Alasdair J. Carmichael

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.

Endothelial Cells as A Source of Oxygen-Free Radicals An Esr Study

Endothelial cells were subjected to anoxia/reoxygenation in order to simulate some of the free radical mechanisms occurring in ischaemia/reperfusion. With ESR and spin trapping using the spin traps 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) and 3,3,5,5-dimethyl-1-pyrroline-1-oxide (M4PO), the results show that upon reoxygenation of endothelial cells, following a period of anoxia, these cells generate superoxide (O2-.). Cytotoxicity of the spin traps was measured by standard trypan blue exclusion methods. Cell injury or death was measured at various times during reoxygenation by lactate dehydrogenase (LDH) release. Experiments using oxyupurinol, SOD, CAT and a combination of SOD and CAT show that while oxypurinol partially prevents spin adduct formation, the combination of SOD and CAT is more effective in doing so. These results suggest that the majority of the oxygen radicals produced by endothelial cells are done so exogenously. The results also suggest that endothelial cells are not only a source of oxygen radicals but also a target.

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-1β), interleukin-6 (IL-6), inter-leukin-8 (IL-8), and tumor necrosis factor-alpha (TNF-α) 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 4.4×106 cells/mL) were exposed to HD (100 and 300 μM) 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 μM of HD. Exposure to 100 μM HD increased release of cytokines. IL-1β (exposed: 1.41×10-5 pg/ cell±1.60×10-6 pg/cell: control 7.10×10-6 pg/ cell±1.20× 10-6 pg/cell), TNF-α (exposed: 1.06× 10-5 pg/cell±7.3× 10-7 pg/cell; control: 4.04×10-6±2.80×10-7 pg/cell) and IL-8 (exposed: 3.71×10-5 pg/ cell±3.26×10-6 pg/cell; control: 2.99×10-6 pg/cell±8.80×10-7 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 μM for 24 h (exposed: 1.47×10-6±1.60×10-7 pg/cell; control: 1.28×10-6± 8.40×10-8 pg/cell). However, cell suspensions of NHEK increased levels of IL-6 after exposure to 300 μM HD (4.67×10-5 pg/cell±3.90×10-6 pg/cell; control: 3.99× 10-6 pg/cell±5.50×10-7 pg/cell). The amount of IL-8 and TNF-α present in cell suspensions increased up to 59-fold and fourfold, respectively, above control levels when NHEK cells 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 other times decreased in cell suspensions. Supernatants were collected from cell culture flasks 24 h after exposure of 100 and 300 μM 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 proinflammatory mediators IL-1β, IL-8, TNF-α 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.

Optimal sample preparation conditions for the determination of uranium in biological samples by kinetic phosphorescence analysis (KPA).

Kinetic phosphorescence analysis (KPA) is a proven technique for rapid, precise, and accurate determination of uranium in aqueous solutions. Uranium analysis of biological samples require dry-ashing in a muffle furnace between 400 and 600 degrees C followed by wet-ashing with concentrated nitric acid and hydrogen peroxide to digest the organic component in the sample that interferes with uranium determination by KPA. The optimal dry-ashing temperature was determined to be 450 degrees C. At dry-ashing temperatures greater than 450 degrees C, uranium loss was attributed to vaporization. High temperatures also caused increased background values that were attributed to uranium leaching from the glass vials. Dry-ashing temperatures less than 450 degrees C result in the samples needing additional wet-ashing steps. The recovery of uranium in urine samples was 99.2+/-4.02% between spiked concentrations of 1.98-1980 ng (0.198-198 microg l(-1)) uranium, whereas the recovery in whole blood was 89.9+/-7.33% between the same spiked concentrations. The limit of quantification in which uranium in urine and blood could be accurately measured above the background was determined to be 0.05 and 0.6 microg l(-1), respectively.

Reaction of Vanadyl with Hydrogen Peroxide. An ESR and Spin Trapping Study

Vanadyl reacts with hydrogen peroxide forming hydroxyl radicals in a Fenton-like reaction. The hydroxyl radicals were spin trapped and identified using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The quantity of hydroxyl radicals spin trapped during the reaction between vanadyl and hydrogen peroxide are equal to half of the hydroxyl radicals spin trapped during the reaction between ferrous ions and hydrogen peroxide. Experiments in the presence of formate show that this hydroxyl radical scavenger effectively competes with DMPO preventing the formation of the DMPO-OH adduct. However, in experiments using ethanol as the hydroxyl radical scavenger it was not possible to completely prevent the formation of DMPO-OH. The formation of this additional DMPO-OH in the presence of ethanol does not depend on the concentration of dissolved oxygen, but does depend on the concentration of hydrogen peroxide added to the vanadyl solution. The results suggest that the additional DMPO-OH formed in the presence of ethanol originates from a vanadium (V) intermediate. This intermediate may oxidize DMPO leading to the formation of DMPO-O2- which rapidly decomposes forming DMPO-OH.

Neutron-induced Free Radicals in Oriented DNA

Samples of oriented DNA containing 30 per cent water were irradiated with neutrons at 77 K. The electron spin resonance (e.s.r.) spectra obtained from these irradiated DNA samples show that the formation of radicals is different when the incident neutrons are parallel or perpendicular to the DNA helix. When the incident neutrons are perpendicular to the DNA helix the e.s.r. spectra of thymine and guanine ionic radicals (T-., G+.) are observed. An additional e.s.r. spectrum corresponding to the hydrogen addition radical on thymine (TH.) is observed when the incident neutrons are parallel to DNA helix. The TH. radical appears to be formed by protonation of T-. .

Trichloroethylene metabolism in vitro: an EPR/SPIN trapping study

Trichloroethylene (TCE) was hypothesized to produce free radicals which could be detected using electron para magnetic resonance spectroscopy with the spin trap, PBN (α-phenyl tert-butyl nitrone). The free radicals detected following incubation of precision cut liver slices in media containing 10 mM PBN had hyperfine coupling constants aN=1.61 mT and aH=0.325 mT. There was a linear increase in free radicals detected in the bathing media when the headspace TCE concentration was increased from 2500- 10 000 p.p.m. The levels of conjugated dienes measured in the slices incubated in PBN supplemented media were less than slices exposed to TCE in incubation media without PBN. The PBN trap may act as a scavenger preventing the propagation of free radicals and inhibiting lipid peroxida tion. The experiments suggest that free radical formation by TCE leads to a concomitant increase in conjugated dienes in liver slices which may contribute to the pathological changes which occur in liver following TCE exposure.

Reaction of disodium cromoglycate with hydrated electrons

A possible mechanism by which disodium cromoglycate (DSCG) prevents a decrease in regional cerebral blood flow but not hypotension in primates following whole body gamma-irradiation was studied. Several studies have implicated superoxide radicals (O2-.) in intestinal and cerebral vascular disorders following ischemia and ionizing radiation, respectively. O2-. is formed during radiolysis in the reaction between hydrated electrons (e-aq) and dissolved oxygen. For this reason, the efficiency of DSCG to scavenge e-q and possibly prevent the formation of O2-. was studied. Hydrated electrons were produced by photolysis of potassium ferrocyanide solutions. The rate constant, k = 2.92 x 10(10) M-1s-1 for the reaction between e-aq and DSCG was determined in competition experiments using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). This spin trap reacts rapidly with e-aq followed by protonation to yield the ESR observable DMPO-H spin adduct. The results show that DSCG is an efficient e-aq scavenger and may effectively compete with oxygen for e-aq preventing the radiolytic formation of O2-..

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.

Trichloroethylene radicals generated by ionizing radiation. An EPR/spin trapping study.

Trichloroethylene (TCE) was exposed in the presence of the spin trap N-tert-butyl-α-phenyl nitrone (PBN, 0.1 M) to ionizing radiation from two different sources in an attempt to determine the origin of the spin-trapped radicals generating the EPR spectra in precision cut liver slices. TCE samples were irradiated with 18 MeV electrons to a total dose of 1000 Gy in a linear accelerator (LINAC) or exposed to 60Co γ-rays to total doses of 100 Gy and 1000 Gy. The results show that three PBN adducts were generated during the LINAC radiations. Two of these spin adducts correspond to the addition of carbon-centered radicals to PBN, and the third adduct is consistent with a decomposition product of PBN. The predominant carbon- entered radical yields a PBN adduct that is more stable, persists for over 24 h and has identical hyperfine coupling constants (a N=1.61 mT, aHβ=0.325 mT) to the PBN adduct obtained when precision-cut liver slices were exposed to TCE. Gamma radiation (100 Gy) of TCE yields PBN adducts with lower primary nitrogen hyperfine coupling constants (aN=1.45 mT and aN=1.54 mT). The results (y- radiation) suggest that the carbon-centered radical is formed on a single TCE carbon that is different than the predominant radical formed during LINAC radiations. This difference is confirmed by experiments using 13C- TCE. The results further suggest that, during γ-radiation of TCE, the radicals are formed by dechlorination at the TCE carbon containing two chlorine atoms. The results obtained during LINAC radiations suggest that the predominant radical is formed by dechlorination at the TCE carbon containing a single chlorine and a single proton. In addition, it is possible that this radical is the initial TCE radical formed during exposure of liver slices to TCE.