G. Benzi

Are reactive oxygen species involved in Alzheimer's disease?

Alzheimers disease has a multifactorial pathogenesis. Among the various factors involved, this review examines, in particular, the possibility of oxidative stress, meaning an imbalance between the formation and spread of reactive oxygen species (ROS) and the antioxidant defenses. This theory is supported by the following observations: (a) the alteration of mitochondrial function, which is likely to lead to the electron leakage in the respiratory chain and the consequent formation of superoxide radicals; (b) the unbalanced high activity of superoxide dismutase and monoamine oxidase B which causes the production of more H2O2; (c) the alteration of iron homeostasis which, in combination with the superoxide and H2O2, gives rise to the most deleterious hydroxyl radicals; (d) the increased lipid peroxidation and membrane alterations; (e) the pro-aggregating effect of ROS on beta/A4 protein and the C-terminal fragment of amyloid precursor (A4CT). Most of these changes are already present in the normal aging brain but are aggravated in AD presumably over a number of years. However, further investigations are needed to confirm these theories particularly regarding the alterations of another target of ROS, the proteins. Peroxidative stress is presumably present in the AD brain. This stress might not be a primary factor in the pathogenesis of AD, but a consequence of the tissue injury. In any case, it could contribute considerably to the pathology, in a vicious cycle of actions and reactions resulting in a critical mass of metabolic errors, responsible in the end for this disease.

Age- and peroxidative stress-related modifications of the cerebral enzymatic activities linked to mitochondria and the glutathione system

The aging brain undergoes a process of enhanced peroxidative stress, as shown by reports of altered membrane lipids, oxidized proteins, and damaged DNA. The aims of this review are to examine: (1) the possible contribution of mitochondrial processes to the formation and release of reactive oxygen species (ROS) in the aging brain; and (2) the age-related changes of antioxidant defenses, both enzymatic and nonenzymatic. It will focus on studies investigating the role of the electron transfer chain as the site of ROS formation in brain aging and the alterations of the glutathione system, also in relation to the effects of exogenous pro-oxidant agents. The possible role of peroxidative stress in age-related neurodegenerative diseases will also be discussed.

The mitochondrial electron transfer alteration as a factor involved in the brain aging

The tissutal concentrations of reduced glutathione (GSH) and the contents of some key components in the electron transfer chain (namely ubiquinone, cytochromes b, c1, c, and aa3) of the intraterminal mitochondria are measured in the forebrains from 20-, 60-, or 100-week-old Wistar rats. Moreover, in 60-week-old rats, the biochemical analyses are performed also 18 h after the induction of a peroxidative stress by cyclohexene-1-one. The rats have been i.p. pretreated for 8 weeks (7 days/week) with agents acting on macrocirculation (papaverine), carbohydrate metabolism (hopanthenate), lipid metabolism (phosphatidylcholine), energy transduction (theniloxazine), and dopaminergic system (dihydroergocriptine). Brain aging is characterized by the decrease in both GSH and mitochondrial cytochrome aa3, without changes in ubiquinone and cytochrome b populations. In the same way, the peroxidative stress induced by cyclohexene-1-one causes both a GSH depletion and an imbalance among the concentrations of the mitochondrial electron transfer carriers. Only cytochrome aa3 retains all the partially-reduced oxygen intermediates tightly bound to its active sites. Therefore, it is possible to hypothesize that an electron leakage at the level of the auto-oxidizing chain components (i.e., cytochrome b and ubiquinone populations) increases the release of activated oxygen species (superoxide radical, hydroxyl radical). The treatment with the quoted pharmacological tools suggests that GSH and mitochondrial electron transfer carriers are functionally linked, but not interdependent one another.

Age-related modifications of cytochrome C oxidase activity in discrete brain regions.

The apparent Km for cytochrome c of cytochrome oxidase does not change but the Vmax decreases in synaptosomes and non-synaptic mitochondria isolated from the cerebral cortex as a whole of 30-month-old rats compared with 4-month-old ones. When the subcellular organelles are submitted to stressful conditions, namely incubation in media of altered osmolality, the percentage of cytochrome oxidase activity released is much higher in senescent rats. The activity of cytochrome oxidase evaluated in non-synaptic mitochondria and synaptosomes isolated from cortical and subcortical regions and cerebellum of rats aged 4 and 30 months shows a highly significant decrease (P less than 0.001) in the parietotemporal cortex of senescent rats (both in non-synaptic mitochondria and synaptosomes) and in the cerebellum (in synaptosomes).

Sequential damage in mitochondrial complexes by peroxidative stress

The biochemical characteristics of the electron transfer chain are evaluated in purified non-synaptic (“free”) mitochondria from the forebrain of 60-week-old rats weekly subjected to peroxidative stress (once, twice, or three times) by the electrophilic prooxidant 2-cyclohexene-1-one. The following parameters are evaluated: (a) content of respiratory components, namely ubiquinone, cytochrome b, cytochrome c1, cytochrome c; (b) specific activity of enzymes, namely citrate synthase, succinate dehydrogenase, rotenone-sensitive NADH: cytochrome c reductase, cytochrome oxidase; (c) concentration of reduced glutathione (GSH). Before the first peroxidative stress induction, the rats are administered for 8 weeks by intraperitoneal injection of vehicle, papaverine, δ-yohimbine, almitrine or hopanthenate. The rats are treated also during the week(s) before the second or third peroxidative stress. The cerebral peroxidative stress induces: (a) initially, a decrease in brain GSH concentration concomitant with a decrease in the mitochondrial activity of cytochrome oxidase of aa3-type (complex IV), without changes in ubiquinone and cytochrome b populations; (b) subsequently, an alteration in the transfer molecule cytochrome c and, finally, in rotenone-sensitive NADH-cytochrome c reductase (complex I) and succinate dehydrogenase (complex II). The selective sensitivity of the chain components to peroxidative stress is supported by the effects of the concomitant subchronic treatment with agents acting at different biochemical steps. In fact, almitrine sets limits to its effects at cytochrome c content and aa3-type cytochrome oxidase activity, while δ-yohimbine sets limits to its effects at the level of tricarboxylic acid cycle (citrate synthase) and/or of intermediary between tricarboxylic acid cycle and complex II (succinate dehydrogenase). The effects induced by sequential peroxidative stress and drug treatment are supportive of the hypothesis that leakage of electrons (as a mandatory side-effect of the normal flux of electrons from both NADH and succinate to molecular oxygen) would be due to alteration in both availability of GSH and the content of components in the respiratory chain associated to energy-transducing system. In this field there is a cascade of derangements involving, at the beginning, the complex IV and, subsequently, other chain components, including cytochrome c and, finally, complexes II and I.

Age-related effect induced by oxidative stress on the cerebral glutathione system.

In the forebrain from male Wistar rats aged 5, 15 and 25 months, age-related putative alterations in the glutathione system (reduced and oxidized glutathione; redox index) were chronically induced by the administration in drinking water of free radical generators (hydrogen peroxide, ferrous chloride) or of inhibitors of endogenous free radical defenses (diethyl-dithio-carbamate, an inhibitor of superoxide dismutase activity). In hydrogen peroxide administered rats, both reduced glutathione and the cerebral glutathione redox index markedly declined as a function of aging, whereas oxidized glutathione consistently increased. In contrast, chronic iron intake failed to modify the reduced glutathione in forebrain from the rats of the different ages tested, whereas the oxidized glutathione was increased in the older brains. The chronic intake of diethyl-dithio-carbamate enhanced the concentrations of reduced glutathione in the forebrains from the rats of the different ages tested, the oxidized glutathione being unchanged. In 15-month-old rats submitted to chronic oxidative stress, ergot alkaloids (and particularly dihydroergocriptine) interfered with cerebral glutathione system, while papaverine was always ineffective. The comprehensive analysis of the data indicates that: (a) both the type of oxidative stress and the age of the animals modulate the cerebral responsiveness to the putative modifiers in the level of tissue free radicals; (b) aging magnifies the cerebral alterations induced by oxidative stress; the (c) cerebral glutathione system may be modified by metabolic rather than by circulatory interferences; (d) a balance between the various cerebral antioxidant defenses is present, the perturbation of an antioxidant system resulting in the compensatory modified activity of component(s) of another system.

Changes induced by aging and drug treatment on cerebral enzymatic antioxidant system

The age-related modifications of the participants to the cerebral enzymatic antioxidant system (superoxide dismutase, glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase) were evaluated in four brain regions from male Wistar rats aged 5, 10, 15, 20, 25, 30, and 35 months. Both the specific enzyme activity and the profile of any enzyme tested markedly differ with age according to the region examined: parieto-temporal cortex, caudate-putamen, substantia nigra and thalamus. This inhomogeneous age-related profile of enzyme activities could explain both the controversial data of literature and the different regional vulnerability of the brain tissue to damage with aging. In rats aged 10, 20, or 30 months, the chronic i.p. treatment for two months with papaverine or ergot alkaloids (dihydroergocristine, dihydroergocornine, dehydroergocriptine) suggests that the antioxidant enzyme activities may be influenced according to the agent utilized, the brain region tested, and the age of the animal. In any case, small differences in the drug structure support marked differences in the type and extent of the intervention on the antioxidant enzymatic system.

Relationship between aging, drug treatment and the cerebral enzymatic antioxidant system

Four different brain regions (parieto-temporal cortex, caudate-putamen, substantia nigra, and thalamus) were examined in rats aged 5, 10, 15, 20, 25, 30, and 35 months. The following enzyme activities related to the antioxidant system were measured: glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glutathione peroxidase, glutathione reductase, and superoxide dismutase (as total). Specific enzyme activities vary markedly with age, according to the various regions studied, indicating nonhomogenous vulnerability of different brain regions to aging. In general, both superoxide dismutase and glutathione reductase tended to decline during the last half of life, while glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase tended to increase slightly with age. In rats of 10, 20, or 30 months, chronic treatment for two months with a vasodilator (papaverine) or a calcium-blocker (nicardipine) indicated that the antioxidant enzyme activities are partially influenced according to the exogenous agent used, the brain region tested, and the age of the animals.

Influence of aging and drug treatment on the cerebral glutathione system

Age-related changes of the components of the glutathione system (reduced and oxidized glutathione) were evaluated in forebrains from male Wistar rats aged 5, 10, 15, 20, 25, 30 and 35 months. The trend of both forms of glutathione and the glutathione redox index markedly differs with age. Reduced glutathione increases during the first third of a rats life and decreases thereafter. In contrast, oxidized glutathione remains relatively constant during the first half of the life-span and increases thereafter. Thus, the glutathione redox index steadily declines with age after an increase during the first third of the rats life-span. In rats aged 10, 20 or 30 months, chronic IP treatment for two months with drugs known to modify cerebral circulation (papaverine) or the cerebral metabolism (ergot alkaloids dihydroergocristine, dihydroergocriptine) indicates that, according to the age, the cerebral glutathione system may be modified by metabolic changes rather than by circulatory events.

Arachidonic acid metabolism and pathophysiologic aspects of subarachnoid hemorrhage in rats.

We studied the ex vivo production of prostaglandin D2, prostaglandin E2, 6-ketoprostaglandin F1 alpha, and leukotriene C4 in the brain tissue of rats subjected to experimental subarachnoid hemorrhage. The ex vivo method allows the study of arachidonic acid metabolites released from brain slices at different times after subarachnoid hemorrhage induction and reflects the residual capacity for arachidonic acid metabolism after the pathologic event. The rats were sacrificed 30 minutes, 1 and 6 hours, and 2 days after subarachnoid hemorrhage was induced by the injection of 0.30 ml autologous arterial blood into the cisterna magna. Concentration of prostaglandin D2 and 6-ketoprostaglandin F1 alpha was increased significantly relative to control 2 days after induction. The concentration of prostaglandin E2 was increased significantly 6 hours after induction, while ex vivo production of leukotriene C4 was increased significantly at 1 and 6 hours and 2 days. The correlation between these results and the occurrence of vasospasm after subarachnoid hemorrhage is discussed. The results obtained from the ex vivo incubation of brain tissue slices after experimental subarachnoid hemorrhage suggest that after the hemorrhage there is a significant modification of brain eicosanoid metabolism, which could be of great importance in interpreting the pathogenesis of subarachnoid hemorrhage-related neuronal impairment.