# Behavioral Alterations in Male Zebrafish After Administration of Androgen Receptor Blockers and an Activator

## TL;DR

This study shows how hormone-blocking drugs and a hormone activator affect the behavior of male zebrafish, highlighting risks to aquatic wildlife and the usefulness of zebrafish in hormone-behavior research.

## Contribution

The study provides new insights into the behavioral effects of androgen receptor blockers and activators in zebrafish, a non-mammalian model.

## Key findings

- Androgen receptor blockers reduced swimming activity and altered social or fear-related behaviors in zebrafish.
- Dihydrotestosterone increased locomotor activity, aggression, and changed exploratory behavior in zebrafish.
- Exposure to these drugs caused significant behavioral changes, including altered shoaling and fear responses.

## Abstract

Male hormones play an important role in shaping animal behavior, including movement, social interaction, fear, and aggression, and are governed by the androgen receptor. Certain drugs used to treat human prostate cancer work by blocking these receptors, and unfortunately, they may enter aquatic environments through wastewater, where they could affect fish. In this study, we examined how long-term exposure to two hormone-blocking drugs, enzalutamide and apalutamide, and one hormone activator, dihydrotestosterone, influenced the behavior of adult male zebrafish. Fish were exposed to each substance for about two weeks and then tested for changes in swimming activity, social grouping, fear responses, and aggression. The hormone-blocking drugs slightly reduced swimming activity and altered social or fear-related behaviors, while the hormone activator increased activity and aggression and changed exploratory behavior. These results show that disrupting male hormone signaling can strongly influence fish behavior in different ways. This research helps improve our understanding of how hormone-related chemicals affect animals and highlights potential risks to aquatic wildlife when such substances enter natural water systems. Additionally, it confirms that zebrafish are an effective model for studying how hormones control behavior, which helps scientists better understand the link between hormones and the brain in all backboned animals, including humans.

Androgen receptor (AR) signaling is crucial for mediating male-typical behaviors across vertebrates. Enzalutamide (ENZ) and apalutamide (APA) are two second-generation androgen receptor inhibitors (SGARIs) that have been primarily used in the treatment of prostate cancer. However, these drugs still possess side effects, and there remains limited information regarding their behavioral and neurophysiological impacts following chronic exposure in non-mammalian animal models, particularly in fish. Thus, this study aimed to evaluate the behavioral alterations in adult male zebrafish (Danio rerio) following exposure to specific AR blockers (ENZ and APA) and an activator, dihydrotestosterone (DHT), to provide a comprehensive comparison between each tested drug. Adult male zebrafish were exposed via aqueous immersion to each compound at a 1 ppm concentration for ~2 weeks and were subjected to a battery of behavioral tests. From the results, both AR blockers were found to slightly compromise fish locomotion, with contrary results observed in DHT-treated fish, which displayed an increased locomotor activity together with slight alterations in fish exploratory behaviors. Furthermore, ENZ also caused a tightened shoal formation in zebrafish, while exposure to APA was observed to slightly diminish the fear response of fish. On the other hand, the DHT-treated group displayed a higher level of aggression compared to the vehicle control group. In conclusion, functional modulation of androgen receptor signaling leads to significant alterations in male zebrafish behavior, particularly affecting fear responses, aggression, and anxiety-related behaviors. We believe that these findings could contribute to a deeper understanding of the relationship between androgens and behaviors in vertebrates, especially zebrafish.