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Research Article

In vitro genotoxicity/antigenotoxicity testing of some conjugated linoleic acid

isomers using comet assay

Francesca Blasi1, Luca Dominici2, Massimo Moretti2, Milena Villarini2, Silvia Maurelli1,

Maria Stella Simonetti1, Pietro Damiani1 and Lina Cossignani1

1 Department of Agricultural Economics and Food Sciences (Section of Food Chemistry),

University of Perugia, Via San Costanzo, 06126 Perugia, Italy 2 Department of Medical-Surgical Specialties and Public Health (Section of Public Health),

University of Perugia, Via del Giochetto, 06122 Perugia, Italy

Running title: Genotoxicity/antigenotoxicity of conjugated linoleic acid

Correspondence: Professor Lina Cossignani, Department of Agricultural Economics and Food

Sciences (Section of Food Chemistry), University of Perugia, Via San Costanzo, 06126 Perugia,

Italy

E-mail: coslina@unipg.it

Phone: +39 075 5857959, Fax: +39 075 5857921

Keywords Conjugated linoleic acid / Genotoxicity / Antigenotoxicity / Comet assay

Abbreviations: Ag+-HPLC, silver-ion high performance liquid chromatography; CLA, conjugated

linoleic acid; CLAc, commercial CLA; CLAg, CLA synthesized from grapestone oil; DMSO,

dimethyl sulfoxide; EMS, ethylmethanesulfonate; FA, fatty acid; GIR, genotoxic inhibition rate;

HRGC, high resolution gas chromatography; LA, linoleic acid; LDH, lactate dehydrogenase;

MEM, minimum essential medium; PBS, phosphate-buffered saline; RGA, remaining genotoxic

activity; TriCLA, homogeneous CLA triacylglycerols; TriCLAc, homogeneous triacylglycerols

synthesized from CLAc; TriCLAg, homogeneous triacylglycerols synthesized from CLAg; 4NQO,

4-nitroquinoline N-oxide

© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Received: February 16, 2012 / Revised: March 19, 2012 / Accepted: April 16, 2012 DOI:10.1002/ejlt. 201200064

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Summary Recently conjugated linoleic acid (CLA) isomers have received considerable attention as potential

anti-cancer agents. The aim of the study was to assess the genotoxicity/antigenotoxicity in vitro of

linoleic acid (LA, c,c-C18:2, ∆-9), CLA isomer mixtures and homogeneous CLA triacylglycerols

(TriCLA) using the comet assay, to evaluate the effects on the extent of DNA injury in human

hepatoma (HepG2) cells. The study was carried out both on commercial CLA (CLAc) and on CLA

synthesized from grapestone oil (CLAg). The CLA isomer mixtures had different isomer profiles,

determined by silver-ion high performance liquid chromatography (Ag+-HPLC), in particular

CLAc was characterized by four main isomers (t8,c10; c9,t11; t10,c12; c11,t13), while CLAg

showed two main isomers (c9,t11; t10,c12). As regard antigenotoxicity testing, LA, TriCLAg and

above all TriCLAc were effective antigenotoxic compounds vs. ethylmethanesulfonate (EMS)

induced genotoxicity, while LA and CLAg were almost equally effective vs. 4-nitroquinoline N-

oxide (4NQO) induced DNA damage. Both TriCLAc and TriCLAg showed an increased

antigenotoxic activity toward EMS and a lower antigenotoxic activity toward 4NQO, with respect

to both CLAc and CLAg. The higher capability of CLAg with respect to CLAc in counteracting the

genotoxicity of 4NQO could be due to the different isomer CLA composition.

Practical applications

CLA isomers have shown many beneficial health effects both on animals and humans. They are

widely used in nutritional supplements, as CLA improves body composition by reducing fat

storage. In this regard it is very important to know, besides the chemical and analytical aspects, also

genotoxic and antigenotoxic effects of different CLA mixtures. To our best knowledge, few results

have been reported on CLA antigenotoxic properties by the comet assay, and no data could be

retrieved in the literature for TriCLA antigenotoxicity testing. The obtained results are interesting in

that they can increase the knowledge on particular fatty acids, recently used in commercial

supplements.

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1 Introduction

Conjugated linoleic acid (CLA) isomers have been reported to have biological activity [1].

However it should be considered that, since CLA is a family of geometric and positional isomers of

linoleic acid (LA, c,c-C18:2, ∆-9) in which the two contiguous double bonds can be in the cis (c) or

trans (t) configuration, the effects of CLA can be isomer dependent. Daily sources of CLA are meat

and dairy products of ruminant animals, in fact the c9,t11-CLA isomer is produced during microbial

hydrogenation in the rumen [2] or by ∆-9 desaturase action on vaccenic acid (t-C18:1, ∆-11) in

most mammalian tissues including man. As the CLA dietary intake is very limited, at present there

is a great interest in production of CLA enriched lipids [3, 4], as functional components in foods

and nutritional supplements.

Diet and other lifestyle factors are correlated with the development of certain types of cancers [5],

in particular many studies have taken into account the fat intake [6]. Dietary CLA reduced

tumorigenesis and metastasis of breast, prostate, skin and colon cancer in experimental animals [7].

Many studies have suggested that CLA isomers possess anti-carcinogenic properties, which exert

their effects at various stages of human cancer development [8]; generally it has been shown that

CLA modulated cell proliferation and apoptosis or altered phospholipid-associated fatty acid (FA)

metabolism and eicosanoid formation [9].

Kelley et al. [7] reviewed the literature regarding the effects of specific CLA isomers on

tumorigenesis in vivo and growth of tumor cell lines in vitro, examining the potential action

mechanisms, but no published reports were found on the effects of purified CLA isomers on human

cancer in vivo. The t10,c12-CLA isomer inhibited cell growth of colon, colorectal, and gastric

cancer cell lines in all studies when assessed, whereas c9,t11-CLA inhibited cell growth only in

some cases; generally t10,c12-CLA was more growth inhibitory than c9,t11-CLA. The strongest

inhibitory effect on human colon cancer cell lines (HT-29 and DLD-1) was shown by t9,t11-CLA,

followed by t10,c12-CLA, c9,c11-CLA and c9,t11-CLA, respectively [10]. A synthetic mixture of

CLA isomers (0.5-1.5 g/100 g, essentially formed from c9t11-CLA and t10c12-CLA, in similar

amount) inhibits chemically induced skin tumor promotion or mammary and colon tumorigenesis

[11]. c9t11-CLA and t10c12-CLA isomers decrease prostate cancer cell proliferation using different

molecular mechanisms; c9t11-CLA affects arachidonic acid metabolism, while t10c12-CLA, that is

the most effective isomer, modulates apoptosis and cell cycle [12].

At present, it remains still unclear whether a diet rich in CLA would be considered healthy as

putative detrimental effects of CLA have been also reported [13, 14]. Moreover, it was found that

mixed isomers of CLA may have equivocal effects, and these variable effects may be due to the

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different isomers present in CLA preparations [15-17]. For these reason, it’s very important to have

reliable and precise analytical techniques for identification and quantification of CLA isomers in

food and nutritional supplements [18, 19]. To this aim, in a previous research an improved high

resolution gas chromatography (HRGC) method for the separation and identification of c,t CLA

isomers has been developed [20].

The purpose of the present study was to extend the research on CLA isomers considering, besides

the chemical and analytical aspects, also genotoxic and antigenotoxic effects of different CLA

mixtures. In the present research the following compounds were subjected to in vitro comet assay:

LA, commercial CLA standard (CLAc), CLA synthesized from grapestone oil (CLAg),

homogeneous CLA triacylglycerols (TriCLAc and TriCLAg). The alkaline single-cell microgel-

electrophoresis (comet) assay [21, 22] was applied. This versatile and sensitive method is widely

used to measure DNA strand breakage. Upon electrophoresis, the DNA of lysed, agarose-embedded

cells extends towards the anode in a structure resembling a comet with the comet tail length or tail

fluorescence content reflecting the frequency of DNA strand breaks and hence DNA damage. The

comet assay is becoming a valuable tool in nutraceutical research, in fact recently, it has been used

for early genotoxicity testing of new pharmaceutical drug candidates because it is rapid and simple

to perform and requires only minute amounts of test substances [22]. Using HepG2 (human

hepatocellular liver carcinoma) cell line, the objectives of the present study were (i) to investigate

LA, CLAc, CLAg, TriCLAc and TriCLAg for cytotoxic effects; (ii) to study the test compounds for

genotoxicity; (iii) to determine if the test compounds possess antigenotoxic capabilities and protect

against EMS- or 4NQO-induced DNA damage.

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2 Materials and Methods

2.1 Chemicals and media

Grapestone oil was acquired in a local market. CLA methyl ester (mixture of cis- and trans-9,11-

and -10,12-octadecadienoic acid methyl esters, <1% LA methyl ester; catalog number O5632), LA

(≥99%), ethidium bromide, low- and normal-melting-point agarose, 4-nitroquinoline N-oxide

(4NQO), tris(hydroxymethyl)aminomethane (Tris-HCl), ethylmethanesulfonate (EMS), and Triton

X-100 were obtained from Sigma-Aldrich Srl, Milan, Italy. Dimethyl sulfoxide (DMSO), ethanol,

ethylenediamine tetraacetic acid disodium salt (Na2EDTA), ethylenediamine tetraacetic acid

tetrasodium salt (Na4EDTA), hydrochloric acid (HCl), sodium chloride (NaCl) and sodium

hydroxide (NaOH) were purchased from Carlo Erba Reagenti Srl, Milan, Italy. Gibco® Minimum

Essential Medium with Earle’s salts and L-glutamine (MEM), antibiotics (i.e. penicillin and

streptomycin), fetal bovine serum, sodium pyruvate, Dulbecco’s phosphate-buffered saline, pH 7.4

(PBS) and trypsin were purchased from Invitrogen Srl, Milan, Italy. Conventional microscope

slides and coverslips were supplied by Knittel-Glaser, Braunschweig, Germany. Lactate

dehydrogenase (LDH) cytotoxicity detection kit was purchased from Takara Bio Inc. (Kyoto,

Japan). Solvents for chromatography were high performance liquid chromatography (HPLC) grade,

purchased from Carlo Erba Reagenti Srl (Milan, Italy), or J.T. Baker, Mallinckrodt Baker B.V.,

(Deventer, Holland).

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2.2 Preparation of CLA isomers from grapestone oil

The alkaline hydrolysis of grapestone oil, the LA purification with urea and the alkaline

isomerization of LA to CLA isomers were carried out following the procedure reported in a

previous work, where sunflower oil was used [3]. Briefly, grapestone oil was reacted for 1 h

at 70 °C with potassium hydroxide, water, ethanol 95% and butylated hydroxytoluene. The

isolated FA were subjected to urea purification using FA, urea and methanol in the

following ratio, 1.0 g : 1.5 g : 4.5 mL; the mixture was maintained at 5 °C overnight and

then subjected to filtration under vacuum. This step was carried out two times. The purified

LA was subjected to the isomerization reaction to CLA isomers with n-butanol and

potassium hydroxide, heating to reflux for 12 h to 160 °C. The FA % composition of the

products of the three steps of the preparation of CLA isomers were determined after

derivatization and HRGC analysis, as reported in a successive Section.

2.3 Synthesis and isolation of TriCLA

The synthesis and the isolation of TriCLA were carried out as reported in a previous

research [4], both using CLAc and CLAg. A solution of glycerol anhydrous, CLA isomers, N,N’-

dicyclohexylcarbodiimide and 4-dimethylaminopyridine in dichloromethane was stirred at 14 °C

for 48 h. The products were purified by thin-layer chromatography (TLC) to isolate the TAG

fraction from trace amounts of reaction reagents and byproducts, using silica gel plates (SIL G-25,

20 × 20 cm, 0.25 mm, Macherey-Nagel, Germany). A mixture of petroleum ether–diethyl ether–

formic acid (70 : 30 : 1, v/v) was used as developing solvent for the TLC. The TAG fraction was

scraped off and extracted from silica with hexane-diethyl ether (50 : 50, v/v); the organic extracts

were pooled and the solvent was evaporated by nitrogen stream. The obtained Tri-CLA fraction

was weighed and the yield was calculated.

2.4 HRGC analysis

Before HRGC analysis, FA were methylated and grapestone oil was transesterified with methanolic

KOH as reported by Christie [23]. A DANI 1000DPC gas-chromatograph (Norwalk, CT, USA),

equipped with a split-splitless injector and with a flame-ionization detector was used. The fused

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silica WCOT capillary column CP-Select CB for fatty acid methyl esters (50 m x 0.25 mm i.d., 0.25

µm f.t.; Varian, Superchrom, Milan, Italy) was used. The chromatograms were acquired and

processed using Clarity integration software (DataApex Ltd., Prague, Czech Republic). The

chromatographic conditions were the following: the injector and detector temperatures were 250

°C; the oven temperature was 130 °C, then increased to 250 °C at 3 °C/min; the final temperature

was held for 10 min. The carrier gas (He) flow rate was 1 mL/min and the split ratio was 1:70.

2.5 Preparation of CLA butyl esters

An aliquot (50 µl) of CLAc or CLAg (2 mg/mL in CH2Cl2) was subjected to sulphuric acid

esterification by reaction with n-buthanol (100 µL) and sulfuric acid (10 µL). The reactions were

carried out in n-pentane (1mL), at 65 °C for 15 min to obtain butyl CLA esters. After the

esterification reaction, the samples were washed five times with water to remove acid residues.

Samples were anhydrificated with sodium sulfate anhydrous, dried, diluted in n-hexane (0.5 mL)

and analyzed by silver-ion HPLC (Ag+-HPLC).

2.6 Ag+-HPLC-UV analysis

The HPLC analysis was carried out using a Shimadzu LC-10AD VP Liquid Chromatography pump

(Kyoto, Japan), an UV 6000LP detector (Spectra System, Thermo Separation Products), operating

at 232 nm and a ChromSpher 5 Lipids analytical silver-impregnated column (5 µm, 250 mm x 4.6

mm i.d.; Chrompack-Varian, Milan, Italy). The chromatograms were acquired and processed using

Class-VP software (Shimadzu, Kyoto, Japan). The Rheodyne injector (7725i Model; Rohnert Park,

CA, USA) had a 20 µL injection loop. The HPLC analyses were performed with isocratic elution

(0.6 mL/min), using n-hexane and acetonitrile (0.1%) as mobile phase.

2.7 Genotoxicity/antigenotoxicity testing

2.7 a) Cell culture

HepG2 cells (ATCC HB 8065) were obtained from Istituto Zooprofilattico Sperimentale della

Lombardia e dell’Emilia Romagna “Bruno Ubertini”, Brescia, Italy. The cells were grown as

monolayer cultures in MEM supplemented with 10% (v/v) fetal bovine serum, 1 mM sodium

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pyruvate, 100 U/mL penicillin and 0.1 mg/mL streptomycin at 37ºC in a humidified atmosphere

containing 5% CO2. HepG2 cells were subcultured by dispersal with 0.05% trypsin in 0.02%

Na4EDTA for a contact time of 5 min and replated at a 1:2 dilution, which maintained cells in the

exponential growth phase. All experiments were performed on HepG2 cells at passages between

101 and 108. Cell stocks were routinely frozen and stored in liquid N2.

2.7 b) Cytotoxicity testing: LDH leakage assay

LA, CLAc, CLAg, TriCLAc and TriCLAg were dissolved in DMSO or ethanol for stock solutions

(0.74 mg/mL) and then stored at +4 °C until use. The highest test concentration (i.e. 7.4 µg/ml) in

the LDH cytotoxicity assay was chosen on the basis of data reported in the literature [24] and

results obtained in a preliminary approach using the tetrazolium MTT viability test and the neutral

red incorporation assay [25]. Cytotoxicity induced by the test compounds was assessed by LDH

release into the culture medium. For cytotoxicity testing, HepG2 cells (1.25×105/well) were seeded

in 96-well tissue culture plates (Orange Scientific, Braine-l’Alleud, Belgium) and were allowed to

attach for 24 h before treatment with extracts. Protocols for cytotoxicity/genotoxicity testing (i.e. 4

h treatment period) were based on observations reported by Sasaki et al. [26]; the medium was then

removed and replaced by fresh MEM containing a range of extracts concentrations (from 0.0 to 7.4

µg/mL) by twofold serial dilutions. The highest concentrations considered in the LDH leakage

assay corresponded to the maximum possible volume of solvent vehicle (i.e. 1%DMSO or ethanol)

that could be possible to add to cell cultures. The cells were then treated for 4 h. Following the

treatment the plates were centrifuged at 250×g for 10 min in order to obtain a cell free supernatant

and the culture medium was aspirated. The activity of LDH in the medium was determined using

Takara's LDH Cytotoxicity Detection Kit according to manufacturer's instructions. LDH leakage

assay is based on the conversion of lactate to pyruvate in the presence of LDH with parallel

reduction of NAD+ on a coupled reaction which converts a yellow tetrazolium salt into a red,

formazan-class dye. The formation of formazan from the above reaction results in a change in

absorbance at 492 nm. Absorbance was recorded using a Tecan Sunrise microplate reader (Tecan

Italia Srl, Milan, Italy). All tests were performed at least in triplicate and repeated twice to calculate

the amount of LDH released. The results of LDH leakage assay determined the choice of

concentration of test compounds to be evaluated afterwards, in the genotoxicity and

antigenotoxicity protocols.

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2.7 c) Genotoxicity/antigenotoxicity testing: comet assay

Treatments for genotoxicity/antigenotoxicity testing were carried out in HepG2 cells subcultured in

6-well tissue culture plates (Orange Scientific, Belgium) inoculated with 5 mL of complete MEM

containing 5×105 cells per well. The overall culture length was 52 h throughout the experiments.

The co-exposure protocol for antigentotoxicity testing was set up on the basis of a previously

published procedure [27]. After seeding for 48 h, the culture medium was replaced by fresh

complete MEM containing 7.4 µg/mL of test compounds. The cells were then incubated further for

4 h according to the following scheme: (a) LA, CLAc, CLAg, TriCLAc or TriCLAg, to check the

absence of genotoxicity of the test compounds; (b1) model mutagen EMS (2.8 mM); (b2) model

mutagen 4NQO (1.5 µM); (c1) simultaneous exposure to EMS and test compounds; (c2)

simultaneous exposure to 4NQO and test compounds. Negative controls (i.e. medium, 1% ethanol

and 1% DMSO) were also included.

At the end of treatments, the cells were washed twice with 5 mL ice-cold PBS, pH 7.4 and detached

with 300 µL of 0.05% trypsin in 0.02% Na2EDTA. After 3 min, trypsinization was stopped by

adding 700 µL complete culture medium. Cells were then collected by centrifugation (70×g, 8 min,

+4 °C). Each treatment protocol was carried out in duplicate.

Immediately after the exposure, HepG2 cells were processed in the comet assay under alkaline

conditions (alkaline unwinding/alkaline electrophoresis, pH > 13), basically following the original

procedure [21, 22], with minor modifications [27, 28]. Briefly, cell pellets were gently resuspended

in low melting point agarose (0.7% in Ca++/Mg++-free PBS, w/v) maintained at 37 °C. Then, 65 µL

of cell suspension in agarose were rapidly layered onto pre-coated (1% normal melting point

agarose in Ca++/Mg++-free PBS) conventional 26×76 mm microscope slides and covered with a

coverslip. After brief agarose solidification at +4 °C, the coverslips were removed and the cell

containing microgels covered with a top layer (75 µL) of 0.7% low melting point agarose. The

coverslips were further removed and the slides immersed in cold, freshly prepared lysing solution

(2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris-HCl and NaOH to pH 10; 1% Triton X-100 added

just before use) for at least 60 min at +4ºC to obtain the lysis of cellular and nuclear membranes of

the cells embedded in agarose microgels. The slides were then drained and placed in a horizontal

electrophoresis box (HU20, Scie-Plas, Cambridge, UK) filled with a freshly prepared

electrophoresis solution (10 mM Na4EDTA, 300 mM NaOH; pH > 13). After 20 min of pre-

electrophoresis to allow DNA unwinding and expression of alkali-labile damage, electrophoresis

runs were performed in an ice bath for 20 min by applying an electric field of 25V (1 V/cm) and

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adjusting the current to 300 mA (Power Supply PS250, Hybaid, Chesterfield, MO, USA). The

microgels were then neutralized with 0.4 M Tris-HCl buffer (pH 7.5). For preservation, the slides

were dehydrated in 70% ethanol (10 min), allowed to air-dry and store in slide boxes at room

temperature until ready to stain and analyze. All the steps of the comet assay were conducted in

yellow light to prevent the occurrence of additional DNA damage.

Immediately before scoring, the air-dried slides were stained with 65 µl of ethidium bromide (20

µg/mL) and was covered with a coverslip. The comets in each microgel were analyzed (blind), at

500× magnification, with an epifluorescent microscope (BX41, Olympus, Japan) under a 100 W

high-pressure mercury lamp (HSH-1030-L, Ushio, Japan), using appropriate optical filters

(excitation filter 510-550 nm and emission filter 590 nm). The microscope, equipped with a high

sensitivity black and white CCD camera (PE2020, Pulnix, UK), was connected to a computerized

analysis system (“Comet Assay III”, Perceptive Instruments, UK) that acquires images, computes

the integrated intensity profile for each cell, estimates the comet cell components, head and tail, and

evaluates a range of derived parameters. These include: tail length (measured from the head centre,

expressed in µm), tail intensity (percent of fluorescence in the comet tail), and tail moment, a

composite parameter in which the migration distance and the amount of migrated DNA (by analogy

with the mechanical term) are expressed as a single value. The mean tail intensity percentage of

DNA migrated in the comet tail was used as a measure of DNA damage. A total of 100 randomly

selected comets (50 cells/replicate slides) were evaluated for each experimental point. For each

independent test, the median tail intensity of 50 cells/slide was assessed and the average of two

replicated slides was calculated as summary statistic [29].

2.8 Statistical analysis

Cytotoxicity and genotoxicity/antigenotoxicity data are presented as the mean ± standard error of

the mean (S.E.M.) of at least triplicate tests. Remaining 4NQO- or EMS-induced genotoxic activity

(RGA%) was calculated according to the following formula [30]:

100(%) ×−−

=CBCARGA

whereas genotoxic inhibition rate (GIR%) was expressed as [31]:

( ) 100100

(%)1% ×⎥⎦

⎤⎢⎣

⎡⎟⎠⎞

⎜⎝⎛−=

RGAGIR

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where A corresponds to the extent of DNA damage observed in cells subjected to the antigenotoxic

treatment (co-exposure to CLA extracts plus a known mutagen), B corresponds to DNA damage

observed in 4NQO or EMS exposed cells and C corresponds to DNA damage extent in the negative

control. Statistical significance was tested by one-way analysis of variance (ANOVA) followed by

LSD (least significant difference) post hoc analysis at significance level p ≤ 0.05.

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3. Results

3.1 CLA samples: preparation and analysis

The used vegetable oil, grapestone oil, simply available and low cost, was subjected initially to

alkaline hydrolysis, then to LA purification with urea and finally to alkaline isomerization of LA to

CLA isomers; the FA% composition of the products of the three steps of the preparation of CLA

isomers, reported in Table 1, was determined after methylation and HRGC analysis. In respect to

sunflower oil, used for CLA production in a previous research [3], grapestone oil is more rich in LA

(69.0% respect to 57.4%) and the purification with urea was carried out only two times and not

three times as in a previous work [4]. The final mixture had 98.4% of CLA with the following

isomer distribution: c9,t11 (47.4% isomer/total CLA), t10,c12 (47.2% isomer/total CLA), t,t

isomers (2.0% isomer/total CLA) and other CLA isomers (1.8% isomer/total CLA).

As the possible CLA isomers are numerous and some isomers are difficult to separate with the

common analysis methods, a deeper chromatographic analysis to verify the real isomer profile of

both CLAc and CLAg was carried out. In fact, initially the HRGC analysis of fatty acid methyl

esters was carried out, then the isomer profiles were confirmed by Ag+-HPLC analysis; the two

mixtures, even if both containing the two principal CLA isomers (c9,t11; t10,c12) showed a

different composition.

The derivatization phase of CLA as butyl esters was carried out to improve the separation of the

various isomers if compared with methyl esters [32]. In Figure 1 (A and B) Ag+-HPLC profiles of

CLA butyl esters from CLAc and CLAg were respectively reported. In the CLAc, differently from

CLA synthesized from grapestone oil, there were four isomers (c11,t13; t10,c12; c9,t11; t8,c10), in

addition to small amounts of t,t and c,c isomers. The CLA isomer identification was carried out

using HRGC coupled with a mass spectrometer, after compound derivatization as Dies-Alder

adducts with 4-methyl-1,2,4-triazolin-3,5-dione [20]. The CLA mixtures, CLAc and CLAg, were

also used to synthesize Tri-CLA; the used chemical approach has shown many advantages, such as

simplicity, speed, low cost and very good yield (about 80%). Tri-CLA had the same fatty acid

composition of the CLA mixture used in the synthesis.

3.2 Cytotoxicity (LDH leakage assay)

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LDH leakage in the medium was determined in order to evaluate injuries in HepG2 cells caused by

test compounds, namely LA, CLAc, CLAg, TriCLAc and TriCLAg. In vitro exposure to test

compounds did not induced any significant cytotoxicity and the reduction in cell viability was

always lower than 5%. Therefore, the highest tested concentration (i.e. 7.4 µg/mL) was chosen for

further evaluation in the genotoxicity and antigenotoxicity protocols.

3.3 Genotoxicity/antigenotoxicity (Comet assay)

Table 2 shows the extent of DNA damage and RGA (%) in HepG2 cells exposed according to

protocols for genotoxicity (i.e. exposure to LA, CLA and TriCLA) or antigenotoxicity testing (i.e.

simultaneous exposure to EMS or 4NQO and test compounds).

The test compounds, namely LA, CLAc, CLAg, TriCLAc and TriCLAg did not induce any DNA

strand breakage at the concentration tested, with mean tail intensity values obtained being similar to

those for the negative control. EMS and 4NQO used as model mutagens demonstrated the

sensitivity of the comet assay and yielded clear positive responses at the used concentrations. As

regard antigenotoxicity testing, simultaneous treatment of cells with EMS and the testing

compounds showed a statistically significant reduction in the extent of DNA damage for cultures

treated with LA, TriCLAc and TriCLAg (vs. EMS treated cells; LSD post hoc test). TriCLAc was

the most effective antigenotoxic compound vs. EMS induced genotoxicity. Whereas simultaneous

treatment of cells with 4NQO and the testing compounds showed a statistically significant

reduction in the extent of DNA damage for cultures treated with LA and CLAg (vs. 4NQO treated

cells; LSD post hoc test), being the testing compounds almost equally effective vs. 4NQO induced

DNA damage.

GIR (%) values are summarized in Figure 2. LA showed a moderate antigenotoxic activity toward

EMS, with GIR (%) of 25.08, and a more evident effect toward 4NQO-induced DNA strand

breakage, with GIR (%) corresponding to 47.60. CLAc and CLAg were moderately to highly

effective in reducing 4NQO-induced genotoxicity, with GIR (%) values of 33.61 and 51.93,

respectively, but their effects toward EMS were instead very low, with GIR (%) values of 10.87 and

12.67, respectively. On the contrary, TriCLAc showed an high antigenotoxic effects toward EMS

and a very low or absent activity toward 4NQO, with GIR (%) corresponding to 41.86 and 11.20,

respectively. TriCLAg was nearly equally effective toward EMS and 4NQO, with GIR (%) values

corresponding to 26.40 and 34.20, respectively.

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4 Discussion

In this study, in vitro genotoxicity/antigenotoxicity testing of LA, CLA and TriCLA was carried

applying the alkaline (alkaline unwinding/alkaline electrophoresis; pH > 13) comet assay. Besides

LA, two CLA mixtures (i.e. CLAc, commercial standard and CLAg, synthesized from grapestone

oil) and two homogeneous triacylglycerols (i.e. TriCLAc and TriCLAg, synthetized from CLAc

and CLAg, respectively) were tested. In the present approach, genotoxicity/antigenotoxicity testing

has been performed on the HepG2 cell line. HepG2 cells retain many of the morphological

characteristics of the liver parenchymal cells from which they originate [33] and present

endogenous bioactivation capacity expressing phase I and phase II enzymes involved in the

activation and/or detoxification of xenobiotics [34, 35]. Thus, the metabolically competent HepG2

cell line is considered to be an excellent model to investigate in vitro the toxicity of drugs, being

this model closer to the in vivo situation as the addition of an exogenous metabolic activation

system (i.e. S9-mix) [36, 37].

For antigenotoxicity testing, we assessed LA, CLA and TriCLA for their protective properties

against the prototypical genotoxic agents, EMS and 4NQO, direct mutagens, widely used in

genotoxicity studies.

4NQO, which binds covalently to DNA, is also an oxidative mutagen that undergoes redox

recycling to generate superoxide radical and other reactive oxygen species [38]. EMS is an

alkylating agent and can produce DNA adducts at virtually every purine or pyrimidine nucleophilic

site (mainly at the O6 position of the guanine base) and at the phosphotriester backbone of the DNA

helix [39]. Therefore, DNA strand-breakage induced by 4NQO or EMS potentially represents a

wide spectrum of DNA lesions (e.g. DNA adducts, apurinic/apirimidinic sites, incomplete excision

repair sites) detectable by the comet assay.

DNA-protective substances can be classified as desmutagens or bioantimutagens [40], being

desmutagenic agent compounds that interact directly with mutagens, or their precursors, in order to

inactivate them. These agents interact with a mutagenic compound in an irreversible way,

inactivating it chemically, usually through a direct link. By applying the simultaneous protocol, the

test compounds showed an antigenotoxic activity toward the model mutagens EMS and 4NQO

likely acting as a desmutagenic agent.

The conjugated diene system of CLA led to a increased capability of counteracting the

genotoxicity of 4NQO, as compared to LA. In particular the higher activity of CLAg with respect to

CLAc could be due to the different isomer CLA composition, since CLAg mainly contains c9,t11

and t10,c12 isomers. CLAc and CLAg showed, with respect to LA, lower antigenotoxic activity

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toward EMS. The reason of the opposite behavior of CLA isomers and LA toward the two mutagen

models could be due to the different mechanism of molecular interaction of the conjugated or

methylene-interrupted diene system with EMS and 4NQO.

Considering the esterified CLA isomers, both TriCLAc and TriCLAg showed an increased

antigenotoxic activity toward EMS and a lower antigenotoxic activity toward 4NQO, with respect

to both CLAc and CLAg. The higher effect showed by TriCLAc toward EMS, as compared to

TriCLAg, might be accountable to c11,t13 and t8,c10 isomers that are not present in TriCLAg;

whereas the absence of antigenotoxic activity toward 4NQO might be ascribed to a reduced amount

of c9,t11 and t10,c12 isomers that are the only CLA isomers present in TriCLAg. These results

confirm that CLA effects depend on the isomer profile and therefore reliable analytical techniques

for CLA isomer separation are extremely important.

To our best knowledge, only in one recent paper [41] CLA have been tested for

antigenotoxic properties by the comet assay, no data could be retrieved in the literature for TriCLA

antigenotoxicity testing. In the mentioned study, the authors did not find any genoprotective effects

of CLA against model mutagens (Caco-2 human colon cells). On the contrary, the tested CLA

isomers were reported to enhance the genotoxic effects of H2O2.

In conclusion, the results of our study showed that LA, CLA and TriCLA possess antigenotoxic

properties, evaluated in vitro using HepG2 cells, mainly acting as desmutagenic agents, and that

qualitative/quantitative effects strongly depend on isomers present in the mixture.

The authors have declared no conflicts of interest.

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Figure legends

Figure 1. Ag+-HPLC profile of CLA isomer mixtures derivatized as butyl esters: [A] CLAc,

commercial standard; [B] CLAg, grapestone oil synthetized CLA.

Figure 2. Genotoxic inhibition rates (GIR%) of LA, CLAc, CLAg, TriCLAc or TriCLAg (7.4

µg/mL) toward model mutagens EMS (2.8 mM) or 4NQO (1.5 µM). Results expressed as the mean

± SEM of at least three independent experiments.

Statistical significance (p ≤ 0.05, Student’s t-test): $ vs. TriCLAc + EMS; # vs. TriCLAc + 4NQO.

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Fig. 1A, Blasi et al.

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Fig. 1B, Blasi et al.

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Fig. 2, Blasi et al.

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Table 1. Fatty acid composition of grapestone oil after alkaline hydrolysis reaction (a), grapestone

oil after two purification processes with urea (b), CLA obtained by isomerization of the LA (c).

Results expressed as mean ± SD (n=3)

a (% mol) b (% mol) c (% mol)

Fatty acids Mean ± SD Mean ± SD Mean ± SD

C14:0 0.1 ± 0.0 - -

C16:0 7.4 ± 0.3 - -

C16:1 (n-9 + n-7) 0.1 ± 0.0 - -

C18:0 4.0 ± 0.2 - -

C18:1 (n-9 + n-7) 18.7 ± 0.3 1.5 ± 0.0 1.4 ± 0.0

C18:2 (n-6) 69.0 ± 0.7 98.0 ± 0.8 0.2 ± 0.0

C18:3 (n-3) 0.4 ± 0.0 0.5 ± 0.0 -

C20:0 0.2 ± 0.0 - -

C20:1 0.2 ± 0.0 - -

c9,t11-CLA - - 47.4 ± 0.5

10t,12c-CLA - - 47.2 ± 0.5

t,t-CLA - - 2.0 ± 0.1

Other CLA isomers - - 1.8 ± 0.0

Total CLA - - 98.4

- not detected

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Table 2. DNA damage in HepG2 liver cells treated in vitro with LA, CLAc, CLAg, TriCLAc or

TriCLAg alone (7.4 µg/mL) (genotoxicity testing) or subjected to simultaneous exposure to test

compounds and model mutagens EMS (2.8 mM) or 4NQO (1.5 µM) (antigenotoxicity testing).

Remaining genotoxic activity, RGA (%), is indicated between brackets. Results expressed as mean

± S.E.M. (n=3)

LA CLAc CLAg TriCLAc TriCLAg

Mean ± S.E.M. Mean ± S.E.M. Mean ± S.E.M. Mean ± S.E.M. Mean ± S.E.M.

Genotoxicity assessment

2.90 ± 0.47 2.68 ± 0.34 2.29 ± 0.37 3.50 ± 1.56 2.74 ± 0.81

Antigenotoxicity assessment

vs. EMS 17.84 ± 0.83* (74.92 ± 4.58)

20.62 ± 0.66 (89.13 ± 6.32)

20.43 ± 1.75 (87.33 ± 5.48)

14.57 ± 1.40* (58.14 ± 4.67)

17.62 ± 1.25* (73.60 ± 5.17)

vs. 4NQO 18.30 ± 2.91* (52.40 ± 3.81)

20.90 ± 4.87 (66.39 ± 7.61)

17.96 ± 3.09* (48.07 ± 6.50)

28.39 ± 7.86 (88.80 ± 9.39)

23.89 ± 4.55 (65.80 ± 9.63)

Statistical significance (p ≤ 0.05, Student’s t-test): * vs. corresponding positive control (model

mutagen alone, EMS or 4NQO)

Negative controls: 1% DMSO 2.29 ± 0.39; 1% Ethanol 2.38 ± 0.49

Positive controls: 2.8 mM EMS 22.89 ± 0.78; 1.5 µM 4NQO 33.66 ± 6.04