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Chronic consumption of a flavanol- and procyanindin-rich diet is associated with reduced levels of 8-hydroxy-2′-deoxyguanosine in rat testes

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Abstract

Cocoa can contain a high concentration of flavanols and procyanidins which have been reported to have strong antioxidative activity. In the present study, male Sprague-Dawley rats were fed diets containing 0, 0.5, 1, or 2% cocoa rich in flavanols for two weeks. Blood, liver, heart and testes were collected and analyzed for markers of oxidative damage. Plasma epicatechin concentrations, 8-hydroxy-2′-deoxyguanosine (8OH2′dG), and oxidized and reduced glutathione were quantitated by HPLC with electrochemical detection. Plasma F2-isoprostanes were measured using an enzyme immunoassay. Plasma epicatechin concentrations increased in a dose-dependant fashion according to the amount of cocoa in the diet (128 nM–790 nM). Cocoa supplementation was associated with lower than normal concentrations of 8OH2′dG in the testes (0.590 + 0.40 vs. 0.328 + 0.29; p < 0.05). Liver and heart 8OH2′dG levels were unaffected by dietary treatment. In erythrocytes, the glutathione pool was significantly less oxidized in the cocoa fed group compared to controls (p < 0.05). In liver and testes, no differences in superoxide dismutase activities were detected. Concentrations of plasma F2-isoprostanes and thiobarbituric acid reactive substances were similar in all groups. These results support the concept that a diet rich in flavanols and procyanidins can improve oxidant defense and reduce tissue markers for oxidative stress, although these effects can be tissue specific.

Introduction

There is increasing interest in the identification of dietary factors that can reduce an individual’s risk for chronic disease. In addition to the well-recognized essential nutrients, food can contain a multitude of other components that can promote health. In this regard, flavonoids represent a class of phytochemicals that has been postulated to have positive health effects, and the consumption of certain foods and beverages rich in these compounds has been reported to be associated with numerous positive health benefits including vessel relaxation, reductions in platelet reactivity, and increases in plasma antioxidant potential [1], [2], [3], [4], [5], [6].

Given the chemical diversity of the flavonoid family, it is reasonable to speculate that the biological effects of these compounds in humans occur through a variety of mechanisms. With the above stated, a number of research groups have suggested that an important role for these nutrients can be to enhance the oxidant defense system. Using a variety of in vitro models, investigators have shown that the flavonoids found in wine, purple grape juice, tea, and cocoa can act as potent antioxidants by scavenging reactive radicals, thus reducing the rate of protein and lipid oxidation [7], [8], [9], [10], [11], [12], [13]. While a substantial body of literature has been developed on the antioxidant effects of flavonoids in vitro, studies documenting the potential of flavonoids to reduce oxidative damage in vivo are more limited. The paucity of in vivo data can be attributed, at least in part, to the difficulty in accurately measuring oxidative damage in vivo. For tissue oxidative damage, two markers that are reputed to be reliable indicators of oxidative damage are F2-isoprostanes, [14] a marker of lipid oxidation, and 8-hydroxy-2′-deoxyguanosine (8OH2′dG) [15] a marker of DNA oxidation. In one of the few studies that have demonstrated an in vivo effect of flavonoids on tissue oxidative damage, the consumption of a flavonoid-rich red wine was associated with a reduction in plasma and urinary F2-isoprostanes in smokers [16]. In addition, a few studies have shown that treating rats with flavonoids isolated from black tea and red wine significantly reduced the amount of chemically induced 8OH2′dG adducts in both the colonic mucosa and liver [17], [18], [19].

Cocoa contains a relatively high concentration of flavonoids, specifically the flavanols epicatechin, catechin, and oligomers of epicatechin known as procyanidins [20]. Purified flavonoids isolated from cocoa have been shown to reduce the rate of LDL oxidation in vitro [8], [11], and LDL isolated from the plasma of subjects who consumed flavonoid-rich cocoa has been reported to be more resistant to oxidation ex vivo [12], [21]. The above work supports the concept that diets rich in certain flavonoids may be associated with improvements in the oxidant defense system. In addition, numerous other studies suggest that the consumption of flavonoids may have the potential to prevent oxidative damage in vivo.

The purpose of the current study, using a rodent model, was to determine if the addition of a flavonoid-rich cocoa to a typical control diet would result in a reduction in markers of oxidative damage in animals fed the diet for two weeks in the absence of an oxidative stress.

Section snippets

Materials

All chemicals used were from Sigma (St. Louis, MO) unless otherwise noted. Alkaline phosphatase and nuclease P1 used for enzymatic hydrolysis of DNA were from Roche (Indianapolis, IN). HPLC solvents were from Fisher (Pittsburg, PA). The flavonoid rich cocoa was generously provided by Mars, Incorporated (Hackettstown, NJ).

Animals

The animal protocol was in accordance with NRC recommendations [22] and was approved by the University of California, Davis Animal Use and Care Committee. Forty-eight adult

Results

Food intake and weight gains were similar among the groups (data not shown). As depicted in Fig. 1, plasma epicatechin concentrations increased in a dose-dependent manner according to the concentration of cocoa in the diet. Plasma catechin and dimer in the plasma were below the limit of detection in the plasma of rats in the low cocoa diet groups. Catechin was detected in plasma obtained from rats fed the 2% cocoa diet (40.2 nM ± 1.23) but at a much lower concentration than epicatechin (790.3

Discussion

The results of this work support the concept that the chronic consumption of a diet rich in certain flavonoids can be associated with improvements in the oxidant defense system. Significantly, there was a strong inverse correlation between plasma epicatechin concentrations and testes 8OH2′dG levels. Previous experiments with flavonoids have shown an inverse association between dietary flavonoid intake and tissue oxidative damage when the study subjects were challenged with an oxidative stress

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    Supported in part by a grant from the National Institutes of Health (DK-35747) and a gift from Mars Incorporated.

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