Sex steroids play a crucial role in the development and differentiation of normal mammary gland as well as in the regulation of breast cancer growth. Local intracrine formation of sex steroids from inactive precursors secreted by the adrenals, namely, dehydroepiandrosterone and its sulfate, may regulate growth and function of peripheral target tissues, including the breast. Both endocrine and paracrine influences on the proliferation of human breast cancer cells are well recognized. Breast tumors harbor tumor-associated macrophages and tumor-infiltrating lymphocytes that secrete a wide spectrum of cytokines. These factors may also contribute to neoplastic cell activity. The present study was designed to investigate the action of cytokines on 3beta-hydroxysteroid dehydrogenase (3beta-HSD) activity, which is an essential step in the biosynthesis of active estrogens and androgens in human breast cancer cell lines and in normal human mammary epithelial cells in primary culture. 3Beta-HSD activity was undetectable in ZR-75-1 and T-47D estrogen receptor-positive (ER)+ cells under basal growth conditions. This activity was markedly induced after exposure to picomolar concentrations of interleukin (IL)-4 or IL-13. The potent stimulatory effect of these cytokines on 3beta-HSD activity was also observed in the ER- MDA-MB-231 human breast cancer cell line and in normal human mammary epithelial cells (HMECs) in primary culture. The stimulation of 3beta-HSD activity by IL-4 and IL-13 results from a rapid increase in 3beta-HSD type 1 mRNA levels as measured by RT-PCR and Northern blot analyses. Such an induction of the 3beta-HSD activity may modulate androgenic and estrogenic biological responses as demonstrated using ZR-75-1 cells transfected with androgen- or estrogen-sensitive reporter constructs and treated with the adrenal steroid 5-androstene-3beta,17beta-diol. The DNA-binding activity of Stat6, a member of the signal transducers and activators of transcription gene family, is activated 30 min after exposure to IL-4 and IL-13 in human breast cancer cell lines as well as in HMECs in primary culture. In these cells, Stat6 activated by IL-4 or IL-13 binds to two regions of the 3beta-HSD type 1 gene promoter, containing Stat6 consensus sequences. IL-4 induction of 3beta-HSD mRNA and activity is sensitive to staurosporine. This protein kinase inhibitor also inhibits IL-4-induced Stat6 DNA-binding activity. Our data demonstrate for the first time that IL-4 and IL-13 induce 3beta-HSD type 1 gene expression, thus suggesting their involvement in the fine control of sex steroid biosynthesis from adrenal steroid precursors in normal and tumoral human mammary cells. Furthermore, aromatase and/or 5alpha-reductase(s) are expressed in the mammary gland and in a large proportion of human breast tumors. An increase in the formation of their substrates, namely, 4-androstenedione and testosterone, may well have a significant impact on the synthesis of active estrogens and androgens in these tissues.

Serum levels of testosterone, dihydrotestosterone, androstenedione, dehydroepiandrosterone sulfate, and sex hormone-binding globulin and urinary levels of testosterone and androstanediol were compared in 75 women with breast carcinoma and 150 age-matched healthy controls. Odds ratios for quartiles of hormones, adjusted for known potential confounders, were computed using conditional logistic regression. Risk of breast cancer was positively associated with levels of all androgens in serum and urine but appeared stronger for testosterone (for trend, P = 0.03) and dehydroepiandrosterone sulfate (for trend, P = 0.06) in serum and for testosterone (for trend, P = 0.001) and androstanediol (for trend, P = 0.04) in urine. The adjusted odd ratios for high versus low quartiles were 2.7 (95% confidence interval, 1.1-6.5) for serum testosterone, 2.8 (1.1-7.4) for dehydroepiandrosterone sulfate, 4.7 (1.8-12.1) for urinary testosterone, and 3.4 (1.4-8.7) for urinary androstanediol. These observations suggest that endogenous androgenic hormones may play an important role in the epidemiology of postmenopausal breast cancer in women.

Depression is characterized by hypothalamo-pituitary-adrenocortical (HPA) axis hyperactivity. In this major mood disorder, neurosteroids and neurotrophins, particularly brain-derived neurotrophic factor (BDNF), seem to be implicated and have some antidepressant effects. BDNF is highly involved in regulation of the HPA axis, whereas neurosteroids effects have never been clearly established. In this systematic in vivo study, we showed that the principal neuroactive steroids, namely dehydroepiandrosterone (DHEA), pregnenolone (PREG) and their sulfate esters (DHEA-S and PREG-S), along with allopregnanolone (ALLO), stimulated HPA axis activity, while also modulating central BDNF contents. In detail, DHEA, DHEA-S, PREG, PREG-S and ALLO induced corticotropin-releasing hormone (CRH) and/or arginine vasopressin (AVP) synthesis and release at the hypothalamic level, thus enhancing plasma adrenocorticotropin hormone (ACTH) and corticosterone (CORT) concentrations. This stimulation of the HPA axis occurred concomitantly with BDNF modifications at the hippocampus, amygdala and hypothalamus levels. We showed that these neurosteroids induced rapid effects, probably via neurotransmitter receptors and delayed effects perhaps after metabolization in other neuroactive steroids. We highlighted that they had peripheral effects directly at the adrenal level by inducing CORT release, certainly after estrogenic metabolization. In addition, we showed that, at the dose used, only DHEA, DHEA-S and PREG-S had antidepressant effects. In conclusion, these results highly suggest that part of the HPA axis and antidepressant effects of neuroactive steroids could be mediated by BDNF, particularly at the amygdala level. They also suggest that neurosteroids effects on central BDNF could partially explain the trophic properties of these molecules.

OBJECTIVE

To compare insulin-like growth factor-I (IGF-I) concentrations in obese and normal subjects, and evaluate the possible relationships between IGF-I concentrations and demographic, anthropometric, metabolic and hormonal variables in obese patients.

METHODS

286 obese outpatients (OB, 234 female and 52 male; age 18-71 y, body mass index (BMI)>> 27 kg/m2) were recruited.

METHODS

BMI, waist-to-hip ratio (WHR), serum basal and oral glucose tolerance test (OGTT)-stimulated glucose and insulin concentrations, IGF-I, basal growth hormone (GH), prolactin (PRL), androgens, thyrotropin (TSH), free triiodothyronine (fT3), free thyroxine (fT4), free fatty acids (FFA), triglycerides, total and high density lipoprotein (HDL)-cholesterol, 24h-urinary cortisol levels and blood pressure (BP) values were measured. IGF-I concentrations were also evaluated in a large population of 326 age-matched controls (controls, 228 women, 98 men; age 20-86 y, BMI < 25 kg/m2).

RESULTS

IGF-I concentrations were lower in OB than in controls (age-adjusted mean: 21.6 vs 23.6 nmol/L, P < 0.03). However, individual IGF-I concentrations in OB were within the age-adjusted normal range. In both groups, IGF-I concentrations were gender-independent, and showed a simple negative correlation with age (r = -0.47). In OB, univariate analysis also shows that IGF-I concentrations were negatively correlated with BMI (r = -0.33), but not WHR, with both basal (r = -0.16) and OGTT-stimulated glucose levels (r = -0.17), as well as FFA levels (r = -0.19), and with both diastolic and systolic BP (both r = -0.17). In OB women, IGF-I concentrations positively correlated with PRL (r = 0.31), testosterone (r = 0.30), androstenedione (r = 0.30), and dehydroepiandrosterone-sulfate (DHEAS) concentrations (r = 0.41). No correlation was found with other variables. The multiple regression analysis showed that IGF-I concentrations were inversely and independently related to age and BMI only.

CONCLUSIONS

In obesity, IGF-I concentrations are slightly reduced, but generally within the age-adjusted normal range. IGF-I concentrations in obesity show independent and negative relationships with age and BMI, but are not associated with fat distribution, insulin secretion, glucose tolerance, BP or risk indices for cardiovascular disease (CVD).

To determine whether endogenous androgens influence bone density in young women, we studied 27 normal women and 19 women with androgen excess, as defined by increased serum bioavailable testosterone (bio T) concentrations. The women ranged from 21-48 yr of age. The 2 groups were comparable with respect to age, anthropomorphic measures, nutrition, gynecological history, and serum cortisol and estradiol levels. Trabecular (lumbar) and cortical (radial) bone density were quantitated by computerized tomography and single photon absorptiometry, respectively. Serum obtained during the follicular phase of the cycle was assayed for bio T, total T, dehydroepiandrosterone sulfate, androstenedione (Adione), and 3 alpha-androstanediol glucuronide (3-Adiol-G). Trabecular bone density was significantly higher in the androgen excess group [172 +/- 7 (+/- SE) vs. 153 +/- 5 mg/mL; P = 0.03]; controlling for serum Adione (but not for serum bio T, total T, dehydroepiandrosterone sulfate, or 3 alpha-androstanediol glucuronide, or 3-Adiol-G) abolished this difference. Similarly, serum Adione correlated more strongly than the other androgens with trabecular bone density (r = 0.31; P = 0.03). Average cortical bone density was not higher in the androgen excess group (0.740 +/- 0.014 vs. 0.722 +/- 0.008 g/cm2; P = 0.27). Among the 27 normal women, cortical density was correlated to serum bio T (r = 0.47; P = 0.01) and total T (r = 0.53; P = 0.004), but not to the other androgens. We conclude that supraphysiological levels of endogenous androgens are associated with increased trabecular bone density in young women. Serum Adione appeared to be the best marker for the impact of androgen on trabecular density. Among normal women, cortical bone density was related to serum T.

Reduced secretion of GH and production of insulin-like growth factor I (IGF-I) contribute to altered body composition in human aging. IGF-binding proteins (IGFBPs) are important modulators of IGF action, yet little is known regarding their role and regulation in aging. Accordingly, we measured levels of IGFBP-1, an important short term modulator of IGF bioavailability that is suppressed by insulin, and levels of IGFBP-3, the major circulating IGF carrier protein, and examined their relationships to insulin, glucose, IGF, and dehydroepiandrosterone sulfate levels and anthropometric measures in old (63-89 yr) and young (23-39 yr) men. Serum levels of IGFBP-1 were increased 3-fold in old vs. young men despite high insulin levels in elders. Nevertheless, IGFBP-1 and insulin levels correlated in old and young men (r = - 0.49; P < 0.002 and r = -0.42; P < 0.025), suggesting that insulin continues to play an important role in the regulation of IGFBP-1 in aging. Glucose levels also were significantly inversely related to IGFBP-1 in old and young men (r = 0.37; P = 0.02 and r = -0.49; P < 0.01), and this relationship was not accounted for by the effect of insulin. IGF-I levels were reduced by 33% in elders (P < 0.001) and correlated with IGFBP-1 levels among old (r = -0.40; P < 0.01), but not young, men, indicating that low GH secretion and/or IGF-I production may contribute to the elevation of IGFBP-1 levels in aging. IGFBP-3 levels were reduced among elders, but not to the same extent as IGF-I, resulting in a relative excess of IGFBP-3 in elders (IGFBP-3/IGF-I ratio, 20.1 +/- 0.9 vs. 15.4 +/- 1.0; P < 0.001). The IGFBP-3/IGF-I ratio correlated with IGF-I levels in young and old men (r = -0.79; P < 0.001 and r = -0.82; P < 0.001), indicating that diminished GH secretion also may contribute to a relative excess of IGFBP-3 among elders. Dehydroepiandrosterone sulfate levels were low in elders, but did not correlate with IGF, IGFBP, insulin, or glucose levels in either age group. Serum levels of IGFBP-1 (but not IGF-I or -II or IGFBP-3) correlated with body mass index and upper arm fat and muscle areas in elders. These relationships were accounted for by the effects of insulin, suggesting that regulation of IGFBP-1 by insulin may play a role in determining body composition in aging. We conclude that insulin remains an important determinant of IGFBP-1 levels in elders, that the fasting glucose level is also a significant determinant of IGFBP-1 in both old and young subjects, and that reduced secretion of GH may contribute to impaired anabolism in aging through multiple mechanisms, including reduced production of IGF-I and alterations in circulating levels of both IGFBP-1 and -3. These findings are consistent with the concept that alterations in IGFBP levels may contribute to changes in IGF bioavailability and body composition in aging.

Our objective was to evaluate the effectiveness of the insulin-sensitizing agent rosiglitazone in obese women with polycystic ovary syndrome (PCOS) and severe insulin resistance. Twelve obese women with PCOS were recruited. All were hirsute and anovulatory with acanthosis nigricans indicating severe insulin resistance. All women were treated with 4 mg of rosiglitazone daily for 6 months. A standard 75-g oral glucose tolerance test with insulin levels was performed before and after the women were treated with rosiglitazone. Glucose and insulin areas under the curve (AUC) were calculated. Serum levels of total and free testosterone, dehydroepiandrosterone sulfate, LH, and 17-hydroxyprogesterone were also measured before and after treatment. The body mass index was determined before and after treatment. There was a highly significant (r = 0.881, P < 0.0001) positive correlation between insulin response during oral glucose tolerance test and basal total testosterone levels. After treatment with rosiglitazone, there were significant decreases in fasting insulin levels (46.0 +/- 6.5 vs. 16.9 +/- 2.0 microU/ml; P < 0.001), insulin AUC (749.3 +/- 136.3 vs. 225.0 +/- 15.7 microU/ml; P = 0.003), fasting glucose levels (90.8 +/- 3.0 vs. 81.8 +/- 1.9 mg/dl; P = 0.003), and glucose AUC (437.9 +/- 25.0 vs. 322.5 +/- 14.7 mg/dl; P < 0.001). Both total testosterone (96.3 +/- 17.3 vs. 56.1 +/- 5.8 ng/dl; P = 0.01) and free testosterone (5.8 +/- 0.6 vs. 3.4 +/- 0.5 pg/ml; P < 0.001) decreased significantly after treatment, although there was no significant change in LH levels. Levels of SHBG increased significantly (18.3 +/- 3.4 vs. 25.8 +/- 6.6 nmol/liter; P = 0.009) after treatment, and dehydroepiandrosterone sulfate levels decreased significantly (P = 0.04). There was no significant change in body mass index (40.4 +/- 2.4 vs. 41.1 +/- 2.7 kg/m(2)). Eleven of the women reverted to regular ovulatory cycles during the treatment period. We conclude that 1) rosiglitazone therapy improves insulin resistance and glucose tolerance in obese women with PCOS; 2) rosiglitazone decreases ovarian androgen production, which appears to be independent of any changes in LH levels; 3) hyperinsulinemia appears to play a key role in the overproduction of ovarian androgens in these women because attenuation of insulin levels is associated with decreased testosterone levels; and 4) short-term rosiglitazone therapy helps restore spontaneous ovulation.

Increased expression of cyclooxygenase-2 (COX-2), the rate-limiting enzyme in prostaglandin synthesis, has been associated with growth regulation and carcinogenesis in several systems. COX-2 is known to be induced by cytokines and the skin tumor promoter 12-tetradecanoylphorbol-13-myristate (TPA). In the present study, we investigated the effects of several non-TPA-type tumor promoters on COX-2 expression in immortalized mouse liver cells. Specifically, we tested peroxisome proliferators (PPs), which are rodent liver tumor promoters that cause gross alterations in cellular lipid metabolism, the rodent liver tumor promoter phenobarbital, and the skin tumor promoters okadaic acid and thapsigargin. The PPs Wy-14643, mono-ethylhexyl phthalate, clofibrate, ciprofibrate ethyl ester, and eicosatetraynoic acid each caused large increases in COX-2 mRNA and protein, with maximal expression seen approximately 10 h after treatment of quiescent cells. COX-2 expression was also induced by thapsigargin, okadaic acid, and calcium ionophore A23187, but not by phenobarbital or the steroid PP dehydroepiandrosterone sulfate. Induction of COX-2 expression generally resulted in increased synthesis of prostaglandin E2 (PGE2). However, the PPs caused little or no increase in PGE2 levels, and they inhibited serum-induced PGE2 synthesis. Unlike non-steroidal anti-inflammatory drugs, the PPs do not directly inhibit cyclooxygenase enzyme activity in vitro. Thus, PPs regulate prostaglandin metabolism via both positive (COX-2 induction) and inhibitory mechanisms. In summary, the strong induction of COX-2 expression by PPs, thapsigargin, and okadaic acid suggests a possible role for COX-2 in the growth regulatory activity of these non-TPA-type tumor promoters.

Serum hormones have been intensively investigated in association with several chronic diseases, but limited information exists on the reliability of a number of hormone determinations. The one-year reproducibility of dehydroepiandrosterone sulfate (DHEAS), total and free testosterone, total estradiol, insulin, C-peptide, and prolactin was studied in 60 premenopausal and 47 postmenopausal women recruited in Varese province, Italy, 1991-1992. The hormonal determinations were made in blood samples collected twice, one year apart, after 12-h fast, in the same month, day, and hour and for premenopausal women on the same day of the luteal phase of the menstrual cycle. Samples from the first drawing were stored at -80 degrees C. Samples from both drawings were assayed simultaneously and in blind fashion. Total estradiol in postmenopause was not evaluated for limitation in the sensitivity of the laboratory method. The intraclass correlation coefficient in premenopausal women was 0.85 for DHEAS, 0.60 for total testosterone, 0.66 for free testosterone, 0.81 for insulin, 0.83 for C-peptide, 0.40 for prolactin, and 0.06 for total estradiol. In postmenopausal women, the coefficient was 0.90 for DHEAS, 0.88 for total testosterone, 0.71 for free testosterone, 0.67 for insulin, 0.73 for C-peptide, and 0.18 for prolactin. These data indicate that total estradiol measured during the luteal phase has a poor intraindividual reproducibility over time, and these findings may have important implications in studies of hormones in the etiology of chronic disease.

Metabolic syndrome (MetS) is a strong risk factor for type 2 diabetes and cardiovascular disease. Conditions associated with hyperandrogenism are often associated with glucose intolerance and other features of MetS in young women. As the prevalence of MetS increases with age and is probably multifactorial, it is reasonable to hypothesize that age-related changes in androgens and other hormones might contribute to the development of MetS in older persons. However, this hypothesis has never been tested in older women. We hypothesized that high levels of testosterone, dehydroepiandrosterone sulfate (DHEA-S), and cortisol and low levels of sex hormone-binding globulin (SHBG) and IGF-I would be associated with MetS in a representative cohort of older Italian women independently of confounders (including inflammatory markers). After exclusion of participants on hormone replacement therapy and those with a history of bilateral oophorectomy, 512 women >>/=65 yr) had complete data on testosterone, cortisol, DHEA-S, SHBG, fasting insulin, total and free IGF-I, IL-6, and C-reactive protein (CRP). MetS was defined according to ATP-III criteria. Insulin resistance was calculated according to HOMA. MetS was found in 145 women (28.3%). Participants with vs. those without MetS had higher age-adjusted levels of bioavailable testosterone (P < 0.001), IL-6 (P < 0.001), CRP (P < 0.001), and HOMA (P < 0.001) and lower levels of SHBG (P < 0.001). After adjustment for potential confounders, participants with decreased SHBG had an increased risk of MetS (P < 0.0001) vs. those with low SHBG. In a further model including all hormones and confounders, log SHBG was the only independent factor associated with MetS (OR: 0.44, 95% CI 0.21-0.91, P = 0.027). In older women, SHBG is negatively associated with MetS independently of confounders, including inflammatory markers and insulin resistance. Further studies are needed to support the notion that raising SHBG is a potential therapeutic target for prevention and treatment of MetS.

BACKGROUND

Recent observations have indicated that reproductive endocrine disorders are common among women taking valproate (VPA) for epilepsy, but it is not known whether respective abnormalities develop in men taking VPA for epilepsy. Carbamazepine (CBZ) may induce endocrine disorders in men with epilepsy, but the endocrine effects of oxcarbazepine (OXC) are not known.

METHODS

Reproductive endocrine function was evaluated in 90 men taking VPA (n = 21), CBZ (n = 40), or OXC (n = 29) as monotherapy for epilepsy and in 25 healthy control men.

RESULTS

Twelve men (57%) taking VPA had increased serum androgen levels. The mean serum level of androstenedione was high in patients taking VPA. Serum levels of dehydroepiandrosterone sulfate were low, and serum concentrations of sex hormone-binding globulin (SHBG) were high in men taking CBZ. The endocrine effects of OXC seemed to be dose-dependent, because serum hormone levels were normal in patients with low OXC doses (< 900 mg/day), but serum concentrations of testosterone, gonadotropins, and SHBG were high in patients with a daily OXC dose>> or = 900 mg.

CONCLUSIONS

VPA increases serum androgen concentrations in men with epilepsy. The endocrine effects of CBZ and OXC were different, because CBZ appears to decrease the bioactivity of androgens, whereas OXC does not.

OBJECTIVE

The role of steroid hormones in the control of human parturition has been a subject of debate. Activation of the fetal hypothalamic-pituitary-adrenal axis leading to an increase in plasma cortisol is followed by the onset of parturition in sheep. In contrast, androgens, specifically, dehydroepiandrosterone sulfate, have been implicated in the control of parturition in nonhuman primates. The purpose of this study was to determine the relationship between human fetal plasma cortisol and dehydroepiandrosterone sulfate and the onset of preterm labor in patients with preterm premature rupture of the membranes.

METHODS

Fetal blood sampling was performed in 51 patients with preterm premature rupture of membranes who were not in labor on admission. Amniotic fluid was cultured for aerobic and anaerobic bacteria and mycoplasmas. Corticosteroids had not been administered before fetal blood sampling. Cortisol and dehydroepiandrosterone sulfate were measured with sensitive and specific immunoassays. Analysis was conducted with nonparametric statistics and survival analysis.

RESULTS

(1) Patients who went into spontaneous labor and delivered within 7 days of cordocentesis had a significantly higher median level of fetal plasma cortisol but not of dehydroepiandrosterone sulfate than those delivered after 7 days (for fetal plasma cortisol: median 8.35 [4.7 to 12.4] micrograms/dL vs median 4.75 [3.0 to 10.4] micrograms/dL, P <.0001; for fetal plasma dehydroepiandrosterone sulfate: median 154.4 [8.6 to 333.8] micrograms/dL vs median 194.6 [96.7 to 402.5] micrograms/dL, P =.09). (2) The cordocentesis-to-delivery interval was significantly shorter in patients with a fetal plasma cortisol value of>>/=7 micrograms/dL (derived by receiver-operating characteristic curve analysis) than in those with fetal cortisol <7 micrograms/dL (median 49 [4 to 1849] hours vs median 325 [11 to 2590] hours, P <.001). (3) Fetal plasma cortisol, but not maternal cortisol, was an independent predictor of the duration of pregnancy after we adjusted for gestational age and the results of amniotic fluid culture (hazards ratio 2.9, P <.05). (4) There was a significant correlation between fetal plasma cortisol and fetal plasma interleukin-6 (r = 0.3, P <.05). (5) A strong relationship was found between the fetal plasma cortisol/dehydroepiandrosterone sulfate ratio and the interval to delivery (P <.005).

CONCLUSIONS

An elevation in fetal plasma cortisol but not dehydroepiandrosterone sulfate was followed by the onset of spontaneous preterm labor in patients with preterm premature rupture of the membranes.

We investigated dehydroepiandrosterone (DHEA) secretion in response to acute psychosocial stress and the relations of DHEA secretion to cortisol secretion, cardiovascular activity, and negative mood changes. Thirty-three male students (mean age 22.6 years) were subjected to the psychosocial stress test "Trier Social Stress Test" (TSST), in which the participants were asked to deliver a speech and perform a mental arithmetic task in front of two audiences. Collections of saliva, measurements of blood pressure and heart rate, and assessments of negative mood by visual analog scales were conducted before, during, and after TSST. Acute psychosocial stress significantly increased salivary DHEA level by an average of 60% immediately after TSST. The peak of DHEA concentration preceded that of cortisol concentration by about 10 min. DHEA response was moderately correlated to cortisol response (r=.34, r(s)=.49) but not to cardiovascular response. Lower DHEA level and elevated cortisol/DHEA ratio during TSST were significantly and moderately correlated with increased negative mood during and after TSST. These results indicated that an acute increase in DHEA concentration under stressful situations might be partly mediated by the activity of hypothalamus-pituitary-adrenal axis and could have some significance in the improvement of negative mood.

A selective and extremely sensitive procedure has been developed and optimized, using high-performance liquid chromatography (HPLC), specific derivatization and gas chromatography-mass spectrometry (GC-MS), to simultaneously quantify very small amounts of different neurosteroids from rat brain. Unconjugated and sulfated steroids in brain extracts were separated by solid-phase extraction. The unconjugated fraction was further purified by HPLC, the steroids being collected in a single fraction, and the sulfated fraction was solvolyzed. All steroids were derivatized with heptafluorobutyric acid anhydride and analyzed by GC-MS (electron impact ionization) using selected-ion monitoring. High sensitivity and accuracy were obtained for all steroids. The detection limits were 1 pg for pregnenolone (PREG), dehydroepiandrosterone (DHEA) and their sulfate esters PREG-S and DHEA-S, 2 pg for progesterone (PROG) and 5 pg for 3alpha,5alpha-tetrahydroprogesterone (3alpha,5alpha-THP). In a pilot study on a rat brain, the concentrations of PREG-S and DHEA-S were 8.26+/-0.80 and 2.47+/-0.27 ng/g, respectively. Those of PREG, DHEA and PROG were 4.17+/-0.22, 0.45+/-0.02 and 1.95+/-0.10 ng/g, respectively. Good linearity and accuracy were observed for each steroid. The methodology validated here, allows femtomoles of neurosteroids, including the sulfates, found in small brain samples (at least equal to 10 mg) to be quantified simultaneously.

Several clinical and experimental studies have demonstrated gender dimorphism in immune and organ responsiveness and in the susceptibility to and morbidity from shock, trauma, and sepsis. In this respect, cell-mediated immune responses have been shown to be depressed in males following trauma-hemorrhage, whereas they were aintained/enhanced in proestrus females. Furthermore, sex hormones have been shown to be responsible for this gender-specific immune response following adverse circulatory conditions. More specifically, studies indicate that androgens produce immunodepression following trauma-hemorrhage in males. In contrast, female sex steroids appear to exhibit immunoprotective properties following trauma and severe blood loss. With regard to the underlying mechanisms, receptors for sex hormones have been identified on various immune cells suggesting direct effects of these hormones on the immune cells. Alternatively, indirect effects of sex hormones, ie, modulation of cardiovascular responses or androgen- and estrogen-synthesizing enzymes, might contribute to gender-specific immune responses. Recent studies indicate that sex hormones, eg, dehydroepiandrosterone (DHEA), also modulate the function of peripheral blood mononuclear cells in surgical patients. Thus, the immunomodulatory properties of sex hormones/receptor antagonists/sex steroid synthesizing enzymes following trauma-hemorrhage suggests novel therapeutic strategies for the treatment of immunodepression in surgical patients.

Both DHEA and GABAA receptor agonists are known to reduce anxiety. Since GABAA receptor agonists are generally thought to elicit their anxiolytic effects by facilitating neuronal uptake of chloride ion, we set out to evaluate whether DHEA elicits its anxiolytic effects by a similar mechanism. The results of the studies show an uneven distribution of basal and GABA-stimulated chloride uptake in different regions (cerebellum, pons-medulla, striatum, hippocampus, mid-brain, hypothalamus and cortex) of rat brain. Contrary to our expectations, however, both DHEA and DHEAS inhibited GABA-mediated chloride uptake with DHEAS being more potent than DHEA. On the other hand, delta 4-androstenedione, another DHEA metabolite, did not have any effect on chloride uptake in any region of the brain. In conclusion, the data presented here, therefore, suggest that DHEA and DHEAS may elicit anxiolysis through mechanisms independent of GABAA receptor-mediated facilitation of neuronal chloride uptake.

BACKGROUND

A syndrome of lipodystrophy, associated with hypertriglyceridaemia, hypercholesterolaemia, hyperinsulinaemia and peripheral insulin resistance has been reported in protease inhibitor (PI)-treated HIV-infected patients. Because lipid metabolism, fat mass distribution and insulin resistance are partly regulated by steroid hormones, we questioned whether lipodystrophy is related to hormonal perturbations.

OBJECTIVE

To evaluate serum lipid and steroid hormone concentrations in HIV-positive men on highly active antiretroviral therapy (HAART) in order to determine whether dyslipidaemia, peripheral loss of fatty tissue and central fat accumulation are related to steroid hormone modifications.

METHODS

A cross-sectional study.

METHODS

Thirty-seven HIV-1-positive men on HAART, 23 of whom had symptoms of lipodystrophy, according to a subjective clinical score of lipodystrophy (SCSL), were tested. Serum concentrations of cholesterol, triglycerides and their subclasses, apolipoproteins and steroid hormones, including cortisol, dehydroepiandrosterone (DHEA), DHEA sulphate, androstenedione, testosterone and dihydrotestosterone were measured.

RESULTS

Serum cholesterol, very low density lipoprotein (VLDL) cholesterol, triglycerides, VLDL triglycerides, high density lipoprotein (HDL) and low density lipoprotein (LDL) triglycerides, apolipoprotein B (ApoB) and atherogenic ratios of cholesterol:HDL cholesterol, LDL cholesterol:HDL cholesterol and ApoB:apolipoprotein A1 (ApoA1) were significantly increased in lipodystrophy-positive compared with lipodystrophy-negative men. The serum cortisol level was similar in lipodystrophy-positive versus lipodystrophy-negative men, but was elevated compared with controls. Serum DHEA was significantly lower in lipodystrophy-positive versus lipodystrophy-negative men and, consequently, the cortisol:DHEA ratio was increased in lipodystrophy-positive patients. A positive correlation was found between the cortisol:DHEA ratio and increased levels of atherogenic lipids. In addition, the SCSL was positively correlated with dyslipidaemia and the cortisol:DHEA ratio.

CONCLUSIONS

This study demonstrates an association between the cortisol:DHEA ratio, lipid alterations and lipodystrophy. This syndrome might result from an imbalance between peripheral lipolysis and lipogenesis, both regulated by cortisol and DHEA.

The metabolism of docetaxel by human liver microsomes was investigated in vitro and compared with that of paclitaxel. A main docetaxel metabolite was generated by human liver microsomes in the presence of NADPH: retention time in high pressure liquid chromatography and its ion fragmentation in mass spectrometry were identical to those of the authentic derivative hydroxylated at the butyl group of the C13 side chain. Kinetic measurements and chemical and immunological inhibitions demonstrated that CYP3A was implicated in the hydroxylation of docetaxel: Km (2 microM) and Vm values of docetaxel for human liver microsomes were comparable to those calculated for the formation of metabolite p-hydroxy-phenyl C3' paclitaxel (M4). Docetaxel hydroxylation correlated only with the CYP3A content of microsomes and with CYP3A-dependent 6 beta-hydroxylation of testosterone and 16-hydroxylation of dehydroepiandrosterone. The formation of hydroxydocetaxel was strongly reduced by CYP3A inhibitors such as ketoconazole, midazolam, erythromycin, testosterone, orphenadrine, and troleandomycin, whereas quinidine (CYP2D6), hexobarbital, tolbutamide, and mephenytoin (CYP2C) had no or little effect. The hydroxylation of docetaxel exhibited a highly positive correlation with the formation of metabolite M4 of paclitaxel (r = 0.929, P < 0.0001, n = 12), but not with its 6-hydroxylation (r = 0.48, P>> 0.15). Docetaxel abolished the hydroxylation of paclitaxel metabolite M4, but was totally inactive on its 6 alpha-hydroxylation. Conversely, paclitaxel reduced significantly the hydroxylation of docetaxel. We examined in vitro the possible interaction among docetaxel, paclitaxel, and drugs which could be associated during chemotherapy. Cisplatin, verapamil, doxorubicin, vinblastine, and vincristine at concentrations usually recommended did not markedly modify taxoid metabolism. Ranitidine and diphenylhydramine had no effect, but 100 microM cimetidine partially inhibited the formation of 6 alpha-hydroxypaclitaxel. Pretreatment of patients with barbiturates strikingly stimulated docetaxel hydroxylation, whereas no acceleration of docetaxel hydroxylation was noticed in a patient receiving steroids.

The rat brain contains large amounts of pregnenolone (P) and dehydroepiandrosterone (D) arising from local biosynthetic pathways. We have devised a procedure for the measurement of both "neurosteroids" either unconjugated or released from their sulfate (S) or fatty acid (L) esters. The measurements were performed at the acrophase of the circadian variation of neurosteroids, and confirmed the large accumulation of P (25 +/- 8 ng/g, mean +/- SD) and of PS (19 +/- 6 ng/g) and DS (2.1 +/- 0.5 ng/g) in the brain of adult male rats. We found that fatty acid esters constitute the major species of neurosteroids in brain (PL 46 +/- 14, and DL 36 +/- 7 ng/g, in adult males). The levels of P and DS were increased by daily injection of vehicle to intact males, whereas castration, without or with testosterone or estradiol supplementation (2 mg daily for 7 days), did not produce a significant change of neurosteroids concentrations. Measurements of neurosteroids had not been previously reported in cyclic females. The levels of P, PL, and DS were identical in proestrous females and in intact males, whereas PS (26 +/- 6 ng/g) and DL (50 +/- 16 ng/g) were increased in females. Compared to proestrous females, diestrous females had lower levels of PS (19 +/- 6 ng/g), DS (1.7 +/- 0.4 ng/g), and PL (43 +/- 19 ng/g). These differences suggested a modulatory role of ovarian secretions on the metabolism of neurosteroids.

Peroxisome proliferators such as clofibric acid, nafenopin, and WY-14,643 have been shown to activate peroxisome proliferator-activated receptor (PPAR), a member of the steroid nuclear receptor superfamily. We have cloned the cDNA from rat that is homologous to that from mouse, which encodes a 97% similar protein. To search for physiologically occurring activators, we established a transcriptional transactivation assay by stably expressing in CHO cells a chimera of rat PPAR and the human glucocorticoid receptor that activates expression of the placental alkaline phosphatase reporter gene under the control of the mouse mammary tumor virus promoter. 150 microM concentrations of arachidonic or linoleic acid but not of dehydroepiandrosterone, cholesterol, or 25-hydroxy-cholesterol, activated the receptor chimera. In addition, saturated fatty acids induced the reporter gene. Shortening the chain length to n = 6 or introduction of an omega-terminal carboxylic group abolished the activation potential of the fatty acid. To test whether a common PPAR binding metabolite might be formed from free fatty acids we tested the effects of differentially beta-oxidizable fatty acids and inhibitors of fatty acid metabolism. The peroxisomal proliferation-inducing, non-beta-oxidizable, tetradecylthioacetic acid activated PPAR to the same extent as the strong peroxisomal proliferator WY-14,643, whereas the homologous beta-oxidizable tetradecylthiopropionic acid was only as potent as a non-substituted fatty acid. Cyclooxygenase inhibitors, radical scavengers or cytochrome P450 inhibitors did not affect activation of PPAR. In conclusion, beta-oxidation is apparently not required for the formation of the PPAR-activating molecule and this moiety might be a fatty acid, its ester with CoA, or a further derivative of the activated fatty acid prior to beta-oxidation of the acyl-CoA ester.

Dehydroepiandrosterone (3 beta-hydroxy-5-androsten-17-one; DHEA) and its conjugates are abundant circulating steroids that originate largely from the adrenal cortex. Their levels decline profoundly with age in human beings of both sexes, as the incidence of most cancers rises. Low levels of these steroids have been associated with the presence and risk of development of cancer. Administration of DHEA to rodents produces protection against spontaneous tumors and chemical carcinogenesis, suppresses weight gain without significantly affecting food intake, ameliorates the severity of diabetes in genetically diabetic mice, and restrains autoimmune processes. DHEA and related steroids also depress the mitogenic effects of carcinogens, tumor promoters and plant lectins, and block viral and carcinogen-induced cell transformations. DHEA and certain congeners are also potent and quite specific inhibitors of mammalian glucose-6-phosphate dehydrogenases. We have observed that the conversion of 3T3-L1 and 3T3-F442A preadipocyte clones to the adipocyte phenotype, in response to appropriate differentiation stimuli (fetal calf serum, insulin, dexamethasone, and 1-methyl-3-isobutylxanthine), is blocked by DHEA and other steroidal inhibitors of glucose-6-phosphate dehydrogenase. The structural requirements for blocking adipocyte differentiation and for inhibiting glucose-6-phosphate dehydrogenase are closely correlated. Evidence is reviewed suggesting that the inhibition of glucose-6-phosphate dehydrogenase is central to the anticarcinogenic and differentiation-blocking actions of DHEA and related steroids. The 3T3 preadipocyte clones provide a valuable system for the analysis of the mechanisms of the effects of DHEA on growth, differentiation and carcinogenesis.

BACKGROUND

Statins have been shown to improve hyperandrogenism in women with polycystic ovary syndrome (PCOS). However, their use has also been associated with impairment of glucose metabolism and an increased risk of type 2 diabetes mellitus. Because women with PCOS are prone to disturbances in glucose metabolism, statin therapy could also have negative effects.

OBJECTIVE

Our objective was to explore the effects of atorvastatin therapy on hormonal and metabolic parameters in women with PCOS.

METHODS

We conducted a randomized, double-blind, placebo-controlled 6-month follow-up study conducted at Oulu University Hospital, Finland.

METHODS

Women with PCOS (Rotterdam criteria) were treated with atorvastatin (20 mg/d, n = 15) or placebo (n = 13) for 6 months.

METHODS

Fasting serum samples were collected at baseline and at 3 and 6 months. Oral and iv glucose tolerance tests were performed at 0 and 6 months.

METHODS

Androgen secretion and glucose metabolism were measured.

RESULTS

Fasting levels and area under the curve of insulin increased significantly and insulin sensitivity (insulinogenic and Matsuda indexes) decreased during 6 months of atorvastatin therapy. Serum levels of dehydroepiandrosterone sulfate decreased in the atorvastatin group, whereas no change was observed in serum testosterone levels. Levels of C-reactive protein, total and low-density lipoprotein-cholesterol, and triglycerides decreased significantly during statin therapy.

CONCLUSIONS

Atorvastatin therapy improves chronic inflammation and lipid profile, but it impairs insulin sensitivity in women with PCOS. Because women with PCOS have an increased risk of developing type 2 diabetes mellitus, the results suggest that statin therapy should be initiated on the basis of generally accepted criteria and individual risk assessment of cardiovascular disease, and not only because of PCOS.

This study concentrated on the initial events triggering the development of nonalcoholic fatty liver disease induced by a high-fat plus fructose (HF-F) diet and on the possibility of delaying nonalcoholic fatty liver disease progression by adding dehydroepiandrosterone (DHEA) to the diet. Sterol regulatory element binding protein-1c (SREBP-1c) activation plays a crucial role in the progression of nonalcoholic fatty liver disease induced by an HF-F diet. This study investigated the protective effects of DHEA, a compound of physiological origin with multitargeted antioxidant properties, against the induction of SREBP-1c and on liver insulin resistance in rats fed an HF-F diet, which mimics a typical unhealthy Western diet. An HF-F diet, fortified or not with DHEA (0.01%, w/w), was administered for 15 weeks to male Wistar rats. After HF-F the liver showed unbalanced oxidative status, fatty infiltration, hepatic insulin resistance, and inflammation. The addition of DHEA to the diet reduced both activation of oxidative-stress-dependent pathways and expression of SREBP-1c and partially restored the expression of liver X-activated receptor-alpha and insulin receptor substrate-2 genes. DHEA supplementation of the HF-F diet reduced de novo lipogenesis and delayed progression of nonalcoholic fatty liver disease, demonstrating a relationship between oxidative stress and nonalcoholic fatty liver disease via SREBP-1c.

A study was conducted in captive baboons to determine (i) the impact of cereal dietary fibre on faecal progestogen excretion, and (ii) whether means of controlling dietary effects could be identified. Blood was collected on 3 days per week and faeces on 5 days per week from four unanesthetized cyclic female baboons, consecutively fed three diets of 5, 10 and 20% fibre for 90 days per diet. A 2 day lag time was detected before progesterone in the blood appeared in the faeces, regardless of diet (mean correlation was 0.62, P = 0.002). Increased dietary fibre had a negative effect on progestogen excretion (P < 0.004). Correspondence between blood and faecal progestogens was consistently greatest and the effect of dietary fibre least when faecal progestogens were expressed g-1 dry faeces. Several means of indexing faecal steroid excretion rates were examined including dehydroepiandrosterone (DHEA) and a number of byproducts of cholesterol metabolism. The cholesterol metabolite, cholestanone, was positively correlated with dietary fibre (r = 0.27; P < 0.04). Multiplying faecal progestogen concentration by the cholestanone g-1 dry faeces concentration increased the correlation between serum and cholestanone-indexed faecal progestogens (r = 0.78, P = 0.0001) compared with nonindexed progestogens (r = 0.71, P = 0.0001). We conclude that expressing faecal progestogens g-1 dry faeces may be sufficient and the most cost-effective method for controlling for most dietary effects when the objective is monitoring longitudinal endocrine status in baboons. However, it may be appropriate to express faecal progestogens by cholestanone concentrations when increased precision is needed to overcome the effects of profound variations in dietary fibre.