Life-time levonorgestrel exposure causes intergenerational alterations of the thyroid-catecholaminergic-GABAergic...

Levonorgestrel (LNG) is a prevalent contraceptive progestin frequently detected in aquatic systems. Although the endocrine disruptive effect of levonorgestrel on parental fish has been documented, its intergenerational effects at environmentally relevant concentrations remain unclear. In this study, zebrafish embryos were exposed to LNG at nominal concentrations of 0.0, 5.0, 50.0, or 500.0 ng L^-1 from 2 h post fertilization (hpf) to 144 days post fertilization (dpf). Chronic exposure to LNG caused sex ratio skew toward males in zebrafish. These F0 males were subsequently crossed with solvent control females to establish the F1 lineages. Integrated transcriptomic and neurochemical analyzes revealed a thyro-catecholaminergic-GABAergic (TCGA) axis dysregulation, characterized by up-regulation of tpo, dio2, DBH and downregulation of gadA/B. This multi-level dysregulation converged on neurotransmitter imbalance: elevated dopamine (DA) and reduced γ-aminobutyric acid (GABA), resulting in elevated triiodothyronine (T3) and reduced thyroxine (T4). These interdependent hormonal shifts were paternally transmitted to F1 offspring, manifesting as heritable hyperlocomotion. Elevated DNA methyltransferase (DNMT) activity and reduced Ten-eleven translocation (TET) activity in the F0 testes and 120 hpf F1 larvae suggested altered DNA methylation maintenance capacity. In addition, paternal exposure led to reduced fecundity, survival, and motor deficits in F1 offspring. Our data indicate that LNG at concentrations from 5 ng L^-1 upward disrupts the integrated TCGA axis, with DNMT/TET alterations suggesting potential paternal epigenetic mechanisms, supporting a framework for progestin-induced intergenerational toxicity.