Free Radicals and Chemiluminescence As Products of the Spontaneous Oxidation of Sulfide in Seawater, and Their Biological Implications

Reference: Biol. Bull. 196: 52-56. (February, 1999)

Free Radicals and Chemiluminescence as Products of the Spontaneous Oxidation of Sulfide in Seawater, and Their Biological Implications

DAVID W. TAPLEY’,*, GARRY R. BUETTNER’, AND J. MALCOLM SHICK’ ’ Department of Zoology and Center for Marine Studies, University of Maine, Orono, Maine 04469-5751; and 2 ESR Facility/EMRB 68, College of Medicine, The University of Iowa, Iowa City, Iowa 52242-1101

The discovery of symbioses between marine invertebrates HS’ + O2 + HOz’ + S (2) and sulfide-oxidizing bacteria at deep-sea hydrothermal vents and in other high-sulfide marine environments has HS’ + 02’- + S + HOz- (3) stimulated research into the adaptations of metazoans to At near-neutral pH, HO,- will immediately protonate: potentially toxic concentrations of sulfide. Most of these studies have focused on a particular action of sulfide-its HO*- + H+ + H20, (4) disruption of aerobic metabolism by the inhibition of mito- chondrial respiration-and on the adaptations of sulfide- The proposed metal-catalyzed mechanism underlying reac- tolerant animals to avoid this toxic effect (I). We propose tion 1 is (4): that sulfidic environments impose another, hitherto over- M”+ + O2 + M’” + I)+ + Q- looked type of toxicity: exposure to free radicals of oxygen, (5) which may be produced during the spontaneous oxidation of M’” + I)+ + HS- AM”+ + HS’ (6) sulfide, thus imposing an oxidative stress. Here we present evidence that oxygen- and sulfur-centered free radicals are Notice that, once either radical is formed, a chain reaction produced during the oxidation of sulfide in seawater, and ensues. The superoxide that is formed can undergo a sub- we propose a reaction pathway~for sulfide oxidation that is sequent reduction or dismutation to form hydrogen peroxide consistent with our observations. We also show that chemi- (H202), a known product of sulfide oxidation (5, 6), which luminescence at visible wavelengths occurs during sulfide is also produced in reactions 3 and 4. In the presence of oxidation, providing a possible mechanism for the unex- transition-metal catalysts, superoxide and H,O, will react to plained light emission from hydrothermal vents (2, 3). form the hydroxyl radical, HO’, perhaps the most oxidizing In the presence of molecular oxygen and trace metal radical that can arise in a biological setting. catalysts, hydrogen sulfide spontaneously oxidizes. Oxida- A similar pathway, in which reaction 2 yields an addition tion-reduction reactions frequently involve free-radical in- product, was proposed later (7): termediates, and a metal-catalyzed pathway in which the HS’ + O2 + HS02’ (7) initial reactions of sulfide oxidation form superoxide and sulfide radicals has been proposed (4). The proposed reac- According to this mechanism, the addition product then tion begins with four steps: reacts with molecular oxygen:

HSO*’ + O2 + HS04’ HS + O2 + HS’ + OZ.- (1) (8) A subsequent series of reactions produces a variety of Received 18 May 1998; accepted 4 December 1998. reactive intermediates, including superoxide, hydrogen per- * Present address: Department of Biology, Salem State College, 352 oxide, and the sulfide radical (7). Lafayette St., Salem, MA 01970-5353. Although these free-radical mechanisms for sulfide oxi-

52 SULFIDE-GENERATED FREE RADICALS 53 dation have been proposed (4, 7), no direct experimental A: Contro support for such a mechanism has been provided to date. We have therefore employed electron paramagnetic resonance (EPR) spin trapping to gather direct evidence that free-radical intermediates are produced during sulfide oxidation. EPR spectrometry is similar to nuclear magnetic B: Sulfide resonance (NMR) spectrometry, but relies on magnetic moments resulting from unpaired electrons instead of those from the atomic nucleus. Samples are exposed to microwave radiation at a fixed wavelength and amplitude while a magnetic field is swept through an appropriate range of field densities. At appropriate combinations of wavelength and C: Simulation pw magnetic field strength, the unpaired electrons will resonate, thereby absorbing microwave energy. This absorbance is recorded as the first derivative. In an EPR spectrum, the relative positions of peaks (lines) are more important than their absolute positions, so spectra typically are plotted with I D: DMSO Control no abscissa, only a scale bar indicating the change in magnetic field strength over a given distance. The ordinate is in arbitrary absorbance units. Spin trapping is a technique for detecting ephemeral radicals by providing a molecule that preferentially reacts with them, forming more stable radical adducts with characteristic spectra. These spectra typically E: Sulfide + DMSO result from a primary peak, due to the radical that was trapped, being split one or more times by adjacent paramagnetic nuclei, typically hydrogen and nitrogen. The splitting of peaks is the result of the magnetic moments of the adduct being oriented either parallel or antiparallel to the magnetic Figure 1. Electron paramagnetic resonance (EPR) spectra of DMPO moments of adjacent nuclei, with either orientation being adducts formed during sulfide oxidation in air-saturated artificial seawater equally likely. The magnetic field of half of the population (ASW) (27) at pH 7.4 and room temperature (=2O”C). (A) Control spec- of adducts will be incrementally increased, while that of the trum of DMPO in ASW with no sulfide added. (B) Spectrum obtained when sulfide (1 mM) is added. (C) Computer simulation of the spectrum in other half will be equally decreased. The values of these B, a composite of DMPO/HO’ (30%) and DMPO/‘SO,- (70%). (D) splittings (aN and aH) for various adducts of spin-trapping Control spectrum of DMPO and dimethyl sulfoxide (DMSO) in ASW with agents are well known and have been tabulated. no sulfide added. (E) Spectrum obtained when 1 mA4 sulfide is added to When sulfide was introduced into artificial seawater DMPO and DMSO in ASW. Computer simulation of this spectrum indi- (ASW) containing the spin-trapping agent dimethylpyrro- cates these relative abundances: DMPO/HO’ (20%), DMPO/‘SO,- (60%) and DMPOI’CH, (20%). Horizontal scale = 10 gauss; since in this type of line-N-oxide (DMPO), a prominent EPR spectrum was ob- spectrometry the relative positions of the lines are more important than tained (Fig. 1A, B). The greater intensity of the high-field their absolute positions, spectra do not usually include an absolute scale. line compared to the low-field line is consistent with ongo- The vertical axis is in arbitrary units. The vertical ticks in (E) mark the ing free radical formation by oxidizing sulfide, since the positions of DMPOKH, peaks. Reaction components, when present, were high-field line is detected by the EPR spectrometer several in the following final concentrations: DMPO, 50 mM, sulfide, 1 mM; seconds after the low-field line. The appearance of a four- DMSO, 0.7 M. Sulfide was added after all the other reactants, and immediately before transferring the sample to the EPR cell. Spectra were line spectrum with a total width of approximately 45 G obtained with a Bruker ESP 300 EPR spectrometer equipped with a TM, ,n suggests that the DMPO-hydroxyl radical adduct (DMPO/ cavity and an aqueous flat cell. Computer simulations of spectra were HO’; UN = uH = 14.9G) is present (8). But the asymmetry carried out using SIMEPR software (28). suggests that there are at least two radical species. The second radical should have hyperfine splittings that would DMPO/HO’ and DMPO/‘SO,- (Fig. 1C) reproduces the produce asymmetries in the two middle lines, yet have a experimental spectrum well. total spectral width near that of DMPO/HO’. Appropriate Chen and Morris (4) postulated superoxide production in candidates are the sulfide radical adduct, DMPO/S’- (aN = their reaction mechanism, but the experiment shown in 16.09G, uH = 16.19G) and the sulfite radical adduct, Figure 1 does not directly support this proposal. However, DMPO/‘SO,~ (aN = 14SG, uH = 16.OG) (8, 9). The trapping superoxide is difficult, first because the kobs for its splittings for DMPO/S’- are inconsistent with the spectra we reaction with DMPO is low (~30 M-’ * s-’ at pH 7.4; c$ obtained; but a computer simulation of the composite of kobs for DMPO/HO’ formation is approximately 3.4 X lo9 54 D. W. TAPLEY ET AL.

M- ’ * s- ‘) (10, 1 l), and also because the superoxide adduct Kinetic data for reaction 2 are not available (18), but the rate (DMPO/‘OOH) can undergo further reactions, including is not likely to exceed that of reaction 7. reductions (12), to form DMPO/HO’ or EPR-silent products Once SO,‘- is formed, it can oxidize to yield SO, and (10, 12). We included DMSO in the reaction mixture to O,‘- (k,, = 1 X 10’ M-’ * ss’ at pH 6.5) (20), which will distinguish between these potential routes by which DMPO/ subsequently hydrate to form HSO,-: HO’ can form (Fig. ID, E). The rate constants for the SOZ’ + 02 + so* + O*‘- reactions of HO’ with DMPO and DMSO are 3.4 X 10” (10) M-1 . s-’ and 7 X 10’ M-’ * ss’, respectively (11, 13). SO2 + H,O + H+ + HSOlm (11) Under our reaction conditions DMSO should have scav- enged about 96% of the HO’ formed; however, the relative If a strongly oxidizing radical is present, HSO,- will be abundance of DMPO/HO’ in the composite spectrum de- oxidized to SO,‘-. For example, it will react with HO’ (k = creased by only 33%, from 30% to 20% of the composite 5.1 X lo9 M-’ * s-’ at pH 11.2) (21): area (Fig. 1E). This implies that some DMPO/HO’ is HSO,- + HO’ + SO?‘- + H,O (12) formed artifactually (10, 14). The DMPO/HO’ adduct can also arise from nucleophilic substitution reactions of spin Our results (Fig. 1) strongly suggest the presence of such an adducts (14 and references therein). For example, DMPO/ oxidizing radical. Therefore, we propose that the reaction ‘OSO,- will hydrolyze to DMPO/HO’ in aqueous solution. sequence 1, 7, and 9-12 occurs during the oxidation of Although formation of SO,‘- is possible in our experiments, sulfide. This sequence produces both oxygen- and sulfur- no evidence for DMPO/‘OSO,- (t,,, = 95s) was seen in the centered radicals, and is consistent with the results of our spectra collected. But our data do not rule out the possibility EPR spin-trapping study (Fig. 1). The oxidation of sulfide is that hydrolysis gave rise to a portion of the DMPO/HO’ catalyzed by trace metals (4) (see below). Therefore we observed. The presence of sulfide, a strong reductant, sug- believe that first-chain initiation is accomplished by reac- gests that direct reduction of DMPOTOOH to DMPO/HO’ tion 6. (12) may have occurred in these experiments. Sulfide-generated free radicals could impose an oxidative The conjecture-that superoxide is produced during sul- challenge to tissues exposed to them, and could represent a fide oxidation, but is not detected by spin trapping-is previously unrecognized type of sulfide toxicity: oxidative supported by investigations into the mechanisms of thiol stress resulting from chronic, subacute exposure to sulfide. Marine animals living in environments where sulfide and oxidation. Superoxide can be produced during the oxidation molecular oxygen coexist are at risk from exogenously of thiols (15-17), but it is difficult to spin trap. In most formed free radicals, as well as from radicals resulting from experiments, superoxide production is demonstrated indi- sulfide oxidation within their own tissues. This is true in rectly by including a molecular probe that is indicative of it. We were unable to find a probe for superoxide that would spite of generally hypoxic conditions in sulfidic environments, since the presence of a strong reductant (sulfide) will not react with sulfide, and were thus unable by this method enhance the production of free radicals (Fig. 1). We have to demonstrate superoxide production; but the results when evidence from studies on protobranch bivalves that sulfide DMSO is included in the reaction mixture, as well as the exposure does impose an oxidative stress on these animals, analogy with thiol oxidation, strongly suggest that superox- and that they possess thermolabile defenses against this ide is produced but not detected in our experiments. (22). Spectra similar to that in Figure 1B are obtained when The reaction mechanism proposed by Chen and Morris sulfide is added to heat-denatured homogenates of tissues postulates the production of the sulfide radical (reaction l), from the protobranch bivalves Solemya velum and Yoldia but no direct evidence for its formation is yet available. The limatula, but are reduced or absent if undenatured homog- sulfide radical has been spin trapped in anoxic conditions enates are used (22). (9), but in our oxic experiments, conversion of the sulfide A free-radical mechanism of sulfide toxicity might ex- radical to oxygenated products appears to be efficient. plain the symptoms associated with subacute sulfide poi- Since the reaction pathways discussed above (reactions soning in humans and laboratory animals. The primary l-8) were first proposed, some of the postulated reactions, symptom of subacute hydrogen sulfide poisoning is local as well as other reactions relevant to the mechanism, have inflammation of moist tissues exposed to the gas (6, 23, 24), been demonstrated and their kinetics quantified. We suggest especially the conjunctivae of the eye and the respiratory that reaction 2 is not likely to predominate; we propose epithelia. In particular, sulfide-induced pulmonary edema is instead that the addition reaction (reaction 7) predominates similar to that which appears under pulmonary oxidative (k6 = 7.5 X lo9 M-’ * s-’ at pH 7) (18, 19) and its product stress (6, 25). Since these symptoms are restricted to moist is then immediately deprotonated at near-neutral pH: tissues, the mechanism of irritation probably involves the aqueous reactions of hydrogen sulfide, including those pro- HSO*’ cs H+ + S02’- (9) ducing radicals. SULFIDE-GENERATED FREE RADICALS 55

600 the vent plumes contain millimolar concentrations of sulfide 1 and are mixing with the ambient oxygenated seawater, the conditions necessary for sulfide-dependent chemiluminescence to occur are present. To determine whether chemilu- minescenceis a sourceof light at the vents, spectrafrom the vent plumes must be compared with those from sulfide oxidation. Sulfide oxidation spectra should preferably be determined at the temperature and pressure found at the vents, and the measurement should be made with a low- resolution, high-sensitivity spectrograph of the type under consideration for future vent work (2). The results presented here have established that both oxygen- and sulfur-centered free radicals are produced dur- 0 20 40 60 80 100 120 140 160 ing the oxidation of sulfide, and that this is accompaniedby Time (min) measurablechemiluminescence at visible wavelengths. Pro- duction of free radicals during sulfide oxidation has the Figure 2. Time course of the disappearance of sulfide from ASW (pH 7.4) continuously sparged with air or nitrogen gas. Sulfide rapidly disap- peared from solution in the aerated treatment (m). When DTPA was 5000 present, the loss of sulfide from aerated solution (A) was reduced to control rates (0: nitrogen-sparged; l : nitrogen-sparged, DTPA present). In the anoxic control experiments, as well as those with DTPA, the loss of sulfide was owing to the degassing of H,S by the nitrogen or air stream, not to the 4000 chemical oxidation of sulfide. In experiments where the pH was more acidic (and the proportion of sulfide as H,S was therefore greater), the loss of sulfide under otherwIse ldentlcal con&Ions was greatly Increased (data not shown). ASW (50 ml) was placed in a glass Erlenmeyer flask and 3000 sparged with air or nitrogen for 1 h before the experiments were begun and continuously thereafter. The experiments were started by addition of suf- 4 ficient sulfide stock, pH 7.4, to result in an initial sulfide concentration of 500 @4. Sulfide concentration was determined at 10.min intervals using 2000 the diamine method (29).

1000 Evidence for metal catalysis of sulfide oxidation is provided by sulfide-depletion studies (Fig. 2) and chemiluminescence studies (Fig. 3). The rate at which sulfide disap- 0 I I , I I I I I I pearsfrom solution (Fig. 2) decreasesto control levels when 0 10 20 30 40 50 60 70 80 the transition metal chelator diethylenetriaminepentaacetic Time (min) acid (DTPA) is included in the system. This is consistent Figure 3. Time course of chemiluminescence, measured as counts per with previous studies demonstrating catalysis of sulfide second (cps), during sulfide oxidation at various initial sulfide concentra- oxidation by trace metals (4). The chemiluminescenceplots tions, with and without chelators. Each point is the average of three for 200 and 500 FM sulfide (Fig. 3) show little or no determinations; error bars are 2 one standard deviation. The amount of induction period. When ethylenediaminetetraacetic acid light emitted was positively correlated with initial sulfide concentration (0: 0 PM, +: 200 FM, A: 500 FM). Luminescence was reduced substantially, (EDTA), which leaves one of the d-orbitals of iron available and there was a 30 min induction period, when the chelator EDTA was for reaction, is included in the system, a distinct induction included in 500 FAN sulfide (7). No chemiluminescence was observed in period is seen. When DTPA, which coordinates all the the presence of DTPA and 500 @f sulfide (+) or with 500 PM sulfide d-orbital electrons of ferric iron, is included, light emission under anoxic conditions (data not shown). Reaction mixtures were pre- is reduced to blank rates. This suggeststhat trace iron is the pared in ASW adjusted to pH 7.4 after the addition of chelators, when they were used. Chemiluminescence was measured in a Packard Tri-Carb model primary transition metal catalyst under the conditions of this 3255 hquid scin~lllarion counter set on the tilrium channel in the out-of- study (10). coincidence mode (30). The photomultiplier in this counter is sensitive to The weak chemiluminescenceat visible wavelengths ob- wavelengths between 380 and 620 nm. Background counts from the empty served during sulfide oxidation (Fig. 3) provides a potential chamber were 172 2 4 cps (mean 2 SD) and of an empty vial were 186 + mechanismfor light production at the deep-seahydrother- 11 cps. For each determination 20 ml of aerated ASW were placed into a vial, and a sufficient amount of sulfide stock was added to obtain the mal vents (2, 3), as suggestedby Hastings (in ref. 2). Pelli desired concentration. Counts were accumulated over 10.min intervals; for and Chamberlain (26) have proposed that thermal black- the experiments employing 500 pM sulfide, counts were accumulated over body radiation is a source of visible light. However, since 1-min intervals for the first 10 min. 56 D. W. TAPLEY ET AL potential for imposing an oxidative stress on organisms 14. Davies, M. J., B. C. Gilbert, J. K. Stell, and A. C. Witwood. 1992. living in sulfidic environments, with ramifications in toxi- Nucleophilic substitution reactions of spin adducts. Implications for the cology, physiology, and clinical medicine. correct identification of reactions intermediates by EPR/spin trapping. J. Chem. Sot. Perkin Trans. 2: 333-335. 15. Misra, H. P. 1974. Generation of superoxide free radical during the Acknowledgments autoxidation of thiols. J. Biol. Chem. 249: 215 I-2155. 16. Saez, G., P. J. Thornalley, H. A. 0. Hill, R. Hems, and J. V. We thank Dr. James North for help with the SIMEPR Bannister. 1982. The production of free radicals during the autoxi- software. Funding was provided by grants from the Asso- dation of cysteine and their effect on isolated rat hepatocytes. Biochim. ciation of Graduate Students and a research assistantship Biophys. Acta 719: 24-3 1. from the Center for Marine Studies at the University of 17. Buettner, G. R., and R. D. Hall. 1987. Superoxide, hydrogen Maine. peroxide, and singlet oxygen in hematoporphyrin derivative, -cysteine, -NADH and light systems. Biochim. Biophys. Acta 823: 501-507. Literature Cited 18. Ross, A. B., W. G. Mallard, W. P. Helman, G. V. Buxton, R. E. Huie, and P. Neta. 1994. NDRL-N/ST Solution Kinetics Database: 1. Childress, J. J., and C. R. Fisher. 1992. The biology of hydrother- Ver. 2.0. National Institute of Standards and Technology, Gaithersburg, mal vent animals: physiology, biochemistry, and autotrophic symbio- Maryland. ses. Oceanogr. Mar. Biol. Annu. Rev. 30: 331-441. 19. Mills, G., K. H. Schmidt, M. S. Matheson, and D. Meisel. 1987. 2. LITE Workshop Participants. 1993. Light in Thermal Environ- Thermal and photochemical reactions of sulfhydryl radicals. Implica- ments (LZTE). National Science Foundation and National Aeronautics tions for colloid photocorrosion. .I. Phys. Chem. 91: 1590-1596. and Space Administration, Woods Hole, MA. 20. Creutz, C., and N. Sutin. 1974. Kinetics of the reactions of sodium 3. Van Dover, C. L., J. Delaney, M. Smith, and J. R. Cann. 1988. dithionite with dioxygen and hydrogen peroxide. Inorg. Chem. 13: Light emission at deep-sea hydrothermal vents. EOS 69: 1498. 2041-2043. 4. Chen, K. Y., and J. C. Morris. 1972. Oxidation of sulfide by 0,: 21. Huie, R. E., and P. Neta. 1987. Rate constants for some oxidations catalysis and inhibition. J. Sunit. Eng. Div. Proc. Am. Sot. Civ. Eng. of S(IV) by radicals in aqueous solution. Atmos. Environ. 21: 1743% 98: 215-227. 1747. 5. Bittersohl, G. 1971. Beitrag zum toxischen Wirkungsmechanismus 22. Tapley, D. W. 1993. SW&de-dependent oxidative stress in marine von Schwefelwasserstoff. Z. Gesamte Hyg. Grenz. 17: 305-308. invertebrates, especially thiotrophic symbioses. Ph.D. thesis, Univer- 6. National Research Council. 1979. Hydrogen Suljide. University sity of Maine, Orono. Park Press, Baltimore. 23. Yant, W. P. 1930. Hydrogen sulphide in industry. Occurrcncc, cf- 7. Kotronarou, A., and M. R. Hoffmann. 1991. Catalytic autoxida- fects, and treatment. Am. J. Public Health 20: 598-608. tion of hydrogen sulfide in wastewater. Environ. Sci. Technol. 25: 24. Lopez, A., M. G. Prior, R. J. Reiffenstein, and I. R. Goodwin. 1989. 1153-l 160. Peracute toxic effects of inhaled hydrogen sulfide and injected sodium 8. Buettner, G. R. 1987. Spin trapping: ESR parameters of spin ad- hydrosulfide on the lungs of rats. Fundam. Appl. Toxicol. 12: 367-373. ducts. Free Radical Biol. Med. 3: 259-303. 25. Halliwell, B., and J. M. C. Gutteridge. 1989. Free Radicals in 9. Bilski, P., C. F. Chignell, J. Szychlinski, A. Borkowski, E. Oleksy, Biology and Medicine. Clarendon Press, Oxford, England. and K. Reszka. 1992. The photo-oxidation of organic and inorganic substrates during UV-photolysis of nitrite anion in aqueous solution. 26. Pelli, D. G., and S. C. Chamberlain. 1989. The visibility of 350°C black-body radiation by the shrimp Rimicaris exoculata and man. J. Am. Chrm. Sot. 114: 549-556. 10. Buettner, G. R., and R. P. Mason. 1990. Spin-trapping methods for Nature 337: 460-461. detecting superoxide and hydroxyl free radicals in vitro and in viva. 27. Cavanaugh, G. M. 1975. Formulae and Methods VI of the Marine Methods Enzymol. 186: 127-l 33. Biological Laboratory Chemical Room. Marine Biological Laboratory, 11. Finkelstein, E., G. M. Rosen, and E. J. Rauckman. 1980. Spin Woods Hole, Massachusetts. trapping. Kinetics of the reaction of superoxide and hydroxyl radicals 28. Duling, D. R. 1994. Simulation of multiple isotropic spin-trap EPR with nitrones. J. Am. Chem. Sot. 102: 4994-4999. spectra. J. Magn. Reson. 104: 105-l IO. 12. Rosen, G. M., and B. A. Freeman. 1984. Detection of superoxide 29. Cline, J. D. 1969. Spectrophotometric determination of hydrogen generated by endothelial cells. Proc. N&l. Acud. Sci. USA 81: 7269- sulfide in natural waters. Limnol. Oceanogr. 14: 454-458. 7273. 30. Gonzalez Flccha, B., S. Llesuy, and A. Boveris. 1991. Hydroper- 13. Dorfman, L. M., and G. E. Adams. 1973. Reactivity qf the Hy- oxide-initiated chemiluminescence: an assay for oxidative stress in droxyl Rudical in Aqueous Solutions. U.S. Government Printing Office, biopsies of heart, liver, and muscle. Free Radical Biol. Med. 10: U.S. National Bureau of Standards, Washington, DC. 93-100.