Food Chemistry 170 (2015) 455–462

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Food Chemistry

journal homepage: www.elsevier .com/locate / foodchem

In vitro genotoxicity of mycotoxins ochratoxin A and fumonisin B1

could be prevented by sodium copper chlorophyllin – Implicationto their genotoxic mechanism

http://dx.doi.org/10.1016/j.foodchem.2014.08.0360308-8146/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Fax: +385 1 6394400.E-mail address: adomijan@pharma.hr (A.-M. Domijan).

Ana-Marija Domijan a,⇑, Goran Gajski b, Ivana Novak Jovanovic c, Marko Geric b, Vera Garaj-Vrhovac b

a Department of Pharmaceutical Botany, Faculty of Pharmacy and Biochemistry, University of Zagreb, A. Kovacica 1, Zagreb, Croatiab Mutagenesis Unit, Institute for Medical Research and Occupational Health, Ksaverska c. 2, Zagreb, Croatiac Toxicology Unit, Institute for Medical Research and Occupational Health, Ksaverska c. 2, Zagreb, Croatia

a r t i c l e i n f o

Article history:Received 18 February 2013Received in revised form 4 July 2014Accepted 10 August 2014Available online 19 August 2014

Keywords:ChlorophyllinMycotoxinsHuman lymphocytesDNA damageOxidative stressProtective effect

a b s t r a c t

The aim of this study was to investigate the possible protective effect of sodium copper chlorophyllin(CHL) against cytotoxicity and DNA damage induced by mycotoxins ochratoxin A (OTA) and fumonisinB1 (FB1). CHL (0.1–100 lg/ml) alone had no impact on cell viability and genome damage in the primaryhuman peripheral blood lymphocytes (HPBLs) and exhibited free radical scavenging activity in the DPPHassay. Both mycotoxins, OTA (4 lmol/l) and FB1 (20 lg/ml), induced DNA damage in HPBLs already after1 h exposure. When the HPBLs were co-exposed to CHL (10 and 100 lg/ml) and OTA (4 lmol/l) or FB1

(20 lg/ml) for 1 h, CHL protected against cell and DNA damage induced by both mycotoxins, implyingthat OTA and FB1 cytogenotoxicity mechanisms function at least partially through oxidative stress. There-fore, CHL could be a perfect candidate for possible use as an antioxidant.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Sodium copper chlorophyllin (CHL) is a semi-synthetic mixtureof water-soluble sodium copper salts derived from chlorophyll,widely used as food dye (Chernomorsky, Rancourt, Virdi,Segelman, & Poretz, 1997; Tumolo & Lanfer-Marquez, 2012). Itsanti-mutagenic and anti-carcinogenic activities against numerousdietary and environmental agents, including the mycotoxinaflatoxin B1, heterocyclic amines, and polycyclic aromatichydrocarbons have been recorded (reviewed in Tumolo & Lanfer-Marquez, 2012). Mechanistic studies have revealed that the anti-mutagenic/anti-carcinogenic properties of CHL may be attributedto its ability to bind carcinogens by forming tight complexes, thusdiminishing its bioavailability (Egner, Muñoz, & Kensler, 2003;Tumolo & Lanfer-Marquez, 2012). However, according to someauthors, the anti-carcinogenic properties of CHL may be due toits antioxidant properties (Ferruzzi, Böhm, Courtney, & Schwartz,2002; Tumolo & Lanfer-Marquez, 2012). For example, CHL wasfound to inhibit the formation of DNA strand breaks caused byreactive oxygen species (ROS) derived from the H2O2/Cu(II) system(Park, Park, Jung, & Chung, 2003). Moreover, CHL was able to

reduce the formation of the major oxidative DNA damage product8-hydroxydeoxyguanosine (8-OH-dG) induced by hydrogen perox-ide (H2O2) and UV or by cyclophosphamide or benzo[a]pyrene(Ibrahim, Elbehairy, Ghoneim, & Amer, 2007; Park et al., 2003). Ithas also been reported that CHL effectively suppresseschemically-, radiation-, and photosensitization-induced lipid per-oxidation (LPO) (Boloor, Kamat, & Devasagayam, 2000; Ibrahimet al., 2007; Kamat, Boloor, & Devasagayam, 2000). It is importantto emphasise that the antioxidant activity of CHL was found to bemuch higher than that of natural chlorophylls, including chloro-phylls a and b, pheophytins and pheophorbids (Ferruzzi et al.,2002; Lanfer-Marquez, Barros, & Sinnecker, 2005).

Ochratoxin A (OTA) and fumonisin B1 (FB1) are mycotoxinsfound as feed and food contaminants world-wide (IPCS, 2001).The nephrotoxicity, hepatotoxicity, genotoxicity and carcinogenic-ity of OTA and FB1 have already been established in experimentalanimals (Domijan & Peraica, 2010). Human exposure to OTA is con-nected to the development of Balkan endemic nephropathy andurinary bladder tumours (Miletic-Medved, Domijan, & Peraica,2005), while human exposure to FB1 is associated with a higherincidence of oesophageal cancer, primary liver tumours and neuraltube defects (Voss, Smith, & Haschek, 2007). Due to a lack of epide-miological data, it is not possible to establish a direct connectionbetween exposure to OTA or FB1 and the development of human

456 A.-M. Domijan et al. / Food Chemistry 170 (2015) 455–462

diseases. Therefore, the International Agency for Research on Can-cer (IARC) classified both OTA and FB1 as Group 2B (possiblehuman carcinogen) (IARC, 1993, 2002).

Although experimental data confirm that both mycotoxins OTAand FB1 are genotoxic, the mechanism of their genotoxicity andcarcinogenicity is still a matter of debate. Based on findings ofOTA-related DNA adducts in cell cultures as well as in animaland human kidneys, some authors support the notion of directOTA genotoxic action after metabolic activation (Pfohl-Leskowicz& Manderville, 2007). However, some studies indicate that OTA isnot able to form reactive intermediates capable of interacting withDNA (Mally et al., 2005), and it has been suggested that OTA caninduce DNA damage through oxidative stress (Arbillaga, Azqueta,Ezpeleta, & Lopez de Cerain, 2007; Domijan, Zeljezic, Kopjar, &Peraica, 2006; Kamp, Eisenbrand, Schlatter, Würth, & Janzowski,2005; Liu et al., 2012). Similarly, some authors have proposed thatoxidative stress may be the underlying mechanism of FB1 genotox-icity (Domijan et al., 2006; Mary, Theumer, Arias, & Rubinstein,2012; Mobio et al., 2000, 2003), while others have failed to findan increase in ROS production together with DNA damage afterFB1 exposure, suggesting oxidative stress is not involved in FB1-induced DNA damage (Galvano et al., 2002).

Since the mechanism of OTA and FB1 genotoxicity as well ascytotoxicity is still controversial, the present study aimed toinvestigate if CHL, as an antioxidant, has the potential to preventcytogenotoxicity induced by these mycotoxins and thus clarifythe role of oxidative stress in cell death and DNA damage inducedby these naturally occurring toxins. First, we evaluated the toxico-logical profile of CHL by measuring the cytotoxic and genotoxicpotential of CHL in primary human peripheral blood lymphocytes(HPBLs). DNA damage was measured with the alkaline cometassay, while further CHL genotoxic activity was assessed with thecytokinesis-block micronucleus (CBMN) assay. Also, CHL free radi-cal scavenging capacity/antioxidant activity was determined withthe DPPH assay. Subsequently, the genotoxic potential of OTAand FB1 in HPBLs was established. The possible protective role ofCHL against OTA and FB1 cytotoxicity and genotoxicity wasfollowed by co-exposing HPBLs to CHL and OTA or FB1. In theseexperiments, H2O2 was used as positive control.

2. Materials and methods

2.1. Chemicals

CHL, acridine orange (AO), cytochalasin-B, disodium EDTA, ethi-dium bromide (EtBr), histopaque, low melting point (LMP) andnormal melting point (NMP) agaroses, RPMI 1640 medium, TritonX-100, L-ascorbic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), (±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trol-ox) and butylated hydroxytoluene (BHT) were purchased fromSigma (St Louis, MO, USA). Giemsa solution was acquired fromMerck (Darmstadt, Germany) and Chromosome kit P fromEuroclone (Milano, Italy). All other chemicals used were labora-tory-grade and were purchased from Kemika (Zagreb, Croatia).

2.2. Blood sampling and treatment

Whole blood samples were taken from a healthy female non-smoking donor (age 32). The donor had not been exposed to ioniz-ing radiation for diagnostic or therapeutic purposes, or vaccinatedand treated with drugs that might have interfered with the resultsof testing for a year before blood sampling. The subject gaveinformed consent to participate in this study. The study was partof the project approved by the institutional ethics committee andobserved the ethical principles of the Declaration of Helsinki.

Whole venous blood was collected under sterile conditions in hep-arinised vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ,USA) containing lithium heparin as anticoagulant.

The comet assay and CBMN assay were conducted on wholeblood, while cytotoxicity was performed on isolated HPBLs. CHLstock solution (1 mg/ml), and CHL dilutions used for treatments(in concentrations 0.1, 0.5, 1, 5, 10, 50 and 100 lg/ml) were pre-pared in sterile redistilled water. Three OTA concentrations (1, 2and 4 lmol/l; dissolved in ethanol) and three concentrations ofFB1 (5, 10 and 20 lg/ml; dissolved in sterile redistilled water) weretested. Also, for each experiment and each exposure time, controlsamples (solvent control) were included. After treatments, individ-ual experiments were conducted according to the standard proto-cols listed below.

2.3. Cell viability (cytotoxicity) test

Cell viability was determined by differential staining with AOand EtBr using a fluorescence microscope (Duke & Cohen, 1992).HPBLs were isolated by histopaque density centrifugation method(Singh, 2000). The slides were prepared using 200 ll of HPBLs and2 ll of stain (AO (100 lg/ml) and EtBr (100 lg/ml, 1:1; v/v), bothdiluted in phosphate-buffered saline, PBS). A total of 100 cellsper repetition were examined with an Olympus BX-51 microscope(Tokyo, Japan) at 400� magnification. The cells were classified asfollows: live cells with functional membrane with uniform greenstaining of the nucleus, and necrotic cells with uniform red stain-ing of the nucleus.

2.4. Alkaline comet (SCGE) assay

The alkaline comet assay was carried out as described by Singh,McCoy, Tice, and Schneider (1988) with minor modifications(Gajski, Garaj-Vrhovac, & Orešcanin, 2008). Briefly, after the expo-sure, 5 ll of whole blood was mixed with 100 ll of 0.5% LMP aga-rose and added to fully frosted slides pre-coated with 0.6% NMPagarose. Once the NMP agarose was solid, the slides were coveredwith 0.5% LMP agarose, and cells lysed (2.5 mol/l NaCl, 100 mmol/lNa2EDTA, 10 mmol/l Tris, 1% sodium sarcosinate, 1% Triton X-100,10% dimethyl sulfoxide; pH 10) over night at 4 �C. After the lysis,the slides were placed in alkaline solution (300 mmol/l NaOH,1 mmol/l Na2EDTA; pH 13) for 20 min at 4 �C to allow DNAunwinding and subsequently electrophoresed for 20 min at 1 V/cm. Finally, the slides were neutralized in 0.4 mol/l Tris buffer(pH 7.5) for 5 min 3 times, stained with EtBr (20 lg/ml) and ana-lysed at 250� magnification using an epifluorescence microscope(Zeiss, Göttingen, Germany) connected through a black and whitecamera to an image analysis system (Comet Assay II; PerceptiveInstruments Ltd., Haverhill, Suffolk, UK). The level of DNA damagewas expressed as percentage of DNA in the tail, and a total of 100randomly captured comets were examined from each slide. Theresults are shown as box plots.

2.5. Cytokinesis-block micronucleus (CBMN) assay

The CBMN assay was performed according to the guidelines ofFenech and Morley (1985) with minor modifications (Gajskiet al., 2008). After the exposure (4 and 24 h) to CHL the wholeblood (500 ll) was incubated in a Euroclone medium (Chromo-some kit P) at 37 �C in an atmosphere of 5% CO2. Cytochalasin-Bwas added at a final concentration of 3 lg/ml 44 h after the culturewas started. The cultures were harvested after 72 h. The lympho-cytes were fixed in methanol–acetic acid (3:1), air-dried andstained with 5% Giemsa solution. All slides were randomised andcoded prior to analysis. The binuclear lymphocytes were analysedunder a light microscope (Olympus CX41, Tokyo, Japan) at 400�

A.-M. Domijan et al. / Food Chemistry 170 (2015) 455–462 457

magnification. Micronuclei (MNi), nucleoplasmic bridges (NPBs)and nuclear buds (NBUDs) were counted in 1000 binucleated cellsand were scored according to the HUMN project criteria publishedby Fenech et al. (2003).

2.6. DPPH assay

The antioxidant activity of CHL was determined spectrophoto-metrically (Cecil 9000 UV/Vis spectrophotometer, Cecil Instru-ments, Cambridge, UK) according to the DPPH radical scavengingmethod described by Šeruga, Novak, and Jakobek (2011). Briefly,50 ll of CHL in different concentrations (1.0–30 lmol/l), freshlyprepared from an aqueous stock solution (6 mmol/l), were mixedwith 120 ll of DPPH�solution (1 mmol/l, in methanol) and1880 ll of methanol. The reaction mixture was vortexed thor-oughly and incubated in the dark at room temperature for15 min and then the absorbance of the mixture (Asample) was mea-sured at 517 nm against the blank sample (blank sample: 50 ll ofCHL solution and 2000 ll of methanol). A fresh DPPH� blank solu-tion (120 ll of 1 mmol/l DPPH� and 1930 ll of methanol) was pre-pared each day and its absorbance was measured daily at 517 nm(ADPPH� ). For the different CHL concentrations, DPPH� free radicalscavenging activity was expressed as percentage inhibition of theinitial concentration of DPPH� and calculated according to the fol-lowing equation:

% Inhibition ¼ ½ðADPPH� � AsampleÞ=ADPPH� � � 100:

In order to obtain the EC50 index, defined as the amount of anti-oxidant (lmol/l) needed to reduce the initial concentration ofDPPH� by 50%, different concentrations of CHL in the reaction mix-ture were plotted against calculated percentage inhibition ofDPPH�. The EC50 index is a measure of the antioxidant activity ofa compound; the lower the EC50 value, the higher the antioxidantactivity of tested compound. BHT, Trolox and ascorbic acid wereused as reference compounds. All tests were carried out intriplicate.

2.7. Statistics

The statistical analyses were performed with Statistica 5.0package (StatSoft, Tulsa, OK, USA). For the comet assay multiplecomparisons between groups were done by means of ANOVA on

Table 1Parameters of the cytokinesis-block micronucleus (CBMN) assay in binucleated human peIncidence of MNi, NPBs and NBUDs was evaluated by analysing 1000 binucleated cells.

Exposuretime (h)

CHLconcentration(lg/ml)

No. of binucleated cellswith MNi

No. ofMNed cells

Total nMNi

1 MN 2 MNi

0 2.00 ± 1.41 0.00 ± 0.00 2.00 ± 1.41 2.00 ± 10.1 1.00 ± 0.00 0.50 ± 0.71 1.50 ± 0.71 2.00 ± 10.5 3.50 ± 0.71 0.50 ± 0.71 4.00 ± 0.00 4.50 ± 0

4 1 3.00 ± 0.00 0.50 ± 0.71 3.50 ± 0.71 4.00 ± 15 4.50 ± 0.71 0.00 ± 0.00 4.50 ± 0.71 4.50 ± 010 4.00 ± 0.00 0.50 ± 0.71 4.50 ± 0.71 5.00 ± 150 3.00 ± 1.41 0.00 ± 0.00 3.00 ± 1.41 3.00 ± 1100 2.00 ± 0.00 0.50 ± 0.71 2.50 ± 0.71 3.00 ± 10 3.50 ± 2.12 0.00 ± 0.00 3.50 ± 2.12 3.50 ± 20.1 5.50 ± 0.71 0.00 ± 0.00 5.50 ± 0.71 5.50 ± 00.5 3.50 ± 0.71 0.00 ± 0.00 3.50 ± 0.71 3.50 ± 0

24 1 5.50 ± 2.83 0.50 ± 0.71 5.50 ± 2.12 6.00 ± 15 5.50 ± 0.71 0.50 ± 0.71 6.00 ± 1.41 6.50 ± 210 4.00 ± 1.41 0.50 ± 0.71 4.50 ± 0.71 5.00 ± 050 4.50 ± 2.12 0.50 ± 0.71 5.00 ± 2.83 5.50 ± 2100 4.00 ± 1.41 1.00 ± 1.41 5.00 ± 0.00 6.00 ± 1

MN – micronuclei; NPB – nucleoplasmic bridge; NBUD – nuclear bud.Note: there were no statistically significant differences between treated samples compa

log-transformed data. Post hoc analyses of differences were doneusing the Scheffé test. As for cell viability, statistical significancewas analysed using the Student’s t-test. The significance of CBMNparameters was tested using the v2-test. The level of statistical sig-nificance was set at P < 0.05.

3. Results

3.1. Toxicological profile of CHL

3.1.1. Cytotoxicity of CHLThe cytotoxicity of CHL was tested on HPBLs exposed to

different CHL concentrations (0.1–100 lg/ml) for 4 or 24 h andestimated by AO and EtBr staining using fluorescence microscopy.CHL at the concentrations tested had no effect on HPBL viability.After 4 h of treatment, viability of the cells was greater than98.77 ± 3.42% and, after 24 h, viability was greater than94.61 ± 2.49%, which was not significantly different from the corre-sponding controls. Taken together, the reduction in cell viabilitywas less than 10% indicating that CHL up to 100 lg/ml for 24 h isnot cytotoxic to HPBLs.

3.1.2. Induction of DNA strand breaks by CHLThe DNA damage in HPBLs after CHL treatment (0.1–100 lg/ml,

incubation for 4 or 24 h) was determined with the alkaline cometassay. CHL at the concentrations tested did not cause DNA damagethat could be detected with the alkaline comet assay. Even thehighest CHL concentration applied (100 lg/ml, for 24 h) had nogenotoxic potential compared to the untreated control.

3.1.3. Induction of micronuclei, nucleoplasmic bridges and nuclearbuds by CHL

The genotoxic activity of CHL was further evaluated using theCBMN assay. The whole blood samples were treated with CHL(0.1–100 lg/ml) for 4 or 24 h, and MNi, NPB and NBUD inductionwas assessed in binucleated lymphocytes. The results arepresented in Table 1. CHL did not induce statistically significantdifference in either CBMN assay parameter compared to untreatedcontrol.

Taken together, results from cell viability, and alkaline cometand CBMN assays indicate CHL exposure to 0.1–100 lg/ml for upto 24 h was not associated with cytotoxic or genotoxic changes

ripheral blood lymphocytes (HPBLs) exposed to CHL (0.1–100 lg/ml) for 4 and 24 h.

o. of Total no. ofNPBs

No. of binucleated cellswith NBUDs

No. of cells withNBUDs

Total no. ofNBUDs

1 NBUD 2 NBUDs

.41 0.50 ± 0.71 4.50 ± 0.71 0.00 ± 0.00 4.50 ± 0.71 4.50 ± 0.71

.41 2.50 ± 0.71 3.50 ± 0.71 0.00 ± 0.00 3.50 ± 0.71 3.50 ± 0.71

.71 1.00 ± 0.00 4.00 ± 1.41 0.00 ± 0.00 4.00 ± 1.41 4.00 ± 1.41

.41 1.50 ± 0.71 3.50 ± 0.71 0.00 ± 0.00 3.50 ± 0.71 3.50 ± 0.71

.71 1.00 ± 1.41 6.00 ± 2.83 0.00 ± 0.00 6.00 ± 2.83 6.00 ± 2.83

.41 2.50 ± 0.71 3.50 ± 2.12 0.50 ± 0.71 4.00 ± 1.41 4.50 ± 0.71

.41 1.50 ± 0.71 5.00 ± 0.00 0.00 ± 0.00 5.00 ± 0.00 5.00 ± 0.00

.41 3.00 ± 2.83 2.50 ± 0.71 0.50 ± 0.71 3.00 ± 1.41 3.50 ± 2.12

.12 2.50 ± 0.71 3.00 ± 1.41 0.00 ± 0.00 3.00 ± 1.41 3.00 ± 1.41

.71 0.50 ± 0.71 3.00 ± 1.41 0.50 ± 0.71 3.50 ± 2.12 4.00 ± 2.83

.71 1.00 ± 1.41 3.50 ± 0.71 0.00 ± 0.00 3.50 ± 0.71 3.50 ± 0.71

.41 2.50 ± 0.71 4.00 ± 1.41 0.50 ± 0.71 4.50 ± 2.12 5.00 ± 2.83

.12 0.50 ± 0.71 2.50 ± 0.71 0.50 ± 0.71 3.00 ± 0.00 3.50 ± 0.71

.00 3.00 ± 2.83 3.50 ± 0.71 0.00 ± 0.00 0.00 ± 0.00 3.50 ± 0.71

.83 1.00 ± 0.00 3.50 ± 0.71 0.50 ± 0.71 4.00 ± 1.41 4.50 ± 2.12

.41 2.00 ± 1.41 3.50 ± 2.12 0.00 ± 0.00 3.50 ± 2.12 3.50 ± 2.12

red to corresponding control (P < 0.05).

Fig. 2. Effect of ochratoxin A (OTA) and fumonisin B1 (FB1) on DNA damage inhuman peripheral blood lymphocytes (HPBLs). The cells were exposed to (A) OTA(1–4 lmol/l) and (B) FB1 (5–20 lg/ml) as well as solvent only (correspondingcontrol) for 1 h. The DNA damage was assessed with the alkaline comet assay and isexpressed as percentage of tail DNA. Data are presented as box plots. The edges ofthe box represents the 25th and 75th percentiles, the median is a square in themiddle of the box, and the error bars represents minimum and maximum values.⁄Statistically significant difference between treated cells and control (P < 0.05).

458 A.-M. Domijan et al. / Food Chemistry 170 (2015) 455–462

in HPBLs. Therefore, 10 and 100 lg CHL/ml, which appeared to nothave any cytotoxic and genotoxic effect on HPBLs, were used tostudy the possible preventive effects of CHL i.e. anti-cytotoxicand anti-genotoxic potential of CHL.

3.2. Antioxidant activity of CHL

The antioxidant activity of CHL was determined using the DPPHmethod. In the DPPH assay, an antioxidant with electron-donatingability reduces the stable 2,2-diphenyl-1-picrylhydrazyl radical(DPPH�) to 2,2-diphenyl-1-picrylhydrazyne (DPPH-H). As can beseen from Fig. 1, CHL is an effective DPPH� scavenger. The radicalscavenging ability of CHL is concentration-dependent; in the con-centration range 1.0–30 lmol/l (equivalent to 0.72–21.72 lg/ml),r2 was 0.994. From the linear regression curve calculated, CHLEC50 (concentration of CHL that reduce DPPH� activity by 50%)was 21.06 lmol/l. By comparing this value with those of certainother established antioxidants, the DPPH� free radical scavengingcapacity of CHL was about 1.6- and 1.9-fold smaller than ascorbicacid and Trolox, respectively, and 4.3-fold higher than that of BHT(Table 2). The antioxidant activity of CHL and reference compoundsdecreased in the following order: ascorbic acid > Trolox >CHL > BHT.

3.3. Induction of DNA strand breaks by OTA and FB1

Only a few studies have dealt with the genotoxicity of mycotox-ins OTA and FB1, and data on their genotoxic potential on HPBLsare lacking. Therefore, at several concentrations, OTA (1, 2 and4 lmol/l) and FB1 (5, 10 and 20 lg/ml) were tested for DNA dam-aging potential on HPBLs after 1 h of exposure. As can be seen fromFig. 2, statistically significant induction of DNA strand breaks wasachieved only after exposure to the highest OTA concentration(4 lmol/l, P < 0.05) as well as only after exposure to the highestFB1 concentration (20 lg/ml) (P < 0.05). These results indicate thatboth mycotoxins OTA and FB1 have the potential to induce DNAdamage in HPBLs. The concentrations of OTA and FB1 that induced

0

20

40

60

80

1 5 10 20 30

inhi

bitio

n (%

)

CHL concentration (µmol/l)

Fig. 1. Antioxidant activity of chlorophyllin (CHL) assessed with the DPPH assay.The antioxidant activity/DPPH free radical scavenging activity of CHL in concen-tration range (1.0–30 lmol/l) was concentration-dependent.

Table 2Values of EC50 index of different antioxidants assessed with the DPPH assay.

Antioxidant EC50 (lmol/l)

Ascorbic acid 11.06Trolox 13.40CHL 21.06BHT 91.23

significant DNA damage were chosen for conducting further cyto-genotoxicity experiments.

3.4. Protective effect of CHL

3.4.1. Prevention of OTA and FB1 cytotoxicity by CHLIn order to establish the possible protective role of CHL on cell

death induced by OTA or FB1, HPBLs were treated with CHL (10and 100 lg/ml), OTA (4 lmol/l), FB1 (20 lg/ml) and co-treatedwith the same concentrations of CHL and OTA or FB1 for 1 h. Inthese experiments, H2O2 was included as a positive control, andHPBLs were treated with CHL (10 and 100 lg/ml) or H2O2

(1 mmol/l), or co-treated with CHL (10 and 100 lg/ml) and H2O2

(1 mmol/l) for 10 min. Results are presented in Fig. 3. As expected,two selected concentrations of CHL did not induce any significantchange in cell viability to HPBLs. However, HPBL exposure toOTA (4 lmol/l) or FB1 (20 lg/ml) induced cell death that was sig-nificantly higher compared to control (Fig. 3B and C; P < 0.05).Exposure of HPBLs to H2O2 also induced significantly higher celldeath compared to controls (Fig. 3A; P < 0.05). However, whenHPBLs were co-exposed to CHL (both concentration) and OTA orFB1 cell viability increased compared to treatment with only OTAor FB1. Also, both concentrations of CHL could prevent cytotoxiceffect of H2O2.

3.4.2. Prevention of OTA and FB1-induced DNA strand breaks by CHLTo examine the possible anti-genotoxic effect of CHL against

DNA damage induced by OTA or FB1, whole blood samples were

A B

C

H2O2 (1 mmol/l)

CHL(10 µg/ml)

CHL(100

µg/ml)

H2O2+CHL (1 mmol/l+ 10 µg/ml)

H2O2+CHL (1 mmol/l+ 100 µg/ml)

control control

control

CHL(10 µg/ml)

CHL(10 µg/ml)

CHL(100

µg/ml)

CHL(100

µg/ml)

OTA (4 µmol/l)

OTA+CHL (4 µmol/l+10 µg/ml)

OTA+CHL (4 µmol/l+100 µg/ml)

FB1+CHL (20 µg/ml+10 µg/ml)

FB1 (20 µg/ml)

FB1+CHL (20 µg/ml+100 µg/ml)

cell

viab

ility

(%)

cell

viab

ility

(%)

cell

viab

ility

(%)

Fig. 3. Protective effect of chlorophyllin (CHL) on H2O2, ochratoxin A (OTA) and fumonisin B1 (FB1) induced cytotoxicity in human peripheral blood lymphocytes (HPBLs). Thecells were exposed to (A) H2O2 for 10 min; or to (B) OTA (4 lmol/l) or (C) FB1 (20 lg/ml) for 1 h; or co-exposed to CHL (10 and 100 lg/ml) and pro-oxidant. The cell viabilitywas determined by differential staining with acridine orange and ethidium bromide. ⁄Statistically significant difference between treated cells and untreated control (P < 0.05).

A.-M. Domijan et al. / Food Chemistry 170 (2015) 455–462 459

treated with CHL (10 and 100 lg/ml), OTA (4 lmol/l), FB1 (20 lg/ml) and co-treated with the same concentrations of CHL and OTAor FB1 for 1 h. Whole blood samples were also treated with H2O2

(1 mmol/l) as positive control and CHL (both concentrations) andco-exposed to the same concentrations of CHL and H2O2 for10 min. As can be seen from Fig. 4, both selected concentrationsof CHL did not induce any significant DNA damage in HPBLs. Whenwhole blood was treated with OTA or FB1, significantly higher DNAdamage was found compared to control sample (Fig. 4B and C;P < 0.05). However, DNA damage induced with OTA or FB1 was pre-vented with both concentrations of CHL indicating that CHL haspotential to prevent DNA damage induced by these two mycotox-ins. Similarly, the effect of H2O2 on DNA integrity was protectedwith CHL (Fig. 4A; P < 0.05).

4. Discussion

CHL is well known for its anti-mutagenic and anti-carcinogenicproperties, however there are some studies reporting its tumour-enhancing and genotoxic effects (Chernomorsky et al., 1997;Tumolo & Lanfer-Marquez, 2012). These contradictory results areattributed to variability in the chemical composition of the prepa-rations of CHL used, concentration and source of CHL, as well as tothe experimental model (in vivo or in vitro) (Chernomorsky et al.,1997; Tumolo & Lanfer-Marquez, 2012). Therefore, to establishthe toxicological profile of CHL, we assessed its cytotoxicity andgenotoxicity on HPBLs using widely accepted biomarkers for theevaluation of genome damage after exposure to different physicaland/or chemical agents as well as to a wide range of naturalproducts (Garaj-Vrhovac & Gajski, 2009). We found that CHL inthe concentration range tested (0.1–100 lg/ml) was not cytotoxicand genotoxic to HPBLs. Additionally, we observed that CHL inthe tested concentration range did not increase malondialdehyde

(MDA) levels (biomarker of LPO), suggesting that CHL has nopotential to induce oxidative damage of lipids (data not shown).These results are consistent with earlier in vitro and in vivo studiesshowing that CHL has no impact on DNA damage and LPO. CHL didnot induce DNA damage detected with the comet assay in HEp-2cells (Botelho et al., 2004) and was not genotoxic to V79 cellsevaluated by the CBMN assay (Bez, Jordão, Vicentini, &Mantovani, 2001). Pietrzak, Halicka, Wieczorek, Wieczorek, andDarzynkiewicz (2008) observed that exposure of human promyelo-cytic (HL-60) cells to CHL had no significant effect on the expres-sion of phosphorylated H2AX (cH2AX), which is considered to bea marker of DNA damage, particularly of DNA double-strandbreaks. In another study, similarly to our results, CHL did notinduce DNA fragmentation or increase liver microsomal MDA levelin albino rats (Ibrahim et al., 2007).

This study used the DPPH assay to check CHL antioxidant activ-ity. The results of the DPPH assay demonstrated that CHL has amarked capacity to scavenge DPPH� free radicals, indicating thatCHL acts as a free radical scavenger probably due to its facileelectron-donating ability. Our observations are in line withLanfer-Marquez et al. (2005) and Ferruzzi et al. (2002), who alsosuggested that CHL may act as an electron-donating antioxidantsince it was able to reduce free radicals such as DPPH� and ABTS�+

(2,20-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)). Bolooret al. (2000) and Kamat et al. (2000) showed that CHL neutralizedhydroxyl radical (�OH) and singlet oxygen (1O2) with a high rate ofefficiency confirming free radical scavenging properties of CHL; therate constants for the scavenging of �OH and 1O2 by CHL are calcu-lated to be in the order of 6.1 � 109 M�1 s�1 and 1.3 � 108 M�1 s�1,respectively. In a study by Park et al. (2003), CHL scavenged hydro-xyl radicals derived from the reaction of H2O2/Fe(III) in a dose-related manner and reduced the formation of superoxide anionsgenerated by 12-O-tetradecanoylphorbol-13-acetate (TPA).

A B

C

control control

control

H2O2(1mmol/l)

CHL(10

µg/ml)

CHL(100

µg/ml)

CHL(100

µg/ml)

CHL(100

µg/ml)

CHL(10

µg/ml)

CHL(10

µg/ml)

H2O2+CHL(1 mmol/l +10 µg/ml)

OTA+CHL (4 µmol/l+10 µg/ml)

H2O2+CHL (1 mmol/l+ 100 µg/ml)

OTA (4 µmol/l)

OTA+CHL (4 µmol/l+100 µg/ml)

FB1+CHL(20 µg/ml+100 µg/ml)

FB1+CHL(20 µg/ml+10 µg/ml)

FB1(20 µg/ml)

% o

f tai

l DN

A

% o

f tai

l DN

A

% o

f tai

l DN

A

Fig. 4. Protective effect of chlorophyllin (CHL) against H2O2, ochratoxin A (OTA) and fumonisin B1 (FB1) induced DNA damage in human peripheral blood lymphocytes(HPBLs). The cells were exposed to (A) H2O2 for 10 min; or to (B) OTA (4 lmol/l) or (C) FB1 (20 lg/ml) for 1 h; or co-exposed to CHL (10 and 100 lg/ml) and pro-oxidant. TheDNA damage was assessed with the alkaline comet assay and is expressed as percentage of tail DNA. Data are presented as box plots. The edges of the box represents the 25thand 75th percentiles, the median is a square in the middle of the box, and the error bars represents minimum and maximum values. ⁄Statistically significant differencebetween treated cells and control (P < 0.05).

460 A.-M. Domijan et al. / Food Chemistry 170 (2015) 455–462

Our results on the genotoxicity of the tested mycotoxins clearlyindicate that OTA and FB1 are genotoxic to HPBLs. In our study, OTA(4 lmol/l) and FB1 (20 lg/ml) induced DNA damage, which wasdetected via comet assay in HPBLs already after 1 h of exposure.Similarly to our results, OTA induced DNA damage assessed withthe comet assay in HPBLs and PK 15 cells (Šegvic Klaric, Daraboš,Rozgaj, Kašuba, & Pepeljnjak, 2010), human peripheral bloodmononuclear cells (HPBMCs) (Liu et al., 2012), HepG2 cells(Ehrlich et al., 2002a), and increased DNA fragmentation in Verocells (Bouslimi, Bouaziz, Ayed-Boussema, Hassen, & Bacha, 2008).The formation of MNi after OTA treatment was found in ovine sem-inal vesicle cells (Degan, Greber, Obrecht-Pflumio, & Dirheimer,1997), HepG2 cells (Ehrlich et al., 2002a), and in PK15 cells(Šegvic Klaric, Pepeljnjak, & Rozgaj 2008). Genotoxicity of OTAwas also confirmed on experimental animals (Zeljezic, Domijan,& Peraica, 2006). Although Dragan et al. (2001) stated that FB1

has no direct DNA reactivity, several in vitro and in vivo studiesdemonstrated that FB1 had the potential to damage DNA. FB1

increased parameters of the comet assay in rat primary astrocytes(Galvano et al., 2002), and in HepG2 cells (Ehrlich et al., 2002b),and formation of MNi in HepG2 cells (Ehrlich et al., 2002b) andPK15 cells (Šegvic Klaric et al., 2008). DNA damage was alsodetected in experimental animals after exposure to FB1 (Domijanet al., 2008; Domijan, Zeljezic, Milic, & Peraica 2007).

Data on the genotoxicity mechanisms of both mycotoxins arestill not fully understood but it is clear that at least some of thisdamage is due to oxidative stress. Oxidative DNA damage afterOTA treatment was detected in V79 cells, CV-1 cells, primary ratkidney cells (Kamp et al., 2005), HK-2 cells (Arbillaga et al., 2007)as well as in rat kidney cells (Domijan et al., 2006). Moreover, ina study on HK-2 cells, OTA-induced oxidative DNA damage wasreduced with N-acetyl-L-cysteine (NAC), an intracellular ROS scav-enger (Arbillaga et al., 2007). OTA has recently been confirmed tocause the formation of 8-OH-dG, the predominant form of oxida-tive DNA lesions, in HPBMCs. In that same study, NAC reducedDNA damage by OTA (detected with the comet assay) (Liu et al.,2012). All this clearly indicates that oxidative stress is implicatedin OTA genotoxicity. Similarly, several studies have demonstratedthat FB1 can generate oxidative DNA damage. FB1-induced forma-tion of 8-OH-dG in C6 cells, MEF cells (Mobio et al., 2003), andSMC cells (Mary et al., 2012), whereas in C6 cells DNA fragmenta-tion caused by FB1 was prevented by pre-incubation with vitamin E(Mobio et al., 2000). Oxidative DNA damage was also found in kid-ney cells of rats treated with FB1 (Domijan et al., 2006). However,in a study by Galvano et al. (2002), FB1 induced DNA damage inastrocytes without a simultaneous increase in ROS level, and there-fore these results could indicate that oxidative stress is notinvolved in DNA damage induced by FB1. Nevertheless, FB1 was

A.-M. Domijan et al. / Food Chemistry 170 (2015) 455–462 461

recently found to induce ROS formation on astrocytes andneuroblastoma cells (Domijan & Abramov, 2011). In this study,dihydroethidium (that detects mostly superoxide radical) wasused for ROS detection, and superoxide radicals are known to causeoxidative DNA damage (Misiaszek, Uvaydov, Crean, Geacintov, &Shafirovich, 2005).

Since literature data indicate that both of the mycotoxins stud-ied here can induce oxidative stress and oxidative DNA damage, inour next series of experiments we tested the ability of CHL as anon-toxic and established antioxidant to prevent cytotoxicity andDNA damage induced by OTA and FB1. To the best of our knowl-edge this is the first study that employed CHL to prevent toxicolog-ical effects of mycotoxins OTA and FB1. In these series ofexperiments, co-exposure to high (100 lg/ml) as well as low(10 lg/ml) CHL concentrations abolished the cytotoxic effect ofOTA and FB1 in HPBLs. Moreover, DNA damage induced by both,OTA and FB1 in HPBLs could be prevented by co-treatment withhigh (100 lg/ml) but also with low (10 lg/ml) CHL concentration.These results confirm that CHL acts as an antioxidant, indicatingthat oxidative stress is involved in OTA and FB1 cytotoxicity andgenotoxicity. It is important to emphasise that in our study CHLprevented cell death and DNA damage generated by H2O2 as well.In biological studies, H2O2 is widely used as an inducer of intracel-lular oxidative stress due to its ability (or its highly reactivederivate hydroxyl radical) to produce oxidative damage of macro-molecules including DNA (Duarte & Jones, 2007). Since in our studyCHL prevented cytotoxicity and genotoxicity of both mycotoxinssimilarly as H2O2, it is possible that OTA and FB1 have a similarmode of action as H2O2, e.g. through oxidative stress. Furthermore,both mycotoxins, OTA and FB1, were found to form predominantROS-induced DNA lesions, 8-OH-dG (Liu et al., 2012; Mary et al.,2012; Mobio et al., 2003). Interestingly, it is found that CHLreduces formation of 8-OH-dG induced by H2O2/UV (Park et al.,2003), and by cyclophosphamide or benzo[a]pyrene (Ibrahimet al., 2007). Therefore, it could be that CHL prevented oxidativestress and consequently oxidative DNA damage generated by bothmycotoxins confirming that DNA damage induced by OTA and FB1

is in fact oxidative DNA damage.Our results are in line with several recent studies that used dif-

ferent naturally occurring antioxidants to prevent, or completelydiminish cytotoxic and genotoxic effects of OTA. In a study byCosta, Utan, Cervellati, Speroni, and Guerra (2007) pre-treatmentwith two catechins, epigallocatechin gallate and epicatechin gal-late, well-known natural occurring antioxidants, prevented OTAinduced cytotoxicity and DNA fragmentation in LLC-PK1 cells.Similarly, pre-treatment with polyphenol enriched cocoa extractsuccessfully reduced cell death and ROS amounts in HepG2 cellstreated with OTA (Corcuera et al., 2012). Also in a study on rats,aqueous extract of Inula crithmoides (rich in flavonoides) protectedagainst oxidative stress and genotoxicity of OTA (Abdel-Wahhab,Abdel-Azim, & El-Nekeety, 2008). Such studies on the protectiveeffect of naturally occurring antioxidants against FB1 cytotoxicityand genotoxicity in available literature are lacking, although in ratstreated with FB1 antioxidant supplementation (consisted of coen-zyme Q10, L-carnitine, vitamin E, and selenium) decreased DNAfragmentation (Atroshi et al., 1999). Also, in the already mentionedstudy by Mobio et al. (2000), pre-treatment with vitamin E pre-vented FB1-induced DNA fragmentation.

In the present study, we have shown that CHL alone has noimpact on cell viability and genome damage and cannot induceLPO in the concentration range tested. Moreover, CHL preventedcell death, DNA damage and possibly carcinogenicity of OTA andFB1 indirectly by preventing oxidative stress, thus confirmingoxidative stress as the mechanism of their cytotoxicity and geno-toxicity. All that is in accordance with accumulating evidence that

CHL possesses antioxidant activity, which makes it a perfect candi-date for a novel antioxidant.

5. Conflict of interests

None.

Acknowledgments

This study received financial support of the Ministry of Science,Education and Sports of the Republic of Croatia (Grant Nos. 022-0222148-2125, 0022-0222148-2142 and 098-0982904-2907).

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