The mode of action (MOA) approach reveals interactive effects of environmental pharmaceuticals on Mytilus galloprovincialis

Aquatic organisms are unintentionally exposed to a large number of pharmaceutical residues in their natural habitats. Ecotoxicological studies have agreed that these compounds are not harmful to aquatic organisms, as their environmental concentrations are typically too low. However, recent reports have shown biological effects at such low concentrations when biological endpoints related to the therapeutic effects are assessed. Therefore, conservation of molecular targets is now addressed as a key aspect for the development of more efficient test strategies for pharmaceutical environmental risk assessment, providing the rationale for the mode of action (MOA) approach. In the present study the MOA approach was used to investigate the interactive effects of fluoxetine (FX) and propranolol (PROP) on the Mediterranean mussels (Mytilus galloprovincialis). Indeed, organisms in the environment are exposed to pharmaceutical mixtures throughout their lifetime, and particular combinations may be of concern. The antidepressant FX increases serotonin (5-HT) levels in the synaptic cleft by inhibiting 5-HT reuptake. PROP, a prototypical β-adrenoceptor antagonist, also blocks 5-HT1 receptors, which are negatively coupled to cAMP-mediated signaling. Cell signaling alterations potentially triggered by 5-HT1 receptor occupation were therefore assessed after a 7-day mussel exposure to FX or PROP, alone or in combination, each at 0.3 ng/L concentration. FX decreased cAMP levels and PKA activities in digestive gland and mantle/gonads, in agreement with an increased occupation of 5-HT1 receptors. PROP caused a decrease in cAMP levels and PKA activities in digestive gland and an increase in cAMP levels in mantle/gonads, consistent with a differential expression of adrenergic and 5-HT receptors in the two tissues. Co-exposure to FX and PROP provides significant indications for antagonistic effects of the pharmaceuticals, consistent with a direct (PROP) and indirect (FX) action on the same molecular target. Interestingly, FX induced over-expression of a 5-HT1 gene product, and PROP counteracted such increase when the mixture was administered, while having per se no effect. Finally, mRNA expression of the ABCB gene encoding the MXR-related transporter P-glycoprotein was reduced by both pharmaceuticals in the digestive gland, while decreased by FX, increased by PROP, and not affected by the mixture in mantle/gonads. Since transcription of this gene product is under cAMP/PKA modulation, the impairment of regulatory pathways triggered by low concentrations of pharmaceuticals have the potential to affect the ability of animals to elaborate strategies of defense or adaptation toward further stress factors. In this specific case, the pharmaceutical mixture limits the detrimental effects of the single compounds.