Three different pesticide formulations were tested for micronuclei production and cell cycle arrest. Micronucleus test represents a suitable method for assessing chromosome damage because both chromosome breakage (clastogenicity) and chromosome loss (aneugenicity) can be measured simultaneously. In the experiments for 24 and/or 48 h exposure, no significant increase (p < 0.05) in MN frequency was found in comparison with negative control. On the contrary, the values of CBPI (cytochalasin blocked proliferative index) were significantly reduced in a dose dependent manner (p < 0.05, p < 0.01, p < 0.001) for both exposure time. Our results indicated an expressive cytotoxic effect of various pesticides in cultivated bovine peripheral lymphocytes

Dianovský, Ján

Holečková, Beáta

Schwarzbacherova, Viera

Šiviková, Katarína

English

Acta Fytotechnica et Zootechnica Online (Faculty of Agrobiology and Food Sciences, Slovak University of Agriculture in Nitra)

Acta fytotechn zootechn, 19, 2016(Special Issue), 93-95 
http://www.acta.fapz.uniag.sk 
© Slovak University of Agriculture in Nitra                                             Faculty of Agrobiology and Food Resources 
93 
          Short Communication 
 
 
Micronucleus assay in genotoxicity assessment 
 
Viera Schwarzbacherová∗, Katarína Šiviková, Beáta Holečková, Ján Dianovský  
University of Veterinary Medicine and Pharmacy, Institute of Genetics, Slovakia  
 
Article Details:  Received: 2016-04-28   |   Accepted: 2016-05-20   |   Available online: 2016-09-01  
http://dx.doi.org/10.15414/afz.2016.19.si.93-95 
 
Three different pesticide formulations were tested for micronuclei production and cell cycle arrest. Micronucleus 
test represents a suitable method for assessing chromosome damage because both chromosome breakage 
(clastogenicity) and chromosome loss (aneugenicity) can be measured simultaneously. In the experiments for 24 
and/or 48 h exposure, no significant increase (P < 0.05) in MN frequency was found in comparison with negative 
control. On the contrary, the values of CBPI (cytochalasin blocked proliferative index) were significantly reduced 
in a dose dependent manner (P < 0.05, P < 0.01, P < 0.001) for both exposure time. Our results indicated an 
expressive cytotoxic effect of various pesticides in cultivated bovine peripheral lymphocytes. 
Keywords: cell proliferation, cytotoxicity, genotoxicity, micronucleus, pesticide 
 
 
1 Introduction 
Pesticides are a significant source of environmental pollution due to their wide spectrum use in 
agriculture and forestry for controlling the spread of pests. Triazole fungicides (e.g. tebuconazole, 
prothioconazole, epoxiconazole etc.) are used as clinical drugs for the treatment and protection of 
cereals, soybeans and a variety of fruits. Their antifungal effect is based on the specific inhibition of 
CYP51, a sterol 14-α-demethylase that catalyzes the demethylation of lanosterol in the ergosterol 
biosynthesis pathway in fungi. Morpholine fungicides (e.g. fenpropimorph) act as inhibitors of the ∆8-
∆7 isomerase and ∆8, 14 sterol reductase, which results in the ergosterol biosynthesis impairment. 
Neonicotinoid insecticides (e.g. thiacloprid) provide effective control of a broad spectrum of sucking 
and chewing pests. They act as agonist of the post-synaptic nicotinic acetylcholine receptors, thus 
disturbing signal transmission in the insect’s nervous system and potentially in mammals. 
In this paper we present the effects of three different pesticide formulations on bovine peripheral 
lymphocytes in vitro, assessed by micronucleus test and cytochalasin-blocked proliferation index 
(CBPI) determination. Analysis of micronuclei (MN) frequencies serves as a biomarker of 
chromosomal damage for genotoxicity testing and biomonitoring studies (Decordier et al., 2009). This 
method allows detection of both clastogenic and aneugenic effects of chemical agents (Norppa and 
Falck, 2003).  
 
2 Material and methods 
2.1 Test samples, cell cultivation and chemicals 
Blood was taken from 2 calves (Holstein crossbreed cattle, 6 months old, 150-200 kg) and was 
cultivated for in vitro MN assay according to Schwarzbacherová et al. (2015). Briefly, the whole blood 
was added to standard cultivation medium and cultivated for 72 h at 37 °C. Lymphocytes were treated 
with the pesticide for the last 24 and/or 48 h for induction of MNs. Two fungicides and an insecticide 
were used in the experiments. Prosaro 250 EC (125 g l-1 tebuconazole, 125 g l-1 prothioconazole), 
Tango Super (84 g l-1 epoxiconazole, 250 g l-1 fenpropimorph)  and Calypso 480 SC (480 g l-1) were 
added in to the cultures at concentrations: 1.5; 3; 7.5; 15; 30 µg ml-1  for Prosaro®, 0.5; 1.5; 3; 6; 
15 µg ml-1 for Tango® and 30; 60; 120; 240; 480 µg ml-1 for Calypso®. Dimethylsulfoxide (DMSO; for 
Prosaro® and Tango®) at a final concentration of 1 % and water (for Calypso®) were used as 
dissolvent and negative controls. 
                                                          
∗  Corresponding Author: Viera Schwarzbacherová. University of Veterinary Medicine and Pharmacy, Institute of Genetics 
Komenského  73, 041 81 Košice, Slovak Republic. E-mail: viera.schwarzbacherova@uvlf.sk  
Acta fytotechn zootechn, 19, 2016(Special Issue), 93-95 
http://www.acta.fapz.uniag.sk 
© Slovak University of Agriculture in Nitra                                             Faculty of Agrobiology and Food Resources 
94 
2.2 MN assay 
To block cytokinesis, cytochalasin-B (Sigma Aldrich, USA) at the final concentration of 6 μg ml−1 was 
added to all cultures, 44 h after initiation of division. One thousand binucleated (BN) cells per donor 
and concentration were scored for micronuclei under the criteria for MN evaluation according to 
Fenech et al. (2003). At least 500 cells were scored for CBPI. The statistical analysis was performed 
using the chi square (χ2) test for MN induction and CBPI. 
 
3 Results and discussion 
The frequencies of MN and CBPI in bovine peripheral blood lymphocytes after exposure to the 
pesticides formulations for 24 and 48 h are presented in Fig. 1A, B and C. Some results have been 
published in works of Schwarzbacherová et al. (2015) and Galdíková et al. (2015). No statistically 
significant increases in the MN formation were found after exposure to all pesticides for both exposure 
times except the highest concentration of Calypso® (480 µg ml-1; Fig. 1C). Similarly, negative results in 
MN frequencies were documented by numerous authors after exposure to several pesticides; e.g. 
after treatment of bovine peripheral lymphocytes with tebuconazole formulation (Orius®) (Šiviková et 
al. 2013); or human lymphocytes and rat bone marrow with imidacloprid formulation (Demsia et al., 
2007), as well. It is assumed that MN frequencies can often be detected at lower levels than those 
seen for chromosome aberrations because not all fragments necessarily form a visible micronucleus 
(Savage, 1988). 
 
 
Figure 1  Induction of MN in bovine peripheral lymphocytes exposed to triazole fungicide Prosaro® 
(A), triazole-morpholine fungicide Tango® (B) and neonicotinoid insecticide Calypso® (C) for 24 and 
48 h. BNMN, binucleated cell with MN; CBPI, cytokinesis block proliferation index; DMSO, 
dimethylsulfoxide, ND, not done, incomplete number of cells. Statistical significant data (χ2 test): 
* P < 0.05; ** P < 0.01; *** P < 0.001. 
 
A reduction of CBPI was observed after exposure to all fungicides tested in a dose-dependent manner 
(P < 0.05; P < 0.01 and P < 0.001) for 24 h. The same results in the inhibition of proliferation was 
obtained after the prolonged time of exposure (48 h) except the results obtained with Tango® (Fig. 1B). 
Acta fytotechn zootechn, 19, 2016(Special Issue), 93-95 
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© Slovak University of Agriculture in Nitra                                             Faculty of Agrobiology and Food Resources 
95 
Moreover, a total inhibition of proliferation activity was obtained after exposure of bovine lymphocytes 
to Prosaro® 250EC at the highest concentrations (15 and 30 µg ml-1; Fig. 1A) 
One explanation for the inhibition of cell proliferation could be the formation of reactive oxygen species 
(ROS) (Kocaman and Topaktas, 2007) as a result of oxidative stress. Another explanation for the 
antimitotic effect of tested fungicides could be in the inhibition of enzymes, which are more directly 
involved in spindle production, assembly and orientation (Hidalgo et al., 1989). Mitotic slippage as well 
as apoptosis and necrosis can change the number of cells that undergo mitosis, and thus lower 
frequency of binucleated cells could be analysed. Disorders of cell division and mitotic spindle 
apparatus could lead to aneuploidy through chromosome gain or loss. Animal aneuploidy is 
associated with reduced fertility/or increased incidence of developmental defects and birth defects. It is 
also one of the most important steps of carcinogenesis. 
 
4 Conclusions  
In conclusion, no genotoxic and clastogenic effect was obtained. Our results indicate that all tested 
pesticides are able to exert a clear cytotoxic effect in cultured bovine peripheral lymphocytes. 
 
Acknowledgments 
This study was supported by the Slovak Scientific Agency VEGA projects 1/0043/15 and 1/0176/16. 
 
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Micronucleus assay in genotoxicity assessment

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