Whether released into the atmosphere, onto land or into rivers, synthetic compounds produced by human activities (e.g. dioxins, detergents, pesticides, herbicides, antifouling paints, pharmaceutics, fertilizers, etc.) often come to rest in the AQUATIC ECOSYSTEM (e.g. estuarine and coastal waters), accumulating in significant amounts in the sediments. There is evidence that some of these compounds disturb the normal activity of endocrine systems (these compounds are referred as endocrine disrupters) by mimicking hormones (or altering their synthesis, metabolism or activity) and adverse biological effects have been noted in exposed animals e.g. decreased fertility, birth deformities, abnormal metabolism / behavior / sexual characters, compromised immune system, cancers, etc. (Colborn and Thayer, 2000; Jobling et al., 2002; Tyler et al., 1998). Many of the physiological changes observed upon chemical exposure are mediated through hormone receptors (e.g. estrogens and aryl hydrocarbon receptors) although subsequent mechanisms of action of endocrine disrupters remain largely unknown.

Despite severe consequences on SKELETAL AND BONE MINERALIZATION (e.g. skeletal deformities, ectopic calcifications, reduced bone mineral density), few studies have investigated the mechanisms of action of aquatic pollutants in bone and no cellular bioassays are available to determine their bone toxicity. Therefore, experimental data on these pollutants and monitoring tools are needed. In some aspects, skeleton/bone tissue acts as an endocrine tissue that regulates mineral homeostasis (Quarles, 2008), and hormones, in particular estrogens, have long been proven to regulate bone growth and mineralization by direct effect on osteoblast, osteocyte and chondrocyte function (Krum and Brown, 2008). Disruption of these mechanisms has been shown to induce severe embryonic skeletal deformities, ectopic calcifications, skeletal fractures, ossification disorders (e.g. osteoporosis), etc. However, only few studies have investigated the effect of endocrine disrupting pollutants on bone tissues (Lind et al., 1999; Naruse et al., 1999; Naruse et al., 2004; Tsukamoto et al., 2004; Tsai et al., 2004; Wejheden et al., 2006). It has been suggested by in vitro and in vivo studies that the effects of some dioxin-like compounds on bone tissue could be due to direct interaction with the aryl hydrocarbon receptor (AhR) in osteoblasts and osteoclasts (Naruse et al., 1999; Naruse et al., 2004). Another in vitro study has proposed osteopontin as a suitable biomarker for dioxin exposure (Wejheden et al., 2006). However, the identification of more bone-related biomarkers is required in order to develop robust and effective testing methods that will withstand the requirements of international validation, and systems in which to address questions regarding the mechanisms of pollutant bone toxicity are also required.

Although teleost fish represent a model of choice for aquatic ecotoxicology research because of their exposure in the aquatic environment, only few studies have been published in that field. Fish embryos exposed to halogenated dioxins such as TCDD exhibited cardiovascular defects triggered by the activation of the aryl hydrocarbon receptor (Peterson et al., 1993; Chen et al., 2008). Novel pathways of toxicity have been recently identified in zebrafish for pesticides (Stehr et al., 2006), metals (Blechinger et al., 2007), and PHAs (Incardona et al., 2005). Future of the zebrafish model in ecotoxicology, may reside in the use of forward genetics to provide insights into pathways that are disrupted by chemical exposure (Carvan et al., 2008) or the use of high-throughput techniques to screen toxicants on embryos (Murphey and Zon, 2006). Despite some limitations which should shortly overcome, fish model can virtually be applied to all ecotoxicological questions (Carvan et al., 2008). In order to reduce/eliminate animal experimentation in fish ecotoxicology research (for scientific, ethical and economic reasons), in vivo exposure should be progressively replaced with in vitro cell systems (i.e. primary cell cultures of established cell lines), which have already demonstrated their suitability to evaluate ecotoxicological hazards of aquatic pollutants (Carvan et al., 2000; Fent, 2001).

Within the scope of AQUATOX project, we will expand from our preliminary data and (1) characterize the effects of various aquatic pollutants on in vitro mineralization (using fish cell lines capable of mineralizing their extracellular matrix previously developed by our group), in vivo formation and mineralization of the skeleton (using fish larvae at early stages of development) and de novo bone formation (using young fish regenerating their caudal fin), then (2) identify the mechanism of action of mineralogenic pollutants through global analysis of gene expression and selective inhibition of various signal transduction pathways. Finally, and taking advantage of the large number of experimental data generated through this project, we propose to develop a dual-component bioassay (a cellular component based on fish cells capable of in vitro mineralization and a molecular component based on a set of biomarkers found to be differentially expressed in the presence of pollutants with bone toxicity) to evaluate and monitor pollution of aquatic ecosystems. We believe that ecotoxicological data/tools generated through AQUATOX project will not only be beneficial for water monitoring schemes and coastal water management but could also be profitable to aquaculture industries where farmed fish are reared using coastal water and exhibit skeletal malformations.