Metabolic stability of superoxide and hydroxyl radical adducts of a cyclic nitrone toward rat liver microsomes and cytosol: A stopped-flow ESR spectroscopy study

The metabolic stability of the spin adducts derived from the reaction of superoxide and hydroxyl radicals with 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BocMPO) in the presence of rat liver microsomes (RLM) and rat liver cytosol (RLC) was studied by using a stopped-flow device coupled to an electron spin resonance (ESR) spectrometer. The kinetics of the disappearance of the BocMPO-OH and BocMPO-OOH radicals could be followed by ESR spectroscopy with treatment of the ESR data by an appropriate computer program. The presence of cytosol led to a 60-fold decrease of the half-life of BocMPO-OOH with the intermediate formation of BocMPO-OH. This effect of cytosol was due to an ascorbate- and thiol-dependent reduction of BocMPO-OOH. RLC only led to a 5-fold decrease of the half-life of BocMPO-OH that was predominantly due to cytosolic ascorbate. RLM led to a 10-fold decrease of the BocMPO-OOH half-life that was mainly related to a direct reaction of the hydroperoxide function of BocMPO-OOH with cytochrome P450 Fe(III) (P450). Other ferric heme proteins, such as methemoglobin (metHb) and horseradish peroxidase (HRP), as well as hemin itself, exhibited a similar behavior. RLM and metHb showed a much weaker effect on BocMPO-OH half-life (2-fold decrease), whereas RLM in the presence of NADPH caused a greater decrease of the BocMPO-OH half-life (≈ 5-fold). The effect of RLM without NADPH was mainly due to a direct reaction with microsomal P450, whereas the RLM- and NADPH-dependent effect was mainly due to flavin-containing reductases such as cytochrome P450 reductase. These data on the effects of liver subcellular fractions on the half-life of the BocMPO-OOH and the BocMPO-OH spin adducts highlight the role of heme as a biological cofactor involved in the disappearance of such spin adducts. They should be helpful for the design of new spin traps that would form more metabolically stable spin adducts in vitro and in vivo.