Resumen de Endocrine disruption and detoxification processes provoked by low levels of pollutants in cultured fish
Water is undoubtedly the most important resource on Earth, since it is an essential element for the development of life. However, human being has exerted a negative impact onto its quality and currently almost no waterbody can be considered absolutely pristine. With the exceptions of those cases of severe contamination, organisms in nature are not exposed to high concentrations of a single pollutant, but on the contrary to mixtures of hundreds or thousands of compounds present at low concentrations (ng/L). These are the so-called micropollutants and currently the assessment of their occurrence and effects represents a major challenge not only to scientists but also to policymakers. Among them, the ¿endocrine disrupting compounds¿ (EDCs), this is, those substances able to interfere with the endocrine system of an organism or its progeny, are of special concern as they can provoke deleterious effects at very low concentrations. Provided the fine-tuning of the hormonal systems, which control key processes such as development and reproduction, any disturbance may lead to dramatic consequences including the collapse of populations. Although any organism is susceptible of being affected by such compounds, fish are especially sensitive as they are immersed throughout their whole lifespan in the aquatic medium, which is the fate of any kind of pollutant (due to direct dumping or spillage, to deposition from the air, or to leakage or runoff from soils). into account that fish are aquatic organisms with long lifecycles, on top of the food chain and with a high economic value, the assessment of the effect of micropollutants and EDCs on their health is of great importance. However, although feral fish offer a great number of advantages in monitoring environmental pollution, they also present a series of disadvantages, including the ability to migrate all along a particular water body or the generation of resistance mechanisms. As an alternative, the use of farmed fish as sentinels of pollution has arisen as a possibility. There, fish are maintained in controlled conditions and continuously exposed to the micropollutants which may be present in the waters. It is necessary to note that in this type of facilities, feeds may represent another potential source of pollutants which needs to be evaluated and characterized.
Therefore, taking into account the above mentioned, the objective of this thesis was to evaluate from an integrative perspective the presence of micropollutants, including EDCs, on water bodies through their effects onto farmed fish. In order to do so, we identified the two main potential sources of exposure (water and feed) and organized the work around these sources. In this way, Chapter I (Research articles I and II) deals with the detection and evaluation of micropollutants and EDCs in waters. To this aim, we performed two sampling campaigns in two rainbow trout (Oncorhynchus mykiss) farms and applied a methodology based on the combination of biological and chemical tools to evaluate the presence and effects of micropollutants in fish. In each sampling, biomarkers of environmental pollution (Cytochromes p450 1A and 3A, CYP1A and CYP3A, respectively) were measured in the liver of sampled fish both at the transcriptional and enzymatic levels. CYP1A, which is induced after ligand-activation of the aryl hydrocarbon receptor (AhR), was measured as ethoxyresorufin-O-deethylase (EROD) activity while CYP3A was assessed by means of benzyloxy-4-[trifluoromethyl]-coumarin-Odebenzyloxylase (BFCOD) activity. The presence of AhR agonists in sediments was assessed in vitro by measuring EROD activity in a fibroblast-like cell line originated from rainbow trout gonadal tissue(RTG-2). In addition, sediment and water samples were collected and analyzed by means of two-dimensional gas chromatography¿time-of-flight mass spectrometry (GCxGC-TOFMS), which evidenced the ubiquitous presence of low levels (ng/L) of polycyclic aromatic hydrocarbons (PAHs) and personal care products (PCPs). A strong and time limited EROD induction in one of the fish farms motivated the use of another approach: Active Biomonitoring (ABM), which consists on the transference of some animals to clean waters monitoring the behavior of the measured biomarkers. After only seven days in a farm with controlled conditions, we observed a significant reduction in the induced enzymatic activities. Collectively these results suggest the presence of low levels of contaminants that can be detected only through the appropriate combination of biological and chemical techniques..
In Chapter II (Research Article III), we aimed to assess the presence of EDCs in commercial fish feeds by means of three in vitro assays. More specifically we first tested the potential AhR agonistic activity by measuring EROD activity in the RTG-2 cell line. Secondly, in order to assess the potential estrogenic activity present in the feed pellets we developed and validated a new reporter assay, named HER-LUC. This assay is based on the use of a cell line (HEK-293) stably transfected with the sea bass estrogen receptor ¿ (sbER¿) and the luciferase gene under the control of estrogen responsive elements (ERE). Thirdly, we evaluated the potential thyromimetic activity of the fish feeds with a cell line, PC-DR-LUC, stably transfected with an avian thyroid receptor (THR) and the luciferase gene as reporter gene under the control of the mentioned receptor (Jugan et al., 2007). Our results showed a relatively low estrogenicity but surprisingly, and for the first time, a high thyromimetic activity which motivated the third part of this work.
Considering this high thyromimetic activity in fish feeds, together with the increasing evidence linking thyroid and immune systems in mammals, we wanted to explore if such relationship also applies to piscine organisms. In this study, presented in Chapter III (Research Article IV) we first characterized the presence of THRs in immune organs and cells of juvenile rainbow trout. We demonstrated that immune organs (head kidney and spleen) and isolated immune cells (from head kidney and peripheral blood) of rainbow trout express both thyroid receptor ¿ (THRA) and ß (THRB), indicating that thyroid signaling is possible in the immune system of this species. The higher expression of THRA in immune-related tissues in comparison to the reference tissue (liver) suggested that this subtype would be the main mediator of thyroid hormones (THs) on trout immune cell function. Thereafter, we performed an in vivo experiment in which juvenile trout were exposed to the active thyroid hormone triiodothryronine (T3) or to the anti-thyroid drug propylthiouracil (PTU) for 15 days. Both treatments altered THR expression in immune organs and cells although in a tissue-dependent manner. Lastly, to explore whether changes in thyroid status affect leukocyte population composition, several marker genes of macrophages, T lymphocytes and B lymphocytes, were measured. Our results showed that the effects of T3 and PTU treatments on leukocyte marker genes were also tissue- and time-dependent.
