Neurotoxicity and inflammation induction in the early life stage of zebrafish in response to harmane exposure.

Abstract

Harmane is a highly lipophilic heterocyclic amine and a particularly potent tremorgenic β-carboline. This study aims to investigate the neurotoxicity of harmane and its underlying mechanisms. Zebrafish embryos at 4 h post-fertilization (hpf) were exposed to harmane. An integrated approach combining phenotypic assessment, neurodevelopmental evaluation, real-time quantitative PCR (RT-qPCR), and RNA sequencing (RNA-seq) with pathway analysis was employed. Results showed that harmane exerted dose-dependent developmental toxicity in zebrafish embryos. Specifically, continuous exposure to 2 μg/mL harmane for 120 hpf resulted in a mortality rate of nearly 50% in larvae, accompanied by various malformations including pericardial edema, yolk sac absorption disorder, abnormal head and eye development, and reduced locomotor activity. Additionally, harmane exposure was associated with alterations in dopaminergic-related neuronal signals and impairment of the central nervous system (CNS). Furthermore, RT-qPCR results showed that harmane treatment led to decreased expression of th1, th2 and dat in zebrafish, along with a significant upregulation of sncga, whereas the expression levels of sncb, syn2b, parkin, pink1 and dj1 were markedly downregulated. Moreover, RNA-seq analysis revealed transcript-level enrichment of the AGE-RAGE signaling pathway, which suggests a potential association between harmane exposure and changes in multiple Parkinson's-disease (PD) associated gene expression. Further investigation demonstrated that harmane exposure induced significant brain inflammation in larvae, with upregulated inflammation-related genes. This effect was associated with neuronal apoptosis, as indicated by acridine orange (AO) staining. This work reveals a potential correlation between harmane-triggered neuroinflammation and neuronal damage. Further dedicated work is required to clarify whether these toxic responses are relevant to human neurodegenerative disease etiology.