Ellagitannins from Rubus idaeus L. Exert Geno- and Cytotoxic Effects

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Ellagitannins from Rubus idaeus L. Exert Geno- and Cytotoxic Effects Against the Human Colon Adenocarcinoma Cell Line Caco-2 Adriana Nowak, Micha# Sójka, El#bieta Klewicka, Lidia Lipi#ska, Robert Klewicki, and Krzysztof Kolodziejczyk J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05387 • Publication Date (Web): 16 Mar 2017 Downloaded from http://pubs.acs.org on March 18, 2017

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Ellagitannins from Rubus idaeus L. Exert Geno- and Cytotoxic Effects Against the

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Human Colon Adenocarcinoma Cell Line Caco-2

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Short title (title running header): Geno- and cytotoxicity of ellagitannins

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Adriana Nowak *1, Michał Sójka 2, Elżbieta Klewicka 1, Lidia Lipińska 1, Robert Klewicki

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Wólczańska 171/173, 90-924 Lodz, Poland

, Krzysztof Kołodziejczyk 2 Institute of Fermentation Technology and Microbiology, Lodz University of Technology,

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4/10, 90-924 Lodz, Poland

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* Corresponding author: Tel.: +48-42-631-34-81; fax: +48-42-636-32-74; e-mail:

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[email protected]

Institute of Food Technology and Analysis, Lodz University of Technology, Stefanowskiego

1 ACS Paragon Plus Environment

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ABSTRACT: Ellagitannins possess several biological activities, including anti-cancer

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properties. The goal of the present study was to investigate the cyto- and genotoxic activity of

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a red raspberry ellagitannin preparation (REP) in the concentration range of 2.5–160 µg/mL,

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as well as that of the main individual raspberry ellagitannins: sanguiin H-6 (SH-6, 12.8–256

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µM) and lambertianin C (LC, 9.3–378 µM), against the human colon adenocarcinoma cell

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line Caco-2. The ellagitannin concentrations used in the study correspond to those found in

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foodstuffs containing raspberry fruit. The REP, SH-6, and LC exhibited strong concentration-

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dependent genotoxic properties, inducing DNA damage ranging from (7.3±1.3)% to

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(56.8±4.3)%, causing double-strand breaks and oxidation of DNA bases. At IC50 (124 µg/mL)

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the REP affected the nuclear morphology and induced the apoptosis of Caco-2 cells. Since

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the REP has been found to possess chemopreventive activity, it can be used as a natural food

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additive to enhance the health benefits of foodstuffs.

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Key words: ellagitannins, genotoxicity, DNA damage, cytotoxicity, raspberry fruit

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INTRODUCTION

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Fruits and vegetables are sources of many phytochemicals, most of which show biological

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activity with different mechanisms of action; they may have anti-oxidative properties, activate

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detoxification enzymes, stimulate the immune system, etc.1 Epidemiological studies have

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shown an association between the consumption of fruit and vegetables and a reduction in the

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risk of some types of cancer.2 Cancers of the digestive tract, and in particular colorectal

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cancer (CRC), are amongst those most responsive to dietary modification as approx. 75% of

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all sporadic cases of CRC are directly influenced by diet.3 Although there are few data on the

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effects of berry consumption on CRC risk, in vitro evidence from models representing CRC

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suggests that berry polyphenols may modulate cellular processes essential for cancer cell

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survival, such as proliferation and apoptosis.4

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Berries are a rich source of antioxidants, including polyphenols such as anthocyanins

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and ellagitannins (ETs). ETs constitute an important class of phytochemicals present in a

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number of edible plants and fruits, including raspberries, strawberries, blackcurrants,

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pomegranates, cranberries, blueberries, and dried nuts.5 Raspberries contain significant

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amounts of ETs (80–160 mg per 100 g of fruit), and in particular lambertianin C, sanguiin H-

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6 (Figure 1), as well as other ETs (especially LC and SH-6 derivatives) in smaller

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quantities.6,7 The daily ET intake in the Western diet is generally low, not exceeding 5

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mg/day, but it increases seasonally.1 ETs are considered to be nutraceuticals due to the health

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benefits they confer, including anti-inflammatory and anti-cancer activity and cardiovascular

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risk reduction.6,8,9

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The aim of this study was to examine the in vitro cyto- and genotoxic effects of a

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raspberry ellagitannin preparation (REP) against the colon cancer cell line Caco-2. The

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novelty of the study lies in demonstrating the genotoxic potency of ellagitannins (sanguiin

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H-6 and lambertianin C) isolated from raspberry fruit (Rubus idaeus L.) and elucidating their


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mechanisms of action in cancer cells (types of DNA damage, changes in nuclear

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morphology). No previous reports on these issues have been published, and so the presented

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findings constitute an important contribution to the field.

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MATERIALS AND METHODS

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Chemicals. Dulbecco’s Modified Eagle’s Medium (DMEM), HEPES, streptomycin

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and penicillin, low and normal melting point agaroses, propidium iodide (PI), 4’,6-diamidino-

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2-phenylindole (DAPI), Tris buffer, NaOH, Triton X-100, ethylenedinitrilotetraacetic acid

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(EDTA), dimethyl sulfoxide (DMSO), thiazolyl blue tetrazolium bromide (MTT), KCl, NaCl,

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and cisplatin were purchased from Sigma-Aldrich, St. Louis, MO, USA. Fetal bovine serum

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(FBS), GlutaMAXTM, TrypLETM Express, PrestoBlue, and acridine orange (AO) were

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purchased from Gibco, Thermo Fisher Scientific, Waltham, MA, USA. Endonuclease III

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(Endo III) and formamidopyrimidine-DNA glycosylase (Fpg) were both purchased from New

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England Biolabs Inc., Germany.

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REP extraction and composition. The extraction method and the composition of

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ellagitannins in the REP were published previously.10 Briefly, ellagitannins were extracted

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from raspberry press cake following juice pressing from fruit pulp (‘Polka’ cultivar). Fresh

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press cake was extracted in two steps using 60% acetone at ambient temperature. The mass

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ratio of the press cake to the extractant was 1:5 and extraction time was 8 h per step. The

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extraction process was augmented with shaking using an orbital shaker. The extracts obtained

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from the two steps were mixed and filtered through a 3.6 mm thick Hobrafilt S40N cellulose

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filter with 5 µm nominal retention (Hobra-Školnik S.R.O., Broumov, Czech Republic).

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Acetone was removed from the raw extract using a rotary vacuum evaporator at 60°C. The

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extract without acetone was passed through a cellulose filter once more, and then purified on

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a 90 × 1.6 cm column packed with Amberlite XAD 1600N resin (DOW, Midland, MI). The



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extract was injected at a rate of approx. 15 mL/min. Elution was carried out at 10 mL/min

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using water/ethanol solutions with ethanol concentrations of 10%, 20%, 30%, 40%, 50%, and

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60%, consecutively. The volume of solutions with each ethanol concentration equaled the

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volume of the column. The eluate obtained using the 40% ethanol solution contained the

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highest concentrations of lambertianin C and sanguiin H-6. Subsequently, ethanol was

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removed and the eluate was concentrated to approx. 5°Brix using a rotary vacuum evaporator

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at 60°C. The concentrated extract was then freeze-dried. The REP yield was 1 g per 1 kg of

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fresh fruit. The preparation obtained in this way consisted of 86% ellagitannins and approx.

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7% flavanols. The polyphenolic composition of the REP and the identification data of the

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ellagitannins it contained are presented in Table 1. The HPLC and LC-MS equipment as well

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as the conditions used for REP characterization are the same as those described in our

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previous article.11

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LC and SH-6 isolation. The isolation methods for lambertianin C and sanguiin H-6

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were published previously.10,11 LC and SH-6 were isolated from an aqueous REP solution

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with a concentration of 4 mg/mL using a Knauer preparative HPLC system (Berlin,

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Germany). The chromatograph was composed of two gradient pumps (Knauer K-501), a

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Phenomenex Luna 10u C18(2) 100A AXIA-packed (250 × 21.2 mm; 10 µm) column

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(Torrance, CA), a UV-Vis detector, a fraction collector (Teledyne ISCO, Lincoln), and

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Eurochrom 2000 chromatographic software. Two eluents were used for separation: eluent A –

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0.1% formic acid in water, eluent B – 75% methanol. The flow rate was 15 mL/min. The

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following gradient was used: 0–5 min 10% B; 5–30 min 10–25% B; 30–50 min 25–35% B;

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50–65 min 35–40% B; 65–70 min 40–10% B; and 70–75 min 10% B. The injection volume

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was 500 µL. The detection parameter was set to 260 nm. Lambertianin C and sanguiin H-6

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peaks were collected from 200 separations. Methanol was removed from the obtained

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solutions using a rotary vacuum evaporator at 60°C. Then, the solutions were freeze-dried,


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producing 96.5 mg of sanguiin H-6 (white-beige powder) and 102 mg of lambertianin C

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(white-creamy powder). The obtained standards of LC and SH-6 were characterized by HPLC

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purity of more than 90% at 210 nm, using the same HPLC equipment and conditions as

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described in our previous work.11

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Caco-2 cell culture. The human colon adenocarcinoma cell line Caco-2 was used as a

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model adherent cell line in the study. The cells were cultured in Roux flasks (Becton,

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Dickinson and Co., Franklin Lakes, NJ, USA) as a monolayer in Dulbecco’s Modified

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Eagle’s Medium supplemented with 10% fetal bovine serum, 4 mM GlutaMAXTM, 25 mM

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HEPES, 100 µg/mL streptomycin and 100 IU/mL penicillin. Cells were cultured for 10 days

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at 37°C under a 5% CO2 atmosphere. They were washed every 3 days with 0.1 M PBS and

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fresh medium was added. After reaching confluence, the cells were detached with TrypLETM

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Express for 10 min at 37°C, suspended in sterile PBS, and aspirated off the plastic flask. The

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cell suspension was centrifuged (187 × g, 5 min). The pellet was re-suspended in fresh

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DMEM. Subsequently, a cell count was performed with the use of a hemocytometer and cell

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viability was determined by trypan blue exclusion. The cells were ready to use if viability was

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at least 90%.

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Single-cell gel electrophoresis assay (comet assay). The final concentration of

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Caco-2 cells in each sample was adjusted to 105 cells/mL. Then, 900 µL of cells in non-

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supplemented DMEM were incubated with 100 µL of ellagitannins (REP), sanguiin H-6

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(SH-6) or lambertianin C (LC) at 37°C for 1 h. All concentrations of the tested compounds

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were freshly prepared in non-supplemented DMEM just before addition to Caco-2 cells. The

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final concentrations of the compounds in the samples were (%):

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a) 160, 80, 40, 20, 10, 5, and 2.5 µg/mL for the REP;

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b) 378, 189, 92.7, 18.9, and 9.3 µM for LC;

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c) 256, 107, 53.4, 26.7, and 12.8 µM for SH-6.



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According to the literature, these concentrations of ellagitannins are typical of foodstuffs containing raspberries and extracts thereof.7,12

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The comet assay was performed under alkaline conditions (pH > 13) according to the

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procedure developed by Błasiak and Kowalik13, as previously described.14 After incubation,

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the cells were centrifuged (182 × g, 15 min, 4°C), decanted, suspended in 0.75% low melting

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point agarose, layered onto slides precoated with 0.5% normal melting point agarose, and

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lysed at 4°C for 1 h in a buffer consisting of 2.5 M NaCl, 1% Triton X-100, 100 mM EDTA,

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and 10 mM Tris, pH 10. After the lysis, the slides were placed in an electrophoresis unit and

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DNA was allowed to unwind for 20 min in an electrophoretic solution containing 300 mM

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NaOH and 1 mM EDTA. Electrophoresis was conducted at 4°C for 20 min at an electric field

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strength of 0.73 V/cm (300 mA). Then, the slides were neutralized with distilled water,

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stained with 2.5 µg/mL PI, and covered with cover slips. The slides were examined at 200×

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magnification under a fluorescence microscope (Nikon, Japan) connected to a video camera

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and a personal computer-based image analysis system – Lucia-Comet v. 7.0 (Laboratory

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Imaging, Prague, Czech Republic). Fifty images were randomly selected from each sample

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and the percentage of DNA in the comet tail was measured.

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Double-strand breaks. The ability of 160, 80, and 40 µg/mL REP to induce DNA

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double-strand breaks (DSBs) was evaluated by the neutral comet assay according to Błasiak

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et al.15 In that case, electrophoresis was run in a buffer consisting of 100 mM Tris and

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300 mM sodium acetate at pH 9.0 (with glacial acetic acid). Electrophoresis was conducted

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for 60 min, after a 20 min equilibration period, at an electric field strength of 0.41 V/cm

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(50 mA), at 4°C. The slides were then processed as described in the “Single-cell gel

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electrophoresis assay” section. The positive control was 50 µM cisplatin.

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Oxidative DNA damage. The comet assay is one of the most accurate methods of

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measuring DNA oxidation.16 Oxidative DNA damage was evaluated with DNA repair 7 ACS Paragon Plus Environment

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enzymes – endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (Fpg).

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The procedure was performed according to Błasiak et al.17 To check the ability of the

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enzymes to recognize oxidized DNA bases, the cells were incubated either with the REP (at

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concentrations of 40, 10, and 2.5 µg/mL), or hydrogen peroxide (50 µM), lysed and post-

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treated with Endo III or Fpg, respectively. After lysis, the slides were washed three times in

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an enzyme buffer (composed of 40 mM HEPES-KOH, 0.1 M KCl, 0.5 mM EDTA, 0.2

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mg/mL bovine serum albumin, pH 8.0) and drained, and the agarose was covered with 25 µL

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of either enzyme buffer or 1 µg/mL enzyme in buffer, sealed with a cover glass and incubated

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for 30 min at 37°C. Further steps were as described above. To determine the net value of

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DNA damage recognized by the enzymes, the DNA damage observed in the absence of the

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enzymes was subtracted from the measurement results.

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Cytotoxicity and IC50 estimation for the REP in MTT and PrestoBlue assays. Cell

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viability was determined using the MTT and PrestoBlue assays. For the experiments, 1 × 104

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Caco-2 cells were seeded in each well of a 96-well plate with black plates used for PrestoBlue

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fluorescence (Becton, Dickinson and Co., Franklin Lakes, NJ, USA) and 100 µL of complete

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culture medium was added into each well. The cells were incubated overnight at 37°C under

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5% CO2. Next, the medium was aspirated and the REP in DMEM without FBS and phenol

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red were added to each well in eight repeats at the following final concentrations: 160, 80, 40,

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20, 10, 5, 2.5, and 1.25 µg/mL. The negative controls contained only cells in DMEM. Cells

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were incubated for 48 h in a CO2 incubator at 37°C under 5% CO2.

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After incubation, the medium with the REP was removed from each well and 100 µL

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of MTT (0.5 mg/mL in PBS, pH 7.2) was added and incubated at 37°C under 5% CO2 for 3 h.

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After that time, MTT was removed and formazan precipitates were solubilized by adding

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50 µL of DMSO. Absorbance was measured at 550 nm with a reference filter of 620 nm,

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using a microplate reader (TriStar² LB 942, Berthold Technologies GmbH & Co. KG, Bad 8 ACS Paragon Plus Environment


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Wildbad, Germany). In the PrestoBlue assay, 100 µL of the PrestoBlue reagent (10% solution

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in PBS, pH 7.2) was added to each well and incubated for 2 h at 37°C under 5% CO2.

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Fluorescence was measured at 560 nm excitation and 590 nm emission using a microplate

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reader. The absorbance/fluorescence of the control sample (untreated cells) represented 100%

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cell

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fluorescence/control OD or fluorescence] × 100%], and cytotoxicity (%) was determined as

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100 – cell viability (%). Results were presented as mean ± standard deviation (SD).

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Experiments with these compounds were conducted with the same cell population.

viability.

Cell

viability

(%)

was

calculated

as follows:

[sample

OD

or

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The value of IC50 (half maximal inhibitory concentration) was read from the dose–

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response curve and determined according to the formula: IC50=(X-Z)/(X-X1)*(CX1-CX)+CX,

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where X represents a 50% decrease in viability; Z is % of viability > Z; X1 is % viability < Z;

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CX is the concentration of the compound for X, and CX1 is the concentration of the

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compound for X1.

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Fluorescent microscopic analysis of nuclear morphology of Caco-2 cells after

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treatment with ETs. Changes in the nuclear morphology of Caco-2 cells after exposure to

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the REP were examined with two different fluorescent staining assays:

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a) Caco-2 cells were seeded with culture medium containing 2.0 × 104 cells/well in a

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Lab-TekTM 8-well chamber slide system (Nunc, Thermo Fisher Scientific, Waltham,

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MA, USA) and left overnight. Next, the cells were treated with the REP (at 124

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µg/mL concentration) for 24 h, washed with PBS, fixed with 70% methanol (J.T.

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Baker) for 15 min at room temperature and air dried. Then cells were stained with

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1 µg/mL of DAPI for 10 min at room temperature in the dark;

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b) 2.5 × 105 of Caco-2 cells, following exposure to the REP (at 124 µg/mL

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concentration) for 24 h, were mixed (1:1) with 100 µg/mL AO and 100 µg/mL PI in


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distilled water and placed on a clean microscope slide.18 The suspension was

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immediately analyzed (within 30 min, before the fluorescent color started to fade).

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Stained cells were observed at 200× and 400× magnification in a fluorescent

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microscope (Nikon Eclipse Ci H600L, Japan) attached to a video camera and a personal

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computer operated with NIS-Elements Advanced Research v. 3.0 software (Nikon).

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Statistical analysis. Comet data were analyzed using two-way analysis of variance

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(ANOVA), while a particular mode of interaction × time was used to compare the effects

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induced by chemicals at this mode of interaction. Differences between samples with normal

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distribution were evaluated by Student’s t-test. Both Student’s t-test and ANOVA were

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performed using OriginPro 6.1 software. Significant differences were accepted at P < 0.05).

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The results are presented as mean ± SEM.

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RESULTS

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Basal endogenous DNA damage induced by the REP, SH-6, and LC. The negative

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control (non-exposed Caco-2 cells) exhibited DNA damage of (4.8±1.0)% (as expressed by

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DNA percentage in the comet tail), while cell treatment with the positive control (50 µM

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hydrogen peroxide) resulted in (61.7±3.3)% damage.

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The genotoxic activity of the REP, SH-6, and LC was tested against the Caco-2 model

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tumor cell line. The genotoxicity of the REP increased in a dose-dependent manner up to the

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concentration of 80 µg/mL (P < 0.05) (Figure 2), resulting in DNA damage ranging from

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(7.3±1.3)% for 2.5 µg/mL to (56.8±4.3)% for 80 µg/mL. Concentrations greater than 80

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µg/mL induced such extensive DNA damage (more than 80%) that comets were no longer

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recognized and could not be measured using the Lucia-Comet analysis program. Moreover,

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the number of comets per slide was much lower than that for concentrations lower than 80



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µg/mL, which may indicate that high ET doses resulted in complete DNA fragmentation in

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numerous cells.

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LC and SH-6 were less genotoxic than the REP, with concentrations of 18.9 µM and

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26.7 µM inducing DNA damage amounting to (12.3±2.0)% and (20.4±2.8)% for LC and SH-

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6, respectively (P < 0.05) (Figures 3 and 4). Concentrations greater than 53.4 µM in the case

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of SH-6 and 189 µM in the case of LC induced very strong DNA damage, which could not be

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measured. Thus, the results for those doses are not included in the graphs (Figures 3 and 4),

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because they would not reflect the actual effects. As was the case with the REP, high doses of

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SH-6 and LC resulted in complete DNA fragmentation in numerous cells as the number of

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comets per slide was much lower than that for lower concentrations.

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The genotoxic activity of SH-6 was higher than that of LC at lower concentrations

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(e.g., 53.4 µM revealed genotoxicity greater than 50%). In the case of LC, DNA damage

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greater than 50% was induced by a 3.5-times higher dose (189 µM). Doses amounting to 12.8

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µM (SH-6) and 9.3 µM (LC) induced weak DNA damage at similar levels (approx. 10%).

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DSBs induced by ETs. The genotoxicity of the REP was assayed in two repeats. Non-

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exposed Caco-2 cells (negative control) exhibited little DNA damage (4.7±1.6%), while

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cisplatin (50 µM, positive control) resulted in (27.4±3.4)% DNA damage. The REP induced

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DSBs at the doses tested (P < 0.05) (Figure 5), with (13.6±3.1)% damage at 160 µg/mL.

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Oxidative DNA bases induced by ETs. This part of the study involved the DNA

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repair enzymes Endo III and Fpg. Endo III converts oxidized pyrimidines into strand breaks,

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which can be detected by the comet assay.16 The results indicate only those DNA base

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modifications which are not alkali-labile. Fpg was used to detect the major purine oxidation

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product, 8-oxoguanine, as well as other altered purines.19 Figure 6 shows DNA base

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modifications in Caco-2 cells after treatment with Endo III and Fpg at 1 µg/mL. The results

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were normalized by subtracting the values for enzyme-specific buffer-only treatments.


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The largest extent of DNA oxidation was recognized by Endo III and Fpg for the

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positive control treated with 50 µM hydrogen peroxide (data not presented), amounting to

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(25.5±3.4)% and (45.8±3.9)%, respectively. Non-exposed Caco-2 cells (negative control)

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exhibited little DNA damage, at (4.1±2.6)% and (4.3±2.0)% for Endo III and Fpg,

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respectively. The REP caused oxidative DNA damage in Caco-2 cells, with the concentration

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of 40 µg/mL exhibiting the highest genotoxicity: (19.8±3.0)% and (15.8±2.9)% as recognized

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by Fpg and Endo III (P < 0.05).

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Cytotoxic activity of the REP. Caco-2 cells were challenged with ETs over a range

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of concentrations from 1.25 to 160 µg/mL. Cell proliferative activity and viability was

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measured by colorimetric MTT and fluorimetric PrestoBlue assays after 48 h, in eight repeats

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for each concentration. The relationship between ET concentration and cytotoxicity (anti-

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proliferative activity) is presented graphically in Figure 7. The viability of Caco-2 cells

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decreased proportionally with increasing REP concentration, starting from 20 µg/mL. In the

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presence of the highest concentration tested (160 µg/mL), REP cytotoxicity exceeded 70%

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and 80% in MTT and PrestoBlue assays, respectively.

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The REP IC50 values estimated in MTT and PrestoBlue assays for the Caco-2 cell line after 48 h of exposure were similar at 124 and 122 µg/mL, respectively.

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Nuclear morphology of Caco-2 cells after treatment with ETs. Following exposure

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to 124 µg/mL REP (IC50 concentration) for 24 h, cells were stained with DAPI and AO/PI,

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and nuclear changes were observed. In the untreated control, cells were spherical,

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homogenously stained, and a pattern of normal chromatin was clearly seen (intact structure)

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(Figures 8 and 9). The majority of REP-treated Caco-2 cells remained attached to the surface

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of the culture slide (Figure 8). Chromatin condensation and nuclear fragmentation were

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considered symptoms of apoptosis because they do not occur in necrotic cells.20 Analysis was

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conducted according to the criteria given by Baskić et al.18 and Salim et al.21 Condensation of



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nuclear material and apoptotic bodies of different sizes (late apoptosis) were observed in the

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treated cells (Figure 8a,b; Figure 9a-d). Blebbing and nuclear margination were also found

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(Figure 9a-f). Noticeable apoptotic morphological changes, including nuclear condensation,

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may suggest the ability of the REP to induce apoptosis.

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DISCUSSION

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Phytochemicals can be defined as non-nutrient bioactive components of fruits and vegetables,

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such as carotenoids, polyphenols, alkaloids, and other nitrogen-containing compounds.3 For

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many years now, plants have been used in pharmaceutical and dietary therapy. ETs are mostly

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present in berries, which are commonly consumed by humans worldwide. The

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chemopreventive activity of ETs and their derivatives is primarily linked to their antioxidant

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capacity. The consumption of natural antioxidants is associated with reduced risk of cancer

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and cardiovascular diseases.22 The chemotherapeutic nature of certain phytochemicals may be

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attributable to their ability to inactivate reactive oxygen species involved in the initiation and

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progression of cancer. Sufficient intake of these phytochemicals with diet may prevent cancer

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by decreasing oxidative DNA damage and enhancing DNA repair.3

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The comet assay is well-suited for investigating nutrient or micronutrient effects on

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DNA damage in humans.19 This is the first report showing the genotoxicity of ellagitannins

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from raspberry fruit and their mechanisms of DNA damage in Caco-2 cells. In our

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experiments, the REP, LC and SH-6 showed high genotoxicity against human colon cancer

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cells (Caco-2). The REP displayed the strongest, while LC the weakest, genotoxic effects.

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Genotoxicity increased in a dose-dependent manner, but only up to a certain concentration,

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which was 53.4 µM for SH-6, 80 µg/mL for the REP, and 189 µM for LC. Higher

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concentrations induced very strong DNA damage. The REP also induced oxidative DNA

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damage against Caco-2 cells as recognized by the Fpg and Endo III repair enzymes.


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Endogenous base oxidation seems to be quite strong, with damage beginning at 10 µg/mL,

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which was 12 times lower than the IC50 value (124 µg/mL) for the REP as evaluated in our

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experiments. Little oxidative damage to DNA was observed only in the presence of 2.5

305

µg/mL REP. This may suggest that at higher concentrations ETs can exhibit pro-oxidant

306

properties against colon cancer cells, which corroborates their chemopreventive properties.

307

Moilanen et al.23 reported deoxyribose and oxidative activity assays showing the pro-oxidant

308

activity of several ETs (e.g., punicalagin, chebulanin, geraniin) extracted from different plant

309

materials. Many of them revealed also anti-oxidant properties. The oxidative activity of ETs

310

was found to be highly variable among the different ET structural features, which include

311

tautomeric forms of the glucose core, the number of nonahydroxytriphenoyl and valoneoyl

312

groups, and the presence of additional glucosyl units.23 Oxidation reactions involving ETs can

313

produce reactive oxygen species and other products, such as quinones, which may cause

314

oxidative stress.23 Diets containing a variety of nutrients lead to changes in oxidative DNA

315

damage. For example, polyunsaturated fatty acids apparently increase oxidative DNA damage

316

in lymphocytes, while the protective effects of antioxidants (or foods containing large

317

quantities thereof) on lymphocytes have been demonstrated as a decrease either in

318

endogenous base oxidation or in sensitivity to H2O2-induced damage in vitro.19

319

Polyphenols can exert both antioxidant and pro-oxidant effects by scavenging and

320

generating free radicals, respectively. Tourino et al.24 suggest that the more effective the

321

antioxidant, the more cytotoxic and antiproliferative properties it exhibits. Moderate

322

generation of reactive oxygen species is selectively toxic to cancer cells and may stimulate the

323

immune system. Indeed, polyphenols may exert beneficial effects by a combination of both

324

radical scavenging and generating mechanisms.24 While the pro-oxidant effects of

325

polyphenols appear to be responsible for the induction of apoptosis in tumor cells, they may

326

also activate endogenous antioxidant systems in normal tissues, offering protection against



327

carcinogenic insult, as it has been found for green tea polyphenols.25 Furthermore, Kallio et

328

al.26 demonstrated that urolithins, the main metabolites of ellagitannins in the gastrointestinal

329

(GI) tract, display both antioxidant and pro-oxidant activities.

330

In the studied REP, the dominant compounds were LC and SH-6, with LC and

331

sanguiin H-10 isomers occurring in smaller amounts.10 Other phenolics present in the REP

332

included flavanols and anthocyanins. Higher concentrations of ellagitannins induced strong

333

DNA damage (exceeding 80–90%). Also “ghost” comets, possibly representing a residue of

334

high-molecular-weight DNA in apoptotic cells, were numerous.19 Some researchers suggest

335

that when almost all DNA is in the tail of a comet and the head is reduced in size, such a

336

comet corresponds to an apoptotic cell. However, according to Collins,19 this is possible, but

337

suppositious. In our research, concentrations greater than 80 µg/mL for the REP, 53.4 µM for

338

SH-6 and 189 µM for LC induced very strong DNA damage, which was strictly correlated

339

with REP cytotoxicity.

340

At concentrations above 80 µg/mL, cytotoxicity sharply increased (for 160 µg/mL it

341

was higher than 70%), whether in MTT or PrestoBlue assays. Krauze-Baranowska et al.27

342

reported the cytotoxic activity of raspberry shoot ellagitannins, the main component of which

343

was SH-6. The IC50 levels of the raspberry extract for cervical adenocarcinoma HeLa cells

344

and promyelocytic leukemia HL-60 cells, as determined in an MTT assay, were 300 µg/mL

345

and 110 µg/mL, respectively, which corresponds to an REP IC50 of 124 µg/mL in the present

346

study. Accordingly, the IC50 levels of SH-6 were 35 µg/mL and 25 µg/mL for HeLa and HL-

347

60 cells, respectively. We did not investigate the cytotoxicity of SH-6. McDougall et al.28

348

showed that different berry extracts were cytotoxic against Caco-2 cells, which were more

349

sensitive at low concentrations. Kreander et al.29 demonstrated that raspberry extract and its

350

various fractions were not cytotoxic at the concentrations used in the MTT assay.


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351

In this study, it was also found that the mechanism of DNA damage caused by the

352

REP involved DSBs, which peaked at 160 µg/mL. DSBs are highly deleterious DNA lesions

353

because they can lead to chromosome aberrations or apoptosis.

354

Since the AO/PI fluorescence staining method provides reliable and reproducible

355

results and is recommended for apoptosis detection,18 AO/PI double staining was applied in

356

the present study to corroborate the results. To assess the morphology of Caco-2 cell nuclei,

357

staining was performed following treatment with 124 µg/mL REP (corresponding to IC50).

358

Untreated cells were observed to have a green intact nuclear structure. Since AO intercalates

359

within fragmented DNA, early apoptosis was characterized by a bright-green nucleus with

360

chromatin condensation. PI permeates through membranes of nonviable cells and binds to

361

DNA, giving orange to red fluorescence. In late apoptotic cells, yellow/orange nuclei with

362

condensed, shrunk, or fragmented nuclei, as well as dense red areas of chromatin

363

condensation and blebbing were observed. Membrane-permeable DAPI staining confirmed

364

chromatin condensation and the presence of apoptotic bodies. Larossa et al.30 reported that

365

ETs and their metabolites induce apoptosis in human colon adenocarcinoma Caco-2 cells, but

366

not in normal colon cells (CCD-112CoN), by using the mitochondrial pathway. Yoshida et

367

al.31 demonstrated that ETs induce apoptosis through a pro-oxidant mechanism in tumor cells

368

(oral squamous cell carcinoma – HSC-2, HSG) to a higher extent than normal fibroblasts.

369

In the presented study, the effects of extracts were tested in one cancer cell line.

370

Whether the cytotoxic effects are specific to cancer cell lines in general has to be elucidated in

371

future studies. A considerable body of research has shown that ETs isolated from different

372

plants (e.g., pomegranate, Terminalia sp., dogwood, raspberry) can induce apoptosis in cancer

373

cells (e.g., HL-60; acute lymphoblastic leukemia CCRF-CEM, CCL-119 and MOLT-3; acute

374

monocytic leukemia THP-1; HT-29 colon cancer cells; prostate cancer cell lines; esophageal

375

epithelial cells).1,32-37 The other major class of phenolics present in the studied REP was



376

identified as anthocyanins,10 which can also give rise to apoptosis in some human cell lines

377

(e.g., Caco-2, HeLa, leukemia U937 cells).38,39 In our preliminary studies, the REP triggered

378

an apoptotic collapse of the mitochondrial membrane potential of cancer cells (unpublished

379

data). It has been suggested that one mechanism by which ETs induce apoptosis involves the

380

mitochondrial route.6

381

The bioavailability of ETs in the human GI tract is poor because of their high

382

molecular weight and polarity; thus, they have never been reported in urine or in the human

383

circulatory system, even after the consumption of considerable amounts of dietary ETs.1,3,5

384

ETs are partly degraded into ellagic acid (EA) in the upper parts of the GI tract, before

385

reaching the colon, probably due to microbial activity during intestinal transit.1,5

386

Lactobacillus strains present in the human GI tract are responsible for transforming ETs to

387

EA due to the activity of the enzyme tannase (tannin acylhydrolase).40,41 The main

388

metabolites of pomegranate juice ETs exposed to a pure culture of Lactobacillus sp. were EA

389

and its glycosyl derivative.42 Coates et al.

390

investigate the cyto- and genotoxic activity of a colon-available raspberry extract (CARE)

391

which contained phytochemicals surviving a digestion procedure mimicking the

392

physicochemical conditions of the upper GI tract. The authors reported that the CARE did not

393

induce significant DNA damage in HT-29 cells as compared to the untreated controls and that

394

the extract was not cytotoxic.

3

used human colon cancer HT-29 cells to

395

Depending on their concentration, the REP, LC and SH-6 demonstrate genotoxic

396

potency towards Caco-2 colon cancer cells in vitro. The mechanisms of DNA damage include

397

the induction of DSBs as well as the oxidation of DNA bases. The REP also shows cytotoxic

398

and antiproliferative activity against Caco-2 cells. An IC50 dose of the REP changes nuclear

399

morphology and induces apoptosis in Caco-2 cells, but this needs to be confirmed in further in

400

vitro studies involving apoptosis detection. Our findings explain the mode of action of


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401

ellagitannins in colon cancer cells.

In summary, the raspberry ellagitannin preparation

402

exhibits chemopreventive activity against the Caco-2 cancer cell line and can be used as a

403

natural food additive conferring major health benefits to foodstuffs.

404 405

ACKNOWLEDGMENTS

406

This study was supported by a grant from the National Science Center, Poland (grant no.

407

2013/09/B/NZ9/01806).

408 409

AUTHOR’S CONTRIBUTION

410

Adriana Nowak designed and performed the experiments, analyzed the data and wrote the

411

manuscript. Michał Sójka prepared and analyzed the REP, isolated the individual ETs, i.e. LC

412

and SH-6, as well as composed Figure 1 in Introduction, “REP extraction and composition”,

413

“LC and SH-6 isolation” in Materials and Methods and revised the manuscript. Elżbieta

414

Klewicka initiated the research concept and revised the manuscript. Lidia Lipińska assisted in

415

preparing samples for REP analysis. Robert Klewicki and Krzysztof Kołodziejczyk analyzed

416

individual ellagitannins: SH-6 and LC.

417 418

CONFLICT OF INTERESTS

419

The authors declare no conflict of interest.



420

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421

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Table 1. Polyphenolic composition and identification of ellagitannins in the raspberry ellagitannin preparation (REP) tR [min]

MS data

MS/MS data

Compound

Mean (mg/100 g)

SD

Ellagitannins 1235, 935, 633, 469, 301 1065 57 2200, 1867, 1567, 1235, 1456 97 15.1 1250-2 933-2, 633, 301 1265, 1103, 935, 933, 633, Sanguiin H-10 isomer 17.7 1567 731 47 469, 301 1869, 1567, 1235, 935, 633, Lambertianin C isomer 19.3 1401-2 2961 332 301 1869, 1567, 1235, 935, 633, Lambertianin C 19.8 1401-2 44156 1705 301 Sanguiin H-6 20.6 1869 1567, 1235, 935, 633, 301 35917 1300 Total ellagitannins 86287 3537 Other phenolics Total anthocyanins 823 6 Total flavanols (mDP) 7003 (1.9) 158 (0.1) Results are given as means of three replicates ± standard deviation (SD); tR – retention time; mDP – mean degree Sanguiin H-10 isomer Lambertianin C without ellagic moiety

14.6

1567

of proanthocyanidin polymerization.



Figure 1.


Page 26 of 36

Page 27 of 36


Figure 2.



Figure 3.


Page 28 of 36

Page 29 of 36


Figure 4.



Figure 5.


Page 30 of 36

Page 31 of 36


Figure 6.



Figure 7.


Page 32 of 36

Page 33 of 36


Figure 8.



Figure 9.


Page 34 of 36

Page 35 of 36


Figure 1. Structures of sanguiin H-6 (SH-6) and lambertianin C (LC), the main Rubus idaeus L. ellagitannins.

Figure 2. Basic DNA damage in Caco-2 cells after exposure to the raspberry ellagitannin preparation (REP), expressed as the mean percentage of DNA in the comet tail in the alkaline comet assay. Fifty cells were analyzed for each treatment. Data given from two independent experiments. Error bars denote S.E.M. The inset on the right shows typical images of 2.5 µg/mL propidium iodide-stained comets: (a) untreated control and (b) sample exposed to 80 µg/mL REP. * Results significantly different from each other, ANOVA (P < 0.05).

Figure 3. DNA damage in Caco-2 cells exposed to lambertianin C (LC), expressed as mean percentage of DNA in the tail in comets in the alkaline version of the comet assay. The number of cells analyzed was 50. Error bars denote S.E.M.

a-c

Results significantly different

from each other, ANOVA (P < 0.05).

Figure 4. DNA damage in Caco-2 cells exposed to sanguiin H-6 (SH-6), expressed as mean percentage of DNA in the tail in comets in the alkaline version of the comet assay. The number of cells analyzed was 50. Error bars denote S.E.M. * Results significantly different from each other, ANOVA (P < 0.05).

Figure 5. Double-strand breaks (DSBs) in Caco-2 cells following exposure to the raspberry ellagitannin preparation (REP), expressed as mean percentage of DNA in the tail of comets in the neutral version of the comet assay. The number of cells analyzed was 100. Error bars denote S.E.M. a Results significantly different, ANOVA (P < 0.05).



Figure 6. Oxidative DNA damage measured as the mean comet tail of Caco-2 cells following exposure to the raspberry ellagitannin preparation (REP) recognized by endonuclease III (Endo III) or formamidopyrimidine-DNA glycosylase (Fpg). The number of cells evaluated for each sample was 50. Net values are presented (after subtracting background values). Error bars denote S.E.M. a, b Results significantly different, ANOVA (P < 0.05).

Figure 7. Cytotoxicity of the raspberry ellagitannin preparation (REP) as determined by MTT and PrestoBlue assays in the Caco-2 cell line after 48 h exposure. Each data point represents a mean of absorbance/fluorescence values for cells from eight individual wells (± SD).

Figure 8. Nuclear morphology of DAPI-stained Caco-2 cells after 24 h exposure to 124 µg/mL raspberry ellagitannin preparation (REP) (a and b). Condensation of nuclear material (arrow heads) and apoptotic bodies (arrows) were observed. Fluorescence microscopy (Nikon, Japan); 200× magnification. Images are representative of one of five similar experiments.

Figure 9. Fluorescent micrographs of acridine orange/propidium iodide-stained Caco-2 cells after 24 h exposure to 124 µg/mL (a–f) raspberry ellagitannin preparation (REP). Membrane blebbing (BL); chromatin condensation (CC); early (EA) and late apoptosis (LA); nuclear fragmentation (NF), and viable cells (VC) were observed. Fluorescence microscopy (Nikon, Japan); 400× magnification. Images are representative of one of five similar experiments.


Page 36 of 36

Ellagitannins from Rubus idaeus L. Exert Geno- and Cytotoxic Effects

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