*3.3. Mitochondrial ROS Generation and Membrane Potential*

In isolated respiring rat liver mitochondria, it has been found that MeJA increases net ROS generation [9]. For this reason, experiments were done in which the action of MeJA treatment on ROS generation by brain mitochondria was measured. Figure 3a shows the results obtained when brain mitochondria isolated from rats under various conditions were incubated in a medium containing succinate as the electron donor. ROS generation was measured as the fluorescence increase due to the formation of the indicator DCF. In mitochondria from arthritic rats, ROS generation was clearly increased by a factor of 1.36. Treatment with MeJA did not reverse this modification although there was some tendency in this direction. There was also a small tendency toward increasing ROS generation in mitochondria from healthy rats treated with MeJA. ROS generation is a dynamic variable which could be dependent on the continuous presence of MeJA. Since the latter is no longer present in mitochondria from treated rats, attempts were made at investigating the short term effects of the compound. The results shown in Figure 3a represent the possible reversible and short-term effects of MeJA on ROS generation in isolated mitochondria from healthy and arthritic rats in the range of up to 10 mM. They show that the compound increased ROS generation in brain mitochondria from both healthy and arthritic rats. The increments relative to the starting conditions were similar for both healthy and arthritic conditions in the range up to 1.25 mM. After this concentration there was a declining tendency, more pronounced in mitochondria from healthy rats.

**Figure 3.** Effects of MeJA on ROS production and membrane potential in brain mitochondria from healthy and arthritic rats. (**a**) ROS production in mitochondria isolated from healthy and arthritic rats, treated or not with different MeJA doses (C, controls; C300, controls treated with 300 mg/kg MeJA; A, arthritic rats; A75, A150 and A300, arthritic rats treated with 75, 150 and 300 mg/kg MeJA, respectively). (**b**) ROS production by brain mitochondria isolated from healthy and arthritic rats and incubated with varying concentrations of MeJA. (**c**) Membrane potential (ΔΨm) of brain mitochondria isolated from healthy and arthritic rats, treated or not with different MeJA doses. (**d**) Membrane potential (ΔΨm) of brain mitochondria isolated from healthy and arthritic rats and incubated with varying concentrations of MeJA. Data are the means ± standard errors of the mean of five animals for each experimental condition. Statistical analysis: ANOVA one-way with Newman–Keuls post-hoc testing. \**p* < 0.05, different from the corresponding controls.

It is generally accepted that ROS generation in mitochondria shows a positive correlation with the membrane potential [51]. The measurements illustrated by Figure 3c,d were done in order the explore this relationship in terms of the effects of arthritis and MeJa. As described in the Materials and Methods section, energization was achieved by the addition of succinate, the same substrate used in the experiments in which ROS generation was measured. Figure 3c reveals a clearly increased membrane energization in mitochondria from arthritic rats. This correlates nicely with the increased rates of ROS generation shown in Figure 3a. The effects of MeJA treatment, however, are unclear, as no statistically significant modifications were detected even though there is a general tendency toward a lower energization. When mitochondria isolated from control and arthritic rats were incubated with varying MeJA concentrations in the range of up to 10 mM in order to explore possible reversible actions (Figure 3d), only minimal modifications were observed. These were restricted to the control rats, in which there was a small increment at high MeJA concentrations.
