© 2014 The Japan Mendel Society Cytologia 79(4): 517–533 Hazards of the Endocrine Disruptors on Mullets (Chelon labrosus) from the Basque Coast (Bay of Biscay) Applying a Biomarker Based Approach Iman Abumourad1,2*, Cristina Bizarro1, Pedro Aragón3, Ángel Maquieira3, Asier Vallejo4, Olatz Zuloaga4, Miren López de Alda5, Bayona Josep5, Damia Barceló5, Miren Cagaraville1 and Maren Ortiz-Zarragoitia1 1 CBET Research Group, UPV/EHU, Basque Country, Spain 2 Deparment of Hydrobiology, Veterinary Division, National Research Center, Cairo, Egypt 3 IRMDT, UPV, Valencia, Spain 4 Kimika Analitikoa Saila, UPV/EHU, Basque Country, Spain 5 Deparment of Environmental Chemistry, IIQABCSIC, Barcelona, Catalunya, Spain Received April 14, 2014; accepted July 31, 2014 Summary Biomonitoring programs are essential tools to evaluate the biological quality status of aquatic environments. Recently, several compounds with endocrine disruption ability have been included in the list of priority substances and therefore, implementation of biomarkers assessing the presence and the effects of such substances are required. In the present work we applied a battery of chemical and biological markers in order to study the effects of endocrine disruptors in four thicklip grey mullet (Chelon labrosus) populations from the Basque Country (Bay of Biscay) inhabiting differently polluted estuaries: Arriluze and Pasaia are marinas located in highly industrialized and densely populated areas, Plentzia is a leisure and touristic town and Gernika is located in the Biosphere Reserve of Urdaibai. Chemical analyses of fish bile were performed in order to determine the uptake of endocrine disruptors. Liver, gonad and brain samples were collected for the study of expression levels of genes associated with reproduction and development such as vtg, cyp19a1 and a2, er and rxr. Histological analysis of gonads was performed to identify possible gametogenic alterations such as intersex gonads. Results indicated clear pollution-dependent responses among four estuaries. Endocrine disruption effects were very marked in mullets from Gernika and Plentzia; these two populations showed high vtg gene expression levels in male mullets together with high alkylphenol metabolites in the bile. Bisphenol A was also present at high concentration in mullets from Gernika. cyp19a2 was upregulated in male mullets from Plentzia. Intersex fish were found in Gernika and Pasaia, the last ones showing high hormone metabolites in bile. The combination of chemical and biomarker approaches in biomonitoring programmes can be a valuable tool to be implemented within the framework of the new water policies. Key words Water pollution, Endocrine disruptor, Intersex gonad, Vitellogenin, Expression of aromatase and nuclear receptors, Thicklip grey mullet, Chelon labrosus. Many different environmental chemicals have the potential to disrupt critical hormone- regulated processes of reproduction and development because the chemical has structural similarities to hormones such as steroids; as a result the endocrine disruptors bind to a hormone receptor or to an enzyme that catalyzes hormone synthesis or degradation and disrupt its function (Atanasov et al. 2005, Baker 2001), even if they existed at low concentrations and transient exposures (Phillips and Harrison 1999). Endocrine disrupting chemicals (EDCs) have been defined * Corresponding author, e-mail: [email protected] DOI: 10.1508/cytologia.79.517 518 I. M. K. Abumourad et al. Cytologia 79(4) early in 1996 by the European Community as “an exogenous substance or mixture that alters function(s) of the endocrine system” and consequently cause adverse effects in an intact organism or its progeny or subpopulations (Baker et al. 2009). EDCs have been linked to a range of effects on reproductive health in humans (Phillips and Harrison 1999) and in animals in the natural environment (Crisp et al. 1998, Phillips and Harrison 1999, McLachlan 2001), and in fishes (Oleksiak 2008). Pollutants are discharged into rivers, lakes and the ocean, where they accumulate in aquatic species. Humans and wildlife are exposed to these compounds directly and through fish and shellfish consumption. In addition, humans are exposed to endocrine disruptors via polluted drinking water. Pollutants are most often present as complex mixtures, with additive, synergistic or antagonistic properties. Neither correlational nor experimental approaches address the ultimate biological consequences of multigenerational exposures for populations of animals living long term in polluted environments, and only rarely have physiological or genetic adaptations that affect reproductive success of these populations been investigated. Xenoestrogens and their degradation products comprise a broad range of widely used and structurally diverse chemicals (Sharpe et al. 1995, Gozzo and Poupaert 1998, Oettel 2002, Yu et al. 2002), and include organochlorine pesticides, fungicides, insecticides, polychlorinated biphenyls (PCBs), detergent derivatives like alkylphenols, plasticizers like phthalates, polycyclic aromatic hydrocarbons (PAHs), polychlorinated dibenzodioxins (PCDDs), surfactants, industrial chemicals, and some natural chemicals such as phytoestrogens and mycoestrogens (Sultan et al. 2001, Starek 2003). At least 45 chemicals or their metabolites have been identified as endocrine disrupters (Colborn et al. 1993). These chemicals share a common mode of action by activating the estrogen receptor (ER) and thereby elicit estrogenic responses (Witorsch 2002). Estrogenic compounds (xenoestrogens) constitute a particular group of EDCs with the ability to mimic natural estrogen (Goksøyr et al. 2003), and also may cause malformation of reproductive organs (Berg et al. 1999), impaired sexual behavior (Halldin et al. 1999), and changes in sexually dimorphic neural circuits (Panzica et al. 2002). One of the most important and sensitive responses to estrogen is the induction of vitellogenin, protein transcription and translation. This makes testing for vitellogenin useful as an indicator of estrogenic activity over a wide range of vertebrate groups (Palmer and Palmer 1995). Vitellogenin (VTG) is a bulky, complex phospholipoglycoprotein bound to calcium, produced by hepatocytes in the liver of females in response to estrogen, secreted in blood and transported to ovaries to form the yolk protein that serves as nutrient to growing oocytes. The estrogen response will be elicited if an estrogenic compound binds to and activates or inhibits the estrogen receptors in hepatocytes. Males normally have no detectable production of vitellogenin due to their normally low levels of endogenous estrogens (Wang et al. 1985, LeGuellec et al. 1988). However, their liver is capable of synthesizing and secreting vitellogenin into the blood in response to exogenous estrogen stimulation (Morales et al. 1991). The response is not as rapid or as strong as in females that are exposed to the same concentrations of estrogen (Danko and Callard 1981). However, since males normally have no vitellogenin, the expression of any vitellogenin serves as an ideal biomarker for xenobiotic estrogenic stimulation. Vitellogenin expression has been used successfully for identification of exposure to environmental estrogens in fish, including wild populations (Goodwin et al. 1992, Purdon et al. 1994), and under laboratory (Chen et al. 1986) and in vitro conditions (Jobling and Sumpter 1993). Xenoestrogens may elicit these effects by direct binding to estrogen receptors (ERs) which regulate VTG synthesis by binding estrogen, or through other nuclear receptors which interact with estrogen response elements (EREs), or the retionoid X receptor (RXR), which has an important role in the regulation of metabolism and development (Arukwe and Goksøyr 2003). Legislation has been implemented world-wide to protect and restore ecological quality in estuarine, coastal and marine systems, for instance Oceans Act in the USA, Australia or Canada, 2014 Hazards of the Endocrine Disruptors on Mullets (Chelon labrosus) 519 the Water Framework Directive (WFD, 2000/60/EC) and Marine Strategy Framework Directive (MSFD, 2008/56/EC) in Europe, and the National Water Act in South Africa (Borja et al. 2008). Since the publication of the MSFD in June 2008 by the European Parliament and Council. Monitoring the quality of marine ecosystems has become an urgent priority. The MSFD states that by 2015 at the latest, “a programme of measures has to be designed to achieve or maintain good environmental status.” This good status should be achieved in 2020 (European Commission 2010). In this context, it is important to measure the concentrations of various pollutants in the marine environment, but it is even more important to describe the possible impact these pollutants may have on the organisms living in the ecosystem. Recently, several compounds with endocrine disruption ability have been included in the list of priority substances of the EU, and therefore, implementation of biomarkers assessing the presence and the effects of such substances is required. The fish species selected for this study is the thicklip grey mullet Chelon labrosus belonging to the family Mugilidae. Mullets are appropriate sentinel species since they can inhabit highly polluted environments and feed on suspended particles and small organisms but also sedimented detritus. It is a euryhaline bottom dweller, inhabiting rivers, estuarine and coastal waters and an abundant species in eastern Atlantic estuaries. It is an omnivorous scavenger