A Sulfonyl Azide-Based Sulfide Scavenger Rescues Mice from Lethal Hydrogen Sulfide Intoxication

Abstract

Exposure to hydrogen sulfide (H2S) can cause neurotoxicity and cardiopulmonary arrest. Resuscitating victims of sulfide intoxication is extremely difficult, and survivors often exhibit persistent neurological deficits. However, no specific antidote is available for sulfide intoxication. The objective of this study was to examine whether administration of a sulfonyl azide-based sulfide-specific scavenger, SS20, would rescue mice in models of H2S intoxication: ongoing exposure and post-cardiopulmonary arrest. In the ongoing exposure model, SS20 (1250 µmol/kg) or vehicle was administered to awake CD-1 mice intraperitoneally at 10 min after breathing 790 ppm of H2S followed by another 30 min of H2S inhalation. Effects of SS20 on survival were assessed. In the post-cardiopulmonary arrest model, cardiopulmonary arrest was induced by an intraperitoneal administration of sodium sulfide nonahydrate (125 mg/kg) in anesthetized mice. After 1 min of cardiopulmonary arrest, mice were resuscitated with intravenous administration of SS20 (250 µmol/kg) or vehicle. Effects of SS20 on survival, neurological outcomes, and plasma H2S levels were evaluated. Administration of SS20 during ongoing H2S inhalation improved 24-h survival (6/6 [100%] in SS20 vs 1/6 [17%] in vehicle; p = .0043). Post-arrest administration of SS20 improved 7-day survival (4/10 [40%] in SS20 vs 0/10 [0%] in vehicle; p = .0038) and neurological outcomes after resuscitation. SS20 decreased plasma H2S levels to pre-arrest baseline immediately after reperfusion and shortened the time to return of spontaneous circulation and respiration. These results suggest that SS20 is an effective antidote against lethal H2S intoxication, even when administered after cardiopulmonary arrest.

Keywords: antidotes; cardiopulmonary resuscitation; heart arrest; hydrogen sulfide; mice.

Figure 1.

Structural formula of SS20, a novel sulfonyl azide-based hydrogen sulfide scavenger, SS20 (Yang et al., 2019). SS20 reacts with hydrogen sulfide to form octasulfur. Abbreviations: H₂S, hydrogen sulfide; N₂, nitrogen; S₈, octasulfur.

Figure 2.

SS20 rescued mice from ongoing hydrogen sulfide breathing (ongoing exposure model). A, A schematic diagram of the ongoing exposure model. SS20 (1250 µmol/kg) or vehicle was administered to awake mice intraperitoneally at 10 min after breathing 790 ppm of H₂S followed by another 30 min of continuous H₂S inhalation. B, Percent survival during the first 24 h after H₂S inhalation. n = 6 for each group. **p < .01 versus vehicle by log-rank test. Abbreviations: H₂S, hydrogen sulfide; O₂, oxygen.

Figure 3.

A mouse model of sulfide intoxication-induced cardiopulmonary arrest and cardiopulmonary resuscitation (post-cardiopulmonary arrest model). A, A schematic diagram of the post-cardiopulmonary arrest model. Na₂S·9H₂O (125 mg/kg) was intraperitoneally administered to anesthetized mice to induce cardiopulmonary arrest. After 30 s of cardiopulmonary arrest, SS20 (250 µmol/kg) or vehicle was intravenously administered. To measure plasma H₂S levels, blood was taken from mice at 4 time points as follows: baseline, respiratory arrest, 2 min after initiation of CPR, and 24 h after CPR. B, Representative ABP change after sulfide intoxication-induced cardiopulmonary arrest and CPR in vehicle-treated and SS20-treated mouse. Abbreviations: ABP, arterial blood pressure; BT, body temperature; CPR, cardiopulmonary resuscitation; ECG, electrocardiogram; Na₂S·9H₂O, sodium sulfide nonahydrate; O₂, oxygen.

Figure 4.

SS20 improved survival and neurological function after sulfide intoxication-induced cardiopulmonary arrest and cardiopulmonary resuscitation (post-cardiopulmonary arrest model). A, Cardiopulmonary arrest was achieved in 22 of 25 mice subjected to sulfide intoxication; 10 were treated with SS20, and 12 were treated with vehicle. ROSC was achieved in 10/10 (100%) of SS20-treated mice and in 10/12 (83%) of vehicle-treated mice. B, Percent survival during the first 7 days after CPR. n = 10 for each group. **p < .01 versus vehicle by log-rank test. C, Neurological function score at 24 h after CPR. n = 10 for each group. ^††p < .01 by Mann-Whitney test. Dead mice (rated as zero) were excluded from analysis. Abbreviations: CPA, cardiopulmonary arrest; CPR, cardiopulmonary resuscitation; ROSC, return of spontaneous circulation.

Figure 5.

Administration of SS20 shortened the time required for cardiopulmonary resuscitation (post-cardiopulmonary arrest model). A and B, The length of time from sulfide intoxication to each time point. Note that the parameters were obtained before treatment with vehicle or SS20. C and D, Parameters during cardiopulmonary resuscitation in mice treated with vehicle or SS20. E and F, The length of time from the initiation of cardiopulmonary resuscitation to each time point in mice treated with vehicle or SS20. n = 10 for each group. *p < .05 by independent t test. ^††††p < .0001 by Mann-Whitney test. Abbreviation: ROSC, return of spontaneous circulation.

Figure 6.

Administration of SS20 increased blood pressure and heart rates after cardiopulmonary resuscitation (post-cardiopulmonary arrest model). A, Changes in mean arterial pressure until 1 h after cardiopulmonary resuscitation. B, Changes in heart rates until 1 h after cardiopulmonary resuscitation. n = 10 for each group. ^†††p < .001 between groups, *p < .05, **p < .01, ****p < .0001 by 2-way repeated-measures ANOVA followed by Bonferroni’s multiple comparisons test. The error bars represent SD. Abbreviations: CPR, cardiopulmonary resuscitation; HR, heart rates; MAP, mean arterial pressure.

Figure 7.

Post-arrest administration of SS20 decreased plasma hydrogen sulfide levels immediately after reperfusion (post-cardiopulmonary arrest model). Blood samples were collected at the following time points: baseline, respiratory arrest, 2 min after initiation of CPR, and 24 h after CPR. Relative H₂S levels in plasma were measured using HSip-1, a highly specific fluoroprobe for detecting H₂S. n = 6 for each time point and treatment. ^††††p < .0001 between groups, *p < .05, **p < .01, ***p < .001, ****p < .0001 by 2-way ANOVA followed by Bonferroni's multiple comparisons test. Abbreviations: CPR, cardiopulmonary resuscitation; H₂S, hydrogen sulfide.

Figure 8.

Histological effects of SS20 on brain injury in mice after sulfide intoxication-induced cardiopulmonary arrest and cardiopulmonary resuscitation (post-cardiopulmonary arrest model). A, A mouse brain atlas with the examined parts of the cerebral cortex. B, The number of Fluoro-Jade B positive cells per mm² in the cerebral cortex of vehicle-treated or SS20-treated mice at 24 h after sulfide intoxication-induced cardiopulmonary arrest and cardiopulmonary resuscitation. n = 6 for each group. **p < .01 by independent t test. C and D, Representative photomicrographs of cerebral cortex sections from mice treated with vehicle or SS20 showing Fluoro-Jade B and DAPI positive cells at 24 h after sulfide intoxication-induced cardiopulmonary arrest and cardiopulmonary resuscitation. Scale bars: C, 500 µm; and D, 100 µm. Abbreviations: FJB, Fluoro-Jade B; DAPI, 4’,6-diamidino-2-phenylindole dihydrochloride.