DSpace at VNU: Microcystin accumulation and biochemical responses in the edible clam Corbicula leana P. exposed to cyanobacterial crude extract

DSpace at VNU: Microcystin accumulation and biochemical responses in the edible clam Corbicula leana P. exposed to cyano...

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4Q1 Thanh-Luu Pham1,5,⁎ , Kazuya Shimizu2, Ayako Kanazawa1, Yu Gao3,

12

14 A R T I C L E I N F O

16 Article history:

17 Received 3 June 2015

18 Revised 10 September 2015

19 Accepted 15 September 2015

20 Available online xxxx

32 tissues The responses of biotransformation, antioxidant enzyme activity to CCE and the

33 fast elimination of MCs during depuration help to explain how the clam can survive for long

34 periods (over a week) during the decay of toxic cyanobacterial blooms in nature

35

© 2016 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences

36 Published by Elsevier B.V

37 Keywords:

38 Bioaccumulation

39 Cyanotoxins

40 Covalently bound microcystins

41 Aqueous extracts

42

43

55 encountered, microcystins (MCs), which are cyclic hepatotoxins

56 composed of seven amino acids with more than 80 structural

57 variants, are the most widespread and occur in up to 75% of CYB

58

59 their cellular uptake requires the activity of organic

anion-60

61 they can accumulate as a free form of MC or specifically

62 interact with protein phosphatases (PP1 and PP2A) in a

J O U R N A L O F E N V I R O N M E N T A L S C I E N C E S X X ( 2 0 1 6 ) X X X – X X X

⁎ Corresponding author

http://dx.doi.org/10.1016/j.jes.2015.09.018

1001-0742/© 2016 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences Published by Elsevier B.V

A v a i l a b l e o n l i n e a t w w w s c i e n c e d i r e c t c o m

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w w w e l s e v i e r c o m / l o c a t e / j e s

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67 (MacKintosh et al., 1990, 1995; Amado and Monserrat, 2010;

68 Lance et al., 2010, 2014)

83 1998; Wiegand et al., 1999; Beattie et al., 2003)

89 Pavagadhi et al., 2012, Sun et al., 2012) in toxicity studies; the

96 Falconer, 2007; Palíková et al., 2007; Smutná et al., 2014) It

111 et al., 2011)

117 (Hwang et al., 2004) During toxic CYBs, it may probably

123

In the present study, we examined the effects of a crude

124 extract of CYBs containing MCs on the freshwater edible clam C

125 leana P., as well as the accumulation and depuration of MCs by

126

127 water column from cyanobacterial cell lysis (often occur at the

128 end of a bloom), effect on aquatic life and to reveal the clam's

129 system of defense against MCs via the activity of the

antioxi-130 dant or detoxification enzymes CAT, SOD, and GST in various

131 organs (gills, foot, mantle, and remaining tissues)

132

1 Materials and methods

134 1.1 Rearing the organisms

135 Freshwater clams were collected at a freshwater fishery

experi-136 mental station in Oita Prefecture, Japan, and transported alive to

137 the laboratory The clams were introduced into sufficient aerated

138 50-L aquatic aquariums containing dechlorinated tap water and

139 with a 5-cm sand layer as the substrate Before the experiments,

140 clams were kept at a density of below 100 individuals per 50 L and

141 acclimatized for 1 month at a photosynthetic photon flux density

142

143 The water temperature was 22°C ± 1°C, pH 7.5 ± 0.3, and the

144 dissolved oxygen concentration7.9 ± 0.6 mg/L All of the

incuba-145 tion water was renewed every 3 days The clams were fed daily

146

147

148 weight of individual clams was 5.22 ± 0.79 g and the shell length

149 was 2.46 ± 0.57 cm

150 1.2 Preparation of cyanobacterial crude extract

151

152 (2001), with minor modifications Briefly, 4 kg wet weight of

153 bloom material (mainly Microcystis spp., collected from Lake

154

155 for 2 days and then thawed at room temperature After the

156 material had thawed completely, it was ice-cooled and sonicated

157 for 1 min This freeze–thaw–sonicate cycle was repeated four

158 times The samples were then centrifuged at 3000 g at 4°C for

159

30 min to remove cell debris The CCE supernatant was collected

160

161 Subsamples of CCE were used for MC analysis Briefly, CCE

162 was centrifuged at 6000 g at 4°C for 15 min The supernatant

163

164 MeOH The samples were analyzed by HPLC for MC

quantifica-165 tion MC-RR, MC-LR, and MC-YR (Wako, Osaka, Japan) were used

166

as standards The HPLC analysis showed that the CCE contained

167 three MC congeners, namely MC-RR (53%) and MC-LR (45%) and

168 the minor congener MC-YR (2%), at a total concentration of

169

170 1.3 Experimental set-up

171 Clams (240 individuals) were placed in eight aquariums

172 (30 clams per aquarium) containing 2 L distilled water and a

173 2-cm sand layer as a substrate, with constant aeration These

174 aquariums were kept at a photosynthetic photon flux density

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203 1.4 Extraction and analysis of MCs in incubation water

219 1.5 Extraction and analysis of unbound MC

221 (2007), with minor modifications Briefly, freeze-dried tissues

231 been preconditioned with 3 mL MeOH 100% and 10 mL ultrapure

232 water The column was first washed with 3 mL MeOH 20% and

233 then eluted with 3 mL MeOH 100% This elution fraction was

234 evaporated to dryness under reduced pressure at below 40°C

235

236

237 with duplicate analyses were used in this determination (n = 4)

238 1.6 Extraction of total MCs

239 Total MC (free- and Co-MC) was extracted as previously reported

240

by Neffling et al (2010), with minor modifications Briefly,

241 freeze-dried tissues were homogenized and trypsinated with

242

243 (pH 7.5) at 37°C for 3 hr; this was followed by oxidation with

244

245 temperature The reaction was quenched with sodium bisulfite

246 solution (40% w/v) until colorless at pH 2 with 10% sulfuric acid

247 After sample centrifugation (2000 g, 30 min, 4°C), the supernatant

248 was collected, diluted five times with ultrapure water, and then

249 applied to an Oasis HLB cartridge (60 mg, Waters Corp.) that had

250 been preconditioned with 3 mL MeOH 100% and 10 mL ultrapure

251 water The column was first washed with 3 mL MeOH 20%, and

252 then the 2-methyl-3-methoxy-4-phenylbutanoic acid (MMPB)

253 fraction, which is the product of MC oxidation, was eluted with

254

3 mL MeOH 80% The eluate fraction was evaporated to dryness

255

256

257

258

259 analysis The Co-MC content was thus estimated by subtracting

260 the free MC content from the total MC content 4-Phenylbutyric

261

262

263 Industries were used as external standards

264 1.7 GC–MS analysis

265

We used a DSQ II mass spectrometer linked to a Trace GC Ultra

266 gas chromatograph system (Thermo Scientific, Waltham, MA,

267 USA) equipped with an Rxi-5 ms column (30 m × 0.25 mm ID,

268 phase thickness 0.25 mm; Restek, Bellefonte, PA, USA) Helium

269 was used as the carrier gas at a flow rate of 1.5 mL/min (splitless

270 mode) The program used for the analysis was 80°C for 1 min

271 followed by an increase to 280°C at 8°C/min The other conditions

272 were as follows: ion source temperature 200°C, injection port

273 temperature 230°C, detector temperature 250°C, and interface

274 temperature 280°C Methylated 4-PB (me4-PB) and meMMPB were

275 detected by using SIM mode Ions at 91 and 104 m/z were selected

276 for me4-PB, and those at 75, 78, 91, 131, and 134 m/z for meMMPB

277 (Suchy and Berry, 2012) Xcalibur software was used for

278 quantitative analysis of these analytes Duplicate samples with

279 duplicate analyses were used (n = 4)

280 1.8 Enzyme extraction

281

282

al (2000), with minor modifications Briefly, samples (gill, foot,

283 mantle, remaining soft tissues) were homogenized in0.1 M

284 sodium phosphate buffer (pH 6.5) (1:5 w/v) containing 20% (V/V)

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299 1.9 Statistical analyses

310 2.1 Microcystin concentrations in incubation water

319 (Fig 2) There was no release of the unmetabolized parent

320 compound into the toxin-free water during the depuration

321 period

322 2.2 Uptake and depuration of free and Co-MC

323 There were no deaths in either of the groups of animals during

324 the experiments The control samples contained no MCs at

325 detectable concentrations (data not shown)

326 Extractable free MC accumulated in the clams during the

327

328 the free MC concentration in the whole clams increased rapidly

329

330 after about 1 day It then gradually declined over the rest of the

331 exposure period The free MC content was well correlated with

332 the concentration of MCs in the incubation water (r = 0.65,

333

P < 0.01) The Co-MC concentration increased slowly during the

334

335 DW) on day 11 It gradually declined thereafter

336 During the depuration period, free MC was quickly eliminated

337 from the clam tissues and below the limit of detection by

338 HPLC In contrast, the Co-MC concentration was enhanced on

339 the first day of depuration and then gradually declined,

340 although Co-MC was still detectable at the end of the depuration

341

342 2.3 Biotransformation enzyme activity

343

We measured GST activity in various tissues of both the exposure

344

345 significantly greater in the exposure group than in the control

346 group, but only on days 0.25, 1, 3, and 11 Significant elevation of

347 GST was also observed at days 10 and 11 in mantle In contrast,

348 GST activity in the foot was significantly lower in the exposure

349 group than in the control group on days 3, 5, 10, and 13, although

350

it had returned to the control level by the end of the experiment

351 GST activity in the remaining tissues did not differ significantly

352 over time between the two groups

acid 2-methyl-3-methoxy-4-phenylbutyric acid (MMPB) and its methyl ester

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353 2.4 Antioxidant enzyme activities

361 the two groups at any time In the remaining tissues SOD

362 activity was significantly greater in the exposure group than in

363 the control group, but only on days 0.25 and 5 Unlike the case

364 with GST, during the depuration period there were no

differ-365 ences in SOD activity between the two groups in any of the

366 tissues

367

368 activity in the gills was significantly greater in the exposure

369 group than in the control group, but only on days 0.25, 1, and

the uptake and depuration periods Arrow indicates the time of renewal of the MC concentration during the uptake period

0 20 40 60 80 100

Foot

0 20 40 60 80 100

Gills Control clams Treated clams

**

**

*

*

0 20 40 60 80 100

Mantle

0 20 40 60 80 100

Remaining tissues

Day

Day

(mg proteins)))

(mg proteins)))

(mg proteins)))

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0 10 20 30 40 50 60

Foot

0 10 20 30 40 50 60

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10

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Control clams Treated clams Gills

*

*** **

*

0

10

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Mantle

**

*

Day

Day

cyano bacterial bloom crude extract Asterisks indicate significant differences compared with controls at the respective time points

0

20

40

60

80

100

120

Control clams Treated clams Gills

0 20 40 60 80 100 120

Foot

**

*

0

20

40

60

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Mantle

0 20 40 60 80 100 120

Remaining tissues

*

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**

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*

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(mg proteins)))

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(mg proteins)))

(mg proteins)))

Day

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*

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388 Grützmacher et al., 2009; Wörmer et al., 2010; Ma et al., 2012;

389 Shimizu et al., 2011, 2012) In our experiment, the concentration

394 et al., 2008) However, we still understand little about the natural

407 (Gkelis et al., 2006), but they were much lower than those in

409 (Amorim and Vasconcelos, 1999, exposed to living cells of

415 (Yokoyama and Park, 2003; Chen and Xie, 2005; Vareli et al.,

428 Martins and Vasconcelos, 2009) By using an oxidation procedure

429

430 Neffling et al., 2010; Suchy and Berry, 2012), we provided evidence

431 for the existence and accumulation of Co-MC in C leana tissues

432 (Fig 2) On average, 0.5% dissolved MCs from incubation water

433 was bound in C leana during the 15-day experiment (data not

434 shown) However, the clam rapidly eliminated the MCs when

435

436 the total MC content in the mussel Mytilus edulis transferred to

437

438

439 shown that MC concentrations significantly decrease within

440

6 days of depuration in the clam Anodonta grandis simpsoniana

441 Also, immediate uptake and rapid release of MCs have been

442

443

444 found here that free MC rapidly began to be released when the

445 clam was transferred to toxin-free water, but the percentage of

446 bound MC increased (and reached 100% of the total MC content)

447

448 occurred due to the enhancement of the free MC binding to PPs

449

At the end of the 5-day depuration period, C leana tissues still

450

451 commonly judged to be rapid in mussel species, it is equally clear

452 that depuration is incomplete, even after a considerable period of

453

454 Co-MC levels should be considered in predictions of risk to higher

455 trophic organisms and humans

456 The long-term effects and accumulation of MCs have been

457

458 Palíková et al., 2003) and other zooplanktonic species (DeMott,

459 1999; Hulot et al., 2012) These studies all showed that MCs had

460

an inhibitory effect, mostly on growth, feeding and generally

461 survival of the experimental animals Continual oral exposures to

462 low doses of MCs have also shown chronic liver injury, but more

463 important is the possibility of carcinogenesis and tumor growth

464

465 that long-term exposure to even very low levels of MCs may be

466 significant, and could ultimately result in liver cancer and other

467 liver diseases in humans The current study revealed that the

468 toxin uptake by C leana from dissolved MCs is possible Despite

469 these relatively low levels, however, our results raise concerns

470 about chronic toxicity from a human health perspective, because

471 humans may be consuming clams contaminated with MCs, and

472 consumption of food contaminated with MCs could promote

473

474 dry weight to wet weight in the case of this clam; our results

475 showed that the total MC content of the clams exceeded the

476

477 Our results therefore suggest that C leana represents a health risk

478

to consumers when aquatic MC concentrations are high

479

It is well known that the family of GST enzymes is the most

480

481 found an elevation in GST activity in the gills during the first

482

3 days of exposure, suggesting that there was an immediate

483 response by the tissue to the CCE This response can be due either

484

to an increase in MC conjugation with GSH or to the detoxification

485

486 al., 2005) The higher GST activity in the exposure group

487 suggested that there was increased MC conjugation capability in

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513 Lushchak, 2011; Paskerová et al., 2012) Exposure of the

528

529

530

ofBurmester et al (2012), who found that SOD activity in two

531 bivalves, D polymorpha and Unio tumidus, was elevated in

532 various tissues after exposure with purified MC-LR or CCE

533

A far more controversial question concerns the adverse

534 effects of pure cyanotoxins, toxic living cells or CCE contains

535 MCs on CAT activity Elevation of CAT activity and other

536 antioxidant enzymes has been observed in the crab

hepato-537

538

539 (Gonçalves-Soares et al., 2012) In contrast, CAT activity was

540 significantly reduced, and SOD activity unchanged, in the crab

541 hepatopancreas after a 7 days' exposure to a high-dose M

542

543 activity in larvae of the bighead carp Hypophthalmichthys nobilis

544

is significantly reduced upon MC-LR exposure, suggesting that

545

546 clam, CAT activity in the mantle was significantly lower in the

547 exposure group than in the control group at the end of the

548 experiment, possibly because at that point the mantle was

549 less efficient than the gills and foot at neutralizing the impact

550

of oxidative stress In contrast, the reduction in CAT activity in

551 the foot toward the end of the exposure period could have

552 been due to the generation of superoxide radicals during

553 oxidative stress; these molecules have been reported to inhibit

554

555 depend not only on the dose and kind of toxin, the route of

556 exposure, and the duration of exposure, but also on the target

557

558 Kestemont, 2006; Pavagadhi et al., 2012; Sun et al., 2012)

559 Contrastingly, multixenobiotic resistance (MXR) in the

560 freshwater mussel D polymorpha is evidence of the insensitivity

561

562

563 (2000), who observed no deaths, malformations, or growth

564 inhibition in Xenopus laevis embryos exposed to purified MCs at

565

566

567

568

be elevated in response to cellular oxidative stress in animal cells

569 (Dias et al., 2009; Turja et al., 2014), and the increased rate of

570 synthesis of these antioxidant enzymes could be a plausible

571 explanation for the insensitivity following MC exposure in some

572

demon-573 strate that biochemical toxic effects are only temporary and that

574 prolonged exposure can lead to adaptation to cope with

575 deleterious effects The significant changes in GST, SOD, and

576 CAT activity that we found in C leana probably reflect

577 adaptation to oxidative conditions However, in toxin-free

578 water, both of the antioxidant enzymes and detoxification

579 enzyme showed adaptive responses at several time points

580 whereby enzyme activity was induced and then returned to

581 control levels The responses of antioxidant and detoxification

582 enzymes might thus contribute to the MC and cyanotoxin

583 tolerance of C leana

584 Many aquatic organisms live and reproduce in contaminated

585 waters, suggesting that they have ways to resist or tolerate

586

587 al., 1995) Exposure to toxins can trigger the MXR mechanism,

day))

Day 0

0.04

0.08

0.12

0.16

0.20

EDI TDI

0.04

Horizontal line indicates the maximum tolerable daily intake

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590 al., 2011).Contardo-Jara et al (2008)point out that the interactions

62 4 R E F E R E N C E S

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649

650 Byrne, M., Phelps, H., Church, T., Adair, V., Selvakumaraswamy, P.,

651 Potts, J., 2000 Reproduction and development of the freshwater

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653

654 Cazenave, J., Bistoni, M.D.L.A., Pesce, S.F., Wunderlin, D.A., 2006

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662 Chernoff, N., Hunter, E.S., Hall, L.L., Rosen, M.B., Brownie, C.F.,

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664

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