RESUMO
Many persistent organic pollutants (POPs) are suspected endocrine disruptors and it is important to investigate their effects at low concentrations relevant to human exposure. Here, the OECD test guideline #456 steroidogenesis assay was downscaled to a 96-well microplate format to screen 24 POPs for their effects on viability, and testosterone and estradiol synthesis using the human adrenocortical cell line H295R. The compounds (six polyfluoroalkyl substances, five organochlorine pesticides, ten polychlorinated biphenyls and three polybrominated diphenyl ethers) were tested at human-relevant levels (1 nM to 10 µM). Increased estradiol synthesis, above the OECD guideline threshold of 1.5-fold solvent control, was shown after exposure to 10 µM PCB-156 (153%) and PCB-180 (196%). Interestingly, the base hormone synthesis varied depending on the cell batch. An alternative data analysis using a linear mixed-effects model that include multiple independent experiments and considers batch-dependent variation was therefore applied. This approach revealed small but statistically significant effects on estradiol or testosterone synthesis for 17 compounds. Increased testosterone levels were demonstrated even at 1 nM for PCB-74 (18%), PCB-99 (29%), PCB-118 (16%), PCB-138 (19%), PCB-180 (22%), and PBDE-153 (21%). The MTT assay revealed significant effects on cell viability after exposure to 1 nM of perfluoroundecanoic acid (12%), 3 nM PBDE-153 (9%), and 10 µM of PCB-156 (6%). This shows that some POPs can interfere with endocrine signaling at concentrations found in human blood, highlighting the need for further investigation into the toxicological mechanisms of POPs and their mixtures at low concentrations relevant to human exposure.
Assuntos
Sobrevivência Celular , Disruptores Endócrinos , Poluentes Orgânicos Persistentes , Bifenilos Policlorados , Testosterona , Humanos , Testosterona/biossíntese , Testosterona/metabolismo , Poluentes Orgânicos Persistentes/metabolismo , Disruptores Endócrinos/toxicidade , Disruptores Endócrinos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Bifenilos Policlorados/toxicidade , Éteres Difenil Halogenados/toxicidade , Estradiol/metabolismo , Estrogênios , Linhagem Celular , Praguicidas/toxicidade , Hidrocarbonetos Clorados/toxicidadeRESUMO
Long-term use of anabolic androgenic steroids (AAS) in supratherapeutic doses is associated with severe adverse effects, including physical, mental, and behavioral alterations. When used for recreational purposes several AAS are often combined, and in scientific studies of the physiological impact of AAS either a single compound or a cocktail of several steroids is often used. Because of this, steroid-specific effects have been difficult to define and are not fully elucidated. The present study used male Wistar rats to evaluate potential somatic and behavioral effects of three different AAS; the decanoate esters of nandrolone, testosterone, and trenbolone. The rats were exposed to 15 mg/kg of nandrolone decanoate, testosterone decanoate, or trenbolone decanoate every third day for 24 days. Body weight gain and organ weights (thymus, liver, kidney, testis, and heart) were measured together with the corticosterone plasma levels. Behavioral effects were studied in the novel object recognition-test (NOR-test) and the multivariate concentric square field-test (MCSF-test). The results conclude that nandrolone decanoate, but neither testosterone decanoate nor trenbolone decanoate, caused impaired recognition memory in the NOR-test, indicating an altered cognitive function. The behavioral profile and stress hormone level of the rats were not affected by the AAS treatments. Furthermore, the study revealed diverse AAS-induced somatic effects i.e., reduced body weight development and changes in organ weights. Of the three AAS included in the study, nandrolone decanoate was identified to cause the most prominent impact on the male rat, as it affected body weight development, the weights of multiple organs, and caused an impaired memory function.
Assuntos
Anabolizantes , Transtornos da Memória , Nandrolona , Ratos Wistar , Testosterona , Animais , Masculino , Testosterona/sangue , Testosterona/análogos & derivados , Ratos , Nandrolona/análogos & derivados , Nandrolona/farmacologia , Anabolizantes/efeitos adversos , Anabolizantes/farmacologia , Transtornos da Memória/induzido quimicamente , Tamanho do Órgão/efeitos dos fármacos , Acetato de Trembolona/farmacologia , Decanoato de Nandrolona/farmacologia , Peso Corporal/efeitos dos fármacos , Corticosterona/sangue , Reconhecimento Psicológico/efeitos dos fármacosRESUMO
Angiotensin IV (Ang IV), a metabolite of Angiotensin II, is a bioactive hexapeptide that inhibits the insulin-regulated aminopeptidase (IRAP). This transmembrane zinc metallopeptidase with many biological functions has in recent years emerged as a new pharmacological target. IRAP is expressed in a variety of tissues and can be found in high density in the hippocampus and neocortex, brain regions associated with cognition. Ang IV is known to improve memory tasks in experimental animals. One of the most potent IRAP inhibitors known today is the macrocyclic compound HA08 that is significantly more stable than the endogenous Ang IV. HA08 combines structural elements from Ang IV and the physiological substrates oxytocin and vasopressin, and binds to the catalytic site of IRAP. In the present study we evaluate whether HA08 can restore cell viability in rat primary cells submitted to hydrogen peroxide damage. After damaging the cells with hydrogen peroxide and subsequently treating them with HA08, the conceivable restoring effects of the IRAP inhibitor were assessed. The cellular viability was determined by measuring mitochondrial activity and lactate dehydrogenase (LDH) release. The mitochondrial activity was significantly higher in primary hippocampal cells, whereas the amount of LDH was unaffected. We conclude that the cell viability can be restored in this cell type by blocking IRAP with the potent macrocyclic inhibitor HA08, although the mechanism by which HA08 exerts its effects remains unclear.
RESUMO
Studies indicate that phthalates are endocrine disruptors affecting reproductive health. One of the most commonly used phthalates, di-n-butyl phthalate (DBP), has been linked with adverse reproductive health outcomes in men, but the mechanisms behind these effects are still poorly understood. Here, adult male mice were orally exposed to DBP (10 or 100 mg/kg/day) for five weeks, and the testis and adrenal glands were collected one week after the last dose, to examine more persistent effects. Quantification of testosterone, androstenedione, progesterone and corticosterone concentrations by liquid chromatography-mass spectrometry showed that testicular testosterone was significantly decreased in both DBP treatment groups, whereas the other steroids were not significantly altered. Western blot analysis of testis revealed that DBP exposure increased the levels of the steroidogenic enzymes CYP11A1, HSD3ß2, and CYP17A1, the oxidative stress marker nitrotyrosine, and the luteinizing hormone receptor (LHR). The analysis further demonstrated increased levels of the germ cell marker DAZL, the Sertoli cell markers vimentin and SOX9, and the Leydig cell marker SULT1E1. Overall, the present work provides more mechanistic understanding of how adult DBP exposure can induce effects on the male reproductive system by affecting several key cells and proteins important for testosterone biosynthesis and spermatogenesis, and for the first time shows that these effects persist at least one week after the last dose. It also demonstrates impairment of testosterone biosynthesis at a lower dose than previously reported.
Assuntos
Dibutilftalato , Testículo , Animais , Dibutilftalato/metabolismo , Humanos , Células Intersticiais do Testículo/metabolismo , Masculino , Camundongos , Espermatogênese , Testículo/metabolismo , Testosterona/metabolismoRESUMO
Anabolic androgenic steroids (AAS) are frequently used to improve physical appearance and strength. AAS are known to affect muscle growth, but many AAS-users also experience psychiatric and behavioral changes after long-term use. The AAS-induced effects on the brain seem to depend on the type of steroid used, but the rationale behind the observed effect is still not clear. The present study investigated and compared the impact of nandrolone decanoate and testosterone undecanoate on body weight gain, levels of stress hormones, brain gene expression, and behavioral profiles in the male rat. The behavioral profile was determined using the multivariate concentric squared field test (MCSF-test). Blood plasma and brains were collected for further analysis using ELISA and qPCR. Nandrolone decanoate caused a reduction in body weight gain in comparison with both testosterone undecanoate and control. Rats receiving nandrolone decanoate also demonstrated decreased general activity in the MCSF. In addition, nandrolone decanoate reduced the plasma levels of ACTH in comparison with the control and increased the levels of corticosterone in comparison with testosterone undecanoate. The qPCR analysis revealed brain region-dependent changes in mRNA expression, where the hypothalamus was identified as the region most affected by the AAS. Alterations in neurotransmitter systems and stress hormones may contribute to the changes in behavior detected in the MCSF. In conclusion, both AAS affect the male rat, although, nandrolone decanoate has more pronounced impact on the physiological and the behavioral parameters measured.
Assuntos
Anabolizantes , Nandrolona , Anabolizantes/farmacologia , Animais , Peso Corporal , Masculino , Nandrolona/farmacologia , Decanoato de Nandrolona , Neurotransmissores/farmacologia , Ratos , Testosterona/análogos & derivados , Testosterona/farmacologiaRESUMO
The important role of mitochondria in maintaining normal brain cell function has been demonstrated in several neurodegenerative diseases where mitochondrial dysfunction is a prominent feature. Accumulating evidence indicates that opioids may induce neuronal cell death and inhibit neurogenesis, two factors that are dependent on normal mitochondrial function. The aim of the present study was to examine the effects of morphine, methadone, and fentanyl on MitoTracker-stained mitochondria. Cells from the neuroblastoma/glioma hybrid cell-line NG108-15 were seeded on 96-well cell culture plates and treated with MitoTracker for 30 min prior to opioid treatment. Morphine, methadone, and fentanyl were added at various concentrations and images of mitochondria were acquired every 30 min for four hours using a high-content imaging device. The parameters total mitochondrial area, mitochondrial network, as well as the number and mean area of mitochondrial objects were analyzed using automated image analysis. Methadone and fentanyl, but not morphine, decreased the mitochondrial network, the number of mitochondrial objects, and increased the mean area of mitochondrial objects. Both methadone and fentanyl altered mitochondrial morphology with no effects seen from morphine treatment. These data suggest that methadone and fentanyl impact mitochondrial morphology negatively, which may be associated with neuronal cell death.
Assuntos
Fentanila/farmacologia , Metadona/farmacologia , Mitocôndrias/efeitos dos fármacos , Morfina/farmacologia , Entorpecentes/farmacologia , Animais , Linhagem Celular Tumoral , Camundongos , Ratos , Imagem com Lapso de TempoRESUMO
The illicit use of anabolic androgenic steroids (AAS) among adolescents and young adults is a major concern due to the unknown and unpredictable impact of AAS on the developing brain and the consequences of this on mental health, cognitive function and behaviour. The present study aimed to investigate the effects of supra-physiological doses of four structurally different AAS (testosterone, nandrolone, stanozolol and trenbolone) on neurite development and cell viability using an in vitro model of immature primary rat cortical cell cultures. A high-throughput screening image-based approach, measuring the neurite length and number of neurons, was used for the analysis of neurite outgrowth. In addition, cell viability and expression of the Tubb3 gene (encoding the protein beta-III tubulin) were investigated. Testosterone, nandrolone, and trenbolone elicited adverse effects on neurite outgrowth as deduced from an observed reduced neurite length per neuron. Trenbolone was the only AAS that reduced the cell viability as indicated by a decreased number of neurons and declined mitochondrial function. Moreover, trenbolone downregulated the Tubb3 mRNA expression. The adverse impact on neurite development was neither inhibited nor supressed by the selective androgen receptor (AR) antagonist, flutamide, suggesting that the observed effects result from another mechanism or mechanisms of action that are operating apart from AR activation. The results demonstrate a possible AAS-induced detrimental effect on neuronal development and regenerative functions. An impact on these events, that are essential mechanisms for maintaining normal brain function, could possibly contribute to behavioural alterations seen in AAS users.
Assuntos
Anabolizantes/química , Anabolizantes/farmacologia , Córtex Cerebral/citologia , Crescimento Neuronal/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Animais , Sobrevivência Celular/efeitos dos fármacos , Córtex Cerebral/embriologia , Relação Dose-Resposta a Droga , Feminino , Nandrolona/química , Nandrolona/farmacologia , Neurônios/metabolismo , Cultura Primária de Células , Ratos Wistar , Receptores Androgênicos/metabolismo , Estanozolol/química , Estanozolol/farmacologia , Testosterona/química , Testosterona/farmacologia , Acetato de Trembolona/química , Acetato de Trembolona/farmacologia , Tubulina (Proteína)/genéticaRESUMO
Although the number of studies that have examined the impact of opioids on cell viability is very limited, it has clearly shown that opioids commonly used in the clinic can both decrease neurogenesis and induce cell death. These negative effects induced by opioids are worrying and there is a need for further in-depth investigations addressing the impact of opioids on cell function and cell viability. A useful in vitro approach for studying the effects of opioids on cellular function and viability is using primary cortical cell cultures obtained from embryonic day 17 (E17) rat embryos. These cell cultures contain both neurons and glial cells that provide a more physiologically relevant culture condition when compared to the use of various commercially available cell lines. The primary cortical cells can be cultivated in 96-well plates, treated with various concentrations of opioids, and cell viability functions such as mitochondrial function and membrane integrity can easily be assessed using specific colorimetric assays.
Assuntos
Analgésicos Opioides/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Cultura Primária de Células/métodos , Analgésicos Opioides/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Células Cultivadas/metabolismo , Córtex Cerebral/citologia , Embrião de Mamíferos/metabolismo , Mitocôndrias/fisiologia , Neuroglia/metabolismo , Neurônios/metabolismo , RatosRESUMO
OBJECTIVE: Growth hormone (GH) is widely known for its peripheral effects during growth and development. However, numerous reports also suggest that GH exert pro-cognitive, restorative, and protective properties in the brain. In in vitro studies, the detection of dendritic spines, small protrusions extending from axons, can act as a marker for cognition-related function as spine formation is considered to be associated with learning and memory. Here we show that an acute 24-hour treatment of GH can increase dendritic spine density in primary hippocampal cell cultures. DESIGN: Primary hippocampal cells were harvested from embryonic Wistar rats and cultured for 14 days. Cells were treated with supra-physiological doses of GH (10-1000 nM) and subjected to a high-throughput screening protocol. Images were acquired and analyzed using automated image analysis and the number of spines, spines per neurite length, neurite length, and mean area of spines, was reported. RESULTS: GH treatment (1000 nM) increased the number of dendritic spines by 83% and spines per neurite length by 82% when compared to control. For comparison BDNF, a known inducer of spine densities, produced statistically non-significant increase in this setting. CONCLUSION: The results was found significant using the highest supra-physiological dose of GH, and the present study further confirms a potential role of the hormone in the treatment of cognitive dysfunction.
Assuntos
Espinhas Dendríticas/efeitos dos fármacos , Hormônio do Crescimento/farmacologia , Hipocampo/citologia , Neuritos/efeitos dos fármacos , Animais , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Ensaios de Triagem em Larga Escala , Técnicas In Vitro , Cultura Primária de Células , RatosRESUMO
The use of anabolic androgenic steroids (AASs) among non-athletes is a public health-problem, as abusers underestimate the negative effects associated with these drugs. The present study investigated the toxic effects of testosterone, nandrolone, stanozolol, and trenbolone, and aimed to understand how AAS abuse affects the brain. Mixed cortical cultures from embryonic rats were grown in vitro for 7â¯days and thereafter treated with increasing concentrations of AASs for 24â¯h (single-dose) or 3â¯days (repeated exposure). Cells were co-treated with the androgen-receptor (AR) antagonist flutamide, to determine whether the potential adverse effects observed were mediated by the AR. Cellular toxicity was determined by measuring mitochondrial activity, lactate dehydrogenase (LDH) release, and caspase-3/7 activity. Nandrolone, unlike the other AASs studied, indicated an effect on mitochondrial activity after 24â¯h. Furthermore, single-dose exposure with testosterone, nandrolone and trenbolone increased LDH release, while no effect was detected with stanozolol. However, all of the four steroids negatively affected mitochondrial function and resulted in LDH release after repeated exposure. Testosterone, nandrolone, and trenbolone caused their toxic effects by induction of apoptosis, unlike stanozolol that seemed to induce necrosis. Flutamide almost completely prevented AAS-induced toxicity by maintaining mitochondrial function, cellular integrity, and inhibition of apoptosis. Overall, we found that supra-physiological concentrations of AASs induce cell death in mixed primary cortical cultures, but to different extents, and possibly through various mechanisms. The data presented herein suggest that the molecular interactions of the AASs with the AR are primarily responsible for the toxic outcomes observed.
Assuntos
Anabolizantes/toxicidade , Androgênios/toxicidade , Morte Celular/efeitos dos fármacos , Córtex Cerebral/efeitos dos fármacos , Anabolizantes/química , Antagonistas de Receptores de Andrógenos/farmacologia , Androgênios/química , Animais , Morte Celular/fisiologia , Células Cultivadas , Córtex Cerebral/metabolismo , Relação Dose-Resposta a Droga , Flutamida/farmacologia , Estrutura Molecular , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Cultura Primária de Células , Ratos Sprague-Dawley , Ratos Wistar , Receptores Androgênicos/metabolismo , Fatores de TempoRESUMO
Evidence to date suggests that opioids such as methadone may be associated with cognitive impairment. Growth hormone (GH) and insulin-like growth factor-1 (IGF-1) are suggested to be neuroprotective and procognitive in the brain and may therefore counteract these effects. This study aims to explore the protective and restorative effects of GH and IGF-1 in methadone-treated cell cultures. Primary cortical cell cultures were harvested from rat fetuses and grown for seven days in vitro. To examine the protective effects, methadone was co-treated with or without GH or IGF-1 for three consecutive days. To examine the restorative effects, methadone was added for the first 24 h, washed, and later treated with GH or IGF-1 for 48 h. At the end of each experiment, mitochondrial function and membrane integrity were evaluated. The results revealed that GH had protective effects in the membrane integrity assay and that both GH and IGF-1 effectively recovered mitochondrial function and membrane integrity in cells pretreated with methadone. The overall conclusion of the present study is that GH, but not IGF-1, protects primary cortical cells against methadone-induced toxicity, and that both GH and IGF-1 have a restorative effect on cells pretreated with methadone.
Assuntos
Hormônio do Crescimento Humano/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Metadona/toxicidade , Substâncias Protetoras/farmacologia , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Ratos Wistar , Proteínas Recombinantes/farmacologiaRESUMO
Steroids are reported to have diverse functions in the nervous system. Enzymatic production of steroid hormones has been reported in different cell types, including astrocytes and neurons. However, the information on some of the steroidogenic enzymes involved is insufficient in many respects. Contradictory results have been reported concerning the relative importance of different cell types in the nervous system for expression of CYP17A1 and 3ß-hydroxysteroid dehydrogenase (3ß-HSD). 3ß-HSD is important in all basic steroidogenic pathways and CYP17A1 is required to form sex hormones. In the current investigation we studied the expression of these enzymes in cultured primary rat astrocytes, in neuron-enriched cells from rat cerebral cortex and in human neuroblastoma SH-SY5Y cells, a cell line often used as an in vitro model of neuronal function and differentiation. As part of this study we also examined potential effects on CYP17A1 and 3ß-HSD by vitamin D, a compound previously shown to have regulatory effects in steroid hormone-producing cells outside the brain. The results of our study indicate that astrocytes are a major site for expression of 3ß-HSD whereas expression of CYP17A1 is found in both astrocytes and neurons. The current data suggest that neurons, contrary to some previous reports, are not involved in 3ß-HSD reactions. Previous studies have shown that vitamin D can influence gene expression and hormone production by steroidogenic enzymes in some cells. We found that vitamin D suppressed CYP17A1-mediated activity by 20% in SH-SY5Ycells and astrocytes. Suppression of CYP17A1 mRNA levels was considerably stronger, about 50% in SH-SY5Y cells and 75% in astrocytes. In astrocytes 3ß-HSD was also suppressed by vitamin D, about 20% at the enzyme activity level and 60% at the mRNA level. These data suggest that vitamin D-mediated regulation of CYP17A1 and 3ß-HSD, particularly on the transcriptional level, may play a role in the nervous system.
Assuntos
17-Hidroxiesteroide Desidrogenases/biossíntese , Encéfalo/enzimologia , Regulação Enzimológica da Expressão Gênica , Esteroide 17-alfa-Hidroxilase/biossíntese , Esteroides/biossíntese , Vitamina D/farmacologia , 17-Hidroxiesteroide Desidrogenases/antagonistas & inibidores , 17-Hidroxiesteroide Desidrogenases/genética , Animais , Encéfalo/efeitos dos fármacos , Linhagem Celular Tumoral , Células Cultivadas , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Ratos , Ratos Sprague-Dawley , Esteroide 17-alfa-Hidroxilase/antagonistas & inibidores , Esteroide 17-alfa-Hidroxilase/genética , Esteroides/antagonistas & inibidoresRESUMO
Human growth hormone (GH) displays promising protective effects in the central nervous system after damage caused by various insults. Current evidence suggests that these effects may involve N-methyl-d-aspartate (NMDA) receptor function, a receptor that also is believed to play a role in opioid-induced neurotoxicity. The aims of the present study were to examine the acute toxic effects of methadone, an opioid receptor agonist and NMDA receptor antagonist, as well as to evaluate the protective properties of recombinant human GH (rhGH) on methadone-induced toxicity. Primary cortical cell cultures from embryonic day 17 rats were grown for 7days in vitro. Cells were treated with methadone for 24h and the 50% lethal dose was calculated and later used for protection studies with rhGH. Cellular toxicity was determined by measuring mitochondrial activity, lactate dehydrogenase release, and caspase activation. Furthermore, the mRNA expression levels of NMDA receptor subunits were investigated following methadone and rhGH treatment using quantitative PCR (qPCR) analysis. A significant protective effect was observed with rhGH treatment on methadone-induced mitochondrial dysfunction and in methadone-induced LDH release. Furthermore, methadone significantly increased caspase-3 and -7 activation but rhGH was unable to inhibit this effect. The mRNA expression of the NMDA receptor subunit GluN1, GluN2a, and GluN2b increased following methadone treatment, as assessed by qPCR, and rhGH treatment effectively normalized this expression to control levels. We have demonstrated that rhGH can rescue cells from methadone-induced toxicity by maintaining mitochondrial function, cellular integrity, and NMDA receptor complex expression.
Assuntos
Hormônio do Crescimento Humano/farmacologia , Metadona/toxicidade , Fármacos Neuroprotetores/farmacologia , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Caspase 3/metabolismo , Caspase 7/metabolismo , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/patologia , Córtex Cerebral/fisiopatologia , Maleato de Dizocilpina/toxicidade , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , L-Lactato Desidrogenase/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Morfina/toxicidade , Naloxona/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Neurônios/fisiologia , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Proteínas Recombinantes/farmacologiaRESUMO
The zinc metallopeptidase insulin regulated aminopeptidase (IRAP), which is highly expressed in the hippocampus and other brain regions associated with cognitive function, has been identified as a high-affinity binding site of the hexapeptide angiotensin IV (Ang IV). This hexapeptide is thought to facilitate learning and memory by binding to the catalytic site of IRAP to inhibit its enzymatic activity. In support of this hypothesis, low molecular weight, nonpeptide specific inhibitors of IRAP have been shown to enhance memory in rodent models. Recently, it was demonstrated that linear and macrocyclic Ang IV-derived peptides can alter the shape and increase the number of dendritic spines in hippocampal cultures, properties associated with enhanced cognitive performance. After screening a library of 10â¯500 drug-like substances for their ability to inhibit IRAP, we identified a series of low molecular weight aryl sulfonamides, which exhibit no structural similarity to Ang IV, as moderately potent IRAP inhibitors. A structural and biological characterization of three of these aryl sulfonamides was performed. Their binding modes to human IRAP were explored by docking calculations combined with molecular dynamics simulations and binding affinity estimations using the linear interaction energy method. Two alternative binding modes emerged from this analysis, both of which correctly rank the ligands according to their experimental binding affinities for this series of compounds. Finally, we show that two of these drug-like IRAP inhibitors can alter dendritic spine morphology and increase spine density in primary cultures of hippocampal neurons.
Assuntos
Cistinil Aminopeptidase/antagonistas & inibidores , Espinhas Dendríticas/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Hipocampo/citologia , Sulfonamidas/farmacologia , Animais , Antígenos CD13/metabolismo , Células Cultivadas , Técnicas de Cocultura , Cistinil Aminopeptidase/metabolismo , Espinhas Dendríticas/enzimologia , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/síntese química , Epóxido Hidrolases/genética , Epóxido Hidrolases/metabolismo , Células HEK293 , Hipocampo/efeitos dos fármacos , Hipocampo/enzimologia , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Ligação Proteica , Ratos Sprague-Dawley , Proteínas Recombinantes/genética , Sulfonamidas/síntese químicaRESUMO
Anabolic androgenic steroids (AAS) are substances that mimic the hormone testosterone, and primarily act via the androgen receptor. In addition to their physiological effect on muscle tissue and growth, research from the last decade has shown that AAS have a pronounced impact on the central nervous system. A large number of studies have demonstrated that AAS affect the mesolimbic reward system in the brain. However, whether the direct effects of AAS on endorphins, dopamine, serotonin and GABA etc. and on the corresponding and related systems lead to dependence needs to be further elucidated. According to recent studies, the prevalence of AAS dependence among AAS users has been estimated to be approximately 30%, and polysubstance use, of both pharmaceutical drugs and narcotics, within this group is common. The present review primarily discusses AAS in the context of addiction and dependence, and further addresses the issue of using multiple substances, i.e. stimulants and opiates in combination with AAS. In addition, aspects of the treatment of AAS dependence, the connection between AAS abuse and cognition, and AAS-induced neurotoxicity are presented. Currently, performance enhancing drugs are frequently used in combination with AAS. Therefore, a large section on growth hormone and insulin-like growth factor is also included.
Assuntos
Hormônio do Crescimento/uso terapêutico , Neurobiologia , Esteroides/efeitos adversos , Transtornos Relacionados ao Uso de Substâncias/tratamento farmacológico , Transtornos Relacionados ao Uso de Substâncias/etiologia , Anabolizantes/efeitos adversos , Androgênios/efeitos adversos , Animais , HumanosRESUMO
Angiotensin IV (Ang IV) and related peptide analogs, as well as nonpeptide inhibitors of insulin-regulated aminopeptidase (IRAP), have previously been shown to enhance memory and cognition in animal models. Furthermore, the endogenous IRAP substrates oxytocin and vasopressin are known to facilitate learning and memory. In this study, the two recently synthesized 13-membered macrocyclic competitive IRAP inhibitors HA08 and HA09, which were designed to mimic the N terminus of oxytocin and vasopressin, were assessed and compared based on their ability to bind to the IRAP active site, and alter dendritic spine density in rat hippocampal primary cultures. The binding modes of the IRAP inhibitors HA08, HA09, and of Ang IV in either the extended or γ-turn conformation at the C terminus to human IRAP were predicted by docking and molecular dynamics simulations. The binding free energies calculated with the linear interaction energy method, which are in excellent agreement with experimental data and simulations, have been used to explain the differences in activities of the IRAP inhibitors, both of which are structurally very similar, but differ only with regard to one stereogenic center. In addition, we show that HA08, which is 100-fold more potent than the epimer HA09, can enhance dendritic spine number and alter morphology, a process associated with memory facilitation. Therefore, HA08, one of the most potent IRAP inhibitors known today, may serve as a suitable starting point for medicinal chemistry programs aided by MD simulations aimed at discovering more drug-like cognitive enhancers acting via augmenting synaptic plasticity.
Assuntos
Cistinil Aminopeptidase/antagonistas & inibidores , Cistinil Aminopeptidase/metabolismo , Espinhas Dendríticas/metabolismo , Dissulfetos/metabolismo , Compostos Macrocíclicos/metabolismo , Animais , Células Cultivadas , Cristalografia , Cistinil Aminopeptidase/análise , Espinhas Dendríticas/química , Dissulfetos/farmacologia , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Feminino , Células HEK293 , Humanos , Compostos Macrocíclicos/farmacologia , Gravidez , Ligação Proteica/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
One of the main treatment challenges in alcohol use disorder (AUD) is the high rate of craving in combination with decreased cognitive functioning including impaired decision making and impulse control that often lead to relapse. Recent studies show that guanfacine, an α-2-adrenoceptor agonist and FDA-approved ADHD medication, attenuates stress-induced relapse of several drugs of abuse including alcohol. Here we evaluated guanfacine's effects on voluntary alcohol intake, the alcohol deprivation effect (ADE), alcohol seeking behavior, and cue/priming-induced reinstatement in Wistar rats that had voluntarily consumed alcohol for at least 2 months before treatment. In addition, guanfacine's ability to regulate glutamatergic neurotransmission was evaluated through electrophysiological recordings in medial prefrontal cortex (mPFC) slices prepared from long-term drinking rats (and alcohol-naive controls) that had received three daily guanfacine (0.6 mg/kg/day) or vehicle injections in vivo. Guanfacine decreased alcohol intake in high, but not low, alcohol-consuming rats and the effects were generally more long lasting than that of the AUD medication naltrexone. Repeated guanfacine treatment induced a long-lasting decrease in alcohol intake, persistent up to five drinking sessions after the last injection. In addition, guanfacine attenuated the ADE as well as alcohol seeking and cue/priming-induced reinstatement of alcohol seeking. Finally, subchronic guanfacine treatment normalized an alcohol-induced dysregulated glutamatergic neurotransmission in the mPFC. These results support previous studies showing that guanfacine has the ability to improve prefrontal connectivity through modulation of the glutamatergic system. Together with the fact that guanfacine appears to be clinically safe, these results merit evaluation of guanfacine's clinical efficacy in AUD individuals.