Effects of Exposure to ED Contaminants (TPT-Cl and Fenarimol

8/8/2019 Effects of Exposure to ED Contaminants (TPT-Cl and Fenarimol

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R E S E A R C H A R T I C L E

Alice Barbaglio Daniela Mozzi Michela SugniPaolo Tremolada Francesco BonasoroRamon Lavado Cinta PorteM. Daniela Candia Carnevali

Effects of exposure to ED contaminants (TPT-Cl and Fenarimol)on crinoid echinoderms: comparative analysis of regenerativedevelopment and correlated steroid levels

Received: 10 February 2005/ Accepted: 2 June 2005/ Published online: 6 January 2006 Springer-Verlag 2006

Abstract Regenerative phenomena reproduce develop-mental processes in adult organisms and are regulated

by neuro-endocrine mechanisms. They can thereforeprovide sensitive tests for monitoring the effects ofexposure to endocrine disrupter contaminants (EDs)which can be bioaccumulated by the organisms causingdysfunctions in steroid hormone metabolism and activ-ities and affecting reproduction and development. Ech-inoderms are prime candidates for this newecotoxicological approach, since (1) they offer uniquemodels to study physiological regenerative processes and(2) in echinoderms vertebrate-type steroids can be syn-thesized and used as terminal hormones along the neuro-endocrine cascades regulating reproductive, growth anddevelopmental processes. We are currently exploring the

effects on the regenerative potential of echinoderms ofdifferent classes of compounds that are well known tohave ED activity. The present paper focuses on thepossible effects of well-known compounds with sus-pected androgenic activity such as TPT-Cl (Triphenyl-tin-chloride) and Fenarimol [()-2,4-dichloro-a-(pyrimidin-5-yl) benzhydryl alcohol]. The selected test-species is the crinoid Antedon mediterranea, a tractableand sensitive benthic filter-feeding species which repre-sents a valuable experimental model for investigation on

the regenerative process from the macroscopic to themolecular level. The present investigation employs an

integrated approach which combines exposure experi-ments and biological analysis utilizing microscopy,immunocytochemistry and biochemistry. The experi-ments were carried out on experimentally induced armregenerations in semistatic controlled conditions withexposure concentrations comparable to those of mod-erately polluted coastal zones. The bulk of results ob-tained so far provide indications of significant sublethaleffects from exposure to TPT-Cl and Fenarimol andmechanisms of toxicity related to developmental physi-ology, which are associated with variations in steroidlevels in the animal tissues. The results indicate thatthese two substances (1) affect growth and development

by interfering with the same basic cellular mechanismsof regeneration, such as cell proliferation, migration anddifferentiation/dedifferentiation, which are possiblycontrolled by steroid hormones; and (2) can induce anumber of significant modifications in the timing,modalities and pattern of arm regeneration, which mayinvolve the activation of cell mechanisms related tosteroid synthesis/metabolism.

Introduction

Endocrine disrupters (EDs) are xenobiotic com-pounds that are persistent and widespread in the envi-ronment, and can be bioaccumulated by exposedorganisms, affecting significantly their physiology, par-ticularly in terms of reproduction, development andgrowth. These contaminants exert their effects by mim-icking the action of natural hormones, particularly ste-roids, interfering synergistically or antagonistically withtheir synthesis, metabolism or activity and interactingwith their nuclear receptors (Cadbury 1998; Colbornet al. 1993; Cooper and Kavlock 1997; Fairley et al.

Communicated by R. Cattaneo-Vietti, Genova

Physical and Chemical Impacts on Marine Organisms, a BilateralSeminar Italy-Japan held in November 2004

A. Barbaglio D. Mozzi M. Sugni P. TremoladaF. Bonasoro M. D. C. Carnevali (&)Dipartimento di Biologia, Universita` degli Studi di Milano,Via Celoria 26, 20133 Milano, ItalyE-mail: [email protected].: +39-02-50314788Fax: +39-02-50314781

R. Lavado C. PorteEnvironmental Chemistry Department, IIQAB-CSIC,C/ Jordi Girona, 18, 08034 Barcelona, Spain

Marine Biology (2006) 149: 6577DOI 10.1007/s00227-005-0205-0


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1996; Gray et al. 1996; Soto et al. 1995). There is a longlist of compounds (including pesticides, fungicides,insecticides, industrial and commercial chemicals anddrugs) known or suspected to act as hormonal modula-tors in human and wildlife populations. There are manycategories of pollutants displaying potential estrogenic/anti-estrogenic activity, as well as androgenic/anti-androgenic activity. However, a complete understandingof their mechanisms of action on different organisms isfar from being achieved. This gap in knowledge affectsparticularly invertebrates which represent more than95% of the extant animal species in natural ecosystems.In recent years aquatic invertebrates have been em-ployed extensively for monitoring environmental haz-ards and as sensitive test models for EDs (CandiaCarnevali 2005; Mu and LeBlanc 2002).

The presence of ED contaminants in the sea has anobvious impact on the physiology of marine inverte-brates (Depledge and Billinghurst 1999), with particularreference to benthic species living in close contact withcontaminated sediments. The phenomena of imposexand intersex described in gastropod molluscs are the

best-documented examples of adverse effects of EDcontaminants in marine animals (Bryan et al. 1986;Gibbs et al. 1988; Horiguchi 1995). On the other hand,with regard to important marine macro-invertebratessuch as echinoderms, the available data are still ratherlimited. Although they offer a wide range of sensitiveand amenable models, they have been only occasionallyemployed in ecotoxicological tests using adult organisms(Anderson et al. 1994; Be kri and Pelletier 2004; Coteuret al. 2001; den Besten 1998; den Besten et al. 1989, 1990,1991a, b; Kobayashi 1984) or developmental stages(Novelli et al. 2002). There are many factors that makeechinoderms the prime candidates for studying the ef-

fects of exposure to ED contaminants. Firstly, echino-derms are benthic animals and are particularlysusceptible to the presence of micropollutants stored inmarine sediments. Primary uptake across external epi-thelia (respiratory surfaces, epidermis, etc.) or secondaryuptake from food, represent important routes of entryfor many dissolved aquatic pollutants which can berapidly bioaccumulated by these organisms (Smith et al.1981; Tremolada et al. 2004). Secondly, regulatory fac-tors and hormones similar to those of vertebrates havebeen detected recently in echinoderms (Dieleman andSchoenmakers 1979; Hines et al. 1994; Janer et al. 2004).In particular, vertebrate-type steroids, both androgens

and estrogens, can be synthesized (Aminin et al. 1995;den Besten et al. 1989; LeBlanc et al. 1999; Voogt et al.1984, 1990, 1991; Shirai and Walker 1988; Schoenmak-ers 1979, 1980; Schoenmakers and Voogt 1980; Shubinaet al. 1998). Current research is focused on echinodermendocrinology and knowledge of the specific mecha-nisms involved, including steroid metabolism, isexpanding rapidly (Janer et al. 2004; Lutz et al. 2004).Echinoderms are deuterostome invertebrates and arephylogenetically closer to chordates than to otherinvertebrate groups. It is not surprising, therefore, that

they possess physiological mechanisms rather similar tothose of vertebrates, in terms of molecules and actions.In some echinoderm classes there is limited but signifi-cant published evidence for the disruptive effects ofcontaminants on steroid metabolism and steroid levelsand on the cytochrome P450 monooxygenase (MO)system (den Besten 1998; den Besten et al. 1989, 1990,1991a, b).

A final important point is that echinoderms havespectacular capacity for regeneration, in addition to thenormal processes of sexual reproduction. Regenerativephenomena, which represent developmental processes inadult organisms, are characterized by enhanced andactive cell proliferation, morphogenesis, differentiationand tissue renewal, which are modulated by endocrineand neurohumoral mechanisms comparable, if notidentical, to those involved in reproductive and devel-opmental processes. Vertebrate-type regulatory factors,including peptides and steroids, are also likely to be in-volved in the regeneration processes (Candia Carnevaliet al. 2001b; Thorndyke and Candia Carnevali 2001).For this reason regenerating echinoderms can be used as

experimental models to test the effects of exposure todifferent types of EDs (Candia Carnevali 2005). Expo-sure to pseudohormonal contaminants has already beenshown to induce variations, in the timing, mechanismsand actions of regenerative development, which isamongst the most sensitive phenomena with respect toenvironmental stress. Previous data obtained from theophiuroids Ophioderma brevispina (Walsh et al. 1986)and Microphiopholis gracillima (DAndrea et al. 1996)showed clearly that exposure to organotin compoundsand metals significantly affects arm regeneration pro-cesses, and demonstrates the usefulness of studyingregenerative development in adult organisms. Regener-

ating echinoderms thus appear to be ideal bioindicatorsfor ED-induced stress at the whole organism, cellularand molecular levels.

An important goal in studying EDs is establishing themost sensitive test-species and the most specific forms ofresponse (endpoints) at which hormonal dysfunction isexpressed unequivocally. Unique endocrine-regulatedprocesses, such as echinoderm regeneration, can providean important target of toxic action and original andquantifiable endpoints which can provide indications forthe specific effects of these persistent pollutants.

The present work is focused on the effects of twopotential endocrine disrupter compounds, TPT-Cl (tri-

phenyltin-chloride) and Fenarimol (()-2,4-dichloro-a-(pyrimidin-5-yl) benzhydryl alcohol), on the regenera-tive potential of crinoid echinoderms, with particularreference to the well-known process of arm regeneration.

TPT-Cl is an organotin compound which is usedextensively in agriculture and in antifouling paints. TPTconcentrations have been detected in terrestrial, fresh-water and coastal/marine environments. This compoundis well known for its androgenic activity (Fait et al. 1994;Matthiessen and Gibbs 1998), although no reliable dataare presently available on its specific mechanisms of

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action. There is some published information on the ef-fects of organotin compounds on marine invertebrates,including a few echinoderms (Depledge and Billinghurst1999; Walsh et al. 1986).

Fenarimol is a halogenated pesticide, particulary afungicide, employed against several plant diseases (IPCS2004). There are no available data about environmentallevels of this pesticide. The presence of Fenarimol in thesediments is suspected to be dangerous for aquatic spe-cies, particularly for benthic organisms.

Our specific aim is to explore the impact of thesepollutans on crucial developmental processes such asrepair, growth and differentiation at the whole organ-ism, tissue and cellular level. The selected experimentalanimal is the crinoid Antedon mediterranea, an echino-derm species representative of marine benthic fauna anda typical microfilter-feeding animal on which persistentsediment-bound micropollutants have an immediateimpact. This experimental model was employed suc-cessfully in our laboratory for pilot ecotoxicologicalstudies. Definitive or preliminary results obtained so faron the effects on crinoid regeneration of a wide range of

different ED compounds, such as PCBs (Polychlorinatedbiphenyls), nonylphenols, organotins (Barbaglio et al.2004; Candia Carnevali 2005; Candia Carnevali et al.2001a, b, c), showed that the regenerative response ofechinoderms is a valuable test for EDs and stronglyencouraged us to develop this applied research field.

This paper focuses on the effects of TPT-Cl andFenarimol on arm regeneration of the echinodermA. mediterranea. It provides a comparative account of(1) the possible damage and alterations to the regener-ative development detectable at whole organism, tissueand cellular levels due to exposure to these suspectedendocrine disrupter compounds, and (2) the correlated

fluctuations of testosterone and estradiol in the animaltissues.

Preliminary results were presented at the 11th Inter-national Echinoderm Conference, Munich 2003 (Bar-baglio et al. 2004).

Materials and methods

Exposure experiments

Specimens of A. mediterranea, collected from the Tyr-rhenian coast of Italy (Giglio Island), were maintained in

aquaria of artificial seawater at 14C, and fed with In-verteMin (Tetra Marin). Exposure tests for both TPT-Cl(Merck) and Fenarimol (Riedel) were performed insemistatic conditions (20% water renewal in 24 h).Groups of 30 specimens (TPT tests) and 35 specimens(Fenarimol tests) were employed in each aquarium(exposure, control and solvent control aquaria). In eachexposed or control specimen, experimental regenerationwas induced by amputating three arms at the autotomyplane. Immediately after amputation, the experimentalanimals were put in the test-aquaria and exposed to

different concentrations of the selected compounds(TPT-Cl: 50, 100, 225, 500, 1,000 ng/l; Fenarimol: 24,240, 2,400 ng/l) for prefixed periods (72 h, 1 and2 weeks): in this way the exposure period correspondedto well defined and established regenerative stage (Can-dia Carnevali and Bonasoro 2001). As far as the selectedTPT-Cl exposure concentrations are concerned, themaximum concentration was close to LC50 experimentalvalues quoted in the literature for molluscs (Rippen1990), the minimum to NOEC experimental valuesknown for echinoderms (O. brevispina, Walsh et al.1986). With regard to Fenarimol, the maximum and theminimum concentrations were chosen on the basis of theminimum EC50 available data (0.18 mg/l for DaphniamagnaBell 1994) reduced by factors of 10 and 10,000.

The TPT-contaminated medium was obtained byadding to the aquaria (50 l artificial seawater) 1.25 mlethanolTPT-Cl solution (TPT-Cl concentrations: 2, 4,9, 20, 40 lg/ml, respectively) at the start of the experi-ment and 0.25 ml ethanolTPT-Cl solution (TPT-Clconcentrations: 2, 4, 9, 20, 40 lg/ml, respectively) day byday. The Fenarimol-contaminated medium was ob-

tained by adding to the aquaria (50 l artificial seawater)1 ml ethanolFenarimol solution (Fenarimol concen-trations: 1.2, 2, 12, 120 lg/ml, respectively) at the startof the experiment and 0.2 ml ethanolFenarimol solu-tion (Fenarimol concentrations: 1.2, 2, 12, 120 lg/ml,respectively) day by day. The final ethanol concentrationin exposure and in solvent control aquaria was 0.025 and0.020 ml/l for TPT-Cl and Fenarimol, respectively: theseconcentrations are much lower than that (0.1 ml/l) offi-cially allowed in long-term ecotoxicity tests with aquaticinvertebrates (see Annex V, Dir67/548/EEC, EEC 1967).

At each selected regenerative stage, chemical analysesof water and echinoderm tissues were performed in or-

der to check the variability of exposure concentrationand bioaccumulation (Tremolada et al. 2005). TPT-Clanalyses were performed by gas-chromatographic sepa-ration and mass-spectrometry detection after derivati-zation of the original compound (TPT-Cl) in theextraction medium. Fenarimol analyses were performedby gas-chromatographic separation. In terms of chemi-cal parameters, a detailed chemical analysis of water andtissue samples from our exposure experiments is still inprogress (Dagnac et al., unpublished; Sakkas et al.,unpublished).

Biological analyses

Histological analysis

Standard methods for morphological analysis by bothstereomicroscope and light microscope, as described inprevious papers (Candia Carnevali et al. 1993), andspecific immunocytochemical protocols for monitoringcell proliferation (BrdU methods) were employed. Astatistical analysis of the quantitative results was per-formed whenever appropriate.

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Exposed and control regenerating arms (three armsfrom each individual) were prefixed with 2% glutaral-dehyde in 0.1 M cacodylate buffer for 45 h, then, afterovernight washing in the same buffer, postfixed with 1%osmium tetroxide in the same buffer. After standarddehydration in an ethanol series, the samples wereembedded in Epon-Araldite 812. In particular, four ex-posed samples per each exposure concentration and fourcontrol samples at all the regenerative stages wereanalysed microscopically. The semithin sections, cutwith a Reichert Ultracut E, were stained by conventionalmethods (crystal violet-basic fuchsin) and then observedin a Jenaval light microscope.

Cell proliferation was monitored using in vivoincorporation of the substituted nucleotide, 5-bromo-deoxyuridine (BrdU), then revealed by a monoclonalantibody against BrdU (Cell proliferation kit: Amer-sham). This same DNA synthesis labelling technique hasbeen previously used successfully to monitor early andadvanced stages of arm regeneration in Antedon (CandiaCarnevali et al. 1995, 1997). For use with semithinEpon-Araldite sections, the standard BrdU-immunocy-

tochemistry protocol for paraffin sections was modifiedas described in detail by Candia Carnevali et al. (1995).In particular, two exposed samples per each exposureconcentration and two control samples at all theregenerative stages were analysed microscopically.

The results of the exposure tests were compared withthose obtained by a parallel analysis of normal regen-erating samples in standard conditions.

Biochemical analysis

Steroid analysis was performed on whole animal sam-

ples, as described in Janer et al. (2004). Briefly, indi-viduals (1.01.5 g wet weight) were homogenized inethanol, and frozen overnight at 80C. Homogenateswere then extracted with ethyl acetate twice. The organicextract was evaporated under nitrogen, and redissolvedin 80% methanol. This solution was then washed withpetroleum ether to remove the lipid fraction and evap-orated to dryness. The dry residue was redissolved in4 ml Milli-Q water and passed through a C18 cartridge(Isolute, 1 g, 6 ml) that had been sequentially precon-ditioned with methanol (4 ml) and milli-Q water (8 ml).After finishing the concentration step, cartridges werewashed with milli-Q water (8 ml), dried, and connected

to a NH2 cartridge (Waters, Sep-Pack

Plus). The C18-NH2 system was then washed with 8 ml hexane, and thesteroids eluted with 9 ml dichloromethane:methanol(7:3). This fraction was collected and evaporated todryness. The efficiency of the extraction and delipidationprocedure was 743% for T, and 803% for E2 (Janeret al. 2004) and 95 (E2) to 97% (T) for the purificationstep (SPE cartridges) (Janer et al. 2004).

Dry extracts were resuspended in 50 mM potassiumphosphate buffer pH 7.6 containing 0.1% gelatin, andassayed for estradiol and testosterone concentration

using commercial RIA kits. Standard curves with thesteroids dissolved in the same phosphate buffer wereperformed in every run. The detection limits were of30 pg/g for E2 and 130 pg/g for T. Intra-assay coeffi-cients of variation were of 6.1 (T) and 3.3% (E2). In-terassay coefficients of variation were 9.3 (T) and 3.5%(E2).

Statistical analysis

Growth

Lengths of regenerating arms were measured using thesoftware ArcView GIS 3.2 and, with regard to TPT-Cltests, comparison of significant difference (P


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2005; Candia Carnevali et al. 2001a, c, 2003). The resultsobtained from the different series of tests performed withTPT-Cl and Fenarimol, respectively, were neither uni-form nor easily interpretable from a qualitative orquantitative point of view. As far as overall growth isconcerned at the advanced stage of 1 and 2 weeks thatcorresponded to long-term exposure periods, both TPT-Cl and Fenarimol samples displayed great variability interms of overall growth in comparison with the uniformgrowth of the controls. With regard to TPT-Cl-exposedsamples alone, a clear effect in terms of enhanced growthcould be detected at the advanced stage of 2 weeks andwith the intermediate-high concentration employed(225 ng/l), as shown by a quantitative analysis carriedout on the measured lengths of all the experimentalregenerates in both exposed and control samples. Fig-ure 1 shows that, according to the statistical analysiscarried out for 1 and 2 weeks p.a. stages, 225 ng/l ap-pears to induce a significant (P


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was characterized by the presence of many differentiatednonblastemal elements, such as myocytes, apparentlymigrating from the distal muscles of the stump (Figs. 2b,5b) and scattered skeletal spicules (Fig. 2c).

Another abnormal feature concerned the presenceand the functional state of granulocytes, which arenormally employed in crinoid repair processes (Heinz-

eller and Welsch 1994). In TPT-Cl-exposed samples alarge number of granulocytes were detected close to boththe coelomic canals and brachial nerve, where they ap-peared to release their cytoplasmic granules (Fig. 3).Similar degranulation phenomena were found also in theadvanced regenerative stages.

As far as Fenarimol-exposed samples are concerned,the atypical features were less conspicuous. At thehighest concentrations employed, the sagittal sections ofthe regenerating arms displayed an unusual empty areadelimited by a thin cicatricial layer, in correspondencewith the brachial nerve end (Fig. 2d).

As far as cell proliferation is concerned, our BrdU

incorporation experiments (not shown) indicated that inboth TPT-Cl and Fenarimol samples, at this early stage,the cell proliferation pattern was only slightly differentfrom that of the controls, the possible differencesbecoming evident only at more advanced stages (seebelow).

Advanced regenerative phase (1 and 2 weeks p.a.)

During the advanced regenerative phases of 1 and2 weeks p.a., which corresponded to medium and long-

term exposure periods, significant anomalies could bedetected particularly in the samples exposed to inter-mediate or higher concentrations of both the com-pounds (Fig. 4).

In sagittal sections the aberrant anatomy of theregenerate could be appreciated even at low magnifica-tion. It was generally characterized by an unusual

developmental pattern of tissues, with particular refer-ence to a pronounced abnormal development of thecomponents of the skeletal tissue (Fig. 4b, c, e, f). AllTPT-Cl- and Fenarimol-exposed samples exhibitedscattered skeletal spicules (Fig. 4b, c). The phenomenonwas particularly evident in Fenarimol samples: in theregenerating arm the ossicles were smaller on the wholethan in the controls (Fig. 4a) and looked much lessdeveloped than other anatomical elements (ambulacralgrooves, coelomic cavities, brachial nerve, etc.) (Fig. 4c).The only exception was the first ossicle of the series(proximal with respect to the amputation surface) whichappeared always to have developed normally. In addi-

tion, in Fenarimol samples (Fig. 4f), the ossicles werecharacterized by a less compact and irregular stereommacro- and microstructure than in the controls(Fig. 4d).

Besides these abnormalities related to the skeleton,TPT-Cl- and Fenarimol-exposed samples showed anextensive rearrangement and/or dedifferentiation ofother differentiated tissues, specifically involving themuscle bundles of the stump (Fig. 5ac). In particular,the 1-week samples exposed to the highest concentra-tions of Fenarimol exhibited also a conspicuous

Fig. 2 Histological sagittal sections of control and exposed

regenerating arms at 72 h p.a.: a Control. The blastema consistsof morphologically undifferentiated cells. b, c TPT-Cl-exposedsamples. b 100 ng TPT-Cl/l. The blastemal bud appears to beformed mostly from ectopic myocytes (arrow head) supplied by theapparent rearrangement of the muscle bundle of the stump.c 225 ng TPT-Cl/l. The blastemal bud consists of ectopic elements,

namely skeletal spicules (arrow) usually not present at this early

stage. d Fenarimol-exposed sample. 240 ng Fenarimol/l. Tissuerepair is still incomplete: in spite of the presence of a thin,continuous external cicatricial layer, a wide empty area (asterisk) isclearly evident at the level of the brachial nerve b blastema; cccoelomic canals; m muscle bundles; n brachial nerve

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bi-directional migration of myocytes between adjacentmuscle bundles (Fig. 5c).

As far as cell proliferation is concerned, our BrdUincorporation experiments showed that in TPT-Clsamples, at both the stages of 1 and 2 weeks, cellproliferation phenomena were always localized in thesame proliferation sites as in the controls (Fig. 6a),namely at the level of the apical blastema, the brachial

nerve and the coelomic epithelium. However, in all theexposed samples, the labelling at these sites appeared tobe less strong and widespread than in the controls(Fig. 6b).

A similar situation was detected in 1-week Fenarimolsamples (Fig. 6c), whereas in 2 weeks samples the pro-liferation pattern appeared to be more similar to thestandard level for this stage.

Tissue steroid levels

Steroid levels were determined in whole animal samplesafter 2 weeks exposure. A significant increase in testos-terone levels was detected in those organisms exposed to225 ng/l TPT. The amount of testosterone detected was440140 pg/g wet weight, double the levels detected inthe control group. Interestingly, this effect was associ-ated with a corresponding significant decrease in estra-diol levels at the same exposure level (Fig. 7a). Thisapparent androgenization effect was more clearly ob-served when the ratio E2/T was calculated. This ratio

ranged from 1.220.44 (control) and 0.830.34 (sol-vent control) to 0.190.04 in individuals exposed to225 ng/l TPT (P


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coastline. This contaminant tends to be accumulated

in soil and sediments (Federoff et al. 1999), but due toits strong affinity for tissues (Kow=3.1; Federoff et al.1999), it can be found at quite a high level inorganisms. This compound is well known for itsandrogenic activity (Fait et al. 1994; Matthiessen andGibbs 1998), although no reliable data are presentlyavailable on its specific mechanisms of action. There issome published information on the effects of organo-tin compounds on marine invertebrates, including afew echinoderms (Depledge and Billinghurst 1999).Particularly relevant are their effects on reproductivebiology (see imposex in gastropod molluscs, Horiguchiet al. 1995), and development (see the benthic filter-

feeding ascidian Styela plicata, Cima et al. 1996; Ya-llapragada et al. 1990). With regard to echinoderms,toxicity tests on the sea urchin Paracentrotus lividusemphasized that TPT can cause critical damage tosperm motility and development (Moschino and Marin2002; Novelli et al. 2002). As previously mentioned,data on the ophiuroid O. brevispina (Walsh et al.1986) showed that exposure to TPT affects signifi-cantly arm regeneration processes.

Fenarimol is a halogenated pesticide extensivelyemployed in agriculture (IPCS 2004). There are no

available data about environmental levels of this pesti-

cide. In spite of this, its Henrys law constant(6.93 s104 Pam3/mol) suggests a moderately lowvolatility (Mackay et al. 1997), implying that it mayreach surface waters quickly by direct overspray or byrun off from treated fields (IPCS 2004). It is rapidlytransferred to the sediments (Jackson and Lewis 1994)where it tends to persist because of its stability (Fitox1999; Kline and Knox 1981; IPCS 2004). For thesereasons the presence of Fenarimol in the sediments issuspected to be dangerous for benthic organisms. Fe-narimol shows moderate values of toxicity in aquaticorganisms: EC50=0.18 mg/l in D. magna (Bell 1994),NOEC=0.113 mg/l again in D. magna (Hoffman et al.

1987). Fenarimol lowers ecdysone levels in this organ-ism, which results in embryo abnormalities (Mu andLeBlanc 2002).

On the whole, our present results confirm that se-lected androgenic compounds (TPT-Cl and Fenarimol)impact strongly on regenerative development and induceobvious malformations in the internal anatomy andhistopathological anomalies involving cell migration,proliferation, histogenesis and differentiation.

Our results indicate that both TPT and Fenarimolstrongly affect skeletal development, as shown by both

Fig. 4 Histological sagittal sections of control and exposedregenerating arm at 1-week p.a. a Control. Normal tissuedifferentiation is in progress in the regenerate. The proximaldistaldevelopment of the brachial ossicles is evident: the proximal andthe distal ossicles are indicated by arrow and arrowheads,respectively. b 225 ng TPT-Cl/l. The regenerating arm showsanomalous histological features: the developing distal ossicles(arrowheads) are similar in size to the proximal one (arrow). c240 ng Fenarimol/l. With the only exception of the proximal ossicleof the series (arrow), all the ossicles (arrowheads) are generally small

and appear to be poorly developed if compared with otherhistological elements (ambulacral grooves, coelomic cavities,brachial nerve). d Detail of a control sample to show the compactstructure of a differentiating ossicle. e Detail of 225 ng TPT-Cl/l-exposed sample. The ossicle structure appears to be poorlydeveloped and rather disorganized. f Detail of 240 ng Fenarimol/l-exposed sample. The development of the skeletal tissue is clearlydelayed. Several skeletal spicules are scattered and disarranged. cccoelomic canals; ga ambulacral grove; m muscle bundle; n rachialnerve

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consequent increase in the relative contribution toregeneration of morphallactic mechanisms in these par-ticular stress conditions. However, the effects of the twocompounds are not identical, since at the stage of2 weeks Fenarimol samples tend to show a conditionanalogous to the normal, in terms of cell proliferationpattern, which is associated with a parallel reduction inthe rearrangement phenomena at the level of the stumptissues. These features suggest that after 2 weeks regen-eration in the Fenarimol-exposed animals was returnedto the normal condition.

Granulocites are normally involved in repairprocesses and discharge their granules in the tissues of

the amputation area. However, in TPT-exposed sam-ples, they appear to be involved in widespread degran-ulation and cell lysis detectable in the connective tissuesof the stump, especially close to the coelomic canals(Fig. 3). This phenomenon could be partly explicable interms of the well-known interference of organotin withCa2+ metabolism and with proteins involved in cellhomeostasis (for instance F1F0 ATPase, Na+K+AT-Pase, Ca2+ATPase as shown in mammals, Powers andBeavis 1991).

Although the histological results of the exposure testswith TPT are similar to those obtained with Fenarimol,these substances have a different chemical composition

Fig. 6 Histological sagittal sections of control and exposedregenerating arms at 1-week p.a. Immunocytochemistry for BrdU.a Control. Many blastemal and coelothelial cells are strongly

labelled. b 225 ng TPT-Cl/l. The immunostaining appears to be lessevident in comparison with the control. c 240 ng Fenarimol/l. Onlya few labelled cells are detectable. cc coelomic canals

Fig. 7 Whole tissue levels of testosterone and estradiol (expressedas pg/g wet weight) in (a) TPT- and (b) Fenarimol-exposedregenerating animals. Data are presented as mean SEM (n=5).Samples exposed to 225 ng TPT-Cl/l show an increase intestosterone levels and a decrease in estradiol levels. An increase

in estradiol levels is evident in samples exposed to 240 ngFenarimol/l. a Significant differences respect to control, b signif-icant differences respect to solvent control. C control, SC solventcontrol

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and therefore could have different mechanisms of actionat different levels. It is not surprising, therefore, that theeffects on steroid levels are quite different for the twocompounds: TPT appears to produce a significant in-crease in testosterone level accompanied by a relevantdecrease in the estrogen levels in samples exposed to225 ng/l; in contrast, Fenarimol induces an increase inestrogen levels in samples exposed to comparable con-centrations (240 ng/l). In agreement with the knownactivity of TBT (Tributyltin), TPT appears to interferewith steroid metabolism, possibly acting on synthesis bycytochrome P450-dependent aromatase or excretion ra-ther than on steroid receptors (Matthiessen and Gibbs1998). While our results seem to confirm that TPT hasan androgenic effect in echinoderms, Fenarimol seems tohave an estrogenic action. The biochemical data showclearly that there are enhanced estradiol levels in Fe-narimol-exposed samples. This is in agreement with theresults of recent in vitro experiments showing that Fe-narimol can interact with both estrogen and androgenreceptors, acting as an estrogen agonist or as anandrogen antagonist (Andersen et al. 2002).

However, since the two compounds cause the sameskeletal anomalies and cell/tissue alterations (decrease incell proliferation and rearrangement/dedifferentiation ofmuscles) it appears that they have a range of differentmechanisms of action at the tissue and cellular level. Onthe one hand, they act differently on hormone metabo-lism and have contrasting effects on the observed hor-mone levels; on the other hand, they appear to affectother physiological mechanisms in a similar way and toinduce a pattern of developmental dysfunctions resultingfinally in anomalous regeneration. They probably affectgrowth and development by interfering with the samebasic cellular mechanisms of regeneration, such as cell

proliferation, migration and differentiation/dedifferenti-ation, which may be controlled, directly or indirectly, bysteroid hormones or their quantitative ratios (Marsh andWalker 1995).

Although definitive results from chemical analysis ofwater and tissue samples from Fenarimol exposureexperiments are not available at the moment, pre-liminary data (Sakkas et al. unpublished) showed thatactual concentration measured in exposure medium at2 weeks were lower than the nominal ones (i.e. 4.5, 128and 1,285 ng/l, respectively, for the nominal concentra-tions of 24, 240, 2,400 ng/l). On the other hand, as far asTPT is concerned, analytical results for TPT exposure

experiments (Tremolada et al. 2004) clearly indicate thatactual concentrations measured in exposure medium aremuch lower than the nominal ones (i.e. 5.53.1, 146.3and 3311 ng/l, respectively, for the nominal concen-tration of 100, 225 and 500 ng/l). Nevertheless, actualTPT exposure concentrations are rather similar to thoserecorded in polluted Mediterranean coastal areas (Tol-osa et al. 1996). This means that TPT concentrationsfound commonly in coastal zones can affect significantlythe physiology of echinoderms, as was already been

found for other marine invertebrates (Depledge andBillinghurst 1999).

In conclusion, the present work further confirms thatcrinoid regeneration is a useful model for evaluatingpossible effects of ED compounds at whole organism,tissue and cellular levels. It demonstrates that thephysiology of sensitive marine animals can be affectedsignificantly by these compounds, even at very lowconcentrations, through their interaction with mecha-nisms regulating development and growth. Finally itprovides valuable indications of the actual threat tomarine ecosystems that is posed by EDs.

Acknowledgements The present work has received financial supportfrom the EU (COMPRENDO Project n EVK1-CT-2002-00129).The authors are particularly grateful to Dr Ulrike Shulte-Oehl-mann for her valuable coordinating activity and to all the partnersof the COMPRENDO project for their direct or indirect supportand advice. Special thanks are addressed to Drs Simona Cerianiand Angelita Doria for their valuable help and technical assistance.All the experiments carried out for the research work are in accordwith the current laws of our country. The authors are grateful tothe anonymous reviewers for their invaluable suggestions andcareful revision of the manuscript.

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Effects of Exposure to ED Contaminants (TPT-Cl and Fenarimol

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