Neonicotinoids induce a promoter-switchin CYP19 expression in breast-cancer cells and alter aromatase activity and hormoneproduction in a feto-placental co-culture model

In estrogen-dependent breast cancer, CYP19 expression is increased via activation of several promoters (PII, I.3, I.7) and by the inhibition of normal I.4 promoter. CYP19 biosynthesizes estrogens, also involved in the development of the placenta and where CYP19 is expressed via I.1 promoter. Little is known about the endocrine disrupting potential of neonicotinoid insecticides on CYP19 expression. Objectives were to develop cell-based assays to identify chemicals that alter promoter-specific expression of CYP19, and to determine the effects of neonicotinoids in these cell assays, including a co-culture model of steroidogenesis in the feto-placental unit. H295R adrenocortical carcinoma cells, Hs578t breast-cancer cells, BeWo trophoblasts and feto-placental co-cultulre (H295R and BeWo) were exposed to atrazine or neonicotinoids (imidacloprid, thiacloprid, thiamethoxam (0.1-30μM)) for 24h. CYP19 expression was measured by RT-qPCR and catalytic activity by tritiated water-release method. Estradiol, estrone, DHEA and -HCG production in co-culture was determined by ELISA. In H295R, atrazine concentration-dependently increased PII- and I.3-mediated CYP19 gene expression (7-fold) and aromatase activity (2-fold). Thiacloprid and thiamethoxam (0.1, 0.3, 10μM) significantly induced PII and I.3-mediated CYP19 expression (14-fold) and aromatase activity. In Hs578t cells, thiacloprid and imidacloprid inhibited I.4 promoter and induced PII-mediated CYP19 expression (up to 50-fold), resulting in elevated CYP19 activity. In BeWo cells, all neonicotinoids inhibited I.1-mediated CYP19 expression. In the co-culture, all neonicotinoids increased estrogen production. Atrazine and neonicotinoids alter CYP19 gene expression, aromatase activity and estrogen biosynthesis in a promoter-specific manner. We are the first to provide a mechanistic basis for the potential of neonicotinoids to alter the biosynthesis of estrogens in placenta or in hormone-dependent breast cancer via the tissue-specific promoters of CYP19.