Fetal Exposure to the Synthetic‐Progesterone Levonorgestrel (LNG) targets the brain resulting in hyperactive behavior using the Zebrafish (Danio rerio) as a model

Abstract

Anxiety‐related disorders are a rising health concern in adolescent and young adults. Evidence suggests that disrupting normal neuronal development (neurogenesis) in the fetal brain could play a role in the manifestation of mental health disorders later in life. Growing literature suggests a role in hormone receptor (HR)‐regulated brain development, particularly in the hypothalamus, the neuroendocrine section of the brain and part of the limbic center. Yet, this link has not been thoroughly investigated. Levonorgestrel (LNG), a synthetic steroid used in contraceptive pills in adults, may also pose a threat to the developing brain by targeting HRs. Furthermore, this endocrine disrupting chemical (EDC) has been found at physiologically relevant levels in potable water sources worldwide providing an alternative route of exposure for unknowing individuals. Therefore, we investigated the effects embryonic exposure to LNG had on neurogenesis and hyperactivity using the zebrafish (Danio rerio) as neurobiological model. First, computational modeling using Quantitative structure‐activity relationships (QSAR), determined the predicted bioavailability, physiological half‐life and overall potential efficacy of LNG as a neuroendocrine disruptor. Next, Zebrafish were bred and fertilized embryos were collected and individually placed in 96‐well plates. Embryos were exposed to LNG levels (at doses ranging from 5–1000ng/L) or a vehicle control starting at 3 hours post fertilization (hpf) to 5 days pf (dpf), the time encompassing neurogenesis. There were no observable differences in fertilization ratios, hatching or morphology between LNG treated groups and vehicle controls. On 5 dpf, all treated and controls were assayed for changes in locomotor activity (i.e. hyperactivity busts). Larval movement were analyzed using computational locomotor activity software. Interestingly, the 5ng dose of LNG significantly (p<0.05) increased hyperactive behavior compared to vehicle controls. HR expression and neuronal marker α‐HuC were used to document neurogenesis. Our data suggests that the observed LNG‐induced hyperactivity represents anxiety‐like behavior as a manifestation of aberrant neurogenesis. This is the first study to show that exposure to low environmentally relevant levels of LNG causes hyperactivity and thus, potentially alters HR regulated neuronal development increasing the risk of other mental issues later in life.Support or Funding InformationThis project was supported in part by UTRGV College of Science (COS) Research Enhancement Seed Grants Program to RKD, the UTRGV Presidential Graduate Research Fellowship to AF and GH, the UTRGV COS Dean's Fellowship to EZ, and the UTRGV Engaged Scholars Award to BHS.