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Environmental Toxicology and Pharmacology 29 (2010) 24–31

Contents lists available at ScienceDirect

Environmental Toxicology and Pharmacology

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eurotoxic effect of the herbicide paraquat on ascidian larvae

iuliana Zegaa, Simona Candianib, Silvia Groppelli a, Fiorenza De Bernardia, Roberta Pennati a,∗

Dipartimento di Biologia, Università di Milano, Via Celoria 26, 20133, Milano, ItalyDipartimento di Biologia, Università di Genova, V.le Benedetto XV 5, 16132 Genova, Italy

r t i c l e i n f o

rticle history:eceived 17 February 2009eceived in revised form 6 July 2009ccepted 2 September 2009vailable online 8 September 2009

a b s t r a c t

Paraquat is an herbicide widely used in agriculture, that proved to have toxic effect on many animal mod-els. Moreover, it is considered a potential etiologic factor of Parkinson’s disease. Ascidians are invertebratechordates, whose larval central nervous system shares basic structural homologies with the vertebrateone. Ascidian larvae exposed to paraquat developed specific alterations of the CNS, that were charac-terized by histological and immunohistochemical analysis. Tyrosine hydroxylase (TH) expression was

eywords:opaminehallusia mammillatayrosine hydroxylasearkinson’s disease

examined by “in situ” hybridization. A decrease of dopamine content in anterior CNS of treated larvaewas observed. In combined treatments with paraquat and l-ascorbic acid, a common anti-oxidant, theseverity of the malformations was significantly reduced, confirming that the oxidative stress is involvedin the toxicity mechanism of paraquat on ascidians. For its sensitivity to paraquat and its simple chordatebody plan, ascidian larva is a promising animal model to further investigate the molecular mechanism of

paraquat neurotoxicity.

. Introduction

Paraquat (1,1′-dimethyl-4,4′-bipyridinium dichloride hydrate)PQ) is a very quick-acting herbicide commonly used in agricul-ure. It is included in a priority list of herbicides of potentialoncern established for the Mediterranean countries by the Euro-ean Union, due to its widespread usage in this area (Barcelò,993). Consequently, it may be present as residues in environment,ood and biological samples. For example, in vineyard-devoted soilsQ was detected in concentration ranging from 53 to 404 �g/kgPateiro-Moure et al., 2008). The toxicity of PQ is essentially relatedo its rapid reduction and subsequent reoxidation to produce theeactive oxygen and nitrogen species (respectively, ROS and RNS)Dinis-Oliveira et al., 2006). In vertebrates, acute exposure to highevel of PQ causes pulmonary toxicity. Chronic exposure to PQas strong effects particularly on dopaminergic neurons of theubstantia nigra (Thrash et al., 2007) that degenerate after releas-ng all their dopamine content. Moreover, several studies provedhe degeneration of dopaminergic neurons in mice exposed toQ (Dinis-Oliveira et al., 2006). Since the loss of these neurons islso the primary neurodegenerative feature of Parkinson’s disease,

echanism of PQ induced cell damage bear implications for the

athogenetic processes of this syndrome. Rodents are routinelysed in the study of molecular mechanisms of PQ toxicity, as toate the exact mechanism through which it could induce selec-

∗ Corresponding author. Tel.: +39 0250314765; fax: +39 0250314802.E-mail address: roberta.pennati@unimi.it (R. Pennati).

382-6689/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.etap.2009.09.001

© 2009 Elsevier B.V. All rights reserved.

tive dopaminergic neuron failure remains unknown (Patel et al.,2006). Nevertheless, there are evidences that tyrosine hydroxylase(TH), the dopamine rate-limiting synthesis enzyme, is inactivatedby RNS generated after exposure to PQ, thus causing a dopaminedepletion in neurons of the substantia nigra (Dinis-Oliveira et al.,2006).

Ascidians are sessile marine organisms belonging to the phylumChordata. They develop through a planctonic larva characterized bya dorsal tubular nervous system and a notochord. Under laboratorycondition, embryos of solitary ascidians, such as Phallusia mammil-lata and Ciona intestinalis, can be easily obtained by adult dissectionand in vitro fertilization. They complete their development after18 h at 18 ◦C and hatched lecitotrophic larvae have a short life. Thesecharacteristics made these animals good experimental models torapidly test toxicity of pollutants, such as heavy metals or pesticides(Bellas et al., 2003, 2004; Groppelli et al., 2007; Pennati et al., 2006).Moreover, as ascidians are invertebrate chordates and are consid-ered the closest living relatives of vertebrates (Delsuc et al., 2006),they represent an interesting model to perform comparative stud-ies on toxicant effects on development. The ascidian tadpole larva isformed by a trunk and a tail. The trunk bears the adhesive papillae,or palps, by which the larva settles, and the sensory vesicle, thatcontains three different sensory organs: the ocellus, a pigmentedcell surrounded by a photoreceptor complex and lying over three

lens cells; the otolith, a round pigmented cell located on the floorvesicle and connected to sensory neurons; the coronet cells, a groupof neurons situated in the posterior sensory vesicle, whose sen-sory function is still debated (Imai and Meinertzhagen, 2007). Thelight sensory organ (ocellus) and the gravity sensory organ (otolith)

G. Zega et al. / Environmental Toxicology a

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AP buffer containing NBT/BCIP substrates. When colour reaction developed, larvaewere washed in PBT, mounted on glass slides with 80% glycerol and observed usingan optical microscope.

ig. 1. Incidence of malformations in larvae developed from embryos treated atwo-cell stage with different concentrations of paraquat (PQ). Data are means ± SD.

re directly implicated in the control of larva swimming behaviourTsuda et al., 2003).

Dopamine has been localized in the central nervous systemCNS) of P. mammillata larvae in cells related to the otolith and itas been shown that this neurotransmitter modulates the onset ofetamorphosis in larvae of this species (Zega et al., 2005). In the

scidian C. intestinalis, transcripts of tyrosine hydroxylase (TH), theopamine synthesis rate-limiting enzyme, were found in some ofhe coronet cells, localized on left portion of the sensory vesicleMoret et al., 2005).

The aim of this work was to study PQ toxicity on ascidian devel-pment and compare it to what it is known on vertebrate modelrganisms. Therefore we tested PQ effects on the development of P.ammillata by characterizing the malformations induced on larvaNS. The effects of combined treatments with PQ and l-ascorbiccid (AA) were also evaluated to determine if a reduction of thexidative stress could reduce the herbicide toxicity on ascidianevelopment. Moreover, we characterized CNS malformations byeans of immunolocalization of �-tubulin, GABA and dopamine. In

. intestinalis, we determined TH expression in control and treatedarvae. We observed some similarities between PQ effects on ascid-an larvae and vertebrate embryos and adults and we discuss theeurotoxic action of this herbicide using ascidians as model organ-

sms.

. Materials and methods

.1. Animals and treatments with paraquat and l-ascorbic acid

Adults of P. mammillata were collected in the gulf of Lerici, La Spezia, whiledults of C. intestinalis were supplied by the “Stazione Zoologica A. Dohrn”, Naples.scidians were reared in aquaria at 16 ◦C, and were dissected to remove male and

emale gametes from the gonoducts. Gametes were used for in vitro fertilization.ertilized eggs were allowed to develop in 9 cm Petri dishes in Millipore-filteredmesh size 0.45 �m) artificial seawater (ASW) at 18 ◦C. One hour post-fertilization,wo-cell stage embryos were collected for treatments.

P. mammillata embryos were exposed to a wide range of PQ concentrationso estimate dose–response effects. We observed that PQ concentration lower than.01 mg/ml did not cause any malformation of ascidian larvae. Therefore we treatedmbryos with the following concentration: 0.01, 0.1, 0.25, 0.5, 1 and 2 mg/ml.wo replicates of each experiment were performed on different culture batchesn different dates. In each replicate, two-cell stage embryos were transferred tocm Petri dishes containing PQ and PQ plus l-ascorbic acid (AA) (Sigma–Aldrich,

taly), diluted to 0.01, 0.1, 0.5 and 1 mM. Both substances were dissolved in ASW.ach treatment for each batch was replicated three times and every replica con-ained 30 ± 3 embryos. Hatched larvae, 18 h post-fertilization, were observed understereo-microscope to score malformations.

Two-cell stage embryos of C. intestinalis were treated with 2 mg/ml PQ, in ordero obtain larvae for “in situ” hybridization experiments.

.2. Histology

Control and PQ treated larvae of P. mammillata were sectioned for detailedbservation of the sensory vesicle structures. Samples fixed in paraformaldehydePFA) were washed twice in phosphate buffer (pH 7.2) (PBS), were dehydrated inn ethanol series and stored at −20 ◦C. Rehydrated specimens were washed again

nd Pharmacology 29 (2010) 24–31 25

in PBS and, after staining in 1% carmin red for 3 h, were embedded in Technovit7100 plastic (Heraeus Kulzer GmbH, Werheim, Germany) and sectioned at 5 �m.Sections were counter stained with 0.5% methylene blue in water for few minutesand mounted in Entellan (Merck, Italy).

2.3. Immunohistochemistry

Control and treated larvae of P. mammillata were fixed in 4% PFA in 0.1 M PBSat room temperature for 2 h. All steps were performed with gentle rocking. Afterrinsing with 0.1 M PBS, the samples were stored in methanol at −20 ◦C. After rehy-dration, specimens were permeabilized with 0.1% Tween-20, 0.25% Triton X-100 inPBS for 1 h, were washed three times in PBS for 10 min each, and incubated for 2 h in50% PBS/50% normal goat serum, previously de-activated at 55 ◦C for 30 min. Then,the samples were incubated with primary antibodies, at 4 ◦C, as following: overnightwith the monoclonal anti-�-tubulin antibody (clone 2-28-33, Sigma, Italy), diluted1:400; overnight with the polyclonal rabbit anti-GABA antibody (Chemicon, Temec-ula, CA), diluted 1:1000; 48 h with the polyclonal rabbit anti-dopamine antibody(AB122S, Chemicon, Italy), diluted 1:400. After several washes in PBS, the sam-ples were incubated in 1% bovine serum albumine (BSA) in PBS for 2 h at roomtemperature, and then incubated at 4 ◦C overnight in PBS, in which goat anti-mouse AlexaFluor 488 or goat anti-rabbit AlexaFluor 488 (Invitrogen, Italy), diluted1:400, were added. Next, the specimens were washed six times in PBS, 20 min each,and mounted in 1,4-diazabicyclo[2,2,2]octane (DABCO, Sigma, Italy) on microscopeslides.

Samples were examined using a confocal laser scanning microscope Leica TCS-NT (Leica Microsystems, Heidelberg, Germany), equipped with laser argon/krypton,75 mW multiline. Series of “optical sections” attained by scanning whole-mountspecimens were projected into one image with greater focal depth. The numberand step size of “optical sections” are given for each image in figure captions. FITCfluorescence was observed using a 488 nm excitation filter and a 530/30 band passfilter.

2.4. In situ hybridization (ISH)

Total RNA from C. intestinalis larvae was extracted using the TRIzol LS reagent(Invitrogen, San Diego, CA). Following extraction, RNA was treated with RNAse-freeDNAse I (Ambion Europe Ltd., UK) according to the manufacturer’s recommen-dations in order to digest contaminating genomic DNA. First-strand cDNA wassynthesized with 5 �g RNA using the SuperScript first-strand synthesis system(Invitrogen, San Diego, CA) and oligo(dT) primers.

A cDNA fragment of 1145 bp corresponding to the tyrosine hydroxylase geneof C. intestinalis (accession no. AJ634597) was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) on RNA samples from larvae. PCR were carriedout in a 50 �l reaction mixture using the Hot Master mix in accordance with the man-ufacturer’s instructions (Eppendorf Srl, Italy) using the following primers: 5′ primerAGTAAGAGCGGATTTTGGAAGA and 3′ primer ACGTTTGGTGCAGTGTTGTG. The PCRproduct were directly cloned using a TOPO TA cloning kit (Invitrogen, San Diego, CA)and then sequenced using a 377 PerkinElmer sequencer. The cDNA corresponding tothe Ci-TH isolated by PCR were used as templates for in vitro transcription by usingBoehringer Mannheim DIG RNA labeling Kit, according to the supplier’s instruction.

Hatched larvae were fixed in fresh 4% paraformaldehyde in 0.5 M NaCl and 0.1 MMOPS at pH 7.5, at room temperature (r.t.) for 90 min. Then, samples were dehy-drated in an ethanol series, and stored at −20 ◦C in 70% ethanol. On the day of theexperiment, samples were rehydrated, wash twice with PBT (0.1% Tween-10% inPBS) and digested with 2 �g/ml Proteinase K for 30 min at 37 ◦C. Next samples werepost-fixed in 4% paraformaldeyde in PBS for 1 h, washed twice in PBT and thenthree times, 10 min each, with 0.25% acetic anhydride in 0.1 M triethanolamine.Samples were then incubated in the hybridization solution (50% formamide, 5×SSC, 50 �g/ml tRNA, 5× Denhardt’s solution, 0.1% Tween-10%, 50 �g/ml heparin)for 1 h. The hybridization was carried out overnight at 50 ◦C with 0.3–0.6 ng/�l Dig-TH probe. On the second day, samples were washed with a descendent series of SSCbuffer in 50% formamide and 0.1% Tween-10% then incubate overnight at 4 ◦C in adilution (1:2000) of alkaline phosphatase-conjugated anti-DIG-antibody in block-ing buffer containing 0.5% blocking reagent and 5% normal sheep serum. On thethird day, samples were washed several times in PBT and afterwards rinsed with

2.5. Statistical analysis

Analysis of variance (ANOVA) was performed to determine the significance ofdifferences among treatments. Tukey’s post hoc test (significant at P < 0.05) was usedto identify specific effects of different concentrations of PQ or of PQ plus l-ascorbicacid on the incidence of the three different phenotypes.

26 G. Zega et al. / Environmental Toxicology and Pharmacology 29 (2010) 24–31

Fig. 2. Morphological analysis of Phallusia mammillata larvae. (A–F) Control larvae. (A) Larvae showing a normal phenotype. (B) Larva trunk in which the sensory vesicle andthe pigmented organs, ocellus (oc) and otolith (ot) are visible. Adhesive papillae (ap) are visible at the rostral tip of the trunk. (C and D) Enlarged views of the sensory organs ofthe vesicle. (C) The ocellus lies above the lens cells (lc) and it is surrounded by the photoreceptor cells (pc). (D) The coronet cells (cc) are recognizable from their bulbous protru-sions (arrow), in the vesicle cavity. (E and F) Histological sections at the level of the sensory vesicle. (E) The fan of photoreceptor cells surrounds the ocellus, in which pigmentgranules are visible, just above the three lens cells. (F) Section at the level of the coronet cells (arrow) in which the cells located under the otolith (asterisks) are clearly visible.

G. Zega et al. / Environmental Toxicology and Pharmacology 29 (2010) 24–31 27

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ig. 3. Effects of different concentrations of paraquat (mg/ml) on the development ofhallusia mammillata larvae. Data are means ± SD. For complete statistic see Table 1.

. Results

.1. Exposure to paraquat

Paraquat (PQ) had no lethal effects on P. mammillata embryosven at highest applied concentrations (Fig. 1). Instead, the herbi-ide proved to induce characteristic malformations to the anteriortructures of the larvae and particularly of the central nervous sys-em (CNS) (Fig. 2). The percentage of malformed larvae developedrom embryos exposed to PQ (from 0.01 to 2 mg/ml) was sig-ificantly different from that of controls (ANOVA: F6,14 = 210.524,< 0.00001) and followed a dose dependent trend (Fig. 1). About0% of developed embryos exposed from 0.25, to 2 mg/ml PQ con-entrations were malformed (Tukey’s post hoc tests: all P ≤ 0.001),ut we observed some differences in the incidence of different typesf malformations (Fig. 3). We identified, in treated larvae, threeifferent induced phenotypes, according to the dimension of theensory vesicle and the shape of the ocellus, which represent threerogressive degrees of severity of CNS malformations (Fig. 2A–H).he phenotypes were named “small sensory vesicle”, “extremelyeduced sensory vesicle” and “big ocellus” (Fig. 2I–K). In controlarvae, the ocellus, located on the right side of the vesicle, is sur-ounded by a fan of sensory neurons, that form the photoreceptoromplex, and lies over three lens cells (Fig. 2C and E). On the leftide, the posterior sensory vesicle houses the coronet cells, neu-ons easily recognizable for their bulbous ends, protruding into theavity (Fig. 2D and F). The otolith is normally located in the mid-le of the vesicle floor connected to at least two neurons, possibly

nvolved in gravity perception (Fig. 2D).In the “small sensory vesicle” (Fig. 2I and L) and in the “extremely

educed sensory vesicle” larvae (Fig. 2J and M), the lens cells werebsent, while photoreceptor cells and coronet cells were misplacedn the vesicle. In “small sensory vesicle” larvae, the otolith was mis-laced on the anterior wall of the vesicle. In the “extremely reducedensory vesicle” larvae, some cells formed an extension from oneide to the other of the cavity (Fig. 2M). The cells located just below

he otolith were always present in these treated larvae (Fig. 2L and).

In the larvae showing the “big ocellus” phenotype, the pigmentranules of the light sensory organ appeared distributed along theosterior dorsal wall of the sensory vesicle, conferring to the ocellus

G–L) PQ treated larvae. (G) Larvae treated with 2 mg/ml PQ: the pigmented organs are mis

henotype. (I–K) Enlarged view of the vesicle of treated larvae showing the three differen

esicle” larvae, some coronet cells are visible in the right ventral portion of the vesicle. Th

extremely reduced sensory vesicle”, the cavity is divided by an extension (arrow) that c

omplex appears disorganized. (K and N) In the “big ocellus” phenotype the vesicle cavity

ell fan is no longer visible as well as the lens cells, while the cells under the otolith are p

dhesive papillae; cc: coronet cells; lc: lens cells; oc: ocellus; ot: otolith; pc: photorecept

Fig. 4. Effects of combined treatments of 2 mg/ml paraquat plus l-ascorbic acid (AA)at different concentrations on the development of Phallusia mammillata larvae. Dataare means ± SD. For complete statistic see Table 2.

a typical elongated shape. Moreover, the sensory vesicle cavity wassmaller than in the other two phenotypes and the otolith appearedvery close to the ocellus (Fig. 2K). The histological analysis revealedthat larvae with the “big ocellus” completely lack the photoreceptorcomplex and the three lens cells. The coronet cells were not clearlyrecognizable while the cells located under the otolith were present(Fig. 2N).

The proportion of the three different PQ induced phenotypeswas significantly different in embryos exposed to 0.5, 1 and 2 mg/mldoses (Fig. 3, Table 1). The percentage of larvae showing a smallsensory vesicle, the less severe phenotype, was about 40% at thelower dose and decreased in higher doses, while the proportion oflarvae developed with an extremely reduced sensory vesicle was35% at 0.5 mg/ml and about 50% at 1 mg/ml of PQ. The incidence ofthe latter phenotype decreased to 37% at the highest tested dose,2 mg/ml. In fact, at this concentration the majority of treated larvae(49.5%) showed a “big ocellus”, the most severe phenotype (Fig. 3,Table 1).

3.2. Combined treatments with PQ and ascorbic acid

To evaluate the effects of an anti-oxidant on PQ toxicity, P. mam-millata embryos were treated with the highest tested concentrationof herbicide (2 mg/ml) plus different concentrations of l-ascorbicacid (AA), from 0.01 to 1 mM (Fig. 4). All larvae exposed to PQplus AA were malformed, but the percentage of the most severephenotype, “big ocellus”, was significantly reduced in combinedtreatments as compared to that conducted with PQ alone. The res-cue effect of AA did not showed a dose dependent trend, mostprobably because at concentrations from 0.5 to 1 mM AA alonecaused significant toxic effects as compared to controls (Table 2).

3.3. Immunolocalization of ˇ-tubulin, dopamine and GABA

To better characterize the malformations induced by PQ, treatedlarvae of P. mammillata were labelled with antibodies against �-tubulin, dopamine and GABA.

placed as compared to control larvae. (H) Trunk of a larva showing the “big ocellus”

t phenotypes, with related histological sections (L–N). (I and L) In “reduced sensory

e otolith hangs upside down from the vesicle frontal wall. (J and M) In larvae with

rosses the vesicle cavity, the ocellus has an irregular shape and the photoreceptor

is strongly reduced and the otolith appears close to the ocellus. The photoreceptor

resent (asterisks). Scale bars: A and G 100 �m; B and H 50 �m; C–F, I–N 25 �m. ap:

or cells.

28 G. Zega et al. / Environmental Toxicology and Pharmacology 29 (2010) 24–31

Fig. 5. Control and 2 mg/ml PQ treated larvae immunolabeled with anti-�-tubulin (A–D), anti-dopamine (E–H) and anti-GABA (I–N) antibodies. (A) Sum of 35 opti-cal sections (step size 1 �m) of the trunk of a control larva. �-Tubulin immunofluoroscence evidences the nervous fibres of the neural tube (nt) that reach thesensory vesicle: some fibres lie dorsally and form the papillary nerves (arrowhead), while others (arrow) lie in the ventrally and are directed to the otolith (dot-ted line). Rostrally, �-tubulin immunofluorescence is present in sensory neurons of the papillae (asterisks). (B) Superimposition of the confocal image with thelight transmission one showing the position of the otolith (arrow). (C) Sum of 35 optical sections (step size 1 �m) of the trunk of a PQ treated larva showing �-tubulin

G. Zega et al. / Environmental Toxicology and Pharmacology 29 (2010) 24–31 29

Table 1Effects of paraquat on the development of Phallusia mammillata: incidence of the three different phenotypes (see the text for description) and statistics. Data are means ± SD.n.s.: not significant.

Normal sensory vesicle Small sensory vesicle Extremely reduced sensory vesicle Big ocellus

Controls 100.0 – – –0.5 mg/ml PQ – 37.2 ± 5.0 35.8 ± 4.0 27.0 ± 2.21 mg/ml PQ – 26.5 ± 4.7 49.2 ± 2.8 24.3 ± 3.82 mg/ml PQ – 12.9 ± 4.4 37.5 ± 2.9 49.5 ± 3.4

F3.8 46.726 170.294 156.837P≤ 0.0001 0.00001 0.00001

Tukey’s post hoc tests P≤0.5 vs. 1 n.s. 0.01 n.s.0.5 vs. 2 0.001 n.s. 0.00011 vs. 2 0.05 0.01 0.0001

Table 2Effects of paraquat (PQ) and l-ascorbic acid (AA) given in combination and alone, on Phallusia mammillata development: incidence of the three different phenotypes andstatistics. Data are means ± SD. n.s.: not significant.

Normal sensory vesicle Small sensory vesicle Extremely reduced sensory vesicle Big ocellus

Controls 96.7 ± 3.6 – 3.33 ± 3.6 –2 mg/ml PQ – 25.4 ± 4.9 22.1 ± 6.7 52.5 ± 10.32 mg/ml PQ + 0.01 mM AA – 40.5 ± 4.6 44.6 ± 3.4 14.8 ± 3.92 mg/ml PQ + 0.1 mM AA – 43.1 ± 6.2 34.3 ± 9.4 22.6 ± 8.62 mg/ml PQ + 0.5 mM AA – 40.0 ± 7.2 46.6 ± 5.3 13.4 ± 3.92 mg/ml PQ + 1 mM AA – 22.7 ± 8.7 51.9 ± 5.4 25.4 ± 11.80.01 mM AA 95.6 ± 4.6 4.4 ± 4.60.1 mM AA 91.3 ± 6.9 8.8 ± 6.90.5 mM AA 91.7 ± 0.8 – 8.3 ± 0.8 –1 mM AA 85.8 ± 3.0 8.5 ± 7.5 5.7 ± 9.9 –

F3.8 918.819 28.130 55.031 13.337P≤ 0.00001 0.00001 0.00001 0.00001

Tukey’s post hoc tests P≤Controls vs. 2 mg/ml PQ 0.00001Controls vs. 2 mg/ml PQ + 0.01 mM AA 0.00001Controls vs. 2 mg/ml PQ + 0.1 mM AA 0.00001Controls vs. 2 mg/ml PQ + 0.5 mM AA 0.00001Controls vs. 2 mg/ml PQ + 1 mM AA 0.00001Controls vs. 0.01 mM AA n.s.Controls vs. 0.1 mM AA 0.05Controls vs. 0.5 mM AA 0.05Controls vs. 1 mM AA 0.0012 mg/ml PQ vs. 2 mg/ml PQ + 0.01 mM AA n.s. n.s. 0.001 0.000112 mg/ml PQ vs. 2 mg/ml PQ + 0.1 mM AA n.s. 0.015 n.s. 0.0022 mg/ml PQ vs. 2 mg/ml PQ + 0.5 mM AA n.s. 0.067 0.001 0.000072 mg/ml PQ vs. 2 mg/ml PQ + 1 mM AA n.s. n.s. 0.0001 0.0052 mg/ml PQ vs. 0.01 mM AA 0.00001 0.003 0.001 0.00001

0.020.01n.s.

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2 mg/ml PQ vs. 0.1 mM AA 0.000012 mg/ml PQ vs. 0.5 mM AA 0.000012 mg/ml PQ vs. 1 mM AA 0.00001

Immunolocalization of �-tubulin reveals larva nervous networkPennati et al., 2003). In control larvae, �-tubulin immunoposi-ive staining was present in the sensory neurons of the palps andn two dorsal papillary nerves, in the nervous fibers of the sen-

ory vesicle and of the neural tube. A bundle of fibers directednder the otolith, most probably towards the sensory cells under

t, was clearly visible in the vesicle floor (Fig. 5A and B). Inreated larvae, these fibers were not detectable, indicating a spe-

mmunofluorescence in fibres of the neural tube (nt) and of the sensory vesicle. At this l

nd adhesive papillae sensory neurons (asterisks) are visible. The otolith is evidenced by

howing the lack of nerve fibres in the sensory vesicle floor (arrow). (E) Immunolocalizat

arrow) located below the otolith (dotted circle), and in the neural tube (nt) (sum of 35

mage. (G) Immunolocalization of dopamine in a trunk of a PQ treated larva where there

n the neural tube (nt) (sum of 35 optical sections, step size 1 �m). (H) Superimposition

I–K) and 2 mg/ml PQ treated larvae (L–N). (I and L) Sum of 3 optical sections (step size 1

ections (step size 1 �m) taken at deeper levels then A and D. (K and N) Superimposition

etectable at more superficial level (L) than in control specimens (I). Scale bars: (A–H) 50

4 0.001 0.000029 0.001 0.00002

n.s. 0.00001

cific malformation of the nervous network connecting the gravitysensory apparatus with the posterior sensory vesicle (Fig. 5C andD).

Immunolocalization experiments with an anti-dopamine anti-

body revealed that this neurotransmitter was present in few cellslocated in the sensory vesicle floor, under the otolith, and in theneural tube (Fig. 5E and F). In treated larvae, this neurotransmit-ter was no longer present in the sensory vesicle (Fig. 5G and H).

evel, ventral fibres are absent (arrow), while rostral papillary nerves (arrowhead)

a dotted line. (D) Superimposition of C with the light transmission image clearly

ion of dopamine in a trunk of a control larva: the neurotransmitter is in some cells

optical sections, step size 1 �m). (F) Superimposition of E with light transmission

is no detectable fluorescence in the sensory vesicle. A faint signal is present only

of G with light transmission image. (I–N) Immunolocalization of GABA in control

�m) taken at level of the right side of sensory vesicle. (J and M) Sum of 30 optical

of J and M with light transmission images. In treated larvae GABA positive cells are

�m, (I–N) 40 �m.

30 G. Zega et al. / Environmental Toxicology and Pharmacology 29 (2010) 24–31

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ig. 6. Expression of Ci-TH (tyrosine hydroxylase) in Ciona intestinalis larvae. (A) Inome of the coronet cells. (B) In 2 mg/ml of PQ exposed larvae, Ci-TH expression app

opamine presence was not detected in the sensory vesicle of lar-ae with all the three different described phenotypes (data nothown).

In larvae of P. mammillata, GABA immunoreactivity was presentithin the CNS in the sensory vesicle, in the neck and in the visceral

anglion (Fig. 5I–K). A superficial optical section, obtained by con-ocal microscopy, showed that, in the region between the posteriorensory vesicle and the visceral ganglion, some GABA positive cellsere differently distributed in treated larvae as compared to con-

rol ones (Fig. 5I and L). Moreover, in PQ exposed larvae (Fig. 5Mnd N), GABA immunoreactivity was detected along the anteriorNS even if the distribution of GABA positive cells in the vesicleppeared different from that of control larvae (Fig. 5J and K).

.4. Expression of tyrosine hydroxylase (TH)

We determined that C. intestinalis embryos exposed to 2 mg/mlQ developed in larvae with phenotypes similar to those describedor P. mammillata ones (data not shown). Since Ciona genome haseen completely sequenced (Dehal et al., 2002), we analyzed thexpression of Ci-TH, the dopamine rate-limiting synthesis enzyme,n larvae showing the “big ocellus” phenotype”. In control larvae,i-TH expression was restricted to the left portion of the sensoryesicle, in some of the coronet cells (Fig. 6A). In PQ exposed larvae,i-TH expression pattern was similar to that of control specimensFig. 6B).

. Discussion

Results from this work evidenced that paraquat exerts aose-dependent teratogenic effect on P. mammillata, inducing char-cteristic and highly reproducible malformations of larval nervousystem. All embryos treated at two-cell stage with PQ (from 0.01o 2 mg/ml) developed in larvae showing a reduction of the sen-ory vesicle cavity, the anterior most region of the central nervousystem, and the malformation of major sensory structures withint. In particular, the pigmented cell of the ocellus had an irregularhape, possibly because both photoreceptor and lens cells, whichormally are in strict contact with it and contribute to form thehotoreceptor complex, were displaced.

The range of PQ concentrations applied in our experiments waset considering previous results obtained with frog embryos andaking into account that development is much faster in ascidi-ns than in Xenopus (24 h vs. several days). Interestingly, no lethalffects on ascidian embryos were observed even at very high con-entrations of PQ. On the contrary, this herbicide was reported toave a strong lethal effect on frog embryos at very low doses (from

.06 to 0.5 mg/l) (Vismara et al., 2000). Therefore, the low sensi-iveness of ascidian embryos to PQ make them interesting modelrganisms to study its toxicity during development.

The immunohistochemical analysis evidenced that the malfor-ations of larval CNS induced by PQ exposure are very specific in P.

ntrol larva, TH transcripts were present in the left portion of the sensory vesicle inn left sensory vesicle.

mammillata. In particular, �-tubulin immunostaining showed thattreated larvae had a typical alteration of the ventral nerve fibersthat connect the otolith to the posterior sensory vesicle. Moreover,dopamine, normally present in the cells connected to the otolith,was no longer detectable in exposed larvae. On the contrary, GABA-positive cells were still present in PQ treated larvae, even if theywere irregularly distributed in the larval trunk.

Taken together, these findings suggest that dopaminergic neu-rons are the selective target of PQ toxicity in ascidian larvae. Thesusceptibility of the dopaminergic neurons to degeneration fol-lowing to the exposure to PQ is well documented in vertebrates(Dinis-Oliveira et al., 2006). Neonatal exposure to PQ induces per-manent changes in striatum dopamine content and behaviour inadult mice (Fredriksson et al., 1993). The toxicity of PQ is essentiallyrelated to its rapid reduction and subsequent reoxidation to pro-duce ROS and RNS. It has been reported that dopaminergic neuronsare much more sensitive to the oxidative stress than other neu-ronal cell types, due to the participation of dopamine in oxidativereactions (Dinis-Oliveira et al., 2006).

To test whether the oxidative stress is implicated in the induc-tion of the observed malformations also in ascidians, we performedcombined treatments with PQ plus ascorbic acid (AA), a well knownantioxidant agent, which proved to be able to rescue the dam-age caused by PQ exposure in Xenopus laevis embryos (Vismaraet al., 2001). Actually, the percentage of larvae developing themost severe phenotype, “big ocellus”, was significantly lower incombined treatments than in treatments with PQ alone. Theseresults suggest that the oxidative stress is involved in the onsetof the observed malformations induced by PQ also in this marineinvertebrate animal model. Moreover, the observed results of dosedependent AA protective effect on ascidians confirm the wellknown fact that anti-oxidants have a defensive role when theyare administered in low concentrations, while when given at highconcentrations they have pro-oxidative effect (Niemiec et al.,2005).

In the ascidian C. intestinalis larva, dopaminergic neuronsexpressing tyrosine hydroxylase (TH), the rate-limiting enzyme inthe synthesis of dopamine, are localized in the ventral sensoryvesicle, the same territory where these neurons are present inP. mammillata larva (Moret et al., 2005). We found that Ci-THexpression pattern was similar in control and PQ treated larvae.These findings indicated that the exposure to the herbicides do notinhibit the differentiation of dopaminergic neurons in ascidians.It is known that catecholaminergic neurons are more sensitive tooxidative stress as compared to other neural cell types (Kanga et al.,2009). Therefore, it is possible to hypothesize that the decrease ofdopamine content observed in treated larvae, expressing TH, was

due to the selective oxidation of this neurotransmitter caused byPQ. Moreover in the mouse brain, there are evidences that depletionin dopamine content in the neurons of the substantia nigra coulddepend on the inactivation of TH by RNS generated consequentlyto PQ exposure (Ara et al., 1998). For that reason, we cannot rule

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ut the hypothesis that dopamine depletion observed in PQ treatedarvae could be due also to TH inactivation.

PQ exposure has been related to the etiology of Parkinson’s dis-ase, the second most common neurodegenerative disorder afterlzheimer’s disease (Dinis-Oliveira et al., 2006). In fact the degen-ration of the dopaminergic neurons of substantia nigra, a nervousentre implicated in movement control, is a key feature of thisyndrome, together with movement impairment such as bradyki-esia (slow movements), tremors, rigidity and postural instabilityBrooks et al., 1999; Thiruchelvam et al., 2002).

Interestingly, ascidian otolith is involved in swimming con-rol (Tsuda et al., 2003) through sensory cells located under thetolith, that are supposed to perceive larva position (Imai andeinertzhagen, 2007). We showed that in P. mammillata dopamin-

rgic neurons are strictly associated to the otolith. Moreover, Moretnd collaborators (2005) suggested that dopamine may modu-ate the neural network involved in swimming behaviour in the C.ntestinalis larva. Thus, it will be of great interest to further inves-igate the effects of PQ exposure on ascidian larvae in order tovidence possible behavioural alterations.

Results from this work evidenced for the first time that paraquatxposure caused a selective depletion in dopamine content in annvertebrate organism, similarly to what already described in ver-ebrates. These observations suggested that dopaminergic neuronsensitiveness to oxidative stress is a common feature of these kindf neurons, regardless of the studied organism.

onflict of interest

The authors declare that there are no conflicts of interest.

cknowledgements

We thank Mr Maioli from the mussel culture of Lerici, Lapezia, for kindly allowing the sampling of P. mammillata animalsnd Paola Cirino from the “Stazione Zoologica A. Dohrn”, Naples,or supplying C. intestinalis adults. We thank Dr. Umberto Fas-io for confocal microscopy images, obtained using the facilitiesf C.I.M.A. (Advanced Microscopy Centre of the University). Thisork was supported by grants from the University of Milan (FIRST

007).

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