Nitroxide radicals are paramagnetic contrast agents, used in magnetic resonance imaging (MRI), that also exert antioxidant effects. Participating in cellular redox reactions, they lose their ability to provide contrast as a function of time after administration. In this study, the rate of contrast loss was correlated to the reducing power of the tissue or the ''redox status.'' The preferential reduction of nitroxides in tumors compared with normal tissue was observed by MRI. The influence of the structure of the nitroxide on the reduction rate was investigated by MRI using two cell-permeable nitroxides, 4-hydroxy-2,2,6,6,-tetramethyl-1-piperidynyloxyl (Tempol) and 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (3CP), and one cell-impermeable nitroxide, 3-carboxy-2, 2,5,5,5-tetramethylpyrrolidine-1-oxyl (3CxP). Pharmacokinetic images of these nitroxides in normal tissue, tumor, kidney, and artery regions in mice were simultaneously obtained using MRI. The decay of Tempol and 3CP in tumor tissue was significantly faster than in normal tissue. No significant change in the total nitroxide (oxidized + reduced forms) was noted from tissue extracts, suggesting that the loss in contrast as a function of time is a result of intracellular bioreduction. However, in the case of 3CxP (membrane impermeable), there was no difference in the reduction rates between normal and tumor tissue. The time course of T 1 enhancement by 3CxP and the total amount of 3CxP (oxidized + reduced) in the femoral region showed similar pharmacokinetics. These results show that the differential bioreduction of cell-permeable nitroxides in tumor and normal tissue is supported by intracellular processes and the reduction rates are a means by which the intracellular redox status can be assessed noninvasively.

Purpose: There is considerable research directed toward the identification and development of functional contrast agents for medical imaging that superimpose tissue biochemical/molecular information with anatomical structures. Nitroxide radicals were identified as in vivo radioprotectors. Being paramagnetic, they can provide image contrast in magnetic resonance imaging (MRI) and electron paramagnetic resonance imaging (EPRI). The present study sought to determine the efficacy of nitroxide radioprotectors as functional image contrast agents. Experimental Design: Nitroxide radioprotectors, which act as contrast agents, were tested by EPRI and MRI to provide tissue redox status information noninvasively. Results: Phantom studies showed that the nitroxide, 3-carbamoyl-PROXYL (3CP), undergoes time-dependent reduction to the corresponding diamagnetic hydroxylamine only in the presence of reducing agents. The reduction rates of 3CP obtained by EPRI and MRI were in agreement suggesting the feasibility of using MRI to monitor nitroxide levels in tissues.The levels of 3CP were examined by EPRI and MRI for differences in reduction between muscle and tumor (squamous cell carcinoma) implanted in the hind leg of C3H mice simultaneously. In vivo experiments showed a T1-dependent image intensity enhancement afforded by 3CP which decreased in a time-dependent manner. Reduction of 3CP was found to be the dominant mechanism of contrast loss.The tumor regions exhibited a faster decay rate of the nitroxide compared to muscle (0.097 min -1 versus 0.067 min -1 , respectively). Conclusions: This study shows that MRI can be successfully used to co-register tissue redox status along with anatomic images, thus providing potentially valuable biochemical information from the region of interest. Magnetic resonance imaging (MRI) provides images withuseful spatial and temporal resolutions and aids in the diagnosis of pathologic conditions in soft tissue. In addition to detailed anatomic information from such scans, suitable contrast agents provide important functional information pertaining to blood flow, perfusion, etc. (1). Most contrast agents used for T 1 -contrast enhancement contain paramagnetic entities such as the Gd 3+ complexes and Mn 2+ complexes. More recently, superparamagnetic iron oxide particles are being used as T 2 * contrast agents in MRI especially in cell tracking (2). Nitroxide radicals are organic molecules that have a single unpaired electron and therefore have the potential to provide T 1 contrast similar to gadolinium complexes. Feasibility of nitroxide radicals as T 1 contrast agents in MRI has been examined (3 -5) before their use for in vivo EPR imaging as probes (6). However, nitroxide spin probes were reported to be not optimal as MRI contrast agents due to their rapid in vivo reduction to the corresponding diamagnetic products (7). In the living body, paramagnetic nitroxide radicals are chemically and/or enzymatically reduced to the diamagnetic hydroxylamine (8 -11). In addition to their biological inst...

The absolute partial pressure of oxygen (pO 2 ) in the mammary gland pad and femoral muscle of female mice was measured using EPR oximetry at 700 MHz. A small quantity of lithium phthalocyanine (LiPc) crystals was implanted in both mammary and femoral muscle tissue of female C3H mice. Subsequent EPR measurements were carried out 1-30 days after implantation with or without control of core body temperature. The pO 2 values in the tissue became stable 2 weeks after implantation of LiPc crystals. The pO 2 level was found to be higher in the femoral muscle than in the mammary tissue. However, the pO 2 values showed a strong dependence on the core body temperature of the mice. The pO 2 values were responsive to carbogen (95% O 2 , 5% CO 2 ) breathing even 44 -58 days after the implantation of LiPc. The LiPc linewidth was also sensitive to changes in the blood supply even 60 days after implantation of the crystals. This study further validates the use of LiPc crystals and EPR oximetry for long-term non-invasive assessment of pO 2 levels in tissues, underscores the importance of maintaining normal body core temperature during the measurements, and demonstrates that mammary tissue functions at a lower pO 2 level than muscle in female C3H mice.