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The expression and inducibility of cytochrome P450 proteins belonging to families CYP2, CYP3 and CYP4 in the aorta of rabbits were investigated immunologically using polyclonal antibodies to the hepatic proteins. Antibodies to CYP2B recognised two proteins, only one of which was inducible by phenobarbitone. One protein was detected by anti-CYP2E1 which was modestly inducible by imidazole. A highly expressed protein was recognised by antibodies to CYP3A1, the levels of which diminished following treatment with rifampicin. Finally, antibodies to CYP4A1 immunoreacted with a single protein in the aorta which was refractive to treatment with ciprofibrate. The data demonstrate that the rabbit aorta expresses a number of cytochrome P450 families which, although inducible in the liver, are not always inducible in the aorta.

The specific binding of the archetypal cytochrome P450 3A subfamily (CYP3A) inducer dexamethasone is examined in microsomal fractions since rat and human liver CYP3A is induced by glucocorticoids through a mechanism which is apparently independent of the cytosolic glucocorticoid receptor. Dexamethasone binds in a specific and saturable manner to microsomes with an affinity constant (K d ) of 59 ± 12.9 nM which compares to a K d of 2.3 ± 0.17 nM in cytosol. The total receptor concentrations ([LR] max e ) in microsomes and cytosol are 9.5 ± 1.67 pmols/mg protein and 410 ± 167 fmol/mg protein respectively. The microsomal binding of dexamethasone is antagonised by several transcriptional and/or post-transcriptional CYP3A inducers with decreasing potency pregnenolone 16α carbonitrile > metyrapone > phenobarbitone. Troleandomycin, which indirectly induces CYP3A1 in vivo and by protein stabilisation, does not antagonise the binding of dexamethasone in microsomes. The transcriptional and/or posttranscriptional induction of CYP3A may therefore be associated with the interaction of inducers with a microsomal protein.

LXR, PXR, and PPAR{alpha} are members of a nuclear receptor family which regulate the expression of genes involved in lipid metabolism. Here, we show the administration of T0901317 stimulates PPAR{alpha} gene expression in the small intestine but not in the liver of both normal and FXR-null mice. The administration of LXR specific ligand GW3965, or PXR specific ligand PCN has the same effect, indicating that ligand-dependent activation of LXR and PXR, but not FXR, is responsible for the increased gene expression of PPAR{alpha} in the mouse small intestine.

The intraperitoneal injection of the well known monooxygenase inducers (phenobarbital, β-naphtoflavone, pregnenolone-16α-carbonitrile, benzo(a)-pyrene, methylcholanthrene) elicits a net decrease in the specific binding of estradiol to its cytosol receptor in female rat livers. Amongst the five chemicals tested, only phenobarbital did not exhibit such a phenomenon, but caused a slight increase. This observation was neither due to a competitive inhibition by these compounds, nor to an enhanced metabolism of [ 3 H]-estradiol. Moreover, when this effect was produced by polycyclic hydrocarbons, it was inversely correlated to the activity of aryl hydrocarbon hydroxylase, induced by these same chemicals.

2-Hydroxyestradiol, 2-hydroxyestrone and 2-hydroxy-17α-ethynylestradiol, oxidation products of naturally occurring estrogens and synthetic estrogens in some oral contraceptives were found to be converted by rat liver microsomes to reactive metabolites that become irreversibly bound to microsomal protein. The irreversible binding required microsomes, oxygen and NADPH. The NADPH could be replaced by a xanthine-xanthine oxidase system which is known to generate superoxide anions. The irreversible binding was substantially inhibited by superoxide dismutase, 30% in those incubations containing NADPH and 98% in those incubations containing the xanthine-xanthine oxidase system. Further studies with 2-hydroxyestradiol showed that microsomal cytochrome P-450 was rate limiting in the NADPH-dependent irreversible binding, because the binding was inhibited 62% by an antibody against NADPH-cytochrome c reductase and 70% in an atmosphere of CO:O2 (9:1) when compared to an atmosphere of N2:O2 (9:1). Phenobarbital, a known inducer of cytochrome P-450, had no effect on the irreversible binding of 2-hydroxyestradiol, whereas another inducer of P-450, pregnenolone-16α-carbonitrile, markedly increased the irreversible binding. In contrast, cobaltous chloride, an inhibitor of the synthesis of cytochrome P-450, decreased both P-450 and the irreversible binding. These results are consistent with a mechanism for irreversible binding of estrogens and 2-hydroxyestrogens to microsomes that requires oxidation of the catechol nucleus by cytochrome P-450-generated superoxide anion.

Steroid-16α-hydroxylase activities have been measured in normal and induced rat livers using four different substrates. The male/female activity ratio as well as the induction factor vary with the substrate indicating that steroid-16α-hydroxylase activity is a heterogenous enzyme. Experiments using specific inhibitors led to the conclusion that steroid-16α-hydroxylase is supported by at least two cytochrome P-450 forms, different from the cytochrome P-448.

Summary NADPH-driven lipid peroxidation was not affected by destruction of cytochrome P450 in microsomes prepared from rats treated with 2-allyl-2-isopropylacetamide. Pretreatment of animals with phenobarbital, pregnenolone-16α-carbonitrile or 3-methylcholanthrene, compounds which markedly increase the microsomal content of specific forms of cytochrome P450, had no subsequent effect upon lipid peroxidation. These results are not consistent with the hypothesis that cytochrome P450 catalyzes the propagation of lipid peroxidation within intact microsomal membranes.

The fate of hydrogen peroxide has been investigated in rat liver microsomes. The net rate of formation of H2O2 appears to be independent of concomitant substrate hydroxylation in microsomes from controls and phenobarbital treated animals. If rats are pretreated with Pregnenolone-16α-carbonitrile, H2O2 formation increases significantly during N-demethylation of aminopyrine. However, H2O2 is consumed in microsomes from 3-Methylcholanthrene treated rats if aminopyrine and NADPH are present. Since the H2O2 formation and consumption are dependent on induction by different agents and on presence of substrates, its fate might be linked to the spin state of cytochrome P-450.

Erythromycin derivatives having lost the cladinose moiety, erythralosamine and its mono- and diacetate, are strong inducers of liver cytochrome P-450, better than troleandomycin, in rats. The major cytochrome P-450 form induced by all these macrolides is electrophoretically and immunologically indistinguishable from the major form induced in rats by pregnenolone carbonitrile. This form is particularly able to metabolize the macrolides and to lead to the corresponding 456 nm absorbing cytochrome P-450 metabolite complexes in vivo and in vitro.