BDE-47 induces oxidative stress, activates MAPK signaling pathway, and elevates de novo lipogenesis in the copepod Paracyclopina nana

BDE-47 induces oxidative stress, activates MAPK signaling pathway, and elevates de novo lipogenesis in the copepod Paracyclopina nana

Aquatic Toxicology 181 (2016) 104–112 Contents lists available at ScienceDirect Aquatic Toxicology journal homepage: www.elsevier.com/locate/aquatox...

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Aquatic Toxicology 181 (2016) 104–112

Contents lists available at ScienceDirect

Aquatic Toxicology journal homepage: www.elsevier.com/locate/aquatox

Research paper

BDE-47 induces oxidative stress, activates MAPK signaling pathway, and elevates de novo lipogenesis in the copepod Paracyclopina nana Min-Chul Lee a,1 , Jayesh Puthumana a,1 , Seung-Hwi Lee b,e,1 , Hye-Min Kang a , Jun Chul Park a , Chang-Bum Jeong a , Jeonghoon Han a , Dae-Sik Hwang a , Jung Soo Seo c , Heum Gi Park d , Ae-Son Om e,∗ , Jae-Seong Lee a,∗ a

Department of Biological Science, College of Science, Sungkyunkwan University, Suwon 16419, South Korea Department of Food and Nutrition, College of Health Science, Honam University, Gwangju 62399, South Korea c Pathology Division, National Institute of Fisheries Science, Busan 46083, South Korea d Department of Marine Bioscience, College of Life Sciences, Gangneung-Wonju National University, Gangneung 25457, South Korea e Department of Food and Nutrition, College of Human Ecology, Hanyang University, Seoul 04763, South Korea b

a r t i c l e

i n f o

Article history: Received 5 October 2016 Received in revised form 21 October 2016 Accepted 24 October 2016 Available online 25 October 2016 Keywords: BDE-47 Paracyclopina nana Oxidative stress MAPK pathways DNL pathways Lipogenesis

a b s t r a c t Brominated flame retardant, 2, 2 , 4, 4 -tetrabromodiphenyl ether (BDE-47), has received grave concerns as a persistent organic pollutant, which is toxic to marine organisms, and a suspected link to endocrine abnormalities. Despite the wide distribution in the marine ecosystem, very little is known about the toxic impairments on marine organisms, particularly on invertebrates. Thus, we examined the adverse effects of BDE-47 on life history trait (development), oxidative markers, fatty acid composition, and lipid accumulation in response to BDE-47-induced stress in the marine copepod Paracyclopina nana. Also, activation level of mitogen-activated protein kinase (MAPK) signaling pathways along with the gene expression profile of de novo lipogenesis (DNL) pathways were addressed. As a result, BDE-47 induced oxidative stress (e.g. reactive oxygen species, ROS) mediated activation of extracellular signal-regulated kinase (ERK) and c-Jun-N-terminal kinase (JNK) signaling cascades in MAPK pathways. Activated MAPK pathways, in turn, induced signal molecules that bind to the transcription factors (TFs) responsible for lipogenesis to EcR, SREBP, ChREBP promoters. Also, the stress stimulated the conversion of saturated fatty acids (SFAs) to polyunsaturated fatty acids (PUFAs), a preparedness of the organism to adapt the observed stress, which could be correlated with the elongase and desaturase gene (e.g. ELO3, 5-DES, 9DES) expressions, and then extended to the delayed early post-embryonic development and increased accumulation of lipid droplets in P. nana. This study will provide a better understanding of how BDE-47 effects on marine invertebrates particularly on the copepods, an important link in the marine food chain. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Brominated flame retardants (BFRs) are a class of flameretardant additives that have been extensively used in domestic and industrial materials to reduce their flammability (Lema et al., 2007; Díaz-Jaramillo et al., 2016). BFRs have been gained much attention due to low production cost and high efficiency, and polybrominated diphenyl ethers (PBDEs) are the most widely used one

∗ Corresponding authors. E-mail addresses: [email protected] (A.-S. Om), [email protected] (J.-S. Lee). 1 These authors equally contributed to this work. http://dx.doi.org/10.1016/j.aquatox.2016.10.025 0166-445X/© 2016 Elsevier B.V. All rights reserved.

(Lema et al., 2007). Though the production of PBDEs has been banned in many countries, these compounds are considered as a new class of persistent organic pollutants (POPs) in the marine environments (Rahman et al., 2001; Yu et al., 2009; Bramwell et al., 2014). The congener of PBDEs, 2, 2 , 4, 4 -tetrabromodiphenyl ether (BDE-47) has elicited great concerns, as a chemical that is highly toxic to aquatic organisms and a suspected link to endocrine abnormalities (Chan and Chan, 2012). Owing to their high lipophilicity and resistance to degradation, BDE-47 resulted in the worldwide distribution in all trophic levels through bioaccumulation and biotransformation across the food web (Meng et al., 2007; USEPA, 2007; Nelson et al., 2015). With these properties, BDE-47 was detected as a dominant chemical in higher trophic level predators such as the porpoise Phocoena phocoena and the seabird Phalacro-

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corax carbo in the marine ecosystems (Law et al., 2002). Moreover, toxicological effects of BDE-47 have been investigated in major taxa of aquatic organisms including fish (Muirhead et al., 2006; Liu et al., 2015), amphibian (Carlsson et al., 2007), crab (Díaz-Jaramillo et al., 2016), polychaete (Díaz-Jaramillo et al., 2016), copepod (Breitholtz and Wollenberger, 2003; Lee et al., 2016a, 2016b, 2016c), rotifer (Wang et al., 2015), and algae (Källqvist et al., 2006). Specifically, decreased ethoxy resorufin-O-deethylase (EROD) activity, leading to impairment of the mixed function oxidase systems, was observed in the fish Oncorhynchus mykiss in response to BDE47 exposure. EROD activity is the important biochemical defence mechanisms for xenobiotic metabolism in the liver (Tjärnlund et al., 1998), suggesting that BDE-47 induces physiological and biochemical damages in aquatic organisms. Thus, elucidating the effect of BDE-47 on marine copepods will be a real asset for a better understanding of mechanistic toxicology and assessment of the potential impacts on the marine ecosystems. As copepods play a major role in the marine ecosystem as a link between producers and high trophic consumers, they have been widely used to study the toxic impairments on aquatic ecosystems (Raisuddin et al., 2007). Considering the several ideal traits for ecotoxicological and environmental genomics research, Paracyclopina nana (cyclopoid copepod) has been used as a model organism to study the mechanistic toxicology of chemicals, which are more prone to evaluate marine pollutants in marine ecosystems (Won and Lee, 2014; Han et al., 2015b; Kim et al., 2016; Dahms et al., 2016). In addition, P. nana is highly sensitive to environmental conditions and produced significant biomarkers towards environmental stressors, which are used as toxicity markers of the aquatic environments and for risk assessment (Lee et al., 2015; Dahms et al., 2016). Recently, biomarkers of carbon nanotubes (CNTs)-induced toxicity were identified from P. nana (Kim et al., 2016). However, no information is available on effects of BDE-47 on molecular signaling pathways and lipid composition along with physiological parameters in P. nana. Thus, this species will be a suitable model for enhancing our understanding of the effects of BDE-47-induced stress on aquatic invertebrates. Due to adverse effects on biota, BDE-47 has been considered as cytotoxic, neurotoxic, genotoxic, endocrine disruptors (EDs), and reactive oxygen species (ROS) inducers (Wang et al., 2012; Díaz-Jaramillo et al., 2016). Coupled with the literary evidence, oxidative stress has identified as an important mechanism, which triggers several signal pathways in the organism and promotes deleterious effects (Wang et al., 2012). For example, Breitholtz and Wollenberger (2003) observed a reduction in development and reproductive capacity of the copepod Nitocra spinipes. Also BDE-47 induced developmental retardation by interacting with the nuclear receptor (NR) genes in the ecdysteroid signaling pathways in the copepod T. japonicus (Hwang et al., 2016), while oxidative stressmediated damages were well explained in response to BDE-47 in T. japonicus (Han et al., 2015a). As all these studies were focused on the copepods belonging to harpacticoids, evaluations of toxic impairments in the copepods belonging to other order are critical to understanding the comparative effects of BDE-47. Thus, more elaborative studies are required to attain better knowledge about the underlying mechanistic aspects of BDE-47 toxicity in different groups of marine copepods. In this paper, we investigated life history trait (development), oxidative markers, fatty acid composition, and lipid accumulation in the cyclopoid copepod in response to BDE-47-induced stress. Also, we measured the expression profile of DNL pathway genes and activation pattern of MAPK pathways in P. nana. This study will provide a better understanding of how BDE-47 affects on aquatic organisms, particularly on cyclopoid copepods.

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2. Materials and methods 2.1. Chemicals All chemicals and reagents used for this study, unless specifically stated otherwise, were purchased from Sigma-Aldrich Co (St. Louis, Missouri, U.S.A). 2.2. Culture and maintenance of Paracyclopina nana The cyclopoid copepod Paracyclopina nana was maintained at the Department of Biological Science, Sungkyunkwan University, Suwon, South Korea. Before experiments, morphometric analysis followed by molecular characterization of the animal was performed to confirm the species identity using mitochondrial DNA cytochrome oxidase 1 (CO1) gene (Ki et al., 2009). P. nana was maintained in filtered artificial seawater (ASW) (TetraMarine Salt Pro,TetraTM, Cincinnati, OH, USA) under confined laboratory conditions (15 psu salinity, 12:12 h [light: dark] photoperiod at 25 ◦ C) and fed with marine microalgae Tetraselmis suecica (∼6 × 104 cells/ml) every 24 h T. suecica were cultivated in two liter transparent glass bottles (DURAN Group, GmbH, Wertheim, Germany) with Walne’s media (Walne, 1970). 2.3. Effects of BDE-47 on development Effects of BDE-47 on post-embryonic developmental stages of P. nana were analysed using 12 h post-hatched nauplii. 50% of the ASW was replaced everyday and animals were fed with 100 ␮g/L of T. suecica (∼6 × 104 cells/ml). For each group, ten nauplii were transferred to 12-well cell culture plate (30012, SPL Life Science Co. Ltd., Seoul, South Korea) containing 4 mL ASW with various concentrations of BDE-47 (0.1 [0.176 nM], 1 [1.76 nM], and 10 ␮g/L [17.6 nM]) in triplicates. Post-embryonic developmental stages, naupliar to copepodid stages and copepodid to adult stages, were observed once every 24 h under a stereomicroscope (SZX-ILLK200, Olympus, Tokyo, Japan) for 14 days. 2.4. Measurement of cellular ROS level and antioxidant enzymatic activities BDE-47-induced reactive oxygen species (ROS) and the subsequent response of antioxidant enzymatic activity of glutathione S-transferase (GST), and glutathione peroxidase (GPx) were measured to evaluate the oxidative stress in P. nana in accordance with our previous study (Kim et al., 2016). Briefly, ∼300 adult copepods were exposed to 0.1, 1, and 10 ␮g/L BDE-47 along with the control group for 24 h in triplicate. For ROS measurement, samples were homogenised in homogenization buffer (pH 7.4) and centrifuged (10,000 × g for 20 min at 4 ◦ C). The supernatants were allowed to react with H2 DCFDA and measured the developed fluorescence (at 485 and 520 nm) using a microplate reader (Thermo Scientific Co., Varioscan Flash, Waltham, MA, USA). For GST activities, samples were homogenised in buffer (pH 8) and centrifuged (13,000 × g for 20 min at 4 ◦ C). Supernatants were allowed for conjugation of GSH with 1-chloro-2,4-dinitrobenzene and the increasing absorbance at 340 nm was measured using a spectrophotometer at 25 ◦ C as the measure of GST activity (Regoli et al., 1997). GPx was measured using glutathione peroxidase (GPx) kit (Cat. no. CGP1, Sigma–Aldrich) following manufacturer’s protocol. Total protein content of the supernatant was determined to normalise ROS contents, GST and GPx activities using the Bradford method (Bradford, 1976). All results were expressed in terms of relative percentage of control to avoid a possible reduction in activity during experiments.

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2.5. Antibodies and western blot analysis BDE-47 induced activation of MAPK signaling pathway were evaluated using Western blot analysis. Polyclonal antibodies to phospho-ERK1/2 (anti-mouse, Thr-202/Tyr-204), and phosphop38 (anti-rabbit, Tyr-182) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Polyclonal antibodies to phospho-SAPK/JNK (anti-rabbit, Thr-183/Tyr-185), was obtained from Cell Signaling Technology (Beverly, MA, USA) and monoclonal antibody to ␤-actin (control) was obtained from Sigma. Using these antibodies phosphorylation patterns of extracellular signal-regulated kinase (ERK), c-Jun-N-terminal kinase (JNK), and p38 were analysed in P. nana by following the protocols from our previous study (Kim et al., 2016). Briefly, ∼500 adult P. nana were exposed to 10 ␮g/L BDE-47 for various time intervals (0, 6, 12, and 24 h), and were homogenized in lysis buffer (40 mM Tris–HCl (pH 8.0), 120 mM NaCl, 0.1% Nonidet-P40) containing protease inhibitor cocktail (Roche, South San Francisco, CA, USA) for total protein extraction. Total protein from the whole animal was separated by gel electrophoresis using 10% sodium dodecyl sulfate-polyacrylamide gel and was transferred to a nitrocellulose membrane (Amersham; Arlington Heights, IL, USA), blocked with 2.5% bovine serum albumin (BSA) in Tris-buffered saline. Blocked proteins were allowed to react with primary antibodies under the humid condition for overnight at 4 ◦ C, treatment with secondary antibody (peroxidase-conjugate, 1:1000). The blots developed were visualised by following enhanced chemiluminescence procedures (Amersham; Arlington Heights, IL, USA) in manufacturer’s protocol. ␤-actin was used as the internal control. 2.6. Nile red fluorescence measurement for the detection of lipid droplets Effect of BDE-47 on DNL pathways and lipid accumulation in vivo was performed using Nile-red staining method for the detection of lipid droplets (LDs) (Greenspan et al., 1985). The experiments were performed with and without the presence of a lipogenesis inhibitor (salicylate) to confirm whether BDE-47 can induce lipogenesis in P. nana (Lee et al., 2016a, 2016b, 2016c). Briefly, P. nana was exposed to 10 ␮g/L BDE-47, 500 ␮M Salicylate, and in combination of 10 ␮g/L BDE-47 and 500 ␮M salicylate along with the control for 24 h. The groups without treatment and with 500 ␮M salicylate were considered as the controls. Fixation and staining were done by placing the specimens in a mixture of formaldehyde (4%) and the Nile red (final conc. 2.5 ␮g/ml) for 5 min. Subsequently, P. nana were viewed under a confocal laser scanning microscope (LSM 510 META; Zeiss, Oberkochen, Germany) at 543, and 560–615-nm excitation and emission wavelengths, respectively. Accumulated LDs in response to BDE-47 were compared with that of the control (Kim et al., 2016). Relative measurements (areas) of LDs were analysed with LAS image acquisition software (ver. 4.3; Leica, Wetzlar, Germany).

the FAMEs was performed in a gas chromatograph (GC-2010, Shimadzu, Kyoto, Japan) with a flame ionisation detector (FID) using a fused silica capillary column (DB-5, 30 m × 0.25 mm i.d., 0.25 ␮m film thickness). Helium was used as a carrier gas. Samples were injected in splitless mode at an initial oven temperature of 40 ◦ C, and raised to 200 ◦ C at 10 ◦ C/min and, finally, to 300 ◦ C at 2 ◦ C/min. FAs were identified from the retention times (RT) of standards and mass spectra from gas chromatograph-mass spectrometer (GCMSQP2010 Plus, Shimadzu, Kyoto, Japan). 2.8. Real-time reverse transcriptase-polymerase chain reaction Transcription profiles (mRNA) of DNL pathway genes in response to BDE-7 exposure (0, 0.1, 1, and 10 ␮g/L) were measured in P. nana over 24 h (6, 12, and 24 h) with the control. Total RNAs were extracted from each experimental group using TRIZOL® reagent (Invitrogen, Paisley, Scotland, UK) in accordance with manufacturer’s instructions. The spectrometric analysis was performed at 230, 260, and 280 nm to measure the quantity and quality of RNA (Ultrospec 2100pro, Amersham Bioscience, Freiburg, Germany). For reverse transcription and first-strand synthesis, two ␮g of total RNA and oligo (dT)20 primer were used and performed using a commercially available kit (SuperScriptTM II RT kit, Invitrogen, Carlsbad, CA, USA). Quantitative real-time polymerase chain reaction (qRTPCR) was conducted under the following conditions; 95 ◦ C/4 min; 35 cycles of 94 ◦ C/30 s, 58 ◦ C/30 s, 72 ◦ C/30 s; 72 ◦ C/10 min using SYBR Green as a probe (Molecular Probes Inc., Eugene, OR, USA) in a CFX96TM real-time PCR system (Bio-Rad, Hercules, CA, USA). To confirm the specific targets, melting curve cycles were run at the following conditions; 95 ◦ C/1 min; 55 ◦ C/1 min; 80 cycles of 55 ◦ C/10 s with a 0.5 ◦ C increase per cycle using RT-PCR F or R primers (Suppl. Table 1). Experiments were performed in triplicates and the mRNA expression levels between samples were normalised using P. nana 18S rRNA gene, as an internal control. The 2−DC T comparative method (Livak and Schmittgen, 2001) was used to calculate the relative fold changes of gene expressions and represented as heat map diagram. 2.9. Statistical analysis All data were expressed as the mean value with standard error (mean ± SE). The Student t-test or one-way analysis of variance (ANOVA) followed by Duncan’s new multiple range test (P <0.05) were performed to analyse the significant differences between experimental groups with the control. All the statistical analyses were performed using SPSS® software (SPSS Inc., Chicago, IL, USA) and differences were considered significant at P < 0.05. 3. Results 3.1. Effects of BDE-47 on post-embryonic development of P. nana

2.7. Fatty acid composition analysis BDE-47-induced effects on fatty acid (FA) compositions in P. nana were analysed by following the protocol of Hama and Handa (1987) and Lee et al. (2016a, 2016b, 2016c) with slight modifications. Briefly, lipids from BDE-47 (10 ␮g/L) exposed groups and the control groups were extracted with dichloromethane/methanol 2:1 (v/v). In all extracts, nonadecylic acid (C19:0) was added as an internal standard. Repeated extraction (thrice) was performed with sonication and the lipid fraction was separated from the watermethanol phase. The separated lipids were converted into fatty acid methyl esters (FAMEs) by saponification using 0.5 M KOHmethanol, followed by methylation with BF3-methanol. Analysis of

The effect of three different concentrations of BDE-47 (0.1, 1.0, and 10 ␮g/L) on P. nana was evaluated from nauplius (N) to copepodid (C) and adult (A) stages and compared with that of control. Except for early developmental stages (N1-C1), there were no significant differences observed between the test groups (Fig. 1). At concentration 10 ␮g/L, a significant reduction (P < 0.05) in developmental time were observed in P. nana, whereas these variations were not observed in N1-A stages. Overall, 10 ␮g/L BDE-47 affected the early post-embryonic stages, while the adaptation to this toxicity during the course of development from nauplius stage to adult stage were demonstrated, suggesting that BDE-47 did not affect the entire developmental time of P. nana.

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A) ROS DCF fluorescence (% control)

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3.2. Measurement of oxidative stress markers To evaluate the levels of oxidative stress markers, intracellular level of ROS, GST and GPx were measured in response to BDE-47 (0, 0.1, 1, and 10 ␮g/L) for 24 h. The intracellular level of ROS was significantly increased (P < 0.05) from the group exposed 0.1 ␮g/L to 10 ␮g/L (Fig. 2A). GST and GPx activities were significantly increased (P < 0.05) at all concentrations compared to control (Fig. 2B and C).

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Fig. 1. Effects of BDE-47 on the life cycle trait of P. nana. A) Developmental stages, B) Post-embryonic developmental time (∼12 days) from nauplius (N1) to copepodite (C1) and nauplius (N1) to adult (A) at various concentrations of BDE-47 (0.1, 1, and 10 ␮g/L) and the controls. Data are the mean ± SD of triplicates. Significant differences from control value are indicated by different letters on the data bar (P < 0.05) analysed by Duncan’s post hoc analysis. Modified from Hwang et al. (2010).

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Control 3.3. Activation of MAPK signaling pathways A time-dependent phosphorylation status of ERK, p38 and JNK were analysed to understand the effects on MAPK signaling pathways of P. nana exposed to 10 ␮g/L BDE-47 over 24 h (0, 6, 12, and 24 h). JNK, the apoptosis-related signal, was found activated at 6 and 12 h post-exposure, but reduced activities were observed at 24 h post exposure. Cell proliferation signaling pathway (e.g. pERK) was activated at 12 and 24 h, while no significant changes were observed in apoptosis-related signal p38 (Fig. 3), suggesting that BDE-47 induced activation of ERK and JNK pathways in MAPK pathways of P. nana.

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Concentration (µg/L) Fig. 2. Oxidative stress markers in P. nana exposed to different concentrations (0.1, 1, and 10 ␮g/L) of BDE-47 for 24 h. A) Reactive oxygen species (ROS) generation. B) Glutathione S-transferase (GST) activity. C) Glutathione peroxidase (GPx) activity. Different alphabetical letters describe significant differences (P < 0.05) in response to different concentrations of BDE-47 exposure after Duncan’s post hoc analysis. Data are the mean ± SD of triplicates.

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Fig. 3. Effects of BDE-47 on mitogen-activated protein kinase (MAPK) signaling pathways of P. nana. Western blot analysis showing time-dependent MAPK protein expression levels of P. nana in response to 10 ␮g/L BDE-47 over 24 h (0, 6, 12, and 24 h). P. nana ␤-actin protein was used as the control. ERK: extracellular signal-regulated kinase. JNK: c-Jun-N-terminal kinases. p38: p38 MAPK kinase.

3.4. Lipid accumulation in response to BDE-47 exposure Effects of BDE-47 (10 ␮g/L) on the accumulation of lipids were evaluated using the confocal microscopic images of Nile red-stained LDs (red fluorescence) (Fig. 4A). The entire areas of LDs in vivo were measured in P. nana exposed to BDE-47 and BDE–47 + SAL compared with that of the unexposed and salicylate (inhibitor) exposed groups (Fig. 4B). Relative area (%) of LDs was measured from the images by giving the lipid area of the control (unexposed) as 100%. The relative areas of LDs were significantly increased (P < 0.05) in P. nana exposed to BDE-47, whereas BDE-47 + SAL and the unexposed group showed a similar pattern, suggesting that BDE-47 exposure increased the accumulation of LDs in P. nana. 3.5. Effect of BDE-47 on fatty acid compositions The composition of saturated fatty acids (palmitic acid [C16:0], stearic acid [C18:0], arachidic acid [C20:0], docosanoic acid [C22:0]) and polyunsaturated fatty acids (PUFAs) such as ␻3PUFAs (eicosatrienoic acid [ETE; C20:3␻3], eicosapentaenoic acid [EPA; C20:5␻3], docosahexaenoic acid [DHA; C22:6␻3]), ␻6-PUFAs (linoleic acid [LA; C18:2␻6], gamma-linolenic acid [GLA; C18:3␻6], dihomo-gamma-linolenic acid [DGLA; C20:3␻6], arachidonic acid [AA; C20:4␻6], and ␻9-PUFAs (hexadecenoic acid [C16:1␻9], oleic acid [C18:1␻9]) were measured in P. nana in response to BDE47 exposure (10 ␮g/L) over 4 days (Fig. 5). Among SFAs, palmitic acid was significantly increased (P < 0.05) at days 1 compared with the control, whereas arachidic acid was decreased significantly (Fig. 5A). All types of PUFAs were significantly high (P < 0.05) in 24 h post-exposed group (Fig. 5B–D). EPA and DHA were the highly increased PUFAs among ␻3-PUFAs. However, almost all SFAs and

Fig. 4. Lipid accumulation in P. nana exposed to BDE-47 for 24 h. A) Confocal photomicrograph of Nile red-stained P. nana exposed to 10 ␮g/L BDE-47, 500 ␮M salicylate, and BDE-47 in combination with the lipogenesis inhibitor salicylate (BDE47 + SAL), and untreated copepods. The red fluorescence indicates the accumulated lipid droplets in vivo. B) The accumulation of lipid droplets in P. nana are represented as a percentage (%) of the relative area using LAS image analysis tool (n = 30). Significant differences from control value are indicated by different letters on the data bar (P < 0.05) analysed by Student’s t-test. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

PUFAs were significantly decreased in the 4th day compared to control. 3.6. Effect of BDE-47 on de novo lipogenesis pathway genes To evaluate the effects of BDE-47 on lipogenesis in P. nana, we measured the expression of transcsription factors (TFs) (EcR, SREBP and ChREBP), ACC, ACLY, KAS, elongases (EL01, EL02 and EL03), and desaturases (4-DES, 5-DES and 6-DES) in response to three concentrations (0.1, 1.0, and 10 ␮g/L) with a control over 24 h (Fig. 6). An illustration of lipogenesis pathway is given in Fig. 6A. All TFs were significantly activated (P < 0.05) with concentration and time-dependent manner with significantly elevated up-regulation (P < 0.05) at 10 ␮g/L for 24 h. Except for KAS gene, ACC and ACLY were elicited almost similar to TFs, suggesting a correlation between TFs and ACC and ACLY (Fig. 6B). 4. Discussion In this study, despite the oxidative stress, no lethal effects were observed in P. nana exposed to BDE-47 at the maximum dissolved concentration (10 ␮g/L). However, early post-embryonic stages (N1-C1) were affected but conformed to that of control during further development (N1-A) (Fig. 1). Recent studies in T. japonicus

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Fig. 5. Heat map diagrams showing a time-dependent variation of fatty acid compositions in P. nana exposed to 10 ␮g/L BDE-47 for 1, 2, and 4 days. A) Saturated fatty acids. B) n-3 (␻3) fatty acids. C) n-6 (␻6) fatty acids. D) n-9 (␻9) fatty acids. Relative concentrations to the control value are represented by the heat map diagrams. C16:0: palmitic acid, C18:0: stearic acid, C20:0: arachidic acid, C22:0: docosanoic acid C20:3␻3: eicosatrienoic acid (ETE), C20:5␻3: eicosapentaenoic acid (EPA), C22:6␻3: docosahexaenoic acid (DHA), C18:2␻6: linoleic acid (LA), C18:3␻6: gamma-linolenic acid (GLA), C20:3␻6: dihomo-gamma-linolenic acid (DGLA), C20:4␻6: arachidonic acid (AA), C16:1␻9: hexadecenoic acid, C18:1␻9: oleic acid.

were corroborating our observations that BDE-47 did not induce lethality despite the developmental retardation and moult inhibition (Han et al., 2015a; Lee et al., 2016a,b,c; Hwang et al., 2016). In T. japonicus, a developmental delay appeared in response to 120 ␮g/L BDE-47 (Han et al., 2015a), whereas in the copepod Nitocra spinipes, 13 ␮g/L BDE-47 induced developmental retardation (Breitholtz and Wollenberger, 2003). Thus, the toxicity of BDE-47 is highly species specific. BDE-47 exposure led to an elevated ROS and activated p-ERK pathways in P. nana. When the production ROS in living systems exceeds their capacity of the antioxidant mechanism (e.g. GST and GPx) is called under “oxidative stress”, an imbalance between oxidants and antioxidant capacity of the cell (Thannickal and Fanburg, 2000; Son et al., 2011). ROS-induced oxidative stress has identified as an important mechanism for BDE-47-mediated toxicity (Wang et al., 2012). As ROS plays an important role in triggering diverse reactions in living organisms that leads to cellular and physiological impairments, BDE-47 has been proved to induce a wide range of toxicities such as cytotoxicity, endocrine disrupting activity, neurotoxicity, and genotoxicity in animals (Valko et al., 2007; He et al., 2008, 2009; Lema et al., 2008; Wang et al., 2012). Similar to the findings on elicited ROS along with GST and GPx in P. nana (Fig. 2),

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BDE-47 evoked ROS levels in the copepod T. japonicus (Han et al., 2015a), mussel Mytilus galloprovincialis (Ji et al., 2013), and the fish O. mykiss (Shao et al., 2008) that led to cellular and physiological impairments. Thus, our observations in P. nana correlate with the above references and further support the concept of BDE-47induced oxidative stress mediates molecular signal cascades, that in turn, cause impairments in animals. Despite the oxidative stress-mediated injury, higher level of ROS activates signaling molecules acting as a “second messenger” in intracellular signaling pathways that regulate cell growth, proliferation, and apoptosis (Son et al., 2011). Though the exact molecular mechanism behind the activation of MAPK pathways is not known, in this study, phosphorylation of ERK and JNK were observed in response to BDE-47 exposure. Thus, BDE-47 induced activation of ERK and JNK in intracellular MAPKs signaling pathways through phosphorylation of MAP3Ks and MAP2Ks by the external stimuli of ROS in P. nana (Fig. 3). Similarly, in the copepods T. japonicus and P. nana, carbon nanotubes (CNTs) led to activation of ERK in MAPK pathways without the production of ROS (Lee et al., 2016a; Kim et al., 2016). In the rotifer Brachionus koreanus, CNTs induced phosphorylation of ERK in the MAPK pathways with ROS induction (Lee et al., 2016b). Also, both JNK and p38 MAPK were phosphorylated in response to 0.05 ␮m microbeads exposure in B. koreanus (Jeong et al., 2016). Taken together, MAPK pathways, irrespective of the phosphorylation of subgroups, are critical to assessing the functional and physiological status of the organism. Indeed, ERKmediated signal transduction in MAPK pathways is essential for mediating the adverse effects that occur in copepods in response to environmental stressors. Confocal microscopic images of Nile-red-stained P. nana revealed that 24 h exposure to BDE-47 (10 ␮g/L) resulted in the elevated accumulation of lipids in the form of LDs (Fig. 4). In T. japonicus, 2.5 ␮g/L BDE-47 exposure significantly increased LDs (Lee et al., 2016a, 2016b, 2016c). Normally, LDs are accumulated in copepods for energy storage and reproduction (Zarubin et al., 2014) and are an adaptive mechanism for maintaining cellular homoeostasis to overcome the expected physiological stress (Gao and Goodman, 2015) via increased turnover rate of the Krebs cycle (Goolish and Burton, 1989). LDs are known to maintain cellular homoeostasis by interacting with the nucleus, endoplasmic reticulum, mitochondria, peroxisomes, and vacuoles (Gao and Goodman, 2015; Barbosa et al., 2015). Despite the increased turnover rate of the Krebs cycle, the transcription factors (TF) and ACC, ACLY, and KAS responsible for DNL pathways were activated in P. nana (discussed below). Furthermore, the area of LDs in salicylate-treated group, an inhibitor of DNL pathway, was reduced compared to BDE47 exposed group. This indicates that BDE-47 led to expression of DNL pathway-related genes and, in turn, accumulation of lipids in P. nana. Apart from the increased level of lipid accumulation, compositional variations in fatty acids and their ratio were observed in P. nana after BDE-47 exposure (Fig. 5). The observed stress led to the elevation of total fatty acid within 24 h, especially the polyunsaturated fatty acids (PUFAs). However, saturated fatty acids (C:20; Arachidic acid) were less in proportion, in comparison with that of the control. In fact, the oxidative stress resulted in the turnover of PUFAs from saturated fatty acids (SFAs), as an early stress tolerance mechanism in copepods. Usually, under stress, copepods produce major ␻3-PUFAs such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from SFAs (Desvilettes et al., 1997; Nanton and Castell, 1998). The selective anabolism of the EPA and DHA suggests that P. nana carefully controls the composition of their lipids ratio to ensure the adaptiveness towards BDE-47 induced stress and to maximize the physiological fitness (Pond, 2012; Mayor et al., 2015). Our observation was in contradictory with the findings from T. japonicus that showed a reduction in arachidonic acid (ARA) dur-

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Fig. 6. A) Diagrammatic representation of the de novo lipogenesis (DNL) pathway in the copepod Paracyclopina nana. EcR: Ecdysone receptor, SREBP: Sterol regulatory element binding protein, ChREBP: Carbohydrate regulatory element binding protein, ACLY: ATP-citrate lyase, ACC: Acetyl-CoA carboxylase, KAS, ␤-ketoacyl-acyl-carrierprotein synthase, PA: Palmitic acid, SA: Stearic acid, OA: Oleic acid, LA: linoleic acid, GLA: ␥-linoleic acid, DGLA: dihomo-␥-linoleic acid, ARA: Arachidonic acid, ALA: ␣-linoleic acid, SDA: Stearidonic acid, ETE: Eicosatrienoic acid, ETA: Eicosatetraenoic acid, EPA: Eicosapentaenoic acid, DPA: Docosapentaenoic acid, DHA: Docosahexaenoic acid. B) Transcription profiles of transcription factors, fatty acid synthases, elongases and desaturases involved in DNL pathway genes in response to different concentrations (0, 1, and 10 ␮g/L) of BDE-47 exposure for 6, 12, and 24 h. P. nana 18S rRNA gene was used as an internal reference to normalize the mRNA expression levels (n = 3). Expression profiles were represented by a heat map. ELO: elongase. DES: desaturase.

ing exposure with BDE-47 for 24 h (Lee et al., 2016a,b,c), suggesting a species-specific distinction in the ratio of fatty acid composition in response to BDE-47, which may regulate the proportion of PUFAs as a mechanism to overcome BDE-47-induced stress. In transcription profiles of DNL pathway genes, BDE-47 induces activation of TFs and upregulation of lipogenesis in P. nana (Fig. 6). TFs (e.g. EcR, SREBP, and two isoforms of ChREBP) for lipogenesis in copepods were evoked in a concentration and time-dependent manner and reached a maximum at 24 h. Moreover, a similar trend was observed in fatty acid synthases ACLY and ACC. This could be correlated with the observed accumulation of lipids in vivo within 24 h post-exposure with BDE-47. These observations are inconsistent with the findings that BDE-47 leads to lipogenesis in the

copepod T. japonicus. Also, a similar observation was reported from various aquatic animals such whale (Dorneles et al., 2015), shellfishes (Munschy et al., 2015), and zooplankton (Peltonen et al., 2014). TFs play a central role in fatty acid synthesis in the DNL pathway by regulating FAs such as ACLY, ACC, and KAS (Strable and Ntambi, 2010; Lee et al., 2016a,b,c). Desaturases (e.g. 4-, 5-, 9desaturases) and elongases are critical in the production of PUFA in copepods through DNL pathways. The biosynthesis of PUFAs involves desaturation in aliphatic chain of a FA and the elongation acyl chain by the sequential action of desaturases and elongases (Uttaro, 2006). Although elongases and desaturases genes were not significantly changed in this study, the induced TFs and synthases (FAs) increased the total lipid content through DNL pathway. These

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results provide a better understanding of expression patterns of fatty acid biosynthesis related genes in response to the obesogen BDE-47, which in turn useful for predicting the effects of BDE-47 on aquatic invertebrates. Overall, in this study, we demonstrated that BDE-47 can induce oxidative stress (e.g. ROS)-mediated activation of ERK and JNK signaling cascades in MAPK pathways. Activated MAPK pathways, in turn, induce the signal molecules that bind to the TFs responsible for de novo lipogenesis (EcR, SREBP, ChREBP). Also, the stress led to the conversion of SFAs to PUFAs, a preparedness of the organism to adapt the observed stress. Taken together, all sequential events explained above led to delayed early post-embryonic development and increased accumulation of lipid droplets in P. nana. Furthermore, these results strengthen our understanding of the mechanistic and biochemical effects of BDE-47 pollution on marine invertebrates especially on the copepods, an important link in the aquatic food chain. Acknowledgements This work was supported by a grant of the Development of Techniques for Assessment and Management of Hazardous Chemicals in the Marine Environment of the Ministry of Oceans and Fisheries, Korea funded to Jae-Seong Lee. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aquatox.2016.10. 025. References Barbosa, A.D., Savage, D.B., Siniossoglou, S., 2015. Lipid droplet-organelle interactions: emerging roles in lipid metabolism. Curr. Opin. Cell Biol. 35, 91–97. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation ofmicro-gram quantities of protein utilizing the principle of protein–dyebinding. Anal. Biochem. 7, 248–254. Bramwell, L., Fernandes, A., Rose, M., Harrad, S., Pless-Mulloli, T., 2014. PBDEs and PBBs in human serum and breast milk from cohabiting UK couples. Chemosphere 116, 67–74. Breitholtz, M., Wollenberger, L., 2003. Effects of three PBDEs on development, reproduction and population growth rate of the harpacticoid copepod Nitocra spinipes. Aquat. Toxicol. 64, 85–96. Carlsson, G., Kulkarni, P., Larsson, P., Norrgren, L., 2007. Distribution of BDE-99 and effects on metamorphosis of BDE-99 and -47 after oral exposure in Xenopus tropicalis. Aquat. Toxicol. 84, 71–79. Chan, W.K., Chan, K.M., 2012. Disruption of the hypothalamic-pituitary-thyroid axis in zebrafish embryo-larvae following waterborne exposure to BDE-47, TBBPA and BPA. Aquat. Toxicol. 108, 106–111. Díaz-Jaramillo, M., Miglioranza, K.S.B., Gonzalez, M., Barón, E., Monserrat, J.M., Eljarrat, E., Barceló, D., 2016. Uptake, metabolism and sub-lethal effects of BDE-47 in two estuarine invertebrates with different trophic positions. Environ. Pollut. 213, 608–617. Dahms, H.-U., Won, E.-J., Kim, H.-S., Han, J., Jeong, C.-B., Park, H.G., Souissi, S., Raisuddin, S., Lee, J.-S., 2016. Potential of the small cyclopoid copepod Paracyclopina nana as an invertebrate model for ecotoxicity testing. Aquat. Toxicol. 180, 282–294. Desvilettes, C., Bourdier, G., Breton, J.C., 1997. On the occurrence of a possible bioconversion of linolenic acid into docosahexaenoic acid by the copepod Eucyclops serrulatus fed on microalgae. J. Plankton Res. 19, 273–278. Dorneles, P.R., Lailson-Brito, J., Secchi, E.R., Dirtu, A.C., Weijs, L., Dalla Rosa, L., Bassoi, M., Cunha, H.A., Azevedo, A.F., Covaci, A., 2015. Levels and profiles of chlorinated and brominated contaminants in Southern hemisphere humpback whales, Megaptera novaeangliae. Environ. Res. 138, 49–57. Gao, Q., Goodman, J.M., 2015. The lipid droplet-a well-connected organelle. Front. Cell. Dev. Biol. 3, 49. Goolish, E.M., Burton, R.S., 1989. Energetics of osmoregulation in an intertidal copepod: effects of anoxia and lipid reserves on the pattern of free amino acid accumulation. Funct. Ecol. 3, 81–89. Greenspan, P., Mayer, E.P., Fowler, S.D., 1985. Nile red: a selective fluorescent stain for intracellular lipid droplets. J. Cell. Biol. 100, 965–973. Hama, T., Handa, N., 1987. Pattern of organic matter production by natural phytoplankton population in a eutrophic lake: I. Intracellular products. Arch. Hydrobiol. 109, 107–120.

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