Fruits and vegetables protect against the genotoxicity of heterocyclic aromatic amines activated by human xenobiotic-metabolizing enzymes expressed in immortal mammalian cells

Mutation Research 703 (2010) 90–98

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Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres

Fruits and vegetables protect against the genotoxicity of heterocyclic aromatic amines activated by human xenobiotic-metabolizing enzymes expressed in immortal mammalian cells K.L. Platt a,∗ , R. Edenharder b , S. Aderhold a , E. Muckel c , H. Glatt c a

Institute of Toxicology, University Medical Center, Johannes Gutenberg-University Mainz, Obere Zahlbacher Str. 67, 55131 Mainz, Germany Department of Hygiene and Environmental Medicine, University Medical Center, Johannes Gutenberg-University Mainz, Obere Zahlbacher Str. 67, 55131 Mainz, Germany c German Institute of Human Nutrition Potsdam-Rehbruecke, Department of Nutritional Toxicology, Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany b

a r t i c l e

i n f o

Article history: Received 29 March 2010 Received in revised form 26 July 2010 Accepted 3 August 2010 Available online 14 August 2010 Keywords: Heterocyclic aromatic amines IQ PhIP Genetically engineered hamster fibroblasts Genotoxicity Protection Fruits Vegetables Comet assay

a b s t r a c t Heterocyclic aromatic amines (HAAs) can be formed during the cooking of meat and fish at elevated temperatures and are associated with an increased risk for cancer. On the other hand, epidemiological findings suggest that foods rich in fruits and vegetables can protect against cancer. In the present study three teas, two wines, and the juices of 15 fruits and 11 vegetables were investigated for their protective effect against the genotoxic effects of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6phenylimidazo[4,5-b]pyridine (PhIP). To closely mimic the enzymatic activation of these HAAs in humans, genetically engineered V79 Chinese hamster fibroblasts were employed that express human cytochrome P450-dependent monooxygenase (hCYP) 1A2 (responsible for the first step of enzymatic activation) and human N(O)-acetyltransferase (hNAT) 2*4 or human sulfotransferase (hSULT)1A1*1 (responsible for the second step of enzymatic activation): V79-hCYP1A2-hNAT2*4 for IQ activation and V79-hCYP1A2hSULT1A1*1 for PhIP activation. HAA genotoxicity was determined by use of the comet assay. Black, green and rooibos tea moderately reduced the genotoxicity of IQ (IC50 = 0.8–0.9%), whereas red and white wine were less active. From the fruit juices, sweet cherry juice exhibited the highest inhibitory effect on IQ genotoxicity (IC50 = 0.17%), followed by juices from kiwi fruit, plum and blueberry (IC50 = 0.48–0.71%). The juices from watermelon, blackberry, strawberry, black currant, and Red delicious apple showed moderate suppression, whereas sour cherry, grapefruit, red currant, and pineapple juices were only weakly active. Granny Smith apple juice and orange juice proved inactive. Of the vegetable juices, strong inhibition of IQ genotoxicity was only seen with spinach and onion juices (IC50 = 0.42–0.54%). Broccoli, cauliflower, beetroot, sweet pepper, tomato, chard, and red-cabbage juices suppressed IQ genotoxicity only moderately, whereas cucumber juice was ineffective. In most cases, fruits and vegetables inhibited PhIP genotoxicity less strongly than IQ genotoxicity. As one possible mechanism of antigenotoxicity, the inhibition of activating enzymes was studied either indirectly with diagnostic substrates or directly by measuring CYP1A2 inhibition. Only sour cherry, blueberry, and black currant juices suppressed the first step of HAA enzymatic activation, whereas most plant-derived beverages inhibited the second step. © 2010 Elsevier B.V. All rights reserved.

1. Introduction

Abbreviations: araC, cytosine 1-␤-d-arabinofuranoside; BaP-7,8-diol, trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene; CYP, cytochrome P450dependent monooxygenase; DMEM, Dulbecco’s modified Eagle medium; FBS, fetal bovine serum; GST, glutathione S-transferase; HAA, heterocyclic aromatic amine; HU, hydroxyurea; IQ, 2-amino-3-methylimidazo[4,5f]quinoline; NAT, N-acetyltransferase; NER, nucleotide excision repair; N-OH-IQ, 2-hydroxyamino-3-methylimidazo[4,5-f]quinoline; N-OH-PhIP, 2-hydroxyamino1-methyl-6-phenylimidazo[4,5-b]pyridine; PAH, polycyclic aromatic hydrocarbon; PBS, phosphate-buffered saline; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5b]pyridine; SULT, sulfotransferase; UGT, UDP-glucuronosyltransferase. ∗ Corresponding author. Tel.: +49 6131 179260; fax: +49 6131 230506. E-mail address: [email protected] (K.L. Platt). 1383-5718/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2010.08.007

Polycyclic aromatic hydrocarbons (PAHs) and heterocyclic aromatic amines (HAAs) can be formed during the cooking and frying of meat and fish at elevated temperatures [1–3]. Many of these xenobiotics are strong mutagens [4], and their dietary intake has been associated with an increased risk for cancers of the breast [5], colon [6,7], pancreas [8], and prostate [9]. On the other hand, epidemiological findings suggest that foods rich in fruits and vegetables can protect against cancer [10–12]. This may be attributable to the phytochemicals in fruits and vegetables that inhibit the genotoxic action of food-borne carcinogens (e.g., PAHs and HAAs) [13]. The protective effects of the juices of

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Fig. 1. Genotoxic compounds used in this study and their enzymatic activation (IQ, 2-amino-3-methylimidazo[4,5-f]quinoline; PhIP, 2-amino-1-methyl-6phenylimidazo[4,5-b]pyridine; BaP-7,8-diol, trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene; BPDE, r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; CYP, cytochrome P450-dependent monooxygenase; UGT, UDP-glucuronosyltransferase; GA, glucuronic acid; NAT, N-acetyltransferase; SULT, sulfotransferase)

numerous fruits and vegetables against HAA mutagenicity in the Ames assay have been demonstrated [14]. This widely applied test system has some limitations concerning its relevance to the human condition, because bacteria are used as indicator cells and rat-liver homogenates for the extra-cellular activation of the mutagens. Both drawbacks are overcome by the use of metabolically active mammalian cells [15]. The detection of mutations in mammalian cells (e.g., HPRT assay) is rather time-consuming, thus hampering highthroughput screening of a large number of fruits and vegetables for antimutagenic activity. Because of its experimental simplicity, the comet assay (single-cell gel electrophoresis, SCGE) is better suited for this purpose compared with the HPRT assay [16,17]. HAAs are enzymatically activated to genotoxic metabolites via a two-step process (Fig. 1) that involves cytochrome P450dependent monooxygenase (CYP) followed by acetylation or sulfonation [18]. In addition to these activating enzymes, UDPglucuronosyltransferase (UGT) (Fig. 1) [19,20] and glutathione S-transferase (GST) [19] are two phase-II enzymes able to reduce the genotoxicity of HAAs by catalyzing the trapping of their reactive metabolites. To mimic the human situation more closely than do rat-liver homogenates and bacteria, we used the human form of the activating enzymes heterologously expressed in V79 Chinese hamster lung fibroblasts [21–23]. Additionally, Hep G2 cells were applied, which are metabolically competent human liver cells derived from a hepatoblastoma [24]. Both these immortalized mammalian cells activated 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to genotoxic species. IQ was selected because of its substantial bacterial

mutagenicity [25]. The less genotoxic PhIP was chosen because of its abundance in cooked food [26,27]. The effects of three teas, two wines, and the juices of 15 fruits and 11 vegetables on HAA genotoxicity were investigated by use of the comet assay. 2. Material and methods 2.1. Chemicals and other materials Chemicals were obtained as follows: IQ (CAS no. 76180-96-6) from Toronto Research Chemicals Inc. (Downsview, ON, Canada), PhIP (CAS no. 105650-23-5) from Biochemical Institute for Environmental Carcinogens (Grosshansdorf, Germany), 2hydroxyamino-3-methylimidazo[4,5-f]quinoline (N-OH-IQ) and 2-hydroxyamino1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP) from Midwest Research Institute (Kansas City, MO, USA), hydroxyurea (HU; CAS no. 127-07-1), cytosine 1-␤-d-arabinofuranoside) (araC; CAS no. 147-94-4), dicumarol (CAS no. 66-762), 7-methoxyresorufin (7-methoxy-3H-phenoxazin-3-one, CAS no. 5725-89-3), and resorufin sodium (7-hydroxy-3H-phenoxazin-3-one sodium salt, CAS no. 34994-50-8) from Sigma–Aldrich (Taufkirchen, Germany). trans-7,8-Dihydroxy7,8-dihydrobenzo[a]pyrene (BaP-7,8-diol; CAS no. 57404-88-3) was prepared as reported previously [28]. Fruits, vegetables and wines were purchased from local suppliers. The preparation of the teas and juices has been described elsewhere [16]. To imitate the cooking process, some vegetable juices were heated on a boiling water bath for 15 min. After cooling, the pH was adjusted to 7.4 with NaOH (10 M) if necessary. Microsomal fractions (SUPERSOMESTM ) containing human CYP1A2 (200 pmol cytochrome P450/mg protein; phenacetin deethylase activity: 26 pmol/min/pmol P450) and human NADPH cytochrome P450 reductase (cytochrome c reductase activity: 2.6 ␮mol/min/mg protein) were supplied by NatuTec (Frankfurt, Germany). Dulbecco’s modified Eagle’s medium (DMEM) containing d-glucose (4.5 g/l) and amino acids was obtained from Gibco Invitrogen (Karlsruhe, Germany). DMEM-1 was used for the cultivation of V79 cells; it contained additionally l-alanyl-lglutamine (862 mg/l) and sodium pyruvate (110 mg/l). DMEM-2 was used for the cultivation of Hep G2 cells; it contained l-glutamine (584 mg/l) but lacked pyruvate.

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DMEM-1 and -2 were both supplemented with penicillin G (100 units/ml) and streptomycin sulfate (100 ␮g/ml), and both were supplied by Gibco Invitrogen (Karlsruhe, Germany). Fetal bovine serum (FBS) and a solution of trypsin (0.05% for the detachment of V79 cells and 0.1% for the detachment of Hep G2 cells) and EDTA (0.02%) in phosphate-buffered saline (PBS) without Ca2+ and Mg2+ were obtained from Biochrom (Berlin, Germany), biochemicals from Roche Diagnostics (Mannheim, Germany), inorganic salts from Roth (Karlsruhe, Germany) and all other chemicals from Merck/VWR International (Darmstadt, Germany). PBS contained Na2 HPO4 (6.5 mM), KH2 PO4 (1.5 mM), NaCl (137 mM), KCl (2.7 mM) in aqueous solution and was adjusted to pH 7.4 with 10 M NaOH. 2.2. Cells and cell cultivation The genetically engineered metabolically active lung fibroblasts of the Chinese hamster V79-hCYP1A2-hNAT2*4 [23], V79-hCYP1A2-hSULT1A1*1 [23], and V79p (control cell line transfected with the puromycin-resistance marker [29]) were cultivated in DMEM-1 as reported previously [16]. The genetically engineered V79 cell lines were only used up to passage 10 in order to avoid loss of enzyme activity. Human-derived Hep G2 cells at passage 77 were generously provided by Dr. P. Steinberg (Institute for Food Toxicology and Analytical Chemistry, University of Veterinary Medicine, Hannover, Germany); they were kept in liquid nitrogen as 1ml aliquots containing approx. 107 cells in FBS with 10% (v/v) DMSO. After thawing, one aliquot of the cryo-preserved Hep G2 cells was added to 9 ml cold DMEM-2 supplemented with 15% FBS and centrifuged (180 × g, 4 ◦ C, 10 min) in order to remove DMSO. The cell pellet was suspended in 5 ml warm (37 ◦ C) medium and cultivated at 37 ◦ C, 5% CO2 and 95% relative humidity in cell-culture flasks (25 cm2 ; Greiner, Frickenhausen, Germany) until sub-confluency had been reached (approx. 3 days). Then the cells were washed with warm PBS and treated with 1 ml warm trypsin/EDTA solution for approx. 60 s. After careful aspiration of the trypsin solution, the cells were incubated (37 ◦ C) for another 5 min. The detached cells were taken up in 6 ml warm medium and pressed twice or thrice through a hypodermic needle (20G; 0.9 mm) to achieve complete dissociation. Cells were sub-cultured in cellculture flasks (25 cm2 ; split ratio 1/6–1/10) with 6 ml medium until sub-confluency had again been reached. Cells were detached by treatment with trypsin/EDTA as described before, suspended in 10 ml warm medium and counted (haemocytometer Neubauer). 2.3. Comet assay On Petri dishes (Ø6 cm; Greiner, Frickenhausen, Germany) 5 × 105 cells of V79hCYP1A2-hNAT2*4, V79-hCYP1A2-hSULT1A1*1 or V79p (control cell line) in 5 ml medium (5% FBS) were incubated for 24 h. The attached cells were then washed with 5 ml warm PBS and incubated for another 2 h with 5 ml medium containing HU (10 mM), araC (1.8 mM), the genotoxin (i.e., IQ, PhIP, N-OH-IQ, N-OH-PhIP, BaP-7,8diol) in 50 ␮l DMSO, and the plant-derived beverages (0–100 ␮l). After the second incubation the cells were washed with 5 ml cold PBS, detached by treatment with 1 ml of trypsin/EDTA solution as described above, suspended in 1 ml cold medium (≥106 cells/ml) and put on ice. Similarly 7.5 × 105 human liver-derived Hep G2 cells in 5 ml medium (15% FBS) were incubated for 24 h on Petri dishes (Ø6 cm). The attached cells were washed with warm PBS and incubated for another 24 h with 5 ml medium containing PhIP (500 ␮M) in 50 ␮l DMSO and the juices (0–150 ␮l) but without HU and araC. Then the medium was removed, the cells were washed with warm PBS and after addition of 5 ml medium containing HU (10 mM) and araC (1.8 mM) again incubated for 2 h. Thereafter the cells were washed with cold PBS, detached by treatment with 1 ml of trypsin/EDTA solution as described above, suspended in 1 ml cold medium (≥106 cells/ml) and put on ice. Cells were only further processed when their viability determined as described [16] exceeded 90%. The comet assay was performed essentially as reported [16]. As parameter for DNA damage the tail moment as defined by Olive et al. [30] was recorded. The tail moment is the product of the amount of DNA in the tail and the mean distance of migration in the tail. All experiments were performed at least twice. Two replicate slides were made from each incubation. Twenty-five nonoverlapping comets in the middle of each slide were analyzed. Thus each data point represents the average tail moment of at least 100 measurements (comets) [16]. The inhibitory activity of the teas, wines and juices is expressed as the IC50 , i.e., the concentration of the plant-derived beverage (%, v/v) required to reduce the genotoxic activity (tail moment) to 50%, calculated as the mean from at least three concentration–response curves. 2.4. Measurement of the enzymatic O-demethylation of 7-methoxyresorufin The O-demethylation of 7-methoxyresorufin, catalyzed by CYP1A2, was determined according to the fluorimetric method of Burke and Mayer [31] modified by Lubet et al. [32]. The incubation mixture contained 7-methoxyresorufin (1.5 ␮M), CYP1A2-microsomal protein (0.06 mg/ml), ␤-NAD (5.8 mM), dicumarol (10 ␮M), plant-derived beverage (0–3%) in 2015 ␮l of Tris (94 mM), MgCl2 (9.4 mM), NaOH (0.5 mM) at pH 7.6. The enzymatic reaction was started by the addition of 0.5 ␮mol ␤-NADPH (10 ␮l) at 20 ◦ C. The formation of resorufin as a function of time was recorded with a spectrophotofluorimeter (FP 750, Jasco, Tokyo, Japan; excitation

Fig. 2. Activation of IQ and PhIP by genetically engineered Chinese hamster V79 cells to genotoxic metabolites determined with the comet assay; values represent mean ± S.D. (n = 3). (§ According to Olive et al. [30]; IQ, 2-amino3-methylimidazo[4,5-f]quinoline; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5b]pyridine; V79p, Chinese hamster V79 fibroblasts expressing the puromycinresistance marker; V79-hCYP1A2-hNAT2*4, V79 cells co-expressing human cytochrome P450-dependent monooxygenase 1A2 and human N-acetyltransferase 2*4; V79-hCYP1A2-hSULT1A1*1, V79 cells co-expressing human cytochrome P450dependent monooxygenase 1A2 and human sulfotransferase 1A1*1.)

 = 522 nm, emission  = 586 nm). After the measurement, two consecutive doses of 1 nmol resorufin sodium in 10 ␮l ethanol were added for calibration and the average value of the responses was taken for calculation.

3. Results All incubations of the HAAs with V79 Chinese hamster fibroblasts were performed in the presence of HU and araC as described previously [16]. Under these conditions IQ was concentrationdependently activated by V79-hCYP1A2-hNAT2*4 to genotoxic metabolites detected with the comet assay (Fig. 2). The control cell line V79p and V79-hCYP1A2-hSULT1A1*1 cells were unable to transform IQ to DNA-reactive products (Fig. 2). The selectivity of both genetically engineered V79 cell lines was less pronounced in PhIP activation than in IQ activation. Both cell lines transformed PhIP to genotoxic metabolites but to different degrees: V79-hCYP1A2-hSULT1A1*1 strongly activated PhIP to genotoxic products whereas V79-hCYP1A2-hNAT2*4 caused considerably weaker genotoxicity (Fig. 2). We then investigated the effects of three teas, two wines, and the juices of 15 fruits and 11 vegetables on the genotoxicity of IQ activated by V79-hCYP1A2-hNAT2*4 and of PhIP activated by V79-hCYP1A2-hSULT1A1*1 (Table 1). Sweet cherry exhibited the highest inhibitory activity on the genotoxicity of IQ, with an IC50 of 0.17%, followed by spinach, kiwi fruit, onion, plum, and blueberry. Moderate suppression of IQ genotoxicity was exerted by the teas, the juices of broccoli, cauliflower and beetroot, the wines, and the

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Table 1 Influence of plant-derived food components on the activity of different genotoxins in the comet assay with metabolically competent V79 cells. Given is the IC50 , i.e., the concentration (%, v/v) of plant-derived beverages required to inhibit the genotoxic activity by 50%, calculated from concentration–response curves (maximal concentration of beverage: 2%, v/v); values represent mean ± S.D. (n = 3). Plant family

Teas and wines Green tea Black tea Rooibos tea White wine Red wine Fruit juices Apple (Granny Smith) Apple (Red delicious) Blackberry Strawberry Sour cherry (Morello) Sweet cherry Plum Black currant Red currant Blueberry Orange Grapefruit Watermelon Kiwi fruit Pineapple Vegetable juices Broccoli Cauliflower Red cabbage Beetroot Spinach Chard (mangold) Tomato Sweet pepper Green beans Cucumber Onion

V79-hCYP1A2-hNAT2*4

V79-hCYP1A2-hSULT1A1*1

IQ (0.1 ␮M)

BaP-7,8-diol (1 ␮M)

N-OH-IQ (20 nM)

Theaceae Theaceae Fabaceae Vitaceae Vitaceae

0.93 ± 0.21 0.83 ± 0.45 0.84 ± 0.07 1.74 ± 0.39 1.06 ± 0.53

73 ± 1%a 61 ± 12% No inhibition 69 ± 21%

0.20 0.30 2.05 0.63

Rosaceae Rosaceae Rosaceae Rosaceae Rosaceae Rosaceae Rosaceae Grossulariaceae Grossulariaceae Ericaceae Rutaceae Rutaceae Cucurbitaceae Actinidiaceae Bromeliaceae

No inhibition 1.76 ± 0.34 1.19 ± 0.01 1.24 ± 0.41 56 ± 4% 0.17 ± 0.02 0.56 ± 0.03 1.39 ± 0.30 65 ± 6% 0.71 ± 0.13 No inhibition 58 ± 8% 1.14 ± 0.14 0.48 ± 0.07 65 ± 5%

No inhibition no inhibition

68 ± 14% 1.20 ± 0.50

Brassicaceae Brassicaceae Brassicaceae Amaranthaceae Amaranthaceae Amaranthaceae Solanaceae Solanaceae Fabaceae Cucurbitaceae Alliaceae

0.92 ± 0.05 0.98 ± 0.01 1.86 ± 0.85 1.04 ± 0.55 0.42 ± 0.31 1.81 ± 0.23 1.62 ± 0.05 1.49 ± 0.33 Toxicb No inhibition 0.54 ± 0.46

± ± ± ±

0.01 0.21 0.39 0.22

PhIP (3–10 ␮M)

N-OH-PhIP (0.3 ␮M)

1.37 ± 0.17 0.37 ± 0.25

71 ± 10% 0.73 ± 0.41

57 ± 9% 0.41 ± 0.36 65 ± 5% 80 ± 11% 0.79 ± 0.57

0.30 0.29 0.19 69

± ± ± ±

0.16 0.13 0.06 14%

0.96 ± 0.58 1.79 ± 0.40 0.73 ± 0.25

59 ± 8% 1.99 ± 0.60 61 ± 1%

0.64 ± 0.06 No inhibition No inhibition

0.46 ± 0.03 80 ± 10% 69 ± 15%

1.62 ± 0.36

68 ± 14%

64 ± 8% 85 ± 1%

No inhibition No inhibition

74 ± 8%

82 ± 2%

0.37 ± 0.33 0.09 ± 0.01

0.83 ± 0.12 0.53 ± 0.10

57 ± 2%

0.85 ± 0.03

0.42 ± 0.24 0.13 ± 0.05

hCYP, human cytochrome P450-dependent monooxygenase; hNAT, human N-acetyltransferase; hSULT, human sulfotransferase; IQ, 2-amino-3-methylimidazo[4,5f]quinoline; BaP-7,8-diol, trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene; N-OH-IQ, N-hydroxy-IQ; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; N-OH-PhIP, N-hydroxy-PhIP. a IC50 was not reached; given is the percentage of genotoxic activity at the maximal concentration (2%, v/v) of the plant-derived beverage. b Viability of cells dropped to 50% with 0.5% juice of green beans, and to 2.4% at the maximal concentration (2%, v/v) of the juice.

juices of watermelon, blackberry, strawberry, black currant, sweet pepper, tomato, the Red delicious cultivar of apple, chard, and red cabbage. The protection against IQ genotoxicity was weak with the juices of sour cherry, grapefruit, red currant and pineapple. No inhibition was found with the Granny Smith cultivar of apple, orange and cucumber juice. The effect of green bean juice on genotoxicity was confounded by its cytotoxicity (Table 1). Inhibition of PhIP genotoxicity was strong with red wine, spinach, and plum, moderate with beetroot, onion, sour cherry, white wine, blueberry, and sweet cherry, and only weak with blackberry and watermelon. Because most vegetables are consumed after cooking, the influence of heating on the protective properties of vegetable juices was investigated (Fig. 3). The inhibitory components of broccoli and cauliflower resisted the elevated temperature, while those in beetroot and spinach were heat-sensitive. A trend toward an increase in protective activity was observed with red cabbage, tomato and sweet pepper, but the difference was statistically significant only with tomato. To elucidate the mechanism by which plants inhibit IQ and PhIP genotoxicity, the effects of various plant-derived beverages on both steps of HAA enzymatic activation were separately investigated. For the first step, BaP-7,8-diol served as diagnostic substrate. For the second step, the N-hydroxylamines of IQ and PhIP were employed. V79-hCYP1A2-hNAT2*4 and V79-hCYP1A2hSULT1A1*1 concentration-dependently activated BaP-7,8-diol to

genotoxic metabolites by almost the same magnitude (Fig. 4). V79p cells were unable to activate BaP-7,8-diol. V79-hCYP1A2-hNAT2*4 cells efficiently transformed N-OH-IQ to reactive species, whereas V79-hCYP1A2-hSULT1A1*1 and V79p cells lacked this enzymatic ability (Fig. 4). V79-hCYP1A2-hSULT1A1*1 cells strongly activated N-OH-PhIP to genotoxic metabolites, V79-hCYP1A2-hNAT2*4 cells were less efficient, but even V79p cells transformed N-OH-PhIP to reactive species (Fig. 4). Sour cherry, blueberry, and black currant juice strongly reduced BaP-7,8-diol genotoxicity (Table 1). Rooibos tea, sweet cherry, red wine, black tea, onion, plum, and kiwi fruit were only weakly active, both apple cultivar, orange, grapefruit, spinach, beetroot, and white wine were inactive. In contrast to BaP-7,8-diol many plant-derived beverages strongly suppressed the genotoxicity of N-OH-IQ, e.g., spinach, plum, black tea, sweet cherry, rooibos tea, sour cherry, beetroot, blueberry, and red wine (Table 1). The protection was less pronounced with the Red delicious cultivar of apple and white wine and only weak with onion, the Granny Smith cultivar of apple, black currant, grapefruit, orange, and kiwi fruit. The DNA reactivity of N-OH-PhIP was efficiently suppressed by spinach, beetroot, and red wine, moderately by sweet cherry, and only weakly by sour cherry, plum, blueberry, and white wine (Table 1). In a further set of experiments, the inhibitory effects of some plant-derived beverages on CYP1A2, responsible for the first step of HAA activation, was investigated. This was achieved by measuring

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Fig. 3. Influence of heating on the protection by vegetable juices against the genotoxicity of IQ (0.1 ␮M) in V79-hCYP1A2-hNAT2*4 cells determined with the comet assay; bars represent mean ± S.D. (n = 3); different from raw juice: **p < 0.01; *p < 0.05. (IQ, 2-amino-3-methylimidazo[4,5-f]quinoline; V79-hCYP1A2-hNAT2*4, V79 cells co-expressing human cytochrome P450-dependent monooxygenase 1A2 and human N-acetyltransferase 2*4.)

enzymatic O-demethylation of 7-methoxyresorufin by microsomes containing human CYP1A2 (Fig. 5). Sour cherry juice and black tea were very efficient with an IC50 of 0.26%. Rooibos tea and black currant juice reduced CYP1A2 activity to 50% at 1.22% and 1.52%, respectively. Spinach and beetroot juices were nearly inactive (Fig. 5). Finally, Hep G2 cells derived from a human hepatoblastoma were employed as metabolic system and as indicator cells rather than genetically engineered V79 hamster fibroblasts to mimic the human condition more closely. Employing the incubation conditions of V79 fibroblasts, PhIP was only weakly activated by Hep

Fig. 5. Influence of plant-derived beverages on the activity of 7-methoxyresorufinO-demethylase in hCYP1A2 SUPERSOMESTM ; values represent mean ± S.D. (n = 3). (100% = 778 pmol resorufin/min/mg protein.)

G2 cells to genotoxic metabolites (data not shown). The genotoxic response increased when PhIP was incubated with Hep G2 cells for 24 h in the absence of HU/araC followed by incubation for 2 h without PhIP in the presence of HU/araC. Under identical conditions IQ was less efficiently activated by Hep G2 cells than PhIP (data not shown). Spinach juice inhibited PhIP genotoxicity more efficiently than beetroot juice (Fig. 6A). In contrast to the results with V79-hCYP1A2-hSULT1A1*1 cells (Fig. 6B), both juices were 10-fold more active when tested with Hep G2 cells

Fig. 4. Activation of BaP-7,8-diol, N-OH-IQ and N-OH-PhIP by genetically engineered Chinese hamster V79 cells to genotoxic metabolites determined with the comet assay; values represent mean ± S.D. (n = 3). (§ According to Olive et al. [30]; BaP-7,8-diol, trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene; N-OH-IQ, 2-hydroxyamino-3methylimidazo[4,5-f]quinoline; N-OH-PhIP, 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine; V79p, Chinese hamster V79 fibroblasts expressing the puromycinresistance marker; V79-hCYP1A2-hNAT2*4, V79 cells co-expressing human cytochrome P450-dependent monooxygenase 1A2 and human N-acetyltransferase 2*4; V79hCYP1A2-hSULT1A1*1, V79 cells co-expressing human cytochrome P450-dependent monooxygenase 1A2 and human sulfotransferase 1A1*1.)

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Fig. 6. Influence of vegetable juices on the genotoxicity of PhIP in Hep G2 (A, 500 ␮M) and in V79-hCYP1A2-hSULT1A1*1 cells (B, 10 ␮M) determined with the comet assay; values represent mean ± S.D. (n = 3). (§ According to Olive et al. [30]; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; V79-hCYP1A2-hSULT1A1*1, V79 cells co-expressing human cytochrome P450dependent monooxygenase 1A2 and human sulfotransferase 1A1*1.)

(Hep G2: IC50(spinach) = 0.04%, IC50(beetroot) = 0.07%; V79-hCYP1A2hSULT1A1*1: IC50(spinach) = 0.54%, IC50(beetroot) = 0.82%). 4. Discussion HAAs require enzymatic activation to exhibit genotoxicity. Thus, V79 Chinese hamster fibroblasts lacking CYP activity [33] were unable to activate IQ or PhIP (Fig. 2). The first step of HAA enzymatic activation is N-oxidation of the exocyclic amino group (Fig. 1), a reaction that is preferentially catalyzed by CYP1A2 [34,35]. The second step consists of either acetylation or sulfonation of the Nhydroxylamine [18] (Fig. 1). The resulting highly unstable esters are spontaneously cleaved to form the reactive nitrenium ion, which represents the ultimate carcinogenic species [18]. Esterification of N-hydroxy-HAAs is carried out most efficiently by the isoenzymes N(O)-acetyltransferase 2 (NAT2) and sulfotransferase 1A1 (SULT1A1) [36]. The wild-type alloenzymes NAT2*4 and SULT1A1*1, associated with particularly high enzyme activity, are expressed in human liver and other tissues [37,38]. Consequently, these allelic variants were used for the construction of the Chinese hamster fibroblasts V79-hCYP1A2-hNAT2*4 and V79hCYP1A2-hSULT1A1*1 [23], which were designed to enzymatically activate IQ and PhIP to genotoxic species. The reactive metabolites covalently bind to DNA to form bulky adducts [18]. The comet assay in the presence of the DNA-repair inhibitors HU and araC [39] was employed to detect these DNA lesions. This analysis is based on the accumulation of single-strand breaks during the repair process. The pivotal role of the acetyltransferase NAT2 in the activation of IQ [22,40,41] and its N-hydroxylamine to genotoxic metabolites was demonstrated (Figs. 2 and 4), whereas sulfonation was inefficient, consistent with previous reports [42–44]. In contrast, PhIP and its N-hydroxylamine were strongly activated by V79-hCYP1A2hSULT1A1*1 cells (Figs. 2 and 4) indicating the significance of

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sulfonation in the case of this HAA [43–45]. The genotoxicity of N-OH-PhIP in V79 cells (Fig. 4), which were found to be deficient in SULT activity [46], could be induced either by spontaneous formation of the nitrenium ion or by another yet unknown enzymatic mechanism [47]. Thus, the weak effect of PhIP activated by V79hCYP1A2-hNAT2*4 (Fig. 2) may represent the intrinsic genotoxicity of N-OH-PhIP and not necessarily the genotoxicity of its acetate. The minor role of acetylation in PhIP activation has already been described [41,43,44]. To elucidate the mechanism of inhibition, the influence of plantderived beverages on HAA genotoxicity was determined not only with IQ and PhIP but also with diagnostic substrates for both steps of their enzymatic activation (Fig. 1). Thus, the second step was characterized by N-OH-IQ and N-OH-PhIP, whereas the activity of CYP1A2 required for the first step was determined by the transformation of BaP-7,8-diol to the dihydrodiol epoxide (Fig. 1). As expected V79-hCYP1A2-hNAT2*4 and V79-hCYP1A2-hSULT1A1*1 cells activated BaP-7,8-diol to similar extent (Fig. 4), in contrast to V79p cells that lack CYP activity [33]. For the moderate reduction of IQ genotoxicity by black and rooibos tea, inhibition of the second activation step appears to be more important than the formation of N-hydroxylamine catalyzed by CYP1A2 (Table 1). Since polyphenols (e.g., (+)-catechin and related flavan-3-ols) are thought to be responsible for the antimutagenicity and anticarcinogenicity of tea [48], it can be envisaged that these compounds either inhibit NAT [49] or reduce the acetate of N-OH-IQ to the parent amine as described for N-acetoxy-PhIP [50]. White wine consistently reduced the genotoxicity of the investigated compounds (IQ, N-OH-IQ, PhIP, N-OH-PhIP, and BaP-7,8-diol) to a lesser extent than red wine (Table 1). Again, phenolic compound (e.g., resveratrol or its analogs) in red wine may be responsible for this effect [51]. Of the fruits, sweet cherry, plum, and blueberry were the most active at inhibiting IQ genotoxicity (Table 1). A similar observation was made when the Salmonella mutagenicity assay was used [14]. The different inhibitory effects of various species (e.g., sour and sweet cherry) or cultivars of the same fruit (e.g., Red delicious and Granny Smith apples) (Table 1), could be an indication of different amounts of phytochemicals found in various species (e.g., cherry [52]), or cultivars (e.g., apple [53]). The weak inhibition by most fruit juices of PhIP genotoxicity activated by V79-hCYP1A2-hSULT1A1*1 cells contrasted with the strong suppression observed with V79 fibroblasts expressing the corresponding rat enzymes [16]. Among the vegetables, spinach and onion were the most efficient at inhibiting IQ genotoxicity not only in mammalian cells (Table 1) but also in bacteria [14]. The distinctly different inhibitory activities of beetroot and spinach juices against DNA damage caused by PhIP were demonstrated not only with V79 cells expressing human hepatic enzymes (Table 1) but also with V79 cells containing hepatic enzymes of rat origin [16]. Fruits and vegetables investigated in this study belong to diverse plant families (Table 1). The botanical origin, however, seems to have no influence on their antigenotoxicity. Most vegetables are consumed after cooking; therefore, the influence of heating was investigated. Remarkable stability of inhibitory activity was demonstrated. Only the inhibitory constituents of beetroot and spinach appeared to be heat-sensitive (Fig. 3). The stronger suppression of genotoxicity by sweet pepper and tomato after cooking could be caused by liberation of inhibitory phytochemicals at elevated temperatures. The toxic effect of green beans on V79 cells (Table 1 and Fig. 3) was very likely caused by the phytohaemagglutinin in the raw vegetable [54]. Because of the thermolability of the lectin, no toxicity was observed after heating (Fig. 3). The use of diagnostic substrates for both HAA activation steps revealed a more pronounced inhibition of the second step (i.e., esterification and DNA binding of the N-hydroxylamines) by most

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plant-derived beverages (Table 1). Only sour cherry, blueberry, and black currant juices exerted a considerable reduction in BaP7,8-diol genotoxicity, which was indirect evidence of CYP1A2 inhibition. Grapefruit juice is known to efficiently inhibit CYP1A2 in vivo [55], but surprisingly did not elicit such inhibition in our cellular system in vitro (Table 1). This observation may be explained by the presence of the glycoside naringin in grapefruit juice, which does not inhibit CYP1A2 but rather is transformed in vivo in the gut by bacterial glycosidases to the strong CYP1A2 inhibitor narigenin [55]. Direct evidence of CYP1A2 inhibition by different plantderived beverages was obtained with microsomes containing the human enzyme (Fig. 5). These results should be compared with the influence on BaP-7,8-diol genotoxicity (Table 1), because this PAH metabolite is transformed by CYP1A2 to the DNA-binding dihydrodiol epoxide. In both systems, sour cherry exhibited a stronger inhibitory effect than black currant. A good correlation was also found with spinach and beetroot with regard to very weak inhibition of CYP1A2 (Fig. 5) and lack of inhibition of BaP-7,8-diol genotoxicity (Table 1). In contrast, rooibos tea and black tea more strongly inhibited CYP1A2 (Fig. 5) than BaP-7,8-diol genotoxicity (Table 1). One reason for this difference could be the limited permeability of the plasma membrane of the V79 cells to the inhibitory phytochemicals in both teas. Although the V79 cells used in this study express the human enzymes required for HAA activation, they are still artificial metabolic systems. The human condition would be more closely mimicked by the use of metabolically competent cells of the human liver, the most important organ for xenobiotic biotransformation in man. We therefore applied Hep G2 cells derived from a human hepatoblastoma [56]. The metabolic capacity of these immortal human cells was demonstrated in a panel of 18 cell lines, among which Hep G2 cells most efficiently metabolized benzo[a]pyrene to mutagens [57]. The suitability of the comet assay using Hep G2 cells for the detection of antimutagenic phytochemicals in food has already been shown [15,58]. In contrast to the metabolically active V79 fibroblasts, an adequate genotoxic response of PhIP in Hep G2 cells was only achieved when applying two incubation periods (i.e., 24 h with PhIP in the absence of HU/araC followed by 2 h without PhIP in the presence of HU/araC). During the first incubation, PhIP is transformed to reactive metabolites that covalently bind to DNA preferentially at the C8 position of deoxyguanosine [18]. These bulky adducts are repaired by nucleotide excision repair (NER), but only those lesions in which the DNA polymerization or the ligation step is not yet completed are detected with the comet assay. Thus, DNA single-strand breaks remaining after the first incubation period represent incomplete NER. During the second incubation period in the absence of PhIP, NER of the stable DNA adducts continues, but because DNA-repair synthesis is inhibited by HU/araC [59], the repair sites remain open, and DNA single-strand breaks accumulate over time. Compared with the genetically engineered V79 cells, the genotoxic response in the comet assay using Hep G2 cells was still lower but may be increased by optimizing the cultivation conditions with respect to the expression of xenobiotic-metabolizing enzymes [60,61]. In a preliminary experiment with Hep G2 cells, the distinctly different inhibitory effect of spinach and beetroot on PhIP genotoxicity could be demonstrated (Fig. 6). In this human cell system, both vegetable juices inhibited PhIP genotoxicity 10-fold more efficiently than in the V79 Chinese hamster system. Whether this greater sensitivity can also be observed with other fruit and vegetable juices, and the relevance of these results to the human condition in vivo, remain to be determined. The vast majority of studies investigating the protective effect of food components against HAA genotoxicity employed bacteria as indicator cells and rat-liver homogenates as metabolizing system

[13]. To our knowledge this is the first study that identified several fruits and vegetables considerably inhibiting HAA genotoxicity by the use of mammalian cells expressing human enzymes. The present data, however, do not provide answers as to the phytochemicals responsible for the antigenotoxicity. One may speculate that anthocyanins in sweet cherries [52], plums [62], and blueberries [63], carotenoids in kiwi fruit and spinach [64], or flavonoids in onions [65] are the active compounds responsible for the observed effects. Furthermore, the mechanism of protection by the phytochemicals remains to be determined. Apart from the inhibition of activating enzymes, investigated in the present study, also inactivating enzymes, e.g., GSTs, could be induced by increasing the nuclear accumulation of the transcription factor Nrf2 [66]. However, the influence of phytochemicals on HAA metabolizing enzymes represents just one of many possibilities [13]. The plant-derived beverages employed in this work were used as prepared. In further investigations these beverages should be analyzed and standardized according to dry weight or phytochemicals to identify the parameters responsible for antigenotoxicity. Particularly, sweet cherry and spinach juice deserve a more thorough exploration. Finally, the metabolizing system employed in our study deserves two critical comments: (i) the response to grapefruit juice revealed the limitation of the genetically engineered V79 cells used to predict the influence of fruits and vegetables on HAA genotoxicity in man in vivo. Thus, a cellular system more closely mimicking the human condition should not only express the human hepatic enzymes necessary to metabolize HAAs, but also the hydrolyzing enzymes (e.g., glycosidases) responsible for the intestinal liberation of active aglycones from their water-soluble conjugates; (ii) metabolically active cellular systems employed in further studies should not only express HAA activating enzymes, e.g., hCYPs, hNATs, hSULTs, but also inducible protective enzymes, e.g., hUGTs, hGSTs. The application of Hep G2 cells in this work was a first, albeit not yet optimal step in this direction. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgements Our sincere thanks are expressed to the European Communities for financial support of this work (Contract No. QLK1-CT99-01197). In this context, we greatly acknowledge the competence and patience of Kerstin Skog, Lund University, Sweden, as coordinator of the funded project “Heterocyclic amines in cooked foods – Role in human health”. References [1] W. Lijinsky, The formation and occurrence of polynuclear aromatic hydrocarbons associated with food, Mutat. Res. 259 (1991) 251–261. [2] T. Sugimura, S. Sato, Mutagens-carcinogens in foods, Cancer Res. 43 (1983) 2415s–2421s. [3] M.G. Knize, C.P. Salmon, P. Pais, J.S. Felton, Food heating and the formation of heterocyclic aromatic amine and polycyclic aromatic hydrocarbon mutagens/carcinogens, Adv. Exp. Med. Biol. 459 (1999) 179–193. [4] R. Goldman, P.G. Shields, Food mutagens, J. Nutr. 133 (2003) 965S–973S. [5] R. Sinha, D.R. Gustafson, M. Kulldorff, W.-Q. Wen, J.R. Cerhan, W. Zheng, 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, a carcinogen in hightemperature-cooked meat, and breast cancer risk, J. Natl. Cancer Inst. 92 (2000) 1352–1354. [6] R. Sinha, M. Kulldorff, M.J. Gunter, P. Strickland, N. Rothman, Dietary benzo[a]pyrene intake and risk of colorectal adenoma, Cancer Epidemiol. Biomarkers Prev. 14 (2005) 2030–2034. [7] L.M. Butler, R. Sinha, R.C. Millikan, C.F. Martin, B. Newman, M.D. Gammon, A.S. Ammerman, R.S. Sandler, Heterocyclic amines, meat intake, and association with colon cancer in a population-based study, Am. J. Epidemiol. 157 (2003) 434–445.

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