Responses to environmental xenobiotics: from endocrine disruption to lipid homeostasis imbalance

Abstract

Many chemicals are continuously entering into the aquatic environment, as a result of their widespread use, disposal and accidental release. Several of these xenobiotics may disrupt reproductive functions of both vertebrates and invertebrates by adversely affecting endocrine signaling pathways, and may also lead to weight gain and metabolic disorders by altering critical pathways involved in lipid homeostasis and metabolism. However, knowledge about the effects of potential endocrine and lipid disrupters, together with information about their metabolism and elimination, in aquatic organisms is still limited. In this context, the present thesis investigated: the metabolism of the synthetic polycyclic musk HHCB and potential effects on the androgen production in the European sea bass (Dicentrarchus labrax); the enzymatic pathways involved in the metabolism of the steroid precursor progesterone (P4) in a model invertebrate species, Mytilus galloprovincialis; and the use of different liver cell lines (RTL-W1 and ZFL) as in-vitro models to assess the ability of known endocrine disrupters (i.e. TBT, TPT, 4-NP, BPA, and DEHP) to disrupt lipid homeostasis in fish cells. We found that HHCB is actively metabolized into a hydroxylated metabolite by sea bass, suggesting the determination of HHCB and/or its hydroxylated metabolite in bile as a tool to assess environmental exposure in wild fish. Furthermore, HHCB inhibited CYP17 and CYP11β catalyzed activities in-vivo, showing the potential to alter to some extent the synthesis of androgens in fish. Then, it was shown the ability of mussels to actively metabolize P4 to 5-alpha-DHP and 3-beta,20-one, with no evidence for the synthesis of 17-aplha-hydroxyprogesterone or androstenedione. Moreover, exposure to high concentrations of P4 (10 micrograms/L) accelerated gamete maturation and release, but had no significant effect on steroid levels or steroid metabolizing enzymes. Thus, environmental concentrations of P4, detected in the ng/L range, are unlikely to have an endocrine action in mussels. The selected endocrine disrupters altered the expression of genes related to lipid metabolism in RTL-W1 cells and produced significant changes in membrane lipids and TAGs. BPA and DEHP significantly induced the intracellular accumulation of TAGs, the effect being more evident after shorter exposure times, while all the compounds tested apart from TPT induced significant changes in membrane lipids –PCs and PC-plasmalogens─, indicating a strong interaction of the toxicants with cell membranes, and possibly with cell signaling. The mode of action of these chemicals is multiple and complex and no clear association between expression of lipid related genes and TAG accumulation was detected in RTL-W1 cells. On the other hand, TBT modified the intracellular lipid profile of ZFL cells but induced triglyceride accumulation only when the culture medium was supplemented with lipids. Finally, being the liver the main site for ‘de novo’ synthesis of lipids, the use of these fish cell lines (i.e. RTL-W1 and ZFL) can be a valuable in-vitro tool to estimate the potential of different compounds and their mixtures to interfere with lipid metabolism in hepatocytes under different exposure conditions (viz. supplementation of cell medium with specific lipids to emulate a lipid reach diet, different exposure lengths and concentrations).

El ambiente acuático es especialmente susceptible a la contaminación, ya que los compuestos químicos pueden llegar a los ríos, lagos y mares, por diferentes vias. Estos xenobióticos pueden afectar las funciones reproductoras, y también perjudicar vías clave involucradas en la adipogénesis y el metabolismo lipídico. Entonces, la necesidad de investigar los efectos de posibles disruptores endocrinos (EDs) y/o lipídicos sobre la fáuna acuática es evidente. El objetivo de esta tesis ha sido evaluar los efectos de estos contaminantes ambientales sobre organismos acuáticos no diana, investigando: el metabolismo y mecanismo de acción del galaxolide (HHCB) en lubinas; posibles alteraciones del sistema endocrino de mejillones expuestos a progesterona (P4) y las vías enzimáticas involucradas en su metabolismo; y la utilización de dos líneas celulares (RTL-W1 y ZFL) para evaluar la capacidad de disruptores endocrinos (TBT, TPT, 4-NP, BPA, y DEHP) de modificar la homeostasis lipídica celular. De este modo, se vio que HHCB puede ser metabolizado por la lubina y que actúa como un inhibidor débil de las actividades CYP17 y CYP11β, pudiendo perjudicar la biosíntesis de esteroides relacionados con la función testicular. Luego, se describió la capacidad de mejillones de metabolizar P4, y el examen histológico de las gónadas mostró que la exposición a 10 μg/L de P4 inducía la maduración y liberación de gametos, aunque las concentraciones de P4 detectadas en el medio ambiente (ng/L) es poco probable que produzcan alteraciones a nivel endocrino en mejillones. En respecto a los efectos obesogenicos, se mostró la abilidad de los EDs estudiados de alterar la expresión de genes relacionados con el metabolismo lipídico en las células RTL-W1 y de causar cambios importantes en los lípidos celulares. Por otro lado, se evidenció la capacidad del TBT de alterar el perfil lipídico de las células ZFL y de inducir acumulación de triglicéridos en hepatocitos sólo cuando el medio de cultivo es suplementado con una mezcla lipídica. En general, la utilización de estas líneas celulares de hígado de pez, RTL-W1 y ZFL, puede ser un modelo in-vitro apto para evaluar la capacidad de contaminantes ambientales de interferir con el metabolismo lipídico en especies acuáticas.