title: In silico prediction and in vivo analysis of antiestrogenic potential of 2-isopropylthioxanthone (2-ITX) in juvenile goldfish (Carassius auratus).
abstract: Previous studies have shown both anti-estrogenic and anti-androgenic activities of 2-isopropylthioxanthone (2-ITX), a well known food contaminant, in in vitro assays. However, no data are available on the anti-estrogenic potentials and risks of 2-ITX in aquatic organisms. This work evaluated the potential endocrine disrupting effects of 2-ITX at the level of estrogen receptor (ER) signaling cascade using juvenile goldfish (Carassius auratus) as model. Firstly, we investigated the ligand binding efficiency of 2-ITX to the ligand binding domains (LBD) of goldfish ER subtypes using a molecular docking approach. Secondly, we assessed the effects of 2-ITX on E2-induced hepatic expression of ERα1, ERβ1, ERβ2, and vitellogenin (VTG) in vivo. Crosstalk between ER-VTG and aryl hydrocarbon receptor 2 (AhR2)-cytochrome P4501A (CYP1A) was also investigated. Fish were injected with increasing doses of 2-ITX ranging from 2 to 10µg/g BW, and results were compared to the effect of tamoxifen, a well-known ER modulator. We observed that compared to ERβ, the interaction potentials of 2-ITX to goldfish ERα1 LBD was more stable in the inactive receptor conformation. The in silico docking simulation analysis also revealed that 2-ITX acted as agonist for the goldfish AhR2 LBDs suggesting the ability of this compound to activate the cross-talk between the ERα- and AhR-signaling pathways. In vivo experiments confirm in silico simulation predictions demonstrating that 2-ITX reduced the estrogenicity of E2 at both transcriptional and post-transcriptional levels, indicating a clear anti-estrogenic effect. Co-exposure of E2 and 2-ITX also resulted in a significant decrease of CYP1A gene expression with respect to 2-ITX alone. Results from these studies collectively revealed that the antiestrogenic property of 2-ITX can be ascribed to a combination of effects on multiple signaling pathways suggesting the potential for this environmental contaminant to affect the hormonal control of reproductive processes in fish.
authors.affiliation: School of Biosciences and Veterinary Medicine, University of Camerino, Via Gentile III Da Varano, I-62032 Camerino, MC, Italy. Electronic address: paolo.cocci@unicam.it.
authors.forename: Paolo
authors.initials: P
authors.lastname: Cocci
authors.affiliation: School of Biosciences and Veterinary Medicine, University of Camerino, Via Gentile III Da Varano, I-62032 Camerino, MC, Italy.
authors.forename: Matteo
authors.initials: M
authors.lastname: Mozzicafreddo
authors.affiliation: School of Biosciences and Veterinary Medicine, University of Camerino, Via Gentile III Da Varano, I-62032 Camerino, MC, Italy.
authors.forename: Mauro
authors.initials: M
authors.lastname: Angeletti
authors.affiliation: School of Biosciences and Veterinary Medicine, University of Camerino, Via Gentile III Da Varano, I-62032 Camerino, MC, Italy.
authors.forename: Gilberto
authors.initials: G
authors.lastname: Mosconi
authors.affiliation: School of Biosciences and Veterinary Medicine, University of Camerino, Via Gentile III Da Varano, I-62032 Camerino, MC, Italy.
authors.forename: Francesco Alessandro
authors.initials: FA
authors.lastname: Palermo
mesh_terms: D000293:Adolescent; D000818:Animals; D003198:Computer Simulation; D052244:Endocrine Disruptors / Q000378:metabolism; D004965:Estrogen Antagonists / Q000633:toxicity*; D047628:Estrogen Receptor alpha / Q000378:metabolism; D015870:Gene Expression; D006054:Goldfish / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D008099:Liver / Q000187:drug effects; D062105:Molecular Docking Simulation; D018336:Receptors, Aryl Hydrocarbon / Q000378:metabolism; D013892:Thioxanthenes / Q000633:toxicity*; D014819:Vitellogenins / Q000378:metabolism
chemical_list: D052244:Endocrine Disruptors; D004965:Estrogen Antagonists; D047628:Estrogen Receptor alpha; D018336:Receptors, Aryl Hydrocarbon; D013892:Thioxanthenes; D014819:Vitellogenins; C518329:isopropyl-9H-thioxanthen-9-one
keywords: Carassius auratus; Docking simulation; Endocrine disruption; Gene transcription; Vitellogenin