Maple syrup urine disease (MSUD) is an inherited neurometabolic disorder biochemically characterized by the accumulation of the branched-chain alpha-
keto acids (BCKA) alpha-ketoisocaproic (KIC), alpha-keto-beta-methylvaleric (KMV) and alpha-ketoisovaleric (KIV) and their respective branched-chain alpha-
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 3h 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),
alpha-tocopherol (
trolox; 10 microM) and Nomega-nitro-
L-arginine methyl ester (
L-NAME; 500 microM). 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.