Carmen M. Arroyo

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‐1β (IL‐1β), IL‐6, IL‐8 and tumor necrosis factor alpha (TNF‐α) release. Commercially available enzyme‐linked immunosorbent assay (ELISA) kits were used to measure the cytokine release in NHEK during exposure to 100 and 300 μM of HD. Exposure to 100 μM HD increased the release of cytokines. The amounts of IL‐8 and TNF‐α present in cell suspensions increased up to 59‐fold and 4‐fold, respectively, above control levels when NHEK were exposed to 300 μM HD. Exposure of NHEK to 300 μM HD had a highly variable effect on the release of IL‐1β, where sometimes the secretion of IL‐1β 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 μM 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‐1 β, IL‐8, TNF‐α 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. Published in 2000 by John Wiley & Sons, Ltd.

DIFFICULTIES ENCOUNTERED IN THE DETECTION OF NITRIC OXIDE (NO) BY SPIN TRAPPING TECHNIQUES. A CAUTIONARY NOTE.

The spin trapping technique was used in an attempt to detect the free radical nitric oxide (NO) in solution. Five different spin traps were examined, alpha-phenyl-N-tert butyl nitrone (PBN), alpha-(4-pyridyl-N-oxide) N-tert-butylnitrone (POBN), 5,5-dimethyl-pyrroline-N-oxide (DMPO), 2-methyl-2-nitrosopropane (MNP) and 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS). Our results suggest that the nitroso spin traps (MNP, DBNBS) are better suited for the identification of NO-related signals, than the nitrones, DMPO, PBN and POBN. In addition, it is shown that spin trapping of NO-related signals with nitroso and nitrone spin traps is subject to many artifacts.

Receptor-mediated generation of an EDRF-like intermediate in a neuronal cell line detected by spin trapping techniques

We have studied receptor-mediated generation of an activator of soluble guanylate cyclase in cultured mouse neuroblastoma cells (clone N1E-115) by ESR/spin trapping spectroscopy. A spin adduct was detected during the activation of muscarinic receptors by carbamylcholine in the presence of the spin trap 3,5-dibromo 4-nitrosobenzene sulphonate (DBNBS). The spin adduct does not correspond to that originating from the free radical nitric oxide or hydroxylamine. The same adduct was generated in cytosol preparations from N1E-115 cells incubated with L-arginine, NADPH, in the presence of calcium. The use of isotopically labelled guanidino-N15-L-arginine supported the generation of a DBNBS spin trapped adduct originating from the guanidino moiety of L-arginine. Superoxide dismutase (SOD) stabilized the precursor of the spin adduct as well as the activator of soluble guanylate cyclase derived from L-arginine. Our results provide direct evidence for the receptor-mediated formation of a diffusible precursor of NO. derived from L-arginine.

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.

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.

Role of cytokine and nitric oxide in the inflammatory response produced by sulfur mustard (HD)

We have determined by immunocytochemistry the levels of interleukin-1 beta; (IL-1β) in cultured human epidermal keratinocytes (NHEK) following exposure to HD. Human skin keratinocytes release significant numbers of IL-1 cytokine as determined by the QuantikineTM Interleukin-1β kit, an enzyme-linked immunosorbent assay (ELISA) procedure. Exposure of NHEK [∼106–107 cells, to HD (2 mM) and preincubation for 3 h at 37xa0°C] results in significant changes in IL-1 activation. In neonatal NHEK exposed to HD, IL-1β is decreased. Conversely, in adult breast NHEK exposed to HD, IL-1β is increased. Nitric oxide (˙NO) has been implicated as the effector molecule that mediates IL-1β. To confirm the involvement of ˙NO in the expression of the IL-1β, electron paramagnetic resonance (EPR) spectroscopy was employed. EPR detectable iron–nitrosyl complex in NHEK exposed to HD (18 h post exposure to 1 mM HD) were measured, and the generation of ˙NO and this induced complex was blocked by N n ω-nitro-L-arginine (L-NOARG), a competitive inhibitor of nitric oxide synthase (NOS). Our results show the release of nitric oxide during IL-1 cytokine expression when keratinocytes are exposed to HD. Based upon this work, it appears possible that IL-1 could be used as a specific marker for epidermal cytoxicity in mechanistic studies of the toxicity of HD and in defining interventive and therapeutic regimens against HD vesication.

Neutralization effects of interleukin-6 (IL-6) antibodies on sulfur mustard (HD)-induced IL-6 secretion on human epidermal keratinocytes.

The proinflammatory cytokine human interleukin-6 (hIL-6) plays an important role in the early and late courses of inflammation, trauma, and wound healing caused by sulfur mustard (HD). Previously, we demonstrated that hIL-6 might be involved in the early event of structural changes of the signal transducer glycoprotein, which indirectly initiates the cascade of events, such as skin irritation and blister formation observed in the pathophysiology of HD injury. In this present work, we focus on the neutralization effect of IL-6 antibodies with regard to the modulation of hIL-6 secretion. Levels of secreted cytokine hIL-6 in normal human epidermal keratinocytes (NHEK) stimulated with HD (10(-4)M) and incubated for 24h at 37°C were determined by enzyme immunoassay, protein immunocytologic assay and reverse-transcriptase-polymerase chain reaction (RT-PCR). The ratio of HD-treated NHEK to constitutive non-stimulated NHEK controls (S/C) on the induction of hIL-6 is reported. S/C was four-fold higher than non-stimulated NHEK controls as determined by ELISA. By using a more sensitive immunocytologic assay, Luminex(100)™, the increment was verified. hIL-6 levels in NHEK stimulated with HD were 21±11ng/mL as measured by Luminex(100)™. The messenger RNA expression of the cytokine (hIL-6) gene was analyzed semiquantitatively. RT-PCR demonstrated that HD induced an increase in the transcription of hIL-6 gene. Selective immunosuppression, using IL-6 neutralizing antibodies, led to a reduction of such expression of HD-induced transcription of hIL-6 in human keratinocytes. The neutralization by pre-incubating NHEK with monoclonal anti-IL6 antibodies decreased hIL-6 secretion by 76%±1.8 ((*)P<0.05).

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 α‐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 aβH = 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 × 107 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. Published in 2000 by John Wiley & Sons, Ltd.

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.