ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 289, No. 1, August 15, pp. 62-70, 1991
Inhibition of Oxygen-Dependent Radiation-Induced Damage by the Nitroxide Superoxide Dismutase Mimic, Tempo1 James B. Mitchell,’ William DeGraff, Dwight Kaufman, Murali C. Krishna, Amram Eli Finkelsteiqt Min S. Ahn, Stephen M. Hahn, Janet Gamson, and Angelo Russo
Samuni,*
Radiobiology Section, Radiation Oncology Branch, Clinical Oncology Program, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892; *Molecular Biology, School of Medicine, Hebrew Uniuersity, Jerusalem, Israel 91010; and TRadiation Oncology Department, Elizabeth General Medical Center, Elizabeth, New Jersey 07202
Received January 31,1991, and in revised form April 18, 1991
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 IIIM Tempo1 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 Tempo1 afforded no protection. Tempo1 treatment under hypoxic conditions did not provide radioprotection. Aerobic X-ray protection by Tempo1 could not be attributed to the induction of intracellular hypoxia, increase in intracellular glutathione, or induction of intracellular SOD mRNA. Tempo1 thus represents a new class of non-thiol-containing radiation protectors, which may be useful in elucidating the mechanism(s) of radiationinduced cellular damage and may have broad applications 0 1991 Academic in protecting against oxidative stress. Press,
Inc.
The deleterious effects of ionizing radiation include cytotoxicity, mutagenesis, and carcinogenesis. The discovery i To whom correspondence should be addressed at Radiation Oncology Branch, National Cancer Institute, NIH, Bldg. 10, Room B3-B69, Bethesda, MD, 20892. ’ Abbreviations used: SOD, superoxide dismutase; DF, desferrioxamine; Tempol, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl; DTPA, diethylenetriaminepentaacedic acid; BSO, L-buthionine sulfoximine; Tempol-H, Tempol-hydroxylamine; RT, room temperature; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate.
in the late 1940s that sulfhydryls (thiols), such as cysteine and cysteamine, provide radiation protection to animals ushered in an era of research aimed at elucidating the mechanism(s) of radiation-induced cytotoxicity and development of more effective protective agents (1, 2). Radiation damages biomolecules, in large part (approximately 80%), through its interaction with water to produce free radicals (H’, OH, e&) and HzOz, or through interaction with oxygen to produce the superoxide anion (‘0;). Thiols, which function by donation of a hydrogen atom to damaged molecules or by “scavenging” radiationproduced free radicals, afford significant protection to animals against whole body radiation (1); however, their use is limited by toxicity. Despite extensive testing and synthetic efforts, no thiol-based radioprotector has been found to be significantly better than cysteamine. Stable nitroxide radicals are useful as NMR imaging contrast agents, EPR spin labels, and pH or oximetry cellular probes (3-7). As part of our studies investigating the chemical and biochemical characteristics of nitroxide radicals, we recently found that they exhibit superoxide dismutase mimetic activity (89) and are capable of protecting mammalian cells from damage by superoxide generated by hypoxanthine/xanthine oxidase and by hydrogen peroxide (10). These findings encouraged us to test if stable nitroxide radicals could protect mammalian cells against ionizing radiation, since approximately 80% of the damaging effects of ionizing radiation is thought to occur through indirect damage caused by radiolysis products of water or through oxygen reacting with radiationinduced carbon-centered free radicals (11). We have found that Tempo1 does protect against ionizing radiation and is the prototypical radiation protector of this group of chemical compounds and, moreover, may provide insight
62 All
0003.9861/91 $3.00 Copyright 0 1991 by Academic Press, Inc. rights of reproduction in any form reserved.
NITROXIDE
into the fundamental cellular damage. EXPERIMENTAL
mechanism(s)
SUPEROXIDE
of radiation
induced
PROCEDURES
Chemicals. Desferrioxamine I(DF) was a gift from Ciba Geigy; 4hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempo]), diethylenetriaminepentaacetic acid (DTPA), and platinum(IV) oxide were purchased from Aldrich Chemical Co. and trioxalato-chromium(II1) KS[Cr(Cz0&]‘3Hz0 (CrOx) from l?faltz and Bauer. Contamination of K3[Cr(C20&]‘3H20 with K&O., necessitated recrystallization twice; concentration was determined spectrophotometrically. Superoxide dismutase (SOD) was purchased from Sigma Chemical Co. and L-buthionine sulfoximine (BSO) was purchased from Schweizerhall, Inc. (South Plainfield, NJ). Oxygen-dependent reoridation of Tempo&hydroxylamine. A 5-ml solution of 50 mM phosphate bufFer, pH 7.8, containing 100 PM DTPA and 100 mM Tempo1 was catalytically reduced [platinum oxide (IV)] by bubbling hydrogen gas through the solution for 2 h. The course of the reduction was followed by removing small aliquots (200 ~1) and observing the loss of the Tempo1 EPR signal. To test if the catalytic reduction of Tempo1 had only proceeded to the one-electron reduction product (Tempo]-hydroxylamine), the nitroxide signal resulting from treatment with ferricyanide (which does not oxidize the amine) was measured and found to be equal to the initial signal before reduction; therefore, only hydroxylamine had been formed. After the reduction had proceeded to greater than 99.9% completion, the platinum catalyst was removed by filtration, and the resulting Tempo]-hydroxylamine (Tempo]-H) was diluted to a final concentration of 1 mM. The rate of oxygen-dependent reoxidation of Tempo]-H (1 mM) in F12 medium at pH 7.3 in a gaspermeable teflon tube positioned in the EPR cavity was then determined. Oxidation of the hydroxylamine was followed while either argon or air was passed across the reaction tube. The rate of oxidation of TempolH in room air was 0.2 rc.M/min. For large-scale preparations, the reduction was essentially as described above except that Tempo1 (5 g; 30 mmol) was dissolved in 75 ml of methanol containing platinum oxide (250 mg). The product after filtration was isolated by flash evaporation and the white residue was stored in the dark under argon at -20°C. The product was characterized by NMR, mass spectroscopy, and ECPR. The compound was 99.8% pure, the impurity being the starting nitroxide. Cell culture. Chinese hamster V79 cells were grown in F12 medium supplemented with 10% fetal calf serum, penicillin, and streptomycin. Survival was assessed in all stud& by the clonogenic assay. The plating efficiency ranged between 80 and 90%. Stock cultures of exponentially growing cells were trypsinized, rinsed, and plated (5 X lo5 cells/dish) into a number of loo-mm petri dishes and incubated 16 h at 37°C prior to experimental protocols. Tempo1 was added to exponentially growing cells in complete F12 medium (final concentrations of 5, 10, 50, or 100 mM) at room temperature (RT) 1.0 min prior to X-irradiation. TempolH was also evaluated under the same conditions at 100 mM. The time required for irradiation (at RT) was approximately 10 min. Immediately after X-ray treatment, cells were rinsed, trypsinized, counted, and plated for macroscopic colony formation. Under these conditions, Tempo1 exerted no cytotoxicity at 5 or 10 mM; however, for 50 and 100 mM treatments there was a reduction in plating efficiency by 20-30%. Radiation survival estimates were corrected to account for Tempo1 cytotoxicity. Tempo]-H exhibited no cytotoxicity. Each dose determination was plated in triplicate, and experiments wlere repeated a minimum of two times. Plates were incubated 7 days; colonies were then fixed with methanol/ acetic acid (3:l) and stained with crystal violet. Colonies containing >50 cells were scored. Error bars shown in the figures represent SE of the mean and are shown when larger than the symbol. Protection factors were calculated by determining the ratio of doses of Tempo]-treated cells to those of control cells at the 10% survival level.
DISMUTASE
MIMIC,
TEMPOL
63
Some studies involved the treatment of cells with Tempo1 and radiation at 4’C. For these studies the medium was removed from the plates and replaced with fresh, chilled (4°C) medium containing 100 mM Tempal; the plates were then placed on top of an ice-bath tray. After a lomin exposure to Tempo], cell cultures were irradiated and processed as described above. Treatment at 4°C with or without Tempo1 did not alter the control plating efficiency. The radiation response of V79 cells exposed to SOD or DF was also assessed. SOD was added (final concentration of 100 pg/ml) to cell cultures 10 min prior to irradiation and DF (final concentration of 500 PM) was added 2 h prior to irradiation at RT. Neither SOD nor DF exerted cytotoxicity alone. Following irradiation cells were plated as described above. Hypoxic irradiation studies were performed by plating cells (2.5 X 106) into specially designed glass flasks (12) and incubating at 37°C overnight. Tempo1 was added to a final concentration of 100 mM and the flasks were sealed with soft rubber stoppers. Needles (19-gauge) were pushed through the rubber stopper to provide entrance and exit ports for a humidified gas mixture of 95% nitrogen/5% COP (Matheson Gas Products). Stoppered flasks were connected in series and mounted on a reciprocating platform and gassed at 37°C for 45 min. The gassing procedure resulted in an equilibrium between the gas and liquid phase and yielded oxygen concentrations in the effluent gas phase of 110 ppm as measured by a Thermox probe (12). After 45 min of deoxygenating, flasks were irradiated and cell survival was assessed as described above. Glutathione depletion was accomplished by exposing exponentially growing cells to 5 mM BSO for 6 h prior to radiation exposure. Control and BSO-treated cells were trypsinized, rinsed twice with phosphatebuffered saline (PBS), and suspended in cold 0.6% sulfosalicylic acid. GSH levels were determined by the glutathione reductase procedure (13). Protein determinations were done by the method of Bradford (14). BSO treatment resulted in GSH levels of
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 289, No. 1, August 15, pp. 62-70, 1991
Inhibition of Oxygen-Dependent Radiation-Induced Damage by the Nitroxide Superoxide Dismutase Mimic, Tempo1 James B. Mitchell,’ William DeGraff, Dwight Kaufman, Murali C. Krishna, Amram Eli Finkelsteiqt Min S. Ahn, Stephen M. Hahn, Janet Gamson, and Angelo Russo
Samuni,*
Radiobiology Section, Radiation Oncology Branch, Clinical Oncology Program, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892; *Molecular Biology, School of Medicine, Hebrew Uniuersity, Jerusalem, Israel 91010; and TRadiation Oncology Department, Elizabeth General Medical Center, Elizabeth, New Jersey 07202
Received January 31,1991, and in revised form April 18, 1991
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 IIIM Tempo1 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 Tempo1 afforded no protection. Tempo1 treatment under hypoxic conditions did not provide radioprotection. Aerobic X-ray protection by Tempo1 could not be attributed to the induction of intracellular hypoxia, increase in intracellular glutathione, or induction of intracellular SOD mRNA. Tempo1 thus represents a new class of non-thiol-containing radiation protectors, which may be useful in elucidating the mechanism(s) of radiationinduced cellular damage and may have broad applications 0 1991 Academic in protecting against oxidative stress. Press,
Inc.
The deleterious effects of ionizing radiation include cytotoxicity, mutagenesis, and carcinogenesis. The discovery i To whom correspondence should be addressed at Radiation Oncology Branch, National Cancer Institute, NIH, Bldg. 10, Room B3-B69, Bethesda, MD, 20892. ’ Abbreviations used: SOD, superoxide dismutase; DF, desferrioxamine; Tempol, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl; DTPA, diethylenetriaminepentaacedic acid; BSO, L-buthionine sulfoximine; Tempol-H, Tempol-hydroxylamine; RT, room temperature; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate.
in the late 1940s that sulfhydryls (thiols), such as cysteine and cysteamine, provide radiation protection to animals ushered in an era of research aimed at elucidating the mechanism(s) of radiation-induced cytotoxicity and development of more effective protective agents (1, 2). Radiation damages biomolecules, in large part (approximately 80%), through its interaction with water to produce free radicals (H’, OH, e&) and HzOz, or through interaction with oxygen to produce the superoxide anion (‘0;). Thiols, which function by donation of a hydrogen atom to damaged molecules or by “scavenging” radiationproduced free radicals, afford significant protection to animals against whole body radiation (1); however, their use is limited by toxicity. Despite extensive testing and synthetic efforts, no thiol-based radioprotector has been found to be significantly better than cysteamine. Stable nitroxide radicals are useful as NMR imaging contrast agents, EPR spin labels, and pH or oximetry cellular probes (3-7). As part of our studies investigating the chemical and biochemical characteristics of nitroxide radicals, we recently found that they exhibit superoxide dismutase mimetic activity (89) and are capable of protecting mammalian cells from damage by superoxide generated by hypoxanthine/xanthine oxidase and by hydrogen peroxide (10). These findings encouraged us to test if stable nitroxide radicals could protect mammalian cells against ionizing radiation, since approximately 80% of the damaging effects of ionizing radiation is thought to occur through indirect damage caused by radiolysis products of water or through oxygen reacting with radiationinduced carbon-centered free radicals (11). We have found that Tempo1 does protect against ionizing radiation and is the prototypical radiation protector of this group of chemical compounds and, moreover, may provide insight
62 All
0003.9861/91 $3.00 Copyright 0 1991 by Academic Press, Inc. rights of reproduction in any form reserved.
NITROXIDE
into the fundamental cellular damage. EXPERIMENTAL
mechanism(s)
SUPEROXIDE
of radiation
induced
PROCEDURES
Chemicals. Desferrioxamine I(DF) was a gift from Ciba Geigy; 4hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempo]), diethylenetriaminepentaacetic acid (DTPA), and platinum(IV) oxide were purchased from Aldrich Chemical Co. and trioxalato-chromium(II1) KS[Cr(Cz0&]‘3Hz0 (CrOx) from l?faltz and Bauer. Contamination of K3[Cr(C20&]‘3H20 with K&O., necessitated recrystallization twice; concentration was determined spectrophotometrically. Superoxide dismutase (SOD) was purchased from Sigma Chemical Co. and L-buthionine sulfoximine (BSO) was purchased from Schweizerhall, Inc. (South Plainfield, NJ). Oxygen-dependent reoridation of Tempo&hydroxylamine. A 5-ml solution of 50 mM phosphate bufFer, pH 7.8, containing 100 PM DTPA and 100 mM Tempo1 was catalytically reduced [platinum oxide (IV)] by bubbling hydrogen gas through the solution for 2 h. The course of the reduction was followed by removing small aliquots (200 ~1) and observing the loss of the Tempo1 EPR signal. To test if the catalytic reduction of Tempo1 had only proceeded to the one-electron reduction product (Tempo]-hydroxylamine), the nitroxide signal resulting from treatment with ferricyanide (which does not oxidize the amine) was measured and found to be equal to the initial signal before reduction; therefore, only hydroxylamine had been formed. After the reduction had proceeded to greater than 99.9% completion, the platinum catalyst was removed by filtration, and the resulting Tempo]-hydroxylamine (Tempo]-H) was diluted to a final concentration of 1 mM. The rate of oxygen-dependent reoxidation of Tempo]-H (1 mM) in F12 medium at pH 7.3 in a gaspermeable teflon tube positioned in the EPR cavity was then determined. Oxidation of the hydroxylamine was followed while either argon or air was passed across the reaction tube. The rate of oxidation of TempolH in room air was 0.2 rc.M/min. For large-scale preparations, the reduction was essentially as described above except that Tempo1 (5 g; 30 mmol) was dissolved in 75 ml of methanol containing platinum oxide (250 mg). The product after filtration was isolated by flash evaporation and the white residue was stored in the dark under argon at -20°C. The product was characterized by NMR, mass spectroscopy, and ECPR. The compound was 99.8% pure, the impurity being the starting nitroxide. Cell culture. Chinese hamster V79 cells were grown in F12 medium supplemented with 10% fetal calf serum, penicillin, and streptomycin. Survival was assessed in all stud& by the clonogenic assay. The plating efficiency ranged between 80 and 90%. Stock cultures of exponentially growing cells were trypsinized, rinsed, and plated (5 X lo5 cells/dish) into a number of loo-mm petri dishes and incubated 16 h at 37°C prior to experimental protocols. Tempo1 was added to exponentially growing cells in complete F12 medium (final concentrations of 5, 10, 50, or 100 mM) at room temperature (RT) 1.0 min prior to X-irradiation. TempolH was also evaluated under the same conditions at 100 mM. The time required for irradiation (at RT) was approximately 10 min. Immediately after X-ray treatment, cells were rinsed, trypsinized, counted, and plated for macroscopic colony formation. Under these conditions, Tempo1 exerted no cytotoxicity at 5 or 10 mM; however, for 50 and 100 mM treatments there was a reduction in plating efficiency by 20-30%. Radiation survival estimates were corrected to account for Tempo1 cytotoxicity. Tempo]-H exhibited no cytotoxicity. Each dose determination was plated in triplicate, and experiments wlere repeated a minimum of two times. Plates were incubated 7 days; colonies were then fixed with methanol/ acetic acid (3:l) and stained with crystal violet. Colonies containing >50 cells were scored. Error bars shown in the figures represent SE of the mean and are shown when larger than the symbol. Protection factors were calculated by determining the ratio of doses of Tempo]-treated cells to those of control cells at the 10% survival level.
DISMUTASE
MIMIC,
TEMPOL
63
Some studies involved the treatment of cells with Tempo1 and radiation at 4’C. For these studies the medium was removed from the plates and replaced with fresh, chilled (4°C) medium containing 100 mM Tempal; the plates were then placed on top of an ice-bath tray. After a lomin exposure to Tempo], cell cultures were irradiated and processed as described above. Treatment at 4°C with or without Tempo1 did not alter the control plating efficiency. The radiation response of V79 cells exposed to SOD or DF was also assessed. SOD was added (final concentration of 100 pg/ml) to cell cultures 10 min prior to irradiation and DF (final concentration of 500 PM) was added 2 h prior to irradiation at RT. Neither SOD nor DF exerted cytotoxicity alone. Following irradiation cells were plated as described above. Hypoxic irradiation studies were performed by plating cells (2.5 X 106) into specially designed glass flasks (12) and incubating at 37°C overnight. Tempo1 was added to a final concentration of 100 mM and the flasks were sealed with soft rubber stoppers. Needles (19-gauge) were pushed through the rubber stopper to provide entrance and exit ports for a humidified gas mixture of 95% nitrogen/5% COP (Matheson Gas Products). Stoppered flasks were connected in series and mounted on a reciprocating platform and gassed at 37°C for 45 min. The gassing procedure resulted in an equilibrium between the gas and liquid phase and yielded oxygen concentrations in the effluent gas phase of 110 ppm as measured by a Thermox probe (12). After 45 min of deoxygenating, flasks were irradiated and cell survival was assessed as described above. Glutathione depletion was accomplished by exposing exponentially growing cells to 5 mM BSO for 6 h prior to radiation exposure. Control and BSO-treated cells were trypsinized, rinsed twice with phosphatebuffered saline (PBS), and suspended in cold 0.6% sulfosalicylic acid. GSH levels were determined by the glutathione reductase procedure (13). Protein determinations were done by the method of Bradford (14). BSO treatment resulted in GSH levels of
