TOXICOLOGY

AND

APPLIED

PHARMACOLOGY

103,482-490

(1990)

Effects of Hydrogen Sulfide Exposure on Lung Mitochondrial Respiratory Chain Enzymes in Rats A. A. KHAN,’ M. M. SCHULER,M. G. PRIOR, S. YONG, R. W. COPPOCK,L. Z. FLORENCE,AND L. E. LILLIE Animal Sciences Division, Alberta Environmental Centre, Vegreville, Alberta, Canada TOE 4L0

Received September 5, I989: accepted January 15, I990 Effects of Hydrogen Sulfide Exposure on Lung Mitochondtial Respiratory Chain Enzymes in Rats. KHAN, A. A., SCHULER, M. M., PRIOR, M. G., YONG, S., COPPOCK, R. W., FLORENCE, L. Z., AND LILLIE, L. E. (1990). Toxicol. Appl. Pharmacol. 103,482-490. Fischer-344 rats were exposed for 4 hr to various concentrations of hydrogen sulfide (HzS) gas and killed either immediately or at 1, 24, or 48 hr after exposure. Mitochondrial fractions from lung tissues were assayed for the activities of respiratory chain enzymes. Exposure of rats to a low concentration (10 ppm) of H2S caused no significant changes in the activities of lung mitochondrial enzymes. However, exposure to sublethal concentrations of H2S (50-400 ppm) produced marked and highly significant depressions in the activities of cytochrome c oxidase and succinate oxidase complexes of the respiratory chain. The inhibition ofcytochrome c oxidase activity in lungs was most severe (>90%) in rats that died from acute exposure to >500 ppm HzS. In rats exposed to 200 and 400 ppm H$, a marked recovery in cytochrome c oxidase activity of lungs was observed at 24 and 48 hr postexposure. Studies in vitro with rat lung mitochondria showed that low concentrations of sulfide also caused a similar and selective inhibition of cytochrome c oxidasc activity. This effect was reversed upon removal of sulfide either by washing or by oxidation with methemoglobin. The nature of sulfide inhibition of cytochrome c oxidase was noncompetitive with respect to ferrocytochrome c. Because the activities of NADH-cytochrome c reductase and succinate-cytochrome c reductase were not significantly altered by HzS exposure and in vitro treatments with low concentrations ofsulfide, it is concluded that under physiological conditions HzS would block the respiratory chain primarily by inhibiting cytochrome c oxidase. Such a biochemical impairment would lead to functional (histotoxic) hypoxia in the lung tiSSUeS.

0 1990 Academic

Press,

Inc.

Natural gas deposits often contain large quantities of hydrogen sulfide (H2S) gas. In Alberta, Canada, exploration and development of such gas (sour gas) fields and operation of associated processing plants have raised concerns for the health and safety of occupational workers, nearby residents, livestock, and wildlife. In general, the presence of toxic concentrations of H2S in sour gas is considered to be a primary factor in occupational fatalities encountered in either acci’ To whom all correspondence should be addressed. 0041-008X/90

$3.00

Copyright 0 1990 by Academic Press, Inc. AH rigbtsofreproduction in any form reserved.

dental blowouts or capping of sour gas wells. High concentrations (>700 ppm) of H2S are known to be acutely lethal for humans and animals; however, lower concentrations can also cause transient health effects (NRCC, 198 1; Beauchamp et al., 1984). Inhalation studies with rats have recently determined that the irritant effects of H2S were caused by cytotoxic and cytopathologic lesions in various regions of the respiratory tract (Lopez et al., 1987, 1988). At a high concentration (400 ppm), these changes were characterized by necrosis and ulceration of nasal respiratory and olfactory epithelial cells (Lopez et al., 482

H2S EFFECTS ON MITQCHONDRIAL

1988) and marked increases in polymorphonuclear leukocytes, total protein, and lactate dehydrogenase activity in nasal and bronchoalveolar lavage fluids. At lower concentrations ( 10 and 200 ppm), HZ!3 caused exfoliation of nasal epithelial cells resulting in a significant increase in total cell counts in nasal lavage fluid (Lopez et al., 1987). It is generally claimed that H2S manifests its lethal effects by inhibiting cytochrome c oxidase, the terminal enzyme in the mitochondrial respiratory chain (NRCC, 198 1; Beauchamp et al., 1984). This concept is supported by the similarities of toxic symptoms produced in animals by H2S, sulfide, and cyanide (Smith, 1986) and by the resemblance of in vitro effects of sulfide and cyanide on cytochrome c oxidase (Gilmour et al., 1967; Nicholls et al.. 1972; Van Buuren et al., 1972; Nicholls, 1975; Nicholls et al., 1976; Smith et al., 1977). Although direct in vitro inhibition of cytochrome c oxidase has been demonstrated upon treatment with sulfide, there are no published reports regarding in vivo effects of gaseous H2S inhalation on cytochrome c oxidase activity in tissues directly involved in respiratory activity, namely the pulmonary tissues. This study was designed to determine the sensitivity of mitochondrial respiratory chain enzymes in rat lung to: (i) H2S inhalation (in vivo effects) and (ii) incubation with sulfide solutions (in vitro effects). MATERIALS

AND

METHODS

Chemicals. Cytochrome c(Type VI, from horse heart), methemoglobin (bovine), NADH (dipotassium salt), and tris(hydroxymethyl)aminomethane were obtained from Sigma Chemical Co. (St. Louis, MO); Bio-Gel P-2 was obtained from Bio-Rad Laboratories (Mississauga, Ontario, Canada): sodium sulfide (Na,S. 9Hz0) was purchased from Fisher Scientific (Edmonton, Alberta, Canada). All other chemicals used were commercially available analytical grade quality. .-lnimals. Male. 8- to IO-week-old, Fischer-344 rats (Charles River, St. Constant, Province of Quebec. Canada) were used. The rats were housed in stainless-steel mesh-bottomed cages and kept in animal rooms maintained at 50 -+ 20% relative humidity, 2 I + 2’C temperature. and a 12-hr artificial light/dark cycle. The animals

ENZYMES

483

had ad libitum access to food (Laboratory Chow No. 5002, Ralston Purina, St. Louis, MO) and reverse osmosis water. The rats were acclimatized for 2 weeks before any treatment. The guidelines of the Canadian Council on Animal Care (CCAC, 1980; 1984) were followed in all stages ofexperimental procedures. For all in vitro studies. rats not exposed to any treatment were used. Gas exposure and monitoring systems. Details of the whole-body inhalation chamber system. gaseous H$!i mixing and monitoring devices. and animal acclimatization, training, and exposure procedures were similar to those described previously (Lopez et ul., 1987: Prior et al., 1988). Exposure. In all experiments. equal numbers of rats (four to six) were randomly assigned to each treatment group and exposed for 4 hr to H2S gas. Because of the limited number of exposure chambers, the exposures to various concentrations of H2S (O-700 ppm) were carried out in separate experiments. In each experiment a paired control group (0 ppm H2S) was used for comparison. Except for rats that died during exposure to lethal H2S concentrations (500-700 ppm), all the treated rats were killed immediately after exposure. Postexposure nature of H2S effects were monitored in rats exposed to atmospheres containing 0. 200. and 400 ppm of H2S gas. The treated rats ( 1Z/group) were transferred to clean air, and 4 rats from each group were killed at I, 24. or 48 hr postexposure. Euthanasia and lung pe&sion. In a preliminary expetiment, rats were euthanized (i) by blunt cranial trauma producing immediate unconsciousness and (ii) under halothane/O*-induced (Stage III, plane 3-4) anesthesia. Because the two methods gave comparable rcsuits. rats in all H2S experiments were routinely anesthetized with halothane prior to euthanasia. The rats were exsanguinated by severing the abdominal aorta; residual blood was removed from the lungs by in situ perfusion. A solution of 0.9% NaCl was passed through the pulmonary vascular system via the left ventricle. exiting through an incision in the right atrium. Simultaneous inflation ofthe lungs by injection ofair through the trachea facilitated good perfusion and rapid blanching of lung tissues. Lungs of rats that died during H2S exposures were not perfused. Preparation qf‘hcng mitochhondria. Excised lung tissues were washed with a chilled solution of 0.98 NaCl and finely minced with a razor. The minced tissue (approrimately 1.5 g) was homogenized with 10 vol (w/v) of‘ a medium containing sucrose (0.25 M), EGTA (I mM). Tris-HC1 (IO mM). pH 7.4. in a Potter-Elvehjem homogenizer. The homogenate was filtered through Miracloth (Calbiochem-Behring Diagnostic, La Jolla, CA) and the filtrate was subjected to differential centrifugation to isolate the mitochondrial fraction (Mustafa el u/ 1977). Preparution c!l’,J~r’rro~~~t(~~~lrarne c. Ferricytochrome (3 (40 mg/ml) WaS dissolved in Tris-HCI buffer. pH 7.0 ( IO

484

KHAN

mM), and then reduced by the addition of a few crystals ofsodium dithionite. This solution was passed through a desalting column of Bio-Gel P-2 (1.5 X 10 cm) to remove any excess sodium dithionite from the ferrocytochrome c. The column was conditioned and eluted with TrisHCl buffer, pH 7.0 (10 mM). Preparation of sulfide solution. Sodium sulfide (Na$. 9H20) crystals were initially rinsed with glass distilleddeionized water to remove any possible oxidation products formed at the surface. Fresh solutions of NaZS (30 mM) were prepared in glass distilled-deionized water and kept at 0-4°C. Immediately before use, the solutions were adjusted to pH 7.0 and diluted appropriately with glass distilled-deionized water. Enzyme assays. Except when otherwise specified, all enzymes were assayed at 25°C under limiting concentrations of enzyme source and standardized assay conditions. Cytochrome c oxidase activity was assayedaccording to the procedure of Wharton and Tzagoloff (1967) with the following modifications. The assay contained potassium phosphate buffer, pH 7.0 (10 mM), ferrocytochrome c (50 PM). distilled water, and mitochondrial suspension. Except for the mitochondrial fraction, all other assaycomponents were preincubated for 1 min in a spectrophotometric cuvette. Enzyme activity was measured routinely by rapidly mixing in the mitochondrial suspcnsion and monitoring the decrease in optical density at 550 nm in a Hewlett Packard 845 1A diode array spectrophotometer (Hewlett-Packard, Palo Alto, CA). The rate ofdecrease during the initial 30 set was used in the calculation of enzyme activity. One milliunit of enzyme caused the oxidation of 1 nmol ferrocytochrome c/min. NADH-cytochrome c reductase and succinate-cytochrome c reductase activities were measured by the methods described by Mustafa et al. (1977). One milliunit ofthese enzymes caused reduction of 1 nmol ferricytochrome c/min. Succinate oxidase activity was determined polarographically at 37°C as described by Mustafa et al. ( 1977). Because of low activity of succinate oxidase in lung mitochondria, this enzyme was more accurately assayed at 37°C than at 25°C. One milliunit of enzyme caused 1 nmol O2 uptake/min. Other analyses. Protein was measured by the procedure of Lowry et al. (195 1) after precipitation with 10% trichloroacetic acid. Bovine serum albumin was used as a reference standard. In vitro studies with sulfide. These studies were conducted with four to five mitochondrial preparations from the lungs of control rats. Activities of mitochondrial enzymes were determined with (specified concentrations) and without (control) added sulfide solution in each assay. All treatment concentrations were based on the amount of Na,S used to prepare the stock solutions. Changes in sulfide concentration due to any enzymatic or nonenzymatic oxidation were not assessedin these experiments. During the period of experimental analysis, the activities of the tested enzymes remained stable in

ET AL. the control samples. Appropriate blanks for each enzyme assay were run and their values were subtracted in the calculation of enzyme activity. The enzyme activities in control and sulfide-treated samples were determined from the linear phase of the reaction rates. In these assays, the mitochondrial suspension and added sulfide were preincubated with the assay buffers. This preincubation period was 2 min for cytochrome c oxidase and succinate oxidase and 1 min for succinate-cytochrome c reductase and NADH-cytochrome c reductase. In these assays,the reactions were initiated by the addition ofsubstrates. In the case of cytochrome c oxidase, the effectsof added sulfide were also measured by directly mixing the mitochondrial suspension with reaction system containing sulfide (no preincubation). Reactivation of sulfide treated cytochrome c oxidase. Lung mitochondrial suspensions were incubated with 0 and 100 pM sulfide for 10 min at 25”C, and analyzed for cytochrome c oxidase activity. These samples were then centrifuged at 10,OOOgfor 10 min at 0-4°C to remove excess sulfide. The mitochondrial pellets were washed three times by suspending them in the homogenizing medium and recentrifuging as above. The suspensions of washed mitochondria were analyzed immediately to check reactivation of cytochrome c oxidase. Reactivation of cytochrome c oxidase in mitochondrial samples treated with 0 and 20 PM sulfide was also checked by incubating the samples with methemoglobin (10 mg/ml) for 5 min at 25°C. Corresponding blanks received the same treatment and their values were subtracted in the calculation of enzyme activity. Kinetic analysis. Concentrations of sulfide required for a 50% inhibition (IC50) ofcytochrome coxidase and succinate oxidasc activities were obtained by linear regression analysis of percentage inhibition vs log of sulfide concentration (Aldridge, 1953). The kinetic nature of cytochrome c oxidase inhibition by sulfide with respect to ferrocytochrome c concentration was analyzed by Lineweaver-Burk plot (Dixon and Webb, 1979). Statistical analysis. The effects of H2S exposures and sulfide incubations on the activities of lung mitochondrial enzymes were compared to the respective control treatments and analyzed for statistical significance using the GLM procedures contained in version 6.03 of SAS for microcomputers (SAS Institute. Inc., 1988). Postexposure effects of H2S with respect to time were based on treatment combinations in a balanced 3 X 3 factorial, completely randomized design. This included 3 postexposure periods (I, 24,48 hr) and 3 treatment concentrations (0,200.400 ppm) of HZS. Means of treatment combinations were compared by orthogonal contrasts.

RESULTS Mortality and clinical efects. Exposure of rats to 10, 50, and 200 ppm H2S caused no

H2S EFFECTS ON MITOCHONDRIAL

485

ENZYMES

TABLE I CHANGES

IN

THE ACTIVITIES OF LUNG MITOCHONDRIAL RESPIRATORY CHAIN ENZYMES FOLLOWING A 4-hr EXPOSURE OF RATS TO VARIOUS CONCENTRATIONS OF HYDROGEN SULFIDE

Concentration of H$i (ppm)

Cytochrome c oxidase

Succinate-cytochrome reductase

Succinate oxidase

c

NADH-cytochrome reductase

500 ppm caused mortalities as described previously (Prior et uf., 1988). Effects of various concentrations of H2S on lung mitochondrial enzymes. The activities of respiratory chain enzymes in lung mitochondria were not altered significantly (p > 0.05) in rats exposed to 10 ppm H2S (Table 1, Expt I). At 50 ppm H$, however, cytochrome c oxidase activity was significantly (p < 0.05) lower than the control; the activities of other enzymes of the respiratory chain remained unaffected (Table 1, Expt 2). Further increases in H,S concentrations (200 and 400 ppm) caused severe and highly significant (p < 0.00 1) inhibition of cytochrome c oxidase and succinate oxidase activities but failed to affect succinate-cytochrome c reductase and

NADH-cytochrome c reductase activities (Table 1, Expt 3). The activity of cytochrome c oxidase was most severely depressed (>90%) in rats which died from exposure to lethal concentrations (500-700 ppm) of H$ (Table 1, Expt 4). Postexposure effects of H2S inhalation on respiratory chain enzymes. Inhalation of H2S (200 and 400 ppm) did not produce any significant (p > 0.05) effects on NADH-cytochrome c reductase and succinate-cytochrome c reductase activities in rats euthanized at various times, postexposure. The activity of cytochrome c oxidase, however, was most severely inhibited in rats exposed to 200 or 400 ppm and killed at 1 hr postexposure, but the inhibition was markedly reduced in rats killed at 24 and 48 hr postexposure (Table 2). As a result, the means of postexposure activities of cytochrome c oxidase were not statistically different (p > 0.05) between control (0 ppm) and 200 ppm H$-exposed rats; however, these activities remained

486

KHAN

ET AL.

TABLE 2 POSTEXPOSUREACTIVITIESOFCYTOCHROMECOXIDASEIN LUNGSOFRATSEXPOSEDTOVARIOUSCONCENTRATIONS

OF HYDROGEN

SULFIDE

FOR 4 hr

Treatment groups

Post-

exposure (W

0

200

400

Enzyme activity (munits/mg protein)” 1 24 48

862 f 14’ (100)b 916+91‘ (100) 748 + 86’.“’ (100)

61Ok 97”/ (70.8) 806 rf~ 65’~~ (88.0) 672 f 1 lo”‘,/ (89.8)

388 + lOOg (45.0) 613 3~ 56d,‘,’ (73.5) 529 + 5@’ (70.7)

a Values are means + SD (n = 4). b Values in parentheses are percentages of respective control group (0 ppm). c-gTreatment combinations sharing the same letter are not statistically different (p > 0.05).

significantly (p < 0.05) depressed in rats exposed to 400 ppm H2S (Table 2). No postexposure effects of H$ were observed in rats exposed to 10 ppm H$.

In vitro efects of suljide on respiratory chain enzymes. Incubation of lung mitochondria with sulfide caused a marked and dose dependent inhibition of cytochrome c oxidase activity (Table 3). Sulfide treatment TABLE 3 EFFECTS OF SULFIDE

Enzyme Cytochrome c oxidase Succinate oxidase

ON LUNG ENZYMES

MIT~CHONDRIAL

Sulfide (PM)

Activity (% of control)”

0.5 1.0 2.0 40.0 80.0

75.4 39.9 22.0 64.0 33.9

” Activities of enzymes were assayed after 2 min preincubation with specified concentrations of sulfide. Control values were: cytochrome c oxidase = 1013 + 223 munits/mg protein (n = 5); succinate oxidase = 28.9 * 5.7 munits/mg protein (n = 4); all treatment values were significantly (p < 0.05) lower than control.

0

0.15 Fe2+Cytochrome

0.30 c, CM

i

I-’

FIG. I. Effect of sulfide on cytochrome c oxidase activity. A double-reciprocal (Lineweaver-Burk) plot of enzyme activity with respect to ferrocytochrome c(Fe’+ cytochrome c) concentration in the presence (0, 2.5 fiM) and absence (0,O PM) of sulfide.

during the assay (no preincubation) also inhibited cytochrome c oxidase activity; however, the effect was less potent (IC50 = 2.6 + 0.6 PM) than observed after incubation with sulfide (IC50 = 1.2 f 0.3 PM). Low concentrations of sulfide also caused marked inhibition of succinate oxidase (Table 3); the respective IC50 was found to be 58.3 + 3.3 PM. At low concentrations (lo-50 PM), sulfide caused either no effect or a 20-30s stimulation of NADH-cytochrome c reductase and succinate-cytochrome c reductase activities. At high concentrations (> 100 PM), sulfide interfered with these assays by causing a rapid nonenzymatic reduction of the electron acceptor (ferricytochrome c). The Lineweaver-Burk plot of cytochrome c oxidase activity at varying concentrations of ferrocytochrome c showed that sulfide inhibited enzyme activity in a noncompetitive manner (Fig. 1). The apparent values of K, and Ki as calculated from the plot were 5.0 and 4.8 PM, respectively. A marked reactivation of sulfide inhibited cytochrome c oxidase activity in the mitochondria was observed upon removal of sulfide from the treated mitochondria by either washing or methemoglobin treatment (Table 4).

H2S EFFECTS TABLE

ON

MITOCHONDRIAL

4

REACTIVATIONOFCYTOCHROMECOXIDASEIN SULFIDE-TREATEDLUNGMITOCHONDRIA Cytochromec oxidase (munits/mg

Assay Control’ Sulfide treated ( 100 PM)’ Control, washed Sulfide treated. washed Control h Sulfide treated (20 FM)" Control + methemoglobin’ Sulfide treated (20 FM) t methemoglobin”

activity protein)”

1020 0 1272 1201 935 231 884 551

U Mean values from two experiments using mitochondria isolated from pooled lungs of eight rats. ’ Enzyme activity was assayed in mitochondrial suspensions incubated for 10 min at 25°C with the specified concentration of sulfide. Corresponding control mitochondrial suspensions were similarly incubated without sulfide. ’ Mitochondrial suspensions (control and sulfide treated) were assayed after incubation for 5 min at 25’C with an equal volume of methemoglobin solution (10 mg/ml).

DISCUSSION Studies of rats have shown that H2S inhalation produces cytotoxic and pathologic alterations in respiratory tract tissues at high concentrations (Lopez et al., 1987, 1988). It is possible that these alterations were caused by primary and secondary biochemical lesions in the exposed tissues. Because relatively little is known about the specific biochemical aspects of H2S toxicity in vivo, we investigated the sensitivity and specificity of H$-related effects on the mitochondrial respiratory chain system in pulmonary tissues. In view of the importance of the respiratory chain system in oxidative and energy metabolism, we monitored the effects of H2S on the activities of enzyme complexes catalyzing electron transport between (i) substrates (NADH/succinate) to cytochrome c (NADH/succinatecytochrome c reductase), (ii) cytochrome c to

ENZYMES

487

oxygen (cytochrome c oxidase), and (iii) succinate to oxygen (succinate oxidase). Inhalation of H2S caused marked and concentration-dependent depression in the activities of cytochrome c oxidase and succinate oxidase in lung mitochondria, but produced no significant effect on succinate-cytochrome c reductase and NADH-cytochrome c reductase activities (Table 1). Postexposure analyses of H2S effects revealed that the inhaled gas was converted to less toxic forms as evidenced by a recovery in cytochrome c oxidase activity (Table 2). This recovery, however, appeared to be influenced by the magnitude of the initial inhibition. In rat lung mitochondria, the biochemical nature of these H2S effects was further assessed in vitro by using aqueous solutions of sulfide. Because of its two dissociation constants (pK, 7.04 and 11.96), the dissolved sulfide at the physiological pH is expected to contain undissociated H2S and hydrosulfide anion (HS) in a 1:3 ratio (Beauchamp et al.. 1984). Our results indicate that in vitro additions of sulfide caused severe inhibition of cytochrome c oxidase and succinate oxidase but did not inhibit succinate-cytochrome c reductase and NADH-cytochrome c reductase in pulmonary mitochondria (Table 3). Previous studies with cytochrome c oxidase had shown that the undissociated form of HIS would exert a more potent inhibition of enzyme than the HS anionic form (Smith et al., 1977). Studies in vitro also showed that cytochrome c oxidase was more sensitive to sulfide (KS0 = 1.2 + 0.3 PM) than succinate oxidase (IC50 = 58.3 f 3.3 PM). Because cytochrome c oxidase is involved in the succinate oxidase system, the difference in the in vitro sensitivity of the two enzyme activities can be attributed to procedural differences between the two enzyme assays. For example, the increased amount (>SOX) of mitochondrial proteins required in the succinate oxidase assay would require a similar increase in the added sulfide concentration to obtain the same inhibition. Furthermore, the increased

KHAN

mitochondrial protein could influence sulfide concentration in the assay due to increased oxidation of sulfide. Oxidation of sulfide has been observed in lung tissues (Bartholomew et al., 1980) and with high concentrations of cytochrome c oxidase (Nicholls and Kim, 198 1). Removal of HzS by these and other physiologic mechanisms (Beauchamp et al., 1984) in the lungs could be responsible for (i) the protection of cytochrome c oxidase and other sensitive enzymes at low exposure concentrations (Table 1) and (ii) the partial reversal of the inhibitory effects during postexposure (Table 2). Studies in vitro have demonstrated that removal of sulfide from the treated mitochondria either by washing or by incubation with methemoglobin restored the activity of severely inhibited cytochrome c oxidase (Table 4). Addition of methemoglobin was shown previously to restore sulfide- and cyanide-inhibited cytochrome c oxidase activity (Smith et al., 1977). In view of the reversible nature of the inhibitory effect and physiological changes in the tissue concentrations of H2S due to oxidation, it is difficult to assess the actual magnitude of the in viva changes. Furthermore, any reactivation during the isolation of mitochondria could affect the magnitude of observable inhibitory effects. It is also important to stress that the magnitude of inhibitory effects in vitro would depend on the actual concentration of sulfide in the assay. Any change in the sulfide concentration due to enzymatic and nonenzymatic mechanisms (Beauchamp et al., 1984) could influence the values of IC50 and other kinetic analyses. At acutely high concentrations, H2S and sulfide would also interfere with some electron carriers of the respiratory chain (e.g., ferricytochrome c). Under physiological conditions such an effect would be of little significance because severe inhibition of cytochrome c oxidase by relatively lower H2S concentrations would block the oxidation of all electron carriers thus leaving them in their reduced state. In lung mitochondria, the noncompetitive nature of cytochrome c oxidase

ET AL.

inhibition by sulfide would suggest that H2S has its own interaction site(s) in the enzyme complex. A similar type of noncompetitive inhibition by sulfide and other mercaptans was reported in earlier studies with purified cytochrome c oxidase from beef and horse heart (Petersen, 1977; Wilms et al., 1980). Alteration in the redox state of the cytochrome c oxidase complex could be responsible for impaired activity. Electron paramagnetic studies with purified cytochrome c oxidase have shown that sulfide and mercaptans indeed caused such redox changes (Wever et al., 1975; Wilms et al., 1980; Nicholls and Kim, 1981). Because cytochrome c oxidase catalyzes the terminal step in aerobic oxidative metabolism, its impairment by H2S would lead to a rapid cessation of tissue respiration and development of pulmonary hypoxia. Since cytochrome c oxidase is a key enzyme in the regulation of cellular energy production in eukaryotic cells (Poyton et al., 1988) its inhibition by H2S would also abolish oxidative phosphorylation, a major source of cellular ATP synthesis. Complete inhibition of ATP production has been reported in rat liver mitochondria upon addition of 20 pM sulfide (Powell and Somero, 1986). Such alterations in energy metabolism of pulmonary tissues would cause vasoconstriction (Voelkel, 1986), swelling of membranes and edema. It has been shown that cellular release of enzymes due to hypoxic and ischemic injuries is associated with decreased ATP, an imbalance in cellular ions, and swelling in cardiac tissues (Tranum-Jensen et al., 1981; Diederichs and Wittenberg, 1988). The results of our study suggest that H2S intoxication of pulmonary oxidative and related energy metabolic processes could be primarily responsible for cytotoxic and edematogenic changes in respiratory tract tissues observed in rats exposed to high sublethal concentrations of H2S (Lopez et al., 1987). Extensive pulmonary edema has been reported in rats (Prior et al., 1988; Lopez et al., 1989) and humans (Burnett et al., 1977) killed by lethal concentrations of H$.

H2S EFFECTS ON MITOCHONDRIAL

The results of this study provide strong evidence that lethal concentrations of H2S do indeed cause almost total inhibition of cytochrome c oxidase (Table 1). This information, together with the pattern of cytochrome c oxidase inhibition at those sublethal concentrations of H2S (50 and 200 ppm, Table 1) which did not produce any evidence of cellular injury in nasal and bronchoalveolar regions (i.e., increases in total protein, lactate dehydrogenase, and alkaline phosphatase) (Lopez et al., 1987) strongly suggest that inhibition of cytochrome c oxidase in pulmonary tissues is a primary biochemical lesion of H2S toxicity. A progressive inhibition of this enzyme with increased H2S concentration would result in histotoxic hypoxia and impairment of ATP homeostasis, thus reducing the probability of survival of affected tissues, and could be responsible for acute injuries and death. Inhibition of cytochrome c oxidase by H2S and sulfide could also result in increased production of superoxide radicals (0;) and Hz02 due to increases in concentration of the reduced form of respiratory chain components (e.g., ubiquinone, flavoprotein dehydrogenases). Such increases in the rate of 02 and Hz02 production have been reported with other respiratory chain inhibitors (Boveris and Chance, 1973; Foreman and Boveris, 1982). In addition, tissue hypoxia could lead to the conversion of xanthine oxidase from a dehydrogenase (non 0; producing) to the oxidase form (Granger et al., 198 1; Roy and McCord, 1983). Recent in vitro studies have shown that sulfide stimulated the production of 0: by xanthine oxidase and inhibited the enzymes involved in the scavenging of reactive oxygen species (superoxide dismutase, glutathione peroxidase, glutathione reductase, and catalase) (Khan et al., 1987). Such impairments in vivo would greatly exacerbate H2S toxicity. ACKNOWLEDGMENTS The authors thank C. Seniuk, J. Kuzyk, C. Miller, B. Kelly, and P. Henry for technical assistance, and G. Flato for preparing the manuscript,

ENZYMES

489

REFERENCES ALDRIDGE. W. N. ( 1953). The differentiation oftrue and pseudo cholinesterase by organo-phosphorus compounds. Biochem. J. 22,62-61. BARTHOLOMEW, T. C., POWELL, G. M., D~DCSON. K. S., AND CURTIS, C. G. ( 1980). Oxidation of sodium sulphide by rat liver, lungs and kidney. Biochrm. Pharmacol. 29,243 I-2437. BEAUCHAMP, R. O., Bus, J. S.. POPP, J. A.. BOREIKO. C. J., AND ANDJELKOVICH, D. A. (1984). A critical review of the literature on hydrogen sulfide toxicity. CRC Crit. Rev. To.uicol. 13, 25-97. BOVERIS, A., AND CHANCE, B. (1973). The mitochondrial generation of hydrogen peroxide: General properties and effects of hyperbaric oxygen. Biochem. J. 134,707-7 16. BURNETT, W. W.. KING, E. G., GRACE. M., AND HALI.. W. F. (1977). Hydrogen sulfide poisoning: A review of 5 years experience. Canad. Med. Assoc. J. 117, 12X-1280. Canadian Council on Animal Care (CCAC) (1980. 1984). Guide to the Cure and L!se c?fEsperimc~ntul,4nlmais, Ottawa. DIEDERICHS,F.. AND WITTENBERG, H. (1988). Enzyme release and changes of intracellular cations. In Ewqmes-Tools and Targets (D. M. Goldberg, D. W. Moss, E. Schmidt. and F. W. Schmidt, Eds.). pp. 77.. 86. Karger, Base]. DIXON. M., AND WEBB, E. C. ( 1979). Ert=~rnt~ Academic Press, New York. FOREMAN, H. J.. AND BOVERIS. A. (1982). Superoxide radical and hydrogen Peroxide in mitochondria. In Free Radicals in Biology (W. A. Pryor, Ed.), Vol. 5, pp. 65-90. Academic Press. New York. GILMOUR, M. V., WILSON, D. F.. AND LEMBERC;, R. (1967). The low-temperature spectral properties of mammalian cytochrome oxidase. II. The enzyme isolated from beef-heart mitochondria. Biochim. f&r,phys. Acta 143,487-499. GRANGER, D. N.. RUTILI. G.. AND MCCORD, J. M. (1981). Superoxide radicals in feline intestinal ischemia. G‘aslroenrerolo~~~81,22-29. KHAN, A. A., SCHULER, M. M.. AND COP~~CK. R. W. (I 987). Inhibitory effects of various sulfur compounds on the activity ofbovine erythrocyte enzymes. .J.Tilticd Environ. MeuIth 22,48 I-490. LOPEZ. A., PRIOR. M.. YONG, S.. ALBASSAM, M.. ANI) LII.LIE, L. E. ( 1987). Biochemical and cytologic alterations in the respiratory tract of rats exposed for 4 hours to hydrogen sulfide. Fundurn. Appl. To.\-i(,ol 9. 753-762. LOPEZ. A., PRIOR, M., YONG. S., AND LILLIE, L. ( 1988). Nasal lesions in rats exposed to hydrogen sulfide forfour hours. Amer. J. lit. Re.7.49, 1107-l I I I, LOPEZ. A.. PRIOR, M. G., REIFFENSTEIN, R. J., AND GOODWIN. L. R. (1989). Peracute toxic effects of in-

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Effects of hydrogen sulfide exposure on lung mitochondrial respiratory chain enzymes in rats.

Fischer-344 rats were exposed for 4 hr to various concentrations of hydrogen sulfide (H2S) gas and killed either immediately or at 1, 24, or 48 hr aft...
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