Morphological alterations and induction of oxidative stress in glial cells caused by the branched-chain α-keto acids accumulating in maple syrup urine disease
Section snippets
Materials
All chemical reagents and cell culture materials were purchased from Sigma (St. Louis, MO, USA), except for Dulbecco's modified Eagle's medium (DMEM) which was purchased from Gibco BRL (Carlsbad, CA, USA), fetal bovine serum (FBS) from Cultilab (Campinas, SP, Brazil) and rhodamine-labelled phalloidin from Molecular Probes (OR, USA).
Maintenance of cell line
The C6 rat glioma cell line was obtained from American Type Culture Collection (Rockville, Maryland, USA). The cells were grown and maintained in DMEM (pH 7.4)
Total antioxidant reactivity (TAR)
TAR, which represents the reactivity or quality of the tissue antioxidants, was determined by measuring the luminol chemiluminescence intensity induced by 2,2′-azo-bis-(2-amidinopropane) (ABAP) (Lissi et al., 1995). The background chemiluminescence was measured by adding 4 ml 2 mM ABAP (in 0.1 M glycine buffer, pH 8.6) into a glass scintillation vial. Ten microliters of luminol (4 mM) were added to each vial and the chemiluminescence was determined. This was considered to be the basal value. Fifty
Effects of the BCKA on C6 cell morphology
C6 cells were treated with different concentrations (1 or 10 mM) of KIC, KMV or KIV (Fig. 1) and morphologically analyzed by phase contrast microscopy after different exposure times (3 and 24 h). Fig. 1A shows that all BCKA-induced morphological alterations in C6 cells in a time-exposure and concentration-dependent manner, leading to a progressively increased cell death. Morphologically altered cells consisting of fusiform or process-bearing cells were already observed at 3 h exposure to the BCKA.
Discussion
Although, the mechanisms responsible for the neurological dysfunction in MSUD are still poorly defined, various reports indicate that excitotoxicity, energy depletion and oxidative stress are probably involved in the brain injury observed in the affected patients (Howell and Lee, 1963, Snyderman et al., 1964, Land et al., 1976, Halestrap et al., 1974, Jouvet et al., 2000, Tavares et al., 2000, Chuang and Shih, 2001, Fontella et al., 2002, Pilla et al., 2003, Sgaravatti et al., 2003, Bridi et
Acknowledgements
This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS) and Pró Reitoria de Pesquisa da Universidade Federal do Rio Grande do Sul (PROPESq-UFRGS).
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