Unique oxidation of imidazolidine nitroxides by potassium ferricyanide: strategy for designing paramagnetic probes with enhanced sensitivity to oxidative stress

Potassium ferricyanide (PF), routinely employed for the oxidation of sterically-hindered hydroxylamines to nitroxides, is considered to be chemically inert towards the latter. In the present study, we report on an unexpected oxidative fragmentation of the imidazolidine nitroxides containing hydrogen atom in the 4-position of the heterocycle (HIMD) by PF resulting in the loss of the EPR signal. The mechanistic EPR, spectrophotometric, electrochemical and HPLC-MS studies support the assumption that the HIMD fragmentation is facilitated by the proton abstraction from the 4-position of the oxoammonium cation formed as a result of the initial one-electron HIMD oxidation. Increase in steric hindrance around the radical fragment by introducing ethyl substituents decreased the rate of ascorbate-induced HIMD reduction by more than 20 times, but did not affect the rate of ferricyanide-induced HIMD oxidation. This preferential sensitivity of HIMDs to oxidative processes has been used to detect peroxyl radicals in the presence of high concentration of the reducing agent, ascorbate. HIMD-based EPR probes capable to discriminate oxidative and reductive processes might find application in biomedicine and related fields for monitoring the oxidative stress and reactive radical species in biological systems.     

Comparative studies with EPR and MRI on the in vivo tissue redox status estimation using redox-sensitive nitroxyl probes: influence of the choice of the region of interest

In vivo decay rates of a nitroxyl contrast agent were estimated by a MR redox imaging (MRRI) technique and compared with the decay rates obtained by the electron paramagnetic resonance spectroscopy (EPRS) and imaging (EPRI). MRRI is a dynamic imaging technique employing T₁-weighted pulse sequence, which can visualise a nitroxyl-induced enhancement of signal intensity by T₁-weighted contrast. EPR techniques can directly measure the paramagnetic nitroxyl radical. Both the squamous cell carcinoma (SCC) tumour-bearing and normal legs of a female C3H mouse were scanned by T₁-weighted SPGR sequence at 4.7 T with the nitroxyl radical, carbamoyl-proxyl (CmP), as the contrast agent. Similarly, the time course of CmP in normal muscle and tumour tissues was obtained using a 700-MHz EPR spectrometer with a surface coil. The time course imaging of CmP was also performed by 300?MHz CW EPR imager. EPRS and EPRI gave slower decay rates of CmP compared to the MRRI. Relatively slow decay rate at peripheral region of the tumour tissues, which was found in the image obtained by MRRI, may contribute to the slower decay rates observed by EPRS and/or the EPRI measurements. To reliably determine the tissue redox status from the reduction rates of nitroxyls such as CmP, heterogenic structure in the tumour tissue must be considered. The high spatial and temporal resolution of T₁-weighted MRI and the T₁-enhancing capabilities of nitroxyls support the use of this method to map tissue redox status which can be a useful biomarker to guide appropriate treatments based on the tumour microenvironment.     

EPR detection of paramagnetic chromium in liver of fish (Anguilla anguilla) treated with dichromate(VI) and associated oxidative stress responses—Contribution to elucidation of toxicity mechanisms

The impact of chromium (Cr) on fish health has been the subject of numerous investigations, establishing a wide spectrum of toxicity, attributed particularly to the hexavalent form [Cr(VI)]. However, reports on the simultaneous assessment of Cr toxicity in fish and its toxico-kinetics, namely involving metal speciation, are scarce. Therefore, keeping in view the understanding of the mechanisms of Cr(VI) toxicity, this work intended to detect the formation of paramagnetic Cr species in liver of Anguilla anguilla following short-term dichromate(VI) intraperitoneal treatment (up to 180 min), assessing simultaneously the pro-oxidant properties. The formation of Cr(V) and Cr(III) was examined by electron paramagnetic resonance (EPR), as an innovative approach in the context of fish toxicology, and related with the levels of total Cr. Cr(V) was successfully detected and quantified by EPR spectrometry, showing a transient occurrence, mostly between 15 and 90 min post-injection, with a peak at 30 min. The limitations of EPR methodology towards the detection and quantification of Cr(III) were confirmed. Although Cr(VI) exposure induced the antioxidant system in the eel's liver, the oxidative deterioration of lipids was not prevented. Overall, the results suggested that Cr(V), as a short-lived species, did not appear to be directly and primarily responsible for the cellular damaging effects observed, since stress responses persisted up to the end of exposure regardless Cr(V) drastic decay. Though further research is needed, ROS mediated pathways (suggested by superoxide dismutase and catalase activity induction) and formation of Cr(III) complexes emerged as the most plausible mechanisms involved in Cr(VI) toxicity.