ABSTRACT: Acetylcholinesterase (AChE) inhibitors are widely used as pesticides and drugs. Their primary effect is the overstimulation of muscular activity. Whether AChE inhibitors and related neurotoxic compound also provoke specific changes in gene transcription patterns that allow to establish a mechanistic link useful for diagnostic purposes has, however, yet not been investigated. Therefore, as a proof of principle, we examined the transcriptomic response of a known AChE inhibitor, the organophosphate azinphos-methyl (APM), in zebrafish embryos and compared the response with two non-AChE inhibiting control compounds, 1,4-dimethoxybenzene (DMB) and 2,4-dinitrophenol (DNP). A highly specific cluster of APM induced gene transcripts was identified and a subset of strongly regulated genes was analyzed in more detail. The small heat shock protein hspb11 was found to be the most sensitive gene in response to AChE inhibitors. Comparison of expression in wild-type, ache and fixe mutant embryos revealed that hspb11 expression was dependent on the nicotinic acetylcholine receptor (nAChR) activity. Furthermore, modulators of intracellular calcium levels led to an hspb11 transcriptional up-regulation which suggest that elevated intracellular calcium levels and the resulting increase in muscular activity triggering hspb11 up-regulation. During early zebrafish development, hspb11 was specifically expressed in muscle pioneers and morpholino-knockdown resulted in defects in slow muscle myosin organization. These findings demonstrate that a combination of transcriptome and functional analyzes in the zebrafish embryo is able to correlate gene expression changes to the mechanism of action of neurotoxic compounds. Zebrafish embryos were exposed for 24 h and 48 h (26-50 and 2-50 hpf) to 5,83 µM of azinphos-methyl (APM), 509 µM of 1,4-dimethoxybenzene (DMB) and 14,2 µM 2,4-dinitrophenol (DNP), the respective LC10 of each compound, in order to determine mode of action specific responding genes, or not exposed and parallel treated as controls. 50 zebrafish embryos were used per treatment, and 4 biological independent replicates were analysed, respectively.
