**1. Introduction**

356 Hepatocellular Carcinoma – Basic Research

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K absence or antagonist-II in the early detection of hepatocellular carcinoma in patients with chronic hepatitis B virus infection. *Scand J Gastroenterol*, Vol. 44, No. 7, Hepatocellular carcinoma is the most frequent form of primary liver cancer, it is one of the most common life threatening solid tumors with global annual diagnosis exceeding one million new cases and remains the third leading cause of cancer death (Ahmedin et al., 2007). Human diet often contains compounds that cause DNA damage. Common dietary mutagens would include N-nitroso compounds (Tricker & Preussmann, 1991), fungal toxins (Gelderblom et al., 2001), or cooked meat carcinogens (Layton et al., 1995). High nitrate levels in processed foods may be a risk factor, possibly through their ability to form Nnitroso compounds in vivo (Ferguson et al., 2004). N-nitroso compounds are known hepatocarcinogenic agents and have been implicated in the etiology of several human cancers Bansal et al., 2005). N-Nitrosamines are mutagenic and carcinogenic compounds widely present in the human diet and have been detected at ppb levels in a wide variety of matrices such as bacon, ham, frankfurters, sausages, cheese, beer, rubber, ground water, smoked tobacco and cosmetics (Filho et al., 2003). N-nitrosopiperidine (NPIP) is a potent extrahepatic carcinogen inducing tumours mainly in the esophagus and the nasal cavity (Gray et al., 1991). N-nitrosodibutylamine (NDBA) produces tumours in the esophagus and urinary bladder in rat, although the liver is its major target tissue for carcinogenesis (Williams et al., 1993). N-nitrosopyrrolidine (NPYR) induce mainly liver tumors in rats (Gray et al., 1991) and is a weak pulmonary carcinogen in mice (Wong et al., 2003) and N-nitrosodimethylamine (NDMA) is the most commonly encountered volatile N-nitrosamine in food samples and is a potent liver, lung and kidney carcinogen (Preussmann & Stewart, 1984).

Heterocyclic amines (HCAs) and polycyclic aromatic hydrocarbons (PAHs) are formed during the high-temperature cooking of meat and fish. To date, more than 20 different heterocyclic amines (HCAs) have been identified in cooked foods and they can be classified into two main groups called carbolines and aminoimidazoazaarenes (AIAs) (Toribio et al., 2007). AIAs are formed at the normal cooking temperatures of 100–225°C while heating

<sup>\*</sup> Corresponding Author

Use of a Human–Derived Liver Cell Line for

induced by food mutagens.

**2. Material and methods** 

**2.1 Chemicals** 

frozen (-80ºC).

**2.2 HepG2 cells** 

oxidized purines and pyrimidines, respectively (Figure 1).

the Detection of Protective Effect of Dietary Antioxidants Against Food Mutagens 359

Several studies established the single cell gel electrophoresis (SCGE) or Comet assay as a suitable method for assessing the ability of phytochemicals to protect cells against genotoxic effect of several xenobiotics (Collins, 2005). In this study, the Comet assay was modified to permit the detection of oxidized bases by including a step in which DNA is digested with formamidopyrimidine-DNA glycosylase (Fpg) or endonuclease III (Endo III) to uncover

Fig. 1. HepG2 cells untreated (A) and treated (B) with N-nitrosopyrrolidine (NPYR) and incubated with Fpg enzyme, visualized under fluorescence microscopy and using comet assay.

As part of our program to evaluate the protective effects of dietary polyphenols with different chemical structure (previously we have evaluated, flavonols and flavanols) we sought to determine whether galic acid (as representative of phenolic acids) or piceatannol (stilbenes) could protect human hepatoma cells (HepG2) from oxidative DNA damage

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

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

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 (IARC, 1993).

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 research on protective dietary factors in chemical carcinogenesis.

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 (Seo et al., 2003).

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 cultures of *Vitis vinifera* (wine grapes).

Several studies established the single cell gel electrophoresis (SCGE) or Comet assay as a suitable method for assessing the ability of phytochemicals to protect cells against genotoxic effect of several xenobiotics (Collins, 2005). In this study, the Comet assay was modified to permit the detection of oxidized bases by including a step in which DNA is digested with formamidopyrimidine-DNA glycosylase (Fpg) or endonuclease III (Endo III) to uncover oxidized purines and pyrimidines, respectively (Figure 1).

Fig. 1. HepG2 cells untreated (A) and treated (B) with N-nitrosopyrrolidine (NPYR) and incubated with Fpg enzyme, visualized under fluorescence microscopy and using comet assay.

As part of our program to evaluate the protective effects of dietary polyphenols with different chemical structure (previously we have evaluated, flavonols and flavanols) we sought to determine whether galic acid (as representative of phenolic acids) or piceatannol (stilbenes) could protect human hepatoma cells (HepG2) from oxidative DNA damage induced by food mutagens.
