Fig. 5. Antioxidants protect against bupivacaine-induced oxidative stress. (A ) Reactive oxygen species (ROS) production was assessed by oxidation of CM-H2DCFDA (5-(and 6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester) derivatives in myotubes treated, or not (Ctl), with the indicated concentrations of bupivacaine for 8 h. Values are the mean ± SEM (n = 4); P < 0.01 with one-way analysis of variance and *P < 0.05 with post hoc unpaired t test versus control (Ctl) with Holm's procedure. (B ) ROS production was measured in myotubes incubated with the indicated concentrations of bupivacaine, N -acetylcysteine (NAC) for 8 h. Values are the mean ± SEM (n = 4), NAC effect is analyzed for the indicated concentrations of bupivacaine, P < 0.01 with one-way analysis of variance; *P < 0.05 with post hoc unpaired t test versus control (Ctl) with Holm's procedure; #P < 0.05 with post hoc unpaired t test versus without NAC (baseline value) for the same bupivacaine concentration, with Holm's procedure. (C ) Oxyblot analysis of carbonylated proteins from myotubes treated with vehicle (Ctl, lane 1 ) or with 1.5 mm of bupivacaine (lanes 2 , 3 , and 4 ) and the indicated concentrations of NAC (lanes 3 and 4 ) for 8 h. Histograms show the quantification of carbonylated proteins. Data are the mean ± SEM (n = 4); *P < 0.05 with unpaired t test versus control (Ctl, line 1); P < 0.01 with one-way analysis of variance and #P < 0.05 with post hoc unpaired t test versus without NAC (baseline value) for the same bupivacaine concentration, with Holm's procedure.