Nitrate and nitrite exposure increases anxiety-like behavior and alters brain metabolomic profile in zebrafish

Introduction 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. Here we tested the hypothesis that nitrate and nitrite treatment would improve indicators of learning and cognitive performance in a zebrafish (Danio rerio) model. We also explored the extent to which nitrate and nitrite treatment affected the brain metabolome in order to understand how nitrate and nitrite supplementation may affect indices of cognitive function. Methods 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, innate predator avoidance, social cohesion, and shuttle box assays were performed. Results 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 significantly increased anxiety-like behavior but did not alter epinephrine, norepinephrine or dopamine levels. Targeted LC-MS/MS analysis revealed no significant increase in brain nitrate or nitrite concentrations with treatment. An untargeted metabolomics analysis found 47 metabolites whose abundance was significantly altered in the brain with nitrate and nitrite treatment including an 18-19% reduction in the neurotransmitter γ-aminobutyric acid (GABA), and 17-22% reduction in its precursor, glutamine, which may contribute to the increased anxiety-like behavior. Conclusion Nitrate and nitrite treatment did not adversely affect multiple parameters of zebrafish health but was associated with mild anxiety-like behavior, changes in the brain metabolome, and caused a short-term decrease in executive function or associative learning.


Introduction 99
Nitrate (NO 3 -), a component of leafy green and root vegetables, including beetroot juice (BRJ) and 100 many green leafy vegetables, has blood pressuring-lowering and ergogenic effects in humans 1 . 101 Nitrate supplementation (either as BRJ or sodium nitrate) has also demonstrated benefits pertaining 102 to cardiovascular health 2 , such as reducing blood pressure, enhancing blood flow, and elevating 103 the driving pressure of O 2 in the microcirculation to areas of hypoxia or exercising tissue 3,4 . These 104 findings are important to cardiovascular medicine and exercise physiology. Indeed, multiple 105 studies support nitrate supplementation as an effective method to improve exercise performance 5,6 . 106 Additionally, it has been reported that dietary nitrate can modulate cerebral blood-flow (CBF), 107 decrease reaction time in neuropsychological tests, improve cognitive performance and suggest 108 one possible mechanism by which vegetable consumption may have beneficial effects on brain 109 function in humans 7,8 . In contrast, other recent studies have found no significant effect of nitrate 110 or nitrite supplementation on cognitive function and this highlights the need for additional studies 111 to clarify the effect of nitrate and nitrite treatment on cognitive function (reviewed in 9,10 ). 112 Nitric oxide (NO) is a gaseous, free radical signaling molecule produced via enzymatic and 113 non-enzymatic pathways. The enzymatic pathways for NO synthesis are produced by three distinct 114 families of nitric oxide synthase (NOS) enzymes in mammals that use L-arginine and numerous 115 co-factors as substrates 11 . NO conveys essential signaling in the cardiovascular, central nervous, 116 and immune systems 12 . NO, through formation of S-nitrosothiols and nitration of alkenes or other 117 nitrated species, is also considered to have hormone-like properties that take part in different 118 metabolic/endocrine disorders such as diabetes and dysglycemia, thyroid disorders, hypertension, 119 heart failure, and obesity 13 . Furthermore, NO plays an important role in regulation of 120 synaptogenesis and neurotransmission in the central and peripheral nervous system 14,15 . NO can 121 also be produced by a NO synthase-independent method through the nitrate-nitrite-nitric oxide 122 pathway. Nitrate present in foods or water is reduced endogenously by lingual nitrate reductases 123 in mammals to nitrite (NO 2 -) and, in the stomach, to nitric oxide (NO) before distribution via blood 124 to tissues 16,17 . Several endogenous enzymes, proteins, and chemical species can reduce nitrite to 125 NO including deoxygenated hemoglobin, xanthine oxidoreductase, deoxymyoglobin, 126 mitochondrial enzymes, ascorbic acid, etc. 18 In spite of the vast amounts of research on NO 127 production, NO-related signaling mechanisms, and the effects of nitrate supplementation on the 128 cardiovascular system; there is still a gap in knowledge regarding whether dietary nitrate 129 supplementation affects the brain metabolome, learning, and other brain functions. 130 In order to determine the physiological and cognitive effects derived from nitrate and nitrite 131 exposure, we carried out a study with the aquatic model organism Danio Rerio (zebrafish). 132 Zebrafish was chosen because it is a complex vertebrate organism that was originally established 133 as a prime model for developmental studies and, is increasingly used for behavioral neuroscience 134 research in part because of standardized and high throughput behavioral performance assays [19][20][21][22][23] . 135 Importantly, as in humans, the nitrate-nitrite-nitric oxide pathway and NOS enzymes play 136 important roles in regulating NO levels, along with cardiac and blood vessel development in 137 zebrafish 24 . In addition, high genetic homology exists between zebrafish and humans for genes 138 associated with disease 25,26 . Furthermore, we established that nitrate treatment in zebrafish 139 improves the oxygen cost of exercise 27 as had been observed in humans. While conducting these 140 experiments we also sought to test the hypothesis that nitrate and nitrite treatment would improve 141 indicators of learning and cognitive performance. We also investigated the effects of nitrate and 142 nitrite treatment on zebrafish behavior and the brain metabolome with the aim of elucidating 143 mechanisms that may contribute to the potential improvement of cognitive performance. To this 144 end, adult zebrafish were exposed to sodium nitrate, sodium nitrite, or control water and tested for 145 changes in learning, memory, and behavior. Furthermore, we utilized targeted and untargeted 146 metabolomics analysis to examine the extent to which treatment resulted in changed nitrate or 147 nitrite concentrations in the brain and altered the brain metabolome.  Experiments contained three treatment groups which were treated for up to 31 days as 1) no 159 treatment (control fish); 2) sodium nitrate-exposed fish (606.9 mg NaNO 3 / L water); and 3) 160 sodium nitrite-exposed fish (19.5 mg NaNO 2 / L of water). The nitrate dose was chosen because it 161 increased blood nitrate and nitrite levels, improved exercise performance, and was non-toxic in 162 zebrafish 27,28 . The nitrite dose was chosen because it increased blood nitrite levels but was not 163 associated with adverse effects at pathology with the exception of some mild irritation of gill 164 epithelium 27,29 . For labeling experiments, a subset of fish was switched to water containing >99% 165 stable isotopes of Na 15 NO 3 , or 100% Na 15 NO 2 (Cambridge Isotope Laboratories, Tewksbury, MA) 166 at day 28 for 3 days of treatment prior to collection. Nitrate and nitrite were dissolved in freshly 167 prepared fish water and, unless otherwise indicated, chemicals were purchased from Sigma-168 Aldrich (St. Louis, MO). The fish water and treatment exposure were replaced every 36 hours 169 throughout the duration of the experiment to maintain low ammonia levels and consistent 170 treatments; pH was held at 6.8-7, total ammonia levels to 0-2.0 ppm, and temperature at 27-29 °C.

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Fish were fed a standard lab diet (Gemma Micro. Skretting, Westbrook, ME) at a volume of ~3% 172 body weight/day. For sample collections fish were euthanized with an overdose of the anesthesia 173 drug, tricaine mesylate, and all efforts were made to minimize suffering. Fish were then dried, 174 weighed, measured for standard length, and brains were collected and snap frozen in liquid 175 nitrogen. Samples were stored in -80°C until used for analysis.  Swimming behavior, startle response, innate predator avoidance, and social cohesion was tested 190 in individual fish between 14-17 days of treatment, using a zebrafish visual imaging system (zVIS) 191 as previously described 32,33 . Briefly, in the free swim assay fish were placed in a tank with 1.7L of 192 water and the data from the first minute was ignored. The location of the fish was then analyzed  Taps were generated by an electric solenoid below each tank. Following a 10-minute acclimation 198 period, a total of five taps were delivered, with 20s following each tap, and the distance moved  For data analysis, the tank was subdivided into three zones in relation to the video projection (close, 205 middle, and far) and the time spent in each zone was calculated.

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Custom-built shuttle boxes were used to test learning with a modified protocol as 207 previously described 32,34 . The programmed protocol of this active avoidance conditioning test was 208 designed to condition the zebrafish to leave the compartment with blue light ("reject side") and 209 swim to the dark side ("accept side", also referred to as the correct side). There were a total of 30 210 trials; each trial consisted of giving the zebrafish 8 seconds to "seek" a dark side of the tank after 211 the blue light came on to avoid a moderate shock. If the fish did not move to the correct side, the 212 16 second (s) shock period was initiated. A moderate pulse of 5 V was delivered at 1 s intervals, 213 for a duration of 500 ms. Fish were removed from the assay when they did not swim to the correct 214 side during 8 consecutive trials and these fish were counted as repeatedly failed. The statistical 215 method remained as previously described 34 , with the data fit using linear regression models to 216 calculate the initial performance of the fish (intercept) and the rate of learning (slopes) for each   In order to quantify nitrate and nitrite uptake into the brain, we used a previously described LC-  To determine significant differences between three treatment group data were analyzed using a

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Effect of nitrate and nitrite treatment on health parameters and learning 316 Treatment increased nitrate or nitrite levels in the fresh and used fish water ( Fig. 1A and B).

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Furthermore, both nitrate and nitrite concentrations in control water were maintained at low levels 318 throughout the treatment period ( Fig. 1A and B). Several parameters of fish health, including fish 319 length and weight, were not significantly changed with nitrate or nitrite treatment ( Fig. 1C and D). 320 Likewise, no significant differences were found between treatment groups for the distance and 321 velocity fish traveled in an unstressed environment ( Fig. 1E and F, P = 0.2089 and 0.2088, 322 respectively). A startle response assay showed that both nitrate-and nitrite-treated fish became 323 habituated to the vibration, similar to control fish, but nitrate-treated fish traveled a small but 324 significant less distance (10%) following the startle (Fig. 1G).

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In order to address if nitrate and nitrite treatments altered learning, fish were tested in a 327 learning and memory assay using custom-built shuttle box, where over 30 consecutive trials they 328 learned to avoid an adverse event (mild shock) by moving when a light came on ( Fig. 2A). As seen 329 from the linear regression calculated from the data, both nitrate and nitrite treated fish initially 330 took longer to make a decision and were shocked longer (Fig. 2B). Over subsequent trials, more 331 nitrate-and nitrite-treated fish (5-7% of the population tested) had to be removed from the assay 332 because they repeatedly failed to learn (Fig. 2C). However, data from all trial periods show that 333 both nitrate and nitrite treated fish were able to learn and had improved decision time and time 334 shocked, as reflected in their rate of learning (Fig. 2D). It should be noted that the rate of learning 335 (a negative slope) has a larger negative value with nitrate and nitrite treatment, relative to control 336 because these fish had greater potential to improve based on their behavior at the beginning of the 337 assay ( Fig. 2B and D). When all fish that were tested are considered, nitrate and nitrite treatment 338 was associated with a significant higher percentage of fish that failed to make a decision and were 339 shocked (Fig. 2E). When the population is filtered to include only fish that could learn (i.e., 340 completed the assay), the nitrite-treated fish were no longer significantly impaired but significant 341 deficits were still present in nitrate-treated fish for time shocked and time to decision (Fig. 2E). The effect of nitrate and nitrite exposure on predator avoidance and social cohesion was tested.

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Unexpectedly, nitrate-treated fish spent a statistically significant more time close to the monitor 346 during the acclimation period (72%), while the nitrite-treated fish spent 37% more time close to 347 the monitor (Fig. 3A). The social video stimulus did not significantly alter fish behavior in any 348 treatment group as compared to the acclimation period (Fig. 3A). Nitrate and nitrite treated fish 349 moved away from the monitor when a predator video was shown, as seen by the significant 350 decrease in time spent in the area close to the monitor (Fig. 3A and Supplemental Figure 1). Fish 351 behavior was also tested in the free swim assay where fish were placed in a novel tank. As 352 expected, control fish spent similar amounts of time at all three depths of the tank, balancing safety 353 from predation and opportunity to find food (Fig. 3B). In contrast, there was a significant 354 difference between the time the nitrate-and nitrite-treated fish spent between the bottom and top 355 zones (Fig. 3B). Nitrate-and nitrite-treated fish spent 22-35% more time in the bottom zone, as 356 compared to control fish. The increase in bottom-dwelling is consistent with anxiety-like behavior.

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Since anxiety can be associated with stress, we measured the levels of some stress hormones and 358 found neither nitrate, nor nitrite treatment significantly increased epinephrine or norepinephrine 359 levels (Fig. 3C). It appeared that nitrite-treated fish experienced lower concentrations of these 360 hormones yet high variability between fish led to no significant differences being detected. Nitrate 361 or nitrite treatment also did not significantly change dopamine concentrations which is the 362 precursor for epinephrine and norepinephrine (Fig. 3C).

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Nitrate and nitrite uptake into the brain 365 For the last three days of the experiment, a subset of fish was treated with 15 N-nitrate or 15 N-nitrite 366 in order to study the uptake of nitrate and nitrite into brain tissue. The resulting percent enrichment 367 results show the proportion of nitrate and nitrite derived from exogenous sources (the treatment in 368 water) versus endogenous sources such as oxidation of NO from NOS-mediated production. We 369 observed a low uptake of nitrate (14%) and almost no uptake of nitrite (0.1%) in the brain, which 370 can be seen by comparing the fish that received labeled nitrate or nitrite as compared to the 371 respective unlabeled nitrate or nitrite treatment conditions. (Fig. 4A and B). Furthermore, no 372 significant changes in nitrate or nitrite concentrations were detected in the brain of animals treated 373 with nitrate or nitrite ( Fig. 4A and B) when compared with the control group. Taken together, these 374 results suggest that the behavioral changes observed with nitrate and nitrite exposure are likely 375 due to indirect effects of treatment on brain metabolism, rather than a direct effect via influx of the 376 nitrate or nitrite into the brain.

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One hundred twenty-four (124) metabolites were annotated using our in-house library (SI Table   380 S1). Of these metabolites, 47 were significantly changed among at least one treatment group, as 381 compared to the others and FDR-corrected P-values (q-values) for all significantly changed 382 metabolites, between all treatment groups, are listed in SI Table S2. For example, deoxyadenosine 383 diphosphate (dADP) was significantly up-regulated (q = 0.018) in fish exposed to nitrate and Notably, nitrate or nitrite treatment resulted in significant differences among multiple 397 metabolites involved in purine metabolism like hypoxanthine, xanthine, inosine, guanine, 398 guanosine, deoxyadenosine diphosphate (dADP) and cyclic adenosine monophosphate (cAMP).

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Interestingly we also observed a significant decline in nicotinamide adenine dinucleotide 400 phosphate (NADP) and NAD. We observed a significant depletion in the annotated fatty acids Here we disproved the hypothesis that nitrate, and nitrite treatment would improve indicators of 419 learning and cognitive performance in a zebrafish model. While nitrate and nitrite treatment did 420 not adversely affect multiple parameters of health, these treatments were associated with mild 421 anxiety-like behavior and an initial deficit in learning, which was consistent with either decreased 422 executive function or associative learning. While we have previously shown the nitrate and nitrite 423 doses used here increased blood and whole body nitrate and nitrite levels, the treatments were not 424 associated with a significant increase in the concentration of nitrate or nitrate in the brain, and only 425 a minor, or almost no uptake of these chemicals into the brain. Nevertheless, some brain 426 metabolites including GABA and glutamine were significantly decreased by nitrate and nitrite 427 treatment suggesting that the changes in behavior and learning may be due to indirect effects of 428 nitrate and nitrite treatment on the nervous system.

429
The anxiety-like behavior we observed with nitrate or nitrite treatment was mild compared anxiety was more similar in scale to zebrafish that were not allowed to exercise 48 .

437
The zebrafish in this study exhibited increased anxiety, as evidenced by staying near the 438 bottom of the novel tank. Nitrate and nitrite treatment also changed the behavior of fish during the 439 acclimation period of the predator and social stimulus assay. We also observed an initial delay in 440 zebrafish decision making following a light stimulus and increased time being shocked initially in 441 the shuttle box task which could represent an initial deficit in associative learning and/or executive 442 function (e.g., decision making). This is inconsistent with literature that showed nitrate, given as 443 BRJ supplement, improved reaction time and cognitive performance 7,49-51 . A plausible mechanism 444 underlying nitrate-induced cognitive improvements is increased vasodilation, yielding improved 445 CBF 7,9,52,53 . This is exemplified by a study in older adults where two days of consuming a high 446 nitrate diet increased regional cerebral perfusion in frontal lobe white matter, particularly between 447 the dorsolateral prefrontal cortex and anterior cingulate cortex 52 . These brain regions participate 448 in executive function, which may have been affected by nitrate and nitrite treatment in our study.

449
In contrast with our results, multiple studies show no significant association with foods containing for GABA production and serves as an important energy source for the nervous system. We also 474 observed changes in brain purine-related metabolites, which is consistent with the known 475 relationship between exogenous and endogenous pathways that generate NO 18,24,27 . The nitrate-476 and nitrite-induced reductions in fatty acids, neurotransmitters, signaling molecules, tricarboxylic 477 acid cycle intermediates, and amino acids are also of interest and warrant future investigation. A 478 significant limitation of this study is the use of whole zebrafish brains to derive metabolomics data, 479 limiting our ability to draw inferences to specific functional structures within the brain, like the 480 zebrafish equivalent of the prefrontal cortex 71 . Interestingly, a study in older adults using a more 481 focused technique measured brain N-acetyl aspartate, creatine, choline, or myo-inositol levels and 482 found no change with 3 day BRJ supplement 57 .

483
It is also possible that the changes in zebrafish behavior we observed were because nitrate 484 and nitrite treatment caused a headache or migraine 72 . Headaches are a predominate side effect 485 from therapeutic use of organic nitrates, which are prodrugs for NO, cause vasodilation of blood 486 vessels in the brain, and "immediate" mild-to-medium severity headaches or "delayed" migraines 487 which involves cGMP or NO dependent S-nitrosylation-mediated changes in ion channel 488 function 73 . Nitroglycerin has been used to model migraines in multiple species including fish 74,75 .

489
Also, headache is the most common side effect in patients taking sildenafil, which promotes blood 490 flow to organs like the brain, through cGMP. Furthermore, consumption of high nitrite foods was in water. To address this potentially toxic metabolite, we regularly measured ammonia and found 518 no effect of nitrate or nitrite treatment on water ammonia levels. Due to the large number of 519 animals needed to conduct the study, we were limited in the number of doses we could test and 520 thus focused on a nitrate dose and exposure duration associated with improvements in exercise 521 performance 27 . More and larger studies are needed to delineate the potential benefits and risks 522 associated with nitrate and/or nitrite treatment on CBF, mood, and cognitive function, particularly 523 in populations of people with differing ages and underlying health status. Importantly, a study in 524 humans is underway to look at the effect of increasing doses of nitrate on cognition-related 525 outcomes 86 . We also cannot differentiate between the direct effects of nitrate or nitrite in the fish, 526 or indirect effects that could be generated by increased NO availability. Nevertheless, we show 527 that nitrate and nitrite treatment in a zebrafish model did not adversely affect multiple parameters 528 of health but was associated with mild anxiety-like behavior, changes in brain metabolome, and 529 an initial decrease in executive function or associative learning.  by an analysis of variance (AOV) followed by a Tukey's post-test where "All" indicates data 574 from all fish analyzed, while "Completed trials" excludes data from fish that repeatedly failed.