Search

ABSTRACTSurfactants have long been known to have microbicidal action and have been extensively used as antiseptics and disinfectants for a variety of general hygiene and clinical purposes. Among surfactants, quaternary ammonium compounds (QAC) are known to be the most useful antiseptics and disinfectants. However, our previous toxicological studies showed that QAC are also the most toxic surfactants for mammalian cells. An understanding of the mechanisms that underlie QAC toxicity is a crucial first step in their rational use and in the design and development of more effective and safer molecules. We show that QAC-induced toxicity is mediated primarily through mitochondrial dysfunction in mammalian columnar epithelial cell cultures in vitro. Toxic effects begin at sublethal concentrations and are characterized by mitochondrial fragmentation accompanied by decreased cellular energy charge. At very low concentrations, several QAC act on mitochondrial bioenergetics through a common mechanism of action, primarily by inhibiting mitochondrial respiration initiated at complex I and, to a lesser extent, by slowing down coupled ADP phosphorylation. The result is a reduction of cellular energy charge which, when reduced below 50% of its original value, induces apoptosis. The lethal effects are shown to be primarily a result of this process. At higher doses (closer to the critical micellar concentration), QAC induce the complete breakdown of cellular energy charge and necrotic cell death.

Due to the increasing number of data demonstrating a connection between diabetes and Alzheimer's disease (AD), efforts have been developed to elucidate the exact mechanism(s) underlying this connection. Although both disorders possess several overlapping features, mitochondrial dysfunction is one of the most relevant rendering mitochondria an important target of scientific research. This review discusses clinical and biochemical features shared by AD and diabetes, giving special attention to the involvement of mitochondria. The realization that mitochondria are at the intersection of cells' life and death has made them a promising target for drug discovery and therapeutic interventions. Here we also discuss in vitro, in vivo and clinical studies that examined the effect of mitochondria-directed therapeutics particularly mitochondrial target antioxidants and Szeto-Schiller peptides.

Oxidative stress occurs early in the progression of Alzheimer disease, significantly before the development of the pathologic hallmarks, neurofibrillary tangles and senile plaques. All classes of macromolecules (sugar, lipids, proteins, and nucleic acids) are affected by oxidative stress leading, inevitably, to neuronal dysfunction. Extensive data from the literature support the notion that mitochondrial and metal abnormalities are key sources of oxidative stress in Alzheimer disease. Furthermore, it has been suggested that in the initial stages of the development of Alzheimer disease, amyloid-β deposition and hyperphosphorylated tau function as compensatory responses to ensure that neuronal cells do not succumb to oxidative damage. However, during the progression of the disease, the antioxidant activity of both agents is either overwhelmed or, according to others, evolves into pro-oxidant activity resulting in the exacerbation of reactive species production.

Berberine [Natural Yellow 18, 5,6-dihydro-9,10-dimethoxybenzo(g)-1,3-benzodioxolo(5,6-a)quinolizinium] is an alkaloid present in plant extracts and has a history of use in traditional Chinese and Native American medicine. Because of its ability to arrest the cell cycle and cause apoptosis of several malignant cell lines, it has received attention as a potential anticancer therapeutic agent. Previous studies suggest that mitochondria may be an important target of berberine, but relatively little is known about the extent or molecular mechanisms of berberine-mitochondrial interactions. The objective of the present work was to investigate the interaction of berberine with mitochondria, both in situ and in isolated mitochondrial fractions. The data show that berberine is selectively accumulated by mitochondria, which is accompanied by arrest of cell proliferation, mitochondrial fragmentation and depolarization, oxidative stress, and a decrease in ATP levels. Electron microscopy of berberine-treated cells shows a reduction in mitochondria-like structures, accompanied by a decrease in mitochondrial DNA copy number. Isolated mitochondrial fractions treated with berberine had slower mitochondrial respiration, especially when complex I substrates were used, and increased complex I-dependent oxidative stress. It is also demonstrated for the first time that berberine stimulates the mitochondrial permeability transition. Direct effects on ATPase activity were not detected. The present work demonstrates a number of previously unknown alterations of mitochondrial physiology induced by berberine, a potential chemotherapeutic agent, although it also suggests that high doses of berberine should not be used without a proper toxicology assessment.

The complex nature and genesis of oxidative damage in Alzheimer disease can be partly answered by mitochondrial and redox-active metal abnormalities. By releasing high levels of hydrogen peroxide, dysfunctional mitochondria propagate a series of interactions between redox-active metals and oxidative response elements. In the initial phase of disease development, amyloid-andamp;#946; deposition and hyperphosphorylated t may function as compensatory responses and downstream adaptations to ensure that neuronal cells do not succumb to oxidative injuries. However, during the progression of the disease, the antioxidant activity of both agents evolves into pro-oxidant activity representing a typical gain-offunction transformation, which can result from an increase in reactive species and a decrease in clearance mechanisms.

Oxidative damage is a major feature of Alzheimer's disease pathophysiology. Instead of succumbing to these oxidative abnormalities, neurons upregulate antioxidant defenses, which suggest a novel balance in oxidant homeostasis in Alzheimer's disease. Evidence indicates that in the initial phase of Alzheimer's disease development, amyloid-β deposition and hyperphosphorylated τ are consequences of oxidative stress and function as a primary line of antioxidant defense. However, during the progression of the disease, the antioxidant activity of both agents evolves into pro-oxidant, representing a typical gain-of-function transformation. This transformation is due to an increase in reactive species and a decrease in clearance mechanisms. However, the notion that amyloid-β and hyperphosphorylated t function as protective components in the early stages of Alzheimer's disease brings into serious question the rationale of current therapeutic strategies aimed to remove both amyloid-β and hyperphosphorylated τ.

Despite the controversial discussion about amyloid-β as a cause or consequence of Alzheimer disease pathophysiology, one of the most devastating neurodegenerative disorders, all researchers working in this field agree that oxidative stress is intimately associated with Alzheimer disease. This review will focus primarily on oxidative stress associated to disturbances in energy metabolism, with special emphasis on the role of mitochondrial dysfunction and the overproduction of reactive oxygen and nitrogen species. These free radical species attack neuronal lipids, proteins and nucleic acids inevitably leading to neuronal dysfunction. These neuronal alterations can be measured using several markers such as protein carbonyls and 3-nitrotyrosine (protein oxidation markers), malondialdehyde, thiobarbituric acid-reactive substances, 4-hydroxynonenal and acrolein (lipid oxidation markers), 8-hydroxyguanosine and 8-hydroxy-2'-deoxyguanosine (nucleic acid oxidation markers) and advanced glycation end products (glyco-oxidation marker). The prompt identification of early signs of oxidative stress and its potential neuronal targets can open a window for the development of new therapeutic strategies envisaged to prevent or, at least, ameliorate the symptoms of Alzheimer disease. In this line, and since oxidative damage occurs due to an imbalance between reactive species production and cell antioxidant defenses, we will discuss in brief the use of antioxidant-based therapies and their effects in the fight against the oxidative stress occurring in Alzheimer disease.

Oxidative stress is a major feature and one of the earliest changes in Alzheimer’s disease. The complex nature and genesis of oxidative damage in Alzheimer’s disease can be partly answered by mitochondrial and redox-active metal abnormalities. Evidence indicates that in the initial phase of Alzheimer’s disease development, amyloid-β deposition and hyperphosphorylated τ, hallmarks of the disease, are consequences of oxidative stress. Therefore, the authors propose that oxidative modifications in early stages of Alzheimer’s disease may actually serve as a homeostatic response or compensatory mechanism against stressful agents resulting in a shift of neuronal priority from normal function to basic survival. Since the cause of Alzheimer’s disease pathophysiology remains controversial, several and distinct therapeutic strategies have been tried to avoid, or at least reduce, the development of this neurodegenerative disorder. In this line, the inherent theoretical limitations of secretase inhibitors and immunotherapy, aimed to avoid the production or clear amyloid-β from the brain is discussed. Promising antioxidant strategies, with the focus on fighting oxidative stress, are highlighted. Currently, antioxidant strategies appear to be the most encouraging therapeutics in reducing the clinical manifestation and evolution of Alzheimer’s disease.

In this work the effect of the neurotoxic amino acid sequence, Aβ25–35, on brain mitochondrial permeability transition pore (PTP) was studied. For the purpose, the mitochondrial transmembrane potential (ΔΨm), mitochondrial respiration and the calcium fluxes were examined. It was observed that Aβ25–35, in the presence of Ca2+, decreased the ΔΨm, the capacity of brain mitochondria to accumulate calcium and led to a complete uncoupling of the respiration. However, the reverse sequence of the peptide Aβ25–35 (Aβ35–25) did not promote the PTP. The alterations promoted by Aβ35–25 and/or Ca2+ could be reversed when Ca2+ was removed by EGTA or when ADP plus oligomycin were present. The pre-treatment with CsA or ADP plus oligomycin prevented the ΔΨm drop and preserved the capacity of mitochondria to accumulate Ca2+. These results suggest that Aβ25–35 can promote the PTP induced by Ca2+.

Increased oxidative stress and changes in antioxidant capacity observed in both clinical and experimental diabetes mellitus have been implicated in the etiology of chronic diabetic complications. Many authors have shown that hyperglycemia leads to an increase in lipid peroxidation in diabetic patients and animals reflecting a rise in reactive oxygen species production. The aim of the study was to compare the susceptibility of mitochondria from brain and liver of Goto-Kakizaki (12-month-old diabetic) rats (GK rats), a model of non-insulin dependent diabetes mellitus, to oxidative stress and antioxidant defenses. Brain and liver mitochondrial preparations were obtained by differential centrifugation. Oxidative damage injury was induced in vitro by the oxidant pair ADP/Fe²⁺ and the extent of membrane oxidation was assessed by oxygen consumption, malondialdehyde (MDA) and thiobarbituric acid reactive substances (TBARS) formation. Coenzyme Q and α-tocopherol contents were measured by high-performance liquid chromatography (HPLC). Brain mitochondria isolated from 12-month-old control rats displayed a higher susceptibility to lipid peroxidation, as assessed by oxygen consumption and formation of MDA and TBARS, compared to liver mitochondria. In GK rats, mitochondria isolated from brain were more susceptible to invitro oxidative damage than brain mitochondria from normal rats. In contrast, liver mitochondria from diabetic rats were less susceptible to oxidative damage than mitochondria from normal rats. This decreased susceptibility was inversely related to their α-tocopherol and coenzyme Q (CoQ) content. The present results indicate that the diabetic state can result in an elevation of both α-tocopherol and CoQ content in liver, which may be involved in the elimination of mitochondrially generated reactive oxygen species. The difference in the antioxidant defense mechanisms in the brain and liver mitochondrial preparations of moderately hyperglycemic diabetic GK rats may correspond to a different adaptive response of the cells to the increased oxidative damage in diabetes. Copyright © 2001 John Wiley & Sons, Ltd.

In the present study, we analyzed how aluminium and oxidative stress induced by ascorbate/Fe2+affect the mechanisms related with the cholinergic system in a crude synaptosomal fraction isolated from rat brain. [3H]Choline uptake, [3H]acetylcholine release, membrane potential and Na+/K+-ATPase activity were determined in the presence or in the absence of aluminium in control conditions and in the presence of ascorbate (0.8 mM)/Fe2+(2.5 μM). The extent of lipid peroxidation was measured by quantifying thiobarbituric acid reactive substances (TBARS). Under oxidizing conditions aluminium increased the formation of TBARS by about 30 %, but was without effect when the synaptosomal preparation was incubated in the absence of oxidants. Additionally, aluminium potentiated the inhibition of the high-affinity [3H]choline uptake observed following lipid peroxidation and had the same effect on the Na+/K+-ATPase activity. [3H]Acetylcholine release induced by 4-aminopyridine, and membrane potential were not significantly affected under oxidizing conditions, either in the absence or in the presence of aluminium. We can conclude that aluminium, by potentiating lipid peroxidation, affects the uptake of choline in nerve endings. This effect, occurring during brain oxidative injury, might contribute to the cholinergic dysfunction and neuronal cell degeneration known to occur in Alzheimer’s disease.

ABSTRACTInsoluble aggregates of the amyloid -peptide (A) are a major constituent of senile plaques found in brains of Alzheimer's disease patients. The -amyloid fragment A140is toxic to rat pheochromocytoma PC12 cells, leading to a concentration-dependent decrease in the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The detrimental effects of A140are enhanced in the presence of 1 mM zinc, whereas 50 Mzinc exerts a protective effect against A140-induced toxicity. Exposure of PC12 cells to low zinc concentrations (50 M)affords a decrease (1.4-fold) in the extent of lipid peroxidation, a decrement in protein oxidation (1.1-fold), and an increase in ATP levels (1.2-fold), although the differences were not statistically significant. However, treatment of cells with high concentrations of zinc (1 mM) led to significant increases in lipid peroxidation (3.7-fold) and protein oxidation (1.5-fold) and to depletion of the ATP pool (21-fold). These data suggest that zinc has a concentration-dependent dual effect, protective and toxic, thus playing an important role in the pathogenesis of Alzheimer's disease.

The herbicide dinitro-o-cresol (DNOC) was evaluated for its effects on bioenergetic activities of potato tuber mitochondria to elucidate its mechanism of action and to compare its toxicological properties with those of the chemically related uncoupler dinitrophenol (DNP). DNOC acts as a typical uncoupler, similarly to the classical uncouplers DNP and carbonylcyanide p-trifluoromethoxyphenyl-hydrazone (FCCP). Low concentrations of DNOC (<100 μM) maximally stimulate succinate-supported respiration of plant mitochondria, with simultaneous collapse of trans-membrane electrical potential, more efficiently than DNP. The herbicide makes the plant mitochondrial membrane more permeable to protons, acting as a protonophore even in non-energized mitochondria. High concentrations of DNOC (>100 μM) act also more efficiently than DNP simultaneously as a protonophore and inhibitor of respiration, especially when respiration is supported by substrates that are transported to the matrix. The efficiency of DNOC is decreased with increase of mitochondrial protein, BSA and exogenous orthophosphate. Although similar effects were observed for animal and plant mitochondria, rat-liver mitochondrial respiration was more sensitive to DNOC than plant mitochondria. Furthermore, in the presence of DNOC, liver mitochondria exhibited a higher state 3 respiratory coupling level than potato tuber mitochondria, as a result of a considerable stimulation (60%) of state 3 respiration. In conclusion, DNOC is a more potent mitochondrial uncoupler and respiratory chain inhibitor than DNP, although their chemical structures are very similar. Apparently, the additional methyl group of DNOC increases its efficiency as an uncoupler and as an inhibitor, as compared to DNP. Plant mitochondria were shown to be as useful as animal mitochondria in evaluating the toxicity of these xenobiotics. © 1998 Society of Chemical Industry.

Extensive literature exists supporting a role for mitochondrial dysfunction and oxidative damage in the pathogenesis of Alzheimer disease. Mitochondria are a major source of intracellular reactive oxygen species and are particularly vulnerable to oxidative stress. It has been recently shown that the immunoreactivity of lipoic acid and cytochrome oxidase-1, two mitochondrial markers, is increased in the cytoplasm of pyramidal neurons in Alzheimer disease cases compared with controls. Furthermore, lipoic acid was found to be strongly associated with granular structures and, by ultrastructure analysis, it was shown to be localized in mitochondria, cytosol and, importantly, in organelles identified as autophagic vacuoles. Lipoic acid was also found associated with the electron dense core of lipofuscin in the brains of Alzheimer disease cases but not in controls, whereas cytochrome oxidase-1 immunoreactivity was limited to mitochondria and cytosol in both Alzheimer and control cases. These data suggest that mitochondria are key targets of increased autophagic degradation in Alzheimer disease. The study of autophagy in Alzheimer disease could clarify the mechanisms underlying this neurodegenerative disorder and, eventually, help in the development of new therapeutic strategies.Addendum to:Autophagocytosis of Mitochondria is Prominent in Alzheimer DiseaseMoreira PI, Siedlak SL, Wang X, Santos MS, Oliveira CR, Tabaton M, Nunomura A, Szweda LI, Aliev G, Smith MA, Zhu X and Perry GJ Neuropathol Exp Neurol 2007; 66:525-32

Mitochondrial abnormalities are prominent in Alzheimer disease. In this study, 2 mitochondrial markers, cytochrome oxidase-1 and lipoic acid, a sulfur-containing cofactor required for the activity of several mitochondrial enzyme complexes, were compared using light and electron microscopic analyses and immunoblot assays. Both lipoic acid and cytochrome oxidase-1 immunoreactivity are increased in the cytoplasm of pyramidal neurons in Alzheimer disease compared with control cases. Of significance, lipoic acid was found to be strongly associated with granular structures, and ultrastructure analysis showed localization to mitochondria, cytosol, and, importantly, in organelles identified as autophagic vacuoles and lipofuscin in Alzheimer disease but not control cases. Cytochrome oxidase-1 immunoreactivity was limited to mitochondria and cytosol in both Alzheimer and control cases. These data suggest that mitochondria are key targets of increased autophagic degradation in Alzheimer disease. Whether increased autophagocytosis is a consequence of an increased turnover of mitochondria or whether the mitochondria in Alzheimer disease are more susceptible to autophagy remains to be resolved.