Cortisol metabolism in vitro—III. Inhibition of microsomal 6β—hydroxylase and cytosolic 4-ene-reductase

doi:10.1016/0960-0760(93)90325-Q

The Journal of Steroid Biochemistry and Molecular Biology

Volume 46, Issue 6, December 1993, Pages 827-832

https://doi.org/10.1016/0960-0760(93)90325-Q Get rights and content

Abstract

The in vitro metabolism of cortisol in human liver fractions is highly complex and variable. Cytosolic metabolism proceeds predominantly via A-ring reduction (to give 3α,5β-tetrahydrocortisol; 3α,5β-THF), while microsomal incubations generate upto 7 metabolites, including 6β-hydroxycortisol (6β-OHF), and 6β-hydroxycortisone (6β-OHE), products of the cytochrome P450 (CYP) 3A subfamily. The aim of the present study was, therefore, to examine two of the main enzymes involved in cortisol metabolism, namely, microsomal 6β-hydroxylase and cytosolic 4-ene-reductase. In particular, we wished to assess the substrate specificity of these enzymes and identify compounds with inhibitory potential. Incubations for 30 min containing [³H]cortisol, potential inhibitors, microsomal or cytosolic protein (3 mg), and co-factors were followed by radiometric HPLC analysis. The K_m value for 6β-OHF and 6β-OHE formation was 15.2 ± 2.1 μM (mean ± SD; n = 4) and the V_max value 6.43 ± 0.45 pmol/min/mg microsomal protein. The most potent inhibitor of cortisol 6β-hydroxylase was ketoconazole (K_i = 0.9 ± 0.4 μM; n = 4), followed by gestodene (K_i = 5.6 ± 0.6 μM) and cyclosporine (K_i = 6.8 ± 1.4 μM). Both betamethasone and dexamethasone produced some inhibition (K_i = 31.3 and 54.5 μ, respectively). However, substrates for CYP2C (tolbutamide), CYP2D (quinidine), and CYP1A (theophylline) were essentially non-inhibitory. The K_m value for cortisol 4-ene-reductase was 26.5 ± 11.2 μM (n = 4) and the V_max value 107.7 ± 46.0 pmol/min/mg cytosolic protein. The most potent inhibitors were androstendione (K_i = 17.8 ± 3.3 μM) and gestodene (K_i = 23.8 ± 3.8 μM). Although both compounds have identical A-rings to cortisol, and undergo reduction, inhibition was non-competitive.

References (24)

S.M. Abel et al.
Cortisol metabolism by human liver in vitro. (I). Metabolite identification and inter-individual variability
J. Steroid Biochem. Molec. Biol.
(1992)
U. Fuhr et al.
Biotransformation of caffeine and theophylline in cell lines genetically engineered for expression of single cytochrome P450 isozymes
Biochem. Pharmac.
(1992)
O.H. Lowry et al.
Protein determination with the Folin phenol reagent
J. Biol. Chem.
(1951)
S. Ward et al.
Metabolism of gestodene in human liver microsomes and cytosol in vitro
J. Steroid Biochem. Molec. Biol.
(1993)
G.M. Tomkins
The enzymatic reduction of Δ⁴-3-ketosteroids
J. Biol. Chem.
(1957)
R.B. Iyer et al.
Human hepatic cortisol reductase activities: enzymatic properties and substrate specificities of cytosolic cortisol Δ⁴-5β-reductase and dihydrocortisol-3α-oxidoreductase(s)
Steroids
(1990)
S. Madden et al.
Metabolism of norgestimate by human gastrointestinal mucosa and liver microsomes in vitro
J. Steroid Biochem. Molec. Biol.
(1991)
F.P. Guengerich
Mechanism-based inactivation of human liver microsomal cytochrome P450IIIA4 by gestodene
Chem. Res. Toxic.
(1990)
R. Bocker et al.
In vitro interactions of contraceptive steroids with human liver cytochrome P450 enzymes
Adv. Contracep.
(1991)
M. Murray
P450 enzymes: Inhibition mechanisms, genetic regulation and effects of liver disease
Clin. Pharmacokin.
(1992)

M. Maurice et al.

Effects of imidazole derivatives on cytochrome P450 from human hepatocytes in primary cultures

FASEB J.

(1992)

M.V. Relling et al.

Tolbutamide and mephenytoin hydroxylation by human cytochrome P450 in the CYP2C subfamily

J. Pharmac. Exp. Ther.

(1990)

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Assessment of electrocatalytic hydroxylase activity of cytochrome P450 3A4 (CYP3A4) by means of derivatization of 6β-hydroxycortisol by sulfuric acid for fluorimetric assay
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Citation Excerpt :
The difference in the values may indicate the contribution of H2O2 generated in the electrochemical system to the formation of metabolites, that was shown in the earlier study with CYP3A4 [58]. The KM value obtained in our work was 10 ± 2 μM of hydrocortisone and was similar to that obtained in the microsomal system (15.2 ± 2.1 μM of hydrocortisone) [59], but different from the KM value in overexpressed CYP3A4 microsomes (148 ± 25 μM of hydrocortisone) [6]. We used ketoconazole, a well-known CYP3A4 inhibitor, to demonstrate the possibility of applying the developed approach to search and investigate inhibitors of steroid-metabolizing enzymes.
We used rapid one-step derivatization of 6β-hydroxylated hydrocortisone by sulfuric acid for fluorimetric determination of CYP3A4-dependent hydroxylase reaction in the electrochemical system. We have shown that CYP3A4 substrate – hydrocortisone – and its 6β-hydroxylated product have different emission wavelengths at an excitation λ_ex = 365 nm after treatment with sulfuric acid:ethanol (3:1) mixture (λ_em = 525 ± 2 nm and λ_em = 427 ± 2 nm, respectively). The detection limit for 6β-hydroxycortisol was estimated to be 0.32 μM (corresponding to 0.095 nmol in 300 μL sample) (S/N = 3). Using the fluorimetric method of 6β-hydroxycortisol detection following the electrolysis of hydrocortisone with CYP3A4 immobilized on a screen-printed graphite electrode modified by didodecyldimethylammonium bromide we have calculated the steady-state kinetic parameters of CYP3A4 for hydrocortisone: the maximal rate of the reaction (V_max) as 89 ± 5 pmol of product per min per pmol of electroactive enzyme and the Michaelis constant (K_M) as 10 ± 2 μM. In our system, ketoconazole inhibited hydroxylase activity of CYP3A4 towards hydrocortisone with the IC₅₀ value of 70 ± 5 nM. The approach proposed for determination of the CYP3A4 electrocatalytic activity can be used for throughput screening of different modulators of this cytochrome P450 isozyme during drug development.
Bile acids and glucocorticoid metabolism in health and disease
2019, Biochimica et Biophysica Acta - Molecular Basis of Disease
Citation Excerpt :
The expression of Cyp3A4 – or Cyp3a11 in rodents – in the liver is induced by elevated concentrations of secondary bile acids via the pregnane X receptor (PXR) and by the primary bile acid CDCA via FXR [46]. CYP3A4, however, is not only crucial for catabolism of bile acids when in excess, but also plays a role in catabolism of cortisol [47]. Cortisol as well as cortisone are broken down into tetrahydrometabolites by 5α-reductase and 5β-reductase as well as 3α-hydroxsteroid dehydrogenase (3α-HSD) in the liver [14].
Glucocorticoids are regulators of stress response essential for survival. Liver disease can alter this homeostatic mechanism in patients with liver cirrhosis – a finding that might mirror the controversially discussed condition of critical illness related corticosteroid insufficiency.
Underlying mechanisms might be shared molecular pathways in both bile acid as well as glucocorticoid metabolism at the level of synthesis, catabolism or the hypothalamus and the pituitary gland. Molecular links include the farnesoid X receptor FXR or the G protein-coupled bile acid receptor TGR5 expressed in the liver and the adrenals.
In this review we sum up knowledge on the regulation of adrenal gland function and steroidogenesis, focussing on bile acids and potential alterations under cholestatic conditions, depict molecular links between glucocorticoid and bile acid metabolism and discuss the difficulties of assessment of adrenal function in humans in general and more specifically in liver diseases.
Two surfaces of cytochrome b<inf>5</inf> with major and minor contributions to CYP3A4-catalyzed steroid and nifedipine oxygenation chemistries
2014, Archives of Biochemistry and Biophysics
Citation Excerpt :
Cytochrome P450 3A4 (CYP3A4) constitutes 30% and 70% of the total CYPs in human liver and intestine, respectively, and metabolizes nearly 60% of the drugs currently in use [1]. In addition to its important function in xenobiotic metabolism, CYP3A4 also metabolizes endogenous steroids, including cortisol, testosterone, 17β-estradiol, progesterone, and androstenedione [2–6]. The ability of CYP3A4 to interact simultaneously with multiple substrate molecules and its conformational heterogeneity leads to non-Michaelis–Menten kinetics [6,7].
Conserved human cytochrome b₅ (b₅) residues D58 and D65 are critical for interactions with CYP2E1 and CYP2C19, whereas E48 and E49 are essential for stimulating the 17,20-lyase activity of CYP17A1. Here, we show that b₅ mutations E48G, E49G, D58G, and D65G have reduced capacity to stimulate CYP3A4-catalyzed progesterone and testosterone 6β-hydroxylation or nifedipine oxidation. The b₅ double mutation D58G/D65G fails to stimulate these reactions, similar to CYP2E1 and CYP2C19, whereas mutation E48G/E49G retains 23–42% of wild-type stimulation. Neither mutation impairs the activity stimulation of wild-type b₅, nor does mutation D58G/D65G impair the partial stimulation of mutations E48G or E48G/E49G. For assays reconstituted with a single phospholipid, phosphatidyl serine afforded the highest testosterone 6β-hydroxylase activity with wild-type b₅ but the poorest activity with b₅ mutation E48G/E49G, and the activity stimulation of mutation E48G/E49G was lost at [NaCl] > 50 mM. Cross-linking of CYP3A4 and b₅ decreased in the order wild-type > E48G/E49G > D58G/D65G and varied with phospholipid. We conclude that two b₅ acidic surfaces, primarily the domain including residues D58–D65, participate in the stimulation of CYP3A4 activities. Our data suggest that a minor population of CYP3A4 molecules remains sensitive to b₅ mutation E48G/E49G, consistent with phospholipid-dependent conformational heterogeneity of CYP3A4.
Corticotropic axis in sepsis
2007, Annales d'Endocrinologie
Theoretical calculation of triazolam hydroxylation and endogenous steroid inhibition in the active site of CYP3A4
2007, Biochimica et Biophysica Acta - Proteins and Proteomics
CYP3A4 has unusual kinetic characteristics because it has a large active site. CYP3A4 produced more 4-hydroxytriazolam than α-hydroxytriazolam at concentrations of more than 60 μM triazolam, and different steroids had different inhibitory effects on the system. To clarify these interesting observations, the interactions between substrate and substrate/steroid were investigated by theoretical calculations. When two triazolam molecules were docked into the active site, the distance between the O-atom and the 4-hydroxylated site was less than the distance to the α-hydroxylated site because of interaction between the two triazolam molecules. Estradiol inhibited both α- and 4-hydroxytriazolam formation by 50%. Dehydroepiandrosterone (DHEA) inhibited α-hydroxylation more than 4-hydroxytriazolam formation, whereas aldosterone had no effect. When one triazolam molecule and one steroid molecule were simultaneously docked, estradiol increased the distance between the O-atom and the two hydroxylated sites, DHEA only increased the distance between the O-atom and α-hydroxylated site, and aldosterone did not change the distances. The relevant angles of Fe–O–C in the hydroxylated site of triazolam also widened, together with increased distance. These findings indicate that formation of a substrate and substrate/effector complex in the active site may be a factor for determining the enzyme kinetic parameters of CYP3A4.
Relative adrenal failure in the ICU: An identifiable problem requiring treatment
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The Journal of Steroid Biochemistry and Molecular Biology

Abstract

References (24)

Cortisol metabolism by human liver in vitro. (I). Metabolite identification and inter-individual variability

J. Steroid Biochem. Molec. Biol.

Biotransformation of caffeine and theophylline in cell lines genetically engineered for expression of single cytochrome P450 isozymes

Biochem. Pharmac.

Protein determination with the Folin phenol reagent

J. Biol. Chem.

Metabolism of gestodene in human liver microsomes and cytosol in vitro

J. Steroid Biochem. Molec. Biol.

The enzymatic reduction of Δ⁴-3-ketosteroids

J. Biol. Chem.

Human hepatic cortisol reductase activities: enzymatic properties and substrate specificities of cytosolic cortisol Δ⁴-5β-reductase and dihydrocortisol-3α-oxidoreductase(s)

Steroids

Metabolism of norgestimate by human gastrointestinal mucosa and liver microsomes in vitro

J. Steroid Biochem. Molec. Biol.

Mechanism-based inactivation of human liver microsomal cytochrome P450IIIA4 by gestodene

Chem. Res. Toxic.

In vitro interactions of contraceptive steroids with human liver cytochrome P450 enzymes

Adv. Contracep.

P450 enzymes: Inhibition mechanisms, genetic regulation and effects of liver disease

Clin. Pharmacokin.

Effects of imidazole derivatives on cytochrome P450 from human hepatocytes in primary cultures

FASEB J.

Tolbutamide and mephenytoin hydroxylation by human cytochrome P450 in the CYP2C subfamily

J. Pharmac. Exp. Ther.

Cited by (32)

Assessment of electrocatalytic hydroxylase activity of cytochrome P450 3A4 (CYP3A4) by means of derivatization of 6β-hydroxycortisol by sulfuric acid for fluorimetric assay

Bile acids and glucocorticoid metabolism in health and disease

Two surfaces of cytochrome b<inf>5</inf> with major and minor contributions to CYP3A4-catalyzed steroid and nifedipine oxygenation chemistries

Corticotropic axis in sepsis

Theoretical calculation of triazolam hydroxylation and endogenous steroid inhibition in the active site of CYP3A4

Relative adrenal failure in the ICU: An identifiable problem requiring treatment