Barbara A. Murry

Mechanisms in tissue-specific regulation of estrogen biosynthesis in humans.

In humans, aromatase P450, which catalyses conversion of C(19)-steroids to estrogens, is expressed in several tissues, including gonads, brain, adipose tissue, skin and placenta, and is encoded by a single-copy gene (CYP19); however, this does not hold true for all species. The human gene is approximately 130 kb and its expression is regulated, in part, by tissue-specific promoters and by alternative splicing mechanisms. Using transgenic mouse technology, it was observed that ovary-, adipose tissue- and placenta-specific expression of human CYP19 is directed by relatively small segments of DNA within 500 bp upstream of each of the tissue-specific first exons. Thus, the use of alternative promoters allows greater versatility in tissue-specific regulation of CYP19 expression. Characterization and identification of transcription factors and crucial cis-acting elements within genomic regions that direct tissue-specific expression will contribute to improved understanding of the regulation of CYP19 expression in the tissues that synthesize estrogens under both physiological and pathophysiological conditions.

Imidazole antimycotics: inhibitors of steroid aromatase

Miconazole and clotrimazole, members of a class of imidazole agents which have broad spectrum antimycotic activity, were shown to be potent inhibitors of steroid aromatase activity of human placental microsomes. The I50 values for the inhibition of aromatase activity by miconazole, clotrimazole, ketoconazole, and aminoglutethimide were 0.6, 1.8, 60 and 44 microM respectively. The most effective compound, miconazole, exhibited competitive kinetics with respect to androstenedione, the aromatase substrate. The apparent inhibitory constant (Ki) was 55 nM, under assay conditions where the apparent Km for androstenedione was 220 nM. The inhibition of aromatase activity by miconazole was shown to be reversible by dilution. Miconazole was a relatively poor inhibitor of the cholesterol side chain cleavage activity of a placental mitochondria-enriched fraction, while both clotrimazole and ketoconazole markedly inhibited this mitochondrial monooxygenase activity. Spectrophotometric studies revealed that miconazole bound to the cytochrome P-450 component of the placental microsomal aromatase complex and had negligible effect on NADPH-cytochrome c (P-450) reductase activity. These results strongly support direct interaction of miconazole with microsomal cytochrome P-450 in human placental microsomes with high affinity resulting in the inhibition of aromatase activity.

Imidazole antimycotics: selective inhibitors of steroid aromatization and progesterone hydroxylation.

Econazole, imazalil, and prochloraz, which have broad spectrum antimycotic activity, are shown to be potent inhibitors of steroid aromatase activity of human placental microsomes. The IC50 values for the inhibition of aromatase activity by econazole, imazalil, miconazole, prochloraz, clotrimazole, ketoconazole, and aminoglutethimide are 0.03, 0.15, 0.6, 0.7, 1.8, 60, and 45 microM, respectively. Econazole and 4-hydroxyandrostenedione also inhibit the steroid aromatase activity of human fetal liver, a finding which suggests that extraplacental aromatase may have many similarities to the placental enzyme. Econazole is a more effective inhibitor of placental aromatization of 19-hydroxyandrostenedione than of androstenedione. This observation is consistent with the competitive nature of the inhibition of aromatase by imidazole antimycotic agents and the reduced affinity of the placental aromatase enzyme for 19-hydroxyandrostenedione compared to androstenedione. The effectiveness of these imidazole antimycotic agents to inhibit the multiple hydroxylations of progesterone which are catalyzed by human fetal adrenal microsomes is also defined. While all of the imidazole antimycotic agents are potent inhibitors of the 16 alpha-, 17 alpha-, and 21-hydroxylations of progesterone, selective inhibitory profiles are apparent. Ketoconazole is a most potent inhibitor of human fetal adrenal progesterone 16 alpha- and 17 alpha-hydroxylases while clotrimazole and imazalil are the most potent inhibitors of progesterone 21-hydroxylase. These results are strongly supportive that imidazole drugs are selective inhibitors not only of steroid aromatase but also of other microsomal steroid hydroxylases.

3β-Hydroxysteroid dehydrogenase/Δ5→4-isomerase expression in rat and characterization of the testis isoform

Abstract The isolation, cloning and expression of a DNA insert complementary to mRNA encoding rat testis 3β-hydroxysteroid dehydrogenase/Δ5→4-isomerase (3β-HSD) is reported. The insert contains an open reading frame encoding a protein of 373 amino acids, which exhibits 73% and 78% identity to the cDNA encoding the human placental form at the amino acid and nucleotide levels respectively. Northern blot analysis of total RNA of rat tissues using as probe a specific radiolabeled cDNA insert encoding rat testis 3β-HSD demonstrated high levels of 1.6 kb mRNA species in ovary, adrenal and Leydig tumor, with lower but detectable message in testis and adult male liver, while the probe also hybridized to a 2.1 kb mRNA species in liver. The cDNA was inserted into a modified pCMV vector and expressed in COS-1 monkey kidney tumor cells. The expressed protein was similar in size to 3β-HSD present in H540 Leydig tumor cell homogenate and human placental microsomal 3β-HSD, as detected by immunoblot analysis, and catalyzed the conversion of pregnenolone to progesterone, 17α-hydroxypregnenolone to 17α-hydroxyprogesterone, and dehydroepiandrosterone to androstenedione. Transfected COS cell homogenates, supplemented with NAD+, but not NADP+, converted pregnenolone to progesterone and dehydroepiandrosterone to androstenedione with apparent Km values of 0.13 and 0.09 μM, respectively. Immunoblot analysis of various rat tissues using a polyclonal antibody directed against human placental 3β-HSD, in addition to immunoreactivity in the adrenal and testis, demonstrated immunoreactive 3β-HSD protein in adult male liver, but not in adult female or fetal liver. We conclude that while one gene product is highly expressed in testicular Leydig cells, and probably adrenal and ovary, accounting for their 3β-HSD content, a 3β-HSD is also expressed in liver in a sex-specific manner.

Practical, enantiospecific syntheses of 14,15-EET and leukotoxin B (vernolic acid)

Abstract Cytochrome P450BM3 and its F87V mutant were exploited for a convenient, laboratory scale (1 mmol) preparation of 14( S ),15( R )-epoxyeicosatrienoic acid [14( S ),15( R )-EET] from arachidonic acid and (+)-leukotoxin B [(+)-12( S ),13( R )-vernolic acid] from linoleic acid, respectively. Their enantiomers were accessed via a four-step chemical inversion.

Nucleotide sequence of a cDNA encoding porcine testis 17α-hydroxylase cytochrome P-450

We describe the isolation and characterization of a cDNA encoding the complete porcine neonatal testis 17α-hydroxylase/C-17,20-lyase cytochrome P -450. The deduced amino acid sequence is 509 amino acids in length.

Ovine steroid 17α-hydroxylase cytochrome P450: characteristics of the hydroxylase and lyase activities of the adrenal cortex enzyme

The steroid 17-hydroxylase cytochrome P450 (CYP17) found in mammalian adrenal and gonadal tissues typically exhibits not only steroid 17-hydroxylase activity but also C-17,20-lyase activity. These two reactions, catalyzed by CYP17, allow for the biosynthesis of the glucocorticoids in the adrenal cortex, as a result of the 17-hydroxylase activity, and for the biosynthesis of androgenic C(19) steroids in the adrenal cortex and gonads as a result of the additional lyase activity. A major difference between species with regard to adrenal steroidogenesis resides in the lyase activity of CYP17 toward the hydroxylated intermediates and in the fact that the secretion of C(19) steroids takes place, in some species, exclusively in the gonads. Ovine CYP17 expressed in HEK 293 cells converts progesterone to 17-hydroxyprogesterone and pregnenolone to dehydroepiandrosterone via 17-hydroxypregnenolone. In ovine adrenal microsomes, minimal if any lyase activity was observed toward either progesterone or pregnenolone. Others have demonstrated the involvement of cytochrome b(5) in the augmentation of CYP17 lyase activity. Although the presence of cytochrome b(5) in ovine adrenocortical microsomes was established, ovine adrenal microsomes did not convert pregnenolone or 17-hydroxypregnenolone to dehydroepiandrosterone. Furthermore the addition of purified ovine cytochrome b(5) to ovine adrenal microsomes did not promote lyase activity. We conclude that, in the ovine adrenal cortex, factors other than cytochrome b(5) influence the lyase activity of ovine CYP17.

The Use of Rat Leydig Tumor (R2C) and Human Hepatoma (Hepg2) Cells to Evaluate Potential Inhibitors of Rat and Human Steroid Aromatase

The efficacies of 10-propargylestr-4-ene-3,17-dione (PED), 4-hydroxyandrostenedione (4-OHA) and the imidazole broad spectrum antimycotic drugs, econazole, imazalil, miconazole and ketoconazole, to inhibit the steroid aromatase activities of rat Leydig tumor (R2C) cells and human hepatoma (HEPG2) cells have been determined. The analysis of inhibition of steroid aromatase activity of intact cells provided further insight into the potential use of such drugs to block cellular estrogen synthesis. The IC50 values for the inhibition of aromatase activity of R2C cells by econazole, imazalil, miconazole, ketoconazole, 4-OHA and PED were 4, 9, 40, 1100, 11 and 10 nM, respectively. These drugs also inhibited the steroid aromatase activity of HEPG2 cells with corresponding IC50 values of 13, 27, 20, 15000, 2 and 2 nM, respectively; these findings were suggestive that the steroid aromatase of rat has many similarities to the human enzyme in its interaction with putative inhibitory compounds. Importantly, however, ketoconazole inhibited the rat aromatase more effectively than it did the human enzyme, while PED and 4-OHA were less effective inhibitors of the rat enzyme compared to that of human. These findings indicate differences in the potencies of various drugs to inhibit estrogen biosynthesis in human and rat cells. These may relate to differences in the two aromatase systems and/or differences in the stability of the drugs in the human hepatoma and rat Leydig tumor cells.

The synthesis and metabolism of [6-3H]-25-hydroxycholesterol in rat adrenal tumor cells

The synthesis of [6-3H]-25-hydroxycholesterol from 26-norcholest-5-en-3 beta-ol-25-one is described. The metabolism of the radiolabeled hydroxysterol in rat adrenocortical carcinoma cells and in mitochondria-enriched preparations of rat adrenal tissue was investigated. We found that [6-3H]-25-hydroxycholesterol was metabolized efficiently to [3H]-pregnenolone in both preparations of rat adrenal tissue.