Dichlorofluorescein Formation Research Articles

Effect of polychlorinated biphenyls on production of reactive oxygen species (ROS) in rat synaptosomes.

In this paper the effect of polychlorinated biphenyls (PCBs) on the production of reactive oxygen species (ROS) in rat synaptosomes is elucidated. The effect of methylmercury (MeHg) on rat synaptosomes was included as a positive control since several studies have investigated the ability of this substance to produce ROS. The exposure of the synaptosomes to the congener 2,2-dichlorobiphenyl (2, 2'-DCB; 12.5 microM) produced a linear increase in the formation of 2',7'-dichlorofluorescein (DCF) as a measure for the production of ROS. The congeners 2,2'-DCB (12.5 microM) and 3,3'-DCB (12.5 microM) stimulated, as expression of ROS production, a significant increase in DCF formation formation compared to the control. The congeners 2-chlorobiphenyl (2-CB) and 2,2',6-trichlorobiphenyl (2,2,6'-TCB) were active at 50 microM, whereas 2,2',4,4',5,5'-hexachlorobiphenyl (2,2',4,4',5,5'-HCB), 4,4'-DCB and 2,2',6,6'-tetrachlorobiphenyl (2, 2',6,6'-TeCB) were not active at this concentration. The increased formation of ROS in response to 2,2'-DCB and MeHg in the synaptosomes was dependent on extracellular Ca(2+). A phospholipase C inhibitor, U73122, was shown to significantly decrease the ROS formation induced by 2,2'-DCB, but did not reduce the ROS formation induced by MeHg. Ethanol (1%), a phospholipase D modulator, reduced the ROS formation induced by MeHg and by 2,2'-DCB by 33 and 52%, respectively. Wortmannin (25 nM), an inhibitor of phosphatidylinositol 3-kinase, completely inhibited the ROS formation induced by MeHg and 2,2'-DCB. It appears that the ROS-stimulating PCBs are the same congeners found to be neuroactive in other types of study. Phospholipase C and D and phosphatidylinositol 3-kinase seem to be involved in the intracellular signalling system that leads to ROS formation during PCB exposure.

Evidence for free radical formation during the oxidation of 2′-7′-dichlorofluorescin to the fluorescent dye 2′-7′-dichlorofluorescein by horseradish peroxidase:: Possible implications for oxidative stress measurements

The oxidation of 2′-7′-dichlorofluorescin (DCFH) to the fluorescent 2′-7′-dichlorofluorescein (DCF) by horseradish peroxidase (HRP) was investigated by fluorescence, absorption, and electron spin resonance spectroscopy (ESR). As has been previously reported, HRP/H 2O 2 oxidized DCFH to the highly fluorescent DCF. However, DCF fluorescence was still observed when H 2O 2 was omitted, although its intensity was reduced by 50%. Surprisingly, the fluorescence increase, in the absence of exogenous H 2O 2, was still strongly inhibited by catalase, demonstrating that H 2O 2 was present and necessary for DCF formation. H 2O 2was apparently formed during either chemical or enzymatic deacetylation of 2′-7′-dichlorofluorescin diacetate (DCFH-DA), probably by auto-oxidation. Spectrophotometric measurements clearly showed that DCFH could be oxidized either by HRP-compound I or HRP-compound II with the obligate generation of the DCF semiquinone free radical (DCF •−). Oxidation of DCF •− to DCF by oxygen would yield superoxide (O 2 •−). ESR spectroscopy in conjunction with the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) revealed the presence of both superoxide and hydroxyl radicals in the DCFH/H 2O 2/HRP system. Both radicals were also detected in the absence of added H 2O 2, although the intensities of the resultant adducts were decreased. This work demonstrates that DCF fluorescence cannot be used reliably to measure O 2 •− in cells because O 2 •− itself is formed during the conversion of DCFH to DCF by peroxidases. The disproportionation of superoxide forms H 2O 2 which, in the presence of peroxidase activity, will oxidize more DCFH to DCF with self-amplification of the fluorescence. Because the deacetylation of DCFH-DA, even by esterases, can produce H 2O 2, the use of this probe to measure H 2O 2 production in cells is problematic.

Measurement of active oxygen species generatedin plantain response to elicitor AVR9 ofCladosporium fulvum

Injection of elicitor preparations containing the Avr9 gene product ofCladosporium fulvum(race 4) into tomato carrying the Cf9 gene induced leaf necrosis and production of active oxygen species (AOS). The nature and temporal development of AOS and its relationship to leaf necrosis was explored usingin plantaprobes for AOS. Generation of O2−was detected in elicitor-injected tomato leaves using a manganese/diaminobenzidine technique. To detect the generation of H2O2, the fluorescent probe, 2′,7′-dichlorofluorescin diacetate (DCFH-DA), was injected into leaf areas previously injected with elicitor. The fluorescent compound, 2′,7′-dichlorofluorescein (DCF), formed from oxidation of DCFH by H2O2in the presence of peroxidases, was produced much faster in interveinal panels injected with elicitor than in those injected with control preparations (lacking the Avr9 product), or with the probe alone. Fluorescence intensity was compared visually under u.v. light and/or measured on a fluorimeter following extraction from the tissue. DCF formation in leaves on injection of known concentrations of H2O2was verified. Elicitor-induced oxidation of DCFH was inhibited by co-injection of elicitor with catalase or an inhibitor of superoxide dismutase. Co-injection of DCFH-DA with the antioxidants,L-ascorbic acid and glutathione, or with KCN, an inhibitor of peroxidases, into leaves previously injected with elicitor also blocked fluorescence development. Oxidation of DCFH was detected in leaves injected with elicitor at concentrations lower than those that cause leaf necrosis. Necrosis development was partially inhibited and/or significantly delayed by injection of elicitor mixed with catalase and several other antioxidants or scavengers of AOS. These results providein plantaevidence for the involvement of AOS in the defence response of tomato to a race specific elicitor ofC. fulvum.

Estimation of oxidative stress in Saccharomyces cerevisae with fluorescent probes

Oxidation of dichlorofluorescin and dihydrofluorescin and transport of their oxidation products were studied in Saccharomyces cerevisiae cells given diacetates of these compounds. It was found that addition of peroxidase significantly accelerates the formation of dichlorofluorescein and fluorescein, indicating that peroxidase activity may be a limiting factor in studies of cellular oxidation using these probes. A significant leakage of dichlorofluorescin and dihydrofluorescin, and of their oxidized forms from the yeast cells, was found, without any apparent lag period. This phenomenon complicates interpretation of data obtained with the fluorescent precursors used but, on the other hand, points to the possibility of using measurements of dichlorofluorescein or fluorescein in the extracellular medium for studies of cellular oxidation processes.

IN VIVO MEASUREMENT OF ACTIVE OXYGEN PRODUCTION IN THE BROWN ALGA FUCUS EVANESCENS USING 2′,7′‐DICHLOROHYDROFLUORESCEIN DIACETATE1

ABSTRACTIntracellular production of active oxygen in the brown alga Fucus evanescens C. Ag. was studied by measuring the capacity for in vivo conversion of 2′,7′‐dichlorohydrofluorescein diacetate (DCFH‐DA) to the fluorescent dye 2′,7′‐dichlorofluorescein (DCF), both in emersed and immersed seaweeds. Algae were incubated in seawater containing DCFH‐DA under a range of conditions, and it was also possible to load algae with DCFH‐DA and then follow subsequent DCF production in emersed tissue. DCF formation was linear for at least 2 h in both darkness and light, with the rate of formation increasing with the light level. DCF formation was temperature dependent. It also increased when algae were treated with H2O2 or methyl viologen (paraquat), which disrupts photosystem 1 electron transport and increases O−2 production. Exogenous catalase reduced in vivo DCF production, presumably by lowering cellular concentrations of H2O2. Hydrogen peroxide was released into the seawater by illuminated algae resulting in external dye conversion to DCF. However, this does not interfere with in vivo measurement of DCF by loaded, washed algae because DCF leakage appeared to be negligible. Internal DCF did not affect photosynthetic oxygen production relative to untreated controls. Overall, our data suggest that DCFH‐DA is a potentially very useful probe for studying active oxygen metabolism in seaweeds subjected to environmental stresses.

Dichlorodihydrofluorescein and Dihydrorhodamine 123 Are Sensitive Indicators of Peroxynitritein Vitro:Implications for Intracellular Measurement of Reactive Nitrogen and Oxygen Species

2,7-Dichlorodihydrofluorescein (DCDHF), commonly known as dichlorofluorescin, and dihydrorhodamine 123 (DHR) are often used to detect the production of reactive nitrogen and oxygen species in cells via oxidation to their respective fluorescent products. To determine which biological oxidants might be involved, DCDHF and DHR were exposed to a number of oxidantsin vitroto determine which are capable of oxidizing these compounds. Formation of dichlorofluorescein (DCF) and rhodamine is typically monitored by measuring their intrinsic fluorescence, however, absorbance can also be utilized (ϵ500 nm= 59,500 and 78,800m−1cm−1for DCF and rhodamine, respectively). Peroxynitrite (ONOO−) readily oxidized both compounds with an efficiency equal to 38% of added ONOO−for DCDHF and 44% for DHR. Addition of nitric oxide (NO) to a superoxide-generating system resulted in DCDHF and DHR oxidation which was inhibitable by superoxide dismutase (SOD). SIN-1-mediated oxidation of DCDHF and DHR was also SOD-inhibitable, suggesting that peroxynitrite is the primary oxidant formed from SIN-1 decomposition. Aerobic addition of NO resulted in DCDHF oxidation in a manner consistent with nitrogen dioxide (·NO2) formation. NO did not oxidize DHR and actually inhibited UV-light-induced DHR oxidation. Simultaneous addition of NO and ONOO−resulted in an apparent inhibition of indicator oxidation; however, subsequent addition of ONOO−alone 20 s later produced a higher than average amount of oxidized indicator. Addition of indicatorafterNO + ONOO−followed by subsequent ONOO−addition gave similar results, suggesting the formation of a relatively stable, oxidant-activated NO/ONOO−adduct. At pH 7.4, hypochlorous acid was 66% efficient at oxidizing DHR but only 9% with DCDHF. Neither H2O2(1 mm) nor superoxide flux alone produced significant indicator oxidation. Oxidation of DCDHF by horseradish peroxidase (HRP) plus H2O2was considerably less efficient than oxidation of DHR. At 20-fold higher concentrations, HRP alone oxidized DHR but the rate was much lower than when H2O2was present. Catalase largely inhibited HRP-mediated oxidation of DHR but not DCDHF, suggesting a direct effect of the peroxidase on DCDHF. These results reveal that peroxynitrite, hypochlorous acid, and H2O2plus peroxidase all oxidize DCDHF and DHR to varying degrees but that neither superoxide, H2O2alone, nor physiological levels of nitric oxide are capable of indicator oxidation. Thus, DCDHF or DHR oxidation in any given cell type may involve more than one oxidant. In cell systems where nitric oxide production occurs, oxidation of either DCDHF or DHR is likely to include a peroxynitrite component. Identification of relevant oxidants will best be achieved with a combined experimental approach which exploits the differential reactivities of DCDHF and DHR and the judicious use of inhibitors and oxidant scavengers.

Spectrophotometric assay for total peroxyl radical-trapping antioxidant potential in human serum

Antioxidants prevent modification of low density lipoprotein (LDL) by free radicals and possibly also atheroma formation. The capacity of human serum to resist attacks by free radicals is measured by the total peroxyl radical-trapping potential (TRAP). Its measurement has thus far required equipment not available in many clinical laboratories such as a thermostated oxygen electrode cell or a luminometer. To develop a simpler method we used a free radical probe, dichlorofluorescin-diacetate (DCFH-DA), described before in studies of respiratory burst in inflammatory cells. Its oxidation by radicals from thermal decomposition of 2,2'-diazobis(2-amidinopropane)dihydrochloride (AAPH) converts this compound to highly fluorescent dichlorofluorescein (DCF). The DCF also has absorbance at 504 nm thus enabling the determination of TRAP either fluorometrically or spectrophotometrically. Increasing the concentration of AAPH enables the measurement of DCF formation and its inhibition by serum samples at room temperature. The intra- and interassay coefficients of variation of this assay are 3.4% and 4.6%, respectively. The mean value for serum TRAP of healthy subjects is 1155 mumol/l (n = 38). The TRAP in human serum can be increased by adding various antioxidant substances to the assay in vitro or by dietary supplementation of healthy subjects with vitamin E in vivo (P < 0.025). An increase was also found in serum vitamin E levels (P < 0.0001) and in the length of the time human LDL is able to resist oxidation (P < 0.05). Thus the determination of TRAP by this method, which requires only commercially available chemicals, can be used for the evaluation of phenomena associated with lipid accumulation in human artery wall.

Oxidation of 2′,7′-Dichlorofluorescin by Peroxynitrite

The simultaneous production of nitric oxide and superoxide anion leads to the formation of peroxynitrite, a potent oxidant which may be an important mediator of cellular injury. Oxidation of dichlorofluorescin to the fluorescent dichlorofluorescein has been used as a marker for cellular oxidant production. The mechanisms of peroxynitrite-mediated oxidation of dichlorofluorescin to dichlorofluorescein were investigated. Chemically synthesized peroxynitrite (50–500 nM) induced the oxidation of dichlorofluorescin to dichlorofluorescein in a linear fashion. In addition, the simultaneous generation of nitric oxide and superoxide anion induced the oxidation of dichlorofluorescin to dichlorofluorescein, while nitric oxide (1–10 μM) alone under aerobic conditions did not. Peroxynitrite-mediated oxidation of dichlorofluorescin was not inhibited by the hydroxyl radical scavengers mannitol (100 mM) or dimethylsulfoxide (100 mM). Moreover, peroxynitrite-mediated oxidation of dichlorofluorescin was not dependent upon metal ion-catalyzed reactions. Furthermore, dichlorofluorescein formation was diminished at alkaline pH. These findings suggest that peroxynitrite-mediated dichlorofluorescein formation results directly from the protonation of peroxynitrite to form the conjugate peroxynitrous acid. L-cysteine was an efficient inhibitor (K1 = 25 μM) of dichlorofluorescin oxidation through competitive oxidation of free sulfhydryls. Urate was a less efficient with a maximum inhibition of only 49%. These results demonstrate that dichlorofluorescin is efficiently oxidized by peroxynitrite.Therefore, under conditions where nitric oxide and superoxide are produced simultaneously, oxidation of dichlorofluorescin may be mediated by the formation of peroxynitrite.

CARCINOGENIC CHROMIUM(VI) INDUCES OXIDATIVE STRESS IN CULTURED HUMAN LEUKEMIC T-LYMPHOCYTES. 1. GENERATIO OF HYDROGEN PEROXIDE DURIN INTRACELLULAR REDUCTION OF CHROMATE

Treatment of human leukemic T-lymphocyte MOLT4 cells with potassium chromate, a chromium (VI) compound, increased the form ation of the oxidized compound dichlorofluorescein (DCF), a highly fluorescent compound, from the parent nonfluorescent compound, 2,7-dichlorofluorescin diacetate (DCF-DA). No such increase in DCF formation was observed when cells were treated with Cr(III) compounds. In cell-free system s, dichlorofluorescin diacetate was also oxidized to DCF by strong oxidants (such as hydrogen peroxide and hydroperoxides) in the presence of peroxidase, suggesting that Cr(VI) treatment increased the intracellular level of these peroxides in MOLT4 cells. The rate of generation of peroxides was found to be both dose and time dependent. These results suggest that accumulation of intracellular peroxides may, at least in part, be associated with chromium-induced genotoxicity and add to the emerging concept that these phenomena may, in part, be mediated via oxidative m echanisms.

Evaluation of reactive oxygen species involvement in amiodarone pulmonary toxicity in vivo and in vitro

Amiodarone (AM) is an effective antidysrhythmic agent, restricted in use by the development of adverse effects, including potentially fatal AM-induced pulmonary toxicity (AIPT). Although the pathogenesis of AIPT is unknown, an oxidant mechanism has been proposed. The present study evaluated the role of reactive oxygen species (ROS) in AM-induced toxicity. The effect of inhibiting lung antioxidant defense on in vivo development of AIPT was evaluated in hamsters. Lung glutathione reductase activity was inhibited by 66%, 6 hours following administration of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) (20 mg/kg i.p.). When AM (1.83 μmol) was administered intratracheally 6 hours after BCNU, toxicity was enhanced, as indicated by lung hydroxyproline content and histological evaluation 21 days later. However, BCNU treatment did not affect AM-induced alterations in lung glutathione, suggesting that the increased toxicity was not due to decreased antioxidant capacity following BCNU. The effect of BCNU on AM cytotoxicity in vitro was evaluated using rabbit lung alveolar macrophages. Incubation with 5 μM BCNU for 2 hours caused greater than 95% inhibition of glutathione reductase activity. However, BCNU treatment had no effect on 146 μM AM-induced cytotoxicity, as assessed by lactate dehydrogenase latency following 12 hours of incubation. Rabbit macrophages loaded with 2′,7′-dichlorofluorescin, which is oxidized by ROS to fluorescent 2′,7′-dichlorofluorescein (DCF), were used to evaluate ROS generation by AM. Incubation of macrophages with AM (73 or 146 μM) for 1 hour, with or without the catalase inhibitor sodium azide (1 mM), did not result in DCF formation. Overall, these results do not support the hypothesis that AIPT is due to ROS action. © 1997 John Wiley & Sons, Inc.

Evaluation of reactive oxygen species involvement in amiodarone pulmonary toxicity in vivo and in vitro.

Amiodarone (AM) is an effective antidysrhythmic agent, restricted in use by the development of adverse effects, including potentially fatal AM-induced pulmonary toxicity (AIPT). Although the pathogenesis of AIPT is unknown, an oxidant mechanism has been proposed. The present study evaluated the role of reactive oxygen species (ROS) in AM-induced toxicity. The effect of inhibiting lung antioxidant defense on in vivo development of AIPT was evaluated in hamsters. Lung glutathione reductase activity was inhibited by 66%, 6 hours following administration of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) (20 mg/kg i.p.). When AM (1.83 mumol) was administered intratracheally 6 hours after BCNU, toxicity was enhanced, as indicated by lung hydroxyproline content and histological evaluation 21 days later. However, BCNU treatment did not affect AM-induced alterations in lung glutathione, suggesting that the increased toxicity was not due to decreased antioxidant capacity following BCNU. The effect of BCNU on AM cytotoxicity in vitro was evaluated using rabbit lung alveolar macrophages. Incubation with 5 microM BCNU for 2 hours caused greater than 95% inhibition of glutathione reductase activity. However, BCNU treatment had no effect on 146 microM AM-induced cytotoxicity, as assessed by lactate dehydrogenase latency following 12 hours of incubation. Rabbit macrophages loaded with 2',7'-dichlorofluorescein, which is oxidized by ROS to fluorescent 2',7'-dichlorofluorescein (DCF), were used to evaluate ROS generation by AM. Incubation of macrophages with AM (73 or 146 microM) for 1 hour, with or without the catalase inhibitor sodium azide (1 mM), did not result in DCF formation. Overall, these results do not support the hypothesis that AIPT is due to ROS action.

Ethanol-induced oxygen radical formation and lipid peroxidation in rat brain: effect of chronic alcohol consumption.

The effect of chronic and in vitro ethanol exposure on brain oxygen radical formation and lipid peroxidation was analyzed. Ethanol induces a dose-dependent increase in lipid peroxidation in brain homogenates. The peroxidative effects of alcohol seem to be related to both cytochrome P450 and the ethanol-inducible form of cytochrome P450 (CYP2E1), because preincubation with metyrapone (an inhibitor of cytochrome P450) or with an antibody against CYP2E1 abolished the ethanol-increased lipid peroxidation. Using the formation of dichlorofluorescein, we also demonstrated that both in vitro and chronic alcohol exposure significantly enhanced the formation of oxygen radical species in synaptosomes. Chronic alcohol treatment also leads to an induction of cytochrome P450 (230%), NADPH cytochrome c reductase (180%), NADPH oxidation (184%), and CYP2E1 in brain microsomes. In addition, this treatment produced a decrease in the GSH/GSSG ratio in brain and significantly enhanced the levels of superoxide dismutase and catalase activities. This mechanism could be involved in the toxic effects of ethanol on brain and membrane alterations occurring after chronic ethanol intake.

Evaluation of the probe 2',7'-dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress.

The use of dichlorofluorescin (DCFH) as a measure of reactive oxygen species was studied in aqueous media. Hydrogen peroxide oxidized DCFH to fluorescent dichlorofluorescein (DCF), and the oxidation was amplified by the addition of ferrous iron. Hydrogen peroxide-induced DCF formation in the presence of ferrous iron was completely inhibited by deferoxamine and partially inhibited by ethylenediaminetetraacetic acid, but was augmented by diethylenetriaminepentaacetic acid. Iron-peroxide-induced oxidation of DCFH was partially inhibited by catalase but not by horseradish peroxidase. Nonchelated iron-peroxide oxidation of DCFH was partially inhibited by several hydroxyl radical scavengers, but was independent of the scavenger concentration, and this suggests that free hydroxyl radical is not involved in the oxidation of DCFH in this system. Superoxide anion did not directly oxidize DCFH. Data suggest that H2O2-Fe(2+)-derived oxidant is mainly responsible for the nonenzymatic oxidation of DCFH. In addition, peroxidase alone and oxidants formed during the reduction of H2O2 by peroxidase oxidize DCFH. Since DCFH oxidation may be derived from several reactive intermediates, interpretation of specific reactive oxygen species involved in biological systems should be approached with caution. However, DCFH remains an attractive probe as an overall index of oxidative stress in toxicological phenomena.

Persistent protein damage despite reduced oxygen radical formation in the aging rat brain

The relation between cerebral oxygen radicals and the aging process was investigated in crude synaptosomal (P2) fractions from rats. The rate of formation of oxygen radicals was measured using the probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA), which is de-esterified and subsequently oxidized by oxygen radicals to a fluorescent product 2′,7′-dichlorofluorescein (DCF). There was a significant agedependent decrease in the formation rate of oxygen radicals, observed by decreased formation of DCF. No difference in oxygen radical formation was apparent between age groups following an in vitro challenge with an ascorbate/FeSO4 mixture. This age-dependent decrease in cerebral oxygen radical generation coincided with age-dependent increases in Superoxide dismutase. No age-related alterations in lipid order in either the hydrophilic or lipophilic membrane regions were observed using fluorescence polarization analysis. Age-dependent losses in cerebral P2 tryptophan fluorescence (a measure of protein degradation), and increased liberation of [14C]protein fragments into the acid-soluble fraction (a measure of overall proteolytic activity) were observed. Results suggest that aging does not proceed as a result of elevated rates of generation of oxygen radicals, a finding that does not support the proposed free radical theory of aging. The observed age-dependent decrease in the formation of oxygen radicals does not effect membrane lipid order. These findings implicate modifications in proteins and activated protein catabolic pathways as major contributing factors in the normal physiological process of senescence.

Sensitive and rapid quantitation of oxygen reactive species formation in rat synaptosomes

The formation of oxygen reactive species in response to oxidative stimuli was measured in rat synaptosomes. Studies employed the non-fluorescent probe 2′,7′-dichlorofluorescin diacetate (DCFH-DA), which after de-esterification is oxidized in the presence of oxygen reactive species to the highly fluorescent 2′,7′-dichlorofluorescein (DCF). Oxygen reactive species formation, as measured by DCF fluorescence, was stimulated by ascorbate and/or FeSO4, and xanthine/xanthine oxidase under various buffering conditions. These agents all increased DCF formation in Tris, HEPES and phosphate buffer. Ascorbate also stimulated the formation of DCF in a concentration-dependent manner. The presence of Ca2+ in HEPES buffer did not enhance or diminish the effects of ascorbate/FeSO4 on DCF formation. Deferoxamine inhibited the ascorbate/FeSO4-induced stimulation of DCF formation, but xanthine/xanthine oxidase-induced stimulation was not affected by pretreatment with superoxide dismutase. Results indicate that DCF fluorescence is a sensitive, quantitative and direct measure of oxygen reactive species formation in synaptosomes, providing a rapid method for investigating early neuronal events that occur during oxidative stress.

Characterization of human mononuclear cells using reduced pyridine nucleotide fluorescence and flow cytometry.

Peripheral blood monocytes undergo an oxidative burst similar to that seen in neutrophils. The basis for this response appears to be an NAD(P)H oxidase that utilizes reduced NAD(P)H to form superoxide anion. We utilized the unique UV-stimulated fluorescence property of reduced pyridine nucleotides to analyze NAD(P)H utilization in monocytes. UV-stimulated fluorescence in mononuclear cell preparations indicated two populations of cells with the highly fluorescent cells having a Coulter volume consistent with that of monocytes. Dual laser analysis with monoclonal antibodies confirmed that these highly fluorescent cells are monocytes by showing them to be OKM1+, Leu DR+, and anti-monocyte 0.2+. Natural killer (NK) cells, as defined by Leu 7, were not found in this highly fluorescent population. Stimulation of mononuclear cells with phorbol myristate acetate caused a fluorescence loss indicative of NAD(P)H oxidation in monocytes but not in lymphocytes. Stimulation with suboptimal concentrations of PMA (1-5 ng/ml) resulted in a dose-dependent fluorescence loss in monocytes that occurred in an all-or-none fashion identical to the pattern observed in neutrophils. Simultaneous measurement of H2O2 production using dichlorofluorescein formation with NAD(P)H fluorescence indicates that oxidant production occurs in a graded manner. This method, then, provides a convenient way to study in single cells the metabolic events involved in depletion and replenishment of NAD(P)H during the oxidative burst and demonstrates an additional means by which to distinguish monocytes from lymphocytes using flow cytometry.

Flow cytometric quantitation of oxidative product formation by polymorphonuclear leukocytes during phagocytosis.

Stimulation of the oxidative metabolic burst of human polymorphonuclear leukocytes (PMNL) may occur by an all-or-none trigger mechanism or by a graded response to increasing stimulation of an individual cell. If the proposed all-or-none mechanism occurred during phagocytosis, a PMNL would expend all of its metabolic potential at once, yet PMNL can proceed to ingest multiple organisms. This study employed dual laser flow cytometry to correlate the number of cell-associated organisms with oxidative product formation in individual PMNL. Intracellular oxidation of nonfluorescent 2',7'-dichlorofluorescein (DCFH) to highly fluorescent 2',7'-dichlorofluorescein (DCF) provided a quantitative assay of H2O2-dependent oxidative product formation generated by the cell's oxidative metabolic burst. Staphylococcus aureus were fixed and stained with Texas red to allow simultaneous monitoring of bacteria (red fluorescence, greater than 580 nm) and DCF (green fluorescence, 510 to 550 nm) content of each cell. Computer correlation of bacterial and DCF fluorescence allowed determination of the DCF formation by PMNL containing specific numbers (0 to 15) of bacteria. Oxidative product formation was directly related to the number of bacteria ingested in a time-dependent manner (mean per cell of 6.4, 12.8, 19.1, and 24.4 attomoles (amol) DCF formed per cell per bacterium after 15, 30, 45, and 60 min, respectively. Opsonization of bacteria with fresh normal serum (primarily C3b opsonization) or with specific IgG demonstrated qualitatively similar responses, except that the response per IgG-opsonized organism was, on the average, more than twice the response to bacteria opsonized with serum. Thus, sequential phagocytosis of multiple bacteria elicits an incremental oxidative response of human PMNL.

Flow cytometric studies of oxidative product formation by neutrophils: a graded response to membrane stimulation.

We have developed a quantitative assay to monitor the oxidative burst (H2O2 production) of polymorphonuclear leukocytes (PMNL) using single cell analysis by flow cytometry, and have examined whether PMNL respond to membrane stimulation with an all-or-none oxidative burst. During incubation with normal neutrophils, dichlorofluorescin diacetate diffused into the cells, was hydrolyzed to 2',7'-dichlorofluorescin (DCFH) and was thereby trapped within the cells. The intracellular DCFH, a nonfluorescent fluorescein analogue, was oxidized to highly fluorescent 2',7'-dichlorofluorescein (DCF) by PMNL stimulated by phorbol myristate acetate (PMA). That the oxidative product was DCF was shown by excitation/emission spectra and by mass spectrometry of the product from PMA-stimulated PMNL. Normal resting and PMA-stimulated PMNL oxidized 6.9 +/- 0.7 and 160 +/- 13 attomoles DCF per cell, respectively, in 15 min. Absence of calcium and magnesium ions and/or addition of 2 mM EDTA did not inhibit DCF formation by PMNL stimulated by 100 ng/ml PMA. Since EDTA prevented aggregation of PMNL (even when stimulated by 100 ng/ml PMA), which would prevent accurate flow cytometric analysis, further experiments were performed with EDTA in the medium. A close correlation between average DCFH oxidation and hexose monophosphate shunt stimulation was demonstrated using cells from patients whose PMNL had oxidative metabolic defects of varying severity. Intracellular DCFH was also oxidized by reagent H2O2 or oxygen derivatives generated by glucose oxidase + glucose or by xanthine oxidase + acetaldehyde; DCFH oxidation by these systems was inhibited by catalase but unchanged by superoxide dismutase. The data indicate that the DCFH oxidation assay is quantitatively related to the oxidative metabolic burst of PMNL, and they strongly suggest that the reaction is mediated by H2O2 generated by the PMNL. Incubation of PMNL with varying concentrations of PMA caused graded responses by all PMNL present; i.e., 1 ng/ml PMA caused a mean response of 34% maximal with a single population of responding PMNL (rather than 66% resting and 34% fully stimulated as predicted by the all-or-none hypothesis). Thus, with these assay conditions, oxidative product formation by PMNL occurs as a graded response to membrane stimulation by PMA.

A sensitive photometric assay for monoamine oxidase

A new spectrophotometric assay for the determination of monoamine oxidase activity is described. This simple and sensitive method is based on a coupled indicator reaction measuring the monoamine oxidase-dependent production of hydrogen peroxide. In this reaction the hydrogen peroxide-dependent oxidation of leuco-2′,7′-dichlorofluorescein to 2′,7′-dichlorofluorescein catalyzed by horseradish peroxidase is followed at 502 nm. Using benzylamine and seven biogenic amines as substrates, linear relationships between 2′,7′-dichlorofluorescein formation rate and monoamine oxidase concentration were found. The assay is especially suitable for determining substrate specificities for physiological amines as well as for inhibitor studies with pargyline or the monoamine oxidase A- and B-specific inhibitors clorgyline and deprenyl.