Microsomes represent a potential source of reactive oxygen species (ROS) via cytochrome P450: the NADPH-dependent reduction of O2 by cytochrome P450 in the presence and absence of substrate to superoxide anion radical and to hydrogen peroxide is well documented for all forms of the enzyme. Hydrogen peroxide and superoxide production occur either by decomposition of the ferrous-dioxygen complex (oxygenated P450) or by release from the peroxy-ferric intermediate (peroxy P450) formed during the catalytic cycle. Compared with other forms of P450,<br/>CYP2E1 exhibits a higher rate of oxidase activity when purified and microsomes from animals pretreated with inducers of CYP2E1 also exhibit much greater rates of NADPH oxidation than microsomes from untreated animals. In this work, we evaluated the generation of superoxide anion by insect microsomes engineered to express CYP2E1 using the spin probe 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CPH) and electron paramagnetic resonance spectrometry (EPR). CYP2E1-expressing microsomes oxidized CPH to the nitroxide CP in the presence of NADPH as a source of electrons. The oxidation of CPH was blocked by superoxide dismutase, but not by hydrogen peroxide or the iron chelator DTPA. Control microsomes (not expressing any cytochrome P450 enzyme) did not oxidize CPH in the presence or absence of NADPH. These results suggest that the spin probe technique is an effective tool to quantify specifically superoxide anion generation by CYP2E1.

Evaluation of the generation of superoxide anion by CYP2E1 using the spin probe CPH

253rd National Meeting (2017)

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