Norethindrone causes oxidative stress, intestinal injury, and disturbed gut microbiota composition in adult female western mosquitofish (Gambusia affinis)

Norethindrone (NET), a widely used synthetic progestin, has been consistently detected in diverse aquatic ecosystems, raising significant concerns about its potential ecological risks. In this study, adult female western mosquitofish (Gambusia affinis) were exposed to environmentally relevant concentrations of NET (5.0, 50.0, and 500.0 ng/L) for 42 days to evaluate the toxicity on gastrointestinal (GI) tract. The results demonstrated that NET exposure significantly induced intestinal damage (i.e., increased villus space in the gut, reduced height of columnar epithelial cells, decreased intestinal muscle layer thickness, and diminished goblet cell density). These pathological alterations might ultimately lead to significant growth inhibition in G. affinis. Furthermore, significant alterations in the antioxidant physiology were observed, including elevated levels of reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione S-transferase (GST), and glutathione peroxidase (GPX). Transcriptomic and protein-protein interaction (PPI) analyses demonstrated that NET exposure induced the activation of oxidative stress-associated pathways, particularly the ferroptosis and PI3K/AKT signaling pathways. Finally, 16S rRNA sequencing analysis revealed that NET exposure significantly altered the structure and function of the microbial community within the GI tract. In conclusion, this study clarifies the mechanisms of NET induced oxidative stress and intestinal tissue damage, highlights the health implications of NET exposure in aquatic organisms, and provides critical insights into its ecotoxicological risks.