The data of Figs. 3(A and B) and 4(A–C) and the effect of the respective classical inhibitors (vs. control) were analyzed by Student’s t Test. Linear regression analysis was performed in order to evaluate the concentration

dependence effect of organochalcogens on mitochondrial complexes. A p < 0.05 value was considered statistically significant. Statistical analysis indicated that Ebs, (PhSe)2 and (PhTe)2 significantly inhibited complex I activity from liver and kidney mitochondria (Fig. 1A and B, respectively). The inhibitory effect was concentration dependent in liver membranes, as revealed by the linear regression analysis (p < 0.05 for all studied organochalcogens). Ebs-induced complex I inhibition was statistically significant from 5 μM onwards, while both (PhSe)2 and (PhTe)2 caused mitochondrial complex I inhibition from 10 μM onwards ( Fig. 1A and B). The IC50 (μM) values for inhibition by organochalcogens Nintedanib in vitro of mitochondrial complex I activity are showed in Ipatasertib Table 1. Rotenone (100 μM), a classical complex I inhibitor, caused a significant inhibition of the mitochondrial complex I activity

( Fig. 1A–B). Fig. 2 shows that Ebs significantly inhibited the complexes I–III activity from liver mitochondrial membranes from 10 μM onwards, with maximal effect at 50 μM (Fig. 2A). The inhibitory effect of Ebs on renal mitochondrial complexes I–III activity was statistically evident only at 50 μM (Fig. 2B). (PhSe)2 and (PhTe)2 did not change the mitochondrial complexes I–III activity from liver (Fig. 2A) or kidney (Fig. 2B). The IC50 (μM) values for inhibition by organochalcogens of mitochondrial complexes

I–III activity are showed in Table 1. In order to better understand the inhibitory effect of different organochalcogens in mitochondrial complexes I–III activity, we carried out experiments using two different conditions. In brief, in the condition 1 the membranes were pre-incubated with the organocompounds (at different concentrations) in the presence of NADH and the reaction was started with cytochrome c3. In the condition 2, the mitochondrial membranes were pre-incubated with different concentrations of oganocompounds and cytochome Cell press c3, and the reaction was started by NADH. Under condition 1, Ebs (5 μM) significantly inhibited complexes I–III activity from liver (Fig. 3A), without affecting renal complexes I–III activity (Fig. 3C). (PhSe)2 and (PhTe)2 did not inhibit the mitochondrial complexes I–III activity from liver or kidney (Fig. 3A and B, respectively). However, under condition 2 Ebs, (PhSe)2 and (PhTe)2 (5 μM) significantly inhibited complexes I–III activity from liver (Fig. 3A) and kidney membranes (Fig. 3B). Rotenone (100 μM) caused a significant inhibition of the mitochondrial complexes I–III activity that varied from 30% to 70% (Fig. 3A–B).