CARYOLOGIA Vol. 64, no. 4: 478-484, 2011

*Corresponding author: cell.: +393473381363; e-mail address: mv.cangialosi@unipa.it

Post-embryonic development effect of Bisphenol A and Tributyltin effects in Ciona intestinalis

Valentina Mansueto1, Maria Vittoria Cangialosi1,* and Ali Said Faqi2

1Department of Animal Biology “G. Reverberi”, Via Archirafi 18, 90123, University of Palermo-Italy; mv.cangialosi@unipa.it; mansuv@libero.it2MPI Research, Mattawan, MI- USA, Alifaqi@mpiresearch.com

Abstract — In the present study we have determined the effects of endocrine disrupting chemicals such as Tributyltin (TBT) and Bisphenol-A (BPA) in juvenile Ciona intestinalis (Ascidiacea, Urochordata). The interest in Urochordata is due to its close phylogenetic relation to vertebrates. Moreover, in the juvenile the organs of a form similar to the adult form are present and they can easily be studied for the eventual morphological altera-tions that can be induced under stress conditions. Juvenile Ciona intestinalis of 4 days post fertilization were incubated for 1 h at increasing concentrations of either TBT or BPA solutions (0.1, 1 and 10 µM). The morphol-ogy of several organs was altered in a concentration-dependent manner in both TBT and BPA treated animals. BPA seems to be more toxic than TBT, destroying the tunic, the gonad cells and inhibiting the rhythmic body contractions. The TBT and BPA induced toxicity on the gonads is in agreement with previous data demonstrat-ing that many chemicals can endanger the reproductive system leading to reproductive failure and consequently a population decline. These results suggest that the juvenile Ciona intestinalis can be used as an alternative or supplemental model for toxicological studies regarding the effects of toxicants not only on organs but also on metamorphosis and on reproductive, defense and nervous systems.

Key words: Bisphenol A (BPA-A), Ciona intestinalis, Developmental Toxicology, Ecotoxicology, Evolution. TBT.

INTRODUCTION

Many chemicals including Tributyltin (TBT) and Bisphenol-A (BPA) are considered endo-crine disrupting chemicals (EDCs). EDCs may be responsible for diverse effects on different bodily systems in invertebrates and vertebrates, including humans by interfering with the normal endocrine function of the organism (Colborn et al. 1993). TBT is widely used as a biocide in a variety of consumer and industrial products. TBT and its degradation products have been isolated from a wide range of marine environ-mental samples. In many cases, a relationship between levels of environmental contamination

and the intensity of shipping traffic can be de-tected. Likewise, BPA is an important industrial chemical that is used primarily to make polycar-bonate plastic and epoxy resins, both of which are used in a wide variety of applications. A recent National Toxicology Program at the US National Institutes of Health raised concerns for the neural and behavioural effects of BPA in fetuses, infants and children at the currently al-lowed human exposures (Update on bisphenol a for Use in food ContaCt appliCations JanU-ary 2010). Toxicological studies on ascidians have increased recently as this taxonomic group is considered phylogenetically related to verte-brates. It has been demonstrated that triorgano-tin compounds, such as TBT, affect the gametes and fertilization (MansUeto et al. 2003; Villa et al. 2003), the embryonic development (Man-sUeto et al. 1989; 1993; 2000; pellerito et al. 1998), and the ultrastructure of embryonic cells of Ciona intestinalis (GianGUzza et al. 1996).

bisphenol a and tribUtyltin effeCts in ciona intestinalis 479

Moreover TBT alters DNA, RNA, protein, glu-cose, Ca2+ contents, protein kinase activity (pUC-Cia et al. 2001), lipids and fatty acids (pUCCia et al. 2005) and inhibits thyroid hormone synthesis in the larva (patriColo et al. 2001). Moreover TBT increases acetylcholinesterase activity (CanGi-alosi et al. 2006; 2009), and alters the steroid pathway in Ciona intestinalis (CanGialosi et al. 2010). In addition BPA affects the embryonic development of Ciona intestinalis (unpublished data). In ascidians after the embryonic develop-ment a swimming larva develops that attaches to a substrate and initiates the metamorphosis process to become a juvenile where the devel-opmental patterns of various adult organs and tissues occur. In the juvenile of C. intestinalis, 4 days after fertilization, all the organs are present: the oral and atrial siphons, for the entry and exit of water, the digestive tract (oesophagus, stom-ach and intestine), the nervous system, the heart, the gonad rudiment and the gill slits. Moreover, a transparent tunic with interspersed cells covers all the body. Many authors have related the role of the tunic to immunity, predation and others (de leo et al. 1981, 1996; de leo 1992; hirose et al. 1994; hirose 2009). A stalk, or stolon, is a peduncle by means of which the juvenile is at-tached to a substratum. The juveniles of ascid-ians are transparent and the cells and organs can be observed clearly making it easy to study the eventual morphological alterations under stress conditions. Because of the potential exposure of humans to these EDCs we studied the toxicity of TBT and BPA in the juvenile C. intestinalis. To our knowledge there is no data available re-garding the toxicity effects of TBT and BPA on ascidian post-embryonic development.

MATERIALS AND METHODS

Test medium and substances - Pasteurized Fil-tered Sea Water (PFSW) was obtained by filter-ing (40 µm diameters) and pasteurizing sea water at 80°C. Tributyltin-chloride (TBT), Bisphenol A (BPA) and ethanol were purchased from Sigma Chemical Co. (St Louis, MO, U.S.A.). The 0.1, 1.0, 10 µM TBT and BPA solutions were pre-pared by dissolving the compounds in 0.0005% ethanol (lipid solvent) containing PFSW.

Biological materials - Ciona intestinalis is one of the most cosmopolitan species of ascidian, a soli-tary tunicate with a cylindrical, gelatinous body, up to 14 cm long, it is a hermaphroditic broad-

cast spawner but cannot self-fertilize. Six hundred adult individuals of C. intestinalis were collected from the Sicily coast (Italy). Animals were removed from their substrate by gently scrapping and were transferred into seawater holding tanks. Prior to the experiments, adult were kept for 24 h under continuous light to aid gametogenesis. Ascidians were held at 18-20°C for no more than 7 days. A mixture of liquid and powdered food developed for aquaculture of larval stages of crustaceans and fish, produced by Salt Creek Inc., South Salt Lake City, Utah, was used for feeding.

Gamete collection and in vitro fertilization - Fe-male and male gametes were obtained by dissec-tion of gonoducts of mature specimens of Ciona intestinalis. Needles were used to carefully break the oviduct without harming the spermiduct. The mature eggs were collected with a Pasteur pipette. Cross fertilization was performed in vitro on eggs pooled from 20 specimens and sperm ex-tracted from a separate group of 10 adults of C. intestinalis. The temperature for the fertilization and embryonic development was maintained at 22±2°C. Fertilization was performed using sterile laboratory equipment. Under this condition, lar-vae hatched about ~22h after fertilization. After metamorphosis, juveniles were cultured in Petri dishes. The seawater was changed every 2 days. C. intestinalis juveniles were examined with Leitz light stereoscope and photographed with a Can-on Power Shot IS camera.

Two different experiments were conducted. Juveniles of Ciona intestinalis were exposed to TBT or BPA. To compare the effects of these two different compounds, we have selected the same concentrations (0.1, 1 and 10 µM) for both BPA and TBT. The number of juveniles was ad-justed to an optimum of 50 juveniles/ml suspen-sion in Pasteurized Filtered Sea Water (PFSW). Two milliliters of juveniles suspension (about 100 juveniles) were introduced in each five Petri dish, containing 18 ml of solutions, in order to obtain: ethanol 0.0005% (solvent control), 0.1, 1 and 10 µM BPA final concentration groups. The second group of experiments with TBT was conducted in the same manner as these previous using the same concentrations. The pH of all so-lutions ranged from 7.8 to 8.2, and the salinity was 37‰. Our experience in the laboratory has shown that after more than 1 h of exposure to 10 µM TBT or BPA, some juveniles die (personal communication), thus in this work the juveniles were exposed to TBT or BPA for only 1 h and the toxicological and morphological changes

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were examined using a Leitz Diaplan stereo-scope. Five replicates were used for each group.

RESULTS

Samples at appropriate stages were identified using the morphological criteria reported by Chiba et al. (2004). Tab. 1 reports the percent of juveniles affected by 0.1, 1.0 and 10 µM TBT or BPA. The morphological alteration increased in concentration-dependent manner from the 0.1 to 1.0 µM and from 1.0 to 10 µM for both TBT and BPA exposed groups. Fig. 1 shows the Ciona intestinalis adult individuals (Fig. 1a), juveniles control (Fig. 1b) and juvenile TBT or BPA treated groups (Fig. 1c-d-e-f-g-h). No mor-tality was observed in both solvent control and TBT or BPA groups. In the solvent control juve-niles of 4 days post fertilization, the organs and tissues were almost similar as those of the adult (Fig. 1b). The detectable gill slits are I and IV, the oral syphon in the anterior part of the body and the atrial siphon, are able to contract for the presence of transverse muscles. Nervous system is in the dorsal side of the body and it is con-stituted of a ganglion and two pigment sensory spots, corresponding to larva otholit and ocellus. The heart at this stage is beating and it is located between the endostyle and the stomach. The di-gestive system is composed of an oesophagus, stomach and intestine. The endostyle on the the ventral side, is homologous to the vertebrate thyroid gland (barrinGton 1964; oGasawara et al. 1999). The gonad arises as a single rudi-ment consisting of a small mass of undifferenti-ated cells (berrill 1975; ishikawa et al. 1988). The stalk originated from the preoral lobe of the larva and is in the posterior side of the body. The tunic, an integumentary tissue, that contains dis-persed cells with morphological characteristics of adult ascidian blood cells (MansUeto, phd

thesis 2009), is around the body. At 4 days from the hatching, the juveniles can feed.

TBT 0.1 µM solution treatment - The oesopha-gus, the endostyle, the tunic and the immature gonad are swollen. The two siphons are con-tracted. The ganglion shows some morphologi-cal alterations. The pigment spots appear fused. The cells of the tunic are present (Fig. 1 c).

TBT 1 µM solution treatment - The endostyle, the tunic and the immature gonad cells appear swollen. The stalk and the siphons are contract-ed. The ganglion structure is disorganized and the pigment spots are fused (Fig.1 d).

TBT 10 µM solution treatment - The oesopha-gus, the stomach, the tunic and the immature gonad are swollen. The two siphons and the stalk are strongly contracted. The ganglion has irregular outlines and an internal structure dis-organized. The pigment spots appear fused. The endostyle is curved and swollen. Many cells of the tunic are absent (Fig.1 e).

BPA 0.1 µM solution treatment - The siphons are enlarged and without contractions. The gan-glion appears slightly altered. The gill slits are altered in their structure. The gonad is reduced and the tunic is destroyed in some parts and no cells are present (Fig. 1 f).

BPA 1 µM solution treatment - The siphons are enlarged and without contractions. The ganglion is destroyed, even if some neurons are present. The gill slits appear altered in their structure. The stomach is swollen and the gonad is reduced. The tunic is almost destroyed (Fig. 1 g).

BPA 10 µM solution treatment - The siphons are enlarged, without contractions and heavily compromised. The ganglion disappears. The gill slits are anomalous, the stomach is swollen, the gonad is reduced. The tunic and the cells are completely absent (Fig. 1 h).

In all BPA solution treatments animals lose the ability to more soon after the incubation.

DISCUSSION

The experiments reported above present evi-dence of deleterious effects of TBT and BPA-A on ascidian juveniles 4 days post-fertilization.

table 1 — Percent of Ciona intestinalis juveniles dam-aged by 0.1, 1.0 and 10 µM TBT or BPA exposure (1h). Values represent mean ± standard deviation of five rep-licates. n.d.= no detected.

µM TBT BPA0 n.d. n.d.

0.1 49.1 ± 8 58.7 ± 13

1.0 67.8 ± 12 78.2 ± 7

10 81.1 ± 9 93.1 ± 15

bisphenol a and tribUtyltin effeCts in ciona intestinalis 481

Several organs including endostyle, intestine, heart, siphons, immature gonad, nervous system and gill slits are altered in a concentration-de-pendent manner.

Fig. 1 — (a) Adult sea-squirt, Ciona intestinalis; (b) juvenile control of C. intestinalis 4 days after fertilization; (c-d-e) juvenile after TBT 0.1, 1, 10 µM exposure; (f-g-h) juvenile after BPA 0.1, 1, 10 µM exposure; Os, oral siphon; As, atrial siphon; Gan, ganglion with pigments spots; E, endostyle; Go, gonad; I-IVGs, Gill slits; Oe, oesophagus; S, stomach; St, stalk; T, tunic with interspersed blood cells (arrows); Scale bar 100 µm.

Metamorphosys - The metamorphosis is the se-quence of morphological events that transform the larva into a sessile, feeding juvenile. This process includes: resorption of the larva tail, a 90° rotation

MansUeto, CanGialosi and faqi482

of the trunk, migration of cells from the haemo-coel to the tunic, retraction of the sensory vesicle and destruction of larval structures (Cloney 1982; Gilbert et al. 1997). Previous studies have demon-strated that the ascidians are protochordates able to concentrate iodide and synthesize the thyroid hormone (ths) in an endostyle that plays a role in metamorphosis (patriColo et al. 1981; 1999). In our research both TBT and BPA-A alter the endo-style morphology of the juvenile Ciona. This result suggests that these compounds can alter the Ciona metamorphosis, in agreement with patriColo et al. (2001), that have demonstrated that ths of ascidia larvae are strongly affected by TBT destroying the thyroid molecule and blocking its neosynthesis in mesenchymal cells of the trunk larva.

Nervous system - The nervous system of the adult is constituted primarily by a ganglion, from which nerves originate. According to Chiba et al. (2004) in the I, IV protostigmata stage, that is the juvenile at 4 days from fertilization, the ganglion can be clearly detected in a compact shape with regular outlines. In TBT-treated individuals, it loses the compactness: the outlines are irregular and in some cases interrupted and the internal structure is disorganized. In the BPA treated ju-veniles the nervous cells are in part destroyed af-ter to the 1 µM solution or completely absent in the 10 µM solution. Our results are in agreement with the findings of other researchers: patriColo el al. (2001) showed that in the histological sec-tions of the larvae of Ciona intestinalis exposed to TBT, there is destruction of the nervous system. oka et al. (2003) showed that BPA-A induces apoptosis in central neural cells during early de-velopment of Xenopus laevis). These studies re-garding the nervous system of C. intestinalis are interesting because the nervous system functions of ascidians indicate strong similarities at the functional levels to that of vertebrates (brown et al. 2005; iMai and MeinertzhaGen 2007 a, b). This suggest that neural mechanisms studied under stress conditions in ascidians may provide information to assist to interpreting some similar neural processes operating in vertebrates.

Digestive system - Our results show that TBT and BPA alter the digestive system. The stomach is more dilated in the TBT-treated individuals and reduced in the BPA-A treated individuals.

Reproductive system - From our results, it is evident that both TBT and BPA are implicated in the alteration of gonad structure suggesting

that the exposure to these chemicals could al-ter the reproductive processes. Previous stud-ies have reported adverse effects on the Ciona steroid pathway (CanGialosi et al. 2010) and a reduction of sperm counts in guppies Poecilia reticulate after TBT and BPA exposure (haU-brUGe et al. 2000) as well a super feminization syndrome induced by BPA, that recorded the superfeminization syndrome in ramshorn snail Marisa cornuarietis (oehlMann et al. 2006).

Defensive system - In a juveniles after TBT and BPA treatment, the tunic is either altered or destroyed respectively. Moreover the cells circulating into the tunic, especially after treat-ment with BPA-A were destroyed. The tunic is an integument covering the epidermis of tuni-cates like a mantle. It is a special tissue where extracellular components fibres and free cells are embedded in a ground substance matrix. A cuticle layer in the exterior tunic can be distin-guished (hirose et al. 1991; 1994; 1996). The tunic as a protective covering and a supporting exoskeleton is also involved in defense reactions and in wound healing (bUriGhel and Clooney, 1997). Previous results have demonstrated that in juvenile cells like blood cells of the adults, are present, indicating a defensive system (Man-sUeto phd thesis 2009). The current data sug-gest that this immune-surveillance in the juvenile of C. intestinalis could be affected by BPA.

Our results indicate that TBT and BPA affect the metamorphosis process, reproductive; de-fense and nervous systems. Based on the results of this study, the Lowest Observable Adverse Effects Levels (LOAEL) for general and repro-ductive toxicity of TBT and BPA is considered to be 0.1 µM.

Acknowledgements — This manuscript is dedi-cated to the memory of Distinguished Professor Sera-fino Mansueto Director of the Department of Internal Medicine and Geriatrics of Policlinico ‘P. Giaccone’ of Palermo, “man of science and of great humanity”.

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Received September 27th 2011; accepted December 2nd 2011