Inhibition of Cytochrome P450 1A2-Mediated Metabolism and Production of Reactive Oxygen Species by Heme Oxygenase-1 in Rat Liver Microsomes

James      R. Reed; George      F. Cawley; Wayne      L. Backes

Abstract

Heme oxygenase-1 (HO-1) is induced in most cell types by many forms of environmental stress and is believed to play a protective role in cells exposed to oxidative stress. Metabolism by cytochromes P450 (P450) is highly inefficient as the oxidation of substrate is associated with the production of varying proportions of hydrogen peroxide and/or superoxide. This study tests the hypothesis that heme oxygenase-1 (HO-1) plays a protective role against oxidative stress by competing with P450 for binding to the common redox partner, the NADPH P450 reductase (CPR) and in the process, diminishing P450 metabolism and the associated production of reactive oxygen species (ROS). Liver microsomes were isolated from uninduced rats and rats that were treated with cadmium and/or β-napthoflavone (BNF) to induce HO-1 and/or CYP1A2. HO-1 induction was associated with slower rates of metabolism of the CYP1A2-specific substrate, 7- ethoxyresorufin. Furthermore, HO-1 induction also was associated with slower rates of hydrogen peroxide and hydroxyl radical production by microsomes from rats induced for CYP1A2. The inhibition associated with HO-1 induction was not dependent on the addition of heme to the microsomal incubations. The effects of HO-1 induction were less dramatic in the absence of substrate for CYP1A2, suggesting that the enzyme was more effective in inhibiting the CYP1A2-related activity than the CPR-related production of superoxide (that dismutates to form hydrogen peroxide).

Keywords: Cytochrome P450, heme oxygenase-1, metabolism, reactive oxygen species, Liver Microsomes, oxidative stress, hydrogen peroxide, super-oxide, cadmium, napthoflavone, 7-ethoxyresorufin, heme catabolism, calcium homeostasis, cytotoxins, cytoprotection, phospholipids, heme, biliverdin, bilirubin, biliverdin reductase, xenobiotics, halothane, acetominophen, carbon tetrachloride, P450-mediated hepatotox-icity, hydroxyl radical, centrifugation, potassium phosphate, carbon-monoxy ferrous complex, western blot densitometry, chemiluminescence, semicarbazide, ammonium ace-tate, formaldehyde, dimethyl sulfoxide, sodium azide, O-dealkylation activity, toxic heavy metal, liver cytosol, metallothionein, lipid, phenacetin, pro-oxidant, CYP1A2-mediated metabolism, xanthine oxidase, cyclooxygenase, oxidative phosphorylation, mitochondrial electron, endoplasmic reticulum, cellular electron transport, electrophilic P450 metabolites