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Characterization of reactive nitrogen species formed during leukocyte peroxidase-catalyzed oxidation of nitrite
Characterization of Reactive Nitrogen Species Formed during Leukocyte Peroxidase-catalyzed Oxidation of Nitrite W&a Wu, Heather Frost*, Harry Ischiropoulos* and Stanley L. Hazen Cleveland Clinic Foundation and *University of Pennsylvania Recent studies suggest a potential role for tbe leukocyte peroxidases. myeloperoxidase (MPO) and eosinophil peroxidase (EPO), in the generation of reactive nitrogen species (RNS) during inflammatory diseases. Peroxidase-catalyzedoxidation of nitrite (NO;), a major end product of NO metabolism, generates a diffusible oxidant capable of promoting aromatic nitration reactions and initiating lipid peroxidation. The chemical identity of the nitrating intermediate formed is unknown, and both one electron (N02. nitrogen dibxide) and two electron (ONOO-, peroxynitrite) oxidation products have been suggested. We performed a series of studies designed to distinguish between these intermediates and report that N02, not ONOO-, is the major RNS formed during MPO- and EPO-catalyzed oxidation of NO?. In the presence of C02, ONOO- forms a more potent nitrating intermediate,
presumably ONOOCO;. Although exposure of tyrosine or 2deoxyguanosine (2-dG) to ONOO‘ in the presence vs. absence of COz resulted in respective 3- and 6-fold increases in nitration of targets, no effect of CO2 was observed on yields of nitration catalyzed by either MPO or EPO. Likewise, under conditions where ONOO- promotes both hydroxylation and nitration of tyrosine, salicylate, Z-dG and phenylalanine, the nitrating intermediate formed during MPO- or EPOcatalyzed oxidation of N02- failed to hydroxylate these targets, despite causing even more extensive nitration than ONOO- in some cases. Finally, NO2 production by peroxidases,was observed in the gas phase of helium-sparged reaction mixtures following gold-catalyzed reduction to NO and detection by chemilmninescence. Thus, NO? should be added to the growing number of gasses formed at sites of inflammation.
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UniuersiQ-Hadassak
Medical
School,
Jerusalem
91120,
ISRAEL
Tetrachlorohydroquinone (TCHQ) has been identified as one of the main metabolites of the widely used wood preservative pentachlorophenol (PCP), and has also been implicated in its genotoxicity. Despite the extensive research on TCHQ biochemistry, the chemistry of the reactive oxygen species (ROS) generated by TCHQ is still unclear. In this study, we try to clarify the nature of ROS formed during TCHQ auto-oxidation. The production of hydroxyl radicals (OH) by TCHQ in the presence of H202 was studied by salicylate hydroxylation method. HPLC with electrochemical detection was used to measure the levels of 2,3- and 2,5-dihydroxybenzoic acid (DHBA) formed when the OH react with salicylate. We found that TCHQ and H202 could produce both 2,3- and 2,5-DHBA when incubated with salicylate. Their production was markedly inhibited by OH scavenging agents such as DMSO, as well as by tetrachlorosemiquinone radical scavengers such as deferoxamine. In contrast, their production was not affected by the non-hydroxamate iron chelators DTPA, phytic acid, as well as the copper-specific chelator bathocuprione. A comparison of product formation and distribution from the reaction of ferrous iron with H202 (Fenton system) strongly suggests that the same OH adducts are formed as in the TCHQ/H202 system. Similar results were also observed with tetrachloro-1,4-benzoquinone (the oxidation product of TCHQ) and H202. The production of OH by TCHQ/H202 was further substantiated by ESR spin trapping experiment with DMPO/DMSO (a characteristic DMPO-methyl radical signal). Based on these results, we suggest that hydroxyl radicals were produced by PCl’ metabolites in the presence of H202, possibly through an organic Fenton reaction. 516
We have previously reported that hyperthermia enhances the resistance of cells to oxyradical damage. The present study aims to assess whether heat pretreatment has the potential to protect cells l?om damage induced by nitrogenderived radicals. Cultured porcine aortic endothelial cells (PAEC) were exposed to 5 mM of 3morpholinosydnonimine-hydrochloride (SIN-l, a nitric oxide donor) at 37°C for 2 hours. The tested group of cells were first placed in 42’C for 2 hours, then exposed to same concentration of SIN-l at 37’C for two more hours. SIN-l toxicity and the hyperthermia protection were determined by the level of lipid peroxidation - the amount of malondialdhyde (MDA) formed by TBA (Thiobarbituric acid) assay. Our data showed that lipid peroxidation occurred within the PAEC exposed to SIN-l was twofold higher than that in the control group (p < 0.01). However, when cells were pretreated with heat, the level of lipid peroxidation significantly reduced by 26% (p < 0.05). These results indicate that hyperthermia such as heat pretreatment protects cells not only from oxyradical damage but also from nitrogen-derived radical cytotoxicity.
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HYDROXYL RADICAL PRODUCTION BY PENTACHLOROPHENOL METABOLITES AND H202: AN ORGANIC FENTON REACTION? Mordeckai Ckevion. Department of Cellular Biochemistry, Ben Zkan 7&and
Hebrew
HYPERTHERMIA PRETREATMENT ATTENUATES NITRIC OXIDE-INDUCED CYTOTOXICITY Line-Hua Zeng Sergiy Jadko, Jun Wu and Tai-Wing Wu (The Toronto Hispital, Toronto, Canada M5T 2S8)
OXYGEN
CHEMICAL REACTIONS OF MENADIONE WITH PROBES FOR SUPEROXIDE: A CAUTIONARY NOTE. Li Zuo Lawrence J Berliner, A John Merola, Valerie P Wright, Thomas LGon. The Ohio State Univ, Pulm. and Crit. Care Med. and Biophysics, Columbus, OH Menadione, a vitamin K derivative, is often used to induce intracellular superoxide (0~~) production. its mechanism is believed to be related to its ability to carry electrons across the inner mitochondrial membrane, resulting in single electron reduction of OZ. In this study, we tested whether menadione interfered with two probes used as indicators of Oz.-, i.e. cytochmme c (cyt c) and hydroethidine (HE). Cyt c can be reduced by 02-, changing its absorbance, a common assay for extracellular 0~~ production. We found that 30pM menadione reduces 56.8+8.6% of cyt c in Ringer’s (95%02:5%CO+ Experiments repeated in the absence of 02 (95%N2:5%CO$ resulted in nearly identical cyl c reduction (55.4*3.4%), confirming that menadione can interact with cyt c directly, even without forming OZ.-. H-E, an intracellular fluorescent probe, can be oxidized by 02) to form ethidium (ET). Addition of menadione (30pM) caused a 13,6+1.1% decrease of HE fluorescence in Ringer’s, but a sharp increase in distilled water (60.7+8.6%). The loss of HE in these differing solutions indicates complex chemistry between HE, menadione and its surrounding media, suggesting that loss of HE fluorescence may not be specific for 0~~ in some conditions. Importantly, however, the fluorescence of ET solutions was m influenced by menadione. These data suggest that menadione can react directly with some probes for Ozc, thus biochemical tests should be undertaken prior to using menadione as a positive control for biological sources of superoxide. Supported by W/LB/ 53333.
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presumably ONOOCO;. Although exposure of tyrosine or 2deoxyguanosine (2-dG) to ONOO‘ in the presence vs. absence of COz resulted in respective 3- and 6-fold increases in nitration of targets, no effect of CO2 was observed on yields of nitration catalyzed by either MPO or EPO. Likewise, under conditions where ONOO- promotes both hydroxylation and nitration of tyrosine, salicylate, Z-dG and phenylalanine, the nitrating intermediate formed during MPO- or EPOcatalyzed oxidation of N02- failed to hydroxylate these targets, despite causing even more extensive nitration than ONOO- in some cases. Finally, NO2 production by peroxidases,was observed in the gas phase of helium-sparged reaction mixtures following gold-catalyzed reduction to NO and detection by chemilmninescence. Thus, NO? should be added to the growing number of gasses formed at sites of inflammation.
I I
11
I
UniuersiQ-Hadassak
Medical
School,
Jerusalem
91120,
ISRAEL
Tetrachlorohydroquinone (TCHQ) has been identified as one of the main metabolites of the widely used wood preservative pentachlorophenol (PCP), and has also been implicated in its genotoxicity. Despite the extensive research on TCHQ biochemistry, the chemistry of the reactive oxygen species (ROS) generated by TCHQ is still unclear. In this study, we try to clarify the nature of ROS formed during TCHQ auto-oxidation. The production of hydroxyl radicals (OH) by TCHQ in the presence of H202 was studied by salicylate hydroxylation method. HPLC with electrochemical detection was used to measure the levels of 2,3- and 2,5-dihydroxybenzoic acid (DHBA) formed when the OH react with salicylate. We found that TCHQ and H202 could produce both 2,3- and 2,5-DHBA when incubated with salicylate. Their production was markedly inhibited by OH scavenging agents such as DMSO, as well as by tetrachlorosemiquinone radical scavengers such as deferoxamine. In contrast, their production was not affected by the non-hydroxamate iron chelators DTPA, phytic acid, as well as the copper-specific chelator bathocuprione. A comparison of product formation and distribution from the reaction of ferrous iron with H202 (Fenton system) strongly suggests that the same OH adducts are formed as in the TCHQ/H202 system. Similar results were also observed with tetrachloro-1,4-benzoquinone (the oxidation product of TCHQ) and H202. The production of OH by TCHQ/H202 was further substantiated by ESR spin trapping experiment with DMPO/DMSO (a characteristic DMPO-methyl radical signal). Based on these results, we suggest that hydroxyl radicals were produced by PCl’ metabolites in the presence of H202, possibly through an organic Fenton reaction. 516
We have previously reported that hyperthermia enhances the resistance of cells to oxyradical damage. The present study aims to assess whether heat pretreatment has the potential to protect cells l?om damage induced by nitrogenderived radicals. Cultured porcine aortic endothelial cells (PAEC) were exposed to 5 mM of 3morpholinosydnonimine-hydrochloride (SIN-l, a nitric oxide donor) at 37°C for 2 hours. The tested group of cells were first placed in 42’C for 2 hours, then exposed to same concentration of SIN-l at 37’C for two more hours. SIN-l toxicity and the hyperthermia protection were determined by the level of lipid peroxidation - the amount of malondialdhyde (MDA) formed by TBA (Thiobarbituric acid) assay. Our data showed that lipid peroxidation occurred within the PAEC exposed to SIN-l was twofold higher than that in the control group (p < 0.01). However, when cells were pretreated with heat, the level of lipid peroxidation significantly reduced by 26% (p < 0.05). These results indicate that hyperthermia such as heat pretreatment protects cells not only from oxyradical damage but also from nitrogen-derived radical cytotoxicity.
El12
I
HYDROXYL RADICAL PRODUCTION BY PENTACHLOROPHENOL METABOLITES AND H202: AN ORGANIC FENTON REACTION? Mordeckai Ckevion. Department of Cellular Biochemistry, Ben Zkan 7&and
Hebrew
HYPERTHERMIA PRETREATMENT ATTENUATES NITRIC OXIDE-INDUCED CYTOTOXICITY Line-Hua Zeng Sergiy Jadko, Jun Wu and Tai-Wing Wu (The Toronto Hispital, Toronto, Canada M5T 2S8)
OXYGEN
CHEMICAL REACTIONS OF MENADIONE WITH PROBES FOR SUPEROXIDE: A CAUTIONARY NOTE. Li Zuo Lawrence J Berliner, A John Merola, Valerie P Wright, Thomas LGon. The Ohio State Univ, Pulm. and Crit. Care Med. and Biophysics, Columbus, OH Menadione, a vitamin K derivative, is often used to induce intracellular superoxide (0~~) production. its mechanism is believed to be related to its ability to carry electrons across the inner mitochondrial membrane, resulting in single electron reduction of OZ. In this study, we tested whether menadione interfered with two probes used as indicators of Oz.-, i.e. cytochmme c (cyt c) and hydroethidine (HE). Cyt c can be reduced by 02-, changing its absorbance, a common assay for extracellular 0~~ production. We found that 30pM menadione reduces 56.8+8.6% of cyt c in Ringer’s (95%02:5%CO+ Experiments repeated in the absence of 02 (95%N2:5%CO$ resulted in nearly identical cyl c reduction (55.4*3.4%), confirming that menadione can interact with cyt c directly, even without forming OZ.-. H-E, an intracellular fluorescent probe, can be oxidized by 02) to form ethidium (ET). Addition of menadione (30pM) caused a 13,6+1.1% decrease of HE fluorescence in Ringer’s, but a sharp increase in distilled water (60.7+8.6%). The loss of HE in these differing solutions indicates complex chemistry between HE, menadione and its surrounding media, suggesting that loss of HE fluorescence may not be specific for 0~~ in some conditions. Importantly, however, the fluorescence of ET solutions was m influenced by menadione. These data suggest that menadione can react directly with some probes for Ozc, thus biochemical tests should be undertaken prior to using menadione as a positive control for biological sources of superoxide. Supported by W/LB/ 53333.
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