RESUMEN
Oxidative stress has been discussed as crucial mechanism of neuronal cell death in the adult brain. However, it was not clear until now whether neurons are more sensitive to oxidative stress than the other cells in the brain, e.g. astrocytes. Therefore both cell types were exposed to oxidative stress provoked by the redox-cycling compound paraquat. Cortical neurons were found to be more sensitive towards paraquat toxicity than astrocytes as shown by MTT and Neutral Red assay, two different cytotoxicity assays. Mitochondrial functions were determined by the mitochondrial membrane potential and intracellular ATP concentrations. Again cortical neurons were more severely impaired (by paraquat than astrocytes). The production of reactive oxygen species after paraquat exposure was much higher in cortical neurons than in astrocytes and correlated with a higher depletion of GSH (intracellular glutathion). Lipid peroxidation could be shown in astrocytes via the breakdown product malondialdehyde (MDA) whereas in cortical neurons 4-hydroxynonenal (4-HNE) was detected as this endpoint. If and how oxidative stress influences the antioxidant defense was determined via changes in the expression of antioxidant enzymes. Paraquat exposure lead to a 2-3 fold increase of catalase, MnSOD and CuZnSOD mRNA expression in astrocytes. In contrast to astrocytes, in cortical neurons catalase and MnSOD mRNA levels were only marginally elevated above 1.5-fold by treatment with paraquat. Expression levels of glutathione peroxidase (GPx) mRNA were the only one that were not changed in both cell types after paraquat exposure. It is concluded that the more marked increase in expression levels of antioxidant enzymes may render astrocytes more resistant to oxidative stress than neuronal cells.
RESUMEN
Oxidative stress has been causally linked to a variety of neurodegenerative diseases. To clarify the role of the antioxidant enzyme (AOE) system in oxidative brain damage primary cultures of rat astroglial cells were exposed to hydrogen peroxide (H2O2). Expression of AOEs and several parameters for cell viability and functionality were measured. In our experiments astrocytes responded to low concentrations of H2O2 exposure with a pronounced generation of ROS which ran parallel with induction of lipid peroxidation. This distinct oxidative stress was not reflected in cell viability or functionality parameters measured. Cytotoxicity, a decrease in glutathione content of astrocytes, and impairment of mitochondrial functions became obvious only for higher concentrations of H2O2. After H2O2 exposure catalase, manganese superoxide dismutase, and glutathione peroxidase expression levels were found to be increased, whereas copper/zinc superoxide dismutase mRNA expression was not affected. These data indicate that the AOE system of astrocytes can be directly regulated by oxidative stress and may thus contribute to protection of cells against oxidative insults.
Asunto(s)
Astrocitos/efectos de los fármacos , Astrocitos/enzimología , Catalasa/metabolismo , Glutatión Peroxidasa/metabolismo , Peróxido de Hidrógeno/toxicidad , Superóxido Dismutasa/metabolismo , Animales , Northern Blotting , Catalasa/genética , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Expresión Génica/efectos de los fármacos , Glutatión/metabolismo , Glutatión Peroxidasa/genética , Peroxidación de Lípido/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/fisiología , ARN Mensajero/metabolismo , Ratas , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/clasificación , Superóxido Dismutasa/genéticaRESUMEN
The cytostatic Adriamycin and the herbicide paraquat form reactive oxygen species during enzymatic activation. Adriamycin, but not paraquat, is also able to intercalate into DNA and to interfere with DNA synthesis and transcription. We investigated the influence of both substances on antioxidant enzyme expression in primary rat hepatocytes. Treatment of hepatocytes with Adriamycin led to an increase in catalase and a decrease in MnSOD mRNA expression. In contrast, exposure of hepatocytes to paraquat resulted in an increase in both catalase and MnSOD message levels. CuZnSOD mRNA was not responsive to either treatment. Adriamycin almost completely inhibited RNA synthesis, but paraquat did not change either RNA or protein synthesis. Both substances induced lipid peroxidation as measured by the accumulation of malondialdehyde in the medium. These findings indicate that catalase and MnSOD are not regulated coordinately in hepatocytes and that ROS-producing agents can differentially influence expression of antioxidant enzymes depending on their capacity to inhibit transcription.
Asunto(s)
Antibióticos Antineoplásicos/toxicidad , Catalasa/biosíntesis , Doxorrubicina/toxicidad , Hepatocitos/efectos de los fármacos , Herbicidas/toxicidad , Paraquat/toxicidad , Superóxido Dismutasa/biosíntesis , Animales , Antibióticos Antineoplásicos/farmacocinética , Catalasa/genética , Cobre/metabolismo , ADN/efectos de los fármacos , ADN/metabolismo , Doxorrubicina/farmacocinética , Expresión Génica/efectos de los fármacos , Hepatocitos/enzimología , Herbicidas/farmacocinética , Sustancias Intercalantes/farmacocinética , Sustancias Intercalantes/toxicidad , Masculino , Manganeso/metabolismo , Paraquat/farmacocinética , ARN/biosíntesis , ARN/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/genética , Zinc/metabolismoRESUMEN
Tumor necrosis factor-alpha is assumed to play a role in toxic liver damage. We examined whether exogenous tumor necrosis factor-alpha must be present for alpha-amanitin cytotoxicity in rat hepatocyte culture. alpha-Amanitin at a concentration of 0.1 microM, which is close to that found in intoxicated patients, inhibits RNA and protein synthesis within 12 h but cytotoxicity only occurs after a latency period and is pronounced at 36 h after the start of treatment. Tumor necrosis factor-alpha is not indispensable for the development of cytotoxicity but aggravates it and leads to a time shift towards earlier times. Lipid peroxidation is low with alpha-amanitin alone even at 36 h but markedly increased by cotreatment with tumor necrosis factor-alpha. The antioxidant silibin prevents the effect of tumor necrosis factor-alpha, indicating an involvement of reactive oxygen species. alpha-Amanitin alone does not increase but dose-dependently inhibits the expression of the antioxidant enzyme manganous superoxide dismutase and decreases the inducing effect of TNF-alpha on the expression of this enzyme. The gene expression of endogenous tumor necrosis factor-alpha in the hepatocytes is not increased but rather inhibited by alpha-amanitin treatment. The results suggest that alpha-amanitin causes delayed cytotoxicity following rapid inhibition of RNA and protein synthesis and that tumor necrosis factor-alpha shortens the latency period and aggravates the cytotoxicity by a mechanism which may involve reactive oxygen species.
Asunto(s)
Amanitinas/toxicidad , Hígado/citología , Silimarina/farmacología , Factor de Necrosis Tumoral alfa/fisiología , Animales , Northern Blotting , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Hígado/efectos de los fármacos , Masculino , Biosíntesis de Proteínas , ARN/análisis , ARN/aislamiento & purificación , Ratas , Ratas Wistar , Superóxido Dismutasa/biosíntesis , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Poly(7-deazaguanylic acid) was enzymatically synthesized by the polymerization of 7-deazaguanosine 5'-diphosphate with polynucleotide phosphorylase from Micrococcus luteus in high yield. The homopolymer showed a similar thermal and total hypochromicity to poly(G) at the long wavelength absorption maximum. No sigmoid melting profile was observed for poly(c7G) as is found for poly(G), implying a single-stranded structure in aqueous solution. From the circular dichroism spectra it can be concluded that the 7-deazapurine nucleotide is much more flexible than the purine nucleotide. In analogy to poly(G), the homopolymer poly(c7G) forms a 1:1 complex with poly(C) under neutral conditions, melting at a similar temperature to the poly(G) complex. However, at pH 2.5, where a poly(G) X 2poly(C) complex is observed, poly(c7G) still binds only one poly(C) strand. This is due to the lack of N-7 in poly(c7G), not allowing Hoogsteen base pair formation, which occurs with poly(G). RNase T1 cleaves poly(c7G), indicating that N-7 of guanosine is not a requirement for nucleotide binding to the enzyme, as has been suggested. Because of the single-stranded structure of poly(c7G), the polynucleotide chain is rapidly hydrolyzed by the single-strand-specific nuclease S1, whereas multistranded poly(G) is completely resistant.