Measurement of Oxidative Stress by Epr Radical-Probe Technique

Abstract

An EPR method for the measurement of the oxidative stress status in biological systems is described. The method is based on the X-band EPR detection of a persistent nitroxide generated under physiological or pseudophysiological conditions by oxidation of a highly lipophylic hydroxylamine probe. The probe employed is bis(lhydroxy-2,2,6,6-tetramethyl-4-piperidinyl)-decandioate which is administrated as hydrochloride salt. This probe is able to give a fast reaction with the majority of radical species involved in the oxidative stress. Furthermore, it crosses cell membranes and distributes in a biological environment without the need to alter or destroy compartmentation. The method is therefore suitable for quantitative measurements of ROS and can be applied to human tissues in real clinical settings. It has been successfully employed in systems of growing complexity and interest, ranging from subcellular fractions to whole animals and human liver.

Modulation of cytochrome P450 and induction of DNA damage in Cyprinus carpio exposed in situ to surface water treated with chlorine or alternative disinfectants in different seasons

“…CYP 2E1, Glutathione S-transferase theta-1) are responsible for the primary oxidation and activation of THMs, alkanes, alkenes, aromatic and halogenated hydrocarbons, to mutagenic and carcinogenic compounds, and several chemical species in the DBP mixture are potential CYP substrates, any perturbation of their activity might result in an increase of highly toxic metabolite production [21]. Moreover, an up-regulation of CYP isoforms is involved in reactive oxygen species (ROS) generation, thus providing further harmful factors for human health [57,58].…”

Perturbation of cytochrome P450, generation of oxidative stress and induction of DNA damage in Cyprinus carpio exposed in situ to potable surface water

“…However, it was recently discovered that the regulation of various CYP isoforms (including CYP1A, CYP2B, CYP3A, CYP4A, and virtually any CYP) generate very large amounts of ROS [48][49][50]. Induction of ROS generation by any of the disinfectants (from +70% for PAA to +280% for NaClO) have some importance, yet these increases are not as massive as variations observed in previous investigations in different animal models treated with other xenobiotics or with other environmental pollutants [42,[51][52][53]. This possibly reflects the very complex activation/suppression pattern observed herein for the many enzymatic activities directly or indirectly involved in ROS generation.…”

“…Microsomes were incubated at 37 • C directly in standard EPR capillary tubes in 0.01 M Na + /K + phosphate buffer (pH 7.4) in the presence of 1 mM NADPH and a 0.5 mM hydroxylamine probe, bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl)decandioate, synthesized as previously described [42]. After mixing, the samples were immediately placed within the spectrometer's EPR cavity.…”

“…The relative rate of ROS generation by liver microsomes obtained from control and 10-day-exposed C. carpio to NaClO, ClO 2 or PAA were obtained, using the EPR radical-probe technique, by monitoring the time evolution of the first EPR spectral line of the nitroxide radical generated by ROS-mediated oxidation of the hydroxylamine probe [42]. The results summarized in Table 3 indicate that fish exposed to the test compounds generally showed significantly higher rates of ROS generation as compared to non-exposed controls (p < 0.05).…”

Determination of mitochondrial reactive oxygen species: methodological aspects

“…EPR spectroscopy has the specific advantage of a direct method for free radical detection (for a recent review see [62]). The unpaired electron confers to free radicals the property of paramagnetic resonance.…”