P-glycoprotein expression in Perna viridis after exposure to Prorocentrum lima, a dinoflagellate producing DSP toxins

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Fish & Shellfish Immunology xxx (2014) 1e9

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P-glycoprotein expression in Perna viridis after exposure to Prorocentrum lima, a dinoflagellate producing DSP toxins Q3

Lu Huang a, b, Jie Wang a, b, Wen-Chang Chen a, b, Hong-Ye Li a, b, Jie-Sheng Liu a, b, Jiang Tao a, b, Wei-Dong Yang a, b, * a b

College of Life Science and Technology, Jinan University, Guangzhou 510632, China Key Laboratory of Aquatic Eutrophication and Control of Harmful Algal Blooms of Guangdong Higher Education Institute, Guangzhou 510632, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 5 January 2014 Received in revised form 6 April 2014 Accepted 23 April 2014 Available online xxx

Bivalves naturally exposed to toxic algae have mechanisms to prevent from harmful effects of diarrheic shellfish poisoning (DSP) toxins. However, quite few studies have examined the mechanisms associated, and the information currently available is still insufficient. Multixenobiotic resistance (MXR) is ubiquitous in aquatic invertebrates and plays an important role in defense against xenobiotics. Here, to explore the roles of P-glycoprotein (P-gp) in the DSP toxins resistance in shellfish, complete cDNA of P-gp gene in the mussel Perna viridis was cloned and analyzed. The accumulation of okadaic acid (OA), a main component of DSP toxins, MXR activity and expression of P-gp in gills of P. viridis were detected after exposure to Prorocentrum lima, a dinoflagellate producing DSP toxins in the presence or absence of P-gp inhibitors PGP-4008, verapamil (VER) and cyclosporin A (CsA). The mussel P. viridis P-gp closely matches MDR/P-gp/ABCB protein from various organisms, having a typical sequence organization as full transporters from the ABCB family. After exposure to P. lima, OA accumulation, MXR activity and P-gp expression significantly increased in gills of P. viridis. The addition of P-gp-specific inhibitors PGP-4008 and VER decreased MXR activity induced by P. lima, but had no effect on the OA accumulation in gills of P. viridis. However, CsA, a broad-spectrum inhibitor of ABC transporter not only decreased MXR activity, but also increased OA accumulation in gills of P. viridis. Together with the ubiquitous presence of other ABC transporters such as MRP/ABCC in bivalves and potential compensatory mechanism in P-gp and MRP-mediated resistance, we speculated that besides P-gp, other ABC transporters, especially MRP might be involved in the resistance mechanisms to DSP toxins. Ó 2014 Published by Elsevier Ltd.

Keywords: Diarrheic shellfish poisoning toxin P-glycoprotein Perna viridis Prorocentrum lima

1. Introduction

Q2

Q1

Harmful algae associate with great negative impacts on marine ecosystems, public health and resort quality, representing a growing problem in coastal waters over the world, as well as in China. One of the biggest headaches is phycotoxins produced by harmful microalgae, which can be ingested by filter-feeding shellfish and accumulate in the tissues, causing a variety of gastrointestinal and neurological illnesses through food chain. The most important phycotoxins are shellfish toxins, including paralytic shellfish poisoning (PSP) toxins, diarrhetic shellfish poisoning (DSP) toxins, amnesic shellfish poisoning (ASP) toxins, neurotoxic

* Corresponding author. College of Life Science and Technology, Jinan University, Guangzhou 510632, China. Tel.: þ86 020 85228470; fax: þ86 020 85225183. E-mail addresses: [email protected], [email protected] (W.-D. Yang).

shellfish poisoning (NSP) toxins and azaspiracid shellfish poisoning (AZP) toxins [1]. The DSP toxins are lipophilic polyether molecules, which include okadaic acid (OA), dinophysistoxin-1 (DTX1), DTX2 and derivative forms [1]. These toxins are produced by some microalgae of the genus Dinophysis and Prorocentrum such as Prorocentrum lima, Prorocentrum concavum, Prorocentrum maculosum, Dinophysis acuminata, Prorocentrum rhathymum, and Dinophysis fortii etc. It is found that OA is a potent selective inhibitor of protein phosphatase types 1 (PP1) and 2A (PP2A), which produces hyperphosphorylation of several proteins, thus leading to various toxic effects [2]. Low concentrations of OA (nanomolar) are shown to have cytotoxic, embryotoxic, mutagenic effects on different cell lines. However, shellfish can survive and grow well during the periods of DSP producing dinoflagellate blooms. Furthermore, it has been shown in laboratory that shellfish accumulate OA and grow without increased mortality when only OA-producing algae is

http://dx.doi.org/10.1016/j.fsi.2014.04.020 1050-4648/Ó 2014 Published by Elsevier Ltd.

Please cite this article in press as: Huang L, et al., P-glycoprotein expression in Perna viridis after exposure to Prorocentrum lima, a dinoflagellate producing DSP toxins, Fish & Shellfish Immunology (2014), http://dx.doi.org/10.1016/j.fsi.2014.04.020

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provided as food source [3,4]. It is likely that organisms naturally exposed to toxic algae have some mechanisms to protect them from harmful effects of OA [5,6]. Some papers have dealt with the molecular effects of OA on bivalves [7,8]. Manfrin et al. [7] for the first time identified several candidate transcripts as OA-stress markers over 35 days of exposure by cDNA microarray. Romero-Geraldo and Hernández-Saavedra [8] observed some noticeable differences in genes expression in juvenile oysters at 14 days of exposure to P. lima. However, very few studies have concerned the resistance mechanisms involved, and the information currently available is still insufficient [5,9], although some papers have suggested the involvement of detoxifying enzymes [10,11]. P-glycoprotein (P-gp) or multidrug resistance 1 (MDR1) belongs to the family of ATP-binding cassette (ABC) transporters responsible for multixenobiotic resistance (MXR) in aquatic organisms [12e14]. Many studies have shown the presence of P-gp protein in bivalves, which exerts important roles in the detoxification of heavy metal ion and organic chemicals [12,15e19]. Luckenbach and Epel [20] first studied its function, gene expression, and localization in mussel gill tissue and presented full-length cDNA sequence of the California mussels (Mytilus californianus). Later, Xu et al. [5] demonstrated the full-length cDNA sequence in the oyster Crassostrea ariakensis. These attempts provide useful information for learning the characters and functions of P-gp in bivalves. However, to the best of our knowledge, only few studies have been conducted so far to assess the role of P-gp in the resistance mechanism of bivalves against DSP toxins [5,9]. In our previous studies, we found that the exposure of C. ariakensis to P. lima increased P-gp mRNA expression in gill tissues, suggesting the potential of P-gp in the resistance mechanism of bivalves against DSP toxins [5]. Mussels, common filter-feeders in estuaries and coastal waters, have been extensively used as models for studies of the MXR in shellfish [9,16,19,21e23]. P. lima is a toxic benthic, epiphytic, photosynthetic dinoflagellate found worldwide in tropical seas [24]. As a known producer of DSP toxins, it has been associated to DSP episodes in different parts of the world, and has been extensively used in aquatic toxicological studies [5,8,21,25]. Here, to further investigate the roles of P-gp in DSP toxins resistance in shellfish, complete cDNA of P-gp gene in the mussel Perna viridis was cloned and analyzed. OA content, MXR activity and P-gp expression in gills of P. viridis were detected after exposure to P. lima in the presence or absence of ABC transporter inhibitors (a broadspectrum MDR modulator CsA, and two P-gp specific inhibitors PGP-4008 and VER). The potential correlation between the OA level and MXR activity were discussed. 2. Materials and methods 2.1. Experimental organisms The mussels, P. viridis (90e110 mm in length), were purchased from Huangsha Seafood Market in Guangzhou, China. Mussel individuals were scrubbed clean to remove sediment and epibionts, then placed in tanks (300 mm  450 mm  300 mm) with natural seawater at 20  C  1  C. The seawater was renewed daily, and the mussels were fed once a day with Platymonas subcordiformis (106 cell L1) for 7 days to acclimate the laboratory condition. P. lima (No. 2579) was kindly provided by the National Center for Marine algae and Microbiota (NCMA, Formerly the CCMP), which has been verified to produce DSP toxins in our former studies. The microalgae were grown as batch cultures in Erlenmeyer flasks containing f/2 medium filter-sterilized by 0.22 mm filters. The cultures were grown at 22  1  C in an artificial climate incubator with an irradiance of 40 mmol photons m2 s1 in a 12/12 h light/dark regime.

2.2. cDNA cloning and sequence analysis of P-gp gene Total RNA was extracted from w30 mg of gill tissue of the mussels with RNeasy Plus Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions. Total RNA was reverse-transcribed to cDNA by using the AMV First Strand cDNA Synthesis Kit (TaKaRa, China). Full-length cDNA sequence of P-gp in P. viridis was obtained with rapid amplification of cDNA ends (RACE). For RACE reactions, 30 -full RACE Core Set Ver.2.0 and 50 -Full RACE Kit (TaKaRa, China) were used. All PCR amplifications were performed with the commercial kit LA TaqÒ with GC Buffer (TaKaRa, China) according to manufacturer’s protocol. Before RACE, partial cDNA sequence of P-gp in P. viridis was obtained using PCR. The primers were designed based on the conserved sequence of MDR gene from Mytilus edulis, Mytilus galloprovincialis, Mytilus sp. KL-2006 (NCBI accession: AF159717.1, EF057747.1 and EF140764.1) by Primer 5.0. The primers for the first PCR and the second PCR were 50 -GAT AGG AGT GGT TAG CCA GGA A-30 /50 -GCA ATG GCT ACT CTT TGT TTC T-30 and 50 -GGA ACC AGT TCT GTT TGC CAC T-30 /50 -CTG AGC ACC ACG ATT CCC TAC T-30 , respectively. The two PCR reactions were all carried out for 30 cycles at 94  C (30 s), 55  C (30 s), and 72  C (1 min). PCR product from the first PCR was used as template of the second PCR reaction, which generated a 285-bp PCR product (CTE510 PCR). Homology of this sequence with ABCB cDNA from other organisms was confirmed by NCBI blastn (data not shown). Based on the CTE510 PCR sequence, primers GAT AGA AAA TGC TGC CAA GAT G and CAA CTA CCA GAG GGT TAT GAA A were designed for 30 -RACE and generated a PCR product (CTE510-3P0). The PCR reactions were conducted for 30 cycles at 94  C (30 s), 55  C (30 s), and 72  C (2 min). Based on the product CTE510-3P0 and homologous sequences (ABS_83556, AET_34454, CAX_46411, XP_001640909, XP_002739992), further nested primers were designed as follows: GGT GAA AAA TCT GCC CTA ATC C/AGT ATG GGA TCA ACG AGG AAA C and TNG TGG ANA GWC GAT GKG CTA/ TGW ATC TAA TGC TGA TGT RGC. The PCR reactions were performed for 30 cycles at 94  C (30 s), 50  C (30 s), and 72  C (2 min), then another partial 30 -sequence (CTE510-T, w1.6 kb) was obtained. Based on the CTE510-T sequence, new primers TCT AAC TAT CCA AGC CAA ACC A/AGA GGT TCT ATG ACC CAG AGG ATG (w1.4 kb product, CTE510-3P3) were designed for the second 30 -RACE, which gave a PCR product (w1.4 kb, CTE510-3P3). The PCR reactions were conducted for 30 cycles at 94  C (30 s), 55  C (30 s), and 72  C (2 min). For 50 -end sequence, based on the homologous sequences (ABS_83556, AET_34454, CAX_46411, XP_001640909, XP_002739992), the nested primers GAG YRG YGA ACT GAS AAC MAG, CGC TGG NDG WGT AGC AGA RGA and TCT TGG CAG CAT TTT CTA TCT C were designed to obtain partial cDNA sequence (CTE511T). The PCR reactions were all conducted for 30 cycles at 94  C (30 s), 50  C (30 s), and 72  C (2 min). As for 50 -RACE, total RNA was treated with CIAP and TAP, then ligated with 50 -RACE adapter using T4 RNA ligase. A nested PCR was conducted with the primers TAA AGT CTC CCC TGG TTC AAA G and TAG CAC TCT CTT TGT TGG CAT C (w1.9 kb, CTE511-5P2). The PCR reactions were all conducted for 30 cycles at 94  C (30 s), 55  C (30 s), and 72  C (2 min). Except for some products that can be directly sequenced, most fragments were purified using the TaKaRa Agarose Fragment Purification Kit Ser. 2.0 (TaKaRa, China), then cloned into the vectors pMD20-T (TaKaRa, China) according to the manufacturer’s instructions, and sequenced. The sequences were assembled and analyzed using tools at the Expert Protein Analysis System (Expasy) Proteomics Server (http:// expasy.org/) and NCBI BLAST search engines. Multiple sequence

Please cite this article in press as: Huang L, et al., P-glycoprotein expression in Perna viridis after exposure to Prorocentrum lima, a dinoflagellate producing DSP toxins, Fish & Shellfish Immunology (2014), http://dx.doi.org/10.1016/j.fsi.2014.04.020

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alignments and determinations of similarity between amino acid sequences of ABC transporters from different organisms were performed using Cluster X2 and BLAST on NCBI. The phylogenetic trees of ABC transporter sequences from different organisms were constructed by neighbor-joining (NJ) method using the program MEGA5.0, and verified by other methods including maximumlikelihood and minimum-evolution methods. The Polyphobius algorithm (http://phobius.sbc.su.se/poly.html) was used to identify transmembrane helices in P-gp mussel. 2.3. Experimental design To learn the relationship between the OA level, MXR activity and P-gp expression, two experiments were designed as described in Table 1. Experiment I. Exposure of mussels to P. lima in the presence or absence of PGP-4008. The compound termed PGP-4008 (N-(1Benzyl-2,3-dihydro-1H-pyrrolo[2,3-b]quinolin-4-yl)-2- phenylacetamide) (Sigma, USA) is a newly developed selective P-gp inhibitor in vitro and in vivo [26]. After preliminary acclimation to the laboratory condition for seven days, the mussels were randomly divided into four treatments fed with algal cultures as described in Table 1. At 6 h, 12 h, 24 h after exposure to P. lima, gill tissues were collected from 36 mussel individuals in each treatment, respectively. Subsequently, tissues from six mussel individuals at each time point within the same treatment were pooled together as one sample for toxins detection. Tissues from three individuals were pooled together for RNA and protein extraction, and tissues from another three ones were pooled together for MXR activity determination. So three samples were obtained at each time point in each treatment for qPCR, MXR activity, toxins and protein analyses. The pooled tissues for RNA and protein extraction were frozen in liquid nitrogen rapidly and stored at 80  C. P-gp activity was measured immediately when the gill tissues were dissected. Experiment II. Exposure of mussels to P. lima and/or verapamil (VER) or cyclosporin A (CsA). After acclimation to the laboratory condition, the mussels were randomly raised in four tanks. To the tanks, P. subcordiformis and/or P. lima were firstly added. After 6 h of P. lima exposure, VER (66.7 mM) or CsA (10 mM) were added as described in Table 1. VER is a model competitive inhibitor of P-gp, which is pumped by P-gp with energy consume [27]. In contrast, CsA is a broad-range inhibitor of MXR by blocking ATPase activity of MXR transporters, which can inhibit P-gp and multidrug resistance-associated proteins (MRPs) etc. [27,28]. At 2 h and 8 h after the addition of inhibitors (at 8 h and 14 h after P. lima exposure), gill tissues were collected from 27 mussels in each treatment, respectively. As described in Experiment I, tissues from six mussel individuals at each time point within the same treatment were pooled together as one sample for toxins detection, while tissues from another three ones were pooled together for MXR activity

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determination. Therefore, three samples were obtained at each time point in each treatment for toxins and MXR activity detection. 2.4. Detection of OA in gill tissue Extraction and analysis of OA in gill tissues were performed by ELISA according to the manufacturer’s instruction Okadaic Acid (DSP) Microtiter Plate (ABRAXIS, USA) [5]. For OA extraction, fresh homogenate (1.0 g) of gill sample was mixed with 6 mL methanol/ deionized water (80/20) and centrifuged at 3000  g at 4  C for 10 min, the supernatant was collected. 2 mL of methanol/deionized water (80/20) was added to the homogenate two times for reextract OA. The extract and re-extract were combined and filtered with 0.45 mm filter (Millex HV, Millipore) for OA detection. 2.5. Real-time RT-PCR Total RNA was extracted from every sample by TRIzol reagent (Invitrogen, Carlsbad, CA), and treated with DNase I (Qiagen, Germany) to remove any possible residual DNA. The concentration and OD260/280 ratio of RNA were determined by NanoDropÒ ND-1000 (NanoDrop, USA). RNA integrity and genome DNA contamination were evaluated by agarose gel electrophoresis. First strand cDNA was synthesized from 4 mg of total RNA using the PrimeScript II 1st Strand cDNA Synthesis Kit (TaKaRa, China). Specific primers for P-gp were designed within the obtained P-gp sequence of P. viridis by Primer 5.0. The primers were 50 -AAA TGG ATG TCA CCC AAG CAG-30 and 50 - TGA GCA CCA CGA TTC CCT ACT-30 . 18S rRNA was used as a housekeeping gene to normalize expression of P-gp [29e32]. The primers were 50 -CGG CGA CGT ATC TTT CAA AT-30 and 50 -CTT GGA TGT GGT AGC CGT TT-30 , which was designed according to the P. viridis sequence in GenBank (NCBI accession numbers: EF613234). The lengths of the amplified fragments were 115 and 136 bp for P-gp and 18S rRNA, respectively. PCR was performed with a LightCyclerÒ 480 Real-Time PCR System using LightCyclerÒ 480 SYBR Green I Master (Roche Applied ScienceÒ) with the following PCR reaction profile: 95  C for 5 min, 44 cycles of 95  C for 10 s, 60  C for 20 s and 72  C for 20 s. The reaction mixture (20 mL) consisted of 10 mL of 2  SYBRÒ Premix Ex TaqÔ, 0.4 mM of each forward and reverse primers and 2 mL of firststrand cDNA template. Amplifications were also performed on equivalent non-transcribed total RNA as negative control to check the absence of DNA contaminants. Amplification products were quantified by 2DDCt with Roche LightCycler 480 Software Version 1.5. 18S rRNA amplification was used as a housekeeping gene for normalization, as it displayed no modulation under experimental treatments. Buratti et al. [29] have studied the changes in expression of P-gp and other genes in M. galloprovincialis naturally exposed to algal toxins, in which 18S rRNA amplification was used as an endogenous control.

Table 1 Summary of experimental treatments and initial algal density. Experiment

Treatment

Experiment I

Control P. lima PGP-4008 P. lima þ PGP-4008 Control P. lima P. lima þ VER P. lima þ CsA

Experiment II

Concentration of ABC transporter inhibitors (mM)

20 20

66.7 10

Initial algal density cells (L1) P. lima

P. subcordiformis

e 3 e 3 e 3 3 3

3 3 3 3 3 3 3 3

105 105 105 105 105

       

106 106 106 106 106 106 106 106

Please cite this article in press as: Huang L, et al., P-glycoprotein expression in Perna viridis after exposure to Prorocentrum lima, a dinoflagellate producing DSP toxins, Fish & Shellfish Immunology (2014), http://dx.doi.org/10.1016/j.fsi.2014.04.020

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Fig. 1. Phylogenetic tree based on multiple alignments (Clustal X) of various ABCB transporter sequences from various vertebrates and invertebrates. Tree was generated using the neighbor-joining method. Percent concordance shown at the nodes is based on 1000 bootstrap iterations. ABCB transporters are as follows: P-gp (accession no. AAN07780.2) for Macaca fascicularis; P-gp (accession no. AAW56424.3) for Oncorhynchus mykiss; P-gp (accession no. CAA46190.1) for Caenorhabditis elegans; P-glycoprotein-like protein (accession no. ABS83556.1) for Mytilus californianus; P-gp 2 (accession no. NP_194326.2) for Arabidopsis thaliana; Mdr 65 (accession no. EFN62270.1) for Camponotus floridanus; Mdr1 (accession no. CAI99869.1) for Brachidontes pharaonis; Mdr1 (accession no. NP_001230917.1) for Cricetulus griseus; Mdr (accession no. CAC86600.1) for Platichthys flesus; Mdr1 (accession no. NP_001075628.1) for Oryctolagus cuniculus; Mdr1 (accession no. NP_990225.1) for Gallus gallus; P-gp (accession no. ADT63773.1) for Lepeophtheirus salmonis; egg permeability glycoprotein (accession no. AAW28777.1) for Strongylocentrotus purpuratus; Mdr1 (accession no. CAM33439.1) for Ovis aries; P-gp (accession no. AAA59575.1) for Homo sapiens; Mdr (accession no. AAA39516.1) for Mus musculus; P-gp (accession no. ACX30417.1) for Trematomus bernacchii; P-gp (accession no. AAA37005.1) for Cricetulus sp.; P-gp (accession no. ADQ20481.1) for Poeciliopsis lucida; Mdr (accession no. EFX86431.1) for Daphnia pulex; P-gp (accession no. CAX46411.2) for Mytilus galloprovincialis; P-gp (accession no. CAB71875.1) for Arabidopsis thaliana; P-gp (accession no. ACM77791.1) for Canis lupus familiaris; P-gp (accession no. AAA28679.1) for Drosophila melanogaster; P-gp (accession no. JN813100) for Crassostrea ariakensis; Mdr (accession no. EKC34962.1) for Crassostrea gigas; Mdr 1A (accession XP_005111509.1) for Aplysia californica; Mdr 1B (accession XP_005089604.1) for Aplysia californica.

2.6. MXR activity in gill tissue

2.7. Western blotting

MXR activity was determined in isolated gill sample of each treatment using a rhodamine B (RhB) transport assay with small modification [33]. Briefly, 110 mg of gill samples were placed into a foil-packaged beaker with 8 mL of artificial seawater (ASW) containing 7.5 mM RhB (Sigma, USA) for 60 min. Then gills were rapidly washed three times with the same volume of ASW to remove RhB adsorbed in the gill surface, and then transferred to another beaker with 8 mL of ASW to eliminate high background noise at later measurement. After a 5 min “pre-efflux” period, the gill fragments were individually transferred to new beakers with 16 mL of ASW on a shaker. RhB efflux was measured during a 60 min exposure period at 20e22  C. 0.2 mL of the media from each vial were collected every 10 min for determination of fluorescence intensity (Ex 535 nm, Em 590 nm) on DTX880 multimode detector (Beckman Coulter, USA). Based on the fluorescence intensity, the standard curve of RhB was drawn and MXR activity was calculated.

Homogenate of gill samples were lysed, then centrifuged at 12,000 g for 30 min at 4  C. The supernatant was used for total protein assays and blot analysis. Proteins were quantified by bicinchoninic acid (BCA) assay with BCA Protein Assay kit (Bio-Rad, Hercules, USA). 70 mg of sample protein per lane was loaded onto 10% SDS polyacrylamide gels and ran at 110 V for 1.5 h at room temperature. Resolved proteins were transferred onto polyvinylidene fluoride membrane in Trans-blot wet buffer. Then the membranes were blocked in Tris-buffered saline with Tween (TBST) containing 5% non-fat milk for 90 min. Immunolabelling was performed using the monoclonal antibody against P-gp (Anti-P-GP, Abcam, USA) overnight at 1: 200 dilutions and a polyclonal secondary anti-mouse IgG antibody diluted to 1:5000 (Sigma, USA) at room temperature for 1 h. To allow for cross-gel comparisons of Pgp expression, a chosen sample was run on all the gels as an internal standard. The protein bands were visualized by using an enhanced chemiluminescence (ECL) system on X-ray film.

Please cite this article in press as: Huang L, et al., P-glycoprotein expression in Perna viridis after exposure to Prorocentrum lima, a dinoflagellate producing DSP toxins, Fish & Shellfish Immunology (2014), http://dx.doi.org/10.1016/j.fsi.2014.04.020

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Quantitation of the signal was performed by densitometry scanning with Image Scanner. Expression levels of P-gp in different samples were calculated relatively to the expression of GAPDH (Proteintech, China).

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secondary structure of P. viridis P-gp predicted by HNN mainly consists of alpha helix (57.08%), b-pleated sheet (10.30%) and random coil (32.65%), similar to P-gp from the other species. 3.2. Expression of P-gp gene in gill tissue

2.8. Statistical analyses Statistical analyses were performed using the software SPSS 10. Duncan’s multiple range test was used to compare differences between means, with significant differences at p ¼ 0.05. All values are expressed as mean  standard deviation. 3. Results 3.1. Sequence analysis of P-gp gene in mussel The sequences obtained by RACE were assembled to a fulllength transcript of P-gp gene, in which the open reading frame is 3936 bp (NCBI accession numbers: JQ696953). The size of predicted protein is 143.93 kDa (1311 aa), and the theoretical pI is 6.62. The P-gp sequence obtained from P. viridis is matched with MDR/P-gp/ABCB gene from various organisms by BLASTx. It exhibits high homology with P-gp mRNA of other bivalve mollusks such as C. ariakensis, Crassostrea virginica, M. californianus, M. galloprovincialis, M. edulis and Dreissena polymorpha. The phylogenetic tree constructed by NJ method reveals that the P-gp from P. viridis clusters together with P-gp protein from C. ariakensis, forms an independent branch, then groups into the other branch with some mollusks (Fig. 1). Similar results were obtained in phylogenetic trees constructed by using other approaches such as maximum-likelihood and minimum-evolution methods (data not shown), validating the present phylogenetic tree. Analyses of the amino acid sequence with Prosite (http://ca. expasy.org/prosite/) and Polyphobius algorithm demonstrated that the P-gp in mussel has typical characteristics of full ABCB transporters, containing 12 putative transmembrane helices with two membrane spanning domains (MSDs) and two nucleotidebinding domains (NBDs) [5,20]. The NBDs contain highly conserved motifs of ABC transporters: the Walker A, Walker B, ABC signatures and so-called A loops upstream of the Walker A regions (Figs. 2 and 3) [34]. Based on the sequon Asn-X-The/Ser [35], there is a N-glycosylation sequon at Asn110-Met111-Thr112 in the MSD1 corresponding to the extracellular loop closest to the NH2-terminal end (Fig. 3). The

The results of real-time RT-PCR of P-gp mRNA in gill tissue of mussel after exposure to P. lima were demonstrated in Fig. 4, where 18S rRNA was used as a normalizing gene. The P-gp/18S rRNA ratio were 2.5-, 3.0- and 3.9-fold higher in P. lima-exposed mussels without PGP-4008 after 6 h, 12 and 24 h, compared with the control counterparts. It is noted that after addition of PGP-4008, P-gp/18S rRNA ratio decreased both in P. lima-exposed mussel and in mussels fed with only P. subcordiformis, suggesting that PGP-4008 can inhibit the expression of P-gp mRNA. 3.3. Expression of P-gp protein in gill tissue Immunoblotting showed a w170 kDa protein specifically reacting with a P-gp antibody in mussel gills from all the experimental treatments (Fig. 5). Protein levels of P-gp in gill tissues did not display any regular changes at 6 h and 12 h after exposure to P. lima, with or without addition of PGP-4008. However, protein levels of P-gp at 24 h after exposure to P. lima in the absence of PGP4008 was higher than that in control (p < 0.05, t-test). In contrast, in the presence of PGP-4008, the P-gp levels both in P. lima-exposed mussel and in mussels fed with only P. subcordiformis were almost the same as control at 24 h, giving further evidence that PGP-4008 can inhibit the expression of P-gp. 3.4. Content of OA in gill tissue As demonstrated in Fig. 6A, after exposure to P. lima, OA concentration in gill tissues significantly increased both in the presence (P. lima þ PGP-4008 group) and absence of PGP-4008, compared with control (p < 0.05). However, there was no significant difference in OA concentration between the P. lima-exposed mussels with PGP-4008 and without PGP-4008 (p > 0.05), implying that Pgp inhibitor PGP-4008 had less effects on the accumulation of OA in gill tissues after exposure to P. lima. The content of OA in control was not zero but 6.3 ng g1, suggesting that mussels might have lived in sea area with OA-producing algae. In another experiment, mussels were exposed to P. lima for 6 h, then VER or CsA was added into the culture systems. After exposure

Fig. 2. Topologies of mussel P-gp protein with membrane-spanning domains (MSDs) as predicted by the Polyphobius algorithm and nucleotide-binding domains (NBDs) indicated by A loop, Walker A and B, and the ABC signature motifs. GS marks the N-glycosylation sites.

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Fig. 3. Alignment of P-gp from P. viridis with human P-gp (GenBank accession no. AAA59575.1). Alignments were performed using Clustal X. MSDs were determined using the Q4 Polyphobius algorithm and are shown in boldface. Underlined sections represent transmembrane helices. GS marks the N-glycosylation sites.

to P. lima, the addition of MXR inhibitors VER and CsA exhibited distinct effects on the accumulation of OA in gill tissues (Fig. 6B). The concentrations of OA in P. lima-exposed mussels with VER (P. lima þ VER group) or CsA (P. lima þ CsA group) and without VER and CsA were all higher than that in control (p < 0.05). The OA concentration in P. lima-exposed mussels with CsA (P. lima þ CsA group) was significantly higher than that in P. lima-exposed mussels without CsA (p < 0.05). In contrast, there was no significant difference in OA level between P. lima-exposed mussels with VER (P. lima þ VER group) and without VER (p > 0.05), suggesting that

the addition of CsA but not VER increased the DSP toxins accumulation in gill of P. viridis after exposure to P. lima. 3.5. MXR activity in gill tissue Changes in MXR activity in gill tissue after P. lima exposure were demonstrated in Fig. 7. It was shown that the total efflux rate were 210.1, 193.4 and 216.6 pmol g1 min1 respectively at 6, 12 and 24 h after exposure to P. lima in the absence of PGP-4008, higher than their control counterparts (126e146 pmol g1 min1, p < 0.05) (Fig. 7A). In contrast, there is no difference between the P. limaexposed mussels with PGP-4008 (P. lima þ PGP-4008 group) and controls (p > 0.05). These results indicated that P. lima exposure can induce MXR activity in gill tissue and that this induction can be inhibited by P-gp specific inhibitor PGP-4008. Fig. 7B gave similar results. The total efflux rate of RhB in P. lima-exposed mussels without MXR inhibitor was considerably higher than that in the control at each time point (p < 0.05). However, in the presence of VER and CsA, the total efflux rates of RhB all decreased, exhibiting similar values with control counterparts (Fig. 7B). These indicated that MXR inhibitors both VER and CsA exhibited potent suppressions on the MXR activity in mussel gills induced by P. lima. 4. Discussion

Fig. 4. P-glycoprotein mRNA expressions in gill tissues from control and P. limaexposed mussels, measured by real-time RT-PCR. Levels of P-gp mRNA were normalized with the 18S rRNA. The values are expressed as mean  S.D. Bars of respective treatment followed by the same letter are not significantly different at p < 0.05 (Duncan’s multiple range test).

To provide more information on P-gp in bivalves, full-length cDNA of P-gp from the gill tissues of P. viridis was obtained. The full-length cDNA is 5216 bp with 3936 bp of open reading frame, in accordance with ABCB in California mussel [20]. The P-gp possesses typical features of full ABCB transport proteins with conserved

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against natural toxins [9,18,40]. In the present study, we found that the level of OA in gills of P. viridis increased significantly after exposure to P. lima, suggesting that P. viridis could promptly accumulate DSP toxins in gill by ingestion of P. lima. However, during the experimental period, the mussel could filter actively and were apparently healthy without death after exposure to P. lima. This insensitivity to DSP toxins has been observed by many studies, implying the tolerance of bivalves to DSP toxins [3,6,41e44]. Corresponding to the accumulation of OA in gill tissue, P-gp gene expression and MXR activity increased at 6, 12 and 24 h, while protein levels of P-gp increased at 24 h after exposure to P. lima. The higher P-gp (gene expression and efflux rate) in mussel gills might justify that P-gp may have an important role in the resistance mechanism of P. viridis against DSP toxins. As for the asynchrony of changes in mRNA and protein level of P-gp, many papers have concerned, which might be ascribed to the post-transcriptional control of P-gp synthesis [33,38,45,46]. Amé et al. [47] found that P-gp mRNA increased 3-fold in gills of Jenynsia multidentata at 12 h, while P-gp protein level increased at 24 h after exposure to MC-LR. Contardo-Jara et al. [48] demonstrated that P-gp expression in gills of the freshwater mussel D. polymorpha was enhanced after 1 h exposure but no changes were detected after longer (72 h) exposure of 100 mg/L MC-LR. To further verify the roles of P-gp in the resistance mechanism against DSP toxins, we observed OA accumulation when the P-gpspecific inhibitor PGP-4008 was present. To our surprise, there in no significant difference in toxin level between the P. lima-exposed mussels with PGP-4008 and ones without PGP-4008. However, the MXR activity and P-gp expression in P. lima-exposed mussels Fig. 5. P-gp expressions in gill tissues from control and P. lima-exposed mussels. (A) Representative western blots for P-gp expression at 6 h, 12 h and 24 h after exposure to P. lima with or without PGP-4008. (B) P-gp levels were measured using immunoblotting and normalized to the GAPDH protein. The values are expressed as mean  S.D. Bars of respective treatment followed by the same letter are not significantly different at p < 0.05 (Duncan’s multiple range test).

nucleotide binding domains (NBD). The size of predicted protein is 143.93 kDa (1311 aa), and the theoretical pI is 6.62. However, based on the sequon Asn-X-The/Ser, we found N-glycosylation sequon existing in P-gp, so the size should be higher than that predicted. As demonstrated by Western blot, the size of the mussel P-gp is about 170 kDa, consistent with C. ariakensis. In keeping with description by Stenham [36], the P-gp protein from P. viridis mainly consists of alpha helix (57.08%), b-pleated sheet (10.30%) and random coil (32.65%). The P-gp mRNA in P. viridis exhibits high homology with P-gp mRNA of other bivalve mollusks such as C. ariakensis, Crassostrea gigas, M. californianus, M. galloprovincialis and M. edulis. Phylogenetic analyses reveal that the mussel P. viridis P-gp protein has the closest relationship with P-gp protein from the oyster C. ariakensis. However, compared to P-gp/MDR/ABCB from other organisms, the P. viridis sequence is more similar to the homolog from the more distantly related Caenorhabditis elegans than to homologs from other bivalves such as M. californianus, M. galloprovincialis and C. gigas. Similarly, Navarro et al. [37] found that the C. ariakensis ABCB sequence is more similar to the homolog from Cyphoma gibbosum than to homolog from M. galloprovincialis. Taken together, it is likely that mussel P. viridis P-gp sequence obtained in our study might be a different paralog than M. californianus, M. galloprovincialis and C. gigas. P-gp, which belongs to ABCB superfamily, has been shown to be ubiquitously present in bivalves [15,16,18,38,39]. However, the roles of P-gp in the resistance mechanism of bivalves against phycotoxins produced by microalgae remain unclear, though some authors have proposed that P-gp in aquatic organisms might confer protection

Fig. 6. MXR activities in gill tissues from control and P. lima-exposed mussels, with or without P-gp inhibitors (PGP-4008, verapamil, cyclosporine A). (A) MXR activities in gill tissues with or without PGP-4008 (20 mM). (B) MXR activities in gill tissues with or without verapamil and cyclosporine A. The values are expressed as mean  S.D. Bars of respective treatment followed by the same letter are not significantly different at p < 0.05 (Duncan’s multiple range test).

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higher than that in P. lima-exposed mussels without CsA, suggesting that the addition of CsA strongly blocked the out-transport of DSP toxins. Furthermore, the MXR activities in P. viridis were inhibited in both treatments added with VER and CsA in RhB efflux model, thus confirming that the two inhibitors depressed transport activity of MXR. However, Qadir et al. [54] demonstrated that CsA is a broad-spectrum MDR modulator, which can prevent multiple ABC protein-mediated resistance with activity against P-gp, MRP-1, BCRP and lung resistance protein (LRP). In contrast, PGP-4008 and VER are only P-gp specific inhibitors. Lin et al. [55] proposed that there is a compensatory mechanism in P-gp and MRPmediated resistance. The loss of one transporter can be functionally compensated by over-expression of the other. As a consequence, the expression of P-gp and MRP may constitute a functional network to defense against xenobiotics [51]. In the present study, when P-gp activity was inhibited by PGP-4008 or VER, MRP will be activated or over-expressed, thus effectively expelling the toxins out of the cells. So the OA accumulation did not increase in the mussels, and tolerability of organisms to DSP toxins still existed. On the other hand, after addition of cyclosporin A, P-gp, MRP-1 and other ABC transporters were all inhibited, thus leading to the toxins accumulation in gill cells and the sensitivity to DSP toxins [51]. It is likely that besides P-gp, other ABC transporters, especially MRP might be involved in the resistance mechanisms, playing significant roles in DSP toxins efflux in bivalves. However, further studies should be addressed to ascertain the expression of MRP response to DSP toxins, especially in the presence of P-gp specific inhibitor, and these works are now underway in our laboratory. 5. Conclusion Fig. 7. OA concentrations in gill tissues measured by ELISA. (A) OA concentration in gills from control and P. lima-exposed mussels with or without the inhibitor PGP-4008. (B) OA concentration in gills from control and P. lima-exposed mussels with or without verapamil and cyclosporine A. The values are expressed as mean  S.D. Bars of respective treatment followed by the same letter are not significantly different at p < 0.05 (Duncan’s multiple range test).

without PGP-4008 was significantly higher than those in P. limaexposed mussels with PGP-4008 (p < 0.05). That’s to say, PGP-4008 could inhibit P-gp expression and MXR activity (accurately, transport activity of P-gp), but had no effects on the OA accumulation induced by P. lima. Many studies have demonstrated that PGP-4008 is a specific inhibitor of P-gp [49,50]. It seems that the roles of P-gp in resistance to DSP toxins should be re-evaluated in mussels. MXR defense in mussels is mediated by at least two ABC transporter types [20]. In addition to P-gp, other important ABC transporter proteins, for example MRPs have been demonstrated to exist in bivalves, which may also provide aquatic organisms with resistance to chemicals [20,37,51]. It appears to be a safety mechanism for organisms that multiple transporters with partly overlapping substrate specificities exist [52]. Luedeking et al. [51] for the first time proved the existence of MRP in M. edulis at the mRNA level. Luckenbach and Epel [20] observed activity and expression of MRP in gill of the California mussels. Navarro et al. [37] demonstrated that P-gp and MRP are constitutively expressed in larvae and adults of zebra mussel, and that they can be induced as cellular stress response. In addition, breast cancer resistance protein (BCRP) subfamily has also been found in the bivalves [53]. To further explore the resistance mechanism, further experiments with other P-gp-specific inhibitors VER and a broad-spectrum inhibitor for ABC transporters, CsA were performed. Similar to PGP-4008, VER had no effect on the DSP toxins accumulation in gill of P. viridis too, when the mussel was exposed to P. lima. However, the OA concentration in P. lima-exposed mussels with CsA was significantly

The mussel P. viridis P-gp closely matches MDR/P-gp/ABCB protein from various organisms. This ABCB transporter has a typical sequence organization as full transporters from the ABCB family. Increases in MXR activity, P-gp expression in gill tissues of P. viridis after exposure to P. lima, suggested that P-gp might play an important role in resistance mechanisms to DSP toxins. However, the addition of the broad-spectrum MDR modulator CsA but not of the specific P-gp inhibitors PGP-4008 or VER increased the DSP toxins accumulation in gill tissues of P. viridis, implying the complexity of resistance mechanisms. It is likely that besides P-gp, other ABC transporter, especially MRP might be involved in the resistance mechanisms. Acknowledgments This work was supported by the National Natural Science Foundation of China (40976065), the National Basic Research Program of China (973 Program) (2010CB428702) and Natural Science Foundation of Guangdong Province (9151063201000012). We also are grateful for the help provided by Neuroscience and Drug Innovation Research Joint Laboratory. References [1] Gerssen A, Pol-Hofstad IE, Poelman M, Mulder PP, Van den Top HJ, De Boer J. Marine toxins: chemistry, toxicity, occurrence and detection, with special reference to the Dutch situation. Toxins 2010;2:878e904. [2] Talarmin H, Droguet M, Pennec J, Schröder H, Muller W, Gioux M, et al. Effects of a phycotoxin, okadaic acid, on oyster heart cell survival. Toxicol Environ Chem 2008;90:153e68. [3] Bauder A, Cembella A. Viability of the toxic dinoflagellate Prorocentrum lima following ingestion and gut passage in the bay scallop Argopecten irradians. J Shell Res 2000;19:321e4. [4] Bauder A, Cembella A, Quilliam MA. Dynamics of diarrhetic shellfish toxins from the dinoflagellate, Prorocentrum lima, in the bay scallop, Argopecten irradians. In: Yasumoto T, Oshima Y, Fukuyo Y, editors. Harmful and toxic algal

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Please cite this article in press as: Huang L, et al., P-glycoprotein expression in Perna viridis after exposure to Prorocentrum lima, a dinoflagellate producing DSP toxins, Fish & Shellfish Immunology (2014), http://dx.doi.org/10.1016/j.fsi.2014.04.020

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