RESUMEN
The rise in popularity of "bath salts" as safe alternatives to MDMA (3,4-methylenedioxymethamphetamine), methamphetamine, and other illicit substances has resulted in increased scrutiny of the contents and toxicology associated with these products. We report a case of sudden death related to the synthetic cathinone methylone (3,4-methylenedioxy-N-methylcathinonmethylone) in a previously healthy 19-year-old man. Although several fatal case reports have been published involving methylone and other synthetic cathinones, this is the first reported case of sudden cardiac death associated with methylone use. Although lack of published data prevented a comparison of blood methylone concentrations between our case and existing reports, the amount of methylone we detected postmortem (0.07 mg/dL) is below those reported in MDMA-related fatalities. Our report suggests that methylone toxicity has been greatly underestimated by users of this synthetic cathinone.
Asunto(s)
Alcaloides/efectos adversos , Estimulantes del Sistema Nervioso Central/efectos adversos , Muerte Súbita Cardíaca/etiología , Drogas de Diseño/efectos adversos , Metanfetamina/análogos & derivados , Estimulantes del Sistema Nervioso Central/sangre , Estimulantes del Sistema Nervioso Central/química , Estimulantes del Sistema Nervioso Central/orina , Toxicología Forense , Cromatografía de Gases y Espectrometría de Masas , Humanos , Masculino , Metanfetamina/efectos adversos , Metanfetamina/sangre , Metanfetamina/química , Metanfetamina/orina , Estructura Molecular , Trastornos Relacionados con Sustancias/complicaciones , Adulto JovenRESUMEN
Exposure to high levels of glucocorticoids (GCs) during development leads to long-term changes in hypothalamic-pituitary-adrenal (HPA) axis regulation, although little is known about the neural mechanisms that underlie these alterations. In this study, we investigated the effects of late gestational (days 18-22) or postnatal (days 4-6) administration of the GC receptor agonist dexamethasone (DEX) on an apoptosis marker in two brain regions critical to HPA axis regulation, the hippocampus and the hypothalamic paraventricular nucleus (PVN). One day after the final DEX injection, male and female rats were sacrificed, and brains were processed for immunohistochemical detection of cleaved caspase-3, an apoptotic cell death indicator. DEX increased cleaved caspase-3 immunoreactivity in the CA1 hippocampal region of both sexes following prenatal but not postnatal treatment. Prenatal DEX also increased caspase-3 immunoreactivity in the CA3 region, an elevation that tended to be greater in females. In contrast, postnatal DEX resulted in a much smaller, albeit significant, induction in CA3 caspase-3 compared with prenatal treatment. Quantitative real-time PCR analysis revealed that prenatal but not postnatal DEX-induced hippocampal cleaved caspase-3 correlated with elevated mRNA of the proapoptotic gene Bad. Few caspase-3-ir cells were identified within the PVN regardless of treatment age, although postnatal but not prenatal DEX increased this number. However, the region immediately surrounding the PVN (peri-PVN) showed significant increases in caspase-3-ir cells following pre- and postnatal DEX. Together these findings indicate that developmental GC exposure increases apoptosis in HPAaxis-associated brain regions in an age- and sex-dependent manner.
Asunto(s)
Apoptosis/efectos de los fármacos , Dexametasona/toxicidad , Hipocampo/efectos de los fármacos , Malformaciones del Sistema Nervioso/patología , Malformaciones del Sistema Nervioso/fisiopatología , Núcleo Hipotalámico Paraventricular/efectos de los fármacos , Efectos Tardíos de la Exposición Prenatal/patología , Efectos Tardíos de la Exposición Prenatal/fisiopatología , Envejecimiento/patología , Animales , Apoptosis/fisiología , Modelos Animales de Enfermedad , Femenino , Glucocorticoides/toxicidad , Hipocampo/embriología , Hipocampo/patología , Malformaciones del Sistema Nervioso/inducido químicamente , Núcleo Hipotalámico Paraventricular/embriología , Núcleo Hipotalámico Paraventricular/patología , Embarazo , Efectos Tardíos de la Exposición Prenatal/inducido químicamente , Ratas , Ratas Sprague-Dawley , Caracteres Sexuales , Factores SexualesRESUMEN
The progressive debilitation of motor functions in Parkinson's disease (PD) results from degeneration of dopaminergic neurons within the substantia nigra pars compacta of the midbrain. Long-term inflammatory activation of microglia and astrocytes plays a central role in the progression of PD and is characterized by activation of the nuclear factor-kappaB (NF-kappaB) signaling cascade and subsequent overproduction of inflammatory cytokines and nitric oxide (NO). Suppression of this neuroinflammatory phenotype has received considerable attention as a potential target for chemotherapy, but there are no currently approved drugs that sufficiently address this problem. The data presented here demonstrate the efficacy of a novel anti-inflammatory diindolylmethane class compound, 1,1-bis(3'-indolyl)-1-(p-t-butylphenyl)methane (DIM-C-pPhtBu), in suppressing NF-kappaB-dependent expression of inducible nitric-oxide synthase (NOS2) and NO production in astrocytes exposed to the parkinsonian neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) through a mechanism distinct from that described for the thiazolidinedione-class compound, rosiglitazone. Chromatin immunoprecipitations revealed that micromolar concentrations of DIM-C-pPhtBu prevented association of the p65 subunit of NF-kappaB with enhancer elements in the Nos2 promoter but had little effect on DNA binding of either peroxisome proliferator-activated receptor-gamma (PPAR-gamma) or the nuclear corepressor NCoR2. Treatment with DIM-C-pPhtBu concomitantly suppressed NO production and protein nitration in MPTP-activated astrocytes and completely protected cocultured primary striatal neurons from astrocyte-dependent apoptosis. These data demonstrate the efficacy of DIM-C-pPhtBu in preventing the activation of NF-kappaB-dependent inflammatory genes in primary astrocytes and suggest that this class of compounds may be effective neuroprotective anti-inflammatory agents in vivo.
Asunto(s)
Apoptosis/efectos de los fármacos , Astrocitos/metabolismo , Indoles/farmacología , Neuronas/metabolismo , Fármacos Neuroprotectores/farmacología , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/farmacología , Animales , Animales Recién Nacidos , Apoptosis/genética , Astrocitos/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/citología , Técnicas de Cocultivo , Cuerpo Estriado/citología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II/genéticaRESUMEN
Reactive astrogliosis is a prominent neuropathologic feature of manganism, a neurodegenerative disorder caused by excessive accumulation of manganese (Mn) in the basal ganglia. Activation of astrocytes has been linked to neuronal injury in manganism resulting from overproduction of inflammatory mediators, including tumor necrosis factor-alpha (TNFalpha), interferon-gamma (IFNgamma), interleukin-1beta (IL-1beta), and nitric oxide (NO), but the signaling mechanisms by which Mn regulates these factors remain poorly understood. We previously reported that Mn enhances production of NO in activated astrocytes that promotes apoptosis in cocultured neuronal cells by a mechanism involving the transcription factor nuclear factor-kappaB (NF-kappaB) (Liu et al., 2005). Because NF-kappaB-dependent expression of inducible nitric oxide synthase (NOS2) can be antagonized by the nuclear orphan receptor peroxisome proliferator-activated receptor-gamma (PPARgamma), we postulated that a novel agonist of this receptor, 1,1-bis(3'-indolyl)-1-(p-trifluoromethylphenyl)methane (cDIM1), would suppress expression of NOS2 in astrocytes and protect cocultured neuronal cells from apoptosis. Submicromolar concentrations of cDIM1 potently suppressed production of NO and expression of NOS2 in cultured astrocytes exposed to Mn and IFNgamma/TNFalpha and prevented apoptosis in cocultures of differentiated PC12 cells, but this neuroprotective effect was lost in the absence of astrocytes. By using fluorescence reporter and chromatin immunoprecipitation (ChIP) assays, we found that cDIM1 prevented activation of NF-kappaB in astrocytes by a mechanism involving stabilization of the nuclear corepressor 2 (NCoR2) on the proximal NF-kappaB binding site of the NOS2 promoter. These data suggest that PPARgamma may be an effective target for limiting inflammatory activation of astrocytes during neurologic injury.
Asunto(s)
Apoptosis/efectos de los fármacos , Astrocitos/metabolismo , Indoles/farmacología , Manganeso/farmacología , Fármacos Neuroprotectores/farmacología , Óxido Nítrico/antagonistas & inhibidores , Células PC12/fisiología , PPAR gamma/agonistas , Animales , Astrocitos/efectos de los fármacos , Sitios de Unión , Técnicas de Cocultivo , Proteínas de Unión al ADN/metabolismo , Interferón gamma/farmacología , FN-kappa B/antagonistas & inhibidores , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II/genética , Co-Represor 2 de Receptor Nuclear , Células PC12/efectos de los fármacos , Regiones Promotoras Genéticas , Ratas , Proteínas Represoras/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Inflammatory activation of glial cells is associated with neuronal injury in several degenerative movement disorders of the basal ganglia, including manganese neurotoxicity. Manganese (Mn) potentiates the effects of inflammatory cytokines on nuclear factor-kappaB (NF-kappaB)-dependent expression of nitric oxide synthase 2 (NOS2) in astrocytes, but the signaling mechanisms underlying this effect have remained elusive. It was postulated in the present studies that direct stimulation of cGMP synthesis and activation of mitogen-activated protein (MAP) kinase signaling pathways underlies the capacity of Mn to augment NF-kappaB-dependent gene expression in astrocytes. Exposure of primary cortical astrocytes to a low concentration of Mn (10 microM) potentiated expression of NOS2 mRNA and protein along with production of NO in response to interferon-gamma (IFNgamma) and tumor necrosis factor-alpha (TNFalpha), which was prevented by overexpression of dominant negative IkappaB alpha. Mn also potentiated IFNgamma- and TNFalpha-induced phosphorylation of extracellular response kinase (ERK), p38, and JNK, as well as cytokine-induced activation of a fluorescent NF-kappaB reporter construct in transgenic astrocytes. Activation of ERK preceded that of NF-kappaB and was required for maximal activation of NO synthesis. Independently of IFNgamma/TNFalpha, Mn-stimulated synthesis of cGMP in astrocytes and inhibition of soluble guanylate cyclase (sGC) abolished the potentiating effect of Mn on MAP kinase phosphorylation, NF-kappaB activation, and production of NO. These data indicate that near-physiological concentrations of Mn potentiate cytokine-induced expression of NOS2 and production of NO in astrocytes via activation of sGC, which promotes ERK-dependent enhancement of NF-kappaB signaling.
Asunto(s)
Astrocitos/fisiología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Guanilato Ciclasa/metabolismo , Manganeso/farmacología , FN-kappa B/fisiología , Óxido Nítrico Sintasa de Tipo II/genética , Transducción de Señal/efectos de los fármacos , Animales , Animales Recién Nacidos , Astrocitos/citología , Astrocitos/efectos de los fármacos , Astrocitos/enzimología , Medios de Cultivo , GMP Cíclico/metabolismo , Activación Enzimática , Genes Reporteros , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Óxido Nítrico/metabolismo , ARN Mensajero/genéticaRESUMEN
Recent advances in understanding the progression of Parkinson's disease (PD) implicate perturbations in astrocyte function and induction of constitutively expressed neuronal nitric oxide synthase (NOS1) in both human PD and in the MPTP model of the disease. Transcriptional regulation of NOS1 is complex but recent data suggest that nuclear factor kappa-B (NF-kappaB) is an important transcription factor involved in inducible expression of the gene. The data presented here demonstrate that mild activation of primary astrocytes with low or 'sub-optimal' concentrations of MPTP (1 microM) and the inflammatory cytokine tumor necrosis factor alpha (10 pg/ml) and interferon gamma (1 ng/ml) results in selective induction of Nos1 mRNA and protein, increased production of nitric oxide (NO), and a significant elevation in global protein nitration. This mild inflammatory stimulus also resulted in activation and recruitment of p65 to a putative NF-kappaB response element located in the Nos1 promoter region flanking exon 1. A role for NF-kappaB in MPTP-dependent induction of NOS1 was confirmed through overexpression of a mutant IkappaBalpha super repressor of NF-kappaB that prevented induction of NOS1. The data presented here thus demonstrate a role for NF-kappaB in selective induction of NOS1 during early inflammatory activation of astrocytes stimulated by low-dose MPTP and inflammatory cytokines.
Asunto(s)
Astrocitos/metabolismo , Regulación Enzimológica de la Expresión Génica , Inflamación/metabolismo , FN-kappa B/metabolismo , Óxido Nítrico Sintasa de Tipo I/biosíntesis , Trastornos Parkinsonianos/metabolismo , Animales , Western Blotting , Técnica del Anticuerpo Fluorescente , Interferón gamma/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Óxido Nítrico Sintasa de Tipo I/genética , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/fisiología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The lipid peroxidation product 4-hydroxynonenal (4-HNE) has been shown to interfere with protein function. The goal of this study was to determine the effects of substrate modification by 4-HNE on protein degradation. Equine liver alcohol dehydrogenase (ADH, EC 1.1.1.1) treated with 2-fold molar excess 4-HNE was degraded by a rabbit reticulocyte lysate (RRL) system approximately 1.5-fold faster than control, while treatment with concentrations up to 100-fold molar excess aldehyde were inhibitory to degradation. Involvement of the 26S proteasome (EC 3.4.99.46) was demonstrated through the use of specific proteasome and ATPase inhibitors, and confirmed by measuring the extent of ADH polyubiquitination. Tryptic digestion and LC/MS analysis of 4-HNE-treated ADH identified modification of two zinc chelating Cys residues. Through molecular modeling experiments a conformational shift in both zinc-containing regions was predicted, with an approximate doubling of the distance between the structural zinc and its respective chelating residues. Modification of residues in the active site zinc binding motif resulted in less pronounced alteration in protein structure. The data presented here demonstrate accelerated ubiquitination and proteasomal degradation of ADH modified with 4-HNE, and suggest a conformational change after 4-HNE docking as a mechanism behind these observations.
Asunto(s)
Alcohol Deshidrogenasa/metabolismo , Aldehídos/farmacología , Hígado/enzimología , Complejo de la Endopetidasa Proteasomal/metabolismo , Alcohol Deshidrogenasa/efectos de los fármacos , Animales , Caballos , Cinética , Complejo de la Endopetidasa Proteasomal/efectos de los fármacosRESUMEN
Estrogen receptors regulate multiple brain functions, including stress, sexual, and memory-associated behaviors as well as controlling neuroendocrine and autonomic function. During development, estrogen signaling is involved in programming adult sex differences in physiology and behavior. Expression of estrogen receptor α changes across development in a region-specific fashion. By contrast, estrogen receptor ß (ERß) is expressed in many brain regions, yet few studies have explored sex and developmental differences in its expression, largely because of the absence of selective reagents for anatomical localization of the protein. This study utilized bacterial artificial chromosome transgenic mice expressing ERß identified by enhanced green fluorescent protein (EGFP) to compare expression levels and distribution of ERß in the male and female mouse forebrain on the day of birth (P0), on postnatal day 4 (P4), and on P21. By using qualitative analysis, we mapped the distribution of ERß-EGFP and found developmental alterations in ERß expression within the cortex, hippocampus, and hypothalamic regions including the arcuate, ventromedial, and paraventricular nuclei. We also report a sex difference in ERß in the bed nucleus of the stria terminalis, with males showing greater expression at P4 and P21. Another sex difference was found in the anteroventral periventricular nucleus of P21, but not P0 or P4, mice, in which ERß-EGFP-immunoreactive cells were densely clustered near the third ventricle in females but not males. These developmental changes and sex differences in ERß indicate a mechanism through which estrogens might differentially affect brain functions or program adult physiology at select times during development.
Asunto(s)
Receptor beta de Estrógeno/biosíntesis , Prosencéfalo/crecimiento & desarrollo , Prosencéfalo/metabolismo , Caracteres Sexuales , Factores de Edad , Animales , Cromosomas Artificiales Bacterianos , Femenino , Inmunohistoquímica , Masculino , Ratones , Ratones TransgénicosRESUMEN
Exposure to high levels of glucocorticoids (GCs) during early development results in lasting disturbances in emotional behavior in rodents. Inhibitory GABAergic neurons, classified by their expression of calcium binding proteins (CBPs), also contribute to stress-related behaviors and may be GC sensitive during development. Therefore, in the present study we investigated the effects of prenatal treatment with the glucocorticoid receptor agonist dexamethasone (DEX) on expression of calbindin and calretinin in brain areas critical to emotional regulation (basolateral/lateral amygdala and hippocampal CA1 and CA3 regions). Late gestational treatment with DEX (gestational days 18-22) significantly decreased the density of calretinin immunoreactive cells in the lateral amygdala of adult female offspring with no differences in the basolateral amygdala, hippocampal CA1, or CA3 regions. Moreover, there were no effects of gestational DEX treatment on calretinin expression in males. Calbindin expression in adulthood was unaltered within either amygdala or hippocampal subregion of either sex following prenatal DEX treatment. Together these findings indicate that late gestational DEX treatment causes a targeted reduction of calretinin within the lateral amygdala of females and this may be one mechanism through which developmental glucocorticoid exposure contributes to lasting alterations in emotional behavior.
Asunto(s)
Amígdala del Cerebelo/metabolismo , Dexametasona/efectos adversos , Glucocorticoides/efectos adversos , Efectos Tardíos de la Exposición Prenatal/metabolismo , Proteína G de Unión al Calcio S100/metabolismo , Animales , Región CA1 Hipocampal/metabolismo , Región CA3 Hipocampal/metabolismo , Calbindina 2 , Calbindinas , Femenino , Masculino , Exposición Materna , Intercambio Materno-Fetal , Neuronas/metabolismo , Embarazo , Ratas , Ratas Sprague-Dawley , Factores SexualesRESUMEN
The neuropeptide thyrotropin-releasing hormone (TRH) is recognized to play an important role in controlling energy balance through direct effects on the CNS, although mechanisms explaining the phenomenon are poorly understood. To begin to understand the effects of TRH on CNS control of energy balance, we first mapped neurons expressing the TRH precursor peptide, prepro-TRH (ppTRH) in the paraventricular nucleus of the rat hypothalamus and the surrounding regions. We identified a population of ppTRH-expressing neurons in the juxtaparaventricular region of the lateral hypothalamus (LHAjp) which were stimulated by the satiety signal leptin (2.5µg/kg, IP). Using a model of fetal glucocorticoid (GC) exposure in which pregnant rats were treated with the synthetic GC dexamethasone (DEX) during gestational days 18-21, it was observed that such exposure resulted in reduced numbers of ppTRH-ir neurons in the LHAjp in adult male and female rats, and was accompanied by increased food intake. Our data provide further insight into the biological role of the LHAjp, as well as the potential involvement of TRH neurons within this region in metabolic disease associated with fetal glucocorticoid exposure.
Asunto(s)
Dexametasona/efectos adversos , Glucocorticoides/efectos adversos , Hipotálamo/patología , Leptina/farmacología , Neuronas/metabolismo , Efectos Tardíos de la Exposición Prenatal/etiología , Efectos Tardíos de la Exposición Prenatal/patología , Precursores de Proteínas/metabolismo , Hormona Liberadora de Tirotropina/metabolismo , Análisis de Varianza , Animales , Animales Recién Nacidos , Peso Corporal/efectos de los fármacos , Ingestión de Alimentos/efectos de los fármacos , Femenino , Masculino , Embarazo , Ratas , Factores SexualesRESUMEN
The clinical use of synthetic glucocorticoids in preterm infants to promote lung development has received considerable attention due to the potential for increased risk of developing metabolic disease in adulthood after such treatment. In this study, we examined the hypothesis that exposure to the synthetic glucocorticoid, dexamethasone (DEX), during late gestation in the rat results in the development of nonalcoholic fatty liver disease in adult offspring. Pregnant Sprague Dawley dams were treated with 0.4 mg/kg DEX beginning on gestational d 18 until parturition (gestational d 23). At postnatal d 21, offspring were weaned onto either a standard chow or high-fat (60% fat-derived calories) diet. In adulthood (postnatal d 60-65), hepatic tissue was harvested and examined for pathology. Liver steatosis, or fat accumulation, was found to be more severe in the DEX-exposed female offspring that were weaned onto the high-fat diet. This finding corresponded with decreased plasma IGF-I concentrations, as well as decreased hypothalamic expression of GHRH mRNA. Morphological measurements on body and long bone length further implicate a GH signaling deficit after fetal DEX exposure. Collectively, these data indicate suppression of GH axis function in the female DEX/high-fat cohort but not in the male offspring. Because deficits in the GH signaling can be linked to the development of nonalcoholic fatty liver disease, our results suggest that the prominent liver injury noted in female offspring exposed to DEX during late gestation may stem from abnormal development of the GH axis at the hypothalamic level.
Asunto(s)
Dexametasona/administración & dosificación , Dexametasona/toxicidad , Hígado Graso/etiología , Factor I del Crecimiento Similar a la Insulina/metabolismo , Efectos Tardíos de la Exposición Prenatal , Animales , Secuencia de Bases , Desarrollo Óseo/efectos de los fármacos , Dieta Alta en Grasa/efectos adversos , Hígado Graso/sangre , Hígado Graso/genética , Hígado Graso/patología , Femenino , Edad Gestacional , Hormona del Crecimiento/metabolismo , Humanos , Hipotálamo/efectos de los fármacos , Hipotálamo/embriología , Hipotálamo/metabolismo , Recién Nacido , Masculino , Enfermedad del Hígado Graso no Alcohólico , Embarazo , Efectos Tardíos de la Exposición Prenatal/sangre , Efectos Tardíos de la Exposición Prenatal/etiología , Efectos Tardíos de la Exposición Prenatal/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Caracteres Sexuales , Transducción de Señal/efectos de los fármacosRESUMEN
Synthetic glucocorticoids (GC) have been used to promote lung development in preterm infants, thereby decreasing respiratory distress syndrome and mortality, yet, concern has arisen from reports that such treatment predisposes individuals to disease in adulthood. Given the variety of preclinical studies that show metabolic and behavioral abnormalities in adulthood following fetal exposure to synthetic GC, we examined the effect of in utero exposure to the synthetic GC, dexamethasone (DEX), on hypothalamic expression of thyrotropin-releasing hormone (TRH) a central neuropeptide involved in mediating behavior and metabolic balance. Pregnant Sprague-Dawley rats were administered 0.4mg/kg DEX on gestational days 18-21. As adults (postnatal day (PD) 60), the offspring were fitted with temperature sensing transmitters allowing real-time monitoring of core body temperature (CBT) across the 24h light dark period. This revealed a significant decrease in CBT throughout the day in prenatal DEX-treated females on estrus and diestrus, but not in male offspring. The reduction in CBT by prenatal DEX exposure was accompanied by a significant decrease in the expression of Trh transcript in the paraventricular nucleus of the hypothalamus (PVN) of female rats at PD 60 and this effect was also present on PD7. There was also a female-specific reduction in the number of preproTRH-immunoreactive (ir) neurons in the PVN, with ppTRH-ir nerve fibers decreases that were present in both male and female offspring. No changes in thyroid hormone (triiodothyronine, T3; thyroxine, T4) were observed in adult offspring, but during development, both males and females (PD14) had lower T3 and T4 levels. These data indicate abnormal expression of TRH results from fetal DEX exposure during late gestation, possibly explaining the decreased CBT observed in the female offspring.
Asunto(s)
Antiinflamatorios/efectos adversos , Temperatura Corporal/efectos de los fármacos , Dexametasona/efectos adversos , Núcleo Hipotalámico Paraventricular/efectos de los fármacos , Efectos Tardíos de la Exposición Prenatal , Precursores de Proteínas/metabolismo , Hormona Liberadora de Tirotropina/metabolismo , Factores de Edad , Análisis de Varianza , Animales , Animales Recién Nacidos , Ritmo Circadiano/efectos de los fármacos , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Edad Gestacional , Núcleo Hipotalámico Paraventricular/metabolismo , Embarazo , Efectos Tardíos de la Exposición Prenatal/inducido químicamente , Efectos Tardíos de la Exposición Prenatal/patología , Efectos Tardíos de la Exposición Prenatal/fisiopatología , Precursores de Proteínas/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Caracteres Sexuales , Factores Sexuales , Hormona Liberadora de Tirotropina/genética , Tiroxina/metabolismo , Triyodotironina/metabolismoRESUMEN
Nitric oxide (NO) is a free radical molecule with a short half-life (<5 s). Because its synthesis from L-arginine by constitutive NO synthase (NOS) is low in many cell types, including neurons and endothelial cells, direct detection of NO in biological systems is a difficult task. During pathological conditions in the CNS, the inducible form of NOS (iNOS or NOS2) is expressed in activated astrocytes and microglial cells and can result in higher levels of NO. However, it may still be difficult to detect NO in these cell types using typical spectrophotometric methods. Of particular note, NO is readily oxidized to nitrite and nitrate (relatively stable products) in cells and medium, which can be measured as a valid indicator of NO synthesis. The conversion of NO to peroxynitrite leads to the formation of stable protein adducts that can be detected by immunohistochemical or immunofluorescence methods. Additionally, intracellular levels of NO can be detected in real time using fluorescence imaging and NO-specific, cell permeable indicator dyes.
Asunto(s)
Astrocitos/metabolismo , Óxido Nítrico/metabolismo , Animales , Células Cultivadas , Cromatografía Líquida de Alta Presión/métodos , Ratones , Microscopía Fluorescente , Óxido Nítrico/química , Ácido Peroxinitroso/metabolismo , Cultivo Primario de Células , Análisis de la Célula Individual/métodos , Técnicas de Cultivo de TejidosRESUMEN
Peroxiredoxin 6 (PRX6) belongs to the 1-Cys class of peroxiredoxins and is recognized as an important antioxidant protein in tissues such as cardiac muscle, skin, and lung. Preliminary in vivo proteomic data have revealed that PRX6 is adducted by 4-hydroxynonenal (4HNE) in the livers of rats chronically fed an ethanol-containing diet. The goals of this study were to evaluate the in vitro effect of aldehyde adduction on PRX6 peroxidase activity, identify specific sites of aldehyde modification using mass spectrometry, and predict conformational changes due to adduction using molecular modeling. PRX6 was found to be resistant to inactivation via aldehyde modification; however, Western blots of adducted protein revealed that both 4HNE and 4-oxononenal (4ONE) caused extensive cross-linking, resulting in high molecular mass species. Tandem mass spectrometry (ESI-LC-MS/MS) analysis demonstrated multiple sites of modification, but adduction of the active site Cys47 was not observed. Molecular modeling simulations indicated that adduction at Cys91 results in a change in protein active site conformation, which potentially restricts access of 4-HNE to Cys47. The Cys91-Lys209 cross-linked adducts could provide the conformational changes required to inactivate the protein by either restricting access to electrophiles or preventing important amino acid interactions within the catalytic triad.
Asunto(s)
Aldehídos/metabolismo , Antioxidantes , Biología Computacional/métodos , Reactivos de Enlaces Cruzados/metabolismo , Peroxiredoxina VI/metabolismo , Aldehídos/química , Alimentación Animal , Animales , Reactivos de Enlaces Cruzados/química , Etanol/administración & dosificación , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Modelos Moleculares , Conformación Molecular , Oxidación-Reducción , Peroxiredoxina VI/química , Ratas , Ratas Sprague-Dawley , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en TándemRESUMEN
4-Hydroxy-2-nonenal (4-HNE) is a major lipid peroxidation (LPO) product formed during oxidative stress. 4-HNE is highly reactive toward cellular nucleophiles and is implicated in the evolution of numerous pathologies associated with oxidative stress and LPO. Recent evidence suggests that chronic prooxidant exposure results in the loss of extracellular signal-regulated kinase (Erk)-1/2 phosphorylation in vivo, a signaling pathway associated with cellular proliferation, survival, and homeostasis. Immunodetection and molecular analysis were used in this study to evaluate the hypothesis that 4-HNE modification of Erk-1/2 inhibits constitutive Erk-Est-like protein (Elk)-1-activating protein (AP)-1 signaling. Primary rat hepatocytes treated with subcytotoxic, pathologically relevant concentrations of 4-HNE demonstrated a concentration-dependent loss of constitutive Erk-1/2 phosphorylation, activity, and nuclear localization. These findings were consistent with iron-induced intracellular LPO, which also resulted in a concentration-dependent decrease in hepatocyte Erk-1/2 phosphorylation and activity. 4-HNE and iron-induced inhibition of Erk-1/2 was inversely correlated with the accumulation of 4-HNE-Erk-1/2 monomer adducts. 4-HNE treatment of hepatocytes decreased nuclear total and phosphorylated Erk-1/2, Elk-1, and AP-1 phosphorylation as well as cFos and cJun activities. The cytosolic modification of unphosphorylated Erk-1/2 was evaluated in vitro using molar ratios of inactive Erk-2 to 4-HNE consistent with increasing oxidative stress in vivo. Liquid chromatography combined with tandem mass spectrometry confirmed monomer adduct formation and identified the major adduct species at the histidine 178 residue within the kinase phosphorylation lip. These novel results show that the formation of 4-HNE-Erk-1/2 monomer-adducts results in the inhibition of Erk-Elk-AP-1 signaling in hepatocytes and implicates the His 178 residue with the mechanism of inhibition.
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Aldehídos/farmacología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Hepatocitos/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Transcripción AP-1/antagonistas & inhibidores , Proteína Elk-1 con Dominio ets/antagonistas & inhibidores , Aldehídos/metabolismo , Secuencia de Aminoácidos , Animales , Ácido Ascórbico/farmacología , Cromatografía Liquida , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Quinasas MAP Reguladas por Señal Extracelular/química , Masculino , Espectrometría de Masas , Datos de Secuencia Molecular , Fosforilación , Ratas , Ratas Sprague-Dawley , Factor de Transcripción AP-1/fisiología , Proteína Elk-1 con Dominio ets/fisiologíaRESUMEN
Atrazine (2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine) is one of the most commonly used herbicides in the United States. Exposures in rodent models have led to a host of biological effects, most notably the suppression of luteinizing hormone surge. Previously, we have reported that diaminochlorotriazine (DACT), an atrazine metabolite, forms a covalent adduct with rat hemoglobin at Cys-125. In the present study, we investigated the formation of a similar covalent adduct at Cys-34 of rat and human albumins following atrazine exposure using MALDI-TOF/TOF MS and adduct-specific immunochemical detection. Using mass spectrometry, a covalent adduct with a mass of 110 Da was located on Cys-34 of albumin from rats exposed to 20, 50, 100, and 200 mg/kg atrazine as well as rat and human albumins exposed in vitro to 90 microg/mL DACT. On the basis of the formation of the adduct in vitro, the adduct structure is a dechlorinated diaminochlorotriazine. To further study this unique protein adduction, we collaborated with Strategic Biosolutions Inc. to generate a polyclonal antibody specific for the DACT adduct and report its use for immunochemical detection. We detected adduct formation in purified serum albumin samples from rats given 5, 10, 20, 50, 100, and 200 mg/kg atrazine as well as rat and human albumins exposed in vitro to 90 microg/mL DACT by using immunochemical analysis. No adducts were detected in control animals or in the in vitro controls using our immunochemical detection method. In summary, these data report the development of a novel immunochemical detection system that could provide a rapid screening methodology for the detection of atrazine in exposed human populations.
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Atrazina/toxicidad , Inmunohistoquímica/métodos , Albúmina Sérica/química , Animales , Western Blotting/métodos , Cisteína/química , Cisteína/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Herbicidas/toxicidad , Humanos , Mediciones Luminiscentes/métodos , Estructura Molecular , Ratas , Ratas Wistar , Reproducibilidad de los Resultados , Albúmina Sérica/análisis , Albúmina Sérica/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Triazinas/química , Triazinas/metabolismoRESUMEN
A proteomic approach was applied to mitochondrial protein isolated from the livers of rats fed a combination high-fat and ethanol diet to identify proteins modified by 4-hydroxynonenal (4-HNE). Using this approach, the endoplasmic reticulum chaperone, protein disulfide isomerase (PDI), which participates in the maturation of newly synthesized proteins through promoting correct disulfide formation, was consistently found to be modified by 4-HNE. Further mass spectral analysis of PDI isolated from the animals revealed modification of an active site Cys residue thought to be involved in client protein binding. To test the hypothesis that 4-HNE inhibits the chaperone, purified bovine PDI was treated with concentrations of 4-HNE ranging from 20 to 200 microM (10-100-fold molar excess aldehyde), resulting in 14-56% inhibition, respectively. Similar treatments with the lipid peroxidation products acrolein (ACR) and 4-oxononenal (4-ONE) resulted in 60 and 100% inhibition, respectively, suggesting inactivation of the chaperone via Cys modification. Thiol sensitivity was confirmed through concentration-dependent inhibition of PDI by the Cys modifier N-ethylmaleimide (NEM). While some degree of sensitivity to these lipid aldehydes is suggested by the data, when compared to inactivation of other proteins by 4-HNE, PDI has demonstrated a relative resistance. It was also observed that physiologic (e.g., 4 mM) concentrations of GSH were capable of removing the 4-HNE adducts, likely serving as a protective mechanism against inactivation by 4-HNE and other lipid peroxidation products. However, because an active site Cys was found to be modified by 4-HNE on PDI in vivo, it is possible that the protective effect of GSH on the chaperone decreases under conditions of sustained oxidative stress, such as during chronic alcohol consumption, as GSH is depleted. The data presented here thus suggest potential impairment of an important molecular chaperone during oxidative stress.
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Cisteína/química , Peroxidación de Lípido , Proteína Disulfuro Isomerasas/antagonistas & inhibidores , Acroleína/química , Aldehídos/química , Secuencia de Aminoácidos , Animales , Western Blotting , Depresores del Sistema Nervioso Central/farmacología , Dieta , Grasas de la Dieta/farmacología , Electroforesis en Gel Bidimensional , Etanol/farmacología , Etilmaleimida/química , Hidrólisis , Insulina/química , Masculino , Espectrometría de Masas , Datos de Secuencia Molecular , Oxidación-Reducción , Ratas , Ratas Sprague-Dawley , Reactivos de Sulfhidrilo , TripsinaRESUMEN
Lipid peroxidation during oxidative stress leads to increased concentrations of thiol-reactive alpha,beta-unsaturated aldehyde, including 4-hydroxy-2-nonenal (4-HNE) and 4-oxo-2-nonenal (4-ONE). These aldehydes have a documented ability to disrupt protein function following adduct formation with specific residues. Therefore, to identify 4-HNE-modified proteins in a model of ethanol-induced oxidative stress, a proteomic approach was applied to liver fractions prepared from rats fed a combination high-fat/ethanol diet. The results revealed that essential 90-kDa heat shock protein (Hsp90) was consistently modified by 4-HNE in the alcohol-treated animals. In vitro chaperoning experiments using firefly luciferase as a client protein were then performed to assess the functional effect of 4-HNE modification on purified recombinant human Hsp90, modified with concentrations of this aldehyde ranging from 23 to 450 microM. Modification of Hsp90 with 4-ONE also led to significant inhibition of the chaperone. Because 4-HNE and 4-ONE react selectively with Cys, a thiol-specific mechanism of inhibition was suggested by these data. Therefore, thiol sensitivity was confirmed following treatment of Hsp90 with the specific thiol modifier N-ethylmaleimide, which resulted in more than 99% inactivation of the chaperone by concentrations as low as 6 microM (1:1 M ratio). Finally, tryptic digest of 4-HNE-modified Hsp90 followed by liquid chromatography/tandem mass spectrometry peptide analysis identified Cys 572 as a site for 4-HNE modification. The results presented here thus establish that 4-HNE consistently modifies Hsp90 in a rat model of alcohol-induced oxidative stress and that the chaperoning activity of this protein is subject to dysregulation through thiol modification.
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Aldehídos/farmacología , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Hepatopatías Alcohólicas/metabolismo , Animales , Modelos Animales de Enfermedad , Peroxidación de Lípido , Masculino , Estrés Oxidativo , Ratas , Ratas Sprague-DawleyRESUMEN
A proteomic approach was applied to liver cytosol from rats fed a diet consisting of high fat and ethanol to identify 4-hydroxy-2-nonenal (4-HNE)-modified proteins in vivo. Cytosolic Hsp72, the inducible variant of the Hsp70 heat shock protein family, was consistently among the proteins modified by 4-HNE. Despite 1.3-fold induction of Hsp72 in the livers of ethanol-fed animals, no increase in Hsp70-mediated luciferase refolding in isolated heptocytes was observed, suggesting inhibition of this process by 4-HNE. A 50% and 75% reduction in luciferase refolding efficiency was observed in rabbit reticulocyte lysate (RRL) supplemented with recombinant Hsp72 which had been modified in vitro with 10 and 100 microM 4-HNE, respectively. This observation was accompanied by a 25% and 50% decrease in substrate binding by the chaperone following the same treatment; however, no effect on complex formation between Hsp72 and its co-chaperone Hsp40 was observed. Trypsin digest and mass spectral analysis of Hsp72 treated with 10 and 100 microM 4-HNE consistently identified adduct formation at Cys267 in the ATPase domain of the chaperone. The role of this residue in the observed inhibition was demonstrated through the use of DnaK, a bacterial Hsp70 variant lacking Cys267. DnaK was resistant to 4-HNE inactivation. Additionally, Hsp72 was resistant to inactivation by the thiol-unreactive aldehyde malondialdehyde (MDA), further supporting a role for Cys in Hsp72 inhibition by 4-HNE. Finally, the affinity of Hsp72 for ATP was decreased 32% and 72% following treatment of the chaperone with 10 and 100 microM 4-HNE, respectively. In a model of chronic alcoholic liver injury, induction of Hsp72 was not accompanied by an increase in protein refolding ability. This is likely the result of 4-HNE modification of the Hsp72 ATPase domain.