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Aquatic Toxicology 154 (2014) 12–18

Contents lists available at ScienceDirect

Aquatic Toxicology

j o ur na l ho me pag e: www.elsev ier .com/ locate /aquatox

,4-Dichlorophenoxyacetic acid alters intracellular pH and ionransport in the outer mantle epithelium of the bivalve Anodontaygnea

arco G. Alves, Pedro F. Oliveira ∗

ICS–UBI–Health Sciences Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6201-506 Covilhã, Portugal

r t i c l e i n f o

rticle history:eceived 19 January 2014eceived in revised form 15 April 2014ccepted 28 April 2014vailable online 6 May 2014

eywords:,4-Dichlorophenoxyacetic acid (2,4-D)nodonta cygnea L.uter mantle epitheliumransepithelial transportntracellular pH

a b s t r a c t

Bivalve molluscs, due to their sedentary mode of life and filter-feeding behavior, are very susceptible topollutant bioaccumulation and used as sentinel organisms in the assessment of environment pollution.Herein we aimed to determine the in vivo, ex vivo and in vitro effects of 2,4-dichlorophenoxyacetic acid(2,4-D), a widely used herbicide, in Anodonta cygnea shell growth mechanisms. For that, we evaluatedthe effect of 2,4-D (100 �M) exposure on the transepithelial short-circuit current (Isc), potential (Vt) andconductance (Gt), as well as on OME ion transport systems and intracellular pH (pHi). In vivo exposureto 2,4-D caused an increase of 50% on the Isc generated by OME and ex vivo addition of that compound tothe apical side of OME also induced an Isc increase. Furthermore, 2,4-D was able to cause a pHi increase inisolated cells of OME. Noteworthy, when 2,4-D was added following the exposure to specific inhibitors ofseveral membrane transporters identified as responsible for pHi maintenance in these cells, no significanteffect was observed on pHi except when the V-type ATPase inhibitor was used, indicating an overlap withthe effect of 2,4-D. Thus, we concluded that 2,4-D is able of enhancing the activity of the V-ATPases present

+

on the OME of A. cygnea and that this effect seems to be due to a direct stimulation of those H transporterspresent on the apical portion of the membrane of OME cells, which are vital for shell maintenance andgrowth. This study allows us to better understand the molecular mechanisms behind 2,4-D toxicity andits deleterious effect in aquatic ecosystems, with particular emphasis on those involved in shell formationof bivalves.

. Introduction

The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) is usedorldwide in forestry and agriculture since the 1940s. Nowadays

t is one of the most used worldwide herbicides. 2,4-D is a chem-cal with putative toxic activity and low biodegradability (Estrellat al., 1993). Besides, it has a high solubility in water and can easilyigrate in aquatic ecosystems (Chu et al., 2004). Thus, its release

nto the environment contributes to the chemical contamination ofhe aquatic ecosystems. Previous studies reported negative effectsf 2,4-D in aquatic animals (Greco et al., 2011; Lindsay et al.,

Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; BCECF-AM, 2′ ,7′-is-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein - Acetoxymethyl Ester; DIDS,,4′-diisothiocyanostilbene disulfonic acid; EPF, Extrapalleal fluid; Gt, Transepithe-

ial conductance; Isc, Transepithelial short-circuit current; SEM, standard error ofhe mean; OME, Outer mantle epithelia; Vt, Transepithelial potential.∗ Corresponding author.

E-mail address: pfobox@gmail.com (P.F. Oliveira).

ttp://dx.doi.org/10.1016/j.aquatox.2014.04.029166-445X/© 2014 Elsevier B.V. All rights reserved.

© 2014 Elsevier B.V. All rights reserved.

2010; Micic et al., 2004; Swinehart and Cheney, 1983). It has beendescribed that oocyte maturation in Xenopus laevis is blocked byexposure to 2,4-D (Stebbins-Boaz et al., 2004). Additionally, expo-sure to low concentrations of 2,4-D alters organ morphogenesis inXenopus laevis (Lenkowski et al., 2010) and high concentrations ofthis herbicide can induce 100% mortality in tadpoles (Morgan et al.,1996). Although it has been reported that in vivo exposure to 2,4-Dcauses serious genetic consequences to mussels (Micic et al., 2004),the molecular mechanisms altered by 2,4-D in aquatic ecosystemsremain largely unknown. For that purpose it is necessary to selectspecies that can act as biological biomarkers and instruments forbiomonitoring possible adverse effects of pollutants. The molluscspresent several prerequisites that make them ideal to be used asbioindicator species and for that reason they are frequently usedin ecotoxicology studies (Patetsini et al., 2013). Anodonta cygnea isa freshwater bivalve that has a wide geographical distribution and

specimens of this species are dominant in their habitat (Robillardet al., 2003). They are sedentary animals that live buried, feed byfiltration and can accumulate pollutants in their tissues becom-ing very valuable in the study of the biochemical effects induced

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y those substances (Hayer and Pihan, 1996; Kadar et al., 2001;obillard et al., 2003). There are several evidences that exposure ofhese animals to several pollutants decreases the time of filtration,hich affects the metabolic activity and the mussel behavior (Cope

t al., 2008; Falfushynska et al., 2012; Kadar et al., 2001).A. cygnea growth is associated and conditioned by the shell

rowth that results from the deposition of calcium carbonate onton organic matrix through a process known as biomineralizationLopes-Lima et al., 2005). The processes of biomineralization occurn the extrapalleal compartment, which is filled with the extrapal-eal fluid (EPF) and delimited by the shell itself and the mantle. The

antle has two distinct epithelia: the outer mantle epithelia (OME)nd the cavity mantle epithelia. The OME covers the metabolicallyctive face of the shell playing a key role in the genesis, growth andepair of the shell. These processes occur through the modulationf Ca2+ and H+ levels (Coimbra et al., 1992, 1993) in the EPF andn the secretion of organic components to the EPF. The ion trans-ort mechanisms present in the OME have been extensively studiedCoimbra et al., 1992, 1993, 1988; da Costa et al., 1999a,b; Machadot al., 1990; Oliveira et al., 2004). More recently, all this informa-ion was computed by Oliveira and collaborators (2008) creating a

athematical model as an analytical tool to elucidate the contri-ution of each ion transporter on the proton balance in the OME of. cygnea.

Herein, we study the effects of 2,4-D, a worldwide used herbi-ide, in the shell biomineralization processes of A. cygnea. For thaturpose, we investigated the in vivo, in vitro and ex-vivo exposure to,4-D and focused our work on the membrane ion transport mecha-isms of the OME cells, particularly on intracellular pH (pHi), sincehis parameter is pivotal to the biomineralization processes thatccur during shell genesis, growth and repair. The concentrationf 100 �M was chosen since it has been reported that freshwaterussels from the Unionidae family are acutely affected by 2,4-D at

oncentrations ranging from micromolar to milimolar concentra-ions (Milam et al., 2005).

. Materials and methods

.1. Chemicals

Pluronic F-127 and 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-arboxyfluorescein–acetoxymethyl ester was purchased fromolecular Probes (Eugene, Oregon, USA).2,4-Dichlorophenoxyacetic acid, concanamycin A, 4,4′-

iisothiocyanostilbene disulfonic acid, ouabain, amiloride andll other chemicals were purchased from Sigma-Aldrich (St. Louis,issouri, USA).

.2. Animals

A. cygnea specimens, collected in the North of Portugal, wereransferred to plastic tanks together with sediments to provideourishment to the animals. The tank water was continuously aer-ted and dechlorinated water was replaced weekly or when turbid.he experiments complied with the “Principles of Animal Care”,ublication n. 86.23, revised in 1985, of the National Institute ofealth and with the current laws of Portugal.

The mussels were acclimatized for at least 15 days before beingsed. After that period, they were considered healthy if the innerurface of the shell was smooth and shiny, and if they closed theiralves when disturbed.

.3. In vivo experiments

After acclimatization the mussels were divided in 2 groups, 12nimals in each, in plastic tanks containing 5 L of dechlorinated

oxicology 154 (2014) 12–18 13

water either with 2,4-D (100 �M) or the solvent used (0.005%ethanol). After 15 days exposure, the OME of A. cygnea was diss-ected and mounted in an Ussing chamber according to standardmethods (Oliveira et al., 2004). The preparations were mounted inpairs from the same animal and bathed in a control solution (inmmol/L: NaCl, 9; KCl, 3; MgCl2, 0.5; CaSO4, 1; NaHCO3, 10). Whengassed with carbogen (95% O2 and 5% CO2) and saturated withwater vapor the solution had a pH of 7.05. After a steady state periodof at least 10 min, the short-circuit current (Isc), transepithelialpotential (Vt) and transepithelial conductance (Gt) were measuredand plotted as average ± standard error of the mean (SEM) of 10readings.

2.4. Ex-vivo experiments on OME

The OME of A. cygnea were dissected and mounted as previ-ously described (Alves and Oliveira, 2013; Oliveira et al., 2004) andthe preparations from each animal were mounted in pairs. The Isc

was measured and, after stabilization, activated as described previ-ously (da Costa et al., 1999a). In brief, the bathing control solutionwas replaced by an activation solution (in mmol/L: NaCl, 9; KCl, 3;MgCl2, 0.5; CaCl2, 1; Sodium succinate, 10; CaSO4, 1; pH 4.0). After20 min, the activation solution was replaced by the control solu-tion. The current was left to stabilize and after a steady state of atleast 10 min, the effect of 2,4-D (100 �M) was studied by indepen-dently adding this compound to both the basolateral (hemolymph)and apical (shell) side, so that they serve as mutual controls. Pre-liminary experiments showed that 0.005% ethanol has no effect inIsc (data not shown), thus 2,4-D stock solutions were prepared inethanol so that the final concentration of solvent in the chamberwould not reach 0.005%. Isc was measured every minute and thenplotted as the average ± SEM of the fractional values calculated bydividing each value by the Isc acquired at time zero. SpontaneousVt and Gt values were calculated as the average of the 10 initialreadings (immediately before addition of 2,4-D) or the average ofthe last 10 readings of each experiment (180 min after addition of2,4-D). In this preparation the I/V curve is linear at least between−100 and +100 mV.

2.5. Cell isolation of OME and viability test

The OME was dissected from the mussels and rinsed twice inAnodonta Ringer (in mM: NaCl 9, KCl 3, MgCl2 0.5, CaCl2 1, HEPES10, Manitol 20, pH 7.4) with Anodonta Ringer and the epitheliumscraped to obtain a cellular suspension. The suspension was cen-trifuged at 500 × g for 5 min and the pellet resuspended in AnodontaRinger.

Cell viability was tested by the Trypan Blue test. Incorporationof the dye Trypan Blue (0.1% w/v in Anodonta Ringer) showed thatonly a minority (5 ± 1%) of cells incorporated the dye.

2.6. Intracellular pH measurements

Intracellular pH was measured using a fluorescent probe.The cellular suspension was incubated in Anodonta Ringer with0.03% (w/v) Pluronic F-127 and 10 �M BCECF-AM (2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein–acetoxymethylester) for 60 min at room temperature. After this period, the cellularsuspension was centrifuged at 500 × g for 5 min and the pellet wasresuspended in Anodonta Ringer. The cells were then transferredto an imaging chamber (Harvard Apparatus model RC-20 H) andthe fluorescence intensities at 490 (F490) and 440 nm (F440), with

an emission wavelength of 515 nm, were measured every 3 s, withan epifluorescence system (DeltaRam, PTI) while the cells wereperfused with the different experimental solutions. The back-ground fluorescence was measured at the end of each experiment

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nd the ratios (F490/F440) were calculated after subtracting theackground fluorescence intensities for each measurement at eachavelength. The calibration procedures were done as described

y Oliveira and collaborators (Oliveira et al., 2009a,b). To studyhe effect on OME pHi of specific membrane transport systems,everal specific inhibitors were used: Concanamycin A (5 �M),

specific inhibitor of the V-type ATPases (Drose and Altendorf,997), 4,4′-diisothiocyanostilbene disulfonic acid (DIDS) (500 �M),n inhibitor of several bicarbonate membrane transporters usuallylassified as acid extruders, Na+-driven and Na+-independentCO3

−/Cl− transporters and Na+/HCO3− co-transporters (Boron,

001; Grassl and Aronson, 1986; Helbig et al., 1988); Ouabain100 �M), a specific inhibitor of the Na+–K+ pump (Schneider et al.,998a; Shimizu et al., 1983); and Amiloride (50 �M), an inhibitorf the Na+/H+ exchangers (Ahearn et al., 1994; Delvaux et al., 1990;ood et al., 1995).

.7. Statistical analysis

The statistical significance between the different experimentalroups was assessed by one-way ANOVA, followed by Bonferroniost-test using GraphPad Prism 5 (GraphPad Software, San Diego,A, USA). For time course experiments (Isc and pHi), the statisticalignificance was assessed by comparing the averages of 10 mea-urements after the steady state was attained. All experimentalata are shown as mean ± SEM (n = 6 for each condition). p < 0.05as considered significant.

. Results

.1. In vivo exposure to 2,4-D increases the transepithelial Isc ofhe OME

When submitted to short-circuit conditions, the OME of A.ygnea is capable of generating an ionic current, largely due tohe action of a V-type H+-ATPase (da Costa et al., 1999a), which isssociated with the calcium movements (deposition/dissolution)hat occur during shell mineralization (Coimbra et al., 1993). In

ur experiments, the OME was mounted on Ussing-type cham-ers generating a spontaneous transepithelial potential differenceVt) of 13.5 ± 1.9 mV and, when short-circuited, an average pos-tive current (Isc) 9.5 ± 1.4 �A/cm2 from the hemolymph to the

ig. 1. Effect of 15-day in vivo exposure to 2,4-D (100 �M) on the short-circuit current (IGt) (panel C) of the OME of Anodonta cygnea. Values are plotted as mean ± SEM of 10 reifferent results (p < 0.05) are as indicated: c—vs. to control.

oxicology 154 (2014) 12–18

shell side (Fig. 1). The transepithelial conductance of the tissuewas 1.01 ± 0.11 mS/cm2. The in vivo exposure to 2,4-D (100 �M)caused a significant increase (One-way ANOVA, p < 0.001) of 50%on the transepithelial Isc generated by the OME (Fig. 1A) and of40% on the transepithelial conductance (One-way ANOVA, p < 0.01)(Fig. 1C). Nevertheless, no significant alteration was observed onthe other electrophysiological parameters measured (Vt) (Fig. 1B).Importantly, no mortality was observed after 2,4-D exposure andmussel survival was not affected during the in vivo treatment.

3.2. Ex-vivo addition of 2,4-D to the apical side of OME inducesan increase on Isc

In the ex-vivo experiments, the OME was mounted on Ussing-type chambers, generating a spontaneous transepithelial potentialdifference (Vt) of 15.9 ± 1.7 mV. After short-circuited and acti-vated, the OME generated an average positive current (Isc)34.4 ± 1.8 �A/cm2 from the hemolymph to the shell side. Thetransepithelial conductance was 1.11 ± 0.09 mS/cm2. When 2,4-D(100 �M) was added to the hemolymph side, no significant effecton the Isc was observed (Fig. 2A). On the other hand, when 2,4-Dwas added to the shell side, the Isc increased significantly about10% (One-way ANOVA, p < 0.05). This effect took nearly 150 min tooccur. Nevertheless, it cannot be due to endogenous alterations ofthe tissue since previous studies reported that the Isc of the prepa-ration is stable for at least 4 h (da Costa et al., 1999b). In theseexperiments, the addition of 2,4-D (100 �M) had no significanteffect on the OME transepithelial conductance, when added to thehemolymph or to the shell side (Fig. 2B).

3.3. 2,4-D causes an increase of intracellular pH in OME cells

The effect of 2,4-D (100 �M) on the pHi of OME cells was assayedusing an intracellular fluorescent probe (BCECF). In our experiment,isolated cells from the OME were equilibrated in Anodonta Ringeruntil a steady state of at least 180 s was reached, and then exposedto Anodonta Ringer plus 2,4-D (100 �M). A significant increase of

pHi (0.09 ± 0.01 units) (Fig. 3) was observed when 2,4-D was addedto the perfusion solution (One-way ANOVA, p < 0.001). This effectwas not reverted when the compound was removed from the exter-nal solution (Fig. 3).

sc) (panel A), spontaneous transepithelial potential (Vt) (panel B) and conductanceadings after the steady state of at least 10 min was observed (n = 12). Significantly

M.G. Alves, P.F. Oliveira / Aquatic Toxicology 154 (2014) 12–18 15

Fig. 2. Effect of 2,4-D (100 �M) on the short-circuit current (Isc) (panel A) and conductance (Gt) (panel B) of the OME of Anodonta cygnea. 2,4-D was added to the hemolymph(n = 6) or shell side (n = 6) of the epithelium after a steady state of 20 min was obtained. Isc

last 10 readings of each experiment. Arrow indicates the instant of addition of 2,4-D. Dashc—vs. control.

Fig. 3. Effect of 2,4-D (100 �M) on intracellular pH (pHi) of OME cells. The firstvertical dotted line represents the addition of 2,4-D and the second vertical dot-ted line represents the removal of 2,4-D of the external perfusion solution. pH isrd

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epresented as mean ± SEM (n = 24). Dashed line represents the SEM. Significantlyifferent results (p < 0.05) are as indicated: c—vs. control.

.4. Inhibition of the OME V-ATPase causes an increase onntracellular pH that is suppressed by the action of 2,4-D

To determine the effect of 2,4-D (100 �M) on the activity ofey transporters responsible for pHi maintenance on isolated OMEells, the combined effect of 2,4-D and of specific membrane trans-ort system inhibitors was tested. Thus, we performed assayshere the addition of this compound followed the use of specific

nhibitors of the most important membrane transporters identifiedn the A. cygnea OME. The cells were first perfused with Anodontainger until a steady state of at least 180 s was reached. After-ard, OME cells were perfused with a solution containing a specific

nhibitor (Concanamycin A, Amiloride, Ouabain or DIDS), until ateady state of at least 60 s was reached, and then they were per-used with a solution containing 2,4-D plus the specific inhibitor inest. The results are presented as the average of at least 10 mea-urements (Fig. 4).

The specific V-ATPase inhibitor Concanamycin A (5 �M) induced significant decrease on pHi of 0.13 ± 0.01 units (Fig. 4A) (One-ay ANOVA, p < 0.001). When OME cells were perfused with 2,4-D

100 �M) in the presence of Concanamycin A no significant alter-tion on pHi was observed (Fig. 4A) (One-way ANOVA, p < 0.001).

he addition of Amiloride (50 �M) caused a significant decrease onHi of 0.12 ± 0.01 units (One-way ANOVA, p < 0.001) (Fig. 4B). A sig-ificant effect was also observed when 2,4-D was added togetherith Amiloride (One-way ANOVA, p < 0.001). The addition of

is plotted as the fractional value (mean ± SEM) and Gt as the average (±SEM) of theed line represents the SEM. Significantly different results (p < 0.05) are as indicated:

2,4-D together with this inhibitor caused a significant recovery inpHi of 0.06 units (Fig. 4B) (One-way ANOVA, p < 0.01). When addedto isolated OME cells, Ouabain (100 �M) diminished the pHi, caus-ing a significant variation of 0.12 ± 0.02 units (Fig. 4C) (One-wayANOVA, p < 0.001). Likewise, when 2,4-D (100 �M) was added inthe presence of Ouabain, the pHi recovered 0.04 units (Fig. 4C)(One-way ANOVA, p < 0.05). DIDS (500 �M) caused the most strik-ing effect on the pHi (a significant decrease of 0.22 ± 0.01 units;One-way ANOVA, p < 0.001) (Fig. 4D), highlighting the involvementof bicarbonate transport systems in the pHi regulation in OME cellsof A. cygnea. When 2,4-D (100 �M) was added in the presence ofDIDS a significant recovery of 0.07 ± 0.01 units of pHi was observed(Fig. 4D) (One-way ANOVA, p < 0.01).

4. Discussion

In shell-forming animals such as A. cygnea, the shell growthresults from calcium carbonate deposition onto an organic matrix,by mechanisms that are finely regulated by OME action. This epithe-lial structure covers the metabolically active surface of the shell andplays a key role on its genesis, growth and maintenance through thecontrol of composition of the fluid that fills the extrapalleal cavity(Coimbra et al., 1992, 1993). A. cygnea must maintain an overallacid–base balance since the deposition of CaCO3 is controlled bythe release of acid equivalents. Alterations in the movements ofH+/HCO3

− across the OME, and consequently in the pHi of OMEcells, affect these processes of shell deposition (Byrne and Dietz,1997; Machado et al., 1988). It has been reported that some envi-ronmental chemicals can disrupt the mechanisms responsible forthe homeodynamics of shell deposition, causing shell thickening ofA. cygnea, due to an imbalance in H+/HCO3

− transport across theOME (Machado et al., 1988, 1989).

2,4-D is one of the most intensively used pesticides/herbicidesin the world in the control of broadleaf weeds and readily dissolvesin water (Environment Canada, 2011). Due to its biodegradability,there may be a great variability in the 2,4-D loading of wastew-aters, as industrial effluents can contain up to 2 mM of 2,4-D(Buenronstro-Zagal et al., 2000; Chin et al., 2005). Data indicatethat bivalves from the Unionidae family are acutely affected byconcentrations of this herbicide within the micromolar to millimo-lar range (Milam et al., 2005). It has also been reported that 2,4-D

might interfere with the activity of specific acid–base transporterspresent in the plasma, vacuolar and mitochondrial membranes(Fernandes et al., 2003; Lohse and Hedrich, 1992; Palmeira et al.,1994), although their influence on proton/bicarbonate membrane

16 M.G. Alves, P.F. Oliveira / Aquatic Toxicology 154 (2014) 12–18

Fig. 4. Variation of intracellular pH (�pHi) of isolated cells of the OME of Anodonta cygnea after exposure to 2,4-D (100 �M) together with Concanamycin A (5 �M) (panelA nel D)a EM (ni the co

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), Amiloride (50 �M) (panel B), Ouabain (100 �M) (panel C) or DIDS (500 �M) (pand a steady state of at least 10 min was observed. �pHi is plotted as the mean ± Snhibitor only. + indicates the presence of the compound; indicates the absence of

ransporters described in the OME of bivalves, and particularly inhe OME of A. cygnea, has never been determined. Interestingly,ecent studies have highlighted that membrane transporters,uch as the Na+/H+ exchanger (NHE), mediate the effects of tox-cants (e.g., metals) on signal transduction pathways in musselsKoutsogiannaki et al., 2006, 2014; Koutsogiannaki and Kaloyianni,011).

Herein, we investigated the effects of in vivo, in vitro and ex-vivoxposure to 2,4-D 100 �M in the shell biomineralization processesccurring on the OME of A. cygnea. This concentration was cho-en based on the reported toxicity of 2,4-D for bivalves from thenionidae family (Milam et al., 2005) and on the availability of

his pesticide in wastewater (Chin et al., 2005). In vitro experi-ents using isolated cells allowed us to investigate the effects

f 2,4-D on specific characteristics and functional activities of theME cells (e.g., ion transport and pHi regulation), with full con-

rol of the experimental conditions. Ex vivo approach using thesolated intact OME facilitated investigations at tissue level, withells maintaining their interactions with the surrounding extracel-ular environment (which may have critical implications for theirehavior and responsiveness), while allowing a tight control of thexperimental conditions. Finally, in vivo approaches provided usith valuable information when evaluating whole-body responses

nd alterations due to the action of 2,4-D, particularly when study-ng more prolonged exposure periods as was the case in ourxperiments. When A. cygnea specimens were exposed in vivo for5 days to 2,4-D, we observed a significant increase of 50% on the Isc

roduced by the OME. This outcome is mainly due to an increase inhe transepithelial flux of H+ ions toward the extrapalleal compart-

ent where shell mineralization occurs, evidencing that exposureo 2,4-D may cause alterations in the calcium deposition equilib-ium, displacing it toward demineralization. The observed increasen Isc corresponds to a rise in H+ transport toward the EPF and aonsequent decrease on the fluid pH, which has been previouslyssociated with an increase of shell reabsorption (Coimbra et al.,993).

It has been reported that 2,4-D is capable of stimulating inivo the V-ATPase present in Saccharomyces cerevisiae vacuolar

embrane, in coordination with a marked activation of plasmaembrane H+-ATPase (Fernandes et al., 2003). Likewise, 2,4-D

timulated the activity of H+-ATPases present in the stoma of Viciaaba, which are responsible for the regulation of its opening (Lohse

. �pHi was calculated as the shift on pHi after the addition of a specific compound = 6). Significantly different results (p < 0.05) are as indicated: *—vs. control; #—vs.mpound.

and Hedrich, 1992). A stimulatory effect of 2,4-D on the activity ofA0A1 synthetases (Lemker et al., 2001; Palmeira et al., 1994), whichhave pronounced functional and structural similarities with the V-ATPases of eukaryotes (Gruber et al., 2001), has also been reported.Thus, it could be expected that 2,4-D could stimulate the V-ATPasepresent in the apical membrane of OME cells and thus provoke anincrease in Isc, similar to what happens in other biological systems.Indeed, our ex vivo results clearly support that assumption, sug-gesting that 2,4-D acts on the V-ATPase, previously identified onthe OME (da Costa et al., 1999a). The ex vivo effects of 2,4-D onOME showed that the Isc generated by that tissue was significantlyhigher when that compound was added to the apical side of thetissue. This stimulatory effect of 2,4-D when added to the apicalside of the OME may be due to one of two distinct mechanisms: (1)increased intracellular H+ concentration or (2) increased V-ATPasefunctioning. Noteworthy, there was an increase in conductance thatmight be explained by an increase in the transcellular ion transportand also in the paracellular pathway conductance, since the totalconductance of an epithelium is the sum of the transcellular and theparacellular pathway conductance. Taking advantage of the math-ematical model, developed by Oliveira and collaborators (2008),that attempts to describe the cellular mechanisms underlying theoperation of the OME, we were able to have a better understand-ing over the 2,4-D action on the Isc. This analytical tool was builtin a modular fashion, enabling the simulation of several distinctexperimental situations, and can be used to show the internalcoherence of the proposed qualitative models based on the exper-imental data. As referred above, 2,4-D stimulated the Isc generatedby the OME in nearly 50%. According to the mathematical model, apartial stimulation (10–15%) of the V-ATPase would fit the kineticsand magnitude of the observed effect. If 2,4-D action resulted inthe direct stimulation of the V-type H+-ATPase activity, the intra-cellular free concentration of H+ would decrease and consequentlythe pHi would increase. On the other hand, if 2,4-D resulted in adirect increase of the free H+ concentration (with the concomitantdecrease on pHi), it would also be followed by an increase of theH+-ATPase activity and consequently of Isc. Combining the in vivoand ex vivo experimental data obtained with the computed effects

of 2,4-D, these are the two most possible and plausible explana-tions for 2,4-D effect in the Isc generated by the OME (Fig. 5). So, inorder to fully disclose such mechanisms, we investigated the effectof 2,4-D on the pHi of isolated cells of OME.

M.G. Alves, P.F. Oliveira / Aquatic T

Fig. 5. Schematic representation of the membrane transport systems present inthe outer mantle epithelium (OME) cells of Anodonta cygnea. Our results provideem

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ology of the mantle of Anodonta cygnea. J. Exp. Biol. 140, 65–88.

vidence that 2,4-D is able to modulate membrane H+ transport and pHi throughodulation of the V-ATPase activity.

When added to isolated OME cells of A. cygnea, 2,4-D caused aignificant increase in pHi. This result supports the hypothesis of V-TPase stimulation of OME cells and not a direct effect on the free H+

oncentration. This is in clear agreement with the kinetics observedor the mathematical simulation of an effect of 2,4-D on V-ATPasectivity, pointing toward a direct effect on that membrane trans-orter. Additionally, to evaluate if this compound exerted its effecty means of any of the transporters described on OME cells, wesed an experimental protocol where 2,4-D effect was evoked afterhe addition of specifics inhibitors and together with that inhibitor.oncanamycin A is a specific inhibitor of the V-type ATPases (Drosend Altendorf, 1997), which when added alone causes a decreasen pHi of OME cells (due to retaining H+ ions inside the cell), bynhibiting the H+-ATPase present on the apical membrane of theseells (da Costa et al., 1999a; Oliveira et al., 2004). In our exper-ments, the addition of 2,4-D after exposure to Concanamycin Aaused no significant effect on OME cells pHi, suggesting an over-ap of the effect of these two compounds on the pHi of OME cells.

e can therefore infer that 2,4-D seems to have a direct effect onhe H+-ATPase inhibited by Concanamycin A.

On the other hand, when using 2,4-D together with Amiloride striking effect on pHi was observed. Amiloride is an inhibitor ofhe Na+/H+ antiporter (Grinstein and Smith, 1987), which is knowno be present on the basolateral membrane of OME cells (Coimbrat al., 1988). This antiporter is directly involved on the movementsf H+ ions and its inhibition causes an intracellular H+ accumula-ion and consequent decrease in pHi. When we further perfusedME cells with 2,4-D and Amiloride, the decrease in pHi caused bymiloride was partly recovered. A similar result was obtained whenerfusing cells with 2,4-D in the presence of a specific inhibitorf the Na+/K+ pump, Ouabain (Schneider et al., 1998b). The pres-nce of the Na+/K+ pump has been described on the basolateralortion of the membrane of OME cells (da Costa et al., 1999b). Theecrease on Na+ efflux from the cell caused by inhibition of thea+/K+ pump, leads to a decrease of the Na+/H+ antiporter trans-ort rate (causing an increase on intracellular H+) and a decrease ona+-dependent HCO3

−/Cl− co-transporter transport rate (causing decrease on HCO3

− influx) (Oliveira et al., 2008). Subsequently,hese two effects result in a rise of free H+ concentration and, hence,

pHi decrease similar to the one observed. When using 2,4-D anduabain, we could also observe that 2,4-D caused a pHi recoveryhen added after this specific inhibitor of the Na+/K+ pump. This

oxicology 154 (2014) 12–18 17

suggests that the transporters inhibited by Ouabain do not suf-fer a 2,4-D-related effect. Finally, when we used DIDS, a specificinhibitor of the Na+-dependent and Na+-independent HCO3

−/Cl−

co-transporters (Boron and Boulpaep, 1983), which have beendescribed on the basolateral portion of the OME by Coimbra et al.(1988) and Barrias (2003), we observed a significant decrease onpHi that should be the result of a decrease on the influx of HCO3

−

to the cell and subsequent pHi decrease (Oliveira et al., 2008).When OME cells were perfused with 2,4-D and DIDS a recoveryof pHi was observed, evidencing an independent effect of thesetwo compounds. Thus, these results indicate that 2,4-D does nothave a direct effect on the Na+/H+ exchangers, Na+/K+ pump orNa+-dependent and Na+-independent HCO3

−/Cl− co-transporterspresent on OME cells membrane, as these specific inhibitors do notabolish the effect of 2,4-D on pHi.

In conclusion, the herbicide 2,4-D is able to modulate mem-brane H+ transport and pHi (via modulation of the activity of aV-ATPase) in the OME of A. cygnea. Those mechanisms are essen-tial for shell maintenance and growth. Thus, our results allow usto further understand the molecular mechanisms underlying 2,4-Dtoxicity and its deleterious effect in aquatic ecosystems, particu-larly on shell formation of bivalves. There is an urgent need for thistype of studies, as the majority of the chemicals in commercial usehave not been systematically tested for environmental safety orscreened for their possible implications in human health.

Acknowledgments

This work was supported by the “Fundac ãopara a Ciên-cia e a Tecnologia”–FCT (PEst-C/SAU/UI0709/2011) co-funded byFundo Europeu de Desenvolvimento Regional–FEDER via ProgramaOperacional Factores de Competitividade–COMPETE/QREN. M.G.Alves (SFRH/BPD/80451/2011) was funded by FCT. P.F. Oliveira wasfunded by FCT through FSE and POPH funds (Programa Ciência2008).

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