Phenol-induced in vivo oxidative stress in skin: evidence for enhanced free radical generation, thiol oxidation, and antioxidant depletion.

A variety of phenolic compounds are utilized in industry (e.g., for the production of phenol (PhOH)-formaldehyde resins, paints and lacquers, cosmetics, and pharmaceuticals). They can be toxic to skin, causing rash, dermal inflammation, contact dermatitis, depigmentation, and cancer promotion. The biochemical mechanisms for the dermal toxicity of phenolic compounds are not well understood. We hypothesized that topical PhOH exposure results in the generation of radicals, possibly via redox-cycling of phenoxyl radicals, which may be an important contributor to dermal toxicity via the stimulation of the induction and release of inflammatory mediators. To test this hypothesis, we (1) monitored in vivo the formation of PBN-spin-trapped radical adducts by ESR spectroscopy, (2) measured GSH, protein thiols, vitamin E, and total antioxidant reserves in the skin of B6C3F1 mice topically treated with PhOH, and (3) compared the responses with those produced by PhOH in mice with diminished levels of GSH. We found that dermal exposure to PhOH (3.5 mmol/kg, 100 microL on the shaved back, for 30 min) caused oxidation of GSH and protein thiols and decreased vitamin E and total antioxidant reserves in skin. The magnitude of the PhOH-induced generation of PBN-spin-trapped radical adducts in the skin of mice with diminished levels of GSH (pretreated with BCNU, an inhibitor of glutathione reductase, or BSO, an inhibitor of gamma-glutamylcysteine synthetase) was markedly higher compared to radical generation in mice treated with PhOH alone. Topical exposure to PhOH resulted in skin inflammation. Remarkably, this inflammatory response was accelerated in mice with a reduced level of GSH. Epidermal mouse cells exposed to phenolic compounds showed the induction of early inflammatory response mediators, such as prostaglandin E 2 and IL-1beta. Since dermal exposure to PhOH produced ESR-detectable PBN spin-trapped signals of lipid-derived radicals, we conclude that this PhOH-induced radical formation is involved in oxidative stress and dermal toxicity in vivo.

Antioxidant balance and free radical generation in vitamin e-deficient mice after dermal exposure to cumene hydroperoxide.

Organic peroxides are widely used in the chemical industry as initiators of oxidation for the production of polymers and fiber-reinforced plastics, in the manufacture of polyester resin coatings, and pharmaceuticals. Free radical production is considered to be one of the key factors contributing to skin tumor promotion by organic peroxides. In vitro experiments have demonstrated metal-catalyzed formation of alkoxyl, alkyl, and aryl radicals in keratinocytes incubated with cumene hydroperoxide. The present study investigated in vivo free radical generation in lipid extracts of mouse skin exposed to cumene hydroperoxide. The electron spin resonance (ESR) spin-trapping technique was used to detect the formation of alpha-phenyl-N-tert-butylnitrone (PBN) radical adducts, following intradermal injection of 180 mg/kg PBN. It was found that 30 min after topical exposure, cumene hydroperoxide (12 mmol/kg) induced free radical generation in the skin of female Balb/c mice kept for 10 weeks on vitamin E-deficient diets. In contrast, hardly discernible radical adducts were detected when cumene hydroperoxide was applied to the skin of mice fed a vitamin E-sufficient diet. Importantly, total antioxidant reserve and levels of GSH, ascorbate, and vitamin E decreased 34%, 46.5%. 27%, and 98%, respectively, after mice were kept for 10 weeks on vitamin E-deficient diet. PBN adducts detected by ESR in vitamin E-deficient mice provide direct evidence for in vivo free radical generation in the skin after exposure to cumene hydroperoxide.

A long-lived tyrosyl radical from the reaction between horse metmyoglobin and hydrogen peroxide.

The reaction between metmyoglobin (metMb) and hydrogen peroxide has been known since the 1950s to produce globin-centered free radicals. The direct electron spin resonance spectrum of a solution of horse metMb and hydrogen peroxide at room temperature consists of a multilined signal that decays in minutes at room temperature. Comparison of the direct ESR spectra obtained from the system under N(2)- and O(2)-saturated conditions demonstrates the presence of a peroxyl radical, identified by its g-value of 2.014. Computer simulations of the spectra recorded 3 s after the mixture of metMb and H(2)O(2) were calculated using hyperfine coupling constants of a(H2,6) = 1.3 G and a(H3,5) = 7.0 G for the ring and a(beta)(H1) = 16.7 G and a(beta)(H2) = 14.2 G for the methylene protons, and are consistent with a highly constrained, conformationally unstable tyrosyl radical. Spectra obtained at later time points contained a mixture of the 3 s signal and another signal that was insufficiently resolved for simulation. Efficient spin trapping with 3, 5-dibromo-4-nitrosobenzenesulfonic acid was observed only when the spin trap was present at the time of H(2)O(2) addition. Spin trapping experiments with either 5,5-dimethyl-1-pyrroline N-oxide (DMPO) or perdeuterated 2-methyl-2-nitrosopropane (MNP-d(9)), which have been shown to trap tyrosyl radicals, were nearly equally effective when the spin trap was added before or 10 min after the addition of H(2)O(2). The superhyperfine structure of the ESR spectra obtained from Pronase-treated MNP-d(9)/*metMb confirmed the assignment to a tyrosyl radical. Delayed spin trapping experiments with site-directed mutant myoglobins in which either Tyr-103 or Tyr-146 was replaced by phenylalanine indicated that radical adduct formation with either DMPO or MNP-d(9) requires the presence of Tyr-103 at all time points, implicating that residue as the radical site.

Electron spin resonance investigation of the cyanyl and azidyl radical formation by cytochrome c oxidase.

Cyanide (CN(-)) is a frequently used inhibitor of mitochondrial respiration due to its binding to the ferric heme a(3) of cytochrome c oxidase (CcO). As-isolated CcO oxidized cyanide to the cyanyl radical ((.)CN) that was detected, using the ESR spin-trapping technique, as the 5,5-dimethyl-1-pyrroline N-oxide (DMPO)/(.)CN radical adduct. The enzymatic conversion of cyanide to the cyanyl radical by CcO was time-dependent but not affected by azide (N(3)(-)). The small but variable amounts of compound P present in the as-isolated CcO accounted for this one-electron oxidation of cyanide to the cyanyl radical. In contrast, as-isolated CcO exhibited little ability to catalyze the oxidation of azide, presumably because of azide's lower affinity for the CcO. However, the DMPO/(.)N(3) radical adduct was readily detected when H(2)O(2) was included in the system. The results presented here indicate the need to re-evaluate oxidative stress in mitochondria "chemical hypoxia" induced by cyanide or azide to account for the presence of highly reactive free radicals.

Potential roles of myoglobin autoxidation in myocardial ischemia-reperfusion injury.

The source(s) of reactive partially reduced oxygen species associated with myocardial ischemia/reperfusion injury remain unclear and controversial. Myoglobin has not been viewed as a participant but is present in relatively high concentrations in heart muscle and, even under normal conditions, undergoes reactions that generate met (Fe3+) species and also superoxide, hydrogen peroxide, and other oxidants, albeit slowly. The degree to which the decrease in pH and the freeing of copper ions, as well as the variations in pO2 associated with ischemia and reperfusion increase the rates of such myoglobin reactions has been investigated. Solutions of extensively purified myoglobin from bovine heart in 50 mM sodium phosphate buffer were examined at 37 degrees C. Sufficiently marked rate increases were observed to indicate that reactions of myoglobin can indeed contribute substantially to the oxidant stress associated with ischemia/reperfusion injury in myocardial tissues. These findings provide additional targets for therapeutic interventions.

An electron spin resonance spin-trapping investigation of the free radicals formed by the reaction of mitochondrial cytochrome c oxidase with H2O2.

The reaction of purified bovine mitochondrial cytochrome c oxidase (CcO) and hydrogen peroxide was studied using the ESR spin-trapping technique. A protein-centered radical adduct was trapped by 5, 5-dimethyl-1-pyrroline N-oxide and was assigned to a thiyl radical adduct based on its hyperfine coupling constants of aN = 14.7 G and abetaH = 15.7 G. The ESR spectra obtained using the nitroso spin traps 3,5-dibromo-4-nitrosobenzenesulfonic acid (DBNBS) and 2-methyl-2-nitrosopropane (MNP) indicated that both DBNBS/.CcO and MNP/.CcO radical adducts are immobilized nitroxides formed by the trapping of protein-derived radicals. Alkylation of the free thiols on the enzyme with N-ethylmaleimide (NEM) prevented 5, 5-dimethyl-1-pyrroline N-oxide adduct formation and changed the spectra of the MNP and DBNBS radical adducts. Nonspecific protease treatment of MNP-d9/.NEM-CcO converted its spectrum from that of an immobilized nitroxide to an isotropic three-line spectrum characteristic of rapid molecular motion. Super-hyperfine couplings were detected in this spectrum and assigned to the MNP/.tyrosyl adduct(s). The inhibition of either CcO or NEM-CcO with potassium cyanide prevented detectable MNP adduct formation, indicating heme involvement in the reaction. The results indicate that one or more cysteine residues are the preferred reductant of the presumed ferryl porphyrin cation radical residue intermediate. When the cysteine residues are blocked with NEM, one or more tyrosine residues become the preferred reductant, forming the tyrosyl radical.

Reexamination of the mechanism of hydroxyl radical adducts formed from the reaction between familial amyotrophic lateral sclerosis-associated Cu,Zn superoxide dismutase mutants and H2O2.

Amyotrophic lateral sclerosis (ALS) involves the progressive degeneration of motor neurons in the spinal cord and motor cortex. Mutations to Cu,Zn superoxide dismutase (SOD) linked with familial ALS are reported to increase hydroxyl radical adduct formation from hydrogen peroxide as measured by spin trapping with 5, 5'-dimethyl-1-pyrrolline N-oxide (DMPO). In the present study, we have used oxygen-17-enriched water and H2O2 to reinvestigate the mechanism of DMPO/.OH formation from the SOD and SOD mutants. The relative ratios of DMPO/.17OH and DMPO/.16OH formed in the Fenton reaction were 90% and 10%, respectively, reflecting the ratios of H217O2 to H216O2. The reaction of the WT SOD with H217O2 in bicarbonate/CO2 buffer yielded 63% DMPO/.17OH and 37% DMPO/.16OH. Similar results were obtained from the reaction between familial ALS SOD mutants and H217O2: DMPO/.17OH (64%); DMPO/.16OH (36%) from A4V and DMPO/.17OH (62%); and DMPO/.16OH (38%) from G93A. These results were confirmed further by using 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide spin trap, a phosphorylated analog of DMPO. Contrary to earlier reports, the present results indicate that a significant fraction of DMPO/.OH formed during the reaction of SOD and familial ALS SOD mutants with H2O2 is derived from the incorporation of oxygen from water due to oxidation of DMPO to DMPO/.OH presumably via DMPO radical cation. No differences were detected between WT and mutant SODs, neither in the concentration of DMPO/.OH or DEPMPO/.OH formed nor in the relative incorporation of oxygen from H2O2 or water.

Site-specific spin trapping of tyrosine radicals in the oxidation of metmyoglobin by hydrogen peroxide.

The reaction between metmyoglobin and hydrogen peroxide produces both a ferryl-oxo heme and a globin-centred radical(s) from the two oxidizing equivalents of the hydrogen peroxide. Evidence has been presented for localization of the globin-centred radical on one tryptophan residue and tyrosines 103 and 151. When the spin-trapping agent 5,5-dimethyl-1-pyrroline N-oxide (DMPO) is included in the reaction mixture, a radical adduct has been detected, but the residue at which that adduct is formed has not been determined. Replacement of either tryptophans 7 and 14 or tyrosines 146 and 151 with phenylalanine has no effect on the formation of DMPO adduct in the reaction with hydrogen peroxide. When tyrosine 103 is replaced with phenylalanine, however, only DMPOX, a product of the oxidation of the spin-trap, is detected. Tyrosine-103 is, therefore, the site of radical adduct formation with DMPO. The spin trap 2-methyl-2-nitrosopropane (MNP), however, forms radical adducts with any recombinant sperm whale metmyoglobin that contains either tyrosine 103 or 151. Detailed spectral analysis of the DMPO and MNP radical adducts of isotopically substituted tyrosine radical yield complete structural determinations. The multiple sites of trapping support a model in which the unpaired electron density is spread over a number of residues in the population of metmyoglobin molecules, at least some of which are in equilibrium with each other.

Nitric oxide trapping of tyrosyl radicals generated during prostaglandin endoperoxide synthase turnover. Detection of the radical derivative of tyrosine 385.

Tyrosyl radicals have been detected during turnover of prostaglandin endoperoxide H synthase (PGHS), and they are speculated to participate in cyclooxygenase catalysis. Spectroscopic approaches to elucidate the identity of the radicals have not been definitive, so we have attempted to trap the radical(s) with nitric oxide (NO). NO quenched the EPR signal generated by reaction of purified ram seminal vesicle PGHS with arachidonic acid, suggesting that NO coupled with a tyrosyl radical to form inter alia nitrosocyclohexadienone. Subsequent formation of nitrotyrosine was detected by Western blotting of PGHS incubated with NO and arachidonic acid or organic hydroperoxides using an antibody against nitrotyrosine. Both arachidonic acid and NO were required to form nitrotyrosine, and tyrosine nitration was blocked by the PGHS inhibitor indomethacin. The presence of superoxide dismutase had no effect on nitration, indicating that peroxynitrite was not the nitrating agent. To identify which tyrosines were nitrated, PGHS was digested with trypsin, and the resulting peptides were separated by high pressure liquid chromatography and monitored with a diode array detector. A single peptide was detected that exhibited a spectrum consistent with the presence of nitrotyrosine. Consistent with Western blotting results, both NO and arachidonic acid were required to observe nitration of this peptide, and its formation was blocked by the PGHS inhibitor indomethacin. Peptide sequencing indicated that the modified residue was tyrosine 385, the source of the putative catalytically active tyrosyl radical.

Nitric oxide trapping of the tyrosyl radical of prostaglandin H synthase-2 leads to tyrosine iminoxyl radical and nitrotyrosine formation.

The determination of protein nitrotyrosine content has become a frequently used technique for the detection of oxidative tissue damage. Protein nitration has been suggested to be a final product of the production of highly reactive nitrogen oxide intermediates (e. g. peroxynitrite) formed in reactions between nitric oxide (NO.) and oxygen-derived species such as superoxide. The enzyme prostaglandin H synthase-2 (PHS-2) forms one or more tyrosyl radicals during its enzymatic catalysis of prostaglandin formation. In the presence of the NO.-generator diethylamine nonoate, the electron spin resonance spectrum of the PHS-2-derived tyrosyl radical is replaced by the spectrum of another free radical containing a nitrogen atom. The magnitude of the nitrogen hyperfine coupling constant in the latter species unambiguously identifies it as an iminoxyl radical, which is likely formed by the oxidation of nitrosotyrosine, a stable product of the addition of NO. to tyrosyl radical. Addition of superoxide dismutase did not alter the spectra, indicating that peroxynitrite was not involved. Western blot analysis of PHS-2 after exposure to the NO.-generator revealed nitrotyrosine formation. The results provide a mechanism for nitric oxide-dependent tyrosine nitration that does not require formation of more highly reactive nitrogen oxide intermediates such as peroxynitrite or nitrogen dioxide.

Peroxidation of a specific tryptophan of metmyoglobin by hydrogen peroxide.

Globin-centered radicals at tyrosine and tryptophan residues and a peroxyl radical at an unknown location have been reported previously as products of the reaction of metmyoglobin with hydrogen peroxide. The peroxyl radical is shown here to be localized on tryptophan through the use of recombinant sperm whale myoglobin labeled with 13C at the indole ring C-3. Peroxyl radical formation was not prevented by site-directed mutations that replaced all three tyrosines, the distal histidine, or tryptophan 7 with non-oxidizable residues. In contrast, mutation of tryptophan 14 prevents peroxyl radical formation, implicating tryptophan 14 as the specific site of the peroxidation.

In vivo production of nitric oxide in rats after administration of hydroxyurea.

The metabolism of nitrovasodilators such as glyceryl trinitrate and nitroprusside provides the active moiety of these drugs (that is, nitric oxide). This process is not limited to the known nitrovasodilators, but also occurs with nitroaromatic antimicrobials. Here we report that the administration of hydroxyurea, an antitumor drug, to rats at pharmacological doses formed detectable nitrosyl hemoglobin, which increased with dose. At higher doses, nitrosyl hemoprotein complexes could also be detected in liver tissue. [15N]hydroxyurea was synthesized and compared with [14N]hydroxyurea. These observations verified that nitric oxide detected as nitrosyl hemoglobin or nitrosyl hemoprotein complexes in rats was the result of the metabolism of hydroxyurea. The time course and dose-dependence of nitric oxide generation were also investigated. Hydroxyurea's antineoplastic activity is caused by its direct action on ribonucleotide reductase, the rate-limiting enzyme in DNA synthesis. Because nitric oxide also inhibits ribonucleotide reductase, this metabolite may supplement this action of hydroxyurea. In addition, the known ability of hydroxyurea to ease the pain of sickle cell anemia patients may be the result of vasodilation by the drug-derived nitric oxide.

ESR spin-trapping of a protein-derived tyrosyl radical from the reaction of cytochrome c with hydrogen peroxide.

The reaction of horse heart cytochrome c with hydrogen peroxide was investigated using the ESR spin-trapping technique and the nitroso spin traps 3,5-dibromo-4-nitrosobenzenesulfonic acid (DBNBS) and 2-methyl-2-nitrosopropane (MNP). The ESR spectra obtained using both spin traps were typical of an immobilized nitroxide and indicated that the adduct was a macromolecule. The intensity of the ESR spectrum corresponding to the DBNBS/*cytochrome c radical adduct was greatly enhanced by performing the reaction under anaerobic conditions, which suggested that the spin trap was competing with O2 for reaction with the radical site(s). Nonspecific proteolysis of either the DBNBS or the MNP adducts revealed isotropic three-line spectra. In addition, a high resolution ESR spectrum for the protease-treated MNP cytochrome c-derived protein radical adduct was obtained. The superhyperfine couplings detected in this spectra were identical to those detected from an authentic MNP/tyrosyl adduct. Carbon-13 labeling of the aromatic ring positions of tyrosine yielded additional hyperfine coupling, demonstrating that the radical site was definitely located on the ring of tyrosine. Mass spectrometry detected as many as four DBNBS/.cytochrome c-derived adducts from the reaction of cytochrome with H2O2. Thus, it would appear four radical sites are formed during the reaction, at least one of which is tyrosine.

Self-peroxidation of metmyoglobin results in formation of an oxygen-reactive tryptophan-centered radical.

In the reaction between hydrogen peroxide and metmyoglobin, the heme iron is oxidized to its ferryl-oxo form and the globin to protein radicals, at least one of which reacts with dioxygen to form a peroxyl radical. To identify the residue(s) that forms the oxygen-reactive radical, we utilized electron spin resonance (ESR) spectroscopy and the spin traps 2-methyl-2-nitrosopropane and 3,5-dibromo-4-nitrosobenzenesulfonic acid (DB-NBS). Metmyoglobin radical adducts had spectra typical of immobilized nitroxides that provided little structural information, but subsequent nonspecific protease treatment resulted in the detection of isotropic three-line spectra, indicative of a radical adduct centered on a tertiary carbon with no bonds to nitrogen or hydrogen. Similar isotropic three-line ESR spectra were obtained by spin trapping the oxidation product of tryptophan reacting with catalytic metmyoglobin and hydrogen peroxide. High resolution ESR spectra of DBNBS/.trp and of the protease-treated DBNBS/.metMb were simulated using superhyperfine coupling to a nitrogen and three non-equivalent hydrogens, consistent with a radical adduct formed at C-3 of the indole ring. Oxidation of tryptophan by catalytic metMb and hydrogen peroxide resulted in spin trap-inhibitable oxygen consumption, consistent with formation of a peroxyl radical. The above results support self-peroxidation of a tryptophan residue in the reaction between metMb and hydrogen peroxide.

Hydroxyl radical formation from cuprous ion and hydrogen peroxide: a spin-trapping study.

Copper toxicity has been presumed to involve catalytic hydroxyl radical (.OH) formation from hydrogen peroxide. Addition of Cu1+ to a solution containing ethanol or dimethylsulfoxide (Me2SO) and the spin-trapping agent alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) results in formation of the alpha-hydroxyethyl radical or methyl radical adduct of 4-POBN, respectively. Adduct formation was prevented by inclusion of catalase, but not by superoxide dismutase. Inclusion of exogenous H2O2 in the reaction mixture increased the yield of ethanol- or Me2SO-derived radical adduct and also enhanced the formation of secondary radical adducts, including 4-POBN/.H and the methyl radical adduct of 2-methyl-2-nitrosopropane. The alpha-hydroxyethyl adduct of 4-POBN is rapidly decomposed in the presence of copper, but not iron salts, whereas the methyl radical adduct is relatively stable in the presence of inorganic copper. The total concentration of radical adduct detected from the reaction between Cu1+ and H2O2, determined by comparison of the integrated spectral intensity with that of the stable 2,2,6,6-tetramethyl-1-piperidinyloxy free radical, was only 1-5% of the maximum amount predicted assuming radical adduct formation from all of the added copper. A variety of copper chelators inhibit formation of carbon-centered radical adducts of 4-POBN, including penicillamine and triethylenetetramine, which are the primary drugs used to treat the copper metabolism disorder Wilson's disease. The results provide clear evidence for hydroxyl radical formation from Cu1+ and H2O2 (either added or formed during the autoxidation of reduced copper.

Variation in hydrogen peroxide sensitivity between different strains of Neisseria gonorrhoeae is dependent on factors in addition to catalase activity.

Catalase, which catalyzes the reduction of hydrogen peroxide to oxygen and water, is considered the primary defense of Neisseria gonorrhoeae against exogenous hydrogen peroxide. Recent reports have demonstrated drastically different sensitivities of the organism to hydrogen peroxide ranging from greater than 80% survival after challenge with 30 mM hydrogen peroxide to less than 0.001% survival after challenge with 10 mM hydrogen peroxide. In this study, we have examined the hydrogen peroxide sensitivities of six clinical gonococcal isolates. The study demonstrates that the variations in gonococcal hydrogen peroxide sensitivities previously reported can be attributed to (i) differences in experimental methods employed or (ii) variation among different gonococcal strains. All of the gonococcal isolates examined generated similar concentrations of catalase, implying that the differences in the H2O2 sensitivity observed may depend on factors in addition to catalase.

Effect of NPC 15669, an inhibitor of neutrophil recruitment and neutrophil-mediated inflammation, on neutrophil function in vitro.

The new anti-inflammatory agent N-[9H-(2,7-dimethylfluorenyl-9-methoxy)carbonyl]-L-leucine (NPC 15669) inhibits inflammation in several animal models dependent upon neutrophil activation and recruitment into the inflammatory lesion. NPC 15669 appears to elicit its pharmacological action by inhibiting the cell surface expression of CD11b/CD18 (Mac-1) on the neutrophil and subsequent adhesion of the neutrophil to the vascular endothelium. The current study sought to further characterize the action of NPC 15669 on neutrophil function. In the range of 1-100 microM, this fluorene enhanced superoxide production in a concentration-dependent fashion. Using spin trapping/ESR spectroscopy, NPC 15669 was found to inhibit myeloperoxidase (MPO)-dependent hydroxyl radical primarily by scavenging hypochlorous acid, and secondarily by inhibiting agonist-stimulated degranulation as assessed by MPO and elastase release. These studies demonstrated that NPC 15669, in addition to inhibiting adhesion, alters other neutrophil functions. Whether the pharmacological activities described for NPC 15669 resulted directly from changes in Mac-1 expression or through some other mechanism is currently under investigation.

Oxidant-scavenging activities of ampicillin and sulbactam and their effects on neutrophil functions.

Luminol-enhanced luminescence is a method used to measure formation of reactive oxygen intermediates important in the ability of neutrophils to kill microbes. Several studies have demonstrated that under some conditions of incubation, ampicillin can inhibit neutrophil-derived luminol-enhanced luminescence. We evaluated the mechanism(s) by which ampicillin inhibited the luminescent response of stimulated neutrophils. We also investigated sulbactam, a beta-lactamase inhibitor which has been given in combination with ampicillin and other beta-lactam antibiotics to increase their spectra, for possible similar effects. Both ampicillin and sulbactam attenuated luminol-enhanced luminescence by approximately 40%. Superoxide production was not prevented by added ampicillin, nor was superoxide scavenged by it. Myeloperoxidase reacts with H2O2 and Cl- to generate OCl-, which is believed to be the oxidizer of luminol that is primarily responsible for enhancement of neutrophil-derived luminescence. Hydroxyl radicals (HO.), which may also oxidize luminol, resulting in luminescence, can be formed from O2- and H2O2 via either myeloperoxidase-dependent (involving intermediate OCl-) or myeloperoxidase-independent (through a metal ion catalyst) reactions. Ampicillin scavenged H2O2 and OCl- and prevented 95% of Fenton reaction-generated HO. from reacting with 5,5-dimethyl-1-pyrroline-N-oxide. Sulbactam was found to scavenge OCl- and HO., but less avidly than ampicillin did. Neither ampicillin nor sulbactam inhibited myeloperoxidase activity. Sublethal concentrations of sulbactam had no significant effect on neutrophil killing of Staphylococcus aureus and Escherichia coli. Our results demonstrate a mechanism(s) by which ampicillin inhibits luminol-enhanced luminescence from stimulated neutrophils, namely, through scavenging of the oxidant(s) primarily responsible for the generation of luminescence.