Abstract
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 receptors (GRs, GRα and GRβ) 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, as well as transcriptional induction of pepck1, fkbp5, sult2st3 and vitellogenin (vtg1) at 24 and/or 48 h post fertilization (hpf). Knockdown of GRs eliminated these effects, while knockdown of AR decreased the ar transcript but caused no expressional changes, 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 GRs eliminated the physiological effects and the up-regulation of vtg1, sult2st3, pepck1, fkbp5 and per1a. Thus, as with CLO, responses in mixtures were regulated by GRs 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 GRs and estrogen receptor (ER). Our findings show that in early embryos GRs cause 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/cross-talk between GRs and ER. These findings have importance for the understanding of molecular effects of steroid mixtures.
Highlights
Fish are exposed to ng/L concentrations of glucocorticoids, androgens, progestins and estrogens in surface water and their high potency may cause adverse effects in fish
We addressed the following questions: How are physiological and expressional responses of steroids affected by knockdown of glucocorticoid receptors (GRs) and androgen receptor (AR)? Which gene transcripts are regulated by GRs and AR, and which genes are regulated by more than one receptor? Can the joint effects in mixtures be assigned to GRs or AR signaling or both? We hypothesize that GR knockdown eliminates glucocorticoid-regulated physiological and transcriptional effects upon exposure to glucocorticoids
Our findings advance current knowledge about effects of environmental steroid mixtures in fish that may exhibited at ng/L concentrations
Summary
Fish are exposed to ng/L concentrations of glucocorticoids, androgens, progestins and estrogens in surface water and their high potency may cause adverse effects in fish. Steroids and their metabolites originate from municipal and hospital wastewater and agricultural runoff, either from excretion as natural hormones including 17β-estradiol (E2), testosterone, progesterone (P4), cortisol and their metabolites, or from medical applications as synthetic hormones (Chang et al, 2009; Weizel et al, 2018; Zhang et al, 2017). Thereby, mostly mixtures of steroids of one class, including estrogens (Brian et al, 2007; Silva et al, 2002), progestins
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