Deborah Coffin

Evaluation of the hydroxylamine tempol-H as an in vivo radioprotector

Nitroxides are stable free radical compounds that protect against the toxicity of reactive oxygen species in vitro and in vivo. Tempol (Aldrich, Milwaukee, WI, USA) is a cell-permeable hydrophilic nitroxide and has been shown to be an in vitro and in vivo radioprotector. The limitations of Tempol as a systemic radioprotector are that it causes substantial reductions in arterial blood pressure when administered intravenously and is associated with seizure activity. Furthermore, Tempol is rapidly reduced to its hydroxylamine form, Tempol-H, which limits the period of time the active form of the nitroxide is available for radioprotection. Based on initial pharmacological and blood pressure experiments performed in mice, we hypothesized that the systemic administration of Tempol-H in vivo would lead to an equilibration between Tempol and Tempol-H that would limit the toxicity of the nitroxide and provide in vivo radioprotection. Tempol-H was administered in increasing doses via an intraperitoneal route to C3H mice. The maximally tolerated dose was found to be 325 mg/kg. The whole-blood pharmacology of Tempol-H was investigated with electron paramagnetic resonance spectroscopy. These studies demonstrated the appearance of Tempol in whole blood immediately after intraperitoneal injection, suggesting that rapid oxidation of Tempol-H to Tempol takes place in vivo. Although the peak concentration of Tempol in whole blood after administration of Tempol-H did not reach the same levels as those observed when Tempol is administered, the whole-blood levels of Tempol were similar by 10 min after injection. Tempol-H provided protection against the lethality of whole-body radiation in C3H mice at 30 d with a dose modification factor of 1.3, which is similar to the results obtained with Tempol. Hemodynamic measurements in C3H mice after intravenous injection showed that Tempol-H produced little effect on blood pressure or pulse compared with Tempol. Tempol-H is a systemic in vivo radioprotector of C3H mice and is associated with less hemodynamic toxicity than Tempol.

Hemodynamic effect of the nitroxide superoxide dismutase mimics

Reactive oxygen species play critical roles in a number of physiologic and pathologic processes. Nitroxides are stable free radical compounds that possess superoxide dismutase (SOD) mimetic activity and have been shown to protect against the toxicity of reactive oxygen species in vitro and in vivo. Tempol, a cell-permeable hydrophilic nitroxide, protects against oxidative stress and also is an in vitro and in vivo radioprotector. In the course of evaluating the pharmacology and toxicity of the nitroxides, Tempol and another nitroxide, 3-carbamoyl-PROXYL (3-CP), were administered intravenously in various concentrations to miniature swine. Tempol caused dose-related hypotension accompanied by reflex tachycardia and increased skin temperature. Invasive hemodynamic monitoring with Swan Ganz catheterization (SGC) confirmed the potent vasodilative effect of Tempol. However, 3-CP had no effect on porcine blood pressure. The hemodynamic effects of Tempol and 3-CP are discussed in the context of differential catalytic rate constants for superoxide disumation that may impact systemic nitric oxide (NO) levels and lead to vasodilation. These findings are consistent with a role for the superoxide ion in the modulation of blood pressure and have potential implications for the systemic use of nitroxides.

Three-dimensional whole body imaging of spin probes in mice by time-domain radiofrequency electron paramagnetic resonance.

Imaging of stable paramagnetic spin probes in phantom objects and in vivo was evaluated using a RF time domain EPR spectrometer/imager operating at 300 MHz. Projections were collected using static magnetic field gradients and images were reconstructed using filtered back‐projection techniques. Results from phantom objects containing approximately 1017 spins of stable paramagnetic probes with single narrow EPR spectra provide three‐dimensional spatial images with resolution better than 2 mm. When the spin probe was administered to mice, the spin probe accumulation was temporally observed in the thoracic, abdominal, and pelvic regions. A three‐dimensional image (from 144 projections) from a live mouse was collected in 5 min. Using fiducial markers, the spin probe accumulation in organs such as liver, kidney, and bladder could be observed. Differences in the oxygen status between liver and kidney were observed from the EPR images from mice administered with spin probe, by treating the time‐domain responses with convolution difference approach, prior to image reconstruction. The results from these studies suggest that, with the use of stable paramagnetic spin probes and time‐domain RF EPR, it is possible to perform in vivo imaging on animals and also obtain important spatially resolved physiologic information. Magn Reson Med 43:375–382, 2000.

DETECTION OF S-NITROSOTHIOLS BY FLUOROMETRIC AND COLORIMETRIC METHODS

Publisher Summary This chapter discusses the detection of S-Nitrosothiols by fluorometric and colorimetric methods. The colorimetric method uses the components of the Griess reaction while the fluorometric method utilizes the conversion of 2, 3-diaminonaphthalene (DAN) to a fluorescent triazole. These methods are conducted at neutral rather than acidic pH that eliminates the interference of contaminating nitrite and allows the detection of nitrosation mediated by the presence of nitric oxide (NO). The colorimetric reaction described herein utilizes the chemistry that occurs among nitrosating species such as those in the NO/O2 reaction and sulfanilamide (SULF) to form a diazonium ion. The fluorometric assay is based on the reaction of DAN with nitrosating intermediates, such as those formed in the NO/O2 reaction, to yield a primary nitrosamine that is converted rapidly to a fluorescent triazole. The colorimetric assay has a detection range of 0.5–100/μM, while the fluorometric assay is effective in the range of 0.05–5μ/M S-nitrosothiol compounds (RSNO). The combination of the two assays provides a detection range from 50 n M to 100μ M RSNO, required for most biological experiments.

Ionizing radiation potentiates the induction of nitric oxide synthase by IFN-gamma and/or LPS in murine macrophage cell lines: role of TNF-alpha.

Macrophages are activated to become cytotoxic by a highly coordinated set of cytokine signals. Ionizing radiation can mimic cytokine signals and lead to enhanced states of activation. We tested the ability of γ‐radiation, alone and with interferon‐γ (IFN‐γ) and/or lipopolysaccharide (LPS), to induce nitric oxide (NO) production in J774.1 and RAW264.7 murine macrophages. NO was induced weakly, moderately, or strongly by IFN‐γ alone, LPS alone, or IFN‐γ + LPS, respectively. Radiation alone (0.5–50 Gy) did not induce NO, but enhanced NO production in a dose‐dependent manner (0.5–5 Gy) when cells were exposed to IFN‐γ or LPS 24 h post‐irradiation. Immunoblots showed parallel induction of nitric oxide synthase (NOS2). Application of antitumor necrosis factor α (TNF‐α) antibody before irradiation blocked induction of NO by IFN‐γ. We conclude (1) that irradiated cells produce more NO in response to either IFN‐γ or LPS and (2) that the increase is mediated by induction of TNF‐α. J. Leukoc. Biol. 64: 459–466; 1998.

Ionizing Radiation Potentiates the Induction of Nitric Oxide Synthase by Interferon‐γ and/or Lipopolysaccharide in Murine Macrophage Cell Lines: Role of Tumor Necrosis Factor‐α

Abstract: Macrophages respond to infection or injury by changing from a “resting” cellular phenotype to an “activated” state defined by the expression of various cytotoxic effector functions. Regulation of the transition from a resting to an activated state is effected by cytokine and/or pathogenic signals. Some signals do not directly induce activation, but instead “prime” the macrophage to respond more vigorously to a second signal. One example of this priming phenomenon involves induction of nitric oxide (NO) synthesis by the enzyme nitric oxide synthase (NOS2). Our experiments indicate that low doses (1–5 Gy) of ionizing radiation can enhance the induction of enzymatically active NOS2 by IFN‐γ or LPS in J774.1 and RAW264.7 murine macrophage cell lines. Radiation alone did not produce this induction, rather, it was effective as a priming signal; cells exposed to radiation produced more NO when a second signal, either IFN‐γ or LPS, was applied 24 h later.

Evaluation of hybridization conditions for spotted oligonucleotide-based DNA microarrays

We compared different hybridization conditions of oligonucleotide-based DNA microarray to acquire optimized and reliable microarray data. Several parameters were evaluated at different hybridization conditions, including signal-to-background (S:B) ratios, signal dynamic range, usable spots, and reproducibility. Statistical analysis showed that better results were obtained when spotted, presynthesized long oligonucleotide arrays were blocked with succinic anhydride and hybridized at 42°C in the presence of 50% formamide.

Protection from radiation-induced alopecia with topical application of nttroxides: Fractionated studies

PURPOSE Hair loss resulting from irradiation of the head and neck or from whole brain irradiation often leads to cosmetic, social, and psychological problems for the radiotherapy patient. Few successful clinical interventions are available. We have shown that nitroxides (stable free radicals) afford radiation protection against single-dose radiation-induced alopecia in a guinea pig model. Here we determine if topical nitroxide application provides protection from fractionated radiation treatment. MATERIALS AND METHODS Two symmetrical and contralateral areas (3 x 5 cm) of skin on the dorsal trunk of guinea pigs were shaved to a hair length of 0.25 cm. A 2 mL solution containing 70 mg/mL nitroxide (Tempo or Tempol) in 70% ethanol was topically applied to the skin surface of one side; 70% ethanol was applied to the contralateral (control) side 10 minutes before irradiation. Animals were placed in a special jig that held skin without decreasing blood flow to the treatment area and fractionated external beam radiation (7 Gy) was delivered daily for eight fractions over 10 days via a 4 MeV linear accelerator. Alopecia (hair density) was scored weekly for 13 to 14 weeks after radiotherapy, using a standardized reference with respect to hair loss and regrowth in the treatment field. RESULTS After radiation treatment, dry desquamation and gradual hair loss were observed for both control and nitroxide-treated skin; however, over weeks 4 to 11 postirradiation hair loss was much more pronounced in control animals when compared with nitroxide-treated animals. Hair density measurements for Tempol treatment over weeks 9 to 13 were approximately 75% compared with measurements in controls of approximately 25%. Tempo-treated animals exhibited hair density values of approximately 90% compared with 12% in controls over weeks 11 to 14. Tempol and Tempo treatments resulted in significant radioprotection. Histologic evaluation showed that radiation treatment alone in ethanol controls resulted in a marked decrease in the number of hair follicles and poor development of remaining follicles; however, nitroxide pretreatment resulted in no appreciable decrease in hair follicles and hair follicles appeared mature. This was also observed in unirradiated ethanol controls. Electron paramagnetic resonance studies revealed that topical nitroxide application did not result in measurable systemic concentrations of either drug. CONCLUSIONS The results of this study suggest that topical application of nitroxides may be useful in a clinical setting to reduce the undesirable toxicity of radiation-induced alopecia.