Adverse effects of methylmercury (MeHg) on life parameters, antioxidant systems, and MAPK signaling pathways in the rotifer Brachionus koreanus and the copepod Paracyclopina nana

Adverse effects of methylmercury (MeHg) on life parameters, antioxidant systems, and MAPK signaling pathways in the rotifer Brachionus koreanus and the copepod Paracyclopina nana

Accepted Manuscript Title: Adverse effects of methylmercury (MeHg) on life parameters, antioxidant systems, and MAPK signaling pathways in the rotifer...

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Accepted Manuscript Title: Adverse effects of methylmercury (MeHg) on life parameters, antioxidant systems, and MAPK signaling pathways in the rotifer Brachionus koreanus and the copepod Paracyclopina nana Authors: Young Hwan Lee, Duck-Hyun Kim, Hye-Min Kang, Minghua Wang, Chang-Bum Jeong, Jae-Seong Lee PII: DOI: Reference:

S0166-445X(17)30199-6 http://dx.doi.org/doi:10.1016/j.aquatox.2017.07.006 AQTOX 4700

To appear in:

Aquatic Toxicology

Received date: Revised date: Accepted date:

31-5-2017 6-7-2017 10-7-2017

Please cite this article as: Lee, Young Hwan, Kim, Duck-Hyun, Kang, Hye-Min, Wang, Minghua, Jeong, Chang-Bum, Lee, Jae-Seong, Adverse effects of methylmercury (MeHg) on life parameters, antioxidant systems, and MAPK signaling pathways in the rotifer Brachionus koreanus and the copepod Paracyclopina nana.Aquatic Toxicology http://dx.doi.org/10.1016/j.aquatox.2017.07.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

-revised

Adverse effects of methylmercury (MeHg) on life parameters, antioxidant systems, and MAPK signaling pathways in the rotifer Brachionus koreanus and the copepod Paracyclopina nana Young Hwan Leea, Duck-Hyun Kima, Hye-Min Kanga, Minghua Wangb, Chang-Bum Jeonga, and Jae-Seong Leea,* a

Department of Biological Science, College of Science, Sungkyunkwan University, Suwon

16419, South Korea b

Key Laboratory of the Ministry of Education for Coastal and Wetland Ecosystems, College

of the Environment & Ecology and State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen 361102, China

____________________________ *

Corresponding author. E-mail: [email protected] (J.-S. Lee)

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Highlights

> MeHg retarded growth in a dose-dependent manner and increased ROS levels in a dosedependent manner in both organisms.

> Antioxidant enzymatic activities revealed different tendencies between P. nana and B. koreanus.

> In B. koreanus, the level of p-ERK was decreased at 1,000 ng/L MeHg, while the levels of p-ERK and p-p38 in P. nana were reduced at 10 ng/L MeHg.

> Antioxidant system in response to MeHg was associated with negative effects on life parameters (e.g. reduced fecundity and survival rate).

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Abstract

To evaluate the adverse effects of MeHg on the rotifer Brachionus koreanus and the copepod Paracyclopina nana, we assessed the effects of MeHg toxicity on life parameters (e.g. growth retardation and fecundity), antioxidant systems, and mitogen-activated protein kinase (MAPK) signaling pathways at various concentrations (1 ng/L, 10 ng/L, 100 ng/L, 500 ng/L, and 1,000 ng/L). MeHg exposure resulted in the growth retardation with the increased ROS levels but decreased glutathione (GSH) levels in a dose-dependent manner in both B. koreanus and P. nana. Antioxidant enzymatic activities (e.g. glutathione S-transferase [GST], glutathione reductase [GR], and glutathione peroxidase [GPx]) in B. koreanus showed more positive responses compared the control but in P. nana, those antioxidant enzymatic activities showed subtle changes due to different no observed effect concentration (NOEC) values among the two species. Expression of antioxidant genes (e.g. superoxide dismutase [SOD], GSTs, glutathione peroxidase [GPx], and catalase [CAT]) also demonstrated similar effects as shown in antioxidant enzymatic activities. In B. koreanus, the level of p-ERK was decreased in the presence of 1,000 ng/L MeHg, while the levels of p-ERK and p-p38 in P. nana were reduced in the presence of 10 ng/L MeHg. However, p-JNK levels were not altered by MeHg in B. koreanus and P. nana, compared to the corresponding controls. In summary, life parameters (e.g. reduced fecundity and survival rate) were closely associated with effects on the antioxidant system in response to MeHg. These observations provide a better understanding on the adverse effects of MeHg on in vivo life parameters and molecular defense mechanisms in B. koreanus and P. nana.

Keywords: MeHg, methyl mercury, reactive oxygen species, rotifer, Brachionus koreanus, copepod, Paracyclopina nana, antioxidant system, MAPK signaling pathway

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1. Introduction

Mercury (Hg) is a widespread toxic environmental contaminant (U.S. EPA, 1997). Mercury is a naturally occurring shiny silver-white odorless liquid metal that is not easily destroyed and is eventually released into the atmosphere or water. In oceans, mercury meets various fates in the forms of inorganic salts, metallic elements, and organic compounds. Methylmercury (MeHg) is the predominant organic form in the ocean and is produced by sulfate-reducing bacteria from precipitated mercury sulfate compounds (Gochfield, 2003). Of all the forms of Hg, MeHg is of the greatest concern due to its potent neurotoxicity (Salonen et al., 1995; Grandjean et al., 1997; Sorensen et al., 1999). The main target for MeHg toxicity is the central nervous system (CNS), where it alters the biochemical and ultrastructural machinery of both astrocytes and neurons (Aschner et al., 2000; Shanker et al., 2003). The generation of reactive oxygen species (ROS) is a major factor in MeHg toxicity (Ou et al., 1999). For example, in the fruit fly Drosophila melanogaster, dietary MeHg was shown to induce intracellular ROS and increase lipid peroxidation in a dose-dependent manner (Chauhan and Chauhan, 2016). In the nematode Caenorhabditis elegans, ROS were induced in response to an 8 h treatment with 25 μM MeHg (Settivari et al., 2009). Oxidative stress induced by MeHg is associated with interaction with intracellular thiols (e.g. glutathione [GSH]) (Shanker et al., 2005; Kaur et al., 2006). Moreover, mercury disrupts the homeostasis of intracellular calcium ions by increasing the permeability of the plasma membrane, leading to calcium ion influx from calcium ion pools (Carratù and Signorile, 2015; Farina et al., 2013). Although marine invertebrates have great ecological importance and their bioaccumulation of MeHg is of interest, only a few studies have investigated the effects of MeHg on these animals. Marine invertebrates occupy a core trophic level in the aquatic ecosystem by transferring energy from producers to consumers among invertebrates (Snell and Janssen, 1995; Raisuddin et al., 2007; Dahms et al., 2011). Thus, zooplankton communities, which serve as the main food for larvae and juvenile fishes, play a key role in aquatic ecosystems by feeding on microalgae and particulate organic matter, including MeHg released by bacteria. Moreover, these organisms are frequently employed in ecotoxicologic assays, as they are one of the most sensitive groups to the effects of toxic chemical products and occupy a central position in the lentic food chain (Hanazato al., 2001). Among the zooplanktons, many efforts with the rotifer and the copepod have been made to establish as a model species for ecotoxicological study due to their advantages for laboratory 4

studies including small size (~150 μm for B. koreanus and ~600 μm for P. nana), short generation cycle (~24 h for rotifer and ~2 weeks for copepod), easy laboratory maintenance, simple structure, and high fecundity (Raisuddin et al., 2007; Dahms et al., 2011; Dahms et al., 2016; Kang et al., 2017). Thus, in the present study, two marine invertebrates, the rotifer Brachionus koreanus and the copepod Paracyclopina nana, were used as an experimental species for a better understanding of adverse effects of MeHg in the marine invertebrates. Here, we present the adverse effects of MeHg on life parameters of copepod and rotifer with further investigation of mitogen-activated protein kinases (MAPKs) signaling transduction that could provide a clue for a better understanding of MeHg toxicity on marine invertebrates. Furthermore, to find out the defense mechanism against MeHg, MeHg-induced oxidative stress and its relevant antioxidant enzymatic activities were measured.

2. Materials and methods

2.1. Culture and maintenance The monogonont rotifer B. koreanus was collected at Uljin (36°58′43.01″ N, 129°24′28.40″ E), South Korea. A single individual rotifer was isolated, reared, and maintained in filtered artificial seawater (ASW) (TetraMarine Salt Pro, Cincinnati, OH, USA). Rotifers were cultured at 25°C under a light: dark 12:12 h photoperiod with 15 practical salinity units (psu). The green microalgae Tetraselmis suecica were used as a live diet (approximately 6×104 cells/mL). Rotifer density was monitored daily under a stereomicroscope (Olympus IX71; Olympus Corp., Tokyo, Japan) and organisms were subcultured during the sigmoidal growth phase. Species identification was confirmed via morphological analysis and sequencing of the mitochondrial DNA cytochrome oxidase 1 (CO1) gene, which served as the barcoding gene for each animal (Hwang et al., 2013; Hwang et al., 2014). The copepod P. nana was maintained under controlled incubator conditions with a 12 h light/12 h dark cycle at a temperature of 25°C. The salinity of the culture medium was 15 psu with a pH of 8.0. P. nana was fed with T. suecica (~ 6×104 cells/ml) once per day, as a dietary source. The species identity of P. nana was verified by morphological characteristics and sequence analysis of the mitochondrial DNA CO1 gene (Ki et al., 2009).

2.2. Acute toxicity tests

The no observed effect concentration (NOEC) and half-lethal concentration (LC50) at 48 h 5

were determined by exposing organisms to MeHg. Methyl mercury chloride (CH3HgCl, analytical grade, molecular weight 251.08, purity > 99.8%; cat. no. 33368) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Acute toxicity testing of methylmercury was performed using B. koreanus neonates (>3 h old) over a period of 24 h. In brief, one B. koreanus neonate was placed each well of a 12well culture plate (SPL Life Sciences, Seoul, South Korea) filled with 4 mL ASW including MeHg (0, 1 [3.983 pM], 10 [39.828 pM], 100 [0.398 nM], 500 [1.991 nM], and 1,000 ng/L [3.983 nM]). Each condition was assessed in triplicate. Toxicity was assessed after examining mortality under a stereomicroscope (M205A, Leica Microsystems Ltd., Wetzlar, Germany). LC50 values were calculated using Probit analysis (ToxRat Ver.2.09, Alsdorf, Germany, GmbH, 2005). The NOEC was determined by Dunnett's test. Ten P. nana adults were exposed to 4 mL ASW containing different concentrations of MeHg (0, 1 [3.983 pM], 10 [39.828 pM], 100 [0.398 nM], 500 [1.991 nM], and 1,000 ng/L [3.983 nM]) for 24 h at 25°C. Each treatment was performed in triplicate in 12-well culture plates. During the experiments, the animals were not fed. Mortality was measured by inspection under a stereomicroscope (SZX-ILLK200, Olympus Corporation) after 24 h. A copepod was considered dead when it showed no movement. Finally, the NOEC, 10% lethal concentration (LC10), and LC50 values were calculated using Probit analysis (ToxRat Ver.2.09).

2.3. Reproduction rate

To determine the effects of different doses of MeHg on the reproduction of B. koreanus and T. japonicus, rotifers and copepods were exposed to various concentrations of MeHg (0, 1, 10, 100, 500, and 1,000 ng/L). Rotifer embryos were collected from a pool of matured rotifers and incubated in ASW for 3 h. Neonates were collected and separated individually into 12-well polystyrene plates and the numbers of newborn rotifers were counted every 12 h as a readout of fecundity over an 8-day period. During the experiment, the 50% test solution was renewed every 24 h with the green algae T. suecica (approximately ~6×104 cells/mL) as a live diet once every 24 h. In the case of P. nana, 10 individual ovigerous females that were grown in different concentration of MeHg exposure (0, 1, 10, 100, 500, and 1,000 ng/L) were transferred to 12well culture plates containing different concentration of MeHg. Fecundity was measured by counting newborn nauplii every 24 h. During the experiments, half of the testing medium was renewed daily with green microalgae T. suecica (~6×104 cells/ml) to maintain the copepods. 6

All experiments were performed in triplicate; temperature was maintained at 25°C.

2.4. Measurement of ROS and GSH levels

To determine whether MeHg induces oxidative stress in the rotifer B. koreanus and/or the copepod P. nana, intracellular ROS levels were measured after MeHg exposure (0, 1, 10, 100, 500, and 1,000 ng/L) using 2’7’-dichlorodihydrofluorescein diacetate (H2DCFDA; Molecular Probes, Eugene, OR, USA). This dye is oxidized to fluorescent dichlorofluorescein (DCF) by intracellular ROS. After MeHg exposure for 24 h, B. koreanus (about 7000 individuals 250 mL) and P. nana (approximately 500 individuals in 250 mL) were homogenized in a buffer containing 0.32 M sucrose, 20 mM HEPES, 1 mM MgCl2, and 0.5 mM PMSF (pH 7.4) using a Teflon homogenizer. The homogenate was then spun by centrifugation at 10,000 × g for 20 min at 4°C, after which the supernatant was collected for measurements. Black 96-well plates (SPL Life Sciences) were filled with phosphate-buffered saline (PBS), probe (H2DCFDA at a final concentration of 40 µM), and the supernatant fraction to a final volume of 200 µL/well. Measurements were obtained at an excitation wavelength of 485 nm and an emission wavelength of 520 nm (ThermoTM Varioskan Flash plate reader, Thermo Electron, Vantaa, Finland). All ROS measurements were normalized to total protein levels and are presented as percent DCF fluorescence relative to control wells. Total protein contents were determined by the Bradford method (Bradford, 1976). Glutathione (GSH) concentrations were determined enzymatically using a GSH assay kit (Sigma-Aldrich). GSH content was measured at an absorbance of 420 nm with a spectrophotometer (ThermoTM Varioskan Flash); standard curves were generated using GSH equivalents (0, 150, and 350 µM).

2.5. Measurement of GSH-related enzymatic activities

After exposure to different concentrations (0, 1, 10, 100, 500, and 1,000 ng/L) of MeHg for 24 h, B. koreanus (about 7000 individuals) and P. nana (approximately 500 individuals) were homogenized in cold buffer (50 mM Tris–HCl, 5 mM EDTA, and 1 mM 2mercaptoethanol, pH 7.5) at a ratio of 1–4 (w/v) with a Teflon homogenizer. The homogenates were centrifuged at 10,000 x g for 10 min at 4°C. The upper aqueous layer containing the enzyme was collected for enzymatic assay according to the manufacturer’s protocol. The glutathione reductase (GR) and glutathione peroxidase (GPx) activity was measured 7

via an enzymatic method using GR and GPx cellular activity assay kits (Sigma-Aldrich), respectively, at an absorbance of 340 nm with a spectrophotometer (ThermoTM Varioskan Flash). In the case of GST, enzymatic activity was measured using 1-chloro-2,4dinitrobenzene (CDNB) as a substrate. The enzymatic assay was monitored by examining the conjugation of CDNB and GSH at 340 nm with a spectrophotometer at 25°C. The total SOD activity was measured by using SOD assay kit (Sigma-Aldrich) at an absorbance level of 440 nm using a spectrophotometer (ThermoTM Varioskan Flash). Enzymatic activity was normalized by total protein and presented as % of control. Total protein was determined using the Bradford method (Bradford, 1976). All the experiments were performed in technical triplicate at 25°C.

2.6. Total RNA extraction and single-stranded cDNA synthesis

Rotifers (approximately 7000 individuals) and copepods (approximately 500 individuals) were homogenized in three volumes of TRIzol® reagent (Invitrogen, Carlsbad, CA, USA) with a tissue grinder. Homogenates were stored at −80°C until use. Total RNA was isolated from tissues according to the manufacturer's instructions. Genomic DNA was removed using DNase I (Sigma-Aldrich). Total RNA was quantified by measuring the absorption of light at A260; the quality of all samples was checked by analyzing the A230/260 and A260/280 ratios using a spectrophotometer (QIAxpert, Qiagen, Hilden, Germany). To check for genomic DNA contamination, total RNA was resolved on a 1% agarose gel that contained ethidium bromide (EtBr). Bands in the gel were visualized with a UV transilluminator (Wealtec Corp., Sparks, NV, USA). To verify total RNA quality, total RNA was analyzed on EtBr-stained 1% formaldehyde/agarose gels. The integrity and band ratio of 18/28S ribosomal RNAs (rRNAs) were analyzed. Single-stranded cDNA was synthesized from total RNA using an oligo(dT)20 primer for reverse transcription (SuperScriptTM III RT Kit, Invitrogen).

2.7. Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR)

In B. koreanus, the mRNA expression levels of GST1, GST2, GST3, GST4, GST5, GST6, GST7, GST8, GPx, catalase (CAT), CuZn-SOD, and Mn-SOD in response to MeHg exposure were examined by qRT-PCR. Organisms were exposed to different concentrations (0, 1, 10, 100, 500, and 1,000 ng/L) of MeHg for 24 h. In P. nana, the mRNA expression levels of GSTO, GST-Z, GST-K, SeGPx, PhGPx, GR, CAT, CuZn-SOD, and Mn-SOD were measured. All real-time qRT-PCR experiments were carried out in unskirted low 96-well clear plates (Bio8

Rad, Hercules, CA, USA). Reaction conditions for detecting specific PCR products were as follows: 94°C/4 min; 35 cycles of 94°C/30 s, 55°C/30 s, 72°C/30 s; and 72°C/10 min. To confirm the amplification of specific products, melting curve analysis was performed as follows: 95°C/1 min, 55°C/1 min, and 80 cycles beginning at 55°C/10 s with a 0.5°C increase per cycle. SYBR Green (Invitrogen) was used to detect specific amplified products. Amplification and detection of SYBR Green-labeled products were performed using a CFX96 real-time PCR system (Bio-Rad). To normalize expression levels between samples, data from each experiment were expressed relative to the expression level of the 18S rRNA gene. The fold change in relative gene expression was calculated by the 2−ΔΔCT method (Livak and Schmittgen, 2001).

2.8. Antibodies

Polyclonal antibodies to phospho-Stress-activated protein kinases (SAPK)/Jun aminoterminal kinases (JNK) (SAPK/JNK) (anti-rabbit, Thr-183/Tyr-185), extracellular signalregulated kinase (ERK) (anti-rabbit), and JNK (anti-rabbit) were obtained from Cell Signaling Technology (Beverly, MA, USA). Polyclonal antibodies to p38 mitogen-activated protein kinase (MAPK) (anti-rabbit), phospho-ERK1/2 (anti-mouse, Thr-202/Tyr-204), and phospho-p38 (anti-rabbit, Tyr-182) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The monoclonal antibody to tubulin was obtained from Sigma-Aldrich. Horseradish peroxidase (HRP)-linked anti-rabbit and anti-mouse antibodies were purchased from Cell Signaling Technology.

2.9. Western blot analysis

B. koreanus (approximately 6000 individuals) and P. nana (approximately 500 individuals) were exposed to different concentrations of MeHg (0, 1, 10, 100, 500, and 1,000 ng/L) for 24 h. Whole animals were homogenized in lysis buffer (40 mM Tris-HCl [pH 8.0], 120 mM NaCl, 0.1% Nonidet-P40) containing a 1×EDTA-free complete protease cocktail (Roche, South San Francisco, CA, USA) to yield total protein extracts. Proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10% gels and were then transferred to a nitrocellulose membrane (Amersham, Arlington Heights, IL, USA). The membrane was blocked with 2.5% bovine serum albumin (BSA) in Tris-buffered saline and incubated with primary antibodies (1:1,000) overnight at 4°C. The blots were next incubated with a peroxidase-conjugated secondary antibody (1:10,000), after which 9

immunoreactive bands were visualized using an enhanced chemiluminescence procedure (Amersham) according to the manufacturer’s protocol.

2.10. Statistical analysis Data are expressed as means ± SDs. Significant differences were analyzed using one-way ANOVA followed by Tukey's test. P<0.05 was considered significant. The SPSS ver. 17.0 software package (SPSS Inc., Chicago, IL, USA) was used for statistical analysis.

3. Results

3.1. In vivo effects of MeHg exposure

The estimated NOEC, LC10, and LC50 values for B. koreanus at 24 h exposure were 1.5, 1.946, and 2.964 μg/L MeHg, respectively (Fig. 1A). In P. nana, the estimated NOEC, LC10, and LC50 values at 24 h exposure were 4.0, 7.804, and 11.502 μg/L MeHg, respectively (Fig. 1B). The fecundity of B. koreanus was decreased by MeHg exposure in the concentrationdependent manner, particularly at 500 and 1,000 ng/L (Fig. 2A). Fecundity of P. nana was significantly decreased (P<0.05) in a concentration-dependent manner (Fig. 2B). Fecundity could not be measured at 1,000 ng/L MeHg since no reproduction was observed.

3.2. ROS and GSH levels

The intracellular ROS level of B. koreanus was significantly increased (P<0.05) in response to MeHg in a concentration-dependent manner (Fig. 3A), whereas the GSH level was decreased in the presence of 500 and 1000 ng/L MeHg (Fig. 3B). However, in P. nana, the intracellular ROS level was increased (P<0.05) in response to MeHg in a concentrationdependent manner (Fig. 3A). In contrast, only a slight variation in the GSH level in P. nana was observed (except in the presence of 100 ng/L MeHg) (Fig. 3B).

3.3. Enzymatic activities of GPx, GR, GST, and SOD

In B. koreanus, the antioxidant activity of GPx was decreased in response to MeHg at concentrations of 1 ng/L and 10 ng/L. However, GR activity was significantly increased 10

(P<0.05) in response to 100 ng/L and 500 ng/L MeHg. GST activity was slightly increased in response to 1 to 500 ng/L MeHg; this increase was significant (P<0.05) in the presence of 1,000 ng/L MeHg (Fig. 4A). In P. nana, the antioxidant activities of GPx and GR were significantly increased (P < 0.05) at 100 ng/L MeHg but recovered to basal levels in response to 500 ng/L MeHg. GST activity showed a significant increase (P<0.05) in response to 1 to 100 ng/L MeHg for 24 h, similar to that of GPx (Fig. 4B).

3.4. Transcription levels of antioxidant-related genes

In B. koreanus, most antioxidant-related genes were up-regulated (P<0.05) at high concentrations of MeHg. In contrast, Cu/Zn-SOD was down-regulated (P<0.05) and its expression showed a negative correlation with MeHg concentration (Fig. 5A). In P. nana, the transcription levels of most antioxidant-related genes were not significantly changed (P < 0.05) (Fig. 5B).

3.5. Phosphorylation status of MAPKs

To examine the activation status of MAPK signaling pathways in B. koreanus and P. nana in response to MeHg, Western blotting was performed. In B. koreanus, the level of p-ERK was decreased after exposure to 1,000 ng/L MeHg (Fig. 6), while the levels of p-ERK and pp38 in P. nana were reduced after exposure to 10 ng/L MeHg. Similar effects were observed for 100, 500, and 1,000 ng/L MeHg exposure. However, p-JNK levels were not affected by MeHg in either B. koreanus or P. nana, compared to their corresponding control groups.

4. Discussion

Acute toxicity and reduced fecundity were observed in B. koreanus rotifers and P. nana copepods after exposure to MeHg. In particular, B. koreanus and P. nana were more sensitive to MeHg than other heavy metals, as evidenced by their low LC50 values; 2.964 μg/L MeHg for B. koreanus and 11.502 μg/L MeHg for P. nana. Metal ions have long been considered very harmful to zoobenthos and zooplankton, even at low concentrations (FernandesLeborans and Novillo, 1995; Monteiro et al., 1995). Particularly, mercury has been shown to persist longer and accumulate more rapidly than other metals (Kerrison et al., 1988), thereby exhibits higher toxicity in invertebrate (Table 1). For example, MeHg has shown 11

approximately 1,000 fold higher toxicity in the copepod T. japonicus compared to other metals (Lee et al., 2017). In the present study, B. koreanus and P. nana also have exhibited much higher sensitivity to MeHg compared to other metals, suggesting that the toxicity of MeHg is particularly distinctive compared to the toxicity of the other metals. In fact, MeHg is lipophilic and highly permeable, resulting in cellular damages and biochemical alterations, as MeHg is able to inhibit enzymatic activities by forming a MeHg-thiol complex (Hastings et al., 1975; Lakowicz and Anderson, 1980). MeHg reacts easily with cysteine to form a conjugate similar to methionine; this conjugate can be taken up by cells through the L-neural amino acid carrier system (Fujiyama et al., 1994). Thus, MeHg is likely to bioaccumulate in marine invertebrates. Consistent with the acute toxicity of MeHg, exposure of B. koreanus and P. nana to MeHg resulted in a deleterious effect on their cumulative offspring. In particular, both organisms had significantly fewer brooding events at 500 ng/L and 1,000 ng/L MeHg. Although the LC50-24 h value (11.502 μg/L MeHg) of P. nana at 24 h exposure was higher than that of B. koreanus (2.962 μg/L MeHg), no P. nana brooding events were detected at 1,000 ng/L MeHg. In

crustaceans,

exposure

to

metals

can

disrupt

biological

processes

including

molting/metamorphosis and reproduction (Defur et al., 1999). For example, exposure to cadmium and chromium can alter the reproductive cycle of the common littoral crab Carcinus maenas, while combinational exposure to these two metals was shown to disrupt the reproductive cycle to an even greater extent. Furthermore, in the three fiddler crabs Uca pugilator, U. pugnax, and U. minax, MeHg exposure inhibited ecdysis and molting behavior (Weis, 1978). Strikingly, growth retardation was found in the early developmental stages (i.e., nauplius to copepodid) of the copepod T. japonicus in response to 1 ng/L MeHg (Lee et al., 2017), even though copepods can tolerate the highest heavy metal concentrations of all zooplanktons (Lee et al., 2007). To investigate the toxicity mechanism of MeHg, oxidative stress induced by MeHg was measured. In MeHg-exposed B. koreanus and P. nana, the intracellular ROS levels were significantly increased in a dose-dependent manner. In general, exposure to metals increases ROS levels and drives subsequent oxidative stress and DNA damage (Tamae, 2010). For example, cadmium-induced cytotoxicity has been implicated in ROS generation due to the depletion of glutathione and protein-bound sulfhydryl groups (Hart et al., 1999; Rikans and Yamano, 2000; Stohs et al., 2000). Copper neurotoxicity has been attributed to mitochondrial toxicity, since copper addition to neuroblastoma cells was shown to markedly stimulate mitochondrial ROS production and to inhibit pyruvate dehydrogenase activity in the mitochondrial Krebs cycle (Mehta et al., 2006). Oxidative stress and ROS production 12

comprise the main mechanism by which MeHg exerts its toxic effects on cells and tissues. For example, MeHg has been shown to increase ROS production in the rodent cerebellum (i.e. isolated rat brain synaptosomes), in cerebellar neuronal cultures (e.g. hypothalamic neuronal cell lines), and in mixed reaggregating cell cultures (Sarafian et al., 1994; Park et al., 1996). Thus, ROS have an important role in initiating and catalyzing diverse radical reactions in living systems (Valko et al., 2007). ROS can also attack various macromolecules (e.g. DNA, lipids, and proteins), leading to such as cellular aging, mutagenesis, and carcinogenesis (Gniadecki et al., 2000). Taken together, MeHg-induced oxidative stress significantly affects the defense mechanisms of rotifers and copepods, providing a potential explanation for the in vivo toxicity of MeHg to marine invertebrates. In B. koreanus, the GSH level was significantly decreased after exposure to 500 and 1,000 ng/L MeHg, implying the formation of a MeHg-GSH (GS-Hg-CH3) complex by interaction of MeHg with the thiol group of GSH, reducing the overall GSH level (Ballatori and Clarkson, 1982). One of the major ROS-mediated mechanisms underlying MeHg-induced toxicity is GSH depletion. The GSH-redox system is an important antioxidant defense for protecting cells against oxidative damage and is also important for detoxification (Griffith et al., 1999). Intracellular GSH content has also been shown to significantly affect anticancer drug-induced apoptosis, indicating that apoptotic effects are inversely related to GSH content (Estrela et al., 2006; Higuchi et al., 2004). Thus, a shift in the cellular GSH-to-GSSG redox balance towards the oxidized species (GSSG) constitutes an important signal that could decide the fate of a cell (Pias et al., 2002). Thus, in B. koreanus, the decreased GSH level is likely linked to dysfunction of the GSH-related defense system, resulting in increased oxidative damage. In contrast to B. koreanus, the GSH level of P. nana was not significantly changed and GSH homeostasis was maintained. Taken together, this difference may explain why rotifers are more sensitive to MeHg-induced oxidative stress than copepods. In MeHg-exposed copepods and rotifers, the antioxidant activities of GST, GR, and GPx were generally increased compared to their levels in the corresponding controls. These GSHrelated enzymes play an important role in the detoxification of byproducts of oxygen utilization via cytochrome P450 (Ketterer, 1983). In the copepod T. japonicus, the antioxidant activities of GPx, GR, and GST were shown to be sensitive to MeHg (Lee et al., 2017). In the present study, in B. koreanus, GR and GST activities were increased in the presence of 10 ng/L MeHg, whereas GPx activity was not; in P. nana, the antioxidant activities were increased in the presence of up to 100 ng/L MeHg and then decreased in the presence of 500 and 1,000 ng/L MeHg. Taken together, these data suggest that the antioxidant-related activities of GST, GR, and GPx are directly related to a defense mechanism against MeHg13

induced oxidative stress. We also analyzed the transcription levels of antioxidant genes in B. koreanus and P. nana. The expression patterns of antioxidant enzymes differed between B. koreanus and P. nana. Specifically, in B. koreanus, the mRNA expression level of GSTs, GPx, and CAT were upregulated by MeHg exposure in the concentration-dependent manner. However, in P. nana, no obvious transcriptional changes were observed. In B. koreanus, Cu/Zn-SOD and Mn-SOD showed different patterns of transcriptional regulation. Specifically, Cu/Zn-SOD was downregulated in all MeHg exposure groups, whereas no significant changes were observed in MnSOD expression. These findings are consistent with previous studies. For example, in the polychaete Neanthes succinea, mRNA expression of Mn-SOD was slightly induced in response to copper exposure, in contrast to Cu/Zn-SOD (Rhee et al., 2011). In the freshwater rotifer B. calyciflorus, Cu/Zn-SOD and Mn-SOD exhibited different expression patterns in response to H2O2 (Yang et al., 2013). In the copepod T. japonicus, Mn-SOD mRNA expression was not induced as strongly as Cu/Zn-SOD by MeHg exposure (Lee et al., 2017). Thus, our findings suggest that the expression of Bk-Cu/Zn-SOD and Bk-Mn-SOD may be regulated differently. Particularly, in B. koreanus, most of the GSTs were up-regulated by MeHg exposure in the concentration-dependent manner; these enzymes were the most sensitive to MeHg exposure. In aquatic invertebrates, the mRNA expression levels of antioxidant-related genes, including GSTs, are generally up-regulated. Therefore, GSTs are potential biomarkers for oxidative stress in response to environmental pollutants such as metals (Hyne and Maher, 2003; Jemec et al., 2010; Rhee et al., 2013; Han et al., 2013). Interestingly, in P. nana, most of the antioxidant genes analyzed showed no significant transcriptional changes in response to MeHg, even though they exhibited increased antioxidant activities. Thus, the correlation between mRNA level and antioxidant activity in P. nana seems to be poor. Previous studies in the frog Xenopus laevis have shown similar results under dehydration stress. Specifically, CAT protein levels were significantly increased, although the mRNA levels did not increase in skeletal muscle (Malik et al., 2014). In MeHgexposed T. japonicus, GST and SOD transcription levels and antioxidant activities had the same tendencies at high concentrations of MeHg (Lee et al., 2017). In the present study, we found no correlation between antioxidant-related mRNA expression and enzymatic activity in MeHg-exposed P. nana, suggesting that the level of mRNA expression does not directly reflect the bioactivity of the corresponding protein. Taken together, our data indicate that antioxidant genes exhibit bell-type transcriptional patterns in B. koreanus, which indicate higher sensitivity to MeHg compared to P. nana. This transcriptional modulation can serve as a good indicator for MeHg-induced oxidative stress, although the expression patterns of 14

antioxidant genes in response to MeHg may vary in different marine organisms. In our study, the activation status of MAPKs was negatively correlated with MeHg concentration, showing decreases in the levels of p-ERK in B. koreanus and of p-ERK and pp38 in P. nana in response to MeHg exposure. This is likely resulted by the generation of oxidative stress by MeHg exposure, as MAPK pathways are known to respond to oxidative stress with higher sensitivity, resulting in the up-stream signaling transduction leading to activation of transcription factors that are involved in many biological processes in terms of cell survival and death (Matsuzawa and Ichijo, 2008; McCubrey et al., 2007; Wada et al., 2004). In particular, ERK signaling pathway is known to be related with cell survival and proliferation (McCubrey et al., 2007). In previous studies, the decreased p-ERK and p-JNK levels (but increased p-p38 levels) were associated with growth retardation and the reduced fecundity in the copepod T. japonicus (Lee et al., 2017). Moreover, in the parasite Schistosoma mansoni, decreased reproduction and worm pairing were observed after treatment with an ERK inhibitor (Ressurreição et al., 2014), indicating that ERK signaling pathway is closely related with reproduction. Thus, these results suggest that the decrease of p-ERK levels in B. koreanus and P. nana in the present study was closely associated with the results of our in-vivo experiments (e.g. reduced fecundity and growth retardation) in response to MeHg exposure. In the case of p-p38, considering its roles in cell death and defense mechanism (Chuang et al., 2000), it is interesting to note that it has shown clear down-regulation tendency after the increase at 1 ng/L of MeHg exposure in P. nana, whereas no changes were observed in B. koreanus. Previously, p38 signaling pathway has been proposed as a key regulatory signaling pathway for defense system in invertebrates (reviewed by Hatanaka et al., 2009). Among the aquatic invertebrates, in the copepod T. japonicus, p38 has been activated by oxidative stress associated with various environmental stressors including ultraviolet B radiation, metals, and nano-sized polystyrene microbeads (Rhee et al., 2013; Kim et al., 2015; Jeong et al., 2017). Moreover, activation of p38 has been shown to be increased by MeHg exposure in the copepod T. japonicus (Lee et al., 2017). On the contrary, the present study has demonstrated opposite results in P. nana. One possible explanation would be related to the inhibition of upstream regulator of p-38 that are involved in redox signaling, for example, apoptosis signal-regulating kinase 1 (ASK1). ASK1 is a MAP3K that is able to activate p38 signaling pathway under oxidative stress condition when it is dissociated from thioredoxin (Trx) (Liu et al., 2000). The mechanism of ASK1 activation is associated with the oxidation of Trx on its thiol groups by ROS. In this regard, MeHg is highly reactive with thiol groups, thereby possibly interrupting the dissociation of ASK1 from Trx, resulting in the inhibition of ASK1 15

activation and then the inhibition of p38 signaling pathway. Although further studies are required to confirm this hypothesis as we cannot eliminateother mechanisms that would be responsible for inhibition of p38 signaling pathway, this result clearly shows the speciesspecific molecular responses and also implies the importance of comparative studies in ecotoxicological studies. In conclusion, in vivo toxicity endpoint analysis revealed that MeHg inflicts adverse effects on the reproduction systems of rotifers and copepods. Moreover, we showed that rotifers and copepods utilize different detoxification processes and antioxidant systems in response to MeHg-induced oxidative stress. Additionally, MAPK signaling pathways were differentially activated in concentration-dependent manners in response to MeHg exposure in rotifers and copepods, indicating the presence of species-specific mechanism. To our knowledge, this is the first study to compare the adverse effects of MeHg on rotifers and copepods. Our findings will enable a better understanding of defense mechanism modes in marine invertebrates.

Acknowledgements

This work was supported by a grant from the Development of Techniques for Assessment and Management of Hazardous Chemicals in the Marine Environment program of the Korean Ministry of Oceans and Fisheries funded to Jae-Seong Lee.

16

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Table 1. Comparison of toxicity of MeHg to other metals in various aquatic invertebrates. Methylmercury Species Brachionus koreanus Paracyclopina nana

Tigriopus japonicus

Daphnia pulex

Other metals

Type of toxicity

Concentration (μg/L)

LC50-24 h

3.0

LC50-24 h LC50-48 h

LC50-48 h LC50-96 h

LC50-48 h LC50-96 h

Type of toxicity

Concentration (μg/L)

Cu (CuSO4) – LC50 (24 h)

1,200

Cu (CuSO4) – LC50 (96 h) Cd (CdCl2) – LC50 (96 h)

800 300

Cu (CuSO4·5H2O) – LC50 (96 h) Zn (ZnCl2) – LC50 (96 h) Cd (CdCl2·5H2O) – LC50 (96 h)

3,900 7,800 25,200

Hg (HgCl2) – LC50 (48 h) Cu (CuSO4·5H2O) – LC50 (48 h) Zn (ZnSO4·7H2O) – LC50 (48 h) Cd (3CdSO4·7H2O) – LC50 (48h) Hg (HgCl2) – LC50 (96 h)

5.2 93 560 1,880 161

11.5 5.5

6.7 4.1

5.7 1.8

Daphnia magna

Mytilus edulis

NOEC (32 d)

0.3

Fathead minnow

LC50-24 h (Embryos) LC50-96 h (Embryos)

221 39

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Reference This study Han et al, 2013 This study This study Hwang et al., 2010 Hwang et al., 2010 Lee et al., 2017 Lee et al., 2017 Lee et al., 2007 Lee et al., 2007 Lee et al., 2007 Chen and McNaught, 1992 Chen and McNaught, 1992 Khangarot and Ray, 1987 Khangarot and Ray, 1987 Khangarot and Ray, 1987 Khangarot and Ray, 1987 Pelletier, 1988 Neslson et al., 1988 Devlin, 2006 Devlin, 2006

Figure legends

Fig. 1. Acute toxicity of MeHg in B. koreanus and P. nana. Survival curves for 24 h are shown along with the corresponding LC50 values. Error bars indicate the standard deviations.

Fig. 2. Effects of different concentrations of MeHg exposure on the fecundity of (A) B. koreanus and (B) P. nana. The black bar indicates the time from the nauplius stage to the copepodid stage (N-C), whereas the grey bar shows the time from the nauplius stage to the adult stage (N-A). Results represent means ± SDs of three replicates (n ~ 10). Differences between groups were analyzed for significance using Tukey’s multiple comparison test. Different letters above columns indicate significant differences (P<0.05).

Fig. 3. Effects of different concentrations of MeHg exposure on (A) intracellular ROS and (B) GSH levels in B. koreanus and P. nana. Levels of ROS and GSH are presented as percentages of controls. Results represent means ± SDs of three replicates (n ~ 1,000). Different letters above columns indicate significant differences (P < 0.05).

Fig. 4. Effects of different concentrations of MeHg exposure on antioxidant enzymes (GST, GPx, and GR) in (A) B. koreanus and (B) P. nana. Enzymatic activities are presented as percentages of controls. Results represent means ± SDs of three replicates (n~1,000). Different letters above columns indicate significant differences (P<0.05).

Fig. 5. Effects of different concentrations of MeHg exposure on antioxidant-related gene expression in (A) B. koreanus and (B) P. nana. Results represent means ± SDs of three replicates (n ~ 500).

Fig. 6. Effects of different concentrations of MeHg exposure on phosphorylation of MAPK signaling proteins in (A) B. koreanus and (B) P. nana.

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