Environmentally relevant exposure to 17alpha-ethinylestradiol affects the telencephalic proteome of male fathead minnows

Abstract

Estrogens are key mediators of neuronal processes in vertebrates. As such, xenoestrogens present in the environment have the potential to alter normal central nervous system (CNS) function. The objectives of the present study were (1) to identify proteins with altered abundance in the male fathead minnow telencephalon as a result of low-level exposure to 17alpha-ethinylestradiol (EE(2)), and (2) to better understand the underlying mechanisms of 17beta-estradiol (E(2)) feedback in this important neuroendocrine tissue. Male fathead minnows exposed to a measured concentration of 5.4 ng EE(2)/L for 48 h showed decreased plasma E(2) levels of approximately 2-fold. Of 77 proteins that were quantified statistically, 14 proteins were down-regulated after EE(2) exposure, including four histone proteins, ATP synthase, H+ transporting subunits, and metabolic proteins (lactate dehydrogenase B4, malate dehydrogenase 1b). Twelve proteins were significantly induced by EE(2) including microtubule-associated protein tau (Mapt), astrocytic phosphoprotein, ependymin precursor, and calmodulin. Mapt showed an increase in protein abundance but a decrease in mRNA expression after EE(2) exposure(,) suggesting there may be a negative feedback response in the telencephalon to decreased mRNA transcription with increasing Mapt protein abundance. These results demonstrate that a low, environmentally relevant exposure to EE(2) can rapidly alter the abundance of proteins involved in cell differentiation and proliferation, neuron network morphology, and long-term synaptic potentiation. Together, these findings provide a better understanding of the molecular responses underlying E(2) feedback in the brain and demonstrate that quantitative proteomics can be successfully used in ecotoxicology to characterize affected cellular pathways and endocrine physiology.

Figure 1

Plasma levels of A) 17β-estradiol (E₂); (control, n=6; EE₂, n=4) and B) testosterone (T) (control, n=9; EE₂, n=8) in male fathead minnow after 48 h exposure to 5.4 ng 17α-ethinylestradiol (EE₂)/L. Steroid levels were analyzed using a non-parametric Mann-Whitney U test but data are presented as mean plasma steroid concentration (±SEM). A significant difference was considered p<0.05.

Figure 2

Pathway analysis of significantly altered proteins in the telencephalon after 48 h exposure to 17α-ethinylestradiol (EE₂). Proteins altered in abundance by EE₂ were involved in cell processes that included proliferation, differentiation, apoptosis, oxidative phosphorylation, neuron network morphology, and long term potentiation. Red indicates an induction in protein abundance and green indicates a reduction in protein abundance. Symbols include extracellular/intracellular proteins and receptors. Except for oxidative phosphorylation, all cell processes shown are affected by >3 of the regulated proteins. Abbreviations are as follows; ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit, isoform 1, ATP5A1; ATP synthase, H+ transporting, mitochondrial F1 complex, beta polypeptide, ATP5B; calmodulin 2, CALM2; cytochrome c oxidase subunit Vb, COX5B; dihydropyrimidinase-like 3, DPYSL3; UCC1; enolase 1, alpha non-neuron, ENO1; ependymin related protein 2, hemoglobin, beta adult minor chain, HBB; histone 1, H2bl, H2BFQ; histone 1, H1c, HIF2; lactate dehydrogenase B, LDHB; malate dehydrogenase 1, NAD (soluble), MDH1; microtubule-associated protein tau, MAPT; myelin basic protein, MBP; myelin protein zero, MPZ; phosphoprotein enriched in astrocytes 15, PEA15; Purkinje cell protein 4, PCP4; similar to CG31613-PA, H3FC; similar to Myristoylated alanine-rich C-kinase substrate (MARCKS) (Protein kinase C substrate 80 kDa protein), MARCKS; stathmin 1, STMN1; tubulin, alpha 1, TUBA3; tubulin, beta 2c, TUBB2.

Figure 3

Real-time PCR (control n=5; EE₂ n=5) for A) Cyp19b mRNA B) B) lactate dehydrogenase B4 (Ldhb4) mRNA C) MAP Tau (Mapt) mRNA D) myelin protein zero (Mpz) mRNA, and E) Cu/Zn superoxide dismutase (Sod1) mRNA. Data are presented as mean copy number (±SEM). A significant difference was considered p<0.05.