Expression, function and regulation of mouse cytochrome P450 enzymes: comparison with human P450 enzymes.

The present review focuses on the expression, function and regulation of mouse cytochrome P450 (Cyp) enzymes. Information compiled for mouse Cyp enzymes is compared with data collected for human CYP enzymes. To date, approximately 40 pairs of orthologous mouse-human CYP genes have been identified that encode enzymes performing similar metabolic functions. Recent knowledge concerning the tissue expression of mouse Cyp enzymes from families 1 to 51 is summarized. The catalytic activities of microsomal, mitochondrial and recombinant mouse Cyp enzymes are discussed and their involvement in the metabolism of exogenous and endogenous compounds is highlighted. The role of nuclear receptors, such as the aryl hydrocarbon receptor, constitutive androstane receptor and pregnane X receptor, in regulating the expression of mouse Cyp enzymes is examined. Targeted disruption of selected Cyp genes has generated numerous Cyp null mouse lines used to decipher the role of Cyp enzymes in metabolic, toxicological and biological processes. In conclusion, the laboratory mouse is an indispensable model for exploring human CYP-mediated activities.

Synthesis and properties of the epimeric 6-hydroperoxyandrostenediones, new substrates/inhibitors of human placental aromatase.

We have recently reported the formation in bovine adrenals and in rat liver of 6 beta-hydroxy-, and 6-oxoprogesterone via the 6 beta-hydroperoxy intermediate. The presence of steroid hydroperoxides in animal tissues, however transient it may be, is not devoid of physiologic significance in view of their characteristic property as potential radical initiators. Since 6-hydroperoxides of androgens have not previously been described, we have synthesized the 2 epimeric 6 alpha-, and 6 beta-hydroperoxy-4-androstene-3,17-diones by oxygenation of 5-androstene-3,17-dione in an aprotic solvent system in the presence of dibenzoyl-peroxide. Their chemical identity and chirality were established by IR, NMR, GC-MS, and by reduction to the known 6 alpha and 6 beta-alcohols. These hydroperoxide stereoisomers could only be separated without decomposition by HPLC using a non-aqueous mobile phase. In our search for a natural, non-estrogenic inhibitor of human placental aromatase, we have studied the effect on this enzyme complex of 6 alpha- and 6 beta-OOH-androstenedione, as well as of their corresponding 6-hydroxy and 6-oxo metabolites. Aromatase activity was measured by a slightly modified version of Thompson and Siiteri's original assay based on 1 beta,2 beta-tritium exchange to 3H water. The C-6 oxygenated androgens were found to competitively inhibit the aromatase reaction in the following descending order: 6-oxo greater than 6 beta-OH greater than 6 alpha-OOH greater than 6 beta-OOH showing Ki values of resp. 2.5, 5.0, 6.5 and 7.5 microM, suggesting that they are interacting with the same active site. Moreover, both 6 alpha- and 6 beta-hydroperoxyandrostenedione are active substrates for the aromatase, giving KM values of 2.8 and 2.5 microM respectively.

The involvement of cytochrome P-450 in hepatic microsomal steroid hydroxylation reactions supported by sodium periodate, sodium chlorite, and organic hydroperoxides.

The mechanism of steroid hydroxylation in rat liver microsomes has been investigated by employing NaIO4, NaClO2, and various organic hydroperoxides as hydroxylating agents and comparing the reaction rates and steroid products formed with those of the NADPH-dependent reaction. Androstenedione, testosterone, progesterone, and 17beta-estradiol were found to act as good substrates. NaIO4 was by far the most effective hydroxylating agent followed by cumene hydroperoxide, NADPH, NaClO2, pregnenolone 17alpha-hydroperoxide, tert-butyl hydroperoxide, and linoleic acid hydroperoxide. Androstenedione was chosen as the model substrate for inducer and inhibitor studies. The steroid was converted to its respective 6beta-, 7alpha, 15-, and 16alpha-hydroxy derivatives when incubated with microsomal fractions fortified with hydroxylating agent. Evidence for cytochrome P-450 involvement in androstenedione hydroxylation included a marked inhibition by substrates and modifiers of cytochrome P-450 and by reagents which convert cytochrome P-450 to cytochrome P-420. The ratios of the steroid products varied according to the type of hydroxylating agent used and were also modified by in vivo phenobarbital pretreatment. It was suggested that multiple forms of cytochrome P-450 exhibiting different affinities for hydroxylating agent are responsible for these different ratios. Horse-radish peroxidase, catalase, and metmyoglobin could not catalyze androstenedione hydroxylation. Addition of NaIO4, NaClO2, cumene hydroperoxide and other organic hydroperoxides to microsomal suspensions resulted in the appearance of a transient spectral change in the difference spectrum characterized by a peak at about 440 nm and a trough at 420 nm. The efficiency of these oxidizing agents in promoting steroid hydroxylation in microsomes appeared to be related to their effectiveness in eliciting the spectral complex. Electron donors, substrates, and modifiers of cytochrome P-450 greatly diminished the magnitude of the spectral change. It is proposed that NaIO4, NaClO2, and organic hydroperoxides promote steroid hydroxylation by forming a transient ferryl ion (compound I) of cytochrome P-450 which may be the common intermediate hydroxylating species involved in hydroxylations catalyzed by cytochrome P-450.