Article pubs.acs.org/est
Hepatic Proteomic Responses in Marine Medaka (Oryzias melastigma) Chronically Exposed to Antifouling Compound Butenolide [5-octylfuran-2(5H)-one] or 4,5-Dichloro-2‑N‑Octyl-4Isothiazolin-3-One (DCOIT) Lianguo Chen,†,⊥ Jin Sun,§,⊥ Huoming Zhang,‡ Doris W. T. Au,∥ Paul K. S. Lam,∥ Weipeng Zhang,† Vladimir B. Bajic,□ Jian-Wen Qiu,§ and Pei-Yuan Qian*,† †
Division of Life Science and Environmental Science Programs, Hong Kong University of Science and Technology, Hong Kong, China ‡ Biosciences Core Laboratory, and □Computational Bioscience Research Center, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia § Department of Biology, Hong Kong Baptist University, Hong Kong, China ∥ State Key Laboratory in Marine Pollution, Department of Biology and Chemistry, City University of Hong Kong, Hong Kong, China S Supporting Information *
ABSTRACT: The pollution of antifoulant SeaNine 211, with 4,5-dichloro-2-n-octyl-4-isothiazolin-3-one (DCOIT) as active ingredient, in coastal environment raises concerns on its adverse effects, including endocrine disruption and impairment of reproductive function in marine organisms. In the present study, we investigated the hepatic protein expression profiles of both male and female marine medaka (Oryzias melastigma) exposed to low concentrations of DCOIT at 2.55 μg/L (0.009 μM) or butenolide, a promising antifouling agent, at 2.31 μg/L (0.012 μM) for 28 days. The results showed that proteins involved in phase I (CYP450 enzyme) metabolism, phase II (UDPGT and GST) conjugation as well as mobilization of retinoid storage, an effective nonenzymatic antioxidant, were consistently up-regulated, possibly facilitating the accelerated detoxification of butenolide. Increased synthesis of bile acid would promote the immediate excretion of butenolide metabolites. Activation of fatty acid β-oxidation and ATP synthesis were consistent with elevated energy consumption for butenolide degradation and excretion. However, DCOIT did not significantly affect the detoxification system of male medaka, but induced a marked increase of vitellogenin (VTG) by 2.3-fold in the liver of male medaka, suggesting that there is estrogenic activity of DCOIT in endocrine disruption. Overall, this study identified the molecular mechanisms and provided sensitive biomarkers characteristic of butenolide and DCOIT in the liver of marine medaka. The low concentrations of butenolide and DCOIT used in the exposure regimes highlight the needs for systematic evaluation of their environmental risk. In addition, the potent estrogenic activity of DCOIT should be considered in the continued applications of SeaNine 211.
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INTRODUCTION Immersed surfaces of man-made structures in the marine environment are usually coated with antifouling paints to prevent undesirable colonization by marine organisms.1 Since the ban of organotin compounds in antifouling paints due to their persistence, accumulation and high toxicity, effective and environmentally friendly substitutes have been in urgent demand by the antifouling industry.2 A number of organic booster biocides, including Irgarol 1051, Diuron and SeaNine 211, have been commonly incorporated into antifouling paints supplemented with cuprous oxide.3,4 However, the toxicity of some booster biocides to marine organisms is comparable to or even higher than organotin.5,6 For example, the commercial antifoulant SeaNine 211 with active biocide of 4,5-dichloro-2-n-octyl-4-isothiazolin-3-one © XXXX American Chemical Society
(DCOIT) (Figure S1 in Supporting Information (SI); Molar mass: 282 g/mol) was considered as an environmentally acceptable alternative of organotin due to DCOIT’s fast degradation.7 However, the concentration of DCOIT in some sheltered coastal areas reached 3.3 μg/L,8 which is substantially higher than 0.1 ng/L, a concentration that significantly delayed the embryonic development of sea urchin.5 The toxic effects of DCOIT on the growth and development of marine organisms are not well understood. Available results demonstrated that DCOIT could inhibit the ATP synthesis,9 suppress the function Received: September 24, 2014 Revised: December 24, 2014 Accepted: January 2, 2015
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DOI: 10.1021/es5046748 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology
frozen in liquid nitrogen and stored at −80 °C for follow-up experiments. Proteomic Analysis. Protein Labeling and Strong Cation Exchange (SCX) Fractionation. Proteins were extracted, quantified and labeled using the TMT Sixplex Isobaric Mass Tagging Kit according to the manufacturer’s instruction (male liver: TMT agent 126 for control group, 127 for butenolide group and 128 for DCOIT group; female liver: 129 for control group, 130 for butenolide group and 131 for DCOIT group). The peptide mixture was fractionated using SCX chromatography depending on their positive charges. The detailed protocol for protein extraction, labeling and fractionation was provided in the SI (Text S1). LC-MS/MS Analysis and Database Search. The peptides of each biological replicate (n = 3) were analyzed at least two times on a TripleTOF 5600 System (AB SCIEX, Concord, ON) coupled with an UltiMate 3000 UHPLC (Dionex Corp., CA). The obtained peptide sequences were searched in the Japanese medaka (Oryzias latipes) protein database (Text S2, SI). Differentially expressed proteins were identified only if the normalized fold change was higher than 1.50 (up-regulation) or less than 0.70 (down-regulation), which was calculated as 95% confidence level based on pairwise analysis between two experimental replicates.15,16 The pathway enrichment in each group was analyzed using the web server KOBAS 2.0.17 The accession numbers of differentially expressed proteins were annotated according to O. latipes proteomics and identified based on KEGG pathways. The statistically significantly enriched pathways were determined using hypergeometric test and Fisher’s exact test. Significantly enriched pathways were identified only if Benjamini and Hochberg’s corrected P value was 99%) was purchased from Waterstone Technology (Carmel, IN). Butenolide (purity >99%) was synthesized by Medicilon, Inc. (Shanghai, China). Vitellogenin primary antibody was obtained from Abcam Inc. (Cambridge, MA). TMT mass tagging kit was purchased from Thermo Scientific (Rockford, IL). The other chemicals used were of analytical grade. Exposure of Marine Medaka to DCOIT and Butenolide. The maintenance and exposure of adult marine medaka (O. melastigma) was carried out according to the previous report.12 Marine medaka (4-month-old) were cultured in fully aerated and charcoal-filtered artificial seawater (salinity: 30‰). The temperature was kept at 24 ± 0.5 °C with 14 h: 10 h light/ dark cycle. The fish were fed once with newly hatched Artemia nauplii and twice with flake food (AX5; Aquatic Ecosystems) daily. After 2 weeks of acclimation in a 13-L tank, the fish were randomly divided into three groups (the DCOIT, butenolide and control groups) and distributed into nine 10 L tanks (12 male and 12 female fish per tank; three tanks per group). The fish were then exposed to nominal 3.0 μg/L DCOIT (0.011 μM) or 3.0 μg/L butenolide (0.015 μM) in a semistatic system. Stock solutions of DCOIT and butenolide were prepared in high performance liquid chromatography (HPLC)-grade dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO). In both the control and exposure tanks, DMSO was kept at a final concentration at 0.001%. The seawater in tanks was halfrenewed daily to maintain appropriate chemical concentrations. The actual concentrations of DCOIT and butenolide in the seawater were measured to be 2.55 μg/L (0.009 μM) and 2.31 μg/L (0.012 μM), respectively.12 The experimental concentration of DCOIT was chosen based on the reported 3.3 μg/L of SeaNine 211 that were detected in the Spain marinas.8 After 28 days of exposure, the fish were anesthetized using 0.03% MS-222, and liver and plasma were collected and immediately B
DOI: 10.1021/es5046748 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology The VTG content in medaka liver was also analyzed using Western blot as previously described (SI Text S4).20 Statistical Analysis. Data were expressed as the mean ± SEM. Normalization and homogeneity of variance of the data from the enzyme activity and ELISA assays were conducted using the Kolmogorov−Smirnov test and Levene’s test, respectively. If necessary, log-transformation was conducted. One-way analysis of variance (ANOVA) was used to identify significant differences between the control and exposure groups followed by the post hoc LSD test in the SPSS 13.0 software package (SPSS, Chicago, IL). A P value
