Giovanna Parenti Castelli

Role of Mitochondria in Oxidative Stress and Aging

The mitochondrial respiratory chain is a powerful source of reactive oxygen species (ROS), considered as the pathogenic agent of many diseases and of aging. We have investigated the role of Complex I in superoxide radical production and found by combined use of specific inhibitors of Complex I that the one‐electron donor in the Complex to oxygen is a redox center located prior to the sites where three different types of coenzyme Q (CoQ) competitors bind, to be identified with an Fe‐S cluster, most probably N2, or possibly an ubisemiquinone intermediate insensitive to all the above inhibitors. Short‐chain coenzyme Q analogues enhance superoxide formation, presumably by mediating electron transfer from N2 to oxygen. The clinically used CoQ analogue idebenone is particularly effective, raising doubts about its safety as a drug. The mitochondrial theory of aging considers somatic mutations of mitochondrial DNA induced by ROS as the primary cause of energy decline; in rat liver mitochondria, Complex I appears to be most affected by aging and to become strongly rate limiting for electron transfer. Mitochondrial energetics is also deranged in human platelets upon aging, as demonstrated by the decreased Pasteur effect (enhancement of lactate production by respiratory inhibitors). Cells counteract oxidative stress by antioxidants: CoQ is the only lipophilic antioxidant to be biosynthesized. Exogenous CoQ, however, protects cells from oxidative stress by conversion into its reduced antioxidant form by cellular reductases. The plasma membrane oxidoreductase and DT‐diaphorase are two such systems: likewise, they are overexpressed under oxidative stress conditions.

The site of production of superoxide radical in mitochondrial Complex I is not a bound ubisemiquinone but presumably iron–sulfur cluster N2

The mitochondrial respiratory chain is a powerful source of reactive oxygen species, considered as the pathogenic agent of many diseases and of aging. We have investigated the role of Complex I in superoxide radical production in bovine heart submitochondrial particles and found, by combined use of specific inhibitors of Complex I and by Coenzyme Q (CoQ) extraction from the particles, that the one‐electron donor in the Complex to oxygen is a redox center located prior to the binding sites of three different types of CoQ antagonists, to be identified with a Fe–S cluster, most probably N2 on the basis of several known properties of this cluster. Short chain CoQ analogs enhance superoxide formation, presumably by mediating electron transfer from N2 to oxygen. The clinically used CoQ analog, idebenone, is particularly effective in promoting superoxide formation.

The Mitochondrial Production of Reactive Oxygen Species in Relation to Aging and Pathology

Abstract: Mitochondria are known to be strong producers of reactive oxygen species (ROS) and, at the same time, particularly susceptible to the oxidative damage produced by their action on lipids, proteins, and DNA. In particular, damage to mtDNA induces alterations to the polypeptides encoded by mtDNA in the respiratory complexes, with consequent decrease of electron transfer, leading to further production of ROS and thus establishing a vicious circle of oxidative stress and energetic decline. This deficiency in mitochondrial energetic capacity is considered the cause of aging and age‐related degenerative diseases. Complex I would be the enzyme most affected by ROS, since it contains seven of the 13 subunits encoded by mtDNA. Accordingly, we found that complex I activity is significantly affected by aging in rat brain and liver mitochondria as well as in human platelets. Moreover, due to its rate control over aerobic respiration, such alterations are reflected on the entire oxidative phosphorylation system. We also investigated the role of mitochondrial complex I in superoxide production and found that the one‐electron donor to oxygen is most probably the Fe‐S cluster N2. Short chain coenzyme Q (CoQ) analogues enhance ROS formation, presumably by mediating electron transfer from N2 to oxygen, both in bovine heart SMP and in cultured HL60 cells. Nevertheless, we have accumulated much evidence of the antioxidant role of reduced CoQ10 in several cellular systems and demonstrated the importance of DT‐diaphorase and other internal cellular reductases to reduce exogenous CoQ10 after incorporation.

Mitochondrial production of oxygen radical species and the role of Coenzyme Q as an antioxidant.

The mitochondrial respiratory chain is a powerful source of reactive oxygen species (ROS), which is considered as the pathogenic agent of many diseases and of aging. We have investigated the role of complex I in superoxide radical production and found by the combined use of specific inhibitors of complex I that the one-electron donor to oxygen in the complex is a redox center located prior to the sites where three different types of Coenzyme Q (CoQ) competitors bind, to be identified with an Fe–S cluster, most probably N2, or possibly an ubisemiquinone intermediate insensitive to all the above inhibitors. Short-chain Coenzyme Q analogs enhance superoxide formation, presumably by mediating electron transfer from N2 to oxygen. The clinically used CoQ analog, idebenone, is particularly effective, raising doubts on its safety as a drug. Cells counteract oxidative stress by antioxidants. CoQ is the only lipophilic antioxidant to be biosynthesized. Exogenous CoQ, however, protects cells from oxidative stress by conversion into its reduced antioxidant form by cellular reductases. The plasma membrane oxidoreductase and DT-diaphorase are two such systems, likewise, they are overexpressed under oxidative stress conditions.

The effect of aging and an oxidative stress on peroxide levels and the mitochondrial membrane potential in isolated rat hepatocytes

We have investigated the effect of ageing and of adriamycin treatment on the bioenergetics of isolated rat hepatocytes. Ageing per se, whilst being associated with a striking increase of hydrogen peroxide in the cells, induces only minor changes on the mitochondrial membrane potential. The adriamycin treatment induces a decrease of the mitochondrial membrane potential in situ and a consistent increase of the superoxide anion cellular content independently of the donor age. The hydrogen peroxide is significantly increased in both aged and adult rat hepatocytes, however, due to the high basal level in the aged cells, it is higher in aged rat cells not subjected to oxidative stress than that elicited by 50 μM adriamycin in young rat hepatocytes. The results suggest that a hydrogen peroxide increase in hepatocytes of aged rats is unable to induce major modifications of mitochondrial bioenergetics. This contrasts with the damaging effect of adriamycin, suggesting that the effects of the drug may be due to the concomitant high level of both superoxide and hydrogen peroxide.

Decreased Pasteur effect in platelets of aged individuals.

We have investigated the mitochondrial energy state in human platelets of young (19-30 years old) and aged individuals (65-87 years old) exploiting the Pasteur effect, i.e. stimulation of lactate production by incubation of the purified platelets with the mitochondrial respiratory chain inhibitor, antimycin A. This assay allows the determination of mitochondrial function with respect to glycolysis, and the ratio of mitochondrial adenosine triphosphate (ATP) to glycolytic ATP. A significant increase of basal, non-stimulated lactate production and decrease of the stimulation by antimycin A were observed in the older individuals, suggesting that the impairment of oxidative phosphorylation detectable in post-mitotic tissues of aged individuals can be observed also in easily collectable blood cells.

Measurement of the lateral diffusion coefficients of ubiquinones in lipid vesicles by fluorescence quenching of 12-(9-anthroyl)stearate

The lateral diffusion coefficients of some ubiquinone homologues have been measured in phospholipid vesicles exploiting the fluorescence quenching of the probe 12‐(9‐anthroyl)stearate by the quinones. Diffusion coefficients higher than 10−6cm2 · s−1 have been found at 25°C, compatible with the localization of the ubiquinones in the low‐viscosity midplane region of the bilayer.

Lack of major changes in ATPase activity in mitochondria from liver, heart, and skeletal muscle of rats upon ageing.

ATP hydrolase activity has been investigated in mitochondria from liver, heart, and skeletal muscle from adult (6 months) and aged (24 months) rats. No significant changes in total ATPase activity were observed in the three tissues, but the oligomycin sensitivity was slightly decreased in heart mitochondria of aged rats. The bicarbonate-induced stimulation of hydrolytic activity was somewhat decreased in mitochondria from aged rats, particularly in liver. No significant change was observed in ATPase activity after release of the endogenous inhibitor protein, IF1. It is concluded that no activity changes to be directly ascribed to the catalytic sector F1 of the enzyme occur upon ageing, but it cannot be excluded that changes in the membrane sector F0 occur as a consequence of mtDNA mutations.

Effect of the oxidative stress induced by adriamycin on rat hepatocyte bioenergetics during ageing.

We have investigated the effect of ageing and of adriamycin treatment on the bioenergetics of isolated rat hepatocytes. Ageing per se, whilst being associated with a striking increase of hydrogen peroxide in the cells, induces only minor changes on mitochondrial functions. The adriamycin treatment induces a decrease of the mitochondrial membrane potential in situ and a consistent increase of the superoxide anion cellular content independently of the donors age, whilst the hydrogen peroxide is significantly higher in aged than in adult rat hepatocytes. Kinetic studies in isolated mitochondria show that the mitochondrial respiratory chain activity (NADH --> O2) of 50 microM adriamycin-treated hepatocytes is lowered both in adult and aged rats. The same adriamycin concentration induces a slight decrease of the maximal rate of ATP hydrolysis in both young and aged rats, without affecting the Km for the substrate. However, at drug concentrations lower than 50 microM, both ATPase and NADH oxidation activities decrease significantly in aged rats only. The results suggest that free radicals increase during ageing in rat hepatocytes but are unable to induce major modifications of mitochondrial bioenergetics. This contrasts with the damaging effect of adriamycin, suggesting that some effects of the drug may be due to other reasons besides oxidative stress.

Fluidizing effect of endogenous ubiquinone in bovine heart mitochondrial membranes

Extraction of endogenous ubiquinone from lyophilized beef heart mitochondria results in increases of both the order parameter of the spin label 5‐NS and of the rotational correlation time of 16‐NS; reconstitution with the pentane extract results in restoration of the original spectral parameters. On the other hand, addition of purified ubiquinone homologs restores the original spectra only in the case of 16‐NS, whereas the order parameter of 5‐NS is restored by addition of mixed phospholipids. The same amounts of ubiquinone homologs incorporated in mixed phospholipid vesicles induce much lower effects. It is suggested that ubiquinone in mitochondria is intercalated with the lipid chains of the membrane in such a way to perturb the fluidity of the hydrophobic core.