Major Testosterone Metabolites Research Articles

Major glucuronide metabolites of testosterone are primarily transported by MRP2 and MRP3 in human liver, intestine and kidney

Testosterone glucuronide (TG), androsterone glucuronide (AG), etiocholanolone glucuronide (EtioG) and dihydrotestosterone glucuronide (DHTG) are the major metabolites of testosterone (T), which are excreted in urine and bile. Glucuronides can be deconjugated to active androgen in gut lumen after biliary excretion, which in turn can affect physiological levels of androgens. The goal of this study was to quantitatively characterize the mechanisms by which TG, AG, EtioG and DHTG are eliminated from liver, intestine, and kidney utilizing relative expression factor (REF) approach. Using vesicular transport assay with recombinant human MRP2, MRP3, MRP4, MDR1 and BCRP, we first identified that TG, AG, EtioG, and DHTG were primarily substrates of MRP2 and MRP3, although lower levels of transport were also observed with MDR1 and BCRP vesicles. The transport kinetic analyses revealed higher intrinsic clearances of TG by MRP2 and MRP3 as compared to that of DHTG, AG, and EtioG. MRP3 exhibited higher affinity for the transport of the studied glucuronides than MRP2. We next quantified the protein abundances of these efflux transporters in vesicles and compared the same with pooled total membrane fractions isolated from human tissues by quantitative LC–MS/MS proteomics. The fractional contribution of individual transporters (ft) was estimated by proteomics-based physiological scaling factors, i.e., transporter abundance in whole tissue versus vesicles, and corrected for inside-out vesicles (determined by 5′-nucleotidase assay). The glucuronides of inactive androgens, AG and EtioG were preferentially transported by MRP3, whereas the glucuronides of active androgens, TG and DHTG were mainly transported by MRP2 in liver. Efflux by bile canalicular transport may indicate the potential role of enterohepatic recirculation in regulating the circulating active androgens after deconjugation in the gut. In intestine, MRP3 possibly contributes most to the efflux of these glucuronides. In kidney, all studied glucuronides seemed to be preferentially effluxed by MRP2 and MDR1 (for EtioG). These REF based analysis need to be confirmed with in vivo findings. Overall, characterization of the efflux mechanisms of T glucuronide metabolites is important for predicting the androgen disposition and interindividual variability, including drug-androgen interaction in humans. The mechanistic data can be extrapolated to other androgen relevant organs (e.g. prostate, testis and placenta) by integrating these data with quantitative tissue proteomics data.

Transient Neonatal-Onset Hypothyroidism Boosts Estradiol Synthesis in the Testis of Adult Wistar Rat

Transient neonatal hypothyroidism is known to boost Sertoli cell (SC), Leydig cell (LC) and germ cell (GC) number at adult age. Existing reports attribute decreased steroidogenic potential of LCs to unaltered/decreased serum testosterone in such rats, despite a boost in the cell number. However, these studies have ignored the status of estradiol in such conditions. In this present study, we tested the hypothesis neonatal-onset hypothyroidism may lead to a temporal shift in adult rat testicular steroidogenesis towards estradiol production. Hypothyroidism was induced in neonates by providing methimazole (MMI) in drinking water (0.05%) to lactating mothers and pups for a transient period from postnatal day 1 (PND 1) to PND 14 or from PND 1 to PND 29. After the experimental period, the pups were provided drinking water free of MMI and sacrificed on PND 91. Coeval rats without MMI exposure served as control. Radioimmunoassay revealed decreased serum titres of luteinizing hormone (LH), follicle stimulating hormone (FSH), growth hormone (GH) and prolactin (PRL); while serum testosterone remained unaltered, its level in testicular interstitial fluid (TIF) decreased. Between the two major metabolites of testosterone, 5α-dihydrotestosterone (DHT) concentration decreased in serum and TIF, whereas estradiol recorded a significant increase in both. Transient neonatal-onset hypothyroidism decreased 3β-hydroxy steroid dehydrogenase (3β-HSD) in LCs but augmented 17β-HSD activity. Radio-receptor assay revealed decreased concentration of LHR and ER in LCs of hypothyroid groups, whereas AR and PRLR increased. While aromatase activity decreased in LCs, it increased along with FSHR in SCs, when compared to control rats. The changes in LHR and FSHR levels and aromatase were consistent with the expression level of the respective genes. The present study supports the proposed hypothesis and suggests that transient neonatal-onset hypothyroidism-induced boost in estradiol in adult rat testis may be due to augmented expression and activity of aromatase, and FSHR content in SCs of these rats.

Systematic comparison of δ13C measurements of testosterone and derivative steroids in a freeze‐dried urine candidate reference material for sports drug testing by gas chromatography/combustion/isotope ratio mass spectrometry and uncertainty evaluation using four different metrological approaches

An alternative calibration procedure for use when performing carbon isotope ratio measurements by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) has been developed. This calibration procedure does not rely on the corrections in-built in the instrument software, as the carbon isotope ratios of a sample are calculated from the measured raw peak areas. The method was developed for the certification of a urine reference material for sports drug testing, as the estimation of measurement uncertainty is greatly simplified. To ensure that the method is free from bias arising from the choice of calibration material and instrument, the carbon isotope ratios of steroids in urine extracts were measured using two different instruments in different laboratories, and three different reference materials (CU/USADA steroid standards from Brenna Laboratory, Cornell University; NIST RM8539 mineral oil; methane calibrated against NIST RM8560 natural gas). The measurements were performed at LGC and the Australian National Measurement Institute (NMI). It was found that there was no significant difference in measurement results when different instruments and reference materials were used to measure the carbon isotope ratio of the major testosterone metabolites androsterone and etiocholanolone, or the endogenous reference compounds pregnanediol, 11- ketoetiocholanolone and 11β-hydroxyandrosterone. Expanded measurement uncertainties at the 95% coverage probability ranged from 0.21‰ to 1.4‰, depending on analyte, instrument and reference material. The measurement results of this comparison were used to estimate a measurement uncertainty of δ(13)C for the certification of the urine reference material being performed on a single instrument using a single reference material at NMI.

Hepatic microsomal cytochrome P450 enzyme activity in relation to in vitro metabolism/inhibition of polychlorinated biphenyls and testosterone in Baltic grey seal (Halichoerus grypus)

Among other factors, cytochrome P450 (CYP) enzyme activity determines polychlorinated biphenyl (PCB) bioaccumulation, biotransformation, and toxicity in exposed species. We measured the oxidative metabolism in vitro of 12 PCB congeners, representing structural groups based on the number and position of the chlorine atoms, by the hepatic microsomes of one Baltic grey seal (Halichoerus grypus). Microsomal metabolism was observed for several PCBs with vicinal H atoms exclusively in the ortho and meta positions and without any ortho-Cl substituents (CB-15 [4,4'-Cl2] and CB-77 [3,3',4,4'-Cl4]), vicinal meta and para-H atoms (CB-52 [2,2',5,5'-Cl4], and -101 [2,2',4,5,5'-Cl5]) or with both characteristics in combination with either only one ortho-Cl (CB-26 [2,3',5-Cl3], CB-31 [2,4',5-Cl3]) or two ortho-Cl substituents (CB-44 [2,2',3,5'-Cl4]). To allocate PCB biotransformation to specific CYPs, the inhibitive effect of compounds with known CYP-specific inhibition properties was assessed on in vitro PCB metabolism and on regio- and stereospecific testosterone hydroxylase activities. Metabolic inhibition was considered relevant at concentrations < or = 1.0 microM because these inhibitors became decreasingly selective at higher concentrations. At < 1.0 microM, ellipticine (CYPIAI/2 inhibitor) selectively inhibited CB-15, -26, -31, and -77 metabolism, with no significant inhibition of CB-44, -52, and -101 metabolism. Inhibition of CB-52 and -101 metabolism by chloramphenicol (CYP2B inhibitor) started at 1.0 microM and maximized at about 100% at 10 microM. Ketoconazole (CYP3A inhibitor) appeared to selectively inhibit CB-26, -31, and -44 metabolism relative to CB-15, -77, and -52 at concentrations < or = 1.0 microM. Major testosterone metabolites formed in vitro were 2beta-(CYP3A), 6beta- (CYP3A, CYPIA), and 16beta- (CYP2B) hydroxytestosterone and androstenedione (CYP2B, CYP2C11). The CYP forms indicated are associated with the specific metabolism of testosterone in laboratory animals. Inhibition of 2beta- and 6beta-hydroxytestosterone formation at ellipticine and ketoconazole concentrations < or = 1.0 microM suggested that both inhibitors were good substrates of CYP3A-like enzymes in grey seal. Chloramphenicol (model for CYP2B) is apparently not a good inhibitor of CYPI A and CYP3A activities in grey seal because the chemical did not inhibit any metabolic route of testosterone at concentrations from 0.1 to 10 microM. Our findings demonstrated that at least CYP1A- and CYP3A-like enzymes in the liver of grey seals are capable of metabolizing PCBs with ortho-meta and/or meta-para vicinal hydrogens. A CYP2B form might also be involved, but this could not be proven by the results of our experiments. Defining the profiles of CYP enzymes that are responsible for PCB biotransformation is necessary to fully understand the bioaccumulation, toxicokinetics, and risk of PCB exposure in seals and other free-ranging marine mammals.

Seasonal Relationships between Plasma and Fecal Testosterone in Response to GnRH in Domestic Ganders

In two groups (eight individuals each) of socially acquainted, outdoor-kept, domestic ganders (male Hungarian white: Anser domesticus), basal and GnRH-stimulated plasma testosterone (T) concentrations were compared with fecal testosterone metabolites (TM) in and between three seasons, spring peak of reproductive activity, summer photorefractoriness, and fall sexual reactivation. Plasma was sampled 90 min following the challenge and T was analyzed by radioimmunoassay following the GnRH challenge. Fecal TM were measured by enzyme immunoassay using two group-specific antibodies against 17β-OH-androgens or a novel antibody against 17-oxo groups, which was found to react with major testosterone metabolites without prior hydrolytic deconjugation. Baseline plasma T and systemic levels were high in spring and fall but low in summer. Plasma T increases in response to GnRH were followed by significantly elevated fecal TM levels 2 to 6 h following the challenge in spring and fall. In fall, at high plasma T levels, fecal TM levels were disproportionally lower than in spring. Variability of TM levels was two to five times higher in feces than in plasma, which explains why correlations between individual plasma T and fecal TM levels generally remained nonsignificant. This points to a low-level short-term relationship between the excreted TM and the plasma T levels. However, the reliability of the method was demonstrated by standard inter- and intraassay variablilities and by a high correspondence between results obtained by the two assays. It is suggested that, with appropriate sample size, fecal TM reflects plasma T increase. However, fecal TM was more variable than the plasma T, and fecal TM responses to GnRH did not always parallel the plasma T response. In addition, seasonal changes in androgen excretion regimes must be taken into account.

In vitro biotransformation of anabolic steroids in canines

Forensic drug testing of anabolic steroids in racing animals is required because of the potential for steroid abuse. Often when the metabolic products of an administered compound have not been identified, the analysis and verification of the administered compound is difficult. The objective of this study was to qualitatively identify the in vitro phase I biotransformation products of anabolic steroids that have a high potential for abuse in canines. The investigated steroids included testosterone, methyltestosterone, mibolerone and boldenone. Steroid biotransformation products were generated using beagle liver microsomes and analysed by high performance liquid chromatography (HPLC)/mass spectrometry (MS) with an electrospray ionization source. Characterization of steroid metabolites was based on HPLC retention, UV and mass spectra. The major testosterone metabolites were identified as androstenedione and 6beta- and 16alpha-hydroxytestosterone. 6beta-Hydroxymethyltestosterone was identified as a major metabolite in the methyltestosterone microsomal incubations. Several mibolerone metabolites were identified as monohydroxylated mibolerones as well as an oxidized mibolerone metabolite. Boldenone metabolites were identified as monohydroxylated boldenones, oxidized boldenone, and testosterone. This information should assist in the determination of anabolic steroid use in canines through the correlation of the urinary metabolites to the administered drug.

Psychoneuroendocrine aspects of temporolimbic epilepsy. Part II: Epilepsy and reproductive steroids.

Reproductive dysfunction is unusually common among men and women with epilepsy. Reproductive endocrine disorders are also common and may be causal. The association between particular reproductive endocrine disorders and the laterality and focality of epileptiform discharges suggests an etiologic role for epilepsy. Gonadal steroids are neuroactive and influence seizure occurrence: estrogen is epileptogenic whereas progesterone has antiseizure effects. Fluctuations in the absolute and relative serum levels of these hormones may play a critical role in establishing three distinct patterns of catamenial epilepsy: 1) perimenstrual and 2) preovulatory in women with ovulatory cycles, and 3) entire luteal phase of the cycle in women with anovulatory cycles. Treatment with progesterone reduces seizure frequency by more than half. In men, testosterone effects may depend on the relative concentrations of two major testosterone metabolites that exert opposing influences on neuronal excitability: estrogen potentiates whereas dihydrotestosterone inhibits NMDA-mediated conductance. Combined therapy using an aromatase inhibitor along with testosterone improves sexual function and may reduce seizures in men with epilepsy.

The use of stable carbon isotope analysis to detect the abuse of testosterone in cattle.

The use of stable carbon isotope analysis to detect the administration of anabolic steroids to cattle was investigated. Samples were extracted by solid-phase extraction on C18 cartridges. Stable isotope ratios (13C:12C) were measured by gas chromatography-isotope ratio mass spectrometry (GC-IRMS) of the underivatised extracts. A programmed temperature vaporiser (PTV) injector was installed in the GC-IRMS system, which conferred a number of advantages. First, it allowed large volumes of sample to be injected whilst the injector liner was cool. The solvent was subsequently vented to the atmosphere prior to transfer of the sample to the GC column. Thus a significantly greater amount of sample could be presented for analysis, thereby increasing the sensitivity. Second, by this means virtually all the solvent could be removed prior to analysis. This eliminates solvent peak tailing, which can be a major problem in GC-IRMS. Finally, the PTV allowed the use of higher initial GC oven temperatures, which in turn facilitated the analysis of underivatised steroids by reducing the GC run time and improving the chromatographic peak shape. The carbon isotope composition of 5 beta-androstane-3 alpha,17 alpha-diol, the major metabolite of testosterone found in bovine bile, was measured in bile samples from untreated cattle and from cattle injected intramuscularly with testosterone or a mixture of testosterone esters. There was considerable inter-animal variation in the values obtained and there was no significant difference between samples from treated and untreated animals. However, when the isotopic composition of the metabolite was normalised with respect to that of an endogenous reference compound (cholesterol) in the same sample, the difference between treated and untreated animals become statistically significant.

A putative 5 α-reductase inhibitor demasculinizes portions of the zebra finch song system

One model of the sexual differentiation of the zebra finch song system holds that both major metabolites of testosterone, dihydrotestosterone (DHT) and estradiol (E 2), act together to masculinize the song system. To test this model, we administered a putative inhibitor of 5 α-reductase (MK-434) to decrease the synthesis of DHT from testosterone (T) in hatchling zebra finches. We tested MK-434's inhibition of 5 α-reductase, 5 β-reductase, and aromatase in vivo and in vitro. In vivo, MK-434 significantly inhibited 5 α-reductase activity but also reduced the activities of 5 β-reductase and aromatase. In vitro, MK-434 was extremely effective in inhibiting 5 α-reductase in the rat prostate but only slightly inhibited 5 α-reductase in the zebra finch telencephalon, where it also reduced aromatase and 5 β-reductase activities. These results suggest that MK-434 might differentially influence the availability of androgenic and estrogenic substrates, depending on the relative abundance of these enzymes in brain. MK-434 demasculinized (decreased) the number and decreased the density of RA neurons but did not significantly affect any other sexually dimorphic aspect of the song system, including the volumes of RA, HVC, and Area X; the size of neural somata in lMAN, HVC, and RA; and the number of neurons in HVC and lMAN. The differential influence of MK-434 on sexually dimorphic characteristics suggests that the various sexually dimorphic characteristics of the song system (1) are sensitive to different hormones, depending on the characteristic; or (2) have different sensitivities to hormone levels, some being easily affected by slightly reduced hormone levels whereas others are not; or (3) have markedly different critical periods depending on the characteristic. Regardless of the reason(s) for differential effects on the sexually dimorphic characteristics of the song system, the data clearly suggest that steroid hormones play a role in the normal masculine development of the song system.

Androgen metabolism in rat L6 myoblast cells; high formation of 5α-androstane-3α,17β-diol from testosterone

We have studied androgen metabolism in L6 rat myoblasts. 4-androstene-3,17-dione (Adione), testosterone, 5α-dihydrotestosterone (DHT), and 5α-androstane-3α,17β-diol (3α-diol) were used for substrates and the amounts of metabolites formed from the respective substrates in the medium were determined. Conversion of Adione to testosterone was dominant over the reverse conversion. DHT formation from testosterone was low and did not change with the duration of incubation, whereas 3α-diol formation increased in a time-dependent manner. Major metabolite of testosterone was not DHT but 3α-diol. A large amount of 3α-diol was formed from DHT, however DHT formation from 3α-diol was very low. These data indicate that L6 cells have high 5α-reductase activity and suggest that DHT formed from testosterone is rapidly metabolized to 3α-diol in these cells.

Production and Action Mechanism of Sex Hormones in Male Dogs

The present study was undertaken to assess the production and the action mechanism of sex hormones in male dogs. The following results were obtained. 1) Plasma concentrations of LH, androstenedione, testosterone, 5 α-dihydrotestosterone (DHT) and estradio1-17 β were measured by the radioimmunoassay method. The level of LH increased with days of age and stayed constant from 8 years through 13 years of age in male dogs. The level of testosterone increased with days of age and stayed constant from 1 year through 10 years of age. Thereafter, the level of testosterone declined gradually. In contrast, there was no age-related increase in the level of estradiol-17β in mature dogs. 2) By measuring androstenedione, testosterone and DHT simultaneously in peripheral and spermatic vein plasma, and determining the temporal relationship between the two vein plasma levels of these hormones, it was suggested that plasma testosterone and DHT were derived mainly from the testis, while plasma androstenedione was derived from the testis and other organs like the adrenal. 3) Plasma concentrations of LH, androstenedione, testosterone and DHT in mature dogs fluctuated episodically, but neither diurnal rhythm nor annual variation of LH and testosterone concentrations was observed. 4) It was suggested that the pathway for activation of steroidogenesis in canine testes by LH was through cAMP formation. 5) In canine testes, major C19-metabolites of pregnenolone were dehydroepiandrosterone, androstenedione and testosterone. Following the addition of a 17α-hydroxylase inhibitor to the testis caused a significant increase in pregnenolone production, indicating that the formation of testosterone from pregnenolone by the Δ 5-pathwaywas dominant in androgen biosynthesis of the testis. 6) Hexestrol dicaprylate (H8), a long-acting estrogen, was used in an attempt to induce temporary sterilization of male dogs. H8 treatment caused significant decreases in the gonadotrophic activities of the pituitary gland and testosterone formation by the testis with the inhibition of spermatogenesis in the dog. It was suggested that H8 interfered with testosterone production by blocking the pituitary function, and also directly inhibited the testicular steroidogenesis. 7) In canine prostates, major metabolites of testosterone were DHT and 5α-androstanediols. DHT had a high binding affinity for androgen receptor in the prostate, while 5α-androstanediols had a low affinity for the receptor. The prostate was shown to contain estrogen receptor. Following injection of estrogen to dogs, it bounded to the estrogen receptor in the prostate gland and caused the enlargement of the gland.

Polyadenylated and nonadenylated messenger RNA and androgen control of sexual behavior and scent marking in male gerbils

To test the hypothesis that testosterone stimulates masculine social behaviors by inducing transcription of messenger ribonucleic acid (mRNA), we studied the effects of cordycepin, an adenosine analog that preferentially impairs synthesis of polyadenylated mRNA. When infused into the medial preoptic area of castrated male gerbils an hour before they received systemic injections of testosterone propionate, cordycepin almost completely blocked recovery of sexual behavior but did not affect recovery of scent marking. In gerbils given saline infusions, both behaviors were restored. When treatments were reversed, sexual activity resumed in males previously exposed to cordycepin and declined in males now receiving the drug. Cordycepin also blocked reinstatement of sexual behavior by the two major metabolites of testosterone, estradiol and dihydrotestosterone. Thus it does not suppress sexual behavior simply by suppressing synthesis of aromatase or 5α-reductase. Again, cordycepin had no effect on scent marking, although this behavior is sensitive to other drugs that inhibit transcription or translation. The data suggest that testosterone may stimulate sexual behavior and scent marking, respectively, by inducing transcription of polyadenylated and nonadenylated mRNAs.

Androgen 5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase activities in ventral prostate epithelial and stromal cells from immature and mature rats.

To study androgen-mediated differentiation in the rat ventral prostate, we separated the two principal cell types (epithelial and stromal) derived from prostates of immature and mature rats on two continuous Percoll gradients. Cells were immediately placed in culture medium. Testosterone metabolism by the two prostatic cell types was evaluated using [3H]testosterone and quantifying the formation of 5 alpha-[3H]dihydrotestosterone (5 alpha-DHT) and 5 alpha-[3H]androstane-(3 alpha or 3 beta), 17 beta-diols. In epithelial cells from both immature and mature rat prostates the major testosterone metabolites were 5 alpha-DHT and 5 alpha-androstane-3 alpha, 17 beta-diol. Stromal cells metabolized less testosterone than did the epithelial cells. Differences in the relative levels of the various metabolites were observed for the two age groups. To examine in more detail the changes in testosterone metabolism observed in vitro both types of cells and unfractionated cells from immature and mature rat prostates were assayed for testosterone 5 alpha-reductase (using testosterone as substrate) and 3 alpha-hydroxysteroid dehydrogenase (using 5 alpha-DHT as substrate) activities (expressed as pmol substrate reduced/min per 10(6) cells). In immature rats both 5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase activities were localized in the epithelial cell fraction (17 and 52 respectively); stromal cells showed lower 5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase activity (4 and 4). Relative to epithelial cells from immature rats epithelial cells from mature rats showed a decrease in 5 alpha-reductase (7) and an increase in 3 alpha-hydroxysteroid dehydrogenase (160) activity while stromal 5 alpha-reductase showed little change (3) and 3 alpha-hydroxysteroid dehydrogenase increased to 22.(ABSTRACT TRUNCATED AT 250 WORDS)

EFFECTS OF TAMOXIFEN ON THE BINDING AND METABOLISM OF TESTOSTERONE BY HUMAN PROSTATIC TISSUE AND PLASMA IN VITRO

The in-vitro metabolism of testosterone in benign and malignant prostatic tissue was examined and distinct quantitative differences between the two types of specimens were observed. The major metabolite of testosterone in the hyperplastic prostate was 5 alpha-dihydrotestosterone and a high 3 alpha(beta)-hydroxysteroid dehydrogenase activity was also detected. In the malignant tissue, 5 alpha-reductase activity was considerably reduced and there was little or no androstanediol formed; the 17 beta-dehydrogenase activity was, however, higher than in the benign tissue. The decrease in 5 alpha-reductase was always followed by a compensatory change in the 3 alpha(beta)-hydroxysteroid dehydrogenase of the malignant prostate. The present study revealed that the ratio of the mean activities of 5 alpha-reductase to 3 alpha(beta)-hydroxysteroid dehydrogenase in the two types of specimen always remained a constant. Although the antioestrogen, tamoxifen, induced an inhibitory effect on the activities of 5 alpha-reductase and 17 beta-hydroxysteroid dehydrogenase in the gland, the present investigation also suggested that tamoxifen stimulated the activity of 3 alpha(3 beta)-hydroxysteroid dehydrogenase. In blood, the action of tamoxifen appeared to be confined to the displacement of androgens from the binding sites on the sex hormone binding globulin.

In Vitro Steroidogenesis and Hormone Levels in Pooled Human Vas Deferens

The ability of human vas deferens to utilize labelled pregnenolone and testosterone for the synthesis of androgens was studied and correlated with the endogenous hormone levels present in the pooled tissue. In vitro incubation of vas tissue with [4‐14C]‐pregnenolone resulted in accumulation of more radioactivity in dehydroepiandrosterone and testosterone compared to that observed in 4‐androstene‐3,17‐dione and progesterone. Labelled [14C]‐testosterone was mainly converted to 4‐androstene‐3,17‐dione and 5α‐dihydrotestosterone, the counts being more in the former than in the latter steroid. The endogenous levels of progesterone, testosterone and 4‐androstene‐3,17‐dione in the vas tissue were higher than the levels of 5α‐dihydrotestosterone and dehydroepiandrosterone. Thus, the vas deferens appears to have all the enzymes necessary for the synthesis of testosterone and the major metabolite of testosterone was not 5α‐dihydrotestosterone as in other male accessory sex organs.

Production and effects of 7α-hydroxytestosterone on testosterone and dihydrotestosterone metabolism in rat testis

1. 1. Testicular 7α-hydroxylation of testerone was assayed in cell extracts of rats between 12 and 79 days of age. Maximal 7α-hydroxylase activity was observed about 60 days, while insignificant activity was obtained prior to 42 days of age. 2. 2. 7α-Hydroxytestosterone, a major metabolite of testosterone in mature rat testis, inhibited 5α-reduction of testosterone in cell extracts of mature but not of immature rat testis. 3. 3. Maximal testicular activity of 3β-hydroxysteroid dehydrogenase using dihydrotestosterone as substrate was obtained in the presence of NAD, while maximal 3α-hydroxysteroid dehydrogenase activity was observed with NADP. Both enzyme activites were reversible. 4. 4. Sensitivity toward testosterone inhibition of 3-hydroxysteroid dehydrogenase varied greatly with stage of testis development being highest at 25–27 days of age. In contrast to testosterone, 7α-hydroxy testosterone was an inhibitor of 3α-hydroxysteroid dehydrogenase only. In the mature rat testis 7α-hydroxytestosterone may be a naturally occurring inhibitor of dihydrotestoster-one and 5α-androstane-3α, 17β-diol formation.

Hair

The psychologic importance of hair to man is in inverse ratio to its physical function. Except for scalp hair and desultory areas of sexual hair, most of man's hair follicles are vestigial. Three problems of hair growth remain to be solved: (1) how the intermittent activity of hair follicles in both animals and man is controlled; (2) how the male hormone alters the hair cycle in human skin; and (3) why larger hairs are produced by testosterone in some areas of the body when in some individuals the hair follicles in the scalp regress. Studies in which skin grafts from rats of different ages were exchanged showed that hair follicles are innately programmed but can be slowly influenced by systemic factors. Steroid hormones, especially estrogens, slow down the moult cycle whereas thyroid hormones accelerate it. What establishes the innate rhythm remains problematical. The fact that plucking out the club hair initiates activity in resting follicles has been explained by the hypothesis that the mitotic inhibitor which accumulates during anagen is normally used up or dispersed during telogen or by wounding. However, contrary to this theory, follicular activity is not prolonged by epilation during anagen. Moreover, if rats are epilated within one or two days of eruption, only club hairs are removed since forceps cannot grasp the tips of the new hairs. Such epilation does not affect the anagen in progress, but remarkedly enough the subsequent resting phase is shortened. Both sexual hair and male-pattern baldness depend on androgenic hormones. Target organs of testosterone convert the hormone to active metabolites, chiefly 5alpha-dihydrotestosterone. In skin, however, 5alpha-dihydrotestosterone may not be the only active tissue androgen. The major metabolite of testosterone incubated with hair roots in androstenedione, and hirsute women without other obvious endocrine abnormality sometimes excrete high levels of androstanediol. Both steroids stimulated the sebaceous glands of hypophysectomized-castrated rats, which, however, showed only a limited response to testosterone. The androgenic steroids, the enzymes that convert them to their active metabolites, and the proteins that bind them are undoubtedly very important to the problems of the growth of sexual hair and male-pattern baldness.

Mode of action of progesterone, gestonorone capronate (depostat) and cyproterone acetate (androcur) on the metabolism of testosterone in human prostatic adenoma: in vitro and in vivo investigations

Selective uptake of H3-testosterone in human benign prostatic hypertrophy (BPH) has been demonstrated by in vitro and in vivo studies. 5α-dihydrotcstosteronc and 5α,3α-androstanediol are by far the major metabolites of testosterone. Progesterone and gestonorone capronate are competitive inhibitors of testosterone metabolism in BPH. These progestagens are taken up in preference to testosterone into prostatic cells. The subsequent formation of dihydrotestostcronc is inhibited, whereas cyproterone acetate does not influence the reduction of testosterone. Animal studies support the concept that this antiandrogcn acts by interfering with the binding of dihydrotestosterone to an intranuclear receptor.

Formation and secretion of 5alpha-androstan-17beta-ol-3-one, 5alpha-androstan-3alpha,17beta-diol and 5alpha-androstan-3beta,17beta-diol by the perfused rabbit testis epididymis.

Rabbit testes epididymides were perfused in vitro at 36.5 C with an artificial medium containing bovine red blooc cells. Three major testosterone metabolites were formed and subsequently elaborated into the venous effluent of testes epididymibdes when testosterone-1,2,6,7-3-H was infused into the artery of the perfused organ. In contrast, testosterone-1,2,6,7-1-H remained unchanged when incubated in the artificial medium under similar conditions. Elution volume from a Sephadex LH-20 column, development in three ascending paper chromatographic (APC) systems, acetylation and development in a fourth APC system followed by gas liquid chromatography (GLC) of the acetylated products tentatively identified the radioactive-metabolites as 5alpha-androstan-17beta-ol-3-one (DHT), 5alpha-androstan-3alpha,17beta-diol (3alphaol) and 5alpha-androstan-3beta,17beta-diol (3beta-ol). The identity of these 5alpha-reduced testosterone metabolites was confirmed by crystallization to constant specific activity in the presence of authentic carrier steroid. These results suggest that DHT and androstanediols formed by perfused rabbit testes epididymides subsequently escape into the venous effluent.

Regulation of human hair growth by steroid hormones. I. Testerone metabolism in isolated hairs.

To investigate the relation between androgens and hair growth, testosterone-1,2-3H metabolism was assessed under standardized conditions in growing (anagen) and resting (telogen) hair roots from 10 anatomical sites from 4 women and 14 men, 6 of whom had varying degrees of male pattern baldness. A micromethod was developed to quantitate androgen metabolism in only a few hair roots. In all hairs examined, 5α-reduced and 17-ketosteroids were the major metabolites of testosterone. No significant relation was found between androgen-mediated growth of hair and the capacity to form 5α-metabolites, e.g., scalp hair of women performed 5α-reduction to approximately the same degree as beard hair from men. The formation of 17-ketometabolites was decreased in telogen hairs from all body sites, whereas the formation of dihydrotestosterone was decreased in telogen hairs only from the scalp. In general a higher formation of 5α-reduced metabolites and 17-ketosteroid metabolites was observed at all sites of the scalp of bald men as compared to hair obtained from the corresponding sites of women and nonbalding men, and a significantly higher rate of metabolism was found at the frontal area of the bald men. It is not clear at present whether these changes are secondary to the balding process or are related causally to the hair loss. On the basis of these studies it is concluded that regional differences in androgen-mediated hair growth cannot be the result of variations in testosterone metabolism in the hair follicles.