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RESEARCH COMMUNICATION |
a Departamento de Biología Celular, Facultad de Ciencias, Universidad de Córdoba, Spain
b Department of Foods and Nutrition, Purdue University, West Lafayette, Indiana, 47907 USA
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: lipid peroxidation • oxidative stress • plasma membrane • ubiquinone • vitamin E and selenium deficiency
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-tocopherol) and ubiquinone (coenzyme Q, CoQ) break the free radical chain reaction (3, 4), avoiding lipid peroxidation and protein carboxylation (5). In addition, cell protection against ROS includes enzyme systems that repair ROS-induced damage such as endonucleases, lipases, and proteases (6, 7).
Humans and various animals cannot synthesize vitamin E and require Se. Rats consuming diets with~out vitamin E and Se for 3 wk become deficient in these nutrients, exhibiting markedly reduced levels of -tocopherol in membranes of fast turnover tissue, such as liver, as well as decreased amounts of the soluble Se-dependent GPX (6). Studies to elucidate the mechanisms to supply antioxidant protection in the absence of these nutrients are extremely important because they can provide insight into the adaptive procedures that cells invoke to defend against chronic oxidative stress. A previous work has documented changes in several enzymes playing important roles in metabolism and detoxification in response to vitamin E deficiency (8). However, changes in other antioxidant mechanisms, likely related to a biochemical adaptation to the oxidative stress, have not been fully studied. One well-documented change in rats deficient in vitamin E and Se involves dramatically enhanced Ca2+-independent phospholipase A2 activity (PLA2) (6, 9). This unique form of PLA2 may play a protective role in cells leading to increased metabolism of fatty acid hydroperoxides (10). However, it is likely that many other uncharacterized systems can participate in the adaptive response that cells develop to overcome the absence of vitamin E and Se.
CoQ has attracted much interest because it is the only lipophilic antioxidant that can be synthesized in all organisms (11). In addition to its well-known role in mitochondrial electron transport, CoQ also mediates trans plasma membrane (PM) electron transport. This activity can drive electrons from cytosolic NADH to extracellular ascorbate free radical (AFR) through cytochrome b5 reductase (12, 13), leading to the maintenance of ascorbate and CoQ in their reduced forms, and can thus protect membranes from peroxidations (14). Also, the cytosolic enzymes DT-diaphorase (15, 16) and NADPH-CoQ reductase (17–19) have been proposed to have a role in the maintenance of the reduced state of CoQ. The interaction between ubiquinol (CoQH2) and -tocopherol as a mechanism for membrane lipids protection is well established (20). In addition to the direct reduction of lipid peroxyl radicals by CoQH2 (21, 22), a significant part of its antioxidant function is based on the regeneration of -tocopherol by reducing -tocopheryl radicals, unusually stable free radicals that, if properly repaired, do not propagate the radical chain reaction (20, 23–25).
We report here the antioxidant protection of rat liver PM carried out by CoQ and its reductases as an adaptation to the oxidative stress induced by vitamin E and Se deficiency. This work demonstrates that deficient PM are very sensitive to lipid peroxidation, and this can be prevented by NAD(P)H. Also, vitamin E and Se deficiency leads to an elevated CoQ concentration and results in the enhancement of quinone reductases (cytochrome b5 reductase and DT-diaphorase) at the PM. These results indicate that CoQ-dependent reductase systems can supply reduc~ing equivalents to the PM that alleviate, at least in part, the effects of the oxidative stress caused by vitamin E and Se deficiency.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-tocopherol equivalents/kg diet) and Se (0.228 mg Se/kg diet). The -E -Se rats were fed the basal diet deficient in both nutrients (<0.1 mg/kg for -tocopherol and <0.025 mg/kg for Se). The composition of both diets was as described by Kuo et al. (9). The animals were housed individually and maintained at 22°C with 12 h light–dark cycles. Rats had free access to food and water at all times and were fed with treatment diets for 7 wk. The protocol for these studies was approved by the Animal Care and Use Committee at Purdue University.
Chemicals and reagents
cis-Parinaric acid (cPN) was from Molecular Probes, Inc. (Eugene, Oreg.). CoQ7, CoQ9, -tocopherol, 
-tocopherol, cytochrome c, ascorbate oxidase, and anti-rabbit antibody conjugated to alkaline phosphatase were obtained from Sigma (Spain). Dextran T 500 was from Pharmacia (Sweden) and PEG from Fisher Scientific (Pittsburgh, Pa.). Purification of pig liver cytochrome b5 reductase and the generation of a specific polyclonal antibody were as described (26).
Preparation of PM fractions
Crude membrane fractions were obtained from rat liver homogenates, and PMs were then purified by the two-phase partition method with a phase system composed of 6.4% dextran T 500, 6.4% PEG 3350, 0.25 M sucrose, and 5 mM potassium phosphate buffer, pH 7.8. Membranes were resuspended in 50 mM Tris-HCl, pH 7.6, containing 10% glycerol, 1 mM PMSF, 1 mM EDTA, and 0.1 mM DTT, and stored at -70°C until needed. PM purity was checked by marker enzymes as described (27).
CoQ and -tocopherol determinations
Lipids were extracted with hexane from purified PM and then a high-performance liquid chromatography separation was performed at 0.8 ml/min using a reverse phase LC-18-DB column (25 cmx5 mm, 5 µm particle size; Supelco, Bellefonte, Pa.). The column was equilibrated in mobile phase (buffer A) composed of 50 mM sodium perchlorate in a mixture of ethanol:methanol:water (9.1:0.4:0.5). -Tocopherol, CoQ9, and CoQ10 were gradient-eluted with 100% ethanol containing 50 mM sodium perchlorate (buffer B) as follows: buffer A 100%, 5 min; gradient to 100% buffer B, 2 min; buffer B 100%, 10 min; gradient to 100% buffer A, 2 min; buffer A 100%, 10 min. Monitoring was carried out simultaneously with an electrochemical detector (amphoteric 0.7 V, BioAnalytical Systems, West Lafayette, Ind.) for -tocopherol (28) and a UV monitor (Waters 484 variable wavelength detector) set at 275 nm for CoQ9 and CoQ10. Eluted compounds were quantified by integration of peak areas and comparison with internal standards (-tocopherol and CoQ7, respectively).
Assay for lipid peroxidation
The loss in fluorescence of cPN previously incorporated into PM was used as an indirect indicator of lipid peroxidation (21). Peroxidation was initiated by thermal decomposition of 2 mM 2,2'-azobis(2-amidinopropane) (AAPH) at 37°C. PMs were assayed for lipid peroxidation at 37°C in an incubation medium consisting of 0.2 mM Tris-HCl buffer, pH 7.6, 300 µg PM, and 3 µM cPN in ethanol made up to a final volume of 3 ml. Fluorescence was recorded continuously in a Kontron SFM 25 spectrofluorometer. Wavelengths were 324 nm (slit of 3 nm) for excitation and 413 nm (slit of 11 nm) for emission. Peroxidation rates were expressed as the decay in fluorescence (in %) per min.
Oxidoreductase activities
All assays were carried at 37°C with constant gentle stirring in 50 mM Tris-HCl, pH 7.6, containing 30–100 µg of PM in a final volume of 1 ml. NAD(P)H-CoQ0 reductase was assayed spectrophotometrically by monitoring CoQ0 reduction at 410 nm in assay medium containing 0.2 mM NAD(P)H and 0.2 mM CoQ0. An extinction coefficient of 0.7 mM-1cm-1 was used to calculate the specific activities. NADH-ferricyanide reductase activity was assayed by measuring the decrease in absorbance at 420 nm in a similar medium but using 0.2 mM potassium ferricyanide as electron acceptor instead of CoQ0. The reaction was started with NADH. An extinction coefficient of 1 mM-1cm-1 was used.
NADH-AFR reductase was assayed by measuring the decrease in absorbance at 340 nm upon addition of 66 x10-3 units of ascorbate oxidase to a reaction mixture containing 0.4 mM fresh ascorbate and 0.2 mM NADH. An extinction coefficient of 6.22 mM-1 cm-1 was used. NADH-cytochrome c oxidoreductase was assayed by measuring the increase in absorbance at 550 nm in a medium containing 0.2 mM NADH, 20 µM cytochrome c, and 1 mM KCN. An extinction coefficient of 29.5 mM-1cm-1 was used. The role of superoxide generated by reaction of semiquinones with molecular oxygen was estimated by including 60 cytochrome c units/ml bovine Cu2+/Zn2+ superoxide dismutase (SOD) in the assay for cytochrome c reductase.
DT-diaphorase activity was assayed measuring the dicumarol (10 µM)-sensitive reduction of cytochrome c in a medium containing 50 mM Tris-HCl, pH 7.5, 0.08% Triton X-100, 0.5 mM NAD(P)H, 10 µM menadione, and 77 µM cytochrome c as described (29). Cytosolic NADPH-CoQ reductase was measured according to Takahashi et al. (17–19).
PLA2, protease, SOD, and GPX activities
PLA2 activity was measured in liver cytosol using L--dipalmitoyl[2-palmitoyl-9,10–3H] phosphatidylcholine (specific activity 1850 GBq/mmol; New England Nuclear, Boston, Mass.) as substrate by the method previously described (6). Protease activity was measured in liver cytosols using the EnzChek protease assay kit from Molecular Probes, Inc. The assay uses BODIPYL FL-casein as substrate and was conducted as described in the literature accompanying the kit. SOD activity in membrane preparations was calculated from the inhibition of light-induced, riboflavin-mediated reduction of nitroblue tetrazolium as described (30). To estimate the Se status of rats used in this study, we assayed two Se-dependent enzymes. Classical GPX, which is a soluble enzyme, was assayed using reduced glutathione and H2O2 as substrates. Phospholipid hy~droperoxide glutathione peroxidase (PHGPX), which can be also found membrane bound (31), was assayed as described by Lei et al. (32) using reduced glutathione and phosphatidylcholine hydroperoxide as substrates.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting
Prior to electrophoresis, PMs were suspended in SDS-dithiothreitol loading buffer [10% (v/v) glycerol, 2% (w/v) SDS, 5% (v/v) mercaptoethanol, 0.00125% (w/v) bromophenol blue and 62.6 mM Tris-HCl, pH 6.8]. Samples were then heated for 15–20 min at 45°C before separation by SDS-PAGE (12.5% acrylamide) and blotting onto nitrocellulose sheets. Blots were then stained either with Ponceau's for visualization of total protein or with a polyclonal antibody raised against cytochrome b5 reductase purified from pig liver PM (26) and a secondary antibody coupled to alkaline phosphatase (Sigma). Densitometric quantification of antibody-stained bands was carried out with a Visilog 4.0 Image Analysis software.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-tocopherol in the liver PM of rats fed without vitamin E and Se was about 5% of that in the rats fed with the control diet. Also, GPX activity was reduced to 1% in liver cytosols and PHGPX activity was reduced to about 38% in membranes of rats fed the deficient diet (
Table 1).
Thus, these membranes are appropriate for studying vitamin E and Se deficiency-induced changes, some of which could be related to a protective response against oxidative damage. Since -tocopherol and CoQ coparticipate as intrinsic components of the antioxidant protection system of membranes, we measured CoQ9 and CoQ10 levels in both control and vitamin E and Se-depleted PM to address the influence of these substances on the pool of CoQ. The decrease in PM-associated -tocopherol was accompanied by a significant increase in CoQ homologues of about 38% CoQ9 and 62% CoQ10 in rat liver PM ( Table 1).
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Redox activities of control and vitamin E and Se-depleted PMs
In addition to its role as antioxidant, CoQ participates as electron carrier in some PM-associated redox activities (12). A significant part of NADH-AFR reductase is transmembrane and mediated by CoQ (13). As observed for CoQ levels in the PM, depletion of vitamin E and Se resulted in a significant increase of NADH-AFR reductase (
Table 1). Also, these membranes displayed higher NADH-CoQ0 reductase activity when compared to control membranes. A lower degree of activation was observed for both the NADH-ferricyanide reductase, which represents the bulk of redox activities at the PM, and the NADH-cytochrome c reductase, a marker for cis-electron transport on the inner side of the membrane through the cytochrome b5. Generation of superoxide by reaction of ubisemiquinones with molecular oxygen was not involved in the reduction of cytochrome c by NADH, since this activity was not sensitive to Cu2+/Zn2+ SOD ( Table 1). This effect was specific to the added enzyme since the PM preparations were SOD-free, as indicated by the results of endogenous SOD activity analysis. NADPH-CoQ0 reductase activity of +E +Se PMs was about 10% of that obtained with NADH. However, NADPH-CoQ0 reductase in -E -Se PMs was dramatically increased to levels comparable to those obtained with NADH ( Table 1).
We have measured the NADPH-dependent CoQ10 reductase according to the method of Takahashi et al. (17–19) in order to ascertain whether this novel activity was involved in the CoQ reduction at the PM. This dicumarol resistant activity was about 7 ± 2 pmol min-1 mg-1, and no significant differences were observed between +E +Se and -E -Se PMs.
Cytochrome b5 reductase and DT-diaphorase levels at the PM are increased as a consequence of vitamin E and Se deficiency
The role of NADH-cytochrome b5 reductase as the CoQ-reductase intrinsic to the PM has been well documented (13, 26, 33, 34). We have used a polyclonal antibody raised against pig liver cytochrome b5 reductase (26) to test the possibility that antioxidant nutrient deficiency could also influence the amount of the enzymes responsible for CoQ reduction at the PM. Equivalent amounts of protein from control and depleted membranes were denatured, separated by SDS-PAGE, and blotted onto nitrocellulose. After antibody staining, a single band corresponding to about 32 kDa was found with control membranes. This is consistent with previous studies of cytochrome b5 reductase purified from rat liver PM (35). After 7 wk consumption of a deficient diet, levels of cytochrome b5 reductase were significantly increased ( Fig. 1).
Densitometric quantification showed that vitamin E and Se deficiency induced a 40% increase of the 32 kDa band.
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The cytosolic NAD(P)H-quinone reductase (DT-diaphorase) is another enzyme that may play an important role in maintaining the CoQH2 levels in extramitochondrial membranes (15, 16). Since both NADH and NADPH were effective as electron donors for CoQ0 reduction with -E -Se PMs, we tested whether DT-diaphorase could also contribute to the enhancement of reductase activity observed under vitamin E and Se deficiency. Little dicumarol-sensitive activity could be measured in control membranes, indicating that quinone-reductase activity in these membranes is supported mainly by the cytochrome b5 reductase. However, significant dicumarol-sensitive activities were measured in PMs from -E -Se rat livers ( Table 1). Salt extraction removed DT-diaphorase activity from -E -Se PMs ( Table 1), indicating that ionic interactions may be important for binding of the enzyme, but had no effect on the cytochrome b5 reductase (26).
A role for NAD(P)H-driven electron transport in antioxidant protection in the absence of -tocopherol
Control and depleted PMs were subjected to AAPH-initiated lipid peroxidation to estimate the particular contribution of -tocopherol and CoQ as antioxidants. As expected, liver membranes from -E -Se rats were very sensitive to lipid peroxidation, and fluorescence of cis-parinaric acid declined very rapidly after addition of the azo initiator to the reaction mixture at 37°C ( Fig. 2B).
Membranes from + E + Se rats were more resistant to lipid peroxidation under the same conditions ( Fig. 2A). Preincubation with NADH protected membranes from peroxidation, an effect that was particularly evident in PMs isolated from -E -Se rats. Consistent with results obtained with the CoQ0 reductase activity (see above), incubation of depleted membranes with both NADH and NADPH restored their ability to withstand the oxidative stimulus, reaching levels of peroxidation even lower than those obtained with membranes isolated from +E +Se rats ( Fig. 2A, B). Both nucleotides were able to deliver reducing equivalents to depleted PMs, thus increasing the antioxidant capacity in the absence of -tocopherol.
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To test the role of increased DT-diaphorase binding to the plasma membrane in the enhanced protection against peroxidation, we also assayed salt-extracted membranes for lipid peroxidation. Salt extraction did not affect significantly the resistance of +E +Se membranes to peroxidation. However, after extraction with high-salt, -E -Se PMs lost their ability to withstand peroxidation in the presence of NADPH, but NADH was still effective. Rates of fluorescence decrease of cPN have been summarized in Table 1.
PLA2, quinone reductase and protease activities of liver cytosol
Consistent with previous reports (6, 9), Ca2+-independent PLA2 activity was more than twofold higher in the -E -Se compared to the control cytosols ( Table 1). Most DT-diaphorase resides in the cytosol; consequently, we also measured this activity in soluble fractions isolated from control and -E -Se rat livers. A slight increase in this activity was observed in liver cytosols after 7 wk consumption of the deficient diet ( Table 1). Cytosolic NADPH-CoQ reductase which is insensitive to low concentrations of dicumarol (17–19), was decreased from 152 ± 13 to 110 ± 10 pmol min-1 mg-1 (n=3, P< 0.05) after vitamin E and Se deprivation. Since proteases have been proposed to play a role in PLA2 activation (9), we also measured general protease activity in liver cytosols from both deficient and control rats using a nonspecific protease assay kit. Nonspecific protease activity was significantly lower (30%) in the -E -Se samples compared to controls ( Table 1).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-tocopherol and Se on the endogenous antioxidants related to the CoQ-dependent reductases in the PM so as to test the hypothesis that this system could be increased as a response to the oxidative stress caused by vitamin E and Se deficiency.
PMs were obtained from rats that had been fed diets either adequate or deficient in vitamin E and Se. Diets deficient in a single nutrient were not chosen because only the double deficiency induces the severe oxidative damage that results in adaptive responses during the time frame of several weeks used in this study (6, 9, 36, 37). PMs from deficient rats contained very low amounts of -tocopherol. A similar degree of vitamin E depletion has been observed in the plasma and liver microsomes of rats that consumed diets deficient in vitamin E and Se for an equivalent period of time (6). Also, decreases in two Se-dependent enzymes—soluble GPX (to about 1%) and membrane-bound PHGPX (to about 38%)—were also similar to those previously described (32).
The decrease in -tocopherol and Se was accompanied by an increase in CoQ associated to the PM. CoQ concentration in highly aerobic tissues is elevated after the increase in oxidative metabolism caused by treatment with thyroid hormone, suggesting an adaptation to the increased oxidative activity (11). Treatment with peroxisome proliferators also leads to increased CoQ levels in several rat organs, an effect that can be attributed to the necessity for a higher antioxidant capacity (38). Also, similar increases in CoQ10 associated with the PM have been found in cultured HL-60 cells upon serum withdrawal (39), which produces an oxidative stress to the cells, increasing lipid peroxidation and programmed cell death (40).
In addition to its well-known role as an intermediate redox carrier in mitochondrial electron transport, CoQ is also required for maintaining PM-associated electron transport (12), which can protect membranes from peroxidation (14, 41). CoQ requirement distinguishes trans-PM redox activities from those activities related to cis-electron transport (13). PMs derived from -E -Se rats, which contained elevated amounts of CoQ, also exhibited increased rates of NADH-CoQ0 and -AFR oxidoreductases. NADH-ferricyanide oxidoreductase (which only requires the cytosolic oriented reductase) and NADH-cytochrome c oxidoreductase (which requires both the reductase and the cytochrome b5) were only slightly stimulated. Cytochrome c reduction in the presence of NADH was not mediated by superoxide generation since it was unaffected by Cu2+/Zn2+ SOD.
Compared to vitamin E, CoQH2 appears to be in a particularly favorable position as a lipophilic antioxidant due to its access to enzymic mechanisms capable of regenerating the antioxidant from its oxidized form (11, 25). It has been demonstrated that the cytochrome b5 reductase acts as a phospholipid-dependent CoQ reductase maintaining the pool of reduced CoQ in the PM (26, 33, 34). Other enzymes functioning as extramitochondrial CoQ-reductases include DT-diaphorase (15, 16), a soluble NADPH-CoQ reductase (17–19), and microsomal NADPH-cytochrome P450 reductase (25, 42, 43). Although the bulk of DT-diaphorase activity resides in the cytosol, a minor portion is usually associated with membranes (29). Furthermore, its assay in vitro requires detergent for optimal activity, which is consistent with the possibility that DT-diaphorase may interact with membrane components at the membrane–cytosol interphase (15).
We evaluated the levels of cytochrome b5 reductase, DT-diaphorase, and NADPH-CoQ reductase to detect possible changes induced by vitamin E and Se deficiency in the enzymes that participate as CoQ reductases in the PM. Cytochrome b5 reductase was present in both control and deficient PMs, its expression being increased in the latter membranes. Little DT-diaphorase was present in control membranes, whereas vitamin E and Se deficiency induced a dramatic increase of PM-bound DT-diaphorase activity, which could be extracted by washing with high-salt buffer. No increase in dicumarol-insensitive NADPH-CoQ reductase was measured in PMs from -E -Se rats; this activity was decreased in liver cytosols from deficient rats, which is consistent with a decrease in NADPH-CoQ reductase in cytosols from rats treated with carbon tetrachloride (44)
As expected, membranes depleted in -tocopherol were very sensitive to AAPH-initiated lipid peroxidation. However, preincubation of membranes with NAD(P)H fully restored their ability to withstand the oxidative stress. It has been reported that NADH incubation of plasma membranes causes the reduction of CoQ10 (12). Since prevention of lipid peroxidation was particularly evident in -E -Se plasma membranes, we can conclude that the antioxidant function of NAD(P)H-driven electron transport does not rely merely on vitamin E recycling from its phenoxy radicals, but implies a direct role for CoQH2 in breaking the reaction chain (21, 22). Our results are in accordance with previous evidence for inhibition of lipid peroxidation by CoQH2 in mitochondria and submitochondrial particles depleted of -tocopherol by pentane-extraction (45).
The protective action of NAD(P)H is in contrast to the prooxidant effect of NAD(P)H-driven electron transport, which is known to initiate lipid peroxidation by activating oxygen in the presence of iron (46) and thus could unmask its antioxidant function (25). In studies of lipid peroxidation with liver microsomes in the presence of metal chelators, NADPH protects microsomes from peroxidation in a process requiring CoQ (42, 43). However, unlike results described here, the antioxidant effect of NADPH is less pro~nounced in microsomes from vitamin E-deficient rats than in microsomes from normal rats, suggesting that vitamin E is required for CoQ-dependent protective effects of NADPH (42). We report here much greater protection by NADH in PMs derived from -E -Se rats than in PMs obtained from control rats. This apparent discrepancy can be explained because rats used in the cited studies were fed a diet deficient only in vitamin E for a similar period, and oxidative stress in rats deficient in vitamin E alone might not be severe enough to induce the protective response (6, 9, 36, 37).
After salt extraction, -E -Se PM lost their ability to withstand peroxidation in the presence of NADPH, but NADH was still effective. Thus, our results support that DT-diaphorase translocation to the PM may have an important role increasing antioxidant protection under oxidative stress, whereas the intrinsic cytochrome b5 reductase can account for constitutive NADH-mediated protection. The cytochrome b5 reductase reduces CoQ through a one-electron mechanism generating both the antioxidant CoQH2 and the ubisemiquinone radical, which can concomitantly reduce the -tocopheryl radical, forming reduced -tocopherol and CoQ (25, 33, 34, 47). Oxidative stimuli and various xenobiotics induce DT-diaphorase and other phase 2 detoxication enzymes that contain antioxidant responsive elements in their promoter regions (48–50). Accumulation of ROS during antioxidant nutrient deficiency could activate transcription of DT-diaphorase and increase enzyme binding to the PM.
As reported previously, PLA2 activity was increased in liver cytosols of -E -Se rats (6, 9). This enzyme may play a protective role, leading to increased metabolism of fatty acid hydroperoxides (6). A previous work has suggested that intracellular forms of PLA2 may undergo activation by proteolysis (51), and it has been proposed that deficiency in vitamin E and Se may lead to proteolytic activation of the form of PLA2 playing a specific role in the metabolism of oxidized phospholipids (9). Thus, it is possible that proteolysis might then be a common mechanism for the activation of protective enzymes induced by oxidative stresses. A decrease in nonspecific protease activity was measured in rat liver cytosols after 7 wk of deficient diet consumption. Our results are consistent with a decrease in intracellular proteolysis in cultured cells under severe oxidative stresses (52). Mild oxidative stress increases intracellular proteolysis by modification of cellular proteins, which results in increased susceptibility to proteolytic degradation, and not by enhanced expression of intracellular proteases (52).
In conclusion, our results show that the CoQ-reductase systems are inducible mechanisms whose activation must be considered as part of the protective cellular response against oxidative injuries.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Abbreviations: AAPH, 2,2'-azobis(2-amidinopropane)hydrochloride; AFR, ascorbate free radical; CoQ, coenzyme Q; CoQH2, ubiquinol; cPN, cis-parinaric acid; DT-diaphorase; cytosolic NAD(P)H-quinone reductase; E, vitamin E; GPX, glutathione peroxidase; PHGPX, phospholipid hydroperoxide glutathione peroxidase; PLA2, phospholipase A2 activity; PM, plasma membrane; ROS, reactive oxygen species; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Se, selenium; SOD, superoxide dismutase;
Received for publication March 6, 1998.
Revision received July 14, 1998.
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