Evaluation of the Adverse Effects of Chronic Exposure to Donepezil (An Acetylcholinesterase Inhibitor) in Adult Zebrafish by Behavioral and Biochemical Assessments

Abstract

Donepezil (DPZ) is an acetylcholinesterase inhibitor used for the clinical treatment of mild cognitive impairment. However, DPZ has been reported to have adverse effects, including causing abnormal cardiac rhythm, insomnia, vomiting, and muscle cramps. However, the existence of these effects in subjects without Dementia is unknown. In this study, we use zebrafish to conduct a deeper analysis of the potential adverse effects of DPZ on the short-term memory and behaviors of normal zebrafish by performing multiple behavioral and biochemical assays. Adult zebrafish were exposed to 1 ppm and 2.5 ppm of DPZ. From the results, DPZ caused a slight improvement in the short-term memory of zebrafish and induced significant elevation in aggressiveness, while the novel tank and shoaling tests revealed anxiolytic-like behavior to be caused by DPZ. Furthermore, zebrafish circadian locomotor activity displayed a higher reduction of locomotion and abnormal movement orientation in both low- and high-dose groups, compared to the control group. Biomarker assays revealed that these alterations were associated with an elevation of oxytocin and a reduction of cortisol levels in the brain. Moreover, the significant increases in reactive oxygen species (ROS) and malondialdehyde (MDA) levels in muscle tissue suggest DPZ exposure induced muscle tissue oxidative stress and muscle weakness, which may underlie the locomotor activity impairment. In conclusion, we show, for the first time, that chronic waterborne exposure to DPZ can severely induce adverse effects on normal zebrafish in a dose-dependent manner. These unexpected adverse effects on behavioral alteration should be carefully addressed in future studies considering DPZ conducted on zebrafish or other animals.

Phenomics Approach to Investigate Behavioral Toxicity of Environmental or Occupational Toxicants in Adult Zebrafish (Danio rerio)

“…Several simple paradigms have been developed to assess alterations in zebrafish behavior, including novel tank, shoaling, aggression, social interaction, and fear response tests. All of these behavior tests are important in order to assess the potential toxicities of test chemicals and enable a comprehensive characterization of zebrafish neurobehavioral phenotypes because all of these tests are complementary to each other, and various toxicants have been demonstrated to affect only specific behaviors in zebrafish (Audira et al., 2021; Audira et al., 2020; Malhotra et al., 2020). Furthermore, these tests are well suited for zebrafish because they are relatively simple and painless (Sousa, Almeida, & Wotjak, 2006).…”

“…Toxicants (e.g., butanol and tetrahydrofuran) are considered to alter zebrafish locomotion if there are statistically significant differences between untreated and treated fish in at least one of four locomotion‐related behavior endpoints, which are average speed, freezing movement time ratio, swimming movement time ratio, and rapid movement time ratio. Meanwhile, the treated zebrafish's exploratory behaviors are considered to be altered by toxicants, e.g., dimethyl sulfoxide (DMSO) and isopropanol, if there are statistically significant differences between untreated and treated fish in at least one of five exploratory behavior‐related endpoints (Audira et al., 2020). These behavior endpoints are time in top percentage, the number of entries to the top, latency to enter the top, total distance traveled in the top, and thigmotaxis.…”

“…Test toxicants are considered to play a role in altering zebrafish fear response if there are statistically significant differences between untreated and treated fish in at least one of two behavior endpoints, which are approaching predator time percentage and average distance to the predator's separator. By utilizing the current fear response test protocol, fewer fear responses toward the stimulus have been observed in acutely ethanol‐treated fish, which might be due to the anxiolytic effect of alcohol (Audira et al., 2018; Audira et al., 2020). In addition, some compounds, such as graphene and graphene oxide nanoparticles at relatively low concentrations, may also affect the fear response of zebrafish, which might be caused by the disruption of its neurotransmission system (Audira et al., 2021).…”

“…In animal models, behavior profiling has become a widely used approach in toxicology and pharmacology. Neurobehavioral studies have been demonstrated to be more sensitive than other whole organism endpoints (e.g., mortality and growth) and exhibit many advantages, such as low cost, robustness, and effectiveness in toxicology research (Audira et al., 2020; Chen, Wang, & Wu, 2011). Several animal models have been used to study behavioral toxicity, especially rodents.…”

Cholinesterase Research

“…Finally, the effect of clinically used donepezil has been investigated: the study of Audira and colleagues [9] is dedicated to donepezil, a currently approved drug for mildto-moderate stages of AD. The study exploits a zebrafish model to analyze potential adverse effects of donepezil on the short-term memory, behavioral and biochemical changes.…”