Antioxidant effects of dihydrocaffeic acid in human EA.hy926 endothelial cells
Introduction
Dietary plant polyphenols and flavonoids have been suggested to prevent colorectal cancer [1] and atherosclerosis [2], at least in part by providing defense against the oxidant stress thought to initiate and perpetuate these diseases. Of the polyphenols, chlorogenic acid (CGA) and its derivatives have long been of interest because of their relatively high content in coffee [3], fruits, vegetables, and wine that characterize the so-called “Mediterranean” diet [4]. Their benefits have been associated with their ability to function as antioxidants. For example, caffeic acid (CA), which corresponds to the hydroxycinnamic acid or “aglycone” portion of of CGA (Fig. 1), is an excellent scavenger of reactive species containing both nitrogen (e.g., peroxynitrite) and oxygen (e.g., superoxide) [5], [6]. At least part of the antioxidant effectiveness of CA may also relate to its ability to chelate transition metals, including iron and copper [7].
In biological systems, CA has shown promise as an antioxidant in vitro and at the cellular level. In a cell-free system, CA at 5 μmol/L completely protected human low-density lipoprotein (LDL) from peroxidative damage due both to a free radical initiator and to copper ions [7]. In addition, this effect was associated with sparing of α-tocopherol (α-TOC) in LDL [8]. The mechanism of the sparing effect of CA on α-TOC was attributed to direct recycling of α-TOC by CA, since the α-tocopheroxyl radical did not appear until after CA was consumed [8]. At the cellular level, CA has been shown to protect cultured human endothelial cells from apoptosis induced by oxidized LDL [9]. The IC50 (8.3 μmol/L) of the effect of CA on apoptosis was similar to that found to protect LDL [9]. Although phenolic derivatives such as ferulic acid (Fig. 1), which contain only one phenolic hydroxyl group, also protected LDL, CA was twice as potent in this regard [9]. In these studies, the protective effect of CA was due both to prevention of LDL oxidation and to a direct effect at the cellular level. We also recently reported that CGA, CA, and its reduced form dihydrocaffeic acid (DHCA) (Fig. 1) decreased lipid peroxidation in plasma and liposomes incubated with human erythrocytes, and that this was accompanied by a sparing effect on erythrocyte α-TOC [10]. In our study, CGA protected plasma and erythrocytes at concentrations as low as 5 μmol/L. Of relevance to this result is that a diet of 0.8% by weight CA in rats resulted in nonfasting CA concentrations in plasma of about 5 μmol/L [11].
CA and related compounds may act as antioxidants in endothelial cells, which would have direct relevance to vascular disease and atherosclerosis. For example, CA and flavonols induce endothelium-dependent vasorelaxation in rat thoracic aorta [12]. At the cellular level, CA at the half-maximal concentration of 8 μmol/L prevented apoptosis in endothelial cells induced by oxidized LDL [9]. This was considered due both to prevention of LDL oxidation and to direct protection of the cells. To further investigate the cellular protection afforded by dietary polyphenolics, we studied whether DHCA can prevent the toxic effects of oxidant stress in EA.hy926 endothelial cells. The latter is a permanent cell line derived from human umbilical vein endothelial cells that expresses factor VIII antigen [13], oxidatively modifies human LDL [14], and shows calcium-dependent stimulation of endothelial nitric oxide synthase (eNOS) [15]. DHCA, which is a metabolic product of CA, was chosen for study since it has been detected in human plasma following coffee ingestion [16], and since it has a similar antioxidant potential in human erythrocytes relative to CA [10]. The present results show 1) that DHCA is taken up by EA.hy926 cells; 2) that it spares α-TOC and decreases membrane lipid peroxidation due to a free radical initiator; 3) that it decreases intracellular oxidant stress due to menadione redox cycling; and 4) that it enhances eNOS activity by increasing expression of the protein.
Section snippets
Materials
Menadione, (d,l)-α-tocopherol (α-TOC) and 3,4-dihydroxyhydrocinnamic acid (DHCA) were obtained from Sigma-Aldrich Chemical Co., (St. Louis, MO). α-TOC was prepared by dissolving it in ethanol to a concentration of 10 mmol/L. cis-Parinaric acid (c-PnA) was obtained from Molecular Probes, Inc. (Eugene, OR) and was prepared by dissolving in ethanol to 180 mmol/L. Protein was measured using the BCA kit from Sigma-Aldrich. The 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) was obtained from
Results
The ability of EA.hy926 cells to take up and to retain DHCA was measured after overnight incubation in culture. As shown in Fig. 2, intracellular DHCA progressively increased to concentrations more than 10-fold those initially present in the incubations. This suggests that the cells are able to concentrate DHCA against a gradient. At concentrations >400 μmol/L, there was cell death related to toxicity of DHCA; consequently, for subsequent studies of antioxidant effects, lower concentrations of
Discussion
In this work we found that human-derived endothelial cells take up DHCA during overnight incubation in culture, and that they retain it under steady-state conditions against a concentration gradient (Fig. 2). This differs from human erythrocytes in short-term (1-hour) incubations, in which neither CA nor DHCA accumulated to concentrations higher than those present in the incubation medium [10]. The present results suggest that DHCA may be transported into endothelial cells via an active
Acknowledgments
This work was supported by National Institutes of Health grant AT01062, and by the Vanderbilt Institute for Coffee Studies. Dr. Huang is a Nestlé International Scholar at the Vanderbilt Institute for Coffee Studies.
References (44)
- et al.
Antioxidant activity of polyphenolics in diets. Rate constants of reactions of chlorogenic acid and caffeic acid with reactive species of oxygen and nitrogen
Biochim. Biophys. Acta
(1997) - et al.
Inhibition of human low-density lipoprotein oxidation by caffeic acid and other hydroxycinnamic acid derivatives
Free. Radic. Biol. Med.
(1995) - et al.
Two related phenolic antioxidants with opposite effects on vitamin E content in low density lipoproteins oxidized by ferrylmyoglobin: consumption vs regeneration
Arch. Biochem. Biophys.
(1995) - et al.
Effect of caffeic acid dietary supplementation on the antioxidant defense system in rat: an in vivo study
Arch. Biochem. Biophys.
(1997) - et al.
Natural dietary polyphenolic compounds cause endothelium-dependent vasorelaxation in rat thoracic aorta
J. Nutr.
(1998) - et al.
Ascorbic acid blunts oxidant stress due to menadione in endothelial cells
Arch. Biochem. Biophys.
(2003) - et al.
Dihydrocaffeic acid: a common contaminant in the liquid chromatographic-electrochemical measurement of plasma catecholamines in man
J. Chromatogr.
(1984) - et al.
Protection and recycling of α-tocopherol in human erythrocytes by intracellular ascorbic acid
Arch. Biochem. Biophys.
(1998) - et al.
A fluorometric method for determination of oxidized and reduced glutathione in tissues
Anal. Biochem.
(1976) - et al.
Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader
Free Radic. Biol. Med.
(1999)
Uptake, recycling, and antioxidant functions of α-lipoic acid in endothelial cells
Free Radic. Biol. Med.
Coordinate regulation of L-arginine uptake and nitric oxide synthase activity in cultured endothelial cells
Free Radic. Biol. Med.
Parinaric acid as a sensitive fluorescent probe for the determination of lipid peroxidation
Biochim. Biophys. Acta
Validation of the peroxidative indicators, cis-parinaric acid and parinaroyl-phospholipids, in a model system and cultured cardiac myocytes
Biochim. Biophys. Acta
Erythrocyte ascorbate recycling: antioxidant effects in blood
Free Radic. Biol. Med.
Flavonoid transport by mammalian endothelial cells
J. Nutr. Biochem.
Effect of vitamin E on the degradation of hydrogen peroxide in cultured human umbilical vein endothelial cells
Life Sci.
Dihydrofluorescein diacetate is superior for detecting intracellular oxidants: Comparison with 2′,7′-dichlorodihydrofluorescein diacetate, 5(and 6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate, and dihydrorhodamine 123
Free Radic. Biol. Med.
The metabolism of menadione (2-methyl-1,4-naphthoquinone) by isolated hepatocytes. A study of the implications of oxidative stress in intact cells
J. Biol. Chem.
Menadione induces endothelial dysfunction mediated by oxidative stress and arylation
Chem. Biol. Interact.
L-ascorbic acid potentiates nitric oxide synthesis in endothelial cells
J. Biol. Chem.
Ascorbic acid enhances endothelial nitric-oxide synthase activity by increasing intracellular tetrahydrobiopterin
J. Biol. Chem.
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