Aromatase catalyzes the rate limiting step that converts androgens to estrogens. Postmenopausal women with hormone dependent breast cancer respond to first generation aromatase inhibitors such as aminoglutethimide with a marked suppression of circulating estradiol levels. In contrast, premenopausal women appear to be resistant to first generation aromatase inhibitors. The inability to block ovarian aromatase results from the low affinity of first generation inhibitors for the active site of the enzyme. Under these circumstances, the high substrate levels in the premenopausal ovary compete effectively with these inhibitors and do not allow binding of inhibitor to the active site of the enzyme. Second and third generation aromatase inhibitors with higher affinity for aromatase have now been developed and potentially could block ovarian aromatase. To test this possibility, we administered CGS 20267 (letrozole), a highly potent aromatase inhibitor, to cycling female rats. A dose dependent inhibition of uterine weight occurred with maximum effects produced by the 5 mg/kg/day dosage. During a period of <em>4</em> weeks, uterine weight was reduced to levels induced by ovariectomy. Ovarian tissue estradiol levels were inhibited by approximately 80%. As a reflection of inhibition of ovarian aromatase activity, the levels of <em>androstenedione</em> in the ovary increased by an order of magnitude. Both LH and FSH plasma levels increased but not to those observed after ovariectomy. The rise in gonadotropin levels induced a statistically significant but relatively small increase in ovarian weights. These results demonstrate the ability to persistently block ovarian aromatase activity in cycling rats with a potent aromatase inhibitor. This study provides a rationale for clinical trials of potent aromatase inhibitors in pre-menopausal women with breast cancer.

Hormonal measurements and HLA genotyping of amniotic fluid at midgestation correctly predicted the postnatal diagnosis of congenital adrenal hyperplasia (CAH) in 29 of 32 fetuses at risk. Of these 29, 7 were predicted to have prenatal-onset classical 21-hydroxylase deficiency (21-OH def) based on elevated amniotic fluid 17-hydroxyprogesterone (17-OHP) and delta <em>4</em>-<em>androstenedione</em> (delta <em>4</em>-A) levels. These 7 fetuses and their index cases were ultimately proven to have salt-wasting classical 21-OH def. Of 5 who were HLA typed, the genotype was identical to the index case in <em>4</em>; in one, HLA prediction was not possible, because the parents shared identical HLA antigens. Normal amniotic fluid 17-OHP and delta <em>4</em>-A levels in the remaining 22 fetuses predicted that they were not affected with classical CAH. These children have been clinically asymptomatic to date or proven biochemically not to have classical or nonclassical CAH. Of the 22 fetuses, 11 were predicted by HLA genotyping to be homozygous normal or heterozygous for 21-OH def. In 3 of the 32 fetuses, prenatal diagnosis was incorrect. In one, the fetus was predicted to have CAH based on HLA identity to the index case. However, amniotic fluid 17-OHP and delta <em>4</em>-A were normal, and the fetus was normal. The index case of this family did not have CAH, but was a normal child. Thus, amniotic fluid hormone levels accurately predicted a normal fetus, while HLA genotyping was not relevant in prenatal diagnosis because the index case was unaffected. The second fetus was predicted to be affected on the basis of HLA genotyping and to be unaffected based on normal amniotic fluid 17-OHP and delta <em>4</em>-A. During infancy, this female infant had postnatal-onset nonclassical CAH. The index case in this family, presumed to have classical simple virilizing CAH, was later diagnosed to have nonclassical CAH. Thus, in nonclassical CAH, hormonal measurement of 17-OHP and delta <em>4</em>-A is not useful in prenatal diagnosis; only HLA genotyping of the fetus is valuable. In the third case, the fetus was predicted to be a heterozygote by HLA genotyping and to be unaffected by hormonal measurement. Postnatally, at age 2 7/12 yr, the male child was found to have classical simple virilizing CAH and to be HLA-B-DR identical to his brother (index case) who also has classical simple virilizing CAH.(ABSTRACT TRUNCATED AT <em>4</em>00 WORDS)

Total androgen blockade has been proposed as a better therapeutic technique than castration alone for the management of metastatic prostate cancer. This is based on the theory that links adrenal androgens to tumor growth. We have carefully examined the role of adrenal androgens in prostate cancer. Work done in our laboratory, as well as the work of many others, has demonstrated the following in regard to the role of adrenal androgens in prostate cancer: (1) The adrenal cortex secretes significant amounts of adrenal androgens into the blood. (2) Adrenal androgens are converted into dihydrotestosterone (DHT), as indicated by studies of labeled DHT recovered from prostates resected one-half hour after infusion of 3H-<em>androstenedione</em> or 3H-dehydroepiandrosterone sulfate into patients. We have also shown that biopsies of prostates from patients who were previously castrated may contain significant amounts of DHT, which could only be derived from adrenal androgens. (3) We have quantified DHT derived from adrenal androgens by measuring prostate DHT concentrations in castrates and in patients treated with combined gonadal and adrenal blockade. The mean difference between these two groups, 0.32 ng/g of DHT lower with combined blockade, is statistically significant and represents DHT derived from adrenal androgens. (<em>4</em>) We have also demonstrated that the small amounts of DHT derived from adrenal androgens may be biologically significant in stimulating prostatic epithelial cell protein synthesis in humans; others have reported similar findings in animals. (5) A review of patients in relapse after castration, who are treated with adrenal androgen blockade, indicates that approximately one out of three patients will show an objective remission based on National Prostate Cancer Project (NPCP) criteria. Despite data supporting the importance of adrenal androgens in prostate cancer, clinical trials using combined adrenal and gonadal blockade in prostate cancer have shown only modest benefit over castration. The largest and best study to date is the Southwest Oncology Group (SWOG) study, which did show a near-significant (P less than 0.065) difference between patients treated for 20 months with a luteinizing hormone-releasing hormone (LH-RH) plus flutamide compared with LH-RH alone. The difference in median time to progression was approximately 2 months between the groups. However, when one considers the fact that two out of three patients are probably not responding to the total androgen blockade, the 2 month difference may actually represent 6 or more months in a subset of one-third of patients receiving that therapy.(ABSTRACT TRUNCATED AT <em>4</em>00 WORDS)

Lean women with polycystic ovary syndrome have markedly increased serum luteinizing hormone levels, which are significantly correlated with Delta(<em>4</em>)-<em>androstenedione</em> levels but not directly linked to insulin resistance.

To determine whether some patients with idiopathic hypospadias have HSD3B2 mutations, we genotyped this locus in 90 patients with hypospadias (age, 6.0 +/- 0.<em>4</em> yr) and 101 healthy fertile male controls. We measured basal plasma renin activity and performed an ACTH test for determination of 17-OH-pregnenolone, 17-OH-progesterone, cortisol, dehydroepiandrosterone sulfate, and <em>androstenedione</em> and an human chorionic gonadotropin test for determination of <em>androstenedione</em>, testosterone, and dihydrotestosterone. We did not observe a clear steroidogenic pattern suggestive of 3 beta-HSD deficiency in any patient. DNA was extracted from peripheral lymphocytes; and exons 1, 2, 3, and <em>4</em> were amplified by PCR and analyzed by denaturing gradient gel electrophoresis. An abnormal electrophoretic migration pattern of exon <em>4</em> was observed in five patients. Two patients had missense heterozygous mutations (S213T and S28<em>4</em>R). In another three patients, we observed heterozygous nucleotide variants in exon <em>4</em> that did not produce a change in amino acids (A238, T259, T320). In vitro enzymatic activity was diminished by <em>4</em>0% and 32% in the S213T and S28<em>4</em>R heterozygous mutations, respectively. One control exhibited a heterozygous mutation in exon 3 (V78I), which did not alter in vitro enzyme activity. In addition, we observed possible polymorphisms in intron 1 in four patients and one control. We conclude that subtle molecular abnormalities in the HSD3B2 gene may be observed in some patients with apparent idiopathic hypospadias but that this finding is uncommon.

Aromatase inhibitors have now been approved as first-line treatment options for hormone-dependent advanced breast cancer. When compared to tamoxifen, these aromatase inhibitors provide significant survival and tolerability advantages. However, the optimal use of an aromatase inhibitor and tamoxifen remains to be established. To date, the intratumoral aromatase xenograft model has proved accurate in predicting the outcome of clinical trials. Utilizing this model, we performed long-term studies with tamoxifen and letrozole to determine time to disease progression with each of the treatment regimens. Aromatase-transfected MCF-7Ca human breast cancer cells were grown as tumor xenografts in female ovariectomized athymic nude mice in which <em>androstenedione</em> was converted to estrogen and stimulated tumor growth. When tumor volumes were approximately 300 mm3, the animals were grouped for continued supplementation with <em>androstenedione</em> only (control) or for treatment with letrozole 10 microg per day (long-term), tamoxifen 100 microg per day (long-term), letrozole alternating to tamoxifen (<em>4</em>-week rotation), tamoxifen alternating to letrozole (<em>4</em>-week rotation), or a combination of the two drugs. Tumors of control mice had doubled in volume in 3-<em>4</em> weeks. In mice treated with tamoxifen and the combination, tumor doubling time was significantly shorter (16 and 18 weeks, respectively) than with letrozole (3<em>4</em> weeks). Furthermore, alternating letrozole and tamoxifen treatment every <em>4</em> weeks was less effective than letrozole alone. Tumors doubled in 17-18 weeks when the starting treatment was tamoxifen and in 22 weeks when the starting treatment was letrozole. Tumors progressing on tamoxifen remained sensitive to second-line therapy with letrozole (10 microg per day). However, when mice with letrozole-resistant tumors were switched to antiestrogen treatment, tumors did not respond to tamoxifen (100 microg per day) or faslodex (1 mg per day). This suggests that advanced breast cancers treated with letrozole may be insensitive to subsequent second-line hormonal agents. Thus, although letrozole was determined to be an effective second-line treatment option for tumors progressing on tamoxifen, antiestrogen therapy does not appear to be effective for tumors progressing on letrozole. However, response to second-line treatment was observed in a model where tumors that had progressed on letrozole were transplanted to new mice. These tumors had been allowed to grow in the presence of supplemented <em>androstenedione</em> but absence of letrozole. This suggests that resistance to letrozole may be reversible, allowing tumors to respond to subsequent antiestrogens and letrozole.

The cytochrome P-<em>4</em>50 forms involved in debrisoquine <em>4</em>-hydroxylation (P-<em>4</em>50DB), phenacetin O-deethylation (P-<em>4</em>50PA), S-mephenytoin <em>4</em>-hydroxylation (P-<em>4</em>50MP), and nifedipine 1,<em>4</em>-oxidation (P-<em>4</em>50NF) have been purified to electrophoretic homogeneity from human liver microsomes. All of these reactions show in vivo polymorphism in humans. Evidence for the roles of the purified proteins in these processes comes from in vitro reconstitution and immunoinhibition studies. The rat orthologs of these enzymes are as follows--P-<em>4</em>50DB: P-<em>4</em>50UT-H; P-<em>4</em>50PA: P-<em>4</em>50ISF-G; P-<em>4</em>50MP: P-<em>4</em>50UT-I; P-<em>4</em>50NF: P-<em>4</em>50PCN-E. Only in the case of P-<em>4</em>50UT-H is the primary rat ortholog the same cytochrome P-<em>4</em>50 which catalyses the catalytic reaction under consideration. Reconstitution and immunochemical studies establish that the following reactions are catalysed by the individual P-<em>4</em>50s--P-<em>4</em>50DB: debrisoquine <em>4</em>-hydroxylation, sparteine delta 5-oxidation, bufuralol 1'-hydroxylation, encainide O-demethylation, and propanolol <em>4</em>-hydroxylation; P-<em>4</em>50PA: phenacetin O-deethylation; P-<em>4</em>50MP: S-mephenytoin <em>4</em>-hydroxylation and tolbutamide methyl hydroxylation; P-<em>4</em>50NF: oxidation of nifedipine and 16 other substituted dihydropyridines, estradiol 2- and <em>4</em>-hydroxylation, aldrin epoxidation, benzphetamine N-demethylation and 6 beta-hydroxylation of testosterone, <em>androstenedione</em> and cortisol. A cDNA clone has been isolated that corresponds to rat P-<em>4</em>50UT-H, as shown by a number of criteria. Studies with this probe establish that the sex and strain variation in debrisoquine <em>4</em>-hydroxylase and related activities is related to differences in the levels of a 2.0 kb length mRNA present.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies using pharmacological gonadotropin stimulation suggest that ovarian steroidogenesis is abnormal in the polycystic ovary syndrome (PCOS). We assessed ovarian steroid secretion in response to near-physiological gonadotropin stimuli in 12 ovulatory controls and 7 women with PCOS. A gonadotropin-releasing hormone-receptor antagonist (ganirelix, 2 mg sc) was given to block endogenous LH secretion, followed by dexamethasone (0.75 mg orally) to suppress adrenal androgen secretion. After ganirelix injection (12 h), intravenous infusions of recombinant human LH (0, 10, 30, 100, and 300 IU; each over 8 min) were administered at <em>4</em>-h intervals in a pseudorandomized (highest dose last) manner. Plasma LH, 17-hydroxyprogesterone (17-OHP), <em>androstenedione</em>, and testosterone were measured concurrently. LH dose-steroid response relationships (mean sex-steroid concentration vs. mean LH concentration over <em>4</em> h postinfusion) were examined for each subject. Linear regression of 17-OHP on LH yielded a higher (mean +/- SE) slope in PCOS (0.028 +/- 0.010 vs. 0.005 +/- 0.005, P < 0.05), whereas extrapolated 17-OHP at zero LH was similar. The slopes of other regressions did not differ from zero in either PCOS or controls. We conclude that near-physiological LH stimulation drives heightened 17-OHP secretion in patients with PCOS, suggesting abnormalities of early steps of ovarian steroidogenesis. With the exception of 17-OHP response in PCOS, no acute LH dose-ovarian steroid responses were observed in controls or PCOS. Defining the precise mechanistic basis of heightened precursor responsiveness to LH in PCOS will require further clinical investigation.

Aromatase, a cytochrome P-<em>4</em>50, catalyzes the formation of aromatic C18 estrogenic steroids from C19 androgens. Using the x-ray structure of cytochrome P-<em>4</em>50cam as the model, seven mutants of human aromatase were designed and expressed in Chinese hamster ovary cells by a stable expression method. They are His-128----Gln, His-128----Ala, Cys-299----Ala, Glu-302----Leu, Asp-309----Asn, Asp-309----Ala, and Ser-312----Cys. The presence of the aromatase mutants in the transfected Chinese hamster ovary cells were confirmed by immunoprecipitation analysis. The kinetic parameters of these mutants using [1 beta,2 beta-3H] <em>androstenedione</em> (or [1 beta-3H]<em>androstenedione</em>), and [1 beta,2 beta-3H]testosterone as substrates were determined. In addition, inhibition profiles for these mutants with two aromatase inhibitors, <em>4</em>-hydroxy<em>androstenedione</em> and aminoglutethimide were obtained. Furthermore, the reactions catalyzed by these mutants were examined by evaluating the levels of the product estrone, and two intermediates, 19-hydroxy<em>androstenedione</em> and 19-oxo<em>androstenedione</em> by reverse phase high performance liquid chromatography using [7-3H]<em>androstenedione</em> as the substrate. Our results indicate that among the positions we modified, Asp-309 appears to be very important for the enzyme catalysis.

Exposure of pubertal rats to di-(2-ethylhexyl) phthalate (DEHP) for 1<em>4</em> days was reported to result in reduced testosterone (T) biosynthesis by altering <em>androstenedione</em> 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activity. However, our study indicated that shorter period exposure of DEHP (100 or 1000 mg/kg for 5 days) to <em>4</em>-week-old male rats did not affect the activity of 17beta-HSD, the rate-limiting enzyme of T biosynthesis in the testis. Testosterone 5alpha-reductase (T5alpha-R) activity in the testis was significantly enhanced, while aromatase mRNA was significantly reduced by increasing doses of DEHP. The expressions of cytochrome P<em>4</em>50 (CYP) isoforms, CYP2C11 and CYP3A, in the testis increased along with their enzymatic activities, T16alpha- and T6beta-hydroxylation, respectively. Thus, the current study clearly indicates that the short period exposure to DEHP alters T metabolism through altering activities of T5alpha-R, aromatase and CYP2C11/3A2 in the testis of prepubertal rats, and that they are more sensitive marker enzymes to the DEHP exposure than those of biosynthetic enzymes of T from <em>androstenedione</em>.

Mutant forms of aromatase cytochrome P-<em>4</em>50 bearing modifications of amino acid residues Pro308 and Asp309 and expressed in transfected Chinese hamster ovary cells were subjected to kinetic analysis and inhibition studies. The Km for <em>androstenedione</em> for expressed wild type (11.0 +/- 0.3 nM SEM, n = 3) increased <em>4</em>-, 25- and 31-fold for mutants Pro308Phe, Asp309Asn and Asp309Ala, respectively. There were significant differences in sensitivity among wild type and mutants to highly selective inhibitors of estrogen biosynthesis. <em>4</em>-Hydroxy<em>androstenedione</em> (<em>4</em>-OHA) a strong inhibitor of wild type aromatase activity (IC50 = 21 nM and Ki = 10 nM), was even more effective against mutant Pro308Phe (IC50 = 13 nM and Ki = 2.8 nM), but inhibition of mutants Asp309Asn and Asp309Ala was considerably less (IC50 = 3<em>4</em>5 and 330 nM and Ki = 55 and 79 nM, respectively). Expressed wild type aromatase and Pro308Phe aromatase were strongly inhibited by CGS 169<em>4</em>9A (IC50 = <em>4</em>.0 and <em>4</em>.6 nM, respectively) whereas mutants Asp309Asn and Asp309Ala were markedly less sensitive (IC50 = 1<em>4</em>0 and 150 nM, respectively). CGS 18320B produced similar inhibition. Kinetic analyses produced Ki = 0.<em>4</em> nM for CGS 169<em>4</em>9A inhibition of wild type versus 1.1, 37 and 58 nM, respectively, against Pro308Phe, Asp309Asn and Asp309Ala. The results demonstrate significant changes in function resulting from single amino acid modifications of the aromatase enzyme. Our data indicate that mutation in Asp309 creates a major distortion in the substrate binding site, rendering the enzyme much less efficient for <em>androstenedione</em> aromatization. The substitution of Pro308 with Phe produces weaker affinity for <em>androstenedione</em> in the substrate pocket, but this alteration favors <em>4</em>-OHA binding. Similarly, mutant Pro308Phe exhibits a slightly greater sensitivity to inhibition by CGS 18320B than does the wild type. These results indicate that residues Pro308 and Asp309 play critical roles in determining substrate specificity and catalytic capability in aromatase.

A- and D-ring <em>androstenedione</em> derivatives were synthesized and tested for their abilities to inhibit aromatase. In one series, C-3 hydroxyl derivatives were studied leading to a very active compound, when the C-3 hydroxyl group assumes 3β stereochemistry (1, IC(50) = 0.18 μM). In a second series, the influence of double bonds or epoxide functions in different positions along the A-ring was studied. Among epoxides, the 3,<em>4</em>-epoxide 15 showed the best activity (IC(50) = 0.1<em>4</em>5 μM) revealing the possibility of the 3,<em>4</em>-oxiran oxygen resembling the C-3 carbonyl group of <em>androstenedione</em>. Among olefins, the <em>4</em>,5-olefin 12 (IC(50) = 0.135 μM) revealed the best activity, pointing out the importance of planarity in the A,B-ring junction near C-5. C-<em>4</em> acetoxy and acetylsalicyloxy derivatives were also studied showing that bulky substituents in C-<em>4</em> diminish the activity. In addition, IFD simulations helped to explain the recognition of the C-3 hydroxyl derivatives (1 and 2) as well as 15 within the enzyme.

The single injection and constant infusion techniques were utilized to study the kinetics of dehydroepiandrosterone (DHEA) metabolism and its peripheral conversion to several other C19-steroids including C19-steroid sulfates. The MCRs (mean +/- SEM) for normal men and normal women were 1866 +/- 1<em>4</em><em>4</em> and 1901 +/- 87 liters/2<em>4</em> h, respectively. The single injection technique yielded values for rate constants (units) and volumes of distribution (1) as follows: K1, <em>4</em>2.6 +/- 7.7 for men and 37.1 +/- 5.0 for women; K2, 6<em>4</em>.3 +/- 11.2 for men and 55.5 +/- 5.0 for women; K2, 6<em>4</em>.3 +/- 11.2 for men and 55.5 +/- 5.0 for women; V1, 38.5 +/- 6.0 for men and 33.7 +/- 2.5 for women; V2, 30.<em>4</em> +/- 7.3 for men and 27.5 +/- 9.9 for women. The constant infusion technique yielded values for the conversion ratios for the transformation of DHEA to several products: delta 5-androstene-3 beta, 17 beta-diol to DHEA of 0.10 +/- 0.01 for men and 0.16 +/- 0.03 for women, delta <em>4</em>-<em>androstenedione</em> to DHEA of 0.0<em>4</em> +/- 0.01 for men and 0.07 +/- 0.02 for women, DHEA sulfate (DHEAS) to DHEA of 6.36 +/- 0.81 for men and 10.09 +/- 0.87 for women, delta 5-androstene-3 beta, 17 beta-diol sulfate to DHEA of 0.<em>4</em>2 +/- 0.06 for men and 0.50 +/- 0.0<em>4</em> for women, and androsterone sulfate to DHEA of 1.11 +/- 0.13 for men and 2.06 +/- 0.18 for women. The ratios for the conversion to DHEA sulfate and androsterone sulfate were significantly higher for women than men. The plasma concentrations of DHEA were 8.50 +/- 0.95 and 8.75 +/- 1.01 ng/ml for men and women, respectively. The calculated production rates for DHEA were 16.3<em>4</em> +/- 2.66 and 16.19 +/- 1.78 mg/2<em>4</em> h for men and women, respectively. There was no sex difference in the binding of DHEA to plasma proteins and this is reflected in the lack of sex difference in the MCRs. Calculations indicate that DHEA is a major precursor of circulating delta 5-diol.

OBJECTIVE

The mechanism of adrenarche is controversial, and there have been competing claims that its origin is in the hypothalamo-pituitary axis or in the adrenal gland itself. ACTH is proposed inducer of adrenarche so patients with ACTH resistance due to be familial glucocorticoid deficiency syndrome provide a model to clarify the degree to which ACTH is involved in the regulation of adrenal androgen secretion during adrenarche.

METHODS

Random analysis of plasma adrenal androgens and urinary adrenal androgen output in treated patients with familial glucocorticoid deficiency.

METHODS

Eleven patients (6 males and 5 females, aged 6.5-21.6 years, <em>4</em> prepubertal, minimum bone age 9 years) with familial glucocorticoid deficiency were studied. In 6, mutations, in the coding region of the ACTH receptor are the cause of their ACTH resistance. In the remaining 5 the receptor was normal.

METHODS

Physical examination, basal serum cortisol, basal plasma ACTH, serum cortisol after stimulation with 250 micrograms ACTH(1-2<em>4</em>), plasma dehydroepiandrosterone-sulphate (DHEAS) and plasma androstenedione (A<em>4</em>), total urinary androgen excretion and single-stranded sequencing of the coding region of the ACTH receptor.

RESULTS

DHEAS was undetectable in 8, and detectable but below the age-matched reference values in 3 patients. A<em>4</em> was measured in 10 patients, and in 3 patients (2 in late puberty) was found to be subnormal whereas in the remaining 7 patients A<em>4</em> was normal for age and pubertal stage consistent with the gonadal contribution to peripheral A<em>4</em> levels. Significantly diminished output of urinary adrenal androgen metabolites in 3 patients confirmed the results found in serum. The lack of adrenarche was independent of the presence of a mutation within the ACTH receptor and of the severity of glucocorticoid deficiency. Despite adequate glucocorticoid replacement therapy ACTH levels remained elevated in 10 of the 11 patients.

CONCLUSIONS

Although reduced adrenocortical inner zone cell number may contribute to the lack of adrenarche, in some patients there appears to be a discrepancy between partial glucocorticoid deficiency and significantly diminished adrenal androgen secretion. These data imply a significant contribution of ACTH to the regulation of adrenarche in normal children either by having a priming effect on the adrenal gland or by acting in concert with other adrenal androgen stimulating factors.

Hyperprolactinemic patients may have increases in plasma dehydroepiandrosterone (DHA) and dehydroepiandrosterone sulfate (DHAS). We examined the effect of lowering serum PRL with bromocriptine or pituitary surgery on the serum concentrations of adrenal androgens and on the production rate (PR) and MCR of DHAS in eight hyperprolactinemic women (HP). We also examined the effect of bromocriptine therapy on adrenal androgens in five normal men. Serum DHAS was elevated in HP compared to normal women (mean +/- SEM, 25<em>4</em> +/- 28 vs. 182 +/- 13 microgram/dl; P less than 0.0<em>4</em>). Serum DHA and <em>androstenedione</em> (delta <em>4</em>) in HP were not significantly different from normal. Serum PRL fell from 160 +/- 16 to 37 +/- 9 ng/ml during or after treatment. Mean 2<em>4</em>-h serum DHAS fell from 198 +/- 30 to 106 +/- 17 micrograms/dl (P less than 0.001) with treatment, without a change in the mean 2<em>4</em>-h serum cortisol concentration (6.2 +/- 0.<em>4</em> vs. 6.6 +/- 0.<em>4</em> micrograms/dl). Thus, the DHAS to cortisol (DHAS/F) ratio fell significantly (32 +/- 5 to 17 +/- <em>4</em>; P less than 0.001). This was also true of the DHAS/F ratio during ACTH stimulation (8 +/- 1 to 6 +/- 1; P less than 0.02). Similar changes were found in basal and ACTH-stimulated DHA/F ratios, whereas the basal and ACTH-stimulated delta <em>4</em>/F ratios did not change significantly with treatment. Treatment lowered the PR of DHAS from 27 +/- 5 to 17 +/- 3 mg/2<em>4</em> h (P less than 0.03) and increased the DHAS MCR from 16 +/- 2 to 21 +/- 3 liters/2<em>4</em> h (P less than 0.01). Bromocriptine treatment of normal men lowered serum PRL from 15 +/- 2 to less than 2.5 ng/ml. There were no significant changes in the basal and ACTH-stimulated serum DHAS/F, DHA/F, or delta <em>4</em>/F ratios or DHAS PR and MCR during bromocriptine therapy. The failure of bromocriptine to significantly alter these steroids in normal men suggests that bromocriptine was not directly responsible for the changes in HP treated with this drug. A mechanism for the increased PR of DHAS in HP was sought by examining the serum concentrations of the steroid biosynthetic intermediates relevant to DHAS production. Lowering serum PRL was associated with a decrease in basal and ACTH-stimulated 17-hydroxypregnenolone/17-hydroxyprogesterone and DHA/delta <em>4</em> ratios, suggesting an increase in 3 beta-hydroxysteroid dehydrogenase/delta <em>4</em>,5-isomerase activity. However, increased gonadal secretion of the delta <em>4</em>-steroids may have occurred with the fall in serum PRL.(ABSTRACT TRUNCATED AT <em>4</em>00 WORDS)

The synthesis and biological evaluation of <em>androstenedione</em> derivatives as inhibitors of estrogen biosynthesis are described. The results show that <em>4</em>-hydroxy analogues are among the most potent in vitro inhibitors of the series. Esterification of the <em>4</em>-hydroxy steroids generally reduced activity. Further conjugation of the 3-keto <em>4</em>-ene system to give <em>4</em>-hydroxy-<em>4</em>,6-androstadiene-3,17-dione caused more rapid inactivation of aromatase in rat ovarian microsomes than <em>4</em>-hydroxy<em>androstenedione</em>. Some compounds exhibited differences in activity when tested for inhibition of human placental microsomes vs. rat ovarian microsomes. The <em>4</em>-hydroxy<em>androstenedione</em> derivatives and their nonbulky esters were generally more potent in vitro and in vivo inhibitors than other substituted steroids in the series. Several of the synthesized compounds markedly reduce (50-81%) estrogen levels in rats on proestrus and/or had antifertility action. To date, none of the compounds surpassed the in vivo inhibitory action of <em>4</em>-hydroxy-<em>4</em>-androstene-3,17-dione or its <em>4</em>-acetate derivative.

R 76713 is a new non-steroidal compound which inhibits aromatase in vitro and in vivo with a potency of at least 1000-fold that of aminoglutethimide. In male cynomolgus monkeys peripheral conversion of labeled <em>androstenedione</em> to estrone is decreased by 85%, <em>4</em>-5 h after a single intravenous dose of 0.003 mg/kg of R 76713, without altering steroid metabolic clearance rates. In rats fed a sodium-depleted diet for 3 weeks, plasma levels of aldosterone and plasma renin activity remain unchanged 2 h after a single oral dose of up to 20 mg/kg of R 76713. This confirms previous data on the selectivity of R 76713 for aromatase inhibition as compared to inhibition of other enzymes involved in steroid biosynthesis. In male volunteers, a single oral dose of 5 or 10 mg of R 76713 lowers median plasma estradiol levels from 70 pM to the detection limit of the assay (30 pM) <em>4</em> and 8 h after intake, whereas no important changes are detected after placebo administration. In 15 premenopausal female volunteers receiving a single oral dose of 20 mg of R 76713, mean plasma estradiol levels decrease from <em>4</em>15 pM (before) to 179, 1<em>4</em>9 and 185 pM respectively <em>4</em>, 8 and 2<em>4</em> h after intake whereas they remain above 380 pM after placebo (n = 7).

The effects of phenobarbital-delayed ovulation on in vitro steroidogenesis and aromatase activity of LH-stimulated preovulatory follicles was ascertained in the cyclic hamster. After 3 days of ovulatory delay, antral follicles exhibited early signs of atresia (degenerating granulosa cells and pyknotic nuclei within oocytes). On days 2 and 3 of delay, LH-stimulated follicles produced more progesterone (P 15--20 ng P/follicle . 90 min) in vitro than control proestrous follicles (2.5 ng P/follicle . 90 min). Concomitant with the increase in LH-stimulated P production was a decline in the in vitro production of <em>androstenedione</em> (A) and estradiol (E2) on day 2 (A, 5 ng/follicle . 90 min; E2, <em>4</em> ng) and day 3 (1 ng A, less than 0.2 ng E2)-proestrous controls: 8 ng A or E2/follicle . 90 min. The steroidogenic profile of 1-day delayed follicles was similar to proestrous controls. A similar E2-P shift was observed in vitro when the steroidogenic profile of preovulatory follicles on the morning of proestrus (2.5 ng P, 8 ng A or E2) was compared with post-LH surge follicles (25 ng P, less than 1 ng A or E2) and with new corpora lutea (36 ng P, less than 0.2 ng A, less than 1 pg E2). A slight increase in the aromatizing activity of follicles was observed on days 1 and 2 of delay. However, atretic antral follicles (day 3 of delay) exhibited an aromatizing capacity similar to that of proestrous controls. Collectively, these results indicate that the loss of E2-synthesizing capacity in atretic antral follicles (induced by ovulatory delay) is due to the lack of A precursor. In addition, the results indicate that phenobarbital-delayed ovulation induces a shift from E2 to P synthesis in 3-day delayed follicles (early atretic). This E2-P shift is similar to that in Graafian follicles on the afternoon of proestrus but occurs at a much slower rate.

Fifty-eight women with polycystic ovarian syndrome (PCO) were treated with electrocautery of the ovarian capsule and then studied by hormone analysis for 12 months. In 72% ovulation appeared to occur within <em>4</em> weeks and in this group mean serum LH and FSH levels showed a statistically significant increase the day after electrocautery, followed by a gradual decline in LH to a level significantly below the pretreatment value. No changes in LH and FSH were seen in those who did not ovulate. The mean levels of testosterone, <em>androstenedione</em> and dihydrotestosterone which were in the upper normal range or slightly above before treatment were significantly reduced after electrocautery. Serum oestradiol levels showed a significant increase after 1 week, when progesterone levels were still unchanged, suggesting that follicular development was already in progress. The mean serum level of sex hormone binding globulin was slightly below the normal range before treatment and then increased gradually. Endocrine responses to electrocautery were similar to those described previously after wedge resection. The simplicity of this treatment and the good response make it an attractive alternative for treating infertility associated with PCO.

Fifteen post-menopausal patients with advanced breast cancer were treated with the LH-RH agonist leuprorelin (D-leu6-des-gly10-Gn-RH-ethylamide) given in a dosage of 7.5 mg as a monthly subcutaneous depot injection, to assess the clinical activity and endocrine response to treatment. None of the 15 patients showed an objective response to treatment, although four patients had stable disease for at least 6 months. No toxicity was demonstrated. Endocrine effects after <em>4</em> weeks' treatment were as follows: mean levels of serum gonadotrophins fell to 10% of their pretreatment values; there were no significant changes in the levels of prolactin on treatment; there was a significant decrease in the levels of serum testosterone in 12 out of 1<em>4</em> patients; there were no significant changes in the levels of oestradiol, <em>androstenedione</em> and oestrone. The lowering of serum testosterone suggests that androgens in post-menopausal women may be partly produced by the ovaries, stimulated by LH and FSH. This fall in testosterone may explain why some post-menopausal breast cancer patients in other studies have been reported to respond to treatment with LH-RH agonists, as it would decrease the substrate for the peripheral synthesis of oestrogens.

Redesign of glutathione transferases (GSTs) has led to enzymes with remarkably enhanced catalytic properties. Exchange of substrate-binding residues in GST A1-1 created a GST A<em>4</em>-<em>4</em> mimic, called GIMFhelix, with >300-fold improved activity with nonenal and suppressed activity with other substrates. In the present investigation GIMFhelix was compared with the naturally-evolved GSTs A1-1 and A<em>4</em>-<em>4</em> by determining catalytic efficiencies with nine alternative substrates. The enzymes can be represented by vectors in multidimensional substrate-activity space, and the vectors of GIMFhelix and GST A1-1, expressed in kcat/Km values for the alternative substrates, are essentially orthogonal. By contrast, the vectors of GIMFhelix and GST A<em>4</em>-<em>4</em> have approximately similar lengths and directions. The broad substrate acceptance of GST A1-1 contrasts with the high selectivity of GST A<em>4</em>-<em>4</em> and GIMFhelix for alkenal substrates. Multivariate analysis demonstrated that among the diverse substrates used, nonenal, cumene hydroperoxide, and <em>androstenedione</em> are major determinants in the portrayal of the three enzyme variants. These GST substrates represent diverse chemistries of naturally occurring substrates undergoing Michael addition, hydroperoxide reduction, and steroid double-bond isomerization, respectively. In terms of function, GIMFhelix is a novel enzyme compared to its progenitor GST A1-1 in spite of 9<em>4</em>% amino-acid sequence identity between the enzymes. The redesign of GST A1-1 into GIMFhelix therefore serves as an illustration of divergent evolution leading to novel enzymes by minor structural modifications in the active site. Notwithstanding low sequence identity (60%), GIMFhelix is functionally an isoenzyme of GST A<em>4</em>-<em>4</em>.

The aim of this study was to clarify the mechanism by which cytochrome P<em>4</em>50 (P<em>4</em>50)-mediated catalytic activity is decreased following interferon (IFN) administration. Microsomal steroid hydroxylation was assessed to test the hypothesis that IFN selectively decreases the activities of individual P<em>4</em>50 isozymes in male rats. Thus, recombinant rat IFN gamma (r-rat IFN gamma) treatment produced <em>4</em>0% and 17% reductions in androst-<em>4</em>-ene-3,17-dione (<em>androstenedione</em>) 6 beta- and 16 beta-hydroxylation, respectively. <em>Androstenedione</em> 16 alpha- and 7 alpha-hydroxylation were unaltered following r-rat IFN gamma treatment. Similar changes in the <em>androstenedione</em> hydroxylation pathways were observed following administration of naturally derived rat IFN alpha/beta. Microsomal levels of P<em>4</em>50IIIA2, the male-specific constitutive steroid 6 beta-hydroxylase, were lower after administration of r-rat IFN gamma (<em>4</em>2% of control fractions). Furthermore, hepatic P<em>4</em>50IIIA2 mRNA was found to be decreased to a similar extent by r-rat IFN gamma. These findings suggest that IFN selectively decreases the content of this isozyme by a mechanism involving altered mRNA regulation. Sex steroids were unlikely to have mediated the decrease in P<em>4</em>50IIIA2 levels since serum estradiol and testosterone levels were unchanged by r-rat IFN gamma. In order to determine whether IFN alters the expression of P<em>4</em>50IIIA1, a steroid-inducible member of the P<em>4</em>50IIIA gene subfamily which is not expressed in untreated rat liver, adult female rats (which lack P<em>4</em>50IIIA2) were coadministered pregnenolone 16 alpha-carbonitrile and r-rat IFN gamma. However, IFN failed to impair the induction of <em>androstenedione</em> 6 beta-hydroxylation produced by pregnenolone 16 alpha-carbonitrile. These findings suggest that although IFN decreases the expression of P<em>4</em>50IIIA2, it may not down regulate the expression of other steroid-inducible P<em>4</em>50IIIA proteins. In view of the existence of human P<em>4</em>50IIIA orthologs which catalyze the metabolism of several important therapeutic agents, the findings of this study may help predict possible drug interactions in patients receiving IFN.

1. The synthesis, both in vivo and in vitro, of various steroid hormones in the adrenals, testes and ovaries of rats was compared at various stages of vitamin A deficiency with the corresponding pair-fed controls. 2. The enzymic conversion of the Delta(5)-3beta-hydroxy steroids into the corresponding Delta(<em>4</em>)-3-oxo steroids was significantly decreased in these tissues even at the mild-deficiency stage, further loss taking place on prolonging the deficiency. 3. In all three tissues the loss in the production of <em>androstenedione</em> from dehydroepiandrosterone could be reactivated in vitro by retinol or retinoic acid, but only at the mild stage of the deficiency. <em>4</em>. Of the various lipoidal materials tried, retinal, retinol, retinoic acid, alpha-tocopherol acetate, menadione, calciferol, lecithin, palmitic acid and cholesterol, only retinol and retinoic acid restored the lost activity. 5. Intraperitoneal injection of retinal, retinol or retinoic acid into the deficient rats, 2<em>4</em>hr. before they were killed, corrected the effect of the deficiency on this reaction. 6. Vitamin A deficiency markedly affected the synthesis of deoxycorticosterone and corticosterone from pregnenolone in the adrenals of rats, even at the mildly deficient stage, with further loss taking place at the acute stage; in vitro, retinol or retinoic acid could restore the loss at the mild-deficiency but not at the acute-deficiency stage. 7. The deficiency had no such effect on the synthesis of deoxycorticosterone from progesterone or of corticosterone from progesterone or deoxycorticosterone. 8. Compared with the pair-fed normals, the adrenals of deficient rats contained smaller amounts of deoxycorticosterone and corticosterone.

We studied the effects of epidermal growth factor (EGF) on steroidogenesis in freshly prepared Percoll-purified Leydig cells from prepubertal and adult rats and mice, and in interstitial cells from immature rats cultured in the presence or absence of LH. It is demonstrated that EGF directly stimulates the output of C19-steroids (testosterone and <em>androstenedione</em>) as well as C21-steroids (progesterone, 17 alpha-hydroxyprogesterone and 20 alpha-hydroxypregn-<em>4</em>-en-3-one) in all systems studied. When combined with LH, EGF has little effect on freshly isolated cells but still stimulates steroidogenesis in cells cultured in the presence of LH. The strong inhibitory effects of EGF on androgen production that have been reported previously are only observed when cells that have lost part of their steroidogenic potential by prolonged culture in the absence of LH are acutely challenged with LH (or cholera toxin or dbcAMP) and EGF. Under the latter conditions EGF blocks the conversion of C21-steroids into C19-steroids. The stimulatory effects of EGF on androgen production are evident within the first hours of incubation and occur at ED50 values of 0.3 up to 2.5 ng/ml. They are not accompanied by any measurable change in the production of cAMP. The effects of EGF are compared to those of LHRH and those of SCF, a putative paracrine factor produced by Sertoli cells. Although there are many similarities between the effects of these three polypeptides on steroidogenesis in Leydig cells, our data indicate that they must act by different mechanisms. Moreover, SCF is the only one of these agonists that markedly stimulates androgen production in the presence of LH.