Determination of H2O2-dependent generation of singlet oxygen from human saliva with a novel chemiluminescence probe

Singlet oxygen (¹O₂) has been shown to play an important role in salivary defense system, but its generation process and level from human saliva remain uncertain due to the lack of a reliable detection method. We have previously reported 4,4′(5′)-bis[2-(9-anthryloxy)ethylthio]tetrathiafulvalene (BAET) as a novel chemiluminescence probe for ¹O₂. In this work, the probe is successfully used to characterize H₂O₂-dependent generation of ¹O₂ from saliva in real time. However, the yield of ¹O₂ is found to be very low, for example, being about 0.13 nmol from 200 μL saliva in the presence of 1 mM of hydrogen peroxide over a 5-s reaction period. The result is also compared with that obtained with another ¹O₂ probe 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one (CLA), demonstrating that, besides ¹O₂, the other reactive oxygen species such as hydroxyl radical may also be involved in the reaction of saliva with H₂O₂. Furthermore, the present study shows that the selectivity of BAET for ¹O₂ is much higher than that of CLA and thus BAET is highly suited for the detection of ¹O₂ in the presence of other reactive oxygen species in biological systems.     

Chemiluminescent detection and imaging of reactive oxygen species in live mouse skin exposed to UVA

The recent increase of ultraviolet (UV) rays on Earth due to the increasing size of the ozone hole is suggested to be harmful to life and to accelerate premature photoaging of the skin. The detrimental effects of UV radiation on the skin are associated with the generation of reactive oxygen species (ROS) such as superoxide anion radical (*O(-)(2)), hydrogen peroxide (H(2)O(2)), hydroxyl radical (*OH), and singlet oxygen ((1)O(2)). However, direct proof of such ROS produced in the skin under UV irradiation has been elusive. In this study, we report first in vivo detection and imaging of the generated ROS in the skin of live mice following UVA irradiation, in which both a sensitive and specific chemiluminescence probe (CLA) and an ultralow-light-imaging apparatus with a CCD camera were used. In addition, we found that *O(-)(2) is formed spontaneously and (1)O(2) is generated in the UVA-irradiated skin. This method should be useful not only for noninvasive investigation of the spatial distribution and quantitative determination of ROS in the skin of live animals, but also for in vivo evaluation of the protective ability of free radical scavengers and antioxidants.     

Resveratrol reduces oxidative stress induced by platinum compounds in blood platelets

The effects of resveratrol (trans-3,4',5-trihydroxystilbene) on the oxidative stress in blood platelets induced by platinum compounds [cisplatin and selenium-cisplatin conjugate] were studied in vitro. The production of thiobarbituric acid reactive substances (TBARS), the level of conjugate diene, the generation of superoxide anion radicals (O2-*) and other reactive oxygen species (O2-*, H2O2, singlet oxygen and organic radicals) were measured by chemiluminescence in blood platelets treated with platinum compounds. Cisplatin at the concentration of 10 microg/ml, as well as selenium-cisplatin conjugate (10 microg/ml) induced oxidative stress in blood platelets: an increase in TBARS, conjugate diene, chemiluminescence and generation of O2-*. In the presence of resveratrol (a natural compound with antioxidant activity) at the concentrations of 1-25 microg/ml, the chemiluminescence, the levels of O2-*, conjugate diene and TBARS were reduced (p < 0.05). We showed that resveratrol at different concentrations (1-25 microg/ml) had a protective effect against oxidative stress in platelets caused by platinum compounds (10 microg/ml) and it diminished platelet lipid peroxidation and reactive oxygen species generation induced by platinum compounds.     

Chemiluminescence probe with Cypridina luciferin analog, 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one, for estimating the ability of human granulocytes to generate O2-

The Cypridina luciferin analog, 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one (CLA), in Hanks' balanced salt solution, emitted a weak luminescence which was not affected by superoxide dismutase or catalase and was not augmented by resting human granulocytes. In contrast, activated granulocytes caused a dramatic increase in the luminescence of CLA. The light emission by CLA in the presence of activated granulocytes was inhibited by superoxide dismutase, but not by catalase or benzoate. Azide at 0.5 mM did not inhibit light emission significantly. These results indicate that O2-, rather than H2O2, HO., singlet oxygen, or HOCl, was the agent responsible for eliciting the chemiluminescence of CLA. Moreover, the intensity of light emission by CLA correlated with the rate of production of O2- either by activated neutrophils or by the xanthine oxidase reaction.     

IL-1beta induces MMP-9 via reactive oxygen species and NF-kappaB in murine macrophage RAW 264.7 cells

IL-1beta increased the production of proenzyme of MMP-9 (pro-MMP-9) in a time- and dose-dependent manner in murine macrophage RAW 264.7 cells. However, the production of MMP-2 was not significantly changed by IL-1beta treatment. The intracellular H(2)O(2) content, as determined with H(2)O(2)-sensitive probe 2('),7(')-dichlorodihydrofluorescein, also increased after IL-1beta treatment (5ng/ml). In addition, exogenous H(2)O(2) (50 microM) was found to increase the production of pro-MMP-9. Transient transfection study using a MMP-9 promoter-reporter construct showed that IL-1beta enhanced the MMP-9 promoter activity. Electrophoretic mobility shift assay and site-directed mutagenesis study on the consensus binding site for NF-kappaB revealed that the activation of NF-kappaB is required for the IL-1beta-induced activation of MMP-9 promoter. N-acetylcysteine, an antioxidant, could abrogate the production of pro-MMP-9, H(2)O(2) generation, and activation of NF-kappaB and MMP-9 promoter. These results suggest that IL-1beta upregulates the MMP-9 expression via production of reactive oxygen species and activation of NF-kappaB in RAW 264.7 cells.     

Oxidative protein cross-linking reactions involving L-tyrosine in transforming growth factor-beta1-stimulated fibroblasts

The mechanisms by which ligand-stimulated generation of reactive oxygen species in nonphagocytic cells mediate biologic effects are largely unknown. The profibrotic cytokine, transforming growth factor-beta1 (TGF-beta1), generates extracellular hydrogen peroxide (H2O2) in contrast to intracellular reactive oxygen species production by certain mitogenic growth factors in human lung fibroblasts. To determine whether tyrosine residues in fibroblast-derived extracellular matrix (ECM) proteins may be targets of H2O2-mediated dityrosine-dependent cross-linking reactions in response to TGF-beta1, we utilized fluorophore-labeled tyramide, a structurally related phenolic compound that forms dimers with tyrosine, as a probe to detect such reactions under dynamic cell culture conditions. With this approach, a distinct pattern of fluorescent labeling that seems to target ECM proteins preferentially was observed in TGF-beta1-treated cells but not in control cells. This reaction required the presence of a heme peroxidase and was inhibited by catalase or diphenyliodonium (a flavoenzyme inhibitor), similar to the effect on TGF-beta1-induced dityrosine formation. Exogenous addition of H2O2 to control cells that do not release extracellular H2O2 produced a similar fluorescent labeling reaction. These results support the concept that, in the presence of heme peroxidases in vivo, TGF-beta1-induced H2O2 production by fibroblasts may mediate oxidative dityrosine-dependent cross-linking of ECM protein(s). This effect may be important in the pathogenesis of human fibrotic diseases characterized by overexpression/activation of TGF-beta1.     

Syntheses, crystal structures, and oxidative DNA cleavage of some Cu(II) complexes of 5-amino-3-pyridin-2-yl-1,2,4-triazole

Three new monomeric Cu(II) complexes of 5-amino-3-pyridin-2-yl-1,2,4-triazole (Hapt), [Cu(Hapt)(H(2)O)(2)(SO(4))] (1), [Cu(Hapt)(2)(H(2)O)(NO(3))](NO(3)) (2), and [Cu(Hapt)(2)(NCS-N)](NCS).H(2)O (3), have been prepared and characterized by single crystal X-ray diffraction. One distorted [CuN(2)O(2)+O(')] square-pyramidal (1), one distorted [CuN(3)O+N(')+O(')] octahedral (2), and one distorted [CuN(4)+N(')] intermediate between square-pyramidal and trigonal-bipyramidal (3) coordination configuration were found and are suggested to be due to the chelating nature of the ligand, which interacts with Cu(II) through the N4(triazole) and N(pyridine) atoms. Spectral properties of these chelates are in accordance with the X-ray structural data. With ascorbate and H(2)O(2) activation, compound 2 exhibits higher nuclease activity than compound 1. The influence on the DNA cleavage process of different scavengers of reactive oxygen species: dimethyl sulfoxide (DMSO), tert-butyl alcohol, sodium azide, 2,2,6,6-tetramethyl-4-piperidone and superoxide dismutase enzyme (SOD), and of the minor groove binder distamycin, is also studied.     

The ApoCorrect assay: a novel, rapid method to determine the biological functionality of radiolabeled and fluorescent Annexin A5

N-[4-(3)H]Benzoylglycylglycylglycine ([(3)H]BzG(3)) was tested as a probe for detecting hydroxyl radicals (*OH). Aerated solutions of l-ascorbate generated *OH, which oxidized [(3)H]BzG(3), yielding hydrophilic (probably hydroxylated) derivatives plus tritiated water. The (3)H(2)O was separated from organic products and remaining [(3)H]BzG(3) on Dowex-1. (3)H(2)O production was much greater with *OH than with other reactive oxygen species (ROS) (e.g., H(2)O(2), superoxide). The slight (3)H(2)O production in the presence of H(2)O(2) or superoxide was blocked by *OH scavengers (e.g., glycerol, mannitol, butan-1-ol) that do not scavenge H(2)O(2) or superoxide. This indicates that (3)H(2)O production was caused by *OH and that other ROS only generated any (3)H(2)O by forming traces of *OH. Doses of *OH that caused detectable nonenzymic polysaccharide scission also caused (3)H(2)O production, indicating that [(3)H]BzG(3) is a sensitive *OH probe in studies of polymer scission. The ability of scavengers and chelators to protect against ascorbate-mediated polysaccharide scission paralleled their ability to inhibit concurrent (3)H(2)O production, indicating that both processes were due to *OH. Thus, [(3)H]BzG(3) is a simple, specific, sensitive, and robust probe for detecting *OH production in vitro. It may have applications for in vivo detection of extracellular *OH in arthritic joints and of apoplastic *OH in plant cell walls.     

Prevention of H2O2 generation by monoamine oxidase protects against CNS O2 toxicity.

Toxicity to the central nervous system (CNS) by hyperbaric oxygen (HBO) presumably relates to increased production of reactive oxygen species. The sites of generation of reactive oxygen species during HBO, however, have not been fully characterized in the brain. We investigated the relationship between regional generation of hydrogen peroxide (H2O2) in the brain in the presence of an irreversible inhibitor of catalase, aminotriazole (ATZ), and protection from CNS O2 toxicity by a monoamine oxidase (MAO) inhibitor, pargyline. At 6 ATA of oxygen, pargyline significantly protected rats from CNS O2 toxicity whereas ATZ enhanced O2 toxicity. In animals pretreated with ATZ, HBO inactivated 21-40% more catalase than air exposure in the six brain regions studied. Because ATZ-mediated inactivation of catalase was H2O2 dependent, the decrease in catalase activity during hyperoxia was proportional to the intracellular production of H2O2. Pargyline, administered 30 min before HBO, inhibited MAO by greater than 90%, prevented ATZ inhibition of catalase activity during HBO, and reversed the augmentation of CNS O2 toxicity by ATZ. These findings indicate that H2O2 generated by MAO during hyperoxia is important to the pathogenesis of CNS O2 toxicity in rats.     

Induction of active forms of oxygen by biotic elicitors in tomato cell cultures

The elicitor-induced generation of two oxygen species in tomato cell culture as well as their involvement into hypersensitive reaction was investigated. Generation of superoxide O2.- was measured by a lucigenin-related chemiluminescence. Accumulation of hydrogen peroxide H2O2 was measured by a fluorescent probe pyranin. Xylanase and chitosan were used as biotic elicitors with different mode of action. It was found that both O2.- and H2O2 had been accumulated in elicitor-treated tomato cells. The results obtained show that reactive oxidants are important signal transduction elements for activation of hypersensitive response in tomato cells.     

Influence of neopterin on generation of reactive species by myeloperoxidase in human neutrophils

Increased neopterin concentrations in human serum indicate activation of cell-mediated immune response. Earlier we have shown that neopterin enhanced generation of singlet oxygen, hydroxyl radical and nitric oxide in human peripheral blood neutrophils by NADPH-independent pathways. To further investigate a participation of neopterin in reactive species production by neutrophils, we studied its influence on myeloperoxidase (MPO) activity. MPO was isolated from human peripheral blood neutrophils from healthy donors. Generation of reactive species by MPO/H(2)O(2) in Earl's solution (pH=7.2) at 37 degrees C was investigated by monitoring of chemiluminescence using luminol as light emitter. In the MPO/H(2)O(2) system, neopterin increased singlet oxygen in a concentration-dependent manner, but it decreased formation of other oxidizing species. Comparing several oxygen scavengers, formation of reactive species was totally blocked by sodium azide (NaN(3)), both in the presence and in the absence of neopterin. Superoxide dismutase (SOD) and d-mannitol insignificantly decreased chemiluminescence of this reaction, but diazabicyclo[2.2.2]octane (DABCO) strongly inhibited it. We conclude that the effects of neopterin on neutrophils' MPO are directed to increase singlet oxygen and to decrease other reactive species via inhibition of MPO and/or scavenging of reactive species.     

Study and application of flow injection spectrofluorimetry with a fluorescent probe of 2-(2-pyridil)-benzothiazoline for superoxide anion radicals

This paper presents an automatic spectrofluorimetric method (flow injection spectrofluorimetry) using a novel fluorescent probe named H. Py. Bzt (2-(2-pyridil)-benzothiazoline) for determining superoxide dismutase (SOD) activity. The fluorescent probe was synthesized in house and fully characterized by elemental analysis and by infrared and (1)H nuclear magnetic resonance spectra. It could specially identify and trap O(2)(*-) and was oxidized by O(2)(*-) to form a strong fluorescence product. Based on this reaction, the flow injection spectrofluorimetric method was proposed and successfully used to determine SOD activity. The proposed method has a better selectivity in the determination of reactive oxygen species because the probe can be oxidized only by O(2)(*-) excluding H(2)O(2). As a kind of simple, rapid, precise, sensitive and automatic technique, it was applied to measurement of SOD activity in scallion, garlic, and onion with satisfactory results.     

Detection of reactive oxygen species in the skin of live mice and rats exposed to UVA light: a research review on chemiluminescence and trials for UVA protection.

The harmful effects of ultraviolet (UV) exposure on the skin are associated with the generation of reactive oxygen species (ROS) such as superoxide anion radical ( O(2)(-)), hydrogen peroxide (H(2)O(2)), hydroxyl radical ( OH), and singlet oxygen ((1)O(2)) as well as with lipid peroxides and their radicals (LOOH and LOO ). To give direct proof that such ROS are generated in UV-exposed skin, we proposed the in vivo detection and imaging method in which both a sensitive and specific chemiluminescence (CL) probe, such as CLA, and an ultralow-light imaging apparatus with a CCD camera were used. With this method we found that O(2)(-) is formed intrinsically and that (1)O(2) and O(2)(-) are generated in the UVA-exposed skin of mice. In addition, we indicated that antioxidative ability against ROS in the skin of hairless rats decreased as age increased. Using these findings, we demonstrated the protective abilities of sodium ascorbate, caffeic acid, essential aroma oils, and zinc(ii) ion and its complexes, which we administered to mice both topically and orally. We present a review for the current state of our research proposing the sensitive CL method as a useful in vivo tool in photobiological research for the detection of oxidative stress as well as for the evaluation of antioxidative agents to the skin.     

Involvement of superoxide generation in salicylic acid-induced stomatal closure in Vicia faba.

Salicylic acid (SA), the known mediator of systemic acquired resistance, induced stomatal closure of Vicia faba L. Application of SA to the epidermal peels evoked an elevation of chemiluminescence of Cripridina lucigenin-derived chemiluminescent reagent (CLA) which is sensitive to superoxide anion (O(2)(.-)). The SA-induced generation of chemiluminescence was suppressed by O(2)(.-)-specific scavengers superoxide dismutase (SOD) and 4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron). These results suggest that O(2)(.-) was generated in epidermal peels by SA-treatment. A peroxidase inhibitor salicylhydroxamic acid (SHAM) inhibited guaiacol peroxidase activity and suppressed the SA-induced CLA chemiluminescence in the epidermal peels, suggesting that O(2)(.-) generation occurred by the peroxidase-catalyzed reaction as proposed for SA-treated tobacco cell suspension culture [Kawano et al. (1998) Plant Cell Physiol. 39: 721]. SOD, Tiron or SHAM suppressed the SA-induced stomatal closure. Moreover, application of superoxide-generating system also induced stomatal closure. These results support the concept of involvement of reactive oxygen species in signal transduction in SA-induced stomatal closure.     

Mechanisms of [Ca2+]i elevation by H2O2 in islets of rats

Activity of reactive oxygen species is elevated in diabetes mellitus and has been implicated in the destruction of cellular components. The toxic effect of reactive oxygen species was investigated by testing the effect of H2O2 on [Ca2+]i in isolated islets of Langehans. H2O2 increased [Ca2+]i in a dose-dependent manner, which was irreversible at high concentrations. The maximum effect of H2O2 on [Ca2+]i was larger than those of KCl, glucose, ATP, carbachol and endothelin-1. The effect of H2O2 was only partially attenuated by removal of external Ca2+ and by the in-organic Ca2+ channel blocker nickel, but was not blocked by voltage-dependent or -independent Ca2+ channel blockers nimodipine, nicardipine, SK&F 96365, econazole and lanthanum. H2O2, disrupted [Ca2+]i homeostasis in islets by affecting both release and influx of Ca2+ and causing dysfunction of Ca2+ clearance systems and may contribute to the pathological process of diabetes.     

植物细胞中蓝光诱导ROS生成的识别和亚细胞定位检测

以2’,7’-二氯二氢荧光素二乙酯(dichlorofluorescein diacetate,H2DCF-DA)为荧光探针孵育拟南芥叶表皮条,利用荧光光谱和激光共聚焦扫描显微技术,对高辐照蓝光诱导下叶肉细胞活性氧(reactive oxygen spe-cies,ROS)的生成,进行了分子识别和亚细胞定位检测。结果表明:植物细胞在蓝光诱导下,可以产生大量的ROS。过氧化氢酶清除实验表明:高辐照蓝光诱导产生的ROS,主要成分是H2O2,并且主要定位在叶绿体和细胞膜上。     

Reactive oxygen species in programmed death of pea guard cells

Hydrogen peroxide potentiates CN(-)-induced apoptosis of guard cells recorded as destruction of cell nuclei in the epidermis from pea leaves. A still stronger effect was exerted by the addition of H2O2 and NADH, which are the substrates of the plant cell wall peroxidase producing O2*- coupled to the oxidation of NADH. The CN(-)-or (CN(-) + H2O2)-induced destruction of guard cell nuclei was completely removed by nitroblue tetrazolium (NBT) oxidizing O2*- and preventing there-by the subsequent generation of H2O2. The reduced NBT was deposited in the cells as formazan crystals. Cyanide-induced apoptosis was diminished by mannitol and ethanol, which are OH* traps. The dyes Rose Bengal (RB) and tetramethylrhodamine ethyl ester (TMRE) photosensitizing singlet oxygen production suppressed the CN(-)-induced destruction of the cell nuclei in the light. This suppression was removed by exogenous NADH, which reacts with 1O2 yielding O2*-. Incubation of leaf slices with RB in the light lowered the photosynthetic O2 evolution rate and induced the permeability of guard cells for propidium iodide, which cannot pass across intact membranes. Inhibition of photosynthetic O2 evolution by 3-(3',4'-dichlorophenyl)-1,1-dimethylurea or bromoxynil prevented CN(-)-induced apoptosis of guard cells in the light but not in the dark. RB in combination with exogenous NADH caused H2O2 production that was sensitive to NBT and estimated from dichlorofluorescein (DCF) fluorescence. Data on NBT reduction and DCF and TMRE fluorescence obtained using a confocal microscope and data on the NADH-dependent H2O2 production are indicative of generation of reactive oxygen species in the chloroplasts, mitochondria, and nuclear region of guard cells as well as with participation of apoplastic peroxidase. Cyanide inhibited generation of reactive oxygen species in mitochondria and induced their generation in chloroplasts. The results show that H2O2, OH*, and O2*- resources utilized for H2O2 production are involved in apoptosis of guard cells. It is likely that singlet oxygen generated by RB in the light, judging from the permeability of the plasmatic membrane for propidium iodide, makes Photosystem II of chloroplasts inoperative and induces necrosis of the guard cells.     

H(2)O(2) generation in Saccharomyces cerevisiae respiratory pet mutants: effect of cytochrome c

Impaired electron transport chain function has been related to increases in reactive oxygen species (ROS) generation. Here we analyzed different pet mutants of Saccharomyces cerevisiae in order to determine the relative contribution of respiratory chain components in ROS generation and removal. We found that the maintenance of respiration strongly prevented mitochondrial H(2)O(2) release and increased cellular H(2)O(2) removal. Among all respiratory-deficient strains analyzed, cells lacking cytochrome c (cyc3 point mutants) presented the highest level of H(2)O(2) synthesis, indicating that the absence of functional cytochrome c in mitochondria leads to oxidative stress. This finding was supported by the presence of high levels of catalase and peroxidase activity despite the lack of respiration. Furthermore, the addition of exogenous cytochrome c to isolated yeast mitoplasts significantly reduced H(2)O(2) detection in a manner enhanced by cytochrome c reduction and the presence of a functional respiratory chain. Together, our results indicate that the maintenance of electron transport by cytochrome c prevents ROS generation by the respiratory chain.     

Generation of oxygen free radicals in thyroid cells and inhibition of thyroid peroxidase

We examined whether superoxide (O(2)(-)) is produced as a precursor of hydrogen peroxide (H(2)O(2)) in cultured thyroid cells using the cytochrome c method and the electron paramagnetic resonance (EPR) method. No O(2)(-) or its related radicals was detected in thyroid cells under the physiological condition. The presence of quinone, 2,3-dimethoxy-l-naphthoquinone (DMNQ), or 2-methyl-1, 4-naphthoquinone (menadione), in the medium produced O(2)(-) and hydroxyl radicals (OH*); the amount of H(2)O(2) generation was also increased. Incubation of follicles with DMNQ or menadione inhibited iodine organification (a step of thyroid hormone formation) and its catalytic enzyme, thyroid peroxidase (TPO). This inhibition should be caused by reactive oxygen species because the two quinones, particularly DMNQ, exert their effect through the generation of reactive oxygen species. It is speculated that the site-specific inactivation of TPO might have occurred at the heme-linked histidine residue of the TPO molecule, a critical amino acid for enzyme activity because OH* (vicious free radicals) can be formed at the iron-linked amino acid. TPO mRNA level and electrophoretic mobility of TPO were not inhibited by quinones. Our study suggests that thyroid H(2)O(2) is produced by divalent reduction of oxygen without O(2)(-) generation. If thyroid cells happen to be exposed to significant amount of reactive oxygen species, TPO and subsequent thyroid hormone formation are inhibited.     

A photochemical study of cells loaded with 2',7'-dichlorofluorescin: implications for the detection of reactive oxygen species generated during UVA irradiation

There have been several attempts to implicate reactive oxygen species in UVA-induced damage by loading cells with 2',7'-dichlorofluorescin (DCFH) and following the appearance of 2',7'-dichlorofluorescein (DCF), its highly fluorescent oxidation product. However, both DCF and DCFH have significant absorption in the 300-400 nm range so it is possible that photochemical reactions will occur in cells containing these dyes when they are irradiated with UVA. HaCaT keratinocytes loaded with DCFH were irradiated with 0, 1, 2, or 4 J/cm(2) UVA and DCF fluorescence was measured. A dose-dependent increase in DCF fluorescence was observed, with the cells exposed to 4 J/cm(2) UVA exhibiting an almost 10-fold increase over dark controls. However, there was no difference in cell viability, as measured by the MTS assay or LDH release, between the dark and the 4 J/cm(2) UVA-exposed groups. Furthermore, a large increase in DCF fluorescence was observed when a cell-free system containing DCF, DCFH, and horseradish peroxidase was UVA irradiated. As a control, keratinocytes loaded with DCFH were incubated in the dark with either exogenously added H(2)O(2) or 5-hydroxy-1,4-naphthoquinone (juglone), which redox cycles to generate superoxide (and H(2)O(2)). In both cases, the cells showed a concentration-dependent increase in DCF fluorescence and a concomitant decrease in viability. Our findings suggest that DCFH can not be used to detect the UVA-induced generation of reactive oxygen species in cells when the dye is present during exposure.