Gene expression profiles in zebrafish brain after acute exposure to domoic acid at symptomatic and asymptomatic doses

Abstract

Domoic acid (DA) is a neuroexcitatory amino acid that is naturally produced by some marine diatom species of the genus Pseudo-nitzschia. Ingestion of DA-contaminated seafood by humans results in a severe neurotoxic disease known as amnesic shellfish poisoning (ASP). Clinical signs of ASP include seizures and neuronal damage from activation of ionotropic glutamate receptors. However, the impacts of DA exposure at levels below those known to induce outward signs of neurobehavioral exicitotoxicity have not been well characterized. To further understand the mechanisms of neurotoxic injury associated with DA exposure, we examined the transcriptome of whole brains from zebrafish (Danio rerio) receiving intracoelomic (IC) injection of DA at both symptomatic and asymptomatic doses. A majority of zebrafish exposed to high-dose DA (1.2 microg DA/g) exhibited clinical signs of neuroexcitotoxicity (EC(50) of 0.86 microg DA/g) within 5-20 min of IC injection. All zebrafish receiving low-dose DA (0.47 microg DA/g) or vehicle only maintained normal behavior. Microarray analysis of symptomatic and asymptomatic exposures collectively yielded 306 differentially expressed genes (1.5-fold, p </= 0.05) predominately represented by signal transduction, ion transport, and transcription factor functional categories. Transcriptional profiles were suggestive of neuronal apoptosis following an overwhelming of protective adaptive pathways. Further, potential molecular biomarkers of neuropathic injury, including the zebrafish homolog of human NDRG4, were identified and may be relevant to DA exposure levels below that causing neurobehavioral injury. In general, DA-modulated gene expression was consistent with other model species thereby validating zebrafish as an appropriate vertebrate model to study mechanisms of DA neurotoxicity. These data provide a basis for identifying pathways of DA-induced injury as well as biomarkers of asymptomatic and symptomatic DA exposure levels.

FIG. 1.

Dose-response relationship of IC injection doses of DA and the percentage of zebrafish (Danio rerio) affected at each dose (n ≥ 4 at each dose). Circular and/or spiral-swimming behavior were used to quantify excitoxicity in fish. Effective concentration (EC₅₀) = the dose at which 50% of the fish tested were affected.

FIG. 2.

Percent survival and the percentage of zebrafish exhibiting normal behavior at 6 h after IC injection exposure to 0.47 μg/g total body weight (low asymptomatic dose) and 1.2 μg/g total body weight (high symptomatic dose) DA as well as controls (mean ± SD). Brain tissues from surviving fish were used for gene expression analyses. Excitotoxic behavior was not observed in any of the control or low-dosed fish during the 6-h observation period. A majority of zebrafish injected with the high dose exhibited excitotoxic behavior.

FIG. 3.

Differential gene expression in zebrafish brain after IC injection exposure to 0.47 μg/g total body weight (low asymptomatic dose) and 1.2 μg/g total body weight (high symptomatic dose) DA. Each biological replicate (n = 3 per treatment) is a pool of four to five individuals. (A) Venn diagram showing number of genes significantly expressed ≥1.5-fold up or down (p < 0.05) in each treatment, with the numbers shown in red indicating upregulated and green downregulated. (B) Comparison of all the genes on the array between the high- and low-dose treatments (values are fold change [log₂] compared to control animals). Genes significantly regulated up or down (≥1.5-fold; p < 0.05) are indicated in red or green, respectively, and correspond to the colored numbers in (A). (C) Bidirectional hierarchical clustering by Pearson correlation of genes significantly expressed ≥1.5-fold up or down (p < 0.05) in at least one treatment group. Red color, upregulation; green color, downregulation; and black, unchanged expression.

FIG. 4.

Functional categorization of genes differentially expressed ≥1.5-fold up or down (p < 0.05) in zebrafish brain after exposure to (A) 0.47 μg/g total body weight (low asymptomatic dose) and (B) 1.2 μg/g total body weight (high symptomatic dose) IC injections of DA. Values are calculated as the percent of differentially expressed genes out of the total number of significant genes in the low or high dose (143 and 70 genes, respectively) with the exclusion of those having unknown function. In total, 37% and 35% of significantly differentially expressed genes were of unknown function in the low- and high-dose treatments, respectively.

FIG. 5.

Comparison of gene expression in zebrafish brain measured by microarray (n = 3 biological replicates, each a pool of four or five animals, gray bars) and real-time qRT-PCR (n = 13–15 individuals, black bars) after exposure to 0.46 μg/g total body weight (low asymptomatic dose) and 1.2 μg/g total body weight (high symptomatic dose) IC injections of DA. Values are expressed as average fold change (log₂) with standard error compared to control animals as indicated for select genes.