RESUMO
Human obesity may be caused by a resistance to circulating leptin. Evidence from rodents and humans suggests that a major component of this resistance is an impairment in the ability of the blood-brain barrier (BBB) to transport leptin from the blood to the brain. One potential way to bypass the BBB is by administering leptin into the intrathecal (i.t.) space. To be effective, i.t. leptin would have to move caudally from the site of injection, enter the cranium, and reach the hypothalamic arcuate nucleus at the base of the pituitary fossa. However, many substances, especially small, lipid-soluble molecules, do not diffuse far from the site of i.t. injection but are resorbed back into blood. To determine whether i.t. leptin can move caudally, we injected leptin conjugated to diethylenetriaminepentaacetic acid (DTPA) and labeled with (68)Ga (G-Ob) into the lumbar space of three baboons. We also studied unconjugated DTPA labeled with (68)Ga, which did not move up the spinal cord but rapidly appeared in blood after i.t. injection. In contrast, G-Ob steadily moved toward the cranium and had reached the hypothalamus 91 and 139 min after i.t. injection in two baboons. We estimated the concentration of leptin in the hypothalamic region to be at least 8 ng/ml, which is about 40 times higher than cerebrospinal fluid levels in normal weight humans and about 4 times higher than the highest level ever recorded after the peripheral administration of leptin. In a third baboon, the leptin neither moved caudally nor appeared in the blood. We conclude that leptin administered i.t. can reach the hypothalamus in therapeutic concentrations, although there is considerable individual variation.
Assuntos
Hipotálamo/diagnóstico por imagem , Leptina/administração & dosagem , Tomografia Computadorizada de Emissão , Animais , Mapeamento Encefálico/métodos , Feminino , Hipotálamo/metabolismo , Injeções Espinhais/métodos , Injeções Espinhais/estatística & dados numéricos , Leptina/farmacocinética , Masculino , Papio , Ácido Pentético/administração & dosagem , Ácido Pentético/farmacocinética , Tomografia Computadorizada de Emissão/métodos , Tomografia Computadorizada de Emissão/estatística & dados numéricosRESUMO
RMP-7 is a bradykinin B2 receptor agonist shown to permeabilize the blood-brain barrier, especially that associated with brain tumors, when administered via both intracarotid and intravenous routes. Both routes of administration are currently being tested in human trials in combination with the chemotherapeutic agent carboplatin as therapy for gliomas. As an essential prerequisite to the initial intracarotid clinical trials, the potential neurotoxicity of intra-arterial administration of RMP-7 (at a high or low dose), alone and in combination with carboplatin, was assessed in anesthetized Red Duroc swine. Five treatment groups were evaluated with each pig receiving a series of alternating, intra-arterial infusions of RMP-7 (or saline) followed by carboplatin (or saline), as follows: (1) vehicle control: saline/saline; (2) carboplatin only control: saline/carboplatin (50 mg total); (3) RMP-7 only control: RMP-7 (750 ng/kg)/saline; (4) low dose combination: RMP-7 (75 ng/kg)/carboplatin (50 mg total); and (5) high dose combination: RMP-7 (750 ng/kg)/carboplatin (50 mg total). For each subject, one of the alternating dosing sequences (above) was repeated four times during a single dosing session which lasted approximately 40 minutes. Assessments during the in-life phase of the study in the pre- and post-treatment periods consisted of heart rate, arterial blood pressure (systolic, diastolic, and mean), blood gases, body weight, general clinical observations (including evaluation for neurological deficit) and clinical pathology (including a comprehensive battery of standard blood coagulation, hematological and serum chemistry tests). In addition, during the time of treatment, heart rate and arterial blood pressure were monitored. The animals were terminated two weeks after dosing and the brain and rete mirabile (distal to site of infusion) were evaluated for gross and histopathological abnormalities. The histopathology analysis included a reader-blinded analysis using low and high power light microscopic examination of both H&E and Kluver-Berrera stained sections through several key cortical and subcortical brain regions. Transient decreases in arterial blood pressure (mean of 10-25 mmHg) were observed in both groups receiving the high dose of RMP-7 (i.e., 750 ng/kg). No other side effects attributable to RMP-7 and/or carboplatin were observed, and clinical observations revealed no evidence of neurologic deficits. Post-mortem examination revealed no evidence of CNS or cerebral vascular pathology attributable to carboplatin and RMP-7. This study demonstrates that intracarotid administration of the maximum tolerated dose of RMP-7 (750 ng/kg) alone, or in combination with carboplatin (50 mg) is not accompanied by any serious adverse effect, apparent cerebrovascular abnormality or neuropathologic consequence and offers further evidence for the safety of this novel therapeutic approach for enhancing delivery of chemotherapeutics to brain tumors.
Assuntos
Antineoplásicos/toxicidade , Barreira Hematoencefálica/efeitos dos fármacos , Bradicinina/análogos & derivados , Carboplatina/toxicidade , Animais , Bradicinina/toxicidade , Permeabilidade Capilar/efeitos dos fármacos , Circulação Cerebrovascular/efeitos dos fármacos , Esquema de Medicação , Hipotensão/induzido quimicamente , Infusões Intra-Arteriais , Masculino , Modelos Biológicos , SuínosRESUMO
To define the kinetics and safety of spinally infused recombinant-methionyl human brain-derived neurotrophic factor (r-metHuBDNF), beagle dogs were prepared with lumbar intrathecal catheters passed through the cisternal membrane to the L1-L4 lumbar level. For kinetic studies, r-metHuBDNF was delivered by bolus or infusion through one catheter and lumbar CSF was sampled periodically through a second. As a lumbar bolus, r-metHuBDNF displayed a biphasic clearance with t(1/2)a = 0.7 hr and t(1/2)b = 7. 9 hr. Lumbar to cisternal concentrations after bolus delivery were approximately 60:1. For safety studies, dogs received continuous intrathecal infusion (2.4 ml/day) for 28 days of saline (n = 6), r-metHuBDNF at 200 (n = 6), 800 (n = 6), or 2000 (n = 7) microg/day. Control dogs showed no changes. Intrathecally infused r-metHuBDNF produced a dose-dependent increase in muscle tone and decreased coordination. Low-dose r-metHuBDNF was associated with moderate increases in muscle tone after 22-28 days of infusion. No clinically important changes were noted in rectal temperature, arterial pressure, respiration and heart rate, body weight, food consumption, stool or urine output, or change in blood chemistries measured throughout the study. Cisternal CSF protein and glucose sampled at 28 days were not different between dose groups and all cultures were negative. Histopathological examination of the spinal cord typically revealed some degree of chronic inflammation around the catheter, including fibrotic adhesions and focal accumulations of lymphoid and plasma cells, but these effects were not dose dependent. In other dogs receiving r-metHuBDNF (2000 or 4000 microg/day), termination of infusion resulted in significant recovery.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/farmacocinética , Tono Muscular/efeitos dos fármacos , Medula Espinal/efeitos dos fármacos , Animais , Comportamento Animal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Fator Neurotrófico Derivado do Encéfalo/administração & dosagem , Fator Neurotrófico Derivado do Encéfalo/líquido cefalorraquidiano , Fator Neurotrófico Derivado do Encéfalo/toxicidade , Cães , Relação Dose-Resposta a Droga , Membro Posterior , Humanos , Infusões Parenterais , Inulina/administração & dosagem , Inulina/líquido cefalorraquidiano , Locomoção/efeitos dos fármacos , Masculino , Músculo Esquelético/efeitos dos fármacos , Distribuição Aleatória , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/líquido cefalorraquidiano , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/toxicidade , Medula Espinal/metabolismo , Medula Espinal/patologiaRESUMO
Recent efforts to evaluate neurobehavioral function in adult rodents as part of preclinical safety studies have typically been directed at evaluating possible environmental neurotoxicants. With considerable basic and clinical research efforts focusing on neurotrophic factors in the treatment of various neurodegenerative diseases, the application of these neurobehavioral evaluations is expected to increase. This report describes a six-month safety study of recombinant-methionyl human brain-derived neurotrophic factor (r-metHuBDNF) in rodents that included a neurotoxicity screening battery and was undertaken to evaluate safety issues that might be anticipated in the clinical setting. R-metHuBDNF was well tolerated by rats over six months of daily subcutaneous administration at doses that exceeded the highest anticipated clinical dose by 100-fold. Although statistically significant behavioral changes were noted, they were isolated and not considered to be associated with r-metHuBDNF treatment. We conclude that the inclusion of a neurotoxicity screening battery was an important study parameter in the assessment of the preclinical safety of this neurotrophin.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/efeitos adversos , Atividade Motora/efeitos dos fármacos , Neurotoxinas/efeitos adversos , Animais , Feminino , Humanos , Masculino , Ratos , Ratos Sprague-Dawley , Recombinação Genética , Segurança , Fatores SexuaisRESUMO
Comparative pharmacotoxicity studies in rats were performed to evaluate the response to r-metHuIL-2[ala-125] following 2 or 4 weeks of daily intravenous or subcutaneous administration, as well as to evaluate pharmacokinetic and pharmacodynamic responses. Pharmacokinetic analysis indicated that r-metHuIL-2[ala-125] showed high bioavailability and nonlinear concentration profiles. Pharmacodynamic responses to intravenous or subcutaneous dosing with r-metHuIL-2[ala-125], as measured by white blood cell counts, were comparable. Preclinical safety studies (6, 30, and 150 micrograms kg-1 day-1) indicated that r-metHuIL-2[ala-125], whether given intravenously or subcutaneously, was associated with increased circulating and infiltrating levels of lymphocytes and eosinophils. Bone marrow lymphoid hyperplasia and splenic extramedullary hematopoiesis were similarly observed in each study. This pattern of effects was considered an exaggerated pharmacodynamic response to r-metHuIL-2[ala-125]. Of further note was a histopathologic finding described as hepatocyte single cell necrosis which was observed following both intravenous and subcutaneous administration and was considered to be a toxic response to high doses of r-metHuIL-2[ala-125]. The no observable adverse effect level (NOAEL) for r-metHuIL-2[ala-125] via intravenous administration was 6 micrograms kg-1 day-1, while that for subcutaneous administration was 30 micrograms kg-1 day-1. Data herein present a form of rHuIL-2 with pharmacokinetic and pharmacodynamic profiles that are similar when given by these two systemic routes. Pharmacotoxic data, based on NOAELs, suggest that subcutaneous administration may be a preferred clinical route of administration.
Assuntos
Medula Óssea/efeitos dos fármacos , Interleucina-2/análogos & derivados , Leucócitos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Baço/efeitos dos fármacos , Absorção , Animais , Disponibilidade Biológica , Células da Medula Óssea , Ensaio de Imunoadsorção Enzimática , Feminino , Meia-Vida , Injeções Intravenosas , Injeções Subcutâneas , Interleucina-2/administração & dosagem , Interleucina-2/farmacocinética , Interleucina-2/toxicidade , Contagem de Leucócitos/efeitos dos fármacos , Fígado/citologia , Pulmão/citologia , Pulmão/efeitos dos fármacos , Linfonodos/efeitos dos fármacos , Masculino , Tamanho do Órgão/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/toxicidadeRESUMO
Many diseases and aging may be associated with oxidative stress in the brain. However, the effects of oxidative stress in the brain should be more clearly described, especially in terms of effects on brain reduced glutathione (GSH). This issue was addressed by intracerebroventricular injection of a direct-acting oxidative stress inducing agent, tert-butylhydroperoxide. Oxidized glutathione (GSSG) levels in the brain increased by as much as 90-fold during tert-butylhydroperoxide-induced oxidative stress. At the same time, brain GSH levels decreased. The brain appears to retain GSSG and not reduce it or export it efficiently. Vitamin E levels in the striatum increased during tert-butylhydroperoxide-induced oxidative stress. Aging alters the ability of the brain to detoxify an oxidative stress, in that 8-month-old mice retain GSSG in their brains much more than 2-month-old mice. Eight-month-old mice were much more susceptible to tert-butylhydroperoxide-induced toxicity than 2-month-old mice. This may indicate that aging makes the brain more susceptible to oxidative damage.
Assuntos
Encéfalo/metabolismo , Oxigênio/metabolismo , Peróxidos/farmacologia , Envelhecimento/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Corpo Estriado/metabolismo , Glutationa/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NADP/metabolismo , Oxirredução , Vitamina E/metabolismo , terc-Butil HidroperóxidoRESUMO
Excitotoxicity and oxidative stress are two phenomena that have been repeatedly described as being implicated in a wide range of disorders of the nervous system. Such disorders include several common idiopathic neurological diseases, traumatic brain injury, and the consequences of exposure to certain neurotoxic agents. While there is evidence that metabolic derangements can lead to these adverse processes, and that these processes may synergize in their damaging effects, the degree of interdependence, and the causal relation between them is not clear. The intent of this review is to delineate potential mechanisms which may unit hyperexcitation to the excessive generation of reactive oxygen species. The degree of linkage between these events appears rather strong. It is likely that excitoxicity frequently leads to a pro-oxidant condition but that high rates of these events appears rather strong. It is likely that excitoxicity frequently leads to a pro-oxidant condition but that high rates of generation of reactive oxygen species are not invariably accompanied by a hyperexcited neuronal state Both excitoxic and 'oxidotoxic' states result from the failure of normal compensatory antiexcitatory and antioxidant mechanisms to maintain cellular homeostasis.
Assuntos
Sistema Nervoso Central/fisiologia , Aminoácidos/metabolismo , Animais , Sistema Nervoso Central/metabolismo , Radicais Livres/metabolismo , Humanos , Degeneração Neural/fisiologia , Doenças do Sistema Nervoso/fisiopatologia , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Estresse Fisiológico/metabolismo , Estresse Fisiológico/fisiopatologiaAssuntos
Envelhecimento/metabolismo , Encéfalo/metabolismo , Animais , Encéfalo/patologia , Radicais Livres , Humanos , OxirreduçãoRESUMO
The use of dichlorofluorescin (DCFH) as a measure of reactive oxygen species was studied in aqueous media. Hydrogen peroxide oxidized DCFH to fluorescent dichlorofluorescein (DCF), and the oxidation was amplified by the addition of ferrous iron. Hydrogen peroxide-induced DCF formation in the presence of ferrous iron was completely inhibited by deferoxamine and partially inhibited by ethylenediaminetetraacetic acid, but was augmented by diethylenetriaminepentaacetic acid. Iron-peroxide-induced oxidation of DCFH was partially inhibited by catalase but not by horseradish peroxidase. Nonchelated iron-peroxide oxidation of DCFH was partially inhibited by several hydroxyl radical scavengers, but was independent of the scavenger concentration, and this suggests that free hydroxyl radical is not involved in the oxidation of DCFH in this system. Superoxide anion did not directly oxidize DCFH. Data suggest that H2O2-Fe(2+)-derived oxidant is mainly responsible for the nonenzymatic oxidation of DCFH. In addition, peroxidase alone and oxidants formed during the reduction of H2O2 by peroxidase oxidize DCFH. Since DCFH oxidation may be derived from several reactive intermediates, interpretation of specific reactive oxygen species involved in biological systems should be approached with caution. However, DCFH remains an attractive probe as an overall index of oxidative stress in toxicological phenomena.
Assuntos
Fluoresceínas/metabolismo , Oxigênio/metabolismo , Catalase/metabolismo , Ácido Edético , Compostos Ferrosos/metabolismo , Fluoresceínas/química , Radicais Livres , Peroxidase do Rábano Silvestre/metabolismo , Peróxido de Hidrogênio/química , Hidróxidos/metabolismo , Radical Hidroxila , OxirreduçãoRESUMO
It has been suggested that methyl mercury may express its neurotoxicity by way of iron-mediated oxidative damage. Therefore, the effect of deferoxamine, a potent iron-chelator, on methyl mercury-induced increases in reactive oxygen species formation was studied in rat brain. The generation rate of reactive oxygen species was estimated in crude synaptosomal fractions using the probes 2',7'-dichlorofluorescin diacetate and dihydrorhodamine 123. The formation rate of the fluorescent oxidation products was used as the measure of reactive oxygen species generation. Seven days after a single injection of methyl mercury (5 mg/kg, ip), the formation rate of reactive oxygen species was significantly increased in the cerebellum. Pretreatment with deferoxamine (500 mg/kg, ip) completely prevented the methyl mercury-induced increase in cerebellar reactive oxygen species generation rates. The oxidative consequences of in vitro exposure to methyl mercury (20 microM) were also inhibited by deferoxamine (100 microM). The formation of the iron-saturated complex ferrioxamine was not affected by a 10-fold excess of methylmercuric chloride or mercuric chloride, suggesting that a deferoxamine-mercurial complex does not form. The findings in this study: (1) provide evidence that iron-catalyzed oxygen radical-producing reactions play a role in methyl mercury neurotoxicity, (2) demonstrate the potential of fluorescent probes as a measure of reactive oxygen species formation, and (3) provide support for iron-chelator therapy in protection against xenobiotic-induced oxidative damage.
Assuntos
Encéfalo/metabolismo , Desferroxamina/farmacologia , Compostos de Metilmercúrio/farmacologia , Superóxidos/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Esterases/efeitos dos fármacos , Esterases/metabolismo , Fluoresceínas/metabolismo , Corantes Fluorescentes/metabolismo , Radicais Livres , Ferro/metabolismo , Masculino , Compostos de Metilmercúrio/antagonistas & inibidores , Ratos , Ratos EndogâmicosRESUMO
Reactive oxygen species (ROS) such as superoxide anion, hydrogen peroxide, and hydroxyl radicals are believed to be initiators of peroxidative cell damage. This paper focused on the use of 2',7'-dichlorofluorescein-diacetate (DCFH-DA) to quantitate cerebral ROS as an index for neurotoxicity. This technique employs an assay of dichlorofluorescein (DCF), the fluorescent product of dichlorofluorescein (DCFH). Data from studies using various free radical generating systems, several iron chelators and hydroxyl radical scavengers suggest that DCFH oxidation may result in several reactive intermediates. In a biological system (synaptosomes isolated from untreated rats) DCF fluorescence was stimulated by ascorbate or FeSO4, while deferoxamine inhibited the ascorbate/FeSO4-induced stimulation of DCF formation. Two organometals, methylmercury (MeHg) and trimethyltin (TMT), known to produce neurotoxicity were tested. In vitro exposure to MeHg (10-20 microM) increased the rate of formation of ROS while TMT (5-40 microM) had no effect. In vivo, 48 hr and 1 week after a single injection of MeHg (1 mg/kg, i.p.) in mice and 1 week after a single injection of MeHg (5 mg/kg, i.p.) in rats, the rate of formation of ROS in both rat and mouse cerebellum was significantly increased. Pretreatment with deferoxamine, a potent iron-chelator, prevented MeHg-induced increase of ROS. In hippocampus and frontal cortex, ROS formation rates were also elevated 48 hr after TMT injection (3 mg/kg, i.p.) in mice. These results demonstrate that DCF fluorescence provides a good measure of overall ROS formation in synaptosomes of both in vitro as well as in vivo systems. Since ROS formation was selectively increased in areas known to be specifically vulnerable to organometals (cerebellum in the case of MeHg and hippocampus in the case of TMT), these studies further support that oxidative damage may be the primary mechanism underlying the neurotoxicity induced by these organometals.
Assuntos
Encéfalo/efeitos dos fármacos , Compostos de Metilmercúrio/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Sinaptossomos/efeitos dos fármacos , Compostos de Trimetilestanho/toxicidade , Animais , Biomarcadores/química , Fluoresceínas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RatosRESUMO
The effects of toluene on lipid peroxidation and rates of reactive oxygen species (ROS) formation have been studied in isolated systems and in vivo. The induction of reactive oxygen species was assayed using the probe 2',7'-dichlorofluorescin diacetate (DCFH-DA). Toluene exposure (1 g/kg, 1 hr, i.p.) did not stimulate cortical lipid peroxidation as evaluated by measurement of conjugated dienes. Exposure to toluene, however, both in vivo and in vitro, caused a significant elevation of ROS formation within cortical crude synaptosomal fractions (P2) and microsomal fractions (P3). The ROS-inducing properties of toluene were blocked in vivo in the presence of a mixed-function oxidase inhibitor, metyrapone. This suggested that a metabolite of toluene may catalyze reactive oxygen formation. Both benzyl alcohol and benzoic acid, in vitro, were found to have free radical quenching properties, while benzaldehyde exhibited significant induction of ROS generation. It appears that benzaldehyde is the metabolite responsible for the effect of toluene in accelerating reactive oxygen production within the nervous system. Benzaldehyde may also contribute to the overall neurotoxicity of toluene.
Assuntos
Encéfalo/metabolismo , Oxigênio/metabolismo , Tolueno/toxicidade , Animais , Encéfalo/efeitos dos fármacos , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Cinética , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Metirapona/farmacologia , Ratos , Tolueno/metabolismoRESUMO
The inherent biochemical, anatomical and physiological characteristics of the brain make it especially vulnerable to insult. Specifically, some of these characteristics such as myelin and a high energy requirement provide for the introduction of free radical-induced insult. Recently, the biochemistry of free radicals has received considerable attention. It also has become increasingly apparent that many drug and chemical-induced toxicities may be evoked via free radicals and oxidative stress. Major points addressed in this work are the regulation of neural free radical generation by antioxidants and protective enzymes, xenobiotic-induced disruption of cerebral redox status, and specific examples of neurotoxic agent-induced alterations in free radical production as measured by the fluorescent probe dichlorofluorescein. This article considers the thesis that free radical mechanisms may contribute significantly to the properties of several diverse neurotoxic agents and proposes that excess production of free radicals may be common phenomena of neurotoxicity.
Assuntos
Encéfalo/metabolismo , Neurotoxinas/toxicidade , Oxigênio/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Radicais LivresRESUMO
The relation between cerebral oxygen radicals and the aging process was investigated in crude synaptosomal (P2) fractions from rats. The rate of formation of oxygen radicals was measured using the probe 2',7'-dichlorofluorescein diacetate (DCFH-DA), which is de-esterified and subsequently oxidized by oxygen radicals to a fluorescent product 2',7'-dichlorofluorescein (DCF). There was a significant age-dependent decrease in the formation rate of oxygen radicals, observed by decreased formation of DCF. No difference in oxygen radical formation was apparent between age groups following an in vitro challenge with an ascorbate/FeSO4 mixture. This age-dependent decrease in cerebral oxygen radical generation coincided with age-dependent increases in superoxide dismutase. No age-related alterations in lipid order in either the hydrophilic or lipophilic membrane regions were observed using fluorescence polarization analysis. Age-dependent losses in cerebral P2 tryptophan fluorescence (a measure of protein degradation), and increased liberation of [14C]protein fragments into the acid-soluble fraction (a measure of overall proteolytic activity) were observed. Results suggest that aging does not proceed as a result of elevated rates of generation of oxygen radicals, a finding that does not support the proposed free radical theory of aging. The observed age-dependent decrease in the formation of oxygen radicals does not effect membrane lipid order. These findings implicate modifications in proteins and activated protein catabolic pathways as major contributing factors in the normal physiological process of senescence.
Assuntos
Envelhecimento/metabolismo , Encéfalo/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Consumo de Oxigênio/fisiologia , Animais , Ácido Ascórbico/farmacologia , Encéfalo/enzimologia , Encéfalo/fisiologia , Córtex Cerebral/metabolismo , Compostos Férricos/farmacologia , Fluoresceínas , Radicais Livres , Técnicas In Vitro , Masculino , Proteínas do Tecido Nervoso/metabolismo , Ratos , Ratos Endogâmicos F344 , Superóxido Dismutase/metabolismo , Triptofano/metabolismoRESUMO
The mechanism by which toluene decreased synaptosomal phosphatidylethanolamine (PE) was investigated by studying degradative and synthetic phospholipid pathways. Toluene stimulated a PE-specific phospholipase (PLase) C both in vivo (44-75%) and in vitro (20-30%) whereas PLase A, PLase D and base exchange enzymes were unchanged. Toluene, in vivo, also increased the synthesis of PE (27%) when expressed as [3H]ethanolamine incorporation into [3H]PE, but had no effect on PE synthesis when administered in vitro. Perhaps this reflects a compensatory mechanism in synaptosomes to replace PE via increasing de novo synthesis. Phospholipid methylation, an event proposed to be related to the transduction of singals across membranes, as well as a measure of membrane function, was studied. Toluene was found to rapidly increase phospholipid methylation (43%, 15 min), followed by a significant decrease (35%, 1 hr). Another measure of membrane, as well as cell function used in these studies was ATPase activity. Toluene, both in vivo and in vitro, stimulated Na+, K(+)-adenosine triphosphatase (ATPase) activity (20-30%, 15-30 min), whereas Mg(++)-ATPase and Ca(++)-ATPase were unaffected, an indication that toluene alters neuronal cell function. Membrane fluidity studies using fluorescence polarization reported that toluene, both in vivo and in vitro, increased the outer synaptosomal membrane fluidity using the probe trimethylammonium-diphenylhexatriene, whereas no effect was observed on the central core fluidity using diphenylhexatriene. These are the first studies to demonstrate that an organic solvent effects only specific membrane region fluidities. One possibility is that early synaptic alterations resulting from toluene exposure may be preceded by increases in outer membrane fluidity.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Fosfolipídeos/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Sinaptossomos/efeitos dos fármacos , Tolueno/farmacologia , Animais , Injeções Intraperitoneais , Masculino , Fluidez de Membrana/efeitos dos fármacos , Metilação , Fosfatidiletanolaminas/biossíntese , Ratos , Ratos Endogâmicos , Sinaptossomos/enzimologiaRESUMO
The effects of the neurotoxic metals methylmercury (MeHg) and trimethyltin (TMT) on oxygen reactive species formation within a crude synaptosomal fraction (P2), using the probe 2',7'-dichlorofluorescin diacetate (DCFH-DA), and intracellular calcium ([Ca2+]i), with the fluorescent indicator fluo-3, have been investigated. Two and seven days after a single injection of MeHg (1 mg/kg) the formation rate of cerebellar oxygen reactive species was significantly increased. Hippocampal and frontocortical oxygen reactive species were elevated 2 days after TMT injection (3 mg/kg). In vitro exposure to MeHg (10-20 microM) increased the formation rate of oxygen reactive species, while TMT (5-40 microM) was without effect. Levels of [Ca2+]i were unaltered in P2 fractions from cerebellum and hippocampus of animals treated with either organometal. The data demonstrate that oxygen reactive species are elevated in brain regions, cerebellum (MeHg) and hippocampus (TMT), believed to be selectively vulnerable to these toxic agents. Findings suggest that oxidative damage may be a mechanism underlying the toxicity of both organometals. The use of DCFH-DA may have potential in the nervous system as an indicator of neurotoxic damage.
Assuntos
Fluoresceínas/metabolismo , Compostos de Metilmercúrio/toxicidade , Sistema Nervoso/efeitos dos fármacos , Oxigênio/fisiologia , Compostos de Trialquitina/toxicidade , Compostos de Trimetilestanho/toxicidade , Animais , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Cálcio/fisiologia , Cerebelo/efeitos dos fármacos , Radicais Livres , Hipocampo/efeitos dos fármacos , Peróxido de Hidrogênio , Hidróxidos , Radical Hidroxila , Masculino , Camundongos , Camundongos Endogâmicos , Mitocôndrias/metabolismo , Superóxidos/metabolismo , Sinaptossomos/efeitos dos fármacosRESUMO
The formation of oxygen reactive species in response to oxidative stimuli was measured in rat synaptosomes. Studies employed the non-fluorescent probe 2?,7?-dichlorofluorescin diacetate (DCFH-DA), which after de-esterification is oxidized in the presence of oxygen reactive species to the highly fluorescent 2?,7?-dichlorofluorescein (DCF). Oxygen reactive species formation, as measured by DCF fluorescence, was stimulated by ascorbate and/or FeSO(4), and xanthine/xanthine oxidase under various buffering conditions. These agents all increased DCF formation in Tris, HEPES and phosphate buffer. Ascorbate also stimulated the formation of DCF in a concentration-dependent manner. The presence of Ca(2+) in HEPES buffer did not enhance or diminish the effects of ascorbate/FeSO(4) on DCF formation. Deferoxamine inhibited the ascorbate/FeSO(4)-induced stimulation of DCF formation, but xanthine/xanthine oxidase-induced stimulation was not affected by pretreatment with superoxide dismutase. Results indicate that DCF fluorescence is a sensitive, quantitative and direct measure of oxygen reactive species formation in synaptosomes, providing a rapid method for investigating early neuronal events that occur during oxidative stress.
RESUMO
This study investigated the effects of toluene (1 g/kg, 1 hr, i.p.) on rat synaptosomal phospholipid methylation (PLM), phospholipid composition, and membrane fluidity. Toluene significantly decreased basal PLM (35%) in studies using [3H]methionine [( 3H]Met) as the methyl donor; this was reflected by similar decreases in phosphatidylmonomethylethanolamine (PME) (30%). No effects were observed in either PLM reactions that used [3H]adenosylmethionine [( 3H]AdoMet) as methyl donor, or AdoMet synthetase, suggesting that toluene preferentially affects PLM reactions that derive methyl groups from [3H]Met. Also, toluene decreased synaptosomal phosphatidylethanolamine (PE) (24%), the initial substrate for PLM, and the addition of PE back to PE-depleted synaptosomes restored methyltransferase activity. Agonist-stimulated PLM using norepinephrine (NE) demonstrated that agonist-receptor coupling returned PLM to control values in synaptosomes from toluene-treated rats. NE-stimulated PLM was also blocked by propranolol (PRO), suggesting a role for toluene in receptor-mediated events. Membrane fluidity studies demonstrated that in vivo administration of toluene increased the outer synaptosomal membrane fluidity, whereas in vitro administration of toluene had no effect. Our observations support a positive relationship between increased PLM activity and increased outer, not core, membrane fluidity. These data demonstrate that specific toluene-phospholipid interactions occur in synaptosomes, resulting in altered membrane composition, function and fluidity.
Assuntos
Encéfalo/metabolismo , Fluidez de Membrana/efeitos dos fármacos , Fosfolipídeos/metabolismo , Sinaptossomos/metabolismo , Tolueno/farmacologia , Animais , Encéfalo/ultraestrutura , Masculino , Metionina/metabolismo , Metionina Adenosiltransferase/metabolismo , Metilação , Norepinefrina/farmacologia , Fosfatidiletanolaminas/metabolismo , Fosfatidiletanolaminas/farmacologia , Propranolol/farmacologia , Ratos , Ratos Endogâmicos , S-Adenosil-Homocisteína/farmacologia , S-Adenosilmetionina/metabolismo , Sinaptossomos/efeitos dos fármacosRESUMO
The effects of dietary vitamin E deficiency on mouse cerebral membrane order and oxygen reactive species were studied. Quantitation of vitamin E levels in several brain regions showed greatest deficiencies in striatum and cerebellum, followed by substantia nigra, and cortex. Vitamin E deficiency increased central-core membrane order in cerebral P2 fraction, but was without effect in the superficial hydrophilic membrane domain. Oxygen radical formation was studied using the probe 2',7'-dichlorofluorescein diacetate. Basal generation rates of oxygen reactive species were 2.5-fold higher when compared to control animals. While hepatic levels of vitamin E are much more reduced than brain levels, in deficient mice, the rate of oxygen radical formation in the liver was unaltered. This implies an special susceptibility of the brain to deficiency of this lipophilic antioxidant vitamin. Data demonstrate that endogenous levels of free radical scavengers, such as vitamin E, may play an important role in maintaining basal oxygen radical levels and membrane integrity. The dietary vitamin E depletion paradigm suggests that a relation exists between elevated levels of oxygen radicals and more rigid hydrophobic central-cores in cerebral membranes, effects that may play a role in mechanisms underlying the neuropathologic lesions observed following vitamin E deficiency.
Assuntos
Encéfalo/metabolismo , Oxigênio/metabolismo , Deficiência de Vitamina E/metabolismo , Animais , Corantes Fluorescentes , Radicais Livres , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Espectrometria de FluorescênciaRESUMO
The relationship between p-xylene's effects on microsomal membranes, cytochrome P-450, and benzo[a]pyrene (BaP) metabolism was studied. p-Xylene (1 g/kg, ip, 1 h) inhibited 3-hydroxy BaP (3-OH) formation and decreased arylhydrocarbon hydroxylase (AHH) activity approximately 40% in rat lung microsomes. BaP dihydrodiol and quinone formation were unchanged by p-xylene administration. Cytochrome P-450 was below the limit of detection in lung microsomes from p-xylene-treated rats. Total phospholipid (PL) and phosphatidylcholine (PC) in microsomal membranes were decreased 28% and 17%, respectively. Cholesterol (CL), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), and sphingomyelin (SM) were unchanged. The net activity of enzymes involved in the synthesis of PC, phosphatidylethanolamine-N-methyltransferase I and II (PMT I and PMT II), was slightly elevated by p-xylene. PL/CL and PC/PE ratios, indicators of membrane fluidity, were decreased 34% and 13%, respectively, in microsomes from p-xylene-treated rats. Analysis of fluidity by fluorescence polarization showed that the actual fluidity of treated microsomes was slightly decreased (5%) as compared to controls. The decrease in P-450, PL, and PC is considered to contribute to the inhibition of BaP metabolism.