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Department of Metabolism and Pathological Biochemistry and * Department of Ultrastructures, Istituto Superiore di Sanità, 00161 Rome, Italy
3To whom correspondence should be addressed. E-mail: masellar{at}iss.it.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-glutamylcisteine synthetase (GCS), GR, and GPx to control values. More importantly, we observed significant overtranscription and increased activities of two antioxidative enzymes, GPx and GR, compared with controls when the biophenols were present in the medium for 2 h and then removed before LDL exposure, or when the cells were exposed to the antioxidants alone for up to 24 h. Our findings suggest that the activation of mRNA transcription of GSH-related enzymes represents an important mechanism in phenolic antioxidative action.
KEY WORDS: • oxidized LDL • biophenols • olive oil • glutathione • oxidative stress
Oxidized LDL (oxLDL)4 have been reported to play an important role in the pathogenesis of atherosclerosis (1). When subjected to oxidative stress, the cells of the arterial wall, i.e., endothelial cells, smooth muscle cells, and macrophages, can oxidize LDL in vitro (2,3). Macrophage-mediated oxidation of LDL depends on the intracellular balance between cellular oxygenase products, i.e., superoxide anions, and antioxidant content such as glutathione (GSH) and its related enzymes glutathione peroxidase (GPx), glutathione reductase (GR), and -glutamilcysteine synthetase (GCS) (4–6).
Phenolic antioxidants, naturally occurring in vegetables, fruits, and beverages (7) such as tea and red wine, may play a key role in the prevention of cardiovascular diseases (8). Phenol intake has been reported to be inversely related to mortality from coronary heart disease and to the incidence of myocardial infarction (9–11). Experimental studies demonstrated that LDL oxidation is substantially reduced by consumption of several phenolic compounds (12,13). Moreover, some flavonoids can modulate the activity of enzymes involved in the cell-mediated oxidation of LDL and in the preservation of GSH (14–17).
Extra virgin olive oil, the typical added fat of the Mediterranean diet, has been associated with a reduced incidence of risk factors for coronary heart disease. This is partly due to the high amount of monounsaturated fatty acids (18), but it is likely to be also the result of the presence of phenolic compounds that have strong antioxidative power. Although several phenolic compounds, e.g., those in green tea and licorice root, were demonstrated to inhibit macrophage cell-mediated oxidation of LDL (19,20), to our knowledge, little information exists concerning similar activity or related mechanisms for the biophenols contained in extra virgin olive oil.
The aim of this study, therefore, was to analyze the effects of two phenolic compounds contained in extra virgin olive oil, i.e., protocatechuic acid and oleuropein, on LDL oxidation mediated by the J774 A.1 murine macrophage-like cells, which express CD14 and are considered to be a useful model system for the study of LDL oxidation processes (21). We examined the effects of phenolic compounds on time-dependent variations in intracellular redox balance during the oxidative process by evaluating modifications in the production of reactive oxygen species (ROS), in GSH content and related enzyme activities and/or gene expression (GPx, GR, and -GCS). We also assessed whether the antioxidants added to the cells have any direct effect on the modulation of intracellular redox balance, and on activity and gene expression of GSH-related enzymes.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Methods
Cell culture. J774 A.1 cells (5 x 105) were seeded in a 25-cm2 flask (Falcon) and grown in RPMI 1640 medium, containing 0.2 mmol/L glutamine, 10 U/mL antibiotics and 10% FCS at 37°C, 5% CO2.
LDL isolation. LDL were isolated from pooled fresh plasma of healthy volunteers by density gradient ultracentrifugation in a vertical rotor (Beckman Vti 50) at 180,000 x g for 2 h at 10°C (22). The isolated LDL were dialyzed with a centrifugal filter device (Millipore) with a molecular weight cut-off of 5000 at 4°C. Briefly, LDL were diluted 1:2 with PBS and centrifuged 5 times at 1300 x g for 1 h at 10°C according to the manufacturer’s instructions.
LDL oxidation by J774 A.1 macrophage-like cells. One milliliter of the cell suspension at a density of 5 x 108 cells/L was added to each well of a 6-well culture plate and incubated at 37°C in 5% CO2 for 2 h. The cells were washed twice with PBS, and 1 mL of phenol red–free DMEM containing 0.2 mmol/L glutamine, 10 kU/L antibiotics, 2% Ultroser G, and LDL (0.2 mg protein) was added to the plate and incubated at 37°C in the presence of 2 µmol/L CuSO4 for 6, 12, and 24 h. The oxidation was stopped by the addition of 1 mmol/L EDTA and 20 µmol/L BHT to prevent artifact formation of LDL oxidation. The culture medium was collected and centrifuged at 200 x g for 10 min to remove detached cells, and used for analytical determinations.
In the experiments that evaluated the antioxidant activity of extra virgin olive oil phenols, protocatechuic acid or oleuropein was added to the cells at different concentrations (range 1.5–25 µmol/L), following two experimental procedures. In procedure A, the biophenols were added to the cells with the LDL and left in the medium throughout the experimental period. In procedure B, the antioxidants were added to the cells for 2 h and then removed; the culture medium was renewed after washing the cells twice with PBS before the addition of LDL.
On the basis of these preliminary experiments (data not shown), the lowest concentrations able to counteract cell-mediated oxidation of LDL were determined. When protocatechuic acid or oleuropein was used, concentrations of 3 and 25 µmol/L were chosen for procedures A and B, respectively. In all experiments, untreated cells were used as a control; as further controls, LDL were incubated in the absence of cells (cell-free LDL) and J774 A.1 cells were incubated with biophenols alone. Experiments were replicated 4 times for each protocol. Each experimental treatment was performed in duplicate for each replication. The duplicates were averaged and this mean was used in the statistical analyses.
TBARS concentration. The extent of lipid peroxidation was determined by measuring the formation of TBARS in the culture medium containing LDL, according to the method of Niehaus and Samuelsson (23). Results were expressed as nmol/mg LDL protein.
Relative electrophoretic mobility (REM). Changes in electrophoretic mobility due to the oxidative modification of LDL were evaluated on 0.5% agarose gel (LipoGel Paragon, Beckman). The agarose gel was electrophoresed in barbital buffer, pH 8.6 (0.05 ionic strength) at 90 V, 40 mA, for 90 min. The electrophoretic mobility was expressed as the ratio between the distance of migration of cell-incubated LDL and that of native LDL. The assay was performed on culture medium containing LDL and on cell-free LDL resuspended in the same medium.
Protein concentration. Protein concentrations in the final LDL and in cell culture preparations were determined with Bradford’s method using bovine serum albumin (BSA) as the standard (24).
Intracellular ROS. Cells (5 x 105) were incubated in 490 µL of HBSS, pH 7.4, with 5 µL of DHE or DHR123 in polypropylene test tubes for 15 min at 37°C as previously described (25). To exclude propidium iodide (PI)-positive cells from the analysis of H2O2 production, we performed a double-staining procedure with DHR123/PI; to estimate the percentage of dead cells in samples stained with DHE (which emits in FL2 channel like PI), we incubated parallel tubes with PI for 15 min at 37°C.
Total intracellular GSH levels. Intracellular GSH content was evaluated using CM-H2DCFDA. Untreated and treated cells (5 x 105) were incubated with 10 µmol/L of CM-H2DCFDA for 15 min at 37°C in HBSS supplemented with 5 g/L BSA. Cells exposed to 7.5 µmol/L L-buthionine-[S,R]-sulfoximine (Sigma), a GSH depleting drug, for 16 h were considered as negative controls (25). PI-positive cells were excluded from our analysis.
Glutathione reductase and glutathione peroxidase activities. Enzymatic activities of GR and GPx were measured by colorimetric assays, based on the oxidation and reduction of NADPH/NADP (GR-340, GPx-340, Oxis International). Samples were analyzed on a Beckman 640 spectrophotometer equipped with 25°C thermostated cuvette holder, measuring the change in absorbance at 340 nm as a function of time.
Extraction of total RNA and semiquantitation by RT-PCR.
Total RNA was extracted by the TRIZOL isolation method (Gibco, BRL). The isolated RNA was used for RT-PCR analysis. PCR was performed using the following couples of primers: 5'-GGA CAG CCC TAC GGA GGA AC-3' and 5'-GGC TTG GAA TGT CAC CTG GA-3' for amplification of GCS; 5'-ATG TGG AAC ACA GCA GTG CA-3' and 5'-GTG CAC TTG GAA CTG ATG AG-3' for GR; 5'-CCT CAA GTA CGT CCG ACC TG-3' and 5'-TAG GAG TTG CCA GAC TGC TG-3' for GPx; 5'-GGA AAG ACT GTT CCA AAA ACA GTG-3' and 5'-GTC TTG GTG CTC TCC ACC TTC CG-3' for amplification of cyclophilin B as a housekeeping gene. The samples were incubated in an automated heat-block (Minicycler, MJ Research) using the following parameters: 94°C for 1 min, 58°C for 1 min, and 72°C for 2 min (using 31 cycles) for GR and GPx amplification; 94°C for 30 s, 65°C for 30 s, and 72°C for 1 min for GCS (using 35 cycles). The PCR products were electrophoresed on a 1.5% agarose gel containing ethidium bromide. Densitometric analysis was performed by a molecular imager FX (Bio-Rad).
Statistical analysis. The results are expressed as means ± SEM of at least 4 experiments performed in duplicate. Comparisons between 2 groups were by Student’s t test. ANOVA and the Student-Newman-Keuls multiple comparison test were used when >2 groups were compared. Differences were considered significant when P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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TBARS production in culture media. J774 A.1 significantly increased the production of TBARS in the medium compared with the cell-free LDL at 12 and 24 h (Table 1). When protocatechuic acid or oleuropein was added with LDL, or when they were added for 2 h and then removed from the medium before LDL addition, cell-mediated oxidation of the lipoproteins was completely inhibited as shown by the lack of increase in TBARS content at 24 h (Table 1).
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Redox imbalance
Intracellular redox balance. During the oxidation of LDL, an increase in O2·- and H2O2 production and a decrease in GSH content were observed in the J774 A.1 cells. After 6 h of incubation, O2·- and H2O2 (Fig. 1, A and B respectively) were already increased (P < 0.001); GSH content (Fig. 1C) did not change within 6 h, but from h 12 on, it decreased progressively, reaching the lowest values at h 24, probably as a consequence of its detoxifying activity in response to ROS overproduction. Protocatechuic acid and oleuropein completely counteracted these adverse effects at every time point tested in both procedures.
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The expression of the enzyme GCS gene in J774 A.1 cells incubated with LDL alone was 
100% greater at 24 h than in control cells and cells treated with the biophenols in both procedures (Fig. 3A).
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The mRNA content for GPx was not influenced by LDL or biophenols in procedure A. Conversely, when the cells were incubated with biophenols 2 h before the addition of LDL (procedure B), protocatechuic acid and oleuropein were increased significantly compared with the control (P < 0.05 and P < 0.01, respectively) (Fig. 3C).
As described in the Materials and Methods, all of the experiments were performed including cells exposed to the biophenols alone for up to 24 h to determine their possible direct influence. The biophenols influenced the variables tested similarly for both procedures, although the strongest effects were observed when they were added for 2 h and then removed from the culture medium. In particular, we observed that protocatechuic acid and oleuropein did not have any affect on ROS production or GSH content compared with control cells (data not shown), whereas they did significantly increase GR and GPx gene expression within 2 h and the related activities within 6 h after treatment with biophenols (Fig. 4A and B). GCS expression did not differ from that of controls (data not shown).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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LDL oxidation is thought to occur in the arterial wall, and it was demonstrated that macrophages, as well as endothelial cells and smooth muscle cells, oxidize LDL in vitro in the presence of catalytic amounts of transition metal ions. Some lines of evidence have also suggested that oxLDL can in turn exert powerful cytotoxic effects on macrophages, thus contributing to the development of cell-poor, lipid-rich cores of advanced atherosclerotic plaques (26). Several phenolic antioxidative compounds contained in extra virgin olive oil were shown to inhibit the LDL oxidation induced by cupric ions in vitro (27). It was demonstrated that LDL isolated from rabbits fed a standard diet modified by the addition of specific extra virgin olive oil phenolic compounds had an increased resistance to oxidation (28,29).
Although the metabolism, bioavailability, and clinical relevance of the different classes of biophenol compounds are subject to debate, phenolic compounds of extra virgin olive oil are absorbed and, as reported (30), bind to plasma lipoproteins after consumption. Moreover, a previous study suggested that the daily consumption of extra virgin olive oil increased the phenolic compounds in plasma and the resistance of LDL against ex vivo oxidation in hyperlipidemic patients (31). The maximum concentration of protocatechuic acid and oleuropein in extra virgin olive oil was estimated to be 19.5 and 5.6 µmol/kg, respectively (32); thus, if we assume a daily consumption of 50 g of olive oil (33), the intake was very low. However, we should also take into account the following: 1) oleuropein normally associates with its derivatives hydroxytyrosol and aglicone (reaching total concentrations of 125 µmol/(kg · d) (32); and 2) protocatechuic acid is recognized as one of the metabolites formed during the absorption of complex polyphenols (34,35). For example, proanthocyanidins and catechins are normally present at high concentrations in vegetables and fruits. This can considerably increase dietary intake. Absorbed compounds could be taken up by the cells, including macrophages, and affect the cellular oxidative status (36).
In the present study, we demonstrated that the phenolic compounds contained in extra virgin olive oil, namely, protocatechuic acid and oleuropein, as previously demonstrated for some polyphenols of red wine (37), inhibit macrophage-mediated oxidation of LDL. Importantly, we provide evidence that the mechanisms underlying this specific activity of biophenols are associated with their "boost" effect on intracellular antioxidizing properties. In fact, these biophenols, together with LDL in the culture medium, counteracted TBARS production and the increase in electrophoretic mobility associated with the oxidation of lipoproteins, thus preventing the peroxidation of their lipid components. These findings could be ascribed to the ability of protocatechuic acid and oleuropein to act as scavengers for radicals and chain-breaking antioxidants, both actions that are related to the ortho- and para-hydroxyl groups present on the phenol ring. On the other hand, lipid peroxidation inhibition seems to depend on the intracellular accumulation of these biophenols and on their capability to in some way block some of the early intracellular events necessary to begin the oxidative process in lipoproteins. In fact, it is worth noting that, when incubated with the macrophage-like J774 A.1 cells for 2 h and then removed from the culture medium before LDL addition, protocatechuic acid and oleuropein exerted a potent inhibitory effect on the macrophage-mediated oxidation of LDL.
LDL oxidation mediated by macrophage cells depends on the intracellular balance between prooxidants and antioxidants, including the glutathione system, (4,6) and involves the activation of NADPH oxidase (38), which produces superoxide anions that are able to convert native LDL to atherogenic oxLDL. Redox imbalance in J774 A.1 cells was already evident after 6 h of incubation with LDL and a catalytic amount of copper, from the increase in ROS production and, after 12 h, from the decrease in GSH content with respect to the control cells. Extra virgin olive oil biophenols inhibited the redox imbalance by scavenging released radicals, thus preserving the intracellular GSH content; they also downregulated the production of superoxide anions, as shown when the biophenols were present before LDL addition and then removed from the medium. These data are in agreement with those recently obtained by Léger et al. (39) in cultured human monocyte THP-1 cells treated with mixtures of wastewaters from olive oil processing. Some questions remain about the effect that the biophenols tested here have on cell activity, e.g., effects on the assembling of the NADPH oxidase complex and on the activation of protein kinase C, shown for other antioxidants (40–42).
Another possible key mechanism in the protective effect exerted by extra virgin olive oil biophenols could involve the sparing of GSH, i.e., the strengthening of intracellular antioxidant defenses. It was demonstrated that macrophage-mediated oxidation of LDL and cellular lipid peroxidation increase in glutathione-depleted cells (4). Reduced glutathione is a critical factor in the cellular antioxidant network as are its related enzymes GPx, which catalyzes the elimination of a variety of hydroperoxides including H2O2 and organic hydroperoxides, and GR, which regenerates GSH from oxidized glutathione (43,44). The functional efficiency of this redox cycle in peroxide catabolism will depend on the optimal activities of the redox enzymes, GPx and GR, and the optimal supply of the reductant GSH. An extensive depletion of the GSH pool will prevent further peroxide degradation, thus compromising tissue cell integrity and representing a stimulus for its de novo synthesis by two sequential ATP-dependent reactions catalyzed by GCS, the rate-limiting enzyme, and by glutathione synthetase (45).
The data we report here suggest that one of the mechanisms by which extra virgin olive oil biophenols inhibited cell-mediated oxidation of LDL was by preventing ROS accumulation through the improvement of the entire GSH redox cycle, as demonstrated for other antioxidative compounds (46). The preservation of enzyme efficiency, and consequently of the normal content of GSH, is also confirmed by the lack of stimulation of mRNA for GCS in the cells treated with biophenols and LDL, compared with the cells treated with LDL alone in which the depletion of GSH was a potent stimulus for GCS gene expression (47). The observed upregulation may indicate a reaction of the cells to meet the increasing demand of glutathione.
The most interesting results were the specific actions exerted by protocatechuic acid and oleuropein on the expression and activity of glutathione peroxidase and glutathione reductase. We demonstrated an increase in mRNA overproduction and enzyme activity in cells pretreated with protocatechuic acid and oleuropein, with and without LDL compared with controls.
It is noteworthy that the two experimental procedures allowed us to distinguish different mechanisms through which the biophenols exerted their protective effects. Following procedure A, we demonstrated their classical antioxidative action as radical scavengers or chain-breakers of peroxidative reaction; with procedure B, we showed an additional intracellular mechanism through the direct activation of mRNA transcription of the detoxifying enzymes. These findings were also confirmed by the potentiated enzyme activity in cells treated with the biophenols alone. The use of in vitro systems can help clarify the mechanisms underlying the strengthening of the cellular defenses exerted by the phenols. Further studies are warranted, however, before these agents can be considered in clinical practice.
We conclude that the protective effect exerted by biophenols may involve more than one mechanism. The observed increase in mRNAs, with no oxLDL-induced alteration (Fig. 4), indicates a direct effect of the biophenols on the DNA transcription of GSH-related enzymes, and suggests that this is one of the mechanisms that improves antioxidative cellular defenses.
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FOOTNOTES |
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2 These authors contributed equally to this work and should thus be considered as first authors.
4 Abbreviations used: CM-H2-DCFDA, 5-chloromethyl-2',7'-dichlorodihydrofluoresceindiacetate; DHE, dihydroetidium; DHR 123, dihydrorhodamine 123; FCS, fetal calf serum; GCS, -glutamilcysteine synthetase; GPx, glutathione peroxidase; GR, glutathione reductase; GSH, glutathione; oxLDL, oxidized LDL; PI, propidium iodide; REM, relative electrophoretic mobility; ROS, reactive oxygen species.
Manuscript received 29 October 2003. Initial review completed 9 December 2003. Revision accepted 15 January 2004.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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