Probiotic Administration Reshapes the Sex Differentiation Trajectory of Zebrafish under Perfluorobutanesulfonate Challenge

Perfluorobutanesulfonate (PFBS) is an emerging pollutant with potent endocrine disrupting effects, while probiotic bacteria can significantly modulate animal endocrinology. However, whether and how probiotics interact with PFBS to maintain the host’s endocrine equilibrium are unknown. Herein, juvenile zebrafish (Danio rerio) were chronically exposed to environmentally relevant concentrations of PFBS during the critical window of gonadal differentiation and maturation (from 21 days postfertilization (dpf) until 150 dpf), with or without dietary supplementation of probiotic Lactobacillus rhamnosus. At 35 dpf, probiotics robustly acted to alleviate PFBS body burden and regulate the toxic fingerprints of PFBS on the sex ratio, estrogen hormones, sex differentiating genes, and estrobolome dynamics. In mature adults, PFBS induced antiestrogenic effects in females, thus forming a male-biased population, accompanied by oogenesis blockade, reduced fecundity, and offspring developmental defects. However, these adverse effects were efficiently counteracted by probiotic supplementation, which was attributed to the holistic activation of the reproductive axis and marked increase of estrogen concentrations. In the gut microenvironment, probiotics mitigated the PFBS-induced disturbance of estrogen-metabolizing bacteria, β-glucuronidase enzyme activity, and critical metabolites along the pentose and glucuronate interconversion pathway, thus directing estrogen gut-liver recycling. Overall, our findings highlight the far-reaching implications of probiotics beyond the gut to mitigate endocrine disrupting effects, suggesting a health intervention strategy based on gut estrobolome remediation. © 2026 American Chemical Society