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C M Arroyo

Publications and source records attributed to C M Arroyo.

At least 19 recordsLinked to original sources
PubMed2004

TNF-alpha expression patterns as potential molecular biomarker for human skin cells exposed to vesicant chemical warfare agents: sulfur mustard (HD) and Lewisite (L).

Studies were conducted to examine the effect of two vesicant chemical warfare agents (VCWA), one of them an arsenical, on cytokine gene expression in normal human epidermal keratinocyte (NHEK) cells. We tested 2,2'-dichlorethylsulfide (sulfur mustard, military designation HD) and 2,chlorovinyldichloroarsine (Lewisite, military designation L), which have significant differences in their chemical, physical, and toxicological properties. Human tumor necrosis factor-alpha (hTNF-alpha) cytokine was detected by using the enzyme-linked immunosorbent assay, a protein multiplex immunoassay, Luminex100, and reverse transcription-polymerase chain reaction (RT-PCR). The messenger RNA expression of hTNF-alpha was determined to provide a semi-quantitative analysis. HD-stimulated NHEK induced secretion of hTNF-alpha in a dose-dependent manner. Dose response effect of Lewisite decreased hTNF-alpha levels. Time-response data indicated that the maximum response for HD occurred at 24 h with an associated cytotoxic concentration of 10(-4) mol/L. NHEK cells stimulated with 10(-4) mol/L HD for 24 h at 37 degrees C increased detectable levels of hTNF-alpha from 5 to 28 ng/ml at an index of cell viability between 85 to 93% as detected by Luminex100. Our results indicated that the increased levels of hTNF-alpha by HD are dependent on the primary cultures, cell densities, and chemical properties of the stimulation. Lewisite under the same conditions as HD caused a reduction of hTNF-alpha from control levels of 1.5 ng/ml to 0.3 ng/ml after stimulation (10(-4) mol/L), with an index of cell viability of reverse similar 34%. We analyzed the transcriptional of hTNF-alpha gene and found that HD (10(-6) to 10(-4) mol/L) activates hTNF-alpha gene in cultured NHEK and that L at 10(-6) to 10(-4) mol/L markedly reduces hTNF-alpha gene. We conclude that the pro-inflammatory mediator, hTNF-alpha, could be a potential biomarker for differentiating between exposure of HD or L.

Adult↗
PubMed2000

Reactivity of chloroethyl sulfides in the presence of a chlorinated prophylactic: a kinetic study by EPR/spin trapping and NMR techniques.

This study reports the kinetic reaction of a chlorinated glycoluril, 1,3,4,6-tetrachloro-7, 8-diphenyl-2,5-diimino glycoluril, also known as S-330, with butyl 2-chloroethyl sulfide (half-sulfur mustard, H-MG) and bis-(2-chloroethyl) sulfide (sulfur mustard, HD) using electron paramagnetic resonance (EPR)/spin trapping and nuclear magnetic resonance (NMR) techniques. Both H-MG and HD are highly reactive in water and are capable of alkylating a variety of critical target molecules. It is well known that compounds containing reactive chlorine are useful neutralizers of HD and other vesicating agents. Organic compounds containing a chloroamide group are generally preferred. Currently, the reactive mechanism of this chlorinated glycoluril with these chloroethyl sulfides has not been documented. The kinetic experiments were performed by adding the monofunctional sulfur mustard (H-MG) directly to the spin trap agent alpha-phenyl-N-tert-butylnitrone (PBN, pH 7.1). The intensity of the EPR spectra obtained from the resulting spin adduct (hyperfine coupling constants aN = 1.45 mT and abetaH = 0.225 mT) was sensitive to the rate at which the spin adduct was formed. Different concentrations of the chloroamide were added to the reaction mixtures of PBN and H-MG. The EPR spectra of separate identical reaction mixtures were recorded with the spectrometer set for kinetic experiments. The rate constant determined by EPR was 1.78 +/- 0.14 x 10(7) M(-1)s(-1). It was found that S-330 reacts 55 times faster than PBN. The results obtained for S-330 by EPR indicate that S-330 is an efficient scavenger of H-MG. Furthermore, a 13C-NMR chemical shift of 0.903 +/- 0.002 ppm was observed for the Cl-N-C-N-Cl carbon in S-330 after exposure to HD (1 mM). In addition, the decay of 13C-NMR resonance at 91.7 ppm chemical shift was observed in the presence of HD. The 13C-NMR data showed that the formation of the ethylene sulfonium ion usually found in the case of HD was not observed in the presence of S-330.

Amides↗
PubMed2000

Response of normal human keratinocytes to sulfur mustard: cytokine release.

Cytokines play a major role in both acute and chronic inflammatory processes, including those produced by sulfur mustard (2,2'-dichlorodiethyl sulfide, HD). This study describes responses of normal human epidermal keratinocytes (NHEK) to HD, defined by interleukin-1beta (IL-1beta), IL-6, IL-8 and tumor necrosis factor alpha (TNF-alpha) release. Commercially available enzyme-linked immunosorbent assay (ELISA) kits were used to measure the cytokine release in NHEK during exposure to 100 and 300 microM of HD. Exposure to 100 microM HD increased the release of cytokines. The amounts of IL-8 and TNF-alpha present in cell suspensions increased up to 59-fold and 4-fold, respectively, above control levels when NHEK were exposed to 300 microM HD. Exposure of NHEK to 300 microM HD had a highly variable effect on the release of IL-1beta, where sometimes the secretion of IL-1beta increased above baseline level and at other times it decreased in cell suspensions. Supernatants were collected from cell culture flasks 24 h after exposure of 100 and 300 microM HD and significantly increased levels of IL-6 were observed. Interleukin-6 was released in a concentration-dependent manner, 3.6-fold up to 8.4-fold, respectively, in supernatant. These pro-inflammatory mediators IL-1beta, IL-8, TNF-alpha and IL-6 may play an important role in HD injury. The present findings suggest that the cytokine changes detected could be used as potential biomarkers of cutaneous vesicant injury.

Biomarkers↗
PubMed1999

Response of normal human keratinocytes to sulfur mustard (HD): cytokine release using a non-enzymatic detachment procedure.

Cytokines play a major role in both acute and chronic inflammatory processes, including those produced by sulfur mustard (HD). This study describes responses of normal human epidermal keratinocyte (NHEK) cells to 2,2'-dichlorodiethyl sulfide, sulfur mustard (HD), defined by interleukin-1 beta (IL-1beta), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-alpha (TNF-alpha) release. A new method for detaching cell to cell adhesion between keratinocytes has been applied. This method permits the characterization of endogenous fluid from cellular content that could be applied for the development of therapeutic intervention. NHEK (typical average cell density 4.4 x 10(6) cells/mL) were exposed to HD (100 and 300 microM) in keratinocyte growth medium (KGM) for 24 h at 37 C in humidified air. Commercially available enzyme-linked immunosorbent assay (ELISA) kits were used to measure the cytokine release in NHEK during exposure to 100 and 300 microM of HD. Exposure to 100 microM HD increased release of cytokines. IL-1beta (exposed: 1.41 x 10(-5) pg/ cell+/-1.60 x 10(-6) pg/cell: control 7.10 x 10(-6) pg/ cell+/-1.20 x 10(-6) pg/cell), TNF-alpha (exposed: 1.06 x 10(5) pg/cell+/-7.3 x 10(-7)pg/cell; control: 4.04 x 10(-6)+/-2.80 x 10(-7) pg/cell) and IL-8 (exposed: 3.71 x 10(-5) pg/ cell+/- 3.26 x 10(-6) pg/cell; control: 2.99 x 10(-6) pg/cell+/-8.80 x 10(-7) pg/cell) were significantly enhanced when NHEK cells were detached from culture flasks by non-enzymatic procedures. Cell suspensions of NHEK released low amounts of IL-6 when exposed to 100 microM for 24 h (exposed: 1.47 x 10(-6)+/-1.60 x 10(-7) pg/cell; control: 1.28 x 10(-6)+/-8.40 x 10(-8) pg/cell). However, cell suspensions of NHEK increased levels of IL-6 after exposure to 300 microM HD (4.67 x 10(-5) pg/cell+/-3.90 x 10(-6) pg/cell; control: 3.99 x 10(-6) pg/cell+/-5.50 x 10(-7) pg/cell). The amount of IL-8 and TNF-alpha present in cell suspensions increased up to 59-fold and fourfold, respectively, above control levels when NHEK cells were exposed to 300 microM HD. Exposure of NHEK to 300 microM HD had a highly variable effect on the release of IL-1beta, where sometimes the secretion of IL-1beta increased above baseline level and other times decreased in cell suspensions. Supernatants were collected from cell culture flasks 24 h after exposure of 100 and 300 microM and significantly increased levels of IL-6 were observed. IL-6 was released in a concentration-dependent manner, 3.6-fold up to 8.4-fold, respectively, in supernatant. These pro-inflammatory mediators IL-1beta, IL-8, TNF-alpha and IL-6 may play an important role in HD injury. The present findings suggest that cytokine changes detected could be used as potential biomarkers of cutaneous vesicant injury.

Cell Adhesion↗
PubMed1997

The chemistry of perfluoroisobutylene (PFIB) with nitrone and nitroso spin traps: an EPR/Spin trapping study.

While applying electron paramagnetic resonance (EPR)/Spin Trapping techniques, several reactive intermediate species were identified in the reaction of perfluoroisobutylene (PFIB) with nitrone and nitroso spin trap agents: the carbon dioxide radical anion (CO2.-), a carbonyl fluoride intermediate (COF), and vinyl carbanions of PFIB. The reaction of PFIB with N-t-butyl-alpha-phenylnitrone (PBN) forms a dipolar ion which undergoes electron transfer reactions generating stable nitrone spin adducts. Nitroso compounds reacted with carbanions derived from PFIB, which raises the possibility that electron transfer reactions of this type might account for the observed nitroxides. Our results suggest that PFIB undergoes some type of electron transfer reaction leading to several reactive intermediate species (RIS). The implications of these observations on pulmonary damage caused by inhalation of PFIB are discussed.

Electron Spin Resonance Spectroscopy↗
PubMed1995

EPR/spin-label technique as an analytical tool for determining the resistance of reactive topical skin protectants (rTSPs) to the breakthrough of vesicant agents.

Ointment formulations of reactive topical skin protectants (rTSPs) or topical skin protectants (TSPs) based on perfluorinated polyether material (PFPE, i.e., fomblin RT-15) were prepared and spin labeled. Four N-oxyl-4-4'-dimethyloxazolidine derivatives of stearic acid, 5-NS, 7-NS, 12-NS, and 16-NS, were used as spin probes. The spin-labeled vehicle, fomblin-RT-15, and vehicle containing chloroamide (S-330, an antivesicant) were exposed to various concentrations of half-mustard gas. The order parameter (S) was dependent on the depth of penetration of the paramagnetic group into the vehicle (fomblin) and on the chemical composition of the reactive antivesicant under investigation. The net change of the viscosity of the vehicle and the chemical composition were seen to affect the penetration profile. This will provide a useful in vitro screening technique to develop antivesicant TSPs.

Alkynes↗
PubMed1995

Activation of alpha-human tumour necrosis factor (TNF-alpha) by human monocytes (THP-1) exposed to 2-chloroethyl ethyl sulphide (H-MG).

Tumour necrosis factor (TNF) is a monokine produced by monocytes and macrophages in response to different stimuli. To determine whether vesicant agents such as half-mustard gas (H-MG; chemical structure: ClCH2CH2SCH2CH3) may induce the release of TNF-alpha in human monocytes (THP-1), ELISA experiments were conducted at different post exposure times. The results indicate that: (1) Significant increases in the TNF-alpha (pg mL-1) concentration were observed as a function of time when THP-1 cells were exposed to 100 microL of 2 M H-MG. A specific serine-type protease inhibitor, N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), led to partial but significant inhibition of TNF activation. (2) Furthermore, this laboratory detected the generation of spin adducts of 2-methyl-2-nitrosopropane (MNP) having a resemblance to MNP-adducts generated from hydrogen atom abstraction of protein constituents. The EPR/Spin Trapping data indicate the trapping of by-products of protein degradation after exposure to H-MG. TNF-alpha may play a role as a biochemical marker for pathophysiological changes induced by H-MG or related agents.

Cell Line↗
PubMed1995

Exercise training generates ascorbate free radical in rat heart.

Exercise generates free radicals and can cause damage to the tissues. This investigation shows the formation of ascorbate radicals during exercise training (ET) which reduce the toxicity of free radicals. Male Fischer-344 rats (n = 8) (77 weeks old) were given exercise training (ET) on a treadmill with a low intensity of exercise that gradually increased from the first to the ninth week resulting in an average increase in respiratory exchange ratio, oxygen consumption rate and heat production. The sedentary control (SC) rats (n = 8) were not exercised and maintained under the same conditions. The heart tissues from different SC and ET rats were analyzed for ascorbate free radical (Asc.-) using electron paramagnetic resonance (EPR). The heart tissue from the ET and not from the SC rat showed the presence of Asc.-. This Asc.- was characterized by an EPR spectrum which showed doublet with a hyperfine coupling constant of 1.89 Gauss (0.189 mT). The benefit of exercise could be attributed to the formation of ascorbate radical in the heart muscle of the old rat. Exercise training can provide protection to the heart tissue against oxidative damage via ascorbate ion and vitamin E.

Animals↗
PubMed1994

The scavenging of hydroxyl radical(.OH) by a prostacyclin analogue, taprostene.

A possible mechanism by which prostacyclin (PGI2) analogues provide beneficial effects including improved survival in shock experimentally induced by endotoxin, polytrauma or hypovolemia was studied. Since several studies have implicated oxygen free radical-mediated tissue damage, we investigated whether PGI2-analogues exert their 'cytoprotective' effects by inhibiting overproduction of oxygen free radicals. For this reason, the efficiency of Taprostene to scavenge hydroxyl radicals (.OH) and to possibly prevent the subsequent formation of reactive oxygen species was studied. Competition experiments were performed in which the .OH generated by H2O2/Fe2+ abstracted a hydrogen from Taprostene (CG-4203) [5Z,13E, 9,11,15S)-2,3,4-trinor-1,5-inter-m-phenylene-6,9-epoxy-11,15-di hyd roxy-15-cyclohexyl-16,17,18,19,20-pentanor-prosta-5,13-dieno ic acid sodium salt], and the resulting carbon-centered radical was trapped with the spin trap 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (M4PO). This spin trap reacted with .OH to yield an M4PO-OH spin adduct observable by Electron Paramagnetic Resonance (EPR) spectroscopy and resulted in the rate constant, k2 = 1.5 x 10(10) M-1s-1, for the reaction between .OH and Taprostene. The results show that Taprostene is an efficient .OH scavenger. In addition, reactions of hypochlorous ion (-OCL) with hydrogen peroxide (H2O2) in the presence of Taprostene were monitored using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and M4PO dissolved in deuterium oxide.

Electron Spin Resonance Spectroscopy↗
PubMed1994

Reactive oxygen species produced in metal-catalyzed oxidation of bis(trifluoromethyl)disulfide and protection by ZE.

Bis(trifluoromethyl)disulfide (TFD), used as an industrial fumigant, was found to generate a thiyl free radical as seen by EPR/spin trapping. Oxygen appears to be an absolute requirement for radical production. The results obtained in this investigation implicate the production of thiyl and reactive oxygen species (ROS), superoxide radical anion and hydroxyl radicals, during TFD autoxidation. The rate of production of these free radical intermediates was found to increase in the presence of iron(III) and copper(II). In addition, the metal ion chelator DETAPAC and ROS scavengers ethanol, mannitol, and PEG-SOD/catalase were found to inhibit free radical production. Reactive oxygen species were not formed when a high-potency zinc plus antioxidant, ZE caps, was present. These results provide support for the pro-oxidation of TFD and a protective role for zinc.

Animals↗
PubMed1993

Autoionization reaction of phosgene (OCCl2) studied by electron paramagnetic resonance/spin trapping techniques.

The reaction of phosgene with nitrone spin traps was investigated using electron paramagnetic resonance (EPR)/spin trapping techniques. Evidence for the intermediacy of a carbamoyl monochloride intermediate was obtained. Isotopic substitution of 13C-phosgene was employed to verify the hyperfine coupling constant assignments. The implications of these observations on pulmonary damage caused by inhalation of phosgene are mentioned.

Electron Spin Resonance Spectroscopy↗
PubMed1993

Reactions of active oxygen and nitrogen species studied by EPR and spin trapping.

The reactions of hydrogen peroxide (H2O2) with nitrite (NO2-) and of superoxide (O2-.) with nitric oxide (NO.) were studied using EPR and spin trapping techniques. These reactions reportedly have a common peroxynitrite (OONO-) intermediate. It has been suggested that this intermediate when protonated rapidly decomposes producing hydroxyl radicals (.OH) and the nitrogen dioxide radical (NO2.). The production of .OH in the reaction between H2O2 and NO2- was confirmed in spin trapping experiments using the spin trap 5,5-dimethyl-1-pyrroline-1-oxide (DMPO). H2O2 and NO2- were mixed at neutral pH and then the pH was decreased to pH 3-3.5 in the presence of DMPO or DMPO and ethanol. In these experiments, the EPR spectrum of the DMPO-OH adduct was obtained in addition to a weak EPR spectrum consisting of a triplet of triplets (a N = 1.415 mT and a N beta = 0.35 mT) indicating the addition of a nitrogen centered radical to DMPO. The formation of .OH was confirmed using ethanol as an .OH scavenger. The DMPO-hydroxyethyl adduct was produced from the reaction of .OH with ethanol. However, in experiments using an excess of ethanol, the formation of DMPO-OH was not prevented. This suggests that the DMPO-OH formed in the decomposition of HOONO does not entirely originate from a direct addition of .OH to DMPO. The reaction of O2-. with NO. was carried out in deaerated and air-saturated solutions at pH 12.3 where the dismutation of O2-. is minimal. The pH was then decreased to pH 3-3.5 in the presence of DMPO or DMPO and ethanol. In these experiments, the most prominent EPR spectrum obtained was a triplet of triplets (aN = 1.415 mT and a beta N = 0.35 mT) suggesting the addition of a nitrogen centered radical to DMPO. The formation of DMPO-OH was minimal and there was no formation of DMPO-hydroxyethyl adducts in the presence of ethanol. The results suggest that NO. in solution yields additional reactive species which act as nitrating agents in the presence of DMPO.

Electron Spin Resonance Spectroscopy↗
PubMed1993

The reactions of 3,5-dibromo-4-nitrosobenzenesulfonate and its biological applications.

Recently, 5,5-dibromo-4-nitrosobenzenesulfonate (DBNBS) has been applied to detect biological free radicals. However, DBNBS has various non-specific reactions which lead to preplexing results. Thus, we investigated some basic reactions of DBNBS in combination of other nitroso spin traps to assign DBNBS spin adducts derived from human platelets which presumably related to the endothelium-derived relaxing factor (EDRF). The collagen activated platelets yielded four spin adducts (ST, LT, SS, and LS) in the presence of DBNBS (40 mM). The broad triplet due to ST was also observed by bubbling NO gas into a DBNBS solution. To identify ST, nitrosobenzene (NB) in dry dioxane was mixed with NO-saturated dioxane. The NB-NO spin adduct was observed but decomposed into diphenyl aminoxyl by the addition of H2O indicating that the primary adduct formed by the reaction of NO and DBNBS is unstable and turns into a dimerization product. Although ST could be eliminated by the inhibitor of EDRF, ST was shown to be produced by non-specific reactions. Another triplet was assigned to an S-centered radical because thiyl radicals which were generated from either the decomposition of S-nitrosothiol, or glutathione oxidation exhibited almost identical triplet signals. The other two sextets were assigned to C-centered radical adducts. Thus, DBNBS detected NQ-related, S-centered, and two C-centered radicals derived from human platelets. Special cautions are necessary for the identification of DBNBS spin adducts in a biological system to exclude artifactual radicals.

Benzenesulfonates↗
PubMed1993

Nitric oxide interaction with lactoferrin and its production by macrophage cells studied by EPR and spin trapping.

The production of nitrate (NO3-) and nitrite (NO2-) from macrophage-derived NO was studied using EPR and spin trapping. The formation of NO3- was determined via EPR in reactions involving the iron-binding protein, lactoferrin. The formation of NO2- was determined via EPR/spin trapping in the reaction between NO2- and H2O2. Dissolved nitric oxide (NO.) was reacted with lactoferrin yielding an EPR spectrum (77 degrees K) different from the normal EPR spectrum obtained for lactoferrin, suggesting that NO. interacts with the ferric ions bound to lactoferrin forming a ferric-nitrosyl type complex. The EPR spectrum (77 degrees K) of this ferric-nitrosyl type complex was also observed in the supernatant fluid of macrophage cell suspensions following their stimulation with lipopolysaccharide (LPS). During LPS stimulation of macrophages, these cells generate NO. which in turn produces NO3- and NO2-. The ferric-nitrosyl type complex is formed in a reaction mixture containing apolactoferrin and bicarbonate following the reaction of Fe+2 with NO3-, generated from macrophage-derived NO(.), to produce Fe+3 and NO(.). Furthermore, in an acidic medium, NO2- reacts with H2O2 forming peroxynitrous acid (HOONO) which rapidly decomposes into hydroxyl radicals (.OH) and the nitrogen dioxide (NO2.) radical. In the supernatant fluid of LPS-stimulated macrophage suspensions, the production of .OH was verified by spin trapping using 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) as the spin trap and ethanol as the .OH scavenger. The EPR spectra corresponding to the DMPO-OH and the DMPO-hydroxyethyl adducts were identified.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Investigation of the existence and biological role of L-arginine/nitric oxide pathway in human platelets by spin-trapping/EPR studies.

The aim of the present study was to apply spin trapping/EPR spectroscopy to investigate the existence and biological role of the L-arginine/nitric oxide pathway in human platelet aggregation. Three different spin traps were used: two nitroso, 3,5-dibromo-4-nitrosobenzenesulfonate (DBNBS) and 2-methyl-2-nitrosopropane (MNP), and a nitrone, 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The effect of spin-trap concentration on the collagen-induced human platelet aggregation was compared to the anti-aggregatory effect caused by L-arginine. The results show that the nitroso spin traps (DBNBS and MNP) are more effective than L-arginine in preventing platelet aggregation. DMPO has virtually no effect on the collagen-induced aggregation except at a high concentration (300 mM). Furthermore, activation of platelets with a low concentration of collagen (17 micrograms/ml) and in the presence of DBNBS or MNP yields several EPR-detectable spin adducts. Some of the observed spin adducts do not correspond to those originating from the interaction of a free radical, nitric oxide (NO.) gas, with the spin traps [Arroyo, C.M. & Kohno, M. (1991) Free Radical Res. Commun. 14, 145-155]. Only one adduct of DBNBS, with a relative intensity of 0.1, observed in the washed-platelet experiment and in the presence of superoxide dismutase, is similar to the EPR spectrum obtained following a reaction of pure NO. gas with DBNBS. This suggests that the EPR spectrum of the DBNBS adduct consisting of a triplet may originate from the production of NO. by these cells. Additional DBNBS and MNP spin adducts were generated during platelet activation in the presence of Ca2+ and of a cytosol-depleted L-arginine preparation from washed platelets to which L-arginine was subsequently added. The formation of these DBNBS and MNP spin adducts were inhibited by N omega-methyl-L-arginine (MeArg, 100 microM), suggesting that these originated from a product of NO synthase. Furthermore, the formation of DBNBS and MNP spin adducts in platelet suspensions was enhanced by the presence of superoxide dismutase; however, their formation was prevented by the endothelial-derived relaxing factor (EDRF) inhibitors methylene blue and hemoglobin. The results from the MeArg and EDRF inhibitor experiments support the existence of the L-arginine/NO pathway in platelets. In addition, the prevention of spin-adduct formation by EDRF inhibitors, suggests that the mechanisms of EDRF formation and the L-arginine/NO pathway in endothelial cells and platelets are similar.(ABSTRACT TRUNCATED AT 400 WORDS)

Arginine↗
PubMed1991

Activation of cyclic GMP formation in mouse neuroblastoma cells by a labile nitroxyl radical. An electron paramagnetic resonance/spin trapping study.

The receptor-mediated generation of an endothelial-derived relaxing factor (EDRF)-free radical intermediate in a neuronal cell line detected by spin trapping techniques has been reported. Here we report the time course of the appearance of the 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS) spin adduct and cyclic GMP formation following addition of carbamylcholine to suspensions of cultured mouse neuroblastoma cells (clone N1E-115). The time course of the appearance of the DBNBS spin adduct shows that spin adduct formation decreases possibly reaching a minimum approximately between 35 and 40 s. This is inversely proportional to cGMP formation which reaches a maximum at approximately 40 s after carbamylcholine activation. In addition, the inhibitory effect of NG-monomethyl-L-arginine (NMMA), potassium ferricyanide, K3Fe(CN)6 and methylene blue in cytosol preparation was investigated. A mechanism is proposed that essentially accounts for the combined results observed by spin trapping/electron paramagnetic resonance (EPR) study providing direct evidence for the muscarinic receptor-mediated formation of a labile, diffusible precursor of nitric oxide (NO.) derived from L-arginine that activates soluble guanylate cyclase.

Animals↗
PubMed1991

The demonstration of DMPO superoxide adduct upon reperfusion using a low non-toxic concentration.

Experiments were conducted in an attempt to circumvent the problem associated with the use of a high concentration of the spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), which has been suggested to have serious toxic effects on tissue. A low concentration of DMPO (10 mM) was used to detect the free radical generation in hearts with ischemia/reperfusion insult. In the effluent immediately after reperfusion, DMPO-OOH, a superoxide spin adduct of DMPO, was obtained. DMPO in that concentration range did not interfere at all with the left ventricular (LV) function during the control perfusion period. Even after reperfusion, LV function was not depressed any more than that occurring in hearts without DMPO, whereas DMPO of the conventional concentration (100 mM) markedly depressed the ventricular function. Enzyme leakage from hearts also supported non-toxicity finding of DMPO at 10 mM; confirming that the DMPO superoxide adduct is real evidence of the generation of superoxide upon reperfusion and is not attributed to an artificial generation due to the cytotoxicity of DMPO.

Animals↗