Hcit significantly inhibited aconitase activity, without altering the other enzymes of the CAC, whereas Orn did not affect any of these activities. Considering that aconitase is highly vulnerable to oxidative damage ( Gardner, 1997) and that Hcit provoked a higher degree of protein oxidative damage compared to Orn, it is possible that aconitase inhibition may have a result of Hcit-induced free radical attack to essential groups of the enzyme. Furthermore, Orn and Hcit significantly Nutlin-3a order reduced the electron transport chain flow by inhibiting the activity of complex I–III. Thus, it is feasible that the inhibition of complex I–III activity by these metabolites and of aconitase

Etoposide mw by Hcit contributed to the inhibition of the CAC. Altogether, these findings indicate that brain bioenergetics associated to energy production is compromised by Hcit and Orn. On the other hand, in vivo administration of Hcit and Orn did not change the activities of creatine kinase (CK) and synaptic Na+, K+-ATPase from cerebral cortex of rats, which are important for cell energy buffering and transfer and to keep the neuronal membrane potential necessary for normal neurotransmission, respectively. Altogether, our present findings indicate that Hcit exerted more significant effects than Orn on most parameters of oxidative stress and bioenergetics here

examined, even though it was administered at a lower dose (1.6 μmol) as compared to Orn (5 μmol), reinforcing that Hcit is relatively a more potent neurotoxin. On the other hand, it seems that the mild to moderate disruption of bioenergetics and oxidative damage induced by Orn could hardly be associated with the neurodegeneration of Plasmin HHH syndrome since this amino acid also accumulates at high amounts in ornithine aminotransferase deficiency, which is characterized by gyrate atrophy of the choroids and retina, with no alteration of the CNS (Javadzadeh and Gharabaghi, 2007, Kaiser-Kupfer et al., 1983 and Simell and Takki, 1973). At the present we cannot determine the pathophysiological relevance of the present

data since to our knowledge brain concentrations of Orn and Hcit are not yet established in HHH syndrome, although blood Orn concentrations may achieve 1 mM during metabolic decompensation in affected patients (Palmieri, 2008 and Valle and Simell, 2001). However, considering that the present in vivo results are in accordance with previous in vitro findings, showing that Orn and particularly Hcit disturb brain bioenergetics ( Viegas et al., 2009) and induce oxidative stress ( Amaral et al., 2009), it is presumed that a dual mechanism, energy deprivation and oxidative damage with reduction of tissue antioxidant defenses, secondary to acute accumulation of Hcit and Orn, may contribute to the neurological dysfunction characteristic of HHH syndrome.