Measuring nitrate reductase activity from human and rodent tongues

Abstract

Reduction of salivary nitrate to nitrite by oral microbes expressing nitrate-reductase has emerged as a crucial pathway in systemic NO homeostasis in humans and other mammals. Selective depletion of oral microbes prevents dietary nitrate-dependent lowering of blood pressure, inhibition of platelet aggregation and ischemic injury. To date, most studies interrogate enterosalivary nitrate reduction by following changes in saliva or plasma nitrite and NO-signaling (functional) end points. Little is known about whether, and if so how, nitrate-reductase enzymatic activity per se (i.e. independent of nitrate levels) is a variable and may account for any individual to individual variation. Here, we describe a minimally invasive protocol that allows for NR activity determination from human, rat and mouse tongue scrapes/swabs. We validate this method using selective application of antiseptic agents to the distal tongue surface which decreased NR activity by >80% and show that bacterial number is a significant variable in measured NR activities between males and females. Also, we show that NR activity is >80% lower in smokers (humans) and after bromine gas exposure (mice), suggesting that exposure to inhaled reactive substances inhibit NR activity identifying a potentially new mechanism by which environmental toxicants promote dysfunction in NO-bioavailability. The described method will facilitate studies testing whether NR specific activity is a variable in different pathophysiologic settings, and in turn how this activity modulates enterosalivary nitrate-reduction.

Keywords: Antiseptic; Chlorohexidine; Halogen; Microbiome; Nitric oxide; Smoking.

Figure 1. Ex-vivo nitrate reductase (NR) activity of human posterior tongue

Panel A: Tongue scrapes were collected from 3 volunteers and incubated for 10min in BHI broth, 37°C. Nitrate (at indicated doses) was then added and nitrite formation measured after 40min. Data are mean ± SEM (n=4, except for nitrate doses 200–800μM, where n=3). Panel B: Average time-dependent nitrite formation (after nitrate addition) in male (□) and female (●) samples. Data are mean ± SEM (n=5 (female) and n=4 (male)), Panel C: Average time-dependent nitrite formation (after nitrate addition) normalized to CFU in male and female samples. Data are mean ± SEM (n=5 (female) and n=4 (male)) *P<0.05 by 2-way ANOVA. Panel D: Nitrate reduction to nitrite was measured in paired tongue scrapes collected from the front and back of the tongue. Data show mean ± SEM (n=5 different donors). *P<0.05 by 2-way ANOVA and ^#P<0.05 by Sidak’s Multiple comparison test. Panel E: data from Panel D normalized to CFU.

Figure 2. Ex-vivo nitrate reductase activity in posterior tongue swabs from mice

Panel A. Nitrate (1mM) was added to tongue swabs immediately after collection or after culturing for the indicating times in BHI at 37°C under aerobic or anaerobic conditions. Panel B: CFU counts from Panel A. Data are mean ± SEM (n=2 for 0 and 6h group; n=4 for 12 and 18h groups) p<0.05 by 1-way ANOVA for significant time dependent changes for aerobic and anaerobic cultures.

Figure 3. Ex-vivo nitrate reductase activity in posterior tongue swabs from male and female mice

Panel A: Nitrate (at indicated doses) was added to 18h aerobic or anaerobic cultures and nitrite formation measured after 40min. Data are mean ± SEM (n=3). Panel B: average nitrite formation versus time traces for aerobic cultures. *P<0.05 by 2-way ANOVA with Sidak’s post-test (n=4 (females), n=5 (males)). Error bars show SEM. Panel C: average nitrite formation versus time traces for anaerobic cultures (n=4 (females), n=4 (males). Panel D: Nitrate formation normalized to CFU for aerobic cultures. Data are mean ± SEM, n=4 (females), n=5 (males). Panel E: Nitrite formation normalized to CFU for anaerobic cultures. Data are mean ± SEM, n=4 (females), n=4 (males).

Figure 4. Effects of chlorohexidine on nitrate reductase activity in mice

Chlorohexidine (0.2%) was administered directly onto the posterior tongue in a 10μl volume either once daily, 7d, or twice daily 3d or 7d and then nitrate reductase activity measured (Panel A), CFU measured after 18h culture in BHI (Panel B) and initial rate of nitrate reductase activity normalized to CFU calculated (Panel C). Data are mean ± SEM (n=3 for 3 days twice daily; n=3 for 7 days once daily; n=6 for 7 days twice daily, n=7 for vehicle control). Panel D shows CFU counts from vehicle (water) or chlorohexidine treated mice (2× day, 7days), upon collection of tongue swabs or after 18 culturing in BHI broth. Data show mean ± SEM (n=5 in each group). *P<0.05 by unpaired t-test. Two data points from 7 days twice daily and one data point from vehicle control were excluded by ROUT analysis.

Figure 5

Male C57Bl/6 male mice received water or water supplemented with nitrate for 2 weeks. Chlorohexidine (0.2%) was administered directly onto the posterior tongue in a 10μl volume twice daily for the last 7d. Panel A: Oral nitrate reductase activity was measured as described in Figure 3. Data show nitrite levels 20min after addition of nitrate to tongue swabs Panel B: CFU measured after 18h culture in BHI. Panel C: Initial rate of nitrate reductase activity normalized to CFU. All data are mean ± SEM (H₂O n=8, CHX n=8, Nitrate-H₂O n=7, Nitrate-CHX n=5). *p<0.05 by unpaired t-test; ^#p<0.05 by 1-way ANOVA with Tukey post-test. One outlier from Nitrate-H₂O and two outliers from nitrate-CHX removed by ROUT analysis. Panel D shows the nitrite levels in plasma. Data are mean ± SEM (n=8 except for H₂O group where n=9). *p<0.05 by 1-way ANOVA with Tukeys post-test.

Figure 6

Scrapes from the posterior tongue were collected from healthy adult non-smokers (n=9; 4 males and 5 females, age range 20–45y as shown in Fig 1), or smokers (n=5; 3 male and 2 female, age range 31–60y). NR activity (Panel A), bacterial count (Panel B), and initial rate of nitrate activity normalized with CFU (Panel C) was calculated. Data are mean ± SEM. ^#p=0.05, *p<0.05 unpaired t-test.

Figure 7

C57/Bl6 mice were exposed to bromine (600 ppm) for 30 min and then brought back to room air. 6h thereafter, tongue swab was collected and cultured in BHI for 18h. NR activity was then determined by adding nitrate (1mM). Panel A shows nitrite formed after 20 min. Panel B shows CFU counts and Panel C corresponding rate of nitrate reductase activity over 20 min normalized to CFU. Data are mean ± SEM (n=7 for Air and n=6 for bromine). ^#p=0.05, *P < 0.05 by unpaired t-test. One outlier from air and two outlier from bromine group removed by ROUT analysis.