The role of estrogen in bone growth and maturation during childhood and adolescence

Twenty years ago it was believed that pubertal growth was stimulated by testicular androgen in boys and by adrenal androgen in girls. Estrogen, which was used to inhibit growth in excessively tall girls, was not thought to have growth-promoting effects. We hypothesized that estrogen has a biphasic effect on epiphyseal growth, with maximal stimulation at low levels. We showed that the administration of low doses of estrogen, corresponding to a serum estradiol level of about 4 pg/ml (15 pmol/l) caused more than a 60% increase over the prepubertal growth rate in both boys and girls. To test the hypothesis that estrogen is the principal mediator of the pubertal growth spurt in boys, we administered the aromatase inhibitor, testolactone, to boys with familial male-limited precocious puberty. Testolactone produced near normalization of both growth velocity and bone maturation, despite levels of serum testosterone that remained within the adult male range. The observation that low levels of estrogen stimulate growth and bone maturation suggested that estrogen might explain the more rapid epiphyseal maturation of prepubertal girls compared to boys. To determine whether prepubertal girls have higher estrogen levels than prepubertal boys, we developed an ultrasensitive recombinant cell bioassay for estrogen with a sensitivity of 0.02 pg/ml (0.07 pmol/l) estradiol equivalents. Prepubertal girls had approximately eight-fold higher levels of serum estradiol than did prepubertal boys (0.6 ± 0.6 pg/ml (SD) (2.2 ± 2.2 pmol/l) vs 0.08 ± 0.2 pg/ml (0.29 ± 0.73 pmol/l), P < 0.05). We concluded that the pubertal growth spurt of both sexes is driven primarily by estrogen, and that the more rapid epiphyseal maturation of prepubertal girls (vs boys) may be explained by their higher estradiol levels.     

Effects of testosterone and estrogen on hepatic levels of cytochromes P450 2C7 and P450 2C11 in the rat.

The effects of exogenous hormone treatment on the expression of cytochromes P450 2C7 and P450 2C11 were studied in neonatally gonadectomized and sham-operated male and female rats. Hepatic levels of cytochrome P450 2C7 were found to be two- to threefold higher in intact adult female versus male rats. Neonatal gonadectomy resulted in a reversal of the relative cytochrome P450 2C7 levels in male and female animals at maturity. Expression of this isozyme was restored in ovariectomized females by estradiol treatment. Furthermore, neonatal and/or pubertal administration of estradiol to intact male rats induced cytochrome P450 2C7 to adult female levels. On the other hand, administration of testosterone at all times examined had no effect in intact female rats, but decreased cytochrome P450 2C7 to normal levels in neonatally castrated males treated during adulthood. Neonatal testosterone treatment also increased hepatic cytochrome P450 2C7 content in both ovariectomized females and intact males. These results indicate that estrogen is required for full expression of cytochrome P450 2C7 while the effect of testosterone is ambiguous. In comparison, neonatal gonadectomy of male rats abolished the adult expression of cytochrome P450 2C11. Normal levels were restored only by treatment with testosterone during adulthood. Neonatal testosterone treatment did not induce cytochrome P450 2C11 levels in gonadectomized rats of either sex. In contrast, neonatal estrogen treatment suppressed cytochrome P450 2C11 expression in intact adult male rats to the same extent as neonatal castration. These results indicate that androgen exposure during the adult, and not the neonatal, phase is essential for full expression of cytochrome P450 2C11.     

The differential regulation of aldosterone output in hamster adrenal by angiotensinII and adrenocorticotropin.

Aldosterone was isolated from hamster adrenal cells and was identified by high performance liquid chromatography and thermospray mass spectroscopy analysis. Basal outputs from adrenal cell suspensions were of the same order of magnitude, 8.4 ± 1.9 ng and 8.0 ± 0.7 ng/2 h/50,000 cells, for aldosterone and corticosteroid, respectively. The outputs of aldosterone and corticosteroid increased with K⁺ concentrations to reach maxima of 3.3- and 1.6-fold at 10 meq/l of K⁺. Angiotensin_II (A_II) produced dose-dependent increases in aldosterone and corticosteroid outputs with maxima of 3- and 4-fold, respectively. In contrast, ACTH induced relatively no changes in aldosterone output, whereas dose-dependent increases in corticosteroid output were found. In time study experiments, with 10⁻⁸ M A_II, aldosterone and corticosteroid outputs were maximally increased after 1 h (6-fold) and 3 h (1.8-fold), respectively. At 10⁻⁸ M, ACTH had a small stimulatory effect on aldosterone output after 6 h, whereas it provoked a gradual increase in corticosteroid output (up to 7-fold after 8 h of incubation). The effects of A_II and ACTH on adrenal cytochrome P-450_11β involved in the last steps of aldosterone formation were evaluated by c combined in vivo andin vitro experiments. The P-450_11β mRNA level was increased by a low sodium intake but not by a 24 h ACTH stimulus. These results taken together indicate that ACTH and A_II differentially regulate P-450_11β. It is postulated that these two regulatory peptides regulate the hamster adrenal steroidogenesis by different P-450 genes.     

Separate induction of the two isozymes of cytochrome P-45011β in rat adrenal zona glomerulosa cells

In the rat adrenal cortex, two isozymes of cytochrome P-450_11β (CYP11B1 and CYP11B2) have been identified. They are encoded by two different genes with a homology much higher in their coding than in their 5′-flanking regions. CYP11B1 is found in all the zones of the gland and catalyzes a single hydroxylation of deoxycorticosterone (DOC) in the 11β- or the 18-position. CYP11B2 is produced exclusively in the zona glomerulosa and catalyzes all three reactions involved in the conversion of DOC to aldosterone. In vivo and in vitro, the expression of the genes encoding CYP11B1 and CYP11B2 is regulated by two separate control systems which appear to operate both independently and interdependently. In vivo, zona glomerulosa expression of CYP11B1 was enhanced by ACTH treatment or potassium depletion and was lowered by potassium repletion. CYP11B2 expression disappeared upon potassium depletion or ACTH treatment, but reappeared during potassium repletion. In vitro, only CYP11B1 activity was detectable and responsive to ACTH treatment in zona glomerulosa cells cultured at a potassium concentration of 6.4 mmol/1. Aldosterone biosynthetic activity and mRNA encoding CYP11B2 could be detected only after at least 1 day of exposure to a high extracellular potassium concentration ( 12 mmol/1).     

Inhibition of adrenal steroid metabolism by administration of 1-aminobenzotriazole to guinea pigs

Prior in vitro investigations demonstrated that the P450 suicide substrate, 1-aminobenzotriazole (ABT), was a potent inhibitor of xenobiotic metabolism but had no effect on steroidogenic enzymes in the guinea pig adrenal cortex. Studies were done to determine if ABT administration to guinea pigs in vivo also selectively inhibited adrenal xenobiotic metabolism. At single doses of 25 or 50 mg/kg, ABT effected rapid decreases in spectrally detectable adrenal P450 concentrations. The higher dose caused approx. 75% decreases in microsomal and mitochondrial P450 levels within 2 h. The decreases in P450 were sustained for 24 h but concentrations returned to control levels within 72 h. Accompanying the ABT-induced decreases in adrenal P450 content were proportionately similar decreases in P450-mediated xenobiotic and steroid metabolism. Microsomal benzo(a)pyrene hydroxylase, benzphetamine N-demethylase, 17-hydroxylase and 21-hydroxylase activities were decreased to 20–25% of control values by the higher dose of ABT. Mitochondrial 11β-hydroxylase and cholesterol sidechain cleavage activities were similarly diminished by ABT treatment. Adrenal 3β-hydroxysteroid dehydrogenase activity, by contrast, was not affected by ABT, indicating specificity for P450-catalyzed reactions. The results demonstrate that ABT in vivo is a non-selective inhibitor of adrenal steroid- and xenobiotic-metabolizing P450 isozymes. The absence of ABT effects on steroid metabolism in vitro suggests that an extra-adrenal metabolite may mediate the in vivo inhibition of steroidogenesis.     

Molecular pathology of steroid 21-hydroxylase deficiency

The molecular pathology of steroid 21-hydroxylase deficiency is attributable to unequal crossover-mediated gene deletion or to large- or small-scale replacement of the functional CYP21B gene sequence by a copy of the analogous CYP21A pseudogene sequence. Because the pathological point mutations originate from the pseudogene which shows only a small number of differences from the functional CYP21B gene sequence, the total number of different pathological point mutations is likely to be small. Mutant P450c21 enzymes carrying specific amino acid substitutions seen in patients with 21-hydroxylase deficiency exhibit activities that correlate with the clinical severity of the disease and with biochemical abnormalities such as 17-hydroxyprogesterone levels after ACTH (corticotropin) stimulation.     

Angiotensin-II-directed glomerulosa cell function in fetal adrenal cells

These studies were undertaken to examine the role of angiotensin II (A-II) in the regulation of adrenal glomerulosa cell differentiation. We were interested particularly in the ability of A-II to support aldosterone production in fetal adrenal cells. Many in vitro studies on acute A-II stimulation of aldosterone synthesis in adrenocortical cells have been documented. However, it is the long-term modification of steroid-metabolizing enzyme expression that leads to the formation and release of specific adrenal steroids. Herein, we used primary cultures of fetal bovine adrenal (FBA) cells to examine the effects of A-II on aldosterone production and the expression of aldosterone synthase cytochrome P450 (P450c18). A-II treatment caused the primary cultures to maintain glomerulosa cell functions. Cells treated for 3 days with A-II increased aldosterone production by 10-fold. A-II stimulation of aldosterone production occurred rapidly (within 30 min) and in a dose-dependent manner. In addition, A-II enhanced the activity of P450c18, the enzyme responsible for conversion of corticosterone to aldosterone. A-II also suppressed ACTH-promoted cortisol production, while increasing ACTH-stimulated release of aldosterone. It appears that these effects of chronic treatment with A-II were mediated through an A-II type 1 (AT₁) receptor since the AT₁ receptor antagonist, Dup753, blocked aldosterone production and the increased P450c18 activity. Receptor binding studies suggest that FBA cells possess approx. 110,000 AT₁ binding sites/cell with K_d = 1.8 × 10⁻⁹ M. Via AT₁ receptors, A-II was able to stimulate both inositol phosphates and cAMP production. The stimulation of cAMP production, however, was much less than seen following ACTH treatment. These data give support to the hypothesis that A-II is involved in the differentiation of fetal adrenal cells into glomerulosa cells. This process appears to be mediated through regulation of steroid-metabolizing enzyme expression and the activation of steroid production.     

Molecular and endocrine characterization of a mutation involving a recombination between the steroid 21-hydroxylase functional gene and pseudogene

The gene encoding steroid 21-hydroxylase activity, P450c21B, is located in the major histocompatibility complex (MHC) class III region, in close proximity to a highly homologous pseudogene, P450c21A. Recombinations between P450c21B and P450c21A have been shown to result in deficiency of 21-hydroxylase activity, the usual cause of congenital adrenal hyperplasia (CAH). A mutant P450c21 gene from a patient with simple virilizing CAH was identified and shown to be consistent with a recombination between P450c21A and P450c21B. Sequence analysis of the mutant gene showed the recombination site to be located between the first exon and the second intron. The mutant gene encodes a leucine instead of the normal proline at codon 31. This mutation resides on a chromosome bearing the HLA-B44 serotype. A comparison of mutation associated with HLA-B44 and that normally found with the HLA-Bw47 serotype suggests that the HLA-B44 mutations are of more ancient origin. The patient's homologous chromosome has a deletion of P450c21B. Endocrinological testing therefore allows for testing of the mutant gene in genetic isolation. Such testing demonstrated that the patient was capable of producing aldosterone and retaining sodium in response to a low-sodium diet, indicating that the mutant gene encodes an enzyme with partial 21-hydroxylase activity.     

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

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

Adult male-specific and neonatally programmed rat hepatic P-450 forms RLM2 and 2a are not dependent on pulsatile plasma growth hormone for expression

Rat hepatic cytochrome P-450 form RLM2 is a testosterone 15 alpha-hydroxylase reported to be male-specific on the basis of purification studies (Jansson, I., Mole, J., and Schenkman, J. B. (1985) J. Biol. Chem. 260, 7084-7093). The sex dependence, developmental regulation, xenobiotic induction, and hormonal control of P-450 RLM2 expression were studied using P-450 form-specific immunochemical and catalytic assays. Polyclonal antibodies raised to rat hepatic P-450 3 (P-450 gene IIA1) were found to cross-react strongly with P-450 RLM2, but not with 10 other rat P-450 forms, suggesting that P-450 3 and P-450 RLM2 are highly conserved in primary structure. Western blotting of liver microsomes under conditions where P-450s 3 and RLM2 are resolved electrophoretically revealed that P-450 RLM2 is markedly induced at puberty in male rats, with no protein detected (less than or equal to 5% of adult male levels) in adult females or immature animals of either sex. A similar developmental dependence was observed for hepatic microsomal testosterone 15 alpha-hydroxylase activity, which was found to be catalyzed primarily by P-450 RLM2. P-450 RLM2 was resistant to induction by several xenobiotics and in the case of phenobarbital and beta-naphthoflavone, was suppressed by 50-60%. Studies on the steroid hormonal regulation of P-450 RLM2 revealed that its adult male-specific expression is imprinted (programmed) in response to neonatal testosterone exposure. Ovariectomy studies demonstrated that suppression by estrogen does not contribute significantly to the absence of P-450 RLM2 in adult female rats. Although the male-specific developmental induction of P-450 RLM2 in response to neonatal testosterone is strikingly similar to that of P-450 2c (testosterone 2 alpha/16 alpha-hydroxylase; gene IIC11), P-450 RLM2 expression is not dependent on the pulsatile pituitary growth hormone secretion required for P-450 2c synthesis. Rather, hypophysectomy of adult male rats increased P-450 RLM2 and its associated testosterone 15 alpha-hydroxylase activity by 50-100%.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of dietary soy and estrous cycle on adrenal cytochrome p450 1B1 expression and DMBA metabolism in adrenal glands and livers in female Sprague-Dawley rats

Cytochrome p450 1B1 (CYP1B1) has been shown to be important in the bioactivation of 7,12-dimethylbenz[a]anthracene (DMBA) to an adrenal toxin in rats. We investigated the effects of diet and stage of estrous cycle on CYP1B1 expression in rat adrenal glands and on DMBA metabolism by rat adrenal and hepatic microsomes. Female Sprague-Dawley (SD) rats were placed on either standard soy-containing NIH-31 rat chow or soy- and alfalfa-free 5K96 diet from postnatal day (PND) 21 until sacrifice at PND50+/-5. Stage of estrous at sacrifice was assessed by vaginal cytology and confirmed by histological examination of the vagina. Dietary soy at the level present in NIH-31 diet did not affect serum estrogen and progesterone levels. Immunohistochemical analysis confirmed that CYP1B1 was exclusively expressed in the zona fasciculata and zona reticularis in adrenal cortex, which are the regions vulnerable to DMBA-induced adrenal necrosis. Adrenal CYP1B1 protein expression, 3H-DMBA depletion, and formation of DMBA-3,4-, and -8,9-dihydrodiols by adrenal microsomes were greater in animals fed 5K96 diet, and the stage of the estrous cycle affected these parameters only in the soy-free 5K96 diet. In hepatic microsomes, the formation of DMBA-3,4-dihydrodiol, 7-hydroxy- and 12-hydroxy-DMBA were lower in animals fed NIH-31 diet than in those fed 5K96 diet. Thus, dietary soy and the estrous cycle appear to regulate adrenal CYP1B1 expression and DMBA metabolism by both adrenal and hepatic microsomes. The use of different basal diets containing variable levels of soy components may affect certain toxicity assessments.     

Hypothalamic excitatory amino acid system during sexual maturation in female rats

The present results indicate that during sexual maturation the APOA-MBH from rats of 30 days of age released significantly higher quantities of GnRH than the tissue from 16-day-old rats (P < 0.01). The addition of NMDA, an agonist of the excitatory amino acids system (EAAs), to the medium after 30 min of incubation significantly increased (P < 0.01) the GnRH release in normal rats of both ages and this increase was significantly (P < 0.01) higher in 30-day-old rats (to 661%) than in rats of 16 days of age (to 273%). The administration of estrogen-progesterone (EP) to rats of 16 days of age did not modify the GnRH release response to NMDA. On the contrary, at 30 days of age EP administration significantly potentiated the GnRH release response to NMDA since while in the control group NMDA increased the GnRH release to 630%, in the EP-pretreated group this was to around 4700% (P < 0.01). EP pretreatment of prepubertal rats decreases the hypothalamic release of aspartate and glutamate, the excitatory amino acids involved in NMDA neurotransmission and glycine but increases EAAs release in peripubertal rats. On the basis of these results it is proposed that the increase in EAAs release by the hypothalamus is directly connected with the onset of puberty and that the maturation of the positive feedback effect of ovarian hormones on gonadotropin secretion is related to the maturation of the capacity of EP to increase hypothalamic EAAs. Before this maturational event EP inhibits EAAs release as well as gonadotropin release (prepubertal rats). NMDA receptor stimulation leads to a positive mechanism which increases the release of Asp and Glu from APOA-MBH both in prepubertal and peripubertal rats, but EP potentiates this mechanism only in peripubertal rats. This could be an additional neuroendocrine mechanism involved in the increase of gonadotropin during sexual maturation which induces the onset of puberty and the preovulatory discharge of these pituitary hormones.