Reduction of 7-alkoxyresorufins by NADPH-cytochrome P450 reductase and its differential effects on their O-dealkylation by rat liver microsomal cytochrome P450.

Abstract Antibody-inhibition experiments established that the induction of cytochrome P450c is largely responsible for the marked increase in liver microsomal 7-ethoxyresorufin O-dealkylation in rats treated with 3-methylcholanthrene, whereas the induction of cytochrome P450b and/or P450e is largely responsible for the marked increase in 7-pentoxy-and 7-benzyloxyresorufin O-dealkylation in rats treated with phenobarbital. When reconstituted with NADPH-cytochrome P450 reductase and lipid, purified cytochrome P450c catalyzed the O-dealkylation of 7-ethoxyresorufin at a rate of ~30 nmol/nmol P450/min, which far exceeded the rate catalyzed by either purified cytochromes P450b and P450e or microsomal cytochrome P450c. In contrast, purified cytochromes P450b and P450e were poor catalysts of the O-dealkylation of 7-pentoxy- and 7-benzyloxyresorufin. However, purified cytochrome P450b is an excellent catalyst of several other reactions, such as the N-demethylation of benzphetamine, the hydroxylation of testosterone, and the O-dealkylation of 7-ethoxycoumarin. The low rate of 7-pentoxyresorufin O-dealkylation catalyzed by purified cytochrome P450b did not reflect a requirement for cytochrome b 5 , and could not be ascribed to an artifact of the method used to measure the formation of resorufin. The catalytic activity of purified cytochrome P450b toward 7-pentoxyresorufin was consistently low over a range of substrate and lipid concentrations, and was not stimulated by sodium deoxycholate (which stimulates the N-demethylation of benzphetamine by purified cytochrome P450b). Evidence is presented which indicates that cytochrome P450c catalyzes the O-dealkylation of both the oxidized and reduced forms of 7-ethoxyresorufin, with perhaps a slight preference for the reduced form. In contrast, cytochrome P450b preferentially catalyzes the O-dealkylation of the oxidized form of 7-pentoxyresorufin. Conditions that favored formation of the reduced form of 7-ethoxyresorufin tended to stimulate its O-dealkylation by purified cytochrome P450c, whereas conditions that favored formation of the reduced form of 7-pentoxyresorufin decreased its rate of O-dealkylation by purified cytochrome P450b. Such conditions included a molar excess of NADPH-cytochrome P450 reductase over cytochrome P450, the presence of superoxide dismutase, and the presence of DT-diaphorase (liver cytosol). Both NADPH-cytochrome P450 reductase and DT-diaphorase increased the rate of formation of the reduced form of the 7-alkoxyresorufins, whereas superoxide dismutase decreased its rate of disappearance (because superoxide anion, which is formed during the redox cycling reactions described in the preceding paper, causes a rapid reoxidation of the reduced form of the 7-alkoxyresorufins). Although a molar excess of NADPH-cytochrome P450 reductase is required for purified cytochrome P450b to exert its full catalytic activity toward benzphetamine, testosterone, and 7-ethoxycoumarin, the O-dealkylation of 7-pentoxyresorufin was greatest when the ratio of NADPH-cytochrome P450 reductase-to-cytochrome P450b was less than one. Inasmuch as a molar excess of NADPH-cytochrome P450 reductase compromised the catalytic activity of cytochrome P450b toward 7-pentoxy- and 7-benzyloxyresorufin, it is significant that the molar ratio of cytochrome P450 to NADPH-cytochrome P450 reductase in liver microsomes is greater than 10:1. We speculate that if rat liver microsomes did not contain a molar excess of cytochrome P450 over NADPH-cytochrome P450 reductase, the O-dealkylation of 7-pentoxy- and 7-benzyloxyresorufin would be of little value in monitoring the induction of cytochromes P450b and P450e by phenobarbital.