Marine Biology (2006) 149: 65–77 DOI 10.1007/s00227-005-0205-0
R ES E AR C H A RT I C L E
Alice Barbaglio Æ Daniela Mozzi Æ Michela Sugni Paolo Tremolada Æ Francesco Bonasoro Ramon Lavado Æ Cinta Porte M. Daniela Candia Carnevali
Effects of exposure to ED contaminants (TPT-Cl and Fenarimol) on crinoid echinoderms: comparative analysis of regenerative development and correlated steroid levels Received: 10 February 2005 / Accepted: 2 June 2005 / Published online: 6 January 2006 Springer-Verlag 2006
Abstract Regenerative phenomena reproduce developmental processes in adult organisms and are regulated by neuro-endocrine mechanisms. They can therefore provide sensitive tests for monitoring the effects of exposure to endocrine disrupter contaminants (EDs) which can be bioaccumulated by the organisms causing dysfunctions in steroid hormone metabolism and activities and affecting reproduction and development. Echinoderms are prime candidates for this new ecotoxicological approach, since (1) they offer unique models to study physiological regenerative processes and (2) in echinoderms vertebrate-type steroids can be synthesized and used as terminal hormones along the neuroendocrine cascades regulating reproductive, growth and developmental processes. We are currently exploring the effects on the regenerative potential of echinoderms of different classes of compounds that are well known to have ED activity. The present paper focuses on the possible effects of well-known compounds with suspected androgenic activity such as TPT-Cl (Triphenyltin-chloride) and Fenarimol [(±)-2,4-dichloro-a(pyrimidin-5-yl) benzhydryl alcohol]. The selected testspecies is the crinoid Antedon mediterranea, a tractable and sensitive benthic filter-feeding species which represents a valuable experimental model for investigation on
the regenerative process from the macroscopic to the molecular level. The present investigation employs an integrated approach which combines exposure experiments and biological analysis utilizing microscopy, immunocytochemistry and biochemistry. The experiments were carried out on experimentally induced arm regenerations in semistatic controlled conditions with exposure concentrations comparable to those of moderately polluted coastal zones. The bulk of results obtained so far provide indications of significant sublethal effects from exposure to TPT-Cl and Fenarimol and mechanisms of toxicity related to developmental physiology, which are associated with variations in steroid levels in the animal tissues. The results indicate that these two substances (1) affect growth and development by interfering with the same basic cellular mechanisms of regeneration, such as cell proliferation, migration and differentiation/dedifferentiation, which are possibly controlled by steroid hormones; and (2) can induce a number of significant modifications in the timing, modalities and pattern of arm regeneration, which may involve the activation of cell mechanisms related to steroid synthesis/metabolism.
Communicated by R. Cattaneo-Vietti, Genova
Introduction
Physical and Chemical Impacts on Marine Organisms, a Bilateral Seminar Italy-Japan held in November 2004
‘‘Endocrine disrupters’’ (EDs) are xenobiotic compounds that are persistent and widespread in the environment, and can be bioaccumulated by exposed organisms, affecting significantly their physiology, particularly in terms of reproduction, development and growth. These contaminants exert their effects by mimicking the action of natural hormones, particularly steroids, interfering synergistically or antagonistically with their synthesis, metabolism or activity and interacting with their nuclear receptors (Cadbury 1998; Colborn et al. 1993; Cooper and Kavlock 1997; Fairley et al.
A. Barbaglio Æ D. Mozzi Æ M. Sugni Æ P. Tremolada F. Bonasoro Æ M. D. C. Carnevali (&) Dipartimento di Biologia, Universita` degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy E-mail:
[email protected] Tel.: +39-02-50314788 Fax: +39-02-50314781 R. Lavado Æ C. Porte Environmental Chemistry Department, IIQAB-CSIC, C/ Jordi Girona, 18, 08034 Barcelona, Spain
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1996; Gray et al. 1996; Soto et al. 1995). There is a long list of compounds (including pesticides, fungicides, insecticides, industrial and commercial chemicals and drugs) known or suspected to act as hormonal modulators in human and wildlife populations. There are many categories of pollutants displaying potential estrogenic/ anti-estrogenic activity, as well as androgenic/antiandrogenic activity. However, a complete understanding of their mechanisms of action on different organisms is far from being achieved. This gap in knowledge affects particularly invertebrates which represent more than 95% of the extant animal species in natural ecosystems. In recent years aquatic invertebrates have been employed extensively for monitoring environmental hazards and as sensitive test models for EDs (Candia Carnevali 2005; Mu and LeBlanc 2002). The presence of ED contaminants in the sea has an obvious impact on the physiology of marine invertebrates (Depledge and Billinghurst 1999), with particular reference to benthic species living in close contact with contaminated sediments. The phenomena of imposex and intersex described in gastropod molluscs are the best-documented examples of adverse effects of ED contaminants in marine animals (Bryan et al. 1986; Gibbs et al. 1988; Horiguchi 1995). On the other hand, with regard to important marine macro-invertebrates such as echinoderms, the available data are still rather limited. Although they offer a wide range of sensitive and amenable models, they have been only occasionally employed in ecotoxicological tests using adult organisms (Anderson et al. 1994; Be´kri and Pelletier 2004; Coteur et 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 make echinoderms the prime candidates for studying the effects of exposure to ED contaminants. Firstly, echinoderms are benthic animals and are particularly susceptible to the presence of micropollutants stored in marine sediments. Primary uptake across external epithelia (respiratory surfaces, epidermis, etc.) or secondary uptake from food, represent important routes of entry for many dissolved aquatic pollutants which can be rapidly bioaccumulated by these organisms (Smith et al. 1981; Tremolada et al. 2004). Secondly, regulatory factors and hormones similar to those of vertebrates have been detected recently in echinoderms (Dieleman and Schoenmakers 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; Schoenmakers 1979, 1980; Schoenmakers and Voogt 1980; Shubina et al. 1998). Current research is focused on echinoderm endocrinology and knowledge of the specific mechanisms involved, including steroid metabolism, is expanding rapidly (Janer et al. 2004; Lutz et al. 2004). Echinoderms are deuterostome invertebrates and are phylogenetically closer to chordates than to other invertebrate groups. It is not surprising, therefore, that
they possess physiological mechanisms rather similar to those of vertebrates, in terms of molecules and actions. In some echinoderm classes there is limited but significant published evidence for the disruptive effects of contaminants on steroid metabolism and steroid levels and 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 have spectacular capacity for regeneration, in addition to the normal processes of sexual reproduction. Regenerative phenomena, which represent developmental processes in adult organisms, are characterized by enhanced and active cell proliferation, morphogenesis, differentiation and tissue renewal, which are modulated by endocrine and neurohumoral mechanisms comparable, if not identical, to those involved in reproductive and developmental processes. Vertebrate-type regulatory factors, including peptides and steroids, are also likely to be involved in the regeneration processes (Candia Carnevali et al. 2001b; Thorndyke and Candia Carnevali 2001). For this reason regenerating echinoderms can be used as experimental models to test the effects of exposure to different types of EDs (Candia Carnevali 2005). Exposure to pseudohormonal contaminants has already been shown to induce variations, in the timing, mechanisms and actions of regenerative development, which is amongst the most sensitive phenomena with respect to environmental stress. Previous data obtained from the ophiuroids Ophioderma brevispina (Walsh et al. 1986) and Microphiopholis gracillima (D’Andrea et al. 1996) showed clearly that exposure to organotin compounds and metals significantly affects arm regeneration processes, and demonstrates the usefulness of studying regenerative development in adult organisms. Regenerating echinoderms thus appear to be ideal bioindicators for ED-induced stress at the whole organism, cellular and molecular levels. An important goal in studying EDs is establishing the most sensitive test-species and the most specific forms of response (endpoints) at which hormonal dysfunction is expressed unequivocally. Unique endocrine-regulated processes, such as echinoderm regeneration, can provide an important target of toxic action and original and quantifiable endpoints which can provide indications for the specific effects of these persistent pollutants. The present work is focused on the effects of two potential endocrine disrupter compounds, TPT-Cl (triphenyltin-chloride) and Fenarimol ((±)-2,4-dichloro-a(pyrimidin-5-yl) benzhydryl alcohol), on the regenerative potential of crinoid echinoderms, with particular reference to the well-known process of arm regeneration. TPT-Cl is an organotin compound which is used extensively in agriculture and in antifouling paints. TPT concentrations have been detected in terrestrial, freshwater and coastal/marine environments. This compound is well known for its androgenic activity (Fait et al. 1994; Matthiessen and Gibbs 1998), although no reliable data are presently available on its specific mechanisms of
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action. There is some published information on the effects of organotin compounds on marine invertebrates, including a few echinoderms (Depledge and Billinghurst 1999; Walsh et al. 1986). Fenarimol is a halogenated pesticide, particulary a fungicide, employed against several plant diseases (IPCS 2004). There are no available data about environmental levels of this pesticide. The presence of Fenarimol in the sediments is suspected to be dangerous for aquatic species, particularly for benthic organisms. Our specific aim is to explore the impact of these pollutans on crucial developmental processes such as repair, growth and differentiation at the whole organism, tissue and cellular level. The selected experimental animal is the crinoid Antedon mediterranea, an echinoderm species representative of marine benthic fauna and a typical microfilter-feeding animal on which persistent sediment-bound micropollutants have an immediate impact. This experimental model was employed successfully in our laboratory for pilot ecotoxicological studies. Definitive or preliminary results obtained so far on the effects on crinoid regeneration of a wide range of different ED compounds, such as PCBs (Polychlorinated biphenyls), nonylphenols, organotins (Barbaglio et al. 2004; Candia Carnevali 2005; Candia Carnevali et al. 2001a, b, c), showed that the regenerative response of echinoderms is a valuable test for EDs and strongly encouraged us to develop this applied research field. This paper focuses on the effects of TPT-Cl and Fenarimol on arm regeneration of the echinoderm A. mediterranea. It provides a comparative account of (1) the possible damage and alterations to the regenerative development detectable at whole organism, tissue and cellular levels due to exposure to these suspected endocrine disrupter compounds, and (2) the correlated fluctuations of testosterone and estradiol in the animal tissues. Preliminary results were presented at the 11th International Echinoderm Conference, Munich 2003 (Barbaglio et al. 2004).
Materials and methods Exposure experiments Specimens of A. mediterranea, collected from the Tyrrhenian coast of Italy (Giglio Island), were maintained in aquaria of artificial seawater at 14C, and fed with InverteMin (Tetra Marin). Exposure tests for both TPT-Cl (Merck) and Fenarimol (Riedel) were performed in semistatic 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 each exposed or control specimen, experimental regeneration was induced by amputating three arms at the autotomy plane. Immediately after amputation, the experimental animals 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 and 2 weeks): in this way the exposure period corresponded to well defined and established regenerative stage (Candia Carnevali and Bonasoro 2001). As far as the selected TPT-Cl exposure concentrations are concerned, the maximum concentration was close to LC50 experimental values quoted in the literature for molluscs (Rippen 1990), the minimum to NOEC experimental values known for echinoderms (O. brevispina, Walsh et al. 1986). With regard to Fenarimol, the maximum and the minimum concentrations were chosen on the basis of the minimum EC50 available data (0.18 mg/l for Daphnia magna—Bell 1994) reduced by factors of 10 and 10,000. The TPT-contaminated medium was obtained by adding to the aquaria (50 l artificial seawater) 1.25 ml ethanol–TPT-Cl solution (TPT-Cl concentrations: 2, 4, 9, 20, 40 lg/ml, respectively) at the start of the experiment and 0.25 ml ethanol–TPT-Cl solution (TPT-Cl concentrations: 2, 4, 9, 20, 40 lg/ml, respectively) day by day. The Fenarimol-contaminated medium was obtained by adding to the aquaria (50 l artificial seawater) 1 ml ethanol–Fenarimol solution (Fenarimol concentrations: 1.2, 2, 12, 120 lg/ml, respectively) at the start of the experiment and 0.2 ml ethanol–Fenarimol solution (Fenarimol concentrations: 1.2, 2, 12, 120 lg/ml, respectively) day by day. The final ethanol concentration in exposure and in solvent control aquaria was 0.025 and 0.020 ml/l for TPT-Cl and Fenarimol, respectively: these concentrations are much lower than that (0.1 ml/l) officially allowed in long-term ecotoxicity tests with aquatic invertebrates (see Annex V, Dir67/548/EEC, EEC 1967). At each selected regenerative stage, chemical analyses of water and echinoderm tissues were performed in order to check the variability of exposure concentration and bioaccumulation (Tremolada et al. 2005). TPT-Cl analyses were performed by gas-chromatographic separation and mass-spectrometry detection after derivatization of the original compound (TPT-Cl) in the extraction medium. Fenarimol analyses were performed by gas-chromatographic separation. In terms of chemical parameters, a detailed chemical analysis of water and tissue samples from our exposure experiments is still in progress (Dagnac et al., unpublished; Sakkas et al., unpublished). Biological analyses Histological analysis Standard methods for morphological analysis by both stereomicroscope and light microscope, as described in previous papers (Candia Carnevali et al. 1993), and specific immunocytochemical protocols for monitoring cell proliferation (BrdU methods) were employed. A statistical analysis of the quantitative results was performed whenever appropriate.
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Exposed and control regenerating arms (three arms from each individual) were prefixed with 2% glutaraldehyde in 0.1 M cacodylate buffer for 4–5 h, then, after overnight washing in the same buffer, postfixed with 1% osmium tetroxide in the same buffer. After standard dehydration in an ethanol series, the samples were embedded in Epon-Araldite 812. In particular, four exposed samples per each exposure concentration and four control samples at all the regenerative stages were analysed microscopically. The semithin sections, cut with a Reichert Ultracut E, were stained by conventional methods (crystal violet-basic fuchsin) and then observed in a Jenaval light microscope. Cell proliferation was monitored using in vivo incorporation of the substituted nucleotide, 5-bromodeoxyuridine (BrdU), then revealed by a monoclonal antibody against BrdU (Cell proliferation kit: Amersham). This same DNA synthesis labelling technique has been previously used successfully to monitor early and advanced stages of arm regeneration in Antedon (Candia Carnevali et al. 1995, 1997). For use with semithin Epon-Araldite sections, the standard BrdU-immunocytochemistry protocol for paraffin sections was modified as described in detail by Candia Carnevali et al. (1995). In particular, two exposed samples per each exposure concentration and two control samples at all the regenerative stages were analysed microscopically. The results of the exposure tests were compared with those obtained by a parallel analysis of normal regenerating samples in standard conditions.
using commercial RIA kits. Standard curves with the steroids dissolved in the same phosphate buffer were performed in every run. The detection limits were of 30 pg/g for E2 and 130 pg/g for T. Intra-assay coefficients of variation were of 6.1 (T) and 3.3% (E2). Interassay coefficients of variation were 9.3 (T) and 3.5% (E2). Statistical analysis Growth Lengths of regenerating arms were measured using the software ArcView GIS 3.2 and, with regard to TPT-Cl tests, comparison of significant difference (P
