nadeem khan | University of Delhi (original) (raw)

Papers by nadeem khan

Biochemistry, 2003

The effect of the cholesterol content of the plasma membrane on the intracellular concentration o... more The effect of the cholesterol content of the plasma membrane on the intracellular concentration of oxygen in Chinese hamster ovary (CHO) cells and their mutants was investigated by EPR oximetry. Total and free cholesterol content was significantly higher in 25 RA CHO cells as compared to wild-type and M 19 CHO cells, with most of the free cholesterol in normal and mutant CHO cells located in the plasma membrane. The plasma membrane cholesterol content also was altered by various biochemical means, and the effect on the oxygen gradient was studied. Comparing the three cell lines, the gradient was larger with increased content of cholesterol in the plasma cell membrane. This result also is supported by an additional increase in the oxygen gradients with the incorporation of additional cholesterol in the plasma membrane and a decrease in the oxygen gradient when the cholesterol was depleted from the plasma membrane. The results indicate that the concentration of cholesterol in the plasma membrane can be an important factor for the magnitude of the oxygen gradient observed across the cell membrane.

Free Radical Biology and Medicine, 2008

In this study, we determined the changes in the intracellular redox environment of the heart duri... more In this study, we determined the changes in the intracellular redox environment of the heart during ischemia and reperfusion and the effects of resveratrol on such changes. Because redox regulation by thioredoxin (Trx) plays a crucial role in signal transduction and cytoprotection against ROS, the effects of resveratrol on the changes in the amounts of thioredoxin were monitored in an attempt to determine the role of intracellular thioredoxin in resveratrol-mediated changes in intracellular redox environment and its role in resveratrol-mediated cardioprotection. Rats were randomly divided into four groups: group I, control (rats were gavaged with vehicle only); group II, rats were gavaged with 2.5 mg/kg body wt resveratrol per day for 10 days; group III, rats were given resveratrol for 10 days, but on the 7th day, they were treated with shRNA against Trx-1; group IV, rats were given resveratrol for 10 days, but were injected (iv) with cisplatin (1 mg/kg body wt) on days 1, 3, 5, 7, and 9. In concert, two groups of mice (Dn-Trx-1) and a corresponding wild-type group were also gavaged with 2.5 mg/kg body wt resveratrol for 10 days. After 10 days, isolated rat and mouse hearts perfused via working mode were made globally ischemic for 30 min followed by 2 h of reperfusion. Ischemia/reperfusion developed an infarct size of about 40% and resulted in about 25% apoptotic cardiomyocytes, which were reduced by resveratrol. Cisplatin, but not shRNA-Trx-1, abolished the cardioprotective abilities of resveratrol. In the experiments with mouse hearts, similar to rat hearts, resveratrol significantly reduced the ischemia/reperfusion-mediated increase in infarct size and apoptosis in both groups. MDA formation, a presumptive marker for lipid peroxidation, was increased in the I/R group and reduced in the resveratrol group, and resveratrol-mediated reduction in MDA formation was abolished with cisplatin, but not with shRNA-Trx-1. I/R-induced reduction in GSH/GSSH ratio was prevented by resveratrol, and resveratrol-mediated preservation of GSH/GSSG ratio was reduced by cisplatin, but not by sh-RNA-Trx-1. RT-PCR revealed an increase in both Trx-1 and Trx-2 transcripts; but only Trx-2 protein, not Trx-1 protein, was enhanced with resveratrol by Western blot analysis. Electron paramagnetic resonance spectroscopic study revealed that resveratrol treatment significantly increased the decay rates of nitroxide radicals compared to control hearts, suggesting that resveratrol can switch into the reduction state more compared to control heart. Finally, resveratrol generated a survival signal by phosphorylation of Akt and increase in induction of Bcl-2 expression, which was inhibited by cisplatin, but not by shRNA-Trx-1. Taken together, the results of this study indicate that resveratrol provides cardioprotection by maintaining intracellular redox environments, and Trx-2 is likely to play a role in switching I/R-induced death signal into survival signal.

Antioxidants & Redox Signaling, 2007

Tissue oxygen plays a crucial role in maintaining tissue viability and in various diseases, inclu... more Tissue oxygen plays a crucial role in maintaining tissue viability and in various diseases, including responses to therapy. Useful knowledge has been gained by methods that can give limited snapshots of tissue oxygen (e.g., oxygen electrodes) or evidence of a history of tissue hypoxia (e.g., EF5) or even indirect evidence by monitoring oxygen availability in the circulatory system (e.g., NMR methods). Each of these methods has advantages and significant limitations. EPR oximetry is a technique for direct measurement of tissue pO 2 , which has several advantages over the other existing methods for applications in which the parameter of interest is the pO 2 of tissues, and information is needed over a time course of minutes to hours, and/or for repetitive measurements over days or weeks or years. The aim of this article is to provide an overview of EPR oximetry using particulates to readers who are not familiar with this technique and its potential in vivo and clinical applications. The data presented here are from the experiments currently being carried out in our laboratory. We are confident that in vivo EPR oximetry will play a crucial role in the understanding and clinical management of various pathologies in the years to come.

Free Radical Biology and Medicine, 2003

We have evaluated the effects of DMPO, CMPO, EMPO, BMPO, and DEPMPO on functioning CHO cells and ... more We have evaluated the effects of DMPO, CMPO, EMPO, BMPO, and DEPMPO on functioning CHO cells and the stability of the radical adducts in the presence of cells. The potential toxic effects of the spin traps were measured by two estimates of cell viability (trypan blue exclusion and colony formation) and one of cell function (rate of oxygen consumption). We also studied the effects of the spin traps on colony formation in a second cell line, 9L tumor cells. Toxicity varied with the type of cell line and the parameter that was measured. In aqueous solutions the order of stability for all spin adducts was SO 3 Ͼ OH Ͼ CH 3 , while in cell suspensions it was SO 3 Ͼ OH Ϸ CH 3 . The radical adducts of the new spin traps have significantly increased stability as compared to DMPO. These results indicate that the new spin traps potentially offer increased stability of spin adducts in functioning cells. It also is clear that it is necessary to carry out appropriate studies of the stability and toxicity in the system that is to be studied for any particular use of these spin traps. It then should be feasible to select the spin trap(s) best suited for the proposed study.

Nmr in Biomedicine, 2004

The development and use of in vivo techniques for strictly experimental applications in animals h... more The development and use of in vivo techniques for strictly experimental applications in animals has been very successful, and these results now have made possible some very attractive potential clinical applications. The area with the most obvious immediate, effective and widespread clinical use is oximetry, where EPR almost uniquely can make repeated and accurate measurements of pO2 in tissues. Such measurements can provide clinicians with information that can impact directly on diagnosis and therapy, especially for oncology, peripheral vascular disease and wound healing. The other area of immediate and timely importance is the unique ability of in vivo EPR to measure clinically significant exposures to ionizing radiation ‘after-the-fact’, such as may occur due to accidents, terrorism or nuclear war. There are a number of other capabilities of in vivo EPR that also potentially could become extensively used in human subjects. In pharmacology the unique capabilities of in vivo EPR to detect and characterize free radicals could be applied to measure free radical intermediates from drugs and oxidative process. A closely related area of potential widespread applications is the use of EPR to measure nitric oxide. These often unique capabilities, combined with the sensitivity of EPR spectra to the immediate environment (e.g. pH, molecular motion, charge) have already resulted in some very productive applications in animals and these are likely to expand substantially in the near future. They should provide a continually developing base for extending clinical uses of in vivo EPR. The challenges for achieving full implementation include adapting the spectrometer for safe and comfortable measurements in human subjects, achieving sufficient sensitivity for measurements at the sites of the pathophysiological processes that are being measured, and establishing a consensus on the clinical value of the measurements. Copyright © 2004 John Wiley & Sons, Ltd.

International Journal of Radiation Oncology Biology Physics, 2004

Purpose: RSR13, an allosteric modifier of hemoglobin, reduces hemoglobin-oxygen binding affinity ... more Purpose: RSR13, an allosteric modifier of hemoglobin, reduces hemoglobin-oxygen binding affinity facilitating oxygen release from hemoglobin, resulting in increases in tissue pO 2 . The purpose of this study was noninvasively to monitor the time course and effect of RSR13 on tumor oxygenation, directly using in vivo electron paramagnetic resonance (EPR oximetry), and indirectly using blood oxygen level dependent magnetic resonance imaging (BOLD MRI). Methods and Materials: The study was performed in transplanted radiation-induced fibrosarcoma tumors (RIF-1) in 18 female C3H/HEJ mice, which had two lithium phthalocyanine (LiPc) deposits implanted in the tumor when the tumors reached about 200 -600 mm 3 . Baseline EPR measurements were made daily for 3 days. Then, for 6 consecutive days and after an initial baseline EPR measurement, RSR13 (150 mg/kg) or vehicle (same volume) was injected intraperitoneally, and measurements of intratumoral oxygen were made at 10-min intervals for the next 60 min. In each mouse, every third day, instead of EPR oximetry, BOLD MRI measurements were made for 60 min after administration of the RSR13. Results: Based on EPR measurements, RSR13 produced statistically significant temporal increases in tumor pO 2 over the 60-min time course, which reached a maximum at 35-43 min postdose. The average time required to return to the baseline pO 2 was 70 -85 min. The maximum increase in tumor tissue pO 2 values after RSR13 treatment from Day 1 to Day 5 (8.3-12.4 mm Hg) was greater than the maximum tumor tissue pO 2 value for Day 6 (4.7 mm Hg, p < 0.01). The maximum increase in pO 2 occurred on Day 2 (12.4 mm Hg) after RSR13 treatment. There was little change in R 2 *, indicating that the RSR13 had minimal detectable effects on total deoxyhemoglobin and hemoglobin-oxygen saturation. Conclusion: The extent of the increase in tumor pO 2 achieved by RSR13 would be expected to lead to a significant increase in the effectiveness of tumor radiotherapy. The lack of a change in the BOLD MRI signal suggests that the tumor physiology was largely unchanged by RSR13. These results illustrate a unique and useful capability of in vivo EPR oximetry and BOLD MRI to obtain repeated measurements of tumor oxygenation and physiology. The dynamics of tumor pO 2 after RSR13 administration may be useful for the design of clinical protocols using allosteric hemoglobin effectors.

Antioxidants & Redox Signaling, 2007

The aim of this article is to provide an overview of how electron paramagnetic resonance (EPR) ca... more The aim of this article is to provide an overview of how electron paramagnetic resonance (EPR) can be used to measure redox-related parameters in vivo. The values of this approach include that the measurements are made under fully physiological conditions, and some of the measurements cannot be made by other means. Three complementary approaches are used with in vivo EPR: the rate of reduction or reactions of nitroxides, spin trapping of free radicals, and measurements of thiols. All three approaches already have produced unique and useful information. The measurement of the rate of decrease of nitroxides technically is the simplest, but difficult to interpret because the measured parameter, reduction in the intensity of the nitroxide signal, can occur by several different mechanisms. In vivo spin trapping can provide direct evidence for the occurrence of specific free radicals in vivo and reflect relative changes, but accurate absolute quantification remains challenging. The measurement of thiols in vivo also appears likely to be useful, but its development as an in vivo technique is at an early stage. It seems likely that the use of in vivo EPR to measure redox processes will become an increasingly utilized and valuable tool.

Biophysical Journal, 2003

Respiratory deficient cell lines are being increasingly used to elucidate the role of mitochondri... more Respiratory deficient cell lines are being increasingly used to elucidate the role of mitochondria and to understand the pathophysiology of mitochondrial genetic disease. We have investigated the oxygen consumption rates and oxygen concentration in wild-type (WT) and mitochondrial DNA (mtDNA) depleted (r 0 ) Molt-4 cells. Wild-type Molt-4 cells have moderate oxygen consumption rates, which were significantly reduced in the r 0 cells. PCMB (p-chloromercurobenzoate) inhibited the oxygen consumption rates in both WT and r 0 cells, whereas potassium cyanide decreased the oxygen consumption rates only in WT Molt-4 cells. Menadione sodium bisulfite (MSB) increased the oxygen consumption rates in both cell lines, whereas CCCP (carbonyl cyanide m-chlorophenylhydrazone) stimulated the oxygen consumption rates only in WT Molt-4 cells. Superoxide radical adducts were observed in both WT and r 0 cells when stimulated with MSB. The formation of this adduct was inhibited by PCMB but not by potassium cyanide. These results suggest that the reactive oxygen species (ROS) induced by MSB were at least in part produced via a mitochondrial independent pathway. An oxygen gradient between the extra-and intracellular compartments was observed in WT Molt-4 cells, which further increased when cells were stimulated by CCCP and MSB. The results are consistent with our earlier findings suggesting that such oxygen gradients may be a general phenomenon found in most or all cell systems under appropriate conditions.

International Journal of Radiation Oncology Biology Physics, 2005

To determine quantitatively the changes in oxygenation of intracranial tumors induced by efaproxi... more To determine quantitatively the changes in oxygenation of intracranial tumors induced by efaproxiral, an allosteric hemoglobin modifier. Efaproxiral reduces hemoglobin-oxygen binding affinity, which facilitates oxygen release from hemoglobin into surrounding tissues and potentially increases the pO(2) of the tumors. The study was performed on 10 male Fisher 344 rats with 9L intracranial tumors. Electron paramagnetic resonance (EPR) oximetry was used to measure quantitatively the changes in the pO(2) in the tumors. Lithium phthalocyanine (LiPc) crystals were implanted in the tumors and in the normal brain tissue in the opposite hemispheres. We monitored the cerebral pO(2) starting 7 to 10 days after the tumor cells were implanted. NMR imaging determined the position and size of tumor in the brain. After an initial baseline EPR measurement, efaproxiral (150 mg/kg) was injected intravenously over 15 minutes, and measurements of tumor and normal brain oxygen tension were made alternately at 10-minute intervals for the next 60 minutes; the procedure was repeated for 6 consecutive days. Efaproxiral significantly increased the pO(2) of both the intracranial tumors and the normal brain tissue on all days. The maximum increase was reached at 52.9 to 59.7 minutes and 54.1 to 63.2 minutes after injection, respectively. The pO(2) returned to baseline values at 106 to 126.5 minutes after treatment. The maximum tumor and normal tissue pO(2) values achieved after efaproxiral treatment from Day 1 through Day 6 ranged from 139.7 to 197.7 mm Hg and 103.0 to 135.9 mm Hg, respectively. The maximum increase in tumor tissue pO(2) values from Day 2 to Day 5 was greater than the maximum increase in normal tissue pO(2). We obtained quantitative data on the timing and extent of efaproxiral-induced changes in the pO(2) of intracerebral 9L tumors. These results illustrate a unique and useful capability of in vivo EPR oximetry to obtain repeated noninvasive measurements of tumor oxygenation over a number of days. The information on the dynamics of tumor pO(2) after efaproxiral administration illustrates the ability of efaproxiral to increase intracranial tumor oxygenation.

Biochemistry, 2003

The effect of the cholesterol content of the plasma membrane on the intracellular concentration o... more The effect of the cholesterol content of the plasma membrane on the intracellular concentration of oxygen in Chinese hamster ovary (CHO) cells and their mutants was investigated by EPR oximetry. Total and free cholesterol content was significantly higher in 25 RA CHO cells as compared to wild-type and M 19 CHO cells, with most of the free cholesterol in normal and mutant CHO cells located in the plasma membrane. The plasma membrane cholesterol content also was altered by various biochemical means, and the effect on the oxygen gradient was studied. Comparing the three cell lines, the gradient was larger with increased content of cholesterol in the plasma cell membrane. This result also is supported by an additional increase in the oxygen gradients with the incorporation of additional cholesterol in the plasma membrane and a decrease in the oxygen gradient when the cholesterol was depleted from the plasma membrane. The results indicate that the concentration of cholesterol in the plasma membrane can be an important factor for the magnitude of the oxygen gradient observed across the cell membrane.

Free Radical Biology and Medicine, 2008

In this study, we determined the changes in the intracellular redox environment of the heart duri... more In this study, we determined the changes in the intracellular redox environment of the heart during ischemia and reperfusion and the effects of resveratrol on such changes. Because redox regulation by thioredoxin (Trx) plays a crucial role in signal transduction and cytoprotection against ROS, the effects of resveratrol on the changes in the amounts of thioredoxin were monitored in an attempt to determine the role of intracellular thioredoxin in resveratrol-mediated changes in intracellular redox environment and its role in resveratrol-mediated cardioprotection. Rats were randomly divided into four groups: group I, control (rats were gavaged with vehicle only); group II, rats were gavaged with 2.5 mg/kg body wt resveratrol per day for 10 days; group III, rats were given resveratrol for 10 days, but on the 7th day, they were treated with shRNA against Trx-1; group IV, rats were given resveratrol for 10 days, but were injected (iv) with cisplatin (1 mg/kg body wt) on days 1, 3, 5, 7, and 9. In concert, two groups of mice (Dn-Trx-1) and a corresponding wild-type group were also gavaged with 2.5 mg/kg body wt resveratrol for 10 days. After 10 days, isolated rat and mouse hearts perfused via working mode were made globally ischemic for 30 min followed by 2 h of reperfusion. Ischemia/reperfusion developed an infarct size of about 40% and resulted in about 25% apoptotic cardiomyocytes, which were reduced by resveratrol. Cisplatin, but not shRNA-Trx-1, abolished the cardioprotective abilities of resveratrol. In the experiments with mouse hearts, similar to rat hearts, resveratrol significantly reduced the ischemia/reperfusion-mediated increase in infarct size and apoptosis in both groups. MDA formation, a presumptive marker for lipid peroxidation, was increased in the I/R group and reduced in the resveratrol group, and resveratrol-mediated reduction in MDA formation was abolished with cisplatin, but not with shRNA-Trx-1. I/R-induced reduction in GSH/GSSH ratio was prevented by resveratrol, and resveratrol-mediated preservation of GSH/GSSG ratio was reduced by cisplatin, but not by sh-RNA-Trx-1. RT-PCR revealed an increase in both Trx-1 and Trx-2 transcripts; but only Trx-2 protein, not Trx-1 protein, was enhanced with resveratrol by Western blot analysis. Electron paramagnetic resonance spectroscopic study revealed that resveratrol treatment significantly increased the decay rates of nitroxide radicals compared to control hearts, suggesting that resveratrol can switch into the reduction state more compared to control heart. Finally, resveratrol generated a survival signal by phosphorylation of Akt and increase in induction of Bcl-2 expression, which was inhibited by cisplatin, but not by shRNA-Trx-1. Taken together, the results of this study indicate that resveratrol provides cardioprotection by maintaining intracellular redox environments, and Trx-2 is likely to play a role in switching I/R-induced death signal into survival signal.

Antioxidants & Redox Signaling, 2007

Tissue oxygen plays a crucial role in maintaining tissue viability and in various diseases, inclu... more Tissue oxygen plays a crucial role in maintaining tissue viability and in various diseases, including responses to therapy. Useful knowledge has been gained by methods that can give limited snapshots of tissue oxygen (e.g., oxygen electrodes) or evidence of a history of tissue hypoxia (e.g., EF5) or even indirect evidence by monitoring oxygen availability in the circulatory system (e.g., NMR methods). Each of these methods has advantages and significant limitations. EPR oximetry is a technique for direct measurement of tissue pO 2 , which has several advantages over the other existing methods for applications in which the parameter of interest is the pO 2 of tissues, and information is needed over a time course of minutes to hours, and/or for repetitive measurements over days or weeks or years. The aim of this article is to provide an overview of EPR oximetry using particulates to readers who are not familiar with this technique and its potential in vivo and clinical applications. The data presented here are from the experiments currently being carried out in our laboratory. We are confident that in vivo EPR oximetry will play a crucial role in the understanding and clinical management of various pathologies in the years to come.

Free Radical Biology and Medicine, 2003

We have evaluated the effects of DMPO, CMPO, EMPO, BMPO, and DEPMPO on functioning CHO cells and ... more We have evaluated the effects of DMPO, CMPO, EMPO, BMPO, and DEPMPO on functioning CHO cells and the stability of the radical adducts in the presence of cells. The potential toxic effects of the spin traps were measured by two estimates of cell viability (trypan blue exclusion and colony formation) and one of cell function (rate of oxygen consumption). We also studied the effects of the spin traps on colony formation in a second cell line, 9L tumor cells. Toxicity varied with the type of cell line and the parameter that was measured. In aqueous solutions the order of stability for all spin adducts was SO 3 Ͼ OH Ͼ CH 3 , while in cell suspensions it was SO 3 Ͼ OH Ϸ CH 3 . The radical adducts of the new spin traps have significantly increased stability as compared to DMPO. These results indicate that the new spin traps potentially offer increased stability of spin adducts in functioning cells. It also is clear that it is necessary to carry out appropriate studies of the stability and toxicity in the system that is to be studied for any particular use of these spin traps. It then should be feasible to select the spin trap(s) best suited for the proposed study.

Nmr in Biomedicine, 2004

The development and use of in vivo techniques for strictly experimental applications in animals h... more The development and use of in vivo techniques for strictly experimental applications in animals has been very successful, and these results now have made possible some very attractive potential clinical applications. The area with the most obvious immediate, effective and widespread clinical use is oximetry, where EPR almost uniquely can make repeated and accurate measurements of pO2 in tissues. Such measurements can provide clinicians with information that can impact directly on diagnosis and therapy, especially for oncology, peripheral vascular disease and wound healing. The other area of immediate and timely importance is the unique ability of in vivo EPR to measure clinically significant exposures to ionizing radiation ‘after-the-fact’, such as may occur due to accidents, terrorism or nuclear war. There are a number of other capabilities of in vivo EPR that also potentially could become extensively used in human subjects. In pharmacology the unique capabilities of in vivo EPR to detect and characterize free radicals could be applied to measure free radical intermediates from drugs and oxidative process. A closely related area of potential widespread applications is the use of EPR to measure nitric oxide. These often unique capabilities, combined with the sensitivity of EPR spectra to the immediate environment (e.g. pH, molecular motion, charge) have already resulted in some very productive applications in animals and these are likely to expand substantially in the near future. They should provide a continually developing base for extending clinical uses of in vivo EPR. The challenges for achieving full implementation include adapting the spectrometer for safe and comfortable measurements in human subjects, achieving sufficient sensitivity for measurements at the sites of the pathophysiological processes that are being measured, and establishing a consensus on the clinical value of the measurements. Copyright © 2004 John Wiley & Sons, Ltd.

International Journal of Radiation Oncology Biology Physics, 2004

Purpose: RSR13, an allosteric modifier of hemoglobin, reduces hemoglobin-oxygen binding affinity ... more Purpose: RSR13, an allosteric modifier of hemoglobin, reduces hemoglobin-oxygen binding affinity facilitating oxygen release from hemoglobin, resulting in increases in tissue pO 2 . The purpose of this study was noninvasively to monitor the time course and effect of RSR13 on tumor oxygenation, directly using in vivo electron paramagnetic resonance (EPR oximetry), and indirectly using blood oxygen level dependent magnetic resonance imaging (BOLD MRI). Methods and Materials: The study was performed in transplanted radiation-induced fibrosarcoma tumors (RIF-1) in 18 female C3H/HEJ mice, which had two lithium phthalocyanine (LiPc) deposits implanted in the tumor when the tumors reached about 200 -600 mm 3 . Baseline EPR measurements were made daily for 3 days. Then, for 6 consecutive days and after an initial baseline EPR measurement, RSR13 (150 mg/kg) or vehicle (same volume) was injected intraperitoneally, and measurements of intratumoral oxygen were made at 10-min intervals for the next 60 min. In each mouse, every third day, instead of EPR oximetry, BOLD MRI measurements were made for 60 min after administration of the RSR13. Results: Based on EPR measurements, RSR13 produced statistically significant temporal increases in tumor pO 2 over the 60-min time course, which reached a maximum at 35-43 min postdose. The average time required to return to the baseline pO 2 was 70 -85 min. The maximum increase in tumor tissue pO 2 values after RSR13 treatment from Day 1 to Day 5 (8.3-12.4 mm Hg) was greater than the maximum tumor tissue pO 2 value for Day 6 (4.7 mm Hg, p < 0.01). The maximum increase in pO 2 occurred on Day 2 (12.4 mm Hg) after RSR13 treatment. There was little change in R 2 *, indicating that the RSR13 had minimal detectable effects on total deoxyhemoglobin and hemoglobin-oxygen saturation. Conclusion: The extent of the increase in tumor pO 2 achieved by RSR13 would be expected to lead to a significant increase in the effectiveness of tumor radiotherapy. The lack of a change in the BOLD MRI signal suggests that the tumor physiology was largely unchanged by RSR13. These results illustrate a unique and useful capability of in vivo EPR oximetry and BOLD MRI to obtain repeated measurements of tumor oxygenation and physiology. The dynamics of tumor pO 2 after RSR13 administration may be useful for the design of clinical protocols using allosteric hemoglobin effectors.

Antioxidants & Redox Signaling, 2007

The aim of this article is to provide an overview of how electron paramagnetic resonance (EPR) ca... more The aim of this article is to provide an overview of how electron paramagnetic resonance (EPR) can be used to measure redox-related parameters in vivo. The values of this approach include that the measurements are made under fully physiological conditions, and some of the measurements cannot be made by other means. Three complementary approaches are used with in vivo EPR: the rate of reduction or reactions of nitroxides, spin trapping of free radicals, and measurements of thiols. All three approaches already have produced unique and useful information. The measurement of the rate of decrease of nitroxides technically is the simplest, but difficult to interpret because the measured parameter, reduction in the intensity of the nitroxide signal, can occur by several different mechanisms. In vivo spin trapping can provide direct evidence for the occurrence of specific free radicals in vivo and reflect relative changes, but accurate absolute quantification remains challenging. The measurement of thiols in vivo also appears likely to be useful, but its development as an in vivo technique is at an early stage. It seems likely that the use of in vivo EPR to measure redox processes will become an increasingly utilized and valuable tool.

Biophysical Journal, 2003

Respiratory deficient cell lines are being increasingly used to elucidate the role of mitochondri... more Respiratory deficient cell lines are being increasingly used to elucidate the role of mitochondria and to understand the pathophysiology of mitochondrial genetic disease. We have investigated the oxygen consumption rates and oxygen concentration in wild-type (WT) and mitochondrial DNA (mtDNA) depleted (r 0 ) Molt-4 cells. Wild-type Molt-4 cells have moderate oxygen consumption rates, which were significantly reduced in the r 0 cells. PCMB (p-chloromercurobenzoate) inhibited the oxygen consumption rates in both WT and r 0 cells, whereas potassium cyanide decreased the oxygen consumption rates only in WT Molt-4 cells. Menadione sodium bisulfite (MSB) increased the oxygen consumption rates in both cell lines, whereas CCCP (carbonyl cyanide m-chlorophenylhydrazone) stimulated the oxygen consumption rates only in WT Molt-4 cells. Superoxide radical adducts were observed in both WT and r 0 cells when stimulated with MSB. The formation of this adduct was inhibited by PCMB but not by potassium cyanide. These results suggest that the reactive oxygen species (ROS) induced by MSB were at least in part produced via a mitochondrial independent pathway. An oxygen gradient between the extra-and intracellular compartments was observed in WT Molt-4 cells, which further increased when cells were stimulated by CCCP and MSB. The results are consistent with our earlier findings suggesting that such oxygen gradients may be a general phenomenon found in most or all cell systems under appropriate conditions.

International Journal of Radiation Oncology Biology Physics, 2005

To determine quantitatively the changes in oxygenation of intracranial tumors induced by efaproxi... more To determine quantitatively the changes in oxygenation of intracranial tumors induced by efaproxiral, an allosteric hemoglobin modifier. Efaproxiral reduces hemoglobin-oxygen binding affinity, which facilitates oxygen release from hemoglobin into surrounding tissues and potentially increases the pO(2) of the tumors. The study was performed on 10 male Fisher 344 rats with 9L intracranial tumors. Electron paramagnetic resonance (EPR) oximetry was used to measure quantitatively the changes in the pO(2) in the tumors. Lithium phthalocyanine (LiPc) crystals were implanted in the tumors and in the normal brain tissue in the opposite hemispheres. We monitored the cerebral pO(2) starting 7 to 10 days after the tumor cells were implanted. NMR imaging determined the position and size of tumor in the brain. After an initial baseline EPR measurement, efaproxiral (150 mg/kg) was injected intravenously over 15 minutes, and measurements of tumor and normal brain oxygen tension were made alternately at 10-minute intervals for the next 60 minutes; the procedure was repeated for 6 consecutive days. Efaproxiral significantly increased the pO(2) of both the intracranial tumors and the normal brain tissue on all days. The maximum increase was reached at 52.9 to 59.7 minutes and 54.1 to 63.2 minutes after injection, respectively. The pO(2) returned to baseline values at 106 to 126.5 minutes after treatment. The maximum tumor and normal tissue pO(2) values achieved after efaproxiral treatment from Day 1 through Day 6 ranged from 139.7 to 197.7 mm Hg and 103.0 to 135.9 mm Hg, respectively. The maximum increase in tumor tissue pO(2) values from Day 2 to Day 5 was greater than the maximum increase in normal tissue pO(2). We obtained quantitative data on the timing and extent of efaproxiral-induced changes in the pO(2) of intracerebral 9L tumors. These results illustrate a unique and useful capability of in vivo EPR oximetry to obtain repeated noninvasive measurements of tumor oxygenation over a number of days. The information on the dynamics of tumor pO(2) after efaproxiral administration illustrates the ability of efaproxiral to increase intracranial tumor oxygenation.