Dihydrotestosterone Glucuronide Research Articles

Potent inhibition of tributyltin (TBT) and triphenyltin (TPT) against multiple UDP-glucuronosyltransferases (UGT): A new potential mechanism underlying endocrine disrupting actions

Organotin compounds (OTs) act as potent endocrine disruptors that are often found in polluted food and water. UDP-glucuronosyltransferases (UGTs) are responsible for termination of multiple endogenous hormones. This study was conducted to investigate the inhibitory effects of two tri-submitted OTs tributyltin (TBT) and triphenyltin (TPT), against activities of UGTs. It is revealed that TBT and TPT act as two potent inhibitors for multiple UGTs. UGT1A8 and -2B15 were coinhibited by the two OTs. UGT1A1 and -1A10 were inhibited by TPT, whereas UGT 2B4 and -2B7 were inhibited by TBT. Kinetic analyses further indicated that TBT and TPT are two competitive nanomolar inhibitors of UGT2B15, with Ki values of 0.45 and 0.46 μM, respectively. Ki values for the other UGTs are determined to be a few micromolars. In addition, the two OTs displayed effective inhibition against UGT2B15 in catalyzing dihydrotestosterone glucuronidation, with IC50 values both in nano-molar range. TPT can additionally inhibit activities of UGT1A1 and -1A10 in estradiol-3-O-glucuronidation, with IC50 values of a few micro-molars. These results indicated that the two OTs can extensively interfere with glucuronidation of endogenous hormones, which may act as a new potential mechanism resulting in endocrine disrupting actions.

Targeted LC–MS/MS analysis of steroid glucuronides in human urine

Conjugation with glucuronic acid is one of the major metabolic reactions in human steroid hormone catabolism. Recently, increasing interest has been raised concerning the biological roles of steroid glucuronides. We have therefore developed and validated a liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for the simultaneous quantification of 15 urinary steroid hormone glucuronides in human urine: androsterone glucuronide (An-G), etiocholanolone glucuronide (Etio-G), epiandrosterone glucuronide (epiAn-G), dihydrotestosterone glucuronide (DHT-G), dehydroepiandrosterone glucuronide (DHEA-G), testosterone glucuronide (T-G), epitestosterone glucuronide (epiT-G), estrone glucuronide (E1−3 G), 17β-estradiol 17-glucuronide (E2−17 G), 17β-estradiol 3-glucuronide (E2−3 G), estriol 16-glucuronide (E3−16 G), pregnenolone glucuronide (Preg-G), tetrahydro-11-deoxycorticosterone 3-glucuronide (THDOC-3 G), cortisol 21-glucuronide (F–G) and pregnanediol glucuronide (PD-G). Sample workup included protein precipitation and solid phase extraction. Internal standards were used to correct for the loss of analytes during sample preparation and analysis. The method showed good linearity (R2≥0.99) and recovery ranged from 89.6 % to 113.8 %. Limit of quantification ranged from 1.9 nmol/L for F–G to 21.4 nmol/L for An-G. Intra-day and inter-day accuracy and precision were below 15 % for all quality controls. The method was successfully applied to 67 urine samples from children and adolescents in whom total concentrations of free and conjugated steroids had been previously determined by GC–MS after enzymatic hydrolysis. Free and sulfated steroids were also measured by LC–MS/MS. In general, the sums of the respective glucuronidated, sulfated and free forms of an analyte corresponded well with its total amount determined after enzymatic hydrolysis by GC–MS. Regarding the most prominent steroid metabolites, the total mean levels of androsterone and etiocholanolone showed an increase up to 5820.0 nmol/L and 4017.8 nmol/L in the group of 15−20 year-old children, respectively. Glucuronide conjugates (4374.3 nmol/L and 3588.5 nmol/L, respectively) dominated. DHEA was excreted mostly as sulfate (0−1 month of age: 184.5 nmol/L; 15−20 years of age: 1618.4 nmol/L) in all age groups. Cortisol was present predominantly as sulfate (mean: 173.8 nmol/L) in newborns. Levels of sulfated cortisol decreased with age, its glucuronidated form increased. The levels of free cortisol were relatively constant throughout childhood. Sex hormones were preferably excreted as glucuronides. In general, steroid hormone metabolites were conjugated to various extents with glucuronic acid or sulfuric acid and their ratio changed over lifetime.

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.

Characterization of Novel Mechanisms of Efflux Transport of the Glucuronides of Testosterone and its Metabolites: a Quantitative Proteomics‐based IVIVE Study and Relevance to Enterohepatic Recirculation of Androgens

Testosterone (T) is primarily eliminated via glucuronidation upon oral administration. As a result, testosterone glucuronide (TG), androsterone glucuronide (AG), etiocholanolone glucuronide (EtioG) and dihydrotestosterone glucuronide (DHTG) are produced as the key circulating metabolites, which are excreted in urine and bile. The goal of this study was to characterize the in vivo fractional contribution of individual hepatic transporters (ft) in the elimination of the glucuronides of T and its metabolites. We first performed vesicular transport screening assay with recombinant human MRP2, MRP3, MRP4, MDR1, and BCRP to identify the transporters that are involved in the efflux of TG, AG, EtioG and DHTG. The transport kinetic analyses were then carried out for the transporters that exhibited activity in the initial screening. The inside‐out vesicle ratio was determined by 5′‐nucleotidase activity assay. We next quantified the protein abundances of these efflux transporters in vesicles, human liver, and intestine by quantitative LC‐MS/MS proteomics using a validated surrogate peptide‐based approach. The ft for each transporter was estimated by proteomics‐based physiological scaling factors, i.e., transporter abundance in whole tissue versus vesicles, and by correction for the inside‐out vesicles. The transport screening assays revealed that TG, AG, EtioG, and DHTG were primarily effluxed by MRP2 and MRP3 with differential contribution of individual transporters. EtioG and DHTG were transported by MDR1 and BCRP, respectively, but at much lower rates than by MRPs. MRP2 and MRP3 exhibited higher intrinsic clearance for TG, followed by DHTG, AG, and EtioG (Figure 1). The Km value for EtioG transport by MRP3 was about 3 to 20‐fold lower than for the other glucuronides. After integrating the transporter abundance data from human tissues, the ft of MRP2 and MRP3 in efflux of TG, EtioG, and DHTG in liver was calculated to be around 0.6 and 0.4, respectively. For AG, MRP3 played a major role (68%) to its efflux in liver [corroborates with our in vivo clinical data (Basit et al., CTS, 11, 2018)]. In conclusion, our results for the first‐time demonstrated that MRP2 and MRP3 are the major transporters that are involved in the efflux of T glucuronide conjugates. Important role of MRP2 in transport of TG, EtioG, and DHTG indicates the potential importance of enterohepatic recirculation in regulating the circulating T, Etio, and DHT after deconjugation in the gut. With this respect, our unpublished data suggest efficient deconjugation of these glucuronides by human relevant β‐glucuronidases. Characterization of the efflux mechanisms of glucuronides of T and DHT is important for predicting the androgen disposition and interindividual variability, including drug‐androgen interaction in humans. The mechanistic transport data can be extrapolated (using quantitative proteomics) to other androgen relevant tissues, such as kidney, prostate, testis and placenta.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Epigenetic regulation of steroid inactivating UDP-glucuronosyltransferases by microRNAs in prostate cancer

Androgens play a central role in prostate cancer progression. Systemic and local androgen bioavailability is controlled by UDP-glucuronosyltransferases conjugating enzymes (UGT), namely UGT2B15, UGT2B17 and UGT2B28. Reporter vector assays in HEK293 cells initially validated in silico-predicted regulatory potential of candidate miRNAs to target UGT transcripts, including miR-376c, miR-409 and miR-494 for UGT2B17, miR-331-5p and miR-376c for UGT2B15 while none were efficient for UGT2B28. miR-376c was shown as the most effective to downregulate UGT2B15 and UGT2B17 through interactions with a site conserved in both UGTs. Ectopic miR-376c expression in prostate cancer cells significantly reduced UGT2B15 and UGT2B17 expression (>32%; P<0.005) with a consequent decrease in dihydrotestosterone glucuronidation (−37%; P<0.001). Consistent with reduced androgen inactivation, ectopic expression of miR-376c changed expression of androgen responsive genes and enhanced cell proliferation with no effect on androgen receptor levels. Sustaining a role of miR-376c in the regulation of androgen-inactivating UGTs, its expression was significantly downregulated in prostatic tumors and further reduced in metastases (P<0.0001), whereas the opposite was observed for UGT2B15/17 (P=0.031). In high-grade tumors (Gleason ≥8), UGT2B15/17 and miR-376c were inversely correlated (r=−0.557; P=0.048) with also a significant relationship in metastases (r=−0.747; P=0.003). In line with a modification in androgen bioavailability, PSA mRNA levels were also negatively correlated to those of UGT2B15/17 (r=−0.573; P=0.01) but positively linked to levels of miR-376c (r=0.577; P=0.039). This study reveals that the androgen-inactivating UGT2B15 and UGT2B17 genes are direct targets of miR-376c and thus may influence steroid metabolism during prostate cancer progression.

Functional Analysis of UGT1A4 P24T and UGT1A4 L48V Variant Enzymes

Aim: To investigate the effects of two nonsynonymous SNPs, UGT1A4*2 (rs#: 6755571, 70C&gt;A, P24T) and UGT1A4*3 (rs#: 2011425, 142T&gt;G, L48V), on the function of UGT1A4 against dihydrotestosterone (DHT), transandrosterone (t-AND), lamotrigine (LTG) and tamoxifen (TAM). Materials &amp; methods: Detailed kinetic experiments were conducted with recombinant UGT1A4wild-type, UGT1A4P24T and UGT1A4L48V, which were overexpressed in HEK293 cell lines. The kinetic profiles and kinetic parameters (Km, Vmax and CLint) obtained with either UGT1A4P24T or UGT1A4L48V were compared with those obtained with the wild-type enzyme. The interaction of TAM on UG1A4-catalyzed DHT glucuronidation was also investigated with the three UGT1A4 polymorphic enzymes. Results: UGT1A4L48V had higher enzyme efficiency (CLint) compared with wild-type UGT1A4 on DHT glucuronidation; UGT1A4P24T and UGT1A4L48V had lower CLint than wild-type UGT1A4 for t-AND and LTG glucuronidation. The TAM CLint with UGT1A4P24T and UGT1A4L48V glucuronidation and the UGT1A4P24T-catalyzed DHT glucuronidation were, on the other hand, similar to those of the wild-type enzyme. With all three enzymes, TAM activated UGT1A4-catalyzed DHT glucuronidation in a concentration-dependent fashion. Conclusion: Decreased CLint of UGT1A4P24T and UGT1A4L48V on LTG glucuronidation may lead to interindividual variations in LTG metabolism in vivo. However, it is less likely that these polymorphisms would have impact on DHT and t-AND metabolism in vivo because these compounds are glucuronidated by multiple enzymes. Original submitted 31 May 2011; Revision submitted 19 July 2011

Glucuronidation of Dihydrotestosterone and trans-Androsterone by Recombinant UDP-Glucuronosyltransferase (UGT) 1A4: Evidence for Multiple UGT1A4 Aglycone Binding Sites

UDP-glucuronosyltransferase (UGT) 1A4-catalyzed glucuronidation is an important drug elimination pathway. Although atypical kinetic profiles (nonhyperbolic, non-Michaelis-Menten) of UGT1A4-catalyzed glucuronidation have been reported occasionally, systematic kinetic studies to explore the existence of multiple aglycone binding sites in UGT1A4 have not been conducted. To this end, two positional isomers, dihydrotestosterone (DHT) and trans-androsterone (t-AND), were used as probe substrates, and their glucuronidation kinetics with HEK293-expressed UGT1A4 were evaluated both alone and in the presence of a UGT1A4 substrate [tamoxifen (TAM) or lamotrigine (LTG)]. Coincubation with TAM, a high-affinity UGT1A4 substrate, resulted in a concentration-dependent activation/inhibition effect on DHT and t-AND glucuronidation, whereas LTG, a low-affinity UGT1A4 substrate, noncompetitively inhibited both processes. The glucuronidation kinetics of TAM were then evaluated both alone and in the presence of different concentrations of DHT or t-AND. TAM displayed substrate inhibition kinetics, suggesting that TAM may have two binding sites in UGT1A4. However, the substrate inhibition kinetic profile of TAM became more hyperbolic as the DHT or t-AND concentration was increased. Various two-site kinetic models adequately explained the interactions between TAM and DHT or TAM and t-AND. In addition, the effect of TAM on LTG glucuronidation was evaluated. In contrast to the mixed effect of TAM on DHT and t-AND glucuronidation, TAM inhibited LTG glucuronidation. Our results suggest that multiple aglycone binding sites exist within UGT1A4, which may result in atypical kinetics (both homotropic and heterotropic) in a substrate-dependent fashion.

Inactivation of androgens by UDP-glucuronosyltransferases in the human prostate

In the human prostate, dihydrotestosterone (DHT) -- the natural androgen having the highest affinity for the androgen receptor -- is not released directly into the systemic circulation from peripheral target tissues but it is rather converted in situ into two metabolites which have a low affinity for the androgen receptor: androsterone (ADT) and androstane-3alpha,17beta-diol (3alpha-DIOL). Several clinical observations indicate that these two androgen metabolites are further inactivated in the prostate by glucuronidation. In the human, the family of UDP-glucuronosyltransferase (UGT) enzymes comprises 18 members in three subfamilies: UGT1A, UGT2A and UGT2B. Identification of the substrates for each member has revealed that three UGT2B enzymes are mainly responsible for DHT, ADT and 3alpha-DIOL glucuronidation: UGT2B7, UGT2B15 and UGT2B17. Tissue distribution and cellular localization of UGT2B transcripts and proteins clearly indicate that only UGT2B15 and UGT2B17 are expressed in the prostate. Using the human prostate carcinoma LNCaP cell line, it was shown that UGT2B expression and activity are negatively regulated by several factors, including androgens. On the other hand, inhibition of UGT2B115/17 expression by small interfering RNA (siRNA) resulted in an induced response to DHT of androgen-receptor target genes such as PSA, KLK4, NKX3.1, TMPRSS2, SLC16A6 and VEGF. It is suggested that the conjugating activity of UGT enzymes in androgen target tissues is a mechanism for modulating the action of steroids and/or protecting the tissues from deleterious high concentrations of androgens.

Isolated Absence of Adrenarche in A Postpubertal Female

Background The development of pubic and axillary hair (adrenarche) is part of the process of normal puberty. We report a case of isolated absence of adrenarche in a post-pubertal female. A review of the literature reveals no previously reported case of isolated absence of adrenarche in a postpubertal female. An investigation was understaken to evaluate the etiology of the lack of sexual hair growth in this patient. Case A 14 year-old postpubertal female presented complaining of heavy menstrual bleeding approximately four months after her first menstrual period. The patient had been previously diagnosed with a developmental delay and speech impediment by her referring physician. Further workup revealed a normal menstrual pattern, yet the physical examination was remarkable for the absence of axillary and pubic hair, as well as short stature. A complete evaluation was performed which included chromosome analysis, serum dehydroepiandrosterone sulfate (DHEA-S), androstenedione, testosterone, dihydrotestosterone (DHT), and 3-alpha androstanediol glucuronide. The serum androstenedione, testosterone, DHT, and 3-alpha androstanediol glucuronide levels were within normal limits. The DHEA-S levels were low yet adrenal function as analyzed by the Adrenocorticotropic hormone (ACTH) stimulation test was normal. Radiologic studies included pelvic ultrasound and bone age assessment with a bone age of 16 years. Skin biopsy of the mons pubis documents the presence of hair follicles. No previous or similar history has been noted in the patient's family history. Also of note, the patient is the product of a consanguineous relationship. Conclusion After review of our investigational findings, it is felt that this unique case likely represents an androgen receptor or post-receptor defect in the hair follicle.

Effect of interleukins on UGT2B15 and UGT2B17 steroid uridine diphosphate-glucuronosyltransferase expression and activity in the LNCaP cell line.

Cytokines are known to modulate the level of both phase 1 and phase 2 drug-metabolizing enzymes in hepatocytes. Although the effects of cytokines on cytochrome P450 (CYP450) enzymes are well understood, there is limited knowledge on how cytokines may affect steroid UDP-glucuronosyltransferase (UGT) phase 2 enzyme activity and expression in different cell types, including hepatocytes and steroid target cells. LNCaP cells, which is a human prostate cancer cell line, is a good model to study the effect of cytokines in steroid target cells because it is known to express steroidogenic enzymes, including UGT2B15 and UGT2B17, which are widely expressed steroid UGT enzymes known to conjugate androgens. In this study, we examined the possible interaction among interleukin-1alpha (IL-1alpha), IL-4, IL-6, and steroid UGT enzymes (UGT2B15 and UGT2B17). Treatment of LNCaP cells with IL-1alpha led to a dose-dependent inhibition of dihydrotestosterone (DHT) glucuronidation. IL-1alpha decreased both UGT activity and LNCaP cell proliferation in the absence and presence of DHT (0.5 nM); a maximal inhibition of 70% was observed. IL-6 inhibited LNCaP cell proliferation as well as the DHT-induced proliferation of these cells. However, neither IL-4 nor IL-6 significantly affected the formation of DHT glucuronide. Ribonuclease protection and Western blot analyses demonstrated a specific reduction of UGT2B17 transcript and protein levels in IL-1alpha-treated LNCaP cells. The level of UGT2B15 was not affected by cytokine treatments, indicating a differential regulation between these two UGT enzymes. Transfection experiments performed with the UGT2B17 gene promoter region indicates that the regulation occurs at the transcription level via putative cis-acting elements. This study indicates that cell proliferation and UGT expression in steroid-responsive cancer cells are differentially regulated depending on the cytokines present in the cell microenvironment.

Characterization and regulation of UDP-glucuronosyltransferases in steroid target tissues

Conjugation of compounds by glucuronidation is a pathway found in all vertebrates studied to date. Although, it is widely recognized that the liver is a major site of glucuronidation, it is now clear that extrahepatic tissues are also involved in the conjugation of compounds to which these tissues are exposed. High levels of androsterone glucuronide and androstane-3 α,17 β-diol glucuronide found in the human prostate, breast cyst fluid and ovary follicular fluid suggest that glucuronidation of 5 α-reduced C 19 steroids occurs in these tissues. Recently, we have reported the tissue distribution of UGT2B15, which can conjugate steroids in several human extrahepatic steroid target tissues including the skin, breast and prostate. We have also isolated a new UGT2B cDNA encoding UGT2B17, that conjugates ADT which is the major 5 α-reduced C 19 steroid glucuronide in the circulation of humans. UGT2B17 is also widely distributed in several human steroid target tissues. This gene was mapped to human chromosome 4q13 and has an exon/intron structure similar to that of rat UGT2B1 and UGT2B2. Both UGT2B15 and UGT2B17, which are able to catalyze the glucuronidation of DHT, are expressed in LNCaP cells. Interestingly, glucuronidation of steroids is markedly regulated by several factors including androgens and growth factors. Treatment of LNCaP cells with dihydrotestosterone (DHT) and epidermal growth factor (EGF) caused a decrease of DHT glucuronidation and UGT2B mRNA levels. RNase protection assays showed a specific decrease of UGT2B17 transcript in LNCaP cells treated with DHT and EGF however, the level of UGT2B15 mRNA was not affected. As well, Western blot analysis demonstrated a diminution of UGT2B17 protein level in response to DHT and EGF. These results demonstrate a differential regulation of different isoforms of steroid conjugating UGTs present in human prostate LNCaP cells. In addition, UGT2B17 was shown to be more labile than UGT2B15 indicating that regulation of UGT2B17 expression would lead to a more rapid change in the level of glucuronidated steroids. Expression of exogenous UGT2B17 in LNCaP cells by gene transfer led to a significant decrease in the androgen response. This result indicates the ability of UGT enzymes to regulate the androgen response by conjugating androgens which abolishes their interaction with their receptor and facilitates their clearance from the cell. The glucuronidation of steroids by UGT enzymes is an important mechanism by which the levels of steroids is regulated in steroid target tissues.

Effect of fibroblastic growth factors (FGF) on steroid UDP-glucuronosyltransferase expression and activity in the LNCaP cell line

It is now widely accepted that factors other than androgens are crucial in the normal and abnormal growth of the prostate. In addition to hormones, many polypeptide growth factors, including the fibroblast growth factor family (FGF), can act as potent mitogens on cell proliferation. The FGF family of growth factors are essential factors for both normal and abnormal proliferation of prostate cells. To study the effect of FGFs on steroid glucuronidation, we used the human prostate cancer LNCaP cell line which is known to be stimulated by FGF resulting in increased cell proliferation. LNCaP cells express steroid metabolizing enzymes including uridine diphosphoglucuronosyltransferases (UGTs). In addition, LNCaP cells treated with dihydrotestosterone (DHT) and epidermal growth factor (EGF) express differential levels of the human UGT2B15 and UGT2B17 transcripts. In the present study, we examined the possible interaction between FGF and steroid UGT enzymes. Results show a dose dependent inhibition of DHT glucuronide (DHT-G) formation following treatment (6 days) with acidic FGF (aFGF) and basic FGF (bFGF). When cells were treated with 10 ng/ml of FGFs, we observed 33 and 51% inhibition of glucuronidation activity using aFGF and bFGF respectively. Ribonuclease protection analyses revealed a 2 and 3 fold increase of UGT2B15 mRNA expression following treatment with aFGF (50 ng/ml) and bFGF (10 ng/ml) respectively. However, a slight decrease in UGT2B17 transcripts was observed, demonstrating a differential regulation. Since a reduction in the glucuronidation of DHT or its 5 α-reduced metabolites may contribute to an increase in intraprostatic androgen levels, down-regulation of UGTs by growth factors such as FGFs may increase the proliferation of androgen-dependent tumors.

Differential regulation of two uridine diphospho-glucuronosyltransferases, UGT2B15 and UGT2B17, in human prostate LNCaP cells.

Although androgens are important regulators in the prostate, other effectors such as growth factors may also act to maintain normal function of the gland. Human prostate and human prostate cancer LNCaP cells express steroid conjugating uridine diphospho-glucuronosyltransferase (UGT) enzymes, and it was shown that the level of UGT activities and transcripts is down-regulated by androgens, especially dihydrotestosterone (DHT). In the present study, we examined the interaction between androgen, epidermal growth factor (EGF), and steroid UGT enzymes. The formation of DHT glucuronide (DHT-G) was inhibited by 47% when LNCaP cells were treated for 6 days with 10 ng/ml of EGF. Northern blot analysis also demonstrated a decrease in the steady-state level of UGT2B transcripts. Treatment with both DHT (0.5 nM) and EGF (10 ng/ml) caused a greater decrease of DHT glucuronidation and UGT2B messenger RNA levels than when the cells were treated with either compound alone. RNase protection assays showed that treatment with DHT and EGF caused a specific decrease of UGT2B17 transcript in LNCaP cells treated; however, the level of UGT2B15 messenger RNA was not affected. As well, Western blot analysis demonstrated a diminution of UGT2B17 protein level in response to DHT and EGF. These results demonstrate a differential regulation of different isoforms of steroid conjugating UGTs present in human prostate LNCaP cells. UGT2B17 was shown to be more labile than UGT2B15, indicating that regulation of UGT2B17 expression would lead to a more rapid change in the level of glucuronidated steroids.

Comparison of plasma androgen glucuronide levels after percutaneous or peroral androgen treatment in men: evidence for important splanchnic contribution to plasma 17 beta-hydroxyandrogen glucuronides.

To investigate whether glucuronides of 17 beta-hydroxyandrogens are formed mainly in peripheral tissues or in splanchnic tissues, we compared androstanediol (AD) glucuronide (ADG) and dihydrotestosterone (DHT) glucuronide (DHTG) levels as well as the ratios of glucuronides over free steroids after transcutaneous DHT gel administration to levels and ratios found after oral administration of testosterone undecanoate. Whereas DHT and AD plasma levels were much higher after transcutaneous DHT gel administration, ADG and DHTG levels as well as glucuronide/free androgen ratios were an order of a magnitude higher after oral TU. This indicates that 17 beta-hydroxyandrogen glucuronides cannot be considered specific metabolites of androgen target tissues and are formed to a large extent in the splanchnic circulation.

Precursors of plasma androstanediol- and androgen-glucuronides in women

Although androstanediol (AD) and androstanediolglucuronide (ADG) are generally considered to be parameters of peripheral androgen action, their plasma levels do not always vary in parallel, suggesting that they may have different precursors. Few hard data being available concerning ADG precursors in women, we studied in postmenopausal women with absent or suppressed adrenal function, the blood conversion rates of testosterone (T), dihydrotestosterone (DHT), androstenedione (A) and dehydroepiandrosterone (DHEA) and its sulfate (DHEAS) to AD and ADG respectively, as well as the conversion of AD to ADG. Moreover conversions of these precursors to testosteroneglucuronide (TG) and dihydrotestosteroneglucuronide (DHTG), respectively, were also studied. Our data show that, whereas plasma A and DHT are the major precursors of AD, plasma DHEAS and A are the major precursors of plasma ADG, accounting for 50 and 15%, respectively, of plasma ADG, A being the major precursor of plasma TG and DHTG, respectively. When the conversion rates, obtained in this study, were applied to the plasma concentration of precursors found in normal young and postmenopausal women, respectively, the calculated concentration of product steroids accounted for almost the totality of the actual plasma levels of ADG, TG and DHTG respectively. The difference in relative importance of their precursors, explains that plasma concentrations of AD and ADG do not always vary in parallel; moreover, the importance of DHEAS as precursor of ADG explains the suppression by dexamethasone and the increase after adrenocortical stimulation of plasma ADG levels.

Structure-activity relationship of the cholestatic activity of dihydrotestosterone glucuronide, allo bile acids and lithocholate

Dihydrotestosterone glucuronide (DHTG), a series of 5α-bile acids, or allo-bile acids (3α-hydroxy-5α-cholanic acid, 3-keto-5α-cholanic acid and 3β-hydroxy-5α-cholanic acid) and their normal bile acid analogues (3α-hydroxy-5β-cholanic acid or lithocholate, 3-keto-5β-cholanic acid and 3β-hydroxy-5β-cholanic acid) were administered intravenously to female rats in order to determine their effects on bile flow. All agents caused a rapid and profound inhibition of bile flow which was dose-dependent. The logarithm of the dose vs the cholestatic response curve for DHTG, the allo-bile acids and lithocholate were all parallel. DHTG was the most potent congener and was two times more potent than 3-keto-5α-cholanic acid and 5 times more potent than lithocholate. These data indicate that the glucuronic acid moiety and the trans configuration of the A and B rings of the steroid nucleus confer the greatest cholestatic potency.

Production of 3 alpha-androstanediol glucuronide in human genital skin.

3 alpha-Androstanediol glucuronide (3 alpha diol-G) is produced extrasplanchnically and is a good clinical marker of androgen action in peripheral tissues. However, the direct formation of androgen glucuronides in peripheral sites such as skin has not been determined in man. Genital skin from 21 premenopausal women and 8 men and foreskin from 6 neonates were incubated with either [14C]testosterone [14C]dihydrotestosterone (DHT) to determine the production of DHT glucuronide and 3 alpha diol-G in skin. After hydrolysis of incubation medium with glucuronidase, followed by extraction and sequential chromatography, constant 3H to 14C ratios of 3 alpha diol confirmed the production of DHT glucuronide and 3 alpha diol-g. The conversion of DHT to 3 alpha diol-G was higher than the conversion from testosterone (P less than 0.05), and conversion was higher in men than in women. These data provide evidence for the direct formation of C19 steroid glucuronides by human skin.

Conjugated and unconjugated C-21, C-19 and C-18 steroid concentrations in human follicular fluid from hyperstimulated follicles.

Concentrations of unconjugated and glucuronated C-21, C-19 and C-18 steroids have been measured in follicular fluid obtained from women hyperstimulated by a combination of clomiphene citrate, pergonal and hCG. Pregnenolone and its glucuronide are found at approximately 5% of the progesterone values (14,395 +/- 2300 ng/ml). Concentrations of dehydroepiandrosterone glucuronide, androst-5-ene-3 beta, 17 beta-diol glucuronide, dihydrotestosterone glucuronide, androstane-3 alpha, 17 beta-diol glucuronide and androstane-3 beta, 17 beta-diol glucuronide are 2-6-fold higher than those of the related unconjugated steroids, while testosterone glucuronide levels are lower than those of testosterone. The concentrations of androsterone glucuronide reach almost 30-fold those of androsterone (2.56 +/- 0.35 ng/ml). By contrast, estradiol and estrone concentrations are 10-20-fold higher than those of conjugated steroids. Measurement of plasma steroid glucuronides indicated that the concentrations in circulation are markedly lower than those detected in follicular fluid. Our data suggest that the ovary in humans may be a site of glucuronidation.

Elevated production and metabolic clearance rates of androgens in morbidly obese women.

Blood production rates of testosterone, dihydrotestosterone (DHT), and 3 alpha-androstanediol (3 alpha-diol) were found to be approximately 2-fold elevated in morbidly obese, nonhirsute, normally menstruating women. Values were intermediate between those found in normal women and those in a group of nonobese normally menstruating women with idiopathic hirsutism. Elevated androgen production rates in obese women were associated with 2- to 3-fold increases in MCRs, presumably due to decreased levels of sex hormone-binding globulin. Thus, increased production rates were offset by increased MCRs, resulting in plasma testosterone, DHT, and 3 alpha-diol concentrations that were similar in the obese and normal women. By contrast, women with hirsutism had increased production rates associated with elevated plasma androgens as well as increased MCRs. Urinary excretion of testosterone glucuronide and 3 alpha-diol glucuronide (3 alpha-diol G) were elevated in both obese and hirsute women, paralleling the increased androgen production rates. Despite increased production rates and excretion of androgens, obese women exhibited no menstrual abnormalities, hirsutism, or other signs of virilism. To explore the apparent ineffectiveness of increased androgen production to produce virilizing symptoms, we measured plasma 3 alpha-diol G levels as a measure of peripheral androgen action. The mean +/- SE plasma 3 alpha-diol G was 53 +/- 8 ng/dl in obese women and 36 +/- 6 in normal women; by contrast, women with idiopathic hirsutism had levels of 440 +/- 99, a 12-fold elevation. Plasma testosterone glucuronide in obese and hirsute women were only 2- to 3-fold elevated, while plasma DHT glucuronide was not increased in obese women and was only 2-fold elevated in hirsute women. Thus, obesity is a state of increased androgen production and accelerated clearance. 3 alpha-diol G levels in obese women were only minimally elevated, in contrast to values in the hirsute women, perhaps reflecting the apparent androgen ineffectiveness.

Origin of plasma androstanediol glucuronide in men.

The role of testosterone (T) and dihydrotestosterone (DHT) as precursors of the peripheral metabolite androstanediol glucuronide (3 alpha diol G) in plasma from normal men was studied. An apparent steady state of both putative precursors and the steroid glucuronides were attained by 8-h constant iv infusions of 3H-labeled steroid after a loading dose. The unconjugated steroids and the steroid glucuronides (after beta-glucuronidase hydrolysis) with 14C indicator were purified by serial microcolumn and paper chromatography steps previously reported to achieve radiochemical purity. The specific activities of 3 alpha diol and 3 alpha diol G in plasma were widely different in each subject, confirming our earlier suggestion that the two peripheral metabolites are formed in different pools. The conversion ratios (CRPre-Prod BB) varied widely. The CRT-3 alpha diol G was generally less than 5%, while the CRDHT-3 alpha diol G was 10 times higher. These results are compatible with the expected model, T----DHT----3 alpha diol G. In some of the studies, T glucuronide (TG) and DHT glucuronide (DHTG) were isolated after T infusions, and DHTG was isolated after DHT infusion. The major conversion product of blood T was DHTG, not TG, and the major conversion product of DHT was 3 alpha diol G. This suggests that metabolism proceeds through a steroid reduction step and glucuronidation. The peripheral pathway to 3 alpha diol G may involve formation of DHTG and then 3 alpha-reduction to 3 alpha diol G. This may also explain why blood levels of unconjugated 3 alpha diol have not been helpful in elucidating disorders of androgen formation, as this androgen mostly arises from sites different from 3 alpha diol G.