RESUMO
The endocrine system is responsible for growth, development, maintaining homeostasis and for the control of many physiological processes. Due to the integral nature of its signaling pathways, it can be difficult to distinguish endocrine-mediated adverse effects from transient fluctuations, adaptive/compensatory responses, or adverse effects on the endocrine system that are caused by mechanisms outside the endocrine system. This is particularly true in toxicological studies that require generation of effects through the use of Maximum Tolerated Doses (or Concentrations). Endocrine-mediated adverse effects are those that occur as a consequence of the interaction of a chemical with a specific molecular component of the endocrine system, for example, a hormone receptor. Non-endocrine-mediated adverse effects on the endocrine system are those that occur by other mechanisms. For example, systemic toxicity, which perturbs homeostasis and affects the general well-being of an organism, can affect endocrine signaling. Some organs/tissues can be affected by both endocrine and non-endocrine signals, which must be distinguished. This paper examines in vitro and in vivo endocrine endpoints that can be altered by non-endocrine processes. It recommends an evaluation of these issues in the assessment of effects for the determination of endocrine disrupting properties of chemicals. This underscores the importance of using a formal weight of evidence (WoE) process to evaluate potential endocrine activity.
Assuntos
Disruptores Endócrinos/farmacologia , Disruptores Endócrinos/uso terapêutico , Sistema Endócrino/diagnóstico por imagem , Animais , Humanos , Medição de RiscoRESUMO
The US Environmental Protection Agency Endocrine Disruptor Screening Program (EDSP) is a tiered screening approach to determine the potential for a chemical to interact with estrogen, androgen, or thyroid hormone systems and/or perturb steroidogenesis. Use of high-throughput screening (HTS) to predict hazard and exposure is shifting the EDSP approach to (1) prioritization of chemicals for further screening; and (2) targeted use of EDSP Tier 1 assays to inform specific data needs. In this work, toxicology data for three triazole fungicides (triadimefon, propiconazole, and myclobutanil) were evaluated, including HTS results, EDSP Tier 1 screening (and other scientifically relevant information), and EPA guideline mammalian toxicology study data. The endocrine-related bioactivity predictions from HTS and information that satisfied the EDSP Tier 1 requirements were qualitatively concordant. Current limitations in the available HTS battery for thyroid and steroidogenesis pathways were mitigated by inclusion of guideline toxicology studies in this analysis. Similar margins (3-5 orders of magnitude) were observed between HTS-predicted human bioactivity and exposure values and between in vivo mammalian bioactivity and EPA chronic human exposure estimates for these products' registered uses. Combined HTS hazard and human exposure predictions suggest low priority for higher-tiered endocrine testing of these triazoles. Comparison with the mammalian toxicology database indicated that this HTS-based prioritization would have been protective for any potential in vivo effects that form the basis of current risk assessment for these chemicals. This example demonstrates an effective, human health protective roadmap for EDSP evaluation of pesticide active ingredients via prioritization using HTS and guideline toxicology information.
Assuntos
Disruptores Endócrinos/toxicidade , Fungicidas Industriais/toxicidade , Testes de Toxicidade/métodos , Triazóis/toxicidade , Bioensaio , Disruptores Endócrinos/classificação , Disruptores Endócrinos/normas , Fungicidas Industriais/classificação , Fungicidas Industriais/normas , Nitrilas/toxicidade , Triazóis/classificação , Triazóis/normas , Estados UnidosRESUMO
Atrazine (ATZ) was administered daily by gavage to pregnant female Sprague Dawley rats at doses of 0, 6.25, 25 or 50 mg/kg/day, either during gestation, lactation and post-weaning (G/L/PW cohort) to F1 generation female offspring or only from postnatal day (PND 21) until five days after sexual maturation (vaginal opening) when the estrogen-primed, luteinizing hormone (LH) surge was evaluated (PW cohort). Additional subgroups of F1 females received the vehicle or ATZ from PND 21-133 or from PND 120-133. Slight reductions in fertility and the percentage of F1 generation pups surviving to PND 21 in the gestationally exposed 50 mg/kg dose group were accompanied by decreased food intake and body weight of dams and F1 generation offspring. The onset of puberty was delayed in of the F1 generation G/L/PW females at doses of 25 and 50 mg/kg/day. F1 generation females in the PW high-dose ATZ group also experienced a delay in the onset of puberty. ATZ had no effect on peak LH or LH AUC in ovariectomized rats 5 days after sexual maturation, irrespective of whether the F1 generation females were treated from gestation onward or only peripubertally. There was no effect of ATZ treatment on the estrous cycle, peak LH or LH AUC of F1 generation females exposed from gestation through to PND 133 or only for two weeks from PND 120-133. These results indicate that developing females exposed to ATZ are not more sensitive compared to animals exposed to ATZ as young adults.
Assuntos
Envelhecimento/efeitos dos fármacos , Atrazina/toxicidade , Exposição Ambiental , Hormônio Luteinizante/metabolismo , Maturidade Sexual/efeitos dos fármacos , Animais , Peso Corporal/efeitos dos fármacos , Cruzamentos Genéticos , Estradiol/farmacologia , Ciclo Estral/efeitos dos fármacos , Comportamento Alimentar/efeitos dos fármacos , Feminino , Ratos , Ratos Sprague-Dawley , Análise de Sobrevida , Fatores de TempoRESUMO
Atrazine (ATR) blunts the hormone-induced luteinizing hormone (LH) surge, when administered by gavage (50-100 mg/kg/day for 4 days), in ovariectomized rats. In this study, we determined if comparable doses delivered either by gavage (bolus dose) or distributed in diet would reduce the LH surge and subsequently affect fertility in the intact female rat. ATR was administered daily to intact female Sprague-Dawley (SD) or Long Evans (LE) rats by gavage (0, 0.75 1.5, 3, 6, 10, 12, 50, or 100 mg/kg/day) or diet (0, 30, 100, 160, 500, 660, or 1460 ppm) during one complete 4-day estrous cycle, starting on day of estrus. Estrous status, corpora lutea, ova, and LH plasma concentrations were evaluated. A second cohort of animals was mated on the fourth treatment day. Fertility metrics were assessed on gestational day 20. A higher portion of LE rats had asynchronous estrous cycles when compared to SD rats both during pretreatment and in response to ATR (≥50 mg/kg). In contrast, bolus doses of ATR (≥50 mg/kg) inhibited the peak and area under the curve for the preovulatory LH surge in SD but not LE animals. Likewise, only bolus-treated SD, not LE, rats displayed reduced mean number of corpora lutea and ova. There were no effects of ATR administered by gavage on mating, gravid number, or fetus number. Dietary administration had no effect on any reproductive parameter measured. These findings indicate that short duration, high-bolus doses of ATR can inhibit the LH surge and reduce the number of follicles ovulated; however, dietary administration has no effect on any endocrine or reproductive outcomes.
Assuntos
Atrazina/toxicidade , Hormônio Luteinizante/sangue , Reprodução/efeitos dos fármacos , Animais , Atrazina/administração & dosagem , Atrazina/sangue , Dieta , Relação Dose-Resposta a Droga , Ciclo Estral/efeitos dos fármacos , Feminino , Herbicidas/administração & dosagem , Herbicidas/toxicidade , Ratos , Ratos Long-Evans , Ratos Sprague-DawleyRESUMO
Although many of the effects of estrogens on the brain are mediated through estrogen receptors (ERs), there is evidence that neuroprotective activity of estrogens can be mediated by non-ER mechanisms. Herein, we review the substantial evidence that estrogens neuroprotection is in large part non-ER mediated and describe in vitro and in vivo studies that support this conclusion. Also, we described our drug discovery strategy for capitalizing on enhancement in neuroprotection while at the same time, reducing ER binding of a group of synthetic non-feminizing estrogens. Finally, we offer evidence that part of the neuroprotection of these non-feminizing estrogens is due to enhancement in redox potential of the synthesized compounds.
Assuntos
Citoproteção/efeitos dos fármacos , Congêneres do Estradiol/farmacologia , Feminização/prevenção & controle , Fármacos Neuroprotetores/farmacologia , Animais , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Congêneres do Estradiol/uso terapêutico , Feminino , Feminização/induzido quimicamente , Ataxia de Friedreich/tratamento farmacológico , Ataxia de Friedreich/patologia , Humanos , Masculino , Modelos Biológicos , Fármacos Neuroprotetores/uso terapêuticoRESUMO
Mitochondria have become a primary focus in our search not only for the mechanism(s) of neuronal death but also for neuroprotective drugs and therapies that can delay or prevent Alzheimer's disease and other chronic neurodegenerative conditions. This is because mitochrondria play a central role in regulating viability and death of neurons, and mitochondrial dysfunction has been shown to contribute to neuronal death seen in neurodegenerative diseases. In this article, we review the evidence for the role of mitochondria in cell death and neurodegeneration and provide evidence that estrogens have multiple effects on mitochondria that enhance or preserve mitochondrial function during pathologic circumstances such as excitotoxicity, oxidative stress, and others. As such, estrogens and novel non-hormonal analogs have come to figure prominently in our efforts to protect neurons against both acute brain injury and chronic neurodegeneration.
Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/prevenção & controle , Estrogênios/uso terapêutico , Mitocôndrias/metabolismo , Neurônios/metabolismo , Fármacos Neuroprotetores/uso terapêutico , Animais , Lesões Encefálicas/tratamento farmacológico , Lesões Encefálicas/metabolismo , Lesões Encefálicas/patologia , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Doença Crônica , Humanos , Mitocôndrias/patologia , Neurônios/patologia , Estresse Oxidativo/efeitos dos fármacosRESUMO
L-type voltage-gated Ca(2+)channels (VGCC) play an important role in dendritic development, neuronal survival, and synaptic plasticity. Recent studies have demonstrated that the gonadal steroid estrogen rapidly induces Ca(2+) influx in hippocampal neurons, which is required for neuroprotection and potentiation of LTP. The mechanism by which estrogen rapidly induces this Ca(2+) influx is not clearly understood. We show by electrophysiological studies that extremely low concentrations of estrogens acutely potentiate VGCC in hippocampal neurons, hippocampal slices, and HEK-293 cells transfected with neuronal L-type VGCC, in a manner that was estrogen receptor (ER)-independent. Equilibrium, competitive, and whole-cell binding assays indicate that estrogen directly interacts with the VGCC. Furthermore, a L-type VGCC antagonist to the dihydropyridine site displaced estrogen binding to neuronal membranes, and the effects of estrogen were markedly attenuated in a mutant, dihydropyridine-insensitive L-type VGCC, demonstrating a direct interaction of estrogens with L-type VGCC. Thus, estrogen-induced potentiation of calcium influx via L-type VGCC may link electrical events with rapid intracellular signaling seen with estrogen exposure leading to modulation of synaptic plasticity, neuroprotection, and memory formation.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Estrogênios/metabolismo , Neurônios/metabolismo , Animais , Canais de Cálcio Tipo L/genética , Linhagem Celular , Estradiol/metabolismo , Estradiol/farmacologia , Estrogênios/farmacologia , Humanos , Mutação , Neurônios/efeitos dos fármacos , Ratos , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
In the present treatise, we provide evidence that the neuroprotective and mito-protective effects of estrogens are inexorably linked and involve the ability of estrogens to maintain mitochondrial function during neurotoxic stress. This is achieved by the induction of nuclear and mitochondrial gene expression, the maintenance of protein phosphatases levels in a manner that likely involves modulation of the phosphorylation state of signaling kinases and mitochondrial pro- and anti-apoptotic proteins, and the potent redox/antioxidant activity of estrogens. These estrogen actions are mediated through a combination of estrogens receptor (ER)-mediated effects on nuclear and mitochondrial transcription of protein vital to mitochondrial function, ER-mediated, non-genomic signaling and non-ER-mediated effects of estrogens on signaling and oxidative stress. Collectively, these multifaceted, coordinated action of estrogens leads to their potency in protecting neurons from a wide variety of acute insults as well as chronic neurodegenerative processes.
Assuntos
Estrogênios/metabolismo , Mitocôndrias/metabolismo , Fármacos Neuroprotetores/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Animais , Calcineurina/metabolismo , Retículo Endoplasmático/metabolismo , Ativação Enzimática , Regulação da Expressão Gênica , Humanos , Toxinas Marinhas , Mitocôndrias/genética , Degeneração Neural/metabolismo , Oxazóis/metabolismo , Oxirredução , Estresse Oxidativo , Fosfoproteínas Fosfatases/antagonistas & inibidores , Transdução de Sinais/fisiologiaRESUMO
Serine/threonine protein phosphatases are important mediators of general cellular function as well as neurodegenerative processes. We have previously shown inhibition of protein phosphatases to be as neurotoxic as glutamate-induced neuronal death but resistant to neuroprotection by estrogens. In this study, the mechanism by which phosphatase inhibition via okadaic acid (OA) induced neurotoxicity is explored. Neurons were exposed to OA or glutamate in the presence or absence of various protein kinases inhibitors, and/or one of four estrogens. Both OA and glutamate induced cell death via increased reactive oxygen species, protein carbonylation, lipid peroxidation, caspase-3 activity, and mitochondrial dysfunction. All estrogens attenuated glutamate-mediated responses, but not OA-induced responses. In addition, inhibition of protein kinase C and mitogen-activated protein kinase pathway was neuroprotective against glutamate but not OA toxicity. Interestingly, inhibition of mitogen-activated protein kinase pathway with PD98096 or U0126 caused a decrease in reactive oxygen species production suggesting that activation of ERK1/2 could further exacerbate the oxidative stress caused by glutamate-induced toxicity; however, these inhibitors had no effect on OA-induced toxicity. Collectively, these results indicate that both glutamate and OA neurotoxicities are mediated by persistent activation of ERK1/2 and/or protein kinase C and a resulting oxidative stress, and that protein phosphatase activity is an important and necessary aspect of estrogen-mediated neuroprotection.
Assuntos
Morte Celular/efeitos dos fármacos , Estrogênios/farmacologia , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores , Ácido Okadáico/toxicidade , Animais , Caspase 3/metabolismo , Caspase 7/metabolismo , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Citosol/efeitos dos fármacos , Citosol/metabolismo , Inibidores Enzimáticos/farmacologia , Feminino , Ácido Glutâmico/toxicidade , Peroxidação de Lipídeos/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Gravidez , Carbonilação Proteica/efeitos dos fármacos , Ratos , Espécies Reativas de Oxigênio , Sais de Tetrazólio , TiazóisRESUMO
It is becoming increasingly clear that protein phosphatases are important modulators of cellular function and that disruption of these proteins are involved in neurodegenerative disease processes. Serine/threonine protein phosphatases (PP) such as protein phosphatase PP1, PP2A, and calcineurin are involved in hyperphosphorylation of tau- as well as beta-amyloid-induced cell death. We have previously shown serine/threonine protein phosphatases to be involved in estrogen-mediated neuroprotection. The purpose of this study was to delineate the role of PP1, PP2A, and calcineurin in the mechanism of estrogen mediated neuroprotection against oxidative stress and excitotoxicity. Treatment with protein phosphatases inhibitor II, endothall, or cyclosporin A, which are specific inhibitors of PP1, PP2A, and calcineurin, respectively, did not have an effect on cell viability. However, in combination, these inhibitors adversely affected cell survival, which suggests the importance of serine/threonine protein phosphatases in maintenance of cellular function. Inhibitors of PP1, PP2A, and calcineurin attenuated the protective effects of estrogen against glutamate-induced -neurotoxicity but did not completely abrogate the estrogen-mediated protection. The attenuation of estrogen-induced neuroprotection was achieved through decrease in the activity of theses serine/threonine phosphatases without the concomitant decrease in protein expression. In an animal model, transient middle cerebral artery occlusion caused a 50% decrease in levels of PP1, PP2A, and PP2B ipsilateral to the lesion in a manner that was prevented by estradiol pretreatment. Therefore, we conclude that in the face of cytotoxic challenges in vitro and in vivo, estrogens maintain the function of PP1, PP2A, and calcineurin.
Assuntos
Calcineurina/metabolismo , Estrogênios/farmacologia , Neurônios/enzimologia , Fármacos Neuroprotetores/farmacologia , Proteína Fosfatase 1/metabolismo , Proteína Fosfatase 2/metabolismo , Animais , Neoplasias Encefálicas , Inibidores de Calcineurina , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Ciclosporina/farmacologia , Ácidos Dicarboxílicos/farmacologia , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Glioma , Ácido Glutâmico/toxicidade , Camundongos , Neurônios/efeitos dos fármacos , Neurotoxinas/toxicidade , Ácido Okadáico/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Proteína Fosfatase 1/antagonistas & inibidores , Proteína Fosfatase 2/antagonistas & inibidores , Ratos , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/prevenção & controleRESUMO
The mechanism of estrogen-mediated neuroprotection is not yet clear. Estrogens have a variety of modes of action, including transducing signaling events such as activation and/or suppression of the mitogen-activated protein kinase (MAPK) pathway. We have previously shown protein phosphatases to be involved in 17beta-estradiol-mediated neuroprotection. In the present study, we assessed the role of estrogen receptors (ERs) in estrogen-mediated neuroprotection from oxidative/excitotoxic stress and the consequential effects on MAPK signaling. Okadaic acid and calyculin A, nonspecific serine/threonine phosphatase inhibitors, were exposed to cells at various concentrations in the presence or absence of 17alpha-estradiol, the enantiomer of 17beta-estradiol, 2-(1-adamantyl)-3-hydroxyestra-1,3,5(10)-trien-17-one (ZYC3; non-ER-binding estrogen analog), and/or glutamate. All three compounds, which we have shown to have little or no binding to ERalpha and ERbeta, were protective against glutamate toxicity but not against okadaic acid and calyculin A toxicity. In addition, in the presence of effective concentrations of these inhibitors, the protective effects of these estrogen analogs were lost. Glutamate treatment caused a 50% decrease in levels of protein phosphatase 1 (PP1), protein phosphatase 2A (PP2A), and protein phosphatase 2B (calcineurin) (PP2B). Coadministration of ZYC3 with glutamate prevented the decreases in PP1, PP2A, and PP2B levels. Furthermore, glutamate treatment caused a persistent increase in phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 that corresponds with the decrease protein levels of serine/threonine phosphatases. ZYC3 blocked this persistent increase in ERK phosphorylation. These results suggest that estrogens protect cells against glutamate-induced oxidative stress through an ER-independent mediated mechanism that serves to preserve phosphatase activity in the face of oxidative insults, resulting in attenuation of the persistent phosphorylation of ERK associated with neuronal death.
Assuntos
Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fármacos Neuroprotetores/farmacologia , Fosfoproteínas Fosfatases/metabolismo , Receptores de Estrogênio/metabolismo , Animais , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Estrogênios/química , Estrogênios/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Fármacos Neuroprotetores/química , Fosfoproteínas Fosfatases/antagonistas & inibidores , Ratos , Receptores de Estrogênio/agonistasRESUMO
Atrazine (ATR) is a commonly used pre-emergence and early postemergence herbicide. Rats gavaged with ATR and its chlorometabolites desethylatrazine (DEA) and deisopropylatrazine (DIA) respond with a rapid and dose-dependent rise in plasma corticosterone, whereas the major chlorometabolite, diaminochlorotriazine (DACT), has little or no effect on corticosterone levels. In this study, we investigated the possible sites of ATR activation of the hypothalamic-pituitary-adrenal (HPA) axis. ATR treatment had no effect on adrenal weights but altered adrenal morphology. Hypophysectomized rats or rats under dexamethasone suppression did not respond to ATR treatment, suggesting that ATR does not directly stimulate the adrenal gland to induce corticosterone synthesis. Immortalized mouse corticotrophs (AtT-20) and primary rat pituitary cultures were treated with ATR, DEA, DIA, or DACT. None of the compounds induced an increase in ACTH secretion or potentiated ACTH release in conjunction with CRH on ACTH release. In female rats gavaged with ATR, pretreatment with the CRH receptor antagonist astressin completely blocked the ATR-induced rise in corticosterone concentrations, implicating CRH release in ATR-induced HPA activation. Intracerebroventricular infusion of ATR, DEA, and DIA but not DACT at concentrations equivalent to peak plasma concentrations after gavage dosing resulted in an elevation of plasma corticosterone concentrations. However, ATR did not induce c-Fos immunoreactivity in the paraventricular nucleus of the hypothalamus. These results indicate that ATR activates the HPA axis centrally and requires CRH receptor activation, but it does not stimulate cellular pathways associated with CRH neuronal excitation.
Assuntos
Atrazina/farmacologia , Corticotrofos/efeitos dos fármacos , Herbicidas/farmacologia , Sistema Hipotálamo-Hipofisário/efeitos dos fármacos , Hipófise/efeitos dos fármacos , Sistema Hipófise-Suprarrenal/efeitos dos fármacos , Glândulas Suprarrenais/efeitos dos fármacos , Glândulas Suprarrenais/patologia , Hormônio Adrenocorticotrópico/efeitos dos fármacos , Hormônio Adrenocorticotrópico/metabolismo , Animais , Atrazina/análogos & derivados , Linhagem Celular , Corticosterona/metabolismo , Corticotrofos/metabolismo , Dexametasona/farmacologia , Feminino , Glucocorticoides/farmacologia , Sistema Hipotálamo-Hipofisário/metabolismo , Camundongos , Técnicas de Cultura de Órgãos , Tamanho do Órgão , Hipófise/metabolismo , Hipófise/cirurgia , Sistema Hipófise-Suprarrenal/metabolismo , Ratos , Triazinas/farmacologiaRESUMO
The signaling pathways that mediate neurodegeneration are complex and involve a balance between phosphorylation and dephosphorylation of signaling and structural proteins. We have shown previously that 17beta-estradiol and its analogs are potent neuroprotectants. The purpose of this study was to delineate the role of protein phosphatases (PPs) in estrogen neuroprotection against oxidative stress and excitotoxicity. HT-22 cells, C6-glioma cells, and primary rat cortical neurons were exposed to the nonspecific serine/threonine protein phosphatase inhibitors okadaic acid and calyculin A at various concentrations in the presence or absence of 17beta-estradiol and/or glutamate. Okadaic acid and calyculin A caused a dose-dependent decrease in cell viability in HT-22, C6-glioma, and primary rat cortical neurons. 17beta-Estradiol did not show protection against neurotoxic concentrations of either okadaic acid or calyculin A in these cells. In the absence of these serine/threonine protein phosphatase inhibitors, 17beta-estradiol attenuated glutamate toxicity. However, in the presence of effective concentrations of these protein phosphatase inhibitors, 17beta-estradiol protection against glutamate toxicity was lost. Furthermore, glutamate treatment in HT-22 cells and primary rat cortical neurons caused a 50% decrease in levels of PP1, PP2A, and PP2B protein, whereas coadministration of 17beta-estradiol with glutamate prevented the decrease in PP1, PP2A, and PP2B levels. These results suggest that 17beta-estradiol may protect cells against glutamate-induced oxidative stress and excitotoxicity by activating a combination of protein phosphatases.
Assuntos
Estrogênios/fisiologia , Neurônios/fisiologia , Fármacos Neuroprotetores/metabolismo , Fosfoproteínas Fosfatases/fisiologia , Animais , Calcineurina/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células/efeitos dos fármacos , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Estradiol/farmacologia , Ácido Glutâmico/farmacologia , Toxinas Marinhas , Neurônios/efeitos dos fármacos , Ácido Okadáico/farmacologia , Oxazóis/farmacologia , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/metabolismo , Ratos , Fatores de TempoRESUMO
The previously-published physiologically based pharmacokinetic model for atrazine (ATZ), deisopropylatrazine (DIA), deethylatrazine (DEA), and diaminochlorotriazine (DACT), which collectively comprise the total chlorotriazines (TCT) as represented in this study, was modified to allow for scaling to humans. Changes included replacing the fixed dose-dependent oral uptake rates with a method that represented delayed absorption observed in rats administered ATZ as a bolus dose suspended in a methylcellulose vehicle. Rate constants for metabolism of ATZ to DIA and DEA, followed by metabolism of DIA and DEA to DACT were predicted using a compartmental model describing the metabolism of the chlorotriazines by rat and human hepatocytesin vitro Overall, the model successfully predicted both the 4-day plasma time-course data in rats administered ATZ by bolus dose (3, 10, and 50 mg/kg/day) or in the diet (30, 100, or 500 ppm). Simulated continuous daily exposure of a 55-kg adult female to ATZ at a dose of 1.0 µg/kg/day resulted in steady-state urinary concentrations of 0.6, 1.4, 2.5, and 6.0 µg/L for DEA, DIA, DACT, and TCT, respectively. The TCT (ATZ + DEA + DIA + DACT) human urinary biomonitoring equivalent concentration following continuous exposure to ATZ at the chronic point of departure (POD = 1.8 mg/kg/day) was 360.6 µg/L.
Assuntos
Atrazina/farmacocinética , Hepatócitos/metabolismo , Modelos Biológicos , Triazinas/farmacocinética , Absorção Fisiológica , Administração Oral , Animais , Atrazina/sangue , Atrazina/urina , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Hepatócitos/efeitos dos fármacos , Humanos , Ratos Sprague-Dawley , Especificidade da Espécie , Distribuição Tecidual , Triazinas/sangue , Triazinas/urinaRESUMO
There is debate over whether the requirements of GLP are appropriate standards for evaluating the quality of toxicological data used to formulate regulations. A group promoting the importance of non-monotonic dose responses for endocrine disruptors contend that scoring systems giving primacy to GLP are biased against non-GLP studies from the literature and are merely record-keeping exercises to prevent fraudulent reporting of data from non-published guideline toxicology studies. They argue that guideline studies often employ insensitive species and outdated methods, and ignore the perspectives of subject-matter experts in endocrine disruption, who should be the sole arbiters of data quality. We believe regulatory agencies should use both non-GLP and GLP studies, that GLP requirements assure fundamental tenets of study integrity not typically addressed by journal peer-review, and that use of standardized test guidelines and GLP promotes consistency, reliability, comparability, and harmonization of various types of studies used by regulatory agencies worldwide. This debate suffers two impediments to progress: a conflation of different phases of study interpretation and levels of data validity, and a misleading characterization of many essential components of GLP and regulatory toxicology. Herein we provide clarifications critical for removing those impediments.
Assuntos
Aprovação de Drogas/legislação & jurisprudência , Disruptores Endócrinos/toxicidade , Formulação de Políticas , Toxicologia/legislação & jurisprudência , Animais , Consenso , Guias como Assunto , Humanos , Controle de Qualidade , Reprodutibilidade dos Testes , Medição de Risco , Toxicologia/normasRESUMO
BACKGROUND AND PURPOSE: Although estrogens are neuroprotective, hormonal effects limit their clinical application. Estrogen analogues with neuroprotective function but lacking hormonal properties would be more attractive. The present study was undertaken to determine the neuroprotective effects of a novel 2-adamantyl estrogen analogue, ZYC3. METHODS: Cytotoxicity was induced in HT-22 cells by 10 mmol/L glutamate. 17beta-Estradiol (E2) or ZYC3 was added immediately before the exposure to glutamate. Cell viability was determined by calcein assay. The binding of E2 and ZYC3 to human alpha (ERalpha) and beta (ERbeta) estrogen receptors was determined by ligand competition binding assay. Ischemia/reperfusion injury was induced by temporary middle cerebral artery occlusion (MCAO). E2 or ZYC3 (100 microg/kg) was administered 2 hours or immediately before MCAO, respectively. Infarct volume was determined by 2,3,5-triphenyltetrazolium chloride staining. Cerebral blood flow was recorded during and within 30 minutes after MCAO by a hydrogen clearance method. RESULTS: ZYC3 significantly decreased toxicity of glutamate with a potency 10-fold that of E2. ZYC3 did not bind to either ERalpha or ERbeta. Infarct volume was significantly reduced to 122.4+/-17.6 and 83.1+/-19.3 mm(3) in E2 and ZYC3 groups, respectively, compared with 252.6+/-15.6 mm(3) in the ovariectomized group. During MCAO, both E2 and ZYC3 significantly increased cerebral blood flow in the nonischemic side, while no significant differences were found in the ischemic side. However, E2 and ZYC3 significantly increased cerebral blood flow in both sides within 30 minutes after reperfusion. CONCLUSIONS: Our study shows that ZYC3, a non-receptor-binding estrogen analogue, possesses both neuroprotective and vasoactive effects, which offers the possibility of clinical application for stroke without the side effects of estrogens. It also suggests that both the neuroprotective and vasoactive effects of estrogen are receptor independent.
Assuntos
Adamantano/farmacologia , Congêneres do Estradiol/farmacologia , Estrona/farmacologia , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Receptores de Estrogênio/química , Adamantano/análogos & derivados , Adamantano/química , Animais , Ligação Competitiva/efeitos dos fármacos , Velocidade do Fluxo Sanguíneo/efeitos dos fármacos , Encéfalo/irrigação sanguínea , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Circulação Cerebrovascular/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Portadores de Fármacos , Estradiol/análogos & derivados , Estradiol/química , Estradiol/farmacologia , Congêneres do Estradiol/química , Receptor alfa de Estrogênio , Receptor beta de Estrogênio , Estrona/análogos & derivados , Estrona/química , Feminino , Ácido Glutâmico/toxicidade , Humanos , Infarto da Artéria Cerebral Média/tratamento farmacológico , Infarto da Artéria Cerebral Média/patologia , Ligantes , Camundongos , Neurônios/citologia , Fármacos Neuroprotetores/química , Ratos , Ratos Sprague-Dawley , Receptores de Estrogênio/metabolismoRESUMO
The protective effects of estrogens have been widely reported in a number of animal and cell culture models, but the molecular mechanisms of this potent neuroprotective activity are not well understood. Both in vitro and in vivo studies indicate that in the central nervous system and peripheral tissues, estrogen treatment reduces cytokine production and inflammatory responses. Nuclear factor-kappa B (NFkappaB) plays an essential role in the regulation of post-ischemic inflammation, which is detrimental to recovery from an ischemic stroke. We investigated the role of NFkappaB in neuronal survival in rats that received transient middle cerebral artery (MCA) occlusion, and observed that this transient cerebral ischemia induced substantial apoptosis and inflammatory responses, including IkappaB phosphorylation, NF-kappaB activation and iNOS over-expression. 17 beta-estradiol (E2) treatment produced strong protective effects by reducing infarct volume, neuronal apoptosis, and inflammatory responses. These findings provide evidence for a novel molecular and cellular interaction between the sex hormone and the immunoresponsive system. These studies also provide evidence that suppression of post-ischemic inflammation may play a critical role in estrogen-mediated neuroprotection.
Assuntos
Estradiol/farmacologia , Ataque Isquêmico Transitório/metabolismo , Ataque Isquêmico Transitório/patologia , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Fármacos Neuroprotetores , Actinas/metabolismo , Animais , Western Blotting , Química Encefálica/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Fragmentação do DNA , Densitometria , Feminino , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Mediadores da Inflamação/metabolismo , Mediadores da Inflamação/fisiologia , Artéria Cerebral Média/fisiologia , Óxido Nítrico Sintase/biossíntese , Óxido Nítrico Sintase Tipo II , Ovariectomia , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismoRESUMO
BACKGROUND: There is a recognized need to improve the application of epidemiologic data in human health risk assessment especially for understanding and characterizing risks from environmental and occupational exposures. Although there is uncertainty associated with the results of most epidemiologic studies, techniques exist to characterize uncertainty that can be applied to improve weight-of-evidence evaluations and risk characterization efforts. METHODS: This report derives from a Health and Environmental Sciences Institute (HESI) workshop held in Research Triangle Park, North Carolina, to discuss the utility of using epidemiologic data in risk assessments, including the use of advanced analytic methods to address sources of uncertainty. Epidemiologists, toxicologists, and risk assessors from academia, government, and industry convened to discuss uncertainty, exposure assessment, and application of analytic methods to address these challenges. SYNTHESIS: Several recommendations emerged to help improve the utility of epidemiologic data in risk assessment. For example, improved characterization of uncertainty is needed to allow risk assessors to quantitatively assess potential sources of bias. Data are needed to facilitate this quantitative analysis, and interdisciplinary approaches will help ensure that sufficient information is collected for a thorough uncertainty evaluation. Advanced analytic methods and tools such as directed acyclic graphs (DAGs) and Bayesian statistical techniques can provide important insights and support interpretation of epidemiologic data. CONCLUSIONS: The discussions and recommendations from this workshop demonstrate that there are practical steps that the scientific community can adopt to strengthen epidemiologic data for decision making.
Assuntos
Exposição Ambiental/análise , Exposição Ambiental/estatística & dados numéricos , Estudos Epidemiológicos , Incerteza , Tomada de Decisões , Humanos , Exposição Ocupacional/análise , Exposição Ocupacional/estatística & dados numéricos , Medição de Risco/métodosRESUMO
Menopause is associated with a precipitous decline in circulating estrogens and a resulting loss of the neuroprotective actions of this steroid hormone. In view of the results of the Women's Health Initiative and the preceding knowledge that orally administered estrogens has a variety of adverse side effects, likely through actions on peripheral estrogen receptor alpha (ERα), we initiated a program of research to synthesis and assess a group of non-feminizing estrogens that lack ability to interact with ERs but retain much of the neuroprotective action of feminizing estrogens. This program of research is aimed at the identification of compounds which do not stimulate ERs but are potentially neuroprotective in vitro and in animal models of neuronal cell death. We discovered that the most effective non-feminizing estrogens were those with large bulky groups in the 2 and/or 4 carbon of the phenolic A ring of the steroid. These compounds were 8- to 114-fold more potent than 17 ß-estradiol (ßE2), but lacked ER binding capacity in vitro and feminizing effects in vivo. The success of this program of research suggests that strategies to optimize non-feminizing estrogens for use in postmenopausal women can be successful.