In vitro, cytochrome b 5 modulates the rate of cytochrome P450-dependent mono-oxygenation reactions. However, the role of this enzyme in determining drug pharmacokinetics in vivo and the consequential effects on drug absorption distribution, metabolism, excretion, and toxicity are unclear. In order to resolve this issue, we have carried out the conditional deletion of microsomal cytochrome b 5 in the liver to create the hepatic microsomal cytochrome b 5 null mouse. These mice develop and breed normally and have no overt phenotype. In vitro studies using a range of substrates for different P450 enzymes showed that in hepatic microsomal cytochrome b 5 null NADH-mediated metabolism was essentially abolished for most substrates, and the NADPH-dependent metabolism of many substrates was reduced by 50 -90%. This reduction in metabolism was also reflected in the in vivo elimination profiles of several drugs, including midazolam, metoprolol, and tolbutamide. In the case of chlorzoxazone, elimination was essentially unchanged. For some drugs, the pharmacokinetics were also markedly altered; for example, when administered orally, the maximum plasma concentration for midazolam was increased by 2.5-fold, and the clearance decreased by 3.6-fold in hepatic microsomal cytochrome b 5 null mice. These data indicate that microsomal cytochrome b 5 can play a major role in the in vivo metabolism of certain drugs and chemicals but in a P450-and substrate-dependent manner.Microsomal cytochrome b 5 is a 15.2-kDa hemoprotein, located in the endoplasmic reticulum with its obligate electron donor, cytochrome b 5 reductase. This protein is involved in fatty acid desaturation, reactivation of methemoglobin to hemoglobin, and electron transfer into the cytochrome P450 system (1-4).The role of cytochrome b 5 in cytochrome P450 monooxygenase reactions has been controversial for nearly 40 years (5, 6). Depending on the cytochrome P450 involved, the experimental conditions, and the substrate utilized, cytochrome b 5 has been shown to stimulate or inhibit cytochrome P450 reactions (2, 6). Indeed, in the case of cytochrome P450 CYP2B4, activity can be both stimulated or inhibited by the presence of the enzyme, depending on the concentration of cytochrome b 5 (7, 8). Cytochrome b 5 has been shown to affect the in vitro activities of a wide range of human P450s, such as CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4, as well as the metabolism of an extensive number of commonly used drugs (9, 10). Although the mechanism(s) of the stimulatory effect of cytochrome b 5 on P450 metabolism appear complex, it is widely accepted that the P450 catalytic cycle involves the sequential transfer of two electrons from NADPH via P450 oxidoreductase (POR).2 Hildebrandt and Estabrook (5) concluded that cytochrome b 5 /cytochrome b 5 reductase was able to supply the second (rate-limiting) electron from NADH and possibly in a more rapid manner. However, recent data from Waskell et al. (8) suggest that cytochrome b 5 and POR are capable of reducing P450s at muc...

This is a report on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the Experimental Biology 2012 meeting in San Diego, California, on April 25, 2012. The symposium speakers summarized and critically evaluated our current understanding of the physiologic, pharmacological, and toxicological roles of NADPH-cytochrome P450 oxidoreductase (POR), a flavoprotein involved in electron transfer to microsomal cytochromes P450 (P450), cytochrome b 5 , squalene mono-oxygenase, and heme oxygenase. Considerable insight has been derived from the development and characterization of mouse models with conditional Por deletion in particular tissues or partial suppression of POR expression in all tissues. Additional mouse models with global or conditional hepatic deletion of cytochrome b 5 are helping to clarify the P450 isoform-and substrate-specific influences of cytochrome b 5 on P450 electron transfer and catalytic function. This symposium also considered studies using siRNA to suppress POR expression in a hepatoma cell-culture model to explore the basis of the hepatic lipidosis phenotype observed in mice with conditional deletion of Por in liver. The symposium concluded with a strong translational perspective, relating the basic science of human POR structure and function to the impacts of POR genetic variation on human drug and steroid metabolism.