Ceramide-dependent caspase 3 activation is prevented by coenzyme Q from plasma membrane in serum-deprived cells

Coenzyme Q (CoQ) is the key factor for the activity of the eukaryotic plasma membrane electron transport chain. Consequently, CoQ is essential in the cellular response against redox changes affecting this membrane. Serum withdrawal induces a mild oxidative stress, which produces lipid peroxidation in membranes. In fact, apoptosis induced by serum withdrawal can be prevented by several antioxidants including CoQ. Also, CoQ can maintain cell growth in serum-limiting conditions, whereas plasma membrane redox system (PMRS) inhibitors such as capsaicin, which compete with CoQ, inhibit cell growth and induce apoptosis. To understand how plasma membrane CoQ prevents oxidative stress-induced apoptosis we have studied the induction of apoptosis by serum withdrawal in CEM cells and its modulation by CoQ. Serum-withdrawal activates neutral sphingomyelinase (N-SMase), ceramide release and caspase-3-related proteases. CoQ addition to serum-free cultures inhibited a 60% N-SMase activation, an 80% ceramide release, and a 50% caspase-3 activity induced by serum deprivation. Caspase activation dependent on ceramide release since C 2 -ceramide was only able to mimic this effect in 10% foetal calf serum cultured cells but not in serum-free cultures. Also, in vitro experiments demonstrated that C 2 -ceramide and ceramide-rich lipid extracts directly activated caspase-3. Taken together, our results indicate that CoQ protects plasma membrane components and controls stress-mediated lipid signals by its participation in the PMRS.     

Relations Between Tocopherol Depletion and Coenzyme Q During Lipid Peroxidation in rat Liver Mitochondria

In order to evaluate different mitochondrial antioxidant systems, the depletion of alpha-tocopherol and the levels of the reduced and oxidized forms of CoQ were measured in rat liver mitochondria during Fe⁺⁺/ascorbate and NADPH/ADP/Fe⁺⁺ induced lipid peroxidation. During the induction phase of malondialdehyde formation, alpha-tocopherol declined moderately to about 80% of initial contents, whereas the total CoQ pool remained nearly unchanged, but reduced CoQ9 continuously declined. At the start of massive malondialdehyde formation, CoQ9 reaches its fully oxidized state. At the same time alpha-tocopherol starts to decline steeply, but never becomes fully exhausted in both experimental systems. Evidently the oxidation of the CoQ9 pool constitutes a prerequisite for the onset of massive lipid peroxidation in mitochondria and for the subsequent depletion of alpha-tocopherol. Trapping of the GSH by addition of dinitrochlorbenzene (a substrate of the GSH transferase), results in a moderate acceleration of lipid peroxidation, but alpha-tocopherol and ubiquinol levels remained unchanged when compared with the controls. Addition of succinate to GSH depleted mitochondria effectively suppressed MDA formation as well as alpha-tocopherol and ubiquinol depletion. The data support the assumption that the protective effect of respiratory substrates against lipid peroxidation in the absence of mitochondrial GSH is mediated by the regeneration of the lipid soluble antioxidants CoQ and alpha-tocopherol.     

Relations Between Tocopherol Depletion and Coenzyme Q During Lipid Peroxidation in rat Liver Mitochondria

In order to evaluate different mitochondrial antioxidant systems, the depletion of alpha-tocopherol and the levels of the reduced and oxidized forms of CoQ were measured in rat liver mitochondria during Fe⁺⁺/ascorbate and NADPH/ADP/Fe⁺⁺ induced lipid peroxidation. During the induction phase of malondialdehyde formation, alpha-tocopherol declined moderately to about 80% of initial contents, whereas the total CoQ pool remained nearly unchanged, but reduced CoQ9 continuously declined. At the start of massive malondialdehyde formation, CoQ9 reaches its fully oxidized state. At the same time alpha-tocopherol starts to decline steeply, but never becomes fully exhausted in both experimental systems. Evidently the oxidation of the CoQ9 pool constitutes a prerequisite for the onset of massive lipid peroxidation in mitochondria and for the subsequent depletion of alpha-tocopherol. Trapping of the GSH by addition of dinitrochlorbenzene (a substrate of the GSH transferase), results in a moderate acceleration of lipid peroxidation, but alpha-tocopherol and ubiquinol levels remained unchanged when compared with the controls. Addition of succinate to GSH depleted mitochondria effectively suppressed MDA formation as well as alpha-tocopherol and ubiquinol depletion. The data support the assumption that the protective effect of respiratory substrates against lipid peroxidation in the absence of mitochondrial GSH is mediated by the regeneration of the lipid soluble antioxidants CoQ and alpha-tocopherol.     

Anti-atherogenic effect of coenzyme Q10 in apolipoprotein E gene knockout mice

Oxidation of low-density lipoprotein (LDL) lipid is implicated in atherogenesis and certain antioxidants inhibit atherosclerosis. Ubiquinol-10 (CoQ10H2) inhibits LDL lipid peroxidation in vitro although it is not known whether such activity occurs in vivo, and, if so, whether this is anti-atherogenic. We therefore tested the effect of ubiquinone-10 (CoQ10) supplemented at 1% (w/w) on aortic lipoprotein lipid peroxidation and atherosclerosis in apolipoprotein E-deficient (apoE-/-) mice fed a high-fat diet. Hydroperoxides of cholesteryl esters and triacylglycerols (together referred to as LOOH) and their corresponding alcohols were used as the marker for lipoprotein lipid oxidation. Atherosclerosis was assessed by morphometry at the aortic root, proximal and distal arch, and the descending thoracic and abdominal aorta. Compared to controls, CoQ10-treatment increased plasma coenzyme Q, ascorbate, and the CoQ10H2:CoQ10 + CoQ10H2 ratio, decreased plasma alpha-tocopherol (alpha-TOH), and had no effect on cholesterol and cholesterylester alcohols (CE-OH). Plasma from CoQ10-supplemented mice was more resistant to ex vivo lipid peroxidation. CoQ10 treatment increased aortic coenzyme Q and alpha-TOH and decreased the absolute concentration of LOOH, whereas tissue cholesterol, cholesteryl esters, CE-OH, and LOOH expressed per bisallylic hydrogen-containing lipids were not significantly different. CoQ10-treatment significantly decreased lesion size in the aortic root and the ascending and the descending aorta. Together these data show that CoQ10 decreases the absolute concentration of aortic LOOH and atherosclerosis in apoE-/- mice.     

A role for reduced coenzyme Q in atherosclerosis?

Substantial evidence implicates oxidative modification of low density lipoprotein (LDL) as an important event contributing to atherogenesis. As a result, the elucidation of the molecular mechanisms by which LDL is oxidized and how such oxidation is prevented by antioxidants has been a significant research focus. Studies on the antioxidation of LDL lipids have focused primarily on alpha-tocopherol (alpha-TOH), biologically and chemically the most active form of vitamin E and quantitatively the major lipid-soluble antioxidant in extracts prepared from human LDL. In addition to alpha-TOH, plasma LDL also contains low levels of ubiquinol-10 (CoQ10H2; the reduced form of coenzyme Q10). Recent studies have shown that in oxidizing plasma lipoproteins alpha-TOH can exhibit anti- or pro-oxidant activities for the lipoprotein's lipids exposed to a vast array of oxidants. This article reviews the molecular action of alpha-TOH in LDL undergoing "mild" radical-initiated lipid peroxidation, and discusses how small levels of CoQ10H2 can represent an efficient antioxidant defence for lipoprotein lipids. We also comment on the levels alpha-TOH, CoQ10H2 and lipid oxidation products in the intima of patients with coronary artery disease and report on preliminary studies examining the effect of coenzyme Q10 supplementation on atherogenesis in apolipoprotein E knockout mice.     

Changes in cerebellar iron metabolism and their prevention by allopurinol in oxidative stress related to acute ethanol administration in rats

An acute ethanol load results in an increased total nonheme iron and cytosolic low molecular weight iron content in rat cerebellum. At the same time the cerebellar susceptibility to lipid peroxidation is enhanced whereas the alpha-tocopherol and ascorbate content is decreased. These changes argue for a cerebellar ethanol-induced oxidative stress, which is prevented by prior allopurinol administration.     

Sensitivity of ATPase-ADPase activities from synaptic plasma membranes of rat forebrain to lipid peroxidation in vitro and the protective effect of vitamin E

The in vitro effects of membrane lipid peroxidation on ATPase-ADPase activities in synaptic plasma membranes from rat forebrain were investigated. Treatment of synaptic plasma membranes with an oxidant generating system (H₂O₂/Fe²⁺/ascorbate) resulted in lipid peroxidation and inhibition of the enzyme activity. Besides, trolox as a water soluble vitamin E analogue totally prevented lipid peroxidation and the inhibition of enzyme activity. These results demonstrate the susceptibility of ATPase-ADPase activities of synaptic plasma membranes to free radicals and suggest that the protective effect against lipid peroxidation by trolox prevents the inhibition of enzyme activity. Thus, inhibition of ATPase-ADPase activities of synaptic plasma membranes in cerebral oxidative stress probably is related to lipid peroxidation in the brain.     

Interaction of ascorbate and alpha-tocopherol enhances antioxidant reserve of erythrocytes during anemia in visceral leishmaniasis

Visceral leishmaniasis (V.L.) is associated with enhanced lipid peroxidation along with impaired function of antioxidant defense system in erythrocytes. The effect of chronic treatment with ascorbate and alpha-tocopherol was studied on erythrocytes in hamsters infected with Leishmania donovani. Combination treatment with both antioxidants proved to be a potential suppressor of lipid hydroperoxide formation as well as hypotonic osmotic lysis during the leishmanial infection. Positive correlations between the depleted levels of erythrocyte ascorbate, GSH and alpha-tocopherol exhibit proportionate alterations in the nonenzymatic antioxidant levels at different stages of infection. Indirect measurement of transmembrane electron transfer as ferricyanide reduction suggests an active participation of endogenous contents of ascorbate and alpha-tocopherol in the protection against oxidative damage of membrane lipids. Cooperative behavior of both antioxidants in the ferricyanide reducing capacity was further evinced by resealing the ghosts in presence of exogenous ascorbate and alpha-tocopherol. Furthermore, intravesicular ascorbate serves in the defense of extravesicular ferricyanide induced oxidation of endogenous alpha-tocopherol. The results suggest an interacting role of ascorbate and alpha-tocopherol in maintaining the antioxidant reserve of erythrocytes during anemia in V.L.     

Protective effect of exogenous coenzyme Q in rats subjected to partial hepatic ischemia and reperfusion.

In a surgical model of liver ischemia lipid peroxidation occurs, as shown by increase of lipid peroxidation end products, endogenous CoQ9 is oxidized and mitochondrial respiration is lowered; however, pre-treatment of the rats by i.p. injection of CoQ10 for 14 days normalizes the above parameters, presumably by way of the observed high extent of reduction of the incorporated quinone; moreover, liver homogenates of the CoQ10-treated rats are more resistant than those of non-treated rats to oxidative stress induced by an azido free radical initiator. This preliminary study suggests that CoQ10 pre-treatment can be of beneficial effect against oxidative damage during liver surgery transplantation.     

Lipid peroxidation associated protein damage in rat brain crude synaptosomal fraction mediated by iron and ascorbate

In crude synaptosomal fractions from rat brain exposed to iron and ascorbate, enhanced lipid peroxidation (more than 3-fold compared to control), loss of protein thiols up to the extent of 40% compared to control, increased incorporation of carbonyl groups into proteins (more than 4.5-fold compared to control) and non-disulphide covalent cross-linking of membrane proteins have been observed. The phenomena are not inhibited by catalase or hydroxyl radical scavengers like mannitol or dimethyl sulphoxide. However, chain breaking antioxidants like alpha-tocopherol and butylated hydroxytoluene prevent both lipid peroxidation and accompanying protein oxidation. It is suggested that in this system lipid peroxidation propagated by the decomposition of preformed lipid hydroperoxides by iron and ascorbate is the primary event and products of the peroxidation process cause secondary protein damage. In view of high ascorbate content of brain and availability of several transition metals, such ascorbate mediated oxidative damage may be relevant in the aetiopathogenesis of several neurodegenerative disorders as well as ageing of brain.     

In vitro lipid peroxidation of human serum catalyzed by cupric ion: antioxidant rather than prooxidant role of ascorbate.

The dual role of ascorbate as an antioxidant and a prooxidant has been clearly documented in the literature. Ascorbate acts as an antioxidant by protecting human serum from lipid peroxidation induced by azo dye-generated free radicals. On the other hand, ascorbate is readily oxidized in the presence of transition metal ions, (especially cupric ion) and accelerates lipid peroxidation in tissue homogenates by producing free radicals. Interestingly, we observed an antioxidant rather than an expected prooxidant role of ascorbate when human serum supplemented with 1.2mmol/L ascorbate underwent lipid peroxidations initiated by 2mmol/L copper sulfate. The antioxidant role of ascorbate was confirmed by studying the conventional thiobarbituric acid reactive substances (TBARS) as well as by observing the protective effect of ascorbate on the copper-induced peroxidation of unsaturated and polyunsaturated fatty acids. The antioxidation protection provided by ascorbate was comparable to that of equimolar alpha-tocopherol when incubated for 24h. However, lipid peroxidation products were lower in serum supplemented with alpha-tocopherol after 48h of incubation. This effect may be attributed to the binding of copper by plpha-tocopherol after serum proteins, thus preventing direct interaction between cupric ions and ascorbate. This proposed mechanism is based on the observation that the concentration of ascorbate decreased more slowly in serum than in phosphate buffer at physiological pH. Our results also indicate an outstanding anti-oxidant property of human serum due to the chelation of transition metal ions (even at high concentrations) by various serum proteins.     

Interaction of nitrogen dioxide with human plasma. Antioxidant depletion and oxidative damage.

Nitrogen dioxide (NO2.) is often present in inhaled air and may be generated in vivo from nitric oxide. Exposure of human blood plasma to NO2. caused rapid losses of ascorbic acid, uric acid and protein thiol groups, as well as lipid peroxidation and depletions of alpha-tocopherol, bilirubin and ubiquinol-10. No increase in protein carbonyls was detected. Supplementation of plasma with ascorbate decreased the rates of lipid peroxidation, alpha-tocopherol depletion and loss of uric acid. Uric acid supplementation decreased rates of lipid peroxidation but not the loss of alpha-tocopherol. We conclude that ascorbic acid, protein -SH groups, uric acid and alpha-tocopherol may be important agents protecting against NO2. in vivo. If these antioxidants are depleted, peroxidation of lipids occurs and might contribute to the toxicity of NO2..     

Coenzyme Q10 supplementation reduces corticosteroids dosage in patients with bronchial asthma

Bronchial asthma is a chronic inflammatory disease of respiratory system, with disturbances in the dynamic balance of oxidant-antioxidant capacity of the lungs. Long-term administration of corticosteroids has been shown to result in mitochondrial dysfunction and oxidative damage of mitochondrial and nuclear DNAs. We previously documented decreased coenzyme Q(10) (CoQ(10)) and alpha-tocopherol concentrations in plasma and blood in corticosteroid-dependent bronchial asthma patients. In the present study we demonstrate that CoQ(10) supplementation reduces the dosage of corticosteroids in these patients. PATIENTS AND METHODS: This was an open, cross-over, randomized clinical study with 41 bronchial asthma patients (13 males, 28 females), ages 25-50 years. All patients suffered from persistent mild to moderate asthma. The patients were divided into two groups, one group receiving standard antiasthmatic therapy and clinically stabilized, and the second group receiving, in addition, antioxidants consisting of CoQ(10) as Q-Gel (120 mg) + 400 mg alpha-tocopherol + 250 mg vitamin C a day. The groups were crossed over at 16 weeks for a total duration of 32 weeks. RESULTS AND CONCLUSIONS: Data show that patients with corticosteroid-dependent bronchial asthma have low plasma CoQ(10) concentrations that may contribute to their antioxidant imbalance and oxidative stress. A reduction in the dosage of corticosteroids required by the patients following antioxidant supplementation was observed, indicating lower incidence of potential adverse effects of the drugs, decreased oxidative stress. This study also demonstrates the significant uptake of CoQ(10) by lung tissue in a rat model using hydrosoluble CoQ(10) (Q-Gel).     

Regeneration of coenzyme Q9 redox state and inhibition of oxidative stress by Rooibos tea (Aspalathus linearis) administration in carbon tetrachloride liver damage

The effect of rooibos tea (Aspalathus linearis) on liver antioxidant status and oxidative stress was investigated in rat model of carbon tetrachloride-induced liver damage. Synthetic antioxidant N-acetyl-L-cysteine (NAC) was used for comparison. Administration of carbon tetrachloride (CCl4) for 10 weeks decreased liver concentrations of reduced and oxidized forms of coenzyme Q9 (CoQ9H2 and CoQ9), reduced -tocopherol content and simultaneously increased the formation of malondialdehyde (MDA) as indicator of lipid peroxidation. Rooibos tea and NAC administered to CCl4-damaged rats restored liver concentrations of CoQ9H2 and alpha-tocopherol and inhibited the formation of MDA, all to the values comparable with healthy animals. Rooibos tea did not counteract the decrease in CoQ9, whereas NAC was able to do it. Improved regeneration of coenzyme Q9 redox state and inhibition of oxidative stress in CCl4-damaged livers may explain the beneficial effect of antioxidant therapy. Therefore, the consumption of rooibos tea as a rich source of natural antioxidants could be recommended as a market available, safe and effective hepatoprotector in patients with liver diseases.     

Protective effect of coenzyme Q10 in simvastatin and gemfibrozil induced rhabdomyolysis in rats

Administration of simvastatin (80 mg/kg, po. evening dose) and gemfibrozil (600 mg/kg, po twice) for 30 days produced significant decrease in the level of reduced glutathione, superoxide dismutase, catalase and increase in the level of lipid peroxidation and various serum parameters (creatine phosphokinase, lactate dehydrogenase, serum glutamate oxaloacetate transaminase, creatinine, urea and blood urea nitrogen). This suggested involvement of oxidative stress in rhabdomyolysis. Increase in the level of reduced glutathione, superoxide dismutase, catalase and decrease in the level of lipid peroxidation and serum parameters after administration of antioxidant CoQ10 (10 mg/kg.ip) proved the protective effect of CoQ10 in rhabdomyolysis.     

Vitamin C suppresses oxidative lipid damage in vivo, even in the presence of iron overload

Ascorbate is a strong antioxidant; however, it can also act as a prooxidant in vitro by reducing transition metals. To investigate the in vivo relevance of this prooxidant activity, we performed a study using guinea pigs fed high or low ascorbate doses with or without prior loading with iron dextran. Iron-loaded animals gained less weight and exhibited increased plasma beta-N-acetyl-D-glucosaminidase activity, a marker of tissue lysosomal membrane damage, compared with control animals. The iron-loaded animals fed the low ascorbate dose had decreased plasma alpha-tocopherol levels and increased plasma levels of triglycerides and F(2)-isoprostanes, specific and sensitive markers of in vivo lipid peroxidation. In contrast, the two groups of animals fed the high ascorbate dose had significantly lower hepatic F(2)-isoprostane levels than the groups fed the low ascorbate dose, irrespective of iron load. These data indicate that 1) ascorbate acts as an antioxidant toward lipids in vivo, even in the presence of iron overload; 2) iron loading per se does not cause oxidative lipid damage but is associated with growth retardation and tissue damage, both of which are not affected by vitamin C; and 3) the combination of iron loading with a low ascorbate status causes additional pathophysiological changes, in particular, increased plasma triglycerides.     

The role of iron in ferritin- and haemosiderin-mediated lipid peroxidation in liposomes.

Ferritin and haemosiderin were shown, by the measurement of malondialdehyde production and loss of polyunsaturated fatty acids, to stimulate lipid peroxidation in liposomes. At pH 7.4 ascorbate was additionally required to achieve peroxidation; however, peroxidation occurred at pH 4.5 in the presence of iron-proteins alone. The damage was completely inhibited by the incorporation of chain-breaking antioxidants (alpha-tocopherol and butylated hydroxytoluene) into the liposomes. Metal chelators (desferrioxamine and EDTA) also completely inhibited lipid peroxidation. These and further results indicate that, at pH 4.5, even in the absence of a reducing agent, iron is released from haemosiderin and can mediate oxidative damage to a lipid membrane.     

Resveratrol inhibition of lipid peroxidation

To define the molecular mechanism(s) of resveratrol inhibition of lipid peroxidation we have utilized model systems that allow us to study the different reactions involved in this complex process. Resveratrol proved (a) to inhibit more efficiently than either Trolox or ascorbate the Fe2+ catalyzed lipid hydroperoxide-dependent peroxidation of sonicated phosphatidylcholine liposomes; (b) to be less effective than Trolox in inhibiting lipid peroxidation initiated by the water soluble AAPH peroxyl radicals; (c) when exogenously added to liposomes, to be more potent than alpha-tocopherol and Trolox, in the inhibition of peroxidation initiated by the lipid soluble AMVN peroxyl radicals; (d) when incorporated within liposomes, to be a less potent chain-breaking antioxidant than alpha-tocopherol; (e) to be a weaker antiradical than alpha-tocopherol in the reduction of the stable radical DPPH*. Resveratrol reduced Fe3+ but its reduction rate was much slower than that observed in the presence of either ascorbate or Trolox. However, at the concentration inhibiting iron catalyzed lipid peroxidation, resveratrol did not significantly reduce Fe3+, contrary to ascorbate. In their complex, our data indicate that resveratrol inhibits lipid peroxidation mainly by scavenging lipid peroxyl radicals within the membrane, like alpha-tocopherol. Although it is less effective, its capacity of spontaneously entering the lipid environment confers on it great antioxidant potential.     

Deficit of coenzyme Q in heart and liver mitochondria of rats with streptozotocin-induced diabetes

Mitochondrial dysfunction and oxidative stress participate in the development of diabetic complications, however, the mechanisms of their origin are not entirely clear. Coenzyme Q has an important function in mitochondrial bioenergetics and is also a powerful antioxidant. Coenzyme Q (CoQ) regenerates alpha-tocopherol to its active form and prevents atherogenesis by protecting low-density lipoproteins against oxidation. The aim of this study was to ascertain whether the experimentally induced diabetes mellitus is associated with changes in the content of endogenous antioxidants (alpha-tocopherol, coenzymes Q9 and Q10) and in the intensity of lipoperoxidation. These biochemical parameters were investigated in the blood and in the isolated heart and liver mitochondria. Diabetes was induced in male Wistar rats by a single intravenous injection of streptozotocin (45 mg x kg(-1)), insulin was administered once a day for 8 weeks (6 U x kg(-1)). The concentrations of glucose, cholesterol, alpha-tocopherol and CoQ homologues in the blood of the diabetic rats were increased. The CoQ9/cholesterol ratio was reduced. In heart and liver mitochondria of the diabetic rats we found an increased concentration of alpha-tocopherol, however, the concentrations of CoQ9 and CoQ10 were decreased. The formation of malondialdehyde was enhanced in the plasma and heart mitochondria. The results have demonstrated that experimental diabetes is associated with increased lipoperoxidation, in spite of the increased blood concentrations of antioxidants alpha-tocopherol and CoQ. These changes may be associated with disturbances of lipid metabolism in diabetic rats. An important finding is that heart and liver mitochondria from the diabetic rats contain less CoQ9 and CoQ10 in comparison with the controls. We suppose that the deficit of coenzyme Q can participate in disturbances of mitochondrial energy metabolism of diabetic animals.     

Stimulation of Antioxidant Enzymes and Lipid Peroxidation by UV-B Irradiation in Thylakoid Membranes of Wheat

In wheat seedlings (Triticum aestivum L. cv. 2329) oxidative stress caused by UV-B radiation led to lipid peroxidation of thylakoid membrane; it was expressed in term of malondialdehyde (MDA) formation. The peroxidation of lipids of thylakoid membrane in isolated chloroplasts was prevented when flavonoids quercetin and rutin were supplied into the incubation medium. The activities of superoxide dismutase, ascorbate peroxidase, and catalase increased during the first hours of UV-B exposure. A comparative study of UV-B and temperature effects showed different profiles of the antioxidant enzymes and MDA, suggesting that these two stresses have distinct sites of action. In addition to quantitative increase in flavonoids, qualitative change in flavonoid composition was also marked during UV-B stress, and a new peak at 330 nm was found as compared to control. This revised version was published online in July 2006 with corrections to the Cover Date.