MILENA RADAKOVIC (1), JEVROSIMA STEVANOVIC (1), NINOSLAV DJELIC (1)*, NADA LAKIC (2), JELENA KNEZEVIC-VUKCEVIC (3), BRANKA VUKOVIC-GACIC (3) and ZORAN STANIMIROVIC (1)
1 : Department of Biology, Faculty of Veterinary Medicine, 2: Department of Statistics, Faculty ofAgriculture, 3: Department of Microbiology, Faculty of Biology, The University of Belgrade, Belgrade, Serbia
- Corresponding author : (Fax, +381-11-2685936; Email, ndjelic à vet.bg.ac.rs)
Amitraz is formamidine pesticide widely used as insecticide and acaricide. In veterinary medicine, amitraz hasimportant uses against ticks, mites and lice on animals. Also, amitraz is used in apiculture to controlVarroadestructor. It this study, the alkaline Comet assay was used to evaluate DNA damaging effects of amitraz in humanlymphocytes. Isolated human lymphocytes were incubated with varying concentrations of amitraz (0.035, 0.35, 3.5,35 and 350μg/mL). The Comet assay demonstrated that all concentrations of amitraz caused statistically significantincrease in the level of DNA damage, thus indicating that amitraz possesses genotoxic potential. The concentration ofamitraz that produced the highest DNA damage (3.5μg/mL) was chosen for further analysis with the antioxidantcatalase. The obtained results showed that co-treatment with antioxidant catalase (100 IU/mL or 500 IU/mL)significantly reduced the level of DNA damage, indicating the possible involvement of reactive oxygen species inDNA damaging effects of amitraz. Flow cytometric analysis revealed increase of the apoptotic index followingtreatment with amitraz. However, co-treatment with catalase reduced the apoptotic index, while treatment withcatalase alone reduced the percentage of apoptotoc cells even in comparison with the negative control. Therefore,catalase had protective effects against ROS-mediated DNA damage and apoptosis.
Radakovic M, Stevanovic J, Djelic N, Lakic N, Knezevic-Vukcevic J, Vukovic-Gacic B and Stanimirovic Z 2013 Evaluation of the DNA damagingeffects of amitraz on human lymphocytes in the Comet assay.J. Biosci.3853–62DOI10.1007/s12038-012-9287-21.
Amitraz N-methylbis (2,4-xylyliminomethyl) amine is for-mamidine pesticide marketed worldwide as an acaricide andinsecticide since 1974 (Croftonet al.1989). It is non-systemic insecticide with contact and respiratory action.In veterinary medicine, amitraz has important uses fordemodectic manage (Demodex canis) in dogs (Farmer andSeawright 1980) and it is also used against the ticks, mitesand lice on cattle, sheep, pigs and goats (Tomlin 1994). Insome countries, diluent form of amitraz is applied in humansto treat pediculosis or scabies (Kalyoncuet al.2002). Also,amitraz is used by beekeepers to controlVarroa destructor. Amitraz exerts toxic effects on ectoparasites by interac-tion with the octopamine receptors of arthropods (Evans andGee 1980; Dudaiet al.1987). Although mammals do not have octopamine receptors, amitraz exerts side effects inmammals through activation ofα2-adrenoceptors (Hsu1996). Amitraz has been shown to induce cytochromeP450-dependent monooxygenases in the liver of treated rats(Uenget al.2004) and decrease hepatic glutathione activityin mouse (Costaet al.1991). In bovine seminal vesicle,amitraz inhibited prostaglandin E2synthesis (Vimet al.1978). At high dose levels, amitraz can cause tumours infemale mice and it is classified as a Group C possible humancarcinogen (US EPA 1996). In dogs, following a topicalapplication, amitraz increased plasma glucose and inhibitedinsulin secretion (Hsu and Schaffer 1988). Evidence fromanimal studies also suggests that amitraz is potential repro-ductive toxicant (Hayes and Laws 1991; Cooperet al.1999).Moreover, amitraz exhibits toxic effects in the human repro-duction cellsin vitroand inhibits the production of the steroid hormone progesterone (Younget al.2005). Inhumans, amitraz intoxication has been reported and exposureeffects include CNS depression, hypothermia, bradycardia,hypotension, hyperglycemia, glycosuria, vomiting and res-piratory failure (Kennelet al.1996; Kalyoncuet al.2002;Yilmaz and Yildizdas2003). Due to its excellent miticidal activity, amitraz is widelyused in apiculture for the obligatory annual control ofVarroadestructor. In beehives, amitraz is used in the form of smokeor vapour (Marchetti and Barbattini1984). The lack ofresidues in honey is related to the instability of amitraz inan acidic medium like honey (Berzas Nevadoet al.1990).According to EMEA (European Agency for the Evaluationof Medicinal Products) residues of amitraz in honey werestableforupto4monthswhensampleswerestored at –20°C. Amitraz also does not remain stable in beeswax(Wallner1999; Kortaet al.2001). It degrades over a periodof 2–4 weeks in honey, but in beeswax it completelydegrades within 1 day (Kortaet al.2001). On the other hand,this acaricide is easily hydrolysed to toxic 2,4-dimethylani-line (2,4-DMA) and various products containing the 2,4-DMA moiety (Jiménezet al.2002). Unlike amitraz, itsdegradation products are stable in honey and can be foundas residues in food and in surface water samples (Cortaet al.1999). Maximum Residue Limit (MRL) in honey is 0.2 mg/kg as defined in No. 2377/90/EC regulationfor amitraz. On the other hand, no MRL is fixed forbeeswaxevenwhenitisused for pharmaceutical pur-poses, food packaging or cosmetics. In honey collectedfrom colonies treated with amitraz, varroacide residuescan be found below the MRL value (Floriset al.2001; Martelet al.2007; Lodesaniet al.2008). Exposure to amitraz would be expected to pose a greaterhazard to pet owners, agricultural workers and beekeepersbecause of the continual exposure to this acaricide. Therefore, it is very important to evaluate possible genotoxiceffects of amitraz in view of health protection. Genotoxicityof the amitraz was explored using different test systems frombacteria to mammals. Osanoet al.(2002) reported genotoxiceffects of amitraz and its metabolite at very low concentra-tions (<0.005 mM) in the Vibrio test. Likewise, amitrazcaused genotoxic effect by induction of chromosomal aber-rations in bone marrow cells of mice (Pejinet al.2006). Incontrast, amitraz and its metabolites were found to benegative for mutation in the Ames test (Tudeket al.1988) and did not induce DNA strand breaks on rat hepato-cytes (Grilliet al.1991). On the basis of available data we cannot obtain a clearidea of the possible genotoxic effects of amitraz. Besides, itis worthwhile to investigate the mechanisms of amitraz ac-tion on DNA. It has been shown that some pesticides mayinduce oxidative stress through the generation of reactiveoxygen species, leading to lipid peroxidation and DNAdamage (Abdollahiet al.2004; Vidyasagaret al.2004; Shadniaet al.2005). There is evidence that free oxygenradicals are produced during AMZ oxidation (Kruk andBounias1992). This data suggest that amitraz may be ableto induce oxidative DNA damage via reactive oxygen spe-cies (ROS).In this study, the Comet assay was used to assess theDNA damaging effect in human lymphocytes exposed toamitraz in order to provide additional genotoxicological datafor this compound. In addition, we used catalase to deter-mine whether the mechanism underlying DNA damage ofamitraz is mediated by ROS. In order to determine an apo-ptotic index we performed the annexin V–propidium iodide(AnnV–PI) staining apoptosis test.
Varamit® (0.2 g/mL of Amitraz dissolved in xylol) waspurchased from Evrotom (Ruma, Serbia; CAS No33089–61–1). The chemical structure of amitraz isshown in figure1. Amitraz stock standard solution wasprepared in propylen glycol to obtain a concentration of100 mg/mL. We used propylen glycol as a solvent insteadof xylol, because pure xylol and amitraz in xylol produceunacceptable levels of cytotoxicity (data not shown). Thestock standard solution of amitraz was appropriately dilutedwith propylen glycol to prepare working solutions to obtain0.035, 0.35, 3.5, 35 and 350μg/mL doses of amitraz.Hydrogen peroxide (100μM) was used as a positive controlto verify the sensitivity of the test system. Negative controlwas composed of all ingredients except an active one (ami-traz). Therefore, in a final volume of 1000μL, the negativecontrol contained 940μL of PBS, 50μL of RPMI 1640 withisolated lymphocytes, 7μL of propylenglycol and 3μLofxylol.
Heparinised blood samples (4 mL) were obtained byvenepuncture from two healthy male donors under25 years of age. The study was approved by the localMedical Ethics Committee,performed in accordancewith Declaration of Helsinki, and informed donor con-sent was also obtained. Lymphocytes were isolated fromwhole blood with Ficoll-Paque medium and centrifugedat 1900g15 min. The lymphocytes forming a layer weredirectly above Ficoll-Paque. The isolated lymphocytes werewashed twice in RPMI 1640 medium, each wash was fol-lowed by a centrifugation 10 min at 1800g. Finally, thesupernatant was removed as carefully as possible withoutdisturbing the pellet. An aliquot of 1 mL of RPMI 1640 wasadded and the pellet was re-suspended. A manual cell countand an estimate of cell viability were performed usingTrypan blue exclusion test.
Alkaline Comet assay was performed according to the Singhet al.(1988) and Ticeet al.(1991) technique with slightmodifications. Microscope slides were precoated with 1%normal melting point agarose and allowed to air-dry at roomtemperature for at least 48 h. After incubation with the testedcompound for 1 h, the cell viability was evaluated usingTrypan blue exclusion test. After centrifugation (5 min at2000 rpm), 100μL of cell suspension was mixed with100μL of 1% low melting point agarose (LMPA). Thesuspension was rapidly pippeted onto the first agarose layerand spread using a coverslip, and placed in the fridge tosolidify. After removal of the coverslip, the 90μL of 0.5% LMPA was added as the third layer, spread using a coverslipand allowed to solidify at 4°C for 5 min. Afterwards, theslides were immersed in cold lysis solution at pH 10 (2.5 MNaCl, 100 mM EDTA, 10 mM Tris pH 10, 1% Triton X–100, 10% DMSO) overnight at 4°C. After lysis, the slideswere placed in a horizontal gel electrophoresis tank toallow DNA unwinding in cold alkaline electrophoresisbuffer (300 mM NaOH, 1 mM EDTA, pH>13) for30 min. Electrophoresis was done at 4°C with electriccurrent of 25 V and 300 mA for 30 min. All these stepswere performed under dimmed light (tank was coveredwith a black cloth) to prevent additional DNA damage. The slides were then neutralized with 400 mM Tris-HCl(pH 7.5) for 5 min. The neutralization was repeatedthree times. Then, the slides were fixed with cold meth-anol, dried and stored. Before analysis, the slides wererehydrated with ice cold distilled water and stained with50μLof20μg/mL ethidium bromide. Various levels of DNA damage on human lymphocytes are presented infigure 2. In the co-treatment we used as an antioxidantcatalase from bovine liver (CAS No 9001–05–2), SigmaChemical Co., St. Louis, USA.
Slides were examined at 400× magnifcation on a fluorescentmicroscope (Leica, UK) and image analysis software (CometAssay IV Image Analysis system, PI, UK). From each rep-licate slide, 50 nuclei were scored (a total of 100 nuclei perdonor) and the percentage of tail DNA was used to evaluatethe extent of DNA migration.
This assay is based on the ability of the protein anexxinV to bind to phosphatidylserine (PS) exposed on theouter membrane leaflet in apoptotic cells (PS alsoappears on the necrotic cell surface). In viable cells,PS is located in the inner membrane leaflet, but uponinduction of apoptosis it is translocated to the outermembrane leaflet and becomes available for annexin Vbinding. The addition of propidium iodide (PI) enabledviable (AnnVneg/PIneg), early apoptotic (AnnVpoz/PIneg) andnecrotic (AnnVneg/PIpoz) cells to be distinguished. Theannexin V assay was performed following the instructionsprovided by the manufacturer of the ANNEXIN V FITC kit(Beckman Coulter, CA, USA). Briefly, after the treatment ofisolated lymphocytes for 1 h, cells (1×106cells/mL) werewashed in cold PBS, suspended in binding buffer and thenadded with 1μL annexin-V FITC and 5μL propidiumiodide (100μg/mL) for 15 min. Finally, 400μL 1× bindingbuffer was added to each tube and cells were read by flowcytometry (Partec Cyflow SL) by differentiation of at least 20,000 cells. Flow cytometric analysis was performed using Partec FloMax software. The apoptotic index (AI) was cal-culated as the percentage of Annexin V positive and PI-negative cells divided by the total number of cells in thegated region.
Statistical analysis of the results obtained in the experimentwas carried out using software STATISTICA v. 6. In the Comet assay, Levene’s test for homogeneity of variance andKolmogorov-Smirnov test for normality of distribution wereused prior to statistical analysis. Considering that the datawere not in line with the requirements for the application ofparametric tests, differences between treatments were testedusing Kruskal-Wallis test and Mann-Whitney U test. As forthe flow cytometric analysis of apoptosis we used thez-testfor proportions.
The viability of lymphocytes treated with amitraz or xylolfor 1 h in Trypan blue exclusion assay was at least 90%. Inexperiments with catalase, viability of lymphocytes was alsoover 90%. In all experimental groups, the viability oflymphocytes treated with the positive control (100μMH2O2) was at least 82%. Therefore, in all our experimentslevel of cytotoxicity was acceptable.
The minimal percentage of tail DNA at all treatments was0%, while maximal DNA damage ranged from 28.09% (at 0.35μg/mL of amitraz) to 100.0% (at 350μg/mL) (table1).DNA damage in all treatments was heterogenous (coeffi-cients of variations ranged from 118.84% at 0.35μg/mL ofamitraz to 158.31% at 350μg/mL of amitraz). Maximalinterquartile range of 14.40% was achieved after the treatmentwith the positive control (H2O2), while a minimum of 3.34%was obtained for the negative control (table1; figure3).Bearing in mind the character of experimental data, we usedmedians to quantify average values. The average percentageof tail DNA ranged from 1.3% in the negative control groupto 4.55 in cells treated with 3.5μg/mL of amitraz. The resultsof Kruskal-Wallis test (H6,1304043.769;p<0.001, figure3)points to very high statistical significance of percentage oftail DNA between various concentrations of amitraz andcontrols (0μg/mL of amitraz and 100μMH2O2). The results of the Mann-Whitney U test (table2) clearly showed thatthis difference is due to statistically highly significant differ-ence between the negative control and all other treatmentswith amitraz, as well as the diffference between treatmentwith 0.35μg/mL of amitraz and treatments with 0.035μg/mLand 3.5μg/mL of amitraz and the positive control (figure3).In experiment with amitraz, 100μMH2O2used as thepositive control gave rise to a significant DNA damage(p<0.001).
In order to understand the mechanism of genotoxic effect ofamitraz, we used the antioxidant catalase. The concentrationof amitraz (3.5μg/mL) that produced the highest DNAdamage was chosen for co-treatment with this antioxidant.In all treatments, the minimal percentage of tail DNA was0% (table3). The maximum percentage of tail DNA was inthe range from 6.82% in the negative control to 96.79% inthe positive control (100μMH2O2). The data obtained inthis experiment was non-homogenous–the coefficients ofvariations were in the range 90.46% (positive control) to143.07% (500 IU/mL catalase). Minimal interquartile rangewas observed in the negative control (1.55%) and the max-imal in the positive control (22.83%) (table3; figure4). Theresults of Kruskal-Wallis test showed very highly statisticalsignificance (H5,12000427.352;p<0.001) (figure4) betweenmedians which were in the range from 0.47% for the treat-ment with 500 IU/mL of catalase to 12.30% for cells treatedwith the positive control. On the other hand, the results ofMann-Whitney U test (table4) showed that percentage of tailDNA in the negative control was significantly lower com-pared to all other treatments, except for the 500 IU/mL ofcatalase alone. In the co-treatment with 3.5μg/mL of amitrazand catalase, the average precentage of tail DNA was lowerin comparison to cultures treated with 3.5μg/mL alone(table4; figures4and5), at both concentrations of catalaseused in this experiment.
The results of evaluation of apoptosis by flow cytometry arepresented in figure6. In the negative control, we observed89.87% of viable cells, 5.42% of cells in early apoptosis and4.72% of necrotic cells. After the tretment with amitraz,viable cells dropped to 70.86% (p<0.001), while therewas a significant (p<0.001) increase of early apoptotic(14.72%) and necrotic cells (14.41%). However, concomi-tant treatment with 3.5μg/mL of amitraz and catalaseresulted in reduction of the apoptotic index (percentage earlyapoptotic cells) in comparison with cells treated only withamitraz, and this effect was more profound with higherconcentration of catalase applied (500 IU/mL). Thus, weobserved 11.34% of early apoptotic cells co-treated with100 IU/mL of catalase (p<0.001) and 8.72% of necroticcells. The co-treatment with 500 IU/mL of catalase causedfurther decrease (p<0.001) of the percentage of early apo-ptotic cells (7.25%) and necrotic cells (6.18%) in comparisonwith the treatment with amitraz alone (3.5μg/mL).Interestingly, lymphocytes treated only with 500 IU/mL ofcatalase had significantly (p<0.05) lower percentage of earlyapoptotic cells (5.13%) in comparison with all other treat-ments including the negative control. This finding indicates that catalase had protective effects against apoptosis. Finally,the positive control (100μMH2O2) produced the mostprofound effect (p<0.001) in comparison with all other treat-ments, with only 67.62% of viable cells and highest percen-tages of early apoptotic (15.74%) and necrotic cells(16.65%).
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