Metabolic and cardiac signaling effects of inhaled hydrogen sulfide and low oxygen in male rats

Abstract

Low concentrations of inhaled hydrogen sulfide (H(2)S) induce hypometabolism in mice. Biological effects of H(2)S in in vitro systems are augmented by lowering O(2) tension. Based on this, we hypothesized that reduced O(2) tension would increase H(2)S-mediated hypometabolism in vivo. To test this, male Sprague-Dawley rats were exposed to 80 ppm H(2)S at 21% O(2) or 10.5% O(2) for 6 h followed by 1 h recovery at room air. Rats exposed to H(2)S in 10.5% O(2) had significantly decreased body temperature and respiration compared with preexposure levels. Heart rate was decreased by H(2)S administered under both O(2) levels and did not return to preexposure levels after 1 h recovery. Inhaled H(2)S caused epithelial exfoliation in the lungs and increased plasma creatine kinase-MB activity. The effect of inhaled H(2)S on prosurvival signaling was also measured in heart and liver. H(2)S in 21% O(2) increased Akt-P(Ser473) and GSK-3β-P(Ser9) in the heart whereas phosphorylation was decreased by H(2)S in 10.5% O(2), indicating O(2) dependence in regulating cardiac signaling pathways. Inhaled H(2)S and low O(2) had no effect on liver Akt. In summary, we found that lower O(2) was needed for H(2)S-dependent hypometabolism in rats compared with previous findings in mice. This highlights the possibility of species differences in physiological responses to H(2)S. Inhaled H(2)S exposure also caused tissue injury to the lung and heart, which raises concerns about the therapeutic safety of inhaled H(2)S. In conclusion, these findings demonstrate the importance of O(2) in influencing physiological and signaling effects of H(2)S in mammalian systems.

Fig. 1.

Diagram illustrating the H₂S exposure system and experimental protocol. A: all gas flow rates were maintained by mass flow controllers. Carrier gases were humidified before converging with the H₂S gas line and the H₂S concentration was confirmed by using a H₂S gas analyzer. Rats were exposed to one of the gas mixtures in an air-tight 6-liter chamber maintained in a chemical safety fume hood. B: physiological parameters were measured 5 min before beginning the exposure experiments and then once per hour for 6 h during the exposure. Note that measurements were made while the animal continued to breathe the appropriate gas mixture through a nose cone. After 6 h, the animal was removed from the exposure chamber and allowed to recover in a separate cage while breathing room air. After 1 h of recovery, a final measurement was recorded for all physiological parameters. Statistical analyses were performed on measurements take before, during (at 5-h time point), and after exposures.

Fig. 2.

Effect of H₂S and/or 10.5% O₂ on body temperature. A: body temperature was measured before, during, and after exposures with an infrared thermometer placed in the ear. The “before” measure was taken ∼5 min before starting the exposure protocol, the “during” measure was taken 5 h into the exposure, and the “after” measure was taken 1 h after the exposure protocol had ended. B: results presented are P values obtained from 2-factor ANOVA with H₂S effect (absent vs. present), O₂ effect (21% vs. 10.5% O₂ tension), and interaction (H₂S × O₂). Results from post hoc statistical analyses: ^aP < 0.001 compared with 10.5% O₂ within during (5 h); ^bP < 0.001 compared with H₂S + 21% O₂ within during (5 h); and ^cP = 0.011 compared with 21% O₂ within after. Data represent means ± SE for n = 4 animals per treatment group.

Fig. 3.

Effect of H₂S and/or 10.5% O₂ on heart rate. A: heart rate was measured before, during, and after exposures with a pulse oximeter. The “before” measure was taken ∼5 min before starting the exposure protocol, the “during” measure was taken 5 h into the exposure, and the “after” measure was taken 1 h after the exposure protocol had ended. B: results presented are P values obtained from 2-factor ANOVA with H₂S effect (absent vs. present), O₂ effect (21% vs. 10.5% O₂ tension), and interaction (H₂S × O₂). Results from post hoc statistical analyses: ^aP = 0.008 compared with 21% O₂ within during (5 h); ^bP < 0.001 compared with 10.5% O₂ within during (5 h); and ^cP = 0.003 compared with 21% O₂ within after (1 h post exposure). Data represent means ± SE for n = 4 animals per treatment group. NS, not significant.

Fig. 4.

Effect of H₂S and/or 10.5% O₂ on ventilation rate. A: ventilation rate was measured before, during, and after exposures with a pulse oximeter. The “before” measure was taken ∼5 min before starting the exposure protocol, the “during” measure was taken 5 h into the exposure, and the “after” measure was taken 1 h after the exposure protocol had ended. B: results presented are P values obtained from 2-factor ANOVA with H₂S effect (absent vs. present), O₂ effect (21% vs. 10.5% O₂ tension), and interaction (H₂S × O₂). Results from post hoc statistical analyses: ^aP < 0.001 compared with 10.5% O₂ within during (5 h) and ^bP < 0.001 compared with H₂S + 21% O₂ within during (5 h). Data represent means ± SE for n = 4 animals per treatment group.

Fig. 5.

Effect of H₂S and/or 10.5% O₂ on hemoglobin saturation. A: hemoglobin saturation was measured before, during, and after exposures with a pulse oximeter. The “before” measure was taken ∼5 min before starting the exposure protocol, the “during” measure was taken 5 h into the exposure, and the “after” measure was taken 1 h after the exposure protocol had ended. B: results presented are P values obtained from 2-factor ANOVA with H₂S effect (absent vs. present), O₂ effect (21% vs. 10.5% O₂ tension), and interaction (H₂S × O₂). Results from post hoc statistical analyses: ^aP = 0.025 compared with 21% O₂ within during (5 h); ^bP = 0.025 compared with 10.5% O₂ within during (5 h); and ^cP < 0.001 compared with H₂S + 21% O₂ within during (5 h). Data represent means ± SE for n = 4 animals per treatment group.

Fig. 6.

Effect of H₂S and/or 10.5% O₂ on lung histopathology. Representative light micrographs of airways from rats exposed to 21% O₂ (A), 10.5% O₂ (B), H₂S + 21% O₂ (C), and H₂S + 10.5% O₂ (D). Lungs were collected 1 h after the cessation of exposures, inflation-fixed with formalin, embedded in paraffin, and stained with hematoxylin and eosin to detect pathological changes. Magnification bar = 30 μm. Histopathology scores for both large (E) and small (F) airways are shown in dot plots with mean provided as lines. Results from statistical analyses: ^aP < 0.005 compared with 21% O₂; ^bP = 0.006 compared with 10.5% O₂; and ^cP = 0.011 compared with H₂S + 21% O₂. Data represent results from n = 3–6 animals per treatment group.

Fig. 7.

Effect of H₂S and/or 10.5% O₂ on heart and liver enzymes. Plasma levels of creatine kinase-MB (CK-MB; A) and alanine aminotransferase (ALT; B) were measured after exposures to assess effects on heart and liver injury, respectively. C: results presented are P values obtained from 2-factor ANOVA with H₂S effect (absent vs. present), O₂ effect (21% vs. 10.5% O₂ tension), and interaction (H₂S × O₂). Results from post hoc statistical analyses: ^aP < 0.005 compared with H₂S + 21% O₂ and ^bP < 0.001 compared with 10.5% O₂. Data represent means ± SE for n = 6 animals per treatment group.

Fig. 8.

Effect of H₂S and/or 10.5% O₂ on Akt and GSK-3β phosphorylation in heart. Representative immunoblots of phosphorylated (top) and total (bottom) Akt (A) and GSK-3β (B) in heart samples from rats exposed to 21% O₂, 80 ppm H₂S + 21% O₂, 10.5% O₂, and 80 ppm H₂S + 10.5% O₂. C, E, and G: densitometry analyses of Akt-P^Ser473, total Akt, and Akt-P normalized to total Akt protein, respectively. D, F, and H: densitometry analyses of GSK-3β-P^Ser9, total GSK-3β, and GSK-3β-P normalized to total GSK-3β protein, respectively. Results from post hoc statistical analyses: ^aP < 0.01 compared with 21% O₂; ^bP < 0.001 compared with 10.5% O₂; and ^cP < 0.05 compared with H₂S + 21% O₂. Data represent means ± SE for n = 4 animals per treatment group. Note that the results from the 2-factor ANOVA for these data are provided in Table 2.