Comparative study of the formation of oxidative damage marker 8-hydroxy-2'-deoxyguanosine (8-OHdG) adduct from the nucleoside 2'-deoxyguanosine by transition metals and suspensions of particulate matter in relation to metal content and redox reactivity

An association between exposure to ambient particulate matter (PM) and increased incidence of mortality and morbidity due to lung cancer and cardiovascular diseases has been demonstrated by recent epidemiological studies. Reactive oxygen species (ROS), especially hydroxyl radicals, generated by PM, have been suggested by many studies as an important factor in the oxidative damage of DNA by PM. The purpose of this study was to characterize quantitatively hydroxyl radical generation by various transition metals in the presence of H₂O₂ in aqueous buffer solution (pH 7.4) and hydroxylation of 2'-deoxyguanosine (dG) to 8-hydroxy-2'-deoxyguanosine (8-OHdG) under similar conditions. The order of metals' redox reactivity and hydroxyl radical production was Fe(II), V(IV), Cu(I), Cr(III), Ni(II), Co(II), Pb(II), Cd(II). Then, we investigated the generation of hydroxyl radicals in the presence of H₂O₂ by various airborne PM samples, such as total suspended particulate (TSP), PM₁₀, PM_2.5 (PM with aerodynamic diameter 10 and 2.5 μm), diesel exhaust particles (DEP), gasoline exhaust particles (GEP) and woodsmoke soot under the same conditions. When suspensions of PMs were incubated with H₂O₂ and dG at pH 7.4, all particles induced hydroxylation of dG and formation of 8-OHdG in a dose-dependent increase. Our findings demonstrated that PM's hydroxyl radical (HO√) generating ability and subsequent dG hydroxylation is associated with the concentration of water-soluble metals, especially Fe and V and other redox or ionizable transition metals and not their total metal content, or insoluble metal oxides, via a Fenton-driven reaction of H₂O₂ with metals. Additionally, we observed, by Electron paramagnetic resonance (EPR), that PM suspensions in the presence of H₂O₂ generated radical species with dG, which were spin-trapped by 2-methyl-2-nitroso-propane (MNP).     

Radical production and DNA damage induced by carcinogenic 4-hydrazinobenzoic acid, an ingredient of mushroom Agaricus bisporus

4-Hydrazinobenzoic acid, an ingredient of mushroom Agaricus bisporus, is carcinogenic to rodents. To clarify the mechanism of carcinogenesis, we investigated DNA damage by 4-hydrazinobenzoic acid using ³²P-labeled DNA fragments obtained from the human p53 and p16 tumor suppressor genes. 4-Hydrazinobenzoic acid induced Cu(II)-dependent DNA damage especially piperidine-labile formation at thymine and cytosine residues. Typical hydroxyl radical scavengers showed no inhibitory effects on Cu(II)-mediated DNA damage by 4-hydrazinobenzoic acid. Bathocuproine and catalase inhibited the DNA damage, indicating the participation of Cu(I) and H₂O₂ in the DNA damage. These findings suggest that H₂O₂ generated by the autoxidation of 4-hydrazinobenzoic acid reacts with Cu(I) to form reactive oxygen species, capable of causing DNA damage. Interestingly, catalase did not completely inhibit DNA damage caused by a high concentration of 4-hydrazinobenzoic acid (over 50 μM) in the presence of Cu(II). 4-Hydrazinobenzoic acid induced piperidine-labile sites frequently at adenine and guanine residues in the presence of catalase. 4-Hydrazinobenzoic acid increased formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a characteristic oxidative DNA lesion, in calf thymus DNA, whereas 4-hydrazinobenzoic acid did not increase the formation of 8-oxodG in the presence of catalase. ESR spin-trapping experiments showed that the phenyl radical was formed during the reaction of 4-hydrazinobenzoic acid in the presence of Cu(II) and catalase. Matrix-assisted laser desorption/ionization time-of-flight mass (MALDI-TOF/mass) spectrometry analysis showed that phenyl radical formed adduct with adenosine and guanosine. These results suggested that 4-hydrazinobenzoic acid induced DNA damage via not only H₂O₂ production but also phenyl radical production. This study suggests that both oxidative DNA damage and DNA adduct formation play important roles in the expression of carcinogenesis of 4-hydrazinobenzoic acid.     

Metabolic activation of carcinogenic ethylbenzene leads to oxidative DNA damage

Ethylbenzene is carcinogenic to rats and mice, while it has no mutagenic activity. We have investigated whether ethylbenzene undergoes metabolic activation, leading to DNA damage. Ethylbenzene was metabolized to 1-phenylethanol, acetophenone, 2-ethylphenol and 4-ethylphenol by rat liver microsomes. Furthermore, 2-ethylphenol and 4-ethylphenol were metabolically transformed to ring-dihydroxylated metabolites such as ethylhydroquinone and 4-ethylcatechol, respectively. Experiment with ³²P-labeled DNA fragment revealed that both ethylhydroquinone and 4-ethylcatechol caused DNA damage in the presence of Cu(II). These dihydroxylated compounds also induced the formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine in calf thymus DNA in the presence of Cu(II). Catalase, methional and Cu(I)-specific chelator, bathocuproine, significantly (P < 0.05) inhibited oxidative DNA damage, whereas free hydroxyl radical scavenger and superoxide dismutase did not. These results suggest that Cu(I) and H₂O₂ produced via oxidation of ethylhydroquinone and 4-ethylcatechol are involved in oxidative DNA damage. Addition of an endogenous reductant NADH dramatically enhanced 4-ethylcatechol-induced oxidative DNA damage, whereas ethylhydroquinone-induced DNA damage was slightly enhanced. Enhancing effect of NADH on oxidative DNA damage by 4-ethylcatechol may be explained by assuming that reactive species are generated from the redox cycle. In conclusion, these active dihydroxylated metabolites would be involved in the mechanism of carcinogenesis by ethylbenzene.     

Hydrogen Peroxide Dependent Oxidative Degradation of DNA by Copper Epinephrine

The hydrogen peroxide dependent oxidation of the epinephrinecopper complex to adrenochrome is mediated by free copper ions. The oxidation is enhanced by chloride ions and by the presence of serum albumin. The reaction is not inhibited by SOD or by hydroxyl radical scavengers.

The 2:1 epinephrine or dopamine:Cu(II) complexes are able to bind to DNA and to catalyze its oxidative destruction in the presence of hydrogen peroxide. The DNA-epinephrine-Cu(II) terenary complex has characteristic spectral properties. It has the capacity to catalyze the reduction of oxygen or H₂O₂ and it preserves the capacity over a wide range of comp1ex:DNA ratios. The rate of DNA cleavage is proportional to the rate of epinephrine oxidation and the rate determining step of the reaction Seems to be the reduction of free Cu(II) ions. The ability to form redox active stable DNA ternary complexes, suggests that under specific physiological conditions, when “free” copper ions are available. catecholamina may induce oxidative degradation of DNA and other biological macromolecules.     

Catechins induce oxidative damage to cellular and isolated DNA through the generation of reactive oxygen species

Green tea catechins have antimutagenic and anticarcinogenic activities. On the other hand, several epidemiological studies have indicated significant positive relationship between green tea consumption and cancer. Catechins enhance colon carcinogenesis in rats initiated with chemical carcinogen. To clarify the mechanism underlying the potential carcinogenicity, we investigated the DNA-damaging ability of catechins in human cultured cells. Catechin increased the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a characteristic oxidative DNA lesion, in human leukemia cell line HL-60 but not in HP100, a hydrogen peroxide (H

2

O

2

)-resistant cell line derived from HL-60. The catechin-induced formation of 8-oxodG in HL-60 cells significantly decreased by bathocuproine. Furthermore, we investigated DNA damage and its site-specificity induced by catechins, using

32

P-labeled DNA fragments. Catechin and epicatechin induced extensive DNA damage in the presence of Cu(II). Catechin caused piperidine-labile sites at thymine and cytosine residues in the presence of Cu(II). Catalase and bathocuproine inhibited the DNA damage, indicating the involvement of H

2

O

2

and Cu(I). NADH enhanced catechins plus Cu(II)-induced 8-oxodG formation in calf thymus DNA, suggesting the redox cycle between catechins and their corresponding quinones, the oxidized forms of catechins. The DNA-damaging ability of epicatechin is stronger than that of catechin, possibly due to the greater turnover frequency of the redox cycle. The difference in their redox properties could be explained by their redox potentials estimated form an ab initio molecular orbital calculation. The present study demonstrated that catechins could induce metal-dependent H

2

O

2

generation during the redox reactions and subsequently damage to cellular and isolated DNA. Therefore, it is reasonably considered that green tea catechins may have the dual function of anticarcinogenic and carcinogenic potentials.     

Bleomycin-Iron Damage To Dna With Formation Of 8-Hydroxydeoxyguanosine and Base Propenals. Indications That Xanthine Oxidase Generates Superoxide From Dna Degradation Products

Bleomycin, in the presence of ferric salts, oxygen and a suitable reductant, degrades DNA with the release of base propenals, detected as thiobarbituric acid (TBA) reactivity, and the formation of 8-hydroxydeo-xyguanosine (80HdG) detected by HPLC. When xanthine oxidase is added to the incubated mixture of DNA degradation products, TBA-reactivity is destroyed but 80HdG formation is increased. EPR Spin trapping experiments show that hydroxyl radicals (OH) are formed in the reaction mixture and can be inhibited by the inclusion of either superoxide dismutase or catalase. These findings suggest that the base propenals and possibly malondialdehyde, formed from them, are aldehydic substrates for xanthine oxidase and, the product of this reaction is superoxide (O₂^-) and hydrogen peroxide (H₂O₂). Thus, TBA reactivity is destroyed in the formation of O₂^- and H₂O₂ which stimulate further oxidative damage to DNA resulting in increased 8OHdG formation.     

Superoxide,Hydrogen Peroxide and Hydroxyl Radical in D1/D2/cytochrome b-559 Photosystem II Reaction Center Complex

Spin-trapping electron spin resonance (ESR) was used to monitor the formation of superoxide and hydroxyl radicals in D1/D2/cytochrome b-559 Photosystem II reaction center (PS II RC) Complex. When the PS II RC complex was strongly illuminated, superoxide was detected in the presence of ubiquinone. SOD activity was detected in the PS II RC complex. A primary product of superoxide, hydrogen peroxide, resulted in the production of the most destructive reactive oxygen species, *OH, in illuminated PS II RC complex. The contributions of ubiquinone, SOD and H(2)O(2) to the photobleaching of pigments and protein photodamage in the PS II RC complex were further studied. Ubiquinone protected the PS II RC complex from photodamage and, interestingly, extrinsic SOD promoted this damage. All these results suggest that PS II RC is an active site for the generation of superoxide and its derivatives, and this process protects organisms during strong illumination, probably by inhibiting more harmful ROS, such as singlet oxygen.     

Nickel (II) Complexes of Histidyl-Peptides as Fenton-Reaction Catalysts

Addition of histidyl-peptides containing the glycyl-glycyl-L-histidyl sequence stimulated the catalysis of Ni(II) hydrogen peroxide reduction. Maximum bleaching of murexide or nitrosodimethylaniline was obtained with glycyl-glycyl-L-histidine. A decrease in the bleaching rates was observed upon addition of SOD or hydroxyl radical scavengers, showing that the hydrogen peroxide/Ni(II)/glycyl-glycyl-L-histidine system generated superoxide anions as well as hydroxyl radicals. In contrast, addition of glycyl-glycyl-L-histidine inhibited the Cu(II) hydrogen peroxide reduction.

When peptides or proteins were exposed to oxygen radicals produced by Ni(II)/glycyl-glycyl-L-histidine catalysis of hydrogen peroxide reduction, the observed effects were similar to those produced by oxygen radicals generated by water radiolysis or by Fe(II) or Cu(II) mediated Fenton-reactions: hydroxylation of phenylalanine, interchange of disulfides, destruction of tryptophans and dityrosine formation.     

Thiourea protects against copper-induced oxidative damage by formation of a redox-inactive thiourea-copper complex

Although thiourea has been used widely to study the role of hydroxyl radicals in metal-mediated biological damage, it is not a specific hydroxyl radical scavenger and may also exert antioxidant effects unrelated to hydroxyl radical scavenging. Thus, we investigated the effects of thiourea on copper-induced oxidative damage to bovine serum albumin (1 mg/ml) in three different copper-containing systems: Cu(II)/ascorbate, Cu(II)/H₂O₂, and Cu(II)/H₂O₂/ascorbate [Cu(II), 0.1 mM; ascorbate, 1 mM; H₂O₂, 1 mM]. Oxidative damage to albumin was measured as protein carbonyl formation. Thiourea (0.1–10 mM) provided marked and dose-dependent protection against protein oxidation in all three copper-containing systems. In contrast, only minor protection was observed with dimethyl sulfoxide and mannitol, even at concentrations as high as 100 mM. Strong protection was also observed with dimethylthiourea, but not with urea or dimethylurea. Thiourea also significantly inhibited copper-catalyzed oxidation of ascorbate, and competed effectively with histidine and 1,10-phenanthroline for binding of cuprous, but not cupric, copper, as demonstrated by both UV-visible and low temperature electron spin resonance measurements. We conclude that the protection by thiourea against copper-mediated protein oxidation is not through scavenging of hydroxyl radicals, but rather through the chelation of cuprous copper and the formation of a redox-inactive thiourea-copper complex.     

Nickel (II) Complexes of Histidyl-Peptides as Fenton-Reaction Catalysts

Addition of histidyl-peptides containing the glycyl-glycyl-L-histidyl sequence stimulated the catalysis of Ni(II) hydrogen peroxide reduction. Maximum bleaching of murexide or nitrosodimethylaniline was obtained with glycyl-glycyl-L-histidine. A decrease in the bleaching rates was observed upon addition of SOD or hydroxyl radical scavengers, showing that the hydrogen peroxide/Ni(II)/glycyl-glycyl-L-histidine system generated superoxide anions as well as hydroxyl radicals. In contrast, addition of glycyl-glycyl-L-histidine inhibited the Cu(II) hydrogen peroxide reduction.

When peptides or proteins were exposed to oxygen radicals produced by Ni(II)/glycyl-glycyl-L-histidine catalysis of hydrogen peroxide reduction, the observed effects were similar to those produced by oxygen radicals generated by water radiolysis or by Fe(II) or Cu(II) mediated Fenton-reactions: hydroxylation of phenylalanine, interchange of disulfides, destruction of tryptophans and dityrosine formation.     

Hydroxyl Radical Generation in the Nadh/Microsomal Reduction of vanadate

ESR spin trapping measurements demonstrate generation of hydroxyl (.OH) radical from reduction of vanadate by rat liver microsomes/NADH without exogenous H₂O₂. Catalase decreases the .OH signal while increasing a vanadium(4+) signal. Addition of superoxide dismutase (SOD) or measurements under an argon atmosphere show decreased .OH radical production. The results suggest that during the one-electron vanadate reduction process by microsomes/NADH, molecular oxygen is reduced to H₂O₂, which then reacts with vanadium (4+) to generate .OH radical via a Fenton-like mechanism.     

Role of Hydroxyl Radicals in Escherzchza Colz Killing Induced by Hydrogen Peroxide

Escherichia coli lethality by hydrogen peroxide is characterized by two modes of killing. In this paper we have found that hydroxyl radicals (OH -) generated by H₂O₂ and intracellular divalent iron are not involved in the induction of mode one lethality (i.e. cell killing produced by concentrations of H₂O₂ lower than 2.5 mM). In fact, the OH radical scavengers, thiourea, ethanol and dimethyl sulfoxide, and the iron chelator, desferrioxarnine, did not affect the survival of cells exposed to 2.5mM H₂O₂. In addition cell vulnerability to the same H₂O₂ concentration was independent on the intracellular iron content. In contrast, mode two lethality (i.e. cell killing generated by concentrations of H₂O₂ higher than 10mM) was markedly reduced by OH radical scavengers and desferrioxamine and was augmented by increasing the intracellular iron content.

It is concluded that OH. are required for mode two killing of E. coli by hydrogen peroxide.     

The effects of chelating agents on radical generation in alkaline peroxide systems, and the relevance to substrate damage

A spin-trapping EPR technique has been employed to explore the generation of hydroxyl radicals from reactions between a series of first row transition metal ions and aqueous hydrogen peroxide at pH 10, and with a range of chelating agents (EDTA, DTPMP and the readily biodegradable ligands S,S-EDDS and IDS). In the absence of these chelating agents only Cu(II) generates a significant level of hydroxyl radicals; in their presence with Cu(II) EDTA and IDS give similar behaviour whereas EDDS and DTPMP inhibit hydroxyl radical generation. For Fe(II), EDTA, DTPMP and IDS significantly enhance ^√OH production under these conditions whereas EDDS does not. Results from model cellulose damage experiments broadly confirm the findings for copper, though experiments with Fe(II) lead to somewhat contrasting results. Our findings are discussed in terms of binding constants and implications for alkaline peroxygen bleaching systems.     

Aspirin potently inhibits oxidative DNA strand breaks: implications for cancer chemoprevention

Epidemiological studies have suggested that the use of aspirin is associated with a decreased incidence of human malignancies, particularly colorectal cancer. Since reactive oxygen species (ROS) are critically involved in multistage carcinogenesis, this study was undertaken to examine the ability of aspirin to inhibit ROS-mediated DNA damage. Hydrogen peroxide (H2O2)+Cu(II) and hydroquinone (HQ) + Cu(II) were used to cause oxidative DNA strand breaks in phiX-174 plasmid DNA. We demonstrated that the presence of aspirin at concentrations (0.5-2 mM) compatible with amounts in plasma during chronic anti-inflammatory therapy resulted in a marked inhibition of oxidative DNA damage induced by either H2O2/Cu(II) or HQ/Cu(II). The inhibition of oxidative DNA damage by aspirin was exhibited in a concentration-dependent manner. Moreover, aspirin was found to be much more potent than the hydroxyl radical scavengers, mannitol and dimethyl sulfoxide, in protecting against the H2O2/Cu(II)-mediated DNA strand breaks. Since the reduction of Cu(II) to Cu(I) is crucially involved in both H2O2/Cu(II)- and HQ/Cu(II)-mediated formation of hydroxyl radical or its equivalent, and the subsequent oxidative DNA damage, we examined whether aspirin could inhibit this Cu(II)/Cu(I) redox cycle. It was observed that aspirin at concentrations that showed the inhibitory effect on oxidative DNA damage did not alter the Cu(II)/Cu(I) redox cycle in either H2O2/Cu(II) or HQ/Cu(II) system. In addition, aspirin was not found to significantly scavenge H2O2. This study demonstrates for the first time that aspirin potently inhibits both H2O2/Cu(II)- and HQ/Cu(II)-mediated oxidative DNA strand breaks most likely through scavenging the hydroxyl radical or its equivalent derived from these two systems. The potent inhibition of oxidative DNA damage by aspirin may thus partially contribute to its anticancer activities observed in humans.     

Fenton反应及其可能的活性产物

活性氧对许多生物分子,如脂质、蛋白质和DNA等均可引起损伤,它与许多疾病过程相联系.由超氧阴离子自由基和过氧化氢所引起的许多损伤被认为与它们转变为反应活性更强的组分有关,这些组分包括羟自由基及可能的高价铁组分.实验材料及理论结果表明,当铁盐与过氧化氢混合时,除羟自由基产生以外,高价铁组分也被认为同时产生.Fenton试剂的活性中间体是一亲核加合物,其反应活性及其产物不同于游离态羟自由基的反应活性及产物.Fenton试剂的产物分布依赖于不同的过渡金属离子、不同的配位体、不同的反应底物以及不同的溶剂基体效应.     

Cleavage of supercoiled circular double-stranded DNA induced by a eukaryotic cambialistic superoxide dismutase from Cinnamomum camphora

Superoxide dismutase (SOD, EC 1.15.1.1) that protectsorganisms from O2?– toxicity is a family of transitionmetal-containing enzyme existing in all oxygen-consu-ming living beings [1,2]. SOD catalyzes the dismutationof the toxic superoxide anion O2?– int…     

8-Hydroxydeoxyguanosine in DNA from TPA-Stimulated Human Granulocytes

8-Hydroxydeoxyguanosine (8-OHdG) is now widely used as a sensitive marker of oxidative damage to DNA. When human granulocytes are stimulated with TPA, they release a large quantity of reactive oxygen species (superoxide, hydrogen peroxide) which might be expected to generate hydroxyl radicals (OH-) which in turn could produce 8-OHdG in the DNA. There had been considerable debate as to whether OH -is detectable in stimulated granulocytes; most workers now agree that none can be detected, unless exogenous iron is added. An earlier report had described that 8-OHdG (a marker of OH -) was increased in the DNA of TPA-stimulated, compared to control, granulocytes. We have repeated this experiment and have been unable to reproduce this Finding. We conclude that the amount of 8-OHdG produced in the DNA of TPA-stimulated human ganulocytes is indistinguishable from that seen in control (unstimulated) cells (less than one 8- OHdG/10⁵ dG).     

Site-specific DNA damage induced by hydrazine in the presence of manganese and copper ions. The role of hydroxyl radical and hydrogen atom.

The mechanism of DNA damage by hydrazine in the presence of metal ions was investigated by DNA sequencing technique and ESR-spin trapping method. Hydrazine caused DNA damage in the presence of Mn(III), Mn(II), Cu(II), Co(II), and Fe(III). The order of inducing effect on hydrazine-dependent DNA damage (Mn(III) greater than Mn(II) approximately Cu(II) much greater than Co(II) approximately Fe(III)) was related to that of the accelerating effect on the O2 consumption rate of hydrazine autoxidation. DNA damage by hydrazine plus Mn(II) or Mn(III) was inhibited by hydroxyl radical scavengers and superoxide dismutase, but not by catalase. On the other hand, bathocuproine and catalase completely inhibited DNA damage by hydrazine plus Cu(II), whereas hydroxyl radical scavengers and superoxide dismutase did not. Hydrazine plus Mn(II) or Mn(III) caused cleavage at every nucleotide with a little weaker cleavage at adenine residues, whereas hydrazine plus Cu(II) induced piperidine-labile sites frequently at thymine residues, especially of the GTC sequence. ESR-spin trapping experiments showed that hydroxyl radical is generated during the Mn(III)-catalyzed autoxidation of hydrazine, whereas hydrogen atom adducts of spin trapping reagents are generated during Cu(II)-catalyzed autoxidation. The results suggest that hydrazine plus Mn(II) or Mn(III) generate hydroxyl free radical not via H2O2 and that this hydroxyl free radical causes DNA damage. A possibility that the hydrogen atom releasing compound participates in hydrazine plus Cu(II)-induced DNA damage is discussed.     

Epr Spectra of Dmpo Spin Adducts of Superoxide and hydroxyl Radicals in Pyridine

Electron spin resonance spectroscopy and the spin trapping technique were used to study the formation of the superoxide radical in pyridine. 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) was employed as a trapping agent. Superoxide radical was generated using chemical (potassium superoxide) and photochemical methods with anthralin, benzanthrone, rose bengal, 1,8-dihydroxyanthraquinone and zinc tetraphenylporphyrine as photoactive pigments. Hyperfine coupling (hf) constants for DMPO/O₂- were determined to be a_N = 12.36 G, a^β_H= 9.85G, a^γ_H = 1.34 G. The a_N and a^β_H constants are in good agreement with values calculated from a previously determined relationship between hf constants and solvent acceptor number (Reszka et al., (1992) Free Radical Res. Commun., in press). When concentrated hydrogen peroxide was added to DMPO in pyridine a similar EPR spectrum was observed. It is suggested that in this case the DMPO/'O₂H adduct is formed by nucleophilic addition of H₂O₂ to DMPO to give a hydroxylamine, followed by oxidation to the respective nitroxide. The EPR spectrum observed when tetrapropylammonium hydroxide and H₂O₂ were added to DMPO in pyridine had hf couplings a_N = 13.53 G, a^β_H = 11.38 G, a^γ_H = 0.79 G and it was assigned to a DMPO/'OH adduct. This assignment was based on similarity of this spectrum to the one produced by UV photolysis of hydrogen peroxide and DMPO in aqueous solution and subsequent transfer to pyridine.     

The Use of Water-Soluble Radical Scavengers to Detect Hydroxyl Radical Formation by Polymorphonuclear Leukocytes

The polymorphonuclear leukocyte secretes both O₂-and H₂O₂ when stimulated by various soluble or particulate stimuli. Since a rcaction involving iron, O₂-, and H₂O₂ could generate the hydroxyl radical (HO.) there has been speculation that the HO-may participate in the bactericidal activity of the neutroph-il. A variety of water-soluble HO. scavengers have been used to test for the participation of HO. and the results imply that HO. might participate. However, other workers have not been able to detect the formation of significant amounts of HO-by the activated neutrophil. We have examined the effect of several commonly used HO. radical scavengers on the ability of the neutrophil to secrete O₂-and H₂O₂. Several of these compounds actively inhibit secretion without affecting the viability of the neutrophil. After considering the various complications inherent in using water soluble radical scavengers, we suggest that they only be used with well defined experimental systems.