Inhibition of oxygen-dependent radiation-induced damage by the nitroxide superoxide dismutase mimic, tempol.

Stable nitroxide radicals have been previously shown to function as superoxide dismutase (SOD)2 mimics and to protect mammalian cells against superoxide and hydrogen peroxide-mediated oxidative stress. These unique characteristics suggested that nitroxides, such as 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol), might protect mammalian cells against ionizing radiation. Treating Chinese hamster cells under aerobic conditions with 5, 10, 50, and 100 mM Tempol 10 min prior to X-rays resulted in radiation protection factors of 1.25, 1.30, 2.1, and 2.5, respectively. However, the reduced form of Tempol afforded no protection. Tempol treatment under hypoxic conditions did not provide radioprotection. Aerobic X-ray protection by Tempol could not be attributed to the induction of intracellular hypoxia, increase in intracellular glutathione, or induction of intracellular SOD mRNA. Tempol thus represents a new class of non-thiol-containing radiation protectors, which may be useful in elucidating the mechanism(s) of radiation-induced cellular damage and may have broad applications in protecting against oxidative stress.     

Evaluation of Tempol Radioprotection in a Murine Tumor Model

Tempol, a stable nitroxide free radical compound, is an in vitro and in vivo radioprotector. Previous studies have shown that Tempol protects C3H mice against whole-body radiation-induced bone marrow failure. In this study, the radioprotection of tumor tissue was evaluated. RIF-1 tumor cells were implanted in female C3H mice 10 d prior to radiation. Groups of mice were injected intraperitoneally with Tempol (275 mg/kg) or PBS followed 10 min later by a single dose of radiation to the tumor bed. Tumor growth curves generated after 10 and 33.3 Gy doses of radiation showed no difference in growth between the Tempol- and PBS-treated animals. A full radiation dose-response experiment revealed a tumor control dose in 50% of the animals in 30 d (TCD_50/30) value of 36.7 Gy for Tempol-treated mice and 41.8 Gy for saline-treated mice suggesting no protection of the RIF-1 tumor by Tempol. Tumor pharmacokinetics were done to determine why Tempol differentially protected bone marrow and not tumor cells. Differential reduction of Tempol in the RIF-1 tumor and bone marrow was evaluated with EPR spectroscopy 10, 20, and 30 min after injection. Bioreduction of Tempol to its corresponding hydroxylamine (which is not a radioprotector) occurred to a greater extent in RIF-1 tumor cells compared to bone marrow. We conclude that the differences in radioprotection may result from enhanced intratumor bioreduction of Tempol to its nonradioprotective hydroxylamine analogue. The nitroxides as a class of compounds may provide a means to exploit the redox differences between normal tissues and tumors. © 1997 Elsevier Science Inc.     

Cellular sites of H2O2-induced damage and their protection by nitroxides

While the exact mechanism of H2O2-induced cytotoxicity is unknown, there is considerable evidence implicating DNA as a primary target. A recent study showed that a cell-impermeable nitroxide protected mammalian cells from H2O2-induced cell killing and suggested that the protection was mediated through cell membrane-bound or extracellular factors. To further define the protective properties of nitroxides, Chinese hamster V79 cells were exposed to H2O2 with or without cell-permeable and impermeable nitroxides and selected metal chelators. EPR spectroscopy and paramagnetic line broadening agents were used to distinguish between intra- and extracellular nitroxide distribution. To study the effectiveness of nitroxide protection, in the absence of a cell membrane, H2O2-mediated damage to supercoiled plasmid DNA was evaluated. Both deferrioxamine and Tempol cross the cell membrane, and inhibited H2O2-mediated cell killing, whereas the cell-impermeable DTPA and nitroxide, CAT-1, failed to protect. Similar protective effects of the chelators and nitroxides were observed when L-histidine, which enhances intracellular injury, was added to H2O2. In contrast, when damage to plasmid DNA was induced (in the absence of a cell membrane), both nitroxides were protective. Collectively, these results do not support a role for membrane-bound or extracellular factors in mediating H2O2 cytotoxicity in mammalian cells.     

The effects of antioxidants on radiation-induced apoptosis pathways in TK6 cells

This study was designed to determine if radiation-mediated activation of the apoptotic pathways would be influenced by antioxidants and if a correlation would be found between radioprotection and changes in transduction pathways. Human lymphoblastoid TK6 cells, known to undergo apoptosis as a result of radiation, were irradiated (6 Gy) with and without antioxidants, and then whole-cell lysates were collected. Parallel studies were conducted to assess the survival (clonogenic assay) and apoptotic index. The impacts of two nitroxide antioxidants, tempol and CAT-1, differing in cell permeability, as well as the sulfhydryl antioxidant N-acetyl-L-cysteine (L-NAC), were estimated. Changes in apoptotic pathway proteins and p53 were assessed by Western blotting. Fraction of apoptotic cells was determined by flow cytometry. Tempol (10 mM), which readily enters cells, partially radioprotected TK6 cells against clonogenic killing, but had no effect on radiation-induced apoptotic parameters such as cleaved caspase 3 or cleaved PARP. Tempol alone did not induce cytotoxicity, yet did increase cleaved PARP levels. The radiation-induced increase in p53 protein was partly inhibited by tempol, but was unaffected by CAT-1 and L-NAC. Both CAT-1 (10 mM), which does not enter cells, and L-NAC (10 mM) had no radioprotective effect on cell survival. Although L-NAC did not protect against radiation-induced cytotoxicity, it completely inhibited radiation-induced increase in cleaved caspase 3 and cleaved PARP. Collectively, the results question the validity of using selected apoptosis pathway members as sole indicators of cytotoxicity.     

Different effectiveness of piperidine nitroxides against oxidative stress induced by doxorubicin and hydrogen peroxide

The piperidine nitroxides Tempamine and Tempace have been studied for their effect on doxorubicin (DOX) and hydrogen peroxide (H₂O₂) cytotoxicity in immortalized B14 cells, a model for neoplastic phenotype. The significance for nitroxide performance of the substituent in position 4 of the piperidine ring was evaluated. The cells were exposed to DOX/H₂O₂ alone or in combination with the nitroxides Tempamine or Tempace. Two other piperidine nitroxides, Tempo and Tempol, were used for comparison. All the nitroxides except Tempamine modestly reduced DOX cytotoxicity. Tempamine evoked a biphasic response: at concentrations lower than 200 μmol/L the nitroxide decreased DOX cytotoxicity, while at concentrations higher than 200 μmol/L, it enhanced DOX cytotoxicity. In contrast to Tempo and Tempol, Tempamine and Tempace ameliorated hydrogen peroxide cytotoxicity, but none of the nitroxides influenced TBARS stimulated by hydrogen peroxide. The cytoprotective effect of Tempace, Tempo and Tempol in DOX-treated cells correlated with the inhibition of DOX-induced lipid peroxidation. The bioreduction rates of the investigated nitroxides differed significantly and were variously affected by DOX depending on the nitroxide substituent. In combination with DOX, Tempo and Tempol were reduced significantly more slowly, while no influence of DOX on Tempamine and Tempace bioreduction was observed. Our results suggest that the structure of the 4-position substituent is an important factor for biological activity of piperidine nitroxides. Among the investigated nitroxides, Tempace displayed the best protective properties in vitro but Tempamine was the only nitroxide that potentiated cytotoxicity of DOX and did not influence DOX-induced lipid peroxidation. However, this nitroxide showed different performance depending on its concentration and conditions of oxidative stress.     

Magnetic resonance imaging of organic contrast agents in mice: capturing the whole-body redox landscape

Nitroxides are a class of stable free radicals that have several biomedical applications including radioprotection and noninvasive assessment of tissue redox status. For both of these applications, it is necessary to understand the in vivo biodistribution and reduction of nitroxides. In this study, magnetic resonance imaging was used to compare tissue accumulation (concentration) and reduction of two commonly studied nitroxides: the piperidine nitroxide Tempol and the pyrrolidine nitroxide 3-CP. It was found that 3-CP was reduced 3 to 11 times slower (depending on the tissue) than Tempol in vivo and that maximum tissue concentration varies substantially between tissues (0.6-7.2mM). For a given tissue, the maximum concentration usually did not vary between the two nitroxides. Furthermore, using electron paramagnetic resonance spectroscopy, we showed that the nitroxide reduction rate depends only weakly on cellular pO(2) in the oxygen range expected in vivo. These observations, taken with the marked variation in nitroxide reduction rates observed between tissues, suggest that tissue pO(2) is not a major determinant of the nitroxide reduction rate in vivo. For the purpose of redox imaging, 3-CP was shown to be an optimal choice based on the achievable concentrations and bioreduction observed in vivo.     

Nitroxide-mediated protection against X-ray- and neocarzinostatin-induced DNA damage.

The stable free radical Tempol (4-hydroxy-2,2,6,6-tetramethyl-piperidinyloxy) has been shown to protect against X-ray-induced cytotoxicity and hydrogen peroxide- or xanthine oxidase-induced cytotoxicity and mutagenicity. The ability of Tempol to protect against X-ray- or neocarzinostatin (NCS)-induced mutagenicity or DNA double-strand breaks (dsb) was studied in Chinese hamster cells. Tempol (50 mM) provided a protection factor of 2.7 against X-ray-induced mutagenicity in Chinese hamster ovary (CHO) AS52 cells, with a protection factor against cytotoxicity of 3.5. Using the field inversion gel electrophoresis technique of measuring DNA dsb, 50 mM Tempol provides a threefold reduction in DNA damage at an X-ray dose of 40 Gy. For NCS-induced damage, Tempol increased survival from 9% to 80% at 60 ng/mL NCS and reduced mutation induction by a factor of approximately 3. DNA dsb were reduced by a factor of approximately 7 at 500 ng/mL NCS. Tempol is representative of a class of stable nitroxide free radical compounds that have superoxide dismutase-mimetic activity, can oxidize metal ions such as ferrous iron that are complexed to DNA, and may also detoxify radiation-induced organoperoxide radicals by competitive scvenging. The NCS chromophore is reduced by sulfhydryls to an active form. Electron spin resonance (ESR) spectroscopy shows that 2-mercaptoethanol-activated NCS reacts with Tempol 3.5 times faster than does unactivated NCS. Thus, Tempol appears to inactivate the NCS chromophore before a substantial amount of DNA damage occurs.     

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.     

γ-Irradiation Damage to Liposomes Differing in Composition and Their Protection by Nitroxides

The present study aims to determine the effect of bilayer composition on oxidative damage and the protection against it in lipid multicomponent membranes. Irradiation damage in 200-nm liposomes and the protection provided by the nitroxide radicals, 2,2,6,6-tetramethylpiperidine-1-oxyl (Tempo) and 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol) were assessed by monitoring several chemical and physical parameters. Liposomes were prepared in four different lipid compositions (mole ratios), DPPC:DPPG 10:1; DPPC:DPPG:cholesterol 10:1:4; EPC:EPG 10:1; and EPC:EPG:cholesterol 10:1:4, and γ-irradiated with a dose of 32 kGy. Lipid degradation was determined by HPLC and GC analyses, whereas size and differential scanning calorimetry measurements were used to monitor physical changes in the liposomal dispersions. The results indicate that: (1) addition of 5 mM Tempo or Tempol, or freezing of the sample inhibited radiation-induced lipid degradation; (2) Tempo and Tempol caused neither physical nor chemical changes in the liposomal dispersions; and (3) both nitroxides prevented or reduced some of the radiation-induced changes in thermotropic characteristics of the liposomes, preventing a shift in the temperature of the maximum of the main phase transition (T_m).     

Yeast DEL assay detects protection against radiation-induced cytotoxicity and genotoxicity: adaptation of a microtiter plate version

The DEL assay in yeast detects DNA deletions that are inducible by many carcinogens. Here we use the colorimetric agent MTS to adapt the yeast DEL assay for microwell plate measurement of ionizing radiation-induced cell killing and DNA deletions. Using the microwell-based DEL assay, cell killing and genotoxic DNA deletions both increased with radiation dose between 0 and 2000 Gy. We used the microwell-based DEL assay to assess the effectiveness of varying concentrations of five different radioprotectors, N-acetyl-l-cysteine, l-ascorbic acid, DMSO, Tempol and Amifostine, and one radiosensitizer, 5-bromo-2-deoxyuridine. The microwell format of the DEL assay was able to successfully detect protection against and sensitization to both radiation-induced cytotoxicity and genotoxicity. Such radioprotection and sensitization detected by the microwell-based DEL assay was validated and compared with similar measurements made using the traditional agar-based assay format. The yeast DEL assay in microwell format is an effective tool for rapidly detecting chemical protectors and sensitizers to ionizing radiation and is automatable for chemical high-throughput screening purposes.     

Stable Nitroxide Radicals Protect Lipid Acyl Chains From Radiation Damage

The present study focused on protective activity of two six-membered-ring nitroxide radicals, 2,2,6,6-tetramethylpiperidine-1-oxyl (Tempo) and 4-hydroxy-Tempo (Tempol), against radiation damage to acyl chain residues of egg phosphatidylcholine (EPC) of small unilamellar vesicles (SUV). SUV were -irradiated (10–12 kGy) under air at ambient temperature in the absence and presence of nitroxides. Acyl chain composition of the phospholipids before and after irradiation was determined by gas chromatography. Both Tempo and Tempol effectively and similarly protected the acyl chains of EPC SUV, including the highly sensitive polyunsaturated acyl chains, C20:4, C22:5, and C22:6. The conclusions of the study are: (a) The higher the degree of unsaturation in the acyl chain, the greater is the degradation caused by irradiation. (b) The fully saturated fatty acids palmitic acid (C16) and stearic acid (C18) showed no significant change in their levels. (c) Both Tempo and Tempol provided similar protection to acyl chain residues. (d) Nitroxides' lipid-bilayer/aqueous distribution is not validly represented by their n-octanol/saline partition coefficient. (e) The lipid-bilayer/aqueous partition coefficient of Tempo and Tempol cannot be correlated with their protective effect. (f) The nitroxides appear to protect via a catalytic mode. Unlike common antioxidants, such as -tocopherol, which are consumed under irradiation and are, therefore, less effective against high radiation dose, nitroxide radicals are restored and terminate radical chain reactions in a catalytic manner. Furthermore, nitroxides neither yield secondary radicals upon their reaction with radicals nor act as prooxidants. Not only are nitroxides self-replenished, but also their reduction products are effective antioxidants. Therefore, the use of nitroxides offers a powerful strategy to protect liposomes, membranes, and other lipid-based assemblies from radiation damage. © 1997 Elsevier Science Inc.     

Differential protection by nitroxides and hydroxylamines to radiation-induced and metal ion-catalyzed oxidative damage

Modulation of radiation- and metal ion-catalyzed oxidative-induced damage using plasmid DNA, genomic DNA, and cell survival, by three nitroxides and their corresponding hydroxylamines, were examined. The antioxidant property of each compound was independently determined by reacting supercoiled DNA with copper II/1,10-phenanthroline complex fueled by the products of hypoxanthine/xanthine oxidase (HX/XO) and noting the protective effect as assessed by agarose gel electrophoresis. The nitroxides and their corresponding hydroxylamines protected approximately to the same degree (33-47% relaxed form) when compared to 76.7% relaxed form in the absence of protectors. Likewise, protection by both the nitroxide and corresponding hydroxylamine were observed for Chinese hamster V79 cells exposed to hydrogen peroxide. In contrast, when plasmid DNA damage was induced by ionizing radiation (100 Gy), only nitroxides (10 mM) provide protection (32.4-38.5% relaxed form) when compared to radiation alone or in the presence of hydroxylamines (10 mM) (79.8% relaxed form). Nitroxide protection was concentration dependent. Radiation cell survival studies and DNA double-strand break (DBS) assessment (pulse field electrophoresis) showed that only the nitroxide protected or prevented damage, respectively. Collectively, the results show that nitroxides and hydroxylamines protect equally against the damage mediated by oxidants generated by the metal ion-catalyzed Haber-Weiss reaction, but only nitroxides protect against radiation damage, suggesting that nitroxides may more readily react with intermediate radical species produced by radiation than hydroxylamines.     

Nitroxides as antioxidants: Tempol protects against EO9 cytotoxicity

Nitroxide free radicals have been shown to be potent antioxidants in a variety of experimental models using diverse means of insults. Among other insults, nitroxides have been shown effective in inhibiting cytotoxicity of quinone-based drugs such as streptonigrin and mitomycin C. These drugs and other chemotherapeutic agents have the potential to undergo bioreductive activation by the normal reducing enzymes within a cell. In the present work we studied the effect of the nitroxide Tempol on the cytotoxicity induced by EO9, a mitomycin C analogue, in HT29 cells under aerobic and hypoxic conditions. The study was aimed to better understand the mechanism of EO9 cytotoxicity and the molecular level of the nitroxide's mode of protection. The reactions of Tempol with activated EO9, and the reactive species formed during EO9 activation were studied in a cell-free solution, using spin-trapping, and electron paramagnetic resonance (EPR) spectrometry. Our results indicate that EO9 induced similar cytotoxicity in HT29 cells under aerobic and hypoxic conditions while Tempol provided similar and almost complete protection to both aerobic and hypoxic cells. The results indicate that EO9 cytotoxicity is due to both 1- and 2-electron reductive activation processes, with aerobic toxicity caused by back-oxidation of the hydroquinone to the semiquinone, EO9.-. Tempol serves both as a useful tool in the study of the mechanisms of quinone-mediated cytotoxicity and as a potent antioxidant against the damaging effects of redox cycling quinones and semiquinones by scavenging of EO9.- or detoxification of O2.- and H2O2.     

A low molecular weight antioxidant decreases weight and lowers tumor incidence

Stable free radical nitroxides are potent antioxidants possessing superoxide dismutase- and catalase-mimetic activity that protect cells and animals against a variety of oxidative insults. Tempol, as a representative nitroxide, was evaluated for its influence on weight maintenance and spontaneous tumor incidence in C3H mice. Tempol administered in either the drinking water or food did not show any untoward effects and prevented animals from becoming obese. Tempol-treated animals' leptin levels were reduced. Long-term treatment with Tempol significantly decreased tumorigenesis when compared to controls (10 vs. 40%, respectively). Selected tissues from Tempol-treated animals exhibited elevated levels of mitochrondrial uncoupling protein-2 (UCP-2) and HSP70. The present data suggest that nitroxides upregulate UCP-2, obviate weight gain, and decrease age-related spontaneous tumor incidence. As a class, nitroxides may provide overall health benefits by contributing to decreased obesity and tumor incidence.     

Nitroxides block DNA scission and protect cells from oxidative damage.

The protective effect of cyclic stable nitroxide free radicals, having SOD-like activity, against oxidative damage was studied by using Escherichia coli xthA DNA repair-deficient mutant hypersensitive to H2O2. Oxidative damage induced by H2O2 was assayed by monitoring cell survival. The metal chelator 1,10-phenanthroline (OP), which readily intercalates into DNA, potentiated the H2O2-induced damage. The extent of in vivo DNA scission and degradation was studied and compared with the loss of cell viability. The extent of DNA breakage correlated with cell killing, supporting previous suggestions that DNA is the crucial cellular target of H2O2 cytotoxicity. The xthA cells were protected by catalase but not by superoxide dismutase (SOD). Both five- and six-membered ring nitroxides, having SOD-like activity, protected growing and resting cells from H2O2 toxicity, without lowering H2O2 concentration. To check whether nitroxides protect against O2.(-)-independent injury also, experiments were repeated under hypoxia. These nitroxides also protected hypoxic cells against H2O2, suggesting alternative modes of protection. Since nitroxides were found to reoxidize DNA-bound iron(II), the present results suggest that nitroxides protect by oxidizing reduced transition metals, thus interfering with the Fenton reaction.     

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.     

Nitroxides as antioxidants: Tempol protects against EO9 cytotoxicity

Nitroxide free radicals have been shown to be potent antioxidants in a variety of experimental models using diverse means of insults. Among other insults, nitroxides have been shown effective in inhibiting cytotoxicity of quinone-based drugs such as streptonigrin and mitomycin C. These drugs and other chemotherapeutic agents have the potential to undergo bioreductive activation by the normal reducing enzymes within a cell. In the present work we studied the effect of the nitroxide Tempol on the cytotoxicity induced by EO9, a mitomycin C analogue, in HT29 cells under aerobic and hypoxic conditions. The study was aimed to better understand the mechanism of EO9 cytotoxicity and the molecular level of the nitroxide's mode of protection. The reactions of Tempol with activated EO9, and the reactive species formed during EO9 activation were studied in a cell-free solution, using spin-trapping, and electron paramagnetic resonance (EPR) spectrometry. Our results indicate that EO9 induced similar cytotoxicity in HT29 cells under aerobic and hypoxic conditions while Tempol provided similar and almost complete protection to both aerobic and hypoxic cells. The results indicate that EO9 cytotoxicity is due to both 1- and 2-electron reductive activation processes, with aerobic toxicity caused by back-oxidation of the hydroquinone to the semiquinone, EO9^–. Tempol serves both as a useful tool in the study of the mechanisms of quinone-mediated cytotoxicity and as a potent antioxidant against the damaging effects of redox cycling quinones and semiquinones by scavenging of EO9^– or detoxification of O₂ ^– and H₂O₂.     

GS-nitroxide (JP4-039)-mediated radioprotection of human Fanconi anemia cell lines

Fanconi anemia (FA) is an inherited disorder characterized by defective DNA repair and cellular sensitivity to DNA crosslinking agents. Clinically, FA is associated with high risk for marrow failure, leukemia and head and neck squamous cell carcinoma (HNSCC). Radiosensitivity in FA patients compromises the use of total-body irradiation for hematopoietic stem cell transplantation and radiation therapy for HNSCC. A radioprotector for the surrounding tissue would therefore be very valuable during radiotherapy for HNSCC. Clonogenic radiation survival curves were determined for pre- or postirradiation treatment with the parent nitroxide Tempol or JP4-039 in cells of four FA patient-derived cell lines and two transgene-corrected subclonal lines. FancG(-/-) (PD326) and FancD2(-/-) (PD20F) patient lines were more sensitive to the DNA crosslinking agent mitomycin C (MMC) than their transgene-restored subclonal cell lines (both P < 0.0001). FancD2(-/-) cells were more radiosensitive than the transgene restored subclonal cell line (? = 2.0 ± 0.7 and 4.7 ± 2.2, respectively, P = 0.03). In contrast, FancG(-/-) cells were radioresistant relative to the transgene-restored subclonal cell line (? = 9.4 ± 1.5 and 2.2 ± 05, respectively, P = 0.001). DNA strand breaks measured by the comet assay correlated with radiosensitivity. Cell lines from a Fanc-C and Fanc-A patients showed radiosensitivity similar to that of Fanc-D2(-/-) cells. A fluorophore-tagged JP4-039 (BODIPY-FL) analog targeted the mitochondria of the cell lines. Preirradiation or postirradiation treatment with JP4-039 at a lower concentration than Tempol significantly increased the radioresistance and stabilized the antioxidant stores of all cell lines. Tempol increased the toxicity of MMC in FancD2(-/-) cells. These data provide support for the potential clinical use of JP4-039 for normal tissue radioprotection during chemoradiotherapy in FA patients.     

Polynitroxyl-albumin (PNA) plus tempol attenuate lung capillary leak elicited by prolonged intestinal ischemia and reperfusion(1)

Stable nitroxyl radicals (nitroxides) are potential antioxidant drugs, and we have previously reported that linking nitroxide to biological macromolecules can improve therapeutic activity in at least two ways. First, polynitroxylated compounds such as polynitroxyl human serum albumin (PNA) are a novel class of high molecular weight, extracellular antioxidants. Second, compounds such as PNA can prolong the half-life of free (unbound, low molecular weight) nitroxides such as 4-hydroxy-2,2,6, 6-tetramethylpiperidine-N-oxyl (Tempol) in vivo. Unlike PNA, Tempol can readily access the intracellular compartment. Thus PNA can act alone in the extracellular compartment, or in concert with Tempol, to provide additional antioxidant protection within cells. In this study, we compared the abilities of PNA, Tempol, and the combination of PNA + Tempol to prevent lung microvascular injury secondary to prolonged gut ischemia (I, 120 min) and reperfusion (R, 20 min) in the rat. Pulmonary capillary filtration coefficient (K(f,c)) and lung neutrophil retention (tissue myeloperoxidase activity, MPO) were measured in normal, isolated rat lungs perfused with blood harvested from I/R rats. Blood donor rats were treated with drug during ischemia. Gut I/R resulted in a marked increase in pulmonary capillary coefficient and lung MPO. PNA + Tempol, but not PNA alone or Tempol alone, at the doses used, prevented the development of lung leak. None of the treatments had an effect on lung neutrophil retention. Anti-inflammatory therapeutic activity appeared to correlate with blood Tempol level: in the presence of PNA, blood Tempol levels were maintained in the 50-100 microM range vs. essentially undetectable levels shortly after Tempol was administered alone. In this model of lung injury secondary to prolonged gut I/R, lung capillary leak was prevented when the membrane-permeable compound Tempol was maintained in its active, free radical state by PNA.     

Cellular metabolism of water-soluble nitroxides: Effect on rate of reduction of cell/nitroxide ratio,oxygen concentrations and permeability of nitroxides

In order to interpret more accurately studies that have used nitroxides and to improve the efficacy of the use of nitroxides in both basic studies of cells and as contrast agents for in vivo NMR, we have initiated a systematic study of the distribution and metabolism of nitroxides in biological systems. Overall, the results provide a reasonably coherent picture of some aspects of the interactions between nitroxides and cells. Reduction of the nitroxides appears to be an intracellular process, so that one of the principal variables that affects the rate of reduction is the ability of a nitroxide to enter cells. The entrance of nitroxides into cells shows considerable variability and ranges from essentially no penetration (e.g., 2,2,6,6-tetramethylpiperidine-N-oxyl-4-trimethylamine) through rates that are comparable to rates of reduction (e.g., 2,2,5,5-tetramethylpyrrolidine-N-oxyl-3-carboxylic acid), to rates that are so fast that there is complete equilibrium between intracellular and extracellular compartments (e.g., Tempone). The presence of a charged group on the nitroxide appears to be the important variable that affects their ability to enter cells. Once a nitroxides enters the cell, the structure of the nitroxide, e.g., piperidine vs. pyrrolidine ring, is major factor that affects the rate of reduction. The rates of reduction increase with increasing concentrations of nitroxides. This indicates that the principal mechanism(s) of reduction do not saturate in the concentration range we studied. We observed no abrupt changes in the rates of reduction over the entire concentration range of cells and nitroxides that we studied, which suggests that the mechanism(s) of nitroxide reduction did not change. The presence of oxygen decreased the observed rate of reduction of many of the nitroxides and this effect was independent of the concentration of nitroxide.