This work presents a methodology for obtaining quantitative oxygen concentration images in the tumor-bearing legs of living C3H mice. The method uses high-resolution electron paramagnetic resonance imaging (EPRI). Enabling aspects of the methodology include the use of injectable, narrow, single-line triaryl methyl spin probes and an accurate model of overmodulated spectra. Both of these increase the signal-to-noise ratio (SNR), resulting in high resolution in space (1 mm) 3 and oxygen concentrations (ϳ3 torr). Thresholding at 15% the maximum spectral amplitude gives leg/tumor shapes that reproduce those in photographs. The EPRI appears to give reasonable oxygen partial pressures, showing hypoxia (ϳ0 -6 torr, 0 -10 3 Pa) in many of the tumor voxels. EPRI was able to detect statistically significant changes in oxygen concentrations in the tumor with administration of carbogen, although the changes were not in- The central role of oxygen in virtually all life processes as the ultimate oxidative substrate for metabolism is well known (1). Oxygenation has a crucial effect on the malignant state (2). Lack of oxygen in a tissue (hypoxia) appears to predispose its surviving cells to mutagenesis, thereby increasing the likelihood that a malignant state will develop (3). Hypoxia affects, most often detrimentally, treatment with conventional anticancer therapies (4). In particular, radiation has been known for nearly a century to be potentiated by oxygen and inhibited by hypoxia (5).Electron paramagnetic resonance imaging (EPRI) can provide a quantitative image of the oxygen concentrations in tissues and tumors of living animals (6,7). The image derives from the EPR spectrum of the unpaired electron from a stable injected spin probe. Oxygen is measured in the distributional compartment of the spin probe. The EPR linewidth is a direct measure of the frequency with which the spin probe encounters molecular oxygen, and is directly proportional to the oxygen concentration (8). One great advantage to imaging the EPR linewidth (and not the line height) is the desensitization to other aspects of the animal or tissue physiology, such as the vasculature. The spectral line height (but not the linewidth) depends on the effectiveness of the delivery of the spin probe to a voxel. Within broad limits, the line height depends on the operating conditions of the imager and the complicated RF distributions in an animal, whereas the linewidth does not.The approach described herein differs from that taken by other groups pursuing in vivo EPRI. Spectral-spatial imaging and in vivo spectral-spatial imaging have been described previously (9,10). In vivo spectral-spatial EPRI for small animals has also been discussed by us and other researchers (6,(11)(12)(13)(14)(15). The present work takes spectralspatial imaging to its logical conclusion: obtaining a full spectrum from each voxel and fitting that spectrum to an accurate spectral shape function with adjustable spectral parameters. These spectral parameters contain the physiologic information fr...

Tumor oxygenation predicts cancer therapy response and malignant phenotype. This has spawned a number of oxymetries. Comparison of different oxymetries is crucial for the validation and understanding of these techniques. Electron paramagnetic resonance (EPR) imaging is a novel technique for providing quantitative high-resolution images of tumor and tissue oxygenation. This work compares sequences of tumor pO 2 values from EPR oxygen images with sequences of oxygen measurements made along a track with an Oxylite oxygen probe. Four-dimensional (three spatial and one spectral) EPR oxygen images used spectroscopic imaging techniques to measure the width of a spectral line in each image voxel from a trityl spin probe (OX063, Amersham Health R&D) in the tissues and tumor of mice after spin probe injection. A simple calibration allows direct, quantitative translation of each line width to an oxygen concentration. These four-dimensional EPR images, obtained in 45 minutes from FSa fibrosarcomas grown in the legs of C3H mice, have a spatial resolution of f1mm and oxygen resolution of f3 Torr. The position of the Oxylite track was measured within a 2-mm accuracy using a custom stereotactic positioning device. A total of nine images that involve 17 tracks were obtained. Of these, most showed good correlation between the Oxylite measured pO 2 and a track located in the tumor within the uncertainties of the Oxylite localizability. The correlation was good both in terms of spatial distribution pattern and pO 2 magnitude. The strong correlation of the two modalities corroborates EPR imaging as a useful tool for the study of tumor oxygenation.

Blood flow and pO2 changes after vascular-targeted photodynamic therapy (V-PDT) or cellular-targeted PDT (C-PDT) using 5,10,15,20-tetrakis(2,6-difluoro-3-N-methylsulfamoylphenyl) bacteriochlorin (F2BMet) as photosensitizer were investigated in DBA/2 mice with S91 Cloudman mouse melanoma, and correlated with long-term tumor responses. F2BMet generates both singlet oxygen and hydroxyl radicals under near-infrared radiation, which consume oxygen. Partial oxygen pressure was lowered in PDT-treated tumors and this was ascribed both to oxygen consumption during PDT and to fluctuations in oxygen transport after PDT. Similarly, microcirculatory blood flow changed as a result of the disruption of blood vessels by the treatment. A novel noninvasive approach combining electron paramagnetic resonance oximetry and laser Doppler blood perfusion measurements allowed longitudinal monitoring of hypoxia and vascular function changes in the same animals, after PDT. C-PDT induced parallel changes in tumor pO2 and blood flow, i.e., an initial decrease immediately after treatment, followed by a slow increase. In contrast, V-PDT led to a strong and persistent depletion of pO2, although the microcirculatory blood flow increased. Strong hypoxia after V-PDT led to a slight increase in VEGF level 24h after treatment. C-PDT caused a ca. 5-day delay in tumor growth, whereas V-PDT was much more efficient and led to tumor growth inhibition in 90% of animals. The tumors of 44% of mice treated with V-PDT regressed completely and did not reappear for over 1 year. In conclusion, mild and transient hypoxia after C-PDT led to intense pO2 compensatory effects and modest tumor inhibition, but strong and persistent local hypoxia after V-PDT caused tumor growth inhibition.

Increased oxidative stresses are implicated in the pathogenesis of Parkinson's disease, and dopaminergic neurons may be intrinsically susceptible to oxidative damage. However, the selective presence of tetrahydrobiopterin (BH 4 ) makes dopaminergic neurons more resistant to oxidative stress caused by glutathione depletion. To further investigate the mechanisms of BH 4 protection, we examined the effects of BH 4 on superoxide levels in individual living mesencephalic neurons. Dopaminergic neurons have intrinsically lower levels of superoxide than nondopaminergic neurons. In addition, inhibiting BH 4 synthesis increased superoxide in dopaminergic neurons, while BH 4 supplementation decreased superoxide in nondopaminergic cells. BH 4 is also a cofactor in catecholamine and NO production. In order to exclude the possibility that the antioxidant effects of BH 4 are mediated by dopamine and NO, we used fibroblasts in which neither catecholamine nor NO production occurs. In fibroblasts, BH 4 decreased baseline reactive oxygen species, and attenuated reactive oxygen species increase by rotenone and antimycin A. Physiologic concentrations of BH 4 directly scavenged superoxide generated by potassium superoxide in vitro. We hypothesize that BH 4 protects dopaminergic neurons from ordinary oxidative stresses generated by dopamine and its metabolites and that environmental insults or genetic defects may disrupt this intrinsic capacity of dopaminergic neurons and contribute to their degeneration in Parkinson's disease. Parkinson's disease (PD)1 is characterized by the selective degeneration of dopaminergic neurons in the substantia nigra (SN) pars compacta. While the etiology and pathogenesis of this cell death in most cases of PD remain unknown, it has been widely hypothesized that increased oxidative stress and mitochondrial dysfunction contribute to dopaminergic neuronal degeneration. Autopsy studies have noted numerous abnormalities indicative of increased oxidative damage in the SN (1-5).In particular, decreased glutathione in the SN has been found in autopsy brains from individuals with incidental Lewy bodies, presumed to represent presymptomatic PD (6). In addition, mitochondrial complex I activity is also decreased in the SN of PD patients (7, 8), and complex I inhibition can increase the formation of reactive oxygen species (9, 10). Although these findings suggest that dopaminergic neurons are susceptible to oxidative stress, studies in vivo have not observed dopaminergic neuronal death following the oxidative stress generated by glutathione depletion (11-13). Furthermore, mesencephalic dopaminergic neurons in primary monolayer (14) and reaggregate (15) cultures are less susceptible to the toxicity of glutathione depletion than nondopaminergic neurons.To explain this unanticipated resistance of dopaminergic neurons to oxidative stress, we hypothesized that dopaminergic neurons may contain additional antioxidants for the metabolism of ROS. One candidate is tetrahydrobiopterin (BH 4 ), which is present abundantly in do...

Purpose-Tumor hypoxia has long been known to produce resistance to radiation. In this study, electron paramagnetic resonance (EPR) oxygen imaging was investigated for its power to predict the success of tumor control depending on tumor oxygenation level and radiation therapy dose.Methods and Materials-Thirty-four EPR oxygen images were obtained from the legs of C3H mice bearing 0.5 ml FSa fibrosarcomas under both normal (air breathing) and clamped tumor conditions. Under the same conditions as those during which the images were obtained, tumors were irradiated to a variety doses near the FSa TCD 50 . Tumor tissue was distinguished from normal tissue using co-registration of the EPR oxygen images with spin-echo MRI images of the tumor and/or stereotactic localization. Tumor voxel statistics in the EPR oxygen image included mean and median pO 2 , and the fraction of tumor voxels below the specified pO 2 values of 3, 6 and 10 torr. Bivariate logistic regression analysis using radiation dose and each of the EPR oxygen image statistics determined which best separated treatment failure from success.Results and Conclusions-TCD 50 measurements were similar to those found in the literature for this syngeneic tumor. Bivariate analysis of 34 tumors demonstrated that tumor cure correlated with dose (p=0.004) and with <10 torr hypoxic fraction (p=0.023). Together, radiation dose and EPR image hypoxic fraction separate the population of FSa fibrosarcomas which are cured from those which fail, thus predicting curability.

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To enhance their therapeutic effectiveness, and decrease cardiotoxicity and other side effects, we intend to conjugate the quinone chelators with monoclonal antibodies and peptide hormones that are specifically targeted to receptors on the cancer cell surface. Some such candidates have already been synthesized. An alternative approach for delivery of our compounds involves the use of specific peptide-based nanoparticles. In addition, our novel approach for treating malignancies is also suitable for photodynamic therapy. Antioxid. Redox Signal. 28, 1394-1403.

Clinical trials to ameliorate hypoxia as a strategy to relieve the radiation resistance it causes have prompted a need to assay the precise extent and location of hypoxia in tumors. Electron Paramagnetic Resonance oxygen imaging (EPR O2 imaging) provides a non-invasive means to address this need. To obtain a preclinical proof of principle that EPR O2 images could predict radiation control, we treated mouse tumors at or near doses required to achieve 50 percent control (TCD50). Mice with FSa fibrosarcoma or MCa4 carcinoma were subjected to EPR O2 imaging and immediately radiated to a TCD50 or TCD50 ±10 Gy.. Statistical analysis was permitted by collection of ~ 1300 tumor pO2 image voxels, including the fraction of tumor voxels with pO2 less than 10 mm Hg (HF10). Tumors were followed for 90 days (FSa) or 120 days (MCa4) to determine local control or failure. HF10 obtained from EPR images showed statistically significant differences between tumors that were controlled by the TCD50 and those that were not controlled for both FSa and MCa4. Kaplan-Meier analysis of both types of tumors showed ~90% of mildly hypoxic tumors were controlled (HF10<10%), and only 37% (FSA) and 23% (MCa4) tumors controlled if hypoxic. EPR pO2 image voxel distributions in these ~0.5 ml tumors provide a prediction of radiation curability independent of radiation dose. These data confirm the significance of EPR pO2 hypoxic fractions. The ~90% control of low HF10 tumors argue that ½ ml subvolumes of tumors may be more sensitive to radiation and may need less radiation for high tumor control rates.