RESUMEN
Post-stroke neurological deficits and mortality are often associated with vascular disruption and neuronal apoptosis. Galectin-3 (Gal3) is a potent pro-survival and angiogenic factor. However, little is known about its protective role in the cerebral ischemia/reperfusion (I/R) injury. We have previously shown significant up-regulation of Gal3 in the post-stroke rat brain, and that blocking of Gal3 with neutralizing antibody decreases the cerebral blood vessel density. Our current study demonstrates that intracerebral local delivery of the Gal3 into rat brain at the time of reperfusion exerts neuroprotection. Ischemic lesion volume and neuronal cell death were significantly reduced as compared with the vehicle-treated MCAO rat brains. Gal3 increased vessel density and neuronal survival after I/R in rat brains. Importantly, Gal3-treated groups showed significant improvement in motor and sensory functional recovery. Gal3 increased neuronal cell viability under in vitro oxygen-glucose deprivation conditions in association with increased phosphorylated-Akt, decreased phosphorylated-ERK1/2, and reduced caspase-3 activity. Gene expression analysis showed down regulation of pro-apoptotic and inflammatory genes including Fas-ligand, and upregulation of pro-survival and pro-angiogenic genes including Bcl-2, PECAM, and occludin. These results indicate a key role for Gal3 in neuro-vascular protection and functional recovery following ischemic stroke through modulation of angiogenic and apoptotic pathways.
Asunto(s)
Inductores de la Angiogénesis/farmacología , Apoptosis/efectos de los fármacos , Caspasas/efectos de los fármacos , Galectina 3/uso terapéutico , Accidente Cerebrovascular Isquémico/prevención & control , Fármacos Neuroprotectores/farmacología , Proteínas Proto-Oncogénicas c-akt/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Animales , Encéfalo , Muerte Celular/efectos de los fármacos , Galectina 3/administración & dosificación , Expresión Génica/efectos de los fármacos , Hipoxia Encefálica/tratamiento farmacológico , Microinyecciones , Neuronas/efectos de los fármacos , Neuronas/patología , Ratas , Ratas Endogámicas SHR , Daño por Reperfusión/prevención & controlRESUMEN
Metabolic dysfunction impacts stroke incidence and outcome. However, the intricate association between altered metabolic program due to aging, and focal ischemia in brain, circulation, and peripheral organs is not completely elucidated. Here we identified locally and systemically altered metabolites in brain, liver, and plasma as a result of normal aging, ischemic-stroke, and extended time of reperfusion injury. Comprehensive quantitative metabolic profiling was carried out using nuclear magnetic resonance spectroscopy. Aging, but healthy rats showed significant metabolic alterations in the brain, but only a few metabolic changes in the liver and plasma as compared to younger rats. But, ischemic stroke altered metabolites significantly in liver and plasma of older rats during early acute phase. Major metabolic changes were also seen in the brains of younger rats following ischemic stroke during early acute phase of injury. We further report that metabolic changes occur sequentially in a tissue specific manner during extended reperfusion time of late repair phase. First metabolic alterations occurred in brain due to local injury. Next, changes in circulating metabolites in plasma occurred during acute-repair phase transition time. Lastly, the delayed systemic effect was seen in the peripheral organ, liver that exhibited significant and persistent changes in selected metabolites during later reperfusion time. The metabolic pathways involved in energy/glucose, and amino acid metabolism, inflammation, and oxidative stress were mainly altered as a result of aging and ischemia/reperfusion. Biomarker analysis revealed citrate, lysine, and tyrosine as potential age-independent blood metabolic biomarkers of ischemia/reperfusion. Overall, our study elucidates the complex network of metabolic events as a function of normal aging and acute stroke. We further provide evidence for a clear transition from local to systemic metabolic dysfunction due to ischemic injury in a time dependent manner, which may altogether greatly impact the post-stroke outcome.
Asunto(s)
Envejecimiento/fisiología , Isquemia Encefálica/metabolismo , Encéfalo/metabolismo , Hígado/metabolismo , Accidente Cerebrovascular/metabolismo , Animales , Espectroscopía de Resonancia Magnética/métodos , Metabolómica/métodos , Estrés Oxidativo/efectos de los fármacos , Ratas , Daño por Reperfusión/metabolismoRESUMEN
Cerebral ischemia evokes abnormal release of proteases in the brain microenvironment that spatiotemporally impact angio-neurogenesis. Dipeptidyl peptidase IV (DPPIV), a cell surface and secreted protease, has been implicated in extracellular matrix remodeling by regulating cell adhesion, migration, and angiogenesis through modifying the functions of the major chemokine stromal-derived factor, SDF1. To elucidate the possible association of DPPIV in ischemic brain, we examined the expression of DPPIV in the post-stroke rat brain and under in vitro ischemia by oxygen glucose deprivation (OGD). We further investigated the effects of DPPIV on SDF1 mediated in vitro chemotactic and angiogenic functions. DPPIV protein and mRNA levels were significantly upregulated during repair phase in the ischemic cortex of the rat brain, specifically in neurons, astrocytes, and endothelial cells. In vitro exposure of Neuro-2a neuronal cells and rat brain endothelial cells to OGD resulted in upregulation of DPPIV. In vitro functional analysis showed that DPPIV decreases the SDF1-mediated angiogenic potential of rat brain endothelial cells and inhibits the migration of Neuro-2a and neural progenitor cells. Western blot analyses revealed decreased levels of phosphorylated ERK1/2 and AKT in the presence of DPPIV. DPPIV inhibitor restored the effects of SDF1. Proteome profile array screening further revealed that DPPIV decreases matrix metalloproteinase-9, a key downstream effector of ERK-AKT signaling pathways. Overall, delayed induction of DPPIV in response to ischemia/reperfusion suggests that DPPIV may play an important role in endogenous brain tissue remodeling and repair processes. This may be mediated through modulation of SDF1-mediated cell migration and angiogenesis.
Asunto(s)
Isquemia Encefálica/enzimología , Movimiento Celular/fisiología , Dipeptidil Peptidasa 4/metabolismo , Neovascularización Patológica/metabolismo , Células-Madre Neurales/enzimología , Accidente Cerebrovascular/enzimología , Animales , Encéfalo/metabolismo , Isquemia Encefálica/patología , Movimiento Celular/efectos de los fármacos , Células Endoteliales/metabolismo , Masculino , Neurogénesis/fisiología , Ratas , Accidente Cerebrovascular/terapiaRESUMEN
Sphingomyelin synthase (SMS) is a key enzyme involved in the generation of sphingomyelin (SM) and regulation of cell growth and survival. However, the effects of SMS on neuronal cell proliferation and cell cycle progression are not completely elucidated. In this study, we examined the direct effects of SMS1 in regulating cell cycle progression and proliferation of Neuro-2a cells that exhibit neuronal characteristics. Neuro-2a cells transfected with SMS-specific small hairpin RNA (shRNA) expressed significantly lower levels of SMS1. RNA interference-mediated depletion of SMS1 in Neuro-2a cells caused a significant decrease in SM levels. Decreased SMS1 levels resulted in reduced proliferation rate and morphological changes including neurite-like outgrowth. Also, silencing of SMS1 induced cell cycle arrest as shown by the increased percentage of cells in G0/G1 and decreased proportion of cells in S phase. These changes were accompanied by upregulation of cyclin-dependent kinase inhibitor p27 and decreased levels of cyclin D1 and phospho-Akt. Nuclear accumulation of p27 was also evident in SMS1-deficient cells. Furthermore, loss of SMS1 inhibited the migratory potential of Neuro-2a cells in association with decreased levels of matrix metalloproteinases. These results indicate that SMS1 plays an important role in mediating the key signaling pathways that are involved in the tight coordination of multiple cellular activities, including neuronal cell proliferation, cell cycle progression, and migration, and therefore may have significant implications in neurodegenerative diseases.
Asunto(s)
Ciclo Celular/fisiología , División Celular/fisiología , Proliferación Celular/fisiología , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismo , Animales , Línea Celular , Ratones , Transducción de Señal/fisiologíaRESUMEN
Tricyclodecan-9-yl-xanthogenate (D609) inhibits phosphatidylcholine (PC)-phospholipase C (PLC) and/or sphingomyelin (SM) synthase (SMS). Inhibiting SMS can increase ceramide levels, which can inhibit cell proliferation. Here, we examined how individual inflammatory and glia cell proliferation is altered by D609. Treatment with 100-µM D609 significantly attenuated the proliferation of RAW 264.7 macrophages, N9 and BV-2 microglia, and DITNC(1) astrocytes, without affecting cell viability. D609 significantly inhibited BrdU incorporation in BV-2 microglia and caused accumulation of cells in G(1) phase with decreased number of cells in the S phase. D609 treatment for 2 h significantly increased ceramide levels in BV-2 microglia, which, following a media change, returned to control levels 22 h later. This suggests that the effect of D609 may be mediated, at least in part, through ceramide increase via SMS inhibition. Western blots demonstrated that 2-h treatment of BV-2 microglia with D609 increased expression of the cyclin-dependent kinase (Cdk) inhibitor p21 and down-regulated phospho-retinoblastoma (Rb), both of which returned to basal levels 22 h after removal of D609. Exogenous C8-ceramide also inhibited BV-2 microglia proliferation without loss of viability and decreased BrdU incorporation, supporting the involvement of ceramide in D609-mediated cell cycle arrest. Our current data suggest that D609 may offer benefit after stroke (Adibhatla and Hatcher, Mol Neurobiol 41:206-217, 2010) through ceramide-mediated cell cycle arrest, thus restricting glial cell proliferation.
Asunto(s)
Hidrocarburos Aromáticos con Puentes/farmacología , Ciclo Celular/efectos de los fármacos , Ceramidas/metabolismo , Tionas/farmacología , Animales , Bromodesoxiuridina/metabolismo , Caspasa 3/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ceramidas/farmacología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Diglicéridos/metabolismo , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Ratones , Norbornanos , Esfingomielinas/metabolismo , Tiocarbamatos , Factores de TiempoRESUMEN
Expressions of cell-cycle regulating proteins are altered after stroke. Cell-cycle inhibition has shown dramatic reduction in infarction after stroke. Ceramide can induce cell-cycle arrest by up-regulation of cyclin-dependent kinase (Cdk) inhibitors p21 and p27 through activation of protein phosphatase 2A (PP2A). Tricyclodecan-9-yl-xanthogenate (D609)-increased ceramide levels after transient middle cerebral artery occlusion (tMCAO) in spontaneously hypertensive rat (SHR) probably by inhibiting sphingomyelin synthase (SMS). D609 significantly reduced cerebral infarction and up-regulated Cdk inhibitor p21 and down-regulated phospho-retinoblastoma (pRb) expression after tMCAO in rat. Others have suggested bFGF-induced astrocyte proliferation is attenuated by D609 due to an increase in ceramide by SMS inhibition. D609 also reduced the formation of oxidized phosphatidylcholine (OxPC) protein adducts. D609 may attenuate generation of reactive oxygen species and formation of OxPC by inhibiting microglia/macrophage proliferation after tMCAO (please also see note added in proof: D609 may prevent mature neurons from entering the cell cycle at the early reperfusion, however may not interfere with later proliferation of microglia/ macrophages that are the source of brain derived neurotrophic factor (BDNF) and insulin-like growth factor (IGF-1) in offering protection). It has been proposed that D609 provides benefit after tMCAO by attenuating hypoxia-inducible factor-1alpha and Bcl2/adenovirus E1B 19 kDa interacting protein 3 expressions. Our data suggest that D609 provides benefit after stoke through inhibition of SMS, increased ceramide levels, and induction of cell-cycle arrest by up-regulating p21 and causing hypophosphorylation of Rb (through increased protein phosphatase activity and/or Cdk inhibition).
Asunto(s)
Antioxidantes , Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/patología , Hidrocarburos Aromáticos con Puentes , Accidente Cerebrovascular/tratamiento farmacológico , Accidente Cerebrovascular/patología , Tionas , Animales , Antioxidantes/farmacología , Antioxidantes/uso terapéutico , Biomarcadores/metabolismo , Isquemia Encefálica/fisiopatología , Hidrocarburos Aromáticos con Puentes/farmacología , Hidrocarburos Aromáticos con Puentes/uso terapéutico , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/metabolismo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Infarto de la Arteria Cerebral Media , Metabolismo de los Lípidos/efectos de los fármacos , Masculino , Norbornanos , Ratas , Ratas Endogámicas SHR , Ratas Sprague-Dawley , Transducción de Señal/fisiología , Accidente Cerebrovascular/fisiopatología , Tiocarbamatos , Tionas/farmacología , Tionas/uso terapéuticoRESUMEN
Today there exists only one FDA-approved treatment for ischemic stroke; i.e., the serine protease tissue-type plasminogen activator (tPA). In the aftermath of the failed stroke clinical trials with the nitrone spin trap/radical scavenger, NXY-059, a number of articles raised the question: are we doing the right thing? Is the animal research truly translational in identifying new agents for stroke treatment? This review summarizes the current state of affairs with plasminogen activators in thrombolytic therapy. In addition to therapeutic value, potential side effects of tPA also exist that aggravate stroke injury and offset the benefits provided by reperfusion of the occluded artery. Thus, combinational options (ultrasound alone or with microspheres/nanobubbles, mechanical dissociation of clot, activated protein C (APC), plasminogen activator inhibitor-1 (PAI-1), neuroserpin and CDP-choline) that could offset tPA toxic side effects and improve efficacy are also discussed here. Desmoteplase, a plasminogen activator derived from the saliva of Desmodus rotundus vampire bat, antagonizes vascular tPA-induced neurotoxicity by competitively binding to low-density lipoprotein related-receptors (LPR) at the blood-brain barrier (BBB) interface, minimizing the tPA uptake into brain parenchyma. tPA can also activate matrix metalloproteinases (MMPs), a family of endopeptidases comprised of 24 mammalian enzymes that primarily catalyze the turnover and degradation of the extracellular matrix (ECM). MMPs have been implicated in BBB breakdown and neuronal injury in the early times after stroke, but also contribute to vascular remodeling, angiogenesis, neurogenesis and axonal regeneration during the later repair phase after stroke. tPA, directly or by activation of MMP-9, could have beneficial effects on recovery after stroke by promoting neurovascular repair through vascular endothelial growth factor (VEGF). However, any treatment regimen directed at MMPs must consider their pleiotropic nature and the likelihood of either beneficial or detrimental effects that might depend on the timing of the treatment in relation to the stage of brain injury.
Asunto(s)
Fibrinolíticos/efectos adversos , Metaloproteinasas de la Matriz/metabolismo , Accidente Cerebrovascular/etiología , Activador de Tejido Plasminógeno/efectos adversos , Animales , Fibrinolíticos/uso terapéutico , Humanos , Modelos Biológicos , Accidente Cerebrovascular/tratamiento farmacológico , Activador de Tejido Plasminógeno/uso terapéuticoRESUMEN
Phosphatidylcholine (PtdCho) is a major membrane phospholipid, and its loss is sufficient in itself to induce cell death. PtdCho homeostasis is regulated by the balance between hydrolysis and synthesis. PtdCho is hydrolyzed by phospholipase A2 (PLA2), PtdChospecific phospholipase C (PtdCho-PLC), and phospholipase D (PLD). PtdCho synthesis is rate-limited by CTP:phosphocholine cytidylyltransferase (CCT), which makes CDP-choline. The final step of PtdCho synthesis is catalyzed by CDP-choline:1,2-diacylglycerol cholinephosphotransferase. PtdCho synthesis in the brain is predominantly through the CDP-choline pathway. Transient middle cerebral artery occlusion (tMCAO) significantly increased PLA2 activity, secretory PLA2 (sPLA2)-IIA mRNA and protein levels, PtdCho-PLC activity, and PLD2 protein expression following reperfusion. CDP-choline treatment significantly attenuated PLA2 activity, sPLA2-IIA mRNA and protein levels, and PtdCho-PLC activity, but did not affect PLD2 protein expression. tMCAO also resulted in loss of CCT activity and CCTalpha protein, which were partially restored by CDP-choline. No changes were observed in cytosolic PLA2 or calcium-independent PLA2 tMCAO. protein levels after Up-regulation of PLA2, PtdCho-PLC, and PLD and regulation of CCT collectively down-resulted in loss of PtdCho, which was significantly restored by CDP-choline treatment. CDP-choline treatment significantly attenuated the infarction volume by 55 +/- 5% after 1 h of tMCAO and 1 day of reperfusion. Taken together, these results suggest that CDP-choline significantly restores Ptd-Cho levels by differentially affecting sPLA2-IIA, PtdCho-PLC, and CCTalpha after transient focal cerebral ischemia. A hypothetical scheme is proposed integrating results from this study and from other reports in the literature.
Asunto(s)
Isquemia Encefálica/enzimología , Citidililtransferasa de Colina-Fosfato/metabolismo , Citidina Difosfato Colina/farmacología , Fosfatidilcolinas/metabolismo , Fosfolipasas A/metabolismo , Animales , Isquemia Encefálica/metabolismo , Citidililtransferasa de Colina-Fosfato/antagonistas & inhibidores , Regulación de la Expresión Génica/fisiología , Masculino , Fosfolipasa D/biosíntesis , Fosfolipasa D/genética , Fosfolipasas A/genética , Fosfolipasas A2 , ARN Mensajero/biosíntesis , Ratas , Ratas Endogámicas Dahl , Ratas Sprague-DawleyRESUMEN
Cytidine-5'-diphosphocholine (CDP-choline, also referred as citicoline), the key intermediate in phosphatidylcholine (PtdCho) synthesis, provided significant benefit in experimental central nervous system (CNS) injury including cerebral ischemia. CDP-choline is synthesized by CTP:phosphocholine cytidylyltransferase (CCT), the key rate-limiting enzyme in PtdCho synthesis. Phospholipase A(2) (PLA(2)) hydrolyzes PtdCho to produce free fatty acids and lyso-PtdCho, an inhibitor of CCT. We investigated the status of CCT and lyso-PtdCho after 10-min transient brain ischemia in gerbils with reperfusion up to 2 days. Ischemia with no reperfusion resulted in loss of CCT activity in cytosol (408 +/- 8 pmol/min/mg protein compared to sham 695 +/- 45; P < 0.01) and membrane (383 +/- 61 compared to sham 532 +/- 54; P < 0.05). CCT activity remained low over 24-hr reperfusion, and returned to sham levels at Day 2 in membrane but remained low in cytosol. CDP-choline significantly increased CCT activity in cytosol at 1 hr reperfusion (saline, 339 +/- 35 compared to CDP-choline, 430 +/- 70; P < 0.05) and in membrane at 6 hr (saline, 381 +/- 32 compared to CDP-choline, 489 +/- 50; P < 0.01) and 24 hr (saline, 417 +/- 24 compared to CDP-choline, 594 +/- 45; P < 0.01), but had no effect on CCT activity at Day 2. Lyso-PtdCho increased at 1-hr reperfusion (219 +/- 5 nmol/g tissue compared to sham, 92 +/- 8; P < 0.01), and remained elevated over 2 days. CDP-choline attenuated lyso-PtdCho levels at 1-hr reperfusion (162 +/- 21, P < 0.01 compared to saline). These data indicate that PtdCho synthesis is impaired after brain ischemia, and CDP-choline may increase PtdCho levels by attenuating the loss of CCT activity and lyso-PtdCho formation.
Asunto(s)
Citidililtransferasa de Colina-Fosfato/metabolismo , Citidina Difosfato Colina/farmacología , Ataque Isquémico Transitorio/enzimología , Lisofosfatidilcolinas/metabolismo , Fármacos Neuroprotectores/metabolismo , Análisis de Varianza , Animales , Citidililtransferasa de Colina-Fosfato/efectos de los fármacos , Citidina Difosfato Colina/metabolismo , Retroalimentación Fisiológica , Gerbillinae , Hipocampo/citología , Hipocampo/enzimología , Neuronas/enzimología , Fármacos Neuroprotectores/farmacologíaRESUMEN
Phospholipid degradation is an important promoter of neuronal death after transient cerebral ischemia. Phospholipid hydrolysis by phospholipase A2 (PLA2) after transient cerebral ischemia releases arachidonic acid. Arachidonic acid metabolism results in formation of reactive oxygen species, lipid peroxides, and toxic aldehydes (malondialdehyde, 4-hydroxynonenal, and acrolein). Citicoline (cytidine-5'-diphosphocholine), an intermediate in phosphatidylcholine synthesis, has undergone 13 phase III clinical trials for stroke, and is being evaluated for treatment of Alzheimer's and Parkinson's diseases. Here we examined the effect of citicoline on PLA2 activity in relationship to attenuating hydroxyl radical (OH*) generation and lipid peroxidation after transient forebrain ischemia in gerbil. High Ca2+ dependency (millimolar range) of PLA2 activity suggests that secretory PLA2 is the predominant isoform in membrane and mitochondria. Citicoline attenuated the increase in PLA2 activity in both membrane and mitochondrial fractions. In vitro, citicoline and its components choline and cytidine had no effect on the PLA2 activity. Thus, citicoline is not a "direct PLA2 inhibitor." Citicoline also significantly attenuated loss of cardiolipin and arachidonic acid release from phosphatidylcholine and phosphatidylethanolamine. Transient cerebral ischemia resulted in significant formation of OH* and malondialdehyde, and citicoline significantly attenuated their formation. These results suggest that citicoline provides neuroprotection by attenuating the stimulation of PLA2.
Asunto(s)
Citidina Difosfato Colina/farmacología , Radical Hidroxilo/metabolismo , Ataque Isquémico Transitorio/fisiopatología , Peroxidación de Lípido/fisiología , Fosfolipasas A/metabolismo , Animales , Ácido Araquidónico/análisis , Calcio/metabolismo , Cardiolipinas/análisis , Membrana Celular/enzimología , Cromatografía Líquida de Alta Presión , Gentisatos/análisis , Gerbillinae , Hipocampo/irrigación sanguínea , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hidroxibenzoatos/análisis , Membranas Intracelulares/enzimología , Masculino , Malondialdehído/metabolismo , Mitocondrias/enzimología , Modelos Biológicos , Fosfatidilcolinas/análisis , Fosfatidiletanolaminas/análisis , Fosfolipasas A2 , Ácido Salicílico/análisis , Ácido Salicílico/farmacologíaRESUMEN
Neuroprotection by citicoline (CDP-choline) in transient cerebral ischemia has been demonstrated previously. Citicoline has undergone several Phase III clinical trials for stroke, and is being evaluated for treatment of Alzheimer's and Parkinson's diseases. Phospholipid degradation and generation of reactive oxygen species (ROS) are major factors causing neuronal injury in CNS trauma and neurodegenerative diseases. Oxidative metabolism of arachidonic acid (released by the action of phospholipases) contributes to ROS generation. We examined the effect of citicoline on phospholipase A(2) (PLA(2)) activity in relation to the attenuation of hydroxyl radical (OH.) generation after transient forebrain ischemia of gerbil. PLA(2) activity (requires mM Ca(2+)) increased significantly (P < 0.05) in both membrane (50.2 +/- 2.2 pmol/min/mg protein compared to sham 35.9 +/- 3.2) and mitochondrial fractions (77.0 +/- 1.2 pmol/min/mg protein compared to sham 33.9 +/- 1.2) after cerebral ischemia and 2 hr reperfusion in gerbil, which was significantly attenuated (P < 0.01) by citicoline (membrane, 39.9. +/- 2.2 and mitochondria, 41.9 +/- 3.2 pmol/min/mg protein). In vitro, citicoline and its components cytidine and choline had no effect on PLA(2) activity, and thus citicoline as such is not a PLA(2) inhibitor. Ischemia/reperfusion resulted in significant OH. generation (P < 0.01) and citicoline significantly (P < 0.01) attenuated their formation (expressed as 2,3-dihydroxybenzoic acid/salicylate ratio; ischemia/24 hr reperfusion, 6.30 +/- 0.23; sham, 2.56 +/- 0.27; ischemia/24 hr reperfusion + citicoline, 4.85 +/- 0.35). These results suggest that citicoline affects PLA(2) stimulation and decreases OH. generation after transient cerebral ischemia.
Asunto(s)
Citidina Difosfato Colina/farmacología , Ataque Isquémico Transitorio/tratamiento farmacológico , Ataque Isquémico Transitorio/metabolismo , Nootrópicos/farmacología , Fosfolipasas A/metabolismo , Animales , Calcio/metabolismo , Muerte Celular/efectos de los fármacos , Gerbillinae , Hipocampo/metabolismo , Hipocampo/patología , Radical Hidroxilo/metabolismo , Ataque Isquémico Transitorio/patología , Peroxidación de Lípido/efectos de los fármacos , Masculino , Neuronas/metabolismo , Neuronas/patología , Fármacos Neuroprotectores/farmacología , Fosfolipasas A2RESUMEN
The soil health index is an approach for assessing the ecological potential of a soil. The index is based on a physical, chemical, and biological characterization and rating of soil conditions. The approach is flexible, permits comparisons amongst soils with widely different properties and contaminant levels, and it can be adapted to site specific conditions. The rationale and development of the index are documented in this report along with sample handling, assessment methods, and quality assurance practices. Standardized reporting formats have also been developed for compiling and presenting the findings. An interpretive guide is included for the reporting formats and how to apply the results to site specific conditions.
Asunto(s)
Microbiología del Suelo/normas , Contaminantes del Suelo/normas , Suelo/normas , Suelo/análisis , Contaminantes del Suelo/análisisRESUMEN
Use of chemical criteria in assessing the potential for adverse toxic effects in contaminated sites can under or overestimate the necessary level of site cleanup required. The use of ecotoxicity testing provides a more direct assessment of adverse environmental impact. A multi-trophic level soil ecotoxicity assessment was done on soil contaminated with crude oil distilled into five different fractions based on hydrocarbon chain lengths. Results indicate that the fraction above C26 was not toxic to microbes, plants, and earthworms, when present in concentrations far above the 1000 mg/kg total petroleum hydrocarbon criterion. Our ecotoxicity test battery results indicate that weathered heavy crude oils can be much less toxic than lighter, freshly spilled diesel oils, yet using a gross measure of total petroleum hydrocarbons would not detect this differences.
Asunto(s)
Determinación de Punto Final , Residuos Peligrosos/análisis , Petróleo/análisis , Petróleo/toxicidad , Contaminantes del Suelo/análisis , Contaminantes del Suelo/toxicidad , Alberta , Animales , Bacterias/efectos de los fármacos , Hidrocarburos/análisis , Hidrocarburos/toxicidad , Lactuca/efectos de los fármacos , Lactuca/crecimiento & desarrollo , Mediciones Luminiscentes , Oligoquetos/fisiología , Suelo/análisis , Microbiología del SueloRESUMEN
DNA adducts formed in human uroepithelial cells (HUC) following exposure to N-hydroxy-4-aminobiphenyl (N-OH-ABP), the proximate metabolite of the human bladder carcinogen 4-aminobiphenyl (ABP), were analyzed by the (32)P-postlabeling method. Two adducts detected by (32)P-postlabeling were previously identified as the 3',5'-bisphospho derivatives of N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP) and N-(deoxyadenosin-8-yl)-4-aminobiphenyl (dA-C8-ABP) (Frederickson S et al. [1992] Carcinogenesis 13: 955-961; Hatcher and Swaminathan [1995b] Carcinogenesis 16: 295-301). In contrast to the dG-C8-ABP adduct, which was 3'-dephosphorylated by nuclease P1, dA-C8-ABP was resistant to nuclease P1, thus providing an enrichment step before postlabeling. Autoradiography of the two-dimensional thin-layer chromatogram of the postlabeled products obtained following nuclease P1 digestion revealed several minor adducts, one of which has been identified in the present study. Postlabeling analyses following nuclease P1 digestion of the products obtained from the reaction of N-acetoxy-4-aminobiphenyl with deoxyguanosine-3'-monophosphate (dGp) demonstrated the presence of this minor adduct. The 3'-monophosphate derivative of the adduct was subsequently chromatographically purified and subjected to spectroscopic analyses. Based on proton NMR and mass spectroscopic analyses of the synthetic product, the chemical structure of the adduct has been identified as N-(deoxyguanosin-N(2)-yl)-4-azobiphenyl (dG-N==N-ABP). (32)P-Postlabeling analysis of the nuclease P1-enriched DNA hydrolysate of HUCs treated with N-OH-ABP or N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) showed the presence of the dG-N==N-ABP adduct. It was also detected in calf thymus DNA incubated with HUC cytosol and N-OH-ABP in the presence of acetyl-CoA, or incubated with HUC microsomes and N-OH-AABP. These results demonstrate that in the target cells for ABP carcinogenesis in vivo, N-OH-ABP and N-OH-AABP are bioactivated by acyltransferases to reactive arylnitrenium ions that covalently interact at the N2 position of deoxyguanosine in DNA.
Asunto(s)
Compuestos de Aminobifenilo/toxicidad , Compuestos Azo/análisis , Compuestos de Bifenilo/análisis , Carcinógenos Ambientales/toxicidad , Aductos de ADN/análisis , ADN/química , Desoxirribonucleótidos/análisis , Urotelio/efectos de los fármacos , Células Cultivadas , Cromatografía Líquida de Alta Presión , Cromatografía en Capa Delgada , Humanos , Espectroscopía de Resonancia Magnética , Radioisótopos de Fósforo , Neoplasias de la Vejiga Urinaria/inducido químicamente , Urotelio/químicaRESUMEN
Polyamines (putrescine, spermidine and spermine) are ubiquitous cellular components, but their specific role in central nervous system (CNS) injury has yet to be characterized. CNS injury results in increased activities of ornithine decarboxylase and spermidine/spermine-N(1)-acetyltransferase, and accumulation of putrescine. The present study determined the polyamine profile in three models of CNS injury, in two different species (gerbil and rat) and two strains of rats (Sprague-Dawley and spontaneously hypertensive): (1) transient focal cerebral ischemia in spontaneously hypertensive rats (SHR); (2) traumatic brain injury in Sprague-Dawley rats; and (3) transient forebrain ischemia in gerbils. While there was a significant increase in putrescine in all three models, spermine and spermidine levels were unaltered in forebrain ischemia and traumatic brain injury. However, transient focal cerebral ischemia shows depletion of spermine and spermidine levels in injured hemisphere compared to contralateral region. Exogenous spermine significantly restored the spermine as well as spermidine levels in the ipsilateral hemisphere after transient focal cerebral ischemia, but did not alter putrescine levels or the ratio of spermidine to spermine. The loss of spermine in particular, may have several consequences that contribute to ischemic injury, including destabilization of chromatin, decreased mitochondrial Ca(2+) buffering capacity, and increased susceptibility to oxidative stress. Based on our and other studies, we propose a tentative antioxidant mechanism of spermine neuroprotection.
Asunto(s)
Lesiones Encefálicas/metabolismo , Encéfalo/metabolismo , Ataque Isquémico Transitorio/metabolismo , Espermidina/metabolismo , Espermina/metabolismo , Animales , Gerbillinae , Putrescina/metabolismo , Ratas , Ratas Endogámicas SHR , Ratas Sprague-DawleyRESUMEN
The DNA adducts were analyzed by 32P-postlabeling method following exposure of human uroepithelial cells (HUC) to N-hydroxy-4-aminobiphenyl (N-OH-ABP), the proximate metabolite of the human bladder carcinogen 4-aminobiphenyl (ABP). TLC of the postlabeled products on the first dimension revealed several products, the majority of which stayed close to the origin and were earlier identified as the 3',5' -bisphospho derivatives of N-(deoxyguanosin-8-yl)-4-aminobiphenyl and N-(deoxyadenosin-8-yl)-4-aminobiphenyl (Carcinogenesis 13 (1993) 955; Carcinogenesis 16 (1995) 295). Here we report characterization of two additional adducts that amounted to less than 5% of the total adducts. Autoradiography of D1 chromatogram of the postlabeled products of calf thymus DNA chemically interacted with N-OH-ABP under acidic conditions revealed two adducts, #1 and #2, with R(f) values of about 0.2 and 0.3, respectively. Two adducts with D1 thin layer chromatographic properties similar to those of adducts #1 and #2 were obtained on postlabeling analyses of products generated by chemical interaction of N-acetoxy-4-aminobiphenyl (N-OAc-ABP) with deoxyguanosine-3' -monophosphate (dGp). Based on proton NMR and mass spectroscopic analyses of the synthetic products derived from N-OAc-ABP, the chemical structures of adducts #1 and #2 have been identified as 3-(deoxyguanosin-N(2)-yl)-4-aminobiphenyl, and N-(deoxyguanosin-N(2)-yl)-4-aminobiphenyl, respectively. Both of these adducts were insensitive to digestion with nuclease P1. 32P-Postlabeling analysis of the nuclease P1 enriched DNA hydrolysate of HUC cells treated with N-OH-ABP showed the presence of adduct #2 but not adduct #1. Adduct #2 was also detected in calf thymus DNA incubated with HUC cytosol and N-OH-ABP in the presence of acetyl CoA. These results suggest that in the target cells for ABP carcinogenesis in vivo, N-OH-ABP is bioactivated by acetyl CoA-dependent acyltransferases to reactive arylnitrenium ions that covalently interact at N(2)-position of deoxyguanosine in DNA.