Margaret Hirst

Ketoconazole blocks adrenal steroidogenesis by inhibiting cytochrome P450-dependent enzymes.

Ketoconazole has recently been shown to interfere with steroidogenesis in patients and rat in vitro systems. In this study we attempted to elucidate the site of inhibition in the adrenal gland. Although ketoconazole impaired adrenocorticotropic hormone stimulated cyclic (c)AMP production, dibutyrl cAMP addition did not bypass the steroidogenic blockade indicating that the critical ketoconazole-inhibited step was distal to cAMP. Addition of radiolabeled substrates to isolated adrenal cells and analysis of products by high performance liquid chromatography demonstrated a ketoconazole block between deoxycorticosterone (DOC) and corticosterone. This 11-hydroxylase step is carried out by a P450-dependent mitochondrial enzyme. No restriction of progesterone or pregnenolone conversion to DOC was detected, steps carried out by non-P450-dependent microsomal enzymes. Inhibition of cholesterol conversion to pregnenolone by mitochondrial fractions indicated a second block at the side chain cleavage step, another mitochondrial P450-dependent enzyme. Adrenal malate dehydrogenase, a non-P450-dependent mitochondrial enzyme was not inhibited while renal 24-hydroxylase, a P450-dependent mitochondrial enzyme in another organ, was blocked by ketoconazole. We conclude that ketoconazole may be a general inhibitor of mitochondrial P450 enzymes. This finding suggests that patients receiving ketoconazole be monitored for side effects relevant to P450 enzyme inhibition. Further, we raise the possibility that this drug action may be beneficially exploited in situations where inhibition of steroidogenesis is a therapeutic goal.

Inhibition of steroidogenic cytochrome P-450 enzymes in rat testis by ketoconazole and related imidazole anti-fungal drugs

Ketoconazole, an imidazole antifungal drug, has previously been shown to diminish testosterone and cortisol production in patients as well as rat and mouse cells in vitro. Inhibition of adrenal mitochondrial cytochrome P-450 enzymes was demonstrated. In this study we tested several imidazole antifungal drugs and examined the individual steps in testicular steroidogenesis to determine which enzymes in the androgen pathway were blocked. In addition, we studied 25-hydroxyvitamin D 24-hydroxylase activity in cultured pig kidney cells (LLC-PK1) to assess a mitochondrial P-450 enzyme in another organ. All imidazoles tested inhibited both total testosterone production and 24-hydroxylase activity but the relative potencies differed. We next studied the individual testicular enzymatic steps between cholesterol and testosterone. Ketoconazole inhibited cholesterol-side-chain-cleavage enzyme (mitochondrial) and C-17,20 lyase (microsomal). The three inhibited enzymes (two testicular and one renal) are all P-450 cytochromes. Testicular 17-hydroxylase, also a P-450 cytochrome, was not inhibited even at high doses of ketoconazole. This is an interesting finding because the testicular hydroxylase and lyase have been shown to be a single protein. Non-cytochrome P-450 enzymes in the androgen pathway were not inhibited. The results demonstrate that several imidazole antifungal drugs all inhibit both microsomal and mitochondrial cytochrome P-450 enzymes in multiple organs.

Glucocorticoids down-regulate the number of 1,25-dihydroxyvitamin D3 receptors in mouse intestine☆

This study examined the hypothesis that glucocorticoids might regulate intestinal receptors for 1,25(OH)2vitamin D3. Mice were treated with saline vehicle or dexamethasone in doses of 50–200 μg/100 gm body weight for 7 days. A dose-dependent fall in the number of receptors was found in dexamethasone treated animals (∼30%); there was no change in the receptor affinity for 1,25(OH)2D3 (0.2 nM). The decline in receptor number required 5–7 days of dexamethasone treatment depending on dose. Adrenalectomy led to a 30% rise in receptor number which could be prevented by the 50 μg dose of dexamethasone. Receptor changes of equal magnitude were exhibited in vitamin D replete or deplete mice.

1,25-Dihydroxyvitamin D3 receptors in mouse colon

Mouse colon possesses a cytoplasmic receptor-like binder for 1,25-dihydroxy vitamin D3 which appears similar to the receptors previously described in other more classical target organs. The properties of this receptor include a sedimentation coefficient of 3.2S in sucrose gradients prepared in 0.3 M KCl, an equilibrium dissociation constant of 0.2 nM and a competitive profile which demonstrates a substantial binding preference for 1,25-dihydroxy vitamin D3 over other vitamin D3 analogues. The concentration of receptor sites was assessed along the entire gastrointestinal tract and found to be in the sequence: duodenum > jejunum > ileum > cecum = proximal colon > distal colon; esophagus and stomach possessed only negligible numbers of binding sites.

Cleavage of the rat intestinal 1,25-dihydroxyvitamin D3 receptor by an endogenous protease to a form with defective DNA binding.

In this report we describe a form of the 1,25(OH)2D3 receptor which no longer binds to DNA. The defective form of the receptor was produced by the action of an endogenous protease. Rat intestinal receptors, obtained by a two-step procedure of a low salt homogenization followed by extraction of the chromatin pellet with high salt, fail to bind to DNA-cellulose. Inclusion of various serine protease inhibitors during the preparation protects against the loss of DNA binding. Sedimentation analysis in sucrose gradients indicates that the defective receptor is measurably smaller than the native receptor and is unable to aggregate normally under low salt conditions. The size difference, as determined by gel chromatography, is approximately 9,000 Da (56,000 for the protected receptor, 47,000 for the cleaved form). The elution from DEAE-cellulose indicates that the overall charge of both intact and cleaved receptor forms is very similar. Cell fractionation and mixing experiments suggest the enzyme may be located in the lysosomal compartment, organelles which are susceptible to breakage during the extraction procedure. The results demonstrate that an endogenous enzyme preferentially cleaves the 1,25(OH)2D3 DNA binding site resulting in a receptor with altered characteristics. Such an enzymatic activity has not been previously described for the 1,25(OH)2D3 receptor from other tissues or species. Since rat intestine is a classically studied target organ, these findings have additional relevance in receptor purification or other studies to characterize the receptor.