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Aquatic Toxicology 142– 143 (2013) 96– 103

Contents lists available at ScienceDirect

Aquatic Toxicology

jou rn al hom epage: www.elsev ier .com/ locate /aquatox

ifferences in retinoid levels and metabolism among gastropodineages: Imposex-susceptible gastropods lack the ability to storeetinoids in the form of retinyl esters

anuel Gestoa,∗, L. Filipe C. Castroa,∗∗, Miguel Machado Santosa,b,∗ ∗ ∗

CIMAR/CIIMAR – Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Rua dos Bragas 289, 4050-123 Porto, PortugalFCUP – Dept. of Biology, Faculty of Sciences, University of Porto, Rua do Campo Alegre, 4169-007 Porto, Portugal

r t i c l e i n f o

rticle history:eceived 22 May 2013eceived in revised form 29 July 2013ccepted 1 August 2013

eywords:etinoidsetabolismastropods

mposexitamin Aetinyl esters

a b s t r a c t

The presence of a complex retinoid system was long believed to be a chordate/vertebrate novelty. How-ever, recent findings indicate otherwise since the gastropod mollusk Osilinus lineatus was found to havethe capacity to store retinoids in the form of retinyl esters (REs), a key feature to maintain a homeostaticcontrol of retinoid levels. Here, we investigated whether such a complex retinoid system is widely dis-tributed among gastropod lineages. Additionally, since one of the most spectacular examples of endocrinedisruption in the wild, the masculinization of female gastropods (imposex) by the retinoid X receptor(RXR) agonist, tributyltin (TBT), has been linked with perturbed retinoid signaling, we also investigatedif retinoid storage mechanisms in the form of retinyl esters were present in imposex-susceptible gas-tropods. Initially, we determined the presence of both polar (active retinoic acid isomers) and nonpolarretinoids (retinol, REs) in selected gastropod species: the limpet Patella depressa and the imposex-susceptible whelks Nucella lapillus and Nassarius reticulatus. Although all species presented active retinoidforms, N. lapillus and N. reticulatus were shown to lack nonpolar retinoids. The absence of REs, which arethe common retinoid storage form found in vertebrates and in O. lineatus suggest that those species are

unable to use them to maintain a homeostatic control of their retinoid levels. In order to further clarify theretinoid metabolic pathways in imposex-susceptible gastropods, a retinoid exposure study was carriedout with N. lapillus. The results demonstrate that although N. lapillus is able to metabolize several retinoidprecursors, it lacks the capacity to store retinoids as REs. Whether the lack of retinoid storage mecha-nisms in the form of REs in imposex-susceptible gastropods plays an important role in the susceptibility

addi

to RXR agonists warrants

. Introduction

Retinoids include a group of compounds chemically and/or func-ionally related to retinol (vitamin A). In vertebrates, they play keyoles in different physiological functions including cell differenti-tion, organogenesis, embryonic growth and development, tissueomeostasis, reproduction, immune function or vision (Gutierrez-

azariegos et al., 2011; Kane, 2012; Theodosiou et al., 2010). Since

nimals cannot synthesize retinoids de novo, these are obtainedrom the diet directly in the form of retinoids (retinol or retinyl

∗ Corresponding author. Permanent address: Laboratorio de Fisioloxía Animal,acultade de Bioloxía, Universidade de Vigo, Campus As Lagoas-Marcosende, 36310igo, Spain. Tel.: +34 986 812386; fax: +34 986 812556.∗∗ Corresponding author. Tel.: +351 223 401856.

∗ ∗Corresponding author. Tel.: +351 223 401812.E-mail addresses: manuelgesto@uvigo.es (M. Gesto), filipe.castro@ciimar.up.pt

L.F.C. Castro), santos@ciimar.up.pt (M.M. Santos).

166-445X/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.aquatox.2013.08.001

tional studies.© 2013 Elsevier B.V. All rights reserved.

esters) or in the form of retinoid precursors such as carotenoids(Theodosiou et al., 2010, for a review). Both lack and excess of activeretinoid forms are deleterious to animals, especially during devel-opment (Maden, 2007). Thus, vertebrates are able to maintain ahomeostatic control of active vitamin A by storing and mobilizing itwhen needed. Hence, different retinoid forms within the organismaccomplish different functions (Theodosiou et al., 2010). Retinoicacid (RA) is the main active form, acting through the specific bind-ing to nuclear receptors (retinoic acid receptors – RARs and retinoidX receptors – RXRs). Retinaldehyde, an intermediate metabolite,also has an active role in the visual cycle. Retinol is the main trans-port form, which travels through circulation to the different tissues.Storage is mainly performed in the form of retinyl esters (REs).

The presence of the RA machinery (i.e., RAR, Aldh1a, Cyp26) wasthought to be a deuterostome novelty (Simões-Costa et al., 2008).

However, a search of the unpublished genomes of lophotrochozoanspecies (e.g., mollusks and annelids) revealed the presence of keymolecular players of the RA machinery in protostomes (Albalatand Canestro, 2009; Campo-Paysaa et al., 2008). Supporting these

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ndings, an increasing body of studies points to a functional rolef retinoids in invertebrates (Sternberg et al., 2008, 2010); addi-ionally, the presence of active retinoids has been demonstrated inome non-chordates (Albalat, 2009; Biesalski et al., 1992; Crétont al., 1993; Dmetrichuk et al., 2008; Gesto et al., 2012a; Nowickyjt al., 2008).

Interestingly, the retinoid signaling pathways seem to be ofxtreme importance from a toxicological point of view since theyre a prime target of the ubiquitous organotins, tributyltin (TBT)nd triphenyltin (TPT). Both compounds have been found to beigh affinity ligands for RXRs in vertebrates and invertebratesNishikawa et al., 2004). The inappropriate modulation of RXR byBT seems to be involved in one of the most spectacular examplesf endocrine disruption: the phenomenon of imposex in femalerosobranch gastropods (superimposition of male secondary sex-al characteristics onto females) (Castro et al., 2007; Lima et al.,011; Nishikawa et al., 2004; Santos et al., 2000, 2006; Stanget al., 2012). Imposex has been linked with population decline andocal extinction of several neogastropod species in areas with highrganotin levels (Gibbs and Bryan, 1986; Hallers-Tjabbes et al.,994), whereas other gastropod groups seem to be less affectedy those compounds. Exploring the nature and complexity of theetinoid pathways among different gastropod clades can shed lighto the specific sensitivity of neogastropods to organotins.

Despite the detection of retinoids in some invertebrate species,t was believed that retinoid metabolism and in particular the

echanisms of retinoid storage in the form of REs were a chor-ate/vertebrate novelty (Albalat, 2009). Such mechanisms are ofxtreme relevance since they provide the capacity to control theevels of active retinoids within the organism. We have recentlyemonstrated for the first time (Gesto et al., 2012a) the presencef a complex retinoid system, including REs-storage capacity, inn invertebrate, the gastropod mollusk Osilinus lineatus (da Costa,778) (Subclass Vetigastropoda, Fam. Trochidae). Our results sug-est that retinoid metabolism and the capacity to maintain aomeostatic control of retinoid levels might have arisen in the com-on ancestor of all Bilateria (Gesto et al., 2012a). It remains unclear

t present if the presence of REs is restricted to O. lineatus or, on thether hand, is a common feature among gastropods.

In this study we assessed the presence of storage and activeetinoids in additional gastropod species, focusing on distinctvolutionary lineages, including the imposex-susceptible groupf Caenogastropoda, as a proxy to investigate whether complexetinoid systems are frequent in this class of invertebrates andlso, whether TBT-sensitive species have in fact different traitsn their retinoid content or metabolism in comparison with otherroups of gastropods. We evaluated the presence of retinoids in theimpet Patella depressa Pennant, 1777 (Subclass Patellogastropoda,am. Patellidae), in the dogwhelk Nucella lapillus (Linnaeus, 1758)Subclass Caenogastropoda, Fam. Muricidae) and in the net-ed dogwhelk Nassarius reticulatus (Linnaeus, 1758) (Subclassaenogastropoda, Fam. Nassariidae). Considering that caenogas-ropods lacked nonpolar retinoids, an in vivo exposure experimentas also carried out with N. lapillus to clarify the retinoid metabolic

haracteristics in this imposex-sensitive species. The overall find-ngs are discussed with respect to the current knowledge on thevolution of retinoid metabolism in invertebrates and to their pos-ible involvement in the sensitivity to RXR agonists.

. Materials and methods

.1. Chemicals

All-trans-retinol (≥95%), all-trans-retinaldehyde (≥98%), all-rans-retinyl acetate (2,600,000–2,940,000 I.U./g), all-trans-retinylalmitate (≈1,800,000 USP units/g), all-trans-RA (≥98%), 9-cis-RA

142– 143 (2013) 96– 103 97

(≥98%) and 13-cis-RA (≥98%) were all purchased from Sigma.Methanol was purchased from VWR-Prolabo.

2.2. Animals

All studied species were collected in the north of Portugal. Adultmales and females of N. lapillus (24.1 ± 2.6 mm shell length) werecollected from intertidal rock crevices at location Homem de Leme,between March and July 2010. N. reticulatus adults of both sexes(21.6 ± 2.2 mm shell length) were captured in November 2010 atlocation Homem de Leme and Póvoa de Varzim. Sampling wasdone at low tide by using mussel baits inside small net traps. Adult(27.9 ± 2.2 mm maximum shell length) specimens of the limpet P.depressa were collected at location Praia da Apúlia between July andNovember 2010. P. depressa individuals were mainly in a maturestate, whereas N. lapillus and N. reticulatus individuals were indiverse maturation stages. Only mature N. lapillus were used in theinjection experiment.

After capture, animals were maintained in 30 L sea water aquariafor 48–72 h. During that period animals were not fed, and temper-ature and photoperiod were set at values corresponding to naturalconditions. All experiments complied with European Guidelines forthe correct use of laboratory animals. In all experiments animalswere narcotized before being sacrificed and treated humanely andwith regard for alleviation of suffering.

2.3. Measurements and experimental design

2.3.1. Retinoids in digestive gland–gonad complex of P. depressa,N. lapillus and N. reticulatus

Before sacrifice and tissue sampling, animals were alwayssedated in a 7% magnesium chloride solution for 30 min. The shellwas measured and then removed (cracked in N. lapillus and N.reticulatus or separated intact in P. depressa). After shell removalthe digestive gland–gonad complex was sampled and immediatelystored at −80 ◦C until retinoid analysis.

Both nonpolar and polar retinoids were assessed in the digestivegland–gonad complex of mature males and females of the threespecies.

Five animals per sex of each species were used for these analyses.The distribution of nonpolar retinoids between gonad and digestivegland was also analyzed in male P. depressa complexes (n = 3).

2.3.2. Retinoids in a battery of tissues of N. lapillusA total of 85 individuals of N. lapillus (33 males and 52 females)

were used in two different sampling days (15 males/27 females thefirst day and 18 males/25 females the second day). The followingtissues were dissected from each animal: digestive gland, kidney,gill, head ganglia (CNS), testis (only males), prostate (only males),ovary (only females) and reproductive glands (including sperm-ingesting gland, albumen gland and capsule gland; only females).Each day, the tissues were pooled separating male from femalestissues (two pools for each sex were made on the second samp-ling day), and finally, three different pools of each tissue wereobtained for each sex. CNS tissue from males and females werepooled together. The pools were stored at −80 ◦C until retinoidanalysis.

2.3.3. In vivo exposure experiment with N. lapillus. Retinoidintramuscular administration

Groups of 10 animals (5 males and 5 females) were distributedin 8 different 30 L aquaria. Each of the following treatments was

assigned to two replicate aquaria: DMSO (vehicle control), all-trans-RA, retinol or retinaldehyde. Treatments were administeredvia intramuscular injection according to Castro et al. (2007). Briefly,animals were sedated by immersion in 7% MgCl2 for 30 min and

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gastropod N. lapillus, including testis, ovary, digestive gland, kid-ney, gill, prostate, sperm-ingesting gland, albumen gland, capsulegland and CNS. Nonpolar retinoids could not be detected in anyof the tissues assessed neither in males nor in females, even after

free ROL RP total ROL

nmol

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8 M. Gesto et al. / Aquatic Toxic

njected into the foot with all-trans-RA, all-trans-retinol or all-rans-retinaldehyde. DMSO was used as a carrier and DMSO aloneas used to inject control (sham) animals. Applied doses were

pproximately 2 �g/g body mass, and the volume of solutionssed for injections was 2 �L in all cases. Applied doses were notxact since all animals were assumed to have a body mass of50 mg wet mass, without the shell. The actual masses of thenimals were 770.5 ± 186.3 mg and 685.5 ± 186.2 mg (mean ± SD)or females and males, respectively. According to that, the aver-ge retinoid doses were 2.33 ± 0.55 �g/g body mass (ranging from.43 to 3.18 �g/g body mass) for males and 2.05 ± 0.49 �g/g bodyass (ranging from 1.35 to 3.07 �g/g body mass) for females. Ani-als were sacrificed 48 h after injection and sampling of digestive

land–gonad complexes was carried out as described above. Theollected tissues were stored at −80 ◦C until used for retinoid anal-sis. Exposure time and doses applied were chosen on the basis ofreliminary injection tests and previously published work (Castrot al., 2007; Gesto et al., 2012a).

.4. Analytical method. Retinoid extraction and HPLC analysis

Tissue extraction and retinoid analysis by HPLC was carried outs described before (Gesto et al., 2012a,b). Briefly, tissues wereomogenized in pure methanol and retinoids were extracted byeans of mixed-mode solid-phase extraction (SPE) with OasisTM

ax 3cc 60 mg cartridges (Waters). After SPE extraction twoliquots were obtained from each sample, one containing nonpo-ar retinoids and the other containing polar bioactive forms. Theliquots containing nonpolar retinoids were divided in three parts.ne to be analyzed directly for free retinol and retinyl palmitate

RP) quantification, and the other two to be further processedor total retinol (after saponification of retinyl esters) and foretinaldehyde (after derivatization with O-ethylhydroxylamine)uantification as described before (Gesto et al., 2012a). Based onpike recovery tests the recoveries of the extraction protocol in P.epressa were 90% for the RA isomers, 85% for retinol, 65% for reti-aldehyde and 58% for RP. In N. lapillus and N. reticulatus samples,he recoveries were 93% for RA isomers, 90% for retinol, 62% foretinaldehyde and 66% for RP.

HPLC analysis of free retinol, total retinol (free + esterified), reti-aldehyde and RP was carried out according to Gesto et al. (2012a).imits of detection were calculated based on signal-to-noise ratio of

and were 1 ng/g wet tissue for retinol and RP (when using 100 mgf tissue). Limits of quantification were about 12 ng/g wet tissue foretinol and retinaldehyde and 7 ng/g wet tissue for RP, when using00 mg of tissue. For analysis of RA isomers, a different HPLC sepa-ation with diode-array detection (DAD) was utilized (Gesto et al.,012b) to analyze samples from the N. lapillus RA-injected groups.imits of detection and quantification of the latter technique were.5 ng/g wet tissue and 10 ng/g wet tissue, respectively, for all threeA isomers. Samples from other groups (the remaining N. lapillusamples and all the P. depressa and N. reticulatus samples) presentedA levels too low to be detected by HPLC–DAD. Subsets of threeamples of those groups were submitted to As Vitas (Oslo, Norway),o be analyzed by LC/MS/MS (Gundersen et al., 2007). When using00 mg tissue, limits of detection and quantification for RA isomersere about 10 pg/g wet tissue and 25 pg/g wet tissue, respectively.

.5. Statistics

Student t-tests were used to compare the retinoid contentetween digestive gland and gonad in P. depressa. One-sample t-

ests were used to analyze the differences between the content ofonpolar retinoids after retinoid injections in N. lapillus, using theouble of the detection limit as reference value for uninjected ani-als. Two-way ANOVA was used to analyze the differences among

142– 143 (2013) 96– 103

the basal levels of RA isomers in males and females of P. depressa,N. lapillus and N. reticulatus, and also to analyze the distributionof RA isomers within N. lapillus tissues. One-way ANOVA followedby Holm–Sidak post hoc tests was used to analyze RA isomers datafrom injection experiment with N. lapillus. All analyses were per-formed using SigmaPlot version 11.0 (Systat Software, Inc.). P < 0.05was considered statistically significant in all analyses.

3. Results

3.1. Retinoids in P. depressa, N. lapillus and N. reticulatusdigestive gland–gonad complex

Among the three assessed species, nonpolar retinoids were onlydetected in the male digestive gland–gonad complex of the limpetP. depressa. Both retinol and RP were detected. Other unidentifiedretinyl esters were also present in the samples since, after saponifi-cation, RP levels were estimated to account for a 19.4% of total REscontent (data not shown). Within the P. depressa complex, the non-polar retinoids were preferentially distributed in the testis (Fig. 1),which contained 17-fold more total retinol than the digestive gland.In female limpets, nonpolar retinoids in the digestive gland–gonadcomplex were below detection limit. Interestingly, polar retinoids,i.e., all-trans-, 9-cis-, and 13-cis-RA isomers were detected in thedigestive gland–gonad complex in the three species (Fig. 2). RAisomers were present at very low levels (especially in the case ofN. lapillus), around three orders of magnitude lower than nonpo-lar retinoids in male P. depressa. RA levels seem to be higher in P.depressa and in N. reticulatus than in N. lapillus. However, in somesamples, the RA levels were below quantification limit. Hence, cau-tion should be taken to establish very clear trends between malesand females or the relative contribution of each isomer to total RAcontent in the different species.

3.2. Retinoids in N. lapillus tissues

Considering that nonpolar retinoids could not be detected inthe digestive gland–gonad complexes of the caenogastropods, wedecided to screen in more detail the presence of both nonpolar andpolar retinoids in a battery of tissues of the imposex-susceptible

Fig. 1. Content of free retinol (ROL), retinyl palmitate (RP) and total (free + esterified)retinol (total ROL) in digestive gland and gonadal tissue of Patella depressa males.Values represent the mean ± SEM (n = 3). Asterisks indicate significant differenceswith respect to digestive gland levels (P < 0.05, Student’s t test). nd: not detected.

M. Gesto et al. / Aquatic Toxicology 142– 143 (2013) 96– 103 99

Patell a depressa

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ig. 2. Content of polar retinoids (13-cis-, 9-cis- and all-trans-retinoic acid isomers;

ucella lapillus and Nassarius reticulatus. Values represent the mean ± SEM (n = 3). n

aponification. Polar retinoids were only analyzed in CNS, and malend female gonads. In some samples, the 9-cis-RA levels were belowuantification limit (Fig. 3).

.3. In vivo injection experiment with N. lapillus

Finally, we performed an injection experiment to investigate if. lapillus, lacking vertebrate-like storage and transport of retinoid

orms, could metabolize retinoid precursors into other retinoidorms, similarly to what has been reported for O. lineatus (Gestot al., 2012a). In the N. lapillus injection experiment, retinol wasetected in both males and females 48 h after all-trans-retinolnd all-trans-retinaldehyde injections (Fig. 4). Those animalseemed to lack detectable amounts of REs, since RP was belowetection limit and no differences were observed between freend total retinol content. Similarly, nonpolar retinoids were notetected in DMSO- (Fig. 4) or all-trans-RA-injected individuals.urthermore, three animals (chosen at random) of DMSO, RA andetinaldehyde treatment groups were assessed for RA isomers.ll-trans, 9-cis and 13-cis isomers were detected in DMSO andll-trans-retinaldehyde-injected animals (Fig. 5). The 9-cis isomerould not be detected in all-trans-RA-injected specimens. Theevels of all-trans and 13-cis-RA in all-trans-RA-injected animals

ere significantly increased in comparison with the control group

n both males and females. Retinaldehyde-injected animals, both

ales and females, also showed increased levels of all the threeA isomers although only some of the increases reached statisticalignificance (Fig. 5). The 9-cis-RA was the predominant polar

testi s ovary CNS

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ig. 3. All-trans- (atRA), 9-cis- (9cRA) and 13-cis-retinoic acid (13cRA) in testis, ovarynd CNS of N. lapillus. Values represent mean ± SEM (n = 3).

free ROL RP total ROL

0

20

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Fig. 4. Levels of free retinol (ROL), retinyl palmitate (RP) and total (free + esterified)retinol (total ROL) in digestive gland–gonad complexes of Nucella lapillus 48 h afterintramuscular injections with DMSO (control), ROL (2 �g/g body mass) or reti-

naldehyde (RAL; 2 �g/g body mass). Values represent mean ± SEM (n = 5). Asterisksindicate significant differences from respective control group (P < 0.05). nd: notdetected.

retinoid in N. lapillus males injected with all-trans-retinaldehyde.In contrast, in retinaldehyde-injected females, 9-cis-RA concen-trations in digestive gland/gonad complex were 4-fold lower incomparison with 13-cis- and all-trans-RA.

4. Discussion

The existence of retinoid metabolism and REs storage to main-tain retinoid homeostasis was previously considered a vertebrate

100 M. Gesto et al. / Aquatic Toxicology

males

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Fig. 5. Levels of all-trans- (atRA), 9-cis- (9cRA) and 13-cis-retinoic acid (13cRA) indigestive gland–gonad complex of Nucella lapillus 48 h after intramuscular injectionwith DMSO (control), all-trans-retinoic acid (2 �g/g body mass) or retinaldehyde(s

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tion of retinaldehyde into RA is irreversible in vertebrates. As

RAL; 2 �g/g body mass). Values represent mean ± SEM (n = 3). Asterisks indicateignificant differences from respective control group (P < 0.05). nd: not detected.

nnovation (Albalat, 2009; Albalat et al., 2011). However, we haveecently demonstrated for the first time the presence of such mech-nisms in the invertebrate gastropod mollusk, O. lineatus (Gestot al., 2012a). Here, we investigated if the occurrence of retinoltorage mechanisms is a trait restricted to this gastropod group or ift is a common feature within additional Gastropoda clades. Hence,

e selected for the present study three different gastropod speciesharing the same habitat as O. lineatus in the rocky intertidal zonend adjacent soft bottoms (N. reticulatus). Whereas O. lineatuselongs to the subclass Vetigastropoda, the species selected forhe present study belong to the subclass Patellogastropoda (P.epressa) and Caenogastropoda (N. lapillus and N. reticulatus). From

toxicological point of view, we had a particular interest in theaenogastropods. Several species of this group are known to beffected by the RXR agonist TBT, with females developing imposext extremely low TBT levels (1–5 ng/L) (Castro et al., 2007; Limat al., 2011). Since strong evidence indicates that the developmentf imposex involves the inappropriate modulation of the retinoidignaling pathways, we aimed as well to investigate if those specieseveloping imposex display different features in their retinoid con-ent and metabolism in comparison to other groups of gastropods.

We demonstrate here that nonpolar retinoids, including retinolnd REs are present in the limpet P. depressa but are absent above

etection limit in N. lapillus and N. reticulatus. Nonpolar retinoidsere detected only in males of P. depressa and were mainly present

n the testis, as has been observed before in O. lineatus (Gesto et al.,

142– 143 (2013) 96– 103

2012a). These results suggest that a specific role could exist forthose compounds in the male gonad. The preferential distributionof the retinoids in gonadal tissue has been reported in other animalslike ascidians (Irie et al., 2004). However, the presence of nonpo-lar retinoids only in males is remarkable since vertebrate oocytescontain relevant levels of these compounds (Irie et al., 2010). Thepresence of retinoid storage forms in P. depressa and O. lineatusdemonstrate that the ability to store retinoids in the form of REs isnot restricted to a single gastropod clade and supports the hypoth-esis that the evolutionary origin of the ability to maintain retinoidhomeostasis through fatty acid esterification could be older thanpreviously believed (Fig. 6A).

Since both caenogastropod snails seem to lack nonpolarretinoids, further studies were done with N. lapillus. Nonpolarretinoids were not detected in any tissue of N. lapillus, suggestingthat this species does not store retinoids in the form of REs. Despitethe absence of nonpolar retinoids in all tissues assessed, RA isomerswere detected in all three tissues used for RA determination (testis,ovary and CNS) at very low concentrations, in the fmol/g wet tis-sue range. Other studies have reported the presence of RA in gonadsand CNS of gastropod mollusks, where they have been suggested toplay a role in gonad maturation and maintenance and in neuronalregeneration and axon pathfinding (Dmetrichuk et al., 2006, 2008;Gesto et al., 2012a). RA levels were in the same range than thoseobserved in O. lineatus digestive gland–gonad complex (Gesto et al.,2012a) but were much lower than the RA content in the CNS andthe hemolymph of the gastropod mollusk Lymnaea stagnalis (Sub-class Heterobranchia) (Dmetrichuk et al., 2008). RA levels were alsolower than those observed in the adult mouse, which were around1–80 pmol/g (Kane et al., 2008). The mechanism by which N. lapil-lus maintain RA levels without detectable levels of retinol or REs isstill unknown.

We then performed an exposure experiment to dissect activeretinoid metabolic routes in N. lapillus. This approach, based onintramuscular injections of retinoid precursors, has been utilizedsuccessfully before in O. lineatus, providing unique insights intothe retinoid metabolic capacity of that species (Gesto et al., 2012a).According to the doses of retinoids injected, the amount of retinoidsfound in the animals were lower than expected, although a clearaccumulation was observed after injection. Processes such asmetabolization and excretion might explain partially the observeddifferences.

Retinol, but not retinyl esters, was detected after retinol andretinaldehyde injections. These results indicate that retinaldehydecan be metabolized to retinol inside the animals but N. lapillus,unlike O. lineatus, seems to lack the required metabolic machin-ery to transform retinol into REs (Fig. 6B). The lack of REs in N.lapillus seems to be justified by the lack of capacity to esterifyretinol. At present, it is unknown which enzyme is responsible forretinol esterification in O. lineatus (or any other mollusk species),but our findings suggest that it is not active in the whole gastro-pod clade. In spite of the lack of retinol esterification capacity, N.lapillus is able to convert retinaldehyde into retinol and into activeRA isomers, suggesting that adequate RA levels in N. lapillus tis-sues could be obtained by synthesizing retinaldehyde from dietarycarotenoids. Supporting this is the fact that N. lapillus feeds mainlyon mussels and barnacles (Hughes and Burrows, 1991), which areknown to contain different carotenes (Herring, 1971; Hertzberget al., 1988).

All-trans-RA-injected animals were used as positive controls forinjection performance: RA was not expected to be transformedinto any nonpolar retinoid after injection, since the transforma-

expected, nonpolar retinoids could not be detected after RA injec-tion. The results demonstrate that part of the all-trans-RA injectedhad suffered isomerization to form 13-cis-RA within the animal.

M. Gesto et al. / Aquatic Toxicology 142– 143 (2013) 96– 103 101

Fig. 6. Summary cladogram and differences in retinoid metabolism within gastropods. Green (solid line) and red (dotted line) arrows represent metabolic steps that areactive or inactive, respectively. Yellow arrows (broken line) indicate unconfirmed metabolic transformations. (A) Cladogram showing the presence/absence of active (RA) orstorage (retinyl esters) retinoid forms in species belonging to different gastropod groups as well as their susceptibility to imposex and their feeding behavior. Hypotheticphylogenetic relationships according to Aktipis et al. (2008). Information about diets and imposex sensitivity based on Barroso et al. (2002), Castro et al. (2007), Crothers(1985, 2001), Hawkins et al. (1989), Nehring (2005) and Stephenson and Lewis (2011). na: not applicable. (B) Differences affecting the main retinoid routes between O. lineatusand the imposex-susceptible gastropod N. lapillus. REs: retinyl esters, ROL: retinol, RAL: retinaldehyde, RA: retinoic acid. (C) Differences in isomerization patterns betweenO. lineatus and N. lapillus after all-trans-retinaldehyde injection. Two possible origins, not mutually exclusive, for 9-cis- and 13-cis-RA isomers are suggested: isomerizationof all-trans-retinaldehyde to 9-cis- and 13-cis-retinaldehyde and subsequent oxidation to 9-cis- and 13-cis-RA isomers or oxidation of all-trans-retinaldehyde to all-trans-RAand posterior isomerization to 9-cis- and 13-cis-RA. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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nterestingly, the isomerization to form 9-cis-RA from injected all-rans-RA does not seem to occur to the same extent in this species,uggesting that an isomer-specific enzyme could be the responsibleor the conversion of all-trans-RA to 13-cis-RA. In contrast, all-trans-etinaldehyde injection led to an increase in the amount of all threeA-isomers, both in males and females. The actual synthetic ori-in of 9-cis- and 13-cis-RA isomers is unknown since they coulde formed by isomerization of all-trans-RA or by oxidation of 9-is-retinaldehyde and 13-cis-retinaldehyde, which could in turn beormed by all-trans-retinaldehyde isomerization (Fig. 6C). Further-

ore, after all-trans-retinaldehyde injection, the RA-isomer profileas gender-dependent, 13-cis-RA and all-trans-RA being dominant

n females and 9-cis-RA in males. These results contrast with thendings in O. lineatus, in which no RA isomerization was detectedfter retinaldehyde injection (Gesto et al., 2012a).

From a toxicological point of view, differences in the retinoidontent and metabolism seen in these species could be of impor-ance. Our results suggest that there are important differences inhe retinoid metabolism between imposex-susceptible gastropodsnd other gastropod groups, which seem to have a retinoid systemith similarities to that of vertebrates. Interestingly, the proposedatural ligands for RXRs and RARs, i.e., 9-cis-RA and all-trans-RA,re known to act as morphogens and participate in the controlf tissue development (Schilling et al., 2012). Furthermore, it isell known that injection of 9-cis-RA, but not all-trans-RA (our

wn unpublished data) led to imposex development in female. lapillus and N. reticulatus and impacts male penis length in N.

apillus (Castro et al., 2007; Lima et al., 2011; Sousa et al., 2010).ence, taken together the results of the present study and previ-us observations that demonstrated an association between RXRodulation and imposex development, we hypothesize that the

nability of N. lapillus and N. reticulatus to divert retinoids to stor-ge forms such as retinyl esters could hamper the maintenance ofhe retinoid homeostasis in the presence of exogenous RXR ago-ists such as TBT. In this regard, it has been shown in vertebrateshat the formation of retinyl esters is an important mechanismmong those involved in the regulation of the retinoid signalingathway (Ross, 2003). This hypothesis warrants additionaltudies.

The capacity to store retinoids in the form of retinyl esters isn important evolutionary transition, since it provides the capac-ty to maintain a retinoid homeostatic control, independently ofhe retinoid/carotenoid sources from recent dietary intake. Thisapacity has been shown to be present in a gastropod species, O.ineatus (Gesto et al., 2012a). Here, we demonstrate that P. depressa,

species from a different gastropod group (Patellogastropoda) alsoisplays the capacity to store REs. Conversely, N. lapillus and N. retic-latus (Caenogastropoda) do not (Fig. 6A). The key factor for REstorage capacity seems to be the enzymatic step that catalyzes theonversion of retinol into REs, which has been shown to be activen O. lineatus but inactive in N. lapillus. Interestingly, we report herehat those assessed species that are known to develop imposexhen exposed to the RXR agonist TBT (N. lapillus and N. reticula-

us) also lack the metabolic capacity to synthesize retinyl esterss retinoid storage forms (Fig. 6A). In contrast, two other gastro-od species (O. lineatus and P. depressa) that share the same habitats N. lapillus and N. reticulatus possess endogenous levels of REs.uture studies should clarify if the inability to form retinyl estersampers the capacity to maintain an adequate retinoid signaling inhe presence of a exogenous RXR agonists. Finally, it is importanto highlight that the two gastropod species in which REs have beenbserved share herbivorism as feeding behavior and the species in

hich REs have not been found are carnivorous (Fig. 6A). Whether

eeding behavior has any relationship with the retinol esterificationapacity should be the aim of future studies involving additionalnvertebrate groups.

142– 143 (2013) 96– 103

Acknowledgements

This work was supported by the project PTDC/MAR/115199/2009 and PTDC/MAR/105199/2008 from Fundac ão para a Ciênciae a Tecnologia (Portugal). M. Gesto was recipient of post-doctoralfellowships from Fundac ão para a Ciência e a Tecnologia (Portugal,SFRH/BPD/47572/2008) and Xunta de Galicia (Spain, ÁngelesAlvarino program), and a research grant (IN809A 2010/360, partlyfunded by European Social Fund) from Xunta de Galicia (Spain).

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