Figure 4

Influence of ROS on CO effect at mitochondrial level. A. Mitochondria were pre-treated or not with β-carotene, followed by CO at 0, 10, 50, 100 or 250 μM or H₂O₂ at 500 μM treatment. ROS quantification using 2',7'-dichlorofluorescein diacetate (H₂DCFDA, λ_exc: 485 nm, λ_em: 530 nm) was done in isolated mitochondria. The values are expressed in relative percentage of 500 μM of H₂O₂ (100%) at 28 minutes after treatment. All values are mean ± SD, n = 3; * p < 0.05 compared to control mitochondria and ** p < 0.05 compared to mitochondria treated with CO at 50 μM. Mitochondria were pre-treated in the presence or absence of 1 μM of β-carotene and/or 10 μM of CO, then atractyloside at 300 μM or calcium at 5 μM was added, followed by swelling measurements (absorbance at 540 nm) for B and depolarization measurements (Rhodamine 123, λ_exc: 485 nm, λ_em: 535 nm) for C. In B and C the values are expressed in relative percentage of 5 μM of Ca²⁺ (100%) measured at 15 minutes after treatment. All values are mean ± SD, n = 3; * p < 0.05 compared to control mitochondria, ** p < 0.05 compared to Ca²⁺-treated mitochondria, *** p < 0.05 compared to Atra-treated mitochondria, # p < 0.05 compared to β-c and Ca²⁺-treated mitochondria and ## p < 0.05 compared to β-c and Atra-treated mitochondria. D. Inner membrane permeabilization was assessed according to [16]. Mitochondria were pre-treated 1 μM of β-carotene and 10 μM of CO, followed by addition of calcium at 5 μM. Data was acquired at 412 nm for 20 minutes at 37°C.