Article pubs.acs.org/est
Adverse Effects, Expression of the Bk-CYP3045C1 Gene, and Activation of the ERK Signaling Pathway in the Water Accommodated Fraction-Exposed Rotifer Eun-Ji Won,†,‡,# Ryeo-Ok Kim,†,# Hye-Min Kang,† Hui-Su Kim,† Dae-Sik Hwang,† Jeonghoon Han,† Young Hwan Lee,† Un-Ki Hwang,§ Bingsheng Zhou,∥ Su-Jae Lee,⊥ and Jae-Seong Lee*,† †
Department of Biological Science, College of Science, Sungkyunkwan University (SKKU), Suwon 16419, South Korea Marine Chemistry and Geochemistry Research Center, Korea Institute of Ocean Science and Technology, Ansan 15627, South Korea § Marine Ecological Risk Assessment Center, West Sea Fisheries Research Institute, National Fisheries Research and Development Institute, Incheon 22383, South Korea ∥ State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, China ⊥ Department of Life Science, College of Natural Sciences, Hanyang University, Seoul 04763, South Korea ‡
S Supporting Information *
ABSTRACT: To examine the deleterious effects of the water accommodated fraction (WAF) of crude oil, the growth curve, fecundity, and lifespan of the monogonont rotifer (Brachionus koreanus) were measured for 24 h in response to three different doses (0.2×, 0.4×, and 0.8×) of WAFs. A higher dose of WAFs significantly reduced the fecundity and lifespan. A rotifer 32K microarray chip showed that the Bk-CYP3045C1 gene had the highest expression. Of the 25 entire CYP genes, the Bk-CYP3045C1 gene showed a significant expression for different doses and times in response to WAFs and chemical components of WAFs (naphthalene and phenanthrene); also, glutathione S-transferase genes, ABC transporter, and other genes showed dose responses upon exposure to 80% WAF over time. Different doses of WAFs increased the oxidative stress with an induction of reactive oxygen species (ROS) and a depletion of glutathione (GSH). Exposure to WAFs did not show toxic effects on survivability in B. koreanus; however, toxicity to WAFs was shown when piperonyl butoxide, a potent inhibitor of cytochrome P450 (CYP) enzymes, was added. This toxicity was dose-dependent. After WAFs exposure, pERK was activated over time in response to WAFs, which suggests that WAFs can be activated by the p-ERK signaling pathway.
1. INTRODUCTION
Analyzing the water accommodated fractions (WAFs) is considered to be an alternative method to evaluate toxic effects of oil exposure.8,11,12 However, limited information is available concerning mechanistic studies of small aquatic invertebrates positioned in the basal levels of the marine food web, which play an important role in marine ecosystems. Rotifers, which are widespread and abundant organisms in marine environments, have received great attention as an ecotoxicological model species.13,14 To date, a wide range of toxicants have been tested on rotifers according to standardized protocols of toxicological assays.15,16 Recently ecotoxicological and ecophysiological studies were highlighted in rotifers; these
Crude oil, contaminating marine environments accidentally or through other anthropogenic activities (e.g., operating ships, oil drilling, and transport of a bunker fuel), may deleteriously affect aquatic ecosystems.1,2 During the past several decades, oil spills have threatened marine environments and are considered to be one of the main factors that cause pollution.3,4 Oil released into aquatic environments can destroy ecosystems throughout the food web by direct physical contact and/or dispersion processes (e.g., evaporation, emulsification, oxidation, biodegradation, and sedimentation), depending on the oil properties, environmental factors, and types of spillage.5 These processes also affect marine organisms due to the acute and chronic toxic effects of oil residues, which cause reproduction impairment, DNA damage, and immunotoxicity in zooplankton, polychaete, and bivalves in the marine environment.3,6−10 © XXXX American Chemical Society
Received: March 15, 2016 Revised: April 28, 2016 Accepted: May 2, 2016
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DOI: 10.1021/acs.est.6b01306 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology
Marine Salt Pro; Tetra, Blacksburg, VA, USA) at 25 °C under a photoperiod cycle of 12 h light/12 h dark. They were fed with the green algae Tetraselmis suecica every 24 h. The B. koreanus used in this study reproduce only by parthenogenesis. Species identification was confirmed by morphological analysis and sequence analysis of the mitochondrial DNA cytochrome oxidase I (COI) as a barcoding gene.25,26 2.2. WAFs Preparation and Exposure. WAFs were made from Iranian heavy crude oil that was supplied by the Korea Institute of Ocean Science & Technology in South Korea. WAF preparation was carried out according to the method described by the Chemical Response to Oil Spills: Ecological Effect Research Forum (CROSERF). Briefly, the crude oil (500 g) and seawater (2 L) were mixed (1:4 = w:v) with a magnetic stirrer for 18 h in a glass carboy, as described in a previous study.27 To obtain WAFs, well-mixed seawater and crude oil were allowed to settle for 6 h at dark conditions. Then, the isolated seawater (containing the soluble parts of crude oil) was diluted with seawater to control the WAFs concentrations. For each test, rotifers were exposed to WAFs with different dilution factors (20, 40, 60, 80, and 100%) for 24 h. 2.3. In vivo Effects on the Growth Retardation, Fecundity, and Lifespan upon Exposure to WAFs. To obtain just-hatched individuals, we isolated eggs from ovigerous individuals, as described by Kim et al.13 Ten newborn neonates (age
