Testosterone Metabolism Research Articles

LC–MS analysis of androgen metabolites in serum and urine from east African chimpanzees ( Pan troglodytes schweinfurthii)

Testosterone regulates a wide variety of behavioral and physiological traits in male vertebrates. It influences reproductive and aggressive behaviors and is used as a marker of gonadal activity. While testosterone is the primary biologically active male gonadal steroid in the blood, it is metabolized into a variety of related steroids when excreted via urine and feces. To monitor endocrinological profiles studies on wild-living animals primarily rely on non-invasively collected samples such as urine or feces. Since a number of androgen metabolites that are found in high concentrations in these matrices do not stem exclusively from gonadal production, but are also produced by the adrenal cortex, the metabolism and excretion pattern of testosterone and its characteristic metabolites have to be investigated. Here, we compare the levels of 11 androgens and their metabolites in serum and urine (after hydrolytic/solvolytic cleavage of conjugates) from female, and intact and castrated male chimpanzees to investigate whether they were of testicular or adrenal origin. For serum, significant differences in concentrations were found only for native testosterone. For urine, testosterone concentrations showed the largest differences between intact and castrated males, and intact males and females, while no differences were seen between females and castrated males. Epitestosterone levels revealed the same pattern. These differences in urinary concentrations could also be seen for 5α-androstane-3α,17β-diol (androstanediol), and less clearly for 5α-dihydrotestosterone (5α-DHT), etiocholanolone, and androsterone. In urine of males, significant correlations were found between the levels of testosterone and 5α-androstane-3α,17β-diol, as well as between testosterone and epitestosterone. Therefore, the clearest urinary markers of gonadal activity in male chimpanzees seems to be testosterone itself.

Abstract B55: Integrative genomic analysis of prostate cancer disparities between African American and Caucasian American populations

Abstract Prostate cancer (PCa) is a disease conferred by gene mutations, numerous alternations in gene expression and aberrant changes in genome composition/architecture. An area of research that continues to garner attention is PCa health disparities, wherein the African American (AA) population exhibits higher incidence and mortality rates compared to Caucasian Americans (CA). To identify the genetic predispositions and oncogenic networks associated with the observed PCa disparities, we applied a systems biology approach by integrating exon, microRNA and SNP information from PCa specimens of AA and CA patients. RNA and DNA purified from PCa (with Gleason score ≥6) and patient-matched normal prostate needle biopsies from AAs and CAs were processed and hybridized onto Affymetrix human Exon 1.0 ST, SNP 6.0 arrays or Agilent human microRNA arrays. A 4-way statistical design (10% FDR, >1.5 fold-change) was employed to identify differentially expressed mRNAs in the following comparisons: AA normal vs. CA normal, AA cancer vs. CA cancer, AA cancer vs. AA normal, and CA cancer vs. CA normal. Pathway analyses of the differentially expressed genes have revealed several critical network-level rewiring of gene interactions accounting for PCa disparities between AA and CA. For example, the mis-regulated testosterone metabolism network in normal AA prostate tissues may represent a genetic predisposition factor in the AA population. Whereas, the inflammatory response (NF-?B network), activated oncogenic signaling pathways (ERK, JNK and p38), and up-regulated cancer promoting genes (RHOA and STAT1) may be associated with the higher recurrence and death rates in AA patients. Moreover, our exon and SNP profiling results have identified hundreds of genes exhibiting differential splicing patterns and/or copy number variations (CNVs) in AA and CA patients. Notably, at least 13 genes residing within the 5 oncogenic signaling pathways have been identified as exhibiting either differential splicing (in FGFR3, PDGFRA, MET, EPHA1, NF1, RASGRP2, GSK3, TSC2, ATM, RAF and RB1) or CNVs (at EPHA, PTEN and APQ between AA and CA PCa specimens. In addition, micro RNA profiling further revealed that 14 micro RNAs (e.g. miR-125b-2∗, miR-34a, miR-100, miR-99a, miR-200a) were differentially expressed in AA and CA cancer samples, potentially related to cell proliferation, anti-apoptosis, cell cycle regulation in AA cancers. Taken together, our data suggest that mRNA splicing, CNVs, deregulated microRNA expression and gene-network rewiring may be critical in PCa health disparities between the AA and CA populations. These findings should advance our knowledge on the molecular mechanisms underlying PCa disparities, and may facilitate the development of novel strategies for PCa detection, diagnosis, prognosis, and therapy in AA population. This work was supported by NCI grant 5U01-CA-116937. Citation Information: Cancer Epidemiol Biomarkers Prev 2010;19(10 Suppl):B55.

Expanding the Use of Fluorogenic Enzyme Reporter Substrates to Imaging Metabolic Flux Changes: the Activity Measurement of 5α-Steroid Reductase in Intact Mammalian Cells

The study of dynamic properties of metabolic and signaling networks is hindered by the lack of methods for imaging metabolic fluxes in individual intact cells. We describe a novel optical approach for measuring the changes of metabolic fluxes in cells, based on a two-substrate competition between a physiological substrate and a fluorogenic reporter substrate. We have constructed a model cell system for a two-step metabolic pathway involved in the metabolism of testosterone. Potent androgen testosterone is converted by steroid 5α-reductase to DHT (5α-dihydrotestosterone), which is subsequently metabolized to 3α-diol (3α,17β-androstanediol) by the reductase AKR1C2 (aldo-ketoreductase 1C2), for which we have previously developed the fluorogenic reporter substrate Coumberone. Despite the medicinal importance of 5α-reductase, there are presently no probes or methods for the continuous activity readout of this enzyme in cells. We show that the activity of 5α-R1 (5α-reductase type 1) can be measured in COS-1 cells via the changes of DHT flux. Our system enables a measurement of 5α-reductase activity in cells, via either fluorimetry or fluorescence microscopy, with a wide dynamic range of activities, and provides a continuous optical assay for evaluation of small molecule inhibitors for this important enzyme. Furthermore, this paper demonstrates a novel optical approach to measuring metabolic flux changes in living cells and expands the utility of fluorogenic enzyme reporter substrates: optical reporters can measure not only the activity of the target enzyme but also the activity of other enzymes upstream in the pathway, for which there are no probes available.

Polymorphisms in Genes Involved in Testosterone Metabolism in Slovak Autistic Boys

Autism spectrum disorders (ASDs) are neurodevelopment disorders which are characterized by impairments in the following core domains: social interaction, language development, verbal/nonverbal communication, and repetitive and restricted behaviors. The androgen theory of autism proposes that autism spectrum disorders develop in part due to elevated fetal testosterone levels, which correlate with a number of autistic traits. The present study evaluates androgen and estrogen levels in saliva as well as polymorphisms in genes for androgen receptor (AR), 5-alpha reductase (SRD5A2), and estrogen receptor alpha (ESR1) in the Slovak population of prepubertal (under 10 years) and pubertal (over 10 years) children with autism spectrum disorders. The examined prepubertal patients with autism, pubertal patients with autism, and prepubertal patients with Asperger syndrome had significantly increased levels of salivary testosterone (P 0.05, P 0.01, and P 0.05, respectively) in comparison with control subjects. We found a lower number of (CAG)n repeats in the AR gene in boys with Asperger syndrome (P 0.001). Autistic boys had an increased frequency of the T allele in the SRD5A2 gene in comparison with the control group. The frequencies of T and C alleles in ESR1 gene were comparable in all assessed groups. The modulating influence of studying genotypes on the effect of testosterone could provide insight into the pathogenesis of autism spectrum disorders.

Testosterone metabolism revisited: discovery of new metabolites

The metabolism of testosterone is revisited. Four previously unreported metabolites were detected in urine after hydrolysis with KOH using a liquid chromatography-tandem mass spectrometry method and precursor ion scan mode. The metabolites were characterized by a product ion scan obtained with accurate mass measurements. Androsta-4,6-dien-3,17-dione, androsta-1,4-dien-3,17-dione, 17-hydroxy-androsta-4,6-dien-3-one and 15-androsten-3,17-dione were proposed as feasible structures for these metabolites on the basis of the mass spectrometry data. The proposed structures were confirmed by analysis of synthetic reference compounds. Only 15-androsten-3,17-dione could not be confirmed, owing to the lack of a commercially available standard. That all four compounds are testosterone metabolites was confirmed by the qualitative analysis of several urine samples collected before and after administration of testosterone undecanoate. The metabolite androsta-1,4-dien-3,17-dione has a structure analogous to that of the exogenous anabolic steroid boldenone. Specific transitions for boldenone and its metabolite 17β-hydroxy-5β-androst-1-en-3-one were also monitored. Both compounds were also detected after KOH treatment, suggesting that this metabolic pathway is involved in the endogenous detection of boldenone previously reported by several authors.

Early exposure to low doses of atrazine affects behavior in juvenile and adult CD1 mice

Environmental exposure to endocrine disrupting chemicals is receiving increasing attention, with particular regard to distinct periods of development where neuroendocrine circuitries are critical for shaping the mammalian brain. Atrazine (ATZ), a widely used herbicide, has been reported to affect steroid hormones and interfere with pathways critical for sex-specific physiological and behavioral development. Aim of the present study was to evaluate effects of perinatal exposure to environmentally relevant subtoxic doses of ATZ, on neurobehavioral development in mice and investigate possible alterations in steroid hormone metabolism. Neurobehavioral development of female and male mice delivered from CD1 dams, and daily exposed from Gestational Day 14 until Postnatal Day 21 (PND 21) to 1 or 100 μg/kg bw ATZ, was investigated. Specifically, locomotor and exploratory activity, social interactions and cognitive performance were evaluated at PND 16, 31 and 60, respectively. Moreover, general toxicity clinical signs, testicular parameters, rate of testosterone metabolism and aromatase activity in F1 male liver were analyzed at adulthood. Changes in exploratory profile and in affiliative/investigative behavior were observed, revealing a feminization of behavioral profile in ATZ-exposed males. Alteration in learning performance at adulthood was also evident. A limited decreased sperm count and concentration, as well as some slight impairment in hepatic testosterone metabolism and in aromatase activity (slightly but not significantly decreased) were observed in both low and high dose exposed animals. In conclusion developmental exposure to non-toxic, environmentally relevant doses of ATZ can produce subtle functional alterations, detectable in juvenile rodents by a detailed behavioral analysis. Behavioral disturbances appeared mainly related with neurodevelopmental disorder affecting the social domain and the emotional/affective repertoire, although further research is needed to elucidate the mechanism through which the effects are induced.

Nanoparticle-rich diesel exhaust-disrupted testosterone biosynthesis and metabolism might be mediated by growth hormone

Nanoparticle-rich diesel exhaust-disrupted testosterone biosynthesis and metabolism might be mediated by growth hormone

Abstract 2227: Genomic study on prostate cancer disparities reveals the differential transcriptomes, microRNA profiles, alternative splicing patterns and copy number variations between Caucasian and African American populations

Abstract Prostate cancer (PCa) is a disease conferred by gene mutations, numerous alternations in gene expression and aberrant changes in genome composition/architecture. An area of research that continues to garner attention is PCa health disparities, wherein the African American (AA) population exhibits higher incidence and mortality rates compared to Caucasian Americans (CA). To identify the genetic predispositions and oncogenetic networks associated with the observed PCa disparities, we applied integrated genomic technologies to investigate RNA and microRNA expressions, alternative splicing events, and DNA copy number variations (CNVs) in AA and CA populations. RNA and DNA purified from PCa (Gleason score > 6) and paired adjacent normal prostate needle biopsies from AAs and CAs were processed and hybridized onto Affymetrix human Exon 1.0 ST and SNP 6.0 arrays. A 4-way statistical design (t-test with 10% false discovery rate) was employed to identify differentially expressed mRNAs in the following comparisons: AA normal vs. CA normal, AA cancer vs. CA cancer, AA cancer vs. AA normal, and CA cancer vs. CA normal. Pathway analyses of the differentially expressed genes (275 to 987 genes in the 4-way comparison) revealed network-level rewiring of gene interactions (e.g. gene networks or circuits) accounting for the PCa disparities between AA and CA. For example, the mis-regulated testosterone metabolism network in normal AA compared with normal CA tissues may represent a predisposition factor in the AA population; whereas the activated inflammatory response (NF-κB network), up-regulated oncogenic signaling pathways (ERK, JNK and p38) and more aggressive cancer invasion and metastasis (highly expressed RHOA and STAT1) in AA carcinomas may associated with the higher recurrence and death rates in AA patients. In addition, our expression and CNV atlas have identified hundreds of genes exhibiting differential splicing patterns or copy number aberrations (deletions or amplifications) between the AAs and CAs, representing candidate genes mediating PCa disparities. Notably, at least 13 genes residing within the 5 oncogenic signaling pathways have been identified as exhibiting either differential splicing (in FGFR3, PDGFRA, MET, EPHA3, NF1, RASGRP2, GSK3, TSC2, ATM, RAF, and RB1) or CNVs (at EPHA, PTEN and APC) between AA and CA PCa specimens. Taken together, our data suggest that gene-network rewiring, mRNA splicing and CNV may play important roles in the PCa health disparities between AA and CA populations. Further identification of these critical genetic elements related to PCa disparities may facilitate the development of biomarkers for screening, early detection, and prediction of clinical outcome. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2227.

CYP2C9-CYP3A4 Protein-Protein Interactions: Role of the Hydrophobic N Terminus

Cytochromes P450 (P450s) interact with redox transfer proteins, including P450 reductase (CPR) and cytochrome b(5) (b5), all being membrane-bound. In multiple in vitro systems, P450-P450 interactions also have been observed, resulting in alterations in enzymatic activity. The current work investigated the effects and mechanisms of interaction between CYP2C9 and CYP3A4 in a reconstituted system. CYP2C9-mediated metabolism of S-naproxen and S-flurbiprofen was inhibited up to 80% by coincubation with CYP3A4, although K(m) values were unchanged. Increasing CYP3A4 concentrations increased the degree of inhibition, whereas increasing CPR concentrations resulted in less inhibition. Addition of b5 only marginally affected the magnitude of inhibition. In contrast, CYP2C9 did not alter the CYP3A4-mediated metabolism of testosterone. The potential role of the hydrophobic N terminus on these interactions was assessed by incubating truncated CYP2C9 with full-length CYP3A4, and vice versa. In both cases, the inhibition was fully abolished, indicating an important role for hydrophobic forces in CYP2C9-CYP3A4 interactions. Finally, a CYP2C9/CYP3A4 heteromer complex was isolated by coimmunoprecipitation techniques, confirming the physical interaction of the proteins. These results show that the N-terminal membrane binding domains of CYP2C9 and CYP3A4 are involved in heteromer complex formation and that at least one consequence is a reduction in CYP2C9 activity.

7α-Hydroxytestosterone Affects 11β-Hydroxysteroid Dehydrogenase 1 Direction in Rat Leydig Cells

The cytochrome P450 2A1 (CYP2A1) is a P450 enzyme that catalyzes the metabolism of testosterone. CYP2A1 has been reported to be present in rat testis. However, its developmental changes and function have not been well characterized. The purpose of this study was to measure the abundance of CYP2A1 (Cyp2a1) mRNA in the developing rat testis and Leydig cells and examine the effects of its product, 7 alpha-hydroxytestosterone (7HT), on an important enzyme, 11 beta-hydroxysteroid dehydrogenase type 1 (11 beta-HSD1) that interconverts active corticosterone and inactive 11-dehydrocorticosterone. As detected by real-time PCR, Cyp2a1 was found to be present exclusively in the Leydig cell. CYP2A1 activity in adult Leydig cells was 5-fold higher than those in progenitor or immature Leydig cells. 7HT competitively suppressed 11 beta-HSD1 oxidase and reductase activities in rat testis microsome with inhibitory constant of 1.2 and 2.9 mum, respectively. In intact Leydig cells, 7HT did not inhibit 11 beta-HSD1 reductase activity, but it stimulated its reductase activity. Thus, at 100 nm and higher concentrations, 7HT significantly switched 11 beta-HSD1 oxidoreductase activities toward reductase. The present data shows that 7HT, the product formed by CYP2A1 from testosterone, regulates the direction of 11 beta-HSD1 activity in rat Leydig cells.

Replacement with GABAergic steroid precursors restores the acute ethanol withdrawal profile in adrenalectomy/gonadectomy mice

The neurosteroid allopregnanolone (ALLO) is a progesterone metabolite that is one of a family of neuroactive steroids (NAS) that are potent positive allosteric modulators of γ-aminobutyric acid A (GABA A) receptors. These GABAergic NAS are produced peripherally (in the adrenals and gonads) and centrally in the brain. Peripherally produced NAS modulate some effects of ethanol intoxication (e.g., anxiolytic, antidepressant, and anticonvulsant effects) in rodents. We have found that NAS also may be involved in the rebound neural hyperexcitability following a high ethanol dose. Removal of the adrenals and gonads (ADX/GDX) increased withdrawal severity following 4 g/kg ethanol, as measured by handling-induced convulsions (HICs) in male and female DBA/2J mice. NAS are produced through the metabolism of progesterone (PROG), deoxycorticosterone (DOC), or testosterone, which can be blocked with the administration of finasteride (FIN), a 5α-reductase enzyme inhibitor. The current investigation was undertaken to clarify the step(s) in the biosynthetic NAS pathway that were sufficient to restore the acute ethanol withdrawal profile in ADX/GDX mice to that seen in intact animals. Male and female DBA/2J mice underwent ADX/GDX or SHAM surgery. After recovery, separate groups of animals were administered PROG, DOC, PROG+FIN, DOC+FIN, FIN, ALLO, ganaxalone (a synthetic ALLO derivative), corticosterone, or vehicle. Animals were then administered a 4 g/kg ethanol dose and allowed to undergo withdrawal. HICs were measured for 12 h and again at 24 h. The results indicate that replacement with PROG and DOC restored the withdrawal profile in ADX/GDX animals to SHAM levels, and that this effect was blocked with co-administration of FIN. Administration of FIN alone increased the withdrawal profile in both SHAM and ADX/GDX males. These findings indicate that the increase in acute withdrawal severity after ADX/GDX may be due to the loss of GABAergic NAS, providing insight into the contribution of endogenous GABAergic NAS to ethanol withdrawal severity.

Association of polymorphisms in CYP19A1 and CYP3A4 genes with lower urinary tract symptoms, prostate volume, uroflow and PSA in a population-based sample

PurposeThe known importance of testosterone for the development of benign prostatic hyperplasia (BPH) prompted us to test the hypothesis whether polymorphisms of two genes (CYP19A1 and CYP3A4) involved in testosterone metabolism are associated with clinical BPH-parameters.MethodsA random sample of the population-based Herne lower urinary tract symptoms cohort was analysed. All these men underwent a detailed urological work-up. Two polymorphisms in the CYP19A1 gene [rs700518 in exon 4 (A57G); rs10046 at the 3′UTR(C268T)] and one in the 3′UTR of CYP3A4 [rs2740574 (A392G)] were determined by TaqMan assay from genomic DNA of peripheral blood. These polymorphisms were correlated to clinical and laboratory BPH-parameters.ResultsA total of 392 men (65.4 ± 7.0 years; 52–79 years) were analysed. Mean International Prostate Symptom Score (IPSS; 7.5), Q max (15.4 ml/s), prostate volume (31 ml) and prostate specific antigen (PSA) (1.8 ng/ml) indicated a typical elderly population. Both polymorphisms in the CYP19A1 gene were not correlated to age, IPSS, Q max, prostate volume and post-void residual volume. Serum PSA was higher in men carrying the heterozygous rs10046 genotype (2.0 ± 0.1 ng/ml) than in those with the CC-genotype (1.7 ± 0.2 ng/ml, P = 0.012). Men carrying one a mutated allele of the CYP3A4 gene had smaller prostates (27.0 ± 2.0 vs. 32 ± 0.8 ml, P = 0.02) and lower PSA levels (1.6 ± 0.3 vs. 1.9 ± 0.1 ng/ml).ConclusionsThe inconsistent associations observed herein and for other gene polymorphisms warrant further studies. In general, the data regarding the association of gene polymorphism to BPH-parameters suggest that this disease is caused by multiple rather than a single genetic variant. A rigorous patient selection based on anatomo-pathological and hormonal profile may possible reduce the number of confounders for future studies thus enabling a more detailed assessment of the association between genetic factors and BPH-parameters.

Toxicogenomics Applied to Cultures of Human Hepatocytes Enabled an Identification of Novel Petasites hybridus Extracts for the Treatment of Migraine with Improved Hepatobiliary Safety

Butterbur extracts (Petasites hybridus) are recommended for the prevention of migraine, but pharmacovigilance reports may be suggestive of rare hepatobiliary toxicity. To evaluate its hepatotoxic potential, a series of in vivo and in vitro studies were carried out. Essentially, there were no signs of hepatocellular toxicity at estimated therapeutic C(max) levels of 60 ng/ml. Nonetheless, in a 28-day toxicity study at approximately 200-fold of therapeutic doses, induced liver transaminases and bilirubin elevations were observed. In a subsequent 6-month chronic toxicity study, the initial hepatobiliary effects were reproduced, but at the end of the study, liver function recovered and returned to normal as evidenced by clinical chemistry measurements. To identify possible mechanisms of hepatotoxicity, we investigated liver function in vitro at > 170-fold of therapeutic C(max) levels, including cytotoxicity (lactate dehydrogenase, MTT, and ATP), transaminase activities (alanine aminotransferase and aspartate aminotransferase), albumin synthesis, urea and testosterone metabolism to assay for cytochrome P450 monooxygenase activity. Only with extracts rich in petasin (37% petasin) and at high and well above therapeutic doses, liver toxicity was observed. A toxicogenomic approach applied to hepatocyte cultures enabled hypothesis generation and was highly suggestive for extracts high in petasin content to impair bile acid transport and lipid and protein metabolism. Importantly, neither chronic rat in vivo nor rat in vitro studies predicted reliably hepatotoxicity, therefore reemphasizing the utility of human-based in vitro investigations for the development of safe medicinal products. Finally, toxicogenomics enabled the characterization of a novel butterbur extract with no signals for hepatotoxicity.

Inhibition of male rat hepatic testosterone metabolism by modulation of androgenic signalling and castration

Inhibition of male rat hepatic testosterone metabolism by modulation of androgenic signalling and castration

Effect of lindane on CYP‐mediated steroid hormone metabolism in male mice following in utero exposure

A wide number of pesticides, including highly persistent organochlorinated compounds, such as lindane (LIN), may induce reproductive and developmental alterations by directly binding to the estrogen/androgen receptors or altering steroid hormone metabolism. In the present work, we have investigated whether LIN in utero exposure of CD1 mice affects the reproductive system in male offspring by causing an impairment of the CYP-dependent steroid hormone metabolism. Dam exposure to 25 mg kg(-1) b.w. LIN occurred during critical developmental periods, from gestational days 9 to 16. Effects on hepatic CYP-mediated testosterone (TST) hydroxylase, aromatase activities and testicular parameters were tested at postnatal days (PND 50, 65-69, 100) that are critical for sexual maturation in CD1 mice. In the adult F1 mice significant changes of male reproductive endpoints (testis weight, spermatid number) as well as dramatic effects on CYP-mediated TST metabolism were observed on PND 65-69, in the absence of any of systemic toxicity. The levels of TST 6beta- and 2alpha-hydroxylation and dehydrogenation showed the highest level of reduction, suggesting CYP 3A and 2C families as the major target of LIN induced effects. All changes were almost recovered on PND 100. No effects on aromatase activity were evidenced. Overall, these findings provide useful information for a better characterization of the LIN mode of action. They suggest that LIN-induced toxicity in males is linked to an impairment of steroid hormone homeostasis, due to CYP-mediated TST catabolism modulation and differs from LIN receptor-mediated mechanism previously reported in females.

Evaluation of the rodent Hershberger bioassay on intact juvenile males—Testing of coded chemicals and supplementary biochemical investigations

Under the auspices of the Organization for Economic Cooperation and Development (OECD) the Hershberger assay on juvenile intact male rats is being validated as a screen for compounds with anti-androgenic potential. We participated in the testing of coded chemicals. Compounds included the positive control flutamide (FLUT, 3 mg/kg), linuron (LIN, 10, 100 mg/kg), p,p′-DDE (16, 160 mg/kg), and two negative substances, 4-nonylphenol (NP, 160 mg/kg) and 2,4-dinitrophenol (DNP, 10 mg/kg). Compounds were administered for 10 consecutive days by gavage to testosterone propionate (TP, 1 mg/kg s.c.)-supplemented rats. Uncoding revealed these results: compared to vehicle controls, treatment with TP resulted in increased androgen-sensitive tissue (AST) weights of ventral prostate (VP), seminal vesicles (SV), levator ani and bulbocavernosus muscles (LABC), Cowper's glands, and epididymides, and in decreased testes weight. When assessing anti-androgenic potential in TP-supplemented rats, FLUT decreased all AST weights, and increased testes weight. p,p′-DDE at the high dose, decreased final body weight and all AST weights, whereas the low dose only affected SV weight. LIN slightly decreased final body weight and decreased absolute SV and LABC and relative SV weights only at the high dose. NP decreased final body weight and only absolute SV weights, DNP was ineffective. Investigations not requested by OECD included measurement of liver enzymes and revealed strong induction of testosterone-metabolizing and phase II conjugating enzymes by p,p′-DDE. Our findings suggest that in principle the juvenile intact male rat can be used in the Hershberger assay to screen for anti-androgenic potential thereby contributing to a refinement of the assay in terms of animal welfare. However, in our hands this animal model was somewhat less sensitive than the peripubertal castrated rat. Final conclusions, however, can only be drawn on the basis of all available validation data. Results obtained with the negative reference compound NP suggest that a treatment-related decrement in body weights may affect AST weights and represent a confounding factor when screening for anti-androgenic properties. Finally, p,p′-DDE may affect AST weights by several mechanisms including enhanced testosterone metabolism.

Metabolism of testosterone in vitro by hypothalamus and other areas of rat brain.

Zusammenfassung Testosteronstoffwechsel in vitro im Hypothalamus und anderen Bezirken des Rattenhirns Verschiedene Hirnareale ausgewachsener mannlicher Ratten wurden in vitro mit 14C markiertem Testosteron inkubiert. Der Hauptmetabolit war 17β-OH-5α-androstan-3-on. Die Gewebe bildeten diesen Metaboliten in der absteigenden Reihenfolge von Mittelhirndach, Hypothalamus, Kleinhirn und dorsaler Hippocampus, Groshirnrinde und Mandelkerne. Es wurden minimale Konzentrationen anderer Metaboliten gefunden.

Thyroid releasing hormone inhibits testosterone metabolism by rat prostate in vitro.

A method involving thin layer chromatography (TLC) was developed to assess 5 alpha-DHT-reductase activity in rat ventral prostate, in terms of the amount of testosterone metabolised to its main 5 alpha-reduced compound, dihydrotestosterone (DHT). Using this method an in vitro study was carried out to determine 5 alpha-DHT reductase activity in the rat ventral prostate as modulated by thyroid releasing hormone (TRH). The study revealed a decrease in the enzyme activity with increasing doses of TRH, as shown by a decrease in the extent of reduction of testosterone to DHT. The ratios of the counts for DHT and testosterone however did not show any dose related effect.

Suppression of myocardial ischemia–reperfusion injury by inhibitors of cytochrome P450 in rats

The effects of inhibitors of cytochrome P450 on myocardial regional ischemia–reperfusion injury were examined in rats . Ischemia–reperfusion injury was evoked by ligation of the left anterior descending coronary artery for 1 h, followed by reperfusion for 24 h. Injuries were evident in causing infarction, decreases in left ventricular systolic pressure and left ventricle (d P/d t max) / P and an increase in left ventricular end-diastolic pressure. Increases in lipid peroxidation and reactive oxygen species levels in the ischemic region were observed. Intravenous injection of the potent cytochrome P450 inhibitor sulfaphenazole at 10 and 30 mg/kg at the time of reperfusion reduced infarct size by 41.7 and 73.2%, respectively; and improved cardiac function accompanied by the decrease in content of lipid peroxide and reactive oxygen species in the area at risk. Cardiac testosterone metabolism was inhibited by sulfaphenazole administration, indicating its inhibitory effects on cardiac cytochrome P450 activity. Another cytochrome P450 inhibitor, cimetidine, given intravenously, had similar effects to sulfaphenazole on reperfusion injury. Taken together, these results indicate that reactive oxygen species derived from cytochrome P450 play an important part in myocardial regional ischemia–reperfusion injury in vivo, and strongly support the hypothesis that cytochrome P450 inhibitors are promising therapeutic agents for cardiac ischemia–reperfusion injury.

Role of mouse CYP2A5 in nicotine clearance and testosterone homeostasis

CYP2A5, a mouse P450 enzyme, is expressed abundantly in the olfactory mucosa (OM), and in other tissues, including the liver and the lateral nasal gland (LNG). To determine the biological and pharmacological functions of CYP2A5 in vivo, we have generated a Cyp2a5‐null mouse. Homozygous Cyp2a5‐null mice are viable and fertile. OM microsomes from Cyp2a5‐null mice had significantly lower activities in the metabolism of testosterone (T) than did OM microsomes from wild‐type (WT) mice. In contrast, hepatic microsomes from Cyp2a5‐null mice showed only small decreases in the overall rates of formation of T metabolites, compared to hepatic microsomes from WT mice. Serum T levels were not altered by the loss of CYP2A5 function in male mice, a result confirming that CYP2A5 does not play a major role in systemic T clearance in vivo. Nevertheless, loss of CYP2A5 was accompanied by significant increases in tissue levels of T in the LNG, a gland important for nasal mucus secretion and production of odorant binding proteins. The Cyp2a5‐null mice also showed decreased clearance of nicotine, with significant increases in the half life and AUC values for serum nicotine, compared with WT mice. These findings indicate that CYP2A5 is the major nicotine oxidase in the mouse liver, and that CYP2A5 plays an important role in the regulation of T levels in the LNG of male mice. (Supported in part by NIH grant ES‐07462)