Nitrate and nitrite exposure leads to mild anxiogenic-like behavior and alters brain metabolomic profile in zebrafish

Abstract

Dietary nitrate lowers blood pressure and improves athletic performance in humans, yet data supporting observations that it may increase cerebral blood flow and improve cognitive performance are mixed. We tested the hypothesis that nitrate and nitrite treatment would improve indicators of learning and cognitive performance in a zebrafish (Danio rerio) model. We utilized targeted and untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis to examine the extent to which treatment resulted in changes in nitrate or nitrite concentrations in the brain and altered the brain metabolome. Fish were exposed to sodium nitrate (606.9 mg/L), sodium nitrite (19.5 mg/L), or control water for 2–4 weeks and free swim, startle response, and shuttle box assays were performed. Nitrate and nitrite treatment did not change fish weight, length, predator avoidance, or distance and velocity traveled in an unstressed environment. Nitrate- and nitrite-treated fish initially experienced more negative reinforcement and increased time to decision in the shuttle box assay, which is consistent with a decrease in associative learning or executive function however, over multiple trials, all treatment groups demonstrated behaviors associated with learning. Nitrate and nitrite treatment was associated with mild anxiogenic-like behavior but did not alter epinephrine, norepinephrine or dopamine levels. Targeted metabolomics analysis revealed no significant increase in brain nitrate or nitrite concentrations with treatment. Untargeted metabolomics analysis found 47 metabolites whose abundance was significantly altered in the brain with nitrate and nitrite treatment. Overall, the depletion in brain metabolites is plausibly associated with the regulation of neuronal activity including statistically significant reductions in the inhibitory neurotransmitter γ-aminobutyric acid (GABA; 18–19%), and its precursor, glutamine (17–22%). Nitrate treatment caused significant depletion in the brain concentration of fatty acids including linoleic acid (LA) by 50% and arachidonic acid (ARA) by 80%; nitrite treatment caused depletion of LA by ~90% and ARA by 60%, change which could alter the function of dopaminergic neurons and affect behavior. Nitrate and nitrite treatment did not adversely affect multiple parameters of zebrafish health. It is plausible that indirect NO-mediated mechanisms may be responsible for the nitrate and nitrite-mediated effects on the brain metabolome and behavior in zebrafish.

Optimization of a liquid chromatography-ion mobility-high resolution mass spectrometry platform for untargeted lipidomics and application to HepaRG cell extracts

“…The authors used a combination of 1 H-NMR for the analysis of monosaccharides, amino acids, and organic acids, GC-MS for cholesterol and fatty acids, and LC-MS for the analysis of lipids. However, more recent zebrafish toxicometabolomics studies (≥2017) reported using LC-HRMS (Orbitrap or QTOF) [15,28,29,31,33,36,48,51,[85][86][87][88], followed by 1 H-NMR [24,30,[89][90][91][92][93] and GC-HRMS [21,94], while multi-analytical platform studies remain scarce.…”

Centella asiatica Alters Metabolic Pathways Associated With Alzheimer’s Disease in the 5xFAD Mouse Model of ß-Amyloid Accumulation

“…Raw data was loaded to PeakView™ with XIC Manager 1.2.0 (ABSciex, Framingham, MA) for peak-picking, retention time correction, and peak alignment. Metabolite identities were confirmed as previously described by matching with an in-house library comprising IROA standards (IROA Technology, Bolton, MA) and other commercially available standards (650 total) ( García-Jaramillo et al, 2020 ). The list of identified peaks was exported to MultiQuant 3.0.2, and chromatograms integrated to obtain peak area values for all the assigned metabolites.…”

Effects of Holding Density on the Welfare of Zebrafish: A Systematic Review

“…43 Accumulation of waste products and other metabolites, such as secreted cortisol, 32 can be problematic in both stationary and recirculating aquaculture systems affecting both development and growth of zebrafish 43,44 and adult zebrafish behavior. 45 The problem is mainly during the start-up period of the aquaria since it takes time to establish an appropriate bacterial culture of nitrifying bacteria. Although few systematic studies have been performed to investigate how water quality is affected by fish density, Pavlidis et al 32 showed a reduction in water quality and dissolved oxygen in their static tanks during the crowding experiment at 40 fish/L.…”