Maria Norlin

Regulation of human vitamin D3 25-hydroxylases in dermal fibroblasts and prostate cancer LNCaP cells

In this study, we examined whether 1α,25-dihydroxyvitamin D3 (calcitriol), phenobarbital, and the antiretroviral drug efavirenz, drugs used by patient groups with high incidence of low bone mineral density, could affect the 25-hydroxylase activity or expression of human 25-hydroxylases in dermal fibroblasts and prostate cancer LNCaP cells. Fibroblasts express the 25-hydroxylating enzymes CYP2R1 and CYP27A1. LNCaP cells were found to express two potential vitamin D 25-hydroxylases—CYP2R1 and CYP2J2. The presence in different cells of nuclear receptors vitamin D receptor (VDR), pregnane X receptor (PXR), and constitutive androstane receptor (CAR) was also determined. Phenobarbital suppressed the expression of CYP2R1 in fibroblasts and CYP2J2 in LNCaP cells. Efavirenz suppressed the expression of CYP2R1 in fibroblasts but not in LNCaP cells. CYP2J2 was slightly suppressed by efavirenz, whereas CYP27A1 was not affected by any of the two drugs. Calcitriol suppressed the expression of CYP2R1 in both fibroblasts and LNCaP cells but had no clear effect on the expression of either CYP2J2 or CYP27A1. The vitamin D3 25-hydroxylase activity in fibroblasts was suppressed by both calcitriol and efavirenz. In LNCaP cells, consumption of substrate (1α-hydroxyvitamin D3) was used as indicator of metabolism because no 1α,25-dihydroxyvitamin D3 product could be determined. The amount of 1α-hydroxyvitamin D3 remaining in cells treated with calcitriol was significantly increased. Taken together, 25-hydroxylation of vitamin D3 was suppressed by calcitriol and drugs. The present study provides new information indicating that 25-hydroxylation of vitamin D3 may be regulated. In addition, the current results may offer a possible explanation for the impaired bone health after treatment with certain drugs.

Oxysterol 7 alpha-hydroxylase activity by cholesterol 7 alpha-hydroxylase (CYP7A)

A 7α-hydroxylation is necessary for conversion of both cholesterol and 27-hydroxycholesterol into bile acids. According to current theories, cholesterol 7α-hydroxylase (CYP7A) is responsible for the former and oxysterol 7α-hydroxylase (CYP7B) for the latter reaction. CYP7A is believed to have a very high substrate specificity whereas CYP7B is active toward oxysterols, dehydroepiandrosterone, and pregnenolone. In the present study, 7α-hydroxylation of various oxysterols in liver and kidney was investigated. Surprisingly, human cholesterol 7α-hydroxylase, CYP7A, expressed as a recombinant in Escherichia coli and COS cells, was active toward 20(S)-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol. This enzyme has previously been thought to be specific for cholesterol and cholestanol. A partially purified and reconstituted cholesterol 7α-hydroxylase enzyme fraction from pig liver showed 7α-hydroxylase activity toward the same oxysterols as metabolized by expressed recombinant human and rat CYP7A. The 7α-hydroxylase activity toward 20(S)-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol in rat liver was significantly increased by treatment with cholestyramine, an inducer of CYP7A. From the present results it may be concluded that CYP7A is able to function as an oxysterol 7α-hydroxylase, in addition to the previously known human oxysterol 7α-hydroxylase, CYP7B. These findings may have implications for oxysterol-mediated regulation of gene expression and for pathways of bile acid biosynthesis. A possible use of 20(S)-hydroxycholesterol as a marker substrate for CYP7A is proposed.

On the substrate specificity of human CYP27A1 implications for bile acid and cholestanol formation

The mitochondrial sterol 27-hydroxylase (CYP27A1) is required for degradation of the C27-sterol side chain in bile acid biosynthesis. CYP27A1 seems, however, to have roles beyond this, as illustrated by patients with a deficient sterol 27-hydroxylase due to mutations of the CYP27A1 gene [cerebrotendinous xanthomatosis (CTX)]. These subjects have symptoms ranging from accumulation of bile alcohols and cholestanol to accelerated atherosclerosis and progressive neurologic impairment. The present work describes a detailed investigation on the substrate specificity of recombinant human CYP27A1. In accordance with some previous work with rat liver mitochondria, the activity in general increased with the polarity of the substrate. An obvious example was the finding that cholesterol was 27-hydroxylated more efficiently than cholesterol oleate but less efficiently than cholesterol sulfate. The oxysterols 24S-hydroxycholesterol and 25-hydroxycholesterol were 27-hydroxylated less efficiently than cholesterol, possibly due to steric hindrance. Surprisingly, sterols with a 3-oxo-Δ4 structure were found to be hydroxylated at a much higher rate than the corresponding sterols with a 3β-hydroxy-Δ5 structure. The rates of hydroxylation of the sterols were: 7α-hydroxy-4-cholesten-3-one>4-cholesten-3-one>7α-hydroxycholesterol>24-hydroxy-4-cholesten-3-one> cholesterol>25-hydroxy-4-cholesten-3-one>24-hydroxycholesterol⩾25-hydroxycholesterol. The possibility is discussed that the findings may have implications for oxysterol-mediated regulation of gene expression. The very high activity of CYP27A1 towards the cholestanol precursor 4-cholesten-3-one may be of importance in connection with the accumulation of cholestanol in patients with CTX.

Estrogen-mediated regulation of CYP7B1: A possible role for controlling DHEA levels in human tissues

The current study examines regulation of CYP7B1, a DHEA 7alpha-hydroxylase, by sex hormones. Transfection with estrogen receptor alpha and treatment with 17beta-estradiol in human embryonic kidney 293 cells significantly increased CYP7B1 catalytic activity and mRNA, and stimulated a human CYP7B1 reporter gene. Transfection with estrogen receptor beta showed similar but less significant effects. In the absence of receptors, 17beta-estradiol suppressed CYP7B1 activity, suggesting that estrogenic effects may be different in cells not expressing receptors. Quantitation of CYP7B1 mRNA in adult and fetal human tissues showed markedly higher CYP7B1 mRNA levels in fetal tissues compared with the corresponding adult ones, except in the liver. This indicates a tissue-specific, developmental regulation of CYP7B1 and suggests an important function for this enzyme in fetal life. DHEA secreted by fetal adrenals is an essential precursor for placental estrogen formation. Since CYP7B1 diverts DHEA from the sex hormone biosynthetic pathway, estrogen receptor-mediated up-regulation of CYP7B1 should lead to less DHEA available for sex hormone synthesis and may help to maintain normal levels of estrogens and androgens in human tissues, especially during fetal development. Regulation by estrogens may also be of importance in other processes where CYP7B1 is involved, including cholesterol homeostasis, cellular proliferation, and CNS function.

FLUTAMIDE METABOLISM IN FOUR DIFFERENT SPECIES IN VITRO AND IDENTIFICATION OF FLUTAMIDE METABOLITES IN HUMAN PATIENT URINE BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY/TANDEM MASS SPECTROMETRY

A new metabolic scheme of flutamide is proposed in this article. Some patients treated with flutamide, a nonsteroidal antiandrogen, have developed severe hepatic dysfunction. Toxic metabolites have been proposed to be responsible for these negative effects. In this study, the qualitative aspects of the in vitro metabolism of flutamide in liver microsomes from human, dog, pig, and rat were evaluated. A direct comparison of the flutamide metabolism in liver and prostate microsomes from pig was made, and the in vivo metabolism of flutamide was investigated in urine from orally treated prostate cancer patients. Liquid chromatography/tandem mass spectrometry was used for analysis. The mass spectrometer was equipped with an electrospray interface and operated in the negative ion mode. In liver microsomes from pig, dog, and rat, extensive hydroxylation of flutamide occurred. One, two, or three hydroxy groups were attached, and isomeric forms were detected for both monohydroxylated and trihydroxylated drug. In pig liver microsomes, isomers of a third metabolite, hydroxylated 4-nitro-3-(trifluoromethyl)-aniline, were also found after incubation with either flutamide or 2-hydroxyflutamide. In human liver microsomes, the pharmacologically active 2-hydroxyflutamide was the only metabolite detected. Several phase I metabolites as well as four intact phase II metabolites could be recovered from the urine samples. For the first time in humans, glucuronic acid conjugates of hydroxylated 4-nitro-3-(trifluoromethyl)-aniline, and mono- and dihydroxylated flutamide were identified, together with hydroxylated 4-nitro-3-(trifluoromethyl)-aniline conjugated with sulfate. In addition, one mercapturic acid conjugate of hydroxylated flutamide, probably formed from flutamide via a reactive intermediate, was detected.

1α,25-Dihydroxyvitamin D3 exerts tissue-specific effects on estrogen and androgen metabolism.

It is well-known that 1α,25-dihydroxyvitamin D(3) and analogs exert anti-proliferative and pro-differentiating effects and these compounds have therefore been proposed to be of potential use as anti-cancer agents. Due to its effects on aromatase gene expression and enzyme activity, 1α,25-dihydroxyvitamin D(3) has been proposed as an interesting substance in breast cancer treatment and prevention. In the present study, we have examined the effects of 1α,25-dihydroxyvitamin D(3) on estrogen and androgen metabolism in adrenocortical NCI-H295R cells, breast cancer MCF-7 cells and prostate cancer LNCaP cells. The NCI-H295R cell line has been proposed as a screening tool to study endocrine disruptors. We therefore studied whether this cell line reacted to 1α,25-dihydroxyvitamin D(3) treatment in the same way as cells from important endocrine target tissues. 1α,25-Dihydroxyvitamin D(3) exerted cell line-specific effects on estrogen and androgen metabolism. In breast cancer MCF-7 cells, aromatase gene expression and estradiol production were decreased, while production of androgens was markedly increased. In NCI-H295R cells, 1α,25-dihydroxyvitamin D(3) stimulated aromatase expression and decreased dihydrotestosterone production. In prostate cancer LNCaP cells, aromatase expression increased after the same treatment, as did production of testosterone and dihydrotestosterone. In summary, our data show that 1α,25-dihydroxyvitamin D(3) exerts tissue-specific effects on estrogen and androgen production and metabolism. This is important knowledge about 1α,25-dihydroxyvitamin D(3) as an interesting substance for further research in the field of breast cancer prevention and treatment. Furthermore, the observed cell line-specific effects are of importance in the discussion about NCI-H295R cells as a model for effects on estrogen and androgen metabolism.

CYP7B1-mediated metabolism of dehydroepiandrosterone and 5α-androstane-3β,17β-diol – potential role(s) for estrogen signaling

CYP7B1, a cytochrome P450 enzyme, metabolizes several steroids involved in hormonal signaling including 5α‐androstane‐3β,17β‐diol (3β‐Adiol), an estrogen receptor agonist, and dehydroepiandrosterone, a precursor for sex hormones. Previous studies have suggested that CYP7B1‐dependent metabolism involving dehydroepiandrosterone or 3β‐Adiol may play an important role for estrogen receptor β‐mediated signaling. However, conflicting data are reported regarding the influence of different CYP7B1‐related steroids on estrogen receptor β activation. In the present study, we investigated CYP7B1‐mediated conversions of dehydroepiandrosterone and 3β‐Adiol in porcine microsomes and human kidney cells. As part of these studies, we compared the effects of 3β‐Adiol (a CYP7B1 substrate) and 7α‐hydroxy‐dehydroepiandrosterone (a CYP7B1 product) on estrogen receptor β activation. The data obtained indicated that 3β‐Adiol is a more efficient activator, thus lending support to the notion that CYP7B1 catalysis may decrease estrogen receptor β activation. Our data on metabolism indicate that the efficiencies of CYP7B1‐mediated hydroxylations of dehydroepiandrosterone and 3β‐Adiol are very similar. The enzyme catalyzed both reactions at a similar rate and the Kcat/Km values were in the same order of magnitude. A high dehydroepiandrosterone/3β‐Adiol ratio in the incubation mixtures, similar to the ratio of these steroids in many human tissues, strongly suppressed CYP7B1‐mediated 3β‐Adiol metabolism. As the efficiencies of CYP7B1‐mediated hydroxylation of dehydroepiandrosterone and 3β‐Adiol are similar, we propose that varying steroid concentrations may be the most important factor determining the rate of CYP7B1‐mediated metabolism of dehydroepiandrosterone or 3β‐Adiol. Consequently, tissue‐specific steroid concentrations may have a strong impact on CYP7B1‐dependent catalysis and thus on the levels of different CYP7B1‐related steroids that can influence estrogen receptor β signaling.

1α,25-Dihydroxyvitamin D3 affects hormone production and expression of steroidogenic enzymes in human adrenocortical NCI-H295R cells

The current study presents data indicating that 1alpha,25-dihydroxyvitamin D(3) affects the production of hormones and expression of crucial steroidogenic enzymes in the human adrenocortical cell line NCI-H295R. This cell line is widely used as a model for adrenal steroidogenesis. Treatment of the cells with 1alpha,25-dihydroxyvitamin D(3) suppressed the levels of corticosterone, aldosterone, DHEA, DHEA-sulfate and androstenedione in the culture medium. In order to study the mechanisms behind this suppression of hormone production, we investigated the effects of 1alpha,25-dihydroxyvitamin D(3) on important genes and enzymes controlling the biosynthesis of adrenal hormones. The mRNA levels were decreased for CYP21A2 while they were increased for CYP11A1 and CYP17A1. No significant changes were observed in mRNA for CYP11B1, CYP11B2 or 3beta-hydroxysteroid dehydrogenase (3betaHSD). In similarity with the effects on mRNA levels, also the endogenous enzyme activity of CYP21A2 decreased after treatment with 1alpha,25-dihydroxyvitamin D(3). Interestingly, the two CYP17A1-mediated activities were influenced reciprocally - the 17alpha-hydroxylase activity increased whereas the 17,20-lyase activity decreased. The current data indicate that the 1alpha,25-dihydroxyvitamin D(3)-mediated decrease in corticosterone and androgen production is due to suppression of the 21-hydroxylase activity by CYP21A2 and the 17,20-lyase activity by CYP17A1, respectively. In conclusion, the current study reports novel findings on 1alpha,25-dihydroxyvitamin D(3)-mediated effects on hormone production and regulation of genes and enzymes involved in steroidogenesis in the adrenocortical NCI-H295R cell line, a model for human adrenal cortex.

CYP7B1-mediated metabolism of 5α-androstane-3α,17β-diol (3α-Adiol): A novel pathway for potential regulation of the cellular levels of androgens and neurosteroids

The current study presents data indicating that 5alpha-androstane-3alpha,17beta-diol (3alpha-Adiol) undergoes a previously unknown metabolism into hydroxymetabolites, catalyzed by CYP7B1. 3alpha-Adiol is an androgenic steroid which serves as a source for the potent androgen dihydrotestosterone and also can modulate gamma-amino butyric acid A (GABA(A)) receptor function in the brain. The steroid hydroxylase CYP7B1 is known to metabolize cholesterol derivatives, sex hormone precursors and certain estrogens, but has previously not been thought to act on androgens or 3alpha-hydroxylated steroids. 3alpha-Adiol was found to undergo NADPH-dependent metabolism into 6- and 7-hydroxymetabolites in incubations with porcine microsomes and human kidney-derived HEK293 cells, which are high in CYP7B1 content. This metabolism was suppressed by addition of steroids known to be metabolized by CYP7B1. In addition, 3alpha-Adiol significantly suppressed CYP7B1-mediated catalytic reactions, in a way as would be expected for substrates that compete for the same enzyme. Recombinant expression of human CYP7B1 in HEK293 cells significantly increased the rate of 3alpha-Adiol hydroxylation. Furthermore, the observed hydroxylase activity towards 3alpha-Adiol was very low or undetectable in livers of Cyp7b1(-/-) knockout mice. The present results indicate that CYP7B1-mediated catalysis may play a role for control of the cellular levels of androgens, not only of estrogens. These findings suggest a previously unknown mechanism for metabolic elimination of 3alpha-Adiol which may impact intracellular levels of dihydrotestosterone and GABA(A)-modulating steroids.

Biochemical characterization of the 7α-hydroxylase activities towards 27-hydroxycholesterol and dehydroepiandrosterone in pig liver microsomes

Microsomal cytochrome P-450 catalyzing the 7alpha-hydroxylation of 27-hydroxycholesterol and dehydroepiandrosterone was partially purified from pig liver. This enzyme fraction also catalyzed 7alpha-hydroxylation of 25-hydroxycholesterol and pregnenolone but did not 7alpha-hydroxylate cholesterol or testosterone. Studies with extrahepatic tissues have suggested the possibility of one common enzyme responsible for the 7alpha-hydroxylation of 27-hydroxycholesterol and dehydroepiandrosterone. A series of experiments was performed to study if there are one or several enzymes 7alpha-hydroxylating these steroids in the liver. The activities towards the two substrates copurified but the ratio between 27-hydroxycholesterol and dehydroepiandrosterone 7alpha-hydroxylation varied considerably in different purification steps and between different preparations. The enzyme inhibitors disulfiram, N-bromosuccinimide, ketoconazole, metyrapone and alpha-naphthoflavone affected the activities in a similar way. Dehydroepiandrosterone inhibited 27-hydroxycholesterol 7alpha-hydroxylation whereas 27-hydroxycholesterol had almost no inhibitory effect on dehydroepiandrosterone 7alpha-hydroxylation. Experiments to examine the nature of inhibition by dehydroepiandrosterone indicated that the two steroids did not compete for the same active site. The results of this study do not rule out the possibility of one single enzyme catalyzing 7alpha-hydroxylation of the two steroids. However, taken together the data suggest that hepatic microsomal 7alpha-hydroxylation of 27-hydroxycholesterol and dehydroepiandrosterone involves at least two, probably closely related, enzymes. (c) 1998 Elsevier Science B. V.