John D. Stringham

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.

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.

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)

Enhanced transdermal delivery of testosterone : a new physiological approach for androgen replacement in hypogonadal men

Abstract This report describes the rationale, development and initial clinical testing of a new modality for the treatment of hypogonadal men — an enhanced transdermal delivery system (TDS) for administering native testosterone. In contrast to the experimental trans-scrotal testosterone patches, the enhanced testosterone TDS can be applied to non-scrotal skin sites, such as the back, chest, arms, etc. A one-month pilot study in six hypogonadal men shows that the nightly application of two patches (for 24 h) delivers ~ 4 to 7 mg of testosterone per day, and produces testosterone plasma levels that closely mimic the magnitude and time course of the normal circadian rhythm seen in healthy young men. Moreover, the enhanced delivery of testosterone across non-scrotal skin, is not associated with any appreciable degree of transdermal first-pass metabolism, and therefore produces physiological levels and patterns of dihydrotestosterone (DHT) and estradiol (E 2 ). In all but one subject, skin tolerability has been acceptable. During 7 months of treatment in two patients, hormone levels have remained within the normal range, tolerability has been good, and subjective improvements in sexual function and well being have been reported. In comparison to other available methods of androgen replacement therapy (i.e. testosterone-ester injections, synthetic oral agents and testosterone pellet implants), we believe that the enhanced transdermal delivery of native testosterone promises to be a more physiological and patient-friendly approach for the treatment of hypogonadal men.

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 Kis 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.

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.

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.

Elevated intranuclear dihydrotestosterone in prostatic hyperplasia of aging dogs.

Abstract 5α-Dihydrotestosterone (DHT) content is several fold higher than normal in whole tissue in hyperplastic prostate of aging men and dogs. The postulate was tested that selective nuclear accumulation of either DHT or 3α-androstanediol (3α-diol) might contribute to the development of benign prostatic hyperplasia (BPH) of aging dogs. The nuclear, mitochondrial, microsomal and cytosolic fractions of both normal ( n = 12) and hyperplastic ( n = 6) prostate were isolated by differential centrifugation and the content of DHT and 3α-diol were measured in each fraction by specific radioimmunoassays. DHT levels were elevated significantly in the nuclei (Wilcoxon rank sum test; 2.4 ± 1.3 ng/g wet weight tissue, mean ± SD, P P P P P P P P P P

Content of 5α-reduced androgens in subcellular compartments of dog prostate

Abstract The distribution of 5α-dihydrotestosterone (DHT), and 3α-androstanediol (3α-diol), 17β-hydroxyandrogens, and 5α-androstanedione, and androsterone, 17-ketoandrogens was determined by measuring their content by radioimmunoassay in subcellular components of prostate from normal adult dogs. DHT followed by 3α-diol was the most abundant 5α-reduced androgen measured in dog prostate. DHT appears to concentrate particularly in the nucleus whereas the other androgens measured do not show such a selective localization.