**2.1 Chemicals**

358 Hepatocellular Carcinoma – Basic Research

foodstuffs in the presence of creatinine, amino-acids and sugars, involving Maillard reaction (Ristic et al., 2004). Among those, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (8- MeIQx), 2-amino- 3,4,8-trimethylimidazo[4,5-f]-quinoxaline (4,8-diMeIQx) and 2-amino-1 methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP) are the most abundant (Skog et al., 1998). Benzo(a)pyrene (BaP), an important PAH, is a potent systemic and local carcinogen known to induce skin, lung, and stomach tumours in animal models (Ueng et al., 2001). NPIP, NDBA, NPYR and HCAs were categorized as Group 2B: possible causative agents in human cancer, while NDMA and B(a)P as Group 2A: probable causative agents in human cancer

N-nitrosamines, HCAs and B(a)P are DNA reactive chemicals that require metabolic activation, usually by various cytochrome P450 (CYP) enzymes for interaction with DNA (Ingelman-Sundberg, 2002). It has been suggested that DNA damage and free radical damage are in part involved in the carcinogenic action induced by N-nitrosamines (Bartsch et al., 1989). Strand breaks or alkali labile sites, including abasic sites, may be results of the action of reactive oxygen species that arise during the metabolism of food mutagens in the cell. In a previous work we showed that N-nitrosamines (Arranz et al., 2007; García et al., 2008a,b), benzo(a)pyrene (Delgado et al., 2009) and heterocyclic amines (Haza et al., 2011) were able to generate DNA strand breaks and oxidized bases. The increasing appreciation of the importance of food mutagens as potential human carcinogens stimulated intense

Current evidence strongly supports a contribution of polyphenols to the prevention of various diseases associated with oxidative stress, such as cancer and cardiovascular, neurodegenerative and age-related diseases (Kanazawa et al., 2006). Phenolics have been reported to have a capacity to scavenge free radicals (Havsteen, 2002). Gallic acid (3,4,5 trihydroxybenzoicacid, GA) is a polyhydroxyphenolic compound, which can be found in various natural products, like gallnuts, tea leaves, bark, green tea, apple-peels, grapes, strawberries, pineapples, bananas, lemons, and in red and white wine (Madlener, et al., (2007). GA is a strong antioxidant that possesses antimutagenic and anticarcinogenic activities (Inoue, et al., 1994; Stich, et al., 1982) and exerts antiproliferative effects on cancer cells by generating hydrogen peroxide (Lapidot, et al., 2002). It inhibits melanogenesis which may be related to GA's antioxidant activity in scavenging reactive oxygen species

Piceatannol (3-hydroxyresveratrol or astringinine, PCA) is a phenolic compound that occurs naturally in grapes and red wine (McDonald et al., 1998). The total amount of PCA in redgrape wine has been reported to be up to 15 mg/l (Cantos et al., 2003), however the biotransformation of the abundant red wine component, resveratrol (*trans*-3,5,4 trihydroxystilbene), contributes to increase PCA concentrations at tissue level (Piver et al., 2004). Both substances are synthesized in plants in response to fungal or other environmental stress, classifying them as phytoalexins. Piceatannol has been identified as the active ingredient of *Melaleucaleucadendron* (white tea tree), *Cassia garretiana* (Asian legume) and *Rheum undulatum* (Korean rhubarb), which are used in traditional herbal medicine (Tsuruga, et al., 1991; Matsuda, et al., 2000) and as the antileukemic compound in the seeds of *Euphorbia lagascae*, which is used in folk medicine to treat cancer, tumors and warts (Ferrigni et al., 1984). Teguo et al. 2001 also detected piceatannol in cell suspension

research on protective dietary factors in chemical carcinogenesis.

(IARC, 1993).

(Seo et al., 2003).

cultures of *Vitis vinifera* (wine grapes).

Galic acid (GA), piceatannol (PCA) and food mutagens used in this study are shown in Figures 2 and 3. N-nitrosodimethylamine (NDMA), N-nitrosodibutylamine (NDBA), Nnitrosopyrrolidine (NPYR), N-nitrosopiperidine (NPIP), benzo(a)pyrene (BaP), dimethyl sulfoxide (DMSO) and low melting point agarose (LMP) were purchased from Sigma-Aldrich (St. Louis, MO). 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (8-MeIQx), 2 amino-3,4,8-trimethyl-3H-imidazo[4,5-f]quinoxaline (4,8-diMeIQx) and 2-amino-1-methyl-6 phenyl- imidazo[4,5-b]pyridine (PhIP), were purchased from Toronto Research Chemicals Inc. (North York, On. Canada). Formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease III (Endo III) were obtained from Trevigen Inc. (Gaithersburg, MD). All other chemicals and solvents were of the highest grade commercially available. Food carcinogens and polyphenols were dissolved in sterile DMSO. The stock solutions were stored deep frozen (-80ºC).

#### **2.2 HepG2 cells**

Human hepatocellular carcinoma (HepG2) cells were purchased from Biology Investigation Center Collection (BIC, Madrid, Spain). Only cells of passage 10-17 were used in the

Use of a Human–Derived Liver Cell Line for

the comet head to the centre of tail distribution.

the Detection of Protective Effect of Dietary Antioxidants Against Food Mutagens 361

with ethidium bromideg/ml) in Tris acetate EDTA (TAE 1X) during 5 minutes and examined in a fluorescence microscope (OLYMPUS BH-2) connected to a computerized image analysis system (Comet Score 5.5). Olive tail moment (OTM) as defined by Olive et al. (1992) was determined and expressed as arbitrary units (AU). OTM= I x L, where I is the fractional amount of DNA in the comet tail (%DNA in the tail) and L is the distance from the centre of

Fig. 3. Chemical structures of food mutagens used in this study: (A) N-nitrosodimethylamine (NDMA),(B) N-nitrosopyrrolidine (NPYR), (C) N-nitrosopiperidine (NPIP), (D) N-nitrosodibuthylamine (NDBA), (E) benzo(a)pyrene (BaP), (F) 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (8-MeIQx), (G) 2-amino-3,4,8-trimethyl-3H-imidazo[4,5-f]quinoxaline

(4,8-diMeIQx) and (H) 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP).

experiments. The cells were cultured as monolayer in Dulbecco's Modified Eagle Medium supplemented with 10% v/v heat inactivated foetal calf serum, 50 U/ml penicillin and 50 g/ml streptomycin and 1% v/v L-glutamine. Culture medium and supplements required for the growth of the cells were purchased from Gibco Laboratories (Life Technologies, Inc., Gaithersburg, MD 20884-9980). Cell cultures were incubated at 37ºC and 100% humidity in a 5% CO2 atmosphere.

Fig. 2. Chemical structures of gallic acid (A) and piceatannol (B).

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

Cell viability was routinely determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) assay in order to select non-toxic concentrations of galic acid and piceatannol. The SCGE assay was carried out according to the protocol of Olive et al. (1992) with minor modifications.

Briefly, HepG2 cells were plated on to multiwell systems at a density of 1.5x105 cells/ml culture medium. 24 hr after seeding, cells were exposed to galic acid (0.1-5 M), or piceatannol (0.1-5 M) or solvent, for another 24 h at 37 ºC and 5% CO2. The solvent concentration in the incubation medium never exceeds 0.1%. After treatments, 10l of a suspension of 1x105 cells were mixed with 70l of LMP agarose type VII (0.75% concentration in PBS), distributed on slides that had been pre-coated with LMP agarose type VII (0.30% concentration in PBS), and left to set on an ice tray. Three slides were prepared for each concentration of the compound tested, one slide for control and the other slides to be treated with Fpg or Endo III. After solidification, the cells were lysed in darkness for 1 hour in a high salt alkaline buffer (2.5M NaCl, 0.1M EDTA, 0.01M Tris, 1% Triton X-100, pH 10). The slides were then equilibrated 3x5 minutes in enzyme buffer (0.04M HEPES, 0.1M of Fpg or Endo III at 1g/ml in enzyme buffer for 30 min at 37ºC in a humid dark chamber. Control slides were incubated with 30l of enzyme buffer only. Following enzyme treatment, the slides were placed in electrophoresis buffer (0.3M NaOH, 1mM EDTA, pH 13, cooled in a refrigerator) in darkness for 40 min. Electrophoresis was performed in a cold-storage room, in darkness, in a Bio-Rad subcell GT unit containing the same buffer, for 30 min at 25V. After electrophoresis, the slides were neutralized using 0.4M Tris pH 7.5 and KCl, 0.5mM EDTA, 0.2 mg/ml BSA, pH 8). After this time, slides were incubated with 30l fixed in methanol. Subsequently, the DNA was stained

experiments. The cells were cultured as monolayer in Dulbecco's Modified Eagle Medium supplemented with 10% v/v heat inactivated foetal calf serum, 50 U/ml penicillin and 50 g/ml streptomycin and 1% v/v L-glutamine. Culture medium and supplements required for the growth of the cells were purchased from Gibco Laboratories (Life Technologies, Inc., Gaithersburg, MD 20884-9980). Cell cultures were incubated at 37ºC and 100% humidity in a

Fig. 2. Chemical structures of gallic acid (A) and piceatannol (B).

**induced by galic acid or piceatannol in the Alkaline Comet assay** 

**2.3 Analysis of DNA damage (strand breaks and oxidized purines/pyrimidines)** 

Cell viability was routinely determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) assay in order to select non-toxic concentrations of galic acid and piceatannol. The SCGE assay was carried out according to the protocol of Olive et al.

Briefly, HepG2 cells were plated on to multiwell systems at a density of 1.5x105 cells/ml culture medium. 24 hr after seeding, cells were exposed to galic acid (0.1-5 M), or piceatannol (0.1-5 M) or solvent, for another 24 h at 37 ºC and 5% CO2. The solvent concentration in the incubation medium never exceeds 0.1%. After treatments, 10l of a suspension of 1x105 cells were mixed with 70l of LMP agarose type VII (0.75% concentration in PBS), distributed on slides that had been pre-coated with LMP agarose type VII (0.30% concentration in PBS), and left to set on an ice tray. Three slides were prepared for each concentration of the compound tested, one slide for control and the other slides to be treated with Fpg or Endo III. After solidification, the cells were lysed in darkness for 1 hour in a high salt alkaline buffer (2.5M NaCl, 0.1M EDTA, 0.01M Tris, 1% Triton X-100, pH 10). The slides were then equilibrated 3x5 minutes in enzyme buffer (0.04M HEPES, 0.1M of Fpg or Endo III at 1g/ml in enzyme buffer for 30 min at 37ºC in a humid dark chamber. Control slides were incubated with 30l of enzyme buffer only. Following enzyme treatment, the slides were placed in electrophoresis buffer (0.3M NaOH, 1mM EDTA, pH 13, cooled in a refrigerator) in darkness for 40 min. Electrophoresis was performed in a cold-storage room, in darkness, in a Bio-Rad subcell GT unit containing the same buffer, for 30 min at 25V. After electrophoresis, the slides were neutralized using 0.4M Tris pH 7.5 and KCl, 0.5mM EDTA, 0.2 mg/ml BSA, pH 8). After this time, slides were incubated with 30l fixed in methanol. Subsequently, the DNA was stained

5% CO2 atmosphere.

(1992) with minor modifications.

with ethidium bromideg/ml) in Tris acetate EDTA (TAE 1X) during 5 minutes and examined in a fluorescence microscope (OLYMPUS BH-2) connected to a computerized image analysis system (Comet Score 5.5). Olive tail moment (OTM) as defined by Olive et al. (1992) was determined and expressed as arbitrary units (AU). OTM= I x L, where I is the fractional amount of DNA in the comet tail (%DNA in the tail) and L is the distance from the centre of the comet head to the centre of tail distribution.

Fig. 3. Chemical structures of food mutagens used in this study: (A) N-nitrosodimethylamine (NDMA),(B) N-nitrosopyrrolidine (NPYR), (C) N-nitrosopiperidine (NPIP), (D) N-nitrosodibuthylamine (NDBA), (E) benzo(a)pyrene (BaP), (F) 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (8-MeIQx), (G) 2-amino-3,4,8-trimethyl-3H-imidazo[4,5-f]quinoxaline (4,8-diMeIQx) and (H) 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP).

Use of a Human–Derived Liver Cell Line for

**acid or piceatannol in the Alkaline Comet assay** 

apoptosis (Henderson et al., 1998).

p≤0.05.

**3. Results** 

the Detection of Protective Effect of Dietary Antioxidants Against Food Mutagens 363

independent experiments. Thus, we compare three means of OTM from 3 different experiments. Cultures without N-nitrosamines or polyphenols were considered as negative controls. In all experiments the following negative controls have been included: cells treated with solvents and treated without enzymes, cells incubated with Endo III and cells incubated with Fpg. Induction of DNA damage by N-nitrosamines was defined as 100% of genotoxicity. The Student's t-test was used for statistical comparison between simultaneous treatments and controls, and differences were considered significant at

**3.1 DNA damage (strand breaks and oxidized purines/pyrimidines) induced by galic** 

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

Table 1. Effect of different concentrations of GA and PCA on DNA strand breaks and on the

**3.2 DNA damage induction by simultaneous treatment of food carcinogens and galic** 

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

formation of Endo III and Fpg sensitive sites of human hepatoma cells.

**acid or piceatannol in the Alkaline Comet assay** 

#### **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**

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 (Figure 4.)

Fig. 4. Comet assay procedure.

### **2.5 Statistical analysis**

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 independent experiments. Thus, we compare three means of OTM from 3 different experiments. Cultures without N-nitrosamines or polyphenols were considered as negative controls. In all experiments the following negative controls have been included: cells treated with solvents and treated without enzymes, cells incubated with Endo III and cells incubated with Fpg. Induction of DNA damage by N-nitrosamines was defined as 100% of genotoxicity. The Student's t-test was used for statistical comparison between simultaneous treatments and controls, and differences were considered significant at p≤0.05.
