Nitrate and nitrite exposure affect cognitive behavior and oxygen consumption during exercise in zebrafish
Authors: Hord Norman Gary , Beaver Laura , Axton Elizabeth , Truong Lisa , St. Marie Lindsey , Tanguay Robert , Logan Christiana , Choi Jaewoo , Spagnoli Sean , Stevens Jan F. 
Published: November 2023

Humans concentrate nitrate from dietary or endogenous sources in the salivary glands, which is then reduced to nitrite, swallowed, and absorbed. Circulating nitrite acts as a reservoir for nitric oxide (NO) with its reduction to NO potentiated in acidic or hypoxic areas, such as contracting skeletal muscle. NO is an important signaling molecule with a short half‐life that regulates cardiovascular function, cellular energetics, and neurotransmission among other things. In humans, consumption of supplemental nitrate reduces blood pressure and decreases the oxygen cost of exercise, but we do not have a complete understanding of how dietary nitrate or nitrite is metabolized particularly in the presence of exercise. We used a stable isotope‐assisted metabolomics approach to assess nitrate and nitrite metabolism with and without exercise, and tested the hypothesis that nitrate and nitrite exposure reduces the cost of exercise and improves cognitive function in a zebrafish model.

Adult zebrafish were exposed to either sodium nitrate (606.9 mg/L), sodium nitrite (19.5 mg/L), or control water for 21 days (n=60–66). A subset of the fish were switched to 100% 15N‐nitrite or 15N‐nitrate for the final 3 days of the exposure. Tissue nitrite and nitrate were quantified using diaminonaphthalene (DAN) derivatization and subsequently analyzed on a 3200 ABSciex QTRAP. Nitrite and nitrate exposure increased tissue nitrite (230% and 340%, respectively) and tissue nitrate (150% and 250%, respectively), and significant increases in nitrate and nitrite in blood were also observed. Nitrate exposure significantly decreased oxygen consumption during exercise in the AutoResp exercise assay. Nitrite treatment increased oxygen consumption with exercise but pathological examination indicated nitrite exposure caused mild gill damage. Metabolomics showed that up to 90% of the tissue nitrite and nitrate can be derived from exogenous sources. The data supports existing data that a feedback mechanism that inhibits endogenous NO production when high levels of nitrite or nitrate are consumed. Nitrite was depleted during exercise in all treatment groups, indicating potential increased utilization of nitrite as a substrate for NO production in hypoxia.

In a free swim test nitrate and nitrite treatment significantly increased the duration of time spent in the bottom of the tank which is a possible indicator of anxiety. Nitrate treated fish also had a significant decline in movement following a startle but this was not detected in nitrite treated fish. In a negative conditioning‐learning assay nitrate treatment significantly impaired decision making time, and caused a 3‐fold increase in the period of time the fish experienced a mild shock. Nitrite treated caused a 2‐fold increase in the period of shock at the beginning of the assay, but the fish were able to learn with time. Taken together the behavioral assays demonstrate that both nitrate and nitrate treatments altered cognitive function in zebrafish and metabolomics on brains is being undertaken to understand the mechanisms contributing to these phenotypes.

Support or Funding Information

Oregon Agricultural Experiment Station, Endowment for the Celia Strickland and G. Kenneth Austin III Endowed Professor of Public Health