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An efficient noninvasive method for in vivo imaging of tumor oxygenation by using a low-field magnetic resonance scanner and a paramagnetic contrast agent is described. The methodology is based on Overhauser enhanced magnetic resonance imaging (OMRI), a functional imaging technique. OMRI experiments were performed on tumor-bearing mice (squamous cell carcinoma) by i.v. administration of the contrast agent Oxo63 (a highly derivatized triarylmethyl radical) at nontoxic doses in the range of 2-7 mmol/kg either as a bolus or as a continuous infusion. Spatially resolved pO 2 (oxygen concentration) images from OMRI experiments of tumor-bearing mice exhibited heterogeneous oxygenation profiles and revealed regions of hypoxia in tumors (<10 mmHg; 1 mmHg ‫؍‬ 133 Pa). Oxygenation of tumors was enhanced on carbogen (95% O 2͞5% CO2) inhalation. The pO2 measurements from OMRI were found to be in agreement with those obtained by independent polarographic measurements using a pO 2 Eppendorf electrode. This work illustrates that anatomically coregistered pO 2 maps of tumors can be readily obtained by combining the good anatomical resolution of water proton-based MRI, and the superior pO 2 sensitivity of EPR. OMRI affords the opportunity to perform noninvasive and repeated pO 2 measurements of the same animal with useful spatial (Ϸ1 mm) and temporal (2 min) resolution, making this method a powerful imaging modality for small animal research to understand tumor physiology and potentially for human applications.A bnormal values of pO 2 (the partial pressure of O 2 ) are linked to many pathophysiological conditions (e.g., ischemic diseases, reperfusion injury, and oxygen toxicity). Approximately one-third of human tumors evaluated for oxygen status have shown significant oxygen deficiency, and oxygen deficiency increases the tumor's resistance toward cancer treatment modalities, including radiation and chemotherapy (1, 2). Additionally, hypoxic microenvironments in tumors are known to promote processes driving malignant progression, such as angiogenesis, elimination of p53 tumor suppressor activity, genetic instability, and metastasis (3-5). Understanding of tumor hypoxia could lead to the discovery of diagnostic and prognostic markers for malignant progression, discovery of novel therapeutic targets, and the development of new constructs for gene therapy applications in human cancer. Hence, a noninvasive technique that could accurately and repetitively measure tissue oxygenation would find broad application in clinical and basic research. Unfortunately, the currently used electrochemical method (6) for in vivo oxygen measurement is an invasive technique applicable only to accessible tumors. Further, the technique is hampered by measurements of only a small part of the total tumor, which cannot be re-evaluated. Several magnetic resonance techniques (7, 8) have been developed for in vivo oximetry, including spin label oximetry (9), MRI (10), and electron paramagnetic resonance imaging (EPRI) (11,12). The blood oxygen level-dependent...

Ferroptosis, a non-apoptotic form of cell death marked by iron-dependent lipid peroxidation1, has a key role in organ injury, degenerative disease and vulnerability of therapy-resistant cancers2. Although substantial progress has been made in understanding the molecular processes relevant to ferroptosis, additional cell-extrinsic and cell-intrinsic processes that determine cell sensitivity toward ferroptosis remain unknown. Here we show that the fully reduced forms of vitamin K—a group of naphthoquinones that includes menaquinone and phylloquinone3—confer a strong anti-ferroptotic function, in addition to the conventional function linked to blood clotting by acting as a cofactor for γ-glutamyl carboxylase. Ferroptosis suppressor protein 1 (FSP1), a NAD(P)H-ubiquinone reductase and the second mainstay of ferroptosis control after glutathione peroxidase-44,5, was found to efficiently reduce vitamin K to its hydroquinone, a potent radical-trapping antioxidant and inhibitor of (phospho)lipid peroxidation. The FSP1-mediated reduction of vitamin K was also responsible for the antidotal effect of vitamin K against warfarin poisoning. It follows that FSP1 is the enzyme mediating warfarin-resistant vitamin K reduction in the canonical vitamin K cycle6. The FSP1-dependent non-canonical vitamin K cycle can act to protect cells against detrimental lipid peroxidation and ferroptosis.

The nitroxyl anion (NO. ), respectively, can lead to oxidation, hydroxylation, nitration, and nitrosation of biomolecules (1). Although a substantial literature exists on the putative biological effects of other RNOS, few studies have focused on nitroxyl (NO Ϫ ), the one electron reduction product of NO. Several reports suggest that NO Ϫ (or its conjugate acid, HNO) can be generated from chemical reactions that occur in vivo (2, 3) including oxidation of L-arginine by tetrahydrobiopterin-free nitric oxide synthase (NOS) (4 -6) and decomposition of Snitrosothiols (7, 8). Taken together, these studies indicate that the chemistry of NO Ϫ is an essential component of the redox chemistry of NO in biological systems.Angeli's salt (AS) is the most commonly used synthetic donor in the study of NO Ϫ effects under biological conditions (9). At physiological pH and temperature, AS spontaneously decomposes to HNO and nitrite with a half-life of 2.5 min,The cytotoxic effects of AS are several orders of magnitude greater than those of other RNOS and are comparable to alkylhydroperoxides (10), suggesting that NO Ϫ formation in vivo could have deleterious consequences. In a myocardial ischemiareperfusion model, treatment with AS markedly increased infarct area (11). In contrast, NO, either from a donor or from oxidation of NO Ϫ in the presence of an electron acceptor, afforded protection in the same model. In the present report, the chemistry of AS is compared with that of ONOO Ϫ and NO/N 2 O 3 to gain insight into the biological mechanisms in which the chemistry of NO Ϫ could be involved. MATERIALS AND METHODSAngeli's salt (Na 2 N 2 O 3 ) was synthesized as described previously (10). The NONOate, DEA/NO (NaEt 2 NN(O)NO), was a generous gift from Dr. Joseph Saavedra (National Cancer Institute, Frederick, MD). Stock solutions (ϳ10 mM) of AS and DEA/NO were prepared in 10 mM NaOH and stored at Ϫ20°C (12). Peroxynitrite was synthesized by mixing solutions of 0.5 M NO 2 Ϫ in 0.5 M HCl and 0.5 M hydrogen peroxide (H 2 O 2 ) followed by rapid quenching in 1 M NaOH, as described previously (13). The resulting basic solution was exposed to MnO 2 to remove excess H 2 O 2 , which was reduced to Ͻ1% per mol of ONOO Ϫ . After filtering, aliquots were stored at Ϫ20°C for less than 2 weeks. Directly prior to use, the concentrations of these RNOS donors in 10 mM NaOH were