RESUMO
Ammonia is a widely used industrial chemical that is recognized as a potent neurotoxin and environmental pollutant. The present study addresses the oxidative stress and tissue pathology caused by 4 weeks of exposure to ammonium acetate (AMA; 100 mg/kg daily; orally) in rats, and their response to oral treatments with alpha-ketoglutarate (A-KG; 1.0 g/kg), a potential cyanide antidote, and/or N-acetyl cysteine (NAC; 10 mg/kg), an antioxidant. The organ-body weight index of brain and liver was significantly increased by AMA but kidney was unaffected. Also, plasma ammonia levels were significantly elevated without any concomitant change in blood gas status and hematology but levels of catalase, superoxide dismutase, glutathione peroxidase, glutathione reductase and reduced glutathione (GSH) in the brain and liver were diminished, accompanied by elevated levels of malondialdehyde. Levels of glutathione disulfide (GSSG) were unaffected, but the ratio of GSH:GSSG was reduced. Plasma alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase and total bilirubin were raised but urea, uric acid and creatinine levels were not altered. AMA also caused temporal, hepatic and renal pathology. However, the renal pathology was not supported by any biochemical alterations. A-KG or NAC alone afforded less protection against AMA as compared to both given together. The protective efficacy of A-KG can be ascribed to its ability to detoxify ammonia and additionally both A-KG and NAC have antioxidant properties as well. The study suggests a new therapeutic regimen for ammonia poisoning.
Assuntos
Acetatos/toxicidade , Acetilcisteína/farmacologia , Ácidos Cetoglutáricos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Amônia/sangue , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Química Encefálica/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Ratos , Ratos Wistar , Estatísticas não Paramétricas , Testes de Toxicidade SubagudaRESUMO
Cyanide is a mitochondrial poison, which is ubiquitously present in the environment. Cyanide-induced oxidative stress is known to play a key role in mediating the neurotoxicity and cell death in rat pheochromocytoma (PC12) cells. PC12 cells are widely used as a model for neurotoxicity assays in vitro. In the present study, we investigated the protective effects of alpha-ketoglutarate (A-KG), a potential cyanide antidote, and N-acetyl cysteine (NAC), an antioxidant against toxicity of cyanide in PC12 cells. Cells were treated with various concentrations (0.625-1.25 mM) of potassium cyanide (KCN) for 4 hours, in the presence or absence of simultaneous treatment of A-KG (0.5 mM) and NAC (0.25 mM). Cyanide caused marked decrease in the levels of cellular antioxidants like superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR). Lipid peroxidation indicated by elevated levels of malondialdehyde (MDA) was found to be accompanied by decreased levels of reduced glutathione (GSH) and total antioxidant status (TAS) of the cells. Cyanide-treated cells showed notable increase in caspase-3 activity and induction of apoptotic type of cell death after 24 hours. A-KG and NAC alone were very effective in restoring the levels of GSH and TAS, but together they significantly resolved the effects of cyanide on antioxidant enzymes, MDA levels, and caspase-3 activity. The present study reveals that combination of A-KG and NAC has critical role in abbrogating the oxidative stress-mediated toxicity of cyanide in PC12 cells. The results suggest potential role of A-KG and NAC in cyanide antagonism.
Assuntos
Acetilcisteína/farmacologia , Ácidos Cetoglutáricos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Cianeto de Potássio/toxicidade , Análise de Variância , Animais , Caspase 3/metabolismo , Catalase/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Eletroforese em Gel de Ágar , Glutationa/metabolismo , Glutationa Peroxidase/metabolismo , Glutationa Redutase/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Malondialdeído/metabolismo , Microscopia de Fluorescência , Células PC12 , Ratos , Superóxido Dismutase/metabolismoRESUMO
Cyanide is a rapidly acting mitochondrial poison that inhibits cellular respiration and energy metabolism leading to histotoxic hypoxia followed by cell death. Cyanide is predominantly a neurotoxin but its toxic manifestations in non-neuronal cells are also documented. This study addresses the oxidative stress mediated cytotoxicity of cyanide in Rhesus monkey kidney epithelial cells (LLC-MK2). Cells were treated with various concentrations of potassium cyanide (KCN) for different time intervals and cytotoxicity was evidenced by increased leakage of intracellular lactate dehydrogenase, mitochondrial dysfunction (MTT assay) and depleted energy status of cells (ATP assay). Cytotoxicity was accompanied by lipid peroxidation indicated by elevated levels of malondialdehyde (MDA), reactive oxygen species (ROS) and reactive nitrogen species (RNS) (DCF-DA staining), diminished cellular antioxidant status (reduced glutathione (GSH), glutathione peroxidase, superoxide dismutase and catalase). These cascading events triggered an apoptotic kind of cell death characterized by oligonucleosomal DNA fragmentation and nuclear fragmentation (Hoechst 33342 staining). Apoptosis was further confirmed by increased caspase-3 activity. Cyanide-induced cytotoxicity, oxidative stress, and DNA fragmentation were prevented by alpha-ketoglutarate (A-KG) and N-acetyl cysteine (NAC). A-KG is a potential cyanide antidote that confers protection by interacting with cyanide to form cyanohydrin complex while NAC is a free radical scavenger and enhances the cellular GSH levels. The study reveals cytotoxicity of cyanide in cells of renal origin and the protective efficacy of A-KG and NAC.
Assuntos
Acetilcisteína/farmacologia , Antioxidantes/farmacologia , Células Epiteliais/efeitos dos fármacos , Ácidos Cetoglutáricos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Cianeto de Potássio/toxicidade , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Células Epiteliais/enzimologia , Células Epiteliais/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Macaca mulattaRESUMO
Kainic acid, an analogue of the excitatory neurotransmitter glutamate, can trigger seizures and neurotoxicity in the hippocampus and other limbic structures in a manner that mirrors the neuropathology of human temporal lobe epilepsy (TLE). However, the underlying mechanisms associated with the neurotoxicity remain unclear. Since amyloid-ß (Aß) peptides, which are critical in the development of Alzheimer's disease, can mediate toxicity by activating glutamatergic NMDA receptors, it is likely that the enhanced glutamatergic transmission that renders hippocampal neurons vulnerable to kainic acid treatment may involve Aß peptides. Thus, we seek to establish what role Aß plays in kainic acid-induced toxicity using in vivo and in vitro paradigms. Our results show that systemic injection of kainic acid to adult rats triggers seizures, gliosis and loss of hippocampal neurons, along with increased levels/processing of amyloid precursor protein (APP), resulting in the enhanced production of Aß-related peptides. The changes in APP levels/processing were evident primarily in activated astrocytes, implying a role for astrocytic Aß in kainic acid-induced toxicity. Accordingly, we showed that treating rat primary cultured astrocytes with kainic acid can lead to increased Aß production/secretion without any compromise in cell viability. Additionally, we revealed that kainic acid reduces neuronal viability more in neuronal/astrocyte co-cultures than in pure neuronal culture, and this is attenuated by precluding Aß production. Collectively, these results indicate that increased production/secretion of Aß-related peptides from activated astrocytes can contribute to neurotoxicity in kainic acid-treated rats. Since kainic acid administration can lead to neuropathological changes resembling TLE, it is likely that APP/Aß peptides derived from astrocytes may have a role in TLE pathogenesis.
Assuntos
Peptídeos beta-Amiloides/fisiologia , Epilepsia do Lobo Temporal/fisiopatologia , Neurônios/efeitos dos fármacos , Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Astrócitos/metabolismo , Astrócitos/fisiologia , Encéfalo/patologia , Células Cultivadas , Modelos Animais de Doenças , Hipocampo/patologia , Humanos , Ácido Caínico/farmacologia , Ácido Caínico/toxicidade , Masculino , Doenças Neurodegenerativas/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Fragmentos de Peptídeos/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/efeitos dos fármacosRESUMO
Cyanide is a rapidly acting neurotoxin that inhibits cellular respiration and energy metabolism leading to histotoxic hypoxia. This results in the dissipation of mitochondrial membrane potential (MMP) accompanied by decreased cellular ATP content which in turn is responsible for increased levels of intracellular calcium ions ([Ca(2+)](i)) and total lactic acid content of the cells. Rat pheochromocytoma (PC12) cells possess much of the biochemical machinery associated with synaptic neurons. In the present study, we evaluated the cytoprotective effects of alpha-ketoglutarate (A-KG) and N-acetylcysteine (NAC) against cyanide-induced cytotoxicity and altered energy metabolism in PC12 cells. Cyanide-antagonism by A-KG is attributed to cyanohydrin formation whereas NAC is known for its antioxidant properties. Data on leakage of intracellular lactate dehydrogenase and mitochondrial function (MTT assay) revealed that simultaneous treatment of A-KG (0.5 mM) and NAC (0.25 mM) significantly prevented the cytotoxicity of cyanide. Also, cellular ATP content was found to improve, followed by restoration of MMP, intracellular calcium [Ca(2+)](i) and lactic acid levels. Treatment with A-KG and NAC also attenuated the levels of peroxides generated by cyanide. The study indicates that combined administration of A-KG and NAC protected the cyanide-challenged PC12 cells by resolving the altered energy metabolism. The results have implications in the development of new treatment regimen for cyanide poisoning.
Assuntos
Acetilcisteína/farmacologia , Cianatos/toxicidade , Metabolismo Energético/efeitos dos fármacos , Sequestradores de Radicais Livres/farmacologia , Ácidos Cetoglutáricos/farmacologia , Células PC12/efeitos dos fármacos , Animais , Cálcio/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Interações Medicamentosas , Ácido Láctico/metabolismo , Peróxidos/metabolismo , Ratos , Fatores de TempoRESUMO
Oral toxicity of several cyanogens and their reversal by alpha-ketoglutarate (A-KG; oral) were studied in rats. LD(50) of acetonitrile (ATCN), acrylonitrile (ACN), malononitrile (MCN), propionitrile (PCN), sodium nitroprusside (SNP), and succinonitrile (SCN) was 4891, 143.3, 69.8, 122.9, 69.8 and 488.7 mg/kg, respectively while the protection index of A-KG (ratio of LD(50) of cyanogens in the presence or absence of A-KG) was>2.0 against MCN (7.6), PCN (2.7) and SNP (3.6) only. We further studied the efficacy of A-KG against acute toxicity of these three cyanogens (0.75 LD(50)) on various hematological and biochemical variables in blood and soft tissues 24h post-exposure. We observed increase in white blood cells (SNP), plasma alanine (PCN, SNP) and aspartate (PCN) aminotransferase, lactate dehydrogenase (MCN, PCN, SNP), Na(+) (MCN, PCN) and cyanide (PCN), and decrease in K(+) (MCN, SNP) accompanied by an increase in brain, kidney and liver malondialdehyde (PCN), decrease in brain glutathione peroxidase, glutathione reductase (PCN, SNP), reduced glutathione (MCN, PCN, SNP) and cytochrome oxidase (PCN), liver rhodanese (PCN, SNP), and kidney cytochrome oxidase (PCN). The study indicates that (i) PCN was most toxic among all the cyanogens and (ii) beside cyanide, A-KG could be considered as an effective antidote for cyanogens.