (0.01 M, pH 6) for antigen retrieval and then blocked in 5% BSA for 1 h at 37 °C to reduce non-specific binding. Finally, tissue sections were incubated with diluted FITC-labeled anti-Lv mAb for 1 h at room temperature and observed under a fluorescence microscope (Nikon Eclipse 50i, Florida). 2.5. Sandwich ELISA Sandwich ELISA was established according to previously reported methods (Li et al., 2018). The plates were firstly coated with anti-Lv mAb (100 μL/well) and then blocked by 1% BSA. After washing, 100 μL of samples or standards (0.95–1000 ng/mL purified Lv) were added into wells and incubated at 37 °C for 1 h. Afterwards, wells were incubated with different dilutions of HRP-labeled anti-Lv IgG solution, and color was developed using TMB substrate (Solarbio, China). The absorbance at 450 nm was measured using a plate reader (Multiskan MK2, Thermo USA). In addition, the precision, sensitivity, and specificity of the assay were analyzed according to methods of Wang et al. (2017).
2. Materials and methods 2.1. Purification and identification of Lv Ovulated eggs were collected from female Japanese flounder and homogenized to prepare an egg homogenate according to the methods of Wang et al. (2015b), and the purification of Lv was conducted using water precipitation method established in our previous study (Zhang et al., 2019). The purity of the obtained protein was analyzed using native polyacrylamide gel electrophoresis (Native-PAGE). Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF/TOF MS) analysis was used to determine the amino-acid sequence of the purified protein (Prasatkaew et al., 2019). Briefly, the protein band was cut from Native-PAGE and digested overnight at 37 °C with 0.02 μg/μL trypsin. Peptide was measured in a MALDI-TOF/TOF MS mass spectrometer (Applied Biosystems, Foster City, USA) in a positive MS reflector mode. The peptide mass fingerprint was analyzed using MASCOT V2.3 search engine (Matrix Science Ltd., London, U.K.), and peptide sequences were identified from the databases at NCBI-bony vertebrates.
2.6. Vtg induction in juvenile Japanese flounder exposed to environmental estrogens Juvenile Japanese flounder (n = 6 per concentration, 18.36 ± 2.94 g, 8.27 ± 1.03 cm) were exposed to nominal concentrations of 0, 2, 10, and 50 ng/L EE2 (Sigma-Aldrich) in 100-L aquaria. Moreover, sexually mature male Japanese flounder (n = 5 per concentration, 800 ± 100 g, 35 ± 5 cm) were exposed to 10 μg/L E2, 10 μg/L EE2, and 100 μg/L BPA. The stock solution of E2, EE2, and BPA was prepared in dimethyl sulfoxide (DMSO) and kept at 4 °C. The DMSO concentration in the control and exposure groups was below 0.01%. To maintain constant exposure concentrations, half of the volume seawater was renewed daily in all tanks. After 7 days, blood was collected from sexually mature Japanese flounder, and after 21 days blood was also collected from the caudal vein of juvenile Japanese flounder. The plasma was obtained by centrifugation, and plasma Vtg was detected by Western blot and sandwich ELISA. The actual concentrations of EE2 in the seawater of 2, 10, and 50 ng/L exposure groups were 0, 7.69 ± 0.72 ng/L, and 45.72 ± 0.99 ng/L, respectively, after 24 h of exposure (Zhang et al., 2019).
2.2. Production of anti-Lv mAb The anti-Lv mAb was prepared according to the method of Li et al. (2005). Briefly, Balb/c mice were immunized with 100 μg of purified Lv for three times in two-week intervals. Then the spleen was removed from the immunized mice, and the splenocytes were fused with the SP2/0 myeloma cells using polyethylene glycol 4000. After three rounds of screening and subcloning, the positive hybridoma cells were injected intraperitoneally into Balb/c mice, and anti-Lv mAb was purified from the ascitic fluid by affinity chromatography on a HiTrap Protein G column (GE Healthcare). The titer, molecular weight, and specificity were determined using conventional procedures (Ling et al., 2015).
2.7. Tissue distribution of Vtg after EE2 exposure After 21 days of exposure to 50 ng/L EE2, the liver, kidney, intestine, spleen, muscle, surface mucus, and terminal tail fin were collected from each fish. Each sample was divided into three parts: (i) for Vtg quantification, the tissues were homogenized in five volumes of ice-cold PBS containing protease inhibitor cocktails (Sigma-Aldrich); (ii) for quantification of Vtg1 and Vtg2 genes, the tissues were used for RNA extraction, reverse transcription and quantitative real-time PCR (qPCR), according to the methods of Yue et al. (2017), details were described in supplementary material; (iii) for histological detection of Vtg, tissues were fixed in 4% paraformaldehyde to prepare paraffin sections. Vtg in the homogenates were measured by Western blot and sandwich ELISA, Vtg1 and Vtg2 from samples were quantified by qPCR, and Vtg in tissue sections were visualized by DIF.
2.3. Preparation of FITC-labeled antibodies Anti-Lv mAb was covalently labeled with fluorescein isothiocyanate (FITC, Sigma-Aldrich) according to the methods of Zhang et al. (2014a) with minor modifications. Briefly, anti-Lv mAb was dialyzed three times in crosslinking agent (7.56 g/L NaHCO3, 1.06 g/L Na2CO3, and 7.36 g/L NaCl, in ddH2O, pH 9.0) at 4 °C. FITC was dissolved in the crosslinking agent (1 mg/mL) and added into the dialyzed mAb solution at a ratio of 1:6, followed by an agitation at 4 °C for 8 h in dark. FITC2
Aquatic Toxicology 216 (2019) 105321
Z. Zhang, et al.
50 ng/L EE2 exposure groups significantly increased to 66.1 ± 5.3 μg/ mL and 433.2 ± 75.9 μg/mL (P < 0.05, Fig. 3B).
2.8. Statistics Data were presented as mean ± standard deviation (SD), and the differences between the control and exposed groups were assessed by one-way analysis of variance followed by a Tukey’s post hoc test. Prior to parametric analysis, data were log-transformed to achieve variance homogeneity. All statistical tests were conducted using SPSS 18.0 software (SPSS Inc., USA), and values were determined as significant when P < 0.05.
3.4. Establishment of DIF for visible detection of Vtg
3. Results
When FITC-labeled anti-Lv mAb was diluted at 1:400, DIF showed the best result (Fig. S8). Vtg was undetected in the liver of Japanese flounder in the control and 2 ng/L EE2 exposure groups, but 10 ng/L EE2 induced clear green fluorescence near sinusoid vessels in the liver (Fig. 4C). In the 50 ng/L EE2 group, strong green fluorescence was observed near the central vein of liver (Fig. 4D).
3.1. Specificity of anti-Lv mAb to Japanese flounder Vtg
3.5. Distribution of Vtg in Japanese flounder
The protein purified from the egg homogenate showed a single band in Native-PAGE (Fig. S1) and matched well with amino acid sequences of Japanese flounder Vtg (XP_019936246.1, Fig. S2, S3). In Western blot analysis, five mAbs were obtained which could all recognize the purified Vtg and Lv, but not the plasma from the control male fish. Specifically, mAb C1F1 detected Vtg subunits with molecular weights of ˜120, ˜100, and ˜92 kDa, while other mAbs (C3F4, F4C4, F4F4 and D3E2) detected Vtg subunits with lower molecular weights (˜64, ˜56, and ˜46 kDa, Fig. 1). In addition, mAb C1F1 and C3F4 had the highest absorption and their titers were 1:256 000 and1:32 000, respectively (Fig. S4, S5).
After exposure to 50 ng/L EE2, positive bands were observed in the tail fin, liver, kidney, intestine, spleen, muscle, and surface mucus by Western blot analysis using anti-Lv mAb as the primary antibody (Fig. 5A). ELISA results showed that the highest Vtg level was detected in the tail fin (174.37 ± 9.31 μg/mg), followed by the liver (93.69 ± 1 4.06 μg/mg), kidney (93.69 ± 14.06 μg/mg), intestine (78.25 ± 15.18 μg/mg), spleen (70.20 ± 43.87 μg/mg), and muscle (44.10 ± 12.00 μg/mg) (Fig. 5B). Surface mucus had the lowest Vtg level (7.80 ± 0.59 μg/mg). Meanwhile, the results of qPCR showed that Vtg1 and Vtg2 mRNA were expressed at different levels in the six tissues. The highest expression levels of Vtg1 and Vtg2 were found in the liver, followed by the spleen and kidney. In contrast, relatively low transcription levels of Vtg1 and Vtg2 were found in the tail fin, intestine, and muscle (Fig. 6). The results of DIF showed that no fluorescent signal was detected in any of the juvenile Japanese flounder tissues from the control group. In the 50 ng/L EE2 groups, strong green fluorescent signals were observed in the kidney and liver, and relatively intense fluorescence signals were found in the spleen and intestine (Fig. 7). Conversely, the muscle showed little fluorescence signal.
3.2. Sandwich ELISA When HRP-labeled anti-Lv pAb was diluted at 1:10000, the sandwich ELISA had a wide working range of 1.95–250 ng/mL (y = 1.3649x-0.7707, R² = 0.98) and a limit of detection (LOD) of 0.75 ng/mL (Fig. 2A, B). Moreover, dilution curves of plasma from E2exposed males were parallel to the Lv standards (Fig. 2C). The intraand inter-assay coefficients of variation (CVs) of sandwich ELISA were 1.32–8.21% and 1.52–8.01%, respectively (Table S2).
4. Discussion
3.3. Vtg induction in plasma of Japanese flounder exposed to E2, EE2 and BPA
This study not only established an ELISA method to quantify Vtg concentration, but also developed a DIF method to locate Vtg in tissues based on anti-Lv mAb. Moreover, these two methods were combined to investigate the distribution of Vtg in marine fish. Fish Vtg is easily degraded during the purification process, and the breakdown products have more immunogenic than Vtg itself, which usually leads to the deviation of quantification of Vtg concentration (Magalhães et al., 2004; Brodeur et al., 2006). Lv, a highly stable Vtg-derived yolk protein, has shown the same immunogenicity as Vtg and was recommended as a better antigen to develop robust immunoassays for Vtg (Wang et al., 2015a). In our current study, Japanese flounder Lv was successfully purified by water precipitation and showed a single band in Native-PAGE, indicative of high purity. The result of mass spectrometry further confirmed that the purified protein was Japanese flounder Lv. Subsequently, using this purified Lv as an antigen, five mAbs were prepared which had good positive reaction with purified Vtg, while no band was observed in the plasma of the control male fish, demonstrating that the prepared mAbs had good specificity to Vtg (Luo et al., 2011; Garnayak et al., 2013). Moreover, mAb C1F1 had a very high titer, which was approximately eight times higher than pAb against zebrafish Lv (1:32,000) (Wang et al., 2017). Thus, our prepared anti-Lv mAbs were highly specific and sensitive for the development of Vtg immunoassays. To date, anti-Vtg pAb-based ELISAs were the most commonly used methods for quantifying plasma Vtg (Selcer and Verbanic, 2014; Gong et al., 2016), and their working ranges were usually 30–1000 ng/mL (Hennies et al., 2003; Roy et al., 2004; Scott et al., 2006). Our study developed a sandwich ELISA using anti-Lv mAb, and this assay had a LOD of 0.75 ng/mL, which was at least nine times lower than the LODs
Vtg concentrations in plasma of sexually mature male Japanese flounder exposed to BPA, E2 and EE2 were 207.26 ± 25.54 ng/mL, 359.12 ± 46.45 ng/mL, and 3501.99 ± 187.65 ng/mL, respectively, which were significantly higher than that in the control group (20.80 ± 0.03 ng/mL, Fig. S7). For juvenile Japanese flounder, no band was observed in either control or 2 ng/L EE2 exposure group, while plasma from 10 and 50 ng/L EE2 exposure groups showed several well-defined bands (Fig. 3A). Compared to the control plasma (0.2 ± 0.026 μg/mL Vtg), concentrations of Vtg in plasma of 10 and
Fig. 1. Western blot analysis of plasma from the control male (lane 1), purified Vtg (lane 2), and purified Lv (lane 3) using anti-Lv mAbs as primary antibodies. 3
Aquatic Toxicology 216 (2019) 105321
Z. Zhang, et al.
Fig. 2. Determination of optimal dilution of antibodies (A) for the sandwich ELISA using Japanese flounder Lv as the standard (B), and the detection of plasma dilution curves of the control male and E2-exposed male fish (C).
of ELISAs for Spotted wolffish (Anarhichas minor, 6.7 ng/mL, Maltais et al., 2014) and sea bass (Lates calcarifer) Vtg (40 ng/mL, Prasatkaew et al., 2019). The working range of the Vtg ELISA established in this study was 1.95–250 ng/mL, which was comparable with values of mAbbased sandwich ELISAs for Japanese medaka (Oryzias latipes) and Crucian carp (Carassius carassius) Vtgs (Nishi et al., 2002; An et al., 2007). Moreover, the robustness and specificity of this assay for quantifying Vtg were confirmed by low intra- and inter-assay CVs and parallelism between the Lv standard curve and plasma dilutions of E2exposed male fish (Roy et al., 2004; Wang et al., 2015a). The practicability and reliability of the method was verified by quantifying the plasma Vtg concentrations in the E2, EE2, and BPA exposure groups. The results showed that the order of estrogenic potency based on the Vtg induction was EE2 > E2 > BPA, which coincides with the results of a previous study (Petersen et al., 2013). These results demonstrated that the developed ELISA for Japanese flounder Vtg could be used to test estrogenic properties of chemicals. In addition, a DIF method was developed to visually detect Vtg induction in tissues using fluoresceinlabeled anti-Lv mAb. The DIF method could detect obvious fluorescence
Fig. 3. Vtg in plasma of juvenile Japanese flounder exposed to 0 (Lane 1), 2 (Lane 2), 10 (Lane 3), and 50 ng/L of 17α-ethinylestradiol (Lane 4) were detected by Western blot (A) and sandwich ELISA (B).
Fig. 4. Direct immunofluorescence analysis of Vtg in the liver of juvenile Japanese flounder exposed to 0 (A), 2 (B), 10 (C), and 50 ng/L 17α-ethinylestradiol (D) for 21 days. Arrows indicate sinusoidal veins. V indicate vein vessels. 4
Aquatic Toxicology 216 (2019) 105321
Z. Zhang, et al.
stronger fluorescence signal and weaker background. Thus, DIF is faster and more sensitive for histological detection of Vtg in tissues. Distribution of Vtg in Japanese flounder were investigated using these developed immunological methods and qPCR. The results of Western blot and sandwich ELISA revealed that Vtg was present not only in the liver, but also in various extrahepatic tissues, including muscle, spleen, intestine, kidney, and tail fin. Surface mucus was found to contain high concentrations of Vtg in goldfish (Carassius auratus) intraperitoneally injected with 3 mg/kg of E2 (Wang et al., 2015b). In our study, Japanese flounder mucus had the lowest Vtg concentration after 50 ng/L EE2 exposure. It is suspected that low induction of Vtg would be easily eliminated through the kidney, but excessive Vtg has to be excreted through the skin. It is interesting to note that tail fin contains the highest concentration of Vtg, but yet relatively low mRNA levels, which might be associated with the abundant blood vessels in this tissue. Due to the ease of sample collection and less damage to fish, the terminal tail fin could be a suitable alternative to plasma and liver for Vtg biomarker analysis. The distribution of Vtg in these tissues of Japanese flounder exposed to 50 ng/L EE2 were also detected by DIF. The results showed that strong fluorescence signals mainly appeared in liver, kidney, intestine, and spleen, which was consist with the results of Western blot and ELISA analysis in our study. Moreover, other studies also found the similar results in Liver (Licata et al., 2018), kidney (Del Giudice et al., 2012), intestine and spleen (Zhang et al., 2014b). In juvenile and male fish exposed to exogenous estrogens, Vtg was considered to be firstly produced in the RER/Golgi region of hepatocytes (Van der Ven et al., 2003; Mortensen and Arukwe, 2007), released into blood circulation by gathering in vein vessels, and then delivered into kidney for Vtg elimination (Del Giudice et al., 2012; Verderame and Scudiero, 2017). Normally, the molecular mass of proteins in glomerular filtrate does not exceed that of albumin (66 kDa, Koger et al., 1999). Since Japanese flounder Vtg has a molecular mass of 520 kDa (Zhang et al., 2019), it could not be resorbed and remained within kidney tubule cells, and thus caused obvious fluorescence signals in kidney tubule. Vtg in extrahepatic tissues, especially the spleen and intestines, were mainly originating from the circulating blood which contained the Vtg produced in the liver. So, strong fluorescent signal was also found in the spleen sinusoidal veins and intestinal epithelial cells (Zhang et al., 2014b). Nevertheless, the kidney showed similar fluorescence intensity to the liver, indicating that both tissues would be suitable for histological detection of Vtg. In conclusion, this study developed a sandwich ELISA and DIF for detection of Vtg in Japanese flounder, and the two methods could effectively detect estrogenic activity of environmentally relevant concentrations of EE2. Furthermore, this study demonstrated that Vtg was not only detected in the liver, but also in extrahepatic tissues. Considering the high concentration of Vtg, the terminal tail fin is suggested as an alternative tissue for Vtg quantification, whereas both kidney and liver are recommended for histological detection of Vtg. These methodological improvements could be used to early detect estrogen pollution in marine environments.
Fig. 5. Detection of Vtg in different tissues of juvenile Japanese flounder expose to 50 ng/L 17α-ethinylestradiol by Western blot (A) and sandwich ELISA (B). Lanes: Mc, Surface mucus; Ms, Muscle; S, Spleen; I, Intestine; K, Kidney; L, liver; F, Tail fin.
Fig. 6. Real-time quantitative PCR analysis of the expression levels of Vtg1 and Vtg2 mRNA in muscle, intestine, fin, kidney, spleen, and liver of juvenile Japanese flounder exposed to 50 ng/L EE2. Values are means ± SD (n = 5), and were normalized against β-actin as a housekeeping gene.
signals in the liver of juvenile Japanese flounder exposed to 10 and 50 ng/L EE2, and this was consistent with the results of plasma Vtg induction detected by Western blot and ELISA. Moreover, the results of DIF showed that Vtg firstly appeared in the sinusoidal veins and then gathered in vein vessels, which was similar to the results of Arukwe and Røe (2007). The above results demonstrated that the established DIF could be used for detection of Vtg induction. To date, indirect IF is normally used for in situ detection of specific protein in tissues (Aoki et al., 2010). For piscine Vtg, indirect IF has been developed for only two species, medaka and bluegill (Lepomis macrochirus) (Kobayashi et al., 2005; Allner et al., 2016). Compared to indirect IF, the DIF method developed in our study detected Vtg antigens in a single step, and did not need additional incubations. Moreover, DIF had a much
Declaration of Competing Interest We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.
Acknowledgements This research was supported by the Fundamental Research Funds for the Central Universities (201964025) and National Natural Science Foundation of China (21607144).
5
Aquatic Toxicology 216 (2019) 105321
Z. Zhang, et al.
Fig. 7. Direct immunofluorescence of Vtg in muscle, spleen, intestine, kidney, and liver of juvenile Japanese flounder exposed to 0 and 50 ng/L 17α-ethinylestradiol for 21 days. V indicate vein vessels. R indicate renal tubules. E indicate epithelial cells of the intestine. S indicate sinusoidal veins.
Appendix A. Supplementary data
development of an ELISA f`or the detection of estrogenic effects. Sci. Total Environ. 309, 93–103. https://doi.org/10.1016/S0048-9697(03)00005-6.lss. Hiramatsu, N., Cheek, A.O., Sullivan, C.V., Matsubara, T., Hara, A., 2005. Chapter 16 vitellogenesis and endocrine disruption. Biochem. Mol. Biol. Fishes. 6, 431–471. https://doi.org/10.1016/S1873–0140(05)80019–0. Hu, S., Zhang, H., Shen, G., Yuan, Z., Xu, T., Ji, R., 2017. Effects of 17β-estradiol and 17αethinylestradiol on the embryonic development of the clearhead icefish (Protosalanx hyalocranius). Chemosphere 176, 18–24. https://doi.org/10.1016/j.chemosphere. 2017.02.094. Huang, W., Cao, L., Ye, Z., Yin, X., Dou, S., 2010. Antioxidative responses and bioaccumulation in Japanese flounder larvae and juveniles under chronic mercury exposure. Comp. Biochem. Physiol. 152C, 99–106. https://doi.org/10.1016/j.cbpc.2010.03. 005. Ihara, M., Kitamura, T., Kumar, V., Park, C.B., Ihara, M.O., Lee, S.J., et al., 2015. Evaluation of estrogenic activity of wastewater: comparison among in vitro ERα reporter gene assay, in vivo vitellogenin induction, and chemical analysis. Environ. Sci. Technol. 49, 6319–6326. https://doi.org/10.1021/acs.est.5b01027. Jin, Y., Wang, W., Sheng, G.D., Liu, W., Fu, Z., 2008. Hepatic and extrahepatic expression of estrogen-responsive genes in male adult zebrafish (Danio rerio) as biomarkers of short-term exposure to 17β-estradiol. Environ. Monit. Assess. 146, 105–111. https:// doi.org/10.1007/s10661-007-0063-y. Kobayashi, K., Tamotsu, S., Yasuda, K., Oishi, T., 2005. Vitellogenin-immunohistochemistry in the liver and the testis of the medaka, Oryzias latipes, exposed to 17beta-estradiol and p-nonylphenol. Zool. Sci. 22, 455–461. https://doi.org/10. 2108/zsj.22.453. Koger, C.S., The, S.J., Hinton, D.E., 1999. Variations of light and temperature regimes and resulting effects on reproductive parameters in medaka (Oryzias latipes). Biol. Reprod. 61, 1287–1293. https://doi.org/10.1095/biolreprod61.5.1287. Li, C.R., Kim, K.T., Kang, Y.J., Kim, A., Kang, S.W., Park, J.S., 2005. A novel enzymelinked immunosorbent assay system for the quantitative analysis of Carassius auratus vitellogenin. Chemosphere 59, 997–1003. https://doi.org/10.1016/j.chemosphere. 2004.11.063. Li, Y., Wang, J., Zheng, M., Zhang, Y., Ru, S., 2018. Development of ELISAs for the detection of vitellogenin in three marine fish from coastal areas of China. Mar. Pollut. Bull. 133, 415–422. https://doi.org/10.1016/j.marpolbul.2018.06.001. Licata, P., Tardugno, R., Pergolizzi, S., Capillo, G., Aragona, M., Colombo, A., Gervasi, T., Pellizzeri, V., Cicero, N., Calò, M., 2018. In vivo effects of PCB-126 and genistein on vitellogenin expression in zebrafish. Nat. Prod. Res. 2, 1–8. https://doi.org/10.1080/ 14786419.2018.1455048. Ling, S., Wang, R., Gu, X., Wen, C., Chen, L., Chen, Z., Chen, Q.A., Xiao, S., Yang, Y., Zhuang, Z., Wang, S., 2015. Rapid detection of fumonisin B1 using a colloidal gold immunoassay strip test in corn samples. Toxicon 108, 210–215. https://doi.org/10. 1016/j.toxicon.2015.10.014. Liu, J., Zhou, N., Fu, R., Cao, D., Si, Y., Li, A., Zhao, H., Zhang, Q., Yu, H., 2017. The polymorphism of chicken-type lysozyme gene in Japanese flounder (Paralichthys olivaceus) and its association with resistance/susceptibility to Listonella anguillarum. Fish Shellfish Immunol. 66, 43–49. https://doi.org/10.1016/j.fsi.2017.05.010. Luo, W., Zhou, Q., Jiang, G., 2011. Development of enzyme-linked immunosorbent assays for plasma vitellogenin in Chinese rare minnow (Gobiocypris rarus). Chemosphere 84, 681–688. https://doi.org/10.1016/j.chemosphere.2011.03.025. Madsen, L.L., Korsgaard, B., Pedersen, K.L., Aagaard, T., Bjerregaard, P., 2013. Vitellogenin as biomarker for estrogenicity in flounder Platichthys flesus in the field and exposed to 17α-ethinylestradiol via food and water in the laboratory. Mar. Environ. Res. 92, 79–86. https://doi.org/10.1016/j.marenvres.2013.09.002.
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.aquatox.2019.105321. References Aljandal, N., Saeed, T., Azad, I., Alsubiai, S., Alzekri, W., Hussain, S., Alhasan, E., 2017. Impact of endocrine disrupting compounds in sewage impacted coastal area on seabream. Ecotoxicol. Environ. Saf. 150, 280–288. https://doi.org/10.1016/j.ecoenv. 2017.12.035. Allner, B., Hennies, M., Lerche, C.F., Schmidt, T., Schneider, K., Willner, M., Stahlschmidt-Allner, P., 2016. Kinetic determination of vitellogenin induction in the epidermis of cyprinid and perciform fishes: evaluation of sensitive enzyme-linked immunosorbent assays. Environ. Toxicol. Chem. 35, 2916–2930. https://doi.org/10. 1002/etc.3475. An, L., Hu, J., Zhu, X., Deng, B., Zhang, Z., Yang, M., 2007. Crucian carp (Carassius carassius) VTG monoclonal antibody: development and application. Ecotoxicol. Environ. Saf. 66, 148–153. https://doi.org/10.1016/j.ecoenv.2005.11.005. Aoki, V., Sousa, J.X.J., Fukumori, L.M., Périgo, A.M., Freitas, E.L., Oliveira, Z.N., 2010. Direct and indirect immunofluorescence. An. Bras. Dermatol. 85, 490–500. https:// doi.org/10.1590/S0365-05962010000400010. Arukwe, A., Røe, K., 2007. Molecular and cellular detection of expression of vitellogenin and zona radiata protein in liver and skin of juvenile salmon (Salmo salar) exposed to nonylphenol. Cell Tissue Res. 331, 701–712. https://doi.org/10.1007/s00441-0070543-y. Brodeur, J., Woodburn, K., Zhang, F., Bartels, M.J., Klecka, G.M., 2006. Plasma sampling and freezing procedures influence vitellogenin measurements by enzyme-linked immunoassay in the fathead minnow (Pimephales promelas). Environ. Toxicol. Chem. 25, 337–348. https://doi.org/10.1897/05-368R.1. Cao, L., Huang, W., Liu, J.H., Yin, X.B., Dou, S.Z., 2010. Accumulation and oxidative stress biomarkers in Japanese flounder larvae and juveniles under chronic cadmium exposure. Comp. Biochem. Physiol. 151C, 386–392. https://doi.org/10.1016/j.cbpc. 2010.01.004. Del Giudice, G., Prisco, M., Agnese, M., Verderame, M., Rosati, L., Limatola, E., Andreuccetti, P., 2012. Effects of nonylphenol on vitellogenin synthesis in adult males of the spotted ray Torpedo marmorata. J. Fish Biol. 80, 2112–2121. https:// doi.org/10.1111/j.1095-8649.2011.03172.X. Flick, R.W., Bencic, D.C., See, M.J., Biales, A.D., 2014. Sensitivity of the vitellogenin assay to diagnose exposure of fathead minnows to 17α-ethynylestradiol. Aquat. Toxicol. 152, 353–360. https://doi.org/10.1016/j.aquatox.2014.04.026. Garnayak, S.K., Mohanty, J., Rao, T.V., Sahoo, S.K., Sahoo, P.K., 2013. Vitellogenin in Asian catfish, Clarias batrachus: Purification, partial characterization and quantification during the reproductive cycle by ELISA. Aquaculture 392–395, 148–155. https://doi.org/10.1016/j.aquaculture.2013.02.020. Gong, B., Murray, K.D., Trimmer, J.S., 2016. Developing high-quality mouse monoclonal antibodies for neuroscience research approaches, perspectives and opportunities. N. Biotechnol. 33, 551–564. https://doi.org/10.1016/j.nbt.2015.11.007. Hara, A., Hiramatsu, N., Fujita, T., 2016. Vitellogenesis and choriogenesis in fishes. Fish. Sci. 82, 187–202. https://10.1007/s12562-015-0957-5. Hennies, M., Wiesmann, M., Allner, B., Sauerwein, H., 2003. Vitellogenin in carp (Cyprinus carpio) and perch (Perca fluviatilis): purification, characterization and
6
Aquatic Toxicology 216 (2019) 105321
Z. Zhang, et al.
vitellogenin. Environ. Toxicol. Chem. 18, 337–347. https://doi.org/10.1002/etc. 5620180234. Van der Ven, L.T., Holbech, H., Fenske, M., Van den Brandhof, E.J., Gielis-Proper, F.K., Wester, P.W., 2003. Vitellogenin expression in zebrafish Danio rerio: evaluation by histochemistry, immunohistochemistry, and in situ mRNA hybridization. Aquat. Toxicol. 65, 1–11. https://doi.org/10.1016/S0166-445X(03)00103-6. Verderame, M., Scudiero, R., 2017. Estrogen-dependent, extrahepatic synthesis of vitellogenin in male vertebrates: a mini-review. C. R. Biol. 340, 139–144. https://doi. org/10.1016/j.crvi.2017.01.005. Wang, H., Tan, J.T., Emelyanov, A., Korzh, V., Gong, Z., 2005. Hepatic and extrahepatic expression of vitellogenin genes in the zebrafish. Danio rerio. Gene. 356, 91–100. https://doi.org/10.1016/j.gene.2005.03.041. Wang, J., Ma, S., Zhang, Z., Zheng, M., Dong, Y., Ru, S., 2017. Vitellogenin induction in caudal fin of guppy (Poecilia reticulata) as a less invasive and sensitive biomarker for environmental estrogens. Sci. Rep. 7, 7647. https://doi.org/10.1038/s41598-01706670-6. Wang, J., Shan, R., Zhang, X., Tian, H., Wang, W., Ru, S., 2015a. Development of a lipovitellin-based sandwich ELISA for quantification of vitellogenin in surface mucus and plasma of goldfish (Carassius auratus). Ecotoxicol. Environ. Saf. 120, 80–87. https://doi.org/10.1016/j.ecoenv.2015.05.010. Wang, J., Wang, W., Zhang, X., Tian, H., Ru, S., 2015b. Development of a lipovitellin–based goldfish (Carassius auratus) vitellogenin ELISA for detection of environmental estrogens. Chemosphere 132, 166–171. https://doi.org/10.1016/j. chemosphere.2015.03.038. Xu, E.G.B., Liu, S., Ying, G.G., Zheng, G.J.S., Lee, J.H.W., Leung, K.M.Y., 2014. The occurrence and ecological risks of endocrine disrupting chemicals in sewage effluents from three different sewage treatment plants, and in natural seawater from a marine reserve of Hong Kong. Mar. Pollut. Bull. 85, 352–362. https://doi.org/10.1016/j. marpolbul.2014.02.029. Yin, N., Jin, X., He, J., Yin, Z., 2009. Effects of adrenergic agents on the expression of zebrafish (Danio rerio) vitellogenin Ao1. Toxicol. Appl. Pharmacol. 238, 20–26. https://doi.org/10.1016/j.taap.2009.04.004. Yue, Z., Yu, M., Zhang, X., Dong, Y., Tian, H., Wang, W., Ru, S., 2017. Semicarbazideinduced thyroid disruption in Japanese flounder (Paralichthys olivaceus) and its potential mechanisms. Ecotoxicol. Environ. Saf. 140, 131–140. https://doi.org/10. 1016/j.ecoenv.2017.02.043. Zhang, C., Zheng, L., Nurnberg, J., Vacari, B.M., Zhou, J., Wang, Y., 2014a. Cleavage of pro-tumor necrosis factor alpha by ADAM metallopeptidase domain 17: a fluorescence-based protease assay cleaves its natural protein substrate. Anal. Biochem. 445, 14–19. https://doi.org/10.1016/j.ab.2013.09.022. Zhang, X., Xiong, L., Liu, Y., Deng, C., Mao, S., 2014b. Histopathological and estrogen effect of pentachlorophenol on the rare minnow (Gobiocypris rarus). Fish Physiol. Biochem. 40, 805–816. https://doi.org/10.1007/s10695–013–9887–2. Zhang, Z., Wang, J., Gao, M., Li, X., Cheng, Y., Zhang, X., Tian, H., Wang, W., Ru, S., 2019. New methods for purification of Paralichthys olivaceus lipovitellin and immunoassay-based detection of vitellogenin. Ecotoxicol. Environ. Saf. 180, 624–631. https://doi.org/10.1016/j.ecoenv.2019.04.087. Zhong, L., Yuan, L., Rao, Y., Li, Z., Zhang, X., Liao, T., Xu, Y., Dai, H., 2014. Distribution of vitellogenin in zebrafish (Danio rerio) tissues for biomarker analysis. Aquat. Toxicol. 149, 1–7. https://doi.org/10.1016/j.aquatox.2014.01.022. Zhou, X., Lian, Z., Wang, J., Tan, L., Zhao, Z., 2011. Distribution of estrogens along Licun River in Qingdao, China. Procedia. Environ. Sci. 10, 1876–1880. https://doi.org/10. 1016/j.proenv.2011.09.293. Zuo, Y., Zhang, K., Deng, Y., 2006. Occurrence and photochemical degradation of 17αethinylestradiol in Acushnet River Estuary. Chemosphere 63, 1583–1590. https:// doi.org/10.1016/j.chemosphere.2005.08.063.
Magalhães, I., Ledrich, M.L., Pihan, J.C., Falla, J., 2004. One-step, non-denaturing purification method of carp (Cyprinus carpio) vitellogenin. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 799, 87–93. https://doi.org/10.1016/j.jchromb.2003.10. 026. Maltais, D., Dupont-Cyr, B.A., Roy, R.L., Le François, N.R., 2014. Purification and partial characterization of vitellogenin from spotted wolffish (Anarhichas minor) and development of an enzyme-linked immunosorbent assay for the determination of gender and sexual maturity. Fish Physiol. Biochem. 40, 279–294. https://doi.org/10.1007/ s10695-013-9843-1. Mortensen, A.S., Arukwe, A., 2007. Effects of 17α-ethynylestradiol on hormonal responses and xenobiotic biotransformation system of Atlantic salmon (Salmo salar). Aquat. Toxicol. 85, 113–123. https://doi.org/10.1016/j.aquatox.2007.08.004. Nishi, K., Chikae, M., Hatano, Y., Mizukami, H., Yamashita, M., Sakakibara, R., Tamiya, E., 2002. Development and application of a monoclonal antibody-based sandwich ELISA for quantification of Japanese medaka (Oryzias latipes) vitellogenin. Comp. Biochem. Physiol. 132C, 161–169. https://doi.org/10.1016/S1532-0456(02) 00058-3. Partridge, C., Boettcher, A., Jones, A.G., 2010. Short-term exposure to a synthetic estrogen disrupts mating dynamics in a pipefish. Horm. Behav. 58, 800–807. https:// doi.org/10.1016/j.yhbeh.2010.08.002. Petersen, K., Fetter, E., Kah, O., Brion, F., Scholz, S., Tollefsen, K.E., 2013. Transgenic (cyp19a1b-GFP) zebrafish embryos as a tool for assessing combined effects of oestrogenic chemicals. Aquat. Toxicol. 138–139, 88–97. https://doi.org/10.1016/j. aquatox.2013.05.001. Prasatkaew, W., Nanthanawat, P., Khongchareonporn, N., Kingtong, S., 2019. A monoclonal antibody against Lates calcarifer vitellogenin and a competitive ELISA to evaluate vitellogenin induction after exposure to xenoestrogen. J. Environ. Sci (China) 75, 325–333. https://doi.org/10.1016/j.jes.2018.05.001. Roy, R.L., Morin, Y., Courtenay, S.C., Robichaud, P., 2004. Purification of vitellogenin from smooth flounder (Pleuronectes putnami) and measurement in plasma by homologous ELISA. Comp. Biochem. Physiol. 139B, 235–244. https://doi.org/10.1016/j. cbpc.2004.07.006. Sawaguchi, S., Koya, Y., Yoshizaki, N., Ohkubo, N., Andoh, T., Hiramatsu, N., Sullivan, C.V., Hara, A., Matsubara, T., 2005. Multiple vitellogenins (Vgs) in mosquitofish (Gambusia affinis): identification and characterization of three functional Vg genes and their circulating and yolk protein products. Biol. Reprod. 72, 1045–1060. https://doi.org/10.1095/biolreprod.104.037895. Scott, A.P., Katsiadaki, I., Witthames, P.R., Hylland, K., Davies, I.M., McIntosh, A.D., Thain, J., 2006. Vitellogenin in the blood plasma of male cod (Gadus morhua): a sign of oestrogenic endocrine disruption in the open sea? Mar. Environ. Res. 61, 149–170. https://doi.org/10.1016/j.marenvres.2005.08.003. Selcer, K.W., Verbanic, J.D., 2014. Vitellogenin of the northern leopard frog (Rana pipiens): development of an ELISA assay and evaluation of induction after immersion in xenobiotic estrogens. Chemosphere 112, 348–354. https://doi.org/10.1016/j. chemosphere.2014.04.073. Segner, H., Casanova-Nakayama, A., Kase, R., Tyler, C.R., 2013. Impact of environmental estrogens on Yfish considering the diversity of estrogen signaling. Gen. Comp. Endocrinol. 119, 191–201. https://doi.org/10.1016/j.ygcen.2013.05.015. Sumpter, J.P., Jobling, S., 1995. Vitellogenesis as a biomarker for estrogenic contamination of the aquatic environment. Environ. Health Perspect. 103, 173–178. https://doi.org/10.1289/ehp.95103s7173. Tollefsen, K.E., Mathisen, R., Stenersen, J., 2003. Induction of vitellogenin synthesis in an Atlantic salmon (Salmo salar) hepatocyte culture: a sensitive in vitro bioassay for the oestrogenic and anti-oestrogenic activity of chemicals. Biomarkers 8, 394–407. https://doi.org/10.1080/13547500310001607827. Tyler, C.R., Aerle, R.V., Hutchinson, T.H., Maddix, S., Trip, H., 1999. An in vivo testing system for endocrine disruptors in fish early life stages using induction of
7