Ultraviolet irradiation induces autofluorescence enhancement via production of reactive oxygen species and photodecomposition in erythrocytes

Ultraviolet (UV) light has a significant influence on human health. In this study, human erythrocytes were exposed to UV light to investigate the effects of UV irradiation (UVI) on autofluorescence. Our results showed that high-dose continuous UVI enhanced erythrocyte autofluorescence, whereas low-dose pulsed UVI alone did not have this effect. Further, we found that H₂O₂, one type of reactive oxygen species (ROS), accelerated autofluorescence enhancement under both continuous and pulsed UVI. In contrast, continuous and pulsed visible light did not result in erythrocyte autofluorescence enhancement in the presence or absence of H₂O₂. Moreover, NAD(P)H had little effect on UVI-induced autofluorescence enhancement. From these studies, we conclude that UVI-induced erythrocyte autofluorescence enhancement via both UVI-dependent ROS production and photodecomposition. Finally, we present a theoretical study of this autofluorescence enhancement using a rate equation model. Notably, the results of this theoretical simulation agree well with the experimental data further supporting our conclusion that UVI plays two roles in the autofluorescence enhancement process.     

Oxygen and hydrogen peroxide enhance light-induced carotenoid synthesis in Neurospora crassa

Previously, we found that intracellular reactive oxygen species (ROS) affect photomorphogenesis in Neurospora crassa. In this study, we investigated the physiological roles of ROS in the response to light and found that the exposure of mycelia to air was important for the light-induced carotenogenesis. Mycelia treated with a high concentration of O(2) gas and H(2)O(2) to release ROS showed an enhancement of light-induced carotenoid accumulation and the expression of gene related to light-inducible carotenogenesis. These results suggested that stimuli caused by the exposure of the mycelia to air containing O(2) gas triggered the light-induced carotenoid synthesis.     

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。     

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.     

Characteristics of alpha-glycerophosphate-evoked H2O2 generation in brain mitochondria

Characteristics of reactive oxygen species (ROS) production in isolated guinea-pig brain mitochondria respiring on alpha-glycerophosphate (alpha-GP) were investigated and compared with those supported by succinate. Mitochondria established a membrane potential (DeltaPsi(m)) and released H(2)O(2) in parallel with an increase in NAD(P)H fluorescence in the presence of alpha-GP (5-40 mm). H(2)O(2) formation and the increase in NAD(P)H level were inhibited by rotenone, ADP or FCCP, respectively, being consistent with a reverse electron transfer (RET). The residual H(2)O(2) formation in the presence of FCCP was stimulated by myxothiazol in mitochondria supported by alpha-GP, but not by succinate. ROS under these conditions are most likely to be derived from alpha-GP-dehydrogenase. In addition, huge ROS formation could be provoked by antimycin in alpha-GP-supported mitochondria, which was prevented by myxothiazol, pointing to the generation of ROS at the quinol-oxidizing center (Q(o)) site of complex III. FCCP further stimulated the production of ROS to the highest rate that we observed in this study. We suggest that the metabolism of alpha-GP leads to ROS generation primarily by complex I in RET, and in addition a significant ROS formation could be ascribed to alpha-GP-dehydrogenase in mammalian brain mitochondria. ROS generation by alpha-GP at complex III is evident only when this complex is inhibited by antimycin.     

Reactive oxygen species in the elongation zone of maize leaves are necessary for leaf extension

The production and role of reactive oxygen species (ROS) in the expanding zone of maize (Zea mays) leaf blades were investigated. ROS release along the leaf blade was evaluated by embedding intact seedlings in 2',7'-dichlorofluorescein-containing agar and examining the distribution of 2',7'-dichlorofluorescein fluorescence along leaf 4, which was exposed by removing the outer leaves before embedding the seedling. Fluorescence was high in the expanding region, becoming practically non-detectable beyond 65 mm from the ligule, indicating high ROS production in the expansion zone. Segments obtained from the elongation zone of leaf 4 were used to assess the role of ROS in leaf elongation. The distribution of cerium perhydroxide deposits in electron micrographs indicated hydrogen peroxide (H(2)O(2)) presence in the apoplast. 2',7'-Dichlorofluorescein fluorescence and apoplastic H(2)O(2) accumulation were inhibited with diphenyleneiodonium (DPI), which also inhibited O*(2)(-) generation, suggesting a flavin-containing enzyme activity such as NADPH oxidase was involved in ROS production. Segments from the elongation zone incubated in water grew 8% in 2 h. KI treatments, which scavenged H(2)O(2) but did not inhibit O*(2)(-) production, did not modify growth. DPI significantly inhibited segment elongation, and the addition of H(2)O(2) (50 or 500 microM) to the incubation medium partially reverted the inhibition caused by DPI. These results indicate that a certain concentration of H(2)O(2) is necessary for leaf elongation, but it could not be distinguished whether H(2)O(2), or other ROS, are the actual active agents.     

Photooxidation of Amplex red to resorufin: Implications of exposing the Amplex red assay to light

The Amplex Red assay, a fluorescent assay for the detection of H(2)O(2), relies on the reaction of H(2)O(2) and colorless, nonfluorescent Amplex Red with a 1:1 stoichiometry to form colored, fluorescent resorufin, catalyzed by horseradish peroxidase (HRP). We have found that resorufin is artifactually formed when Amplex Red is exposed to light. In the absence of H(2)O(2) and HRP, the absorption and fluorescence spectra of Amplex Red changed during exposure to ambient room light or instrumental excitation light, clearly indicating that the fluorescent product resorufin had formed. This photochemistry was initiated by trace amounts of resorufin that are present in Amplex Red stock solutions. ESR spin-trapping studies demonstrated that superoxide radical was an intermediate in this process. Oxygen consumption measurements further confirmed that superoxide and H(2)O(2) were artifactually produced by the photooxidation of Amplex Red. The artifactual formation of resorufin was also significantly increased by the presence of superoxide dismutase or HRP. This photooxidation process will result in a less sensitive assay for H(2)O(2) under ambient light exposure and potentially invalid measurements under high energy exposure such as UVA irradiation. In general, precautions should be taken to minimize exposure to light during measurement of oxidative stress with Amplex Red.     

Low-power laser irradiation activates Src tyrosine kinase through reactive oxygen species-mediated signaling pathway

Low-power laser therapy in medicine is widespread but the mechanisms are not fully understood. It has been suggested that low-power laser irradiation (LPLI) could induce photochemical reaction and activate several intracellular signaling pathways. Reactive oxygen species (ROS) are considered to be the key secondary messengers produced by LPLI. Here, we studied the signaling pathway mediated by ROS upon the stimulation of LPLI. Src tyrosine kinases are well-known targets of ROS and can be activated by oxidative events. Using a Src reporter based on fluorescence resonance energy transfer (FRET) and confocal laser scanning microscope, we visualized the dynamic Src activation in Hela cells immediately after LPLI. Moreover, Src activation by LPLI was in a dose-dependent manner. The increase of Src phosphorylation at Tyr416 was detected by Western blotting. In the presence of vitamin C, catalase alone, or the combination of catalase and superoxide dismutase (SOD), the activation of Src by LPLI is significantly abolished. In contrast, G?6983 loading, a PKC inhibitor, did not affect this response. Treatment of Hela cells with exogenous H(2)O(2) also resulted in a concentration-dependent activation of Src. These results demonstrated that it was ROS that mediated Src activation by LPLI. Cellular viability assay revealed that laser irradiation of low doses (相似文献   

Reactive oxygen and targeted antioxidant administration in endothelial cell mitochondria

We used fluorescent probes and EPR to study the mechanism(s) underlying reactive oxygen species (ROS) production by endothelial cell mitochondria and the action of mitoquinol, a mitochondria-targeted antioxidant. ROS measured by fluorescence resulted from complex I superoxide released to the matrix and converted to H(2)O(2). In contrast, EPR largely detected superoxide generated at complex III and effluxed outward. ROS fluorescence by mitochondria fueled by the complex II substrate, succinate, was substantial but markedly inhibited by rotenone. Superoxide, detected by EPR, in succinate-fueled mitochondria was not inhibited by rotenone and likely derived from semiquinone formation at complex III. Mitoquinol decreased H(2)O(2) fluorescence by succinate-fueled mitochondria but had little effect on the EPR signal for superoxide. This was not associated with a detectable decrease in membrane potential. Mitoquinol markedly enhanced ROS fluorescence in mitochondria fueled by the complex I substrates, glutamate and malate. Inhibitor studies suggested that this occurred in complex I, at one or more Q binding pockets. The above effects of mitoquinol were determined in mitochondria isolated and subsequently exposed to the targeted antioxidant. However, similar effects were observed in mitochondria after antecedent exposure to mitoquinol/mitoquinone in culture, suggesting that the agent is retained after isolation of the organelles. In conclusion, ROS production in bovine aortic endothelial cell mitochondria results largely from reverse transport to complex I and through the Q cycle in complex III. Mitoquinol blocks ROS from reverse electron transport but increases superoxide production derived from forward transport. These effects likely occur at one or more Q binding sites in complex I.     

Variation of 1-pyrenebutyric acid fluorescence lifetime in single living cells treated with molecules increasing or decreasing reactive oxygen species levels

1-Pyrenebutyric acid (PBA) is a fluorescent probe whose fluorescence lifetime depends on local oxygen and free radical concentrations. We propose to use PBA fluorescence lifetime to quantify reactive oxygen species (ROS) in biological samples. Time-resolved microfluorimetry was used to record the fluorescence decay of single living cells loaded with this probe. We measured intracellular PBA fluorescence lifetimes and reduced nicotinamide adenine dinucleotide phosphate intensities under various oxygen concentrations. To confirm the feasibility of the new method, CCRF-CEM cells were treated with drugs that are known to increase or decrease ROS production. After treatment with adriamycin, we observed a decrease of PBA fluorescence lifetime. This corresponded to an increase of ROS concentration (80%). After treatment with cysteamine, we observed a reduction of the ROS concentration by 67%. Moreover, addition of exogenous H(2)O(2) solution resulted in a decrease of PBA fluorescence lifetime due to a raising of the intracellular ROS concentration. These results support our hypothesis linking a part of PBA fluorescence lifetime variations to intracellular fluctuation of ROS.     

Electrochemical measurement of the flux of hydrogen peroxide releasing from RAW 264.7 macrophage cells based on enzyme-attapulgite clay nanohybrids

Determination of cellular ROS (reactive oxygen species) could lead to a better understanding of the clinical consequences of the enhancement in ROS concentration, and assisting in studies of the biological effect of ROS in cells. This work developed an electrochemical approach for measuring the flux of H(2)O(2) (a major ROS in living organisms) releasing from RAW 264.7 macrophage cells. This approach is based on the electrocatalytic reduction of the releasing H(2)O(2) at the biosensor of HRP-attapulgite/GC, which was fabricated by depositing the horseradish peroxidase-attapulgite nanohybrids on the glassy carbon (GC) electrode. The biosensor exhibited a rapid response, a wide linear range, a high sensitivity, a low detection limit, as well as good stability and repeatability due to using the natural mineral (attapulgite) as the enzyme immobilization substrate. In addition, some common coexisting ROS and compounds in biological system such as hypochlorite (OCl(-)), nitric oxide (NO), peroxynitrite (ONOO(-)), and ascorbic acid (AA) etc., did not cause any interference due to the use of a low operating potential (-400mV, versus SCE). Moreover, the developed approach can also be used for studying the effects of the stimulator loading and a variety of stimuli on the generation of H(2)O(2) in cells and the release flux of H(2)O(2) from cells. Therefore, this work has demonstrated a simple and effective sensing platform for detection of cellular H(2)O(2) released from cells such as RAW 264.7 cells, which has potential utility to cellular biology and pathophysiology.     

Intracellular GSH levels rather than ROS levels are tightly related to AMA-induced HeLa cell death

Antimycin A (AMA) inhibits succinate oxidase and NADH oxidase, and also inhibits mitochondrial electron transport between cytochromes b and c. We investigated the involvement of ROS and GSH in AMA-induced HeLa cell death. AMA increased the intracellular H(2)O(2) and O(2)(*-) levels and reduced the intracellular GSH content. ROS scavengers (Tempol, Tiron, Trimetazidine and NAC) did not down-regulate the production of ROS and inhibit apoptosis in AMA-treated cells. Treatment with NAC and N-propylgallate showing the enhancement of GSH depletion in AMA-treated cells significantly intensified the levels of apoptosis. Calpain inhibitors I and II (calpain inhibitor III) and Ca(2+)-chelating agent (EGTA/AM) significantly reduced H(2)O(2) levels in AMA-treated HeLa cells. However, treatment with calpain inhibitor III intensified the levels of O(2)(*-) in AMA-treated cells. In addition, calpain inhibitor III strongly depleted GSH content with an enhancement of apoptosis in AMA-treated cells. Conclusively, the changes of ROS by AMA were not tightly correlated with apoptosis in HeLa cells. However, intracellular GSH levels are tightly related to AMA-induced cell death.     

Control of Ascorbate Peroxidase 2 expression by hydrogen peroxide and leaf water status during excess light stress reveals a functional organisation of Arabidopsis leaves

In Arabidopsis leaves, high light stress induces rapid expression of a gene encoding a cytosolic ascorbate peroxidase (APX2), whose expression is restricted to bundle sheath cells of the vascular tissue. Imaging of chlorophyll fluorescence and the production of reactive oxygen species (ROS) indicated that APX2 expression followed a localised increase in hydrogen peroxide (H2O2) resulting from photosynthetic electron transport in the bundle sheath cells. Furthermore, leaf transpiration rate also increased prior to APX2 expression, suggesting that water status may also be involved in the signalling pathway. Abscisic acid stimulated APX2 expression. Exposure of ABA-insensitive mutants (abi1-1, abi2-1) to excess light resulted in reduced levels of APX2 expression and confirmed a role for ABA in the signalling pathway. ABA appears to augment the role of H2O2 in initiating APX2 expression. This regulation of APX2 may reflect a functional organisation of the leaf to resolve two conflicting physiological requirements of protecting the sites of primary photosynthesis from ROS and, at the same time, stimulating ROS accumulation to signal responses to changes in the light environment.     

Imaging of NPQ and ROS formation in tobacco leaves: heat inactivation of the water-water cycle prevents down-regulation of PSII

Non-photochemical chlorophyll fluorescence quenching (NPQ) plays a major role in the protection of the photosynthetic apparatus against damage by excess light, which is closely linked to the production of reactive oxygen species (ROS). The effect of a short heat treatment on NPQ and ROS production was studied with detached tobacco leaves by fluorescence imaging of chlorophyll and of the ROS sensor dye HO-1889NH. NPQ was stimulated >3-fold by 3 min pre-treatment at 44 degrees C, in parallel with suppression of CO(2) uptake, while no ROS formation could be detected. In contrast, after 3 min pre-treatment at 46 degrees C, NPQ was suppressed and ROS formation was indicated by quenching of HO-1889NH fluorescence. After 3 min pre-treatment at 46 degrees C and above, partial inactivation of ascorbate peroxidase and light-driven accumulation of H(2)O(2) was also observed. These data are discussed as evidence for a decisive role of the Mehler ascorbate peroxidase or water-water cycle in the formation of the NPQ that reflects down-regulation of PSII.     

Increase of accessibility of murine lens protein fluorophores to water in the development of radiation-induced cataract

The accessibility of tryptophanyl fluorophores in crystalline proteins to water molecules was estimated by measuring the enhancement of the fluorescence of lens homogenates in 70% D2O as compared to 100% H2O. Assuming that two sorts of fluorophores exist in the proteins, one entirely accessible to H2O and D2O and the other--absolutely not, we have calculated the portion of either group in the protein fluorescence (alpha and 1-alpha, correspondingly). Measurement of murine lens homogenates fluorescence at different stages of radiation-induced cataract, initiated with total gamma irradiation in a dose 5 Gy have shown an increased accessibility of tryptophanyl for water with cataract development. At earlier stages of cataract (appearance of scattered dot opacities) the portion of water-accessible tryptophanyl increased from 0.14 to 0.18, i.e. by a factor of 1.3. The data obtained suggest that protein globules unfold in the coarse of cataract development.     

Differential role of hydrogen peroxide in UV-induced signal transduction

The present study investigated the differential requirement of ROS in UV-induced activation of these pathways. Exposure of the mouse epidermal C141 cells to UV radiation led to generation of ROS as measured by electron spin resonance (ESR) and by H2O2 and O2. fluorescence staining assay. Treatment of cells with UV radiation or H2O2 also markedly activated Erks, JNKs, p38 kinase and led to increases in phosphorylation of Akt and p70(S6k) in mouse epidermal JB6 cells. The scavenging of UV-generated H2O2 by N-acety-L-cyteine (NAC, a general antioxidant) or catalase (a specific H2O2 inhibitor) inhibited UV-induced activation of JNKs, p38 kinase, Akt and p70(S6k), while it did not show any inhibitory effects on Erks activation. Further, pretreatment of cells with sodium formate (an .OH radical scavenger) or superoxide dismutase (O2-. radical scavenger) did not inhibit any of these pathways. These results demonstrate that H2O2 generation is required for UV-induced phosphorylation of Akt and p70(S6k), and involved in activation of JNKs and p38 kinase, but not Erks.     

Hydrogen peroxide is critical for UV-induced apoptosis inhibition

Apoptosis is an important cell death system that deletes damaged and mutated cells, preventing the induction of cancer. We previously have reported that UV irradiation inhibited the apoptosis induced by serum starvation and cell detachment. This phenomenon is suitable for clarifying the relationship between cancer and the dysregulation of apoptosis by UV irradiation. Here, we have studied the factors responsible for this inhibition of apoptosis, focusing on reactive oxygen species (ROS) and DNA damage. Treatment with xanthine oxidase in the presence of hypoxanthine, which is known to produce superoxide anion (O2*-) and hydrogen peroxide (H2O2), inhibited the induction of apoptosis. The xanthine oxidase-induced anti-apoptotic effect was suppressed in the presence of an H2O2-eliminating enzyme, catalase, but not in the presence of an O2*--eliminating enzyme, superoxide dismutase. Treatment with H2O2 itself significantly inhibited the induction of apoptosis. Furthermore, the effect of the inhibition of cell death by UVB irradiation and by H2O2 treatment decreased in H2O2-resistant cells. Although both UVB and H2O2 are known to induce DNA damage, other DNA damaging agents, like gamma-irradiation and treatment with cisplatin and bleomycin, showed no inhibition of apoptosis. These findings suggested that H2O2 was essential to the inhibition of apoptosis, in which DNA damage had no role.     

H2O2致WB-F344细胞内活性氧的产生及机理

以双氢罗丹明123（DHR123）作为荧光探针,采用激光共聚焦扫描显微镜研究小剂量（800nmol/L)H2O2诱导大鼠肝卵细胞株WB－F344细胞内活性氧产生的动态变化过程及其机理。结果发现：（1）小剂量H2O2的一次作用可以引起胞内活性氧的产生;（2）胞内活性氧清除剂N－乙酰－L－半胱氨酸（NAC）处理2h时后,再加入小剂量H2O2,发现胞内活性氧的产生明显减少;（3）用广谱的蛋白激酶抑制剂2－氨基嘌呤（2－AP）、Ca^2 依赖性蛋白激酶（PKC）抑制剂Bisindolylmaleimide Ⅰ、酷氨酸蛋白激酶（TPK）抑制剂Tyrphostin25分别预处理15min后,H2O2诱导的胞内活性氧的产生现象均消失;（4）细胞在无外钙环境下,小剂量H2O2诱导的胞内活性氧的产生明显减少;（5）细胞在无外钙环境下用NAC预处理后,H2O2诱导的胞内活性氧的产生现象消失。结果表明,H2O2可以通过胞内信号转导系统诱使WB细胞胞内活性氧产生,这可能与小剂量H2O2调控细胞生物学功能（如增殖、转化）相关。