Murali C. Krishna

Hypoxia: importance in tumor biology, noninvasive measurement by imaging, and value of its measurement in the management of cancer therapy.

PURPOSE The Cancer Imaging Program of the National Cancer Institute convened a workshop to assess the current status of hypoxia imaging, to assess what is known about the biology of hypoxia as it relates to cancer and cancer therapy, and to define clinical scenarios in which in vivo hypoxia imaging could prove valuable. RESULTS Hypoxia, or low oxygenation, has emerged as an important factor in tumor biology and response to cancer treatment. It has been correlated with angiogenesis, tumor aggressiveness, local recurrence, and metastasis, and it appears to be a prognostic factor for several cancers, including those of the cervix, head and neck, prostate, pancreas, and brain. The relationship between tumor oxygenation and response to radiation therapy has been well established, but hypoxia also affects and is affected by some chemotherapeutic agents. Although hypoxia is an important aspect of tumor physiology and response to treatment, the lack of simple and efficient methods to measure and image oxygenation hampers further understanding and limits their prognostic usefulness. There is no gold standard for measuring hypoxia; Eppendorf measurement of pO(2) has been used, but this method is invasive. Recent studies have focused on molecular markers of hypoxia, such as hypoxia inducible factor 1 (HIF-1) and carbonic anhydrase isozyme IX (CA-IX), and on developing noninvasive imaging techniques. CONCLUSIONS This workshop yielded recommendations on using hypoxia measurement to identify patients who would respond best to radiation therapy, which would improve treatment planning. This represents a narrow focus, as hypoxia measurement might also prove useful in drug development and in increasing our understanding of tumor biology.

Pharmacologic doses of ascorbate act as a prooxidant and decrease growth of aggressive tumor xenografts in mice

Ascorbic acid is an essential nutrient commonly regarded as an antioxidant. In this study, we showed that ascorbate at pharmacologic concentrations was a prooxidant, generating hydrogen-peroxide-dependent cytotoxicity toward a variety of cancer cells in vitro without adversely affecting normal cells. To test this action in vivo, normal oral tight control was bypassed by parenteral ascorbate administration. Real-time microdialysis sampling in mice bearing glioblastoma xenografts showed that a single pharmacologic dose of ascorbate produced sustained ascorbate radical and hydrogen peroxide formation selectively within interstitial fluids of tumors but not in blood. Moreover, a regimen of daily pharmacologic ascorbate treatment significantly decreased growth rates of ovarian (P < 0.005), pancreatic (P < 0.05), and glioblastoma (P < 0.001) tumors established in mice. Similar pharmacologic concentrations were readily achieved in humans given ascorbate intravenously. These data suggest that ascorbate as a prodrug may have benefits in cancers with poor prognosis and limited therapeutic options.

Ascorbate in pharmacologic concentrations selectively generates ascorbate radical and hydrogen peroxide in extracellular fluid in vivo

Ascorbate (ascorbic acid, vitamin C), in pharmacologic concentrations easily achieved in humans by i.v. administration, selectively kills some cancer cells but not normal cells. We proposed that pharmacologic ascorbate is a prodrug for preferential steady-state formation of ascorbate radical (Asc•−) and H2O2 in the extracellular space compared with blood. Here we test this hypothesis in vivo. Rats were administered parenteral (i.v. or i.p.) or oral ascorbate in typical human pharmacologic doses (≈0.25–0.5 mg per gram of body weight). After i.v. injection, ascorbate baseline concentrations of 50–100 μM in blood and extracellular fluid increased to peaks of >8 mM. After i.p. injection, peaks approached 3 mM in both fluids. By gavage, the same doses produced ascorbate concentrations of <150 μM in both fluids. In blood, Asc•− concentrations measured by EPR were undetectable with oral administration and always <50 nM with parenteral administration, even when corresponding ascorbate concentrations were >8 mM. After parenteral dosing, Asc•− concentrations in extracellular fluid were 4- to 12-fold higher than those in blood, were as high as 250 nM, and were a function of ascorbate concentrations. By using the synthesized probe peroxyxanthone, H2O2 in extracellular fluid was detected only after parenteral administration of ascorbate and when Asc•− concentrations in extracellular fluid exceeded 100 nM. The data show that pharmacologic ascorbate is a prodrug for preferential steady-state formation of Asc•− and H2O2 in the extracellular space but not blood. These data provide a foundation for pursuing pharmacologic ascorbate as a prooxidant therapeutic agent in cancer and infections.

Chemical biology of nitric oxide: Regulation and protective and toxic mechanisms

Publisher Summary This chapter discusses the important aspects of the solution chemistry of nitrogen oxide (NO) and reactive nitrogen oxide species (RNOS), biochemical targets of NO and intermediates in the autoxidation (NO X ), and the effect of NO in the presence of other toxic molecules, such as reactive oxygen species (ROS). There are two types of nitric-oxide synthase: constitutive (cNOS) and inducible (iNOS). Since cNOS generates low levels of NO, direct effects rather than indirect effects of NO would be particularly relevant. In case of iNOS, considerably higher concentrations of NO are formed for longer periods of time; therefore, both direct and indirect effects could be relevant. This chapter discusses, from a chemical perspective, those processes that are involved in the interactions with key cellular components as well as detoxification and control of NO in vivo . Defining the chemical, biochemical, and cellular pathways of NO quantitatively can provide insights into the role that NO plays in the etiology of various diseases that in turn can provide a basis for the development of new therapeutic agents. The chemical biology of NO will provide the understanding as to how NO can be regulatory, toxic, and protective in biological systems.

Radioprotectors and Mitigators of Radiation-Induced Normal Tissue Injury

The article reviews agents in clinical use or in development as radioprotectors and mitigators of radiation-induced normal tissue injury.

Overhauser enhanced magnetic resonance imaging for tumor oximetry: Coregistration of tumor anatomy and tissue oxygen concentration

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 pO2 (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% O2/5% CO2) inhalation. The pO2 measurements from OMRI were found to be in agreement with those obtained by independent polarographic measurements using a pO2 Eppendorf electrode. This work illustrates that anatomically coregistered pO2 maps of tumors can be readily obtained by combining the good anatomical resolution of water proton-based MRI, and the superior pO2 sensitivity of EPR. OMRI affords the opportunity to perform noninvasive and repeated pO2 measurements of the same animal with useful spatial (≈1 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.

Inhibition of oxygen-dependent radiation-induced damage by the nitroxide superoxide dismutase mimic, Tempol

Stable nitroxide radicals have been previously shown to function as superoxide dismutase (SOD)2 mimics and to protect mammalian cells against superoxide and hydrogen peroxide-mediated oxidative stress. These unique characteristics suggested that nitroxides, such as 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol), might protect mammalian cells against ionizing radiation. Treating Chinese hamster cells under aerobic conditions with 5, 10, 50, and 100 mM Tempol 10 min prior to X-rays resulted in radiation protection factors of 1.25, 1.30, 2.1, and 2.5, respectively. However, the reduced form of Tempol afforded no protection. Tempol treatment under hypoxic conditions did not provide radioprotection. Aerobic X-ray protection by Tempol could not be attributed to the induction of intracellular hypoxia, increase in intracellular glutathione, or induction of intracellular SOD mRNA. Tempol thus represents a new class of non-thiol-containing radiation protectors, which may be useful in elucidating the mechanism(s) of radiation-induced cellular damage and may have broad applications in protecting against oxidative stress.

Superoxide Modulates the Oxidation and Nitrosation of Thiols by Nitric Oxide-derived Reactive Intermediates CHEMICAL ASPECTS INVOLVED IN THE BALANCE BETWEEN OXIDATIVE AND NITROSATIVE STRESS

Thiol-containing proteins are key to numerous cellular processes, and their functions can be modified by thiol nitrosation or oxidation. Nitrosation reactions are quenched by O2, while the oxidation chemistry mediated by peroxynitrite is quenched by excess flux of either NO or O2. A solution of glutathione (GSH), a model thiol-containing tripeptide, exclusively yielded S-nitrosoglutathione when exposed to the NO donor, Et2NN(O)NONa. However, when xanthine oxidase was added to the same mixture, the yield of S-nitrosoglutathione dramatically decreased as the activity of xanthine oxidase increased, such that there was a 95% reduction in nitrosation when the fluxes of NO and O2 were nearly equivalent. The presence of superoxide dismutase reversed O2-mediated inhibition, while catalase had no effect. Increasing the flux of O2 yielded oxidized glutathione (GSSG), peaking when the flux of NO and O2 were approximately equivalent. The results suggest that oxidation and nitrosation of thiols by superoxide and NO are determined by their relative fluxes and may have physiological significance.

Nitric oxide (NO) protects against cellular damage by reactive oxygen species

Since the discovery of nitric oxide (NO) as an endogenously formed radical, its effect on numerous physiological processes has been intensively investigated. Some studies have suggested NO to be cytotoxic while others have demonstrated it protective under various biological conditions. Though NO shows minimal cytotoxicity to a variety mammalian cell cultures, it does modulate the toxicity of some agents such as reactive oxygen species. Often, NO is generated in the presence of these reactive oxygen species in response to foreign pathogens or under various pathophysiological conditions. We will show that NO can play a protective role under oxidative stress resulting from superoxide, hydrogen peroxide and alkyl peroxides. It was found by measuring the time-concentration profiles of NO released from various NO donor compounds that only microM levels of NO were required for protection against the toxicity of these reactive species. It was found that there are several chemical reactions which may account for these protective effects such as NO preventing heme oxidation, inhibition of Fenton-type oxidation of DNA, and abatement of lipid peroxidation. Taken together, NO at low concentrations clearly protects against peroxide-mediated toxicity.

Direct detection and time-locked subsampling applied to pulsed electron paramagnetic resonance imaging

The application of direct time-locked subsampling (TLSS) to Fourier transform electron paramagnetic resonance (FT-EPR) spectroscopy at radio frequencies (rf) is described. With conventional FT-EPR spectroscopy, the high Larmor frequencies (L(f)) often necessitate the use of intermediate frequency (IF) stages to down convert the received free induction decay (FID) signal to a frequency that can be acquired with common data acquisition technology. However, our research focuses on in vivo studies, and consequently utilizes a FT-EPR system with a L(f) of 300 MHz. This relatively low frequency L(f), in conjunction with the advent of bandpass sampling analog-to-digital conversion and signal processing technologies, has enabled us to omit the IF stage in our FT-EPR system. With this in mind, TLSS techniques have been developed to directly sample the 300 MHz FID signal at a sampling rate of 80 MHz providing a signal bandwidth of 20 MHz. The required modifications to the data acquisition and processing system specific to this application are described. Custom software developed to control the EPR system setup, acquire the signals, and post process the data, is outlined. Data was acquired applying both coherent averaging and stochastic excitation sequences. The results of these experiments demonstrate digital down conversion of the 300 MHz FID signal to quadrature baseband. Direct FID TLSS eliminates many noise sources common in EPR systems employing traditional analog receiver techniques, such as the IF mixer stage in single channel systems, and the quadrature baseband mixer stage in dual channel systems.