Abstract
Recently, we proposed that singlet oxygen (1O2) plays an essential role in microsomal cytochrome P450 (P450)-dependent p-hydroxylation of aniline and O-deethylation of 7-ethoxycoumarin. We then examined whether the role of 1O2 is general in the P450-dependent substrate oxidations. In the present study, we examined ω- and (ω-1)-hydroxylations of lauric acid, O-demethylation of p-nitroanisole, and N-demethylation of aminopyrine in rat liver microsomes. The addition of β-carotene and NaN3 significantly suppressed these reactions in a concentration-dependent manner, and 1O2 during the reactions was detected by ESR spin-trapping using 2,2,6,6-tetramethyl-4-piperidone (TMPD) as a 1O2-spin trapping reagent, where the addition of 1O2 quenchers, SKF-525A as a P450 inhibitor, or p-nitroanisole decreased ESR signal intensities due to TMPD-1O2 adduct. Next, we examined the effect of 1O2 quenchers on P450-dependent reactions in the human liver microsomes, and 1O2 was also indicated to be an active species in substrate hydroxylations and dealkylations such as nifedipine oxidation by CYP3A4. On the basis of the results, we concluded that 1O2 is an essentially important active oxygen species in both rat and human P450-dependent substrate oxidations.
