The presence of a dehydroepiandrosterone-specific receptor binding complex in murine T cells.

We have investigated the ability of dehydroepiandrosterone (DHEA) to alter the production of interleukin-2 (IL-2) and to bind to a specific binding complex in antiCD3 epsilon activated T cells. Binding activity correlated with the presence of a specific DHEA binding complex in the cytosol and nuclei of DHEA-responsive T-cell hybridomas, as well as in CD4+ and CD8+ cells isolated from peripheral lymph nodes of normal mice. Scatchard analysis determined that intact lymphocytes and cytosolic fractions contained high affinity binding for [3H]DHEA (approx. 2.6 nM) with 1000-7000 binding sites existing per cell. Five of the T-cell hybridomas tested both responded to DHEA treatment with increased production of IL-2 and also contained specific high affinity [3H]DHEA binding. Four additional T-cell hybridomas were found to contain no specific [3H]DHEA binding and were also unresponsive to DHEA influences on IL-2 production. Sucrose density gradients demonstrated a 3-4s [3H]DHEA binding complex in high salt and a 7-8s binding complex in low salt. Specific binding was inhibited by preincubation of the cytosol fractions with either trypsin or chymotrypsin, or by heating to 60 degrees C for 1 h (less than 15% of control). [3H]DHEA binding was unaffected by preincubation of the cytosol fractions with ribonuclease, deoxyribonuclease, or phospholipase A. The DHEA-protein complexes bound to DNA-cellulose with the amount of binding being slightly increased by preincubation at 25 degrees C as compared to 4 degrees C. As expected, [3H]DHEA binding was inhibited by the addition of unlabeled DHEA, but was also modestly inhibited by dihydrotestosterone and cortisol. Binding of DHEA was unaffected by progesterone, dexamethasone, estradiol, androsterone, DHEAS, and beta-etiocholanolone at all concentrations tested. DHEA was incapable of inhibiting the binding of [3H]DHT to the androgen receptor or [3H]dexamethasone to the glucocorticoid receptor. Collectively, these findings suggest that murine T cells contain a specific DHEA receptor. We believe that DHEA is a steroid hormone that is directly involved in the regulation of IL-2 production by both normal and some T-cell hybridomas.

Enhanced transdermal delivery of testosterone across nonscrotal skin produces physiological concentrations of testosterone and its metabolites in hypogonadal men.

None of the current or experimental androgen treatment modalities for male hypogonadism has been reported to produce physiological concentrations or circadian variations in testosterone (T) and its metabolites, dihydrotestosterone (DHT) and estradiol (E2). This investigation describes a novel transdermal dosage form designed to enhance the delivery of native T across nonscrotal skin. The main objective was to determine whether the nightly application of two experimental transdermal patches to different sites on the body (e.g. back, chest, arms, etc.) would result in normal plasma levels of T, DHT, and E2 for men and mimic the normal circadian variation. Six hypogonadal males (aged 24-66 yr) were studied 4 weeks after stopping T ester treatment. After single application of two patches, T levels increased from a pretreatment baseline of 5.8 +/- 0.94 nmol/L (mean +/- SE; 167 +/- 27 ng/dL) to an average peak concentration of 44.1 +/- 4.8 nmol/L (1273 +/- 138 ng/dL) 5.7 +/- 0.6 h after application and reached a 24-h level of 16.9 +/- 2.9 nmol/L (488 +/- 85 ng/dL). DHT and E2 levels exhibited parallel variations within the normal reference ranges. During 4 weeks of daily evening application to various sites on the torso, the mean delivery of T from two patches was 5.2 +/- 0.1 mg/day (approximately 20% of the patch content), and morning T levels were within the normal limits. On day 28 of treatment, the 24-h plasma profiles of T, DHT, and E2 (obtained with two patches on the back) approximately mimicked the normal circadian variations reported in healthy young men. The time-averaged T level was 21.8 +/- 2.9 nmol/L (629 +/- 84 ng/dL), and the plasma concentration ratios of DHT/T (0.07 +/- 0.01) and E2/T (0.005 +/- 0.001) were within the normal range. SHBG concentrations were not significantly altered over the 4 weeks of treatment. The patches were well tolerated, except for one patient who developed a local reaction to an excipient during the third week of treatment. Two of the patients (one with Klinefelter's syndrome) completed several months of continuous therapy. T, DHT, and E2 have remained in the range of normal, and plasma LH levels in the patient with Klinefelter's syndrome became normal. Subjective improvement in symptoms has continued, and tolerability has been good in both patients. These results indicate that the enhanced transdermal delivery of T across nonscrotal skin is a patient-friendly androgen replacement modality and produces physiological concentrations of T and its metabolites, which are unattainable with other treatment modalities.

Extracellular calcium and luteinizing hormone effects on 22-hydroxycholesterol used for testosterone production in mouse Leydig cells.

The effect of extracellular calcium on testosterone synthesis in response to luteinizing hormone (LH) or 22-hydroxycholesterol (22-OH-C) by isolated adult mouse Leydig cells was studied. Leydig cells were isolated by linear density gradient centrifugation. The cells were incubated in minimum essential medium with or without calcium (1.36 mmol/L) in an atmosphere of 95% air and 5% carbon dioxide at 37 degrees C for 3 hours with or without LH (10 ng/sample), or with or without 22-OH-C (10 mumol/L). Testosterone production in response to LH was significantly lower (P less than 0.02) in the absence of extracellular calcium and in the presence of verapamil (10 mumol/L), a calcium channel blocking agent. Extracellular calcium did not significantly (P greater than 0.05) affect testosterone production in cells incubated with 22-OH-C in either the presence or absence of LH. The results suggest that steps in steroidogenesis from 22-OH-C to testosterone are unaffected by extracellular calcium content and that extracellular calcium affects the use of intracellular cholesterol by the cholesterol side-chain cleavage enzyme.

Effects of a fat-containing meal on sex hormones in men.

The effect of a fat-containing meal on plasma sex steroid concentrations was investigated in normal men. After an overnight fast on two separate occasions, subjects ingested a liquid meal containing either a nonnutritive sweetener (control), or isocaloric meals of mixed calorie sources with either high-fat content or mixed carbohydrate and protein with minimal fat. The order of the meals was alternated. Blood samples were collected at 15-minute intervals and pooled each hour. Sampling began at 7:00 AM and the test meal was ingested at 8:00 AM. Sex steroids, including estrone, estradiol, testosterone, and dihydrotestosterone (DHT), sex hormone-binding globulin (SHBG) capacity, free testosterone concentration, and luteinizing hormone (LH) were determined by either specific radioimmunoassay or dialysis. The fat-containing meal, but not the nonnutritive or mixed carbohydrate and protein meal, resulted in a significant (P less than .01) reduction in total and free testosterone. Estrogens and luteinizing hormone were unaffected by either meal. This is the first documentation, to our knowledge, of the acute effect of a fat-containing meal on sex steroid concentrations in blood. Our observations suggest that a fat-containing meal reduces testosterone concentrations without affecting luteinizing hormone. This might indicate that fatty acids modulate testosterone production by the testes.

Nicotine and cotinine inhibit steroidogenesis in mouse Leydig cells.

Cigarette smoking alters plasma testosterone concentrations in men. The objectives of this study were to determine if nicotine and cotinine, two alkaloid products of cigarettes, affect luteinizing hormone(LH)-stimulated steroidogenesis in isolated adult mouse Leydig cells. Leydig cells from adult Swiss-Webster mice were isolated by linear density gradient and incubated (95% O2, 5% CO2) in minimum essential medium at 37 C for 3 hours with LH (10 ng) and with or without nicotine or cotinine (10(-5)-10(-7) M). Both nicotine and cotinine produced dose response inhibition (P less than 0.05) of LH-stimulated testosterone production (50-70%). The addition of 8-bromo-3',5'-cyclic monophosphate (cAMP, 500 uM) stimulated steroidogenesis comparable to LH in the absence of the alkaloids, but both nicotine and cotinine significantly (P less than 0.05) reduced testosterone production in response to cAMP, suggesting that the alkaloids inhibit testosterone production in response to LH distal to the formation of cAMP. In MEM without calcium, LH-stimulated testosterone synthesis was decreased, and neither nicotine nor cotinine significantly affected steroidogenesis. The addition of a calcium ionophore in MEM with normal calcium content enhanced (P less than 0.05) the inhibitory effects of nicotine and cotinine on LH-responsive steroidogenesis. A calcium channel blocking agent, verapamil, at 10uM significantly (P less than 0.05) reversed the inhibition of LH-stimulated testosterone production produced by both alkaloids when incubated in the medium with a normal calcium concentration. These results suggest that nicotine and cotinine either affect intracellular calcium content or block the effects of calcium on steroidogenesis in mouse Leydig cells.

Nonesterified fatty acids modulate steroidogenesis in mouse Leydig cells.

The effects of nonesterified fatty acids (NEFA) in modulating testosterone synthesis stimulated by luteinizing hormone (LH, 10 ng/sample) were investigated in isolated adult mouse Leydig cells. LH-stimulated testosterone production was inhibited by triglycerides (0-500 mg/dl, 50 mg/dl, 94% of the control and 500 mg/dl, 40%) and by a mixture of NEFA (100 microM, 70% of control, 200 microM, 54%, and greater than 200 microM, less than 50%). Oleic acid was a more potent inhibitor than linoleic, stearic, or palmitic acids. 8-Bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP, 5 mM) stimulated testosterone production comparable to LH but failed to reverse the inhibition of steroidogenesis produced by the NEFA. The inhibition produced by NEFA was dependent on extracellular Ca2+; and a Ca2+ channel antagonist, verapamil (10 microM), enhanced the inhibition of chylomicrons and fatty acids. 22(R)-hydroxycholesterol (10 microM) reversed the inhibition produced by NEFA. The inhibitory effects of NEFA were reversible by removal of the fatty acids. The results indicate that NEFA are potent modulators of testosterone synthesis in Leydig cells stimulated with either LH, cAMP, or intracellular Ca2+. NEFA inhibit steroidogenesis at one of the steps preceding conversion of cholesterol to pregnenolone.

Estradiol and testosterone metabolism and production in men with prostatic cancer.

We recently observed a familial influence on the plasma concentration of sex-steroids and the metabolic clearance in men with prostatic cancer. We have now determined, by isotope dilution techniques, the blood estradiol and testosterone production and clearance rates in men with prostatic cancer and in unrelated controls. Thirty-eight men had a diagnosis of prostatic cancer before the age of 63, and 22 controls matched for age were randomly selected from the general population. None of the patients or controls had received endocrine therapy. The plasma content of testosterone, dihydrotestosterone, estrone, estradiol, 3 alpha-androstanediol glucuronide, dehydroepiandrosterone sulfate, sex-hormone binding globulin, apparent free testosterone concentration, follicle stimulating hormone and luteinizing hormone were not significantly different between the groups. The metabolic clearance and production rates of testosterone were significantly (P = 0.008 and P = 0.013, respectively) higher in patients [447 +/- 26 L/day/body surface area(m2) and 2.21 +/- 0.17 mg/day/m2, n = 38] than in controls [346 +/- 20 L/day/m2 and 1.70 +/- 0.11 mg/day/m2, n = 22]. The PR and MCR of estradiol were not significantly different between patients with prostatic cancer (n = 19) and controls (n = 12). These results indicate that men with prostatic cancer have elevated clearance and production rates of testosterone without an alteration of estradiol production or clearance.

Relationship between body mass index, cigarette smoking, and plasma sex steroids in normal male twins.

Smoking has been observed to affect plasma sex hormones and body mass index. The relationship between smoking, body mass index, and plasma concentration of sex hormones was studied in normal adult male twins. The analyses were performed for between 150 and 159 twin pairs for whom hormonal data were available on both twins. With bivariate analysis, neither body mass index nor smoking affected estrone, luteinizing hormone, follicle-stimulating hormone, ratio of testosterone to estradiol, or ratio of testosterone to dihydrotestosterone. Body mass index significantly (P less than 0.05) affected sex hormone binding globulin, whereas smoking had no effect. The plasma contents of testosterone and dihydrotestosterone and the luteinizing hormone/testosterone ratio were affected by both body mass index and smoking, although, after allowing for body mass, smoking was less significant (0.05 less than P less than 0.10). A path model was formulated to examine the relationship of body mass and sex steroid levels. Our results suggest that body mass index affects sex steroids, since common environmental factors do not account for the strength of the relationship. The bivariate analysis suggests that the smoking effect on sex hormones (except perhaps for dihydrotestosterone) is secondary to an effect on body mass index.

Quantitating genetic and nongenetic factors influencing androgen production and clearance rates in men.

Both hereditary and nonhereditary factors have a decided influence on plasma sex steroid concentrations in men. We studied the relative contributions of genetic and nongenetic factors on the production rate (PR) and MCR of testosterone and dihydrotestosterone (DHT) and their conversion ratios to other metabolites in monozygotic (MZ; n = 22) and dizygotic (DZ; n = 24) male twins. Zygosity was determined by measurement of 10 blood proteins and enzymes. The kinetic studies were conducted with isotope dilution techniques. The genetic effect was determined from the equation: 2[rMZ - rDZ], where r is intraclass correlation. A heritability of over 40% was found for the PRs of DHT/body surface area and of testosterone/body surface area. Nongenetic factors accounted for 50% or more of the variation of the conversion ratios for testosterone/3 alpha-androstanediol and DHT/3 alpha-androstanediol. The results suggest that genetic factors markedly influence the PRs of testosterone and DHT, suggesting that the PR of these potent androgens is under genetic control despite the decided influence of environmental factors on their clearance.

Heritability of variation of plasma cortisol levels.

Heritability of the variation of the plasma concentrations of total and unbound cortisol, cortisol binding globulin (CBG), and dehydroepiandrosterone sulfate (DHEA-S) was investigated in 20 monozygotic (MZ) and 20 dizygotic (DZ) male twin pairs. Three plasma samples collected between 8 AM and 9:30 AM were pooled for the assays. Heritability was calculated from the intraclass correlation [2(rMZ - rDZ)]. The mean age, total and unbound cortisol, CBG, and DHEA-S were not significantly different between the MZ and DZ groups of twins. The heritability index for variability of the plasma content of steroids was 45.4% (p less than .05) for total cortisol, 50.6% (P less than .05) for unbound plasma cortisol, 57.8% (P less than .05) for DHEA-S, and 32.4% (P greater than .05) for CBG. The data were analyzed by factor analysis, and heritability estimates were corrected for factors including age, smoking, drinking, exercise, and degree of obesity. These factors did not account for the variation in hormone values in twin pairs. Factor analysis of the three quantitative measurements, cortisol, percent free cortisol, and DHEA-S, provides no evidence for shared factors. The correlation coefficients between age and CBG and total and unbound plasma cortisol concentrations were insignificant. The correlation coefficient between total plasma cortisol levels and CBG was 0.57, which indicates that CBG accounts for 32% of the variation of plasma cortisol concentrations. The results suggest that genetic factors have a decided influence on the variation of the concentration of cortisol of DHEA-S in normal adult men.

Hereditary and environmental influences on the variation of thyroid hormones in normal male twins.

Heritability of the variation of the plasma total and unbound T4 (free T4), T3, T4-binding globulin (TBG), and TSH concentrations was investigated in 15 monozygotic and 15 dizygotic male twin pairs. The variability in plasma of total (58%) and free T4 (72%) concentrations was significantly less (P less than 0.01) in the twin pairs than in unrelated men. Half of the variability of T3 (P less than 0.05), TBG (P less than 0.05), and TSH (P less than 0.05) was affected by influences shared by the twin pairs in both monozygotic and dizygotic twin pairs. The heritability index for variability of the plasma total T4 and free T4 was greater than 28% (P less than 0.05), and it was 25% or less for T3 and TBG. Variation in TBG accounted for less than 20% (P less than 0.001) of the variation in thyroid hormone concentrations. The results indicate that familial factors, which are affected by genetic and/or environmental factors, influence the variation of plasma TBG, TSH, T4, free T4, and T3 concentrations among normal men. Genetic factors influenced the variation in plasma T4 and free T4 levels.

Nicotine and cotinine effects on 3 alpha hydroxysteroid dehydrogenase in canine prostate.

We have recently observed that cigarette smoking affects plasma androgen concentrations. The effects of nicotine and cotinine, two products of cigarette smoking, on testosterone metabolism were determined. The activity of delta 4 steroid 5 alpha-reductase, which converts testosterone to 5 alpha-dihydrotestosterone (DHT) was measured in isolated dog prostate nuclei using testosterone (0-200 nM) as substrate and NADPH as cofactor. Activity of 3 alpha-hydroxysteroid dehydrogenase (HSD), which converts DHT to 3 alpha-androstanediol (3 alpha-diol) and is a reversible enzyme, was measured in isolated dog prostate microsomes with DHT (0-20 microM) as substrate and NADPH as cofactor. When microsomal fractions were incubated for 1 hour with and without nicotine (0-50 microM) and cotinine (0-100 microM), enzyme activity of HSD was significantly suppressed (p less than 0.001). The Vmax was not affected significantly (p greater than 0.60) and Km increased with increasing concentrations of nicotine and cotinine (p less than 0.05). Both nicotine and cotinine are competitive inhibitors of HSD in dog prostate microsomes with Ki's of 61 and 89 microM, respectively. The apparent 5 alpha-reductase activity was unaffected by nicotine and cotinine. The inhibitors produced a marked effect on activity of HSD when used in concentrations achieved in humans who smoke cigarettes. The results suggest that nicotine and cotinine are competitive inhibitors of the HSD, an important enzyme involved in the metabolism of DHT and produce an accumulation of DHT. These products of cigarette smoking could alter androgen action in tissue such as skin and prostate.

The effect of nutritional factors on sex hormone levels in male twins.

Dietary intake has been hypothesized as being associated with several hormonally related cancers including prostate, breast, ovarian, and endometrial cancer. Because diet may affect hormones directly, it is logical to examine the effects of dietary factors on hormone production and levels. Therefore, a set of 72 male MZ and 83 male DZ twin pairs was ascertained from the Utah birth certificates. A quantitative food frequency questionnaire was administered and blood samples were drawn for hormonal assays. Heritability estimates for hormonal levels were calculated indicating a range from no heritability for sex hormone binding globulin (SHBG), estrone, and testosterone glucuronide to 70% for androstanediol glucuronide and luteinizing hormone. To examine nutritional factors, the difference in hormone and SHBG levels between each MZ twin and his co-twin were correlated with the difference in nutrient intake. Weight and obesity were significantly correlated with plasma testosterone and follicle stimulating hormone. Fat intake showed a significant association with testosterone. Androstanediol glucuronide, a steroid that reflects tissue formation of dihydrotestosterone, was inversely correlated with caloric intake, theobromine and caffeine. Testosterone glucuronide exhibited significant correlations with calories and vitamin A. This study suggests that dietary intake affects plasma sex-steroid levels in men.

Production, clearance, and metabolism of testosterone in men with prostatic cancer.

It was previously unknown whether the production and metabolism of testosterone was altered in men with prostatic cancer. We recently observed a familial influence on the plasma concentration of sex steroids in men with the cancer. We have now determined, by isotope dilution techniques, the blood testosterone production and clearance rates and testosterone metabolism to potent androgen metabolites in men with prostatic cancer, their brothers, and unrelated controls. Nineteen men had a diagnosis of prostatic cancer before age 63 (probands), 23 were brothers of these index cases, and nine controls matched for age were selected randomly from the general population. None had received endocrine therapy. The plasma content of testosterone, dihydrotestosterone, sex hormone binding globulin, apparent free testosterone concentration, follicle-stimulating hormone, and luteinizing hormone were not significantly different between the groups. The metabolic clearance rate of testosterone was significantly (P = .04) higher in probands (458 liters/day/body surface area, median) than in controls (306 liters/day/body surface area). The conversion ratios of both testosterone (1.8%) and dihydrotestosterone (16.9%) to 3 alpha-androstanediol were significantly greater (P = .04 and P = .004, respectively) in probands than in controls (0.95%, 7.8%). These results indicate that men with prostatic cancer have elevated clearance rates of testosterone and an increased conversion ratio of testosterone to its potent 5 alpha-reduced metabolites.

Quantitating genetic and nongenetic factors that determine plasma sex steroid variation in normal male twins.

We have observed that familial factors have a decided influence on the plasma content of sex steroids in men both in the general population and in men of families with prostatic cancer. The contribution of genetic and nongenetic familial factors on the variation of plasma sex steroid content and action has now been investigated in 75 pairs of normal male monozygotic (MZ) twins and 88 pairs of dizygotic (DZ) twins. Zygosity was determined by measuring ten blood proteins and enzymes. The mean plasma values for testosterone (T), dihydrotestosterone (DHT), estradiol (E2), estrone (E1), and 3 alpha-androstanediol glucuronide (3 alpha-diol G), free T, LH, FSH, SHBG, age, and degree of adiposity were all similar between the groups of twins. Familial factors (P less than 0.01) accounted for 50% or more of the variation in plasma hormone levels in MZ twins (3 alpha-diol G, 84%; T/DHT, 70%; T, 63%; E1, 63%; free T, 61%; E2, 57%; DHT, 56%; LH, 55%; and FSH, 54%) except for SHBG, which was 30%. The familial influence was greater in MZ twins than in DZ twins for all measurements except for SHBG. The heritability of the variation of hormone levels in plasma was determined from the equation: 2[rMZ(intraclass correlation) - rDZ]. Genes regulate 25% to 76% of the total variation of plasma content of the hormones except for DHT (12%) and SHBG (less than 1%). Genetic regulation of tissue DHT formation was suggested by observing a 48% genetic effect on the plasma content of 3 alpha-diol G.(ABSTRACT TRUNCATED AT 250 WORDS)

Subnormal tissue 3 alpha-androstanediol and androsterone in prostatic hyperplasia.

We investigated the role of 3 alpha-androstanediol (3 alpha-diol) in the development of benign prostatic hyperplasia (BPH) and the apparent equilibrium of enzymes which metabolize it in normal and hyperplastic prostatic tissue of humans. We determined the endogenous concentrations of 3 alpha-diol, androsterone, its 3 alpha-17-keto metabolite or precursor, and 5 alpha-dihydrotestosterone (DHT), its 3-keto,17 beta-hydroxy product of precursor, by RIA after extraction and paper chromatography of the androgens from normal and hyperplastic prostate glands. The mean concentrations of 3 alpha-diol and androsterone were about one-third of normal in BPH. The mean ratio of the concentration of DHT to 3 alpha-diol was significantly higher (P less than 0.005) than normal in BPH, whereas no statistical difference was observed for the mean ratio of the tissue levels of 3 alpha-diol to androsterone in the two groups. Our data do not support the postulate that 3 alpha-diol is causally related to the development of BPH. However, they indicate that the apparent equilibrium of the 3 alpha-hydroxysteroid oxidoreductase favors the formation of the 3-keto-oxidized product, DHT, which may have relevance to the occurrence of the renewed growth of the prostate of aging men.