Dichlorofluorescein Formation Research Articles

Homocysteine May Decrease Glucose Uptake and Alter the Akt/GSK3β/GLUT1 Signaling Pathway in Hippocampal Slices: Neuroprotective Effects of Rivastigmine and Ibuprofen.

Homocysteine (Hcy) is a risk factor for neurodegenerative diseases, such as Alzheimer's Disease, and is related to cellular and tissue damage. In the present study, we verified the effect of Hcy on neurochemical parameters (redox homeostasis, neuronal excitability, glucose, and lactate levels) and the Serine/Threonine kinase B (Akt), Glucose synthase kinase-3β (GSK3β) and Glucose transporter 1 (GLUT1) signaling pathway in hippocampal slices, as well as the neuroprotective effects of ibuprofen and rivastigmine alone or in combination in such effects. Male Wistar rats (90 days old) were euthanized and the brains were dissected. The hippocampus slices were pre-treated for 30min [saline medium or Hcy (30 µM)], then the other treatments were added to the medium for another 30min [ibuprofen, rivastigmine, or ibuprofen + rivastigmine]. The dichlorofluorescein formed, nitrite and Na+, K+-ATPase activity was increased by Hcy at 30 µM. Ibuprofen reduced dichlorofluorescein formation and attenuated the effect of Hcy. The reduced glutathione content was reduced by Hcy. Treatments with ibuprofen and Hcy + ibuprofen increased reduced glutathione. Hcy at 30 µM caused a decrease in hippocampal glucose uptake and GLUT1 expression, and an increase in Glial Fibrillary Acidic Protein-protein expression. Phosphorylated GSK3β and Akt levels were reduced by Hcy (30 µM) and co-treatment with Hcy + rivastigmine + ibuprofen reversed these effects. Hcy toxicity on glucose metabolism can promote neurological damage. The combination of treatment with rivastigmine + ibuprofen attenuated such effects, probably by regulating the Akt/GSK3β/GLUT1 signaling pathway. Reversal of Hcy cellular damage by these compounds may be a potential neuroprotective strategy for brain damage.

Experimental Conditions That Influence the Utility of 2'7'-Dichlorodihydrofluorescein Diacetate (DCFH2-DA) as a Fluorogenic Biosensor for Mitochondrial Redox Status.

Oxidative stress has been causally linked to various diseases. Electron transport chain (ETC) inhibitors such as rotenone and antimycin A are frequently used in model systems to study oxidative stress. Oxidative stress that is provoked by ETC inhibitors can be visualized using the fluorogenic probe 2′,7′-dichlorodihydrofluorescein-diacetate (DCFH2-DA). Non-fluorescent DCFH2-DA crosses the plasma membrane, is deacetylated to 2′,7′-dichlorodihydrofluorescein (DCFH2) by esterases, and is oxidized to its fluorescent form 2′,7′-dichlorofluorescein (DCF) by intracellular ROS. DCF fluorescence can, therefore, be used as a semi-quantitative measure of general oxidative stress. However, the use of DCFH2-DA is complicated by various protocol-related factors that mediate DCFH2-to-DCF conversion independently of the degree of oxidative stress. This study therefore analyzed the influence of ancillary factors on DCF formation in the context of ETC inhibitors. It was found that ETC inhibitors trigger DCF formation in cell-free experiments when they are co-dissolved with DCFH2-DA. Moreover, the extent of DCF formation depended on the type of culture medium that was used, the pH of the assay system, the presence of fetal calf serum, and the final DCFH2-DA solvent concentration. Conclusively, experiments with DCFH2-DA should not discount the influence of protocol-related factors such as medium and mitochondrial inhibitors (and possibly other compounds) on the DCFH2-DA-DCF reaction and proper controls should always be built into the assay protocol.

Phenolic compounds from the stems of Fissistigma polyanthoides and their anti-oxidant activities

The stems of Fissistigma polyanthoides (A.DC.) Merr. are traditionally used for the treatment of rheumatism and for recuperating women after childbirth. In our continuous phytochemical investigation of this plant, four new (1, 2, 5, and 19) and fifteen known (3, 4, and 6–18) phenolic compounds were isolated. The structures of all compounds were elucidated based on extensive spectroscopic analyses (1D-, 2D-NMR, and MS), and in comparison with reported literature data. The new natural products showed to be two poly-methoxylated chalcones (1 and 2) and two flavonoid glycosides, with 19 containing an uncommon sugar moiety (quinovose). Compounds with sufficient amount were tested for their anti-oxidant activity in a cell-based assay using the human bronchial epithelial cell line BEAS-2B. The compounds' capacity to inhibit the peroxyl radical triggered formation of dichlorofluorescein (DCF) was investigated in a dose-dependent manner. Both, anti-oxidant (3, 4, 6, 8–12, and 14) and pro-oxidative (5 and 16) properties were found for the investigated substances. The half maximal concentrations (IC50) for the inhibition of ROS formation ranged between 18.8 μM and 63.5 μM. Compounds, which acted protectively in the cellular antioxidant activity (CAA) assay and did not negatively affect cell viability, could be interesting targets for further investigations.

NADPH oxidase inhibitor inhibits methylglyoxal‐induced podocyte apoptosis (LB729)

Methylglyoxal (MG) is one of uremic toxins produced by glycolysis that may results in diabetic nephropathy. In this study, we investigated the effect and mechanism of MG on apoptosis in podocytes. Conditionally immortalized podocytes for differentiation were induced by culturing the cells at 37°C for 13~15 days and treated MG with various concentration (0, 50,100, 200, 400 μM). Our data show that MG induced a decrease in cell viability in a dose‐dependent manner and increased formation of dichlorofluorescein (DCF)‐sensitive intracellular reactive oxygen species (ROS). This enhanced ROS formation was significantly reduced by NADPH oxidase inhibitor (DPI, 5 μM). Furthermore, MG treatment induced apoptosis in podocyte and NADPH oxidase inhibitor inhibited MG‐induced podocytes apoptosis via cleavage inhibition of poly (ADP‐ribose) polymerase (PARP). MG treatment also induced phosphorylation of extracellular‐regulated kinase 1/2 (ERK1/2), p38 mitogen‐activated protein kinases (MAPKs), protein kinase C‐alpha and ‐ delta/zeta, and phosphoinositide‐3 kinase (PI3K). Pretreatment with these inhibitors inhibited MG‐induced phosphorylation of these molecules in podocytes. These results suggest that MG‐induced apoptosis may be mediated by signaling of ERK, MAPK, PKC and PI3K in podocyte.Grant Funding Source: KIOM13040

Modulation of the cellular redox status by the Alternaria toxins alternariol and alternariol monomethyl ether

The mycotoxin alternariol (AOH) has been reported to possess genotoxic properties, inducing enhanced levels of DNA damage after only 1h of incubation. In the present study we addressed the question whether the induction of oxidative stress might contribute to the genotoxic effects of AOH or its naturally occurring monomethylether (AME). In the dichlorofluorescein (DCF) assay, treatment of HT29 cells for 1h enhanced the formation of dichlorofluorescein, indicative for ROS formation. The total glutathione (tGSH) was transiently decreased. In accordance with the results of the DCF assay, AOH and AME enhanced the proportion of the transcription factor Nrf2 in the nucleus. Concomitantly, the Nrf2/ARE-dependent genes γ-glutamylcysteine ligase (γ-GCL) and glutathione-S-transferase (GSTA1/2) showed enhanced transcript levels. After 24h of incubation this effect was also reflected on the protein level by an increase of GST activity. However, in spite of the positive DCF assay and the activation of the redox-sensitive Nrf2/ARE-pathway, the level of oxidative DNA damage, measured in the comet assay by the addition of formamidopyrimidine-DNA-glycosylase (fpg) remained unaffected. Of note, after 3h of incubation no significant DNA damaging potential of AOH and AME was detectable, indicating either inactivation of the compounds or enhanced DNA repair. In summary, the mycotoxins AOH and AME were found to modulate the redox balance of HT29 cells but without apparent negative effect on DNA integrity.

Antioxidant properties of propargylamine derivatives: assessment of their ability to scavenge peroxynitrite

A series of arylpropargylamines, variously substituted in the hydrogen in p-position and in the propargyl moiety, were studied as potential peroxynitrite scavengers. The scavenging activity of these compounds was evaluated through peroxynitrite (ONOO-)-mediated oxidation of dichlorofluorescin and linoleic acid by measuring the dichlorofluorescein formation and oxygen consumption, respectively. Among tested compounds, only 1-phenylpropargylamine (AP3) promoted concentration-dependent inhibition of ONOO(-)-induced dichlorofluorescin and linoleic acid oxidation with IC50 values of 637 and 63 microM, respectively. The AP3 spectral changes in UV-visible absorbance properties in the presence of peroxynitrite suggested the formation of a new compound. This was identified by gas-chromatograph-mass spectrometer analysis as phenylpropargyl alcohol. Structure-activity relationship analysis indicated that the scavenging activity of AP3 was due to the aminopropargyl moiety and availability of the nitrogen electron pair. This data suggested that AP3 could be considered a lead compound for the synthesis of new ONOO- scavenger derivatives.

Cytoprotection against hydrogen peroxide-induced cell death in cultured mouse mesangial cells by erigeroflavanone, a novel compound from the flowers of Erigeron annuus

Hyperglycemia-induced oxidative stress has been suggested as a mechanism underlying diabetic complications. Oxidative stress triggers cell death in various cell types, including glomerular mesangial cells which play important roles in diabetic nephropathy. In the present study, we investigated the potential cytoprotective effect of erigeroflavanone, a novel flavanone derivative from the flowers of Erigeron annuus, in cultured mouse mesangial cells using hydrogen peroxide (H 2O 2) as an oxidative stress inducer. Our data show that hydrogen peroxide induced a decrease in cell viability that was attenuated by erigeroflavanone. Hydrogen peroxide treatment increased formation of dichlorofluorescein (DCF)-sensitive intracellular reactive oxygen species (ROS). This enhanced ROS formation was significantly reduced by pretreatment with erigeroflavanone in a dose-dependent manner. Hydrogen peroxide treatment also induced phosphorylation of the mitogen-activated protein kinases (MAPKs), c-Jun terminal kinase (JNK), extracellular-regulated kinase (ERK) and p38, and activated caspase-3. Pretreatment with erigeroflavanone inhibited hydrogen peroxide-induced activation of MAPKs and caspase-3. From these data we conclude that erigeroflavanone provides a protective effect against oxidative stress-induced cell death in mesangial cells that is associated with its antioxidant action and inhibition of MAPKs and caspase-3. These results suggest that erigeroflavanone has potential as a therapeutic agent in the treatment of renal diabetic complications.

Singlet Oxygen Reacts with 2′,7′‐Dichlorodihydrofluorescein and Contributes to the Formation of 2′,7′‐Dichlorofluorescein

There are controversial reports in the literature concerning the reactivity of singlet oxygen ((1)O(2)) with the redox probe 2',7'-dichlorodihydrofluorescein (DCFH). By carefully preparing solutions in which (1)O(2) is quantitatively generated in the presence of DCFH, we were able to show that the formation rate of the fluorescent molecule derived from DCFH oxidation, which is 2',7'-dichlorofluorescein (DCF), increases in D(2)O and decreases in sodium azide, proving the direct role of (1)O(2) in this process. We have also prepared solutions in which either (1)O(2) or dication (MB(2+)) and semi-reduced (MB) radicals of the sensitizer and subsequently super-oxide radical (O(2)(-)) are generated. The absence of any effect of SOD and catalase ruled out the DCFH oxidation by O(2)(-), indicating that both (1)O(2) and MB(2+) react with DCFH. Although the formation of DCF was 1 order of magnitude larger in the presence of MB(2+) than in the presence of (1)O(2), considering the rate of spontaneous decays of these species in aqueous solution, we were able to conclude that the reactivity of (1)O(2) with DCFH is actually larger than that of MB(2+). We conclude that DCFH can continue to be used as a probe to monitor general redox misbalance induced in biologic systems by oxidizing radicals and (1)O(2).

Cellular Antioxidant Activity (CAA) Assay for Assessing Antioxidants, Foods, and Dietary Supplements

A cellular antioxidant activity (CAA) assay for quantifying the antioxidant activity of phytochemicals, food extracts, and dietary supplements has been developed. Dichlorofluorescin is a probe that is trapped within cells and is easily oxidized to fluorescent dichlorofluorescein (DCF). The method measures the ability of compounds to prevent the formation of DCF by 2,2'-azobis(2-amidinopropane) dihydrochloride (ABAP)-generated peroxyl radicals in human hepatocarcinoma HepG2 cells. The decrease in cellular fluorescence when compared to the control cells indicates the antioxidant capacity of the compounds. The antioxidant activities of selected phytochemicals and fruit extracts were evaluated using the CAA assay, and the results were expressed in micromoles of quercetin equivalents per 100 micromol of phytochemical or micromoles of quercetin equivalents per 100 g of fresh fruit. Quercetin had the highest CAA value, followed by kaempferol, epigallocatechin gallate (EGCG), myricetin, and luteolin among the pure compounds tested. Among the selected fruits tested, blueberry had the highest CAA value, followed by cranberry > apple = red grape > green grape. The CAA assay is a more biologically relevant method than the popular chemistry antioxidant activity assays because it accounts for some aspects of uptake, metabolism, and location of antioxidant compounds within cells.

The roles of thiol-derived radicals in the use of 2′,7′-dichlorodihydrofluorescein as a probe for oxidative stress

2′,7′-Dichlorodihydrofluorescein (DCFH2) is one of the most widely used probes for detecting intracellular oxidative stress, but requires a catalyst to be oxidized by hydrogen peroxide or superoxide and reacts nonspecifically with oxidizing radicals. Thiyl radicals are produced when many radicals are “repaired” by thiols, but are oxidizing agents and thus potentially capable of oxidizing DCFH2. The aim of this study was to investigate the reactivity of thiol-derived radicals toward DCFH2 and its oxidized, fluorescent form 2′,7′-dichlorofluorescein (DCF). Thiyl radicals derived from oxidation of glutathione (GSH) or cysteine (CysSH) oxidized DCFH2 with rate constants at pH 7.4 of ∼4 or ∼2×107 M−1 s−1, respectively. Both the rates of oxidation and the yields of DCF were pH-dependent. Glutathione-derived radicals interacted with DCF, resulting in the formation of DCFH absorbing at 390 nm and loss of fluorescence; in contrast, cysteine-derived radicals did not cause any depletion of DCF fluorescence. We postulate that the observed apparent difference in reactivity between GS and CysS toward DCF is related to the formation of carbon-centered, reducing radicals from base-catalyzed isomerization of GS. DCF formation from interaction of DCFH2 with GS was inhibited by oxygen in a concentration-dependent manner over the physiological range. These data indicate that in applying DCFH2 to measure oxidizing radicals in biological systems, we have to consider not only the initial competition between thiols and DCFH2 for the oxidizing radicals, but also subsequent reactions of thiol-derived radicals, together with variables—including pH and oxygen concentration—which control thiyl radical chemistry.

Effects of protein malnutrition on oxidative status in rat brain

Objectives This study evaluated the effects of protein malnutrition on oxidative status in rat brain areas. Methods We investigated various parameters of oxidative status, free radical content (dichlorofluorescein formation), indexes of damage to lipid (thiobarbituric acid-reactive substances assay), and protein damage (tryptophan and tyrosine content) in addition to total antioxidant reactivity levels and antioxidant enzyme activities of superoxide dismutase, glutathione peroxidase, and catalase in different cerebral regions (cortex, hippocampus, and cerebellum) from rats subjected to prenatal and postnatal protein malnutrition (control 25% casein and protein malnutrition 7% casein). Results Protein malnutrition altered various parameters of oxidative stress, especially damage to macromolecules. Free radical content was unchanged by protein malnutrition. There was an increase in levels of thiobarbituric acid-reactive substances, the index of lipid peroxidation, in the cerebellum and cerebral cortex ( P < 0.05) from protein-malnourished rats. Moreover, significant decreases in tryptophan and tyrosine in all tested brain structures ( P < 0.05) were observed. Catalase activity was significantly decreased in the cerebellum ( P < 0.05). In addition, a significant decrease in total antioxidant reactivity levels ( P < 0.05) was observed in the cerebral cortex from protein-malnourished rats. Conclusions The present data indicated that protein malnutrition increased oxidative damage to lipids and proteins from the studied brain areas. These results may be an indication of an important mechanism for changes in brain development that are caused by protein malnutrition.

Nitric oxide down‐regulates the expression of the catalytic NADPH oxidase subunit Nox1 in rat renal mesangial cells

Glomerular mesangial cells can produce high amounts of nitric oxide (NO) and reactive oxygen species (ROS). Here we analyzed the impact of NO on the ROS-generating system, particularly on the NADPH oxidase Nox1. Nox1 mRNA and protein levels were markedly decreased by treatment of mesangial cells with the NO-releasing compound DETA-NO in a concentration- and time-dependent fashion. By altering the cGMP signaling system with different inhibitors or activators, we revealed that the effect of NO on Nox1 expression is at least in part mediated by cGMP. Analysis of a reporter construct comprising the 2547 bp of the nox1 promoter region revealed that a stimulatory effect of IL-1beta on nox1 transcription is counteracted by an inhibitory effect of IL-1beta-evoked endogenous NO formation. Moreover, pretreatment of mesangial cells with DETA-NO attenuated platelet-derived growth factor (PDGF)-BB or serum stimulated production of superoxide as assessed by real-time EPR spectroscopy and dichlorofluorescein formation. Transfection of mesangial cells with siRNAs directed against Nox1 and Nox4 revealed that inhibition of Nox1, but not Nox4 expression, is responsible for the reduced ROS formation by NO. Obviously, there exists a fine-tuned crosstalk between NO and ROS generating systems in the course of inflammatory diseases.

Spectrofluorometric determination of intracellular levels of reactive oxygen species in drug-sensitive and drug-resistant cancer cells using the 2′,7′-dichlorofluorescein diacetate assay

This article examines a non-invasive spectrofluorometric method using the 2′,7′-dichlorofluorescein diacetate (DCHF-DA) assay for quantifying the intracellular reactive oxygen species (ROS i) produced in four cultured cancer cell lines: drug-sensitive (K562) and drug-resistant (K562/ adr) human erythromyelogenous leukemia cell lines, and drug-sensitive (GLC4) and drug-resistant (GLC4/ adr) human small cell lung carcinoma cell lines. The oxidation of the probe to the fluorescent dichlorofluorescein (DCF) was continuously monitored by following the DCF fluorescence intensity as a function of time using a standard spectrofluorometer in the presence of an extracellular DCF fluorescence quencher (Co 2+). By fitting the spectrofluorometric data to a kinetic model based on the following two reactions: (i) deacetylation of DCHF-DA to the oxidant-sensitive compound 2′,7′-dichlorofluorescein (DCHF) by cellular esterase enzymes (pseudo-first-order rate constant: k e) and (ii) oxidation of DCHF by ROS i (second-order rate constant: k 2), the parameters intervening in DCF formation, k e and the product of k 2 by the ROS i concentration, were quantitatively determined for the different cell lines studied. The results revealed that the intracellular esterase content or activity is similar in K562, K562/ adr, and GLC4 cells, but 5-fold higher in GLC4/ adr cells. The product k 2[ROS i] was found to be similar in the four cell lines considered, with a mean value of (5.3±0.9)×10 −7 cell −1 s −1. Assuming that H 2O 2 (in combination with peroxidases) is the primary responsible species for DCHF oxidation in intact cells, and using the rate constant value k 2 = 790 ± 62 M - 1 s - 1 established in our laboratory for the reaction of DCHF with H 2O 2 in the presence of horseradish peroxidase, the mean value of the intracellular levels of ROS i in those cells was estimated to be 0.67±0.16 nM per cell. Such a value compares favorably to H 2O 2 intracellular steady-state concentrations that have been estimated in the literature for a few other cell types.

Direct oxidation of 2′,7′-dichlorodihydrofluorescein by pyocyanin and other redox-active compounds independent of reactive oxygen species production

Formation of dichlorofluorescein (DCF), the fluorescent oxidation product of 2′,7′-dichlorodihydrofluorescein (DCFH 2), in cells loaded with the latter compound is often used to detect ROS formation. We previously found that exposure of DCFH 2-loaded A549 cells to the Pseudomonas aeruginosa secretory product pyocyanin results in DCF formation, consistent with ROS production. However, since pyocyanin directly accepts electrons from NAD(P)H, we hypothesized that pyocyanin might directly oxidize DCFH 2 to DCF without an ROS intermediate. Incubation of DCFH 2 with pyocyanin rapidly resulted in DCF formation, the rate of which was proportional to the [pyocyanin] and was not inhibited by SOD or catalase. Phenazine methosulfate, a pyocyanin analog, was more effective than pyocyanin in generating DCF. Mitoxantrone and ametantrone also produced DCF. However, menadione, paraquat, plumbagin, streptonigrin, doxorubicin, daunorubicin, and 5-iminodaunorubicin did not. Pyocyanin, phenazine methosulfate, mitoxantrone, and ametantrone also oxidized dihydrofluorescein and 5- (and 6-) -carboxy-2′,7′-dichlorodihydrofluorescein, whereas dihydrorhodamine was oxidized only by pyocyanin or phenazine methosulfate. Under aerobic conditions, the interaction of DCFH 2 with pyocyanin or phenazine methosulfate (but not mitoxantrone or ametantrone) produced superoxide, as detected by spin trapping. Direct oxidation of the fluorescent probes needs to be controlled for when employing these compounds to assess ROS formation by biological systems exposed to redox active compounds.

Photosensitized oxidation of 2',7'-dichlorofluorescin: singlet oxygen does not contribute to the formation of fluorescent oxidation product 2',7'-dichlorofluorescein.

2',7'-Dichlorofluorescin (DCFH) is often employed to assess oxidative stress in cells by monitoring the appearance of 2',7'-dichlorofluorescein (DCF), its highly fluorescent oxidation product. We have investigated the photosensitized oxidation of DCFH in solution and elucidated the role played by singlet molecular oxygen (1O(2)) in this reaction. We used rose bengal (RB), protoporphyrin, and DCF as photosensitizers. Irradiation (550 nm) of RB (20 microM) in 50 mM phosphate (pH 7.4) in the presence of DCFH (50 microM) resulted in the rapid formation of DCF, measured as an increase in its characteristic absorbance and fluorescence. The oxidation rate was faster in deoxygenated solution, did not increase in D(2)O, and even increased in the presence of sodium azide. The presence of antioxidants that react with 1O(2), thus removing oxygen, accelerated DCF formation. Such results eliminate any potential direct involvement of 1O(2) in DCF formation, even though DCFH is an efficient (physical) quencher of 1O(2) (k(q) = 1.4 x 10(8) M(-1)s(-1) in methanol). DCF is also a moderate photosensitizer of 1O(2) with a quantum yield of circa phi = 0.06 in D(2)O and phi = 0.08 in propylene carbonate, which unequivocally indicates that DCF can exist in a triplet state upon excitation with UV and visible light. This triplet can initiate photo-oxidization of DCFH via redox-and-radical mechanism(s) similar to those involving RB (vide supra). Our results show that, upon illumination, DCF can function as a moderate photosensitizer initiating DCFH oxidation, which may prime and accelerate the formation of DCF. We have also shown that, while 1O(2) does not contribute directly to DCF production, it can do so indirectly via reaction with cellular substrates yielding peroxy products and peroxyl radicals, which are able to oxidize DCFH in subsequent dark reactions. These findings suggest that DCFH should not be regarded as a probe sensitive to singlet molecular oxygen, and that care must be taken when using DCFH to measure oxidative stress in cells as a result of both visible and UV light exposure.

NF-kappa B binding activity and cyclooxygenase-2 expression in persistent CCl(4)-treated rat liver injury.

The involvement of NF-kappa B binding activity is known to be important in the mechanism of acute liver injury and in the induction of cyclooxygenase (COX-2). This study was performed to evaluate NF-kappa B binding activity and the expression of COX-2 in chronic liver injury induced by carbon tetrachloride (CCl(4)). Liver tissues from Sprague-Dawley rats were collected at 1, 3, 5, and 7th week after intraperitoneal injection of 0.1 mL of CCl(4)/100 g body weight twice a week. Reactive oxy-gen species (ROS) were measured in the postmitochondrial fraction by dichlorofluorescein formation with a fluorescent probe. An electrophoretic mobility shift assay was performed for NF-kappa B binding activity. Western blot was performed to measure the level of COX-1, COX-2, p65, p50, and I B proteins. ROS and NF-kappa B activity increased during the CCl4-induced chronic liver injury. The expression of nuclear p65 protein and p50 protein increased compared with that of the control, while the cytoplasmic I B protein decreased as the inflammation persisted. The expression of COX-2 in CCl(4)-treated rat liver increased compared with that of the control. It could be suggested that ROS produced by CCl(4) treatment increased NF-kappa B binding activity and thereby COX-2 expression, and these might be implicated in the progress of chronic liver damage.

Cytochrome c Is a Potent Catalyst of Dichlorofluorescin Oxidation: Implications for the Role of Reactive Oxygen Species in Apoptosis

The generation of reactive oxygen species has been suggested to occur at increased rates during apoptosis, but the validity and significance of this remain contentious. In several key studies levels of reactive oxygen species have been monitored using the intracellular probe dichlorofluorescin (DCFH2), which undergoes oxidation to the fluorescent dichlorofluorescein (DCF). We report here that cytochrome c, which is released from mitochondria during cell death, is a potent catalyst of DCF formation. In a model system using xanthine oxidase to generate superoxide radicals, the rate of DCF formation was insensitive to changes in the rate of superoxide production over a 17-fold range, but extremely sensitive to nanomolar concentrations of cytochrome c. Thus we conclude that the DCF fluorescence observed in dying cells is a reflection of increased cytosolic cytochrome c. Moreover, we suggest that the suppression of DCF formation by the anti-apoptotic oncoprotein Bcl-2, which has been suggested to have antioxidant properties, can be explained on the basis of its prevention of mitochondrial cytochrome c release.

Enlarged and astaxanthin-accumulating cyst cells of the green alga Haematococcus pluvialis

The cyst cells of Haematococcus pluvialis were separated into fractions of relatively uniform size by sucrose density gradient centrifugation. The fraction at the bottom of the centrifuge tube with the largest specific gravity from density gradients of mature cysts mainly consisted of enlarged, red cyst cells and had the highest astaxanthin content. To examine the relationship between cell size and astaxanthin content of cysts, formation of the fluorescent dichlorofluorescein (DCF) from 2′,7′-dichlorohydrofluorescein diacetate of cyst cells in each fraction from density-gradient centrifugation under oxidative stress caused by methyl viologen (1.0 mM) was studied. The formation of DCF in cyst cells was decreased with larger cell diameter. This decrease was also correlated with increases in astaxanthin content. Therefore, both cell diameter and the fluorescent DCF content of cyst cells would be good parameter to select astaxanthin-hyperproducing strains from native populations of H. pluvialis.

Enlarged and Astaxanthin-Accumulating Cyst Cells of the Green Alga Haematococcus pluvialis

The cyst cells of Haematococcus pluvialis were separated into fractions of relatively uniform size by sucrose density gradient centrifugation. The fraction at the bottom of the centrifuge tube with the largest specific gravity from density gradients of mature cysts mainly consisted of enlarged, red cyst cells and had the highest astaxanthin content. To examine the relationship between cell size and astaxanthin content of cysts, formation of the fluorescent dichlorofluorescein (DCF) from 2',7'-dichlorohydrofluorescein diacetate of cyst cells in each fraction from density-gradient centrifugation under oxidative stress caused by methyl viologen (1.0 mM) was studied. The formation of DCF in cyst cells was decreased with larger cell diameter. This decrease was also correlated with increases in astaxanthin content. Therefore, both cell diameter and the fluorescent DCF content of cyst cells would be good parameter to select astaxanthin-hyperproducing strains from native populations of H. pluvialis.

In vivo antioxidant role of astaxanthin under oxidative stress in the green alga Haematococcus pluvialis.

Intracellular production of active oxygen in the green alga Haematococcus pluvialis was studied by measuring the capacity for in vivo conversion of 2',7'-dichlorohydrofluorescein diacetate to the fluorescent dye dichlorofluorescein in different algal cell types (i.e., vegetative, immature cyst and mature cyst cells). The increase in formation of dichlorofluorescein by methyl viologen (superoxide anion radical generator) was linear for 2 h in immature cyst cells (low astaxanthin) in a methyl viologen-concentration-dependent manner, while no production was detected in mature (high astaxanthin) cysts. Compared to cyst cells, formation of dichlorofluorescein in vegetative cells (no astaxanthin) was markedly increased by methyl viologen. The formation of dichlorofluorescein in cyst cells was decreased with higher astaxanthin content under excessive oxidative stress. All of the active oxygen species tested (singlet oxygen, superoxide anion radical, hydrogen peroxide and peroxy radical) at 10(-3) M increased the intracellular dichlorofluorescein formation in immature cysts, but did not increase the dichlorofluorescein content of mature cysts. Therefore, astaxanthin in cyst cells appeared to function as an antioxidant agent against oxidative stress.