Effects of environmental steroid mixtures are regulated by individual steroid receptor signaling

Fish are exposed to steroids of different classes in contaminated waters, but their effects are not sufficiently understood. Here we employed an anti-sense technique using morpholino oligonucleotides to knockdown the glucocorticoid receptor (GR) and androgen receptor (AR) to investigate their role in physiological and transcriptional responses. To this end zebrafish embryos were exposed to clobetasol propionate (CLO), androstenedione (A4) and mixtures containing different classes of steroids. CLO caused a decrease of spontaneous muscle contraction and increase of heart rate and transcriptional induction of pepck1, fkbp5, sult2st3 and vitellogenin (vtg1) at 24 and/or 48 hours post fertilization (hpf). Knockdown of GR eliminated these effects, while knockdown of AR decreased the ar transcript but caused no expressional change, except induction of sult2st3 after exposure to A4 at 24 hpf. Exposure to a mixture of 6 steroids comprising progesterone (P4) and three progestins, cyproterone acetate, dienogest, drospirenone, 17β-estradiol (E2) and CLO caused a significant induction of pepck1, sult2st3, vtg1 and per1a. Knockdown of GR eliminated the physiological effects and up-regulation of vtg1, sult2st3, pepck1, fkbp5 and per1a. Thus, as with CLO, responses in mixtures were regulated by GR independently from the presence of other steroids. Exposure to a mixture comprising A4, CLO, E2 and P4 caused induction of vtg1, cyp19b, sult2st3 and fkbp5. Knockdown of AR had no effect, indicating that regulation of these genes occurred by the GR and estrogen receptor. Our findings show that in early embryos GR causes vtg1 and sult2st3 induction in addition to known glucocorticoid target genes. Each steroid receptor regulated its own target genes in steroid mixtures independently from other steroids. However, enhanced expressional induction occurred for vtg1 and fkbp5 in steroid mixtures, indicating an interaction between receptors. These findings have importance for the understanding of molecular effects of steroid mixtures.