**4. Discussion**

The aim of the present study was to evaluate the protective effect of galic acid and piceatannol towards food mutagens-induced oxidative DNA damage (strand breaks and oxidized purines/pyrimidines) in human hepatoma cells (HepG2), using the single-cell gel electrophoresis (SCGE) assay. There is speculation that oxidative DNA damaged is involved in cancer development (Mastaloudis et al., 2004). Otherwise, metabolism of food mutagens in cells could generate reactive oxygen species that arise in DNA strand breaks or alkali labile sites, including abasic sites. Previous studies in our research group showed that NDBA, NPIP (García et al., 2008), NDMA and NPYR (Arranz et al., 2007) were able to generate oxidized bases. In addition, during the metabolic process, BaP also produces reactive oxygen species (ROS) via cytochrome P4501A1 (Burczynski and Penning 2000). In a previous investigation we also showed that HepG2 cells treated with BaP or HCAs induced Fpg and Endo III sensitive sites, indicating the presence of oxidized purines and pyrimidines, respectively (Delgado et al., 2009, Haza et al., 2010).

Polyphenols have an important activity as antioxidants and also a remarkable role on carcinogen activation in vivo and on carcinogenesis (Dolara et al., 2005; Lambert et al., 2005). However, some flavonoids such as galic acid, piceatannol, resveratrol, quercetin and myricetin showed cytotoxicity for a number of cell lines or even induced oxidative DNA strand breakage in human lymphotcytes or in HepG2 cells at concentrations higher than 100

Table 4. Protective effect of GA and PCA on DNA strand breaks, the formation of Endo III

The aim of the present study was to evaluate the protective effect of galic acid and piceatannol towards food mutagens-induced oxidative DNA damage (strand breaks and oxidized purines/pyrimidines) in human hepatoma cells (HepG2), using the single-cell gel electrophoresis (SCGE) assay. There is speculation that oxidative DNA damaged is involved in cancer development (Mastaloudis et al., 2004). Otherwise, metabolism of food mutagens in cells could generate reactive oxygen species that arise in DNA strand breaks or alkali labile sites, including abasic sites. Previous studies in our research group showed that NDBA, NPIP (García et al., 2008), NDMA and NPYR (Arranz et al., 2007) were able to generate oxidized bases. In addition, during the metabolic process, BaP also produces reactive oxygen species (ROS) via cytochrome P4501A1 (Burczynski and Penning 2000). In a previous investigation we also showed that HepG2 cells treated with BaP or HCAs induced Fpg and Endo III sensitive sites, indicating the presence of oxidized purines and

Polyphenols have an important activity as antioxidants and also a remarkable role on carcinogen activation in vivo and on carcinogenesis (Dolara et al., 2005; Lambert et al., 2005). However, some flavonoids such as galic acid, piceatannol, resveratrol, quercetin and myricetin showed cytotoxicity for a number of cell lines or even induced oxidative DNA strand breakage in human lymphotcytes or in HepG2 cells at concentrations higher than 100

and Fpg sensitive sites of human hepatoma cells induced by BaP and PhIP.

pyrimidines, respectively (Delgado et al., 2009, Haza et al., 2010).

**4. Discussion** 

M (Hadi et al., 2007; Johnson and Loo, 2000). The mechanism by which these flavonoids induce DNA damage at higher concentrations might be due to the pro-oxidant properties of these compounds (Wu et al., 2004). Thus, it is important to evaluate whether the adverse effect of GA and PCA on DNA in human hepatoma cells, as shown in Table 1. Our results indicate that none of the dietary polyphenols (GA, PCA) concentrations tested (0.1-5 M) caused DNA strand breaks, or oxidized purine or pyrimidine bases per se in HepG2 cells (Table 1), although at concentrations higher than 5 M induced DNA strand breaks and oxidative DNA damage in HepG2 cells (data not shown). Approximately, people in the Unites States ingest each day 1g of tannic acid (TA) (Sanyal et al., 1997). As one of the food additives, TA is probably hydrolyzed in the acidic pH in the stomach, releasing the 10 potentially reactive GA residues (Brune et al., 1989). GA concentration in the stomach could achieve a maximum of 1.5mmol/L. GA from tablets and tea was rapidly absorbed, but the highest GA concentration observed in plasma was only 1.83 mol/L and 2.09 mol/L, respectively (Shahrzad et al., 2001). Thus, considering the uptake of hydrolysable TA, the concentrations of GA used in this study would not be absurd.

In the present study, we observed that, GA was less efficient that piceatannol to reduce DNA damage induced by food mutagens tested. The presence of three or four hydroxyl groups present in GA and PCA respectively, results in differing protective effects against food mutagens.


Table 5. Protective effect of GA and PCA on DNA strand breaks, the formation of Endo III and Fpg sensitive sites of human hepatoma cells induced by MeIQx and diMeIQx.

Our results revelaled that GA only prevented the DNA strand breaks induced by BaP. However, it protected cells against oxidative DNA damage-induced by food mutagens. GA

Use of a Human–Derived Liver Cell Line for

NPYR, NPIP, PhIP, MeIQx and diMeIQx.

*Toxicolog*y, 45: 1662-1669.

*Nutrition*, 81: 261S-267S.

*Biological Interactions*, 156: 101-111.

*Biochemistry and. Biophysics*, 455: 197-203.

*European Journal of Nutrition*, 47: 479-490.

*Mechanisms of Mutagenesis*, 591: 237-246.

**6. Acknowledgments** 

547-558.

(UCM).

**7. References** 

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NDBA, NDMA, NPYR, NPIP, MeIQx and diMeIQx.. However, GA at the concentrations tested only protects human hepatoma cells against DNA strand breaks induced by BaP, oxidized pyrimidines induced by NDMA and BaP and oxidized purines induced by NDMA,

This work has been supported by Grant ALI2005-01517 from the Ministerio de Educación y Ciencia (Spain) and by Grant 910177 from the BSCH and the Universidad Complutense

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removed oxidized pyrimidines induced by NDBA and BaP and oxidized purines induced by NDMA, NPYR, NPIP, MeIQx, diMeIQx and PhIP. This may be due to the antioxidant property of GA (Gali et al., 1992). Phenolic hydroxyl groups are known to be potent in scavenging free radicals and the OH group at the para position to the carboxylic group is especially effectual for the antioxidant activity (Son and Lewis, 2002). Thus the three hydroxyl groups present in GA may be responsible for its antioxidant activity. Although, Lu et al (2006) showed that the protective effect of GA derivatives seemed to depend more on their molecular polarities rather than antioxidant activities.

Our results also showed that DNA strand breaks induced by NDMA, NPYR, BaP and PhIP were reduced by PCA. In addition, PCA also removed the oxidized pyrimidines induced by NDMA, NPYR, NPIP, BaP and PhIP and the oxidized purines induced by NDMA, NPYR, NDBA, NPIP, MeIQx and diMeIQx. Supporting that, an additional hydroxyl group in the chemical structure of the PCA would significantly affect the biological activity against the mutagen. Shahidi and Wanasundara (1992) and Makena and Chung (2007) reported that the position and number of hydroxyl groups are crucial in the inhibitory effects of polyphenols. Moreover, it has been found that the biological activity of trans-Resveratrol (t-RES) and its analogues (PCA) significantly depends on the structural determinants, which are i) the number and position of hydroxyl groups(Wolter et al., 2002) ii) intramolecular hydrogen bonding (Fang et al., 2002), iii) stereoisomery and iv) double bond (Wright et al., 2001). Thus, the protective effect of GA and PCA against food mutagens may vary with the structure and dose of the individual compounds and the mutagenic compound.

Taking together our results PCA was more efficient against DNA strand breaks induced by NDMA, NPYR, BaP and PhIP that GA .GA only prevent DNA strand breaks induced by BaP. Comparing the protective effect of both compounds against BaP, PCA showed higher protective effect (60-65%) than GA (18-36%). In addition PCA was also the most active against oxidized pyrimidines induced by NDMA, NPYR, NPIP, BaP and PhIP. GA only showed reduction of the oxidized pyrimidines induced by NDMA and BaP. This reduction was higher (23%, NDMA and 80-63%, BaP) than the observed by PCA against these two compounds. On the contrary, we also showed that GA and PCA at all concentrations increased oxidized purines induced by BaP. This effect could be attributed to the excess of reactive oxygen species (ROS) produced by BaP. They might cause irreparable oxidative DNA damage (Johnson and Loo, 2000). In addition, phenolic compounds have both antioxidant and prooxidant effects depending on the experimental conditions [28]. However, GA and PCA were very efficient to remove oxidized purines induced by all the mutagens tested with the exception of NDBA (GA) and PhIP (PCA). Therefore, although there is a general structure-activity relationship (López-Lázaro, 2002) that shows that some subclasses of polyphenols can be more potent antimutagens, these structural considerations can change depending on the substitution pattern of the molecule and/or the kind of compound used to induce DNA damage.
