Morphological alterations and induction of oxidative stress in glial cells caused by the branched-chain α-keto acids accumulating in maple syrup urine disease

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Abstract

Maple syrup urine disease (MSUD) is an inherited neurometabolic disorder biochemically characterized by the accumulation of the branched-chain α-keto acids (BCKA) α-ketoisocaproic (KIC), α-keto-β-methylvaleric (KMV) and α-ketoisovaleric (KIV) and their respective branched-chain α-amino acids in body fluids and tissues. Affected MSUD patients have predominantly neurological features, including cerebral edema and atrophy whose pathophysiology is not well established. In the present study we investigated the effects of KIC, KMV and KIV on cell morphology, cytoskeleton reorganization, actin immunocontent and on various parameters of oxidative stress, namely total antioxidant reactivity (TAR), glutathione (GSH) and nitric oxide concentrations, and on the activities of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) in C6 glioma cells. We initially observed that C6 cultivated cells exposed for 3 h to the BCKA (1 and 10 mM) changed their usual rounded morphology to a fusiform or process-bearing cell appearance, while 24 h exposure to these organic acids elicited massive cell death. Rhodamine-labelled phalloidin analysis revealed that these organic acids induced reorganization of the actin cytoskeleton with no modifications on total actin content. It was also observed that 3 h cell exposure to low doses of all BCKA (1 mM) resulted in a marked reduction of the non-enzymatic antioxidant defenses, as determined by TAR and GSH measurements. In addition, KIC provoked a reduced activity of SOD and GPx, whereas KMV caused a diminution of SOD activity. In contrast, CAT activity was not modified by the metabolites. Furthermore, nitric oxide production was significantly increased by all BCKA. Finally, we observed that the morphological features caused by BCKA on C6 cells were prevented by the use of the antioxidants GSH (1.0 mM), α-tocopherol (trolox; 10 μM) and Nω-nitro-l-arginine methyl ester (l-NAME; 500 μM). These results strongly indicate that oxidative stress might be involved in the cell morphological alterations and death, as well as in the cytoskeletal reorganization elicited by the BCKA. It is presumed that these findings are possibly implicated in the neuropathological features observed in patients affected by MSUD.

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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