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Mitochondrial Medicine – Molecular Pathology of Defective Oxidative Phosphorylation

Address correspondence to Egil Fosslien, M.D., Department of Pathology (M/C 847), College of Medicine, University of Illinois at Chicago, 1819 West Polk Street, Chicago IL 60612; tel 312 996 7323; fax 312 996 7586, e-mail efosslie{at}uic.edu

	Abstract

Top Abstract Introduction Classification of Defects Mitochondrial Dysfunction in... Molecular Pathology Mitochondrial Genetics Therapy Conclusions References

 
Different tissues display distinct sensitivities to defective mitochondrial oxidative phosphorylation (OXPHOS). Tissues highly dependent on oxygen such as the cardiac muscle, skeletal and smooth muscle, the central and peripheral nervous system, the kidney, and the insulin-producing pancreatic ß-cell are especially susceptible to defective OXPHOS. There is evidence that defective OXPHOS plays an important role in atherogenesis, in the pathogenesis of Alzheimer’s disease, Parkinson’s disease, diabetes, and aging. Defective OXPHOS may be caused by abnormal mitochondrial biosynthesis due to inherited or acquired mutations in the nuclear (n) or mitochondrial (mt) deoxyribonucleic acid (DNA). For instance, the presence of a mutation of the mtDNA in the pancreatic ß-cell impairs adenosine triphosphate (ATP) generation and insulin synthesis. The nuclear genome controls mitochondrial biosynthesis, but mtDNA has a much higher mutation rate than nDNA because it lacks histones and is exposed to the radical oxygen species (ROS) generated by the electron transport chain, and the mtDNA repair system is limited. Defective OXPHOS may be caused by insufficient fuel supply, by defective electron transport chain enzymes (Complexes I - IV), lack of the electron carrier coenzyme Q10, lack of oxygen due to ischemia or anemia, or excessive membrane leakage, resulting in insufficient mitochondrial inner membrane potential for ATP synthesis by the F₀F₁-ATPase. Human tissues can counteract OXPHOS defects by stimulating mitochondrial biosynthesis; however, above a certain threshold the lack of ATP causes cell death. Many agents affect OXPHOS. Several nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit or uncouple OXPHOS and induce the [topical] phase of gastrointestinal ulcer formation. Uncoupled mitochondria reduce cell viability. The Helicobacter pylori induces uncoupling. The uncoupling that opens the membrane pores can activate apoptosis. Cholic acid in experimental atherogenic diets inhibits Complex IV, cocaine inhibits Complex I, the poliovirus inhibits Complex II, ceramide inhibits Complex III, azide, cyanide, chloroform, and methamphetamine inhibit Complex IV. Ethanol abuse and antiviral nucleoside analogue therapy inhibit mtDNA replication. By contrast, melatonin stimulates Complexes I and IV and Gingko biloba stimulates Complexes I and III. Oral Q10 supplementation is effective in treating cardiomyopathies and in restoring plasma levels reduced by the statin type of cholesterol-lowering drugs.

(received 15 September 2000; accepted 30 October 2000)

Keywords: mitochondria, oxidative phosphorylation, apoptosis, atherosclerosis, diabetes mellitus, gastrointestinal disease, Alzheimer’s disease, Parkinson’s disease, Helicobacter pylori, ethanol, statin, melatonin, cancer

	Introduction

Top Abstract Introduction Classification of Defects Mitochondrial Dysfunction in... Molecular Pathology Mitochondrial Genetics Therapy Conclusions References

 
Historical perspective,  In 1956 Harman introduced the free radical theory of aging suggesting that over time, free radicals cause damage to macromolecules (DNA, lipids, proteins) [1]. Mitochondrial medicine was initiated six years later with the report on Luft’s syndrome, a mitochondrial functional disorder involving loose coupling and a greatly elevated basic metabolic rate despite normal thyroid metabolism [2].

In 1963, threadlike structures that could be digested by DNAase were discovered in mitochondria of chick embryos [3,4]. The following year, mtDNA was isolated from purified yeast mitochondria. Phosphorylating submitochondrial particles were first isolated from yeast in 1966 [5].

In 1970, the first respiratory chain deficiency without elevated basal oxygen consumption that was due to a reduced activity of a specific enzyme, cytochrome b, was identified. The symptoms included dementia, cerebellar ataxia, and proximal muscle weakness [6]. Next came reports on a defect of pyruvate dehydrogenase [7] and two years later the identification of reduced cytochrome oxidase c in Menkes’ syndrome [8]. Another year later, myopathies due to a defect in muscle carnitine [9] and defects in carnitine palmitoyl transferase were reported [10].

In 1981, the first complete (Cambridge) sequence of mitochondrial DNA (mtDNA) was published [11]. In 1984, Miquel and Fleming emphasized that the mitochondrion is the major site of oxidative damage and that oxidative damage to mitochondria plays an important role in aging [12]. The first neurodegenerative disease associated with a mutation of mtDNA was described in 1988 [13].

The following year Linnane et al expanded on the mitochondrial theory of aging: somatic accumulation of mitochondrial genome mutations during life is a major cause of human aging and degenerative disease [14]. Their theory was based upon the following observations: (a) high frequency of gene mutation in mitochondrial DNA, (b) the small size of the mitochondrial genome, (c) lack of effective repair mechanism for mtDNA, and (d) somatic segregation of individual mtDNA during eukaryotic cell division.

Mitochondrial DNA damage may be a biomarker of oxygen damage over time. Mutations in mtDNA, whether inborn or acquired, accelerate mitochondrial dysfunction that induces further damage, and additional mutations and deletions [15].

Presently, it is realized that a gradual decline in ATP generation with age induces loss of stamina, memory, vision, and hearing and contributes to aging-related diseases such as cardiovascular disease and diabetes [16]. In addition, many exogenous agents, even therapeutic ones such as acidic nonsteroidal anti-inflammatory drugs (NSAIDs), can inhibit the mitochondrial energy system [17] and cause disease at any age.

Oxidative phosphorylation.  An efficient mitochondrial oxidative phosphorylation (OXPHOS) system is essential since it provides most of the adenosine triphosphate (ATP), the chemical energy required for a cell’s metabolism [18]. Therefore, each step of the mitochondrial energy conversion system must function efficiently for cell survival. Auspiciously, it is a redundant system, since each cell carries up to a few thousand mitochondria. Furthermore, when a cell with a few defective mitochondria divides, one daughter cell might receive the defective mitochondria and perish, while the other daughter cell is restored to normal. However, in post-mitotic cells this option is not available. It has been estimated that over 90% of mitochondria are contained in postmitotic cells [19].

Because of differences in the threshold values of different tissues, only some tissues may be affected and exhibit pathology as a result of an inherited mitochondrial DNA mutation, even if that mutation is present in all tissues [20]. Similarly, exogenous substances that affect OXPHOS may affect different tissues differently.

	Classification of Defects

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Structural defects.  1. Inherited.  Primary mitochondrial diseases are caused by inherited defects in the mitochondrial structure, which lead to defective function of the mitochondria. They involve oxidative phosphorylation or other mitochondrial pathways like the urea cycle and fatty acid oxidation [21]. They cause a wide range of diverse clinical manifestations; such as ataxia, cardiomyopathy, dementia, epilepsy, myopathy, polyneuropathy, and retinal pigment anomaly [22,23].

2. Acquired.   (a) Ischemia and deletion. In ischemic heart disease, a 40-45-fold increase in the common (4,799 bp) mtDNA deletion in the myocardium has been reported. Transcripts of mtDNA-coded oxidative phosphorylation enzyme subunits were increased, pointing to an association between ischemia and OXPHOS deficiency, enhanced ROS generation and mtDNA deletions [24]. Furthermore, animal models point to ischemia as the primary event. For instance, myocardial ischemia produced by ameroid constriction of canine coronary arteries resulted in the development of myocardial mtDNA deletions similar to deletions observed in human ischemic hearts [25].

 (b) Organ preservation. Prevention of serious impairment of oxidative phosphorylation from ischemia reperfusion injury is critical to transplantation and open heart surgery. It has been suggested that mitochondrial oxidative phosphorylation observed in the isolated rat heart after 8–12 hr of hypothermic ischemia [26].

Functional defects.  This intricate energy conversion system is susceptible to malfunction by many causes occurring either alone or in various combinations. A few causes are illustrated in Figure 1. Many NSAIDs that inhibit oxidative phosphorylation inhibit the enzyme activities rather than their synthesis.

View larger version (20K): [in this window] [in a new window]   Fig. 1. Illustration shows defective mitochondrial oxidative phosphorylation (OXPHOS). Numbers in brackets refer to numbers in square boxes. Defective OXPHOS impairs the electron flow from metabolites (fuel) [1] through the electron transport chain [2] (enzyme Complexes I - IV) to oxygen. The generation of the inner mitochondrial membrane potential (m) [3] is lowered and phosphorylation [4] reduced, leading to insufficient generation of adenosine triphosphate (ATP) by F0F1-ATPase (OXPHOS complex V) for proper cellular function. Inhibition of any of the enzyme complexes of oxidative phosphorylation (for example by an acid NSAID) lowers generation. Ischemia caused by atherosclerosis or anemia reduces respiration. Inhibition of phosphorylation or loss of potential without ATP generation by passive or induced membrane proton leaks causes uncoupling of oxidative phosphorylation. During the mitochondrial permeability transition (MPT) mitochondrial pores open, causing membrane depolarization, loss of membrane potential, cessation of oxidative phosphorylation, and initiation of apoptosis. The text in italics indicates exogenous sources of mitochondrial dysfunction.

2. Dysfunction of the electron transport chain.  Electrons from the metabolites must be efficiently carried via coenzyme-Q10 (Q10) to Complex III, and then via cytochrome c and Complex IV to oxygen. An ample supply of oxygen as electron acceptor must be provided. Uninhibited, effective proton pumping by Complexes I, III, and IV must deposit the released energy as an electrical charge across the mitochondrial inner membrane. Salicylate and indomethacin can inhibit the electron transport chain. The electron transport may be reduced or blocked by an inadequate supply of oxygen. Blood flow reduction due to atherosclerosis, reduction in oxygen carrying capacity due to anemia or smoking, or a reduction in the oxygen partial pressure at high altitude or reduced airplane cabin pressure can all impair the function of the electron transport chain. Furthermore, defective ATP generation may be due to lack of substrates, essential cofactors, or may be caused by inefficient use of oxygen [29]. A virus [30,31] may induce OXPHOS dysfunction. Conditions of oxidative stress, chronic alcohol abuse, a large number of drugs, or toxin can also lead to a decline of mitochondrial function [21].

3. Abnormal coupling and uncoupling.  The inner mitochondrial membrane potential serves as a convertible energy currency of the cell [32] (Figure 1). Therefore, the majority of the stored energy must be efficiently coupled to Complex V to phosphorylate adenosine diphosphate (ADP) to adenosine triphosphate (ATP). Uncoupling in the form of inhibition of phosphorylation must be limited. Uncoupling in the form of losses due to passive membrane permeability, altered membrane lipids, or uncoupling proteins must be controlled. The amount of heat generated when some hydrogen ions passively leak back across the membrane must be restricted, and the heat released by active channeling of protons back through the membrane by uncoupling proteins (UCP) must be efficiently regulated. Induction of the membrane permeability transition (MPT) by NSAIDs (eg, diclofenac sodium,mefenamic acid [33]), or by toxic endogenous or exogenous agents, must be limited.

As an example of membrane leakage, in Luft’s disease the loose coupling causes increased mitochon-drial respiration but reduced phosphorylation with low ATP generation [34]. The excess metabolic turnover produces heat instead of ATP, resulting in a steady elevation of body temperature. Muscle fibers show evidence of attempts to compensate for loose coupling in the form of aggregates of large mitochondria full of cristae [35], the primary location of OXPHOS enzymes. Compensatory increase of mitochondrial synthesis may occur in the presence of other causes of defective oxidative phosphorylation [36,37].

Damage and repair.  1. Reactive oxygen metabolites.  The reactive oxygen metabolites (ROM) generated during mitochondrial respiration can damage membranes, DNA, and mitochondrial oxidative phosphorylation enzymes, causing a vicious cycle of declining mitochondrial function [38]. For instance, in the cultured endothelial cell and the vascular smooth muscle cell reactive species preferentially damage mtDNA compared with the transcriptionally inactive nuclear ß-globin gene. The mitochondrial mRNA transcripts, protein synthesis, and ATP synthesis decreased. Smooth muscle cells suffered less impairment than the endothelial cells [39].

Diseases of oxidative phosphorylation often occur together with impaired ß-oxidation. Reduced flux through the respiratory chain increases the NADH/NAD⁺ ratio. Beta-oxidation is inhibited and produces secondary carnitine deficiency. Generation of reactive oxygen species increases and -tocopherol is depleted probably because of consumption due to lipid peroxidation [40].

2. Antioxidants.  Experimentally, the oxidative stress can be enhanced by gene inactivation of the antioxidant enzyme glutathione peroxidase-1 (Gpx1) in the mutant mouse. It is normally highly expressed in the mouse liver. The presence of the mutated Gpx1-gene causes a significant increase in hydrogen peroxide release by the liver mitochondria. Moreover, it markedly degrades the efficiency of mitochondrial oxidative phosphorylation as evidenced by a significant reduction in the respiratory control ratio [41].

	Mitochondrial Dysfunction in Major Diseases

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Cardiovascular diseases.  1. Early suggestions of OXPHOS involvement in atherogenesis.  Atherosclerosis is the major cause of ischemic heart disease. From the 1960s, Whereat emphasized that abnormalities of mitochondrial oxidative phosphorylation might play an essential role in atherogenesis [42,43]. He suggested that anoxia or carbon monoxide or "other mitochondrial electron transport chain inhibitors that contaminate our hazardous environment" could initiate atherosclerosis [44].

Moreover, hypoxia and mitochondrial dysfunction might help explain the cause of lipid accumulation in the atheromatous plaque. For instance, local arterial hypoxia might increase endothelial permeability. This would in part explain the lipid insudation theory that increased permeability promotes mural insudation of plasma lipid. Experiments show that reduced oxygen tension or the presence of carbon monoxide accelerates the development of atherosclerosis in the rabbit fed a high fat diet. In humans, the increase in blood carbon monoxide in tobacco smokers might be the link between tobacco smoke and atherosclerosis [44].

As an alternative to this insudation hypothesis it has been postulated that hypoxia enhances mural lipid accumulation in atherosclerosis through fatty acid synthesis by mitochondria in the aorta. Whereas normally fatty acid synthesis in the aorta is minimal, a block in the respiratory chain, for instance by hypoxia, impairs the oxidation of NADH and causes NADH accumulation that initiates mitochondrial fatty acid synthesis [44,45].

Involvement of defective oxidative phosphorylation in atherogenesis is supported by experiments showing lack of mitochondrial ATPase and succinate dehydrogenase activity in cultured aortic vacuolated smooth muscle cells from the atherosclerosis-susceptible pigeon [46]. Furthermore, there is evidence that reduced intimal mitochondrial energy generation at lesion sites contributes to lesion formation in susceptible compared with the resistant pigeon [45].

2. Inflammation and mitochondrial dysfunction.   (a) Cyclooxygenase induction. Recent experimental evidence, particularly using gene knockout animal models, not only corroborates the idea that dysfunction of oxidative phosphorylation plays an important part in atherogenesis but also points to a close association between inflammatory components in the atherosclerotic lesion and mitochondrial dysfunction. In an inflammatory environment oxidized lipids are generated by the activity of lipoxygenases and cyclooxygenases. Mitochondria release radical oxygen species that deplete the cellular content of reduced glutathione. Oxidized fatty acids and oxidized forms of cholesterol accumulate. These alterations are associated with apoptosis and necrosis as seen in the necrotic core of the advanced atherosclerotic lesion [47].

The expression of cyclooxygenase-2 (COX-2) and other pro-inflammatory mediators has been demonstrated in macrophages, smooth muscle cells, and endothelial cells of small vessels in the atherosclerotic plaque [48]. Others described finding an immuno-reactive 70-kDa COX-1 protein and a smaller, 50-kDa COX-2 protein. The COX-2 isoform was located together with macrophages primarily at the perimeter of the lipid core and the shoulder area of the atheroma and secondarily in the microvascular endothelium of the lesion. By comparison, the normal artery only expresses the constitutive COX-1 isoform [49].

 (b) Prostaglandin synthesis. COX-2 converts arachidonic acid to prostaglandin-E2 (PGE2), which induces interleukin-6 (IL-6) [50]. PGE2 inhibits cholesterol esterification in vitro, and might do the same in the arterial wall [51]. PGE2 extracted from lesions of rabbits rendered atherosclerotic by supplementing their diet with 1% cholesterol inhibited cholesterol esterification in vitro [51]. The levels of PGE2 and IL-6 in atheromas vary greatly but are significantly different from normal mural levels. For instance, atherosclerotic aneurysms of the abdominal aorta of 13 patients contained on average 29 times more PGE2 and almost 8 times more IL-6, compared to 16 normal abdominal aortas [52].

IL-6 induces haptoglobin (Hp) and both contribute to inflammation and angiogenesis [50]. Haptoglobin binds hemoglobin [53] and serves as an important antioxidant. Its angiogenic activity was first demonstrated in 1993 [54]. Both the in vitro Matrigel model of angiogenesis and in vivo models showed that purified haptoglobin stimulated angiogenesis [54]. It was suggested that haptoglobin might be important for angiogenesis as part of tissue repair in chronic inflammatory conditions [54]. A marked increase in the haptoglobin was detected in protein extracts from human aortic fibro-fatty lesions but not in aortic intima from patients without atherosclerosis [55]. Haptoglobin enhances cholesterol crystallization in the bile [56], but a similar mechanism has not yet been demonstrated for the cholesterol crystal formation in the atherosclerotic lesion.

 (c) The need for cholic acid in atherogenic diets. High fat diets that are commonly used to induce atherosclerotic lesions in animal models of atherogenesis contain cholic acid [57]. It induces nitric oxide (NO) synthesis in endothelial cells and reduces apoA-I [58]. Dihydrocholic acid induces COX-2 [59]. Whereas high-fat, high-cholesterol diets without cholic acid resemble human diets more closely and elevate LDL and HDL, they do not induce atherosclerosis in animals that are not genetically prone to develop atherosclerosis. Addition of cholic acid elevates LDL but lowers HDL and overcomes the resistance to the development of atherosclerosis. Findings obtained in the mouse model of diet-induced atherosclerosis partly explain the reduction in the plasma levels of HDL. The addition of cholic acid to the diet induces the expression of apoA-I regulatory protein-1 and decreases the expression of apoA-I [60].

 (d) Inhibition of electron transport by interferon-gamma. CD4⁺ and CD8⁺ T-cells in the atherosclerotic lesion of the apoE null mouse secrete interferon-gamma (INF-). Targeted disruption of the IFN- receptor in such mice reduces lesion size, cellularity, and lipid content and indicates that IFN-contributes to lesion formation [61]. In vitro discoveries corroborate these observations. IFN- induced the inducible nitric oxide (NO) synthase (iNOS) of the endotoxin-activated J-774 macrophage cell. The NO inhibited the mitochondrial respiration of coincubated L-929 fibroblasts [62]. It competed with oxygen at Complex IV of cultured L-929 fibroblasts and reversibly inhibited Complex IV. Others have shown that Trolox, a lipid-soluble vitamin E analogue, but not ascorbate, can protect against NO-induced damage of cytochrome oxidase, suggesting that the damage is mediated through lipid peroxidation [63] (Figure 2). Gene knockout and in vitro studies indicate that the nitric oxide secreting, activated macrophage in the atherosclerotic lesion might very likely inhibit respiration, not only of its own mitochondria, but also of mitochondria in other lesion cells, such as the smooth muscle cell [62].

View larger version (18K): [in this window] [in a new window]   Fig. 2. Links between inflammation and inhibition of electron transport and proton-pumping Complexes I and IV in atherogenesis. The illustration is based upon in vitro findings that interferon-gamma (INF-) released by CD4 and CD8 cells induces the inducible nitric oxide synthase (iNOS) in the activated macrophage (Mø]. Nitric oxide inhibits Complex IV. Secondly, INF- inhibits expression of the nuclear gene NDUFV1 that codes for a subunit of Complex I. MIM: mitochondrial inner membrane; SMC: smooth muscle cell.

3. Infectious agents.  Chlamydia pneumoniae infection can damage mitochondria; they become swollen and their cristae become fragmented [65]. Chlamidia, other bacteria and several types of viruses have repeatedly been detected in the human atherosclerotic lesion, suggesting that some infectious agents initiate atherosclerosis [66]. Moreover, infection with cytomegalovirus (CMV) is significantly associated with coronary heart disease, particularly in patients with diabetes [67]. And in the New Zealand White rabbit fed a 2% atherogenic diet the intravenous inoculation with the Herpesvirus type-4 (BHV-4) accelerated the development of atherosclerotic lesions [68]. However, experiments with germ-free apoE(-/-) mice showed that a bacterial or viral infection is not a sine qua non for the development of atherosclerosis [69].

4. Observations in gene knockout and transgenic mice.   (a) The apoE knockout mouse. The apoE(-/-) mouse develops massive atherosclerosis on a normal diet. Surprisingly, the presence of just a small amount of systemic apoE, less than 2% of the wild type, prevents lesion development. When transgenic apoE-knockout mice that had been engineered to express apoE strictly in the adrenal gland were fed an atherogenic diet, it was found that such low levels of systemic apoE blocked atherosclerotic lesion development in the aorta even in the presence of hypercholesterolemia [70]. Four weeks after bone marrow transplantation to generate macrophages expressing various forms of apoE in the murine apoE null mouse, the macrophages expressing murine apoE significantly reduced the size of atherosclerotic lesions and lowered the serum cholesterol level. However, the macrophages expressing human apoE3-Leiden or apoE2 did not curtail lesion size [71].

Infection with the murine gamma-herpesvirus-68 (MHV-68) accelerated atheroma formation in infected apoE(-/-) mice compared with control mice. [72]. By contrast, addition of oral Q10 to a high-fat diet given to uninfected apoE null mice significantly reduced the size of atherosclerotic lesions in the aorta [73].

 (b) The LDL-receptor knockout mouse. The LDL-receptor knockout mouse, LDLR(-/-), develops atherosclerotic lesions of the thoracic and abdominal aorta after 12 weeks on a diet not supplemented with cholic acid. Two groups of mice were fed either a high-fat diet or a high-fat diet supplemented with sodium cholate. Similar lesions developed in both groups demonstrating that the receptor defect overcomes the resistance to develop atherosclerosis on a high-fat diet not supplemented with cholic acid [74].

Overexpression of LPL can protect against atherosclerosis in the LDL-receptor (LDLR) deficient mouse. At the end of an 8-week atherogenic diet the LDLR(-/-) mouse with transgenic overexpression of human LPL had 18-fold smaller lesion areas, compared with the LDLR(-/-) mouse not possessing the LPL transgene. It was suggested that the LPL-induced reduction in plasma levels of remnant lipoproteins caused the reduction in lesion area in the mice that overexpressed LPL [75].

In ovariectomized LDLR(-/-) mice, aortic lesion size was reduced by physiologic amounts of exogenous 17ß-estradiol. The effect was independent of alterations of the cholesterol concentration in the plasma [75].

 (c) Lipoprotein lipase overexpression. Observations in the muscle-specific LPL transgenic mouse suggest that the lipoprotein lipase activity might be a rate-limiting step in the triglyceride-derived supply of free fatty acids to mitochondria and that LPL expression participates in the regulation of the biogenesis of mitochondria. The overexpression of LPL in cardiac and skeletal muscle increased the uptake of free fatty acids and induced muscle fiber degeneration, glycogen storage, and marked mitochondrial proliferation [77].

5. Defective fuel supply.   (a) Defect in the cellular uptake of fuel. As noted above, the apoE knockout mouse develops severe atherosclerosis. LDL from aortic atherosclerotic lesions (A-LDL) is chemically modified and is not taken up as much as normal LDL by rabbit aortic smooth muscle cells (SMC) in vitro. Both lipoproteins down-regulate the cell surface LDL receptor and both failed to induce cellular cholesterolester accumulation in the cell [78]. However, incubation of human arterial SMC with human chylomicron remnants increases cell cholesterol and suppresses LDL-receptor activity [79].

The LDL-receptor down-regulation can also be bypassed in vitro by covalent linkage of N,N-dimethyl-1,3-propanediamine (DMPA) to LDL, leading to vast uptake and extensive accumulation of intracellular lipid inclusions containing liquid crystals of cholesteryl esters. These in vitro features resemble the alterations that are found in smooth muscle cells in vivo during atherogenesis [80]. HIPDM (N-N-N’-trimethyl-N’-(2-hydroxy-3-methyl-5-iodobenzyl)-1,3 propanediamine) is structurally similar to DMPA. It accumulated preferentially in mitochondria when given intravenously to rabbits, and its distribution was similar to succinate cytochrome c reductase. It remained in mitochondria for at least five hours after injection [81].

 (b) Intracellular defect in fuel transport and delivery to mitochondria. A study of human atherosclerotic plaques material removed percutaneously showed many smooth muscle cells, mainly of the intermediate phenotype, having abundant perinuclear fat and glycogen deposits and many mitochondria [36]. The increase in the number of mitochondria in the atherosclerotic lesion indicates enhanced synthesis in an attempt to compensate for dysfunction of the mitochondrial bioenergetics system. The perinuclear lipid accumulation is similar to that in Reye’s syndrome. There is evidence suggesting that the mitochondrial permeability transition (MPT) is involved in mitochondrial injury of the liver in this rare disorder. It is strongly associated with viral infection and concomitant aspirin ingestion and appears primarily [82] but not always [83] in childhood.

6. Defective oxidative phosphorylation in atherogenesis.   (a) Inherited structural defects. Direct evidence of a deficiency of respiratory enzymes in atherogenesis was provided by two cases of metabolic disease with premature atherosclerosis considered of mitochondrial origin. The premature atherosclerosis was diagnosed in two brothers whose parents and two sisters were free of symptoms. The brothers, who died prematurely in their third decade of life, also suffered from diabetes mellitus, sensorineural deafness, photomyoclonic epilepsy, and progressive renal failure. The mitochondrial abnormalities consisted of partial deficiencies of Complexes III and IV. The enzyme defect could be ascertained in the kidney and in fibro-blasts, but not in muscle. Whether the mutation was located in the mtDNA or the nDNA coding for mitochondrial enzyme subunits was not resolved [84].

 (b) Acquired structural defects. Somatic mtDNA damage accumulates with age, degrades oxidative phosphorylation and accelerates aging. In the normal human heart the 'common' 4,977 nucleotide pair (nt) deletion (mtDNA4,977) and the mtDNA7,436 and mtDNA10,422 deletions accumulate with age above 40. In atherosclerosis of the coronary arteries this somatic damage is significantly accelerated. In human hearts with coronary artery disease (CHD), the 'common' deletion is up to over 200 times more frequent than in age-matched control hearts free of CHD [85].

The 'common' deletion abolishes the mitochondrial synthesis of subunits for Complexes I, IV, and V. Significantly higher levels of this deletion were detected by the polymerase chain reaction (PCR) in subjects aged 73–95 compared with subjects aged 60–72 years old in mtDNA extracted from smooth muscle cells of aortic atherosclerotic lesions from 18 surgical and 9 autopsy cases. These findings support the theory that defective oxidative phosphorylation contributes to the degenerative cell phenotype and atherosclerosis during aging [86].

 (c) Reperfusion injury. Cardiac mitochondria isolated after reperfusion are structurally abnormal, carry large amounts of ionized calcium, and generate a disproportionate quantity of oxygen free radicals that cause deletions of mtDNA. The oxidative phosphorylation is irreversibly damaged [87]. Myocardial recovery after ischemia and reperfusion of isolated perfused hearts from rats was not improved by prior Q10 supplementation and myocardial Q10 content was unaffected [88].

In the dog model of reperfusion, the PGE2 level in vivo in the great cardiac vein and ex vivo in heart mitochondria changed significantly after 20 minutes of left artery occlusion. Pretreatment with indomethacin inhibited the PGE2 increase [89] suggesting cyclooxygenase-2 induction or activation by the reperfusion. When isolated mitochondria were exposed to PGE2 in the presence of ionized calcium in vitro, proton conductivity increased, leading to uncoupling, inhibition of respiration, and lowering of ATP synthesis [90].

 (d) Inhibition of the electron transport chain results in intracellular lipid accumulation. The essential role of Q10 in the electron transport system has been demonstrated using chloroquine, a Q10 analog. Chloroquine reversibly inhibits electron transport and cell growth [91]. In an in vitro model for lipid accumulation in atherosclerosis, smooth muscle cells from pig aortas were incubated with LDL, chloroquine, or both. LDL supplemented cells showed normal morphology save for a few cells containing large lipid droplets. By comparison, chloroquine was toxic to the cells, which developed large autophagic vacuoles. LDL and chloroquine together induced autophagic vacuoles, large lipid droplets, and a marked increase in esterified cholesterol [92]. Treating the remnant cells with chloroquine (30 mM) increased the content of cholesterol by 90% and cholesterol ester by 370% [79].

 (e) Cellular regulation of intracellular cholesterol synthesis. It has been postulated that the protective effect of estrogen against heart disease is due to its tissue-specific regulation of 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase, the main regulator of the isoprenoid metabolic pathway. It converts 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) to mevalonate. Its promoter region harbors a sequence element that differs from the consensus sequence of the estrogen-responsive element (ERE) by only one mismatch in each half of the palindrome. It is unresponsive to estrogen in the liver but responds to estrogen in other tissues that require enhanced cell proliferation [93]. Lipoprotein-deficient serum from women using oral contraceptives induced HMG-CoA reductase in the cultured human aortic smooth muscle cell [94]. ATP inhibits HMG-CoA reductase [95,96].

7. Speculations; filling gaps in present knowledge.  This review of atherosclerosis analyzes and correlates present experimental evidence of defective oxidative phosphorylation in its etiology. A few suggestions are introduced here to fill the gaps in our understanding, as present experimental data are insufficient to establish a unified theory of the pathophysiology involved.

 (a) Reduced binding of liposomes to mitochondria reduces fuel transfer. The apoE null mouse develops atherosclerotic lesions even when fed a diet free of cholic acid supplementation. The common explanation is that the lack of apoE inhibits removal of lipo-proteins by the liver, leading to accumulation of atherogenic lipoproteins in the circulation. ApoE binds to the cellular surface low-density lipoprotein (LDL) receptor, also known as the apo B,E(LDL) receptor, and the LDL-related protein (LRP). In addition, it binds to a 59-kDA intracellular apoE binding protein identified to contain the - and ß-subunits of F₁-ATPase. It was detected in the canine and human liver cell [97].

As an alternative explanation of intracellular lipid accumulation, I suggest that the intracellular lipid-transport system utilizes liposomes that bind via their surface apoE to associate with mitochondria. This idea is derived from the above reports of intracellular apoE-binding proteins that are imported into the mitochondrion. A lack of liposome binding via apoE to the mitochondrial protein in the apoE-null mouse would lead to a defect in the lipid fuel for mitochondrial energy conversion and to intracellular lipid accumulation. Similarly, in humans, such reduced binding by apoproteins like apo4 might contribute not only to atherogenesis, but also to other diseases such as Alzheimer’s disease.

 (b) Lack of LPL. Furthermore, one might speculate that intracellular LPL induces increased release of fatty acids from mitochondrial-bound liposomes for oxidative phosphorylation and thus protects intimal cells and reduces the lesion areas in the LDL-receptor null mouse. A defect in LPL synthesis would therefore not only interfere with intravascular but also with intracellular lipolysis (Figure 3).

View larger version (25K): [in this window] [in a new window]   Fig. 3. Illustration showing evidence-based and hypothetical role of dysfunctional oxidative phosphorylation (OXPHOS) in atherogenesis. A combination of inflammation (COX-2 induction) and dysfunction of OXPHOS causes the atherosclerotic lesion. The latter induces HMG-CoA reductase and increases the synthesis of Q10 as well as cholesterol as they share the same pathway via mevalonate and farnesyl. Hypercholesterolemic diets used to induce experimental atherosclerosis contain bile acids, which inhibit OXPHOS and induce COX-2. Abbreviations: LP: lipoprotein; LDLR: low-density lipoprotein receptor; LRP: low-density lipoprotein receptor related protein; LPL: lipoprotein lipase; CHOL: cholesterol; FA: fatty acid; apoE: apolipoprotein E; SMC: smooth muscle cell; A: atherosclerotic artery, cross section view; COX-2: cyclooxygenase-2; PGE2: prostaglandin E2; IL-6: interleukin-6; Hp: haptoglobin; CoQ10: coenzyme Q10 (ubiquinone), CE: cholesteryl ester. For further explanation see text.

The gene for COX-2 is located at chromosome region 1q25, the same region as the gene for cytosolic phospholipase (cPLA2), and it has been suggested that the two genes are co-regulated [98]. cPLA synthesizes arachidonic acid from membrane phospholipids. Arachidonic acid selectively inhibits Complexes I and III and significantly elevates mitochondrial hydrogen peroxide production. Unsaturated acid inhibits more than saturated acid [99]. These findings suggest another link between inflammation and OXPHOS.

The co-localization of HIPDM with succinate cytochrome c reductase suggests that the enzyme might be inhibited by HIPDM, causing accumulation of intracellular lipids, reduced ATP synthesis, less inhibition of HMG-CoA reductase, increased Q10 synthesis and simultaneous increase in cellular cholesterol synthesis.

 (d) Combined defects. The detrimental effects of reduced fuel supply and inhibition of oxidative phosphorylation on cellular energy metabolism is demonstrated by Reye’s syndrome. Aspirin has been shown to inhibit ß-oxidation and thus the fuel supply, and certain viruses induce the MPT. Additional damage could be induced by salicylate, a metabolite of aspirin. Applying the same reasoning to atherogenesis, it appears reasonable to assume that the inhibition of the availability of lipid fuel to mitochondria combined with drug- or virus-induced direct inhibition of oxidative phosphorylation would significantly reduce ATP generation and be highly detrimental for cell survival. For instance, as noted above, the Herpesvirus accelerates experimental atherosclerosis. A way for estrogen to protect intimal cells would be the increased synthesis of Q10 by stimulation of HMG-CoA-reductase. The findings that ovariectomized rabbits given estrogen do not develop atherosclerosis even in the presence of a high blood cholesterol level suggest that the atherogenic defect is mural, and that local cellular regulation is most important in atherogenesis. This is also supported by the results showing that when Q10 is blocked by chloroquine, the low ATP level induces an increase in receptor uptake.

8. Cardiomyopathy.  Several mtDNA mutations and reduced respiratory enzymes activities have been associated with hypertrophic cardiomyopathy. For instance, deficiencies in respiratory chain enzyme activities were detected in half of a series of 32 endocardial biopsies. The activity of either Complex I, IV, or both, was significantly reduced relative to the combined activities of Complexes II and III [100].

Diabetes.  Mitochondrial DNA with large deletions has been detected in patients with diabetes associated with deafness [101]. Diabetes causes impairment of glucose uptake into cells. It is an independent risk factor for atherosclerosis [102]. The pancreatic ß-cell depleted of mtDNA does not secrete insulin [103]. Beta cells with mtDNA mutations show a defective mitochondrial inner membrane potential and induce diabetes. Insulin resistance is closely related to the regulation by uncoupling proteins and other energy regulators [103]. The high-glucose environment induces glycation of proteins. The concurrent ROS-generation induces apoptosis in vascular cells involved in complications of diabetes mellitus [103].

Maternally inherited diabetes and deafness (MIDD) is a rare disease found only in 1% to 2% of individuals with diabetes. Enzyme activities of less than five percent of the tolerance levels of Complexes I, I+III, and IV have been detected in skeletal muscle biopsies. Modification of diet and increased exercise has provided only temporary improvement [104].

In the rat model of diabetes induced by streptozotocin (STZ) the mitochondrial oxidative phosphorylation is significantly reduced. However, the ATP generation can be completely restored by physical training even if the plasma glucose or insulin levels remain essentially unaltered [105]. Others have reported that endurance exercise training increased oxidative capacity of the muscle and doubled the number of mitochondria in rat muscle [106]. Reduced oxygen consumption per mitochondrion might lead to less oxygen radical generation per mitochondrion with less damage to individual mitochondria [107].

IL-1ßis an acknowledged mediator of dysfunction of the pancreatic ß-cell in Type I diabetes mellitus. Differential-mRNA display reveals that IL-1ß induces ANT1 expression in cultured, purified rat ß-cells [108], suggesting an altered ANT isoform expression.

There is in vitro evidence that sulphonylureas might impair mitochondrial function. For instance, tolbutamide can depolarize the mitochondrial potential of the cultured pancreatic ß-cell [109].

Gastrointestinal disease.  1. The "topical" effect in ulcer formation.   (a) Nonsteroidal anti-inflammatory drugs: Local inference with mucosal mitochondrial oxidative phosphorylation by nonsteroidal anti-inflammatory drugs (NSAIDs) is pivotal in the development of gastrointestinal ulcers in patients treated with them. Indeed it has been postulated that this "topical" phase is a sine qua non of NSAID-induced gastrointestinal damage [110].

Like bile acids, nonsteroidal anti-inflammatory drugs can inhibit oxidative phosphorylation [111]. Lipophilic NSAIDs are readily transported through the cell wall and are enriched within the mitochondrion [112]. Whereas the anti-inflammatory effects of NSAIDs are due to inhibition of COX-2, the concurrent inhibition of COX-1 by non-selective NSAIDs and their interference with mitochondrial oxidative phosphorylation can cause serious gastrointestinal side effects. NSAIDs such as indomethacin are very effective analgesic and antiphlogistic agents and are widely used, resulting in a large number of patients that experience side effects of the treatment. For instance, it has been estimated that NSAID-induced hemorrhage and ulcerations cause up to 20,000 deaths yearly in the United States [17].

Indomethacin uncouples oxidative phosphorylation at micromolar concentrations and inhibits respiration at higher concentration in vitro [113]. Oral administration of indomethacin in the rat significantly lowered ex vivo jejunal ATP and -tocopherol levels [114]. Electron microscopic examination of the mucosa revealed that it caused dose-dependent mitochondrial changes comparable to those inducible in vivo by the classical mitochondrial uncoupler dinitrophenol [113]. Other NSAIDs such as nimesulide, meloxicam, and piroxicam also uncouple mitochondria and stimulate respiration in vitro [115]. They stimulate basal and uncoupled respiration in mitochondria incubated in the presence of either glutamate plus malate or succinate [115]. Diclofenac, but not naproxen, also slightly inhibits ATPase activity. Furthermore, nimesulide and diclofenac, but not naproxen, reduce ATP synthesis by blocking the activity of the adenine nucleotide translocase [115].

Diphenylamine is a shared structure of several NSAIDs that are able to uncouple mitochondrial oxidative phosphorylation. For instance, diphenylamine, mefenamic acid, and diclofenac produce mitochondrial swelling of rat liver mitochondria obtained from freshly isolated hepatocytes and decrease cellular ATP content, mainly through uncoupling of the mitochondrial oxidative phosphorylation [116]. An important finding was that fructose, an effective glycolytic substrate, provided partial protection against the ATP depletion and cell injury induced by these compounds [117].

Direct evidence that uncoupling of oxidative phosphorylation is important in the pathogenesis of NSAID-induced gastrointestinal side effects was obtained from studies of intestinal damage in rats caused by parenteral administration of either aspirin, the uncoupling agent 2,4-dinitrophenol, or indomethacin. Aspirin given orally inhibited intestinal mucosal cyclooxygenase without causing a topical effect: it increased mucosal permeability and reduced mucosal prostanoid levels but did not alter mitochondrial morphology. In these experiments, aspirin caused neither significant inflammation nor ulcer formation. However, the mitochondrial uncoupler 2,4-dinitrophenol increased intestinal permeability, but had no effect on intestinal prostanoid levels. In contrast, the two agents administered together induced alterations similar to those induced by oral indomethacin administration such as altered mitochondrial morphology, increased permeability, decreased prostanoid levels and the formation of intestinal ulcers [118].

Five hours after application directly to the human gastric mucosa, aspirin induces dilatation of mitochondria and the endoplasmic reticulum, rupturing of apical membranes, intercellular edema, and widening of capillary fenestrae [119]. These findings resulted from a controlled clinical trial involving 5 healthy volunteers as probands and 5 other healthy subjects as controls. All volunteers were free of Helicobacter pylori (H. pylori) infection (a significant point, vide infra). It has also been suggested that aspirin at relative concentrations similar to human antipyretic and anti-inflammatory pharmacological doses can uncouple oxidative phosphorylation of rat mitochondria in vivo and in vitro [120]. However, these in vivo experiments might not have differentiated between the effect of aspirin and its salicylate metabolite.

Importantly, even if an agent itself does not affect oxidative phosphorylation, one of its metabolites might do so. As an example, salicylate, a main metabolite of aspirin, but not aspirin itself, uncouples oxidative phosphorylation. A close correlation between the uncoupling activity of salicylate and its congeners and their anti-inflammatory potency had been noted almost forty years ago [112]. Acetylsalicylic acid in the low millimolar range both uncoupled and inhibited oxidative phosphorylation of rat renal cortex mitochondria in vitro; in comparison, the NSAID dipyrone only uncoupled OXPHOS [121]. Other investigators reported that salicylate induced the mitochondrial permeability transition (MPT), depleted the mitochondrial inner membrane potential and uncoupled oxidative phosphorylation [27].

Different NSAIDs differ in their effect on mitochondria, and their effects on mitochondria may be concentration dependent. A number of NSAIDs and acidic prodrugs stimulate mitochondrial respiration in vitro and uncouple mitochondria. Modification or removal of the ionizable group can eliminate the adverse effect on mitochondria. For instance, dimeroflurbiprofen, a modified flurbiprofen, non-acidic prodrugs such as nabumetone, and non-acidic selective COX-2 inhibitors do not cause in vitro uncoupling [122].

 (b) Helicobacter pylori. NSAIDs that do not induce the "topical" phase of gastrointestinal damage, such as the selective COX-2 inhibitors celecoxib and rofecoxib, cause fewer gastric ulcers than indomethacin in animal experiments and are better tolerated in clinical trials [123]. However, infection with the gastrophilic Gram-negative H. pylori might increase the risk of ulcer formation in patients receiving such NSAID therapy because the vacuolating cytotoxin (VacA) released by the bacterium might induce the "topical" effect. Support for this assumption has evolved from in vitro observations: vacuolating cytotoxin prepared from H. pylori decreases ATP levels and increases the number of cultured gastric cells in the G₀/G₁ phase. The cytotoxin decreases the potential m across the mitochondrial inner membranes of the cultured gastric AZ-521 epithelial cell [31].

 (c) Non-acidic and COX-2 selective NSAIDs. One might presume that non-acidic NSAIDs such as nabumetone and NSAIDs that have no enterohepatic circulation do not induce a [topical] phase. Nevertheless, it has been reported that nabumetone can inhibit mitochondrial respiration. It limited the mitochondrial inner membrane potential and reduced ATP synthesis via specific inhibition of Complex I of the respiratory chain [115]. Nabumetone reduced ATP synthesis via specific inhibition of Complex I of the respiratory chain of cultured cells, and in the whole heart nabumetone reduced the oxygen uptake [115,113]. Moreover, cultured cells and the whole heart exposed to nabumetone showed a reduction in oxygen uptake indicating in vivo inhibition of the respiratory chain [115]. Nonetheless, clinical studies indicate that the COX-2 selective inhibitor celecoxib, which does not affect oxidative phosphorylation, and nabumetone are somewhat similar in their rate of ulcer induction. Both drugs show about a 4-fold reduction in ulcer complications vs comparator NSAIDs. [124,125].

 (d) The effect of DMSO. In many comparison animal studies of the gastrointestinal effects of various NSAIDs, eg indomethacin versus celecoxib, the drugs are diluted in dimethyl sulphoxide (DMSO). When DMSO is used as a vehicle for suspension of lipophilic NSAIDs in animal or clinical studies it might alter the effect of the drugs on oxidative phosphorylation. An important, sometimes overlooked, variable in studies of NSAIDs is the effect of DMSO itself. As an example, isolated rat kidney mitochondria exposed in vitro to cyclosporin A with and without DMSO were minimally affected. However, when exposed to DMSO alone there was an increase of about 20% in spontaneous ATP generation [126]. The NSAID imidazole causes hyperpolarization of the mitochondrial membrane in cultured mouse erythroleukemia cells exposed to DMSO as opposed to membrane depolarization observed with DMSO alone [127]. In addition, the vehicle can affect different drugs differently [128]. It has been suggested that DMSO itself may be an anti-inflammatory agent [129]. DMS, a known metabolite of DMSO, but not DMSO, produces mitochondrial uncoupling in vitro [130]. It might explain the observation that DMSO administration to rats induces structural alterations in mitochondrial membranes leading to enhancement of cytochrome oxidase activity in liver mitochondria [131].

2. Restoration of apoptosis in neoplasia.  The early observation that lipophilic NSAIDs are readily transported through the cell wall and are enriched within the mitochondrion [112] has attracted much recent attention. This has happened because acidic NSAIDs can initiate apoptosis in many tumors, for instance colorectal neoplasms. The fact that some NSAIDs exhibit significant anti-tumor effects was first detected in epidemiological studies and later verified in animal studies and in clinical trials. A major part of this effect is due to the NSAID-induced inhibition of cyclooxygenase-2 (COX-2) in the tumor, and selective COX-2 inhibitors have been shown to prevent or inhibit the growth of certain tumors [123]. In comparison, a significant part of tumor growth inhibition by non-selective, acidic NSAIDs is due to the NSAID-induced depolarization of the mitochondrial inner membrane potential that brings about the MPT and restores apoptosis [50].

Ethanol-induced disease.  Ethanol depresses the oxidative phosphorylation of hepatic mitochondria from rats fed ethanol chronically [132]. Such mitochondria display a reduced capacity to incorporate ³⁵S-methionine into mitochondrial polypeptide gene products in vitro. The ethanol exposure reduces the steady-state concentration of every mitochondrial gene product. However, below a threshold, ethanol enhances mitochondrial DNA replication, for instance in the ethanol-treated embryo [133].

The rat liver mitochondrion exposed to ethanol in vitro exhibits malfunction of the transporter of glutathione (GSH). As a result, the mitochondrial matrix is depleted of GSH that normally metabolizes hydrogen peroxide. The mitochondrion becomes more susceptible to alcohol-induced oxidative stress. However, the effect can be prevented by supplementation with S-adenosyl-L-methionine [134].

Alcoholics may show abnormalities of mitochondria even after cessation of ethanol intake [135]. The investigation of oxidative phosphorylation of mice that were fed ethanol might suggest an explanation. The studies revealed that the GSH levels were reduced and the extent of oxidation of mitochondria DNA was significantly increased. These alterations combined with the lack of an effective mtDNA repair system might result in permanent damage to the mitochondria of alcoholic subjects [135].

Mitochondria from rats fed ethanol chronically show a reduction of total phospholipids (except for cardiolipin) in the inner membrane, alteration in the cytochrome content, and a decline of the ability to produce ATP [136]. It was also demonstrated that chronic ethanol consumption caused a marked decrease in ATPase activity in the rat liver mitochondrion [137]. In addition, it has been found that chronic ingestion of ethanol significantly reduced the concentration of two mtDNA-encoded polypeptides, subunits 8 and 6, of the F₀F₁-ATPase in the rat liver. In contrast, ethanol consumption had neither an effect on the nuclear encoded subunits of F₀F₁-ATPase nor on the nuclear-encoded adenine nucleotide transporter [138].

It has been suggested that a myocardial metabolite of ethanol, fatty acid ethyl ester (FAEE), might be a link between chronic ethanol abuse and myocardial dysfunction. In the rabbit, FAEE binds to myocardial mitochondria in vivo and in vitro and can be hydrolyzed to fatty acid, an uncoupler of oxidative phosphorylation [139].

Ethanol at a concentration of 1 mM and 10 mM lightly reduces cell viability of cultured rat hepatocytes and increases the combined ROS generation at Complexes I and III about 70% and 150% respectively [140]. Whereas ethanol consumption significantly increases ROS generation by the rat hepatocytes in such in vitro studies, the loss of hepatocyte viability is mostly correlated with a decrease in the cellular ATP level [141]. Moreover, the amount of dietary fat (high-fat or low-fat diet) has no significant effect on these ethanol-induced alterations [141]. Remarkably, supplementation with fructose [140] or pretreatment with the alcohol dehydrogenase inhibitor 4-methy-lpyrazole (4-MP) [142] prevented in vitro ethanol-induced cytotoxicity. In a rat model of the fetal alcohol syndrome, ethanol exposure in utero reduces perinatal activities of Complexes II and IV and depresses respiration of mitochondria from fetal hearts [143].

Neurological disorders.  1. Primary and secondary defects of OXPHOS.  Recent evidence suggests that defective oxidative phosphorylation contributes to neurodegenerative disorders. Primary defects in the electron transport chain have been detected in Alzheimer’s disease (AD) and Parkinson’s disease (PD). In comparison, in Friedreich’s ataxia and Huntington’s disease, mutations of the nuclear DNA, probably induced by free radicals, result in low levels of aconitase that cause secondary defects in oxidative phosphorylation. Lack of ATP that lowers the threshold to undergo apoptosis may be a central mechanism in the pathogenesis of these neurodegenerative diseases [144].

2. Alzheimer’s disease.   (a) The effect of aging and oxidative damage. Until recently the association of any mitochondrial defect with the etiology and pathogenesis of Alzheimer’s disease had been doubtful [145]. However, a few reports suggest that oxidative stress and mitochondrial dysfunction play a significant role in the pathogenesis [146]. It is probable that the age-related impairment of mitochondrial function induced by oxygen reactive metabolites might predispose or cause diseases such as Alzheimer’s disease [147]. Importantly, it has been demonstrated that brains from AD-patients show more nicking and fragmentation of both the nuclear and mitochondrial DNA. In addition, the amount of mtDNA was reduced and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-OH-dG) was detected by immunostaining indicating oxidative damage. These alterations might predispose to further neuronal damage by exposure to other endogenous or environmental factors [147].

 (b) The effect of apoE4. The role of mitochondrial dysfunction might depend on the presence of apoE4 [148]. The apo4 allele is associated with increased H₂O₂-induced lipid peroxidation in the frontal cortex of patients with Alzheimer’s disease [148]. Clinically, the presence of the 4 allele of the apoE gene increases the risk of dementia on average 5-fold and is independent of its effect on plasma lipids and atherogenesis [149].

In AD patients who carried the 4 allele the clinical dementia rating (CDR) score correlated with diminished activity of the mitochondrial enzyme -ketoglutarate dehydrogenase. By contrast, in AD patients without the 4 allele the CDR correlated significantly better with the densities of neuritic plaques and tangles [150]. Reduced levels of pyruvate dehydrogenase and cytochrome oxidase have also been detected in AD brain tissue [146].

 (c) Amyloid precursor protein. Transfection of the gene for amyloid precursor protein (ßAPP) into cultured normal human muscle fibers caused overexpression of the protein and induced structural abnormalities of mitochondria and reduced cytochrome-c oxidase activity. Abnormal accumulation of ßAPP and similar mitochondrial abnormalities with reduced activity of this enzyme complex have been observed in the brain of patients with Alzheimer’s disease and in patients with inclusion-body myositis suggesting that ßAPP is an important factor in the pathogenesis of the two diseases [151].

3. Parkinson’s disease.  Mitochondrial DNA with large 5kb deletions, eliminating genes encoding protein subunits essential for mitochondrial ATP synthesis, have been found in the striatum of patients with Parkinson’s disease. However, such deletions were also found in aged subjects without the disease [152]. It has been suggested that the significant increase of Complex IV defects found in neurons of the substantia nigra are most likely due to accelerated aging [153]. Reduced activity of Complex I has been implicated in both idiopathic Parkinson’s disease and Parkinsonism induced by the mitochondrial permeability transition [154]. In vitro, the apoptosis-inducing neurotoxin N-methyl-4-phenylpyridinium (MPP⁺) inhibits Complex I, depletes ATP, and induces release of cytochrome c by opening the membrane pores [155].

Reduced availability of coenzyme Q10 may also play a role in the etiology of Parkinson’s disease. In the mouse model, oral therapy with Q10 protected against the detrimental effects of PMTP on the dopaminergic system of the substantia nigra [156].

Neoplasia.  There is evidence that somatic mutations of the mitochondrial genome may be involved in the etiology of cancer and that aggregation of non-somatic mutants may play a role in their progression. An analysis using 2-dimensional gene scanning of mtDNA from 21 papillary carcinomas of the thyroid gland revealed 3 different somatic mutations in 5 of the tumors, which mainly affected genes encoding enzyme Complex I of the respiratory chain [157].

A reduced activity of Complex IV and V, the latter due to a decrease of the F₁–ß moiety, was detected in mitochondria from hepatocellular carcinoma [158]. The F₀ moiety of the F₀F₁-ATPase of the rat liver mitochondrion contains a distinct binding site for diethylstilbestrol (DES), and at low concentrations, it is a potent F₀F₁-ATPase inhibitor [159]. DES is associated with the development of clear cell adenocarcinoma of the vagina and cervix in women exposed to it in utero.

Neoplastic transformation can be associated with markedly altered expression of both the nuclear and mitochondrial DNA encoded oxidative phosphorylation genes [160]. For instance, an analysis of human diploid fibroblasts and their SV 40-transformed counterparts revealed that the mtDNA number declined. However, the mRNA levels for the mtDNA-encoded 12 S rRNAs, ND2, ATPase6+8, COIII, ND5+6, and cytochrome b (CYTB) genes and the mRNAs for the nuclear-encoded ATP-synthase-ß were increased. In addition, the adenine nucleotide translocator (ANT) isoform 1 and 2 genes were markedly induced. A different study found that the ATPase activity in mitochondrial particles from Zajdela hepatoma and Yoshida sarcoma was substantially lower than the control mitochondrial particles isolated from rat heart and rat liver [161]. Tumor mitochondrial particles contained 2 to 3 times more ATPase inhibitor than particles from control mitochondria.

A number of findings support a role for oxidative stress and defective oxidative phosphorylation in carcinogenesis. For instance, biochemical alterations in biopsy tissue from 59 patients with gastric cancer were compared with tumor-free, adjacent mucosa. All tumors exhibited a significant impairment of function as evidenced by reduction in tetrazolium dye staining and of the antioxidant enzyme superoxide dismutase (SOD), and glutathione S-transferase (GST) consistent with oxidative stress. Positive staining with H. pylori antibody significantly decreased the reduction in tetrazolium staining [162].

Gastric cancer is associated with defective mitochondrial function in the tumor and the adjacent tumor-free mucosa [162] and H. pylori infection might be involved in the pathogenesis of gastric cancer: 10% to 20% of infected cases develop ulcers and many develop atrophic gastritis [163].

Mitochondrial dysfunction of Complexes I and IV has been observed in HIV-1-negative children whose mothers had been administered a prophylactic combination of the nucleoside analogue zidovudine and lamivudine or zidovudine alone during pregnancy to prevent mother-to-child HIV-1 transmission [164]. It has been suggested that AZT-induced oxidative damage in nuclear DNA of fetal tissues might be the reason why it can be a perinatal carcinogen. For example, treatment of CD-1 Swiss pregnant mice and the pregnant patas monkey (Erythrocebus patas) with AZT induces a significant increase in 8-oxo-2'-deoxyguanosine (8-oxo-dG) in fetal tissues such as the liver and kidney [165,166].

It has been suggested that fragments of mitochondrial DNA released during mtDNA damage and incorporated into nDNA might cause cancer [167]. Recent experimental evidence supports this hypothesis: it was demonstrated that in the mouse and rat mitochondrial-DNA-like inserts were much more abundant in tumors than in normal tissue [168].

	Molecular Pathology

Top Abstract Introduction Classification of Defects Mitochondrial Dysfunction in... Molecular Pathology Mitochondrial Genetics Therapy Conclusions References

 
Mitochondrial synthesis and turnover.  1. Nuclear DNA.  The biosynthesis of mitochondria is controlled by the nucleus. Nuclear DNA codes for most mitochondrial proteins needed for the synthesis of mitochondria and encodes most of the estimated 1000 proteins required for proper OXPHOS function [169] (Figure 4). Remarkably, mitochondria in the ⁰-cell lack mtDNA but the cell still synthesizes mitochondria in such cells, showing that nuclear DNA alone controls mitochondrial synthesis.

View larger version (37K): [in this window] [in a new window]   Fig. 4. The synthesis of mitochondria is controlled by the nucleus. An individual mitochondrion may have up to ten copies of mtDNA in its matrix compartment. Most significantly, the vital proton pumping subunits of the respiratory complexes (I, III, IV) are all coded by the mtDNA. In addition, mtDNA encodes the two proton-conductive subunits of complex V, the F0F1-ATPase. In contrast, the respiratory chain enzyme complex II is entirely coded by nDNA and does not pump protons across the inner mitochondrial membrane. The circular, bacterial-plasmid-like mtDNA is densely packed and lacks introns and an effective repair system. Located in the matrix, it is exposed to radical oxygen species (ROS) generated during mitochondrial respiration. ROS can cause point mutations in mtDNA leading to deletions and release of mtDNA fragments, some of which are incorporated into nDNA. It has been proposed that such fragments might cause cancer. Extra-mitochondrial DNA can enter mitochondria via porin, a possible entry point for introducing therapeutic DNA. Transfection of old or uncoupled mitochondria reduces cell viability in vitro. NCL = neuronal ceroid lipofuscinosis.

2. Regulation and coordination of mitochondrial biosynthesis.  By stimulating mitochondrial biogenesis, human tissues seek to counteract defective oxidative phosphorylation, which might be caused by a mtDNA mutation [172]. The exact molecular mechanism that regulates the coordinated mitochondrial and nuclear gene expression for the complete synthesis of mitochondria is uncertain [173]. Some evidence suggests that the control region of the F₀F₁-ATPase genes coordinate the expression of the 2 genomes depending on the energy demands of the cells, especially in muscle [174].

Experiments using the transgenic fruit fly Drosophila megaloblaster have provided evidence that the ubiquitous DNA binding transcription factor Sp1 regulates the expression of nuclear genes involved in mitochondrial biogenesis [175]. In these experiments, promoter regions for the mitochondria transcription factor (mtTFA), cytochrome c1, adenine nucleotide translocator 2, and F₁-ATPase ß subunit were transfected into the Drosophila cell lines. All 4 promoters harbor multiple, proximal Sp1-activating elements that account for half or more of transcription activation by Sp1, regulating both positive and negative expression of the nuclear genes that code for OXPHOS subunits.

A study of human tissues containing 12% or less non-mutant mtDNA revealed increased transcript levels of a variety of oxidative phosphorylation and related bioenergetic genes. The patients suffered either from myopathies, encephalopathy, lactic acidosis, stroke-like episodes (MELAS), or other pathogenic mtDNA mutations. The level of mtDNA transcripts was increased. Nuclear OXPHOS gene transcripts were also increased including the ATP synthase ß subunit, the heart-muscle isoform of the adenine nucleotide translocator, and subunits for Complex I. Besides, ancillary nuclear gene transcripts were increased including muscle mitochondrial creatine phosphokinase, hexokinase I, the E1 subunit of pyruvate dehydrogenase, muscle glycogen phosphorylase, and phosphofructokinase [172]. There is evidence that transcription of nuclear-encoded molecules that support mtDNA replication is unaltered even by substantial variations in mtDNA levels [176].

3. Mitochondrial DNA.   (a) Inhibition of mtDNA replication. Azidothymidine (3'-azido-3'-deoxythy-midine, AZT, zidovudine) and other antiviral nucleoside analogue drugs inhibit mitochondrial DNA replication [177]. They are toxic to muscle mitochondria. AZT is a DNA chain terminator, which inhibits the mitochondrial -DNA polymerase that is required for mitochondrial DNA replication [178,179]. Long-term treatment with AZT induces delayed and sometimes severe mitochondrial toxicity [180]. For instance, Southern blotting of muscle biopsy specimens of AZT-treated patients with myopathy revealed an up to two-thirds reduction in mitochondrial DNA compared with normal adult controls. However, cessation of the treatment reversed the depletion of mtDNA [181].

It has been argued that either infection with the human immunodeficiency virus (HIV) or azidothymidine treatment can cause myopathy [182,179]. One study postulated that HIV infection rather than the AZT (ZDV) caused the myopathies in the majority of the patients [183]. However, several other studies indicate that typical mitochondrial abnormalities are observed only in biopsies of AZT-treated patients but not in mitochondria from the non-treated HIV positive patients [182,179]. In one such study, the abnormal mitochondria contained paracrystalline inclusions and were found in "ragged-red" fibers of the biopsy specimen [182].

Azidothymidine has also been employed to develop a rat model of the mitochondrial energy decline associated with overt mitochondrial diseases and the aging process [184]. Treatment of the rats with AZT induced a decline in soleus muscle function in vivo and ex vivo and decreased bioenergetic capacity of heart sub-mitochondrial particles. When such particles were prepared from heart mitochondria of young and aged rats treated with AZT, the mitochondrial particles derived from the aged rats were less able to maintain the membrane potential, compared to those prepared from the young rats. Remarkably, treatment with Q10 improved soleus muscle function in vivo and significantly improved cardiac mitochondrial membrane potential capacity in vitro [184].

Damage to mtDNA by oxygen radicals is the primary cause of mitochondrial myopathy with AZT therapy. A 4-week period of administration of low doses of AZT to mice caused the mouse liver mitochondria to convert one quarter of the total deoxyguanosine (dG) to 8-OH-dG, indicating ROS damage. It was suggested that lack of a mitochondrial DNA-repairing system enhanced the damage to mtDNA caused by AZT. The hypothesis was that the combined effect resulted in impaired mitochondrial respiratory chain function causing oxygen radicals that were responsible for 8-OH-dG formation [185].

Azidothymidine reduces the activities of Complexes I and III of the mitochondrial respiratory chain. After supplementation with AZT of human muscle cells in vitro their mitochondria were enlarged and contained electron-dense deposits in the matrix and abnormal cristae. The respiratory control ratio was decreased indicating uncoupling [186]. Rats treated with AZT showed increased serum lactate and glucose levels and 100-fold elevation of creatine kinase. The highest tissue concentration of AZT was found in the skeletal muscle and the heart [186]. Other findings indicate that inhibition of electron transport chain enzyme complexes is tissue-specific. In vitro, AZT inhibited Complex I in mitochondria isolated from liver, skeletal muscle, and brain. In addition, in this study it inhibited Complex II of mitochondria isolated from the muscle [187].

4. Abnormal mitochondrial degradation.  Lipofuscin, the "wear-and-tear" aging pigment found in the heart and other organs of older individuals, probably develops from dead mitochondria. With increasing age, the yellow brown pigment accumulates around the nucleus of the postmitotic cell. In vitro experiments using isolated mitochondria suggest that lipofuscin can develop from mitochondria by lipid peroxidation: the conversion could be prevented by addition of an antioxidant to the incubation medium [188]. Furthermore, electron microscopy findings of old myocardium have identified mitochondrial membrane fragments in lipofuscin granules. The fragments have been tentatively identified as remnants of mitochondrial cristae, supporting the notion that the lipofuscin is derived from dead mitochondria in the postmitotic cell [189]. In vitro exposure to 40% ambient oxygen or inhibition of proteases with the thiol protease inhibitor leupeptin caused accumulation of fluorescent material consistent with ceroid and lipofuscin within the secondary lysosome of cultured AG-1518 human fibroblasts. The findings suggest that ceroid and lipofuscin form through peroxidative damage of autophagocytosed material [190,191].

Mitochondrial energy conversion.  1. The membrane.   (a) Conductivity. Membrane conductivity depends upon the type of lipid in the membrane as well as the type and concentration of membrane protein [192] (Figure 5). The composition of fatty acids of the membrane phospholipids affects the permeability of the membrane [193]. The effect of dietary lipids and mitochondrial function are of particular interest, since it has been found that mitochondrial enzyme activities are affected both by alterations of mitochondrial membrane lipids as well as mitochondrial enzymes.

View larger version (28K): [in this window] [in a new window]   Fig. 5. Illustration showing causes of dysfunction of the inner mitochondrial membrane. Its bilipid structure with the hydrophobic center and hydrophilic outsides provides capacitance that permits it to be charged with an electric potential. The interaction between membrane lipids and proteins has been studied using alternating current at 1 Hz and 1KHz [PDB] to investigate in vitro both the resistance and capacitance of a lipid bilayer membrane made from either phosphatidylinositol or oxidized cholesterol. Such measurements have shown that membrane conductivity depends upon the type of lipid in the membrane as well as the type and concentration of membrane proteins. For details see text.

 (b) Hyperpolarization. Hyperpolarization of the inner mitochondrial lipid-bilayer membrane leads to a large, non-linear increase in proton permeability [195], similar to the proton permeability of pure phospholipid bilayers. In vitro findings suggest that the lipid-dependent proton leak accounts for only about 5% of the total mitochondrial proton leak [196]. In addition, the proton leak is significantly modified by the presence of proteins embedded in the mitochondrial membrane [197]. Membrane hyperpolarization through ATP hydrolysis by F₀F₁-ATPase enhances generation of reactive oxygen species. For instance, isoprenoid farnesol induces ATP hydrolysis in mitochondria in cells of Saccharomyces cerevisiae. It inhibits oxygen consumption and induces the proton pumping function of F₀F₁-ATPase, which results in mitochondrial inner membrane hyperpolarization. The F₀F₁-ATPase inhibitor oligomycin and the F₁-ATPase inhibitor sodium azide abolish the effect [198].

 (c) Cardiolipin. Cardiolipin is synthesized in mitochondria and is localized almost exclusively within the inner mitochondrial membrane. It is a phospholipid (disphospatidylglycerol). Cardiolipin interacts with membrane-bound proteins to orient and activate them. It affects matrix proteins, mitochondrial membrane receptors, and leader peptides. Cardiolipins, especially those with high linoleic acid (18:2) content, strongly bind many mitochondrial carrier proteins and oxidative phosphorylation enzymes. They exhibit an especially high affinity for cytochrome oxidase. Whereas cardiolipins are not absolutely essential for activation of this enzyme complex in vitro, maximal activities of cytochrome oxidase are only obtained when cardiolipins are present [199,200].

In vitro, saturated cardiolipins form membrane bilayers while unsaturated cardiolipins form nonlamellar phases. Cardiolipins are capable of participating in proton conductive pathways along bilipid membranes when embedded in an ordered bilipid matrix containing phosphatidyl choline and phosphatidylethanolamine, such as the inner membrane of the mitochondrion [201].

Anti-cardiolipin antibodies are independent risk factors for atherosclerotic vascular disease [202]. Infection with the herpes simplex virus (HSV) can markedly inhibit the synthesis of cardiolipins while leaving the synthesis of other phospholipids relatively unaffected [203]. Extracts of diesel exhaust particles decrease mitochondrial cardiolipin, induce uncoupling, lower the membrane potential and ATP levels, and induce apoptosis [204].

2. The electron transport chain (Figure 6).   (a) The enzyme complexes of the mitochondrial inner membrane. Most of Complexes I and III and up to 4 copies of Complex IV are arranged together in supramolecular structures referred to as respirasomes [205] or supra-complexes [206]. Complex I (NADH:CoQ-oxidoreductase) is a pyridine nucleotide transhydrogenase and a proton pump. It couples the transfer of hydride between NADP⁺ and NAD⁺ to proton translocation across the mitochondrial membrane [207]. Complex IV, cytochrome c oxidase, is the third respiratory chain proton pump. It catalyzes the reduction of oxygen to water. Its active site consists of a heme group with a binuclear center of a copper ion [208].

View larger version (23K): [in this window] [in a new window]   Fig. 6. Schematic illustration of the energy flow in the electron transport chain. The proton-pumping complexes I, III, and IV generate the inner mitochondrial membrane potential,,m, typically measuring about 150 mV. Coenzyme Q10 (Q10) accepts electrons from complexes I and II and transfers them to complex III. Cytochrome c transfers electrons from complex III to complex IV where the electrons are transferred to oxygen. The mammalian respiratory chain enzymes are not randomly distributed in the mitochondrial inner membrane but arranged in supramolecular clusters referred to as supramolecular structures or respirasomes. Listed in Italics are agents that inhibit (below membrane) or stimulate (above membrane). AA: arachidonic acid. For details see text.

Human cocaine abuse is associated with heart and liver toxicity. In vitro exposure of neonatal rat cardiomyocytes to cocaine induced slight leakage of lactate dehydrogenase and significantly inhibited glutamate/malate-mediated respiration of isolated mitochondria, suggesting inhibition of Complex I [210]. Norcocaine, norcocaine nitroxide, and N-hydroxynorcocaine, the N-oxidative metabolites of cocaine, but not cocaine, deplete ATP of isolated mouse liver mitochondria in vitro. Norcocaine could completely inhibit mitochondrial respiration [211].

A mutation at base pair 3,243 in MELAS disrupts transcription of a termination sequence located with the tRNA (Leu) [UUR] gene leading to the synthesis of an abnormal 16S ribosomal RNA. It causes defective translation of ND1-7, the mtDNA-encoded subunits of respiratory Complex I, and alters its affinity for the NADH substrate and reduces the mitochondrial membrane potential. Increased mitochondrial NADH might partially compensate for the defect but requires an intact membrane potential for transport of hydrogen from cytosolic NADH into the mitochondrion. Interestingly, a 5-month course of oral nicotinamide administered to a patient with this MELAS mutation resulted in a 50% fall in blood lactate plus pyruvate concentration [212].

 (b) Coenzyme Q10 - the electron carrier. Coenzyme Q10 (Q10), also known as ubiquinone-10, is an endogenously synthesized vitamin-like lipid (Figure 7), an antioxidant, and an essential electron carrier in the mitochondrial respiratory chain [213]. Orally administered it is absorbed to a significant degree. As an example, supplementation in a randomized crossover study by Q10 at concentration of 30 mg/day resulted in significant increases in serum Q10 levels whether mixed with the food or administered as a capsule [214].

View larger version (19K): [in this window] [in a new window]   Fig. 7. This illustration shows the chemical structure of Coenzyme (Co) Q10 (ubiquinone) and part of the synthetic pathway. The polyisoprenoid lateral chain of CoQ10 originates from acetyl-CoA that is converted by 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase to mevalonate, which is then converted to farnesyl. Next, farnesyl is converted either to cholesterol or CoQ10. Administration of a statin-type cholesterol-lowering druginhibits HMG-CoA reductase and reduces the synthesis of cholesterol and ubiquinone. For details see text.

The daily uptake of Q10 differs depending upon the type of food in the diet. For instance, the daily uptake from the average Danish diet has been estimated at only 3.5 mg of Q10 per day, and was mainly derived from meat and poultry. A recent study of a group of Greenland Eskimos showed that their serum Q10 levels were significantly higher than the Danish population. These Eskimos live in a most remote area of Greenland and have a low prevalence of ischemic heart disease. It is possible that their high serum levels of Q10 are due to the high Q10 levels in their diet, which is derived primarily from sea mammals and fish [215].

 (c) Coenzyme Q10 - structure and synthesis. Coenzyme Q10 regulates oxidative phosphorylation and prevents lipid peroxidation [216]. Its polyisoprenoid lateral chain originates from acetyl-CoA via mevalonate and isopentenylpyrophosphate sharing its biosynthetic pathway with cholesterol [216]. Mevalonate is converted to farnesyl, which is converted to either cholesterol or Q10 [216]. Because of the shared pathway from acetyl-CoA via farnesyl, the administration of a statin-type of cholesterol-lowering drug that inhibits HMG-CoA reductase reduces the synthesis not only of cholesterol, but also of Q10, and lowers the blood level of Q10 [216].

The results from studies on the regulation of expression and activity of HMG-CoA-reductase, a 97-kDa-protein [217], and the rate-limiting enzyme of the cholesterol biosynthetic pathway are conflicting. As examples, one study found that ATP and insulin stimulate rat hepatic microsomes HMG-CoA-reductase activity, and the activity was 4-fold higher at night compared to daytime activity [218]. However, others report that ATP inhibits [95] or inactivates [96] the enzyme. ATP at physiological concentration causes swift and irrevocable inactivation of the enzyme activity in the cultured digitonin-permeabilized ovary cell of the Chinese hamster [217].

 (d) Coenzyme Q10 - inhibition by statins. The statin types of cholesterol-lowering drugs that inhibit HMG-CoA reductase also reduce synthesis of ubiquinone and lower the blood level of Q10 [216,219] (Figure 8). The serum Q10 level is also decreased by low-density lipoprotein (LDL) apheresis, and in patients with non-insulin dependent diabetes mellitus (NIDDM) and normal cholesterol levels, but elevated in diabetic patients with hypercholesterolemia [220]. When a group of NIDDM patients with hypercholesterolemia were treated with daily doses of 20 mg of simvastatin, their Q10 blood levels declined significantly. In contrast, Q10 blood levels in a similar group of 30 patients supplemented with 100 mg Q10 per day increased significantly [216] (Figure 8).

View larger version (38K): [in this window] [in a new window]   Fig. 8. Serum (top and bottom) and blood (middle) Q10 levels of patients with hypercholesterolemia (top and middle) and NIDDM (bottom). Statin therapy lowers Q10 levels. However, oral Q10 supplementation restores blood Q10 levels. Data from the following references: De Pinieux et al [219] (top), Bargossi et al [216] (middle), and Miyake et al [220] (bottom).

Experiments with brief ischemia and reperfusion in dog myocardium suggest that lipophilic but not hydrophilic HMG-CoA-reductase inhibitors enter mitochondria, inhibit Q10 biosynthesis and thereby mitochondrial electron transport, and depress ATP generation [221]. In the rat model, administration of the water-soluble pravastatin to rats age 35 and 55 weeks significantly accelerated the age-related decline in the activity of Complex I of mitochondria from the diaphragm and the psoas major muscles. In comparison, pravastatin had no significant effect on mitochondria from the rat heart and liver, which did not show any age-related diminution of respiratory function up to age 55 weeks. Based upon these findings it was suggested that proper diaphragmatic function should be carefully considered when prescribing pravastatin for the elderly patient [222].

In contrast, others found evidence of simvastatin and pravastatin inhibition of oxidative phosphorylation in the rat heart after experimentally induced ischemia. The ATP production per unit oxygen in rat heart mitochondria ex vivo was decreased after one hour of prior in vivo ischemia. Pretreatment of the rats by statins enhanced the decline in ATP synthesis. The lipid-soluble simvastatin caused a greater decline than the water-soluble pravastatin [223]. The effect on myocardial Q10 levels was not determined.

However, in a series of similar experiments, but using only 30 minutes of ischemia, pretreatment with the lipid-soluble simvastatin, but not with pravastatin, significantly reduced the myocardial level of Q10. The ex vivo ADP/O ratio with succinate was significantly reduced in mitochondria only from the simvastatin-treated, and not from the pravastatin-treated rats. Thus the decrease of myocardial Q10 levels induced by cholesterol-lowering therapy with a lipid-soluble, but not a water-soluble, statin might cause worsening of heart mitochondrial respiration during ischemia [224].

A significant, progressive deficiency in blood Q10 levels was reported in patients infected with HIV, with ARC, and with AIDS. Treatment with Q10 led to appreciable and sometimes marked clinical improvement [225]. After interventional cardiac procedures, old patients show inferior recovery compared to young patients. In the rat model of stress induced by rapid electrical pacing of isolated working hearts, pre-stress work performance of senescent hearts was inferior to that of young hearts. Remarkably, pretreatment with Q10 abolished the difference between the young and old rat hearts [226].

In rabbits, simvastatin induced mitochondrial swelling, autophagic vacuoles, and muscle fiber necrosis in all 6 rabbits, pravastatin (300 mg) in 2, and pravastatin (100 mg) in none of the rabbits. However, surprisingly, in spite of the decrease in muscle Q10 content, more so in the pravastatin group, and the mitochondrial swelling observed particularly in the simvastatin group, the mitochondrial respiratory chain enzymes were normal in all groups in this study [227].

 (e) Coenzyme Q10 - regulation of Q10 synthesis. A study of cultured human skin fibroblasts failed to detect any feedback regulation by Q10 of 3-hydroxy-3-methylglutaryl-Coenzyme A (HMG-CoA) reductase. Whereas supplementation of the cells with Q10 in the medium increased the cellular content of Q10 about 10-fold, it did not suppress HMG-CoA reductase nor the incorporation rate of ¹⁴C- acetate into Q10 [228].

This would appear to rule out any involvement of Q10 in the regulation of HMG-CoA. However, if regulation of HMG-CoA occurs via ATP, an alternate interpretation is possible. OXPHOS might have been fully saturated with Q10 due to an ample endogenous supply of Q10. The ATP supply would then have been adequate before supplementation, and there might have been no reason for a compensatory increase in the expression of the reductase to enhance further Q10 synthesis, even after the cellular uptake of a significant amount of exogenous Q10.

 (e) Other inhibitors. The poliovirus can significantly alter mitochondrial function as demonstrated by inhibition of Complex II of cultured COS-1 and T47D cells [30]. Ceramide mediates the effects of tumor necrosis factor-alpha (TNF-). It might be involved in TNF- induced apoptosis since exposure to N-acetylsphingosine, a C2-ceramide analog, rapidly inhibits Complex III in isolated mitochondria [229]. In the rat model, methamphetamine or 3,4-methylene-dioxymethamphetamine inhibits expression of Complex IV as evidenced by a rapid decrease in cytochrome c oxidase staining in the substantia nigra, the nucleus accumbens, and the striatum [230].

A sufficient supply of iron is necessary to prevent dysfunction of iron-containing OXPHOS enzymes. ³¹P magnetic resonance spectra of the gastrocnemius muscle of iron-deficient Wistar rats revealed slow recovery of intracellular phosphate and phosphocreatine concentration after exercise. The alteration in the muscle bioenergetics persisted beyond treatment with iron and complete correction of the anemia suggesting impaired oxidative phosphorylation [231].

 (f ) Aging. Neither Sprague-Dawley rats nor C57/B17 mice lived longer on a diet supplemented daily with 10mg/kg of Q10 compared to control animals not receiving Q10 supplementation. In the rat, plasma and liver Q10 levels were significantly elevated by the supplementation. However, tissue levels of Q10 in the heart, brain, or kidney were unaffected [232].

3. Phosphorylation.   (a) Complex V - the F₀F₁-ATPase. The final step of the mitochondrial energy conversion is the phosphorylation of ADP (Figure 9). Complex V, the F₀F₁-ATPase, provides approximately 95% of the adenosine triphosphate (ATP) produced by the cell [18]. It has been estimated that 3 protons are used by F₀F₁-ATPase for each ATP molecule that is synthesized [233]. Complex V is inhibited by phenothiazines and similar compounds [234], and by di-(2-ethylhexyl)phthalate (DEHP), a plasticizer [235]. The anesthetic n-butanol and tetracaine can inhibit ATPase activity. Irradiation with ultraviolet light results in a conformational change of amino acid residues in the active site of ATPase and inhibition of ATPase in activity in both membrane-bound and soluble F₁ vitro [236]. Succinate may partially protect against these ultraviolet light-induced effects [236].

View larger version (31K): [in this window] [in a new window]   Fig. 9. A schematic illustration of phosphorylation, uncoupling protein (UCP), and the mitochondrial permeability transition pore complex (PTPC). Complex V, the F0F1-ATPase, utilizes the membrane potential to phosphorylate adenosine diphosphate (ADP) to adenosine triphosphate (ATP) and provides most of the ATP produced by the cell. The adenine nucleotide transporter (ANT) exchanges the ADP and ATP across the membrane. Agents that interfere with normal functions are indicated in italics. For details see text.

Mutations of a gene coding for an OXPHOS enzyme subunit may alter the sensitivity of the enzyme to inhibitors. As an example, the Sprague-Dawley and the BHE/Cdb rat differ in their mtDNA sequence for a subunit of Complex V. Base substitutions in the ATPase 6 gene result in the substitution of aspartate for asparagine at position 101 and the substitution of leucine for serine at position 129. As a consequence, isolated mitochondria from the rat with the base substitution are more sensitive to oligomycin inhibition of OXPHOS compared with mitochondria isolated from the Sprague-Dawley rat. These results are consistent with results from human fibroblasts having a mutated ATPase-6 gene [237].

 (b) The adenine nucleotide transporter (ANT). The transmembrane transport of adenine nucleotides by ANT can be inhibited by morphine [238]. Specific antibodies against ANT can limit oxidative phosphorylation by inhibiting transmembrane nucleotide transport. Recent findings suggest that a virus might induce cardiac mitochondrial dysfunction via an antibody-mediated attenuation of the capacity of ANT to exchange nucleotides. First, immunizing guinea pigs with ANT disturbs heart mitochondria and reduces heart function. Second, infecting mice with the Coxsackie B3 virus induces specific anti-ANT antibody production in over 70% of the mice. Perfused, isolated hearts from the animals that produced the anti-ANT antibody showed significantly reduced mitochondrial oxidative phosphorylation and over 50% reduction in left ventricular pressure [239]. Bile acids reduce ANT in vitro [240].

The carboxyl end of the HIV-1 viral protein R binds to purified ANT and rapidly dissipates the membrane potential of isolated mitochondria and induces apoptosis in cultured cells. Addition of Bcl-2, which inhibits the opening of the permeability transition complex pore, prevents the apoptosis [241].

 (c) Regulation of oxidative phosphorylation. Oxidative phosphorylation is controlled differently in different tissues. In muscle and heart, control of respiration is the primary control, whereas in brain, liver, and kidney, control is through regulation of the ATP synthase and the phosphate carrier [242].

One theory of the regulation of oxidative phosphorylation is that breakdown products of ATP diffuse freely to the mitochondria to stimulate OXPHOS. However, calcium entry into the mitochondria cannot explain the first fast phase of oxidative phosphorylation activation, and it has been proposed that delay of the energy-related signal in the cytoplasm dominates the response time of OXPHOS [243].

Thyroid hormone enhances oxidative phosphorylation as shown by thyroidectomy in the rat model of hypothyroidism. Removal of the thyroid reduces ATPase activity by over 30% in the liver mitochondria [244]. In the rat, thyroid hormone up-regulates the expression of selected nuclear genes that encode OXPHOS complex sub-units [245]. Even subunits of the same enzyme complex may be differentially regulated by thyroid hormone. For instance, the mRNA for cytochrome c1 is increased as much as 20-50-fold by thyroid hormone. However, other nuclear OXPHOS genes such as the F₁-ATPase ß-subunit and the core protein 1 of Complex III do not respond to thyroid hormone [245]. Mitochondria from thyroxin-treated rats incubated with succinate as substrate showed an ex vivo increase in the respiration rate of almost 50%, and there was a 10% increase in the membrane potential compared with mitochondria from normal, non-treated rats [246].

In hyperthyroidism, triiodothyronine induces increased ATP consumption in the heart. However, relatively more membrane energy is used for heat production and less directed toward ATP production and muscle contraction [247].

Anabolic-androgenic steroids can affect respiratory chain enzymes. For instance, there was a significant decrease in the activities of Complexes I, III, and IV in rats treated with either fluoxymesterone, methylandrostanolone, or stanozolol [248]. Others have reported that glucocorticoids such as hydrocortisone, prednisolone, dexamethasone, and triamcinolone, inhibit Complex IV [249].

In the animal model the effect of cannabinoids is different for single use compared with prolonged use. A single dose (10 mg/kg) of (9)-tetrahydrocannabinol increased both oxidative phosphorylation of rat brain mitochondria and cerebral lipoperoxidation ex vivo. When the same dose was administered twice daily for 4.5 days, it resulted in uncoupling of brain oxidative phosphorylation and induced neuronal damage [250].

4. Uncoupling.   (a) Effects on cell survival. Mitochondrial transfer experiments have demonstrated that damaged mitochondria can reduce the viability of a cell [251]. For instance, 20% of young human fibroblasts injected with isolated mitochondria from old rats exhibited signs of degeneration after a few days [252]. In contrast, only 5% of cells microinjected with fresh mitochondrial preparations from young rats showed signs of degeneration. Young mitochondria have a high respiratory control ratio: The energy released during mitochondrial respiration is tightly coupled to phosphorylation of ADP. However, if young mitochondria are uncoupled by exposure to an uncoupler such as 2,4-dinitrophenol, the synthesis of ATP is reduced. Remarkably, young mitochondria that are partially uncoupled induce changes in the recipient cells similar to those induced by old mitochondria [252]. In such mitochondrial transfer experiments, the proportion of dead cells is dependent on the state of uncoupling of the injected mitochondria. On the other hand, supplying the recipient cells with a substrate that is easily metabolized protects the cells. For instance, supplementation with D(-)-beta-hydroxybutyrate sodium salt in a dose-dependent manner prevents degeneration induced by micro-injection of uncoupled mitochondria.

Fatty acids cause non-shivering thermogenesis in larger mammals by inducing uncoupling proton flow across the inner mitochondrial membrane, only above a threshold membrane potential of 125 mV. [253].

The branched-chain phytanic acid, 3,7,11,15-tetra-methylhexadecanoic acid, which accumulates throughout the body in Refsum disease, increases the mobility of inner membrane phospholipids, alters the conformational state and mobility of transmembrane proteins, and induces uncoupling in vitro [254].

 (b) Passive proton leak. Transmembrane outward pumping of protons from the mitochondrial matrix to the intermembranous space and inward transmembrane proton leak results in a futile proton cycle. It dissipates the mitochondrial redox energy, which consumes approximately 15% of the standard metabolic rate of working muscle and liver in vivo [255,256]. The proton leak is a basic component present in all mitochondria.

Hyperthermia can increase proton conductance of the mitochondrial inner membrane and degrade oxidative phosphorylation. The resulting proton leak is caused by alteration of the membrane order. An in vitro study of intact female rat mitochondria using fluorescent measurements to determine membrane phospholipid polarization revealed that the transition occurred between 40 and 43ºC [257].

In addition, an augmentative component present in some mitochondria induces a significant flow of protons across the mitochondrial inner membranes into the mitochondrial matrix. Some suggest that in the rat resting hepatocyte almost one-third of its resting oxygen consumption is dissipated as heat due to proton leaks [258]. The proton leak in rat tissues varies depending upon the tissue examined [259,255].

 (c) Active proton leak. Uncoupling proteins regulate the dissipation of the membrane potential formed through respiration. Instead of being used for ATP synthesis the mitochondrial membrane energy is converted to heat [260]. The heat production not only protects against cold environments but also regulates the mitochondrial energy balance [261]. Through the regulation of uncoupling [262], mitochondria can adjust their metabolism to the supply of substrates and the cellular ATP requirement, while minimizing ROS production by lowering the membrane potential [103].

The first uncoupling protein (UCP) that was discovered is a classical example of an augmentative component. It is a 32-kDa membrane-protein that is specifically induced in rat brown adipose tissue [263]. It was later renamed UCP1, as additional protein homologues, namely UCP2, widely expressed in rodent and human tissues [263], and UCP3 were identified. UCP1 uncouples mitochondria of brown adipose tissue [264]. Purine nucleotides on the cytosolic side of UCP1 inhibit its proton conductance. Fatty acids increase UCP1-induced energy dissipation [265] and regulate the mitochondrial energy system by tuning the degree of coupling of oxidative phosphorylation [266]. Retinoids induce UCP1 transcription in transgenic mice [263].

In the mouse, LPS, IL-1ß, and TNF induce the expression of UCP2 mRNA. However, the effect is tissue dependent and apparently differently regulated. For instance, LPS strongly induces the UCP2 expression in muscle and liver, but prior administration of indomethacin inhibits expression only in liver [267].

The UCP3 gene is expressed in rodent skeletal muscle and brown adipose tissue and might also play a role in the mitochondrial degradation of fatty acids [261]. Two UCP3 RNA transcripts produce 2 isoforms, one long, full length (UCP3L) and one short, truncated (UCP3S) isoform, that are highly expressed in skeletal muscle. Both strongly impair the mitochondrial coupling efficiency and increase thermogenesis, with the short isoform being the most active [268]. A mutation of the UCP3 gene that increases the proportion of the short isoform slows its insertion into the mitochondrial inner membrane, decreases fat oxidation, and enhances the susceptibility to obesity [269].

The absence of UCP-1 in the UCP1-ablated mouse leads to high expression levels of UCP2 and UCP3 and induces low cold tolerance. Interestingly, the mice do not become obese, and in these experiments, UCP2 and UCP3 were not associated with any inherent uncoupling or with an increased basal metabolism [264]. In comparison, a 24-hour starvation period in the regular rat increased UCP2 and UCP3 in the skeletal muscle more that 4-fold and doubled the UPC3 protein levels while mitochondrial proton conductance remained constant [270].

The adenine nucleotide translocator (ANT) and the voltage-dependent anion channel (VDAC) are part of the permeability transition pore complex [241]. During a mitochondrial permeability transition (MPT), the complex opens the high conductance pore that increases membrane permeability to solutes of molecular mass up to 1.5 kDa [27]. The mitochondria swell, the membrane depolarizes and its potential dissipates; oxidative phosphorylation is uncoupled.

The onset of a mitochondrial permeability transition can be observed with confocal fluorescence microscopy by using red fluorescing tetramethylrhodamine methylester as a membrane potential-indicating fluorophore in combination with cytoplasmic calcein [271]. As pores open, green-fluorescing calcein moves into mitochondria. There is a simultaneous release of the red dye. Remarkably, cyclosporin A blocks the opening of the pores and prevents cell death. In vitro, salicylate induces the mitochondrial permeability transition. It kills cultured rat hepatocytes at concentrations of 0.3–5 mM in a concentration-dependent manner [27,272].

Similarly, other compounds, such as 3-mercapto-propionic and 4-pentenoic acids, adipic, benzoic, isovaleric, and valproic acids, and Neem oil that have been implicated in the pathogenesis of Reye’s syndrome can induce the onset of the mitochondrial permeability transition in freshly isolated hepatic mitochondria in vitro. Surprisingly, the induction of the MPT in these experiments did not substantially reduce the membrane potential. This might be due to a rapid increase in respiration that temporarily maintains the membrane potential [273]. Apparently, an intact, rapidly responding OXPHOS regulatory system might maintain the inner membrane potential temporarily, even in the presence of some degree of MPT. Such a mechanism might be very important to cell survival, since these findings suggest that uncoupling alone does not always induce complete depolarization. It has also been suggested that onset of MPT might be involved in chemical toxicity and Jamaican vomiting sickness which, like Reye’s syndrome, are characterized by hyper-ammonemia, hypoglycemia, microvesicular steatosis, and encephalopathy [273].

Low temperatures markedly decrease the resting respiration due to membrane leak; however, the respiration due to intrinsic proton pump uncoupling increases [274]. In rat liver mitochondria, the glycoside antibiotic sporaviridin uncouples oxidative phosphorylation. It increases the permeability of the inner membrane of the mitochondria [275]. Regulating the proton leak may rapidly permit mitochondria to channel energy flux to or from ATP synthesis [193].

5. Functional equivalency diagram.  The function of the oxidative phosphorylation system can be simplified by reference to a schematic diagram where the function of the electron transport chain is compared to that of a fuel cell (Figure 10). The input to the fuel cell consists of the food metabolites and oxygen; the energy released charges a capacitor (the membrane). The energy stored temporarily in the capacitor is either coupled to an ATP converter or uncoupled due to inhibition of the converter or due to heat loss caused by fixed or variable discharge of the capacitor. The mitochondrial permeability transition (MPT) is analogous to a switch that rapidly discharges the capacitor. If the switch is closed only momentarily the fuel cell might rapidly respond and recharge the capacitor. However, if the fuel cell is damaged, complete discharge might occur.

View larger version (16K): [in this window] [in a new window]   Fig. 10. Simplified schematic diagram of oxidative phosphorylation. The electron transport chain is compared to a fuel cell with an input side consisting of food metabolites and oxygen (not shown) and an output side that charges a capacitor (the membrane) up to about 150 mV. The voltage is coupled either to (a) an ATP converter or (b) dissipated as heat. Uncoupling is the reduction of ATP generation due to either (a) inhibition of the converter or (b) fixed or variable heat loss. Activation of the mitochondrial permeability transition (MPT) is compared to a switch that rapidly discharges the capacitor. When closure of the MPT switch starts to discharge the capacitor, a fully operational fuel cell might rapidly respond to prevent complete discharge. However, if the fuel cell response is inadequate, complete discharge follows (apoptosis).

	Mitochondrial Genetics

Top Abstract Introduction Classification of Defects Mitochondrial Dysfunction in... Molecular Pathology Mitochondrial Genetics Therapy Conclusions References

The energy-converting metabolism, originally bound to the plasma membrane, was relocated to the intracellular space. This endosymbiosis led to the transfer of most genes from the symbiont to the nuclear genome of the host [280]. Evolution might have favored the transfer of bacterial DNA to the more protected environment of the nuclear genome to prevent defective mitochondria from flourishing to the detriment of an organism, particularly in its syncytial tissue [281].

A few genes essential to OXPHOS remained in the mitochondrion during phylogenesis. Some of these genes code for the proton-pumping subunits of electron transport chain enzyme complexes. As hydrophobic proteins they could not be synthesized in the cytoplasm and imported into mitochondria. Many additional bacterial genes present in the nuclear genome may have originated from bacteria that were simply taken up and processed as food [282].

Some sequence data suggest that the formation of the mitochondrion occurred simultaneously with the formation of the nuclear genome of the eukaryotic cell [277]. Phylogenetic DNA sequence comparisons suggest that extensive lateral gene transfer occurred between early bacteria, archaea, and ancestral eukaryotes, resulting in "chimeric" genomes containing genes from multiple sources [283].

2. The structure.  The Cambridge sequence was mainly derived from a single human placenta with a few regions coming from HeLa cell mtDNA and bovine mtDNA, which were used to establish the base for several ambiguous nucleotides. The Cambridge sequence consists of 16,569 base pairs encoding hydrophobic protein subunits of the mitochondrial electron-transport system and ATPase: seven subunits of complex I, apocytochochrome b of Complex III, three subunits of Complex IV, and two subunits of ATPase (Complex V). The remaining mtDNA genes specify 2 mitochondrial ribosomal RNA (RNA), 22 organelle-specific transfer RNA (tRNA) and genes regulating transcription and replication (D-loop) [11]. All other mitochondrial proteins are coded for by the nuclear genome.

There are 10³ to 10⁵ circular mitochondrial DNA molecules in the human cell [284]. Heteroplasmy is the simultaneous presence of both normal and mutated mtDNA [20].

3. The high mutation rate of mtDNA.  The stability of the mitochondrial genome and its efficiently regulated expression is essential for maintaining the membrane potential and a functional oxidative phosphorylation pathway [285]. However, the rate of mutation of mtDNA, caused, for instance, by radical oxygen metabolites produced by the respiratory chain, is 10 to 20 times higher than the mutation rate of nuclear DNA. Besides, the mtDNA lacks effective repair and contains no histones.

Mutations of the mitochondrial genome are the most important causes of known inherited and acquired genetic OXPHOS deficiency [145]. A comparison of the evolutionary rate of the nDNA-encoded ß subunit with that of the mtDNA encoded ATPase 6- and 8-subunits, 7 other mtDNA OXPHOS genes, and a number of nuclear genes revealed significant differences in their synonymous substitution rate. The rate for the ATPase6 and 8 genes was 12 times greater than the rate of the nuclear gene for the ß subunit. Even more remarkable was the finding that the mutation rate of the average mtDNA gene was 17-fold that of the nuclear ß subunit gene. These high substitution mutation rates and strong selective constraints of mammalian mtDNA proteins explain why mtDNA mutations result in a disproportionately large number of human hereditary diseases of OXPHOS [286].

Propagation.  1. Mitochondria of the male gamete.  The fate of mitochondrial DNA differs significantly in male and female gametes. Analysis of the entire mitochondrial genome in sperm donors revealed that most of the spermatozoan mitochondria from patients with oligoasthenospermia had multiple mtDNA deletions. Surprisingly, multiple deletions were found in normal sperm as well [287].

The Darwinian competition to fertilize the ovum should favor the sperm with the most efficient OXPHOS and therefore the highest motility. There is evidence that factors reducing the mitochondrial energy production are responsible for some cases of male infertility. For instance, there is a direct correlation between sperm mitochondria respiratory chain enzyme activities and sperm motility. Some findings argue that particular cases of asthenozoospermia are caused by defective mitochondrial energy production. For instance, the activities of Complexes I, II, and IV in sperm samples from asthenozoospermic subjects were significantly lower compared with those from the control individuals [288]. The sperm motility depended primarily on the mitochondrial volume and therefore the total energy available generated by OXPHOS in the sperm midpiece.

Flow cytometry findings using the cationic dye JC-1 to measure the sperm mitochondrial membrane potential corroborate a correlation between the potential and sperm motility [289]. Moreover, cytometry revealed a correlation between mitochondrial uncoupling and immature spermatozoa. The degree of uncoupling also correlated with spermatozoa defective for nuclear maturity. A higher percentage of immature spermatozoa was found in men from barren couples compared to donors of proven fertility.

In a patient with inherited mitochondrial disease caused by reduced activity of Complexes I and IV the sperm motility was reduced compared to normal control [290]. Remarkably, in vitro supplementation of the medium with pyruvate that enters the respiratory chain at Complex I and succinate that enters at Complex II tripled the sperm motility compared with only 12% motility when supplemented with glucose alone. Ultrastructural changes of the mitochondria were found not only in the spermatozoa but in spermatids as well, suggesting that the abnormalities were due to a primary mitochondrial defect rather than to secondary degeneration of the spermatozoa.

Paternal mitochondrial DNA has not been detected in human somatic cells of the offspring, at least not at the limits of detection of present technology. It is not known how the paternal mitochondria are eliminated from the human morula; however, the process in humans might be analogous to the mechanism in the mouse, which rejects the sperm-derived mitochondria at the 4- to 8-cell stage [291].

2. Mitochondria and the female gamete.  Delayed motherhood is characterized by a higher risk of conceiving an offspring suffering from a mitochondrial DNA disorders [292]. And while the mitochondria from oocytes collected from twelve women showed few mtDNA rearrangements [287], there was considerable mutational heterogeneity in the individual oocyte donor. However, oocytes have an efficient mtDNA repair system, which is basically independent of maternal age [292]. It protects and perpetuates the maternal mitochondrial genome.

In addition, the process of oogenesis, follicle formation, and loss of the less fit purifies the female germ-line mitochondrial DNA [293]. This culling revives the mitochondrial genome as it passes, by way of the oocyte cytoplasm, from one generation to the next. This process provides the bottleneck that refines the haploid mitochondrial genome in the oocyte and maintains the integrity of maternal mitochondrial inheritance.

Cloning.  The loss of non-oocyte mitochondria has also been detected in mammalian cloning that fused a somatic cell by electroporation to an enucleated oocyte from the same animal. It might be expected that the cloned progeny should contain mtDNA from both the donor and recipient, resulting in heteroplasmy. However, somatic cells in cloned sheep contain only the female germ-line mitochondrion [292]. How the donor cell mitochondria disappear just like sperm mitochondria is unknown. Possibly, contrary to the somatic cell mitochondria, oocyte mitochondria might carry some kind of recognition signal sequence that protects them from expulsion or rapid degradation.

As the first cloned sheep showed advanced biological age, it was suggested that this might be due to free radical-induced cellular damage to their inherited somatic mitochondria [294]. However, the premature aging of the cloned sheep is probably related to their shortened telomeres. Remarkably, the opposite was observed in cloned cows, where the telomeres in several cases were elongated, suggesting that cloning can also result in a prolonged life span.

Genetic diseases.  1. The problem with limited diversity.  An important point in inherited mitochondrial diseases is the fact that populations with limited genetic diversity are at risk for diseases that are rare elsewhere. A case in point is the so-called "Finnish disease heritage" [295]. An analysis of mitochondrial mutations that have accumulated revealed that only a small number of men and women contributed to the genetic lineage present in the Finnish population. The mitochondrial genes examined were those of the mtDNA nucleotide positions that evolve slowly in the mitochondrial control region. For the nuclear genes, the Y-chromosomal haplotype population of the peroxisome proliferator-activated receptor gamma (PPAR)-coactivator-1 (PGC-1) was analyzed [295]. PGC-1 coordinates the expression of both mitochondrial and nuclear encoded OXPHOS enzyme subunits and the uncoupling protein [103].

2. Heteroplasmy.  In diseases caused by inherited mutations of mtDNA, the link between genotype and phenotype varies so that the same mtDNA mutation may give rise to a variety of phenotypes. Moreover, the same phenotype may be seen with different mtDNA mutations [145]. Differing points of view have been introduced to explain this phenomenon. For instance, it has been proposed that sporadic stem cell mutation during embryogenesis or mitotic segregation might result in different degrees of heteroplasmy in various tissues [20]. Heteroplasmy can also result from acquired mutations of mtDNA.

3. Mutations and deletions.  A defect in the germline mtDNA affects all mtDNA plasmids in all mitochondria of all somatic cells and explains why inherited mtDNA diseases can be very serious. Mitochondrial DNA mutations causing overt mitochondrial diseases are characterized by a decline in mitochondrial respiratory function [184]. This has been demonstrated in cases of progressive kidney disease or maternally inherited diabetes and deafness, both caused by a single point mutation of the mitochondrial tRNA. Cybrid cells were prepared by inserting donor mitochondria into ⁰ cells, which lack mtDNA. The mitochondria were derived from fibroblasts of a patient carrying an A to G transition at nucleotide position 3,243 of the mtDNA (Figure 11). The heteroplasmy in the cybrid cells varied from none to 100%. The cybrid cells containing predominantly mutant mtDNA showed marked defects in mitochondrial morphology, poor respiratory chain Complex I and IV activities, and lactic acidosis [296].

View larger version (41K): [in this window] [in a new window]   Fig. 11. Mitochondrial deletions (top) of the mitochondrial genome (bottom), which are illustrated in nucleotides (nt) in linear form along the x-axis. For clarity, the mitochondrial genes coding for subunits of oxidative phosphorylation (OXPHOS) enzyme Complexes I, III, IV, and V are shown in separate lanes. Genes that are partially or completely deleted by the 'common' deletion are highlighted. The map was prepared from numerical data downloaded from "MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, GA, USA, http://www.gen.emory.edu/mitomap.html, 1999".

More than 100 primary defects in the mitochondrial genome have been associated with encephalomyopathies and the majority has been linked to defective oxidative phosphorylation [298]. For instance, several mtDNA mutations cause rare encephalomyopathies such as Leigh and Leigh-like syndromes, fatal infantile lactic acidosis, neonatal cardiomyopathy with lactic acidosis, and macrocephaly with progressive leukodystrophy [209]. Knowledge of the normal mtDNA variation in a specific human population is essential to understand the pathophysiology of diseases caused by an mtDNA aberration [299].

By comparison, mutated nDNA genes encode mitochondrial OXPHOS proteins that cause Friedreich’s ataxia and hereditary spastic paraplegia [145]. In the case of amyotrophic lateral sclerosis and Huntington’s disease, the defect of oxidative phosphorylation is secondary to events induced by a mutation in a nuclear gene encoding a non-mitochondrial protein [145].

	Therapy

Top Abstract Introduction Classification of Defects Mitochondrial Dysfunction in... Molecular Pathology Mitochondrial Genetics Therapy Conclusions References

 
Several approaches have been used to treat mitochondrial diseases, ranging from dietary measures to administration of redox agents, vitamins, coenzymes, and enzyme activator [212].

Antioxidants limit lipid peroxidation and decrease prostaglandin synthesis [300]. The risk of oxygen-derived free radical damage can be significantly reduced by improved diet, particularly fruits, vegetables, and grains, by reasonable exercise, and by reduced alcohol consumption [301].

Q10.  Therapeutic use of Q10 can benefit patients with cardiomyopathy. Determination of Q10 tissue levels in myocardial biopsies from a series of 45 patients with cardiomyopathy using high performance liquid chromatography (HPLC) showed significantly lower levels of the coenzyme in the cases of more advanced than in the milder cases of heart failure. Oral supplementation with 100 mg of Q10 daily benefited nearly two-thirds of the patients. Those with dilated cardiomyopathy showed the most clinical improvement [29].

Administration of Q10 significantly reverses exercise-induced abnormalities of oxidative phosphorylation of the brain of patients with known mitochondrial enzyme defects. Magnetic resonance spectroscopy studies of such patients show that exercise increases the level of ADP and inorganic phosphate and reduces the level of phosphocreatine in the brain. Treatment with Q10 significantly shortens the rate of brain phosphocreatine recovery after the exercise and corroborates in vitro observations that Q10 concentration in the inner mitochondrial membrane regulates the efficiency of oxidative phosphorylation [302].

The liver mitochondria of the diet-induced atherosclerotic rabbit showed a significant increase in hydroperoxides and a serious drop in the content of Q10. Virgin olive oil or vitamin E-stabilized fish oil, but not sunflower oil, added to the diet in part reversed the diet-induced alterations [303]. Coenzyme Q10 (3 mg/kg/day) administered to rabbits fed an atherogenic diet significantly reduced aortic cholesterol and aortic as well as coronary artery plaque size [304]. When applied to the epidermis, Q10 penetrates into the viable layers. Weak photon emission measurements indicate that it reduces the level of epidermal oxidation. It prevents photoaging, suppresses the expression of collagenase, and reduces the wrinkle depth [305].

An adequate supply of vitamin B6 is essential for the endogenous synthesis of the quinone nucleus of coenzyme Q10 (Q10) from tyrosine. It should therefore be recommended that patients treated with Q10 should receive concurrent supplementation with B6 to enhance endogenous synthesis of Q10 [306].

Melatonin.  A pineal hormone melatonin, N-acetyl-5-methoxytryptamine [307], lowers plasma cholesterol levels in genetically hypercholesterolemic rats. It also reduces the fatty changes in their liver [308]. Most importantly, melatonin concentrates in mitochondria. In rats fed a 1% cholesterol + 0.5% cholic acid diet daily, treatment with ip injections (4 mg) of the antioxidant melatonin for periods of up to 4 weeks lowered the diet-induced increases in plasma levels of VLDL and LDL. It decreased plasma HDL and diminished diet-induced fatty change of the liver [307].

Melatonin enhances the activity of Complexes I and IV and prevents ruthenium-red-induced reduction in the activities of these complexes in vivo, suggesting a therapeutic use of melatonin in drug-induced mitochondrial damage [309]. Melatonin can also attenuate ethanol-induced mitochondrial DNA depletion, which occurs after an alcohol binge [310].

However, the lack of purity of commercial melatonin preparations limits their use. For instance, 6 impurities detected in 3 commercial melatonin preparations are structural analogs of contaminants found in the dietary supplement L-tryptophan. These contaminants were implicated as etiologic agents in the eosinophilia-myalgia syndrome epidemic that occurred a few years ago [311].

Carnitine.  Carnitine is essential for long-chain fatty acid oxidation and for shuttling accumulated acyl groups out of the mitochondria [312]. In the chronic alcohol-fed rat, oral L-carnitine by itself, but more so if administered with oral Q10, protects against alcohol-induced hepatic lipid infiltration [313].

In humans, therapy with carnitine ameliorates the symptoms of claudication of peripheral arterial disease. It restores skeletal muscle function of patients on dialysis who suffer from dialysis-induced reduction of muscle carnitine. However, while carnitine supplementation improves exercise performance in certain disease states, any benefit in healthy individuals to support the high metabolic demands of heavy exercise is uncertain [312].

Gingko biloba.  By scavenging the superoxide anion, the Ginkgo biloba Egb761 extract provides postischemic protection against re-oxygenation injury to rat liver mitochondria in vitro [314]. Besides, treatment with Egb761 extract in part prevents the development of peroxide formation in the mitochondrion of the old rat [315]. Extract of Ginkgo biloba is anti-ischemic mainly due to the presence of bilobalide in the terpenoid fraction. Bilobalide delays hypoxia-induced decrease in ATP content of endothelial cells in vitro [316]. It increases the activity of Complex I, protects Complexes I and III activities and delays the onset of ischemia-induced damage. It permits respiratory activity and ATP regeneration by mitochondria under ischemic conditions as long as some oxygen is present [317]. These insights were obtained using mitochondria isolated from rats treated with bilobalide (2–8 mg/kg). There was a dose-dependent increase in the respiratory control ratio, by reason of lower oxygen consumption during state 4. Bilobalide reduced the sensitivity of oxygen consumption to inhibition of Complex I by amytal or Complex III by myxothiazol or antimycin A. In Alzheimer’s disease, therapy with recombinant human apo3/3, apoe3/e4, dehydroepiandrosterone (DHEA), or Ginkgo biloba extract (EGb 761) protected against the lipid peroxidation [148].

Dietary fats.  Consumption of fish oil lowers plasma triacylglycerol levels. There is evidence that the effect is due to increased mitochondrial fatty acid oxidation. As an example, in rat liver parenchymal cells and in purified mitochondria, eicosapentaenoic acid (EPA) increases mitochondrial fatty acid oxidation. It also increases carnitine palmitoyltransferase-I [318]. Supplementation of the diet with virgin olive oil to enrich the mitochondrial membranes with mono-unsaturated fatty acids and Q10 prevents free radical damage to heart mitochondria of male rats [319].

Genetic engineering.  Genetic material can enter mitochondria in vivo under both physiological and pathological conditions. In vitro, double-stranded DNA crosses lipid bilayers doped with isolated mitochondrial porin that serves as a voltage-dependent anion channel. DNA crossing requires application of an electrical field to the membrane and is blocked by addition of anti-porin antibody [320]. This discovery opens up the possibility of mitochondrial genetic engineering by introducing new DNA into the mitochondrion to repair mtDNA defects. Besides, it might be possible to introduce DNA sequences that might bestow resistance to adult-onset diseases by preventing obesity and atherosclerosis or premature cognitive decline. For instance, a mitochondrial genotype, mt5178A, was recently identified in Japanese centenarians, suggesting that some mtDNA sequences might be more resistant to mutation than others [321].

To cure mitochondrial diseases due to mutated mtDNA, it has been proposed to introduce anti-mtDNA sequence-specific molecules into the mitochondrion, selectively inhibiting replication of mutated genomes and restoring health by permitting the propagation of only the wild-type mitochondrial DNA [322].

	Conclusions

Top Abstract Introduction Classification of Defects Mitochondrial Dysfunction in... Molecular Pathology Mitochondrial Genetics Therapy Conclusions References

 
Mitochondrial medicine began with the description of Luft’s syndrome in 1962. The field developed rapidly after the complete sequence of mitochondrial DNA was determined in 1981 and the methods of molecular biology were applied to study the role of the mitochondrion in the etiology of human diseases.

This unique organelle developed from endocytosed bacteria. Most of the bacterial genes ended up in the host cell nucleus that controls the biosynthesis of the present day mitochondrion. Only 37 genes remained as maternally-inherited mitochondrial DNA. A few of these genes code for the lipophilic, proton-pumping subunits of Complexes I, III, and IV of the electron transport chain, which establish the mitochondrial transmembrane potential that powers the synthesis of ATP. Hence, the expression of both genomes is essential for the proper biosynthesis and function of the mitochondrial oxidative phosphorylation system.

Populations with limited genetic diversity have high risk for rare mitochondrial diseases. Mitochondrial DNA has a much higher mutation rate than nuclear DNA, because it lacks histones and is exposed to radical oxygen species (ROS) generated by the electron transport chain, and the mitochondrial DNA repair system is limited. ROS-induced deletion of fragments of mtDNA promotes premature aging, and migration of the deleted fragments into the nuclear genome has been linked to carcinogenesis. When the amount of mtDNA with mutation or deletion in a cell reaches a tissue-dependent threshold, the cellular metabolism becomes critical and the cell undergoes necrosis.

In specialized tissues, mitochondria perform a number of diverse functions, but the common function in all mitochondria is the generation of ATP. The electron transport chain carries electrons from metabolites, which acts as a fuel, to oxygen, and stores the extracted energy as a membrane potential that is used as needed for ATP or heat generation. However, the oxidative phosphorylation system is vulnerable to structural and functional damage. For instance, a temporary lack of oxygen caused by ischemia and reperfusion increases the generation of ROS. The membrane energy stores can be depleted by excessive membrane leakage due to damage by ROS or due to inappropriate membrane lipid content. Inhibition of enzymes by endogenous or exogenous compounds or lack of or inhibition of the electron carrier Q10, ANT, or other transporters can reduce ATP generation.

Acidic NSAIDs inhibit or uncouple oxidative phosphorylation and induce the "topical phase" of gastrointestinal ulcer formation. In vitro, vacuolating cytotoxin prepared from H. pylori decreases the mitochondrial inner membrane potential of the cultured gastric epithelial cell. If the bacterium causes uncoupling in vivo as well, it might induce the "topical phase," leading to ulcer formation even when a selective COX-2 inhibitor is administered. However, it has not yet been shown that H. pylori uncouples mitochondria in vivo. Aspirin inhibits ß-oxidation, but not electron transport. However, the aspirin metabolite salicylate decouples mitochondria by inducing the mitochondrial permeability transition. Cocaine inhibits Complex I, the poliovirus inhibits Complex II, ceramide inhibits Complex III, and azide, cyanide, chloroform, and methamphetamine inhibit Complex IV. By contrast, melatonin stimulates Complexes I and IV and Gingko biloba stimulates Complexes I and III.

Up to a certain limit, a cell has the ability to counteract reduced synthesis of ATP by synthesizing more mitochondria, as is the case with moderate ethanol use. However, abuse of ethanol or therapy with AZT leads to inhibition of mtDNA replication.

Defective oxidative phosphorylation contributes to atherogenesis, diabetes, Alzheimer’s disease, Parkinson’s disease, and aging. In classical animal models of atherogenesis, cholic acid added to the diet promotes lesion formation. Cholic acid, like INF-, induces the formation of nitric oxide, which inhibits Complex IV. ApoE-null and LDL-receptor-null mice develop atherosclerosis on a diet not supplemented with cholic acid. In the latter case, overexpression of LPL reduces atherosclerosis.

In humans, the presence of apoE4 increases the risk of both atherosclerosis and Alzheimer’s disease. A common feature is the reduced availability of high energy fuel to the oxidative phosphorylation system because of lack of (a) cellular uptake or (b) possibly the binding of apoE4 on intracellular liposome to the imported, apoE-binding part of Complex V. However, the second option has not been demonstrated experimentally.

Oral therapy with -tocopherol and Q10 helps prevent atherosclerosis by protecting the LDL particle, the major carrier of Q10 in the plasma, from peroxidation. Lipoprotein uptake and intracellular synthesis provide Q10 for regulation of oxidative phosphorylation where Q10 is essential as it transfers electrons from Complexes I and II to Complex III. Importantly, statins that inhibit HMG-CoA-reductase lower plasma levels of cholesterol and Q10 because Q10 shares part of its isoprenyl side-chain synthesis pathway with cholesterol. Oral therapy with Q10 normalizes its plasma levels and as therapy can also improve cardiac function in cardiomyopathies.

Much useful knowledge has been revealed by recent research on oxidative phosphorylation. A few therapeutic alternatives are available. Avoiding substances that damage mitochondria and supplementation with compounds that protect mitochondrial structure and function are presently most important. There is a need to develop genetic engineering methods to repair or replace damaged mitochondrial DNA.

	References

Top Abstract Introduction Classification of Defects Mitochondrial Dysfunction in... Molecular Pathology Mitochondrial Genetics Therapy Conclusions References

 

1. Harman D. Aging; a theory based on free radical and radiation chemistry. J Gerontol 1956;11:298–300.[Free Full Text]
2. Luft R, Ikkos D, Palameri G, Ernster L, Afzelius B. A case of severe hypermetabolism of non-thyroid origin with a defect in the maintenance of mitochondrial respiratory control: a correlated clinical, biochemical, and morphological study. J Clin Invest 1962;41:1776–1804.
3. Nass MMK, Nass S. Intramitochondrial fibers with DNA characterisitics. I. Fixation and electron staining reactions. J Cell Biol 1963;19:593–612.[Abstract/Free Full Text]
4. Nass S, Nass MMK. Intramitochondrial fibers with DNA characterisitics. II. Enzymatic and other hydrolytic treatments. J Cell Biol 1963;19:593–612.
5. Schatz G, Racker E. Stable phosphorylating submitochondrial particles from baker’s yeast. Biochem Biophys Res Commun 1966;22:579–584.[Medline]
6. Spiro AJ, Moore CL, Prineas JW, Strasberg PM, Rapin I. A cytochrome-related inherited disorder of the nervous system and muscle. Arch Neurol 1970; 23:103–112.[Abstract/Free Full Text]
7. Jope R, Blass JP. A comparison of the regulation of pyruvate dehydrogenase in mitochondria from rat brain and liver. Biochem J 1975;150:397–403.[Medline]
8. Kunz WS, Kuznetsov AV, Clark JF, Tracey I, Elger CE. Metabolic consequences of the cytochrome c oxidase deficiency in brain of copper-deficient Mo(vbr) mice. J Neurochem 1999;72:1580–1585.[Medline]
9. Engel AG, Angelini C. Carnitine deficiency of human skeletal muscle with associated lipid storage myopathy: a new syndrome. Science 1973;179:899–902.[Abstract/Free Full Text]
10. DiMauro S, DiMauro PM. Muscle carnitine palmityltransferase deficiency and myoglobinuria. Science 1973; 182:929–931.[Abstract/Free Full Text]
11. Anderson S, Bankier AT, Barrell BG, de Bruijn MH, Coulson AR, Drouin J, Eperon IC, Nierlich DP, Roe BA, Sanger F, Schreier PH, Smith AJ, Staden R, Young IG. Sequence and organization of the human mitochondrial genome. Nature 1981;290:457–465.[Medline]
12. Miquel J, Fleming JE. A two-step hypothesis on the mechanisms of in vitro cell aging: cell differentiation followed by intrinsic mitochondrial mutagenesis. Exp Gerontol 1984;19:31–36.[Medline]
13. Wallace DC, Singh G, Lott MT, Hodge JA, Schurr TG, Lezza AM, Elsas LJ 2d, Nikoskelainen EK. Mitochondrial DNA mutation associated with Leber’s hereditary optic neuropathy. Science 1988;24:1427–1430.
14. Linnane AW, Marzuki S, Ozawa T, Tanaka M. Mitochondrial DNA mutations as an important contributor to ageing and degenerative diseases. Lancet 1989;1:642–645.[Medline]
15. Hayakawa M, Hattori K, Sugiyama S, Ozawa T. Age-associated oxygen damage and mutations in mitochondrial DNA in human hearts. Biochem Biophys Res Commun 1992;189:979–985.[Medline]
16. Knight JA. The biochemistry of aging. Adv Clin Chem 2000;35:1–62.[Medline]
17. Fosslien E. Adverse effects of nonsteroidal anti-inflammatory drugs on the gastrointestinal system. Ann Clin Lab Sci 1998;28:67–81.[Abstract]
18. Schoffner JM. Mitochondrial myopathy diagnosis. Neurol Clin 2000;18:105–123.[Medline]
19. Kohn RR. Intrinsic aging in postmitotic cells. In: Aging Gametes (Blandau RJ et al, Eds), Karger, New York, 1975, pp 1–18.
20. Rossignol R, Malgat M, Mazat JP, Letellier T. Threshold effect and tissue specificity. Implication for mitochondrial cytopathies. J Biol Chem 1999;274:33426–33432.[Abstract/Free Full Text]
21. Treem WR, Sokol RJ. Disorders of the mitochondria. Semin Liver Dis 1998;18:237–253.[Medline]
22. Farmer P. Commentary: Human mitochondrial cytopathies. Ann Clin Lab Sci 2000;30:159–162.[Abstract]
23. Elverland HH, Torbergsen T. Audiologic findings in a family with mitochondrial disorder. Am J Otol 1991;12:459–465.[Medline]
24. Corral-Debrinski M, Stepien G, Shoffner JM, Lott MT, Kanter K, Wallace DC. Hypoxemia is associated with mitochondrial DNA damage and gene induction. Implications for cardiac disease. JAMA 1991;266: 1812–1816.[Abstract/Free Full Text]
25. Marin-Garcia J, Goldenthal MJ, Ananthakrishnan R, Mirvis D. Specific mitochondrial DNA deletions in canine myocardial ischemia. Biochem Mol Biol Int 1996;40:1057–1065.[Medline]
26. Kuwabara M, Takenaka H, Maruyama H, Onitsuka T, Hamada M. Effect of prolonged hypothermic ischemia and reperfusion on oxygen consumption and total mechanical energy in rat myocardium: participation of mitochondrial oxidative phosphorylation. Transplantation 1997;64:577–583.[Medline]
27. Trost LC, Lemasters JJ. Role of the mitochondrial permeability transition in salicylate toxicity to cultured rat hepatocytes: implications for the pathogenesis of Reye’s syndrome. Toxicol Appl Pharmacol 1997;147:431–441.[Medline]
28. Pessayre D, Mansouri A, Haouzi D, Fromenty B. Hepatotoxicity due to mitochondrial dysfunction. Cell Biol Toxicol 1999;15:367–373.[Medline]
29. Mortensen SA. Perspectives on therapy of cardiovascular diseases with coenzyme Q10 (ubiquinone). Clin Investig 1993;71(8 Suppl):S116–123.[Medline]
30. Koundouris A, Kass GE, Johnson CR, Boxall A, Sanders PG, Carter MJ. Poliovirus induces an early impairment of mitochondrial function by inhibiting succinate dehydrogenase activity. Biochem Biophys Res Commun 2000;271:610–614.[Medline]
31. Kimura M, Goto S, Wada A, Yahiro K, Niidome T, Hatakeyama T, Aoyagi H, Hirayama T, Kondo T. Vacuolating cytotoxin purified from Helicobacter pylori causes mitochondrial damage in human gastric cells. Microb Pathog 1999;26:45–52.[Medline]
32. Skulachev VP. Membrane electricity as a convertible energy currency for the cell. Can J Biochem 1980;58:161–175.[Medline]
33. Uyemura SA, Santos AC, Mingatto FE, Jordani MC, Curti C. Diclofenac sodium and mefenamic acid: potent inducers of the membrane permeability transition in renal cortex mitochondria. Arch Biochem Biophys. 1997;342:231–235.[Medline]
34. Sjostrand FS. Molecular pathology of Luft disease and structure and function of mitochondria. J Submicrosc Cytol Pathol. 1999;31:41–50.[Medline]
35. DiMauro S, Bonilla E, Lee CP, Schotland DL, Scarpa A, Conn H Jr, Chance B. Luft’s disease. Further biochemical and ultrastructural studies of skeletal muscle in the second case. J Neurol Sci 1976;27:217–232.[Medline]
36. Dartsch PC, Bauriedel G, Schinko I, Weiss HD, Hofling B, Betz E. Cell constitution and characteristics of human atherosclerotic plaques selectively removed by percutaneous atherectomy. Atherosclerosis 1989; 80:149–157.[Medline]
37. Vielhaber S, Feistner H, Schneider W, Weis J, Kunz WS. Mitochondrial complex I deficiency in a female with multiplex arthrogryposis congenita. Pediatr Neurol 2000;22:53–56.[Medline]
38. Tanaka M, Kovalenko SA, Gong JS, Borgeld HJ, Katsumata K, Hayakawa M, Yoneda M, Ozawa T. Accumulation of deletions and point mutations in mitochondrial genome in degenerative diseases. Ann NY Acad Sci 1996;786:102–111.[Medline]
39. Ballinger SW, Patterson C, Yan CN, Doan R, Burow DL, Young CG, Yakes FM, Van Houten B, Ballinger CA, Freeman BA, Runge MS. Hydrogen peroxide- and peroxynitrite-induced mitochondrial DNA damage and dysfunction in vascular endothelial and smooth muscle cells. Circ Res 2000;86:960–966.[Abstract/Free Full Text]
40. Infante JP, Huszagh VA. Secondary carnitine deficiency and impaired docosahexaenoic (22:6n-3) acid synthesis: a common denominator in the pathophysiology of diseases of oxidative phosphorylation and beta-oxidation. FEBS Lett 2000;468:1–5.[Medline]
41. Esposito LA, Kokoszka JE, Waymire KG, Cottrell B, MacGregor GR, Wallace DC. Mitochondrial oxidative stress in mice lacking the glutathione peroxidase-1 gene. Free Radic Biol Med 2000;28:754–766.[Medline]
42. Whereat AF. Oxygen consumption of normal and atherosclerotic intima. Circulation Res 1961;9:571–575.[Abstract/Free Full Text]
43. Whereat AF. Recent advances in experimental and molecular pathology - Atherosclerosis and metabolic disorder in the arterial wall. Exp Mol Path 1967;7:2332–47.
44. Whereat AF. Is atherosclerosis a disorder of intramitochondrial respiration? Ann Intern Med 1970;73: 125–127.
45. Hajjar DP, Smith SC. Focal differences in bioenergetic metabolism of atherosclerosis-susceptible and -resistant pigeon aortas. Atherosclerosis 1980;36:209–222.[Medline]
46. Smith SC, Strout RG, Dunlop WR, Smith EC. Mitochondrial involvement in lipid vacuole formation in cultured aortic cells from white Carneau pigeons. J Atheroscler Res 1966;6:489–496.[Medline]
47. Rosenfeld ME. Inflammation, lipids, and free radicals: lessons learned from the atherogenic process. Semin Reprod Endocrinol 1998;16:249–261.[Medline]
48. Stemme V, Swedenborg J, Claesson H, Hansson GK. Expression of cyclo-oxygenase-2 in human atherosclerotic carotid arteries. Eur J Vasc Endovasc Surg 2000;20:146–152.[Medline]
49. Schonbeck U, Sukhova GK, Graber P, Coulter S, Libby P. Augmented expression of cyclooxygenase-2 in human atherosclerotic lesions. Am J Pathol 1999;155:1281–1291.[Abstract/Free Full Text]
50. Fosslien E. Molecular pathology of cyclooxygenase-2 in neoplasia. Ann Clin Lab Sci 2000;30:3–21.[Abstract]
51. Berberian PA, Ziboh VA, Hsia SL. Inhibition of cholesterol esterification in rabbit aorta by prostaglandin E2. Atherosclerosis 1977;27:213–220.[Medline]
52. Reilly JM, Miralles M, Wester WN, Sicard GA. Differential expression of prostaglandin E2 and interleukin-6 in occlusive and aneurysmal aortic disease. Surgery 1999;126:624–627.[Medline]
53. Delanghe J, Allcock K, Langlois M, Claeys L, De Buyzere M. Fast determination of haptoglobin phenotype and calculation of hemoglobin binding capacity using high pressure gel permeation chromatography. Clin Chim Acta 2000;291:43–51.[Medline]
54. Cid MC, Grant DS, Hoffman GS, Auerbach R, Fauci AS, Kleinman HK. Identification of haptoglobin as an angiogenic factor in sera from patients with systemic vasculitis. J Clin Invest 1993;91:977–985.
55. Stastny JJ, Fosslien E. Quantitative alteration of some aortic intima proteins in fatty streaks and fibro-fatty lesions. Exp Mol Pathol 1992;57:205–214.[Medline]
56. Yamashita G, Ginanni Corradini S, Secknus R, Takabayashi A, Williams C, Hays L, Chernosky AL, Holzbach RT. Biliary haptoglobin, a potent promoter of cholesterol crystallization at physiological concentrations. J Lipid Res 1995;36:1325–1333.[Abstract]
57. Nishina PM, Verstuyft J, Paigen B. Synthetic low and high fat diets for the study of atherosclerosis in the mouse. J Lipid Res 1990;31:859–869.[Abstract]
58. Nakajima T, Okuda Y, Chisaki K, Shin WS, Iwasawa K, Morita T, Matsumoto A, Suzuki Ji, Suzuki S, Yamada N, Toyo-Oka T, Nagai R, Omata M. Bile acids increase intracellular Ca⁺⁺ concentration and nitric oxide production in vascular endothelial cells. Br J Pharmacol 2000;130:1457–1467.[Medline]
59. Zhang F, Subbaramaiah K, Altorki N, Dannenberg AJ. Dihydroxy bile acids activate the transcription of cyclooxygenase-2. J Biol Chem 1998;273:2424–2428.[Abstract/Free Full Text]
60. Srivastava RA, Srivastava N, Averna M. Dietary cholic acid lowers plasma levels of mouse and human apolipoprotein A-I primarily via a transcriptional mechanism. Eur J Biochem 2000;267:4272–4280.[Medline]
61. Gupta S, Pablo AM, Jiang Xc, Wang N, Tall AR, Schindler C. IFN- potentiates atherosclerosis in ApoE knockout mice. J Clin Invest 1997;99:2752–2761.[Medline]
62. Brown GC, Foxwell N, Moncada S. Transcellular regulation of cell respiration by nitric oxide generated by activated macrophages. FEBS Lett 1998;439:321–324.[Medline]
63. Heales SJ, Bolanos JP, Land JM, Clark JB. Trolox protects mitochondrial complex IV from nitric oxide-mediated damage in astrocytes. Brain Res 1994;668: 243–245.[Medline]
64. Schuelke M, Loeffen J, Mariman E, Smeitink J, van den Heuvel L. Cloning of the human mitochondrial 51 kDa subunit (NDUFV1) reveals a 100% antisense homology of its 3'UTR with the 5'UTR of the gamma-interferon inducible protein (IP-30) precursor: is this a link between mitochondrial myopathy and inflammation? Biochem Biophys Res Commun 1998; 245:599–606.[Medline]
65. Todd WJ, Doughri AM, Storz J. Ultrastructural changes in host cellular organelles in the course of the chlamydial developmental cycle. Zentralbl Bakteriol [Orig A] 1976;236:359–373.
66. Morre SA, Stooker W, Lagrand WK, van den Brule AJ, Niessen HW. Microorganisms in the aetiology of atherosclerosis. J Clin Pathol 2000;53:647–654.[Abstract/Free Full Text]
67. Sorlie PD, Nieto FJ, Adam E, Folsom AR, Shahar E, Massing M. A prospective study of cytomegalovirus, herpes simplex virus 1, and coronary heart disease: the atherosclerosis risk in communities (ARIC) study. Arch Intern Med 2000;160:2027–2032.[Abstract/Free Full Text]
68. Lin TM, Jiang MJ, Eng HL, Shi GY, Lai LC, Huang BJ, Huang KY, Wu HL. Experimental infection with bovine herpesvirus-4 enhances atherosclerotic process in rabbits. Lab Invest 2000;80:3–11.[Medline]
69. Wright SD, Burton C, Hernandez M, Hassing H, Montenegro J, Mundt S, Patel S, Card DJ, Hermanowski-Vosatka A, Bergstrom JD, Sparrow CP, Detmers PA, Chao YS. Infectious agents are not necessary for murine atherogenesis. J Exp Med 2000; 191:1437–1442.[Abstract/Free Full Text]
70. Thorngate FE, Rudel LL, Walzem RL, Williams DL. Low levels of extrahepatic nonmacrophage ApoE inhibit atherosclerosis without correcting hypercholesterolemia in ApoE-deficient mice. Arteriosclerosis Thromb Vasc Biol 2000;20:1939–1945.[Abstract/Free Full Text]
71. Van Eck M, Herijgers N, Van Dijk KW, Havekes LM, Hofker MH, Groot PH, Van Berkel TJ. Effect of macrophage-derived mouse ApoE, human ApoE3-Leiden, and human ApoE2 (Arg158—>Cys) on cholesterol levels and atherosclerosis in ApoE-deficient mice. Arterioscler Thromb Vasc Biol 2000;20:119–127.[Abstract/Free Full Text]
72. Alber DG, Powell KL, Vallance P, Goodwin DA, Grahame-Clarke C. Herpesvirus infection accelerates atherosclerosis in the apolipoprotein E-deficient mouse. Circulation 2000;102:779–785.[Abstract/Free Full Text]
73. Witting PK, Pettersson K, Letters J, Stocker R. Anti-atherogenic effect of coenzyme Q10 in apolipoprotein E gene knockout mice. Free Radic Biol Med 2000;29:295–305.[Medline]
74. Lichtman AH, Clinton SK, Iiyama K, Connelly PW, Libby P, Cybulsky MI. Hyperlipidemia and atherosclerotic lesion development in LDL receptor-deficient mice fed defined semipurified diets with and without cholate. Arterioscler Thromb Vasc Biol 1999;19:1938–1944.[Abstract/Free Full Text]
75. Shimada M, Ishibashi S, Inaba T, Yagyu H, Harada K, Osuga JI, Ohashi K, Yazaki Y, Yamada N. Suppression of diet-induced atherosclerosis in low density lipoprotein receptor knockout mice overexpressing lipoprotein lipase. Proc Natl Acad Sci USA 1996;93:7242–7246.[Abstract/Free Full Text]
76. Marsh MM, Walker VR, Curtiss LK, Banka CL. Protection against atherosclerosis by estrogen is independent of plasma cholesterol levels in LDL receptor-deficient mice. J Lipid Res 1999;40:893–900.[Abstract/Free Full Text]
77. Levak-Frank S, Radner H, Walsh A, Stollberger R, Knipping G, Hoefler G, Sattler W, Weinstock PH, Breslow JL, Zechner R. Muscle-specific overexpression of lipoprotein lipase causes a severe myopathy characterized by proliferation of mitochondria and peroxisomes in transgenic mice. J Clin Invest 1995; 96:976–986.
78. Hoff HF, Pepin JM, Morton RE. Modified low density lipoprotein isolated from atherosclerotic lesions does not cause lipid accumulation in aortic smooth muscle cells. J Lipid Res 1991;32:115–214.[Abstract]
79. Floren CH, Albers JJ, Bierman EL. Uptake of chylomicron remnants causes cholesterol accumulation in cultured human arterial smooth muscle cells. Biochim Biophys Acta 1981;663:336–349.[Medline]
80. Goldstein JL, Anderson RG, Buja LM, Basu SK, Brown MS. Overloading human aortic smooth muscle cells with low density lipoprotein-cholesteryl esters reproduces features of atherosclerosis in vitro. J Clin Invest 1977;59:1196–1202.
81. Miniati M, Paci A, Cocci F, Ciarimboli G, Monti S, Pistolesi M. Mitochondria act as a reservoir for the basic amine HIPDM in the lung. Eur Respir J 1996;9:2306–2312.[Abstract]
82. Larsen SU. Reye’s syndrome. Med Sci Law 1997; 37:235–241.[Medline]
83. Duerksen DR, Jewell LD, Mason AL, Bain VG. Coexistence of hepatitis A and adult Reye’s syndrome. Gut 1997;41:121–124.[Abstract/Free Full Text]
84. Feigenbaum A, Bergeron C, Richardson R, Wherrett J, Robinson B, Weksberg R. Premature atherosclerosis with photomyoclonic epilepsy, deafness, diabetes mellitus, nephropathy, and neurodegenerative disorder in two brothers: a new syndrome? Am J Med Genet 1994;49:118–124.[Medline]
85. Corral-Debrinski M, Shoffner JM, Lott MT, Wallace DC. Association of mitochondrial DNA damage with aging and coronary atherosclerotic heart disease. Mutat Res 1992; 275:169–180.[Medline]
86. Bogliolo M, Izzotti A, De Flora S, Carli C, Abbondandolo A, Degan P. Detection of the [4977 bp] mitochondrial DNA deletion in human atherosclerotic lesions. Mutagenesis 1999;14:77–82.[Abstract/Free Full Text]
87. Ferrari R. The role of mitochondria in ischemic heart disease. J Cardiovasc Pharmacol 1996;28:S1–10.
88. Lonnrot K, Tolvanen JP, Porsti I, Ahola T, Hervonen A, Alho H. Coenzyme Q10 supplementation and recovery from ischemia in senescent rat myocardium. Life Sci 1999; 64:315–323.[Medline]
89. Miyazaki Y, Kotaka K, Ogawa K, Satake T, Sugiyama S, Ozawa T. Mechanism of mitochondrial damage after coronary reperfusion. Jpn Circ J 1982;46:974–979.[Medline]
90. Cherkasskaia MD, Matulis AA, Iasaitis AA. Effect of prostaglandin E2 on energy metabolism in isolated rat liver mitochondria.Vopr Med Khim 1982;28:110–114.
91. Crane FL, Sun IL, Crowe RA, Alcain FJ, Low H. Coenzyme Q10, plasma membrane oxidase and growth control. Mol Aspects Med 1994;15:1–11.[Medline]
92. Leake DS, Peters TJ. Lipid accumulation in arterial smooth muscle cells in culture. Morphological and biochemical changes caused by low density lipoproteins and chloroquine. Atherosclerosis 1982; 44:275–291.[Medline]
93. Di Croce L, Vicent GP, Pecci A, Bruscalupi G, Trentalance A, Beato M. The promoter of the rat 3-hydroxy-3-methylglutaryl coenzyme A reductase gene contains a tissue-specific estrogen-responsive region. Mol Endocrinol 1999;13:1225–1236.[Abstract/Free Full Text]
94. Subbaiah PV, Bagdade JD. Oral contraceptive steroids and atherosclerosis: lipogenesis in human arterial smooth muscle cells and dermal fibroblasts in presence of lipoprotein-deficient serum from oral contraceptive users. Artery 1980; 6:437–457.[Medline]
95. Chow JC, Higgins MJ, Rudney H. The inhibitory effect of ATP on HMGCoA reductase. Biochem Biophys Res Commun 1975;63:1077–1084.[Medline]
96. Ursini F, Valente M, Ferri L, Gregolin C. Inactivation of soluble 3-hydroxy-3-methylglutaryl CoA reductase by ATP. FEBS Lett 1977;82:97–101.[Medline]
97. Beisiegel U, Weber W, Havinga JR, Ihrke G, Hui DY, Wernette-Hammond ME, Turck CW, Innerarity TL, Mahley RW. Apolipoprotein E-binding proteins isolated from dog and human liver. Arteriosclerosis 1988;8:288–297.[Abstract/Free Full Text]
98. Vane JR, Bakhle YS, Botting RM. Cyclooxygenases 1 and 2. Ann Rev Pharmacol Toxicol 1998;38:97–120.[Medline]
99. Cocco T, Di Paola M, Papa S, Lorusso M. Arachidonic acid interaction with the mitochondrial electron transport chain promotes reactive oxygen species generation. Free Radicals Biol Med 1999;27:51–59.[Medline]
100. Zeviani M, Mariotti C, Antozzi C, Fratta GM, Rustin P, Prelle A. OXPHOS defects and mitochondrial DNA mutations in cardiomyopathy. Muscle Nerve 1995;3:S170–174.[Medline]
101. Ballinger SW, Shoffner JM, Hedaya EV, Trounce I, Polak MA, Koontz DA, Wallace DC. Maternally transmitted diabetes and deafness associated with a 10.4 kb mitochondrial DNA deletion. Nat Genet 1992;1:11–15.[Medline]
102. Wu LL. Review of risk factors for cardiovascular diseases. Ann Clin Lab Sci 1999;29:127–133.[Abstract]
103. Kagawa Y, Cha SH, Hasegawa K, Hamamoto T, Endo H. Regulation of energy metabolism in human cells in aging and diabetes: Biochem F₀F₁, mtDNA, UCP, and ROS. Biophys Res Commun 1999;266:662–676.[Medline]
104. Gebhart SS, Shoffner JM, Koontz D, Kaufman A, Wallace D. Insulin resistance associated with maternally inherited diabetes and deafness. Metabolism 1996;45:526–531.[Medline]
105. Mokhtar N, Lavoie JP, Rousseau-Migneron S, Nadeau A. Physical training reverses defect in mitochondrial energy production in heart of chronically diabetic rats. Diabetes 1993;42:682–687.[Abstract]
106. Davies KJ, Packer L, Brooks GA. Biochemical adaptation of mitochondria, muscle, and whole-animal respiration to endurance training. Arch Biochem Biophys 1981;209:539–554.[Medline]
107. Cottrell DA, Blakely EL, Borthwick GM, Johnson MA, Taylor GA, Brierley EJ, Ince PG, Turnbull DM. Role of mitochondrial DNA mutations in disease and aging. Ann N Y Acad Sci 2000;908:199–207.[Medline]
108. Chen MC, Schuit F, Eizirik DL. Identification of IL-1beta-induced messenger RNAs in rat pancreatic beta cells by differential display of messenger RNA. Diabetologia 1999;42:1199–203.[Medline]
109. Smith PA, Proks P, Moorhouse A. Direct effects of tolbutamide on mitochondrial function, intracellular Ca⁺⁺ and exocytosis in pancreatic beta-cells. Pflugers Arch 1999;437:577–588.[Medline]
110. Bjarnason I, Hayllar J. Early pathogenic events in NSAID-induced gastrointestinal damage. Ital J Gastroenterol. 1996;28 Suppl 4:19–22.
111. Famaey JP, Whitehouse MW. About some common biochemical and pharmacological properties of bile salts and non steroidal anti-inflammatory drugs. Arch Int Physiol Biochim 1978;86:577–951.[Medline]
112. Whitehouse MW. Biochemical properties of anti-inflammatory drugs: Uncoupling of oxidative phosphorylation in a connective tissue (cartilage) and liver mitochondria by salicylate analogues: relationship of structure to activity. Biochem Pharmacol 1964;13: 319–336.[Medline]
113. Somasundaram S, Rafi S, Hayllar J, Sigthorsson G, Jacob M, Price AB, Macpherson A, Mahmod T, Scott D, Wrigglesworth JM, Bjarnason I. Mitochondrial damage: a possible mechanism of the "topical" phase of NSAID induced injury to the rat intestine. Gut 1997;41:344–353.[Abstract/Free Full Text]
114. Somasundaram S, Hayllar J, Bjarnason I. Effect of indomethacin on rat jejunal ATP, antioxidants as well as structure and function of T84 cell line. Gastroenerol 2000;118:A248 (Abstract # 1446).
115. Moreno-Sanchez R, Bravo C, Vasquez C, Ayala G, Silveira LH, Martinez-Lavin M. Inhibition and uncoupling of oxidative phosphorylation by nonsteroidal anti-inflammatory drugs: study in mitochondria, submitochondrial particles, cells, and whole heart. Biochem Pharmacol 1999;57:743–52.[Medline]
116. Masubuchi Y, Yamada S, Horie T. Possible mechanism of hepatocyte injury induced by diphenylamine and its structurally related nonsteroidal anti-inflammatory drugs. J Pharmacol Exp Ther 2000;292:982–987.[Abstract/Free Full Text]
117. Nieminen AL, Saylor AK, Herman B, Lemasters JJ. ATP depletion rather than mitochondrial depolarization mediates hepatocyte killing after metabolic inhibition. Am J Physiol 1994;267:C67–74.
118. Somasundaram S, Sigthorsson G, Simpson RJ, Watts J, Jacob M, Tavares IA, Rafi S, Roseth A, Foster R, Price AB, Wrigglesworth JM, Bjarnason I. Uncoupling of intestinal mitochondrial oxidative phosphorylation and inhibition of cyclooxygenase are required for the development of NSAID-enteropathy in the rat. Aliment Pharmacol Ther 2000;14:639–650.[Medline]
119. McCarthy CJ, Sweeney E, O’Morain C. Early ultrastructural changes of antral mucosa with aspirin in the absence of Helicobacter pylori. J Clin Pathol 1995;48:994–997.[Abstract/Free Full Text]
120. Petrescu I, Tarba C. Uncoupling effects of diclofenac and aspirin in the perfused liver and isolated hepatic mitochondria of rats. Biochim Biophys Acta 1997; 1318:385–394.[Medline]
121. Mingatto FE, Santos AC, Uyemura SA, Jordani MC, Curti C. In vitro interaction of nonsteroidal anti-inflammatory drugs on oxidative phosphorylation of rat kidney mitochondria: respiration and ATP synthesis. Arch Biochem Biophys 1996;334:303–308.[Medline]
122. Mahmud T, Rafi SS, Scott DL, Wrigglesworth JM, Bjarnason I. Nonsteroidal antiinflammatory drugs and uncoupling of mitochondrial oxidative phosphorylation. Arthritis Rheum 1996;39:1998–2003.[Medline]
123. Fosslien E. Biochemistry of cyclooxygenase (COX)-2 inhibitors and molecular pathology of COX-2 in neoplasia. Crit Rev Clin Lab Sci 2000;37:431–502.[Medline]
124. Emery P, Zeidler H, Kvien TK, Guslandi M, Naudin R, Stead H, Verburg KM, Isakson PC, Hubbard RC, Geis GS. Celecoxib versus diclofenac in long-term management of rheumatoid arthritis. Lancet 1999;354:2106–2111.[Medline]
125. Scott DL, Palmer RH. Safety and efficacy of nabumetone in osteoarthritis: emphasis on gastro-intestinal safety. Aliment Pharmacol Ther 2000;14: 443–452.[Medline]
126. Nassberger L. Effect of cyclosporin A and different vehicles on ATP production in mitochondria isolated from the rat kidney cortex. Pharmacol Toxicol 1990; 67:147–150.[Medline]
127. Wong W, Robinson SH, Tsiftsoglou AS. Relationship of mitochondrial membrane potential to hemoglobin synthesis during Friend cell maturation. Blood 1985; 66:999–1001.[Abstract/Free Full Text]
128. Uribe S, Rangel P, Pena A. Molecular association enhances the toxic effects of non-substituted monoterpene suspensions on isolated mitochondria. Xenobiotica 1991;21:679–688.[Medline]
129. Famaey JP, Whitehouse MW. About some biochemical propertiesof dimethylsulfoxide and three of its homologues: is the acidic function essential for nonsteroidal anti-inflammatory activities? Agents Actions 1974;4:259–263.[Medline]
130. Mhatre SS, Chetty KG, Pradhan DS. Uncoupling of oxidative phosphorylation in rat liver mitochondria following the administration of dimethyl sulphoxide. Biochem Biophys Res Commun 1983;110:325–331.[Medline]
131. Desai SD, Chetty KG, Pradhan DS. Dimethyl sulfoxide elicited increase in cytochrome oxidase activity in rat liver mitochondria in vivo and in vitro. Chem Biol Interact 1988;66:147–55.[Medline]
132. Coleman WB, Cunningham CC. Effects of chronic ethanol consumption on the synthesis of polypeptides encoded by the hepatic mitochondrial genome. Biochim Biophys Acta 1990;101:142–150.
133. Kennedy JM, Kelley SW, Meehan JM. Ventricular mitochondrial gene expression during development and following embryonic ethanol exposure. J Mol Cell Cardiol 1993;25:117–131.[Medline]
134. Fernandez-Checa JC, Kaplowitz N, Garcia-Ruiz C, Colell A, Miranda M, Mari M, Ardite E, Morales A. GSH transport in mitochondria: defense against TNF-induced oxidative stress and alcohol-induced defect. Am J Physiol 1997;273:G7–17.
135. Wieland P, Lauterburg BH. Oxidation of mitochondrial proteins and DNA following administration of ethanol. Biochem Biophys Res Commun 1995; 213:815–819.[Medline]
136. Schilling RJ, Reitz RC. A mechanism for ethanol-induced damage to liver mitochondrial structure and function. Biochim Biophys Acta 1980;603:266–277.[Medline]
137. Montgomery RI, Coleman WB, Eble KS, Cunningham CC. Ethanol-elicited alterations in the oligomycin sensitivity and structural stability of the mitochondrial F₀F₁-ATPase. J Biol Chem 1987;262:13285–13289.[Abstract/Free Full Text]
138. Coleman WB, Cahill A, Ivester P, Cunningham CC. Differential effects of ethanol consumption on synthesis of cytoplasmic and mitochondrial encoded subunits of the ATP synthase. Alcohol Clin Exp Res 1994;18:947–950.[Medline]
139. Bora PS, Farrar MA, Miller DD, Chaitman BR, Guruge BL. Myocardial cell damage by fatty acid ethyl esters. J Cardiovasc Pharmacol 1996;27:1–6.[Medline]
140. Bailey SM, Pietsch EC, Cunningham CC. Ethanol stimulates the production of reactive oxygen species at mitochondrial complexes I and III. Free Radic Biol Med 1999;27:891–900.[Medline]
141. Bailey SM, Cunningham CC. Effect of dietary fat on chronic ethanol-induced oxidative stress in hepatocytes. Alcohol Clin Exp Res 1999;23:1210–1218.[Medline]
142. Bailey SM, Cunningham CC. Acute and chronic ethanol increases reactive oxygen species generation and decreases viability in fresh, isolated rat hepatocytes. Hepatology 1998; 28:1318–1326.[Medline]
143. Nyquist-Battie C, Freter M. Cardiac mitochondrial abnormalities in a mouse model of the fetal alcohol syndrome. Alcohol Clin Exp Res 1988;12:264–267.[Medline]
144. Schapira AH. Mitochondrial involvement in Parkinson’s disease, Huntington’s disease, hereditary spastic paraplegia and Friedreich’s ataxia. Biochim Biophys Acta 1999; 1410:159–170.[Medline]
145. Schapira AH, Cock HR. Mitochondrial myopathies and encephalomyopathies. Eur J Clin Invest 1999; 29:886–898.[Medline]
146. Gibson GE, Sheu KF, Blass JP. Abnormalities of mitochondrial enzymes in Alzheimer disease. J Neural Transm 1998;105:855–870.
147. de la Monte SM, Luong T, Neely TR, Robinson D, Wands JR. Mitochondrial DNA damage as a mechanism of cell loss in Alzheimer’s disease. Lab Invest 2000;80:1323–1335.[Medline]
148. Ramassamy C, Averill D, Beffert U, Bastianetto S, Theroux L, Lussier-Cacan S, Cohn JS, Christen Y, Davignon J, Quirion R, Poirier J. Oxidative damage and protection by antioxidants in the frontal cortex of Alzheimer’s disease is related to the apolipoprotein E genotype. Free Radic Biol Med 1999;27:544–553.[Medline]
149. Prince M, Lovestone S, Cervilla J, Joels S, Powell J, Russ C, Mann A. The association between ApoE and dementia does not seem to be mediated by vascular factors. Neurology 2000;54:397–402.[Abstract/Free Full Text]
150. Gibson GE, Haroutunian V, Zhang H, Park LC, Shi Q, Lesser M, Mohs RC, Sheu RK, Blass JP. Mitochondrial damage in Alzheimer’s disease varies with apolipoprotein E genotype. Ann Neurol 2000; 48:297–303.[Medline]
151. Askanas V, McFerrin J, Baque S, Alvarez RB, Sarkozi E, Engel WK. Transfer of beta-amyloid precursor protein gene using adenovirus vector causes mitochondrial abnormalities in cultured normal human muscle. Proc Natl Acad Sci USA 1996;93:1314–1319.[Abstract/Free Full Text]
152. Ikebe S, Tanaka M, Ohno K, Sato W, Hattori K, Kondo T, Mizuno Y, Ozawa T. Increase of deleted mitochondrial DNA in the striatum in Parkinson’s disease and senescence. Biochem Biophys Res Commun 1990;17:1044–1048.
153. Itoh K, Weis S, Mehraein P, Müller-Hocker J. Defects of cytochrome c oxidase in the substantia nigra of Parkinson’s disease: an immunohistochemical and morphometric study. Mov Disord 1997;12:9–16.[Medline]
154. Greenamyre JT, MacKenzie G, Peng TI, Stephans SE. Mitochondrial dysfunction in Parkinson’s disease. Biochem Soc Symp 1999;66:85–97.[Medline]
155. Cassarino DS, Parks JK, Parker WD Jr, Bennett JP Jr. The Parkinsonian neurotoxin MPP⁺ opens the mitochondrial permeability transition pore and releases cytochrome c in isolated mitochondria via an oxidative mechanism. Biochim Biophys Acta 1999; 1453:49–62.[Medline]
156. Shults CW, Haas RH, Beal MF. A possible role of coenzyme Q10 in the etiology and treatment of Parkinson’s disease. Biofactors 1999;9:267–272.[Medline]
157. Yeh JJ, Lunetta KL, van Orsouw NJ, Moore FD Jr, Mutter GL, Vijg J, Dahia PL, Eng C. Somatic mitochondrial DNA (mtDNA) mutations in papillary thyroid carcinomas and differential mtDNA sequence variants in cases with thyroid tumours. Oncogene 2000;19:2060–2066.[Medline]
158. Capuano F, Varone D, D’Eri N, Russo E, Tommasi S, Montemurro S, Prete F, Papa. Oxidative phosphorylation and F₀F₁-ATP synthase activity of human hepatocellular carcinoma. Biochem Mol Biol Int 1996; 38:1013–1022.[Medline]
159. McEnery MW, Pedersen PL. Diethylstilbestrol. A novel F₀-directed probe of the mitochondrial proton ATPase. J Biol Chem 1986;261:1745–1752.[Medline]
160. Torroni A, Stepien G, Hodge JA, Wallace DC. Neoplastic transformation is associated with coordinate induction of nuclear and cytoplasmic oxidative phosphorylation genes. J Biol Chem 1990; 265:20589–20593.[Abstract/Free Full Text]
161. Luciakova K, Kuzela S. Increased content of natural ATPase inhibitor in tumor mitochondria. FEBS Lett 1984;177:85–88.[Medline]
162. Eapen CE, Madesh M, Balasubramanian KA, Pulimood A, Mathan M, Ramakrishna BS. Mucosal mitochondrial function and antioxidant defences in patients with gastric carcinoma. Scand J Gastroenterol 1998;33:975–981.[Medline]
163. Karlsson KA. The human gastric colonizer Helicobacter pylori: a challenge for host-parasite glycobiology. Glycobiology 2000;10:761–771.[Abstract/Free Full Text]
164. Blanche S, Tardieu M, Rustin P, Slama A, Barret B, Firtion G, Ciraru-Vigneron N, Lacroix C, Rouzioux C, Mandelbrot L, Desguerre I, Rotig A, Mayaux MJ, Delfraissy JF. Persistent mitochondrial dysfunction and perinatal exposure to antiretroviral nucleoside analogues. Lancet 1999;354:1084–1089.[Medline]
165. Bialkowska A, Bialkowski K, Gerschenson M, Diwan BA, Jones AB, Olivero OA, Poirier MC, Anderson LM, Kasprzak KS, Sipowicz MA. Oxidative DNA damage in fetal tissues after transplacental exposure to 3'-azido-3'-deoxythymidine (AZT). Carcinogenesis 2000;21:1059–1062.[Abstract/Free Full Text]
166. Olivero OA, Anderson LM, Diwan BA, Haines DC, Harbaugh SW, Moskal TJ, Jones AB, Rice JM, Riggs CW, Logsdon D, Yuspa SH, Poirier MC. Transplacental effects of 3'azido-2',3'-dideoxythymidine (AZT): tumorigenicity in mice and genotoxicity in mice and monkeys. J Natl Cancer Inst 1997;89:1602–1608.[Abstract/Free Full Text]
167. Hadler HI. Comment: mitochondrial genes and cancer. FEBS Lett 1989;256:230–232.[Medline]
168. Hadler HI, Devadas K, Mahalingam R. Selected nuclear LINE elements with mitochondrial-DNA-like inserts are more plentiful and mobile in tumor than in normal tissue of mouse and rat. J Cell Biochem 1998;68:100–109.[Medline]
169. Shoffner JM. Oxidative phosphorylation disease diagnosis. Ann NY Acad Sci 1999;893:42–60.[Medline]
170. Pfanner N, Neupert W. Transport of F₁-ATPase subunit beta into mitochondria depends on both a membrane potential and nucleoside triphosphates. FEBS Lett 1986;209:152–156.[Medline]
171. Koehler CM. Protein translocation pathways of the mitochondrion. FEBS Lett. 2000;476:27–31.[Medline]
172. Heddi A, Stepien G, Benke PJ, Wallace DC. Coordinate induction of energy gene expression in tissues of mitochondrial disease patients. J Biol Chem 1999;274: 22968–22976.[Abstract/Free Full Text]
173. Enriquez JA, Fernandez-Silva P, Montoya J. Autonomous regulation in mammalian mitochondrial DNA transcription. Biol Chem 1999;380:737–747.[Medline]
174. Kagawa Y, Hamamoto T, Endo H, Ichida M, Shibui H, Hayakawa M. Genes of human ATP synthase: their roles in physiology and aging. Biosci Rep 1997;17:115–146.[Medline]
175. Zaid A, Li R, Luciakova K, Barath P, Nery S, Nelson BD. On the role of the general transcription factor Sp1 in the activation and repression of diverse mammalian oxidative phosphorylation genes. J Bioenerg Biomembr 1999;31:129–135.[Medline]
176. Moraes CT, Kenyon L, Hao H. Mechanisms of human mitochondrial DNA maintenance: the determining role of primary sequence and length over function. Mol Biol Cell 1999;10:3345–3356.[Abstract/Free Full Text]
177. Simpson MV, Chin CD, Keilbaugh SA, Lin TS, Prusoff WH. Studies on the inhibition of mitochondrial DNA replication by 3'-azido-3'-deoxythymidine and other dideoxynucleoside analogs which inhibit HIV-1 replication. Biochem Pharmacol 1989;38:1033–1036.[Medline]
178. Naviaux RK, Markusic D, Barshop BA, Nyhan WL, Haas RH. Sensitive assay for mitochondrial DNA polymerase gamma. Clin Chem 1999;45:1725–1733.[Abstract/Free Full Text]
179. Pezeshkpour G, Illa I, Dalakas MC. Ultrastructural characteristics and DNA immunocytochemistry in human immunodeficiency virus and zidovudine-associated myopathies. Hum Pathol 1991;22:1281–1288.[Medline]
180. Lewis W, Dalakas MC. Mitochondrial toxicity of antiviral drugs. Nat Med 1995;1:417–422.[Medline]
181. Arnaudo E, Dalakas M, Shanske S, Moraes CT, DiMauro S, Schon EA. Depletion of muscle mitochondrial DNA in AIDS patients with zidovudine-induced myopathy. Lancet 1991;337:508–510.[Medline]
182. Dalakas MC, Illa I, Pezeshkpour GH, Laukaitis JP, Cohen B, Griffin JL. Mitochondrial myopathy caused by long-term zidovudine therapy. N Engl J Med 1990; 322:1098–1105.[Abstract]
183. Simpson DM, Citak KA, Godfrey E, Godbold J, Wolfe DE. Myopathies associated with human immunodeficiency virus and zidovudine: can their effects be distinguished? Neurology 1993;43:971–976.[Abstract/Free Full Text]
184. Linnane AW, Degli Esposti M, Generowicz M, Luff AR, Nagley P. The universality of bioenergetic disease and amelioration with redox therapy. Biochim Biophys Acta 1995;1271:191–194.[Medline]
185. Hayakawa M, Ogawa T, Sugiyama S, Tanaka M, Ozawa T. Massive conversion of guanosine to 8-hydroxy-guanosine in mouse liver mitochondrial DNA by administration of azidothymidine. Biochem Biophys Res Commun 1991;176:87–93.[Medline]
186. Lamperth L, Dalakas MC, Dagani F, Anderson J, Ferrari R. Abnormal skeletal and cardiac muscle mitochondria induced by zidovudine (AZT) in human muscle in vitro and in an animal model. Lab Invest 1991;65:742–751.[Medline]
187. Modica-Napolitano JS. AZT causes tissue-specific inhibition of mitochondrial bioenergetic function. Biochem Biophys Res Commun 1993;194:170–177.[Medline]
188. Chio KS, Reiss U, Fletcher B. Peroxidation of subcellular organelles: formation of lipofuscin fluorescent pigments. Science 1969;166:1535–6.[Abstract/Free Full Text]
189. Koobs DH, Schultz RL, Jutzy RV. The origin of lipofuscin and possible consequences to the myocardium. Arch Pathol Lab Med 1978;102:66–68.[Medline]
190. Harman D. Lipofuscin and ceroid formation: the cellular recycling system. Adv Exp Med Biol 1989; 266:3–15.[Medline]
191. Terman A, Brunk UT. Ceroid/lipofuscin formation in cultured human fibroblasts: the role of oxidative stress and lysosomal proteolysis. Mech Ageing Dev 1998;104:277–291.[Medline]
192. Gallucci E, Micelli S, Monticelli G. Pore formation in lipid bilayer membranes made of phosphatidyl-inositol and oxidized cholesterol followed by means of alternating current. Biophys J 1996; 71:824–831.[Medline]
193. Brand MD, Chien LF, Ainscow EK, Rolfe DF, Porter RK. The causes and functions of mitochondrial proton leak. Biochim Biophys Acta 1994;1187:132–139.[Medline]
194. Shigenaga MK, Hagen TM, Ames BN. Oxidative damage and mitochondrial decay in aging. Proc Natl Acad Sci USA 1994;91:10771–10778.[Abstract/Free Full Text]
195. Brown GC, Brand MD. Changes in permeability to protons and other cations at high proton motive force in rat liver mitochondria. Biochem J 1986;234:75–81.[Medline]
196. Brookes PS, Rolfe DF, Brand MD. The proton permeability of liposomes made from mitochondrial inner membrane phospholipids: comparison with isolated mitochondria. J Membr Biol 1997;155:167–174.[Medline]
197. Brown GC, Brand MD. On the nature of the mitochondrial proton leak. Biochim Biophys Acta 1991;1059:55–62.[Medline]
198. Machida K, Tanaka T. Farnesol-induced generation of reactive oxygen species dependent on mitochondrial transmembrane potential hyperpolarization mediated by F₀F₁-ATPase in yeast. FEBS Lett 1999;462:108–112.[Medline]
199. Abramovitch DA, Marsh D, Powell GL. Activation of beef-heart cytochrome c oxidase by cardiolipin and analogues of cardiolipin. Biochim Biophys Acta 1990;1020:34–42.[Medline]
200. Hoch FL. Cardiolipins and biomembrane function. Biochim Biophys Acta 1992;1113:71–133.[Medline]
201. Hübner W, Mantsch HH, Kates M. Intramolecular hydrogen bonding in cardiolipin. Biochim Biophys Acta 1991;1066:166–174.[Medline]
202. Glueck CJ, Lang JE, Tracy T, Sieve-Smith L, Wang P. Evidence that anticardiolipin antibodies are independent risk factors for atherosclerotic vascular disease. Am J Cardiol 1999;83:1490–1494.[Medline]
203. Tucker T, Steiner MR, Steiner S. Inhibition of cardiolipin synthesis following infection with herpes simplex virus. Intervirology 1974;4:249–256.[Medline]
204. Hiura TS, Li N, Kaplan R, Horwitz M, Seagrave JC, Nel AE. The role of a mitochondrial pathway in the induction of apoptosis by chemicals extracted from diesel exhaust particles. J Immunol 2000;165:2703–2711.[Abstract/Free Full Text]
205. Schagger H, Pfeiffer K. Supercomplexes in the respiratory chains of yeast and mammalian mitochondria. EMBO J 2000;19:1777–1783.[Medline]
206. Arnold I, Pfeiffer K, Neupert W, Stuart RA, Schagger H. Yeast mitochondrial F₁F₀-ATP synthase exists as a dimer: identification of three dimer-specific subunits. EMBO J 1998;17:7170–7178.[Medline]
207. Bragg PD, Hou C. Effect of NBD chloride (4-chloro-7-nitrobenzo-2-oxa-1,3-diazole) on the pyridine nucleotide transhydrogenase of Escherichia coli. Biochim Biophys Acta 1999;1413:159–171.[Medline]
208. Hunter DJ, Oganesyan VS, Salerno JC, Butler CS, Ingledew WJ, Thomson AJ. Angular dependences of perpendicular and parallel mode electron paramagnetic resonance of oxidized beef heart cytochrome c oxidase. Biophys J 2000; 78:439–450.[Medline]
209. Loeffen JL, Smeitink JA, Trijbels JM, Janssen AJ, Triepels RH, Sengers RC, van Den Heuvel LP. Isolated complex I deficiency in children: clinical, biochemical and genetic aspects. Hum Mutat 2000;15:123–134.[Medline]
210. Yuan C, Acosta D Jr. Effect of cocaine on mitochondrial electron transport chain evaluated in primary cultures of neonatal rat myocardial cells and in isolated mitochondrial preparations. Drug Chem Toxicol 2000;23:339–348.[Medline]
211. Boess F, Ndikum-Moffor FM, Boelsterli UA, Roberts SM. Effects of cocaine and its oxidative metabolites on mitochondrial respiration and generation of reactive oxygen species. Biochem Pharmacol 2000; 60:615–623.[Medline]
212. Majamaa K, Rusanen H, Remes A, Hassinen IE. Metabolic interventions against complex I deficiency in MELAS syndrome. Mol Cell Biochem 1997; 174:291–296.[Medline]
213. Artuch R, Colome C, Vilaseca MA, Pineda M, Campistol J. Ubiquinone: metabolism and functions. Ubiquinone deficiency and its implication in mitochondrial encephalopathies. Treatment with ubiquinone. Rev Neurol 1999;29:59–63.[Medline]
214. Weber C, Bysted A, Holmer G. Coenzyme Q10 in the diet: daily intake and relative bioavailability. Mol Aspects Med 1997;18(Suppl):S251–4.
215. Pedersen HS, Mortensen SA, Rohde M, Deguchi Y, Mulvad G, Bjerregaard P, Hansen JC. High serum coenzyme Q10, positively correlated with age, selenium and cholesterol, in Inuit of Greenland. Biofactors 1999;9:319–323.[Medline]
216. Bargossi AM, Battino M, Gaddi A, Fiorella PL, Grossi G, Barozzi G, Di Giulio R, Descovich G, Sassi S, Genova ML, et al. Exogenous CoQ10 preserves plasma ubiquinone levels in patients treated with 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors. Int J Clin Lab Res 1994; 24:171–176.[Medline]
217. Leonard DA, Chen HW. ATP-dependent degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in permeabilized cells. J Biol Chem 1987;262:7914–7919.[Abstract/Free Full Text]
218. Ozasa S. Studies on the regulatory factors of 3-hydroxy-3-methylglutaryl CoA reductase(HMG CoA reductase) activity. Hokkaido Igaku Zasshi 1976;51:313–325.[Medline]
219. De Pinieux G, Chariot P, Ammi-Said M, Louarn F, Lejonc JL, Astier A, Jacotot B, Gherardi R. Lipid-lowering drugs and mitochondrial function: effects of HMG-CoA reductase inhibitors on serum ubiquinone and blood lactate/pyruvate ratio. Br J Clin Pharmacol 1996;42:333–337.[Medline]
220. Miyake Y, Shouzu A, Nishikawa M, Yonemoto T, Shimizu H, Omoto S, Hayakawa T, Inada M. Effect of treatment with 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors on serum coenzyme Q10 in diabetic patients. Arzneimittelforschung 1999;49:324–329.[Medline]
221. Ichihara K, Satoh K, Yamamoto A, Hoshi K. Are all HMG-CoA reductase inhibitors protective against ischemic heart disease? Nippon Yakurigaku Zasshi 1999;114(Suppl 1):142P–149.
222. Sugiyama S. HMG CoA reductase inhibitor accelerates aging effect on diaphragm mitochondrial respiratory function in rats. Biochem Mol Biol Int 1998;46:923–931.[Medline]
223. Satoh K, Ichihara K. Effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors on mitochondrial respiration in ischemic rat hearts. Eur J Pharmacol 1995; 292:271–275.[Medline]
224. Satoh K, Yamato A, Nakai T, Hoshi K, Ichihara K. Effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors on mitochondrial respiration in ischaemic dog hearts. Br J Pharmacol 1995;116:1894–1898.[Medline]
225. Folkers K, Langsjoen P, Nara Y, Muratsu K, Komorowski J, Richardson PC, Smith TH. Biochemical deficiencies of coenzyme Q10 in HIV-infection and exploratory treatment. Biochem Biophys Res Commun 1988;153:888–896.[Medline]
226. Rosenfeldt FL, Pepe S, Ou R, Mariani JA, Rowland MA, Nagley P, Linnane AW. Coenzyme Q10 improves the tolerance of the senescent myocardium to aerobic and ischemic stress: studies in rats and in human atrial tissue. Biofactors 1999;9:291–299.[Medline]
227. Nakahara K, Kuriyama M, Sonoda Y, Yoshidome H, Nakagawa H, Fujiyama J, HiguchiI, Osame M. Myopathy induced by HMG-CoA reductase inhibitors in rabbits: a pathological, electrophysiological, and biochemical study. Toxicol Appl Pharmacol 1998;152:99–106.[Medline]
228. Maltese WA, Aprille JR, Green RA. Activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase does not respond to ubiquinone uptake in cultured cells. Biochem J 1987;246:441–447.[Medline]
229. Gudz TI, Tserng KY, Hoppel CL. Direct inhibition of mitochondrial respiratory chain complex III by cell-permeable ceramide. J Biol Chem 1997;272:24154–24158.[Abstract/Free Full Text]
230. Burrows KB, Gudelsky G, Yamamoto BK. Rapid and transient inhibition of mitochondrial function following methamphetamine or 3,4-methylenedioxymethamphetamine administration. Eur J Pharmacol 2000;398:11–18.[Medline]
231. Thompson CH, Green YS, Ledingham JG, Radda GK, Rajagopalan B. The effect of iron deficiency on skeletal muscle metabolism of the rat. Acta Physiol Scand 1993; 147:85–90.[Medline]
232. Lonnrot K, Holm P, Lagerstedt A, Huhtala H, Alho H. The effects of lifelong ubiquinone Q10 supplementation on the Q9 and Q10 tissue concentrations and life span of male rats and mice. Biochem Mol Biol Int 1998;44:727–737.[Medline]
233. Sorgato MC, Galiazzo F, Panato L, Ferguson SJ. Estimation of H⁺-translation stoichieometry of mitochondrial ATPase by comparison of proton-motive forces with clamped phosphorylation potentials in submitochondrial particles. Biochim Biophys Acta 1982;682:184–188.[Medline]
234. Ruben L, Rasmussen H. Phenothiazines and related compounds disrupt mitochondrial energy production by a calmodulin-independent reaction. Biochim Biophys Acta 1981:63:415–422.
235. Kora S, Sado M, Terada. Effect of the plasticizer di-(2-ethylhexyl)phthalate on oxidative phosphorylation in rat liver mitochondria: modification of the function of the adenine nucleotide translocator. J Pharmacobiodyn 1988;11:773–778.[Medline]
236. Chavez E, Cuellar A. Inactivation of mitochondrial ATPase by ultraviolet light. Arch Biochem Biophys 1984;230:511–516.[Medline]
237. Kim SB, Berdanier CD. Oligomycin sensitivity of mitochondrial F₁F₀-ATPase in diabetes-prone BHE/Cdb rats. Am J Physiol 1999;277:E702–707.[Medline]
238. Gegenava GP, Chistiakov VV. Effect of morphine in vitro on the oxidative phosphorylation in rat liver mitochondria. Biull Eksp Biol Med 1975;80:77–79.
239. Schulze K, Witzenbichler B, Christmann C, Schultheiss HP. Disturbance of myocardial energy metabolism in experimental virus myocarditis by antibodies against the adenine nucleotide translocator. Cardiovasc Res 1999;44: 91–100.[Abstract/Free Full Text]
240. Konstantinov IuM, Filippova SN, Vavilin VA, Panov AV, Liakhovich VV. Changes in oxidative phosphorylation in rat liver mitochondria during the development of cholestasis. Vopr Med Khim 1980; 26:498–502.[Medline]
241. Jacotot E, Ravagnan L, Loeffler M, Ferri KF, Vieira HL, Zamzami N, Costantini P, Druillennec S, Hoebeke J, Briand JP, Irinopoulou T, Daugas E, Susin SA, Cointe D, Xie ZH, Reed JC, Roques BP, Kroemer G. The HIV-1 viral protein R induces apoptosis via a direct effect on the mitochondrial permeability transition pore. J Exp Med 2000;191:33–46.[Abstract/Free Full Text]
242. Rossignol R, Letellier T, Malgat M, Rocher C, Mazat JP. Tissue variation in the control of oxidative phosphorylation: implication for mitochondrial diseases. Biochem J 2000; 347:45–53.
243. van Beek JH, van Wijhe MH, Eijgelshoven MH, Hak JB. Dynamic adaptation of cardiac oxidative phosphorylation is not mediated by simple feedback control. Am J Physiol 1999;277:H1375–1384.
244. Clot JP, Baudry M. Effect of thyroidectomy on oxidative phosphorylation mechanisms in rat liver mitochondria. Mol Cell Endocrinol 1982;28:455–469.[Medline]
245. Nelson BD, Luciakova K, Li R, Betina S. The role of thyroid hormone and promoter diversity in the regulation of nuclear encoded mitochondrial proteins. Biochim Biophys Acta 1995;1271:85–91.[Medline]
246. Shears SB, Bronk JR. The influence of thyroxine administered in vivo on the transmembrane protonic electrochemical potential difference in rat liver mitochondria. Biochem J 1979;178:505–507.[Medline]
247. Mohr-Kahaly S, Kahaly G, Meyer J. Cardiovascular effects of thyroid hormones. Z Kardiol 1996;85(Suppl 6):219–231.
248. Molano F, Saborido A, Delgado J, Moran M, Megias A. Rat liver lysosomal and mitochondrial activities are modified by anabolic-androgenic steroids. Med Sci Sports Exerc 1999;31:243–250.[Medline]
249. Simon N, Jolliet P, Morin C, Zini R, Urien S, Tillement JP. Glucocorticoids decrease cytochrome c oxidase activity of isolated rat kidney mitochondria. FEBS Lett 1998;435: 25–28.[Medline]
250. Costa B, Colleoni M. Changes in rat brain energetic metabolism after exposure to anandamide or (9)tetra-hydrocannabinol. Eur J Pharmacol 2000;395:1–7[Medline]
251. Corbisier P, Remacle J. Involvement of mitochondria in cell degeneration. Eur J Cell Biol 1990;51:173–182.[Medline]
252. Corbisier P, Remacle J. Influence of the energetic pattern of mitochondria in cell ageing. Mech Ageing Dev 1993;71: 47–58.[Medline]
253. Wrong O. Distal renal tubular acidosis: the value of urinary pH, pCO₂ and NH⁴⁺ measurements. Pediatr Nephrol 1991;5:249–255.[Medline]
254. Schonfeld P, Struy H. Refsum disease diagnostic marker phytanic acid alters the physical state of membrane proteins of liver mitochondria. FEBS Lett 1999;457:179–183.[Medline]
255. Rolfe DF, Brand MD. Contribution of mitochondrial proton leak to skeletal muscle respiration and to standard metabolic rate. Am J Physiol 1996;271:C1380–1389.
256. Rolfe DF, Newman JM, Buckingham JA, Clark MG, Brand MD. Contribution of mitochondrial proton leak to respiration rate in working skeletal muscle and liver and to SMR. Am J Physiol 1999;276:C692–699.
257. Willis WT, Jackman MR, Bizeau ME, Pagliassotti MJ, Hazel JR. Hyperthermia impairs liver mitochondrial function in vitro. Am J Physiol Regul Integr Comp Physiol 2000; 278:R1240–1246.[Abstract/Free Full Text]
258. Brand MD. The proton leak across the mitochondrial inner membrane. Biochim Biophys Acta 1990;1018:128–133.[Medline]
259. Stuart JA, Brindle KM, Harper JA, Brand MD. Mitochondrial proton leak and the uncoupling proteins. J Bioenerg Biomembr 1999;31:517–525.[Medline]
260. Watanabe A, Nakazono M, Tsutsumi N, Hirai A. AtUCP2: a novel isoform of the mitochondrial uncoupling protein of Arabidopsis thaliana. Plant Cell Physiol 1999;40:1160–1166.[Abstract/Free Full Text]
261. Muzzin P, Boss O, Giacobino JP. Uncoupling protein 3: its possible biological role and mode of regulation in rodents and humans. J Bioenerg Biomembr 1999;31:467–473.[Medline]
262. Ricquier D, Bouillaud F. The uncoupling protein homologues: UCP1, UCP2, UCP3, StUCP and AtUCP. Biochem J 2000;345:161–179.
263. Ricquier D, Miroux B, Cassard-Doulcier AM, Levi-Meyrueis C, Gelly C, Raimbault S, Bouillaud F. Contribution to the identification and analysis of the mitochondrial uncoupling proteins. J Bioenerg Biomembr 1999;31:407–418.[Medline]
264. Nedergaard J, Matthias A, Golozoubova V, Jacobsson A, Cannon B. UCP1: the original uncoupling protein - and perhaps the only one? New perspectives on UCP1, UCP2, and UCP3 in the light of the bioenergetics of the UCP1-ablated mouse. J Bioenerg Biomembr 1999;31:475–491.[Medline]
265. Nicholls DG, Rial E. A history of the first uncoupling protein, UCP1. J Bioenerg Biomembr 1999;31:399–406.[Medline]
266. Jezek P. Fatty acid interaction with mitochondrial uncoupling proteins. J Bioenerg Biomembr 1999;31: 457–466.[Medline]
267. Faggioni R, Shigenaga J, Moser A, Feingold KR, Grunfeld C. Induction of UCP2 gene expression by LPS: a potential mechanism for increased thermogenesis during infection. Biochem Biophys Res Commun 1998;244:75–78.[Medline]
268. Hinz W, Gruninger S, De Pover A, Chiesi M. Properties of the human long and short isoforms of the uncoupling protein-3 expressed in yeast cells. FEBS Lett 1999;462:411–415.[Medline]
269. Renold A, Koehler CM, Murphy MP. Mitochondrial import of the long and short isoforms of human uncoupling protein-3. FEBS Lett 2000;465:135–140.[Medline]
270. Cadenas S, Buckingham JA, Samec S, Seydoux J, Din N, Dulloo AG, Brand MD. UCP2 and UCP3 rise in starved rat skeletal muscle but mitochondrial proton conductance is unchanged. FEBS Lett 1999;462:257–260.[Medline]
271. Lemasters JJ, Qian T, Bradham CA, Brenner DA, Cascio WE, Trost LC, Nishimura Y, Nieminen AL, Herman B. Mitochondrial dysfunction in the pathogenesis of necrotic and apoptotic cell death. J Bioenerg Biomembr 1999; 31:305–319.[Medline]
272. Al-Nasser IA. Salicylate-induced kidney mitochondrial permeability transition is prevented by cyclosporin A. Toxicol Lett 1999;105:1–8.[Medline]
273. Trost LC, Lemasters JJ. The mitochondrial permeability transition: a new pathophysiological mechanism for Reye’s syndrome and toxic liver injury. J Pharmacol Exp Ther 1996;278:1000–1005.[Abstract/Free Full Text]
274. Canton M, Luvisetto S, Schmehl I, Azzone GF. The nature of mitochondrial respiration and discrimination between membrane and pump properties. Biochem J 1995;310: 477–481.
275. Miyoshi H, Tamaki M, Harada K, Murata H, Suzuki M, Iwamura H. Uncoupling action of antibiotic sporaviridins with rat-liver mitochondria. Biosci Biotechnol Biochem 1992;56:1776–1779.[Medline]
276. Paquin B, Laforest MJ, Forget L, Roewer I, Wang Z, Longcore J, Lang BF. The fungal mitochondrial genome project: evolution of fungal mitochondrial genomes and their gene expression. Curr Genet 1997; 31:380–395.[Medline]
277. Lang BF, Gray MW, Burger G. Mitochondrial genome evolution and the origin of eukaryotes. Annu Rev Genet 1999;33:351–97.[Medline]
278. Vellai T, Vida G. The origin of eukaryotes: the difference between prokaryotic and eukaryotic cells. Proc R Soc Lond (B Biol Sci) 1999;266:1571–1577.[Medline]
279. Karlin S, Brocchieri L, Mrazek J, Campbell AM, Spormann AM. A chimeric prokaryotic ancestry of mitochondria and primitive eukaryotes. Proc Natl Acad Sci USA 1999;96: 9190–9195.[Abstract/Free Full Text]
280. Brown JR, Doolittle WF. Archaea and prokaryoteto-eukaryote transition. Microbiol Mol Biol Rev 1997; 61:456–502.[Abstract/Free Full Text]
281. Clarke A. Mitochondrial genome: defects, disease, and evolution. J Med Genet 1990;27:451–456.[Abstract/Free Full Text]
282. Doolittle WF. You are what you eat: a gene transfer ratchet could account for bacterial genes in eukaryotic nuclear genomes. Trends Genet 1998;14:307–311.[Medline]
283. Doolittle WF. Phylogenetic classification and the universal tree. Science 1999;284:2124–2129.[Abstract/Free Full Text]
284. Graff C, Clayton DA, Larsson NG. Mitochondrial medicine: recent advances. J Intern Med 1999; 246:11–23.[Medline]
285. Clayton DA. Vertebrate mitochondrial DNA: a circle of surprises. Exp Cell Res 2000;255:4–9.[Medline]
286. Wallace DC, Ye JH, Neckelmann SN, Singh G, Webster KA, Greenberg BD. Sequence analysis of cDNAs for the human and bovine ATP synthase beta subunit: mitochondrial DNA genes sustain seventeen times more mutations. Curr Genet 1987;12:81–90.[Medline]
287. Reynier P, Chretien MF, Savagner F, Larcher G, Rohmer V, Barriere P, Malthiery Y. Long PCR analysis of human gamete mtDNA suggests defective mitochondrial maintenance in spermatozoa and supports the bottleneck theory for oocytes. Biochem Biophys Res Commun 1998;252:373–377.[Medline]
288. Ruiz-Pesini E, Diez C, Lapena AC, Perez-Martos A, Montoya J, Alvarez E, Arenas J, Lopez-Perez MJ. Correlation of sperm motility with mitochondrial enzymatic activities. Clin Chem 1998;44:1616–1620.[Abstract/Free Full Text]
289. Troiano L, Granata AR, Cossarizza A, Kalashnikova G, Bianchi R, Pini G, Tropea F, Carani C, Franceschi C. Mitochondrial membrane potential and DNA stainability in human sperm cells: a flow cytometry analysis with implications for male infertility. Exp Cell Res 1998;241: 384–393.[Medline]
290. Folgero T, Bertheussen K, Lindal S, Torbergsen T, Oian P. Mitochondrial disease and reduced sperm motility. Hum Reprod 1993;8:1863–1868.[Abstract/Free Full Text]
291. Cummins JM, Kishikawa H, Mehmet D, Yanagimachi R. Fate of genetically marked mitochondrial DNA from spermatocytes microinjected into mouse zygotes. Zygote 1999;7:151–156.[Medline]
292. Tarin JJ, Brines J, Cano A. Long-term effects of delayed parenthood. Hum Reprod 1998;13:2371–2376.[Abstract/Free Full Text]
293. Jansen RP, de Boer K. The bottleneck: mitochondrial imperatives in oogenesis and ovarian follicular fate. Mol Cell Endocrinol 1998;145:81–88.[Medline]
294. Allen JF, Allen CA. A mitochondrial model for premature ageing of somatically cloned mammals. IUBMB Life 1999; 48:3693–72.
295. Sajantila A, Salem AH, Savolainen P, Bauer K, Gierig C, Paabo S. Paternal and maternal DNA lineages reveal a bottleneck in the founding of the Finnish population. Proc Natl Acad Sci U S A 1996;93:12035–12039.[Abstract/Free Full Text]
296. van den Ouweland JM, Maechler P, Wollheim CB, Attardi G, Maassen JA. Functional and morphological abnormalities of mitochondria harbouring the tRNA(Leu)-(UUR) mutation in mitochondrial DNA derived from patients with maternally inherited diabetes and deafness (MIDD) and progressive kidney disease. Diabetologia 1999; 42:485–492.[Medline]
297. Raha S, Merante F, Shoubridge E, Myint AT, Tein I, Benson L, Johns T, Robinson BH. Repopulation of rho0 cells with mitochondria from a patient with a mitochondrial DNA point mutation in tRNA(Gly) results in respiratory chain dysfunction. Hum Mutat 1999;13:245–254.[Medline]
298. Hanna MG, Nelson IP. Genetics and molecular pathogenesis of mitochondrial respiratory chain diseases. Cell Mol Life Sci 1999;55:691–706.[Medline]
299. Torroni A, Wallace DC. Mitochondrial DNA variation in human populations and implications for detection of mitochondrial DNA mutations of pathological significance. J Bioenerg Biomembr 1994;26:261–271.[Medline]
300. Knight JA. Free radicals, antioxidants, and the immune system. Ann Clin Lab Sci 2000;30:145–158.[Abstract]
301. Knight JA. Diseases related to oxygen-derived free radicals. Ann Clin Lab Sci 1995;25:111–121.[Abstract]
302. Barbiroli B, Iotti S, Lodi R. Aspects of human bioenergetics as studied in vivo by magnetic resonance spectroscopy. Biochimie 1998;80:847–853.[Medline]
303. Ramirez-Tortosa MC, Quiles JL, Gil A, Mataix J. Rabbit liver mitochondria coenzyme Q10 and hydroperoxide levels: an experimental model of atherosclerosis. Mol Aspects Med 1997;18(Suppl):S233–236.
304. Singh RB, Shinde SN, Chopra RK, Niaz MA, Thakur AS, Onouchi Z. Effect of coenzyme Q10 on experimental atherosclerosis and chemical composition and quality of atheroma in rabbits. Atherosclerosis 2000;148:275–282.[Medline]
305. Hoppe U, Bergemann J, Diembeck W, Ennen J, Gohla S, Harris I, Jacob J, Kielholz J, Mei W, Pollet D, Schachtschabel D, Sauermann G, Schreiner V, Stab F, Steckel F. Coenzyme Q10, a cutaneous antioxidant and energizer. Biofactors 1999;9:371–378.[Medline]
306. Willis R, Anthony M, Sun L, Honse Y, Qiao G. Clinical implications of the correlation between coenzyme Q10 and vitamin B6 status. Biofactors 1999;9:359–363.[Medline]
307. Mori N, Aoyama H, Murase T, Mori W. Anti-hypercholesterolemic effect of melatonin in rats. Acta Pathol Jpn 1989;39:613–618.[Medline]
308. Aoyama H, Mori N, Mori W. Effects of melatonin on genetic hypercholesterolemia in rats. Atherosclerosis 1988; 69:269–272.[Medline]
309. Martin M, Macias M, Escames G, Reiter RJ, Agapito MT, Ortiz GG, Acuna-Castroviejo D. Melatonin-induced increased activity of the respiratory chain complexes I and IV can prevent mitochondrial damage induced by ruthenium red in vivo. J Pineal Res 2000;28:24224–24228.
310. Mansouri A, Gaou I, De Kerguenec C, Amsellem S, Haouzi D, Berson A, Moreau A, Feldmann G, Letteron P, Pessayre D, Fromenty B. An alcoholic binge causes massive degradation of hepatic mitochondrial DNA in mice. Gastroenterology 1999;117:181–190.[Medline]
311. Williamson BL, Tomlinson AJ, Mishra PK, Gleich GJ, Naylor S. Structural characterization of contaminants found in commercial preparations of melatonin: similarities to case-related compounds from L-tryptophan associated with eosinophilia-myalgia syndrome. Chem Res Toxicol 1998; 11:234–240.[Medline]
312. Brass EP, Hiatt WR. The role of carnitine and carnitine supplementation during exercise in man and in individuals with special needs. J Am Coll Nutr 1998;17:207–215.[Abstract/Free Full Text]
313. Bertelli A, Cerrati A, Giovannini L, Mian M, Spaggiari P, Bertelli AA. Protective action of L-carnitine and coenzyme Q10 against hepatic triglyceride infiltration induced by hyperbaric oxygen and ethanol. Drugs Exp Clin Res 1993; 19:65–68.[Medline]
314. Du G, Willet K, Mouithys-Mickalad A, Sluse-Goffart CM, Droy-Lefaix MT, Sluse FE. EGb 761 protects liver mitochondria against injury induced by in vitro anoxia/reoxygenation. Free Radic Biol Med 1999; 27:596–604.[Medline]
315. Sastre J, Millan A, Garcia de la Asuncion J, Pla R, Juan G, Pallardo, O’Connor E, Martin JA, Droy-Lefaix MT, Vina J. A Ginkgo biloba extract (EGb 761) prevents mitochondrial aging by protecting against oxidative stress Free Radic Biol Med 1998;24:298–304.[Medline]
316. Janssens D, Michiels C, Delaive E, Eliaers F, Drieu K, Remacle J. Protection of hypoxia-induced ATP decrease in endothelial cells by Ginkgo biloba extract and bilobalide. Biochem Pharmacol 1995;50:991–999.[Medline]
317. Janssens D, Remacle J, Drieu K, Michiels C. Protection of mitochondrial respiration activity by bilobalide. Biochem Pharmacol 1999;58:109–119.[Medline]
318. Madsen L, Rustan AC, Vaagenes H, Berge K, Dyroy E, Berge RK. Eicosapentaenoic and docosahexaenoic acid affect mitochondrial and peroxisomal fatty acid oxidation in relation to substrate preference. Lipids 1999;34:951–963.[Medline]
319. Huertas JR, Martinez-Velasco E, Ibanez S, Lopez-Frias M, Ochoa JJ, Quiles J, Parenti Castelli G, Mataix J, Lenaz G. Virgin olive oil and coenzyme Q10 protect heart mitochondria from peroxidative damage during aging. Biofactors 1999;9:337–343.[Medline]
320. Szabo I, Bathori G, Tombola F, Coppola A, Schmehl I, Brini M, Ghazi A, De Pinto V, Zoratti M. Double-stranded DNA can be translocated across a planar membrane containing purified mitochondrial porin. FASEB J 1998;12:495–502.[Abstract/Free Full Text]
321. Tanaka M, Gong J, Zhang J, Yamada Y, Borgeld H, Yagi K. Mitochondrial genotype associated with longevity and its inhibitory effect on mutagenesis. Mech Ageing Dev 2000; 116:65–76.[Medline]
322. Taylor RW, Chinnery PF, Turnbull DM, Lightowlers RN. In vitro genetic modification of mitochondrial function. Hum Reprod 2000 (Suppl 15);2:79–85.

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