Fig. 1B shows that MEL totally

prevent Prist-induced in vitro lipoperoxidation [F(7,24) = 8.805; P < 0.001]. FG 4592 These results indicate that reactive species are involved in Prist-induced increase of lipid peroxidation. The next set of experiments was carried out to evaluate the in vitro effect of Prist on carbonyl and sulfhydryl content in cortical supernatants from young rats, which are parameters that evaluate protein oxidative damage. Prist significantly increased carbonyl formation at 100 μM and higher concentrations (up to 87%) [F(4,19) = 10.409; P < 0.01] ( Fig. 2A). This branched-chain fatty acid also provoked an enhancement of sulfhydryl oxidation (up to 33%) [F(4,25) = 18.877; P < 0.001] in a dose-dependent manner [β = −0.860; P < 0.001] ( Fig. 2B). Considering that carbonyl originates from the attack of free radicals to proteins and the sulfhydryl groups are oxidized by these reactive radicals, it is therefore presumed that Prist induces protein oxidative damage. The non-enzymatic antioxidant defenses were also investigated by assessing the concentrations of GSH, the naturally occurring antioxidant found in the brain, in the presence of Prist in cortical supernatants. It can be seen in Fig. 3A that Prist significantly Trametinib solubility dmso diminished GSH levels (up to 28%) in a dose-dependent manner [F(4,19) = 19.489; P < 0.001] [β = −0.845; P < 0.001]. G protein-coupled receptor kinase It is therefore

concluded that Prist reduces the major brain antioxidant defense.

It was also tested whether the antioxidants MEL (1000 μM), TRO (10 μM), combination of SOD plus CAT (20 mU/mL each) or L-NAME (750 μM) could prevent Prist-induced decrease of GSH levels in cortical supernatants. Fig. 3B shows that MEL [F(5,24) = 30.334; P < 0.001] fully prevented and TRO [F(5,24) = 30.334; P < 0.001] attenuated Prist-induced decrease of GSH levels. The data indicate that Prist-elicited diminution of GSH concentrations occurred via reactive oxygen species. In order to evaluate whether Prist could directly affect thiol groups in a cell free medium, we exposed a commercial GSH solution (150 μM) to 200 μM Prist for 1 h in the absence of brain supernatants. Fig. 4 shows that Prist per se did not modify GSH levels, whereas N-ethylmaleimide (NEM, 150 μM) (positive control) markedly oxidized GSH. The data clearly indicate that Prist does not behave as a direct oxidant. Finally, we assessed whether nitrogen reactive species were involved in Prist pro-oxidant effects by investigating the effect of Prist on nitrate and nitrite production. It can be seen in Table 1 that Prist did not induce nitrogen reactive species generation in cerebral cortex from young rats. Taken together these observations suggest that the pro-oxidant effects of Prist were mainly due to reactive oxygen species.