Analysis of reactive oxygen species generated by neutrophils using a chemiluminescence probe L-012.

Reactive oxygen species (ROS) play important roles in the defense mechanism against infection and in the pathogenesis of various diseases. Although chemical properties of ROS generated by leukocytes have been studied extensively, methods available for their analysis are not sufficiently sensitive. We found that 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4-(2H,3H)dione (L-012) reacted with various types of ROS generated by activated neutrophils in human blood and oral cavity, and from peritoneal cavity of the rat, and developed strong chemiluminescence (CHL). Under physiological conditions, opsonized zymosan-dependent CHL intensity of L-012 in human blood and rat peritoneal neutrophils was about 100 and 10 times higher than that of luminol and luciferin analog MCLA, respectively. Phorbol ester-activated CHL of oral neutrophils was also higher with L-012 than that with luminol and MCLA. The presence of either superoxide dismutase, catalase, uric acid, deferoxamine, or azide decreased CHL intensity of L-012 by 52, 57, 57, 63, and 91%, respectively. Kinetic analysis revealed that L-012 developed CHL predominantly by reacting with hydroxyl radical and hypochlorite. Thus, highly sensitive L-012 permits studies on ROS generation by complex biological systems, such as leukocytes, and on the role of ROS in the pathogenesis of various diseases.     

Production of reactive oxygen species in peripheral blood is increased in individuals with Helicobacter pylori infection and decreased after its eradication

BACKGROUND: Helicobacter pylori infection has been reported to cause gastroduodenal ulcer, mucosa-associated lymphoid tissue lymphoma, and gastric cancer. Recent studies have suggested that H. pylori infection may also associate with other diseases, including hematologic and dermatologic disorders, and cardiovascular injury, by unknown mechanisms. METHODS: Production of reactive oxygen species (ROS) was determined in peripheral blood samples from 86 patients (34 H. pylori-negative and 52 H. pylori-positive subjects) using a highly sensitive chemiluminescence probe, L-012 (8-amino-5-chloro-7-phenylpyrido(3,4-d) pyridazine-1 and 4 (2H, 3H) dione). Eleven H. pylori-positive individuals were also analyzed their ROS production in peripheral blood after H. pylori eradication. RESULTS: ROS production was significantly higher in individuals with H. pylori infection than in those without infection. Enhanced production of ROS was decreased significantly after eradication of H. pylori. No correlation was found between the extent of ROS production and sex, age, smoking status, alcohol ingestion, use of medications, or serum level of C-reactive protein. CONCLUSION: These findings suggest that ROS production was enhanced in peripheral blood by H. pylori infection. Chemiluminescence analysis of blood samples using L-012 permits evaluation of systemic oxidative stress in patients with H. pylori infection.     

In vivo imaging of reactive oxygen and nitrogen species in inflammation using the luminescent probe L-012

Production of reactive oxygen and nitrogen species (ROS/RNS) is an important part of the inflammatory response, but prolonged elevated levels of ROS/RNS as under chronic inflammation can contribute to the development of disease. Monitoring ROS/RNS in living animals is challenging due to the rapid turnover of ROS/RNS and the limited sensitivity and specificity of ROS/RNS probes. We have explored the use of the chemiluminescent probe L-012 for noninvasive imaging of ROS/RNS production during inflammation in living mice. Various inflammatory conditions were induced, and L-012-dependent luminescence was recorded with an ultrasensitive CCD camera. Strong luminescent signals were observed from different regions of the body corresponding to inflammation. The signal was reduced by administration of the SOD mimetic tempol, the NADPH oxidase inhibitor apocynin, and the inhibitor of nitric oxide synthesis L-NAME, signifying the requirement for the presence of ROS/RNS. Additionally, the L-012 signal was abolished in mice with a mutation in the Ncf1 gene, encoding a protein in the NADPH oxidase complex 2, which generates ROS/RNS during inflammation. In conclusion, L-012 is well distributed in the mouse body and mediates a strong ROS/RNS-dependent luminescent signal in vivo and is useful for monitoring the development and regulation of inflammation in living organisms.     

Priming Effect of Pseudomonal Leukocidin on Chemiluminescence Response of Rabbit Polymorphonuclear Leukocytes

To clarify effects of pseudomonal leukocidin (42.5 kd) on chemiluminescence (CL) production of polymorphonuclear leukocytes (PMNs), rabbit PMNs were stimulated by zymosan or phorbol myristate acetate (PMA) after pretreatment with the leukocidin, which by itself stimulated little chemiluminescence response. The extent of CL responses stimulated by zymosan or PMA was respectively 5.3- or 3.5-fold greater in leukocidin (1.5 μg/ml)-pretreated PMNs than in non-pretreated ones. The priming effect of the leukocidin was greater than that of G-CSF and related to some steps before NADPH oxidase activation. The increased CL productions might be related to tissue damages caused by pseudomonal infections in vivo.     

In vitro evaluation of the behaviour of human polymorphonuclear neutrophils in direct contact with chitosan-based membranes

Several novel biodegradable materials have been proposed for wound healing applications in the past few years. Taking into consideration the biocompatibility of chitosan-based biomaterials, and that they promote adequate cell adhesion, this work aims at investigating the effect of chitosan-based membranes, over the activation of human polymorphonuclear neutrophils (PMNs). The recruitment and activation of polymorphonuclear neutrophils (PMNs) reflects a primary reaction to foreign bodies. Activation of neutrophils results in the production of reactive oxygen species (ROS) such as O(2)(-) and HO(-) and the release of hydrolytic enzymes which are determinant factors in the inflammatory process, playing an essential role in the healing mechanisms. PMNs isolated from human peripheral blood of healthy volunteers were cultured in the presence of chitosan or chitosan/soy newly developed membranes. The effect of the biomaterials on the activation of PMNs was assessed by the quantification of lysozyme and ROS. The results showed that PMNs, in the presence of the chitosan-based membranes secrete similar lysozyme amounts, as compared to controls (PMNs without materials) and also showed that the materials do not stimulate the production of either O(2)(-) or HO(-). Moreover, PMNs incubated with the biomaterials when stimulated with phorbol 12-myristate 13-acetate (PMA) or formyl-methionyl-leucyl-phenylalanine (fMLP) showed a chemiluminescence profile with a slightly lower intensity, to that observed for positive controls (cells without materials and stimulated with PMA), which reflects the maintenance of their stimulation capacity. Our data suggests that the new biomaterials studied herein do not elicit activation of PMNs, as assessed by the low lysozyme activity and by the minor detection of ROS by chemiluminescence. These findings reinforce previous statements supporting the suitability of chitosan-based materials for wound healing applications.     

Oxidative activity of human polymorphonuclear leukocytes stimulated by the long-chain phosphatidic acids

It has already been suggested that phosphatidic acids (PAs) play an important role in the regulation of signaling pathways involved in the production of reactive oxygen species (ROS) by human polymorphonuclear leukocytes (PMNs). The present study was performed to elucidate the effects of extracellularly added PA-- 1,2-distearoyl- (DSPA) and 1-stearoyl-2-arachidonoyl-sn-glycero-phosphate (SAPA)--on the ROS production and on the elastase release by human PMNs. ROS production was monitored by luminol-amplified chemiluminescence and the elastase activity was measured in the supernatant of the PA-stimulated human PMNs by colorimetric assay. Obtained effects were compared with those of cells stimulated by either a chemotactic tripeptide, phorbol ester or calcium ionophore. Our results show that long-chain PAs at concentrations higher than 3 x 10(-5) mol/l stimulate the ROS production by human PMNs, whereas they were ineffective in promoting the elastase release. The chemiluminescence pattern of the SAPA-stimulated cells exhibited a biphasic curve, whereas cell stimulation with DSPA resulted in a monophasic chemiluminescence curve. Stimulation of the ROS production by PAs in dependence of the fatty acid composition required the activity of protein kinases.     

Detection of Superoxide and Peroxynitrite in Model Systems and Mitochondria by the Luminol Analogue L-012

In the present study we investigated the specificity and sensitivity of the chemiluminescence (CL) dye and luminol analogue 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4-(2H,3H) dione (L-012) to detect reactive oxygen species (ROS) such as superoxide, peroxynitrite and hydrogen peroxide in cell free systems as well as in isolated mitochondria. The results obtained by L-012 were compared with other CL substances such as luminol, lucigenin, coelenterazine and the fluorescence dye dihydroethidine. The results indicate that the L-012-derived chemiluminescence induced by superoxide from hypoxanthine/xanthine oxidase (HX/XO) or by 3-morpholino sydnonimine (SIN-1)-derived peroxynitrite largely depends on the incubation time. Irrespective of the experimental conditions, L-012-derived CL in response to HX/XO and SIN-1 was 10–100 fold higher than with other CL dyes tested. In a cell-free system, authentic peroxynitrite yielded a higher L-012-enhanced CL signal than authentic superoxide and the superoxide-induced signal in cell-free as well as isolated mitochondria increased in the presence of equimolar concentrations of nitrogen monoxide (NO). The superoxide signal/background ratio detected by L-012-enhanced CL in isolated mitochondria with blocked respiration was 7 fold higher than that obtained by the superoxide sensitive fluorescence dye dihydroethidine. We conclude that L-012-derived CL may provide a sensitive and reliable tool to detect superoxide and peroxynitrite formation in mitochondrial suspensions.     

Detection of superoxide and peroxynitrite in model systems and mitochondria by the luminol analogue L-012

In the present study we investigated the specificity and sensitivity of the chemiluminescence (CL) dye and luminol analogue 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4-(2H,3H) dione (L-012) to detect reactive oxygen species (ROS) such as superoxide, peroxynitrite and hydrogen peroxide in cell free systems as well as in isolated mitochondria. The results obtained by L-012 were compared with other CL substances such as luminol, lucigenin, coelenterazine and the fluorescence dye dihydroethidine. The results indicate that the L-012-derived chemiluminescence induced by superoxide from hypoxanthine/xanthine oxidase (HX/XO) or by 3-morpholino sydnonimine (SIN-1)-derived peroxynitrite largely depends on the incubation time. Irrespective of the experimental conditions, L-012-derived CL in response to HX/XO and SIN-1 was 10-100 fold higher than with other CL dyes tested. In a cell-free system, authentic peroxynitrite yielded a higher L-012-enhanced CL signal than authentic superoxide and the superoxide-induced signal in cell-free as well as isolated mitochondria increased in the presence of equimolar concentrations of nitrogen monoxide (NO). The superoxide signal/background ratio detected by L-012-enhanced CL in isolated mitochondria with blocked respiration was 7 fold higher than that obtained by the superoxide sensitive fluorescence dye dihydroethidine. We conclude that L-012-derived CL may provide a sensitive and reliable tool to detect superoxide and peroxynitrite formation in mitochondrial suspensions.     

Measurement of NAD(P)H oxidase-derived superoxide with the luminol analogue L-012

In the present study we sought to determine the ability of the chemiluminescence dye 8-amino-5-chloro-7-phenylpyridol[3,4-d]pyridazine-1,4-(2H,3H)dione sodium salt (L-012) to detect superoxide in different biological systems. In human whole blood or isolated leukocytes, the sensitivity of the luminol analogue L-012 to detect superoxide was higher as compared with luminol, lucigenin, coelenterazine, and the fluorescence dye dihydroethidine. In isolated leukocytes as well as aortic rings from control (New Zealand White) and hyperlipidemic (Watanabe heritable hyperlipidemic) rabbits, L-012-enhanced chemiluminescence was successful in detecting differences in superoxide formation under basal conditions and on stimulation with the direct activator of protein kinase C, phorbol 12,13-dibutyrate (PDBu). The effects of PDBu were abrogated by gliotoxin and inhibitors of protein kinase C such as chelerythrine, identifying NAD(P)H oxidase as the significant superoxide source. Experiments using electron paramagnetic resonance and the spin trap 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide revealed that in contrast to lucigenin, L-012 is not subject to redox cycling. These findings indicate that L-012-enhanced chemiluminescence represents a sensitive and reliable probe to detect superoxide in whole blood, inflammatory cells, and vascular tissue.     

Oxidative stress underlies the mechanism for Ca(2+)-induced permeability transition of mitochondria

Recent studies demonstrated that the generation of intracellular reactive oxygen species (ROS) was enhanced prior to the onset of mitochondrial membrane permeability transition (MPT), a critical step for the induction of DNA fragmentation and apoptosis. Although Ca₂₊ induces typical MPT that involves depolarization and swelling of mitochondria and finally releases cytochrome c into cytosol, the mechanism by which ROS induce MPT remains unclear. In the presence of inorganic phosphate, Ca₂₊ increased the oxygen consumption and ROS production by isolated mitochondria as determined by a chemiluminescence (CHL) method using L-012. Ca₂₊ increased the generation of H₂O₂ by some mechanism that was inhibited by cyclosporin A but not by superoxide dismutase (SOD) and trifluoperazine. Ca₂₊ decreased the content of free thiols in adenine nucleotide translocase (ANT) in mitochondrial membranes with concomitant increase in ROS generation. The presence of cyclosporin A, trifluoperazine, or SOD inhibited the Ca₂₊-induced increase of L-012 CHL and decrease in the free thiols of ANT. These results indicate that Ca₂₊ increases the generation of ROS which oxidize the free thiol groups in mitochondrial ANT, thereby inducing MPT to release cytochrome c.     

ABC transporters affect the detection of intracellular oxidants by fluorescent probes

Intracellular production of reactive oxygen species (ROS) plays an important role in the control of cell physiology. For the assessment of intracellular ROS production, a plethora of fluorescent probes is commonly used. Interestingly, chemical structures of these probes imply they could be substrates of plasma membrane efflux pumps, called ABC transporters. This study tested whether the determination of intracellular ROS production and mitochondrial membrane potential by selected fluorescent probes is modulated by the expression and activity of ABC transporters. The sub-clones of the HL-60 cell line over-expressing MDR1, MRP1 and BCRP transporters were employed. ROS production measured by luminol- and L-012-enhaced chemiluminescence and cytochrome c reduction assay showed similar levels of ROS production in all the employed cell lines. It was proved that dihydrorhodamine 123, dihexiloxocarbocyanine iodide, hydroethidine, tetrachloro-tetraethylbenzimidazolocarbo-cyanine iodide and tetramethylrhodamine ethyl ester perchlorate are substrates for MDR1; dichlorodihydrofluoresceine, hydroethidine and tetramethylrhodamine ethyl ester perchlorate are substrates for MRP1; dichlorodihydrofluoresceine, dihydrorhodamine 123, hydroethidine and tetrachloro-tetraethylbenzimidazolocarbo-cyanine iodide are substrates for BCRP. Thus, the determination of intracellular ROS and mitochondrial potential by the selected probes is significantly altered by ABC transporter activities. The activity of these transporters must be considered when employing fluorescent probes for the assessment of ROS production or mitochondrial membrane potential.     

Oxidative Stress Underlies the Mechanism for Ca2+-induced Permeability Transition of Mitochondria

Recent studies demonstrated that the generation of intracellular reactive oxygen species (ROS) was enhanced prior to the onset of mitochondrial membrane permeability transition (MPT), a critical step for the induction of DNA fragmentation and apoptosis. Although Ca²⁺ induces typical MPT that involves depolarization and swelling of mitochondria and finally releases cytochrome c into cytosol, the mechanism by which ROS induce MPT remains unclear. In the presence of inorganic phosphate, Ca²⁺ increased the oxygen consumption and ROS production by isolated mitochondria as determined by a chemiluminescence (CHL) method using L-012. Ca²⁺ increased the generation of H²O² by some mechanism that was inhibited by cyclosporin A but not by superoxide dismutase (SOD) and trifluoperazine. Ca²⁺ decreased the content of free thiols in adenine nucleotide translocase (ANT) in mitochondrial membranes with concomitant increase in ROS generation. The presence of cyclosporin A, trifluoperazine, or SOD inhibited the Ca²⁺-induced increase of L-012 CHL and decrease in the free thiols of ANT. These results indicate that Ca²⁺ increases the generation of ROS which oxidize the free thiol groups in mitochondrial ANT, thereby inducing MPT to release cytochrome c.     

Functional Activity of Blood Polymorphonuclear Leukocytes as an Oxidative Stress Biomarker in Human Subjects

In the present work, we studied the role of polymorphonuclear leukocytes (PMN) in aged individuals and coronary heart disease (CHD)-bearing patients, two physiopathological processes associated with overproduction of reactive oxygen species (ROS). The effects of antioxidant supplementation on the functional activity of PMN from CHD patients were also determined. The function of PMNs was evaluated by measuring of phagocytosis, killing activity, and ROS production. Luminol amplified chemiluminescence (CL) was used to estimate ROS production by stimulated PMNs. Total cholesterol and the LDL-cholesterol fraction from CHD patients were found to be higher than those recommended, returning to normal levels after antioxidant therapy. PMN CL of CHD patients was found to be higher than the associated control groups. Antioxidant therapy administrated to CHD patients lead to an increase in the killing activity accompanied by a decrease in PMN CL of these subjects. The study also showed that killing activity of PMN from human subjects over 60 years was significantly lower than the activity measured in younger subjects. PMN CL produced after stimulation was found to be positively correlated with the increasing age of human subjects (r = .946, p < .01).     

Effects of endotoxin on oxidative metabolism of polymorphonuclear leukocytes

The influence of endotoxin on rat polymorphonuclear leucocytes (PMN) ability to generate oxygen free radicals (OFR) has been studied by chemiluminescence method. PMNs derived from intact animals were used as a control. PMNs derived from animals with 1.5 h endotoxemia increased OFR production after stimulation by latex. In contrast, PMNs derived from intact animals and preincubated with endotoxin for 1.5 h decreased OFR production after stimulation bw latex. It was proposed that stimulating effect of endotoxin on PMNs in vivo was mediated by plasma components.     

Some dinophycean red tide plankton species generate a superoxide scavenging substance

Recent studies indicate that some raphidophycean red tide flagellates produce substances able to scavenge superoxide, whereas there have been no reports on superoxide scavenger production by dinophycean red tide flagellates. In this study, we examined the superoxide-scavenging activity of aqueous extracts from dinophycean red tide flagellates, Gymnodinium spp., Scrippsiella trochoidea, and Karenia sp., by a luminol analog L-012-dependent chemiluminescence (CL) method and an electron spin resonance (ESR)-spin trapping method, and compared the activity to that of raphidophycean red tide flagellates, Chattonella spp., Heterosigma akashiwo, and Fibrocapsa japonica. In the experiment applying the L-012-dependent CL method, only the aqueous extracts from raphidophycean red tide flagellates showed superoxide-scavenging activity. On the other hand, applying the ESR-spin trapping method, we found that the aqueous extracts from dinophycean red tide flagellates also showed superoxide-scavenging activity. This is the first report on the production of a superoxide-scavenger by dinophycean red tide flagellates.     

Production of reactive oxygen species (ROS) by various marine fish species during the larval stage

Previous studies have indicated that the devil stinger produces reactive oxygen species (ROS) during early development from fertilized egg to larva. To determine whether ROS generation is a common feature in marine fish species, we conducted chemiluminescence analysis using ROS specific probe (L012) on larvae of six marine fish species. Marbled rockfish, black rockfish, and devil stinger showed higher levels of chemiluminescence response (CR), whereas the levels of CR of sevenband grouper, tiger puffer, and red seabream were fairly lower. These CRs were inhibited by the addition of superoxide dismutase. Hypersensitive photon-counting microscopic observation of black rockfish suggested that ROS production was concentrated in the head area. Our results suggest that the larvae of these six marine fishes produce ROS to considerably different extents depending on species, and that rockfish species, belonging to ovoviviparous fish, tend to produce much higher levels of ROS especially at the later larval stage.     

Phospholipase A2 modulates respiratory burst developed by neutrophils in patients with rheumatoid arthritis

Activated by bacterial peptides, phorbol esters, calcium ionophores and other agonists, neutrophils (PMNs) release the proinflammatory mediator, arachidonic acid (AA) via the intervention of phospholipase A(2) (PLA(2)). AA may play an essential role in activation of NADPH-oxidase, which is involved in the generation of superoxide anion by neutrophils. The present study is focused on the involvement of PLA(2) in the respiratory burst developed by PMNs isolated from patients with rheumatoid arthritis (RA). PLA(2) exists in very high levels in diseases such as rheumatoid arthritis and may cause acute inflammatory and proliferative changes in synovial structures. The respiratory burst was evaluated as superoxide anion release, using an amplified chemiluminescence method. The assays were performed using PMNs untreated or treated with different doses of stimulatory reagents (phorbol 12-myristate-13-acetate (PMA), calcium ionophore (A23187)). Our data suggested that PMA stimulated the production of superoxide anion in a dose-response manner, as compared with A23187, which did not induce a significant release of superoxide anion in PMNs-RA. The exogenous addition of AA significantly amplified the superoxide anion release by PMNs-RA stimulated with PMA and to a lesser extent, by PMNs stimulated with A23187. AA has also reversed the inhibitory effect of arachidonyl-trifluorometylketone and E-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)2H-pyran-2-one (BEL) on the superoxide anion release by PMNs-RA. In conclusion, the differential responses to these two agents suggested that different isoforms of PLA(2) were activated by A23187 or PMA, and support the idea that activation of these different PLA(2) served distinct functions of PMNs. Therefore, the inhibition of PLA(2) enzymes might be of great importance in the immunotherapy of rheumatoid arthritis.     

Some Dinophycean Red Tide Plankton Species Generate a Superoxide Scavenging Substance

Recent studies indicate that some raphidophycean red tide flagellates produce substances able to scavenge superoxide, whereas there have been no reports on superoxide scavenger production by dinophycean red tide flagellates. In this study, we examined the superoxide-scavenging activity of aqueous extracts from dinophycean red tide flagellates, Gymnodinium spp., Scrippsiella trochoidea, and Karenia sp., by a luminol analog L-012-dependent chemiluminescence (CL) method and an electron spin resonance (ESR)-spin trapping method, and compared the activity to that of raphidophycean red tide flagellates, Chattonella spp., Heterosigma akashiwo, and Fibrocapsa japonica. In the experiment applying the L-012-dependent CL method, only the aqueous extracts from raphidophycean red tide flagellates showed superoxide-scavenging activity. On the other hand, applying the ESR-spin trapping method, we found that the aqueous extracts from dinophycean red tide flagellates also showed superoxide-scavenging activity. This is the first report on the production of a superoxide-scavenger by dinophycean red tide flagellates.