Toxicology in Vitro xxx (2014) xxx–xxx

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Toxicology in Vitro

journal homepage: www.elsevier .com/locate / toxinvi t

Evaluation of cytogenetic and DNA damage in human lymphocytestreated with adrenaline in vitro

http://dx.doi.org/10.1016/j.tiv.2014.08.0010887-2333/� 2014 Elsevier Ltd. All rights reserved.

Abbreviations: CAT, catalase; CBPI, cytokinesis-block proliferation index; EPI,adrenaline; FPG, fluorescence-plus-Giemsa; MN, micronucleus; ROS, reactiveoxygen species; SCE, sister chromatid exchange.⇑ Corresponding author. Address: Department of Biology, Faculty of Veterinary

Medicine, University of Belgrade, 11000 Belgrade, Serbia. Tel.: +381 11 26 58 894;fax: +381 11 2685 936.

E-mail address: ndjelic@sbb.rs (N. Djelic).

Please cite this article in press as: Djelic, N., et al. Evaluation of cytogenetic and DNA damage in human lymphocytes treated with adrenaline in vitricol. in Vitro (2014), http://dx.doi.org/10.1016/j.tiv.2014.08.001

Ninoslav Djelic a,⇑, Milena Radakovic a, Biljana Spremo-Potparevic b, Lada Zivkovic b, Vladan Bajic c,Jevrosima Stevanovic a, Zoran Stanimirovic a

a Department of Biology, Faculty of Veterinary Medicine, University of Belgrade, Oslobodjenja Blvd. 18, Belgrade, Serbiab Department of Biology and Human Genetics, Institute of Physiology, Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, Belgrade, Serbiac Institute for Pharmaceutical Research and Development, Galenika, Pasterova 2, 11000 Belgrade, Serbia

a r t i c l e i n f o

Article history:Received 17 December 2013Accepted 10 August 2014Available online xxxx

Keywords:Sister chromatid exchangeMicronucleiDNA damageOxidative stress

a b s t r a c t

Catechol groups can be involved in redox cycling accompanied by generation of reactive oxygen species(ROS) which may lead to oxidative damage of cellular macromolecules including DNA. The objective ofthis investigation was to evaluate possible genotoxic effects of a natural catecholamine adrenaline incultured human lymphocytes using cytogenetic (sister chromatid exchange and micronuclei) and the sin-gle cell gel electrophoresis (Comet) assay. In cytogenetic tests, six experimental concentrations of adren-aline were used in a range from 0.01–500 lM. There were no indications of genotoxic effects ofadrenaline in sister chromatid exchange and micronucleus tests. However, at four highest concentrationsof adrenaline (5 lM, 50 lM, 150 lM and 300 lM) we observed a decreased mitotic index and cell-cycledelay. In addition, in the Comet assay we used adrenaline in a range from 0.0005–500 lM, at two treat-ment times: 15 min or 60 min. In contrast to cytogenetic analysis, there was a dose-dependent increase ofDNA damage detected in the Comet assay. These effects were significantly reduced by concomitant treat-ment with quercetin or catalase. Therefore, the obtained results indicate that adrenaline may exhibitgenotoxic effects in cultured human lymphocytes, most likely due to production of reactive oxygenspecies.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Adrenaline has been called a hormone of ‘‘fight or flight’’ due toits immediate release under the influence of various stressors.Actually, there is increasing evidence that stress hormones andneurotransmitters may represent a link between the immune,endocrine and central nervous systems (Kin and Sanders, 2006).The physiological effects of adrenaline prepare the body forextraordinary physical and mental exertion. It is well establishedthat, at a molecular level, specific binding of adrenaline to mem-brane adrenergic receptors coupled to heterotrimeric glycopro-teins initiates a cascade of biochemical responses inside the cellleading to change of cellular activity (Molenaar et al., 2007).

It has been demonstrated that adrenaline has the highest affin-ity for b-adrenoceptors. Binding of adrenaline to these receptorsactivates Gs protein to stimulate adenylate cyclase (Namiecinskaet al., 2006). Pharmacological and stereochemical investigationsrevealed that the aromatic catechol moiety of adrenaline is essen-tial for its agonist activity (Liapakis et al., 2004).

Although the molecular mechanisms of the effects of adrenalineand other catecholamines in various mammalian tissues are stud-ied in detail, possible genotoxic and mutagenic effects are notinvestigated enough. Interestingly, it has been revealed that dopa-mine induces DNA strand breaks in human skin fibroblasts andgene mutations in mouse lymphoma cells (Moldeus et al., 1983).However, dopamine was not genotoxic in Salmonella/mamma-lian-microsome mutagenicity test, sex linked recessive lethal testin Drosophila melanogaster, sister-chromatid exchange (SCE) testin human lymphocytes and micronucleus assay in mouse and rat(Moldeus et al., 1983). In addition to these results, mutageniceffects of various catecholamines (including adrenaline) wererevealed on mouse lymphoma L5178Y cell thymidine kinase locus(McGregor et al., 1988). Most likely, the molecular mechanism

o. Tox-

2 N. Djelic et al. / Toxicology in Vitro xxx (2014) xxx–xxx

underlying the genotoxic effects of catecholamines in mouse lym-phoma cells implies creation of superoxide anion (Moldeus et al.,1983; McGregor et al., 1988). In addition, there are interestingexperimental findings on plasmid DNA that catechol derivatives,including adrenaline, induce DNA strand breakage by ferryl spe-cies, whereas the induction of 8-hydroxyguanine (8OHG) is dueto hydroxyl radical (OH�) (Miura et al., 2000).

More recent studies have shown that noradrenaline induces pri-mary DNA damage in the Comet assay on human lymphocytes(Djelic and Anderson, 2003) and sperm (Dobrzynska et al., 2004).Since the antioxidant enzyme catalase reduces the effect of nor-adrenaline in the Comet assay, it has been concluded that theDNA damage resulted mainly from reactive oxygen species (ROS).This result is in accordance with experimental findings that adren-aline and other catecholamines can be involved in redox cyclingunder the influence of the superoxide anion (Genova et al., 2006).Namely, adrenaline may undergo oxidation and cyclisation to adre-nochrome which is reduced to the corresponding semiquinone byNADPH in liver microsomes and by NADH and mitochondrial com-plex I in bovine heart submitochondrial particles. Finally, the sem-iquinone can react with molecular oxygen therefore producingsuperoxide anion and regenerating adrenochrome (Genova et al.,2006).

It is worth noting that superoxide anion may induce chromo-some breakage and SCEs in human lymphocytes in vitro(M’Bemba-Meka et al., 2007). Considering that literature data con-cerning the genotoxic and mutagenic effects of adrenaline areincomplete and equivocal, the aim of the present study was toevaluate genotoxic effects of a wide range of concentrations ofadrenaline using two cytogenetic endpoints – SCEs and micronu-clei, as well as evaluation of primary DNA damage in the single cellgel electrophoresis (Comet) assay.

2. Materials and methods

2.1. Blood samples, culture conditions and treatment

The study was approved by the local Medical Ethics Committee,performed in accordance with Declaration of Helsinki, andinformed donor consent was also obtained. Human peripheralblood lymphocyte cultures were set up according to a standardprotocol (Rooney and Czepulkowski, 1986). Briefly, heparinisedwhole blood samples (0.8 mL) obtained by venipuncture fromthree healthy men under 30 years of age, were added to vials with9.2 mL of RPMI 1640 (Gibco, Grand Island, NY), supplemented with15% of heat-inactivated foetal calf serum (Gibco, Eggenstein,Germany), 1% of antibiotics (penicillin and streptomycin, Galenika,Belgrade, Serbia) and 5 lg/ml of phytohaemagglutinin (MurexDiagnostics Ltd., Dartford, England). Duplicate cultures from eachdonor were incubated in the dark for 72 h at 37 �C.

In cytogenetic tests, exactly 48 h after the beginning of incuba-tion adrenaline (Adrenaline HCl, Jugoremedija, Zrenjanin, Serbia,CAS No. 51-43-4) was added to the cultivation vials in amountsto obtain six final experimental concentrations (range from0.01 lM to 300 lM). In order to determine experimental concen-trations of adrenaline we consulted textbooks on pharmacology(Reynolds, 1996; Varagic and Miloševic, 2008). Thus, in cytogenetictests the concentration of 1 lM corresponds to average therapeuticdose of adrenaline in human medicine and 5 lM to the maximaltherapeutic dose. The concentration of 0.01 lM is comparable toadrenaline plasma level during intensive stress in humans(Zouhal et al., 2008), while 50, 150 and 300 lM represent 10-fold,30-fold and 60-fold higher doses than maximal therapeutic dose.The acetone solution of N-methyl-N0-nitro-N-nitrosoguanidine(MNNG) (CAS No. 70-25-7, Sigma Chemical Co., St. Louis, MO) at

Please cite this article in press as: Djelic, N., et al. Evaluation of cytogenetic andicol. in Vitro (2014), http://dx.doi.org/10.1016/j.tiv.2014.08.001

a final concentration of 1 lM was the positive control. The negativecontrol was prepared as placebo in Jugoremedija (Zrenjanin) as asolution of all compounds in adrenaline-HCl except an active one– adrenaline.

In the Comet assay, after the isolation on ficoll gradient, lym-phocytes resuspended in RPMI 1640 were treated with appropriateconcentrations of adrenaline from 0.0005 lM (comparable tophysiological blood level of adrenaline in humans) to 500 lM, for15 min or 1 h, whereas H2O2 as the positive control, was addedto Eppendorf tubes to final concentration of 100 lM. Finally, theconcentration of 300 lM of adrenaline was chosen for furtherco-treatment with catalase or quercetin.

2.2. Sister chromatid exchange (SCE) test

In the SCE test, in order to obtain visible SCEs 5-bromo-20-deoxyuridine (BrdUrd, Sigma Chemical Co., St. Louis, MO, USA, finalconcentration 20 lM) was added in each culture one hour after thebeginning of incubation. Two hours before harvesting colcemid(Ciba, Basel, Switzerland) was added to the cultures to achieve a finalconcentration of 0.5 lg/mL. After hypotonic treatment (0.075 MKCl) followed by three repetitive cycles of fixation in methanol/acetic acid solution (3:1, v/v), centrifugation, and resuspension,the cell suspension was dropped on chilled, grease-free codedmicroscopic slides, air-dried over flame, aged, and then differentiallystained according to the fluorescence-plus-Giemsa (FPG) procedure(Perry and Wolff, 1974). For each donor, 60 well-spread mitoses (30mitoses per duplicate culture) were scored, and the values obtainedwere calculated as SCEs per cell. At least 1000 cells per culture werescored in order to calculate the mitotic index (MI). With an aim tocalculate the cell proliferation index (CPI), at least 500 metaphasesper culture were examined to determine the proportion of cells inthe first, second and third mitotic division (Istifli and Topaktas,2013).

2.3. Micronucleus assay

In the cytokinesis-block micronucleus assay cytochalasin-B(Sigma Chemical Co., St. Louis, MO, USA, final concentration 6 lg/mL) was added 44 h after the beginning of incubation. After 72 h,the cells were gently rinsed in serum free RPMI 1640 medium, thenexposed to short hypotonic treatment (3 min) with 0.075 M KCl atroom temperature. After standard procedure of preparation (threecycles in methanol-acetic acid solution, 3:1, v/v) the staining wasperformed in 2% Giemsa (Merck, Darmstadt, Germany) solutionin Gurr buffer (pH = 6.8). At least 1000 binucleated cells per donorwere analysed for the frequency of MN at 400� magnification,according to the criteria described by Fenech (1993). The cytokine-sis-block proliferation index (CBPI) was calculated according toSurallés et al. (1994).

2.4. Isolation of lymphocytes

Heparinised blood samples (4 mL) were obtained by venepunc-ture from three healthy male donors under 30 years of age. Lym-phocytes were isolated from whole blood with Ficoll-Paquemedium and centrifuged at 1900 g 15 min. The lymphocytesforming a layer were directly above Ficoll-Paque. The isolated lym-phocytes were washed twice in RPMI 1640 medium, each wash wasfollowed by a centrifugation 10 min at 1800 g. Finally, the superna-tant was removed as carefully as possible without disturbing thepellet. An aliquot of 1 ml of RPMI 1640 was added and the pelletwas resuspended. A manual cell count and an estimate of cellviability were performed using the Trypan blue exclusion test.

DNA damage in human lymphocytes treated with adrenaline in vitro. Tox-

N. Djelic et al. / Toxicology in Vitro xxx (2014) xxx–xxx 3

2.5. The Comet assay

Before each experiment, microscope slides were precoated with1% normal melting point agarose (Sigma, St. Louis, MO) in doubledistilled water and left at room temperature to allow the agaroseto dry. The alkaline single cell gel electrophoresis (Comet) assaywas performed on isolated human peripheral blood lymphocytesaccording to the method of Singh et al. (1988) with slight modifica-tions (Tice et al., 2000; Speit and Rothfuss, 2012). The cell suspensionin PBS was treated with adrenaline at concentrations from0.0005 lM to 500 lM for 15 min or 1 h at 37 �C. The cell viabilitywas checked by Trypan blue exclusion (Pool-Zobel et al., 1993). Aftertreatment, the cell suspensions were centrifuged at 2000 rpm for5 min, and the cell pellet was mixed with an equal amount of 1%low melting point agarose (Sigma), rapidly placed on precoatedmicroscopic slides covered with a coverslip and allowed to solidifyfor 5 min at 4 �C. Then the coverslips were gently removed, andthe 0.5% agarose was placed, covered with a coverslip, left for5 min at 4 �C, then the coverslip was removed and the slides wereplaced overnight in a lysis solution (2.5 M NaCl, 100 mM EDTA,10 mM Tris, 1% Triton X-100 and 10% DMSO, pH 10 adjusted withNaOH). After lysis, the slides were placed in electrophoresis buffer(10 M NaOH, 200 mM EDTA, pH 13) for 30 min at 4 �C in the darkto allow DNA unwinding. Electrophoresis was carried out for30 min at 25 V and 300 mA at 4 �C. Finally, the slides were gentlyrinsed with neutralising solution (0.4 M Tris base, pH 7.5) threetimes, 5 min each time. Staining of DNA was accomplished with50 lL of ethidium bromide (20 lg/mL) per each slide. The cometswere observed and analysed using Olympus X 50 microscope (Olym-pus Optical Co., Gmbh Hamburg, Germany), equipped with thedevice for fluorescence recording at 100�magnification. Evaluationof DNA damage was performed as described by Anderson et al.(1994). Namely, cells were graded by eye into five categories corre-sponding to the following amounts of DNA in the tail: (A) no damage,<5%; (B) low level damage, 5–20%; (C) medium level damage, 20–40%; (D) high level damage, 40–95%; (E) total damage, >95%. In orderto obtain semi-quantitative analysis of data, the score of DNA dam-age (so-called total comet score, TCS) was calculated as follows:TCS = 1 � B + 2 � C + 3 � D + 4 � E, where B to E represents percent-ages of cells within above mentioned categories B to E. Therefore, theoverall score for 100 of comets is between 0 and 400 arbitrary units.

With an aim to ascertain possible mechanisms of genotoxiceffects of adrenaline in the Comet assay, we also performedco-treatments with 300 lM of adrenaline and antioxidantscatalase (final concentrations of 100 and 500 IU/mL) or quercetin(final concentrations of 100 and 500 lM). In these experimentsco-treatment lasted for 15 min or 1 h.

Table 1Evaluation of the effects of adrenaline in SCE test in cultured human lymphocytes: results

Concentration of adrenaline SCE range Mean SCE

Negative control 3–9 6.50Adrenaline0.01 lM 2–10 6.541 lM 4–14 6.685 lM 4–10 6.2750 lM 3–11 6.53150 lM 3–12 6.60300 lM 2–15 6.74Positive control (MNNG) 6–31 14.20***

A total of 60 second division metaphases per donor were analysed for sister chromatid exthe negative control; MI, mitotic index; CPI, cell proliferation index, CPI = (M1 + M2 + 3M3

third and higher mitotic divisions; DMSO – dimethylsulfoxide; MNNG – N-methyl-N0

(ANOVA, Student’s t-test).* p < 0.05 (ANOVA, Student’s t-test, Dunnet’s t-test for SCE, v2 test for MI and CPI).

** p < 0.01(ANOVA, Student’s t-test, Dunnet’s t-test for SCE, v2 test for MI and CPI).*** p < 0.001 (ANOVA, Student’s t-test, Dunnet’s t-test for SCE, v2 test for MI and CPI).

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2.6. Statistical analysis

The statistical analysis of experimental values in SCE test was per-formed by one-way analysis of variance (ANOVA) and, subsequently,by Student’s t-test and post hoc Dunnet’s t-test using the Statgraph4.2 software. The statistical analysis of MN frequencies, MI, CPI andCBPI were performed byv2 test. Data from the Comet assay were eval-uated by the non-parametric Kruskal–Wallis ANOVA followed by theDunn’s multiple comparison test. A p value of 60.05 was consideredas indicative of statistical significance for all tests used.

3. Results

3.1. Sister chromatid exchanges

All three donors were of good health, similar age, under nomedication and statistical analysis (ANOVA, Student’s t-test)showed no clear differences in their response to adrenaline.

Experimental values of SCEs, mitotic index (MI) and cell prolif-eration index (CPI) are presented in Table 1. The mean SCE per cellfrequency in adrenaline-treated cultures ranges from 6.27 to 6.73.Statistical analysis by ANOVA showed significant variationbetween the tested groups (negative, positive controls and eightexperimental concentrations of adrenaline) (F = 220,732;p < 0.0001). On the other hand, comparisons of negative controlwith treated groups (eight groups with experimental concentra-tions of adrenaline) showed no significant (p > 0.05) difference inrelation to any of treated groups (Dunnet’s t-test and Student’st-test). Therefore, adrenaline has not caused a significant depar-tures in the mean SCE per cell frequency from the negative controlvalue (6.50). On the basis of SCE analysis there were no indicationsof genotoxic effect of adrenaline under the experimental condi-tions described in this investigation. Only the positive controlhad significantly elevated mean SCE per cell in comparison to thenegative control (Dunnet’s t-test and Student’s t-test). The positivecontrol (MNNG) increased mean SCE per cell frequency by 118%,therefore confirming that conditions were suitable for detectionof genotoxic effects in SCE test.

Apart from valuable cytogenetic data, it is possible to determineproliferative activity of lymphocytes on the same microscopicslides used in SCE test. The MI as a percentage of cells in mitosisis a parameter used to evaluate possible cytotoxic/cytostatic ormitogenic effects. In this investigation, after the treatment of cul-tures with the negative control there were 5.91% of cells in mitosis.The positive control exhibited cytotoxic effects and caused adecrease in MI to 3.04%. In cultures treated with adrenaline MI

from three donors.

SD Xk MI (%) CPI

1.40 100.00 5.91 1.84

1.70 100.62 5.11 1.801.84 102.77 5.69 1.731.53 96.46 3.60* 1.69**

1.51 100.46 2.67*** 1.62***

1.65 101.54 1.86*** 1.47***

1.69 103.06 1.77*** 1.38***

4.95 218.31 3.04*** 1.58***

changes (SCE); SD, standard deviation; Xk – percentage of the mean value of SCE of+)/100, where M1 is percentage of cells in the first, M2 in the second and 3M3+ in the-nitro-N-nitrosoguanidine. There were no significant differences between donors

DNA damage in human lymphocytes treated with adrenaline in vitro. Tox-

A

B

C0.0

005

0.001 0.0

1 0.2 1 5 50 150

300

500

0

50

100

150 *** *********

**************

concetrations (µM)

TCS

C0.0

005

0.001 0.0

1 0.2 1 5 50 150

300

500

0

50

100

150

*

***** ***

concetrations (µM)

TCS

Fig. 1. Effect of various adrenaline concentrations on purified human lymphocytesat two treatment times: (A) 15 min, (B) 60 min; C – negative control; TCS – totalcomet score. The positive control (100 lM H2O2) caused an increase of TCS (data notshown in the graph) to 199.0 ± 8.0 after 15 min, and 188.0 ± 1.0 after 60 min,respectively. �P < 0.05, ��P < 0.01, ���P < 0.001.

4 N. Djelic et al. / Toxicology in Vitro xxx (2014) xxx–xxx

ranges were from 1.86% to 5.69%. Actually, the four highest concen-trations of adrenaline (5 lM, 50 lM and 150 lM and 300 lM)caused a significant decrease in MI when compared to the negativecontrol level. In addition, there was a significant cell-cycle delaydetected by decrease in cell proliferation index (CPI) at four highestconcentrations of adrenaline, and also after the treatment with thepositive control (MNNG).

3.2. Micronuclei

The results of cytokinesis-block micronucleus assay are pre-sented in Table 2. This assay was performed on blood samples fromthe same three male donors. In cultures treated with the negativecontrol average frequency of micronuclei was 12.8‰. Treatmentwith adrenaline has not caused significant changes in MN fre-quency in relation to the negative control. Only the positive controlproduced a significant (p < 0.001) increase in MN frequency to thevalue of 36.5‰, therefore confirming that experimental conditionswere suitable for detection of aneugenic and/or clastogenic effects.Cytokinesis-block proliferation index (CBPI) and percentage ofbinucleated (BN) cells were decreased at the four highest concen-trations of adrenaline (Table 2).

3.3. DNA damage in the Comet assay

At all experimental points, the cell viability in Trypan blueexclusion test was acceptable (over 83%). We found that adrenalineinduces primary DNA damage at all tested concentrations after thetreatment for 15 min, except at the lowest concentration(0.0005 lM), which approximately corresponds to a physiologicallevel of adrenaline in the human blood (Fig. 1). After 1 h, the levelof DNA damage in lymphocytes treated with 5 lM of adrenalineand all other higher concentrations gave rise to a significant DNAdamage (Fig. 1). However, the effects were slightly lower in com-parison with 15 min treatment. Therefore, we assume that withprolonged treatment, the DNA repair reduced the level of primaryDNA damage which could be expressed as increased DNA migra-tion in the Comet assay.

In order to study the mechanisms involved in DNA damageinduced by adrenaline, we had further studies with antioxidantscatalase and quercetin (Fig. 2). We used both treatment times(15 min and 1 h) for our study with antioxidants. In each experi-ment with antioxidants, we chose the concentration of 300 lM ofadrenaline which induces high levels of DNA damage but still withacceptable level of cytotoxicity (below 20%). The concomitanttreatment with an antioxidant catalase efficiently reduced DNA

Table 2Evaluation of the effects of adrenaline in cytokinesis-blocked micronucleus assay on hum

Treatment BN cells scored Distribution of BN cells accord

0 1

Negative control 3194 3156 35Adrenaline0.01 lM 3085 3050 291 lM 3308 3262 445 lM 3061 3027 3350 lM 3043 3004 36150 lM 3086 3041 39300 lM 3022 2978 40Positive control (MNNG) 3013 2922 74

At least 1000 binucleated (BN) cells per donor were analysed for the frequency oCBPI = [MI + 2MII + 3(MIII+IV)]/100, where MI to MIV represent the percentage of cells wnitrosoguanidine.

* p < 0.05 (v2 test).** p < 0.01 (v2 test).

*** p < 0.001 (v2 test).

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damage at 100 IU/mL, and this effect was even more profound at500 IU/mL of catalase, both at 15 min and 1 h of incubation at37 �C. Likewise, the flavonoid quercetin reduced DNA damage atboth concentrations used (100 lM and 500 lM) at 15 min, butafter 60 min produced a significant (p < 0.01) antioxidant effectonly at the higher concentration (500 lM). Interestingly, the anti-oxidant effect of both catalase and quercetin had higher levels ofstatistical significance at 15 min in comparison to co-treatmentfor 60 min.

an peripheral blood lymphocytes: results from three donors.

ing to No. of MN MN/103 cells BN (%) CBPI

2 3

3 0 12.8 50.1 1.57

5 1 13.6 45.9 1.522 0 14.5 42.7 1.481 0 11.4 33.5* 1.38***

3 0 13.8 32.1** 1.36***

4 2 17.2 28.5*** 1.31***

2 2 16.5 29.2*** 1.34***

15 2 36.5*** 30.6*** 1.42***

f micronuclei (MN); CBPI – cytokinesis-block proliferation index, calculated asith one to four nuclei; DMSO – dimethylsulfoxide; MNNG – N-methyl-N0-nitro-N-

DNA damage in human lymphocytes treated with adrenaline in vitro. Tox-

A

C A

A/CAT10

0

A/CAT50

0

A/Q10

0

A/Q50

00

50

100

150

*** ****** ***

negative controladrenaline (300 µM)adrenaline + catalase100 or 500 IU/mLadrenaline + quercetin100 or 500 µM

Concentration of A (µM),CAT (IU/mL) and Q (µM)

TCS

B

C A

A/CAT10

0

A/CAT50

0

A/Q10

0

A/Q50

00

50

100

150

*** **

negative controladrenaline (300 µM)adrenaline + catalase100 or 500 IU/mLadrenaline + quercetin100 or 500 µM

Concentration of A (µM), CAT (IU/mL) and Q (µM)

TCS

Fig. 2. The effects of catalase (CAT) and quercetin (Q) on reduction of DNA damagecaused by adrenaline (A) at two treatment times: (A) 15 min, (B) 60 min; C –negative control; TCS – total comet score. The positive control (100 lM H2O2)caused an increase of TCS (data not shown in the graph) to 182.5 ± 4.2 after 15 min,and 177.7 ± 1.7 after 60 min, respectively. �P < 0.05, ��P < 0.01, ���P < 0.001.

N. Djelic et al. / Toxicology in Vitro xxx (2014) xxx–xxx 5

4. Discussion

Experimental and epidemiological data strongly support theinfluence of hormones in the processes of carcinogenesis (Schielet al., 2006; Kabbarah et al., 2006; Pruthi et al., 2012). Apart frombeing involved in the processes of tumor promotion, it has beenshown that some hormones, especially estrogens, may also act asendogenous mutagens (Liehr, 2001; Cavalieri and Rogan, 2004),therefore having the dual role both in the initiation and in the pro-motion of carcinogenesis.

Overwhelming evidence indicates that the metabolic conver-sion of estrogens to catecholestrogens and subsequent redoxcycling of phenolic groups in the catechol ring leads to the creationof ROS and oxidative stress in target cells (Liehr, 2001; Li et al.,2004). Interestingly, there are also experimental indices that cate-chol groups of some non-steroidal hormones (e.g. adrenaline) andneurotransmitters (dopamine, noradrenaline) may be involved inredox cycling accompanied by generation of ROS which favors oxi-dative stress (Moldeus et al., 1983; Djelic and Anderson, 2003;Dobrzynska et al., 2004; Zahid et al., 2010, 2011). Therefore, itseems that the creation of oxidative stress is the key mechanismof genotoxic effects of both steroidal and non-steroidal hormoneswith catechol groups. Moreover, McGregor et al. (1988) investi-gated various catecholamines and related substances and observedthat although the phenolic moiety was not mutagenic, the additionof a second hydroxyl group, which forms catechol, was sufficient toform a mutagenic agent.

Although the literature data about genotoxic effects of adrena-line are scarce, there are some equivocal findings of weak muta-genic effects of adrenaline in Salmonella typhimurium TA 100strain, both with and without liver S-9 metabolic activation

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(Deitz, 1990). Interestingly, Martínez et al. (2000) have classifiedadrenaline and other tested catecholamines as potent oxidativemutagens in WP2 Mutoxitest in Escherichia coli tester strainsIC203. In SCE test on Chinese hamster ovary (CHO) cells adrenalinegave equivocal response without S9 activation, and negativeresponse in the presence of S9 activation. On the other hand,adrenaline did not induce chromosome aberrations in CHO cells,with or without S9 mix (Deitz, 1990).

Some published data shown that noradrenaline causes DNAdamage in human lymphocytes (Djelic and Anderson, 2003) andsperm (Dobrzynska et al., 2004), probably by inducing the oxida-tive stress.

In this investigation, we evaluated cytogenetic effects of adren-aline using two different endpoints – sister chromatid exchangeand micronuclei. Although the biological meaning and molecularmechanisms underlying SCE formation remain unclear, there isample evidence that genotoxic agents can give rise to SCEs(Djelic and Djelic, 2002; Pingarilho et al., 2013). Despite findingsof DNA-damaging effects of adrenaline in the Comet assay, underthe experimental conditions of this investigation we did notobserve a significant change of SCE per cell frequency in culturedhuman lymphocytes. This is in accordance with experimental find-ing that dopamine, as a compound with catechol group, has notinfluenced SCE per cell frequency in cultured human lymphocytes(Moldeus et al., 1983).

Keeping in mind that the SCE test is considered as an ancillarytest in genetic toxicology (Speit and Henderson, 2005), we alsoused the cytokinesis block micronucleus assay in order to evaluatepossible clastogenic or aneugenic effects of adrenaline. However,changes in frequency of micronuclei have not reached a statisti-cally significant level in this investigation. It should be mentionedthat results of dopamine testing in bone marrow micronucleusassay were also negative (Moldeus et al., 1983). Therefore, we con-clude that, under the experimental conditions described in thisinvestigation, there was no cytogenetically detectable genotoxiceffect of adrenaline.

In addition to the investigation of genotoxic effects, we deter-mined mitotic and cell proliferation indices for each experimentalconcentration and controls. There was a significant decrease of theMI at three highest concentrations of adrenaline. As expected, thepositive control caused a more profound decrease of the mitoticindex, possibly due to the arrest of mitosis because of the repairof genetic damage. Additionally, in cells with a relatively high levelof genetic damage cytotoxic effects occur.

It is generally accepted that short-term cultures of phytohae-magglutinin-activated lymphocytes are suitable for the analysisof cell-cycle kinetics. Actually, the presence of lymphocyte subpop-ulations in which blastogenesis started at different times aftermitogenic stimulation allows the chemical agent to influence cellkinetics (Sobol et al., 2013). The cell-cycle delay observed in thepresent study is in accordance with findings that theophyllineinhibits proliferation of lymphocytes stimulated with phytohae-magglutinin (Kimura et al., 2003). Namely, the molecular mecha-nism of signal transduction underlying the action of bothadrenaline and theophylline implies increased concentration ofintracellular cAMP, so we assume that increased cAMP may havecontributed to the cell-cycle delay observed in this investigation.This assumption is supported by experimental findings thatnaturally occurring catecholamines inhibit the proliferation ofT-lymphocytes (Jiang et al., 2009). These inhibitory effects can beabolished by non-selective b-blocker propranolol or by b2-blockerbutoxamine (Edgar et al., 2003).

The level of primary DNA damage in isolated human lympho-cytes was evaluated by using the in vitro Comet assay. Althoughin this investigations we used visual scoring for the analysis ofcomets, a recent study has shown that there is an acceptable level

DNA damage in human lymphocytes treated with adrenaline in vitro. Tox-

6 N. Djelic et al. / Toxicology in Vitro xxx (2014) xxx–xxx

of agreement between various scoring methods (Azqueta et al.,2011) and, therefore, our results should be considered as valid.Despite being somewhat subjective, the visual scoring of cometsgives reliable, quantitative results providing that a well trainedand experienced microscopist performs the analysis (Azquetaet al., 2011). The observed increase of DNA damage was more pro-found after 15 min of incubation compared to 1 h of treatmentwith adrenaline. Probably, the repair processes were not fullyactive after 15 min, and after 1 h they had enough time for moreefficient repair of DNA damage. Chiaramonte et al. (2001) showedthat significant repair of DNA damage occurred in cells 30 min to1 h after the exposure to oxidative agent.

The most interesting experimental observation in this investi-gation is that we found no genotoxic effects in cytogenetic tests,whereas there was a significant DNA damage in the Comet assay.Namely, the alkaline version of Comet assay is very sensitivemethod for evaluation of DNA damage, and it is able to detectDNA strand breaks and alkali labile sites (Singh et al., 1988). How-ever, it measures genomic damage which comprises all damage toDNA including gene and chromosome type changes, whereas thecytogenetic tests measure only chromosome type changes(Wagner et al., 2003). The absence of cytogenetic effects of adren-aline is consistent with our previous finding that adrenaline doesnot induce chromosome aberrations in cultured human lympho-cytes (Djelic et al., 2003). It is already established (Suggitt et al.,2003) that some compounds exert genotoxic effects primarily atgene level (e.g. EMS), and others primarily induce chromosomechanges (e.g. mitomycin C).

With an aim to indirectly examine molecular mechanisms ofDNA damage under the influence of adrenaline we used theantioxidants catalase and quercetin. The enzyme catalase convertshydrogen peroxide into water and oxygen, whereas quercetin is awell-known hydroxyl radical scavenger (Cemeli et al., 2009). Theexperimental findings in this investigation have shown that antiox-idants catalase and quercetin significantly decrease DNA damage inlymphocytes exposed to 300 lM of adrenaline. Therefore, most ofthe DNA damage induced by adrenaline is mediated by ROS. Thisfinding is consistent with study of Djelic and Anderson (2003)showing that the antioxidant catalase reduced DNA damagingeffects of noradrenaline in the Comet assay. Since the antioxidantscatalase and quercetin did not reduce DNA damage to the negativecontrol level, the stimulation of adrenergic receptors was partiallyresponsible for the genotoxic effects of adrenaline, as previouslysuggested (Flint et al., 2007; Hara et al., 2011).

Although the concentrations of adrenaline that induced DNAdamage in our investigation were much higher than physiologicallevel of adrenaline in human blood, elevated concentrations of cir-culating catecholamines are found in hypoglycemia, hemorrhagichypotension (Goldstein, 2003), ischemia (Akiyama and Yamazaki,2001), pheochromocytoma (Gerlo and Sevens, 1994) and drugabuse (Carvalho et al., 1996). The recorded values of plasma leveladrenaline in patients with severe heart failure is 20 and 58 nM(Raymondos et al., 2000) and 11 nM in pheochromocytomapatients (Hegedus, 2000). The results obtained in our experimentssuggest that persons with high plasma levels of adrenaline can beprone to increased DNA damage.

In conclusion, although the genotoxic effects of adrenaline werenot detected at cytogenetic level, we observed that adrenalineexhibited genotoxic effects expressed as primary DNA damage inthe Comet assay, even at concentrations of 0.01 lM and 0.2 lMwhich mimic adrenaline blood concentrations in an acute stress inhumans (Zouhal et al., 2008). The DNA damaging effects ofadrenaline suggest its role in the processes of the initiation of carci-nogenesis. In addition to possible genotoxic effects which may leadto cancer initiation, stress and adrenaline itself can also compromisethe immune system and influence other stages of carcinogenesis.

Please cite this article in press as: Djelic, N., et al. Evaluation of cytogenetic andicol. in Vitro (2014), http://dx.doi.org/10.1016/j.tiv.2014.08.001

Namely, chronic psycho-emotional stress can strengthen carcino-genic effects of physical, chemical and biological carcinogens(Bukhtoyarov and Samarin, 2009). Moreover, it has been shown thatadrenaline can reduce the effects of chemotherapy of malignantcells. Thus, adrenaline upregulates mdr1 gene expression in MCF-7breast cancer cells by stimulation of a2-adrenoceptors (Su et al.,2005). The mdr1 gene codes the plasma membrane ATPase capableto expel cytostatics from malignant cells. Adrenaline can alsoincrease the cell proliferation and decrease cisplatin induced apop-tosis in colon cancer HT29 cells (Pu et al., 2012). Therefore, it wouldbe interesting to further examine relevance of adrenaline in the pro-cesses of mutagenesis and carcinogenesis both in vitro and in vivousing various test systems, cell types and treatment conditions.

Conflict of Interest

The authors declare that there are no conflicts of interest.

Transparency Document

The Transparency document associated with this article can befound in the online version.

Acknowledgements

The authors are grateful to ‘‘Jugoremedija’’ (Zrenjanin, Serbia)for preparation of the negative control (placebo). Grant Sponsor:Serbian Ministry of Education and Science (Grant Numbers III46002 and OI 173034).

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