Radiation therapy is one of the main modalities to treat cancer/tumor. The response to radiation therapy, however, can be influenced by physiological and/or pathological conditions in the target tissues, especially by the low partial oxygen pressure and altered redox status in cancer/tumor tissues. Visualizing such cancer/tumor patho-physiological microenvironment would be a useful not only for planning radiotherapy but also to detect cancer/tumor in an earlier stage. Tumor hypoxia could be sensed by positron emission tomography (PET), electron paramagnetic resonance (EPR) oxygen mapping, and in vivo dynamic nuclear polarization (DNP) MRI. Tissue oxygenation could be visualized on a real-time basis by blood oxygen level dependent (BOLD) and/or tissue oxygen level dependent (TOLD) MRI signal. EPR imaging (EPRI) and/or T₁-weighted MRI techniques can visualize tissue redox status non-invasively based on paramagnetic and diamagnetic conversions of nitroxyl radical contrast agent. ¹³C-DNP MRI can visualize glycometabolism of tumor/cancer tissues. Accurate co-registration of those multimodal images could make mechanisms of drug and/or relation of resulted biological effects clear. A multimodal instrument, such as PET-MRI, may have another possibility to link multiple functions. Functional imaging techniques individually developed to date have been converged on the concept of theranostics.

Significance: In vivo assessment of paramagnetic and diamagnetic conversions of nitroxyl radicals based on cyclic redox mechanism can be an index of tissue redox status. The redox mechanism of nitroxyl radicals, which enables their use as a normal tissue-selective radioprotector, is seen as being attractive on planning radiation therapy. Recent Advances: In vivo redox imaging using nitroxyl radicals as redox-sensitive contrast agents has been developed to assess tissue redox status. Chemical and biological behaviors depending on chemical structures of nitroxyl radical compounds have been understood in detail. Polymer types of nitroxyl radical contrast agents and/or nitroxyl radical-labeled drugs were designed for approaching theranostics. Critical Issues: Nitroxyl radicals as magnetic resonance imaging (MRI) contrast agents have several advantages compared with those used in electron paramagnetic resonance (EPR) imaging, while support by EPR spectroscopy is important to understand information from MRI. Redox-sensitive paramagnetic contrast agents having a medicinal benefit, that is, nitroxyl-labeled drug, have been developed and proposed. Future Directions: A development of suitable nitroxyl contrast agent for translational theranostic applications with high reaction specificity and low normal tissue toxicity is under progress. Nitroxyl radicals as redox-sensitive magnetic resonance contrast agents can be a useful tool to detect an abnormal tissue redox status such as disordered oxidative stress. Antioxid. Redox Signal . 36, 95–121.

Questions about which reactive oxygen species (ROS) or reactive nitrogen species (RNS) can escape from the mitochondria and activate signals must be addressed. In this study, two parameters, the calculated dipole moment (debye, D) and permeability coefficient (Pm) (cm s⁻¹), are listed for hydrogen peroxide (H₂O₂), hydroxyl radical (•OH), superoxide (O₂^•−), hydroperoxyl radical (HO₂•), nitric oxide (•NO), nitrogen dioxide (•NO₂), peroxynitrite (ONOO⁻), and peroxynitrous acid (ONOOH) in comparison to those for water (H₂O). O₂^•− is generated from the mitochondrial electron transport chain (ETC), and several other ROS and RNS can be generated subsequently. The candidates which pass through the mitochondrial membrane include ROS with a small number of dipoles, i.e., H₂O₂, HO₂•, ONOOH, •OH, and •NO. The results show that the dipole moment of •NO₂ is 0.35 D, indicating permeability; however, •NO₂ can be eliminated quickly. The dipole moments of •OH (1.67 D) and ONOOH (1.77 D) indicate that they might be permeable. This study also suggests that the mitochondria play a central role in protecting against further oxidative stress in cells. The amounts, the long half-life, the diffusion distance, the Pm, the one-electron reduction potential, the pK_a, and the rate constants for the reaction with ascorbate and glutathione are listed for various ROS/RNS, •OH, singlet oxygen (¹O₂), H₂O₂, O₂^•−, HO₂•, •NO, •NO₂, ONOO⁻, and ONOOH, and compared with those for H₂O and oxygen (O₂). Molecules with negative electrical charges cannot directly diffuse through the phospholipid bilayer of the mitochondrial membranes. Short-lived molecules, such as •OH, would be difficult to contribute to intracellular signaling. Finally, HO₂• and ONOOH were selected as candidates for the ROS/RNS that pass through the mitochondrial membrane.

Imaging tumor microenvironments such as hypoxia, oxygenation, redox status, and/or glycolytic metabolism in tissues/cells is useful for diagnostic and prognostic purposes. New imaging modalities are under development for imaging various aspects of tumor microenvironments. Electron Paramagnetic Resonance Imaging (EPRI) though similar to NMR/MRI is unique in its ability to provide quantitative images of pO₂ in vivo. The short electron spin relaxation times have been posing formidable challenge to the technology development for clinical application. With the availability of the narrow line width trityl compounds, pulsed EPR imaging techniques were developed for pO₂ imaging. EPRI visualizes the exogenously administered spin probes/contrast agents and hence lacks the complementary morphological information. Dynamic nuclear polarization (DNP), a phenomenon that transfers the high electron spin polarization to the surrounding nuclear spins (¹H and ¹³C) opened new capabilities in molecular imaging. DNP of ¹³C nuclei is utilized in metabolic imaging of 13C-labeled compounds by imaging specific enzyme kinetics. In this article, imaging strategies mapping physiologic and metabolic aspects in vivo are reviewed within the framework of their application in cancer research, highlighting the potential and challenges of each of them.

The impact of the site of the Fenton reaction, i.e., hydroxyl radical (^•OH) generation, on cytotoxicity was investigated by estimating cell lethality in rat thymocytes. Cells were incubated with ferrous sulfate (FeSO₄) and hydrogen peroxide (H₂O₂), or pre-incubated with FeSO₄ and then H₂O₂ was added after medium was replaced to remove iron ions or after the medium was not replaced. Cell lethality in rat thymocytes was estimated by measuring cell sizes using flow cytometry. High extracellular concentrations of FeSO₄ exerted protective effects against H₂O₂-induced cell death instead of enhancing cell lethality. The pre-incubation of cells with FeSO₄ enhanced cell lethality induced by H₂O₂, whereas a pre-incubation with a high concentration of FeSO₄ exerted protective effects. FeSO₄ distributed extracellularly or on the surface of cells neutralized H₂O₂ outside cells. Cytotoxicity was only enhanced when the Fenton reaction, i.e., the generation of ^•OH, occurred inside cells. An assessment of plasmid DNA breakage showed that ^•OH induced by the Fenton reaction system did not break DNA. Therefore, the main target of intracellularly generated ^•OH does not appear to be DNA.

The amounts of reactive oxygen species generated in aqueous samples by irradiation with X-ray or clinical carbon-ion beams were quantified. Hydroxyl radical (^•OH), hydrogen peroxide (H₂O₂), and the total amount of oxidation reactions, which occurred mainly because of ^•OH and/or hydroperoxy radicals (HO₂^•), were measured by electron paramagnetic resonance-based methods. ^•OH generation was expected to be localized on the track/range of the carbon-ion beam/X-ray, and mM and M levels of ^•OH generation were observed. Total ^•OH generation levels were identical at the same dose irrespective of whether X-ray or carbon-ion beam irradiation was used, and were around 0.28–0.35 µmol/L/Gy. However, sparse ^•OH generation levels decreased with increasing linear energy transfer, and were 0.17, 0.15, and 0.09 µmol/L/Gy for X-ray, 20 keV/µm carbon-ion beam, and >100 keV/µm carbon-ion beam sources, respectively. H₂O₂ generation was estimated as 0.26, 0.20, and 0.17 µmol/L/Gy, for X-ray, 20 keV/µm carbon-ion beam, and >100 keV/µm carbon-ion beam sources, respectively, whereas the ratios of H₂O₂ generation per oxygen consumption were 0.63, 0.51, and 3.40, respectively. The amounts of total oxidation reactions were 2.74, 1.17, and 0.66 µmol/L/Gy, respectively. The generation of reactive oxygen species was not uniform at the molecular level.