Acrylamide acute neurotoxicity in adult zebrafish

Abstract

Acute exposure to acrylamide (ACR), a type-2 alkene, may lead to a ataxia, skeletal muscles weakness and numbness of the extremities in human and laboratory animals. In the present manuscript, ACR acute neurotoxicity has been characterized in adult zebrafish, a vertebrate model increasingly used in human neuropharmacology and toxicology research. At behavioral level, ACR-treated animals exhibited "depression-like" phenotype comorbid with anxiety behavior. At transcriptional level, ACR induced down-regulation of regeneration-associated genes and up-regulation of oligodendrocytes and reactive astrocytes markers, altering also the expression of genes involved in the presynaptic vesicle cycling. ACR induced also significant changes in zebrafish brain proteome and formed adducts with selected cysteine residues of specific proteins, some of them essential for the presynaptic function. Finally, the metabolomics analysis shows a depletion in the monoamine neurotransmitters, consistent with the comorbid depression and anxiety disorder, in the brain of the exposed fish.

Figure 1

Behavioral effects of 3 days exposure to 0.75 mM acrylamide (ACR) on zebrafish tested in the novel tank. (a) Behavioral parameters assessed in standard 6-min novel tank test (NTT), as well as a cartoon of the experimental tank divided into two equal virtual zones, top and bottom and representative traces of control and ACR-treated zebrafish.

Figure 2

Behavioral effects of 3 days exposure to 0.75 mM acrylamide (ACR) on zebrafish: erratic movements and freezing bouts. Erratic movements, number and duration (s) of freezing bouts manual and ethograms allowing to visualize the occurrence of these behaviors and the transitions between them, with the diameter of each circle reflecting the frequency of the behavioral activity, and the width and direction of each arrow representing the frequency of the transitions between behaviors. Data reported as mean ± SEM (n = 23–24), *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t-test). Data from 3 independent experiments.

Figure 3

Behavioral effects of 3 days exposure to 0.75 mM acrylamide in the open field. Behavioral parameters assessed in standard open field test (OFT), as well as representative traces of control and ACR-treated zebrafish, showing the two virtual zones, center and periphery, in the arena.

Figure 4

Effects of 3 days exposure to 0.75 mM acrylamide on the shoaling test, cortisol levels and skin coloration. (a) Behavioral parameters of zebrafish shoaling behavior in control and ACR-treated zebrafish. Data reported as mean ± SEM (n = 23–25 for OFT and n = 16–18 for shoaling test), *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t-test). Data from 3 independent experiments. (b) Whole-body cortisol levels and skin coloration in control (n = 16–18) and ACR-treated (n = 14–16) adult zebrafish. Data from 3 independent experiments.

Figure 5

Expression of selected transcripts related with axonal damage, synaptic vesicle cycling and neurotoxicity in the brain of adult zebrafish exposed to 0.75 mM acrylamide (ACR) for 3 days. Results are plotted as log2 ΔΔCt, in order to see both the up regulation and the down regulation. *p < 0.05 (Student’s t-test). Data from 3 independent experiments (n = 14–17).

Figure 6

Effects of acrylamide exposure on the adult zebrafish brain proteome. (a) Unsupervised heat map of the differential proteins. Hierarchical euclidean clustering was performed on the intensities of the differential proteins after Z-score normalization. It was done using the Perseus software. Left panel represents the down-regulated proteins and the right panel the up-regulated proteins following the ACR treatment. (b) ACR-modified cysteine residues. Left panel represent the expression levels of the 191 proteins containing ACR-modified cysteine residues, and the right panel the Log-2 intensities of the peptides with ACR –adducts in cysteine residues. CN: control pool (n = 5 pools; 3 brains each); ACR: pool ACR (n = 5 pools; 3 brains each).

Figure 7

Changes in the profile of neurochemicals in the brain of adult zebrafish control and exposed to 0.75 mM acrylamide (ACR) for 3 days. (a,b) Results from the PLS-DA analysis. (a) Bi-plot of sample scores (circles) and parameter loadings (in black characters, only VIPs); green and red symbols correspond to control and acrylamide-treated samples. (b) List of parameters showing VIPs scores above 1. Green and red shadows indicate parameters associated to control and treated groups, respectively. Short (key) and complete names are indicated. (c) Changes in the profile of the main neurotransmitter systems in zebrafish brains exposed for 3 days to 0.75 mM ACR. Values are represented as log2 of fold change to control; green and purple columns correspond to decreased and increased levels of neurochemicals respect to the controls. Statistical analysis performed using Student’s t-test, *p < 0.05. Data from 2 independent (n = 12 per group).