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1.
Acta Neuropathol ; 146(5): 685-705, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37740734

RESUMO

Oxidative stress plays an essential role in the development of Parkinson's disease (PD). 8-oxo-7,8-dihydroguanine (8-oxodG, oxidized guanine) is the most abundant oxidative stress-mediated DNA lesion. However, its contributing role in underlying PD pathogenesis remains unknown. In this study, we hypothesized that 8-oxodG can generate novel α-synuclein (α-SYN) mutants with altered pathologic aggregation through a phenomenon called transcriptional mutagenesis (TM). We observed a significantly higher accumulation of 8-oxodG in the midbrain genomic DNA from PD patients compared to age-matched controls, both globally and region specifically to α-SYN. In-silico analysis predicted that forty-three amino acid positions can contribute to TM-derived α-SYN mutation. Here, we report a significantly higher load of TM-derived α-SYN mutants from the midbrain of PD patients compared to controls using a sensitive PCR-based technique. We found a novel Serine42Tyrosine (S42Y) α-SYN as the most frequently detected TM mutant, which incidentally had the highest predicted aggregation score amongst all TM variants. Immunohistochemistry of midbrain sections from PD patients using a newly characterized antibody for S42Y identified S42Y-laden Lewy bodies (LB). We further demonstrated that the S42Y TM variant significantly accelerates WT α-SYN aggregation by cell and recombinant protein-based assays. Cryo-electron tomography revealed that S42Y exhibits considerable conformational heterogeneity compared to WT fibrils. Moreover, S42Y exhibited higher neurotoxicity compared to WT α-SYN as shown in mouse primary cortical cultures and AAV-mediated overexpression in the substantia nigra of C57BL/6 J mice. To our knowledge, this is the first report describing the possible contribution of TM-generated mutations of α-SYN to LB formation and PD pathogenesis.


Assuntos
Doença de Parkinson , Humanos , Animais , Camundongos , Doença de Parkinson/patologia , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , 8-Hidroxi-2'-Desoxiguanosina , Camundongos Endogâmicos C57BL , Mutagênese , DNA
2.
Innov Clin Neurosci ; 15(7-8): 27-31, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30254797

RESUMO

Cognitive, affective, and sleep disturbances can be found in patients with Huntington's disease (HD), and medications used to treat these HD-related sequela can also impact HD-related movement disorders. We present the case of a 52-year-old Caucasian man with previously undiagnosed HD who exhibited significant choreoathetoid movements that improved with discontinuation of fluoxetine and lisdexamfetamine upon hospital admission. Following diagnosis of HD through genetic testing, he was administered 5mg of oral melatonin on two consecutive evenings, which resulted in worsening choreoathetosis. We calculated Naranjo adverse event scores of 5, 5, and 2 for fluoxetine, lisdexamfetamine, and melatonin, respectively, based on our assessment, review of outpatient medical records, and available literature. We review the literature surrounding these possible adverse drug events and their mechanisms regarding dopaminergic modulation in early-middle stages of HD. Our report indicates that caution should be exercised when initiating psychostimulants, fluoxetine, and melatonin in patients with early-middle stage HD. Screening for HD might be warranted for patients who develop choreoathetosis after initiation of the aforementioned medications. We recommend ascertaining baseline level of chorea before initiating these medications in patients with known HD and closely monitoring for exacerbation during therapy.

3.
DNA Repair (Amst) ; 49: 26-32, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27865706

RESUMO

Somatic instability of CAG repeats has been associated with the clinical progression of CAG repeat diseases. Aging and DNA repair processes influence the somatic stability of CAG repeat in disease and in mouse models. However, most of the studies have focused on genetically engineered transgenic repeats and little is known about the stability of naturally polymorphic CAG repeats. To study whether age and/or DNA repair activity have an effect on the somatic stability of CAG repeats, we analyzed variations of the length of naturally polymorphic CAG repeats in the striatum of young and aged WT and ogg1 KO mice. Some multiple and long polymorphic CAG repeats were observed to have variable length in the striatum of aged mice. Interestingly, a low level of repeat variability was detected in the CAG repeat located in tbp, the only mouse polymorphic CAG repeat that is associated with a trinucleotide disease in humans, in the striatum of aged mice and not in young mice. We propose that age may have an effect on the somatic stability of polymorphic CAG repeats and that such an effect depends on intrinsic CAG repeat characteristics.


Assuntos
Envelhecimento , DNA Glicosilases/metabolismo , Reparo do DNA , Expansão das Repetições de Trinucleotídeos , Animais , Corpo Estriado/metabolismo , DNA/metabolismo , Camundongos , Camundongos Knockout
4.
Neurotoxicology ; 57: 258-269, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27771255

RESUMO

Pb exposure is associated with cognitive deficits including Attention Deficit Hyperactivity Disorder (ADHD) in children and alters auditory temporal processing in humans and animals. Serotonin has been implicated in auditory temporal processing and previous studies from our laboratory have demonstrated that developmental Pb decreases expression of serotonin (5-HT) in the adult murine lateral superior olive (LSO). During development, certain non-serotonergic sensory neurons, including auditory LSO neurons, transiently take up 5-HT through the serotonin reuptake transporter (SERT). The uptake of 5-HT is important for development of sensory systems. This study examines the effect of Pb on the serotonergic system in the LSO of the early postnatal mouse. Mice were exposed to moderate Pb (0.01mM) or high Pb (0.1mM) throughout gestation and postnatal day 4 (P4) and P8. We found that Pb exposure prolongs the normal developmental expression of 5-HT by LSO neurons and this is correlated with expression of SERT on LSO cell bodies. The prolonged expression of 5-HT by postnatal LSO neurons is correlated with decreased synaptic immunolabeling within the LSO. This Pb-associated decrease in synaptic density within the LSO could contribute to the auditory temporal processing deficits and cognitive deficits associated with developmental Pb exposure.


Assuntos
Chumbo/farmacologia , Neurônios/efeitos dos fármacos , Serotonina/metabolismo , Complexo Olivar Superior/citologia , Fatores Etários , Análise de Variância , Animais , Animais Recém-Nascidos , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Chumbo/sangue , Camundongos , Camundongos Endogâmicos CBA , Monoaminoxidase/metabolismo , Proteínas de Ligação a RNA/metabolismo , Complexo Olivar Superior/efeitos dos fármacos , Sinaptofisina/metabolismo , Fatores de Tempo , Triptofano Hidroxilase/metabolismo
5.
J Sports Sci ; 33(16): 1692-701, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25620316

RESUMO

The purpose of this study was to determine the effects of repeated bouts of long-duration endurance exercise on both muscle and urinary levels of oxidative DNA damage in moderately trained individuals. Seven moderately trained male cyclists participated in this study. All participants repeated two sessions consisting of a 5-h cycling period (equivalent to approximately 52%[Formula: see text]O2peak) followed by a 15-h rest, then a 40-km time trial. During the sessions, participants were instructed to take water ad libitum and to consume a standard sports drink consisting of 0.12 g·kg(-1) body weight·hr(-1) of carbohydrates. For each session, 24 h urine output was collected on the day before the 5-h exercise, and also between the 5-h exercise and 40-km time trial, in addition to between days 1-5 post-exercise. Subsequently, muscle and urinary levels of 8-hydroxy-2'- deoxyguanosine (8-OHdG) were determined using high performance liquid chromatography with electrochemical detection. No significant alterations were observed between two sessions at the muscle or urinary levels of 8-OHdG. These results suggest that repeated bouts of exercise with a 7-day washout period may not lead to an accumulation of DNA damage products after a second 5-h stationary cycling bout.


Assuntos
Desoxiguanosina/análogos & derivados , Exercício Físico/fisiologia , Músculo Esquelético/metabolismo , Resistência Física/fisiologia , 8-Hidroxi-2'-Desoxiguanosina , Adulto , Apoptose , Ciclismo/fisiologia , Dano ao DNA , Desoxiguanosina/metabolismo , Desoxiguanosina/urina , Humanos , Masculino , Estresse Oxidativo/fisiologia , Adulto Jovem
6.
PLoS Genet ; 10(2): e1003974, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24516391

RESUMO

The accumulation of somatic mitochondrial DNA (mtDNA) mutations is implicated in aging and common diseases of the elderly, including cancer and neurodegenerative disease. However, the mechanisms that influence the frequency of somatic mtDNA mutations are poorly understood. To develop a simple invertebrate model system to address this matter, we used the Random Mutation Capture (RMC) assay to characterize the age-dependent frequency and distribution of mtDNA mutations in the fruit fly Drosophila melanogaster. Because oxidative stress is a major suspect in the age-dependent accumulation of somatic mtDNA mutations, we also used the RMC assay to explore the influence of oxidative stress on the somatic mtDNA mutation frequency. We found that many of the features associated with mtDNA mutations in vertebrates are conserved in Drosophila, including a comparable somatic mtDNA mutation frequency (∼10(-5)), an increased frequency of mtDNA mutations with age, and a prevalence of transition mutations. Only a small fraction of the mtDNA mutations detected in young or old animals were G∶C to T∶A transversions, a signature of oxidative damage, and loss-of-function mutations in the mitochondrial superoxide dismutase, Sod2, had no detectable influence on the somatic mtDNA mutation frequency. Moreover, a loss-of-function mutation in Ogg1, which encodes a DNA repair enzyme that removes oxidatively damaged deoxyguanosine residues (8-hydroxy-2'-deoxyguanosine), did not significantly influence the somatic mtDNA mutation frequency of Sod2 mutants. Together, these findings indicate that oxidative stress is not a major cause of somatic mtDNA mutations. Our data instead suggests that somatic mtDNA mutations arise primarily from errors that occur during mtDNA replication. Further studies using Drosophila should aid in the identification of factors that influence the frequency of somatic mtDNA mutations.


Assuntos
Envelhecimento/genética , DNA Mitocondrial/genética , Mutação/genética , Estresse Oxidativo , Envelhecimento/patologia , Animais , DNA Glicosilases/genética , Reparo do DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Humanos , Mitocôndrias/genética , Mitocôndrias/patologia , Modelos Animais , Taxa de Mutação , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/genética
7.
J Pharmacol Exp Ther ; 348(2): 336-45, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24297779

RESUMO

Genetic variation in the multidrug resistance gene ABCB1, which encodes the efflux transporter P-glycoprotein (P-gp), has been associated with Parkinson disease. Our goal was to investigate P-gp transport of paraquat, a Parkinson-associated neurotoxicant. We used in vitro transport models of ATPase activity, xenobiotic-induced cytotoxicity, transepithelial permeability, and rhodamine-123 inhibition. We also measured paraquat pharmacokinetics and brain distribution in Friend leukemia virus B-type (FVB) wild-type and P-gp-deficient (mdr1a(-/-)/mdr1b(-/-)) mice following 10, 25, 50, and 100 mg/kg oral doses. In vitro data showed that: 1) paraquat failed to stimulate ATPase activity; 2) resistance to paraquat-induced cytotoxicity was unchanged in P-gp-expressing cells in the absence or presence of P-gp inhibitors GF120918 [N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide] and verapamil-37.0 [95% confidence interval (CI): 33.2-41.4], 46.2 (42.5-50.2), and 34.1 µM (31.2-37.2)-respectively; 3) transepithelial permeability ratios of paraquat were the same in P-gp-expressing and nonexpressing cells (1.55 ± 0.39 and 1.39 ± 0.43, respectively); and 4) paraquat did not inhibit rhodamine-123 transport. Population pharmacokinetic modeling revealed minor differences between FVB wild-type and mdr1a(-/-)/mdr1b(-/-) mice: clearances of 0.47 [95% confidence interval (CI): 0.42-0.52] and 0.78 l/h (0.58-0.98), respectively, and volume of distributions of 1.77 (95% CI: 1.50-2.04) and 3.36 liters (2.39-4.33), respectively; however, the change in clearance was in the opposite direction of what would be expected. It is noteworthy that paraquat brain-to-plasma partitioning ratios and total brain accumulation were the same across doses between FVB wild-type and mdr1a(-/-)/mdr1b(-/-) mice. These studies indicate that paraquat is not a P-gp substrate. Therefore, the association between ABCB1 pharmacogenomics and Parkinson disease is not attributed to alterations in paraquat transport.


Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Células Epiteliais/efeitos dos fármacos , Herbicidas/farmacocinética , Paraquat/farmacocinética , Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Animais , Transporte Biológico/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Permeabilidade Capilar/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células Epiteliais/metabolismo , Corantes Fluorescentes/metabolismo , Herbicidas/administração & dosagem , Herbicidas/metabolismo , Herbicidas/farmacologia , Masculino , Moduladores de Transporte de Membrana/farmacologia , Camundongos , Camundongos Knockout , Paraquat/administração & dosagem , Paraquat/metabolismo , Paraquat/farmacologia , Doença de Parkinson/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Rodamina 123/metabolismo , Sus scrofa , Distribuição Tecidual , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATP
8.
Neurochem Int ; 61(5): 721-30, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22743193

RESUMO

Cumulative damage to cellular macromolecules via oxidative stress is a hallmark of aging and neurodegenerative disease. Whether such damage is a cause or a subsequent effect of neurodegeneration is still unknown. This paper describes the development of an age-associated mild parkinsonian model in mice that lack the DNA repair enzyme 8-oxoguanine glycosylase 1 (Ogg1). Aged OGG1 knock-out (OGG1 KO) mice show a decreased spontaneous locomotor behavior and evidence a decrease in striatal dopamine levels, a loss of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra (SN), and an increase in ubiquitin-positive inclusions in their remaining SN neurons. In addition, young OGG1 KO mice are more susceptible to the dopaminergic toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) than their wild-type (WT) counterparts. Age-associated increases in 7,8-dihydro-2'-deoxyguanine (oxo(8)dG) have been reported in brain regions and neuronal populations affected in Parkinson's disease (PD), toxin-induced parkinsonian models, and mice harboring genetic abnormalities associated with PD. Because of these increased oxo(8)dG levels, the OGG1 KO mouse strain could shed light on molecular events leading to neuronal loss as a consequence of cumulative oxidative damage to DNA during aging and after toxicological challenge.


Assuntos
Envelhecimento/genética , Corpo Estriado/metabolismo , DNA Glicosilases/deficiência , Intoxicação por MPTP/genética , Substância Negra/metabolismo , Envelhecimento/efeitos dos fármacos , Envelhecimento/patologia , Animais , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/patologia , DNA Glicosilases/genética , Feminino , Intoxicação por MPTP/patologia , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Vias Neurais/efeitos dos fármacos , Vias Neurais/metabolismo , Vias Neurais/patologia , Substância Negra/efeitos dos fármacos , Substância Negra/enzimologia
10.
Neuropharmacology ; 61(4): 677-86, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21635908

RESUMO

High doses of methamphetamine induce the excessive release of dopamine resulting in neurotoxicity. However, moderate activation of dopamine receptors can promote neuroprotection. Therefore, we used in vitro and in vivo models of stroke to test the hypothesis that low doses of methamphetamine could induce neuroprotection. We demonstrate that methamphetamine does induce a robust, dose-dependent, neuroprotective response in rat organotypic hippocampal slice cultures exposed to oxygen-glucose deprivation (OGD). A similar dose dependant neuroprotective effect was observed in rats that received an embolic middle cerebral artery occlusion (MCAO). Significant improvements in behavioral outcomes were observed in rats when methamphetamine administration delayed for up to 12 h after MCAO. Methamphetamine-mediated neuroprotection was significantly reduced in slice cultures by the addition of D1 and D2 dopamine receptor antagonist. Treatment of slice cultures with methamphetamine resulted in the dopamine-mediated activation of AKT in a PI3K dependant manner. A similar increase in phosphorylated AKT was observed in the striatum, cortex and hippocampus of methamphetamine treated rats following MCAO. Methamphetamine-mediated neuroprotection was lost in rats when PI3K activity was blocked by wortmannin. Finally, methamphetamine treatment decreased both cleaved caspase 3 levels in slice cultures following OGD and TUNEL staining within the striatum and cortex in rats following transient MCAO. These data indicate that methamphetamine can mediate neuroprotection through activation of a dopamine/PI3K/AKT-signaling pathway.


Assuntos
Metanfetamina/administração & dosagem , Fármacos Neuroprotetores/administração & dosagem , Fosfatidilinositol 3-Quinase/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Transdução de Sinais/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Hipocampo/efeitos dos fármacos , Hipocampo/enzimologia , Hipocampo/patologia , Masculino , Técnicas de Cultura de Órgãos , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Transdução de Sinais/fisiologia , Acidente Vascular Cerebral/enzimologia , Acidente Vascular Cerebral/patologia , Acidente Vascular Cerebral/prevenção & controle
11.
Am J Respir Cell Mol Biol ; 42(5): 537-44, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-19541843

RESUMO

Although use of methamphetamine (MA) by smoking is the fastest growing method of administration, very limited data are available describing the effects of smoked MA. Using a murine inhalation exposure system, we explored the pulmonary effects of low-dose acute inhalation exposure to MA vapor (smoke). Inhalation of MA vapor resulted in transiently reduced pulmonary function, as measured by transpulmonary resistance, dynamic compliance, and whole-body plethysmography compared with unexposed control animals. These changes were associated with an approximately 34% reduction in serotonin (5-hydroxytryptamine [5-HT]) metabolism/inactivation to 5-hydroxyindolacetic acid, and a nearly 40% reduction in monoamine oxidase (MAO)-A activity in the lung. Pretreatment of mice with a selective 5-HT reuptake inhibitor completely ablated the MA-induced changes in pulmonary function, confirming a key role for the 5-HT transporter (serotonin transporter [SERT]) and the serotonergic system in this effect. Immunofluorescent staining of mouse lung tissue confirmed high expression of SERT in airway epithelial cells. Using mouse airway epithelial cell line, LA-4, and purified human MAO-A, it was demonstrated that MA impedes 5-HT metabolism through direct inhibition of MAO-A activity in vitro. Together, these data demonstrate that low-dose exposure to MA results in reduced pulmonary function mediated via SERT and subsequent perturbation of 5-HT metabolism in the lung. This supports a role for the serotonergic system in MA-mediated pulmonary effects.


Assuntos
Pulmão/efeitos dos fármacos , Pulmão/fisiologia , Metanfetamina/administração & dosagem , Metanfetamina/farmacologia , Serotonina/metabolismo , Animais , Citalopram/administração & dosagem , Citalopram/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Pulmão/citologia , Camundongos , Camundongos Endogâmicos BALB C , Modelos Biológicos , Monoaminoxidase/metabolismo , Testes de Função Respiratória , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Fatores de Tempo
12.
Redox Rep ; 14(2): 82-92, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19389276

RESUMO

Levels of oxidized guanosine base in DNA have become a hallmark biomarker in assessing oxidative stress implicated in a variety of disease and toxin-induced states. However, there is evidence that the guanosine in the nucleotide triphosphate pool (GTP) is more susceptible to oxidation than guanosine residues incorporated into nucleic acids and this causes a substantial amount of the oxidized product, 8-oxoguanosine 5'-triphosphate (oxo(8)GTP), to accumulate in cell-free and in cell-culture preparations. Electron paramagnetic resonance (EPR) spectroscopy and direct EPR analysis of free radical production by copper sulfate and L-ascorbic acid demonstrates that the hydroxyl radical (HO(*)) is produced via oxidation of Cu(+) to Cu(2+) while in a complex with GTP. This HO(*) production is dependent on the availability of oxygen and the presence of GTP in the reaction milieu. Verification of free radical-mediated production of oxo(8)GTP is presented using HPLC with electrochemical detection and matrix-assisted laser desorption/ionization linear time-of-flight mass spectrometry (MALDI-LTOF-MS). The sum of these results is presented in a novel mechanism of GTP oxidation by Cu(2+) and L-ascorbic acid. A better understanding of the chemistry involved in this oxidative modification of GTP facilitates a more comprehensive understanding of its potential physiological consequences.


Assuntos
Cobre/química , Guanosina Trifosfato/química , Radical Hidroxila/química , Ácido Ascórbico/química , DNA/química , DNA/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Concentração de Íons de Hidrogênio , Oxirredução , Estresse Oxidativo , Consumo de Oxigênio , Espécies Reativas de Oxigênio/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
13.
Free Radic Biol Med ; 46(9): 1241-9, 2009 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-19245828

RESUMO

Alzheimer disease (AD) is a progressive neurodegenerative disorder whose clinical manifestations appear in old age. The sporadic nature of 90% of AD cases, the differential susceptibility to and course of the illness, as well as the late age onset of the disease suggest that epigenetic and environmental components play a role in the etiology of late-onset AD. Animal exposure studies demonstrated that AD may begin early in life and may involve an interplay between the environment, epigenetics, and oxidative stress. Early life exposure of rodents and primates to the xenobiotic metal lead (Pb) enhanced the expression of genes associated with AD, repressed the expression of others, and increased the burden of oxidative DNA damage in the aged brain. Epigenetic mechanisms that control gene expression and promote the accumulation of oxidative DNA damage are mediated through alterations in the methylation or oxidation of CpG dinucleotides. We found that environmental influences occurring during brain development inhibit DNA-methyltransferases, thus hypomethylating promoters of genes associated with AD such as the beta-amyloid precursor protein (APP). This early life imprint was sustained and triggered later in life to increase the levels of APP and amyloid-beta (Abeta). Increased Abeta levels promoted the production of reactive oxygen species, which damage DNA and accelerate neurodegenerative events. Whereas AD-associated genes were overexpressed late in life, others were repressed, suggesting that these early life perturbations result in hypomethylation as well as hypermethylation of genes. The hypermethylated genes are rendered susceptible to Abeta-enhanced oxidative DNA damage because methylcytosines restrict repair of adjacent hydroxyguanosines. Although the conditions leading to early life hypo- or hypermethylation of specific genes are not known, these changes can have an impact on gene expression and imprint susceptibility to oxidative DNA damage in the aged brain.


Assuntos
Doença de Alzheimer/etiologia , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Epigênese Genética , Estresse Oxidativo , Receptores de Superfície Celular/metabolismo , Fatores Etários , Doença de Alzheimer/genética , Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/genética , Precursor de Proteína beta-Amiloide/genética , Animais , Dano ao DNA , Metilação de DNA , Exposição Ambiental/efeitos adversos , Regulação da Expressão Gênica/fisiologia , Predisposição Genética para Doença , Humanos , Chumbo/efeitos adversos , Oxirredução , Nexinas de Proteases , Receptores de Superfície Celular/genética
14.
Free Radic Biol Med ; 46(6): 828-35, 2009 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-19167482

RESUMO

The guanine base is prone to oxidation by free radicals regardless of the cellular moiety it is bound to. However, under conditions of oxidative stress, 8-oxoguanosine triphosphate (oxo(8)GTP) formation has been shown to occur without oxidation of the guanine base in DNA. In vitro studies have suggested that oxo(8)GTP could impact G-protein signaling and RNA synthesis. Whether increased levels of oxo(8)GTP translate into cellular malfunction is unknown. Data presented herein show that oxo(8)GTP is formed in cell-free preparations as well as in PC12 cells after exposure to physiologically relevant oxidative conditions generated with 10 microM copper sulfate and 1 mM L-ascorbic acid (Cu/Asc). We also determined that oxo(8)GTP has biological activity as a potent inhibitor of nitric oxide-stimulated soluble guanylyl cyclase (sGC). The increase in oxo(8)GTP formation in purified GTP and PC12 cells exposed to Cu/Asc caused a significant reduction in the product of sGC activity, cGMP. This oxidation of GTP was attenuated by the addition of reduced glutathione under these same Cu/Asc conditions, thus preventing the decrease in sGC activity. This suggests that oxo(8)GTP is produced by free radicals in vivo and could have significant impact on cell functions regulated by sGC activity such as synaptic plasticity in the central nervous system.


Assuntos
Sistema Nervoso Central/enzimologia , Ativação Enzimática/efeitos dos fármacos , Guanosina Trifosfato/análogos & derivados , Guanilato Ciclase/antagonistas & inibidores , Feocromocitoma/enzimologia , Animais , Ácido Ascórbico/farmacologia , Extratos Celulares , Linhagem Celular , Sistema Nervoso Central/patologia , Cromatografia Líquida de Alta Pressão , Sulfato de Cobre/farmacologia , GMP Cíclico/metabolismo , Radicais Livres/metabolismo , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Técnicas In Vitro , Óxido Nítrico/metabolismo , Oxirredução/efeitos dos fármacos , Estresse Oxidativo , Feocromocitoma/patologia , Ratos
15.
J Neurosci ; 28(28): 7219-30, 2008 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-18614692

RESUMO

Folate deficiency and resultant increased homocysteine levels have been linked experimentally and epidemiologically with neurodegenerative conditions like stroke and dementia. Moreover, folate deficiency has been implicated in the pathogenesis of psychiatric disorders, most notably depression. We hypothesized that the pathogenic mechanisms include uracil misincorporation and, therefore, analyzed the effects of folate deficiency in mice lacking uracil DNA glycosylase (Ung-/-) versus wild-type controls. Folate depletion increased nuclear mutation rates in Ung-/- embryonic fibroblasts, and conferred death of cultured Ung-/- hippocampal neurons. Feeding animals a folate-deficient diet (FD) for 3 months induced degeneration of CA3 pyramidal neurons in Ung-/- but not Ung+/+ mice along with decreased hippocampal expression of brain-derived neurotrophic factor protein and decreased brain levels of antioxidant glutathione. Furthermore, FD induced cognitive deficits and mood alterations such as anxious and despair-like behaviors that were aggravated in Ung-/- mice. Independent of Ung genotype, FD increased plasma homocysteine levels, altered brain monoamine metabolism, and inhibited adult hippocampal neurogenesis. These results indicate that impaired uracil repair is involved in neurodegeneration and neuropsychiatric dysfunction induced by experimental folate deficiency.


Assuntos
Encefalopatias/etiologia , Deficiência de Ácido Fólico/complicações , Degeneração Neural/etiologia , Uracila-DNA Glicosidase/deficiência , Análise de Variância , Animais , Comportamento Animal , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Técnicas de Cultura de Células/métodos , Células Cultivadas , Córtex Cerebral/citologia , Nucleotídeos de Desoxiuracil/metabolismo , Embrião de Mamíferos , Comportamento Exploratório/fisiologia , Deficiência de Ácido Fólico/patologia , Glutationa/metabolismo , Hipocampo/citologia , Homocisteína/sangue , Aprendizagem em Labirinto/fisiologia , Metionina/sangue , Camundongos , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Neurônios/fisiologia , Neurotransmissores/metabolismo , Natação
16.
J Neurosci ; 28(1): 3-9, 2008 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-18171917

RESUMO

The sporadic nature of Alzheimer's disease (AD) argues for an environmental link that may drive AD pathogenesis; however, the triggering factors and the period of their action are unknown. Recent studies in rodents have shown that exposure to lead (Pb) during brain development predetermined the expression and regulation of the amyloid precursor protein (APP) and its amyloidogenic beta-amyloid (Abeta) product in old age. Here, we report that the expression of AD-related genes [APP, BACE1 (beta-site APP cleaving enzyme 1)] as well as their transcriptional regulator (Sp1) were elevated in aged (23-year-old) monkeys exposed to Pb as infants. Furthermore, developmental exposure to Pb altered the levels, characteristics, and intracellular distribution of Abeta staining and amyloid plaques in the frontal association cortex. These latent effects were accompanied by a decrease in DNA methyltransferase activity and higher levels of oxidative damage to DNA, indicating that epigenetic imprinting in early life influenced the expression of AD-related genes and promoted DNA damage and pathogenesis. These data suggest that AD pathogenesis is influenced by early life exposures and argue for both an environmental trigger and a developmental origin of AD.


Assuntos
Envelhecimento , Doença de Alzheimer , Exposição Ambiental , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Chumbo/toxicidade , Fatores Etários , Doença de Alzheimer/etiologia , Doença de Alzheimer/patologia , Doença de Alzheimer/fisiopatologia , Secretases da Proteína Precursora do Amiloide , Peptídeos beta-Amiloides/análise , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Células Cultivadas , Córtex Cerebral , Modelos Animais de Doenças , Embrião de Mamíferos , Epigênese Genética , Feminino , Imunoglobulinas/metabolismo , Macaca fascicularis , Camundongos , Camundongos Endogâmicos C57BL , Neurônios , Fragmentos de Peptídeos/análise
17.
J Chromatogr B Analyt Technol Biomed Life Sci ; 856(1-2): 121-30, 2007 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-17581804

RESUMO

Oxidation of the guanosine moiety in DNA has become a hallmark biomarker in assessing oxidative stress. The oxidation of guanosine in the nucleotide triphosphate pool has been overlooked due to the lack of a reliable methodology. This method describes a sample processing and high performance liquid chromatography with electrochemical detection protocol for the analysis of the cellular pool of guanosine triphosphates and oxidized guanosine triphosphates. Validation of this method is demonstrated along with evaluation of these analytes in control and oxidizing conditions in vitro and in HEK 293T cells. Oxidation of this triphosphate pool occurred independently of oxidation to DNA.


Assuntos
Biomarcadores/análise , Cromatografia Líquida de Alta Pressão/métodos , Eletroquímica/métodos , Guanosina Trifosfato/análise , Guanosina/análise , Estresse Oxidativo , Calibragem , Linhagem Celular , Humanos , Fosforilação , Padrões de Referência
18.
Toxicol Sci ; 99(1): 277-88, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17578862

RESUMO

The community members of Libby, MT, have experienced significant asbestos exposure and developed numerous asbestos-related diseases including fibrosis and lung cancer due to an asbestos-contaminated vermiculite mine near the community. The form of asbestos in the contaminated vermiculite has been characterized in the amphibole family of fibers. However, the pathogenic effects of these fibers have not been previously characterized. The purpose of this study is to determine the cellular consequences of Libby amphibole exposure in macrophages compared to another well-characterized amphibole fiber; crocidolite asbestos. Our results indicate that Libby asbestos fibers are internalized by macrophages and localize to the cytoplasm and cytoplasmic vacuoles similar to crocidolite fibers. Libby asbestos fiber internalization generates a significant increase in intracellular reactive oxygen species (ROS) as determined by dichlorofluorescein diacetate and dihydroethidine fluorescence indicating that the superoxide anion is the major contributing ROS generated by Libby asbestos. Elevated superoxide levels in macrophages exposed to Libby asbestos coincide with a significant suppression of total superoxide dismutase activity. Both Libby and crocidolite asbestos generate oxidative stress in exposed macrophages by decreasing intracellular glutathione levels. Interestingly crocidolite asbestos, but not Libby asbestos, induces significant DNA damage in macrophages. This study provides evidence that the difference in the level of DNA damage observed between Libby and crocidolite asbestos may be a combined consequence of the distinct chemical compositions of each fiber as well as the activation of separate cellular pathways during asbestos exposure.


Assuntos
Amiantos Anfibólicos/toxicidade , Macrófagos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Animais , Amiantos Anfibólicos/metabolismo , Asbesto Crocidolita/metabolismo , Asbesto Crocidolita/toxicidade , Linhagem Celular , Dano ao DNA , DNA Glicosilases/metabolismo , Relação Dose-Resposta a Droga , Macrófagos/metabolismo , Macrófagos/ultraestrutura , Camundongos , Montana , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo
19.
J Environ Prot Sci ; 1: 23-28, 2007 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-20463849

RESUMO

Reduced glutathione (GSH) is an intracellular molecule essential for many aspects of cell physiology and defense. Determination of GSH has been used to identify potential anti-cancer drugs and for the assessment of drug toxicity via generation of oxidative stress. The described protocol was designed to modify existing protocols for the fluorescent detection of intracellular GSH in a high throughput 96-well microplate format. Dibromobimane was used to label intracellular GSH, and an additional dye, Hoechst 33342 was used to measure cell density for data normalization. Cell density curves were performed using HEK 293T cells to determine the optimal starting cell density, (< 8.0 × 10(4) cells/well) for fluorescent analysis. Fluorescent dyes were also analyzed for compatibility and spectral overlap. The method was further validated by exposing HEK 293T cells to GSH modulating agents; tert-butylhydroquinone a potent inducer of GSH, and L-buthionine-(SR)-sulfoximine a potent inhibitor of GSH. This study provides a fast, simple method for the high throughput screening of GSH in a widely available 96-well format. It also addresses the pitfalls associated with fluorescent compounds in cell culture and proper data normalization.

20.
FASEB J ; 20(6): 788-90, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16484331

RESUMO

Oxidative damage to DNA has been associated with neurodegenerative diseases. Developmental exposure to lead (Pb) has been shown to elevate the Alzheimer's disease (AD) related beta-amyloid peptide (Abeta), which is known to generate reactive oxygen species in the aging brain. This study measures the lifetime cerebral 8-hydroxy-2'-deoxyguanosine (oxo8dG) levels and the activity of the DNA repair enzyme 8-oxoguanine DNA glycosylase (Ogg1) in rats developmentally exposed to Pb. Oxo8dG was transiently modulated early in life (Postnatal day 5), but was later elevated 20 months after exposure to Pb had ceased, while Ogg1 activity was not altered. Furthermore, an age-dependent loss in the inverse correlation between Ogg1 activity and oxo8dG accumulation was observed. The effect of Pb on oxo8dG levels did not occur if animals were exposed to Pb in old age. These increases in DNA damage occurred in the absence of any Pb-induced changes in copper/zinc-superoxide dismutase (SOD1), manganese-SOD (SOD2), and reduced-form glutathion (GSH). These data suggest that oxidative damage and neurodegeneration in the aging brain could be impacted by the developmental disturbances.


Assuntos
Envelhecimento/fisiologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Dano ao DNA/efeitos dos fármacos , Chumbo/toxicidade , Estresse Oxidativo/efeitos dos fármacos , 8-Hidroxi-2'-Desoxiguanosina , Animais , Animais Recém-Nascidos , Encéfalo/patologia , DNA Glicosilases/genética , DNA Glicosilases/metabolismo , Reparo do DNA/efeitos dos fármacos , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Feminino , Glutationa/metabolismo , Masculino , Regiões Promotoras Genéticas , Ratos , Ratos Long-Evans , Superóxido Dismutase/metabolismo
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