Androgen receptor gene polymorphism influence fat accumulation: A longitudinal study from adolescence to adult age.

To determine the influence of androgen receptor CAG and GGN repeat polymorphisms on fat mass and maximal fat oxidation (MFO), CAG and GGN repeat lengths were measured in 128 young boys, from which longitudinal data were obtained in 45 of them [mean ± SD: 12.8 ± 3.6 years old at recruitment, and 27.0 ± 4.8 years old at adult age]. Subjects were grouped as CAG short (CAGS ) if harboring repeat lengths ≤ 21, the rest as CAG long (CAGL ); and GGN short (GGNS ) if GGN repeat lengths ≤ 23, or long if > 23 (GGNL ). CAGS and GGNS were associated with lower adiposity than CAGL or GGNL (P < 0.05). There was an association between the logarithm of CAG repeats polymorphism and the changes of body mass (r = 0.34, P = 0.03). At adult age, CAGS men showed lower accumulation of total body and trunk fat mass, and lower resting metabolic rate (RMR) and MFO per kg of total lean mass compared with CAGL (P < 0.05). GGNS men also showed lower percentage of body fat (P < 0.05). In summary, androgen receptor CAG and GGN repeat polymorphisms are associated with RMR, MFO, fat mass, and its regional distribution in healthy male adolescents, influencing fat accumulation from adolescence to adult age.

Association of Taq 1B CETP polymorphism with insulin and HOMA levels in the population of the Canary Islands.

BACKGROUND AND AIMS: Cholesteryl ester transfer protein (CETP) is an enzyme with a key role in lipoprotein metabolism. A common genetic polymorphism, the Taq 1B, influences CETP activity and HDL-cholesterol levels, with individual homozygotes for the B1 allele exhibiting higher enzyme activity and lower HDL-cholesterol levels than carriers of at least one B2 allele. Our aim was to analyze the influence of Taq 1B CETP polymorphism on cardiovascular risk factors in a representative sample of adult subjects from Canary population. METHODS AND RESULT: A total of 518 adult subjects from the Canary Islands, enrolled in a nutritional survey (the ENCA study), were included. The Taq 1B polymorphism was analyzed by PCR-RFLP. Compared with individuals with at least one B2 allele, and after adjusting for age, sex, BMI, waist perimeter, smoking and alcohol intake, carriers of the B1B1 genotype showed lower HDL-cholesterol levels (geometric mean (95% CI): 46.6 (44.5-48.8) vs. 50.6 (49.1-52.9)mg/dl; P=0.003); and higher insulin (geometric mean (95% CI): 11.1 (10.5-11.9) vs. 10.0 (9.5-10.5µU/ml; P=0.008) and HOMA levels (geometric mean (95% CI): 2.3 (2.1-2.5) vs. 2.1 (1.9-2.1); P=0.009). In addition, the B1B1 genotype was more frequent in individuals who had low levels of HDL-cholesterol according to the National Cholesterol Education Program Adult Treatment Panel III (NCEP-ATP III) criteria (Odds Ratio (OR): 1.563; 95% CI: 1.04-2.34; P=0.030), and in those included in the upper quartile of insulinemia (OR: 1.90; 95% CI: 1.20-3.03; P=0.007) and HOMA (OR: 1.61; 95% CI: 1.02-2.57; P=0.043). CONCLUSION: The observed influence of Taq 1B polymorphism on insulin levels and HOMA highlights the possible role of CETP in the regulation of glucose homeostasis.

Gene polymorphisms in TYMS, MTHFR, p53 and MDR1 as risk factors for breast cancer: a case-control study.

Breast cancer (BC) is a complex disease influenced by environmental and genetic factors. The disease has important genetic and environmental components, most of them are still unknown. An important role of gene polymorphisms related to the risk of developing BC has been reported. However, the results have been controversial. We investigated the association of TSER, MTHFR C677T, p53 codon 72 and MDR1 C3435T gene polymorphisms with breast carcinoma in women from Canary Islands (Spain). Blood samples collected from 135 patients with BC and 304 healthy controls all of them Caucasian, were analyzed through polymerase chain reaction-restriction fragment length polymorphism. Subsequently, a structured questionnaire including patient history and risk factors in relation to BC development was filled out. Allelic frequencies of these genetic variations were: TSER, (2) 0.55 and (3) 0.45 in cases, 0.49 and 0.51 respectively in controls (P=0.240); MTHFR C677T, (C) 0.63 and (T) 0.37 in cases, 0.60 and 0.40 respectively in controls (P=0.568); p53 Arg72Pro, (Arg) 0.74 and (Pro) 0.26 in cases and controls (P=0.910); MDR1 C3435T, (C) 0.52 and (T) 0.48 in cases, 0.55 and 0.45 respectively in controls (P=0.523). We did not observe any gene polymorphism as a risk factor to develop BC. A statistical association was observed between p53 codon 72 polymorphism and family history of breast cancer in both groups, as well as between MDR1 C3435T and smoking habits in cases (P<0.05). Gene polymorphisms vary by regions. The present study contributes to the characterization of the genetic pattern of the Canary population.

Serum resistin and polymorphisms of androgen receptor GAGn and GGNn and aromatase TTTAn.

There is evidence that androgens are regulators of insulin resistance (IR), and may be involved in the regulation of resistin, a cytokine that has been related with IR. Earlier studies found that androgen receptor length polymorphisms CAGn and GGNn and the aromatase polymorphism TTTAn may influence receptor or enzyme activity and serum concentrations of androgens. This study was designed to determine whether polymorphism length was related to serum resistin concentration and to other variables related with IR. In 1,580 persons chosen randomly from the general population of the Canary Islands (Spain), we measured polymorphism length, waist circumference, waist/hip ratio, BMI, and serum glucose concentration. In smaller subgroups, we also measured C-peptide (n = 677), resistin (n = 583), and leptin concentration (n = 754) and estimated IR (homeostasis model assessment-IR (HOMA2-IR)). In men, polymorphism length correlated with resistin concentration (CAGn, r = 0.13, P = 0.031; TTTAn, r = 0.15, P = 0.005; GGNn, r = -0.15, P = 0.026), and the correlations were confirmed in multivariate regression models. The length of CAGn and TTTAn correlated inversely with C-peptide (r = -0.13, P = 0.016 and r = -0.21, P < 0.001, respectively) and with estimated IR (r = -0.12, P = 0.032 and r = -0.19, P = 0.001, respectively). In men, length of the CAGn, GGNn, and TTTAn was associated with serum resistin concentration. These results support the hypothesis that androgens may be involved in the regulation of resistin. Resistin may be a link between IR and androgens.

Steroid binding sites in liver membranes: interplay between glucocorticoids, sex steroids, and pituitary hormones.

Steroid hormones activate target cells through specific receptors that discriminate among ligands based upon recognition of distinct structural features. For most known steroids, membrane and nuclear receptors co-exist in many target cells. However, while the structure of the nuclear receptors and their function as transcriptional activators of specific target genes is generally well understood, the identity of the membrane receptors remains elusive. Using pharmacological and biochemical approaches, we are beginning to characterize receptors for glucocorticoids and anabolic-androgenic steroids in male rat liver membranes. Male rat liver endoplasmic reticulum contains two steroid binding sites which are functionally related and associated with a 90-134 kDa oligomeric protein: (1) the low-affinity glucocorticoid binding site (LAGS), composed at least in part of two peptides (37 and 53 kDa) that bind glucocorticoids and (2) the stanozolol binding protein (STBP), composed at least in part of three peptides (22, 31, and 55 kDa) that bind the synthetic androgen stanozolol. These steroid binding proteins have many properties different from those of classical nuclear receptors, with the salient differences being a failure to recognize "classical" ligands for nuclear receptors together with marked differences in biochemical properties and physiological regulation. The mechanism of interaction of glucocorticoids with the LAGS can be clearly distinguished from that with STBP. Moreover, STBP shows an extremely narrow pharmacological profile, being selective for ST and its analog, danazol, among more than 100 steroids and non-steroidal compounds that were assayed, including those that are able to displace glucocorticoids from the LAGS. The level of LAGS activity undergoes dramatic variations following changes from the physiological serum levels of thyroid hormones, glucocorticoids, GH, vitamin A, and E2. However, neither thyroid hormones nor GH have a critical role on STBP activity. The STBP is functionally related to LAGS. We have suggested a novel mechanism for STBP whereby membrane-associated glucocorticoid binding activity is targeted by stanozolol (and 16beta-hydroxylated stanozolol): stanozolol modulates glucocorticoid activity in the liver through negative allosteric modulation of the LAGS resulting in an effective increase in classical GR-signaling by increasing glucocorticoid availability to the cytosolic GR.

Solubilization and photoaffinity labeling identification of glucocorticoid binding peptides in endoplasmic reticulum from rat liver.

Steroid-binding proteins unrelated to the classical nuclear receptors have been proposed to play a role in non-genomic effects of steroid hormones. We have previously described that the low-affinity glucocorticoid binding protein (LAGS), present in the endoplasmic reticulum of the male rat liver, has pharmacological and biochemical properties different from those of nuclear receptors. The LAGS is under multihormonal regulation and binds glucocorticoids, progestins, and synthetic steroids but is unable to bind either estradiol, testosterone, or triamcinolone acetonide. In this study, we have solubilized the LAGS and investigated their pharmacological and hydrodynamic properties and their peptide composition. We found that LAGS is an integral protein bound to the endoplasmic reticulum. CHAPS provided its optimal solubilization without changes in its pharmacological properties. Hydrodynamic properties of LAGS showed that it has a molecular mass of at least 135 kDa. SDS-PAGE of covalently-labeled LAGS showed that [3H]dexamethasone binds two peptides of 53 and 37 kDa, respectively. Thus, the LAGS appears as an oligomeric protein under multihormonal regulation. The availability of solubilized LAGS and the fact that it can be induced in vivo represent major steps toward purification and understanding the functional significance of this unique steroid-binding protein.

Prevention of dexamethasone-induced lymphocytic apoptosis in the intestine and in Peyer patches by enteral nutrition.

BACKGROUND: Apoptosis is a programmed cell death, genetically controlled, that can be activated by physiological and pathophysiological events and by the administration of several drugs, including the exogenous administration of corticosteroids. The aim of this study was to develop a rat model of intestinal lymphocytic apoptosis induced by dexamethasone to determine if apoptosis could be prevented by enteral or parenteral nutrition. METHODS: Male Sprague-Dawley rats were used. On day 0, the right internal jugular vein was catheterized for parenteral nutrition, and a silicone tube was inserted into the duodenum for enteral feeding. Animals (n = 6/group) were randomly assigned to one of the following 3 feeding regimens: an immune-enhancing enteral diet; its placebo (the same formula without immunonutrients); and isocaloric isonitrogenous parenteral nutrition. On the seventh day, 200 microg of dexamethasone or vehicle were administered by i.v. bolus, and the diets were continued for 1 more day. Intestinal Peyer patches and thymic lymphocytic apoptosis were determined both by flow-cytometry and immunohistochemistry. RESULTS: A single dexamethasone dose (200 microg/rat) administered to surgically treated rats fasted for 18 hours, 24 hours later, caused massive intestinal and Peyer patches lymphocytic apoptosis (96 +/- 2% and 85 +/- 5%, respectively; p < .0001 versus vehicle in both kinds of tissue). Lymphocytic apoptosis was reduced to almost indetectable levels in intestinal and lamina propia lymphocytes (from 96 +/- 2% to <0.6%; p < .0001). Peyer patches lymphocytic apoptosis was reduced as well (from 85 +/- 5% to 15 +/- 7.1%; p < .001) in animals prefed the 2 enteral nutrition formulas. Those prefed parenteral nutrition only showed a partial decrease of lymphocytic apoptosis in the intestine (from 96 +/- 2% to 53 +/- 23%; p < .001). Nutrition had no effect on the dexamethasone-induced thymus involution. CONCLUSIONS: Enteral nutrition prevents intestinal intraepithelial and lamina propia lymphocytic apoptosis due to dexamethasone. These findings support the use of early enteral nutrition in critically ill patients treated with corticosteroids.

Photoaffinity labeling identification of a specific binding protein for the anabolic steroids stanozolol and danazol: an oligomeric protein regulated by age, pituitary hormones, and ethinyl estradiol.

We have demonstrated previously that both rat and human liver microsomes contain a highly specific binding protein for the anabolic steroids stanozolol (ST) and danazol (DA). In this study we solubilized the male rat liver ST-binding protein (STBP) and investigated the following parameters: 1) pharmacological properties, 2) hydrodynamic properties, 3) peptidic composition, 4) the effects of age and hypophysectomy, and 5) inducibility by 17alpha-ethinyl estradiol. We found that STBP is an integral protein bound to the endoplasmic reticulum. 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) provided its optimal solubilization without changes in its pharmacological properties, i.e. high specificity for ST and danazol, between natural steroids and ligands of low affinity glucocorticoid-binding sites or of progesterone-binding sites. Hydrodynamic properties of the STBP showed that it has a molecular mass of at least 118 kDa. SDS-PAGE of covalently labeled STBP under nonreducing conditions showed that [3H]ST binds to a 110-kDa protein. The STBP was resolved under reducing conditions into three peptides of 55, 31, and 22 kDa, respectively. STBP increased from immature to adult rats, and it dramatically decreased after hypophysectomy. Unlike the 22-kDa peptide, both the 55- and 31-kDa peptides drastically decreased in both immature and hypophysectomized rats. 17alpha-Ethinyl estradiol administration to immature or hypophysectomized rats induced the 55- and 31-kDa [3H]STBP to a greater extent than the 22-kDa peptide. Thus, STBP appears as an oligomeric protein composed of hormone-regulated peptides. The availability of solubilized STBP and the fact that it can be induced in vivo represent major steps toward the purification and functional significance of this unique steroid-binding protein.

Thyroid hormone regulation of rat hepatocyte proliferation and polyploidization.

The liver of adult mammals contains various classes of polyploid hepatocytes produced by a process that is partially regulated by hormones. However, it is not well understood how the hormones affect the rate of hepatocyte proliferation under physiological conditions. Here we have studied the specific roles of 3,5,3'-triiodothyronine (T3), growth hormone (GH), and sex steroids on the percentage of diploid nuclei in S phase and on the population of liver tetraploid (4C) cell nuclei in several rat model systems. Gonadal steroids had no effect on the S phase but account for gender differences in the 4C nuclei. Hypophysectomy in adult male rats produced a moderate decrease in 4C nuclei that was reversed by treatment with 25 micrograms T3. kg-1. day-1, whereas treatment with 200 micrograms human recombinant GH (hGH). kg-1. day-1 was ineffective. Rats made hypothyroid by methimazole treatment of dams and pups until death showed a low S phase and only 5% of 4C nuclei at 70 days of age. T3 significantly increased the S phase 24 h after administration and restored the adult normal level of 4C nuclei after 10 days of treatment. hGH did not affect the 4C nuclei or the S phase in the hypothyroid rats. These results suggest that the processes of hepatocyte proliferation and polyploidization of the rat liver are under endocrine control, with thyroid hormones playing the essential regulatory role.

Evaluation of acute and chronic hepatotoxic effects exerted by anabolic-androgenic steroid stanozolol in adult male rats.

Stanozolol (ST) is a 17alpha-alkyl anabolic-androgenic steroid (17alpha-AAS) often misused by athletes and bodybuilders. The use of anabolic-steroids by sportsmen and teenagers has increased dramatically, thus raising the question about their hepatotoxicity, specially those such as ST which are orally administered. Previously, we have reported diverse in vivo effects exerted by this steroid and published the existence of a highly specific ST-binding site in male rat liver microsomes. The existence of this binding site, the reported hepatic effects exerted in humans, and the very limited information about its potential hepatotoxicity led us to treat adult male rats acutely and chronically with ST and study different parameters that could indicate liver damage: serum levels of transaminases, concentration of monooxygenase enzymes in liver, liver membrane lipid peroxidation products, liver histopathology, and cell cycle/ploidy status of liver cells. In our study, no changes in serum transaminases or lipid peroxidation levels were obtained. However, acute stanozolol treatment significantly decreased the levels of cytochrome P450 (Cyt. P450) and cytochrome b5 (Cyt. b5) during the first 48 h of treatment, while subsequently, at 72 and 96 h, these microsomal enzymes underwent a significant increase in their levels. In sharp contrast with this response to acute treatment, the content of these two enzymes during chronic treatment showed an important decrease. Interestingly, acutely and chronically ST-treated livers showed slight to moderate inflammatory or degenerative lesions in centrilobular hepatocytes. Flow cytometric analysis demonstrated that both acute and chronic ST treatment were capable of increasing the percentage of S-phase fraction (%SPF) of liver cells. These findings taken together clearly show that this steroid is capable of altering the liver capacity for metabolizing xenobiotics and indicate that high doses of ST could exert a proliferative effect on liver cells. Such data should be considered in risk evaluations for this compound.

Tamoxifen aziridine binding to cytosolic proteins from human breast specimens is negatively associated with estrogen receptors, progesterone receptors, pS2, and cathepsin-D.

[3H]Tamoxifen Aziridine ([3H]TAZ) is a derivative of the antiestrogen tamoxifen that covalently labels the Estrogen Receptor (ER), and perhaps other uncharacterized proteins. In a previous article we described that [3H]TAZ binds to a cytosolic protein from human uterine tissues that shares some, but not all, the ER properties. Here we have extended these studies to [3H]TAZ binding to cytosol proteins from human breast cancer specimens, and studied its quantitative association with other molecular markers and clinico-pathological variables. Cytosols were obtained in hypotonic buffer containing 20 mM molybdate and protease inhibitors, incubated with [3H]TAZ, and subjected to Sucrose Gradient Analysis (SGA). A [3H]TAZ labeled peak that consistently migrated with the 4S fractions was found in most of the assayed cytosols (range of 0 to 1278 fmol/ mg p.). The 4S peak of [3H]TAZ was partially inhibited by both estrogens and antiestrogens. When [3H]E2 was used instead of [3H]TAZ, only an 8S peak was detected. [3H]TAZ was covalently bound to a protein with an apparent MW of 65 kDa, as determined by SDS-PAGE and fluorography. The mean of [3H]TAZ binding was significantly higher in the subgroups of samples classified as ER-, PR-, pS2- or cathepsin D-, than in the respective positive subgroups (P < 0.01 in all the cases). [3H]TAZ binding was not associated with clinico-pathological variables, except that its mean was significantly larger in tumors larger than 5 cm than in smaller tumors. These results, and those previously reported, suggest that: 1) [3H]TAZ labels a cytosolic protein present in human breast cancers and uterine tissues that does not share all the ER properties, and 2) the [3H]TAZ binding by breast cancer cytosols is negatively associated with markers of estrogenic dependency, and its quantification may provide valuable information on antiestrogen responsiveness of a given tumor.

The two native estrogen receptor forms of 8S and 4S present in cytosol from human uterine tissues display opposite reactivities with the antiestrogen tamoxifen aziridine and the estrogen responsive element.

We have investigated the capability of the different native ER forms, present in cytosols from human uterine tissues, of reacting with the antiestrogen [3H]Tamoxifen aziridine ([3H]TA) and with the Estrogen Responsive Element (ERE). Cytosols from uterine leiomyoma (myoma) prepared in buffer containing 40 mM molybdate and protease inhibitors, labelled with [3H]estradiol and analyzed in low-salt sucrose gradient showed 8S and 4S ER forms. The same cytosols labelled with [3H]TA only showed a 4S ER form, whereas the ERE only reacted with fractions from the 8S peak. The band of ERE reaction in the EMSA assay showed a lower relative mobility than the band labelled with [3H]TA, but both bands contained immunoreactive ER of 65 kDa. Electrophoretic mobility of the [3H]TA-labelled band in that system was not affected by cytosol treatment with cross-linkers or SDS, which suggests that it is a monomeric protein. The [3H]TA-binding 4S ER form was found in all studied myoma samples, as well as in human endometrium or myometrium, but not in rat tissues. These results suggest that the 8S and 4S ER form were already present before cytosol from human uterine tissues comes into contact with the molybdate buffer. They both contain the same ER molecule of 65 kDa, either in the free form or as an oligomer. Only the ER dimers, which have been described both in the cytosolic 8S form and in the nuclear 4-5S form, react with the ERE. [3H]TA only binds to the 4S ER monomer probably because its binding site is concealed in the 8S form under these experimental conditions. The opposite reactivity of the 8S and 4S ER forms with [3H]TA and the ERE support the hypothesis that they may constitute separate entities with a different physiological role.

Estrogen antagonism on T3 and growth hormone control of the liver microsomal low-affinity glucocorticoid binding site (LAGS).

Male rat liver microsomes contain a low-affinity glucocorticoid binding site (LAGS) capable of binding all natural glucocorticoids and progesterone with a Kd from 20 to 100 nM. The LAGS level is under endocrine control by T3, glucocorticoids and GH. These hormones act synergistically at physiological concentrations to increase the LAGS level. Since female rats show a LAGS level that is much lower than the males (0.15 vs 23 pmol/mg protein, respectively), here we investigated whether estradiol could decrease the LAGS in the male rat. Orchiectomized (OX) male rats showed a higher LAGS level than intact rats. This effect was reversed by implanting a Sylastic capsule containing testosterone. When the OX rats were implanted for 20 days with estrogen capsules that provided an estradiol level in serum of 40 pg/ml, their LAGS level decreased from 23 to 0.2 pmol/mg protein. This effect was not observed in intact male rats and can be partially reversed by testosterone implants into OX rats. Both hypophysectomized male rats and hypothyroid-orchiectomized male rats showed very low levels of LAGS. Administration of physiological doses of GH and/or T3 to these rats greatly increased their LAGS level (from 0.3 to 15 and 16 pmol/mg protein, respectively). Implantation of estrogen capsules to these rats two weeks prior to starting treatment completely inhibited the increase in the LAGS level in response to T3, and significantly decreased the response to hGH, and to a combination of hGH and T3. These results suggest that physiological estradiol levels can antagonize the LAGS induction by T3 and hGH in the male rat, and could be responsible for the low level of LAGS in the female rat. Moreover, estrogen capsules also inhibited the increase in the body and hepatic weights observed after hGH treatment, which suggests a powerful inhibitory effect of low estradiol levels on the male rat liver functions under regulation by T3 and/or GH.

Identification of a specific binding site for the anabolic steroid stanozolol in male rat liver microsomes.

Male rat liver microsomes contain a [3H]dexamethasone binding site, capable of binding glucocorticoids and progesterone. We have shown previously that the 17 alpha-alkylated androgen, stanozolol, can inhibit the [3H]dexamethasone binding to microsomes through a negative allosteric mechanism, which gives rise to the possibility of its interaction with a different binding site. In this study, the existence of a single-saturating binding site, capable of binding the radioactive steroid with a maximum number of the specific binding site of 49 +/- 2 pmol/mg of protein and a Kd of 37 +/- 1.3 nM was demonstrated by using [3H]stanozolol. In competition experiments, only stanozolol and danazol were able to compete with [3H]stanozolol for its binding to microsomes, among more than 60 steroids and other compounds tested. The binding of [3H]stanozolol was depressed after protease treatment of the microsomes, or after the administration of cycloheximide to adult male rats for 24 hr, which suggest its proteic nature. The [3H]stanozolol binding site was detected in many tissues of the rat, with the highest concentrations being found in the liver. It was detected from birth, increasing afterward in concentration and reaching a peak at 2 to 3 months of age. This is the first experimental verification of the existence in liver microsomes of a specific binding site for some 17 alpha-alkylate androgens, such as stanozolol and danazol, different from the androgen receptor or the [3H]dexamethasone binding site.

Validation of a differential PCR and an ELISA procedure in studying HER-2/neu status in breast cancer.

HER-2/neu oncogene status and total cellular p185HER-2 content were simultaneously analyzed in 415 invasive breast-cancer specimens by differential PCR and ELISA respectively. Mathematical analysis of the data led us to establish a cut-off value of 1.7 for the ratio between the intensity of the HER-2/neu gene band and the reference gene band, to consider the HER-2/neu gene amplified, and of 260 fmol/mg protein, to consider p185HER-2 over-expressed. Of the 415 tumors studied, 15% showed a diverse degree of HER-2/neu gene amplification. Of these tumors, 87% showed over-expression of the p185HER-2. Of the remaining 352 specimens that did not display HER-2/neu gene amplification, 97% showed no p185HER-2 over-expression (p < 0.0001). In 40 selected samples with a p185HER-2 level lower than 260 fmol/mg protein, the degree of p185HER-2 phosphorylation was very low or undetectable. Conversely, 38 of 46 selected tumors with a p185HER-2 level higher than 260 fmol/mg protein exhibited a considerable degree of p185HER-2 phosphorylation (p < 0.0001). Our data suggest that: (i) differential PCR and ELISA, which are relatively simple procedures, give similar information on HER-2/neu status in breast cancer; and (ii) given the large series analyzed, the cutoff values established can be considered as safe values for determining whether, in a given tumor, the HER-2/neu oncogene is amplified or p185HER-2 is over-expressed.

[3H]dexamethasone binding activity in liver microsomes is modulated differently by 17 alpha-alkylated androgens and testosterone in vivo.

Rat liver microsomes contain a single class of steroid binding sites, capable of binding various glucocorticoids and progesterone. In a previous article, we have described the in vitro interaction of several androgens with this binding site. Unlike natural androgens, the 17 alpha-alkyl derivatives stanozolol and danazol were capable of interacting with this binding site through a negative allosteric pattern. Now, the effects these steroids exert on the microsomal [3H]dexamethasone binding site have been studied in vivo. The administration of a single dose of stanozolol to rats provoked a significant reduction in the microsomal [3H]dexamethasone binding capacity. This effect was maximal two hr after stanozolol administration and persisted for six hr. The restoration of the [3H]dexamethasone binding level after stanozolol administration was dependent on protein synthesis, since it was blocked by the concomitant administration of cycloheximide. None of the other androgens tested (danazol, methyltestosterone, fluoxymesterone, and testosterone propionate) was capable of provoking a similar effect when administered 2 or 24 hr prior to sacrifice. In rats treated for seven days with a daily dose of diverse androgens and sacrificed 24 hr after the last treatment, none of the 17 alpha-alkyl androgens assayed provoked significant changes in the microsomal [3H]dexamethasone binding level, although stanozolol, danazol, and methyltestosterone provoked a significant increase in glucocorticoid receptor concentration. In contrast, the administration of testosterone propionate provoked a 50% reduction in the [3H]dexamethasone binding level without causing changes in the glucocorticoid receptor concentration. These results provide new evidence on the existence of different effects on the liver of 17 alpha-alkyl androgens, compared to the effects produced by natural androgens.

Age-related changes in the induction of tyrosine aminotransferase by dexamethasone: correlation with the low-affinity glucocorticoid binding sites.

Rat liver membranes contain Low-affinity glucocorticoid binding sites (LAGS), capable of binding with low affinity (Kd approximately 100 nM) endogenous glucocorticoids. Unlike the glucocorticoid receptor (GR), the LAGS level undergoes abrupt changes throughout life. The investigation of these changes may be useful in determining whether the LAGS are involved in the cellular response to glucocorticoids. For this purpose, we have studied glucocorticoid induction of tyrosine aminotransferase (TAT), and its relationship with the LAGS level in adrenalectomized and fasted rats of different ages. No significant differences in the GR level, or in its Kd and activation, were observed among rats of 1, 3, and 12 months of age. On the other hand, the LAGS level showed an important variation with age, from almost undetectable in 1-month-old rats, to a maximum value in 3-month-old rats. With respect to TAT activity, an increase with age in the threshold of response to dexamethasone (DEX) administration was observed. The smallest dose of DEX capable of provoking a significant TAT induction rose from 0.1 microgram/kg body wt. in 1-month-old rats to 10 micrograms/kg body wt. in 12-month-old rats. However, the smallest dose of DEX able to elicit the maximal response was 10 micrograms/kg body wt. in all the assayed ages. This dose provoked a 40% decrease in the GR level, but did not significantly modify the LAGS content. From these results, we conclude that there is an age-related change in the threshold of response to DEX that cannot be explained by the GR-glucocorticoid interaction. The possibility that the LAGS modulate the cell response to glucocorticoids arises from the coincidence of this change with that observed in the LAGS concentration throughout life.

Ethinylestradiol interacts with liver microsomes and induces binding sites for steroid hormones in the male rat liver.

The present work focuses on the interaction of 17 alpha-ethinyl estrogen derivatives with the [3H]dexamethasone ([3H]DEX) binding site from male rat liver microsomes and the induction of this site by the in vivo administration of natural and synthetic estrogens. [3H]DEX binds to a single-saturating binding site (Kd = 100 nM; maximal binding = 13 pmol/mg of protein) in the liver microsomes. In competition experiments, ethinylestradiol (EE2) and mestranol were able to inhibit [3H]DEX binding to microsomes, whereas natural estrogens, tamoxifen or estrogen sulfates were ineffective. Saturation analysis performed by incubating [3H]EE2 with liver microsomes revealed the existence of a low-affinity (Kd = 280 +/- 30 nM) and high capacity (maximal binding = 16 +/- 2 pmol/mg of protein) binding site. Saturation, competition and dissociation experiments suggest that [3H]DEX and [3H]EE2 interact with the same microsomal entity. Synthetic and natural estrogens increased the hepatic expression of the [3H]DEX binding site in immature, hypothyroid and hypophysectomized male rats. This induction required at least 2 days of treatment, and could only be achieved by pharmacological doses of estrogens.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of growth hormone in regulation of low affinity glucocorticoid-binding sites from male rat liver microsomes.

GH participates in the regulation of the expression of several hepatic proteins, some of which are subject to multihormonal control. We have previously shown the participation of glucocorticoids and thyroid hormones in the regulation of the hepatic low affinity glucocorticoid-binding sites (LAGS). Here, we provide evidence that also implicates GH in the endocrine control of the LAGS through the use of several animal models, all of them having a very low or undetectable plasma GH level: the hypothyroid (TX), the hypophysectomized, and the GH-deficient Lewis-derived dwarf rat. In dwarf rats, the level of LAGS was only 35% of that found in normal Lewis rats. Treatment of these rats with human (h) GH significantly increased the LAGS level in a dose-response manner. In TX rats, hGH treatment provoked a significant increase in the LAGS level (from 0.9 +/- 0.2 to 7.2 +/- 0.8 pmol/mg protein), so that it represented about 65% of the level found in intact animals. In both hypothyroid-adrenalectomized and hypophysectomized rats, the isolated effect of hGH was not as pronounced as in TX or dwarf rats; however, a potentiation of the effect of hGH was observed when this hormone was injected together with corticosterone acetate. On the other hand, when hGH, T3, and corticosterone acetate were given in combination to hypophysectomized rats, hGH and T3 behaved as agonists of the LAGS induction at T3 doses lower than or equal to 0.1 microgram/100 g BW and as antagonists at T3 doses higher than this. When T4 was used instead of T3, this hormone was capable of potentiating the effect of hGH at doses lower than or equal to 1.5 micrograms/100 g BW. From these results we conclude that 1) GH as well as thyroid and glucocorticoid hormones participate in the endocrine regulation of the LAGS; and 2) under physiological conditions, it is conceivable that GH, thyroid hormones, and glucocorticoids act synergistically in the endocrine regulation of the LAGS.

Stanozolol and danazol, unlike natural androgens, interact with the low affinity glucocorticoid-binding sites from male rat liver microsomes.

Some 17 alpha-alkylated androgens used as anabolic agents, such as stanozolol (ST) and danazol (DA), have specific effects on the liver that are not exerted by testosterone. This gives rise to the possibility that a steroid-binding protein, other than the androgen receptor, could modulate the intracellular actions of these agents. Male rat liver microsomes contain a homogeneous population of [3H]dexamethasone ([3H]DEX)-binding sites which we have denominated low affinity glucocorticoid-binding sites (LAGS). Because glucocorticoids, progestagens, and the synthetic estrogen ethynyl estradiol compete with [3H]DEX for binding to the LAGS, we aimed to study the possible interactions between androgens and the LAGS. To investigate whether several androgens had the capability of interacting with the LAGS, we performed competition experiments. The LAGS had no affinity for testosterone or methyltrienolone (R1881). However, some 17 alpha-alkylated androgens (DA (IC50, 116 nM) > ST >> fluoxymesterone > mestaline > methandriol >> methandrostenolone > methyltestosterone) were able to compete with [3H]DEX binding to liver microsomes. ST and DA were potent inhibitors of [3H]DEX binding to liver microsomes. They decreased both the affinity and the number of [3H]DEX-binding sites, increased the dissociation rate of [3H]DEX from the LAGS, and provoked a time- and dose-dependent inactivation of the [3H]DEX-binding site. These results strongly suggest that ST and DA exert a negative allosteric modulation on [3H]DEX binding to the LAGS. The in vivo administration of ST (but not other androgens) to male rats provoked a time- and dose-dependent decrease in the LAGS level. Full recovery of the LAGS concentration required at least 8 h and was blocked by protein synthesis inhibitors. Such results suggest that ST irreversibly inactivates the [3H]DEX-binding site in vivo as it does in vitro. Taken together, these observations are indicative of an irreversible interaction between some 17 alpha-alkylated androgens and the LAGS both in vitro and in vivo and suggest that ST may be an important pharmacological tool that can be used in the elucidation of the molecular structure of the LAGS. These results also mean that the LAGS are a steroid-binding entity able to distinguish between natural androgens and 17 alpha-alkylated testosterone derivatives used as anabolic agents.