Effetto dei pesticidi sulla attività della aromatasi umana e del recettore degli estrogeni

Estrogens are a class of steroid hormones that control a wide variety of functions, from reproduction to regulation of cardiovascular, musculoskeletal and central nervous system. They are synthetized by the enzyme human aromatase (HA), member of the cytochrome P450s superfamily, which is responsible of the conversion of androgens into estrogens in the last step of steroidogenesis. Estrogens action is mediated by estrogen receptors (ERs) which activates the genomic or the non-genomic signalling. The genomic estrogen-dependent pathway is considered the classical mechanism of action where the ER dimer translocates in the nucleus, binds the consensus estrogen responsive elements (EREs) and promotes the activation of gene transcription. As a consequence of human activity, new chemicals are released into the environment with the potential to cause adverse effects for ecosystems and/or human health. Some of them have already been identified and they are considered as emerging pollutants (EPs). Many of them act by interfering with the endocrine system and they are known as endocrine disrupting chemicals (EDCs). Therefore, in this project, a group of pesticides in the list of EPs were tested for their interference with the estrogen synthesis and/or signalling in order to test if they can be considered as EDCs. The pesticides tested were glyphosate, oxadiazon, methiocarb, imidacloprid, thiacloprid, clothianidin, acetamiprid and thiamethoxam. First, the molecular interaction between aromatase and the pesticides was investigated. To do that, a recombinant form of the aromatase (rArom) was used and the activity of the enzyme in the presence of these pesticides measured. Among the pesticides tested, only glyphosate resulted to inhibit rArom. The herbicide acts as a non-competitive inhibitor when tested at 1 µM of concentration and as a mixed-inhibitor at 5 µM. Therefore, at the lowest concentration tested, glyphosate binds to an allosteric site, that was identified by molecular dynamics (MD) simulations. When increasing its concentration, it became able to bind also to another site, possible the active site. Moreover, glyphosate was found to partially inhibit rArom activity by a maximum of 30%. Then, three pesticides were selected and tested to probe if they could induce an estrogenic activity in MELN cells (MCF-7 cells stably transfected with a vector containing the ERE gene). The pesticides tested were glyphosate, thiacloprid and imidacloprid. Among them, the two neonicotinoids thiacloprid and imidacloprid induce an estrogenic activity at the highest concentration tested (2.50x10-4M and 5x10-4M for thiacloprid, 5x10-4M and 1x10-3M for imidacloprid). The overall data demonstrate that some of the pesticides studied are potential endocrine disrupting chemicals and they act by interfering with the estrogen pathways at different levels. In particular, glyphosate inhibits aromatase enzyme while thiacloprid and imidacloprid promote the ER-dependent estrogenic activity. The approach used in this work can be useful for a first screening of new chemical entities for their potential endocrine disrupting activity on estrogen system.