Toxicol. Sci. 2002 Dorman 18 25

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Cytochrome Oxidase Inhibition Induced by Acute Hydrogen SulfideInhalation: 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

Hydrogen sulfide (H2S) is an important brain, lung, and nose

toxicant. Inhibition of cytochrome oxidase is the primary biochem-

ical effect associated with lethal H 2S exposure. The objective of

this study was to evaluate the relationship between the concentra-

tion of sulfide and cytochrome oxidase activity in target tissues

following acute exposure to sublethal concentrations of inhaledH2S. 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 H2S (10, 30, 80, 200, and 400 ppm). We also deter-

mined 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 H2S expo-

sure 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 H2S exposure. Decreased cytochrome oxidase ac-

tivity was observed in the olfactory epithelium following exposure

to > 30 ppm H 2S. Increased olfactory epithelial sulfide concen-

trations were observed following exposure to 400 ppm H2S. Hind-brain and nasal respiratory epithelial sulfide concentrations were

unaffected by acute H2S exposure. Nasal respiratory epithelial

cytochrome oxidase activity was reduced following acute exposure

to > 30 ppm H2S. Liver sulfide concentrations were increased

following exposure to > 200 ppm H2S and cytochrome oxidase

activity was increased following inhalation exposure to > 10 ppm

H2S. Our results suggest that cytochrome oxidase inhibition is a

sensitive biomarker of H2S 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 H2S.

Key Words: hydrogen sulfide; pharmacokinetics; cytochrome

oxidase; nasal toxicity; rat; inhalation.

Hydrogen sulfide (H2S) is a colorless gas with a character-

istic rotten-egg odor. In nature, H 2S is produced primarily by

the decomposition of organic matter and is found in natural

gas, petroleum, volcanic, and sulfur-spring emissions. Hydro-

gen 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 con-

tainment (Donham et al., 1982; Hall and Rumack, 1997; Hoi-

dalet al., 1986; Jaakkolaet al., 1990; Wattet al., 1997). Lethal

human exposure to H2S is usually associated with individuals

working within heavily contaminated confined spaces (e.g.,

sewers, manure pits) and with the oil and sour gas industries

(Arnoldet 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 mito-

chondrial 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 H2S (Ammann, 1986). Human exposure to H2S results inconcentration-dependent toxicity in the respiratory, cardiovas-

cular, and nervous systems. Acute human exposure to rela-

tively low concentrations ( 50 ppm) of H2S 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 character-

istic odor associated with H 2S, many exposed individuals are

unaware of its presence because their sense of smell is severely

impaired following exposure to 150 ppm H2S. Acute human

exposure to high concentrations of H2S (e.g., 500 ppm)

results in a rapid onset of respiratory paralysis and uncon-

sciousness that can result in death within minutes (Beauchampet al., 1984). Persistent sequelae of H2S 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 espe-

cially sensitive to H2S inhalation. Acute exposure of rats to

moderately high concentrations of H2S ( 80 ppm) resulted in

regeneration of the nasal respiratory mucosa and full thickness

1 To whom correspondence should be addressed. Fax: (919) 558-1300.

E-mail: [email protected] Present address: Bristol-Myers Squibb Pharmaceutical Research Institute,

PO Box 5400, Princeton, NJ 08543-5400.

TOXICOLOGICAL SCIENCES65, 1825 (2002)

Copyright 2002 by the Society of Toxicology

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H2S exposure. Methods used to generate and characterize the H2S expo-

sure 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 approxi-

mately 0.5 l/min per animal port. Hydrogen sulfide was metered through mass

flow controllers (MKS Instruments, Andover, MA) and mixed with the nose-

only unit air supply to provide the desired target H 2S concentration. ExposureH2S concentrations were determined by gas chromatography-FPD (Hewlett-

Packard 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 mldistilled 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/A565 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 Wey-

ant 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 homog-

enized using an ultrasonic sonifier. Tissue homogenates were centrifuged

(3000 g for 10 min at 4C). The supernatant was removed and added to an

equivalent amount of 0.25 M sucrose buffer. The sample was then recentri-

fuged (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 (50150 mg) were sec-

tioned 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 tetraethylammo-

nium 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 H2S due to volatilization. Samples were centrifuged for 5 min

at 3000

g(4C) and were then kept at room temperature for 24 h to completethe 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, Wilming-

ton, 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 AS15 analytical column (4

250 mm, Dionex Corporation, Sunnyvale, CA) with an IONPACAG15 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 po-

tentials 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 conductivitydetector (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 work-

station (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 (r2 0.95) of a calibration curve based

on aqueous samples within a range of 550 ppb for sulfide and 110 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 35) 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 compar-

ison with the preexposure (control) group using Dunnetts 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 H2S 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 ex-

posure to 400 ppm H 2S, reaching peak concentrations at the

end of the 3-h H 2S exposure (Fig. 2). Lung sulfide concentra-

tions rapidly decreased to preexposure levels within 15 min

after the end of the H2S exposure (Fig. 2). Significantly in-

creased end-of-exposure lung sulfide concentrations were ob-

served following exposure to 80 ppm H 2S (Table 1). Asignificant positive nonlinear relationship between the H2S

exposure concentration and the end-of-exposure lung sulfide

concentration was observed. Significantly increased end-of-

exposure liver sulfide concentrations were also observed fol-

lowing exposure to 200 ppm H 2S (Table 1). End-of-expo-

sure hindbrain sulfide concentrations were unaffected by H 2S

exposure (Table 2). Although end-of-exposure nasal respira-

tory epithelium sulfide concentrations were elevated following

20 DORMAN ET AL.


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exposure to 400 ppm H 2S (Table 2), the observed increase wasnot statistically significant (p 0.065). In contrast, end-of-

exposure olfactory epithelium sulfide concentrations were sig-

nificantly increased following exposure to 400 ppm H 2S (Table

2). Neither lung nor hindbrain sulfide concentration was in-

creased following subchronic exposure to 80 ppm H2S (Ta-

ble 3).

Lung Sulfite, Sulfate, and Thiosulfate Concentrations

Mean preexposure lung sulfite, sulfate, and thiosulfate con-

centrations were 359 18, 140 7, and 505 27 g/g,respectively. Significantly increased lung sulfite, sulfate, and

thiosulfate concentrations were observed in rats exposed to 400

ppm H2S and occurred 15 min after the end of the 3-h H 2S

exposure (Fig. 3). Lung sulfite, sulfate, and thiosulfate concen-

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 0.05).

TABLE 1

Mean ( SEM) Lung and Liver Sulfide Concentrations and

Cytochrome Oxidase Activity Immediately After the End of a

3-Hour H2S Exposure

H2S exposure Sulfide concentration Cytochrome oxida se

Lung

0 0.54 0.03 1.76 0.02

10 0.43 0.03 1.65 0.06

30 0.64 0.05 1.56 0.06*

80 0.78 0.04* 1.46 0.04*

200 0.97 0.02* 1.43 0.06*

400 0.88 0.10* 1.16 0.04*

Liver0 0.55 0.11 2.06 0.10

10 1.11 0.22 2.74 0.10*

30 0.74 0.08 3.04 0.11*

80 0.86 0.06 2.79 0.23*

200 1.55 0.20* 2.85 0.19*

400 1.55 0.42* 2.87 0.12*

Note. H2S exposure is given in ppm, sulfide concentration in g/g, cyto-

chrome oxidase in U/mg protein.

*Indicates statistically different from control values (p 0.05).

TABLE 2

Mean ( SEM) Hindbrain and Nasal Epithelium Sulfide Con-

centrations Immediately After the End of a Single 3-Hr H2S

Exposure and Cytochrome Oxidase Activity Following One or

Five 3-Hour H2S Exposures

H2S exposure

Single exposure

Five

exposures

Sulfide

concentration

Cytochrome

oxidase

Cytochrome

oxidase

Hindbrain

0 1.21 0.05 2.14 0.12 ND

200 1.12 0.05 2.55 0.26 ND

400 1.14 0.04 2.28 0.31 ND

Respiratory epithelium

0 1.73 0.14 1.23 0.05 1.02 0.17

30 ND 0.60 0.08* 0.83 0.11

80 ND 0.50 0.08* 0.60 0.19

200 1.37 0.11 0.92 0.02* 0.74 0.07

400 2.73 0.77 0.94 0.02* 0.86 0.19

Olfactory epithelium

0 1.42 0.11 1.20 0.06 1.13 0.0930 ND 1.01 0.07* 0.75 0.13*

80 ND 0.99 0.07* 0.84 0.11*

200 1.25 0.06 0.92 0.03* 0.70 0.12*

400 2.07 0.33* 0.92 0.04* 0.51 0.01*


chrome oxidase in U/mg protein. ND, not determined.

*Indicates statistically different from control values (p 0.05).

TABLE 3

Mean ( SEM) Hindbrain and Lung Sulfide Concentrations

Immediately After the End of a Subchronic (70-day) H 2S Expo-

sure

H2S exposure (ppm)

Sulfide

concentration (g/g)

Cytochrome oxidase

(U/mg protein)

Hindbrain

0 2.89 0.21 2.48 0.23

10 2.80 0.26 2.21 0.14

30 2.72

0.30 2.17

0.0480 2.98 0.22 2.12 0.02

Lung

0 1.01 0.14 1.04 0.04

10 0.98 0.12 1.05 0.03

30 0.96 0.12 0.95 0.02

80 0.90 0.15 0.87 0.02*


chrome oxidase in U/mg protein.

*Significantly different from control value (p 0.05).

21HYDROGEN SULFIDE TOXICOKINETICS AND TOXICODYNAMICS


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trations rapidly decreased to preexposure levels within minutesafter this transient increase (Fig. 3).

Cytochrome Oxidase Activity

Decreased lung cytochrome oxidase activity was observed

following exposure to 30 ppm H2S (Table 1). End-of-

exposure lung cytochrome oxidase activity was inversely lin-

early correlated (r2 0.65) with lung sulfide concentration

(p 0.0001, ANOVA). Liver cytochrome oxidase activity was

significantly increased to approximately 130168% of preex-

posure 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 epitheliumcytochrome oxidase activities were observed following a single

3-h exposure to 30 ppm H 2S (Table 2). Repeated (5-day)

exposure to H2S also resulted in significant cytochrome oxi-

dase inhibition in the olfactory epithelium (Table 2). Repeated

(5-day) exposure to H2S did not affect cytochrome oxidase

activity in the nasal respiratory nasal epithelium (Table 2).

Subchronic exposure to 80 ppm H 2S resulted in reduced cyto-

chrome oxidase activity in the lung but not the hindbrain

(Table 3).

DISCUSSION

This study examined the toxicokinetics of H2S in rats fol-lowing acute exposure to sublethal concentrations of the gas.

The highest H2S 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; Bren-

nemanet 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 at-

mospheric H2S 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

H2S (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 forendogenous 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

(Goodwinet al.,1989; Kageet al.,1988; Mitchellet 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 simul-

taneously detect and quantify sulfide and sulfide metabolites in

the same sample of tissue. Endogenous tissue sulfide concen-

trations 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. Back-

ground 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 portal-

of-entry effects associated with H 2S inhalation and the known

relationship between H2S inhalation and pulmonary edema and

fibrinocellular alveolitis (Lopezet al.,1988a). As expected, weobserved that end-of-exposure lung sulfide concentrations were

highly correlated with the amount of H2S in the exposure

atmosphere, and elevated lung sulfide concentrations were

observed following a single 3-h H2S exposure to 80 ppm.

End-of-exposure lung sulfide concentrations were not in-

creased in rats exposed subchronically to 80 ppm. In con-

trast, decreased cytochrome oxidase activity was observed in

the lung after a 3-h exposure to 30 ppm H2S as well as

following subchronic exposure to 80 ppm H 2S. This finding

indicates that cytochrome oxidase inhibition is a more sensitive

biomarker of H2S exposure than tissue sulfide concentrations.

Lung sulfide concentrations rapidly returned to preexposure

levels within minutes after the end of a 3-h exposure to 400ppm H2S, suggesting that rapid pulmonary elimination or me-

tabolism of sulfide occurs. An accumulation of sulfide metab-

olites was not observed in the lung during the 3-h H 2S expo-

sure. Transient increases in lung sulfite, sulfate, and thiosulfate

concentrations were observed, however, immediately after the

end of the 400-ppm H2S exposure. This increase in sulfide

metabolite concentrations occurred coincidentally with the

rapid decrease in lung sulfide concentration. This observation

FIG. 3. Mean ( SEM) lung sulfite, thiosulfate, and sulfate concentrations

before, during, and after a 3-h inhalation exposure to 400 ppm H2S (n 6

rats/time point). *Indicates statistically increased lung sulfite, thiosulfate, and

sulfate concentrations when compared with preexposure (control) values (p

0.05).

22 DORMAN ET AL.


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suggests that the detoxification of sulfide to sulfate may be-

comes less effective as the concentration of sulfide increases in

blood and other tissues due to H2S 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 an-

other metabolite (hydroquinone) is delayed until after the ben-

zene 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 H2S inhalation.

We also observed increased sulfide concentrations and cy-

tochrome oxidase inhibition in the upper respiratory tract from

H2S-exposed rats. Although our data showed that olfactory, but

not respiratory, epithelial sulfide concentrations were signifi-

cantly elevated following exposure to 400 ppm H2S, the ob-

served differences in end-of-exposure tissue sulfide concentra-

tions are unlikely to be toxicologically significant. For

example, end-of-exposure olfactory epithelium sulfide concen-

trations following a 3-h exposure to 400 ppm H 2S were 146%

of levels observed in unexposed animals, whereas end-of-

exposure respiratory epithelium sulfide concentrations ob-

served in the same animals were 158% of control levels.

Cytochrome oxidase activity was significantly decreased

within the olfactory and respiratory nasal epithelium immedi-

ately after a single 3-h exposure to 30 ppm H2S. Olfactory

cytochrome oxidase activity was also significantly decreased to

4566% of control levels following 5 consecutive days of

exposure to 30 ppm H2S. Repeated (5-day) H2S 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 ne-crosis of the olfactory mucosa increased in severity (Brenne-

man et al., 2001). Our data suggest that the regenerated respi-

ratory epithelium becomes resistant to H2S-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, H2S-

induced 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 200 ppm H2S. This

unusual dose-response relationship may indicate that some

compensatory changes are occurring in the epithelium in re-

sponse to H2S exposure. Furthermore, regional differences in

sulfide delivery within the olfactory epithelium are correlatedwith the development of nasal pathology at this site (Moulin et

al., 2001). Our sampling procedure does not allow us to detect

regional differences in H2S delivery to the olfactory epithe-

lium, 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 pa-

thology. Our laboratory has shown that acute inhalation expo-

sure of male rats to 400 ppm H 2S results in severe mitochon-

drial swelling in degenerating olfactory neurons within the

olfactory epithelium (Brenneman et al.,2001). This ultrastruc-

tural lesion is consistent with, but not specific for, H2S-induced

anoxic cell injury due to cytochrome oxidase inhibition. These

data provide strong evidence that cytochrome oxidase inhibi-

tion may indeed play a critical role in H2S-induced olfactory

pathology. When considered together, our data suggest that theolfactory neuroepithelium is intrinsically more sensitive than

the nasal respiratory epithelium to H2S-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 concentra-

tions in acutely or subchronically H 2S-exposed animals. Ware-

nycia et al. (1989) showed that accumulation of sulfide oc-

curred in the hindbrain of male Sprague-Dawley rats exposed

to lethal quantities of sodium hydrosulfide (NaHS), an alkaline

salt that liberates H2S 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

H2S-exposed animals. Savolainen and coworkers (1980) like-

wise showed that a single 2-h exposure of mice to 100 ppm

H2S 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 cyto-

chrome oxidase inhibition in this tissue. Indeed, rats exposed

to 10 ppm H2S 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 H2S. A similar observation wasnoted 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 subchroni-

cally exposed (8 h/day, 5 days/week, for 5 weeks) to 100 ppm

H2S. 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 H2S de-

toxification 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 H2S exposure concentration

and resulting sulfide concentrations and cytochrome oxidaseactivities in the hindbrain, lung, and nose, each of which is a

critical target for H2S-induced toxicity. Our results suggest that

acute exposure to low concentrations ( 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 H2S exposure than is tissue sulfide concentration.



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It is not unexpected that measurement of total tissue sulfide

concentrations is a relatively insensitive biomarker of H2S

exposure, as most tissues contain high endogenous levels of

sulfide and this metabolite is highly volatile when unbound.

More refined studies using radiolabeled H2S with evaluation of

total and mitochondrial sulfide could better elucidate the dose-

response relationship between tissue sulfide concentration and

H2S exposure. Despite the limitations in our experimentaldesign, 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 environ-

mental 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, Greg-

ory Kedderis, and Barbara Kuyper for their reviews of the manuscript.

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