RESUMEN
N-acetyltransferase 1 (NAT1) is an enzyme that metabolizes carcinogens, which suggests a potential role in breast carcinogenesis. High NAT1 expression in breast tumors is associated with estrogen receptor α (ERα+) and the luminal subtype. We report that NAT1 mRNA transcript, protein, and enzyme activity were higher in human breast tumors with high expression of ERα/ESR1 compared with normal breast tissue. There was a strong correlation between NATb promoter and NAT1 protein expression/enzyme activity. High NAT1 expression in tumors was not the result of adipocytes, as evidenced by low perilipin (PLIN) expression. ESR1, NAT1, and XBP1 expression were associated in tumor biopsies. Direct regulation of NAT1 transcription by estradiol (E2) was investigated in ERα (+) MCF-7 and T47D breast cancer cells. E2 did not increase NAT1 transcript expression but increased progesterone receptor expression in a dose-dependent manner. Likewise, NAT1 transcript levels were not increased by dihydrotestosterone (DHT) or 5α-androstane-3ß, (3ß-adiol) 17ß-diol. Dithiothreitol increased levels of the activated, spliced XBP1 in ERα (+) MCF-7 and T47D breast cancer cells but did not affect NAT1 or ESR1 expression. We conclude that NAT1 expression is not directly regulated by E2, DHT, 3ß-adiol, or dithiothreitol despite high NAT1 and ESR1 expression in luminal A breast cancer cells, suggesting that ESR1, XBP1, and NAT1 expression may share a common transcriptional network arising from the luminal epithelium associated with better survival in breast cancer. Clusters of high-expression genes, including NAT1, in breast tumors might serve as potential targets for novel therapeutic drug development.
Asunto(s)
Arilamina N-Acetiltransferasa/genética , Neoplasias de la Mama/patología , Receptor alfa de Estrógeno/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Isoenzimas/genética , Androstano-3,17-diol/farmacología , Neoplasias de la Mama/genética , Dihidrotestosterona/farmacología , Estradiol/farmacología , Humanos , Células MCF-7 , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
BACKGROUND: There is increasing evidence showing that chronic non-bacterial prostatic inflammation is involved in the pathogenesis of benign prostatic hyperplasia (BPH) and male lower urinary tract symptoms (LUTS). It has also been reported that estrogen receptor ß (ERß) could have an immunoprotective role in prostatic tissue. Therefore, we investigated the effect of ERß-activation on not only prostatic inflammation, but also bladder overactive conditions in a rat model with nonbacterial prostatic inflammation. METHODS: Male Sprague-Dawley rats (8 weeks, n = 15) were divided into three groups: sham-saline group (n = 5), formalin-vehicle group (n = 5), and formalin-treatment group (n = 5). The sham-saline group had sham operation and 50 µl normal saline injected into each ventral lobe of the prostate. The formalin-vehicle group had 50 µl 5% formalin injection into bilateral ventral lobes of the prostate. The formalin-treatment group was treated with 3α-Adiol (a selective ERß agonist precursor) at a dose of 3 mg/kg daily from 2 days before induction of prostatic inflammation, whereas formalin-vehicle rats received vehicle (olive oil). In each group, conscious cystometry was performed on day 28 after intraprostatic formalin injection or sham treatment. After cystometry, the bladder and prostate were harvested for evaluation of mRNA expression and histological analysis. RESULTS: In cystometric investigation, the mean number of non-voiding contractions was significantly greater and voiding intervals were significantly shorter in formalin-vehicle rats than those in sham-saline rats (P < 0.05). In RT-qPCR analysis, mRNA expression of NGF, P2X2, and TRPA1 receptors was significantly increased in the bladder mucosa, and mRNA expression of TNF-α, iNOS and COX2 in the ventral lobes of prostate was significantly increased in formalin-vehicle rats compared with sham-saline rats (P < 0.05). In addition, relative mRNA expression ratio of ERß to ERα (ERß/ERα) in the ventral lobes of prostate was significantly decreased in formalin-vehicle rats compared with sham-saline rats (P < 0.05). These changes were ameliorated by 3α-Adiol administration in formalin-treatment rats. CONCLUSIONS: These results indicate that ERß activation by 3α-Adiol administration, which normalized the ERß/ERα expression ratio in the prostate, can improve not only prostatic inflammation, but also bladder overactivity. Therefore, ERß agonists might be useful for treating irritative bladder symptoms in patients with symptomatic BPH associated with prostatic inflammation. Prostate 77:803-811, 2017. © 2017 Wiley Periodicals, Inc.
Asunto(s)
Androstano-3,17-diol/farmacología , Receptor beta de Estrógeno , Síntomas del Sistema Urinario Inferior , Hiperplasia Prostática , Prostatitis , Vejiga Urinaria/metabolismo , Animales , Receptor beta de Estrógeno/agonistas , Receptor beta de Estrógeno/antagonistas & inhibidores , Estrógenos/farmacología , Síntomas del Sistema Urinario Inferior/diagnóstico , Síntomas del Sistema Urinario Inferior/inmunología , Síntomas del Sistema Urinario Inferior/fisiopatología , Masculino , Próstata/inmunología , Próstata/patología , Hiperplasia Prostática/inmunología , Hiperplasia Prostática/patología , Hiperplasia Prostática/fisiopatología , Prostatitis/diagnóstico , Prostatitis/inmunología , Prostatitis/fisiopatología , Factores Protectores , Ratas , Ratas Sprague-Dawley , Resultado del Tratamiento , Factor de Necrosis Tumoral alfa/metabolismo , Vejiga Urinaria/patología , UrodinámicaRESUMEN
BACKGROUND: Estrogen receptor (ER) ß has been suggested to affect ovarian carcinogenesis. We examined the effects of four ERß agonists on proliferation and gene expression of two ovarian cancer cell lines. METHODS: OVCAR-3 and OAW-42 ovarian cancer cells were treated with the ERß agonists ERB-041, WAY200070, Liquiritigenin and 3ß-Adiol and cell growth was measured by means of the Cell Titer Blue Assay (Promega). ERß expression was knocked down by transfection with specific siRNA. Additionally, transcriptome analyses were performed by means of Affymetrix GeneChip arrays. To confirm the results of DNA microarray analysis, Western blot experiments were performed. RESULTS: All ERß agonists tested significantly decreased proliferation of OVCAR-3 and OAW-42 cells at a concentration of 10 nM. Maximum antiproliferative effects were induced by flavonoid Liquiritigenin, which inhibited growth of OVCAR-3 cells by 31.2% after 5 days of treatment, and ERB-041 suppressing proliferation of the same cell line by 29.1%. In OAW-42 cells, maximum effects were observed after treatment with the ERß agonist WAY200070, inhibiting cell growth by 26.8%, whereas ERB-041 decreased proliferation by 24.4%. In turn, knockdown of ERß with specific siRNA increased cell growth of OAW-42 cells about 1.9-fold. Transcriptome analyses revealed a set of genes regulated by ERß agonists including ND6, LCN1 and PTCH2, providing possible molecular mechanisms underlying the observed antiproliferative effects. CONCLUSION: In conclusion, the observed growth-inhibitory effects of all ERß agonists on ovarian cancer cell lines in vitro encourage further studies to test their possible use in the clinical setting.
Asunto(s)
Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Receptor beta de Estrógeno/agonistas , Estrógenos/farmacología , Neoplasias Ováricas/tratamiento farmacológico , Androstano-3,17-diol/farmacología , Androstano-3,17-diol/uso terapéutico , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Estrógenos/uso terapéutico , Femenino , Flavanonas/farmacología , Flavanonas/uso terapéutico , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Ováricas/genética , Neoplasias Ováricas/fisiopatología , Oxazoles/farmacología , Oxazoles/uso terapéutico , Fenoles/farmacología , Fenoles/uso terapéuticoRESUMEN
Maternal social stress during late pregnancy programs hypothalamo-pituitary-adrenal (HPA) axis hyper-responsiveness to stressors, such that adult prenatally stressed (PNS) offspring display exaggerated HPA axis responses to a physical stressor (systemic interleukin-1ß; IL-1ß) in adulthood, compared with controls. IL-1ß acts via a noradrenergic relay from the nucleus tractus solitarii (NTS) to corticotropin releasing hormone neurons in the paraventricular nucleus (PVN). Neurosteroids can reduce HPA axis responses, so allopregnanolone and 3ß-androstanediol (3ß-diol; 5α-reduced metabolites of progesterone and testosterone, respectively) were given subacutely (over 24 h) to PNS rats to seek reversal of the "programmed" hyper-responsive HPA phenotype. Allopregnanolone attenuated ACTH responses to IL-1ß (500 ng/kg, i.v.) in PNS females, but not in PNS males. However, 3ß-diol normalized HPA axis responses to IL-1ß in PNS males. Impaired testosterone and progesterone metabolism or increased secretion in PNS rats was indicated by greater plasma testosterone and progesterone concentrations in male and female PNS rats, respectively. Deficits in central neurosteroid production were indicated by reduced 5α-reductase mRNA levels in both male and female PNS offspring in the NTS, and in the PVN in males. In PNS females, adenovirus-mediated gene transfer was used to upregulate expression of 5α-reductase and 3α-hydroxysteroid dehydrogenase mRNAs in the NTS, and this normalized hyperactive HPA axis responses to IL-1ß. Thus, downregulation of neurosteroid production in the brain may underlie HPA axis hyper-responsiveness in prenatally programmed offspring, and administration of 5α-reduced steroids acutely to PNS rats overrides programming of hyperactive HPA axis responses to immune challenge in a sex-dependent manner.
Asunto(s)
Androstano-3,17-diol/farmacología , Sistema Hipotálamo-Hipofisario/efectos de los fármacos , Sistema Hipófiso-Suprarrenal/efectos de los fármacos , Pregnanolona/farmacología , Estrés Psicológico/tratamiento farmacológico , 3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/genética , 3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/metabolismo , 3-alfa-Hidroxiesteroide Deshidrogenasa (B-Específica)/genética , 3-alfa-Hidroxiesteroide Deshidrogenasa (B-Específica)/metabolismo , Androstano-3,17-diol/uso terapéutico , Animales , Femenino , Sistema Hipotálamo-Hipofisario/crecimiento & desarrollo , Interleucina-1beta/farmacología , Masculino , Núcleo Hipotalámico Paraventricular/efectos de los fármacos , Núcleo Hipotalámico Paraventricular/crecimiento & desarrollo , Núcleo Hipotalámico Paraventricular/metabolismo , Sistema Hipófiso-Suprarrenal/crecimiento & desarrollo , Embarazo , Complicaciones del Embarazo/tratamiento farmacológico , Pregnanolona/uso terapéutico , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Factores Sexuales , Núcleo Solitario/efectos de los fármacos , Núcleo Solitario/crecimiento & desarrollo , Núcleo Solitario/metabolismoRESUMEN
The brain has high energy requirements to maintain neuronal activity. Consequently impaired mitochondrial function will lead to disease. Normal aging is associated with several alterations in neurosteroid production and secretion. Decreases in neurosteroid levels might contribute to brain aging and loss of important nervous functions, such as memory. Up to now, extensive studies only focused on estradiol as a promising neurosteroid compound that is able to ameliorate cellular bioenergetics, while the effects of other steroids on brain mitochondria are poorly understood or not investigated at all. Thus, we aimed to characterize the bioenergetic modulating profile of a panel of seven structurally diverse neurosteroids (progesterone, estradiol, estrone, testosterone, 3α-androstanediol, DHEA and allopregnanolone), known to be involved in brain function regulation. Of note, most of the steroids tested were able to improve bioenergetic activity in neuronal cells by increasing ATP levels, mitochondrial membrane potential and basal mitochondrial respiration. In parallel, they modulated redox homeostasis by increasing antioxidant activity, probably as a compensatory mechanism to a slight enhancement of ROS which might result from the rise in oxygen consumption. Thereby, neurosteroids appeared to act via their corresponding receptors and exhibited specific bioenergetic profiles. Taken together, our results indicate that the ability to boost mitochondria is not unique to estradiol, but seems to be a rather common mechanism of different steroids in the brain. Thus, neurosteroids may act upon neuronal bioenergetics in a delicate balance and an age-related steroid disturbance might be involved in mitochondrial dysfunction underlying neurodegenerative disorders.
Asunto(s)
Metabolismo Energético/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Neuronas/efectos de los fármacos , Neurotransmisores/farmacología , Adenosina Trifosfato/metabolismo , Envejecimiento/metabolismo , Androstano-3,17-diol/farmacología , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Deshidroepiandrosterona/farmacología , Relación Dosis-Respuesta a Droga , Estradiol/farmacología , Estrona/farmacología , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Mitocondrias/metabolismo , Mitocondrias/fisiología , Enfermedades Neurodegenerativas/metabolismo , Neuronas/metabolismo , Neurotransmisores/metabolismo , Consumo de Oxígeno , Pregnanolona/farmacología , Progesterona/farmacología , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/metabolismo , Testosterona/farmacologíaRESUMEN
Estrogen receptor ß (ERß) is activated in the prostate by 5α-androstane-3ß,17ß-diol (3ß-Adiol) where it exerts antiproliferative activity. The proliferative action of the androgen receptor is activated by 5α-dihydrotestosterone (DHT). Thus, prostate growth is governed by the balance between androgen receptor and ERß activation. 3ß-Adiol is a high-affinity ligand and agonist of ERß and is derived from DHT by 3-keto reductase/3ß-hydroxysteroid dehydrogenase enzymes. Here, we demonstrate that, when it is expressed in living cells containing an estrogen response element-luciferase reporter, 17ß-hydroxysteroid dehydrogenase type 6 (17ßHSD6) converts the androgen DHT to the estrogen 3ß-Adiol, and this leads to activation of the ERß reporter. This conversion of DHT occurs at concentrations that are in the physiological range of this hormone in the prostate. Immunohistochemical analysis revealed that 17ßHSD6 is expressed in ERß-positive epithelial cells of the human prostate and that, in prostate cancers of Gleason grade higher than 3, both ERß and 17ßHSD6 are undetectable. Both proteins were present in benign prostatic hyperplasia samples. These observations reveal that formation of 3ß-Adiol via 17ßHSD6 from DHT is an important growth regulatory pathway that is lost in prostate cancer.
Asunto(s)
Receptor beta de Estrógeno/metabolismo , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/patología , Racemasas y Epimerasas/metabolismo , Transducción de Señal , Androstano-3,17-diol/farmacología , Proliferación Celular/efectos de los fármacos , Dihidrotestosterona/farmacología , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Epitelio/patología , Receptor beta de Estrógeno/genética , Células HEK293 , Humanos , Hiperplasia , Inmunohistoquímica , Masculino , Modelos Biológicos , Próstata/efectos de los fármacos , Próstata/metabolismo , Próstata/patología , Neoplasias de la Próstata/genética , Transducción de Señal/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/genéticaRESUMEN
Aromatase inhibitors (AIs) have been reported to exert their antiproliferative effects in postmenopausal women with hormone receptor-positive breast cancer not only by reducing estrogen production but also by unmasking the inhibitory effects of androgens such as testosterone (TS) and dihydrotestosterone (DHT). However, the role of androgens in AI-resistance mechanisms is not sufficiently understood. 5α-Androstane-3ß,17ß-diol (3ß-diol) generated from DHT by 3ß-hydroxysteroid dehydrogenase type 1 (HSD3B1) shows androgenic and substantial estrogenic activities, representing a potential mechanism of AI resistance. Estrogen response element (ERE)-green fluorescent protein (GFP)-transfected MCF-7 breast cancer cells (E10 cells) were cultured for 3 months under steroid-depleted, TS-supplemented conditions. Among the surviving cells, two stable variants showing androgen metabolite-dependent ER activity were selected by monitoring GFP expression. We investigated the process of adaptation to androgen-abundant conditions and the role of androgens in AI-resistance mechanisms in these variant cell lines. The variant cell lines showed increased growth and induction of estrogen-responsive genes rather than androgen-responsive genes after stimulation with androgens or 3ß-diol. Further analysis suggested that increased expression of HSD3B1 and reduced expression of androgen receptor (AR) promoted adaptation to androgen-abundant conditions, as indicated by the increased conversion of DHT into 3ß-diol by HSD3B1 and AR signal reduction. Furthermore, in parental E10 cells, ectopic expression of HSD3B1 or inhibition of AR resulted in adaptation to androgen-abundant conditions. Coculture with stromal cells to mimic local estrogen production from androgens reduced cell sensitivity to AIs compared with parental E10 cells. These results suggest that increased expression of HSD3B1 and reduced expression of AR might reduce the sensitivity to AIs as demonstrated by enhanced androgen metabolite-induced ER activation and growth mechanisms. Androgen metabolite-dependent growth of breast cancer cells may therefore play a role in AI-resistance.
Asunto(s)
Andrógenos/metabolismo , Inhibidores de la Aromatasa/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Resistencia a Antineoplásicos , Receptores de Estrógenos/metabolismo , Androstano-3,17-diol/metabolismo , Androstano-3,17-diol/farmacología , Neoplasias de la Mama/metabolismo , Dihidrotestosterona/metabolismo , Dihidrotestosterona/farmacología , Femenino , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Letrozol , Células MCF-7/efectos de los fármacos , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Nitrilos/farmacología , Progesterona Reductasa/genética , Progesterona Reductasa/metabolismo , Receptores Androgénicos/metabolismo , Elementos de Respuesta/genética , Transducción de Señal , Esteroide Isomerasas/genética , Esteroide Isomerasas/metabolismo , Testosterona/metabolismo , Testosterona/farmacología , Triazoles/farmacologíaRESUMEN
RM, a novel aminosteroid synthesized by our research group, shows a broad spectrum of antitumor activity against nine cancer cell lines and limited toxicity against two normal cell lines. However, its related mechanism of action has not yet been elucidated. In this study, we investigated the cellular and molecular events underlying the cytotoxicity of RM in human acute promyelocytic leukemia HL-60 cells. RM was found to induce a G0/G1 cell cycle block of HL-60 cells but not terminal myeloid differentiation. Interestingly, typical apoptotic morphological changes were exhibited by HL-60 cells treated with RM stained with Hoechst 33342 and examined by fluorescence microscopy. Apoptotic death assay using annexin-V/propidium iodide dual staining flow cytometry demonstrated a dose-dependent apoptotic effect of RM on HL-60 cells. In addition, RM induced the cleavage of caspase-3, caspase-8 and PARP, but not the cleavage of caspase-9. Our findings suggest that RM reduces HL-60 cells survival through a caspase-dependent death receptor pathway.
Asunto(s)
Androstano-3,17-diol/farmacología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Leucemia Promielocítica Aguda/patología , Caspasas/metabolismo , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Células HL-60 , Humanos , Microscopía Fluorescente , Poli(ADP-Ribosa) Polimerasas/metabolismo , Fase de Descanso del Ciclo Celular/efectos de los fármacos , Factores de TiempoRESUMEN
This study investigated the antineoplasic potential of a new family of aminosteroids. The antiproliferative activity of seven 5α-androstane-3α,17ß-diol derivatives selected from a screening study was measured on nine cancerous cell lines (HL-60, K-562, LNCaP, PC-3, Shionogi, MCF-7, MDA-MB-231, BT-20, and OVCAR-3) and two normal cell lines (peripheral blood lymphocytes and WI-38). The aminosteroids efficiently inhibited the cell growth of seven cancer cell lines [inhibitory concentration (IC(50)) values=0.2-6.4 µmol/l] and showed weak toxicity on normal cell lines. Two representative aminosteroids were tested and found to induce apoptosis and a G0/G1 cell cycle block in HL-60-treated cells, but not terminal myeloid differentiation. By a nuclear morphology analysis with fluorescence microscopy, typical apoptotic morphological changes were exhibited by treated cells. One aminosteroid tested in vivo (xenograft model) reduced the breast cancer (MCF-7 cells) tumor growth induced in nude mice. Furthermore, the information gathered suggests that this family of aminosteroids induced growth inhibition cells by arresting the cell cycle and triggering apoptosis.
Asunto(s)
Androstano-3,17-diol/farmacología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Androstano-3,17-diol/química , Animales , Antineoplásicos/química , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Línea Celular , Línea Celular Tumoral , Femenino , Fase G1/efectos de los fármacos , Células HL-60 , Humanos , Concentración 50 Inhibidora , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Microscopía Fluorescente , Neoplasias/patología , Fase de Descanso del Ciclo Celular/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The influence of 3α-androstanediol (3α-diol) on twitch and electroencephalogram (EEG) of the epileptic rats induced by pentylenetetrazole (PTZ) has been observed in this experiment in order to comprehensively explore the role of 3α-diol on epileptic attack from the aspects of behavior and EEG. Thirty-two male Sprague-Dawley rats were evenly and randomly divided into 4 groups: the normal and supplied with oil epileptic (N+oil+PTZ) group, the normal and supplied with 3α-diol epileptic (N+3α-diol+PTZ) group, the gonadectomized and supplied with oil epileptic (GDX+oil+PTZ) group and the gonadectomized and supplied with 3α-diol epileptic (GDX+3α-diol+PTZ) group. The changes of the behavior and EEG of epileptic rats in every group were recorded and analyzed. The results of behavior observation showed that the latency to clonic seizure and tonic-clonic seizure was shortened and the number of tonic-clonic seizure was increased significantly in the GDX+oil+PTZ group in comparison with N+oil+PTZ group (P < 0.05); comparing GDX+3α-diol+PTZ group with GDX+oil+PTZ group, or N+3α-diol+PTZ group with N+oil+PTZ group, we found that the latency to clonic seizure and tonic-clonic seizure became prolonged significantly, and the number of clonic seizure and tonic-clonic seizure was decreased significantly (P < 0.05). The results of EEG showed that the latency to epileptic waves was cut and the number of epileptic waves was augmented significantly in the GDX+oil+PTZ group in comparison with N+oil+PTZ group (P < 0.05); comparing GDX+3α-diol+PTZ group with GDX+oil+PTZ group, or N+3α-diol+PTZ group with N+oil+PTZ group, we found that the latency to epileptic waves became lengthened significantly, the number of epileptic waves was reduced significantly and the percentage of change of TP (total power of spectrum) was lessened significantly (P < 0.05). These results indicate that 3α-diol has an antiepileptic activity in the gonadectomized and normal epileptic rats.
Asunto(s)
Androstano-3,17-diol/análogos & derivados , Anticonvulsivantes/farmacología , Epilepsia/tratamiento farmacológico , Pentilenotetrazol/efectos adversos , Convulsiones/tratamiento farmacológico , Androstano-3,17-diol/farmacología , Animales , Electroencefalografía , Epilepsia/inducido químicamente , Masculino , Ratas , Ratas Sprague-Dawley , Convulsiones/inducido químicamenteRESUMEN
Most of the many biological effects of estrogens are mediated via the estrogen receptors ERalpha and beta. The current study examines the role of CYP7B1-mediated catalysis for activation of ER. Several reports suggest that CYP7B1 may be important for hormonal action but previously published studies are contradictory concerning the manner in which CYP7B1 affects ERbeta-mediated response. In the current study, we examined effects of several CYP7B1-related steroids on ER activation, using an estrogen response element (ERE) reporter system. Our studies showed significant stimulation of ER by 5-androstene-3beta,17beta-diol (Aene-diol) and 5alpha-androstane-3beta,17beta-diol (3beta-Adiol). In contrast, the CYP7B1-formed metabolites from these steroids did not activate the receptor, indicating that CYP7B1-mediated metabolism abolishes the ER-stimulating effect of these compounds. The mRNA level of HEM45, a gene known to be stimulated by estrogens, was strongly up-regulated by Aene-diol but not by its CYP7B1-formed metabolite, further supporting this concept. We did not observe stimulation by dehydroepiandrosterone (DHEA) or 7alpha-hydroxy-DHEA, previously suggested to affect ERbeta-mediated response. As part of these studies we examined metabolism of Aene-diol in pig liver which is high in CYP7B1 content. These experiments indicate that CYP7B1-mediated metabolism of Aene-diol is of a similar rate as the metabolism of the well-known CYP7B1 substrates DHEA and 3beta-Adiol. CYP7B1-mediated metabolism of 3beta-Adiol has been proposed to influence ERbeta-mediated growth suppression. Our results indicate that Aene-diol also might be important for ER-related pathways. Our data indicate that low concentrations of Aene-diol can trigger ER-mediated response equally well for both ERalpha and beta and that CYP7B1-mediated conversion of Aene-diol into a 7alpha-hydroxymetabolite will result in loss of action.
Asunto(s)
Neoplasias de la Mama/metabolismo , Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/metabolismo , Riñón/metabolismo , Microsomas Hepáticos/metabolismo , Esteroide Hidroxilasas/metabolismo , Adyuvantes Inmunológicos/farmacología , Anabolizantes/farmacología , Androstano-3,17-diol/farmacología , Animales , Western Blotting , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Catálisis , Células Cultivadas , Familia 7 del Citocromo P450 , Deshidroepiandrosterona/farmacología , Receptor alfa de Estrógeno/genética , Receptor beta de Estrógeno/genética , Exonucleasas/genética , Exonucleasas/metabolismo , Exorribonucleasas , Humanos , Riñón/citología , Riñón/efectos de los fármacos , Luciferasas/metabolismo , Microsomas Hepáticos/efectos de los fármacos , ARN Mensajero/genética , Elementos de Respuesta , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esteroide Hidroxilasas/genética , PorcinosRESUMEN
Testosterone modulates seizure susceptibility, but the underlying mechanisms are obscure. Recently, we demonstrated that testosterone affects seizure activity via its conversion to neurosteroids in the brain. Androstanediol (5alpha-androstan-3alpha,17beta-diol) is an endogenous neurosteroid synthesized from testosterone. However, the molecular mechanism underlying the seizure protection activity of androstanediol remains unclear. Here, we show that androstanediol has positive allosteric activity as a GABA(A) receptor modulator. In whole-cell recordings from acutely dissociated hippocampus CA1 pyramidal cells, androstanediol (but not its 3beta-epimer) produced a concentration-dependent enhancement of GABA-activated currents (EC(50) of 5 microM). At 1 microM, androstanediol produced a 50% potentiation of GABA responses. In the absence of GABA, androstanediol has moderate direct effects on GABA(A) receptor-mediated currents at high concentrations. Systemic doses of androstanediol (5-100 mg/kg), but not its 3beta-epimer, caused dose-dependent suppression of behavioral and electrographic seizures in mouse hippocampus kindling, which is a model of temporal lobe epilepsy. The ED(50) value for antiseizure effects of androstanediol was 50 mg/kg, which did not produce sedation/motor toxicity. At high (2x ED(50)) doses, androstanediol produced complete seizure protection that lasted for up to 3 h after injection. The estimated plasma concentrations of androstanediol producing 50% seizure protection in the kindling model (10.6 microM) are within the range of concentrations that modulate GABA(A) receptors. These studies suggest that androstanediol could be a neurosteroid mediator of testosterone actions on neuronal excitability and seizure susceptibility via its activity as a GABA(A) receptor modulator and that androstanediol may play a key role in men with epilepsy, especially during the age-related decline in androgen levels.
Asunto(s)
Androstano-3,17-diol/farmacología , Moduladores del GABA , Receptores de GABA-A/efectos de los fármacos , Antagonistas de Andrógenos/farmacología , Androstano-3,17-diol/toxicidad , Animales , Región CA1 Hipocampal/citología , Región CA1 Hipocampal/efectos de los fármacos , Interpretación Estadística de Datos , Potenciales Evocados/efectos de los fármacos , Flutamida/farmacología , Hipocampo/fisiología , Técnicas In Vitro , Excitación Neurológica , Masculino , Ratones , Ratones Endogámicos C57BL , Desempeño Psicomotor/efectos de los fármacos , Células Piramidales/efectos de los fármacos , Convulsiones/prevención & control , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
Gonadotropins and testosterone are important regulators of spermatogenesis, even though gonadotropin receptors and the androgen receptor are not expressed by germ cells. However, a functional role for estrogens in connection with male reproduction has been postulated on the basis of the phenotypes of mice lacking estrogen receptor (ER) and cytochrome P-450 aromatase. This has further support by findings of ER expression in the testis, including that of ERbeta in spermatogonia. 5alpha-Androstane-3beta, 17beta-diol (3betaAdiol), a metabolite of testosterone produced via the intermediate potent androgen 5alpha-dihydrotestosterone (DHT), has been reported to selectively bind ERbeta rather than EpsilonRalpha, but not androgen receptor. Here, we have characterized the influence of 17beta-estradiol (E), the major physiological estrogen, 3betaAdiol, and DHT on DNA synthesis in vitro by segments of the seminiferous epithelium at different stages of the seminiferous epithelial cycle in the rat. E and 3betaAdiol exerted similar stimulatory effects on premitotic DNA synthesis in stage I segments, whereas other stages tested (V, VIIa, and XIII-IX) remained unresponsive. In contrast, DHT had no effect on this process. 5-bromo-2'-deoxyuridine labeling of stage I segments revealed a 30-fold higher labeling index in the presence than in the absence of E, and the labeled cells were identified as spermatogonia. Moreover, high levels of 3betaAdiol were found in the testis of intact rats as well as in primary cultures of rat Leydig cells in response to human chorionic gonadotropin. We suggest that 3betaAdiol may serve as a growth factor for germ cells stimulating premitotic DNA synthesis in connection with spermatogenesis via an ERbeta-dependent pathway.
Asunto(s)
Androstano-3,17-diol/farmacología , Células Epiteliales/fisiología , Receptor beta de Estrógeno/fisiología , Túbulos Seminíferos/fisiología , Espermatogonias/fisiología , Animales , Células Cultivadas , Replicación del ADN/efectos de los fármacos , Dihidrotestosterona/farmacología , Células Epiteliales/efectos de los fármacos , Masculino , Ratas , Ratas Sprague-Dawley , Túbulos Seminíferos/citología , Túbulos Seminíferos/efectos de los fármacos , Espermatogonias/efectos de los fármacosRESUMEN
Androgen and androgen receptor (AR) are involved in growth of normal prostate and development of prostatic diseases including prostate cancer. Androgen deprivation therapy is used for treating advanced prostate cancer. This therapeutic approach focuses on suppressing the accumulation of potent androgens, testosterone and 5alpha-dihydrotestosterone (5alpha-DHT), or inactivating the AR. Unfortunately, the majority of patients with prostate cancer eventually advance to androgen-independent states and no longer respond to the therapy. In addition to the potent androgens, 5alpha-androstane-3alpha,17beta-diol (3alpha-diol), reduced from 5alpha-DHT through 3alpha-hydroxysteroid dehydrogenases (3alpha-HSDs), activated signaling may represent a novel pathway responsible for the progression to androgen-independent prostate cancer. Androgen sensitive human prostate cancer LNCaP cells were used to compare 5alpha-DHT and 3alpha-diol activated androgenic effects. In contrast to 5alpha-DHT, 3alpha-diol regulated unique patterns of beta-catenin and Akt expression as well as Akt phosphorylation in parental and in AR-silenced LNCaP cells. More significantly, 3alpha-diol, but not 5alpha-DHT, supported AR-silenced LNCaP cells and AR negative prostate cancer PC-3 cell proliferation. 3alpha-diol-activated androgenic effects in prostate cells cannot be attributed to the accumulation of 5alpha-DHT, since 5alpha-DHT formation was not detected following 3alpha-diol administration. Potential accumulation of 3alpha-diol, as a result of elevated 3alpha-HSD expression in cancerous prostate, may continue to support prostate cancer growth in the presence of androgen deprivation. Future therapeutic strategies for treating advanced prostate cancer might need to target reductive 3alpha-HSD to block intraprostatic 3alpha-diol accumulation.
Asunto(s)
Androstano-3,17-diol/farmacología , Neoplasias de la Próstata/patología , Transducción de Señal/efectos de los fármacos , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Citoplasma/efectos de los fármacos , Dihidrotestosterona/farmacología , Progresión de la Enfermedad , Ensayos de Selección de Medicamentos Antitumorales , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes Relacionados con las Neoplasias , Humanos , Masculino , Modelos Biológicos , Fosforilación/efectos de los fármacos , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Interferente Pequeño/metabolismo , Receptores Androgénicos/metabolismo , Factores de Tiempo , beta Catenina/metabolismoRESUMEN
The gender difference in behavioral and hormonal response to stress is well known, but the underlying mechanism remains elusive. Arginine-vasopressin (AVP) and corticotrophin-releasing hormone (CRH) are two major regulatory peptides in the brain involved in stress regulation. Their response to stress has been shown to be modulated by sex hormones. The androgen metabolite, 5alpha-androstane-3beta, 17beta-diol (3beta-diol), has been identified as an estrogenic hormone. It binds to estrogen receptors (ERs) and modulates estrogen response element mediated promoter activities via the ER pathway. The present study involved in vitro transfection assays to examine whether 3beta-diol can directly modulate CRH and AVP promoter activity. Our results demonstrate that in CHO-K1 cell lines, when ERs were over-expressed, 3beta-diol could significantly stimulate CRH and AVP promoter activity through an ER pathway. The effect of 3beta-diol on the behavioral, the CRH and the AVP response to stress in the rat was also investigated. We found that chronic, but not acute administration of 3beta-diol significantly decreased the immobile duration in the forced swim test. In rats exposed to the forced swim test, CRH mRNA expression in the hypothalamus was enhanced by chronic 3beta-diol administration, while the AVP mRNA expression was not affected. These results suggest that 3beta-diol may play an anti-depressive role in affective behavior and may have a direct effect on CRH expression.
Asunto(s)
Androstano-3,17-diol/farmacología , Conducta Animal/efectos de los fármacos , Hormona Liberadora de Corticotropina/metabolismo , Estrógenos , Estrés Psicológico/metabolismo , Estrés Psicológico/psicología , Animales , Arginina Vasopresina/farmacología , Células CHO , Hormona Liberadora de Corticotropina/genética , Cricetinae , Cricetulus , Antagonistas de Estrógenos/farmacología , Hipotálamo/efectos de los fármacos , Hipotálamo/metabolismo , Luciferasas/genética , Masculino , Regiones Promotoras Genéticas/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Natación/psicología , Tamoxifeno/farmacología , TransfecciónRESUMEN
Even though few steroids are used for the treatment of leukemia, 2beta-(4-methylpiperazinyl)-5alpha-androstane-3alpha,17beta-diol (1) was recently reported for its ability to inhibit the proliferation of human leukemia HL-60 cells. With an efficient procedure that we had developed for the aminolysis of hindered steroidal epoxides, we synthesized a series of 2beta-amino-5alpha-androstane-3alpha,17beta-diol N-derivatives structurally similar to 1. Hence, the opening of 2,3alpha-epoxy-5alpha-androstan-17beta-diol with primary and secondary amines allowed the synthesis of aminosteroids with diverse length, ramification, and functionalization of the 2beta-side chain. Sixty-four steroid derivatives were tested for their capacity to inhibit the proliferation of HL-60 cells; thus obtaining first structure-activity relationship results. Ten aminosteroids with long alkyl chains (7-16 carbons) or bulky groups (diphenyl or adamantyl) have shown antiproliferative activity over 78% at 10microM and superior to that of the lead compound. The 3,3-diphenylpropylamino, 4-nonylpiperazinyl and octylamino derivatives of 5alpha-androstane-3alpha,17beta-diol inhibited the HL-60 cell growth with IC(50) of 3.1, 4.2 and 6.4microM, respectively. They were also found to induce the HL-60 cell differentiation.
Asunto(s)
Androstano-3,17-diol/síntesis química , Androstano-3,17-diol/farmacología , Leucemia Promielocítica Aguda/tratamiento farmacológico , Esteroides/síntesis química , Esteroides/farmacología , Androstano-3,17-diol/análogos & derivados , Androstano-3,17-diol/química , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Células HL-60 , Humanos , Concentración 50 Inhibidora , Conformación Molecular , Estereoisomerismo , Esteroides/química , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Androgen ablation is the standard of care prescribed to patients with advanced or metastatic prostate cancer (PCa) to slow down disease progression. Unfortunately, a majority of PCa patients under androgen ablation progress to castration-resistant prostate cancer (CRPC). Several mechanisms including alternative intra-prostatic androgen production and androgen-independent androgen receptor (AR) activation have been proposed for CRPC progression. Aldo-keto reductase family 1 member C3 (AKR1C3), a multi-functional steroid metabolizing enzyme, is specifically expressed in the cytoplasm of PCa cells; and positive immunoreactivity of the type A γ-aminobutyric acid receptor (GABAAR), an ionotropic receptor and ligand-gated ion channel, is detected on the membrane of PCa cells. We studied a total of 72 radical prostatectomy cases by immunohistochemistry, and identified that 21 cases exhibited positive immunoreactivities for both AKR1C3 and GABAAR. In the dual positive cancer cases, AKR1C3 and GABAAR subunit α1 were either expressed in the same cells or in neighboring cells. Among several possible substrates, AKR1C3 reduces 5α-dihydrotesterone (DHT) to form 5α-androstane-3α, 17ß-diol (3α-diol). 3α-diol is a neurosteroid that acts as a positive allosteric modulator of the GABAAR in the central nervous system (CNS). We examined the hypothesis that 3α-diol-regulated pathological effects in the prostate are GABAAR-dependent, but are independent of the AR. In GABAAR-positive, AR-negative human PCa PC-3 cells, 3α-diol significantly stimulated cell growth in culture and the in ovo chorioallantoic membrane (CAM) xenograft model. 3α-diol also up-regulated expression of the epidermal growth factor (EGF) family of growth factors and activation of EGF receptor (EGFR) and Src as measured by quantitative polymerase chain reaction and immunoblotting, respectively. Inclusion of GABAAR antagonists reversed 3α-diol-stimulated tumor cell growth, expression of EGF family members, and activation of EGFR and Src to the level observed in untreated cells. Results from the present study suggest that 3α-diol may act as an alternative intra-prostatic neurosteroid that activates AR-independent PCa progression. The involvement of AKR1C3-mediated steroid metabolisms in modulating GABAAR activation and promoting PCa progression requires continued studies.
Asunto(s)
Miembro C3 de la Familia 1 de las Aldo-Ceto Reductasas/metabolismo , Anabolizantes/farmacología , Androstano-3,17-diol/farmacología , Neoplasias de la Próstata/patología , Receptores de GABA-A/metabolismo , Miembro C3 de la Familia 1 de las Aldo-Ceto Reductasas/genética , Proliferación Celular , Progresión de la Enfermedad , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Masculino , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Receptores de GABA-A/genética , Transducción de Señal , Células Tumorales CultivadasRESUMEN
Estrogen receptor beta (ERbeta) and androgen receptor (AR) are found in high levels within populations of neurons in the hypothalamus. To determine whether AR or ERbeta plays a role in regulating hypothalamo-pituitary-adrenal (HPA) axis function by direct action on these neurons, we examined the effects of central implants of 17beta-estradiol (E2), 5alpha-dihydrotestosterone (DHT), the DHT metabolite 5alpha-androstan-3beta, 17beta-diol (3beta-diol), and several ER subtype-selective agonists on the corticosterone and adrenocorticotropin (ACTH) response to immobilization stress. In addition, activation of neurons in the paraventricular nucleus (PVN) was monitored by examining c-fos mRNA expression. Pellets containing these compounds were stereotaxically implanted near the PVN of gonadectomized male rats. Seven days later, animals were killed directly from their home cage (nonstressed) or were restrained for 30 min (stressed) before they were killed. Compared with controls, E2 and the ERalpha-selective agonists moxestrol and propyl-pyrazole-triol significantly increased the stress induced release of corticosterone and ACTH. In contrast, central administration of DHT, 3beta-diol, and the ERbeta-selective compound diarylpropionitrile significantly decreased the corticosterone and ACTH response to immobilization. Cotreatment with the ER antagonist tamoxifen completely blocked the effects of 3beta-diol and partially blocked the effect of DHT, whereas the AR antagonist flutamide had no effect. Moreover, DHT, 3beta-diol, and diarylpropionitrile treatment significantly decreased restraint-induced c-fos mRNA expression in the PVN. Together, these studies indicate that the inhibitory effects of DHT on HPA axis activity may be in part mediated via its conversion to 3beta-diol and subsequent binding to ERbeta.
Asunto(s)
Andrógenos/farmacología , Androstano-3,17-diol/farmacología , Dihidrotestosterona/farmacología , Receptor beta de Estrógeno/metabolismo , Sistema Hipotálamo-Hipofisario/metabolismo , Núcleo Hipotalámico Paraventricular/metabolismo , Sistema Hipófiso-Suprarrenal/metabolismo , Antagonistas de Andrógenos/farmacología , Andrógenos/administración & dosificación , Andrógenos/metabolismo , Androstano-3,17-diol/administración & dosificación , Androstano-3,17-diol/antagonistas & inhibidores , Animales , Dihidrotestosterona/administración & dosificación , Dihidrotestosterona/antagonistas & inhibidores , Implantes de Medicamentos , Estradiol/farmacología , Antagonistas de Estrógenos/farmacología , Receptor beta de Estrógeno/agonistas , Flutamida/farmacología , Sistema Hipotálamo-Hipofisario/efectos de los fármacos , Inmovilización , Masculino , Neuronas/metabolismo , Núcleo Hipotalámico Paraventricular/citología , Núcleo Hipotalámico Paraventricular/enzimología , Sistema Hipófiso-Suprarrenal/efectos de los fármacos , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ratas , Ratas Sprague-Dawley , Estrés Fisiológico/metabolismo , Tamoxifeno/farmacologíaRESUMEN
Arginine vasopressin (AVP) is a neuropeptide involved in the regulation of fluid balance, stress, circadian rhythms, and social behaviors. In the brain, AVP is tightly regulated by gonadal steroid hormones in discrete regions with gonadectomy abolishing and testosterone replacement restoring normal AVP expression in adult males. Previous studies demonstrated that 17beta-estradiol, a primary metabolite of testosterone, is responsible for restoring most of the AVP expression in the brain after castration. However, 5alpha-dihydrotestosterone (DHT) has also been shown to play a role in the regulation of AVP expression, thus implicating the involvement of both androgen and estrogen receptors (ER). Furthermore, DHT, through its conversion to 5alpha-androstane-3beta,17beta-diol, has been shown to modulate estrogen response element-mediated promoter activity through an ER pathway. The present study addressed two central hypotheses: 1) that androgens directly modulate AVP promoter activity and 2) the effect is mediated by an estrogen or androgen receptor pathway. To that end, we overexpressed androgen receptor, ERbeta, and ERbeta splice variants in a neuronal cell line and measured AVP promoter activity using a firefly luciferase reporter assay. Our results demonstrate that DHT and its metabolite 5alpha-androstane-3beta,17beta-diol stimulate AVP promoter activity through ERbeta in a neuronal cell line.
Asunto(s)
Arginina Vasopresina/genética , Dihidrotestosterona/farmacología , Receptor beta de Estrógeno/genética , Neuronas/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Andrógenos/química , Andrógenos/metabolismo , Androstano-3,17-diol/farmacología , Arginina Vasopresina/metabolismo , Secuencia de Bases , Línea Celular Tumoral , Receptor beta de Estrógeno/metabolismo , Expresión Génica/efectos de los fármacos , Vectores Genéticos/genética , Humanos , Inmunohistoquímica , Luciferasas/genética , Luciferasas/metabolismo , Datos de Secuencia Molecular , Estructura Molecular , Mutagénesis Sitio-Dirigida , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
To gain insight into the mechanism by which steroidal hormones influence the development of canine prostatic hyperplasia, nuclear and cytosolic androgen- and estrogen-receptor content, as measured under exchange conditions by the binding of [(3)H]R1881 (methyltrienolone) and [(3)H]estradiol, respectively, were quantitated in the prostates of purebred beagles of known age. In young dogs with spontaneously arising and experimentally induced (androstanediol plus estradiol treatment) prostatic hyperplasia, nuclear, but not cytosolic, prostatic androgen-receptor content was significantly greater than that determined in the normal prostates of age-matched dogs (3,452+/-222 and 4,035+/-274 fmol/mg DNA vs. 2,096+/-364 fmol/mg DNA, respectively). No differences were observed between the androgen-receptor content of the normal prostates of young dogs and the hyperplastic prostates of old dogs. The cytosolic and nuclear estrogen-receptor content of spontaneously arising prostatic hyperplasia in both young and old animals was similar to that found in normal prostates. The administration of estradiol plus androstanediol to castrate dogs significantly increased the prostatic nuclear androgen-receptor content over that found in dogs treated only with androstanediol. This estradiol-associated increase in nuclear androgen-receptor content was accompanied by the development of benign prostatic hyperplasia. Estradiol treatment of castrate dogs resulted in an increase in prostatic nuclear estrogen-receptor content, in the appearance of a putative prostatic cytosolic progesterone receptor, and in an alteration of the epithelium of the prostate to one characterized by squamous metaplasia. Treatment of castrate dogs with both estradiol and androstanediol resulted in a reduction in prostatic nuclear estrogen-receptor content, disappearance of the progesterone receptor, and loss of squamous metaplasia. An increase in nuclear androgen-receptor content, thus, appears to be an important event in the development of both spontaneously arising and experimentally induced canine prostatic hyperplasia. The mechanism of androgen-estrogen synergism in the experimental induction of canine benign prostatic hyperplasia may be explained by estradiol-mediated increases in nuclear androgen-receptor content. Because androstanediol blocked certain estradiol-mediated events within the prostate, a negative feedback mechanism may exist in which the response of the canine prostate to estrogens is modulated by rising levels of androgen.