Genotoxicity of heterocyclic amines (HCAs) on freshly isolated human peripheral blood mononuclear cells (PBMC) and prevention by phenolic extracts derived from olive, olive oil and olive leaves

Accepted Manuscript Genotoxicity of heterocyclic amines (HCAs) on freshly isolated human peripheral blood mononuclear cells (PBMC) and prevention by phenolic extracts derived from olive, olive oil and olive leaves Raffaela Fuccelli, Patrizia Rosignoli, Maurizio Servili, Gianluca Veneziani, Agnese Taticchi, Roberto Fabiani PII:

S0278-6915(18)30766-X

DOI:

10.1016/j.fct.2018.10.033

Reference:

FCT 10138

To appear in:

Food and Chemical Toxicology

Received Date: 18 July 2018 Revised Date:

9 October 2018

Accepted Date: 11 October 2018

Please cite this article as: Fuccelli, R., Rosignoli, P., Servili, M., Veneziani, G., Taticchi, A., Fabiani, R., Genotoxicity of heterocyclic amines (HCAs) on freshly isolated human peripheral blood mononuclear cells (PBMC) and prevention by phenolic extracts derived from olive, olive oil and olive leaves, Food and Chemical Toxicology (2018), doi: https://doi.org/10.1016/j.fct.2018.10.033. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Genotoxicity of heterocyclic amines (HCAs) on freshly isolated human peripheral blood mononuclear cells (PBMC) and prevention by

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phenolic extracts derived from olive, olive oil and olive leaves Raffaela Fuccellia, Patrizia Rosignolia, Maurizio Servilib,

Gianluca Venezianib, Agnese Taticchib*, Roberto Fabiania*

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Department of Chemistry, Biology and Biotechnology (Biochemistry and Molecular Biology Unit), via del Giochetto, 06126 Perugia, University of Perugia, Italy Department of Agricultural, Food and Environmental Science (Food Science and Technology Unit), via S. Costanzo, 06126 Perugia, University of Perugia, Italy

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*Corresponding authors:

Roberto Fabiani (email: [email protected])

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Agnese Taticchi (email: [email protected])

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Abstract In this study we investigated the genotoxic potential of 2-amino-1-methyl-6-phenylimidazo[4,5b]pyridine, (PhIP); 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline, (IQ); 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, (MeIQx) and 2-amino-3,4,8-trimethyl-3H-imidazo[4,5-f]quinoxaline

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(DiMeIQx) on human freshly isolated peripheral blood mononuclear cells (PBMCs) by the comet assay. The preventive ability of three different phenolic extracts derived from olive (O-PE), virgin olive oil (OO-PE) and olive leaf (OL-PE) on PhIP induced DNA damage was also investigated.

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PhIP and IQ induced a significant DNA damage at the lowest concentration tested (100 µM), while the genotoxic effect of MeIQx and DiMeIQx become apparent only in the presence of DNA repair

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inhibitors Cytosine b-D-arabinofuranoside and Hydroxyurea (AraC/HU). The inclusion of metabolic activation (S9-mix) in the culture medium increased the genotoxicity of all HCAs tested. All three phenolic extracts showed an evident DNA damage preventive activity in a very low concentration range (0.1 – 1.0 µM of phenols) which could be easily reached in human tissues “in

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vivo” under a regular intake of virgin olive oil. These data further support the observation that consumption of olive and virgin olive oil may prevent the initiation step of carcinogenesis. The leaf

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or supplements.

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waste could be an economic and simple source of phenolic compounds to be used as food additives

Keywords: Heterocyclic aromatic amines; PBMC; gentoxicity; comet assay; olive oil; phenols

Abbreviations PhIP: 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; IQ: 2-amino-3-methyl3H-imidazo[4,5-f]quinoline; MeIQx: 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline; DiMeIQx: 2-amino-3,4,8-trimethyl-3H-imidazo[4,5-f]quinoxaline; PBMC: peripheral blood mononuclear cells; O-PE: olive phenolic extract; OO-PE: olive oil phenolic extract; OL-PE: olive leaf phenolic extract.

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1. Introduction Heterocyclic aromatic amines (HCAs) are potent mutagen and carcinogen compounds produced during cooking of protein rich foods such as red meat, poultry and fish (Sugimura et al., 2004). The amount of HCAs produced depends upon the type of meat, cooking method,

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temperature and duration of cooking. Pan-frying, grilling, or barbecuing at high temperature produces the highest amounts of HCAs (Ni et al., 2008). More than 20 different HCAs have been identified which are produced by either a pyrolysis of amino acids or a complicated series of

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reactions involving pyridine or pyrazine (derived from heat degradation products of amino acids and hexoses) and creatinine (Murkovic, 2004; Turesky, 2007). In this way are produced the so

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called “aminoimidazoarenes” which includes 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline (IQ), 2-amino-3,8-dimethyl-imidazo[4,5f]quinoxaline (MeIQx), and 2-amino-3,4,8-trimethyl-3H-imidazo[4,5-f]quinoxaline (DiMeIQx) (Kizil et al., 2011). PhIP is the most abundant HCA in the human diet while IQ, MeIQx and

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DiMeIQx are among the most potent mutagen compounds ever tested in the Ames/Salmonella assay (Sugimura et al., 2004). Several studies have shown an association between HCAs intake and cancer risk. A recent meta-analysis has demonstrated that human exposure to PhIP, MeIQx and

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DiMeIQx is associated to an increment of colorectal cancer risk (Chiavarini et al., 2017). In

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addition, HCAs are able to induce tumours in different sites in experimental rodent models (Zheng and Lee, 2009). The International Agency for Research on Cancer (IARC) has included IQ in the group 2A (probably carcinogenic to humans) and the others (PhIP, MeIQx and DiMeIQx) in the group 2B (possibly carcinogenic to human) (IARC, 1993). HCAs are pro-carcinogens acting after metabolic activation to form reactive intermediates that bind to DNA leading to DNA-adducts, DNA strand breaks and mutation (Turesky and Le Marchand, 2011). Different methods have been used to test the genotoxicity of some HCAs considering cytogenetic endpoints such as sister chromatid exchange, chromosomal aberrations and micronuclei (Katic et al., 2010; Hewitt et al., 2007; Wu et al., 1997). However, these methods have

ACCEPTED MANUSCRIPT the limit that they can be carried out only on proliferating cells. Instead, a simple and reproducible way to measure the DNA damage in not proliferating cells is the “comet assay” (Singh, 2016). Performed under alkaline conditions (pH >13) this test is able to detect DNA double-strand breaks, single-strand breaks, alkali-labile sites, DNA-DNA/DNA-protein cross-linking, and incomplete

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excision repair sites (Pu et al., 2015). The comet assay has been previously used to measure the DNA damage induced by HCAs in different human tumour cell lines derived from intestine (Nowak et al., 2014), liver (Pezdirc, et al., 2013; Viegas et al., 2012; Haza and Morales, 2011) and breast

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(Jain et al., 2015) essentially in the absence of external metabolic activation (S9). Although, the use of cultured proliferating cells to investigate DNA damage is very useful, it may have some

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drawbacks. In particular, highly proliferating cells may be less susceptible to genotoxic insults since they are more active in repairing the DNA damage comparing to resting cells (Collins et al., 1995; Duthie and Collins, 1997). In addition, the growth state of the cells may deeply influence the activity of different detoxifying enzymes, in particular those related to oxidative stress (Kruszewski

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et al., 2012).

Surprisingly, very little is known about the genotoxicity of HCAs on freshly isolated normal (non proliferating) human peripheral blood mononuclear cells (PMBC). Recently, two studies have

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investigated the genotoxic activity of both PhIP and IQ on human cryopreserved lymphocytes (Baumgartner et al., 2012; Kurzawa-Zegota et al., 2012). No data are available on the genotoxic

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effect of the highly mutagenic MeIQx and DiMeIQx on PBMC. In the last few years, several in vitro, animal, and epidemiological studies have demonstrated the importance of healthy nutrition and dietary components on cancer prevention. In this context, great attention has been paid to the Mediterranean diet which has been associated with a lower risk of all-cause cancer mortality and cancer incidence in different organs (Schwingshackl and Hoffmann, 2015). The more distinctive characteristic of this diet is the presence of olive oil as the main fat source. In addition to the oleic acid, olive oil contains a myriad of other compounds possessing distinct biological activity (Servili et al., 2009). Among them great attention has be

ACCEPTED MANUSCRIPT given to the “secoiridoid” phenols and their derivatives such as hydroxytyrosol (3,4dyhydroxyphenylethanol; 3,4-DHPEA) and tyrosol (p-hydroxyphenylethanol; pHPEA) which have been shown to have anti-cancer activities in vivo (Fabiani, 2016) and to prevent the DNA damage induced in vitro by different compounds in a variety of cellular systems (Fuccelli et al., 2014).

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On the base of the above reported considerations, the aim of this study was to investigate the genotoxicity of the selected HCAs (PhIP, IQ, MeIQx and DiMeIQx) on human freshly isolated peripheral blood mononuclear cells (PBMC). In addition, we tested the ability of three different

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phenolic extracts derived from olive, virgin olive oil, and olive leaf to prevent the DNA damage

2. Material and methods 2.1. Chemicals and reagents

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induced by PhIP on PBMC.

Hydroxytyrosol (3,4-DHPEA) and tyrosol (p-HPEA) were supplied respectively by Cabru s.a.s. (Arcore, Milan, Italy) and Sigma-Aldrich (Milan, Italy), while verbascoside, oleuropein and rutin

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were obtained from Extrasynthese (Genay, France) The dialdehydic forms of elenolic acid linked to 3,4-DHPEA and p-HPEA (3,4-DHPEA-EDA and p-HPEA-EDA), the isomer of oleuropein and ligstroside aglycon (3,4-DHPEA-EA and p-HPEA-EA) and lignans ((+)-1-acetoxypinoresinol and

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(+)-pinoresinol) were obtained as previously described (Montedoro et al. 1993).

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RPMI 1640 medium, L-glutamine, penicillin/streptomycin and heat-inactivated foetal calf serum were obtained from Microtech (Microtech Research Product, Italy), low melting-point and normal melting-point agarose were from Gibco (Gibco BRL, Life Technolo-gies, Paisley, Scotland). Conventional microscope slides were from Knittel Glaser (Steroglass, Perugia, Italy). PhIP, IQ, MeIQx and DiMeIQx were acquired from Toronto Research Chemicals, Inc. (Downsview, Toronto, Canada). Stock solutions of the different HCAs were prepared by dissolving 25 mg of each compounds in a variable volume of DMSO to obtain the following concentrations: PhIP 50 mM, IQ 130 mM, MeIQx 100 mM and DiMeIQx 25mM. Aliquots, stored at -20 °C, were thawed and diluted just before use with RPMI 1640 medium to the desired concentrations. The

ACCEPTED MANUSCRIPT amount of DMSO in the final treatment medium never exceeded 0.1%. Aroclor 1254-induced S9 fraction from Sprague-Dawley rats was provided by Molecular Toxicology, Inc. (Trinova Biochem, Germany). The S9-mix (containing both the liver extract and co-factors) was freshly prepared by adding 20 mL of deionized water to obtain a 10% suspension (10% Mutazyme) as suggested by the

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supplier. The inclusion of 50-100 µL of this suspension in a total volume of 1 mL resulted in a final concentration of S9-mix corresponding to 0.5-1.0 %. Histopaque 1077 and all other reagents were

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purchased from Sigma (Sigma-Aldrich, Irvine, UK).

2.2. Olive, olive oil and olive leaves phenolic extracts preparation

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Olive were obtained from cultivar Leccino. The olive phenolic extract (O-PE) was obtained as follows: 10 grams of olive pulp were homogenised by an Ultraturrax T50 (IKA Labortechnik, Staufen, Germania) with 100 mL of 80% methanol for 2 minutes a 9000 rpm; the homogenate has been centrifuged at 3000 rpm for 10 minutes and the extraction was performed in triplicate. The

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recovered supernatants were collected and the solvent evaporated under nitrogen flow. Virgin olive oil was obtained from olives (Olea europaea L.) from cultivar Leccino, processed using an industrial plant TEM 200 system (Toscana Enologica Mori, Tavarnelle Val di Pesa,

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Florence, Italy) consisting of a hammer mill, a malaxer (applying a malaxation for 40 min at 25 °C) with a gas controller system and a working capacity of 200 kg of olives, and a two-phase decanter;

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a UVPX 305 AGT 14 centrifuge (Alfa Laval S.p.A., Tavarnelle Val di Pesa, Florence, Italy) was used to separate the olive oil. The virgin olive oil phenolic extract (OO-PE) was obtained from 400 g of OO as previously reported (Montedoro et al., 1993). The olive leaves were obtained from cultivar Leccino. The olive leaves phenolic extract (OLPE) was obtained following the same procedure above described for obtaining the O-PE. The phenolic extracts (O-PE, OO-PE and OL-PE) were dissolved in a solution of ethanol/water (1/2, v:v) at a concentration of total phenol corresponding to 200 mg/mL, divided into aliquots and stored at -20 °C in the dark. Just before use the samples were thawed and diluted in RPMI 1640

ACCEPTED MANUSCRIPT medium to obtain a total concentration of 3,4-DHPEA + 3,4-DHPEA-containing compounds (3,4DHPEA-EDA, 3,4-DHPEA-EA, verbascoside and oleuropein) corresponding to 100 µM. All of the solutions were sterilised by filtration on 0.22 µm filters (Celbio S.r.l., Milan, Italy) before use. 2.3. Chemical analysis of O-PE, OO-PE and OL-PE

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The HPLC analyses of different phenolic extracts were conducted according to previously published methods (Selvaggini et al., 2006). The OO-PE, O-PE and OL-PE were analyzed by HPLC with an Agilent Technologies system (Santa Clara, CA, USA) model 1100 composed of a

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vacuum degasser, a quaternary pump, an autosampler, a thermostatted column compartment, a diode array detector, and a fluorescent detector. The C18 column used was a Spherisorb ODS-1 250

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x 4.6 mm with a particle size of 5 µm (Waters, Milford, MA, USA); the injected sample volume was 2 mL. The mobile phase was 0.2% acetic acid (pH 3.1) in water (A)/methanol (B) at a flow rate of 1 mL/min. The total running time was 73 min and the gradient changed as follows: 95% A/5% B for 2 min, 75% A/25% B in 8 min, 60% A/40% B in 10 min, 50% A/50% B in 16 min, 0% A/100%

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B in 14 min and then maintained for 10 min, return to initial conditions in 13 min. All phenolic compounds were detected by DAD at 278 nm with the only exception of lignans detected by FLD, activated at an excitation wavelength of 280 nm and emission at 339 nm (Selvaggini et al. 2006).

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2.4. Isolation of peripheral blood mononuclear cells Peripheral blood mononuclear cells (PBMC) were isolated from leucocyte-enriched (buffy

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coats) human peripheral blood of healthy donors on a density gradient as previously reported (Fabiani et al., 2007). Briefly, blood samples (2 ml) diluted to 8 ml with RPMI 1640 without serum, were layered over 2 ml of Histopaque 1077 and centrifuged at 1600 rpm for 20 min. The layer containing the mononuclear cells at the interface between the plasma and the Histopaque was recovered and washed twice with RPMI 1640. The viable PBMC obtained were counted by the trypan-blue exclusion technique and the density was adjusted to 1 × 106 cells/ml with RPMI 1640. The cells were then used for the different experiments. 2.5. Treatment of PBMC with HCAs.

ACCEPTED MANUSCRIPT The PBMC suspensions were exposed to increasing concentrations of HCAs in RPMI 1640 medium supplemented with 10% FCS, 2.0 mM L-glutamine, 100 U/ml penicillin and 100 U/ml streptomycin (complete RPMI medium). After incubation for 30 min at 37 °C and 5% CO2, the cell viability and the DNA damage were evaluated by the trypan-blue exclusion technique and the

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comet assay, respectively. In some experiments the treatment of PBMC with HCAs was carried out in different experimental conditions regarding the following parameters: ì) incubation time (15 min - 24 h), medium (simple RPMI 1640 and PBS) and temperature (4 °C); ìì) stimulation of PBMC by

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PMA (phorbole 12-myristate 13-acetate, 2 µM); ììì) PBMC density (0.25-0.5 × 106 cells/ml); ìv) inclusion of DNA repair inhibitors cytosine 1-β-d-arabinofuranoside (AraC) and hydroxyurea (HU);

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v) inclusion of external metabolic activation (S9-mix); vì) inclusion of the different phenolic extracts (O-PE, OO-PE and OL-PE). The concentration ranges of the different HCAs used to expose the cells were determined on the basis of cell viability after the treatment, which was always higher than 85%.

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2.6. Single-cell gel electrophoresis (comet assay).

The single-cell gel electrophoresis assay was performed essentially as previously described (Singh, et al., 1988). The cell viability was measured by the trypan blue exclusion method in all

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experimental conditions reported in the different experiments both in control and treated PBMC. The results showed that the viability was always above 85% and the differences between the treated

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cells and the respective controls resulted always statistically not significant. After treatment, aliquots of the cell suspension (50–100 µl, 0.5–1.0 ×105 cells) were transferred to 1.5-ml Eppendorf tubes and centrifuged at 1300 rpm for 6 min. The supernatant was discarded and the pellet was mixed with 75 µl of low melting-point agarose (0.7% in PBS), which was then distributed into conventional microscope slides pre-coated with normal melting-point agarose (0.5% in PBS), and dried at 50 °C. After the agarose had solidified (4 °C for 10 min), a third layer of normal meltingpoint agarose was applied similarly to the second. The slides were then immersed in the lysis solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris–HCl, pH 10, containing freshly added 1%

ACCEPTED MANUSCRIPT Triton X100 and 10% DMSO) for 1 h at 4 °C and then placed into a horizontal electrophoresis apparatus filled with freshly made buffer (1 mM Na2EDTA. 300 mM NaOH). After 20 min of preincubation (unwinding of DNA), the electrophoresis was run for 20 min at a fixed voltage of 25 V (0.83 V/cm) and 300 mA, adjusted by raising or lowering the level of the electrophoresis buffer in

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the tank. At the end of the electrophoresis, the slides were washed three times with neutralisation buffer (0.4 M Tris–HCl, pH 7.5), stained with 50µl ethidium bromide (20 µg/ml), and kept in a

under red light to prevent any additional DNA damage. 2.7. Comet detection and statistical analysis.

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moisture chamber in the dark at 4 °C until analysis. All steps described above were carried out

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The cells (100 comets for each sample, twice, in blind) were analysed 24h after staining at 400X magnification with a fluorescence microscope (Zeiss, R.G.) equipped with a 50-W mercury lamp. The extension of each comet was analysed by means of a computerised image-analysis system (Comet assay II, Perceptive Instruments, UK) which, amongst several other parameters, gave the

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“Tail Intensity %”, which represents the percentage of fluorescence intensity in the tail relative to the total intensity of the comet (Collins et al., 1995). Significant differences of the results of each experiment, repeated at least four times with different PBMC preparations (n=4), were assessed

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using both Student’s t-test and one-way ANOVA. When a significant (p < 0.05) treatment effect was detected, the mean values were compared using Tukey’s post hoc comparisons.

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3. Results and discussion 3.1 Effect of HCAs on DNA damage of PBMC Treatment of freshly isolated PBMC for 30 min at 37°C in complete RPMI medium with increasing concentrations of the different HCAs (PhIP, IQ, MeIQx and DiMeIQx) caused a dose-dependent increment of DNA damage (Figure 1). The data of control samples were within our historical control range of DNA damage in PBMC (% of DNA in the tail: 3.19 ± 0.61, n=24). At the end of incubation, the cell viability determined by the trypan blue exclusion method, was higher than 85% in all cases and the differences between the treated cells and the respective controls resulted always

ACCEPTED MANUSCRIPT statistically not significant demonstrating the absence of any cytotoxic activity by HCAs (data not shown). In the case of PhIP and IQ, a statistically significant effect was observed already at the lowest tested dose of 100 µM (Figure 1A and 1B) while for MeIQx and DiMeIQx the genotoxicity was significant only at the higher concentration of 1000 µM (Figure 1C and 1D). The genotoxic

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potential of PhIP resulted very similar to that showed by IQ. This observation is in sharp contrast with the mutagenicity of these two compounds since in Ames salmonella test PhIP (1.800 TA98 revertants/µg) was 240 times less potent than IQ (433.000 TA98 revertants/µg) (Sugimura et al.,

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2004). Similarly, the low genotoxic potential demonstrated by MeIQx and DiMeIQx is not according to their high mutagenicity, which was found to be 145.000 and 183.000 TA98

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revertants/µg, respectively (Sugimura et al., 2004). These data suggest that the strand-breaking activity of the four HCAs tested was not directly related to their mutagenic potencies on bacteria. In any case, our genotoxicity results are in part similar to those obtained by other authors on different cell systems. In particular, on metabolically competent human liver HepG2 cells derived

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from a hepatoblastoma both PhIP and MeIQx were found to be genotoxic after 24h of incubation at 200 µM and 250 µM, respectively (Pezdirc et al., 2013; Viegas et al., 2012). Instead, DiMeIQx at 200 µM resulted not effective on HepG2 cells in one study (Pezdirc et al., 2013) whereas it resulted

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genotoxic in another study (Haza and Morales, 2011). In addition, treatment of human hepatoma HepaRG cells with MeIQx for 24 h resulted in a null genotoxic effect at a concentration of up 1000

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µM (Dumont et al., 2010). In cryopreserved lymphocytes, both PhIP and IQ were able to induce a DNA damage in the absence of external metabolic activation in the concentration range of 10-100 µM and 25-183 µM, respectively (Baumgartner et al., 2012; Kurzawa-Zegota et al., 2012). 3.2 Effect of different experimental conditions on HCAs induced DNA damage Two different mechanisms may be primarily involved in the HCAs genotoxicity. The first can be related to the formation of DNA-adducts caused by HCAs reactive intermediate metabolites and the consequent appearance of excision-repair-induced strand breaks (Turesky and Le Marchand, 2011). On the other hand, several evidences suggest that HCAs can also induce an oxidative stress

ACCEPTED MANUSCRIPT resulting in the formation of oxidised DNA bases, apurinic/apyrimidinic sites (AP sites) and DNA strand breaks (Carvalho et al., 2015; Jain et al., 2015). In both cases, the activation of xenobioticmetabolizing enzymes and DNA repair systems may deeply influence the DNA damage induced by HCAs as evidenced by the comet assay. On the base of these considerations, and with the aim of

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improving the sensibility of the comet assay we investigated the effect of different experimental conditions on the DNA damage induced by HCAs. The results reported in Table 1 demonstrated that both lowering the incubation temperature to 4°C and reducing the incubation time to 15 min

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(both parameters can reduce the DNA repair activity of the cells) (Duthie & Collins, 1997) did not influence the genotoxicity of the four HCAs tested. Nevertheless, prolonging the PBMC exposure

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time to 2 and 24 h did not increased the HCAs genotoxicity (results not shown). Similarly, reducing the cell density to 0.25 x 106 cells/ml or replacing the exposure RPMI complete medium with a phosphate buffer (PBS) or a simple RPMI (without serum) did not enhance the genotoxicity of HCAs (results not shown).

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To investigate further whether DNA repair systems could influence the HCAs genotoxicity we exposed PBMC to PhIP, IQ, MeIQx and DiMeIQx (100 µM) in the presence of two inhibitors of DNA resynthesis during nucleotide excision repair AraC (1.8 mM) and HU (10 mM) (Fram &

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Kufe, 1985). The results reported in Figure 2 indicate that inclusion of AraC/HU significantly increased the DNA damage in PBMC not treated with HCAs (37.4 %, p=0.00782) and in PBMC

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treated with MeIQx (63.5 %, p=0.00197) and DiMeIQx (72.4 %, p=0.00015). Instead, in PBMC treated with PhIP and IQ the slightly increase of genotoxicity (PhIP: 12.5 %, p=0.253; IQ: 10.1 %, p=0.503) was not statistically significant (Figure 2). These results are in contrast with previous data showing that AraC/HU did not significantly increase the DNA damage induced by MeIQx in metabolically competent MCL-5 cells (Martin et al., 1999). Worthy of note is also the observation that the inclusion of AraC/HU makes both MeIQx and DiMeIQx significantly genotoxic toward PBMC at a concentration (100 µM) much lower than that observed in metabolically competent MCL-5 cells which resulted 2130 µM and 410 µM, respectively (Pfau et al., 1999). In disagreement

ACCEPTED MANUSCRIPT with our data are also the results obtained on genetically engineered V79 cells (Chinese hamster lung fibroblasts) to express rat cytochrome P450 1A2 and rat sulfotransferase 1C1 (V79-rCYP1A2rSULT1C1) in which a genotoxic activity of PhIP could be evidenced only in the presence of

3.3 Effect of metabolic activation on HCAs-induced DNA damage

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AraC/HU (Edenharder et al., 2002).

It is known that human lymphocytes express several CYPs enzymes in response to activation by different stimuli (Siest et al., 2008). A recent study has shown that activated T lymphocytes

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(which are the predominant cells present in PBMC fraction) were able to bioactivate HCAs bringing to the detection of higher DNA adducts (Bellamri et al., 2016). Therefore, we tested whether

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activation of PBMC fraction (which also contains monocytes) with PMA could increase the HCAs induced DNA damage. However, the results obtained suggest that exposure of PBMC to HCAs at 100 µM in the presence of PMA did not significantly increase their genotoxicity (results not shown). Interestingly, we noticed a genotoxic effect by PMA itself. This phenomenon can be the

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related to the PMA-induced activation of the NADPH oxidase present in the monocytes with the consequent production of reactive oxygen species and DNA damage (Fabiani et al., 2001). A common strategy to activate pro-mutagens is to include in the experimental system an

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exogenous metabolic activation fraction (S9-mix). We investigated the genotoxicity of the different HCAs treating the PBMC in the presence of two doses (0.5 and 1.0 %) of S9-mix obtained from

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Aroclor induced rats. As shown in Figure 3 the metabolic activation dose-dependently increased the genotoxicity of all HCAs tested. On the other hand, we observed a reduction of DNA damage in the presence of S9-mix in the PBMC not exposed to the HCAs. The 1% dose of S9-mix was more effective than the lower dose of 0.5%, this makes both the MeIQx and DiMeIQx significantly genotoxic even at the lowest tested dose of 100 µM (Figure 3). In addition, the inclusion of 1% of S9-mix make the genotoxic activity of the higher doses of HCAs (500 and 1000 µM) not much more effective than the lower dose (100 µM) (Figure 3). Our results are in agreement with those obtained on cryopreserved lymphocytes showing that the inclusion of human S9 increased the

ACCEPTED MANUSCRIPT genotoxicity of both PhIP and IQ (Baumgartner et al., 2012). Instead, different results were obtained on bovine colonocytes where both PhiP and IQ were not genotoxic in the absence of an external metabolizing system while in the presence of S9 mix, a significant increase in the median tail length was noted for PhiP but not for IQ even a highest used concentration of 300 µM

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(Follmann & Birkner, 2008). To the best of our knowledge, no data are present in the literature regarding the effect of external metabolic activation on genotoxicity of both MeIQx and DiMeIQx. 3.5 Preventive effect of phenolic extracts (OO-PE, O-PE and OL-PE) on HCAs-induced DNA

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damage

The phenolic composition of extracts derived from virgin olive oil (OO-PE), olive (O-PE) and

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olive leaves (OL-PE) used for testing the DNA damage preventive activity is shown in Table 2. As expected the richest phenolic fraction (total phenols: 65.2%) was obtained from olive oil which contained high amounts of aglycon secoiridoids (3,4-DHPEA-EDA, p-HPEA-EDA, 3,4-DHPEAEA and p-HPEA-EA) their derivatives (3,4-DHPEA and p-HPEA) and lignans (acetoxypinoresinol

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and pinoresinol). On the other hands, extracts obtained from olive and olive leaves evidenced high amount of 3,4-DHPEA-EDA but undetectable level of most of the other aglycon secoiridoids’ derivatives and lignans but substantial amounts of oleuropein, verbascoside and rutin (Table 2).

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Inclusion of equimolar concentrations of the different extracts, in term of more bioactive 3,4DHPEA containing compounds (3,4-DHPEA-EDA, 3,4-DHPEA-EA, oleuropein and verbascoside),

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in the PhIP exposure medium resulted in a dose-dependent prevention of DNA damage (Figure 4). A reduction of PhIP-induced DNA damage was evidenced for all three extracts (OO-PE: 4A; O-PE: 4B and OL-PE: 4C) in a very low concentration range (0.1-1.0 µM) both in the absence and in the presence of the S9-mix. The extract derived from olive oil (OO-PE) was the most active among the others since a preventive effect resulted statistically significant at the lower dose tested 0.1 µM (Figure 4A). Our results are in agreement with literature data, which have demonstrated the ability of olive oil phenols to inhibit DNA damage caused by different agents on several cell systems. In particular, previous studies have focused on the “in vitro” prevention of oxidative DNA damage

ACCEPTED MANUSCRIPT caused by either hydrogen peroxide (Rosignoli et al., 2016; Erol et al., 2012; Warleta et al., 2011) or various environmental pollutants such as alkene epoxides and TCDD (Fuccelli et al., 2014; Ilavarasi et al., 2011). Whether the PhIP-induced DNA damage in PBMC is mediated by reactive oxygen species and the olive oil phenols act as antioxidants remains to be determined.

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It is important to underline that the low concentrations of phenols able to reduce the DNA damage can be reached in human tissues in vivo under a regular intake of olive oil in the Mediterranean countries (30-50 g/d). Indeed, olive oil phenols are efficiently absorbed in humans

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(Mirò-Casas et al., 2003). In addition, intervention studies have shown that the intake of 40 mL of olive oil, containing a medium amount of phenols (164 mg/kg), can result in a plasma concentration

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of 3,4-DHPEA in the low µM range during the first 4 h after ingestion (Covas et al., 2006). Based on these considerations, our data support the hypothesis that the DNA damage preventive activity of phenols may be a crucial mechanism that explains epidemiological data indicating an inverse correlation between olive oil consumption and cancer risk in different sites (Psaltopoulou et al.,

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2011). In the case of digestive system, which represents the first target site for HCAs genotoxicity, a high intake of olive oil was associated to a significant 30% reduction of cancer risk (Psaltopoulou et al., 2011). Although the chemopreventive activities of olive oil phenols may be involved in the

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inhibition of both promotion and progression steps of cancerogenesis, preclinical studies have demonstrated that intake of olive oil enriched with phenols caused a reduction of oxidative DNA

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damage in human (Weinbrenner et al., 2004; Salvini et al., 2004). Further studies are needed to clarify whether and to what extent the anti-genotoxic characteristics of phenols are responsible for the cancer preventive activity of olive oil. Similar to our results, previous studies have demonstrated a preventive activity on HCAsinduced DNA damage by other food compounds such as flavonoids, diallyl sulphide, xanthohumol and curcumin on different cell systems. However, generally these compounds were active at concentrations much higher than those used in the present investigation. In particular, a reduction of DNA damage induced in vitro by IQ and PhIP on lymphocytes was observed by supplementation

ACCEPTED MANUSCRIPT with flavonoids quercetin and rutin at 250 µM (Kurzawa-Zegota et al., 2012). Similarly, the PhIPinduced DNA damage on breast cell line MCF-10 was significantly prevented by diallyl sulphide at 100 µM (Aboyade-Cole et al., 2008) and curcumin at 150 µM (Jain et al., 2015). On the other hand, a protective effect of xanthohumol on DNA damage induced by MeIQx and PhIP in HepG2 cells

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was observed in a concentration range (0.01-10 µM) similar to that showed to be effective with our phenolic extracts (Viegas et al., 2012).

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4. Conclusions

Our data showed that all HCAs tested (PhIP, IQ, MeIQx and DiMeIQx) caused a DNA damage

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in PBMC which was increased by a metabolic activation. In the case of MeIQx and DiMeIQx the genotoxicity was also enhanced by DNA repair inhibitors AraC and HU. Our data, demonstrating a genotoxic effect of HCAs on human PBMC, could be preliminary for further molecular epidemiological studies aimed to investigate both the individual susceptibility and the relationships

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between HCAs exposure and DNA damage. The genotoxicity of PhIP was efficiently prevented by very low concentrations of phenolic extracts derived from virgin olive oil, olive and olive leaves. These data further support the observation that consumption of olive and virgin olive oil may

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prevent the initiation step of carcinogenesis. The leaf waste could be an economic and simple

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source of phenolic compounds to be used as food additives or supplements.

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Legend to the figures Figure 1. Genotoxic effect of increasing concentrations of HCAs (A: PhIP; B: IQ; C: MeIQx; D: DiMeIQx) on freshly isolated human PBMC. The cells were exposed to HCAs in complete RPMI

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medium at 37 °C for 30 min. The DNA damage was measured by the comet assay and the results are expressed as the mean ± S.D. of the values (Tail Intensity, % DNA) of four experiments

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performed with different cell preparations. Means without a common letter differ, p < 0.05 (Tukey post-hoc test).

Figure 2. Effect of the inclusion of two inhibitors of DNA resynthesis during nucleotide excision repair AraC (1.8 mM) and HU (10 mM) on DNA damage induced by HCAs. The cells were

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exposed either in the presence or in the absence of AraC/HU to the different HCAs at 100µM for 30 min at 37°C for 30 min and then the genotoxicity was quantified by the comet assay. The results are the mean ± S.D. of the values (Tail Intensity, % DNA) of four experiments performed with different

t-test).

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cell preparations. *P <0.05, **P<0.01 compared to the PBMC not treated with inhibitors (Student’s

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Figure 3. Induction of DNA damage on freshly isolated human PBMC by exposure to different HCAs (A: PhIP; B: IQ; C: MeIQx; D: DiMeIQx) either in the absence or in the presence of S9-mix at two doses of 0.5 and 1.0 %. The cells were incubated at 37 °C with increasing concentrations of HCAs for 30 min in RPMI medium enriched or not with S9-mix. The DNA damage was measured by the comet assay and the results are expressed as the mean ± S.D. of the values (Tail Intensity, % DNA) of four experiments performed with different cell preparations. Statistical analysis was carried out considering all “twelve” groups, therefore the letters indicate comparison both within and between groups. Means without a common letter differ, p < 0.05 (Tukey post-hoc test).

ACCEPTED MANUSCRIPT Figure 4. Effect of increasing concentrations of phenolic extracts obtained from olive oil (A: OOPE) olive (B: O-PE) and olive leaves (C: OL-PE) on PhIP induced DNA damage in PBMC. The cells were exposed to PhIP (1000 µ) for 30 min at 37°C in RPMI medium either enriched or not with the different phenolic extracts. The DNA damage was measured by the comet assay and the

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results are expressed as the mean ± S.D. of the values (Tail Intensity, % DNA) of four experiments performed with different cell preparations. Means without a common letter differ, p < 0.05 (Tukey post-hoc test).

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Acknowledgements

This work was supported by grants from MIUR ITALY PRIN 2015 “Olive phenols as

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multifunctional bioactives for healthier foods: evaluation of simplified formulation to obtain safe meat products and new foods with higher functionality” (Project No. 20152LFKAT_001).

Conflict of interest

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The authors declare that they have no conflict of interest.

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ACCEPTED MANUSCRIPT Table 1. Effect of temperature and exposure time on HCAs induced DNA damage (Tail intensity, % DNA) in PBMC.

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HCAs, 100 µM Control IQ PhIP MeIQx DiMeIQx

Temperature and time of exposure 37°C 4°C 30 min 15 min 30 min 15 min a,A a,A a,A 3.02 ± 0.98 2.17 ± 1.17 3.15 ± 1.02 3.86 ± 2.34 a,A 7.86 ± 1.57 b,B 5.38 ± 1.68 a,b,B 7.59 ± 2.35 b,B 7.07 ± 3.83 a,B b,C b, C b,C 7.96 ± 2.70 7.39 ± 3.76 6.65 ± 1.88 6.72 ± 2.45 a,C a,b,D a,D a,D 6.17 ± 2.37 5.55 ± 1.67 5.89 ± 2.93 5.15 ± 2.54 a,D 5.91 ± 1.70 a,b,E 6.01 ± 1.45 a,b,E 5.68 ± 2.23 a,b,E 5.57 ± 3.24 a,E

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Values are means ± SD, n = 4. Means without a common letter differ, p < 0.05 (Tukey post-hoc test). Capital letters refer to the comparisons made within the horizontal rows while the small letters refer to the comparisons performed within the vertical columns.

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Table 2 Phenolic composition of extracts obtained from virgin olive oil (OO-PE), olive (O-PE) and olive leaves (OL-PE) expressed as mg/g of extract.

OO-PE

O-PE

OL-PE

3,4-DHPEA

2.0 ± 0.1

7.5 ± 0.4

1.8 ± 0.1

p-HPEA

2.2 ± 0.2

1.0 ± 0.1

0.6 ± 0.2

3,4-DHPEA-EDA

440.7 ± 2.7

362.5 ± 9.1

p-HPEA-EDA (Oleocanthal)

70.6 ± 1.0

N.D.

3,4-DHPEA-EA (Oleuropein aglicon)

102.3 ± 3.8

N.D.

p-HPEA-EA (Ligstroside aglicon)

12.8 ± 0.3

(+)-1-acetoxypinoresinol

11.4 ± 0.1

(+)-pinoresinol

10.1 ± 0.9

Verbascoside

N.D.

Oleuropein

N.D.

Rutin

N.D.

N.D.: not detected

49.2 ± 1.9

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N.D.

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Total phenols

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Phenolic compounds

N.D.

N.D.

N.D.

N.D.

N.D.

N.D.

N.D.

14.4 ± 0.1

4.0 ± 0.1

30.3 ± 2.3

60.9 ± 2.0

1.6 ± 0.1

14.0 ± 0.3

417.4 ± 9.4

130.5 ± 2.8

c

15 b

10 5

b a

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500

B

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Tail Intensity, % DNA

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1000

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Tail Intensity, % DNA

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100 500 MeIQx, µM

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Figure 1

100 500 DiMeIQx, µM

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Heterocyclic amines, 100 µM

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Figure 2

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Tail Intensity, %DNA

Without AraC/HU With AraC/HU

DiMeiQx

15

Control 100 µM 500 µM 1000 µM

d

d,e

a

a

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e

d,e

5

a,b,c

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0,5 S9 mix, %

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A

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2 0

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6 4 2 0

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The metabolic activation (S9-mix) increased the genotoxicity of all HCAs tested

Three phenolic extracts clearly prevent the PhIP induced DNA damage in a very low concentration

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range easily reached in humans under a regular intake of virgin olive oil.