Increased production of reactive oxygen species (ROS) by mitochondria is involved in oxidative damage to the organelle and in committing cells to apoptosis or senescence, but the mechanisms of this increase are unknown. Here we show that ROS production by mitochondrial complex I increases in response to oxidation of the mitochondrial glutathione pool. This correlates with thiols on the 51-and 75-kDa subunits of complex I forming mixed disulfides with glutathione. Glutathionylation of complex I increases superoxide production by the complex, and when the mixed disulfides are reduced, superoxide production returns to basal levels. Within intact mitochondria oxidation of the glutathione pool to glutathione disulfide also leads to glutathionylation of complex I, which correlates with increased superoxide formation. In this case, most of this superoxide is converted to hydrogen peroxide, which can then diffuse into the cytoplasm. This mechanism of reversible mitochondrial ROS production suggests how mitochondria might regulate redox signaling and shows how oxidation of the mitochondrial glutathione pool could contribute to the pathological changes that occur to mitochondria during oxidative stress.

Summary Purpose:  Long‐term therapy with antiepileptic drugs (AEDs) has been associated with metabolic consequences that lead to an increase in risk of atherosclerosis in patients with epilepsy. We compared the long‐term effects of monotherapy using different categories of AEDs on markers of vascular risk and the atherosclerotic process. Methods:  One hundred sixty adult patients who were receiving AED monotherapy, including two enzyme‐inducers (carbamazepine, CBZ; and phenytoin, PHT), an enzyme‐inhibitor (valproic acid, VPA), and a noninducer (lamotrigine, LTG) for more than 2 years, and 60 controls were enrolled in this study. All study participants received measurement of common carotid artery (CCA) intima media thickness (IMT) by B‐mode ultrasonography to assess the extent of atherosclerosis. Other measurements included body mass index, and serum lipid profile or levels of total homocysteine (tHcy), folate, uric acid, fasting blood sugar, high sensitivity C‐reactive protein (hs‐CRP), or thiobarbituric acid reactive substances (TBARS). Key Findings:  Long‐term monotherapy with older‐generation AEDs, including CBZ, PHT, and VPA, caused significantly increased CCA IMT in patients with epilepsy. After adjustment for the confounding effects of age and gender, the CCA IMT was found to be positively correlated with the duration of AED therapy. Patients with epilepsy who were taking enzyme‐inducing AED monotherapy (CBZ, PHT) manifested disturbances of cholesterol, tHcy or folate metabolism, and elevation of the inflammation marker, hs‐CRP. On the other hand, patients on enzyme‐inhibiting AED monotherapy (VPA) exhibited an increase in the levels of uric acid and tHcy, and elevation of the oxidative marker, TBARS. However, no significant alterations in the markers of vascular risk or CCA IMT were observed in patients who received long‐term LTG monotherapy. Significance:  Patients with epilepsy who were receiving long‐term monotherapy with CBZ, PHT, or VPA exhibited altered circulatory markers of vascular risk that may contribute to the acceleration of the atherosclerotic process, which is significantly associated the duration of AED monotherapy. This information offers a guide for the choice of drug in patients with epilepsy who require long‐term AED therapy, particularly in aged and high‐risk individuals.

Mitochondria play a central role in redox-linked processes in the cell through mechanisms that are thought to involve modification of specific protein thiols, but this has proved difficult to assess. In particular, specific labeling and quantitation of mitochondrial protein cysteine residues have not been achieved due to the lack of reagents available that can be applied to the intact organelle or cell. To overcome these problems we have used a combination of mitochondrial proteomics and targeted labeling of mitochondrial thiols using a novel compound, (4-iodobutyl)triphenylphosphonium (IBTP). This lipophilic cation is accumulated by mitochondria and yields stable thioether adducts in a thiol-specific reaction. The selective uptake into mitochondria, due to the large membrane potential across the inner membrane, and the high pH of the matrix results in specific labeling of mitochondrial protein thiols by IBTP. Individual mitochondrial proteins that changed thiol redox state following oxidative stress could then be identified by their decreased reaction with IBTP and isolated by two-dimensional electrophoresis. We demonstrate the selectivity of IBTP labeling and use it to show that glutathione oxidation and exposure to an S-nitrosothiol or to peroxynitrite cause extensive redox changes to mitochondrial thiol proteins. In conjunction with blue native gel electrophoresis, we used IBTP labeling to demonstrate that thiols are exposed on the matrix faces of respiratory Complexes I, II, and IV. This novel approach enables measurement of the thiol redox state of individual mitochondrial proteins during oxidative stress and cell death. In addition the methodology has the potential to identify novel redox-dependent modulation of mitochondrial proteins.Changes in the thiol redox state of mitochondrial proteins are significant in a number of cellular processes including the permeability transition, cell death due to calcium loading and oxidative stress, the response of cells to nitric oxide, tumor necrosis factor signaling, commitment to apoptosis, and in regulating respiratory chain function (1-9). However the detailed mechanisms and the proteins involved are uncertain. This is partly because of the technical challenges presented by determining thiol modifications of proteins in general and the difficulties inherent in mitochondrial proteomics. Potential protein thiol alterations include formation of mixed disulfides or internal disulfides from vicinal dithiols, S-nitrosation, and the formation of higher oxidation states (10 -15). The differential reactivity of individual protein thiols and the range of lifetimes of altered redox states can act as signal sensors or transducers to influence mitochondrial function (13-17). Nitric oxide may be a particularly important regulator of mitochondrial protein thiols because it diffuses easily into mitochondria and partitions selectively into the lipid bilayer where it can modify otherwise inaccessible thiols (18 -20). Modification of protein thiols by nitric oxide most likely occurs...

SUMMARYPurpose: Long-term antiepileptic drug (AED) therapy has been associated with an increase in risk of atherosclerosis. At issue is whether this risk is related to the duration of AED therapy. We evaluated the hypothesis that the cumulative effect of long-term exposure to AEDs plays a pivotal role in the pathogenesis of atherosclerosis in patients with epilepsy. Methods: One hundred ninety-five patients under long-term AED therapy and 195 healthy age-and sex-matched control subjects received measurement of intima media thickness (IMT) at the far wall of the common carotid artery (CCA) by Bmode ultrasonography to assess the extent of atherosclerosis. Other measurements included body mass index (BMI) and blood lipid profile or homocysteine, folic acid, uric acid, fasting blood sugar, high sensitivity C-reactive protein (hs-CRP), thiobarbituric acid reactive substances (TBARS), and total reduced thiols.Results: CCA IMT was significantly increased in patients with epilepsy, with male subjects exhibiting thicker IMT than their female counterparts. Whereas BMI, homocysteine, hs-CRP, and TBARS were significantly elevated, folic acid and thiols were significantly reduced in patients with epilepsy. Multiple linear regression analysis further revealed that duration of AED therapy, age, gender, and TBARS level (index for oxidative stress) were independently associated with CCA IMT. In addition, the log-transformed CCA IMT increased linearly with duration of AED therapy after adjustments for age, gender, and TBARS level. Discussion: The duration of AED therapy is significantly associated with the acceleration of atherosclerosis in patients with epilepsy, alongside independent contributions of age, gender, and oxidative stress to the atherosclerotic process.