65, 18 –25 (2002)
Copyright © 2002 by the Society of Toxicology
TOXICOLOGICAL SCIENCES
Cytochrome Oxidase Inhibition Induced by Acute Hydrogen Sulfide
Inhalation: Correlation with Tissue Sulfide Concentrations in the Rat
Brain, Liver, Lung, and Nasal Epithelium
David C. Dorman, 1 Frederic J.-M. Moulin, 2 Brian E. McManus, Kristen C. Mahle, R. Arden James, and Melanie F. Struve
CIIT Centers for Health Research, 6 Davis Drive, PO Box 12137, Research Triangle Park, North Carolina 27709-2137
Received June 28, 2001; accepted October 4, 2001
gas, petroleum, volcanic, and sulfur-spring emissions. Hydrogen sulfide is associated with more than 70 types of industries,
including artificial fiber synthesis, food production, paper and
pulp manufacture, roofing, sewage treatment, and swine containment (Donham et al., 1982; Hall and Rumack, 1997; Hoidal et al., 1986; Jaakkola et al., 1990; Watt et al., 1997). Lethal
human exposure to H 2S is usually associated with individuals
working within heavily contaminated confined spaces (e.g.,
sewers, manure pits) and with the oil and sour gas industries
(Arnold et al., 1985; Guidotti, 1994; Kilburn, 1993). The toxic
effects of sublethal doses have been much less characterized. A
recent review of the adverse health effects from H 2S exposure
is available (ATSDR, 1999).
The primary mechanism for the toxic action of H 2S is direct
inhibition of cytochrome oxidase, an enzyme critical for mitochondrial respiration (Khan et al., 1990; Nicholls and Kim,
1982). Tissues with high oxygen demand (e.g., brain and heart)
are especially sensitive to disruption of oxidative metabolism
by H 2S (Ammann, 1986). Human exposure to H 2S results in
concentration-dependent toxicity in the respiratory, cardiovascular, and nervous systems. Acute human exposure to relatively low concentrations (ⱕ 50 ppm) of H 2S results in ocular
and respiratory mucous membrane irritation leading to nasal
congestion, pulmonary edema, and a syndrome known as gas
eye, which is characterized by corneal inflammation (ATSDR,
1999; Reiffenstein et al., 1992). Despite the strong characteristic odor associated with H 2S, many exposed individuals are
unaware of its presence because their sense of smell is severely
impaired following exposure to ⱖ 150 ppm H 2S. Acute human
exposure to high concentrations of H 2S (e.g., ⱖ 500 ppm)
results in a rapid onset of respiratory paralysis and unconsciousness that can result in death within minutes (Beauchamp
et al., 1984). Persistent sequelae of H 2S poisoning are often
related to the olfactory system and may include hyposmia,
dysosmia, and phantosmia (Hirsch and Zavala, 1999; Kilburn,
1997).
Animal studies confirm that the olfactory system is especially sensitive to H 2S inhalation. Acute exposure of rats to
moderately high concentrations of H 2S (ⱖ 80 ppm) resulted in
regeneration of the nasal respiratory mucosa and full thickness
Hydrogen sulfide (H 2S) is an important brain, lung, and nose
toxicant. Inhibition of cytochrome oxidase is the primary biochemical effect associated with lethal H 2S exposure. The objective of
this study was to evaluate the relationship between the concentration of sulfide and cytochrome oxidase activity in target tissues
following acute exposure to sublethal concentrations of inhaled
H 2S. Hindbrain, lung, liver, and nasal (olfactory and respiratory
epithelial) cytochrome oxidase activity and sulfide concentrations
were determined in adult male CD rats immediately after a 3-h
exposure to H 2S (10, 30, 80, 200, and 400 ppm). We also determined lung sulfide and sulfide metabolite concentrations at 0, 1.5,
3, 3.25, 3.5, 4, 5, and 7 h after the start of a 3-h H 2S exposure to
400 ppm. Lung sulfide concentrations increased during H 2S exposure and rapidly returned to endogenous levels within 15 min after
the cessation of the 400-ppm exposure. Lung sulfide metabolite
concentrations were transiently increased immediately after the
end of the 3-h H 2S exposure. Decreased cytochrome oxidase activity was observed in the olfactory epithelium following exposure
to > 30 ppm H 2S. Increased olfactory epithelial sulfide concentrations were observed following exposure to 400 ppm H 2S. Hindbrain and nasal respiratory epithelial sulfide concentrations were
unaffected by acute H 2S exposure. Nasal respiratory epithelial
cytochrome oxidase activity was reduced following acute exposure
to > 30 ppm H 2S. Liver sulfide concentrations were increased
following exposure to > 200 ppm H 2S and cytochrome oxidase
activity was increased following inhalation exposure to > 10 ppm
H 2S. Our results suggest that cytochrome oxidase inhibition is a
sensitive biomarker of H 2S exposure in target tissues, and sulfide
concentrations are unlikely to increase postexposure in the brain,
lung, or nose following a single 3-h exposure to < 30 ppm H 2S.
Key Words: hydrogen sulfide; pharmacokinetics; cytochrome
oxidase; nasal toxicity; rat; inhalation.
Hydrogen sulfide (H 2S) is a colorless gas with a characteristic rotten-egg odor. In nature, H 2S is produced primarily by
the decomposition of organic matter and is found in natural
1
To whom correspondence should be addressed. Fax: (919) 558-1300.
E-mail: [email protected].
2
Present address: Bristol-Myers Squibb Pharmaceutical Research Institute,
PO Box 5400, Princeton, NJ 08543-5400.
18
HYDROGEN SULFIDE TOXICOKINETICS AND TOXICODYNAMICS
19
chrome oxidase inhibition could account for the increased
sensitivity of the rodent olfactory epithelium to H 2S inhalation.
MATERIALS AND METHODS
Chemicals. Unless otherwise indicated, all chemical reagents were purchased from Sigma Chemical Co. (St. Louis, MO). Purified cytochrome c
oxidase (from bovine heart) was purchased from Worthington Biochemical
Corporation (Lakewood, NJ).
FIG. 1. Schematic representation of the metabolism of H 2S (adapted from
Beauchamp et al., 1984).
necrosis of the olfactory mucosa (Brenneman et al., 2001;
Lopez et al., 1988b). Subchronic exposure of rats to 30 or 80
ppm H 2S resulted in nasal pathology that was limited to the
olfactory epithelium (Brenneman et al., 2000a). The olfactory
mucosal lesions observed in rats following H 2S inhalation are
not unique. For example, similar patterns of nasal injury were
observed following exposure to chlorine, dimethylamine, and
other irritant gases (Buckley et al., 1985; Jiang et al., 1983;
Morgan, 1991). Comparable olfactory lesions were also observed following parenteral administration of iminodipropionitrile, thus suggesting that systemic delivery may play a role
in the nasal toxicity induced by certain chemicals (Genter et
al., 1995). Our laboratory recently demonstrated that systemic
delivery of H 2S was not an important consideration for this
nasal toxicant (Brenneman et al., 2000b). Regional differences
in H 2S delivery or uptake, local clearance processes, or differences in cellular sensitivity to the gas more likely contribute to
the site-specific distribution of H 2S-induced lesions within the
rat nose (Morgan and Monticello, 1990).
Hydrogen sulfide metabolism occurs through three pathways: oxidation, methylation, and reaction with cytochrome c
and other metallo- or disulfide-containing proteins
(Beauchamp et al., 1984). The major metabolic pathway for
H 2S is the rapid multistep hepatic oxidation of sulfide to sulfate
(Fig. 1) and subsequent elimination of sulfate in the urine
(Bartholomew et al., 1980; Beauchamp et al., 1984). Several
investigators have examined the toxicokinetics of H 2S following inhalation. Kage et al. (1992) reported elevated blood and
urinary thiosulfate concentrations in rabbits exposed to 100 –
200 ppm H 2S for 60 min. Kangas and Savolainen (1987)
likewise reported elevated urinary thiosulfate levels in human
volunteers exposed to 8, 18, or 30 ppm H 2S for 30 – 45 min.
Few studies have examined sulfide concentrations in lung,
brain, or other target tissues following H 2S inhalation.
The objective of this study was to evaluate the relationship
between the concentration of sulfide and cytochrome oxidase
activity in target tissues following acute exposure to sublethal
concentrations of inhaled H 2S. We also determined whether
increased sulfide deposition or enhanced sensitivity to cyto-
Animals. A total of 266 9- to 10-week old male CD rats were obtained
from Charles River Laboratories, Inc. (Raleigh, NC) and maintained in the
CIIT animal facility, which is accredited by the Association for Assessment
and Accreditation of Laboratory Animal Care. Animals were individually
housed in polycarbonate cages with cellulose fiber chip bedding (ALPHAdri™, Shepherd Specialty Papers, Kalamazoo, MI) and provided NIH-07
pelleted rodent chow (Zeigler Brothers, Gardners, PA) and deionized filtered
tap water ad libitum except during inhalation exposures. Animal rooms were
ventilated with HEPA-filtered air and maintained at 18.5–21.5°C and 40 –70%
humidity on a 12-h light– dark cycle. The study was conducted according to
federal guidelines for the care and use of laboratory animals (NRC, 1996) and
under the supervision of the CIIT Institutional Animal Care and Use Committee.
Experimental design. Our first objective was to evaluate the relationship
between the concentration of sulfide and cytochrome oxidase activity in known
target tissues (i.e., lung and hindbrain) following acute exposure to inhaled
H 2S. In this experiment, animals (n ⫽ 6 rats/exposure concentration) were
exposed to 0, 10, 30, 80, 200, or 400 ppm H 2S for 3 h. End-of-exposure sulfide
concentrations and cytochrome oxidase activity were determined in hindbrain,
lung, and liver samples from rats exposed to 200 or 400 ppm H 2S. Tissue
samples from animals in other H 2S exposure groups were evaluated for sulfide
concentration or cytochrome oxidase activity if the end-of-exposure value for
the end point in the 200-ppm H 2S exposure group was significantly different
from that observed in an unexposed (control) group. We were also interested
in evaluating the time course of the sulfide concentration in a target tissue. This
evaluation was performed on lung tissues, as it was the only target organ that
demonstrated a clear dose-response relationship between tissue sulfide concentration and cytochrome oxidase inhibition. Lung sulfide concentration was
determined at 0, 1.5, 3, 3.25, 3.5, 4, 5, and 7 h after the start of a 3-h exposure
(n ⫽ 6 rats/exposure concentration/time point) to 400 ppm H 2S. Rats from this
first experiment were killed by CO 2 asphyxiation followed by abdominal
exsanguination.
Our second objective was to evaluate whether increased sulfide deposition
or enhanced sensitivity to cytochrome oxidase inhibition could account for the
increased sensitivity of the rodent olfactory epithelium to H 2S inhalation. In
this experiment, rats (n ⫽ 6 rats/exposure concentration/time point) were
exposed to 0, 30, 80, 200, or 400 ppm for 3 h/day for either 1 day or 5
consecutive days. Immediately following the end of the H 2S exposure, rats
were killed by decapitation, and the head was sectioned sagitally on the bridge
of the nose. The nasal respiratory and olfactory mucosa were removed with the
supporting ethmoturbinates and flash-frozen in liquid nitrogen. These samples
were used for tissue sulfide evaluations (single exposure only) or cytochrome
oxidase activity (1- and 5-day exposures).
We were also interested in determining whether subchronic H 2S exposure
could result in altered lung or hindbrain sulfide concentrations or cytochrome
oxidase activity. In order to assess this possibility, we evaluated lung and
hindbrain samples for sulfide concentration and cytochrome oxidase activity
from male rats exposed for 6 h/day for 70 consecutive days to 0, 10, 30, or 80
ppm H 2S. These animals had been used in a reproductive and developmental
toxicity study (Dorman et al., 2000) and were subsequently evaluated for
H 2S-induced nasal pathology (Brenneman et al., 2000a). Samples were collected immediately after the end of the last H 2S exposure and flash-frozen in
liquid nitrogen within several minutes after euthanasia.
20
DORMAN ET AL.
H 2S exposure. Methods used to generate and characterize the H 2S exposure atmospheres have been previously described (Struve et al., 2001). Briefly,
gas cylinders containing 5% (50,000 ppm) H 2S in nitrogen were purchased
from Holox Gases (Cary, NC). Nose-only exposures were conducted using rat
nose-only tubes and a nose-only system with 52 exposure ports (Cannon et al.,
1983). Total air flow in the nose-only units was adjusted to provide approximately 0.5 l/min per animal port. Hydrogen sulfide was metered through mass
flow controllers (MKS Instruments, Andover, MA) and mixed with the noseonly unit air supply to provide the desired target H 2S concentration. Exposure
H 2S concentrations were determined by gas chromatography-FPD (HewlettPackard model 6890 with a GS-Q 30 meter ⫻ 0.53 ␮m Alltech column) at least
six times during each 3-h exposure. The generation system was operated by the
Andover Infinity control system (Andover Controls Corporation, Andover,
MA). Animals were exposed to 0, 10, 30, 80, 200, or 400 ppm H 2S for 3 h.
Cytochrome oxidase activity. Cytochrome oxidase activity was evaluated
by determining the rate of oxidation of reduced ferricytochrome c using
methods described by Weyant et al. (1988). Bovine-derived ferricytochrome c
was initially dissolved (10 mg/ml) in a 0.01 M sodium phosphate buffer (pH ⫽
7.0) and then reduced by the addition of ascorbic acid (2.4 mg/ml) for 24 h.
Excess ascorbate was removed by equilibrium dialysis in a 0.01 M sodium
phosphate buffer (pH ⫽ 7.0) using 3,500 molecular weight cutoff tubing
(Spectrum Medical Industries, Los Angeles, CA). Three changes of buffer
were performed over a 24-h period. The assay reagent contained 0.7 ml (7 mg)
reduced cytochrome c, 1 ml sodium phosphate buffer (pH ⫽ 7.0), and 8.3 ml
distilled water. The degree of reduction of the final assay mix was measured
using a Beckman DV 650 UV/VIS spectrophotometer (Fullerton, CA), and the
assay reagent was considered fully reduced if the A 550/A 565 ratio was greater
than 6.5, as specified by the product insert from Worthington Biochemical
Corporation (Lakewood, NJ).
The following modifications were made to the methods described by Weyant et al. (1988) to accommodate the use of a COBAS FARA II analyzer
(Roche Diagnostic System, Somerville, NJ). Representative 50- to 200-mg
tissue samples were diluted 10-fold with a 0.25 M sucrose buffer and homogenized using an ultrasonic sonifier. Tissue homogenates were centrifuged
(3000 ⫻ g for 10 min at 4°C). The supernatant was removed and added to an
equivalent amount of 0.25 M sucrose buffer. The sample was then recentrifuged (3000 ⫻ g for 10 min) and the resulting supernatant used for the
cytochrome oxidase assay. Enzyme activity was measured by monitoring the
oxidation of reduced cytochrome c at 550 nm. Absorbance readings were taken
following a 10-s incubation time and at 5-s intervals for 90 s. Total protein
concentration within each sample was analyzed with the COBAS FARA II
spectrophotometer using commercially available reagents (Roche Diagnostic
System, Somerville, NJ).
Determination of tissue sulfide concentrations. Hindbrain, liver, lung,
and nasal epithelium samples from control rats and animals exposed to H 2S
were evaluated for sulfide content. Tissue samples (50 –150 mg) were sectioned directly from frozen tissues, weighed, and placed into a clear glass
crimp-top vial with molded conical bottom (Sun International, Wilmington,
NC). The vials were sealed using a teflon septum, and 1 ␮l tetraethylammonium hydroxide (TEAH, 35% aqueous solution, SACHEM, Austin, TX) per
milligram of sample was added to the vial to digest the sample. The TEAH was
added to the vial with a gastight syringe (Hamilton, Reno, NV) in order to
minimize loss of H 2S due to volatilization. Samples were centrifuged for 5 min
at 3000 ⫻ g (4°C) and were then kept at room temperature for 24 h to complete
the sample digestion. After 24 h, 8 ␮l of 28 mM NaOH per milligram sample
were injected into the vial, and the samples were centrifuged again at 3000 ⫻
g for 5 min. A 100-␮l sample of the supernatant was then added to 400 ␮l of
28 mM NaOH in a polypropylene ConSert vial (Sun International, Wilmington, NC) to complete a 50-fold dilution of the tissue sample. The diluted
supernatant sample was then injected into the liquid chromatography system.
Sulfide and its metabolites were separated by high-performance liquid
chromatography (HPLC) using methods adapted from Mitchell et al. (1993)
and Rocklin and Johnson (1983). The liquid chromatogram consisted of a
Model 580 dual-piston solvent delivery module (ESA Inc., Chelmsford, MA),
a pulse-dampener, a Waters 717 plus refrigerated autosampler (Millipore
Corporation, Milford, MA), and an IONPAC威 AS15 analytical column (4 ⫻
250 mm, Dionex Corporation, Sunnyvale, CA) with an IONPAC威 AG15 guard
column. Sulfide was detected using a Coulochem II electrochemical detector
(ESA Inc., Chelmsford, MA) equipped with a model 5020 guard cell and a
model 5040 amperometric analytical cell with silver target. The applied potentials of the guard cell and analytical cell were 584 and 50 mV, respectively.
The output range of the ESA 5040 analytical cell was set at 20 nA/V.
Lung sulfide metabolites were detected by a Dionex CD20 conductivity
detector (Dionex Corporation, Sunnyvale, CA). The conductivity detector was
equipped with an anion self-regenerating suppressor (ASRS-Ultra 4 mm,
Dionex Corporation) in recycle mode. The range of the conductivity detector
was 3 ␮s, and the anion self-regenerating suppressor was set at 300 mA. The
data were acquired and integrated by a Baseline 810 chromatography workstation (Waters, Millipore Corporation, Milford, MA).
Analytical-grade reagents were used, and all standards and eluents were
prepared using distilled, deionized water with a specific resistance of 17.8
megohm-cm. Sulfide, sulfite, sulfate, and thiosulfate were eluted isocratically
at a flow rate of 1.5 ml/min using helium-degassed 28 mM NaOH as the mobile
phase. Under these conditions, elution times for sulfide, sulfite, sulfate, and
thiosulfate were 4, 8, 10, and 35 min, respectively. Tissue concentrations were
determined from the linear regression (r 2 ⫽ 0.95) of a calibration curve based
on aqueous samples within a range of 5–50 ppb for sulfide and 1–10 ppm for
sulfite, sulfate, and thiosulfate. The assay detection limit for sulfide was 1
ng/ml, corresponding to 0.05 ␮g/g tissue. Recovery rates for sulfide, sulfite,
sulfate, and thiosulfate were 88 ⫾ 5%, 85 ⫾ 5%, 71 ⫾ 10%, and 102 ⫾ 8%,
respectively, based on recovery rates obtained from control rat liver samples
(n ⫽ 3–5) spiked with known amounts of each analyte of interest.
Statistics. Unless otherwise noted, data are reported as means ⫾ SEM. All
statistical analyses were performed using a standard statistical package (JMP,
SAS Institute Inc., Cary, NC). Time-course and dose-response data were
evaluated by one-way analysis of variance (ANOVA) followed by a comparison with the preexposure (control) group using Dunnett’s test. The distribution
of all data was tested for normality using the Shapiro-Wilk test before analysis.
Linear regression correlations were performed according to standard statistical
procedures and tested by analysis of variance. For all tests, a p value of 0.05
or less was considered significant.
RESULTS
Tissue Sulfide Concentrations following H 2S Exposure
Sulfide was observed in all control (preexposure) tissue
samples. Mean lung and liver sulfide concentrations in control
animals were 0.54 ⫾ 0.03 and 0.55 ⫾ 0.11 ␮g/g, respectively.
As expected, lung sulfide concentrations increased during exposure to 400 ppm H 2S, reaching peak concentrations at the
end of the 3-h H 2S exposure (Fig. 2). Lung sulfide concentrations rapidly decreased to preexposure levels within 15 min
after the end of the H 2S exposure (Fig. 2). Significantly increased end-of-exposure lung sulfide concentrations were observed following exposure to ⱖ 80 ppm H 2S (Table 1). A
significant positive nonlinear relationship between the H 2S
exposure concentration and the end-of-exposure lung sulfide
concentration was observed. Significantly increased end-ofexposure liver sulfide concentrations were also observed following exposure to ⱖ 200 ppm H 2S (Table 1). End-of-exposure hindbrain sulfide concentrations were unaffected by H 2S
exposure (Table 2). Although end-of-exposure nasal respiratory epithelium sulfide concentrations were elevated following
21
HYDROGEN SULFIDE TOXICOKINETICS AND TOXICODYNAMICS
TABLE 2
Mean (ⴞ SEM) Hindbrain and Nasal Epithelium Sulfide Concentrations Immediately After the End of a Single 3-Hr H 2S
Exposure and Cytochrome Oxidase Activity Following One or
Five 3-Hour H 2S Exposures
Five
exposures
Single exposure
H 2S exposure
FIG. 2. Mean (⫾ SEM) lung sulfide concentrations before, during, and
after a 3-h inhalation exposure to 400 ppm H 2S (n ⫽ 6 rats/time point).
*Indicates statistically different from preexposure control values (p ⱕ 0.05).
exposure to 400 ppm H 2S (Table 2), the observed increase was
not statistically significant (p ⫽ 0.065). In contrast, end-ofexposure olfactory epithelium sulfide concentrations were significantly increased following exposure to 400 ppm H 2S (Table
2). Neither lung nor hindbrain sulfide concentration was increased following subchronic exposure to 80 ppm H 2S (Table 3).
Hindbrain
0
200
400
Respiratory epithelium
0
30
80
200
400
Olfactory epithelium
0
30
80
200
400
Sulfide
concentration
Cytochrome
oxidase
Cytochrome
oxidase
1.21 ⫾ 0.05
1.12 ⫾ 0.05
1.14 ⫾ 0.04
2.14 ⫾ 0.12
2.55 ⫾ 0.26
2.28 ⫾ 0.31
1.73 ⫾ 0.14
ND
ND
1.37 ⫾ 0.11
2.73 ⫾ 0.77
1.23 ⫾ 0.05
0.60 ⫾ 0.08*
0.50 ⫾ 0.08*
0.92 ⫾ 0.02*
0.94 ⫾ 0.02*
1.02 ⫾ 0.17
0.83 ⫾ 0.11
0.60 ⫾ 0.19
0.74 ⫾ 0.07
0.86 ⫾ 0.19
1.42 ⫾ 0.11
ND
ND
1.25 ⫾ 0.06
2.07 ⫾ 0.33*
1.20 ⫾ 0.06
1.01 ⫾ 0.07*
0.99 ⫾ 0.07*
0.92 ⫾ 0.03*
0.92 ⫾ 0.04*
1.13 ⫾ 0.09
0.75 ⫾ 0.13*
0.84 ⫾ 0.11*
0.70 ⫾ 0.12*
0.51 ⫾ 0.01*
ND
ND
ND
Note. H 2S exposure is given in ppm, sulfide concentration in ␮g/g, cytochrome oxidase in U/mg protein. ND, not determined.
*Indicates statistically different from control values (p ⱕ 0.05).
Lung Sulfite, Sulfate, and Thiosulfate Concentrations
Mean preexposure lung sulfite, sulfate, and thiosulfate concentrations were 359 ⫾ 18, 140 ⫾ 7, and 505 ⫾ 27 ␮g/g,
TABLE 1
Mean (ⴞ SEM) Lung and Liver Sulfide Concentrations and
Cytochrome Oxidase Activity Immediately After the End of a
3-Hour H 2S Exposure
H 2S exposure
Lung
0
10
30
80
200
400
Liver
0
10
30
80
200
400
Sulfide concentration
Cytochrome oxidase
0.54 ⫾ 0.03
0.43 ⫾ 0.03
0.64 ⫾ 0.05
0.78 ⫾ 0.04*
0.97 ⫾ 0.02*
0.88 ⫾ 0.10*
1.76 ⫾ 0.02
1.65 ⫾ 0.06
1.56 ⫾ 0.06*
1.46 ⫾ 0.04*
1.43 ⫾ 0.06*
1.16 ⫾ 0.04*
0.55 ⫾ 0.11
1.11 ⫾ 0.22
0.74 ⫾ 0.08
0.86 ⫾ 0.06
1.55 ⫾ 0.20*
1.55 ⫾ 0.42*
2.06 ⫾ 0.10
2.74 ⫾ 0.10*
3.04 ⫾ 0.11*
2.79 ⫾ 0.23*
2.85 ⫾ 0.19*
2.87 ⫾ 0.12*
Note. H 2S exposure is given in ppm, sulfide concentration in ␮g/g, cytochrome oxidase in U/mg protein.
*Indicates statistically different from control values (p ⱕ 0.05).
respectively. Significantly increased lung sulfite, sulfate, and
thiosulfate concentrations were observed in rats exposed to 400
ppm H 2S and occurred 15 min after the end of the 3-h H 2S
exposure (Fig. 3). Lung sulfite, sulfate, and thiosulfate concen-
TABLE 3
Mean (ⴞ SEM) Hindbrain and Lung Sulfide Concentrations
Immediately After the End of a Subchronic (70-day) H 2S Exposure
H 2S exposure (ppm)
Hindbrain
0
10
30
80
Lung
0
10
30
80
Sulfide
concentration (␮g/g)
Cytochrome oxidase
(U/mg protein)
2.89 ⫾ 0.21
2.80 ⫾ 0.26
2.72 ⫾ 0.30
2.98 ⫾ 0.22
2.48 ⫾ 0.23
2.21 ⫾ 0.14
2.17 ⫾ 0.04
2.12 ⫾ 0.02
1.01 ⫾ 0.14
0.98 ⫾ 0.12
0.96 ⫾ 0.12
0.90 ⫾ 0.15
1.04 ⫾ 0.04
1.05 ⫾ 0.03
0.95 ⫾ 0.02
0.87 ⫾ 0.02*
Note. H 2S exposure is given in ppm, sulfide concentration in ␮g/g, cytochrome oxidase in U/mg protein.
*Significantly different from control value (p ⱕ 0.05).
22
DORMAN ET AL.
FIG. 3. Mean (⫾ SEM) lung sulfite, thiosulfate, and sulfate concentrations
before, during, and after a 3-h inhalation exposure to 400 ppm H 2S (n ⫽ 6
rats/time point). *Indicates statistically increased lung sulfite, thiosulfate, and
sulfate concentrations when compared with preexposure (control) values (p ⱕ
0.05).
trations rapidly decreased to preexposure levels within minutes
after this transient increase (Fig. 3).
Cytochrome Oxidase Activity
Decreased lung cytochrome oxidase activity was observed
following exposure to ⱖ 30 ppm H 2S (Table 1). End-ofexposure lung cytochrome oxidase activity was inversely linearly correlated (r 2 ⫽ 0.65) with lung sulfide concentration
(p ⬍ 0.0001, ANOVA). Liver cytochrome oxidase activity was
significantly increased to approximately 130 –168% of preexposure levels in all H 2S-exposed animals (Table 1). Hindbrain
cytochrome oxidase activity was unaffected by H 2S inhalation
(Table 2). Decreased olfactory and nasal respiratory epithelium
cytochrome oxidase activities were observed following a single
3-h exposure to ⱖ 30 ppm H 2S (Table 2). Repeated (5-day)
exposure to H 2S also resulted in significant cytochrome oxidase inhibition in the olfactory epithelium (Table 2). Repeated
(5-day) exposure to H 2S did not affect cytochrome oxidase
activity in the nasal respiratory nasal epithelium (Table 2).
Subchronic exposure to 80 ppm H 2S resulted in reduced cytochrome oxidase activity in the lung but not the hindbrain
(Table 3).
DISCUSSION
This study examined the toxicokinetics of H 2S in rats following acute exposure to sublethal concentrations of the gas.
The highest H 2S exposure concentration used in our study (400
ppm) occurs with acute accidental human poisonings and is
associated with olfactory paralysis in humans and olfactory
epithelial necrosis in laboratory animals (ATSDR, 1999; Brenneman et al., 2001; Lopez et al., 1988b). The lowest exposure
concentration used in our study (10 ppm) equals the current
TLV-TWA recommended by the American Conference of
Governmental Industrial Hygienists. In contrast, ambient atmospheric H 2S concentrations range from 0.01 to 50 ppb,
depending on the proximity of the sampling site to tidal flats,
marshes, anaerobic soils, and other environmental sources of
H 2S (Warneck, 1988; Graedel et al., 1986).
Hydrogen sulfide is normally present in mammalian tissues,
and some evidence suggests that it is required for certain types
of nerve transmission (Kimura, 2000). Literature values for
endogenous levels of sulfide are variable and depend on the
procedures used to extract the sulfide from the tissue and the
analytical chemical methods used to quantify this metabolite
(Goodwin et al., 1989; Kage et al., 1988; Mitchell et al., 1993).
Special care must be taken to minimize the loss of free sulfide
from the tissue sample due to the volatility of this gas. We used
a strong base (TEAH) not only to digest our tissue samples, but
also to minimize evaporative losses of H 2S. Another advantage
of our analytical method is that the use of an amperometric
analytical cell and a conductivity detector allowed us to simultaneously detect and quantify sulfide and sulfide metabolites in
the same sample of tissue. Endogenous tissue sulfide concentrations determined with our analytical methods are similar to
those reported in the literature. For example, brain sulfide
concentrations observed in naive rats (1.21 ⫾ 0.05 ␮g/g) from
our acute study are comparable to values (1.94 ⫾ 0.24 ␮g/g)
reported by Warenycia and coworkers (1990), although the
results from our 70-day exposure are somewhat higher. Background lung tissue sulfate concentrations observed in our study
(140 ⫾ 6.5 ␮g/g) were approximately 2.5-fold higher than
those observed by Rozman and coworkers (1992).
Our interest in the lung was stimulated by possible portalof-entry effects associated with H 2S inhalation and the known
relationship between H 2S inhalation and pulmonary edema and
fibrinocellular alveolitis (Lopez et al., 1988a). As expected, we
observed that end-of-exposure lung sulfide concentrations were
highly correlated with the amount of H 2S in the exposure
atmosphere, and elevated lung sulfide concentrations were
observed following a single 3-h H 2S exposure to ⱖ 80 ppm.
End-of-exposure lung sulfide concentrations were not increased in rats exposed subchronically to ⱕ 80 ppm. In contrast, decreased cytochrome oxidase activity was observed in
the lung after a 3-h exposure to ⱖ 30 ppm H 2S as well as
following subchronic exposure to 80 ppm H 2S. This finding
indicates that cytochrome oxidase inhibition is a more sensitive
biomarker of H 2S exposure than tissue sulfide concentrations.
Lung sulfide concentrations rapidly returned to preexposure
levels within minutes after the end of a 3-h exposure to 400
ppm H 2S, suggesting that rapid pulmonary elimination or metabolism of sulfide occurs. An accumulation of sulfide metabolites was not observed in the lung during the 3-h H 2S exposure. Transient increases in lung sulfite, sulfate, and thiosulfate
concentrations were observed, however, immediately after the
end of the 400-ppm H 2S exposure. This increase in sulfide
metabolite concentrations occurred coincidentally with the
rapid decrease in lung sulfide concentration. This observation
HYDROGEN SULFIDE TOXICOKINETICS AND TOXICODYNAMICS
suggests that the detoxification of sulfide to sulfate may becomes less effective as the concentration of sulfide increases in
blood and other tissues due to H 2S exposure (Fischer et al.,
2000). A similar pharmacokinetic pattern has been observed in
mice exposed to benzene, where competitive inhibition of an
intermediate metabolite (phenol) occurs and formation of another metabolite (hydroquinone) is delayed until after the benzene inhalation ends (Medinsky et al., 1996; Rickert et al.,
1979). Additional studies will be required to confirm our
hypothesis that competitive inhibition of sulfide metabolism
occurs during H 2S inhalation.
We also observed increased sulfide concentrations and cytochrome oxidase inhibition in the upper respiratory tract from
H 2S-exposed rats. Although our data showed that olfactory, but
not respiratory, epithelial sulfide concentrations were significantly elevated following exposure to 400 ppm H 2S, the observed differences in end-of-exposure tissue sulfide concentrations are unlikely to be toxicologically significant. For
example, end-of-exposure olfactory epithelium sulfide concentrations following a 3-h exposure to 400 ppm H 2S were 146%
of levels observed in unexposed animals, whereas end-ofexposure respiratory epithelium sulfide concentrations observed in the same animals were 158% of control levels.
Cytochrome oxidase activity was significantly decreased
within the olfactory and respiratory nasal epithelium immediately after a single 3-h exposure to ⱖ 30 ppm H 2S. Olfactory
cytochrome oxidase activity was also significantly decreased to
45– 66% of control levels following 5 consecutive days of
exposure to ⱖ 30 ppm H 2S. Repeated (5-day) H 2S exposure
did not change cytochrome oxidase activity in the respiratory
nasal epithelium. This observation is consistent with our recent
studies that showed that regeneration of the nasal respiratory
mucosa occurs rapidly during this 5-day period, whereas necrosis of the olfactory mucosa increased in severity (Brenneman et al., 2001). Our data suggest that the regenerated respiratory epithelium becomes resistant to H 2S-induced
cytochrome oxidase inhibition. It should be noted that our
dose-response data for cytochrome oxidase activity in the nasal
tissue demonstrates some inconsistencies. For example, H 2Sinduced inhibition of nasal respiratory epithelial cytochrome
oxidase activity was greater in rats exposed to either 30 or 80
ppm than in rats acutely exposed to ⱖ 200 ppm H 2S. This
unusual dose-response relationship may indicate that some
compensatory changes are occurring in the epithelium in response to H 2S exposure. Furthermore, regional differences in
sulfide delivery within the olfactory epithelium are correlated
with the development of nasal pathology at this site (Moulin et
al., 2001). Our sampling procedure does not allow us to detect
regional differences in H 2S delivery to the olfactory epithelium, as we pooled the entire olfactory epithelium into one
sample.
It is reasonable to question whether cytochrome oxidase
inhibition is a mode of action for H 2S-induced olfactory pathology. Our laboratory has shown that acute inhalation expo-
23
sure of male rats to 400 ppm H 2S results in severe mitochondrial swelling in degenerating olfactory neurons within the
olfactory epithelium (Brenneman et al., 2001). This ultrastructural lesion is consistent with, but not specific for, H 2S-induced
anoxic cell injury due to cytochrome oxidase inhibition. These
data provide strong evidence that cytochrome oxidase inhibition may indeed play a critical role in H 2S-induced olfactory
pathology. When considered together, our data suggest that the
olfactory neuroepithelium is intrinsically more sensitive than
the nasal respiratory epithelium to H 2S-induced cytochrome
oxidase inhibition. This result is not unexpected, as neurons are
known to be exquisitely sensitive to chemical-induced hypoxic
damage (Nicklas et al., 1992).
We did not observe increased hindbrain sulfide concentrations in acutely or subchronically H 2S-exposed animals. Warenycia et al. (1989) showed that accumulation of sulfide occurred in the hindbrain of male Sprague-Dawley rats exposed
to lethal quantities of sodium hydrosulfide (NaHS), an alkaline
salt that liberates H 2S in vivo. Our inability to detect an
increase in hindbrain sulfide concentrations probably reflected
the lower (sublethal) doses of H 2S used in our study. We also
did not observe altered brain cytochrome oxidase activity in
H 2S-exposed animals. Savolainen and coworkers (1980) likewise showed that a single 2-h exposure of mice to 100 ppm
H 2S did not result in inhibition of brain cytochrome oxidase
activity. We observed increased liver sulfide concentrations in
rats exposed for 3 h to 200 ppm. Despite the presence of
elevated liver sulfide concentrations, we did not observe cytochrome oxidase inhibition in this tissue. Indeed, rats exposed
to ⱖ 10 ppm H 2S for 3 h had significantly elevated hepatic
cytochrome oxidase activity. For example, liver cytochrome
oxidase activity was 136% of preexposure levels following a
single 3-h exposure to 400 ppm H 2S. A similar observation was
noted by Khan and coworkers (1998), who also observed a
small increase in liver cytochrome oxidase activity (to 109% of
control values, not statistically significant) in rats subchronically exposed (8 h/day, 5 days/week, for 5 weeks) to 100 ppm
H 2S. The biological significance of this observation is unclear,
but it implies that respiration in the liver is not inhibited by H 2S
at these treatment concentrations, and possibly, that H 2S detoxification processes in this organ require additional energy
demands, as reflected by increased respiratory cytochrome
activity.
The results of our study provide important new information
defining the relationship between H 2S exposure concentration
and resulting sulfide concentrations and cytochrome oxidase
activities in the hindbrain, lung, and nose, each of which is a
critical target for H 2S-induced toxicity. Our results suggest that
acute exposure to low concentrations (ⱖ 30 ppm) of H 2S is
associated with cytochrome oxidase inhibition in the lung and
nose. Inhibition of cytochrome oxidase often occurred in the
absence of elevated tissue sulfide concentration. These data
suggest that cytochrome oxidase inhibition is a more sensitive
biomarker of H 2S exposure than is tissue sulfide concentration.
24
DORMAN ET AL.
It is not unexpected that measurement of total tissue sulfide
concentrations is a relatively insensitive biomarker of H 2S
exposure, as most tissues contain high endogenous levels of
sulfide and this metabolite is highly volatile when unbound.
More refined studies using radiolabeled H 2S with evaluation of
total and mitochondrial sulfide could better elucidate the doseresponse relationship between tissue sulfide concentration and
H 2S exposure. Despite the limitations in our experimental
design, our data should prove useful in the development of
biologically based dosimetry and pharmacodynamic models
for this chemical. The development of dosimetry based models
should improve the risk assessment for this important environmental contaminant.
ACKNOWLEDGMENTS
This study was funded in part by a grant from the American Petroleum
Institute (API). The authors thank Drs. Susan Borghoff, Jeffrey Everitt, Gregory Kedderis, and Barbara Kuyper for their reviews of the manuscript.
Dorman, D. C., Brenneman, K. A., Struve, M. F., Miller, K. L., James, R. A.,
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