In vitro study of reactive oxygen species production during photodynamic therapy in ultrasound-pretreated cancer cells

Several recent studies bring evidence of cell death enhancement in photodynamic compound loaded cells by ultrasonic treatment. There are a number of hypotheses suggesting the mechanism of the harmful ultrasonic effect. One of them considers a process in the activation of photosensitizers by ultrasonic energy. Because the basis of the photodynamic damaging effect on cells consists in the production of reactive oxygen species (ROS), we focused our study on whether the ultrasound can increase ROS production within cancer cells. Particularly, we studied ROS formation in ultrasound pretreated breast adenocarcinoma cells during photodynamic therapy in the presence of chloroaluminum phthalocyanine disulfonate (ClAlPcS2). Production of ROS was investigated by the molecular probe CM-H2DCFDA. Our results show that ClAlPcS2 induces higher ROS production in the ultrasound pretreated cell lines at a concentration of 100 microM and light intensity of 2 mW/cm2. We also observed a dependence of ROS production on photosensitizer concentration and light dose. These results demonstrate that the photodynamic effect on breast cancer cells can be enhanced by ultrasound pretreatment.     

Sea urchin spermatozoa generate at least two reactive oxygen species; the type of reactive oxygen species changes under different conditions

Reactive oxygen species (ROS) cause oxidative stress and act as signal transduction molecules in many cells. Spermatozoa from several mammals generate ROS, which are involved in male infertility and signaling during capacitation. In the present study, we investigated ROS generation by sea urchin spermatozoa at the initiation of motility, during dilution with seawater, and following egg jelly treatment. In seawater containing an ROS indicator, 5-(and 6-)chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H(2)DCFDA), fluorescence increased after the addition of spermatozoa. The ROS generation rate was dependent upon the dilution ratio and respiratory rate of the spermatozoa. Spermatozoa in sodium-free seawater did not increase fluorescence, but fluorescence did increase with the addition of NaCl. Sodium chloride also led to the initiation of sperm motility and respiration. Using the indicator MitoSOX Red, ROS generation was detected from spermatozoa exposed to egg jelly dissolved in seawater, but not in normal seawater. Moreover, the respiratory inhibitor antimycin A prevented CM-H(2)DCFDA-detectable ROS and increased MitoSox-detectable ROS at a higher concentration. These findings revealed that the ROS generated were of different species, possibly hydrogen peroxide (H(2)O(2)) and superoxide anion (O(-)(2)), and their detected levels were altered by egg jelly. We concluded that sea urchin spermatozoa generate at least two species of ROS depending on the physiological conditions to which they are exposed. It is possible that the major ROS from sea urchin spermatozoa changes during the course of fertilization.     

A modified fixed staining method for the simultaneous measurement of reactive oxygen species and oxidative responses

The generation of reactive oxygen species (ROS) in a live-cell system is routinely measured using the oxidation-sensitive fluorescent probe dichlorofluorescein (DCF). However, it is difficult to simultaneously monitor cellular oxidative responses and ROS generation in cells, and analyses of cellular oxidative responses are typically performed after ROS generation has been evaluated. In this study, we developed a modified fixed staining method that allows the simultaneous analysis of ROS generation and oxidative responses using standard immunostaining techniques. A microplate reader-based assay showed that of the fixatives tested, only methanol did not alter the hydrogen peroxide (H₂O₂)-mediated oxidation of the responsive dye 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA), a chloromethyl derivative of H₂DCFDA, or the fluorescence of oxidized DCF in vitro. Further in vivo assays using flow cytometry showed that both methanol and acetic acid maintained the fluorescence of oxidized DCF in H₂O₂-, antimycin A-, and serum starvation-treated human lung adenocarcinoma A549 cells and human microvascular endothelial HMEC-1 cells. Following acetic acid-based fixation, the ROS generation in starved HMEC-1 cells could be evaluated by flow cytometric analysis while simultaneously monitoring the phosphorylation status of p38 mitogen-activated protein kinase. Immunostaining also revealed the synchronization of ROS generation and the H₂O₂-induced phosphorylation of Src homology-2 domain-containing phosphatase2. This study describes a modified method that may be used in future biomedical investigations to simultaneously measure intracellular ROS production and cellular oxidative responses.     

Dynamic determination of Ox-LDL-induced oxidative/nitrosative stress in single macrophage by using fluorescent probes

Increased oxidative/nitrosative stress, resulting from generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) appears to play an important role in the inflammatory responses to atherosclerosis. By using MitoTracker Orange CM-H(2)TMRos, CM-H(2)DCFDA (DCF-DA), Dihydrorhodamine 123 (DHR123), DAF-FM, Dihydroethidium (DHE) and JC-1 alone or in all combinations of red and green probes, the present study was designed to monitor the ROS and RNS generation in acute exposure of single monocyte U937-derived macrophage to oxidized low density lipoprotein (Ox-LDL). Acute Ox-LDL (100 microg/ml) treatment increased time-dependently production of intracellular nitric oxide (NO), superoxide (O2*-), hydrogen peroxide (H(2)O(2)) and peroxynitrite (ONOO(-)), and decreased mitochondrial membrane potential (Deltapsi) in single cell. Pretreatment of aminoguanidine (an inhibitor of inducible nitric oxide synthase (iNOS), 10 microM) and vitamin C (an antioxidant agent, 100 microM) for 2h, reduced significantly the Ox-LDL-induced increase of NO and O2*-, and vitamin C completely inhibited increase of intracellular NO and O2*-. In contrast to aminoguanidine, Vitamin C pretreatment significantly prevented Ox-LDL-induced overproduction of NO and O2*- (P<0.01), indicating that antioxidant may be more effective in therapeutic application than iNOS inhibitor in dysfunction of ROS/RNS. By demonstrating a complex imbalance of ROS/RNS via fluorescent probes in acute exposure of single cell to Ox-LDL, oxidative/nitrosative stress might be more detected in the early atherosclerotic lesions.     

Respiratory chain components involved in the glycerophosphate dehydrogenase-dependent ROS production by brown adipose tissue mitochondria

Involvement of mammalian mitochondrial glycerophosphate dehydrogenase (mGPDH, EC 1.1.99.5) in reactive oxygen species (ROS) generation was studied in brown adipose tissue mitochondria by different spectroscopic techniques. Spectrofluorometry using ROS-sensitive probes CM-H2DCFDA and Amplex Red was used to determine the glycerophosphate- or succinate-dependent ROS production in mitochondria supplemented with respiratory chain inhibitors antimycin A and myxothiazol. In case of glycerophosphate oxidation, most of the ROS originated directly from mGPDH and coenzyme Q while complex III was a typical site of ROS production in succinate oxidation. Glycerophosphate-dependent ROS production monitored by KCN-insensitive oxygen consumption was highly activated by one-electron acceptor ferricyanide, whereas succinate-dependent ROS production was unaffected. In addition, superoxide anion radical was detected as a mGPDH-related primary ROS species by fluorescent probe dihydroethidium, as well as by electron paramagnetic resonance (EPR) spectroscopy with DMPO spin trap. Altogether, the data obtained demonstrate pronounced differences in the mechanism of ROS production originating from oxidation of glycerophosphate and succinate indicating that electron transfer from mGPDH to coenzyme Q is highly prone to electron leak and superoxide generation.     

Effects of ethanol on pancreatic beta-cell death: interaction with glucose and fatty acids

Western lifestyle plays an important role in the prevalence of type 2 diabetes by causing insulin resistance and pancreatic β-cell dysfunction, a prerequisite for the development of diabetes. High fat diet and alcohol are major components of the western diet. The aim of the present study was to investigate the effects of ethanol and fatty acids on β-cell survival and metabolism. We treated the rat β-cell line RINm5F with ethanol, a mixture of palmitic and oleic acids, or both. Reactive oxygen species (ROS) were determined by (5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate) (CM-H2DCFDA) fluorescence assay, and mitochondrial activity was assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) reduction assay and by determining ATP production. Cell viability was assessed with a cell counter and trypan blue exclusion, and the mode of cell death by Hoechst33342 and propidium iodide staining. With both ethanol and fatty acid treatments, MTT reduction and ATP production decreased, whereas ROS production increased. Ethanol treatment had no effect on cell number, whereas fatty acid treatment reduced the cell number. Cell incubation with ethanol, fatty acids, or both increased the number of Hoechst 33342-positive nuclei. However, the majority of nuclei from fatty acid-treated cells were stained with propidium iodide, indicating a loss of plasma membrane integrity. We conclude that both ethanol and fatty acids generate cellular oxidative stress, and affect mitochondrial function in RINm5F β-cells. However, ethanol causes β-cell death by apoptosis, whereas fatty acids cause cell death predominantly by necrosis. It is not known whether these results are applicable to human β-cells.     

N-Propargylic β-enaminones in breast cancer cells: Cytotoxicity,apoptosis, and cell cycle analyses

Breast cancer is one of the most common cancers worldwide and the discovery of new cytotoxic agents is needed. Enaminones are regarded to be a significant structural motif that is found in a variety of pharmacologically active compounds however the number of studies investigating the anticancer activities of N-propargylic β-enaminones (NPEs) is limited. Herein we investigated the potential cytotoxic and apoptotic effects of 23 different NPEs (1-23) on human breast cancer cells. Cytotoxicity was evaluated via MTT assay. Apoptotic cell death and cell cycle distributions were investigated by flow cytometry. CM-H2DCFDA dye was used to evaluate cellular ROS levels. Expression levels of Bcl-2, Bax, p21, and Cyclin D1 were measured by quantitative real-time PCR. ADME properties were calculated using the ADMET 2.0 tool. NPEs 4, 9, 16, and 21 showed selective cytotoxic activity against breast cancer cells with SI values >2. NPEs induced apoptosis and caused significant changes in Bcl-2 and Bax mRNA levels. The cell cycle was arrested at the G0/G1 phase and levels of p21 and Cyclin D1 were upregulated in both breast cancer cells. ROS levels were significantly increased by NPEs, suggesting that the cytotoxic and apoptotic effects of NPEs were mediated by ROS. ADME analysis revealed that NPEs showed favorable distributions in both breast cancer cell lines, meaning good lipophilicity values, low unfractionated values, and high bioavailability. Therefore, these potential anticancer compounds should be further validated by in vivo studies for their appropriate function in human health with a safety profile, and a comprehensive drug interaction study should be performed.     

Analysis of mitochondrial free radical generation in animal models of neuronal disease

Mitochondria, the power plant of all eukaryotic cells, produce cellular energy in the form of ATP via electron transport and oxidative phosphorylation. However, the mitochondria leak electrons that can act as major sources of oxidative stress, and their dysfunction, have been proposed as causative events underlying neurodegeneration in stroke and neurodegenerative diseases. We examined whether MitoTracker Red CM-H(2)XRos, a rosamine derivative used to detect mitochondrial free radicals in vitro, would be applied to analyze the mitochondrial free radicals in various models of neurological diseases in vivo. The injections of MitoTracker Red CM-H(2)XRos revealed generation of mitochondrial free radicals primarily in vulnerable neurons following focal cerebral ischemia as well as administration of Fe(2+) or 3-nitropropionic acid. MitoTracker Red CM-H(2)XRos was retained after fixation, compatible with immunocytochemistry or nuclear staining, and can be applied to study roles of mitochondrial free radicals in the process of neurodegeneration in vivo.     

Xanthine oxidase-generated hydrogen peroxide is a consequence, not a mediator of cell death

Oxidative stress has been associated with a wide range of diseases including atherosclerosis, cancer and Alzheimer's disease. When present in excessive concentrations, reactive oxygen species (ROS) can cause deleterious effects. This has led to the notion that the anticancer effects of various chemotherapeutics may be mediated, at least in part, by an increase in ROS. To investigate the role of xanthine oxidase (XO), a source of hydrogen peroxide, in cell death, MCF7, HeLa and 293T cells were treated with various cell-death-inducing drugs in the presence and absence of allopurinol, a specific inhibitor of XO. In the absence of allopurinol, each drug led to a time- and concentration-dependent increase in percent DNA fragmentation and ROS levels, regardless of the mechanism of cell death incurred, i.e. caspase dependent and caspase independent. By contrast, pretreatment with allopurinol led to dramatically lower ROS levels in all cases, suggesting that XO is a major contributor to oxidative stress. However, allopurinol did not exhibit a protective effect against cell death. Similarly, the administration of siRNA against XO also did not exhibit a protective effect against cell death. The level of oxidative stress was recorded using the ROS sensor CM-H(2) DCFDA and a ratiometric bioluminescent assay that takes advantage of the increased sensitivity of Firefly luciferase to hydrogen peroxide, compared with a stable variant of Renilla luciferase (RLuc), RLuc8. Overall, these findings suggest that XO-generated hydrogen peroxide, and perhaps hydrogen peroxide in general, is a consequence, but not a mediator of cell death.     

Prevention of intracellular oxidation in yeast: the role of vitamin E analogue, Trolox (6-hydroxy-2,5,7,8-tetramethylkroman-2-carboxyl acid)

Reactive oxygen species (ROS) are not only generated in conditions of cellular stress but are also constitutively produced in most cell types by specific metabolic processes. This research focused on a potential antioxidant Trolox (model compound for alpha-tocopherol), with the aim to establish exact mechanisms of Trolox intracellular oxidation prevention on model organism Saccharomyces cerevisiae. Measuring intracellular oxidation of Trolox-treated yeast cells revealed that Trolox decreased intracellular oxidation during normal metabolism. Trolox treatment decreased cyto- and geno-toxicity of treated yeast cells in MES buffer, lowered intracellular oxidation, decreased intracellular peroxides formation, and increased H(2)O(2) degradation and superoxide quenching yeast extract ability. This study suggests that Trolox treatment provides prevention against intracellular ROS formation. Trolox application as therapeutic agent against intracellular ROS formation would be worth considering. Additionally, results indicate that yeasts are good model organisms for studying intracellular oxidation and oxidative stress. The obtained results on yeast cells might be useful to direct further human-related search for the Trolox evaluation as a human supplement used for protecting cells against intracellular free radical formation.     

Production of reactive oxygen species after photodynamic therapy by porphyrin sensitizers

The objectives of this study was to investigate the production of reactive oxygen species (ROS) after photodynamic therapy (PDT) in vitro. We examined second generation sensitizers, porphyrines (TPPS4, ZnTPPS4 and PdTPPS4) and compared their effectivity on ROS generation in G361 cell line. Used porphyrines are very efficient water-soluble aromatic dyes with potential to use in photomedicine and have a high propensity to accumulate in the membranes of intracellular organelles like lysosomes and mitochondria. Interaction between the triplet excited state of the sensitizer and molecular oxygen leads to produce singlet oxygen and other ROS to induce cell death. Production of ROS was verificated by molecular probe CM-H2DCFDA and viability of cells was determined by MTT assay. Our results demonstrated that ZnTPPS4 induces the highest ROS production in cell line compared to TPPS4 and PdTPPS4 at each used concentration and light dose. These results consist with a fact that photodynamic effect depends on sensitizer type, its concentration and light dose.     

Beyond LDL oxidation: ROS in vascular signal transduction

The notion that oxidative stress contributes to the pathogenesis of vascular disease was originally driven by observations that low-density lipoprotein (LDL) modification is a prominent feature of atherosclerosis. More recently, it has become clear that the relation between oxidative stress and vascular disease goes beyond LDL oxidation and involves cellular production of reactive oxygen species (ROS). Considerable data now indicate that ROS represent an important means of cellular signaling, although the precise mechanisms whereby ROS accomplish this function remain unclear. Emerging data point to protein thiol groups as important targets for post-translational protein modification by ROS. In this review, the data linking ROS to cell signaling is discussed and the notion that ROS mediate a vascular "injury" response is proposed.     

Protein oxidation and cellular homeostasis: Emphasis on metabolism

Reactive oxygen species (ROS) are generated as the result of a number of physiological and pathological processes. Once formed ROS can promote multiple forms of oxidative damage, including protein oxidation, and thereby influence the function of a diverse array of cellular processes. This review summarizes the mechanisms by which ROS are generated in a variety of cell types, outlines the mechanisms which control the levels of ROS, and describes specific proteins which are common targets of ROS. Additionally, this review outlines cellular processes which can degrade or repair oxidized proteins, and ultimately describes the potential outcomes of protein oxidation on cellular homeostasis. In particular, this review focuses on the relationship between elevations in protein oxidation and multiple aspects of cellular metabolism. Together, this review describes a potential role for elevated levels of protein oxidation contributing to cellular dysfunction and oxidative stress via impacts on cellular metabolism.     

Quantitation of intracellular oxidation in a renal epithelial cell line

We quantitated the presence of intracellular oxidizing species in response to oxidative stimuli using fluorescent cell analytic techniques. The studies were performed with a laser-activated flow cytometry system using 2',7'-dichlorofluorescin diacetate (DCFDA) as a probe for intracellular oxidation events. Oxygen radical formation was initiated by the addition of FeCl2 or xanthine oxidase to the culture media. Xanthine oxidase and FeCl2 both increased intracellular DCFDA oxidation over control (p less than .001). Increases in intracellular DCFDA oxidation in response to xanthine oxidase exposure were inhibited by extracellular superoxide dismutase, catalase and dimethyl sulfoxide (p less than 0.001), implicating the superoxide anion, hydrogen peroxide, and the hydroxyl radical in producing the changes in intracellular dichlorofluorescein fluorescence. Increases in intracellular DCFDA oxidation in response to xanthine oxidase correlated with loss of cellular viability, as established by decreased plating efficiency. We conclude that relative intracellular oxidation can be quantitated within the cultured renal cell and that some extracellularly generated radicals may be capable of traversing the intact cell membrane to oxidize DCFDA in the cell interior.     

Generation of ROS in response to CCK-8 stimulation in mouse pancreatic acinar cells

In the present study we have studied the changes in the intracellular reduction-oxidation state in mouse pancreatic acinar cells following stimulation with cholecystokinin octapeptide (CCK-8) and its dependence on Ca2+ mobilization. In our investigations cytosolic Ca2+ concentration and reactive oxygen species (ROS) production were determined by loading of cells with fura-2 and CM-H2DCF-DA, respectively. Changes in these parameters were determined by following changes in fluorescence in the cuvette of a spectrofluorimeter. The results show that stimulation of cells with CCK-8 and/or the sarco-endoplasmic reticulum Ca2+ pump inhibitor, thapsigargin (Tps), both induced changes in cytosolic free Ca2+ concentration and led to an increase in fluorescence of CM-H2DCF-DA, reflecting an increase in oxidation. In the presence of Tps, addition of CCK-8 did not significantly increase fluorescence compared to that evoked by the SERCA inhibitor. Similar results were obtained in the absence of extracellular Ca2+ and in the presence of EGTA. When the cells were challenged in the presence of the intracellular Ca2+ chelator BAPTA and in the absence of extracellular Ca2+ the responses to both CCK-8 and Tps were reduced although not completely inhibited. The mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxy-phenylhydrazone and the inhibitor of the electron transport chain, antimycin, evoked a marked increase in CM-H2DCF-DA fluorescence and completely inhibited CCK-8 and Tps-evoked responses, indicating that ROS are generated in the mitochondria. In summary, stimulation of mouse pancreatic acinar cells with CCK-8 leads to generation of ROS, and this effect may be derived from Ca2+ mobilization from intracellular stores and involves mitochondrial metabolism.     

Sensitization of U937 cells to heat shock by oxalomalate, a competitive inhibitor of NADP+-dependent isocitrate dehydrogenase

Heat shock may increase oxidative stress due to increased production of reactive oxygen species (ROS) and/or the promotion of cellular oxidation events. Cytosolic NADP

+

-dependent isocitrate dehydrogenase (ICDH) in U937 cells produces NADPH, an essential reducing equivalent for the antioxidant system. The protective role of ICDH against heat shock in U937 cells was investigated in control and cells treated with oxlalomalate, a competitive inhibitor of ICDH. Upon exposure to heat shock, the viability was lower and the protein oxidation, lipid peroxidation and oxidative DNA damage were higher in oxalomalate-treated cells as compared to control cells. We also observed the significant increase in the endogenous production of ROS, as measured by the oxidation of 2'7'-dichlorodihydrofluorescin in U937 cells treated with oxalomalate. These results suggest that ICDH plays an important role as an antioxidant defense enzyme in cellular defense against heat shock through the removal of ROS.     

Palmitate-induced apoptosis of microvascular endothelial cells and pericytes

BACKGROUND: Recent observations in the EURODIAB Complications Study demonstrated that markers of insulin resistance are strong risk factors for retinopathy incidence in patients with diabetes. However, the molecular mechanism underlying this remains to be elucidated. In this study, we investigated the influence of palmitate, a major saturated free fatty acid in plasma, on the apoptotic cell death of cultured microvascular endothelial cells (EC) and retinal pericytes. MATERIALS AND METHODS: The intracellular formation of reactive oxygen species (ROS) was detected using the fluorescent probe CM-H(2)DCFDA. DNA synthesis was determined by measuring [(3) H]-thymidine incorporation into cells. DNA fragmentations of EC were quantitatively analyzed in an enzyme-linked immunosorbent assay, and DNA laddering was evaluated on agarose gel electrophoresis. RESULTS: Palmitate increased ROS generation in microvascular EC. Furthermore, palmitate significantly inhibited DNA synthesis and induced apoptotic cell death in EC, which were completely prevented by an antioxidant, N-acetylcysteine. Palmitate up-regulated pericyte mRNA levels of a receptor for advanced glycation end products (AGE), and thereby potentiated the apoptotic effects of AGE on pericytes. CONCLUSIONS: The results suggest that palmitate could induce apoptotic cell death in microvascular EC and pericytes through the overgeneration of intracellular ROS, and thus be involved in the development of diabetic retinopathy.     

Altered intracellular redox status in Gaucher disease fibroblasts and impairment of adaptive response against oxidative stress

Gaucher disease (GD) is a lysosomal storage disorder, due to glucosylceramide (GlcCer) accumulation in several body tissues, which causes cellular failure by yet unidentified mechanisms. Several evidence indicates that GD pathogenesis is associated to an impairment in intracellular redox state. In fibroblast primary cultures, reactive oxygen species (ROS) levels and protein carbonyl content resulted significantly increased in GD patients compared to healthy donors, suggesting that GD cells, facing a condition of chronic oxidative stress, have evolved an adaptive response to survive. The ROS rise is probably due to NAD(P)H oxidase activity, being inhibited by the treatment with diphenylene iodonium chloride. Interestingly, GD cells are more sensitive to H(2)O(2) induced cell death, suggesting a dysregulation in the adaptive response to oxidative stress in which APE1/Ref-1 plays a central role. We found that the cytoplasmic amounts of APE1/Ref-1 protein were significantly higher in GD fibroblasts with respect to controls, and that GD cells failed to upregulate its expression upon H(2)O(2) treatment. Both ROS and APE1/Ref-1 increases are due to GlcCer accumulation, being prevented by treatment of GD fibroblasts with Cerezyme and induced in healthy fibroblasts treated with conduritol-beta-epoxide. These data, suggesting that GD cells display an impairment in the cellular redox state and in the adaptive cellular response to oxidative stress, may open new perspectives in the comprehension of GD pathogenesis.     

Two Distinct Sources of Elicited Reactive Oxygen Species in Tobacco Epidermal Cells

Reactive oxygen species (ROS) play a prominent role in early and later stages of the plant pathogenesis response, putatively acting as both cellular signaling molecules and direct antipathogen agents. A single-cell assay, based on the fluorescent probe dichlorofluorescein, was used to scrutinize the generation and movement of ROS in tobacco epidermal tissue. ROS, generated within cells, quickly moved apoplastically as H2O2 into neighboring cells. Two classes of rapidly elicited intracellular ROS, originating from distinct sources, were distinguished. Cryptogein, the fungal elicitor from Phytophthora cryptogea, induced ROS from a flavin-containing oxidase source. ROS accumulation could be inhibited by a number of pharmacological agents, suggesting induction through an active signal transduction pathway. The insensitivity of the increase in ROS to the external addition of enzymes that dissipate ROS suggests that this oxidative increase is primarily intracellular. In contrast, amines and polyamines, compounds that form during wounding and pathogenesis, induced ROS at an apoplastic site from peroxidase- or amine oxidase-type enzyme(s). Salicylic acid, a putative inhibitor of cellular catalases and peroxidases, did not induce cellular ROS, as measured by dichlorofluorescein fluorescence. The physiological relevance of ROS-generated signals was indicated by the rapid alteration of the epidermal cell glutathione pool and the cellular redox state. In addition, induction of ROS by all elicitors was correlated with subsequent cell death.     

The Use of Fluorescent Probes to Assess Oxidative Processes in Isolated-Perfused Rat Heart Tissue

The formation of reactive oxygen species (ROS) in intact heart tissue has been assessed by direct ESR measurements, and indirectly by the formation of characteristic tissue products and the protective effects of various antioxidants. The development of lipid soluble esters of compounds which can be trapped intra-cellularly after hydrolysis, and which fluoresce after oxidation, has provided a new tool to investigate ROS in vitro. The utility of 2',7'-dichlorofluorescin diacetate (DCFDA) in isolated-perfused rat heart tissue was investigated in the present study. DCFDA and its deacetylated form were incubated with various levels of hydrogen peroxide or t-butylhydroperoxide (tBOOH). Conversion of the diacetate form to a fluorescent product required 4-5 h with hydrogen peroxide and up to 24 h with tBOOH. In contrast, the deacetylated form fluoresced at 80% of maximum levels 1 h after the addition of 100 mM tBOOH. DCFDA was loaded into heart tissue by infusing for lO min at a final concentration of 10,aM in Krebs-Henseleit bicarbonate buffer. After a lO min washout period, analysis of freeze-clamped heart tissue revealed that the trapped material was readily converted to a fluorescent product by tBOOH, indicating hydrolysis had occurred. Fluorescence of material trapped in heart tissue was approximately 24% of the maximum achieved after oxidation with lOOmM tBOOH. This value decreased to 18 and 13% when the loading and washout periods were from 0 to 20 or 10 to 30min of hypoxia, respectively. Similar results were obtained with the less readily oxidized dicarboxy derivative of DCFDA. Infusion of 500μM tBOOH increased the oxidation of DCFDA in heart tissue from 24 to 31%. These data demonstrate that DCFDA can be loaded into heart tissue and is capable of reflecting relative changes in the oxidative state of this organ.