In vivo MRI evaluation of anabolic steroid precursor growth effects in a guinea pig model

Anabolic steroids are widely used to increase skeletal muscle (SM) mass and improve physical performance. Some dietary supplements also include potent steroid precursors or active steroid analogs such as nandrolone. Our previous study reported the anabolic steroid effects on SM in a castrated guinea pig model with SM measured using a highly quantitative magnetic resonance imaging (MRI) protocol. The aim of the current study was to apply this animal model and in vivo MRI protocol to evaluate the growth effects of four widely used over-the-counter testosterone and nandrolone precursors: 4-androstene-3 17-dione (androstenedione), 4-androstene-3β 17β-diol (4-androsdiol), 19-nor-4-androstene-3β-17β-diol (bolandiol) and 19-nor-4-androstene-3 17-dione (19-norandrostenedione). The results showed that providing precursor to castrated male guinea pigs led to plasma steroid levels sufficient to maintain normal SM growth. The anabolic growth effects of these specific precursors on individual and total muscle volumes, sexual organs, and total adipose tissue over a 10-week treatment period, in comparison with those in the respective positive control testosterone and nandrolone groups, were documented quantitatively by MRI.     

The importance of SRS-1 residues in catalytic specificity of human cytochrome P450 3A4

The structural basis for the regioselective hydroxylation of Delta-4-3-ketosteroids by human CYP3A4 was investigated. Prior studies had suggested that the chemical reactivity of the allylic 6beta-position might have a greater influence than steric constraints by the enzyme. Six highly conserved CYP3A residues from substrate recognition site 1 were examined by site-directed mutagenesis. F102A and A117L showed no spectrally detectable P450. V101G and T103A exhibited a wild-type progesterone metabolite profile. Of five mutants at residue N104, only N104D yielded holoenzyme and exhibited the same steroid metabolite profile as wild-type. Of four mutants at position S119 (A, L, T, V), the three hydrophobic ones produced 2beta-OH rather than 6beta-OH progesterone or testosterone as the major metabolite. Kinetic analysis showed S(50) values similar to wild-type for S119A (progesterone) and S119V (testosterone), whereas the V(max) values for 2beta-hydroxysteroid formation were increased in both cases. All four mutants exhibited an altered product profile for 7-hexoxycoumarin side-chain hydroxylation, whereas the stimulation of steroid hydroxylation by alpha-naphthoflavone was similar to the wild-type. The results indicate that the highly conserved residue S119 is a key determinant of CYP3A4 specificity and reveal an important role of the active site topology in steroid 6beta-hydroxylation.     

Glucocorticoid-specific gene activation by the intact human glucocorticoid receptor expressed in yeast. Glucocorticoid specificity depends on low level receptor expression.

In the presence of appropriate reporter genes mammalian nuclear receptors are competent to transactivate gene expression when expressed in yeast cells. Thus yeast genetics could be used to identify determinants of steroid specificity for these mammalian proteins. However, unlike the estrogen, progesterone, vitamin D3, and thyroid hormone receptors, the glucocorticoid receptor shows an apparently abnormal steroid specificity in yeast (Schena, M., and Yamamoto, K. (1988) Science 241, 965-967), suggesting that the expressed protein might be incorrectly folded. We show here that the glucocorticoid receptor does exhibit a normal steroid specificity in yeast cells, but only at low levels of expressed receptor protein. Thus, at least under these conditions, genetic studies on steroid specificity are possible. At least part of the abnormal specificity that is sometimes observed for the glucocorticoid receptor in yeast appears to result from an artifact of the assay system and is not due to an abnormal receptor structure. This mechanism could account for all our data and so could provide the sole explanation of the abnormal specificity observed. However, it is also possible that part of the abnormal specificity could result from structural or other changes in receptor function, which occur when the receptor expression level is increased.     

Male copulatory behavior in the female rat after perinatal treatment with an antiandrogenic steroid

Adult male copulatory behavior was studied in female rats treated with an antiandrogenic steroid (cyproterone) perinatally. A time-sampling method was used to obtain a detailed record of all behavior occurring during testing. Females were treated with cyproterone either prenatally, pre- and postnatally, or not at all. They were ovariectomized and given testosterone prior to the mating tests. Treated animals engaged in significantly less female-directed activity and fewer of them engaged in copulatory acts in early tests. By the last test there were no differences between the groups. Only untreated animals ever displayed the ejaculatory pattern and only rarely.     

Reconstructed epidermis and full-thickness skin for absorption testing: influence of the vehicles used on steroid permeation

A protocol for percutaneous absorption studies has been validated, based on the use of reconstructed human epidermis (RHE) and aqueous solutions of test substances. However, it is often the case that it is more-complex formulations of drugs or chemicals which will make contact with the skin surface. To investigate whether RHE and the reconstructed full-thickness skin model (FT-model) can be used to predict uptake from formulations, we compared the permeation of hydrocortisone and testosterone when applied in emulsion form and as a solution containing the penetration enhancer, ethanol. Human and pig skin and a non-cornified alveolar model served as references. The results were compared with steroid release from the formulations. The permeation rates of the steroids were ranked as: alveolar model > RHE > FT-model, pig skin > human skin. In accordance with the rapid hydrocortisone release from the formulations, the permeation rates of this steroid exceeded those of testosterone. Only minor differences were observed when comparing the testosterone formulations, in terms of release and permeation. However, the ranking of the permeation of the hydrocortisone formulations was: solution > w/o emulsion > o/w emulsion, which permitted the elucidation of penetration enhancing effects, which is not possible with drug release studies. Differences in penetration were most obvious with native skin and reconstructed tissues, which exhibited a well-developed penetration barrier. In conclusion, RHE and skin preparations may be useful in the development of topical dermatics, and in the framework of hazard analysis of toxic compounds and their various formulations.     

Time-course and steroid specificity of aromatase induction in rat hypothalamus-preoptic area

We studied the time-course and steroid specificity for aromatase induction in the hypothalamus-preoptic area (HPOA) of the adult male rat. Aromatase activity (AA) was measured in tissue homogenates by using a radiometric assay that quantifies the stereospecific production of 3H2O from [1 beta-3H] androstenedione. We found that by 48 h after administration of testosterone, HPOA AA was significantly (p less than 0.01) greater than control values in castrated rats. In contrast, AA was significantly (p less than 0.01) reduced 12 h after castration, and reached its lowest levels by 4 days after castration. Several other steroids, administered in 3-cm Silastic capsules for 7 days, were tested for their capacity to induce hypothalamic AA. In addition to testosterone, only 5 alpha-dihydrotestosterone and 5 alpha-androstane-3 alpha, 17 beta-diol were effective. Neither the stereoisomers of these compounds nor several other steroids, including estradiol, progesterone, and corticosterone, were active. This profile of activity indicates that the induction of HPOA AA is androgen-specific and, together with the demonstrated time-course of induction, lends further support to the hypothesis that androgens regulate AA through a receptor mechanism and the synthesis of new protein.     

In vitro evolution of the binding specificity of neocarzinostatin,an enediyne-binding chromoprotein

Neocarzinostatin is the most studied member of the enediyne-chromoprotein family, and is clinically used as an antitumoral agent. Neocarzinostatin could be a promising drug delivery vehicle if new binding specificities could be conferred to its protein scaffold. We used in vitro evolution methods to demonstrate that this approach is feasible. We created large libraries containing between 1.7 x 10(8) and 1.4 x 10(9) independent clones, where up to 13 side chains pointing toward the binding crevice were randomly substituted. We then used phage display to select variants that bind to a model ligand (testosterone) which is unrelated to the natural ligand of neocarzinostatin. Several different binders were selected from each library. The corresponding proteins were expressed in Escherichia coli and their affinities and specificities were characterized in detail. K(D) values of about 20 nM were obtained for streptavidin-bound testosterone. The K(D) of selected proteins for free soluble testosterone are between 7 and 55 microM and therefore higher than the K(D) for streptavidin-bound testosterone. The spacer and streptavidin used during selection contributed to the high affinity of the selected binders for the target. Binding studies of 15 different steroids related to testosterone allowed us to determine that C3, 4, 5, 6, and 7 on cycles A and B and the conjugated 3 oxo group of the steroid molecule were essential for molecular recognition. Other testosterone analogues substituted on C1, 2, 9, 11, 15, and 17 were not discriminated from testosterone. These results demonstrate that the binding specificity of this protein family can be extended to compounds that are completely unrelated to the natural enediyne chromophore family. This type of highly expressed, stable proteins with tailored binding properties have a wide potential range of applications.     

Use of a new radioactive ligand, 7 alpha, 17 alpha-dimethyl[17 alpha-methyl 3H]19-nortestosterone for the estimation of androgen receptors in rat liver cytosol

The use of the testosterone derivative, 7 alpha, 17 alpha-dimethyl[17 alpha-methyl 3H] 19-nortestosterone for the estimation of cytosolic androgen receptors in male rat liver is described. Use of this compound demonstrates binding which has a similar dissociation constant, maximum binding and steroid specificity to that seen with other synthetic testosterone derivatives. In contrast to previous data significant binding to the progesterone receptor also occurs and future studies with this ligand should employ triamcinolone acetonide to block such binding.     

Effect of oxidizing adulterants on human urinary steroid profiles

Steroid profiling is the most versatile and informative technique adapted by doping control laboratories for detection of steroid abuse. The absolute concentrations and ratios of endogenous steroids including testosterone, epitestosterone, androsterone, etiocholanolone, 5α-androstane-3α,17β-diol and 5β-androstane-3α,17β-diol constitute the significant characteristics of a steroid profile. In the present study we report the influence of various oxidizing adulterants on the steroid profile of human urine. Gas chromatography–mass spectrometry analysis was carried out to develop the steroid profile of human male and female urine. Oxidants potassium nitrite, sodium hypochlorite, potassium permanganate, cerium ammonium nitrate, sodium metaperiodate, pyridinium chlorochromate, potassium dichromate and potassium perchlorate were reacted with urine at various concentrations and conditions and the effect of these oxidants on the steroid profile were analyzed. Most of the oxidizing chemicals led to significant changes in endogenous steroid profile parameters which were considered stable under normal conditions. These oxidizing chemicals can cause serious problems regarding the interpretation of steroid profiles and have the potential to act as masking agents that can complicate or prevent the detection of the steroid abuse.     

Effects of three types of resisted sprint training devices on the kinematics of sprinting at maximum velocity

Resisted sprint running is a common training method for improving sprint-specific strength. For maximum specificity of training, the athlete's movement patterns during the training exercise should closely resemble those used when performing the sport. The purpose of this study was to compare the kinematics of sprinting at maximum velocity to the kinematics of sprinting when using three of types of resisted sprint training devices (sled, parachute, and weight belt). Eleven men and 7 women participated in the study. Flying sprints greater than 30 m were recorded by video and digitized with the use of biomechanical analysis software. The test conditions were compared using a 2-way analysis of variance with a post-hoc Tukey test of honestly significant differences. We found that the 3 types of resisted sprint training devices are appropriate devices for training the maximum velocity phase in sprinting. These devices exerted a substantial overload on the athlete, as indicated by reductions in stride length and running velocity, but induced only minor changes in the athlete's running technique. When training with resisted sprint training devices, the coach should use a high resistance so that the athlete experiences a large training stimulus, but not so high that the device induces substantial changes in sprinting technique. We recommend using a video overlay system to visually compare the movement patterns of the athlete in unloaded sprinting to sprinting with the training device. In particular, the coach should look for changes in the athlete's forward lean and changes in the angles of the support leg during the ground contact phase of the stride.     

Testosterone in saliva: application to the follow-up of prostate cancer

The basic aspects of steroid hormones transport, their tissular release and the interpretation of salivary testosterone values as a reflect of the free hormone in serum are reviewed in this article, as well as the salivary testosterone applications in several disorders, with a special emphasis on prostatic carcinoma. The usefulness of salivary testosterone in the short-term (n = 4) and long-term (n = 13) follow-up of patients affected by prostatic carcinoma after medical or surgical orchiectomy has also been studied. Our results show a correlation (r = 0.62) between salivary and serum concentration values, as well as a significative decrease (p less than 0.001) in both quantities after treatment. Both these findings and the advantages inherent to the sampling obtaining, lead to the conclusion that salivary testosterone is a good alternative to serum testosterone.     

Aromatase activity of steroid sulphatase-deficient placentae

Aromatase activity was measured in homogenates from steroid sulphatase deficient and steroid sulphatase positive placentae. The activity of the aromatase complex was determined from the rate of formation of [14C] oestrone plus [14C] oestradiol when [14C] testosterone was incubated with a rate-limiting quantity of homogenate. A reduced level of aromatase activity was found in vitro in 70% of steroid sulphatase-deficient placentae tested, but the deficiency was much less complete than that of steroid sulphatase. The mean (+/- SD) aromatase activity of steroid sulphatase-deficient placentae was 380 +/- 180 pmol product/h/g tissue (n = 10), significantly lower (P less than 0.001, t-test) than the mean aromatase activity of steroid sulphatase positive placentae (701 +/- 70 pmol product/h/g tissue, n = 10). Seventy per cent of the steroid sulphatase deficient placentae showed lower aromatase activity than that of normal placentae stored for a comparable period of time. Assay imprecision and the sex of the foetus were excluded as reasons for the diminished aromatase activity found. It may arise through an abnormal gene product and consequent alterations in the structure of the microsomal membrane in which both aromatase complex and steroid sulphatase enzymes are retained.     

Infection intensity-dependent accuracy of reagent strip for the diagnosis of Schistosoma haematobium and estimation of treatment prevalence thresholds

BackgroundReagent strip to detect microhematuria as a proxy for Schistosoma haematobium infections has been considered an alternative to urine filtration for individual diagnosis and community-based estimates of treatment needs for preventive chemotherapy. However, the diagnostic accuracy of reagent strip needs further investigation, particularly at low infection intensity levels.MethodsWe used existing data from a study conducted in Tanzania that employed urine filtration and reagent strip testing for S. haematobium in two villages, including a baseline and six follow-up surveys after praziquantel treatment representing a wide range of infection prevalence. We developed a Bayesian model linking individual S. haematobium egg count data based on urine filtration to reagent strip binary test results available on multiple days and estimated the relation between infection intensity and sensitivity of reagent strip. Furthermore, we simulated data from 3,000 hypothetical populations with varying mean infection intensity to infer on the relation between prevalence observed by urine filtration and the interpretation of reagent strip readings.Principal findingsReagent strip showed excellent sensitivity even for single measurement reaching 100% at around 15 eggs of S. haematobium per 10 ml of urine when traces on reagent strip were considered positive. The corresponding specificity was 97%. When traces were considered negative, the diagnostic accuracy of the reagent strip was equivalent to urine filtration data obtained on a single day. A 10% and 50% urine filtration prevalence based on a single day sampling corresponds to 11.2% and 48.6% prevalence by reagent strip, respectively, when traces were considered negative, and 17.6% and 57.7%, respectively, when traces were considered positive.Conclusions/SignificanceTrace results should be included in reagent strip readings when high sensitivity is required, but excluded when high specificity is needed. The observed prevalence of reagent strip results, when traces are considered negative, is a good proxy for prevalence estimates of S. haematobium infection by urine filtration on a single day.     

Detection of epileptogenic cortical malformations with surface-based MRI morphometry

Magnetic resonance imaging has revolutionized the detection of structural abnormalities in patients with epilepsy. However, many focal abnormalities remain undetected in routine visual inspection. Here we use an automated, surface-based method for quantifying morphometric features related to epileptogenic cortical malformations to detect abnormal cortical thickness and blurred gray-white matter boundaries. Using MRI morphometry at 3T with surface-based spherical averaging techniques that precisely align anatomical structures between individual brains, we compared single patients with known lesions to a large normal control group to detect clusters of abnormal cortical thickness, gray-white matter contrast, local gyrification, sulcal depth, jacobian distance and curvature. To assess the effects of threshold and smoothing on detection sensitivity and specificity, we systematically varied these parameters with different thresholds and smoothing levels. To test the effectiveness of the technique to detect lesions of epileptogenic character, we compared the detected structural abnormalities to expert-tracings, intracranial EEG, pathology and surgical outcome in a homogeneous patient sample. With optimal parameters and by combining thickness and GWC, the surface-based detection method identified 92% of cortical lesions (sensitivity) with few false positives (96% specificity), successfully discriminating patients from controls 94% of the time. The detected structural abnormalities were related to the seizure onset zones, abnormal histology and positive outcome in all surgical patients. However, the method failed to adequately describe lesion extent in most cases. Automated surface-based MRI morphometry, if used with optimized parameters, may be a valuable additional clinical tool to improve the detection of subtle or previously occult malformations and therefore could improve identification of patients with intractable focal epilepsy who may benefit from surgery.     

Predicting end-of-test semen quality in bulls prior to performance testing

The first objective of this study was to determine if serum concentrations of specific hormones (testosterone, progesterone and androstenedione) in bulls at the start of performance testing could predict semen quality at the end-of-test when used in a multivariate model. The second objective was to evaluate other clinical measurements (breed, age, body weight, hip height and scrotal circumference) for predicting end-of-test semen quality. End-of-test semen quality was related to steroid concentrations and several pre-testing measurements, including age, body weight, hip height and scrotal circumference (SC). Combining the 3 steroid concentrations into a predictive test had a sensitivity of 0.6 and specificity of 0.5 at its most accurate point. The repeatability of the test result was extremely low (r(2) = 0.16; P < 0.05). In multivariate analyses, breed and start-of-test SC remained significant predictors of end-of-test semen quality (P < 0.05) while the other variables were nonsignificant (P > 0.1), suggesting that start-of-test SC was the most accurate predictor of end-of-test semen quality. Removing bulls at the start-of-test that had scrotal measurements of less than 20 cm, 24 cm, 28 cm or 32 cm resulted in sensitivities and specificities of 0.19, 0.94; 0.41, 0.81; 0.64, 0.56; and 0.94, 0.12, respectively. No cut-point had both adequate sensitivity and specificity. Because clinical tests were correlated, combining the tests to improve accuracy was not justified.     

Semiconductor fault detection and classification for yield enhancement and manufacturing intelligence

Process monitoring and profile analysis are crucial in detecting various abnormal events in semiconductor manufacturing, which consists of highly complex, interrelated, and lengthy wafer fabrication processes for yield enhancement and quality control. To address real requirements, this study aims to develop a framework for semiconductor fault detection and classification (FDC) to monitor and analyze wafer fabrication profile data from a large number of correlated process variables to eliminate the cause of the faults and thus reduce abnormal yield loss. Multi-way principal component analysis and data mining are used to construct the model to detect faults and to derive the rules for fault classification. An empirical study was conducted in a leading semiconductor company in Taiwan to validate the model. Use of the proposed framework can effectively detect abnormal wafers based on a controlled limit and the derived simple rules. The extracted information can be used to aid fault diagnosis and process recovery. The proposed solution has been implemented in the semiconductor company. This has simplified the monitoring process in the FDC system through the fewer key variables. The results demonstrate the practical viability of the proposed approach.     

Elevated testosterone levels during nonbreeding-season territoriality in a fall-breeding lizard,Sceloporus jarrovi

Summary In many vertebrates, seasonal activation of sexual and territorial behaviors coincides with seasonal gonadal activation and is caused by the increase in sex steroid hormones. Both male and femaleSceloporus jarrovi are territorial, but in this species territorial behavior is seasonally activated in late April, months before seasonal gonadal maturation, which occurs in August prior to the fall mating season. Measurements of seasonal changes in circulating levels of the sex steroid hormones testosterone, progesterone, and estradiol indicated that testosterone levels in both sexes are elevated when territorial behavior is expressed, even during the period of nonbreeding-season territoriality during the summer. This suggests that a nonbreeding season behavior is activated by a sex steroid hormone in this species.     

On the regulation of rat testicular steroidogenesis. Short term effect of luteinizing hormone and cycloheximide in vivo and Ca2+ in vitro on steroid production in cell-free systems.

An attempt has been made to correlate the rapid effect of luteinizing hormone on testicular steroid production in vivo with testicular steroid concentrations and in vitro steroid production rates in testis tissue preparations. Within 20 min after intravenous administration of 25 mug luteinizing hormone, increases were observed in testosterone concentrations in testicular venous plasma and in whole testis tissue and in pregnenlone concentrations isolated testis mitochondrial fractions. Testosterone production by whole testis homogenates and pregnenolone production by isolated mitochondrial fractions were significantly increased within 5 min after in vivo administration of luteinizing hormone. Injection of cycloheximide 10 min prior to luteinizing hormone prevented the stimulating effect of luteinizing hormone to steroid levels in testicular venous plasma and testis tissue and on steroid production rates by preparations of rat testis tissue. Cycloheximide treatment of control animals did not significantly alter testosterone concentrations and testosterone production rates vitro, although mitochondrial pregnenolone concentrations and production rates were decreased. Testosterone production by whole testis homogenates as well as the pregnenolone production by isolated mitochondrial fractions obtained from luteinizing hormone treated testes and control glands showed a biphasic time curve A period (5-10 min) of high steroid production was followed by a period lower steroid production. Addition of 25 mug luteinizing hormone or 10(-8)--10(-5) M adenosine 3':5'-monophosphate (cyclic AMP) to the incubation medium had no effect pregnenolone production by isolated mitochondrial fractions. Administration of leuteinizing hormone in vivo markedly enhance the stimulating effect of Ca2+ on testosterone production by whole testis homogenates and on pregnenolone production by isolated mitochondrial fractions.     

Steroid Binding by Mouse Salivary Proteins

The goals of this study were to determine thesteroid-binding specificity of the mouse salivaryandrogen-binding protein (ABP) family and to ascertainwhether there might be other proteins in mouse saliva capable of binding steroids. The optimalconditions for testosterone binding by mouse salivaryproteins were determined using a small-scalechromatography system to separate bound from unboundsteroid. Testosterone binding appeared to be biphasicbut was directly proportional to saliva concentration,with an optimum temperature of 37 C in the second phase.These results were used to develop a steroid-binding protocol to study the steroid specificity ofsalivary proteins separated by electrophoresis. The ABPfamily bound testosterone and progesterone well andHO-progesterone and DHT to a lesser extent but did not bind either cholesterol or estradiol.Steroid structural comparisons suggest that binding byABP is governed by the A ring of the steroid. Anotherprotein that is not a member of the ABP family bound cholesterol specifically but no protein thatspecifically bound estradiol was observed.     

Robust combination of multiple diagnostic tests for classifying censored event times

Recent advancement in technology promises to yield a multitude of tests for disease diagnosis and prognosis. When there are multiple sources of information available, it is often of interest to construct a composite score that can provide better classification accuracy than any individual measurement. In this paper, we consider robust procedures for optimally combining tests when test results are measured prior to disease onset and disease status evolves over time. To account for censoring of disease onset time, the most commonly used approach to combining tests to detect subsequent disease status is to fit a proportional hazards model (Cox, 1972) and use the estimated risk score. However, simulation studies suggested that such a risk score may have poor accuracy when the proportional hazards assumption fails. We propose the use of a nonparametric transformation model (Han, 1987) as a working model to derive an optimal composite score with theoretical justification. We demonstrate that the proposed score is the optimal score when the model holds and is optimal "on average" among linear scores even if the model fails. Time-dependent sensitivity, specificity, and receiver operating characteristic curve functions are used to quantify the accuracy of the resulting composite score. We provide consistent and asymptotically Gaussian estimators of these accuracy measures. A simple model-free resampling procedure is proposed to obtain all consistent variance estimators. We illustrate the new proposals with simulation studies and an analysis of a breast cancer gene expression data set.