The term “redox environment” is usually used when dealing with biological systems because they have numerous linked redox cou-ples (50). Normally, the biological environment of an organism is maintained in redox homeostasis regulated by several factors such as level of O2, thiols, and enzymatic pathways. Alteration in any of these factors due to disease will cause changes in the redox environment of the cells and tissues. It is important to be able to detect and visualize such changes, especially in systems like tumors, where the redox environment plays a role in the efficacy of treatment. The development of low-frequency EPR (1.0 GHz and lower) has enabled mapping and imaging of the tumor redox environment (51–53).
4.1. Nitroxyls as Redox Probes for EPR Imaging
Low-molecular weight stable nitroxyl free radicals are useful as spin probe contrast agents in functional imaging studies (54, 55). EPR imaging, using the nitroxyls, can provide functional information on the global redox status in experimental animal models of pathological conditions, in vivo. Two critical prop-erties of nitroxyls provide such capability: (i) nitroxyl labels participate in redox reactions (Fig. 1.6) where the nitroxyl (paramagnetic, EPR detectable) is reduced to the correspond-ing hydroxylamine (diamagnetic, EPR “silent”), and vice versa,
Imaging of Free Radicals In Vivo 17
Fig. 1.6. Structure of 3-CP and its tissue metabolite. The “EPR active” 3-CP (3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl, or 3-carbamoyl-proxyl) nitroxyl probe undergoes one-electron reduction in tissues to “EPR-silent” hydroxylamine form.
establishing equilibrium between the two species (56, 57); (ii) in cells, the nitroxyls undergo reduction to the correspond-ing hydroxylamines more efficiently under hypoxic conditions than under aerobic conditions via intracellular enzymatic pro-cesses (58). When nitroxyls are administered in vivo, a rapid equilibrium is established between the levels of nitroxyl and hydroxylamine. The level of the nitroxyl, detectable in vivo, is independent of whether the nitroxyl or the hydroxylamine is administered. Based on the above-mentioned properties, several studies were carried out using stable nitroxyls as probes in EPR spectroscopy and imaging experiments to obtain tissue/tumor morphology and redox status.
4.2. Imaging of the Nitroxyl Pharmacokinetics in Tumor
Since tumor tissues are characterized by significant hetero-geneities in terms of redox status and oxygenation, it is desirable to obtain spatially resolved images of nitroxyl distribution and clearance simultaneously within the tumor volume. Figure 1.7 shows a few selected images from a series of 2D spatial maps of nitroxyl content in normal and buthionine sulfoximine (BSO)-treated tumor tissues obtained as a function of time after 3-CP infusion. Each time-course image within the series, as shown in the figure, was normalized with respect to the maximum inten-sity obtained within that series, usually in the images collected in about 4–6 min after infusion. BSO-treated animals demon-strated a slower rate of reduction of nitroxyl in the tumor com-pared to untreated mice. The measurements clearly suggest that the nitroxyl is reduced more efficiently in the tumor than in nor-mal tissue. However, the tumor may contain variable regions of redox state, depending on a variety of factors including differences in reducing equivalents, oxygenation, and pH.
18 Vikram, Rivera, and Kuppusamy
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Fig. 1.7. Spatially resolved clearance of nitroxyl in RIF-1 tumor tissue. After tail vein infusion of 3-CP, a series of two-dimensional images of the nitroxyl from tumor (untreated and BSO-treated) were measured using L-band EPRI method. A few selected images and the corresponding approximate time after infusion are shown. The images represent the mean nitroxyl concentration in a two-dimensional projection of the tissue volume (10× 10 mm2; depth, 5 mm) averaged over 1.5–2.0 min. The image data were acquired using a magnetic field gradient of 15 G/cm at 16 orientations in the two-dimensional plane. Each image within a series was normalized with respect to the maximum intensity in that series. The nitroxyl in the tumor of BSO-treated mouse persisted longer, compared with that in the untreated mouse.
4.3. Redox Mapping of Tumor
In order to obtain spatially resolved information regarding the reduction rate constants of nitroxyls in the tumor, we developed algorithms for direct measurement and mapping of the rate con-stants (redox mapping) (53, 59). The “redox mapping” method is based on the following principle (Fig. 1.8). Spatial imaging of
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Fig. 1.8. Reconstruction of redox image. The procedure of obtaining redox image from a 2D pharmacokinetic image data is illustrated. Nitroxyl intensity from a given voxel within the image is followed as a function of time to obtain the reduction profile (plot) versus t. The profile is modeled with an appropriate kinetic expression, usually a pseudo first-order decay process, to obtain rate constant at each voxel. The computation is repeated over all the voxels to reconstruct the redox image.
Imaging of Free Radicals In Vivo 19
nitroxyl in the tumor is performed to obtain time-series images.
The time-dependent intensity of the nitroxyl in a given voxel is obtained from the images and fitted to a pseudo first-order kinetic rate equation to compute the rate constant.
The procedure will be repeated for all voxels in the image to obtain rate constants for each of the individual voxels. The result-ing array of rate constants is then displayed on a color scale or frequency (histogram) plot. An appropriate threshold of intensity level is defined to exclude those regions where the nitroxyl con-centration is too small to make calculations. This means that areas with nitroxyl intensity above the threshold level provide spatially resolved visualization (mapping) of the rate constants in the tis-sue.
4.4. Redox Mapping of RIF-1 Tumor:
Effect of BSO Treatment
The rate constants for the clearance of the probe in each pixel of the pharmacokinetic images in Fig. 1.7 were computed and displayed in the form a color-coded image and frequency his-togram in Fig. 1.9 (52). The plot shows the presence of a range
Fig. 1.9. Redox mapping of tumor. Two-dimensional spatial mapping of pseudo first-order rate constants (left panels) and frequency plot (right panels) of the nitroxide reduc-tion rate constants in the RIF-1 tumors of untreated (air-breathing), BSO-treated, and carbogen-breathing mice were obtained from the time-course image data similar to those shown in Fig. 1.7.
20 Vikram, Rivera, and Kuppusamy
of rate constants, which is a measure of heterogeneity in the redox status within each tissue. The redox data show that the BSO treatment significantly decreases the rate constant of nitroxyl reduction in the tumor. It is known that BSO inhibits the ␥-glutamylcysteine synthetase enzyme that is responsible for GSH synthesis (60). Studies have shown that tumor tissue levels of GSH can be depleted to very low levels without any toxicity (61).
The observed effect of BSO treatment on the redox constants is, therefore, attributed to alterations in the tissue GSH levels.
In order to investigate the correlation between the observed rate constants and tissue GSH, the tissue levels of GSH were measured in the tissues of normal (control) and BSO-treated (same dose as for the EPR measurements) mice using EC-HPLC. The BSO-treated tissue data showed a significant reduction in the GSH lev-els in the tumor tissue when compared to the untreated control (52). On the other hand, no significant differences were observed between treated and untreated normal tissues. Thus, it is evident that BSO causes a differential depletion among normal and tumor tissues.
4.5. Redox Mapping of RIF-1 Tumor:
Effect of
Carbogen-Breathing
Figure 1.9also shows redox images obtained from RIF-1 tumor-bearing mice subjected to carbogen (95% O2 + 5% CO2 breath-ing (51)). The redox map indicates the spatially resolved rates of nitroxyl depletion within the tumor and the differences in the intensities noticed in the map indicate the spatial heterogeneity in the tumor redox activity. From this plot, it is clear that the rate constants in the tumor of an air-breathing mouse are dis-tributed widely with a median value of 0.055 min–1. Similar mea-surements of EPR imaging pharmacokinetics were made when the animal was breathing carbogen. The carbogen-breathing data revealed marked differences in the magnitude and distribution of the rate constants with a median value of 0.042 min–1. The dis-tribution of the rate constants is significantly narrowed and less variable when compared to the air-breathing animal. These results indicate that the redox rate constants are significantly heteroge-neous in the case of air-breathing animal. On the other hand, the distribution is significantly narrowed during carbogen-breathing indicating that carbogen-breathing decreases the tumor redox activity with less heterogeneity in the distribution of the redox state.
The heterogeneity in the redox status may arise as a conse-quence of the heterogeneity in perfusion or O2 concentration in the tumor. The diminished heterogeneity of the tumor redox sta-tus in the case of carbogen-breathing may be due to elimination of diffusion-limited hypoxia, thereby rendering the tumor more homogeneously oxygenated.