Steroid hormones and their combinations affect circadian rhythm pathway in F0 Gambusia affinis using transcriptome sequencing: validation by life-long exposure of F1 offspring and molecular dynamics

Estrogens and androgens are widely distributed in aquatic environments, yet the molecular mechanisms underlying their potential interactive effects in fish remain poorly understood. This study assessed the hepatic effects of 17α-ethinylestradiol (EE2), 17α-methyltestosterone (MT) at low (MTL) and high (MTH) concentrations, and their mixtures (EE2+MTL and EE2+MTH) in two generations of Gambusia affinis. RNA sequencing revealed treatment-dependent transcriptional changes in the liver of F0 female, with more unique than overlapping differentially expressed genes across treatments. Both individual and combined EE2 and MT exposure disrupted circadian rhythm-related pathways in F0 female. Moreover, long-life exposure of F1 offspring to EE2, MT, and their mixtures significantly altered the transcriptional levels of circadian rhythm-related genes (e.g., nr1d1) in the liver. The precise tertiary structure of G. affinis NR1D1 was constructed, and molecular docking demonstrated that EE2, MT, and their complexes bind to NR1D1. Molecular dynamics simulations revealed that the binding free energies of EE2 and MT complexes with NR1D1 were lower than those of EE2 or MT alone. These findings suggest that both hormones and their mixtures have significant potential to disrupt circadian rhythm in G. affinis.