Concordant up-regulation of cytochrome P450 Cyp3a11, testosterone oxidation and androgen receptor expression in mouse brain after xenobiotic treatment

Inactivation of testosterone by specific hydroxylations is a main function of cytochrome P450 (P450, CYP) in the brain. Recent data imply that induction of brain P450s by neuroactive drugs alters steroid hormone levels and endocrine signalling, giving rise to endocrine disorders. In this study, we investigated this drug–hormone crosstalk in mouse brain. Phenytoin led to a significant increase of 2α-, 2β-, 6β-, 16α- and 16β-hydroxytestosterones, while 6α- and 15α-hydroxytestosterones showed no significant alteration of their metabolism compared with untreated controls. Inhibition of testosterone hydroxylation using the chemical inhibitors orphenadrine, chloramphenicol, ketoconazole and nifedipine as well as antibodies against CYP3A- and 2B-isoforms pointed to major role of Cyp3a11 and an only minor function of Cyp2b9/10 in mouse brain. Cyp3a11 revealed to be the major isoform affected by phenytoin. There was considerable overlap of Cyp3a11 and AR expression in neuronal structures of the limbic system, namely the hippocampus, amygdala, hypothalamus and thalamus. Phenytoin treatment led to an increase of both, Cyp3a11 and AR expression in the limbic system. Additionally, the coherence between CYP3A and AR expression was analysed in PC-12 cells. Inhibition of phenytoin-induced endogenous CYP3A2 and AR by ketoconazole led a reduction of their expression to basal levels. We conclude that Cyp3a11 plays a crucial role in directing drug action to hormonal response within the limbic system of mouse brain in a so-called drug–hormone crosstalk.     

Autologous down-regulation of androgen receptor messenger ribonucleic acid

Autoregulation of androgen receptor (AR) mRNA was investigated using Northern blot analysis with AR cDNA fragments as probes. The amount of AR mRNA increased 2- to 10-fold with androgen withdrawal and decreased below control levels after androgen stimulation in rat ventral prostate, coagulating gland, epididymis, seminal vesicle, kidney, and brain, and in a human prostate cancer cell line, LNCaP. In rat ventral prostate, AR mRNA increased 2- to 3-fold within 24 h after castration and remained elevated for 4 days. Treatment with testosterone propionate beginning 24 h after castration reduced ventral prostate AR mRNA 4-fold within 8 h of androgen replacement. Administration of estradiol 24 h after castration had no significant effect on prostatic AR mRNA. Androgens, including testosterone and the synthetic androgen methyltrienolone (R1881), or the antiandrogen cyproterone acetate down-regulated AR mRNA in vitro in LNCaP cells, whereas estradiol was without effect. Administration of testosterone propionate to rats with androgen insensitivity did not decrease AR mRNA. Down-regulation of AR mRNA by androgen is therefore a receptor-mediated process which occurs in vivo in rat tissues that differ in androgen responsiveness and in cultured human prostate cells.     

Induction of liver cytochrome P-450 (CYP) 3A in male and female rats by a steroidal androgen receptor antagonist,Zanoterone

The cytochrome P-450 (CYP) mediated hydroxylation of testosterone to 6β-, 7α-, and 16α-hydroxytestosterone (6β-, 7α-, and 16α-OHT) and the de-alkylation of ethoxycoumarin to 7-hydroxycoumarin (ECOD) and ethoxyresorufin to resorufin (EROD) were used to probe changes in CYP monooxygenase activities in liver microsomes form rats treated with the androgen receptor antagonist, zanoterone (Z). Phenobarbital (PB) and β-naphthoflavone (β-NF) were used as comparators. There were sex-related differences in the constitutive CYP activities and in the responses of CYP activities to Z. The greatest effect of Z administration was on 6β-OHT activity: It was increased up to 5.2-fold in males and 13.9-fold in females (Z high dose). The effect was larger than that produced by PB or β-NF (⊆threefold increases). Z (high dose), PB, and β-NF increased ECOD to a similar extent, e.g., about 1.3-fold in males and 1.2–2.9-fold in females. β-NF increased EROD (11.2-fold males, 6.2-fold females) more than PB (3.4- to 4.6-fold) or Z (1.3- to 1.7-fold). Since hydroxylation of testosterone at the 6β position in rats and humans is catalyzed primarily by CYP isoforms from the 3A subfamily, the increase in 6β-OHT suggests that Z induced CYP 3A activity. Theses findings were confirmed with Western immunoblots with probes for rat CYP 1A1, 2B1/2, 2E1, 3A, and 4A. Z produced a three-to fourfold increase in the 3A isoform for both male and female rats. Results from this study suggest that in a clinical setting Z therapy has the potential to induce CYPs of the 3A subfamily and in so doing alter the metabolism and clearance of drugs that are substrates for the 3A subfamily. © 1997 John Wiley & Sons, Inc.     

Effects of steroidal and non-steroidal antiandrogens on the androgen binding properties of the rat ventral prostate androgen receptor

Steroidal (cyproterone acetate) and non-steroidal (RU23908 and hydroxyflutamide) antiandrogens are able to block testosterone-induced increases in nuclear androgen receptor (AR) in the prostate of 1-day orchidectomized rats, but when given alone, RU23908 and hydroxyflutamide increase nuclear AR (RU23908 greater than hydroxyflutamide) in the same animal model. The increases in nuclear AR induced by antiandrogen alone or with testosterone alone are blocked by cycloheximide 1 h after administration, suggesting that androgen or antiandrogens induce de novo AR synthesis. Concomitant to nuclear AR accumulation, testosterone is able to induce depletion of cytosol and microsomal AR. Blockade of testosterone-induced depletion of microsomal AR, but not of cytosol AR, occurs in the presence of antiandrogens. Cyproterone acetate has a higher relative binding affinity (RBA) for microsomal AR and cytosol AR than RU23908 or hydroxyflutamide. This phenomenon is in good agreement with the degree of inhibition by these compounds of the association rate of androgen for the microsomal AR. This correlation between RBA and inhibition of the initial rate of hormone binding to the receptor is not found for cytosol AR. The results show that antiandrogens are not 'pure' antagonists of androgen action and they are potent agonists in the absence of testosterone. Furthermore, testosterone alone or antiandrogens per se regulate AR levels acutely by protein-synthesis dependent mechanisms of action, in rat ventral prostate.     

食蚊鱼CYP19b基因克隆及环境激素暴露对其表达的影响

为探讨17-雌二醇(E2)、17-甲基睾酮(MT)和雄烯二酮(AED)暴露对食蚊鱼脑组织CYP19b表达的影响,首次克隆和分析了食蚊鱼(Gambusia affinis) CYP19b cDNA的全系列。采用静水暴露方法, 分别设置2、10、50和100 ng/L E2、MT和AED各4个实验组, 并设置对照组和平行组, 定量测定了1、3、5和7d脑组织中的CYP19b mRNA表达水平的变化。成功克隆了食蚊鱼CYP19b基因全长, 获得基因片段总长1670 bp, ORF(OpenReading Frame)是从52 bp到1554 bp, 共编码501个氨基酸。通过与其他相关鱼类CYP19b基因编码的氨基酸序列相似性作比较, 食蚊鱼CYP19b基因氨基酸序列的同源性很高。结果显示, 短时间的暴露, E2较低浓度组(2和10 ng/L)对食蚊鱼作用不明显, 但E2相对较高浓度组(50和100 ng/L)能显著地上调食蚊鱼CYP19b基因的表达(P0.01)。暴露早期MT对CYP19b表达无明显影响, 随着暴露时间的持续延长, 除了MT较高浓度组(50 ng/L)外, 其他各浓度组MT对CYP19b的表达有显著的抑制作用(P0.01), 并呈一定的剂量相关关系。AED作为雄激素类似物与MT的作用效果相似, 相对较高浓度(50和100 ng/L)的AED抑制CYP19b的表达(P0.01)。研究结果初步表明, 3种典型环境激素对食蚊鱼CYP19b基因表达的影响十分显著, 但不同的暴露浓度及不同的取样时间点可能表现为促进或抑制CYP19b基因的表达。     

Sex differences,laterality, and hormonal regulation of androgen receptor immunoreactivity in rat hippocampus

Sex differences, laterality, and hormonal regulation of androgen receptor (AR) immunoreactivity in rat hippocampal CA1 pyramidal cells were examined using the PG21 antibody. Adult male rats were either castrated or sham-operated at least 2 weeks prior to sacrifice. Gonadally intact females were sacrificed on the day of proestrus. Animals received an injection of either testosterone propionate (TP) or vehicle 2 h prior to sacrifice. Within CA1, both the intensity of staining and the number of AR+ cells were assessed. AR immunostaining was detected in all the groups with marked variation among them. The overall ranking of staining intensity was: gonadally intact males > females given TP > castrated males given TP > females > castrated males given vehicle. The number of AR+cells within subregions of CA1 showed the same basic pattern: among control-treated animals, gonadally intact males have more than females, but castrated males have the least, and acute TP treatment increases the number in both sexes. The increased level of AR immunoreactivity in CA1 of castrated males following acute TP treatment suggests that testicular androgens in adulthood normally increase AR immunoreactivity there, producing a sex difference favoring males in gonadally intact animals. We also found a higher number of AR+ CA1 cells on the left than on the right, but only in gonadally intact males and in females given TP. These results suggest that a laterality of AR distribution in the rat hippocampus may lead to lateralities in hippocampal structure and function.     

Parallel evolution between aromatase and androgen receptor in the animal kingdom

There are now many known cases of orthologous or unrelated proteinsin different species that have undergone parallel evolutionto satisfy a similar function. However, there are no reportedcases of parallel evolution for proteins that bind a commonligand but have different functions. We focused on two proteinsthat have different functions in steroid hormone biosynthesisand action but bind a common ligand, androgen. The first protein,androgen receptor (AR), is a nuclear hormone receptor and thesecond one, aromatase (cytochrome P450 19 [CYP19]), convertsandrogen to estrogen. We hypothesized that binding of the androgenligand has exerted common selective pressure on both AR andCYP19, resulting in a signature of parallel evolution betweenthese two proteins, though they perform different functions.Consistent with this hypothesis, we found that rates of aminoacid change in AR and CYP19 are strongly correlated across themetazoan phylogeny, whereas no significant correlation was foundin the control set of proteins. Moreover, we inferred that genomictoolkits required for steroid biosynthesis and action were presentin a basal metazoan, cnidarians. The close similarities betweenvertebrate and sea anemone AR and CYP19 suggest a very ancientorigin of their endocrine functions at the base of metazoanevolution. Finally, we found evidence supporting the hypothesisthat the androgen-to-estrogen ratio determines the gonadal sexin all metazoans.     

Sodium acetate and androgen receptor blockade improve gestational androgen excess-induced deteriorated glucose homeostasis and antioxidant defenses in rats: roles of adenosine deaminase and xanthine oxidase activities

Nutritional challenges and androgen excess have been implicated in the development of gestational diabetes and poor fetal outcome, but the mechanisms are not well delineated. The effects of short chain fatty acid (SCFA) on glucose dysmetabolism and poor fetal outcome induced by gestational androgen excess is also not known. We tested the hypothesis that blockade of androgen receptor (AR) and suppression of late gestational androgen excess prevents glucose dysmetabolism and poor fetal outcome through suppression of adenosine deaminase (ADA)/xanthine oxidase (XO) pathway. Twenty-four pregnant Wistar rats were treated (sc) with olive oil, testosterone propionate (0.5 mg/kg) singly or in combination with SCFA (sodium acetate; 200 mg/kg; p.o.) or AR blocker (flutamide; 7.5 mg/kg; p.o.) between gestational days 14 and 19. The results showed that late gestational androgen excess led to glucose deregulation, poor fetal outcome, increased plasma and hepatic free fatty acid and lactate dehydrogenase, liver function marker enzymes, malondialdehyde, uric acid, ADA and XO activities. Conversely, gestational androgen excess resulted in reduced body weight gain, visceral adiposity, plasma and hepatic anti-oxidant defenses (glutathione peroxidase, reduced glutathione/glutathione disulphide ratio, glucose-6-phosphate dehydrogenase, adenosine and nitric oxide). However, all these effects were ameliorated by either sodium acetate or flutamide treatment. The study demonstrates that suppression of testosterone by SCFA or AR blockade protects against glucose deregulation and poor fetal outcome by improvement of anti-oxidant defenses and replenishment of hepatic oxidative capacity through suppression of ADA/XO pathway. Hence, utility of SCFA should be encouraged for prevention of glucose dysmetabolism and poor fetal outcome.     

Proteomic changes in rat spermatogenesis in response to in vivo androgen manipulation; impact on meiotic cells

The production of mature sperm is reliant on androgen action within the testis, and it is well established that androgens act on receptors within the somatic Sertoli cells to stimulate male germ cell development. Mice lacking Sertoli cell androgen receptors (AR) show late meiotic germ cell arrest, suggesting Sertoli cells transduce the androgenic stimulus co-ordinating this essential step in spermatogenesis. This study aimed to identify germ cell proteins responsive to changes in testicular androgen levels and thereby elucidate mechanisms by which androgens regulate meiosis. Testicular androgen levels were suppressed for 9 weeks using testosterone and estradiol-filled silastic implants, followed by a short period of either further androgen suppression (via an AR antagonist) or the restoration of intratesticular testosterone levels. Comparative proteomics were performed on protein extracts from enriched meiotic cell preparations from adult rats undergoing androgen deprivation and replacement in vivo. Loss of androgenic stimulus caused changes in proteins with known roles in meiosis (including Nasp and Hsp70-2), apoptosis (including Diablo), cell signalling (including 14-3-3 isoforms), oxidative stress, DNA repair, and RNA processing. Immunostaining for oxidised DNA adducts confirmed spermatocytes undergo oxidative stress-induced DNA damage during androgen suppression. An increase in PCNA and an associated ubiquitin-conjugating enzyme (Ubc13) suggested a role for PCNA-mediated regulation of DNA repair pathways in spermatocytes. Changes in cytoplasmic SUMO1 localisation in spermatocytes were paralleled by changes in the levels of free SUMO1 and of a subunit of its activating complex, suggesting sumoylation in spermatocytes is modified by androgen action on Sertoli cells. We conclude that Sertoli cells, in response to androgens, modulate protein translation and post-translational events in spermatocytes that impact on their metabolism, survival, and completion of meiosis.     

Differential regulation of androgen receptors in the separate rat prostate lobes: androgen independent expression in the lateral lobe

In order to understand the hormonal regulation of androgen receptors (AR) in the separate lobes of the rat prostate gland, the present study examined AR levels in the ventral, dorsal and lateral prostate lobes as a function of androgen withdrawal to complete prostatic regression and subsequent testosterone replacement. In the intact rat, the 3 prostate lobes contained significantly different amounts of androgen binding sites. Mean number of total cellular AR in the ventral, dorsal and lateral lobes was 7370, 1690, and 1015 fm/mg DNA, respectively. These receptors were primarily localized within the nuclear fraction of homogenized tissue: ventral, 86%; dorsal, 83%; and lateral, 100% nuclear localization. Androgen withdrawal was initiated via castration and rats were sacrificed 1, 2, 3, 5, 7, 10 and 14 days thereafter. Nuclear AR levels fell rapidly to 5, 24 and 30% of intact values by 48 h in the ventral, dorsal and lateral lobes, respectively. Levels of nuclear AR continued to decline in the ventral and dorsal lobes to undetectable levels by Day 10. In marked contrast, lateral lobe nuclear AR began to increase on Day 3 postcastration, reaching intact values by Day 7 and 133% intact levels by Day 14. Cytosolic AR in the ventral and dorsal lobes initially increased following castration, but subsequently declined to low levels by Day 14. Cytosolic AR were not detectable in the lateral prostate at any time point following castration. To determine the nuclear AR response to testosterone at this time, 14 day castrate rats were given 2 cm testosterone implants and sacrificed 1, 3, 5, 7, 10 and 14 days thereafter. As expected, nuclear AR rapidly returned in the ventral and dorsal lobes by Day 1 and reached a plateau by Day 5. A short term response to androgen exposure occurred in the lateral lobe where an immediate 9-fold increase in nuclear AR quantity was observed; however, these levels rapidly declined to pre-implant values by Day 5 and remained at that level despite continued exposure to testosterone. These f findings indicate that while nuclear AR levels in the ventral and dorsal prostate are primarily regulated by androgens, a testosterone-independent component exists within the lateral lobe.     

Mutations in the helix 3 region of the androgen receptor abrogate ARA70 promotion of 17beta-estradiol-induced androgen receptor transactivation

The influence of estrogen on the development of the male reproductive system may be interrupted in a subset of partial androgen insensitivity syndrome (PAIS) patients. PAIS describes a wide range of male undermasculinization resulting from mutations in the androgen receptor (AR) or steroid metabolism enzymes that perturb androgen-AR regulation of male sex organ development. In this study, we are interested in determining if PAIS-derived AR mutants that respond normally to androgen have altered responses to estrogen in the presence of ARA70, a coregulator previously shown to enhance 17beta-estradiol E2-induced AR transactivation. The wild-type AR (wtAR) and two PAIS AR mutants, AR(S703G) and AR(E709K), all bind to androgen and E2 and subsequently translocate to the nucleus. Whereas ARA70 functionally interacts with the wtAR and the PAIS AR mutants in response to androgen, E2 only promotes the functional interaction between ARA70 and the wtAR but not the PAIS AR mutants. ARA70 increases E2 competitive binding to the wtAR in the presence of low level androgen and also retards E2 dissociation from the wtAR. ARA70 is present in both the cytoplasm and the nucleus of various mouse testicular cells during early embryogenesis day 16, at postpartum day 0 during estradiol synthesis and in the Leydig cells at postpartum day 49. ARA70 may be unable to modulate the PAIS AR mutants-E2 binding, diminishing the effect of E2 via AR during male reproductive system development in patients with such mutations. Therefore, the presence of ARA70 in the testosterone and E2-producing Leydig cells may enhance the overall activity of AR during critical stages of male sex organ development.     

Direct biphasic modulation of gonadotropin-stimulated testicular androgen biosynthesis by prolactin

Prolactin (PRL) exerts both stimulatory and inhibitory effects upon testicular steroidogenesis in vivo. The direct effects of PRL on biosynthesis of testicular androgen were studied in primary cultures of testicular cells obtained from adult, hypophysectomized or neonatal, intact rats. In cells from adult animals, treatment with human chorionic gonadotropin (hCG) (10 ng/ml) significantly increased testosterone and progesterone production relative to their respective controls. In contrast, neither steroid was increased by treatment with rat PRL (rPRL) or ovine PRL (oPRL) alone. Upon addition of 0.1-3 ng/ml of either rPRL or oPRL to the hCG-treated cultures, testosterone production was progressively increased up to a maximum of 70% greater than with hCG alone. However, when PRL exceeded 3 ng/ml, the testosterone response began to decline and was 39 or 24% less than from cells treated with hCG alone at 300 ng/ml of rPRL or oPRL, respectively. A similar biphasic response pattern was observed in cells from neonatal animals. In contrast to the biphasic effect of PRL on production of androgen, PRL treatment enhanced hCG-stimulated production of progesterone in a dose-related manner without exerting an inhibitory effect. At 3 and 300 ng/ml, rPRL augmented hCG action by 2.5- and 8-fold, respectively. Similarly, in the presence of inhibitors of pregnenolone metabolism, rPRL also enhanced hCG-stimulated production of pregnenolone. Quantitation of steroid intermediates in the testosterone biosynthetic pathway revealed that the stimulatory effect of 3 ng/ml rPRL on testosterone production was associated with 1.3- and 2.8-fold increases in accumulation of androstenedione and 17 alpha-hydroxyprogesterone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anabolic-androgenic steroid interaction with rat androgen receptor in vivo and in vitro: a comparative study

Anabolic steroids are synthetic derivatives of testosterone and are characterized by their ability to cause nitrogen retention and positive protein metabolism, thereby leading to increased protein synthesis and muscle mass. There are disagreements in the literature in regards to the interaction of anabolic steroids with the androgen receptor (AR) as revealed by competitive ligand binding assays in vitro using cytosolic preparations from prostate and skeletal muscle. By use of tissue extracts, it has been shown that some anabolic steroids have binding affinities for the AR that are higher than that of the natural androgen testosterone, while others such as stanozolol and methanedienone have significantly lower affinities as compared with testosterone. In this study we show that stanozolol and methanedienone are low affinity ligands of the rat recombinant AR as revealed by a ligand binding assay in vitro, however, based on a cell-based AR-dependent transactivation assay, they are potent activators of the AR. We also show that a single injection of stanozolol and methanedienone causes a rapid cytosolic depletion of AR in rat skeletal muscle. Based on these results, we conclude that anabolic steroids with low affinity to AR in vitro, can in fact in vivo act on the AR to cause biological responses.     

Inhibition of androgen binding in human foreskin fibroblasts by antiandrogens

Antiandrogen effects on androgen receptor binding and androgen metabolism were studied in cultured human newborn foreskin fibroblasts. Three different antiandrogens were tested in this system: (a) cyproterone acetate (CA); (b) RU23908; and (c) R2956. CA and R2956 were equipotent inhibitors of androgen binding to its intracellular receptor. The magnitude of this action was nearly twice as great against the endogenous androgen ligands, dihydrotestosterone (DHT) or testosterone (T), than with the synthetic ligand, methyltrienolone (R1881). Whereas the relative binding affinities of CA and R2956 were approximately 5-10 times less than T or DHT, RU23908 was another order of magnitude less effective as an inhibitor of androgen binding. The lower relative binding affinity determined for RU23908 could not be explained on the basis of a requirement for metabolic activation. Subcellular fractionation studies and sucrose density gradient analysis further confirmed the rank order of antiandrogenic potency. None of the antiandrogens influenced the rate or profile of metabolites from cellular metabolism of T or DHT. We propose that cultured human genital skin fibroblasts may serve as a valuable system for the future evaluation of antiandrogens in intact ells under physiologic conditions.     

Regulation of androgen receptor protein and mRNA concentrations by androgens in rat ventral prostate and seminal vesicles and in human hepatoma cells

The effects of androgen withdrawal and replacement on the concentrations of androgen receptor (AR) protein and AR mRNA were investigated in rat ventral prostate and seminal vesicles and in cultured human hepatoma (HepG2) cells. AR mRNA concentrations were determined by Northern blotting with single stranded AR cRNA as the hybridization probe, whereas antibodies raised against two synthetic 17-amino acid long peptides corresponding to the N-terminal and steroid-binding regions of the AR were employed in immunological receptor assays. AR mRNA levels in both prostate and seminal vesicles increased about 2-fold within 24 h after castration and continued to rise within the next 48 h to values that were 9- to 11-fold higher than those in intact controls. Administration of pharmacological doses of testosterone (400 micrograms steroid/day) to 1-day castrated animals for 24-48 h brought about a decrease in AR mRNA levels in accessory sex organs to levels in intact controls. Similar results were obtained in cultured HepG2 cells where a switch to serum- and steroid-free medium elicited a rapid increase (approximately 4-fold in 10 h) in the AR mRNA level, which was prevented by inclusion of 10(-7) M testosterone in culture medium. Similar, but quantitatively less marked, changes occurred in the AR protein concentration in prostate, seminal vesicles, and HepG2 cells, as determined by immunoblotting using antibodies against AR peptides. In addition, immunohistochemical studies showed that AR is a nuclear protein of the prostatic epithelial cells in both intact and castrated rats, and suggested that short term castration increases the concentration of nuclear AR in the prostate. Taken together, these data indicate that androgens down-regulate the concentration of AR protein and AR mRNA in a variety of target tissues.