Differential sensitivities of plant and animal mitochondria to the herbicide paraquat

Paraquat herbicide is toxic to animals, including humans, via putative toxicity mechanisms associated to microsomal and mitochondrial redox systems. It is also believed to act in plants by generating highly reactive oxygen free radicals from electrons of photosystem I on exposure to light. Paraquat also acts on non-chlorophyllous plant tissues, where mitochondria are candidate targets, as in animal tissues. Therefore, we compared the interaction of paraquat with the mitochondrial bioenergetics of potato tuber, using rat liver mitochondria as a reference. Paraquat depressed succinate-dependent mitochondrial Delta(psi), with simultaneous stimulation of state 4 O2 consumption. It also induced a slow time-dependent effect for respiration of succinate, exogenous NADH, and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD)/ascorbate, which was more pronounced in rat than in potato mitochondria. However, with potato tuber mitochondria, the Delta(psi) promoted by complex-I-dependent respiration is insensitive to this effect, indicating a protection against paraquat radical afforded by complex I redox activity, which was just the reverse of to the findings for rat liver mitochondria. The experimental set up with the tetraphenyl phosphonium (TPP+)-electrode also indicated production of the paraquat radical in mitochondria, also suggesting its accessibility to the outside space. The different activities of protective antioxidant agents can contribute to explain the different sensitivities of both kinds of mitochondria. Values of SOD activity and alpha-tocopherol detected in potato mitochondria were significantly higher than in rat mitochondria, which, in turn, revealed higher values of lipid peroxidation induced by paraquat.     

Light-dependent spin trapping of hydroxyl radical from human erythrocytes.

The generation of reactive oxygen species from human erythrocytes has previously been demonstrated. Furthermore, erythrocytic protoporphyrin IX has been shown to generate superoxide and singlet oxygen when exposed to light. These findings suggest that a component of erythrocytic reactive oxygen species production may be light-dependent. By inhibiting erythrocyte superoxide dismutase, catalase, and glutathione peroxidase with N,N-diethyldithiocarbamate or sodium cyanide, we demonstrate the light-dependent generation of hydroxyl radical in human erythrocytes using spin trapping/Electron Spin Resonance spectroscopy. This finding may be significant in tissues where blood is exposed to light, such as in the eye.     

The adjustment of photosynthetically grown cells of Rhodospirillum rubrum to aerobic light conditions

Summary The growth of photosynthetically precultured cells of Rhodospirillum rubrum under aerobic condition in light is investigated. Special emphasis is given to the question of whether the photosynthetic electron transport chain is influenced under these conditions. Light-induced absorbance changes under anaerobic conditions show that although in whole cells a variation can be noted, the reactions of isolated membranes decrease only very slowly and parallel to each other. The photophosphorylation activity remains constant on a bacteriochlorophyll basis. On a cell mass basis this activity decreases parallel to the decreasing bacteriochlorophyll content. Light-dependent NAD⁺ reduction by ascorbate-DCPI remains constant on a bacteriochlorophyll basis, whereas succinate supported NAD⁺ reduction in light increases. On a cell mass basis the activity of succinate supported NAD⁺ reduction stays nearly constant, thus showing similar responses to the presence of oxygen in light as the NADH oxidase system. NADH oxidase activity increases on a bacteriochlorophyll basis and does not change on a cell mass basis. Parallel to the NADH-oxidase system, oxygen uptake in the dark by whole cells does not change after aerobiosis in light. Light inhibits respiration even after several generations of growth in the presence of oxygen; however, the inhibition decreases slowly. Light inhibition of respiration can be totally overcome by the addition of the uncoupler CCCP. These results indicate that light-dependent electron transport is not influenced significantly by the presence of oxygen. Although the respiratory system is formed, cells preferentially grow photosynthetically. Respiration takes over when the amount of bacteriochlorophyll reaches very low values.Abbreviations ADP adenosine diphosphate - ATP adenosine triphosphate - BChl bacteriochlorophyll - CCCP carbonylcyanide-m-chlorophenyl hydrazone - DCPI Na-2,6-dichlorophenol-indophenol - NAD(P)⁺ nictotinamide-adenine-di; nucleotide (phosphate) - NADPH reduced NAD(P)⁺ - TMPD N,N,N-N-tetra; methyl-p-phenyldiamine     

Effects of Acifluorfen on Endogenous Antioxidants and Protective Enzymes in Cucumber (Cucumis sativus L.) Cotyledons

The herbicide acifluorfen (2-chloro-4-(trifluoromethyl)phenoxy-2-nitrobenzoate) causes strong photooxidative destruction of pigments and lipids in sensitive plant species. Antioxidants and oxygen radical scavengers slow the bleaching action of the herbicide. The effect of acifluorfen on glutathione and ascorbate levels in cucumber (Cucumis sativus L.) cotyledon discs was investigated to assess the relationship between herbicide activity and endogenous antioxidants. Acifluorfen decreased the levels of glutathione and ascorbate over 50% in discs exposed to less than 1.5 hours of white light (450 microeinsteins per square meter per second). Coincident increases in dehydroascorbate and glutathione disulfide were not observed. Acifluorfen also caused the rapid depletion of ascorbate in far-red light grown plants which were photosynthetically incompetent.

Glutathione reductase, dehydroascorbate reductase, superoxide dismutase, ascorbate oxidase, ascorbate free radical reductase, peroxidase, and catalase activities rapidly decreased in acifluorfen-treated tissue exposed to white light. None of the enzymes were inhibited in vitro by the herbicide. Acifluorfen causes irreversible photooxidative destruction of plant tissue, in part, by depleting endogenous antioxidants and inhibiting the activities of protective enzymes.

     

Lucigenin-enhanced chemiluminescence of the animal tissues

Lucigenin-enhanced chemiluminescence (LcCL) allows one to investigate the reactions of superoxide anion radical (*O2-) generated by mitochondria and is applied to study the superoxide production in enzymatic and membrane systems by isolated mitochondria and cells, and in whole organs. The application of lucigenin-enhanced chemiluminescence to estimate the respiration of human tissues involves the use of small tissue pieces, which can be obtained, for instance, by biopsia; however, no systematic investigations have been performed on these objects. In the present paper, a comparative study of lucigenin-enhanced chemiluminescence of tissues isolated from different organs of the rat was carried out to elucidate its dependence on the extent of tissue defragmentation, storage time, and access for oxygen. It was shown that the addition of lucigenin to a piece of tissue, a suspension of fine tissue fragments, and homogenates greatly enhanced chemiluminescence, and a whole piece of tissue possessed a much lesser (by 1-1.5 order of magnitude) intensity of chemiluminescence than homogenate or gruel. In the absence of stirring of the surrounding solution, the lucigenin-enhanced chemiluminescence of tissue quickly decreased, apparently due to a decrease in the level of oxygen in the tissue, as the result of its consumption. The chemiluminescence consisted of two components: a lucigenin-dependent and lucigenin-independent one (intrinsic chemiluminescence). Thus, the tissue was a source of lucigenin-enhanced chemiluminescence, and this luminescence was observed only at a sufficient access for oxygen. The lucigenin-independent component did not practically depend on oxygen and was determined by the components coming out of the tissue into the surrounding solution. Nitric oxide (NO) inhibited chemiluminescence as its concentration increased and did not affect considerably the rate of oxygen consumption by the tissue. The results obtained allow one to conclude that lucigenin can be used as a rather effective chemiluminescent probe for the production of superoxide radicals by tissue pieces.     

The one-electron reduction of uroporphyrin I by rat hepatic microsomes

Uroporphyrin I, which accumulates in body tissues of congenital erythropoietic porphyria patients, can undergo an enzymatic one-electron reduction to the porphyrin anion radical when a suitable reducing cofactor is present. We have demonstrated, in the absence of light, that anaerobic microsomal incubations containing NADPH and uroporphyrin I give an electron spin resonance spectrum consistent with the enzymatic formation of a porphyrin anion free radical. This radical undergoes a second-order decay (k2 approximately 10(5) M-1 s-1) due to nonenzymatic disproportionation of the radical. Aerobic microsomal incubations were also investigated for the reduction of oxygen to superoxide by monitoring oxygen consumption and the spin-trapping of superoxide. These experiments demonstrate that electron transfer from the porphyrin radical to molecular oxygen does occur, but due to the slow formation of the radical anion, no oxygen consumption above the basal level could be detected in the microsomal incubations. The photoreduction of uroporphyrin I in aerobic and anaerobic incubations was also investigated.     

The generation of singlet oxygen (o(2)) by the nitrodiphenyl ether herbicide oxyfluorfen is independent of photosynthesis

The mechanism of action of the p-nitrodiphenyl ether herbicides has remained ambiguous because of conflicting reports in the literature. The diphenyl ether herbicide oxyfluorfen causes a light induced consumption of oxygen which resembles the electron acceptor reaction of paraquat. However, this reaction is not linked to the transport of electrons through photosystem I. This conclusion is based on the observation that the rate of oxygen consumption, in the presence of oxyfluorfen, does not demonstrate a first order rate dependence on light intensity. Using the bleaching of N,N-dimethyl p-nitrosoaniline as a specific detector of singlet oxygen, we demonstrate that oxyfluorfen is a potent generator of this toxic radical. The production of singlet oxygen occurs in the presence of inhibitors of photosynthetic electron transport (oxyfluorfen at 10⁻⁴ molar and paraquat) and also under temperature conditions (3°C) which prevent electron transport. This light induced reaction results in oxygen consumption and is the primary cause of lethality for oxyfluorfen. The production of singlet oxygen occurs rapidly and at low herbicide concentrations (10⁻⁹ molar). The reaction occurs without photosynthetic electron transport but does require an intact thylakoid membrane.     

Role of reactive oxygen species and Cr(VI) in Ras-mediated signal transduction

Previous studies have shown that a constitutively active isoform of Ras is able to produce superoxide radical (O2(-)). The present study investigate the mechanisms by which O2(-) radical mediates signals from Ras protein to the nucleus, leading to cellular responses such as apoptosis in Cr(VI)-stimulated cells. Two human prostate tumor cell lines, Ras(+), which overexpresses Ras, and Ras(-), which has a normal Ras level, were utilized. Compared to Ras(-) cells, Ras(+) cells exhibited higher susceptibility to apoptosis induced by Cr(VI). Catalase, sodium formate, and deferoxamine inhibited Cr(VI)-induced apoptosis. Similar differences were observed in both cellular DNA damage and the activation of p53 protein. The differences in Cr(VI)-induced cell responses in Ras(+) and Ras(-) cells were due to differences in the generation of free radicals between these two cells. ESR spin trapping measurements showed that Ras(+) cells generated more hydroxyl radical ((.)OH), O2(-) radical, and Cr(V) than Ras(-) cells following Cr(VI) stimulation. The generation of the reactive oxygen species (ROS) can be abolished by the addition of superoxide dismutase (SOD) or if the experiment were carried out in an argon atmosphere. Catalase inhibited spin adduct signals but was much less potent than SOD. The mechanism of ROS generation in Cr(VI)-stimulated Ras(+) cells involves the reduction of molecular oxygen to O2(-) radical by a flavoenzyme-containing NADPH oxidase complex as shown by oxygen consumption and diphenylene iodonium (DPI) inhibition. Results shown above support the following conclusions: (a) Ras protein mediates O2(-) radical generation through reduction of molecular oxygen by NADPH oxidase in Cr(VI)-stimulated cells. (b) The O2(-) radical and Cr(VI) produce other reactive species, including H2O2, OH radical, and Cr(V) through O2(-) dismutation and Haber-Weiss type of reactions. (c) Among these reactive species, (.)OH radical is responsible for the further transduction of signals from Ras to the nucleus, leading to various cell responses.     

Active oxygen species in blue light mediated signal transduction in coleoptile tips

Coleoptile tip is a blue-light sensitive tissue possessing a "blue light receptor" which, upon activation, elicits a signal cascade resulting in phototropic curvature of the coleoptile. In this context, the nature of the photoreceptors and the exact mechanism through which the photoreceptors transduces the signal across the membrane are not clear. In this study, we attempted to examine whether the blue light receptor perturbs redox status of the coleoptile tip and sensitizes molecular oxygen as part of the signal reactions. Coleoptile tips of Sorghum bicolor and wheat (Triticum vulgare) grown in the dark showed pronounced ascorbate free radical signal, which diminished upon illumination with weak blue light for one minute. Concomitantly, the generation of superoxide radical by the coleoptile tip was augmented upon illumination with blue light. Various thiol blockers tested in this study caused powerful inhibition of blue light induced superoxide anion radical generation. Treatment with these thiol blockers, with the exception of NEM, resulted in marked increase in the levels of ascorbic acid free radical in the blue light irradiated coleoptiles. The blue light stimulated O*-2-generation by the coleoptile tip homogenate is also inhibited by the inhibitors of blue light responses viz phenylacetic acid, potassium iodide, and sodium azide. Based on our observations, we postulate that the activated blue light receptor present in the coleoptile tip sensitizes molecular oxygen to superoxide anion radical in the tip initializing the blue light signal cascade reactions.     

The effect of the herbicide 2,4,5 trichlorophenoxy acetic acid (245T) on the growth and metabolism of Tetrahymena pyriformis

The herbicide 2,4,5 trichlorophenoxy acetic acid (245T) at concentrations from 0.5 to 0.9 mM, was found to inhibit respiration and then growth in exponentially growing cultures of Tetrahymena pyriformis. Cell division was stopped for periods up to 60 minutes after which the cells recovered and division resumed. Recovery of oxygen utilization and cell division occurred in the presence of 245T. 245T was shown to inhibit mitochondrial oxygen utilization. Mitochondria from cells that had recovered from 245T treatment lost their sensitivity to low concentrations of the herbicide and sedimented deeper in a sucrose gradient than mitochondria from control cells.     

Effects of Optically Active 1-(α-Methylbenzyl)-3-(3,4-dichlorophenyl)urea on Reactions of Mitochondria and Chloroplasts

Effects of the R- and S-isomers and racemate of 1-(alpha-methylbenzyl)-3-(3,4-dichlorophenyl)urea (MBPU) were measured on phosphorylation and electron transport in mung bean (Phaseolus aureus L.) mitochondria and spinach (Spinacia oleracea L.) chloroplasts.In chloroplasts, S-MBPU inhibited basal and methylamine-uncoupled electron transport with ferricyanide as the oxidant, both photoreduction and coupled photophosphorylation with water as the electron donor and with ferricyanide and nicotinamide adenine dinucleotide phosphate (NADP) as oxidants, and cyclic photophosphorylation with phenazine methosulfate as the electron mediator under an argon gas phase. With ascorbate 2,6-dichloro-phenolindophenol as the electron donor, phosphorylation coupled to NADP reduction was inhibited, but the reduction of NADP was not inhibited. The R-isomer of MBPU, like the S-isomer, inhibited all of the photophosphorylation reactions studied. However, unlike the S-isomer, the R-isomer either did not inhibit or was a very weak inhibitor of all photoreduction reactions. The effects of the MBPUs on the chloroplast reactions can be explained by action at two different sites: an optically specific site near photosystem II and the oxygen evolution pathway, and a second optically nonspecific site associated with the generation of ATP.In mitochondria, both the R- and S-isomers stimulated state 4 respiration, inhibited state 3 respiration, and released oligomycin-inhibited respiration with malate, succinate, and NADH as substrates. Both enantiomers were equally active in all studies with malate and succinate as substrates. However, with NADH as substrate, R-MBPU was a stronger inhibitor of state 3 respiration and a weaker stimulator of state 4 respiration than S-MBPU.     

One electron reduction of CCl4 in oxygenated aqueous solutions: a CCl3O2-free radical mediated formation of Cl- and CO2

Product yields have been determined after one-electron-induced reduction of CCl4 in aqueous solutions containing t-butanol and various concentrations of oxygen. It was shown that CCl3 radicals add oxygen to form CCl3O2 radicals, which eventually yield three chloride ions and CO2. A constant ratio of G(Cl-)/G(CO2) = 4 is found in solutions containing 1.5 X 10(-4) M or more oxygen. Competing reactions of the CCl3 radical increase this ratio at lower oxygen concentrations. The rate constant for the oxygen addition to CCl3 radicals was determined by pulse radiolysis to 3.3 X 10(9) M-1 s-1. Possible reaction mechanisms leading to the observed end products are discussed.     

Interactions of the anesthetic nitrous oxide with bovine heart cytochrome c oxidase. Effects on protein structure, oxidase activity, and other properties

The interactions of nitrous oxide with cytochrome c oxidase isolated from bovine heart muscle have been investigated in search of an explanation for the inhibition of mitochondrial respiration by the inhalation anesthetic. Oxidase activity of the isolated enzyme is partially and reversibly reduced by nitrous oxide. N2O molecules are shown by infrared spectroscopy to occupy sites within the oxidase. Occupancy of sites within the protein by N2O has no observed effects on visible Soret spectra or on the O2 reaction site; no evidence is found for N2O serving as a ligand to a metal. The anesthetic does not substitute for O2 as an oxygen atom donor in either the cytochrome c oxidase or carbon monoxide dioxygenase reactions catalyzed by the enzyme. N2O appears to affect oxidase activity by reducing the rate of electron transfer from cytochrome c to the O2 reaction site rather than by interfering directly with the reduction of O2 to water. Cytochrome c oxidase represents a target site for nitrous oxide and possibly other anesthetics, and the inhibition of oxidase activity may contribute significantly to the anesthetic and/or toxic effects of these substances.     

Ecological implications of recently discovered and poorly studied sources of energy for the growth of true fungi especially in extreme environments

Rhodopsin transmembrane proton pumps (fuelled by visible light which is absorbed by retinal (carotenoid) chromophores) exist in all three domains of living species and in all groups of true fungi studied. Light driven proton and sodium pumps are likely to be essential for some marine fungi, especially hypersaline tolerant and endolithic species. Rhodopsin macromolecular machines, using visible light, drive metabolic reactions in addition to those provided by aerobic respiration, providing extra energy needed for the maintenance and growth of fungi, especially in euphotic environments where oxygen concentration is limited. In addition, dissimilatory nitrate and metal oxide reduction can provide sources of energy for fungi in the absence of oxygen, for example, in fungal species growing in marine sediments. Finally, the oxidation of elemental sulphur, iron and manganese can be a source of energy. Some fungi are, therefore, lithotrophs and photoheterotrophs. The ecological implications of these latter processes are discussed.     

Degradation of 2,4-Dichlorophenoxyacetic Acid (2,4-D) by a Hypersaline Microbial Mat and Related Functional Changes in the Mat Community

Microbial mats possibly possess degradation capacities for haloorganic pollutants because of their wide range of different functional groups of microorganisms combined with extreme diurnal changes in pH, oxygen, and sulfide gradients. In this study, 20 mg/l of the chlorinated herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was applied to a pristine hypersaline cyanobacterial mat from Guerrero Negro, Mexico, under a light regime of 12 h dark/12 h light (600 mol photons/m²s). The loss of 2,4-D was followed by chemical GC analysis; functional changes within the mat were determined with microelectrodes for oxygen, photosynthesis, pH, and sulfide. The depletion of 2,4-D due to photooxidation or sorption processes was checked in control experiments. Within 13 days, the light/dark incubated mats degraded 97% of the herbicide, while in permanent darkness only 35% were degraded. Adsorption of 2,4-D to the mat material, agar, or glass walls was negligible (4.6%), whereas 21% of the herbicide was degraded photochemically. The 2,4-D removal rate in the light/dark incubations was comparable to values reported for soils. The phototrophic community of the mat was permanently inhibited by the 2,4-D addition by 17% on average. The sulfate reduction in the entire mat and the respiration in the photic zone were inhibited more strongly but returned to original levels. Since at the end of the experiment the photosynthetic and respiratory activity of the mats were almost as high as in the beginning and 2,4-D almost completely disappeared, we conclude that the examined mats represent a robust and effective system for the degradation of the herbicide where probably the aerobic heterotrophic population is a major player in the degradation process.This revised version was published online in November 2004 with corrections to Volume 48.     

Mechanism(s) for the metabolism of mitoxantrone: electron spin resonance and electrochemical studies

Mitoxantrone has been reported to lack certain properties that characterize quinone containing antitumor agents that undergo enzymatic reduction. These properties are the stimulation of NADPH oxidation, the stimulation of oxygen consumption by microsomes and reductases and, the absence of oxygen free radicals during these reactions. Having these properties implies the presence of a futile redox cycle that requires the generation and the oxidation of a semiquinone free radical. It would follow that if mitoxantrone does not redox cycle in the presence of reductases, then the semiquinone free radical is not produced or, if it is formed, it reacts quickly to form diamagnetic products. However, using liver microsomes, there are reports of the formation of the mitoxantrone free radial anion. In this paper we investigated the mitoxantrone free radical anion generated electrochemically and found that in the presence of oxygen it behaved like other semiquinones. That is, it is oxidized to the parent compound (presumably generating oxygen free radicals), indicating the ability to redox cycle. The reduction potential to generate such free radical in aqueous medium is very high (-0.79 V) when compared to diaziquone (-0.36 V) and Adriamycin (-0.6 V). This suggests that mitoxantrone may not be a substrate for reductases. Under reductive conditions with purified NADPH cytochrome P-450 reductase which very easily reduces diaziquone and Adriamycin, mitoxantrone was not reduced. However, under the same conditions, mitoxantrone was oxidized by the prototype oxidase horseradish peroxidase with the production of a mitoxantrone free radical. This oxidation was accompanied by a drastic change in color and the formation of a dark precipitate. Because microsomes contain a variety of enzymes, we suggest that the previously observed free radical in microsomes is probably due to the oxidation of mitoxantrone. In this theory, this product is probably a polymer which would not require oxygen to be formed. Thus, under oxidative conditions, the mitoxantrone free radical cation will also display impaired redox activity.     

Investigations into the Mechanism of Action of a Novel Nitric Oxide Generator on Cellular Respiration

Abstract: Nitric oxide may regulate cellular respiration by competition with oxygen at mitochondrial cytochrome oxidase. Using an astrocyte-derived cell line, we have compared the mechanism of action of the nitric oxide-generating compound Roussin's black salt with that of sodium nitroprusside on cellular oxygen consumption. Intense light exposure induced the release of large quantities of nitric oxide from both of the donor compounds. However, in room light only Roussin's black salt generated low levels of the radical. Simultaneous measurement of oxygen consumption and of nitric oxide production demonstrated that sodium nitroprusside only had inhibitory actions when exposed to intense light (nitric oxide release), whereas Roussin's black salt had inhibitory actions in room light. Extracellular haemoglobin did not prevent the inhibition of respiration rate induced by Roussin's black salt even though stimulation of nitric oxide release on light exposure was markedly reduced. Preincubation of cells with Roussin's black salt and subsequent measurement of levels of light-liberated nitric oxide demonstrated that the compound was rapidly internalised. The uptake of sodium nitroprusside was minimal. These data suggest that, in contrast to sodium nitroprusside, the cellular internalisation of Roussin's black salt allows site-directed nitric oxide release and very effective inhibition of cellular respiration.     

Induction of ethylene synthesis and lipid peroxidation in damaged or TMV-infected tobacco leaf tissues by light

The effect of light on ethylene and ethane production in damaged leaf tissues was investigated. When whole leaves of tobacco cv. Samsun NN were damaged with liquid nitrogen, the ethylene formation was the highest, if 100?% of leaves were injured and were kept in the light, the lowest when leaves after 100?% injury were kept in darkness. Ethane production (lipid peroxidation) could be detected only in damaged, but not in control leaves, and was much higher in light than in darkness. In addition, there was a strong degradation of chlorophyll of damaged leaves kept in light. In light aminoethoxy-vinylglycine (AVG) inhibited ethylene formation in control, non-damaged whole leaves effectively, but in leaves with 100?% damage the inhibitory effect was much weaker and similar to the effect of propyl gallate (PG), a free radical scavenger. Both AVG and PG treatments decreased ethylene formation by control leaf discs and discs with 100?% damage. Ethane production was significantly inhibited by PG and slightly by AVG in the case of 100?% damage. Tiron, another free radical scavenger gave similar results on leaf discs as PG did. Paraquat (methylviologen, Pq), as a photosynthesis inhibiting and reactive oxygen species (ROS) producing herbicide produced a large amount of ethylene and ethane in light but very small amount in darkness. In accordance, tobacco mosaic virus (TMV) infection on the necrotic host resulted in significantly larger amount of ethylene and ethane formation in light than in darkness. We conclude that ethylene and ethane production of damaged plant tissues is strongly induced by light and ROS that are involved in this induction.     

Metabolic activation of sulfur mustard leads to oxygen free radical formation

We recently published electron paramagnetic resonance (EPR) spin trapping results that demonstrated the enzymatic reduction of sulfur mustard sulfonium ions to carbon-based free radicals using an in vitro system containing sulfur mustard, cytochrome P450 reductase, NADPH, and the spin trap α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) in buffer (A.A. Brimfield et al., 2009, Toxicol. Appl. Pharmacol. 234:128-134). Carbon-based radicals have been shown to reduce molecular oxygen to form superoxide and, subsequently, peroxyl and hydroxyl radicals. In some cases, such as with the herbicide paraquat, a cyclic redox system results, leading to magnified oxygen free radical concentration and sustained tissue damage. Low mustard carbon radical concentrations recorded by EPR in our in vitro system, despite a robust (4.0mM) sulfur mustard starting concentration, led us to believe a similar oxygen reduction and redox cycling process might be involved with sulfur mustard. A comparison of the rate of mustard radical-POBN adduct formation in our in vitro system by EPR at atmospheric and reduced oxygen levels indicated a sixfold increase in 4-POBN adduct formation (0.5 to 3.0 μM) at the reduced oxygen concentration. That result suggested competition between oxygen and POBN for the available carbon-based mustard radicals. In parallel experiments we found that the oxygen radical-specific spin trap 5-tert-butoxycarbonyl-5-methylpyrroline-N-oxide (BMPO) detected peroxyl and hydroxyl radicals directly when it was used in place of POBN in the in vitro system. Presumably these radicals originated from O(2) reduced by carbon-based mustard radicals. We also showed that reactive oxygen species (ROS)-BMPO EPR signals were reduced or eliminated when mustard carbon radical production was impeded by systematically removing system components, indicating that carbon radicals were a necessary precursor to ROS production. ROS EPR signals were completely eliminated when superoxide dismutase and catalase were included in the complete in vitro enzymatic system, providing additional proof of oxygen radical participation. The redox cycling hypothesis was supported by density functional theory calculations and frontier molecular orbital analysis.