Comparison of the Stimulatory and Inhibitory Effects of Steroid Hormones and α-Naphthoflavone on Steroid Hormone Hydroxylation Catalyzed by Human Cytochrome P450 3A Subfamilies

Abstract

The inhibitory and stimulatory effects of steroid hormones and related compounds on the hydroxylation activity at the 6β-position of two steroid hormones, progesterone and testosterone, by CYP3A4, polymorphically expressed CYP3A5, and fetal CYP3A7 were compared to clarify the catalytic properties of the predominant forms of the human CYP3A subfamily. Hydroxylation activities of progesterone and testosterone by CYP3A4, CYP3A5, and CYP3A7 were estimated using HPLC. The Michaelis constants (K_m) for progesterone 6β-hydroxylation by CYP3A5 were markedly decreased in the presence of dehydroepiandrosterone (DHEA) and α-naphthoflavone (ANF), whereas progesterone and DHEA competitively inhibited testosterone 6β-hydroxylation mediated by CYP3A4, and progesterone competitively inhibited CYP3A5-mediated activity, which was weaker than that for CYP3A4. ANF noncompetitively inhibited testosterone 6β-hydroxylation mediated by both CYP3A4 and CYP3A5. Progesterone and testosterone 6β-hydroxylation mediated by CYP3A7 was inhibited or unaffected by DHEA, pregnenolone, and ANF. These results suggested that DHEA and ANF stimulated progesterone 6β-hydroxylation by CYP3A5 but not by CYP3A4 and CYP3A7; however, progesterone, DHEA, and ANF inhibited testosterone 6β-hydroxylation mediated by all CYP3A subfamily members. The inhibitory/stimulatory pattern of steroid–steroid interactions is different among CYP3A subfamily members and CYP3A5 is the most sensitive in terms of activation among the CYP3A subfamily members investigated.

INTRODUCTION

Cytochrome P450 (CYP or P450) 3A4, one of most important human P450 enzymes, accounts for approximately 30% of all P450 enzymes in the human liver microsomes,^1,2) and is responsible for more than 50% of the oxidation of clinically used pharmaceuticals.³⁾ CYP3A4 and polymorphically expressed CYP3A5⁴⁾ are 83% homologous in terms of amino acid sequences, however, we previously demonstrated that some differences in the reported parameters including the Michaelis constant (K_m), maximal velocity (V_max or k_cat), intrinsic clearance (CL_int, calculated by dividing k_cat by K_m) and inhibition constant (K_i) values for reactions mediated by CYP3A4 and CYP3A5 can be identified, although CYP3A4 and CYP3A5 have overlapping substrate specificities^5,6); findings that are also consistent with those reported in other reviews.^7,8) CYP3A7 is the major form of P450 expressed in the human fetal liver (>36% of the total P450 in fetal liver); however, CYP3A7 presents in only trace amounts in the adult liver.^9,10) The amino acid sequence of CYP3A7 shows 88% similarity to that of CYP3A4.¹¹⁾

Various endogenous steroid hormones are metabolized by drug-metabolizing P450s such as CYP2C, CYP2D, and CYP3A subfamilies as well as steroidogenic P450s, including CYP11B1, CYP11B2, CYP17A1, CYP19A1, and CYP27A1.^12,13) In terms of 6β-hydroxylation, testosterone is predominantly metabolized by the CYP3A subfamily^12,13) and is recommended as a preferred reaction of CYP3A for in vitro studies in accordance with the guidelines for new drug applications regarding in vitro experiments of drug–drug interactions regulated by the U.S. Food and Drug Administration, European Medicines Agency, and Japanese Ministry of Health, Labour, and Welfare.^14–16) Progesterone, a progestational hormone, is hydroxylated not only at the C17 and C21 positions by steroidogenic CYP17A1 and CYP21A2, respectively, but also at the C2β, C6, C16α, and C21 positions by drug-metabolizing P450s, including CYP3A4.^12,13) Because cortisol is a stress hormone biotransformed by the adrenal glands and is released for various reasons including the physiological response to stress,¹⁷⁾ this steroid hormone is used as a biomarker of psychophysiological stress.^18,19) Furthermore, the ratio of 6β-hydroxycortisol to cortisol in urine or plasma is proposed as an in vivo endogenous marker of CYP3A4 metabolic activity.^20–22) CYP3A5 and CYP3A7 as well as CYP3A4 catalyze the oxidation of various compounds, including the 6β- and 16α-hydroxylation of endogenous steroid hormones such as testosterone, cortisol, and dehydroepiandrosterone (DHEA).^10,23,24) We recently compared the enzymatic kinetic parameters, such as K_m and k_cat values, for the 6β-hydroxylation of progesterone, testosterone, and cortisol mediated by CYP3A4, CYP3A5, and CYP3A7.^25,26)

We previously found that steroid hormones, such as progesterone, testosterone, and α-naphthoflavone (ANF), stimulate CYP3A4-mediated metabolism, including nifedipine oxidation and/or 7-benzyloxyresorufin O-debenzylation.²⁷⁾ In addition, many studies have demonstrated the activation of CYP3A4-mediated metabolic reactions, including the hydroxylation of steroid hormones such as progesterone.^28,29) ANF is a famous activator of various CYP3A4-mediated reactions including aflatoxin B₁ 8,9-epoxidatin, 17β-estradiol 2-hydroxylation, and phenanthrene 9,10-dihydrodiol formation in addition to the reactions described above.²⁸⁾ The activation has similarly been demonstrated for other P450s, including CYP1A2, CYP2C9, CYP2D6, and CYP3A7,^28,29) However, few studies have reported the activation of metabolic activities mediated by CYP3A5 and CYP3A7, although it has been reported that the activation of sulfate conjugates of steroid hormones, such as DHEA and pregnenolone, stimulates CYP3A7-mediated carbamazepine 10,11-epoxidation.²⁹⁾ In addition, we recently found that testosterone stimulated progesterone 6β-hydroxylation mediated by CYP3A5 and CYP3A7 as well as CYP3A4, although progesterone inhibited testosterone 6β-hydroxylation.²⁶⁾

Pregnenolone is biotransformed from cholesterol by CYP11A1, and DHEA is formed from pregnenolone via 17α-hydroxypregnenolone by CYP17A1.¹³⁾ As mentioned above, ANF is a reported activator of CYP3A4.²⁷⁾ Thus, in the present study, we compared the effects of these steroid hormones and ANF on the 6β-hydroxylation of progesterone and testosterone mediated by CYP3A4, CYP3A5, and CYP3A7. These results would be a useful resource for clarifying the effect of CYP3A5 polymorphism and fetal CYP3A7 expression on the drug–drug interaction (especially, cooperation) with various compounds including steroid hormones.

MATERIALS AND METHODS

Materials

Easy CYP CYP3A4R, CYP3A5R, and CYP3A7R, expressed in recombinant Escherichia coli, which were co-expressed with reduced form of nicotinamide adenine dinucleotide phosphate (NADPH)-P450 reductase (high reductase Bactosomes) but not with cytochrome b₅, were obtained from Cypex Limited (Dundee, U.K.).³⁰⁾ 6β-Hydroxyprogesterone and 6β-hydroxytestosterone were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.) and Steraloids Inc. (Newport, RI, U.S.A.), respectively. Progesterone, pregnenolone, DHEA, and ANF were obtained from Tokyo Chemical Industry (Tokyo, Japan). Cortisol and deoxycorticosterone (21-hydroxyprogesterone) were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan), and testosterone was obtained from Nacalai Tesque (Kyoto, Japan). All other chemicals used were of the highest quality commercially available.

Hydroxylation Activities of Steroid Hormones

Progesterone and testosterone 6β-hydroxylation activities were determined as described previously.^{25,26,31–33)} Briefly, the incubation mixtures (final volume, 0.5 mL) contained 10 nM CYP3A4, CYP3A5, or CYP3A7; 1 mM NADPH; and 100 mM phosphate buffer (pH 7.4). In the preliminary experiments, the linearity of the reaction with incubation time and P450 concentration was confirmed for each CYP3A subfamily member.^{25,26,31–33)} All data were analyzed using the average of duplicate or triplicate measurements. K_m, k_cat, and K_i values were estimated by fitting to Michaelis–Menten kinetics by means of nonlinear least squares regression analysis (MULTI³⁴⁾).

RESULTS

The effects of steroid hormones and ANF on progesterone and testosterone 6β-hydroxylation activities by CYP3A4, CYP3A5, and CYP3A7 were estimated at substrate concentrations of 10 µM (for CYP3A4 and CYP3A5) or 20 µM (for CYP3A7) for progesterone and 100 µM for testosterone (Table 1). The substrate concentrations were approximately equal to or below the K_m values for each CYP3A subfamily member.^25,26) Interestingly, DHEA markedly increased progesterone 6β-hydroxylation activities mediated by CYP3A5 but not those by CYP3A4 and CYP3A7, although DHEA inhibited testosterone 6β-hydroxylation mediated by all CYP3A subfamilies investigated. ANF inhibited the 6β-hydroxylation of progesterone and testosterone mediated by all CYP3A subfamilies, except for an increase of 40% in the CYP3A5-mediated progesterone 6β-hydroxylation activity by ANF at 1 µM. Pregnenolone inhibited CYP3A4-mediated 6β-hydroxylation of both progesterone and testosterone and CYP3A7-mediated 6β-hydroxylation of progesterone, whereas the CYP3A5-mediated reaction was not inhibited. In all experiments, 21-hydroxyprogesterone was not detected from progesterone when incubated with all CYP3A subfamilies (data not shown).

Table 1. Effect of Steroid Hormones and ANF on the 6β-Hydroxylation of Progesterone and Testosterone Mediated by CYP3A4, CYP3A5, and CYP3A7

Substrate	Effector	CYP3A	% of the velocity in the absence of effector
			1 µM*	10 µM*	25 µM*	100 µM*	250 µM*
Progesterone	DHEA	CYP3A4	109.3	120.4	—	51.1	—
		CYP3A5	115.1	136.0	—	156.0	—
		CYP3A7	100.3	63.0	—	33.2	—
	ANF	CYP3A4	90.8	59.0	—	27.7	—
		CYP3A5	140.3	66.1	—	12.9	—
		CYP3A7	83.1	38.8	—	22.4	—
	Pregnenolone	CYP3A4	87.5	63.4	—	40.5	—
		CYP3A5	120.9	107.4	—	118.4	—
		CYP3A7	90.2	64.7	—	55.7	—
Testosterone	DHEA	CYP3A4	94.2	79.6	—	41.5	—
			—	—	69.9	58.0	37.8
		CYP3A5	—	—	92.0	78.5	70.5
		CYP3A7	101.9	95.2	—	48.7	—
	ANF	CYP3A4	81.5	45.4	—	6.8	—
		CYP3A5	86.5	41.7	—	4.2	—
		CYP3A7	106.7	78.6	—	38.8	—
	Pregnenolone	CYP3A4	95.1	83.5	—	51.6	—
		CYP3A5	104.3	103.2	—	82.6	—
		CYP3A7	113.7	87.7	—	105.1	—

The effect of steroid hormones on CYP3A4, CYP3A5, and CYP3A7 activities were determined at substrate concentrations of 10 µM (for CYP3A4 and CYP3A5) or 20 µM (for CYP3A7) for progesterone and 100 µM for testosterone. DHEA: dehydroepiandrosterone. ANF: α-naphthoflavone, HLM: human liver microsomes. *Concentrations of steroid hormones and ANF as effectors (inhibitors and/or activators). Underline: more than 30% increase.

Therefore, the effects of DHEA and ANF on the progesterone 6β-hydroxylation mediated by CYP3A4 and/or CYP3A5 were investigated in detail (Fig. 1). The K_m values for CYP3A5 decreased to 31% that of the control in the presence of DHEA at concentrations below 10 µM, whereas the k_cat value gradually decreased with increasing DHEA. Thus, k_cat/K_m values for CYP3A5 increased by a maximum of 188%. For ANF, the K_m values for both CYP3A4 and CYP3A5 decreased to 63 and 33% that of the control, respectively, at concentrations of 2.5–5 µM. The k_cat value for both CYP3A subfamilies were decreased to 44–67% in the presence of DHEA. Thus, k_cat/K_m values for CYP3A5, but not for CYP3A4, increased by a maximum of 132%.

Fig. 1. Effect of DHEA and ANF on Progesterone 6β-Hydroxylation Mediated by CYP3A4 and CYP3A5

V_max and K_m values are means ± standard deviation of the data set using a nonlinear kinetic analysis from mean values obtained in duplicate or triplicate at each substrate/effector concentration.

In contrast, progesterone and DHEA competitively inhibited testosterone 6β-hydroxylation mediated by CYP3A4 with K_i values of 6.5 and 10.6 µM, respectively (Fig. 2). Progesterone competitively inhibited CYP3A5-mediated activity with a K_i value of 42.2 µM, which was four-fold higher than that for CYP3A4. ANF noncompetitively inhibited the activities mediated by CYP3A4 and CYP3A5 with K_i values of 6.1  and 5.4 µM, respectively (Fig. 2).

Fig. 2. Effect of Progesterone, DHEA, and ANF on Testosterone 6β-Hydroxylation Mediated by CYP3A4 and CYP3A5

For progesterone and DHEA, ●, 0 µM; ▲, 5 µM; ■, 20 µM; ◆, 100 µM. For ANF, ●, 0 µM; ■, 2.5 µM; ▲, 10 µM. Results were estimated by nonlinear regression analysis from mean values obtained in duplicate or triplicate at each substrate/effector concentration.

DISCUSSION

The present results and the previously reported findings in terms of the effects (activation and inhibition) on metabolic reactions^26–29) are summarized in Table 2. We previously demonstrated that steroid hormones, including progesterone and testosterone, stimulate CYP3A4-mediated metabolism such as nifedipine oxidation and 7-benzyloxyresorufin O-debenzylation.²⁷⁾ In addition, the activation of CYP3A4-catalyzed reactions, including progesterone 6β-hydroxylation, carbamazepine 10,11-epoxidation, and midazolam 4-hydroxylation, has been investigated by several laboratories.^27,28) Furthermore, the activation phenomena have been demonstrated for other P450s including CYP3A7.^28,29) CYP3A7-catalyzed carbamazepine 10,11-epoxidation was reported to be activated by sulfate conjugates of steroid hormones such as DHEA and pregnenolone²⁹⁾; however, there are few reports on the activation of metabolic activities catalyzed by CYP3A5 and CYP3A7. We recently reported that K_m values of progesterone 6β-hydroxylation catalyzed by CYP3A4, CYP3A5, and CYP3A7 were decreased, and k_cat values for CYP3A5, but not for other CYP3A subfamily members, were gradually increased with increasing testosterone concentrations.²⁶⁾ In the present study, the K_m values of progesterone 6β-hydroxylation mediated by CYP3A5, rather than that mediated by CYP3A4, decreased in the presence of DHEA and ANF, suggesting that the activation behavior differs among CYP3A4, CYP3A5, and CYP3A7. On the other hand, testosterone 6β-hydroxylation activities catalyzed by these CYP3A subfamily members were decreased or unaffected in the presence of DHEA, ANF, and pregnenolone as well as the previously reported progesterone and cortisol,²⁶⁾ indicating that no activation occurred. Progesterone and DHEA competitively inhibited, whereas ANF noncompetitively inhibited. In addition, the K_i value of progesterone in comparison to testosterone 6β-hydroxylation mediated by CYP3A5 was higher than that for CYP3A4, whereas the K_i values of ANF were similar between CYP3A4 and CYP3A5, indicating that the differences in inhibition strength between these CYP3A subfamily members were inhibitor-dependent. However, activation reactions were observed in various CYP3A-mediated reactions such as progesterone 6β-hydroxylation, 7-benzyloxyresorufin O-debenzylation, carbamazepine 10,11-epoxidation, and/or nifedipine oxidation, but not testosterone 6β-hydroxylation, as summarized in Table 2.^26,27,29) Thus, these results support the validity of the recommendation by the guidelines that testosterone 6β-hydroxylation is a preferred reaction of CYP3A4/5 for in vitro drug interaction study.^14–16) On the other hand, we used CYP3A enzymes expressed in recombinant Escherichia coli in the present study and our recent report,²⁸⁾ whereas our previous findings²⁷⁾ and the results reported by Nakamura et al.²⁹⁾ were obtained using CYP3A enzymes expressed in baculovirus-infected insect cells. In addition, our present and all of the previous study^27,28) was conducted by use of CYP3A enzymes not expressed with cytochrome b₅, although CYP3A enzymes coexpressed with cytochrome b₅ was used in the experiment by Nakamura et al.²⁹⁾ Selection of the enzyme source (liver microsomes or recombinant P450s) and/or the coexpression of cytochrome b₅ would affect the activation.²⁸⁾ To compare quantitatively the strength of activation among CYP3A subfamily members in detail, further studies using various enzyme source would be necessary.

Table 2. Effect of Steroid Hormones and Related Compounds on the Steroid Hydroxylation Mediated by CYP3A4, CYP3A5, and CYP3A7

Effector	CYP	Metabolic reaction
		PROG6β	TS6β	CORT6β	7-BR	NF	CBZ
DHEA	CYP3A4	ME	I (C)	—	—	—	A29)
	CYP3A5	A (Km↓), I (kcat↓)	I	—	—	—	—
	CYP3A7	I	I	—	—	—	—
Pregnenolone	CYP3A4	I	I	—	—	—	—
	CYP3A5	ME	ME	—	—	—	—
	CYP3A7	I	ME	—	—	—	—
ANF	CYP3A4	A (Km↓), I (Km↑, kcat↓)	I (N)	—	A27	I 27	—
	CYP3A5	A (Km↓), I (kcat↓)	I (N)	—	—	—	—
	CYP3A7	I	I	—	—	—	—
Progesterone	CYP3A4	X	I (C)	I26)	A27)	A (Km↓)27)	—
	CYP3A5	X	I (C)	ME26)	—	—	—
	CYP3A7	X	I26)	I26)	—	—	—
Testosterone	CYP3A4	A (Km↓)26)	X	ME26)	A27)	A (Km↓)27)	A29)
	CYP3A5	A (Km↓, kcat↑)26)	X	ME26)	—	—	—
	CYP3A7	A (Km↓)26)	X	I26)	—	—	—
Cortisol	CYP3A4	ME26)	ME26)	X	—	—	—
	CYP3A5	ME26)	ME26)	X	—	—	—
	CYP3A7	ME26)	ME26)	X	—	—	—

A: Activation, I: inhibition, I (C): competitive inhibition; I (N): noncompetitive inhibition, ME: minor effect (weak inhibition with less than 20% decrease) or no effect, —: no reports, DHEA: dehydroepiandrosterone, ANF: α-naphthoflavone, PROG6β: progesterone 6β-hydroxylation, TS6β: testosterone 6β-hydroxylation, CORT6β: cortisol 6β-hydroxylation,7-BR: 7-benzoyloxyresorufin O-debenzylation, NF: nifedipine oxidation, CBZ: carbamazepine 10,11-epoxidation.

Considering CYP3A4, it is well known that the large binding pocket in CYP3A4 is responsible to accommodate substrates of diverse structure and size, and the size of the active site might allow the cooperativity phenomena.³⁵⁾ Although cooperativity in CYP3A4 catalysis is interpreted by mechanisms leading to simultaneous occupation of the active site by multiple ligands and/or binding to nearby allosteric sites,^28,35–38) the mechanisms are not fully elucidated. Lewis et al.³⁹⁾ demonstrated the correlation of lipophilicity (log P) to substrate affinity (in the form of –log K_m) for CYP2B6. We investigated X log P computed by X Log P3 3.0 (PubChem release) as reported previously⁴⁰⁾; the X log P values of ANF, pregnenolone, progesterone, testosterone, 7-benzyloxyresorufin, carbamazepine, and nifedipine were 4.8, 4.2, 3.9, 3.3, 2.9, 2.5, and 2.2, respectively, indicating no significant relationship between lipophilicity and the presence or absence of activation.

Interestingly, in the present study, we observed that testosterone stimulated V_max of progesterone 6β-hydroxylation mediated by CYP3A5 but not that by CYP3A4 and CYP3A7, in addition to the decrease in K_m.²⁶⁾ In addition, ANF increased the affinity (decrease of K_m values) for progesterone 6β-hydroxylation mediated by CYP3A4 and CYP3A5 but not that by CYP3A7. These results suggest that CYP3A5 seems to be the most sensitive in terms of activation among these CYP3A subfamily members. Testosterone activates progesterone 6β-hydroxylation, 7-benzyloxyresorufin O-dealkylation, and nifedipine oxidation but not cortisol 6β-hydroxylation, indicating that testosterone is the most extensively investigated activator.^26–29) Using a docking simulation program for the binding of CYP3A4 and CYP3A5 with some substrates, including steroid hormones, we recently demonstrated that molecular docking simulations using docking energies (U values) could partially explain the differences in substrate affinity (the K_m values).²⁵⁾ Further quantitative analytical studies including CYP3A7 in addition to CYP3A4 and CYP3A5, for instance, using the docking simulation, might be of interest, although drastic differences in the K_m values of steroid hormone hydroxylation among CYP3A subfamily members were not observed.²⁶⁾

In conclusion, steroid–steroid interactions were different among CYP3A subfamily members. Interestingly, the remarkable activation of progesterone 6β-hydroxylation mediated by CYP3A4 and/or CYP3A5 but not by CYP3A7 with DHEA and ANF were observed. Further studies including molecular docking simulations would be necessary to explain the differences in the accessibility of substrates/effectors to the heme moiety and conformational changes in CYP3A subfamily members.

Acknowledgments

This work was supported in part by a Grant-in-Aid from Wesco Scientific Promotion Foundation.

Conflict of Interest

The authors declare no conflict of interest.

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