Antagonism of Acute Sulfide Poisoning in Mice by Nitrite Anion without Methemoglobinemia

Abstract

There are currently no FDA-approved antidotes for H2S/sulfide intoxication. Sodium nitrite, if given prophylactically to Swiss Webster mice, was shown to be highly protective against the acute toxic effects of sodium hydrosulfide (∼LD40 dose) with both agents administered by intraperitoneal injections. However, sodium nitrite administered after the toxicant dose did not detectably ameliorate sulfide toxicity in this fast-delivery, single-shot experimental paradigm. Nitrite anion was shown to rapidly produce NO in the bloodstream, as judged by the appearance of EPR signals attributable to nitrosylhemoglobin and methemoglobin, together amounting to less than 5% of the total hemoglobin present. Sulfide-intoxicated mice were neither helped by the supplemental administration of 100% oxygen nor were there any detrimental effects. Compared to cyanide-intoxicated mice, animals surviving sulfide intoxication exhibited very short knockdown times (if any) and full recovery was extremely fast (∼15 min) irrespective of whether sodium nitrite was administered. Behavioral experiments testing the ability of mice to maintain balance on a rotating cylinder showed no motor impairment up to 24 h post sulfide exposure. It is argued that antagonism of sulfide inhibition of cytochrome c oxidase by NO is the crucial antidotal activity of nitrite rather than formation of methemoglobin.

Figure 1

Prophylactically administered NaNO₂ ameliorates NaHS toxicity in mature and juvenile mice. (A) Injection paradigm. (B) Mice (Swiss Webster males, 16–18 weeks of age) were given NaHS in saline (16 mg/kg, ip), and times until death were recorded. The duration of survival (breathing cessation) was measured from the time of the sulfide injection (t = 0). Survival quotients are shown with surviving mice/total mice written above the bar. NaNO₂ (12 or 24 mg/kg, ip) was given 5 min prior to NaHS injection. Supplemental oxygen (100% O₂) was administered for either 15 min or until death immediately after NaHS injections (* p ≤ 0.05 vs NaHS injection alone). (C) Juvenile mice (Swiss Webster males, 6–8 weeks old) were injected (ip) with either 16, 18, or 20 mg/kg NaHS, and survival was recorded as for adults. In addition, 24 mg/kg NaNO₂ was given 5 min before 18 mg/kg NaHS (* p ≤ 0.05 vs 18 mg/kg NaHS injection).

Figure 2

RotaRod testing of neuromuscular coordination following NaHS/NaCN/NaNO₂ exposures in adult Swiss Webster mice. (A) RotaRod testing paradigm: arrows indicate RotaRod testing times; lines with bars indicate injection times (all ip). Mice were trained on the RotaRod 24 h before injection, and a baseline performance was obtained 1 h before injection (Preip). Mice were tested every 15 min after injections for 1 h to assess recovery. (B) Comparison of performance for injections of 16 mg/kg NaHS (●), 24 mg/kg, NaNO₂ (□) and 24 mg/kg NaNO₂ injected 5 min prior to 16 mg/kg NaHS dose (▲). (C) Comparison of performance for injections of 16 mg/kg NaHS (●), 6.4 mg/kg NaCN (◆), or 24 mg/kg NaNO₂ injected 5 min prior to 6.4 mg/kg NaCN (□). Numbers of animals (in parentheses) used in each set of experiments: NaHS (6), NaNO₂ (8), NaNO₂ + NaHS, (8), NaCN (9), NaNO₂ + NaCN (10). (*, p ≤ 0.05 vs controls.)

Figure 3

EPR spectra (X-band, 10 K) of whole mouse blood. (A) EPR spectrum of drawn blood following dose of 24 mg/kg NaNO₂ administered ip 5–10 min prior to sacrifice showing clear evidence for the generation of nitric oxide. Signal (1) at ~1100 gauss: metHb; signal (2) at ~3300 gauss: HbNO. The combined intensities of the metHb plus HbNO signals represents <5% of the total heme present in the blood (~9 mM). (B) Spectrum of blood following dose of 16 mg/kg NaHS. This dose of NaHS roughly amounts to a maximal concentration of ~2.8 mM in the blood. (C) Spectrum of blood following dose of 24 mg/kg of NaNO₂ (t = 0) and 16 mg/kg of NaHS at 2 min. The signals at ~2700, 3060, and 3700 gauss all arise from metHbSH (designated signal 3) and represent <3% (~0.2 mM) of the total Hb (~200 mM). (D) Spectrum of blood following NaHS injection 2 min prior to NaNO₂. (metHbSH signals not detected.)

Figure 4

EPR spectra (X-band, 10 K) of mouse heart tissue. (A) EPR spectrum of minced heart tissue following dose of 24 mg/kg NaNO₂ administered ip 5–10 min prior to sacrifice showing clear evidence for the generation of nitric oxide. Signal (4) at ~1100 gauss: metMb; signal (5) at ~3300 gauss: MbNO. The combined intensities of the metMb plus MbNO signals approach 100% of the total heme (~260 μM) present in the heart. (B) Spectrum of heart tissue following dose of 16 mg/kg NaHS. (C) Spectrum of heart tissue following dose of 24 mg/kg of NaNO₂ (t = 0) and 16 mg/kg of NaHS at 2 min. No signals due to metMbSH were observed. (D) Spectrum of heart tissue following NaHS injection 2 min prior to NaNO₂. (metHbSH signals not detected.)

Figure 5

Electronic absorption spectra of cytochrome c oxidase derivatives showing displacement of HS⁻ by NO. Samples were prepared in 100 mM aqueous potassium phosphate buffer, pH 7.4, 0.05% lauryl maltoside, 25 °C, 1.00 cm pathlength. (Dash-dot trace) Cytochrome c oxidase as isolated (oxidized, resting), 5 μM in enzyme; (dotted trace) partially reduced sulfide adduct 5 μM in enzyme, 0.2 mM in NaHS; (dashed trace) partially reduced sulfide adduct plus NO, 5 μM in enzyme, 0.2 mM in NaHS, 1.9 mM (1.0 atm) NO; (solid trace) partially reduced sulfide adduct plus NO, 5 mM in enzyme, 0.2 mM in NaHS, 1.9 mM (1.0 atm) NO, plus ~1 mM in Na₂S₂O₄. (A) Soret region 380–470 nm. (B) Q (or α) band region 570–650 nm.

Figure 6

Resistance of bovine pulmonary artery endothelial cells (BPAEC) to sulfide toxicity is increased in the presence of sodium nitrite. Effect of NaNO₂ on BPAEC and BPAEC treated with NaHS. BPAEC were plated and then covered with Parafilm just prior to experiments. Aqueous solutions (in media) of both NaNO₂ (1 mM) and NaHS (5 mM) were injected through the Parafilm into the cell media of wells while shaking the plates gently. Plates were incubated at 37 °C for 1 h and then treated with both SYBR Green and propidium iodide dyes. Cell counts were taken using a Zeiss IM 35 fluorescent microscope and an Infinity 2 camera with the Infinity Analyze software.