Use of a cocktail of spin traps for fingerprinting large range of free radicals in biological systems.

It is well established that the formation of radical species centered on various atoms is involved in the mechanism leading to the development of several diseases or to the appearance of deleterious effects of toxic molecules. The detection of free radical is possible using Electron Paramagnetic Resonance (EPR) spectroscopy and the spin trapping technique. The classical EPR spin-trapping technique can be considered as a "hypothesis-driven" approach because it requires an a priori assumption regarding the nature of the free radical in order to select the most appropriate spin-trap. We here describe a "data-driven" approach using EPR and a cocktail of spin-traps. The rationale for using this cocktail was that it would cover a wide range of biologically relevant free radicals and have a large range of hydrophilicity and lipophilicity in order to trap free radicals produced in different cellular compartments. As a proof-of-concept, we validated the ability of the system to measure a large variety of free radicals (O-, N-, C-, or S- centered) in well characterized conditions, and we illustrated the ability of the technique to unambiguously detect free radical production in cells exposed to chemicals known to be radical-mediated toxic agents.

Assessment of liver phagocytic activity using EPR spectrometry and imaging.

The aim of the present study was to evaluate the usefulness of electron paramagnetic resonance (EPR) spectroscopy and imaging in assessing the phagocytic activity of the liver after administration of India ink. We conducted experiments on livers from control rodents and from rodents in which the Kupffer cell population had been depleted by pretreatment with gadolinium chloride. The EPR signal intensity recorded in liver homogenates was about two times lower in GdCl(3) treated rats than in control rats. EPR imaging carried out on precision-cut liver slices indicated a good correlation between the depletion of Kupffer cells and the EPR signal intensity.

Synthesis of two persistent fluorinated tetrathiatriarylmethyl (TAM) radicals for biomedical EPR applications.

Tetrathiatriarylmethyl radicals are attractive spin probes extensively used in biomedical magnetic resonance applications. We report a straightforward synthesis of two original tetrathiatriarylmethyl radicals incorporating, respectively, 15 and 45 fluorine atoms, and thus possessing a high affinity to fluorous media. F15T-03 and F45T-03 exhibit a single sharp EPR spectrum and their EPR line broadening is highly sensitive to molecular oxygen. These spin probes are specially designed for assessment of tumor oxygenation using perfluorocarbon formulations.

The acidic tumor microenvironment promotes the reconversion of nitrite into nitric oxide: towards a new and safe radiosensitizing strategy.

PURPOSE: The biological status of nitrite recently evolved from an inactive end product of nitric oxide catabolism to the largest intravascular and tissue storage of nitric oxide (NO). Although low partial O(2) pressure favors enzymatic reconversion of nitrite into NO, low pH supports a nonenzymatic pathway. Because hypoxia and acidity are characteristics of the tumor microenvironment, we examined whether nitrite injection could preferentially lead to NO production in tumors and influence response to treatments. EXPERIMENTAL DESIGN: The effects of nitrite were evaluated on arteriole vasorelaxation, tumor cell respiration and tumor blood flow, oxygenation, and response to radiotherapy. RESULTS: We first showed that a small drop in pH (-0.6 pH unit) favored the production of bioactive NO from nitrite by documenting a higher cyclic guanosine 3',5'-monophosphate-dependent arteriole vasorelaxation. We then documented that an i.v. bolus injection of nitrite to tumor-bearing mice led to a transient increase in partial O(2) pressure in tumor but not in healthy tissues. Blood flow measurements failed to reveal an effect of nitrite on tumor perfusion, but we found that O(2) consumption by nitrite-exposed tumor cells was decreased at acidic pH. Finally, we showed that low dose of nitrite could sensitize tumors to radiotherapy, leading to a significant growth delay and an increase in mouse survival (versus irradiation alone). CONCLUSIONS: This study identified low pH condition (encountered in many tumors) as an exquisite environment that favors tumor-selective production of NO in response to nitrite systemic injection. This work opens new perspectives for the use of nitrite as a safe and clinically applicable radiosensitizing modality.

Molecular electron paramagnetic resonance imaging of melanin in melanomas: a proof-of-concept.

The incidence of malignant melanoma is increasing at an alarming rate. As the clinical outcome of the disease strongly depends on the localization of the lesion, early detection at the initial stages of development is critical. Here, we suggest spatial characterization of melanoma based on the presence of endogenous stable free radicals in melanin pigments. Taking into account the abundance of these naturally occurring free radicals in proliferating melanocytes and their localization pattern, we hypothesized that electron paramagnetic resonance (EPR) imaging could be a unique tool for mapping melanomas with high sensitivity and high resolution. The potential of EPR to image melanoma samples was demonstrated in vitro in animal and human samples. Using EPR systems operating at low frequency, we were also able to record in vivo EPR spectra and images from the melanin present in a subcutaneous melanoma implanted in a mouse. In addition to the proof-of-concept and the achievement of providing the first non-invasive image of an endogenous radical, this technology may represent a key advance in improving the diagnosis of suspected melanoma lesions.

Evaluation of lipid-based carrier systems and inclusion complexes of diethyldithiocarbamate-iron to trap nitric oxide in biological systems.

The success of spin trapping techniques in vivo hinges on whether spin traps with high trapping efficiency and biocompatibility can be developed. Currently, two iron chelates based on the dithiocarbamate structure (hydrophilic ferro-di(N-methyl-D-glucamine-dithiocarbamate, or Fe(II)-MGD, and lipophilic ferro-di(diethyldithiocarbamate), or Fe(II)-DETC), are used for spin trapping of nitric oxide (NO) in biologic systems. However, detection efficiency is hampered by a complex redox chemistry for Fe(II)-MGD and by the insolubility of Fe(II)-DETC in water. To circumvent these problems, two new spin trap formulations based on Fe(II)-DETC were developed: a lipid-based carrier system stabilized by lecithin and inclusion complexes in hydroxypropyl-beta-cyclodextrin. The capability of these two systems to trap NO was determined and compared to the standard spin traps in vitro (in the presence of an NO donor) and in vivo (after induction of septic shock in mice). The sensitivity of the detection of NO was significantly increased (by a factor of 4) using the lipid-based carrier systems or inclusion complexes compared to the standard spin trap agents.

Development and evaluation of biocompatible inks for the local measurement of oxygen using in vivo EPR.

In vivo EPR oximetry is a powerful minimally invasive method that allows the measurement of oxygen in tissues through the use of a paramagnetic probe. In the present study, we investigated new strategies for preparing biocompatible inks containing carbon black particles (Printex U), which could be used as oxygen sensors. The carbon black particles were dispersed in solutions of biocompatible polymers of carboxy methyl cellulose (CMC), hydroxypropyl methyl cellulose (HPMC) or polyvinyl pyrrolidone (PVP). A total of 12 polymers with different molecular weights were tested. A physico-chemical characterization of the inks was carried out to assess the sedimentation of the particles, the rheological behavior of these inks, and the relative diffusion of the inks. The preparations with CMC and PVP had the highest viscosity and stability. The presence of the polymers did not modify the calibration curves (EPR linewidth as a function of the pO2) of the carbon black. In vivo, the oxygen sensors were stable for at least one month in muscles as the EPR linewidth remained fully sensitive to induced ischemia or carbogen challenge. The calibration curve was not modified after this period of implantation. A first study of biocompatibility was carried out in vitro (hemolysis and cytotoxicity assay) and in vivo (histological examination). No sign of toxicity was observed using these inks. These preparations are good candidates for future in vivo studies including clinical trials.

Carbon blacks as EPR sensors for localized measurements of tissue oxygenation.

New electron paramagnetic resonance (EPR) oximetry probes were identified in the class of carbon black materials. These compounds exhibit very high oxygen sensitivity and favorable EPR characteristics for biological applications. At low pO(2), the linewidth is particularly sensitive to changes in oxygen tension (sensitivity of 750 mG/mmHg). The application of the probes for oximetry was demonstrated in vivo: the pO(2) was measured in muscle in which the blood flow was temporarily restricted as well as in tumor-bearing mice during a carbogen breathing challenge. The responsiveness to pO(2) was stable in muscle for at least 3 months. No toxicity was observed using these materials in cellular experiments and in histological studies performed 2, 7, and 28 days after implantation. In view of their EPR characteristics (high sensitivity) as well as the well-characterized production procedure that make them available on a large scale, these probes can be considered as very promising tools for future developments in EPR oximetry.