**3. Results**

362 Hepatocellular Carcinoma – Basic Research

Induction of DNA damage (strand breaks and oxidative DNA damage) by NDMA, NPYR (Arranz et al., 2007) NPIP, NDBA (García et al., 2008), B(a)P (Delgado et al., 2008), 8-MeIQx, 4,8-diMeIQx and PhIP (Haza and Morales, 2010) have been previously evaluated by our laboratory. HepG2 cells were plated on to multiwell systems at a density of 1.5x105 cells/ml culture medium. 24 h after seeding, the corresponding galic acid or piceatannol concentrations were added to the wells and plates were incubated for 24hr at 37ºC and 5% CO2. After incubation, cells were simultaneously treated with the concentrations of food mutagens that caused a significant increased on DNA damage and previously evaluated by our laboratory. NPYR (50mM without enzymes and 5mM with EndoIII or Fpg enzymes), NDMA (135mM without enzymes and 27mM with EndoIII or Fpg enzymes), NDBA (3 mM), NPIP (44 mM), BaP (50 M), Me IQx (500 M), 4,8-diMeIQx (200 M) or PhIP (300 M), and different concentrations of galic acid (0.1-5M) or piceatannol (0.1-5M) for another 24 hours at 37ºC and 5% CO2. After the treatments, the cells were processed as described above

Images of 50 randomly selected cells per concentration were evaluated and the test was carried out three times. The reported OTM is the mean ± standard deviation (S.D.) of three

**2.4 Analysis of DNA damage (strand breaks and oxidized purines/pyrimidines) induced by a simultaneous treatment of food mutagens and galic acid or piceatannol** 

**in the Alkaline Comet assay** 

Fig. 4. Comet assay procedure.

**2.5 Statistical analysis** 

(Figure 4.)

#### **3.1 DNA damage (strand breaks and oxidized purines/pyrimidines) induced by galic acid or piceatannol in the Alkaline Comet assay**

No cytotoxicity has been previously found at the concentrations of galic acid or piceatannol tested (data not shown). Cell viability was always above 80% of control viability. At noncytotoxic concentrations (0.1-5M) piceatannol and gallic acid did not induce DNA strand breaks and oxidative DNA damage (**Table 1**). For this reason this concentration range was used in subsequent studies. DNA damage was not measured at cytotoxic concentrations (> 5 M) because under these conditions DNA damage is caused as a consequence of necrosis or apoptosis (Henderson et al., 1998).


Table 1. Effect of different concentrations of GA and PCA on DNA strand breaks and on the formation of Endo III and Fpg sensitive sites of human hepatoma cells.

#### **3.2 DNA damage induction by simultaneous treatment of food carcinogens and galic acid or piceatannol in the Alkaline Comet assay**

Protection afforded by piceatannol and gallic acid towards NDBA and NPIP-induced oxidative DNA damage was shown in **Table 2**. No protective effect was shown by piceatannol and gallic acid against NDBA or NPIP-induced DNA strand breaks in HepG2 cells. Gallic acid, but not piceatannol, weakly reduced the Endo III sensitive sites induced by NDBA (28.5%, 0.1 M). However, piceatannol reduced the NPIP-induced Endo III sensitive sites at all concentrations tested (28-36%, 0.1-5 M) and no effect was shown by

Use of a Human–Derived Liver Cell Line for

and 5 mM with Endo III or Fpg enzymes).

concentrations (MeIQx, 25.4% and diMeIQx, 27.4%).

damage was shown in **Table 4**.

the Detection of Protective Effect of Dietary Antioxidants Against Food Mutagens 365

The effect of piceatannol and gallic acid against BaP and PhIP-induced oxidative DNA

Table 3. 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 NDMA (135 mM without enzymes and 27 mM with Endo III or Fpg enzymes) and NPYR (50 mM without enzymes

Piceatannol protected against DNA strand breaks induced by BaP and PhIP at all concentrations tested (0.1-5 M, 60.0-65.3% and 34.7-12.5%, respectively). However, gallic acid only exerted protection against BaP-induced DNA strand breaks at the highest concentrations (1-5M, 18.-36%). An important decrease of the formation of BaP-induced Endo III sensitive sites was also shown by piceatannol at the lowest concentration (0.1 M, 60.5%), whereas gallic acid drastically reduced the formation of Endo III sensitive sites at all the concentrations tested (0.1-5 M, 79.6-63.9%). On the other hand, only gallic acid showed a weakly protective effect against the PhIP-induced Fpg sensitive sites (0.1-1 M, 17.7%).

The protective effect of gallic acid and piceatannol against MeIQx and diMeIQx-induced oxidative DNA damage was shown in **Table.5**. No effect was shown by piceatannol and gallic acid against DNA strand breaks and the formation of Endo III sensitive sites induced by MeIQx and diMeIQx. Fpg sensitive sites induced by MeIQx (41.1-31.3%) and diMeIQx (41.1-23.5%) were prevented by gallic acid at all the concentrations tested (0.1-5 M). However, piceatannol only reduced the formation of Fpg sensitive sites at the lowest

gallic acid. The maximum reduction of Fpg sensitive sites induced by NDBA was found at the highest concentration of piceatannol (5 M, 56%). However, the maximum reduction of Fpg sensitive sites induced by NPIP was at the lowest concentration (0.1 M, 34.2%). Gallic acid only exerted its protective effect against NPIP-induced Fpg sensitive sites (42.1-23.6%, 0.1-5 M).


Table 2. 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 3 mM of NDBA and 44 mM of NPIP.

**Table 3** shows the effect of piceatannol and gallic acid against NPYR and NDMA-induced oxidative DNA damage. Results revealed that piceatannol at the lowest concentration reduced the DNA strand breaks induced by NPYR and NDMA (0.1 M, 32.2% and 47.6%, respectively). On the contrary, gallic acid did not shown any protective effect against NPYR or NDMA-induced DNA strand breaks. The formation of Endo III sensitive sites induced by NPYR was prevented only by piceatannol at all the concentrations (0.1-5 M, 12.5-25%), whereas, both compounds, PCA (0.1-5 M, 30.7-19.2%) and GA (0.1-1M, 23%) protected against the formation of Endo III sensitive sites induce by NDMA, respectively. On the other hand, the formation of Fpg sensitive sites induced by NPYR and NDMA were reduced by PCA and GA. At a dose of 0.1 M, PCA exhibited the maximum reduction (38.8%) on Fpg sensitive sites induced by NPYR, whereas GA exhibited it at 5.0 M (18.5%). PCA and GA also reduced the formation of Fpg sensitive sites induced by NDMA at concentrations of 0.1- 1 M, respectively (30.9% and 23.8-14.2%).

gallic acid. The maximum reduction of Fpg sensitive sites induced by NDBA was found at the highest concentration of piceatannol (5 M, 56%). However, the maximum reduction of Fpg sensitive sites induced by NPIP was at the lowest concentration (0.1 M, 34.2%). Gallic acid only exerted its protective effect against NPIP-induced Fpg sensitive sites

Table 2. 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 3 mM of NDBA and 44

**Table 3** shows the effect of piceatannol and gallic acid against NPYR and NDMA-induced oxidative DNA damage. Results revealed that piceatannol at the lowest concentration reduced the DNA strand breaks induced by NPYR and NDMA (0.1 M, 32.2% and 47.6%, respectively). On the contrary, gallic acid did not shown any protective effect against NPYR or NDMA-induced DNA strand breaks. The formation of Endo III sensitive sites induced by NPYR was prevented only by piceatannol at all the concentrations (0.1-5 M, 12.5-25%), whereas, both compounds, PCA (0.1-5 M, 30.7-19.2%) and GA (0.1-1M, 23%) protected against the formation of Endo III sensitive sites induce by NDMA, respectively. On the other hand, the formation of Fpg sensitive sites induced by NPYR and NDMA were reduced by PCA and GA. At a dose of 0.1 M, PCA exhibited the maximum reduction (38.8%) on Fpg sensitive sites induced by NPYR, whereas GA exhibited it at 5.0 M (18.5%). PCA and GA also reduced the formation of Fpg sensitive sites induced by NDMA at concentrations of 0.1-

(42.1-23.6%, 0.1-5 M).

mM of NPIP.

1 M, respectively (30.9% and 23.8-14.2%).

The effect of piceatannol and gallic acid against BaP and PhIP-induced oxidative DNA damage was shown in **Table 4**.


Table 3. 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 NDMA (135 mM without enzymes and 27 mM with Endo III or Fpg enzymes) and NPYR (50 mM without enzymes and 5 mM with Endo III or Fpg enzymes).

Piceatannol protected against DNA strand breaks induced by BaP and PhIP at all concentrations tested (0.1-5 M, 60.0-65.3% and 34.7-12.5%, respectively). However, gallic acid only exerted protection against BaP-induced DNA strand breaks at the highest concentrations (1-5M, 18.-36%). An important decrease of the formation of BaP-induced Endo III sensitive sites was also shown by piceatannol at the lowest concentration (0.1 M, 60.5%), whereas gallic acid drastically reduced the formation of Endo III sensitive sites at all the concentrations tested (0.1-5 M, 79.6-63.9%). On the other hand, only gallic acid showed a weakly protective effect against the PhIP-induced Fpg sensitive sites (0.1-1 M, 17.7%).

The protective effect of gallic acid and piceatannol against MeIQx and diMeIQx-induced oxidative DNA damage was shown in **Table.5**. No effect was shown by piceatannol and gallic acid against DNA strand breaks and the formation of Endo III sensitive sites induced by MeIQx and diMeIQx. Fpg sensitive sites induced by MeIQx (41.1-31.3%) and diMeIQx (41.1-23.5%) were prevented by gallic acid at all the concentrations tested (0.1-5 M). However, piceatannol only reduced the formation of Fpg sensitive sites at the lowest concentrations (MeIQx, 25.4% and diMeIQx, 27.4%).

Use of a Human–Derived Liver Cell Line for

food mutagens.

concentrations of GA used in this study would not be absurd.

the Detection of Protective Effect of Dietary Antioxidants Against Food Mutagens 367

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

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

Table 5. Protective effect of GA and PCA on DNA strand breaks, the formation of Endo III

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

and Fpg sensitive sites of human hepatoma cells induced by MeIQx and diMeIQx.


Table 4. 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 BaP and PhIP.
