ISJ 11: 204-212, 2014 ISSN 1824-307X RESEARCH REPORT Exposure to tributyltin chloride induces penis and vas deferens development and increases RXR expression in females of the purple snail (Plicopurpura pansa) D Domínguez-Ojeda1, AE Rojas-García2, ML Robledo-Marenco2, BS Barrón-Vivanco2, IM Medina-Díaz2 1Escuela Nacional de Ingeniería Pesquera, Universidad Autónoma de Nayarit, Bahía de Matanchén Km. 12, San Blas, 63740 Nayarit, México 2Laboratorio de Contaminación y Toxicología Ambiental, Universidad Autónoma de Nayarit, Av. de la Cultura s/n, Col. Los Fresnos, 63155 Tepic, Nayarit, México Accepted June 26, 2014 Abstract Tributyltin (TBT) and its derivatives are widely used as antifouling paints for ships, resulting in their being released into the marine environment. Aquatic invertebrates, particularly marine gastropods, are extremely sensitive to TBT and undergo changes in the imposition of male secondary sex characteristics in response to exposure. This study aimed to evaluate the development of imposex and the expression of the retinoid X receptor (RXR) in tissues of Plicopurpura pansa (males and females) exposed to tributyltin chloride (TBTCl). The histological results showed a penis-like structure in imposexed female and an undeveloped vas deferens that lacked circular muscular layers. TBTCl treatment increased the messenger RNA (mRNA) of RXR in females with imposex. The highest level of mRNA RXR was found in the digestive gland and penis-forming area in females under in vivo exposure compared with control females. These results indicate that TBTCl modulates mRNA levels of RXR in females. mRNA RXR in imposex females and females exposed to TBTCl only was similar to that of males, indicating that RXR might contribute to the development of imposex. To our knowledge, this study is the first to show that TBTCl induces imposex and biphallia in this snail species, and that this effect is accompanied by an increase in RXR expression. Key Words: tributyltin; imposex; retinoid X receptor (RXR); purple snail Introduction Organotin compounds have been widely used activity (the enzyme that converts androgens to as antifouling paints for ships and fishing nets since estrogens) (Matthiessen and Gibbs, 1998; McAllister the 1960s, resulting in their continuous release into and Kime, 2003). This worldwide phenomenon the marine environment (De Mora, 1996; Fent, represents one of the most detrimental 1996; Champ, 2000). Tributyltin (TBT) and its consequences of pollution by anthropogenic derivatives are endocrine disruptors that induce chemicals and has led these compounds to being deregulation of vertebrate and invertebrate banned from antifouling paints in a number of endocrine systems (Golub and Doherty, 2004). countries; however, organotin compounds remain in Aquatic invertebrates, particularly marine the environment (Nishikawa, 2006). gastropods, are extremely sensitive to organotin To date, very low concentrations of TBT have compounds such as TBT. Consequently, they been shown to induce imposex in marine undergo changes in imposition of male secondary gastropods (Smith, 1980, 1981; Bryan et al., 1993; sex characteristics in response to exposure Horiguchi et al., 1997; Matthiesen and Gibbs, 1998; (Maguire, 2000; Oetken et al., 2004; Sternberg et al., Nishikawa, 2006; Chacón et al., 2007; Oehlmann et 2010), which is caused by a decrease in aromatase al., 2007). These abnormalities are the result of a ___________________________________________________________________________ masculinization process by which male sex organs develop a notable penis and vas deferens. In certain Corresponding author: Irma Martha Medina Díaz species, the growth of the vas deferens disrupts the Laboratorio de Contaminación y Toxicología Ambiental structure and function of the oviducts, preventing Secretaría de Investigación y Posgrado normal breeding activity and causing population Universidad Autónoma de Nayarit decline (Bryan et al., 1986; Gibbs and Bryan, 1986; 63155 Tepic, Nayarit, México Nakanishi, 2008). At present, the effects associated E-mail: [email protected] with TBT have been reported in 268 species of 204 Table 1 Body size and weight of female and male purple snails used in flow-through exposure experiments (October to March, 2011) Control females Control males TBTCl-exposed females TBTCl-exposed males Shell height (cm) 3.14±0.346 3.16±0.249 3.085±0.33 3.185±0.27 Shell weight (g) 4.87±1.61 5.6±1.19 4.85±1.8 6.22±1.40 Mean ± Standard deviation (SD). There was no statistical differences between size and weight with exposure to tributyltin chloride (TBTCI). intertidal gastropods worldwide, belonging to the and Mason, 1996); 3) an increase in the level of an orders Vetigastropoda, Mesogastropoda, and alanine-proline-glycine-tryptophan amide Neogastropoda. Neogastropoda is the most affected neuropeptide in response to TBT, and 4) TBT order, which contains 214 species, of which 101 are interference in the release of penis part of the Muricidae family (Titley-O'Neal et al., morphogenetic/retrogressive factor from the 2011). The purple snail (Plicopurpura pansa) is part pedal/cerebropleural ganglia (Féral and Le Gall, of this affected group of species. This snail is an 1983). intertidal carnivorous gastropod that inhabits rocky Another important mechanism proposed to intertidal beaches exposed to strong wave action. It induce imposex development is through the retinoid is distributed in the Pacific Ocean, from the X receptor (RXR). This receptor is a member of the northwestern Mexican coast to northern Peru (Keen, nuclear receptor superfamily of ligand-activated 1971). In addition, this gastropod possesses both transcription factors that have been characterized in economic and cultural importance. a wide variety of metazoan phyla and is highly Alternative mechanisms by which TBT induces conserved (Bouton et al., 2005). RXR play a central imposex in gastropods include the following: 1) role in a variety of nuclear signaling pathway, have increase in androgen levels as a result of the TBT- multiple physiological functions, and play key roles mediated inhibition of aromatase (Spooner et al., in embryo patterning and organogenesis in 1991; Bettin et al., 1996); 2) TBT-mediated inhibition mammals. RXR is known to act both as a ligand- of the excretion of androgen sulfate conjugates, with dependent transcription factor and as a common a consequent increase in androgen levels (Ronis hetero- or homodimer partner for many non-steroid Fig. 1 Hematoxylin and eosin semi-thin cross-section of the male penis of the purple snail 2 months after treatment with 10 μg Sn/L of Tributyltin chloride (TBTCl). Abbreviations: it, interstitial tissue; ml, muscle layer; vd, vas deferens. 205 Fig. 2 Hematoxylin and eosin cross-section of the penis-like structure (2 mm in length) that developed behind the right tentacle of a female purple snail 2 months after treatment with 10 μg Sn/L of Tributyltin chloride (TBTCl). Abbreviations: it, interstitial tissue; ml, muscle layer; vd, vas deferens. nuclear receptors. Because 9-cis retinoic acid Materials and Methods effectively induces imposex, RXR may play an important role in the development of imposex in Collection of specimens gastropods (Mangelsdorf and Evans 1995; Morris- Experiments were performed with a Kay 1997; Bouton et al., 2005; Stenberg et al., gonochoristic prosobranch species, the purple snail, 2008). Recently, Horiguchi et al. (2010) suggested Plicopurpura pansa Neogastropoda: Thaididae. In that RXR might be involved in organotin-mediated March 2010, live female and male purple snails induction of male-type genitalia (penis and vas were collected by hand at low tide at Olas Altas, deferens) in female rock shells (Thais clavigera). Mazatlán, Sinaloa, Mexico (23°12’ 29.87’’ N, Although imposex studies have been completed 106°25’ 43.67’’ W). The specimens were in several gastropods, the development of imposex immediately transported to the laboratory in plastic by TBT in the purple snail (Plicopurpura pansa) has cage containing seawater from the collection site. not been previously described. The aim of the Once at the laboratory, the specimens were reared present study was to evaluate the development of for 2 weeks in a laboratory aquarium to acclimate to imposex and expression in the RXR gene (mRNA) artificial seawater (Red Sea Salt®, Israel). The of female and male P. pansa exposed to Tributyltin purple snails were fed with pieces of squid (Loligo chloride (TBTCl). spp.). Fig. 3 Temporal change in the incidence of imposex in female purple snails exposed to 10 μg Sn/L of Tributyltin (TBTCl) for 6 months in a flow-through system. 206 Fig. 4 Temporal change in the average penis length of female purple snails exposed to 10 μg Sn/L of Tributylin (TBTCl) for 6 months in a flow-through system. Histological procedure control males, and exposed males) with a flow- The soft tissues (penis and penis-like structure) through system of artificial seawater (Read Sea of males and imposexed females were extracted Salt®, Israel) by means of an aeration pump. The from the shell and were fixed with 10% temperature of the experimental seawater was formaldehyde for 12 h. After dehydration with maintained at 25 ± 1 °C and a salinity of 38 ± 2 Salt ethanol (70, 80, 90, 96, and 100%), the tissues were per unit (SPU). The purple snails in the two immersed in xylene and then embedded in paraffin exposure groups were removed each month for and sliced into 7 µm sections. The paraffin- imposex examination (presence or absence of embedded