Identification of microsomal, organic hydroperoxide-dependent catechol estrogen formation: Comparison with nadph-dependent mechanism

Exogenous aromatic polycyclic hydrocarbons, notably benzo[a]pyrene, can be metabolized by both NADPH-dependent monooxygenase(s) and an organic hydroperoxide (OHP)-dependent mechanism. To determine whether phenolic estrogens, endogenous aromatic polycyclic hydrocarbons, can also be hydroxylated by these alternative pathways, conversion of estradiol (E₂) to 2 and 4-hydroxylated catecholestrogens (2 and 4-OH-CEs) by human placental microsomes was examined under conditions previously shown to support CE formation by NADPH and OHP-dependent mechanisms. CEs were formed under both conditions. Properties of OHP-dependent activity were similar to CE formation by peroxidases and distinct from the NADPH-dependent monooxygenase. NADPH supported only 2-hydroxylation, whereas cumene hydroperoxide supported 2 and 4-hydroxylation equally. Monooxygenase-mediated activity had characteristics of a high-affinity, low-capacity enzyme system (apparent K_m for E₂ = 0.3 μM, and V_max = 31 pmol/mg protein/30 min). Whereas peroxidatic activity had properties of low affinity and high capacity (apparent K_m for E₂ = 55 μM, V_max = 666 pmol/mg protein/10 min). The requirement of peroxidatic activity for oxidizing co-substrate could be met by OHPs but not by H₂O₂. Peroxidatic CE formation could have special functional significance for physiological and pathological consequences of estrogen action since it generates 4-OH-CEs which are both effective catechols and potent, long-acting estrogens. Moreover, it could provide a link through co-oxygenation between estrogens and diverse cellular mechanisms involving generation of OHPs.