Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 72(2): 21-27, 2019 Firenze University Press www.fupress.com/caryologiaCaryologia International Journal of Cytology, Cytosystematics and Cytogenetics ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/caryologia-698 Citation: A. Özkara (2019) Assess- ment of cytotoxicity and mutagenicity of insecticide Demond EC25 in Allium cepa and Ames Test. Caryologia 72(2): 21-27. doi: 10.13128/caryologia-698 Published: December 5, 2019 Copyright: © 2019 A. Özkara. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distri- bution, and reproduction in any medi- um, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Assessment of cytotoxicity and mutagenicity of insecticide Demond EC25 in Allium cepa and Ames Test Arzu Özkara Department of Molecular Biology and Genetic, Faculty of Arts and Sciences, Afyon Kocatepe University, Afyonkarahisar/TURKEY E-mail: ozkara@gmail.com Abstract. The mutagenicity and cytotoxicity of Demond EC25, a synthetic pyrethroid insecticide, was assessed using two standard genotoxicity assays of the Salmonella typhimurium mutagenicity assay (Ames test) and Allium cepa test. Cytogenetic effects of Demond EC25 were evaluated in the root meristem cells of Allium cepa. The test concentrations of compounds were selected by determining EC50 of the Allium root growth and onion seeds were exposed to Demond EC25 (50, 100, and 200 ppm) for 24, 48, and 72 hours. The concentrations Demond EC25 was compared with the value for the negative control using Dunnet-t test, 2 sided. The results indicated that mitot- ic index was clearly decreased with increasing the concentration of Demond EC25 in each treatment group as compared to the controls. Demond EC25 was tested for muta- genicity in bacterial reversion assay systems with two strains (TA98 and TA100) of Sal- monella typhimurium absence and presence of S9 fraction. The doses of Demond EC25 were 50, 100, 200, 400, 800 µg/plate and test materials were dissolved in DMSO. Our results show that Demond EC25 was found to be mutagenic in 800 and 400 μg/plate doses of TA98 in the without S9 mix and 800 μg/plate in the with S9 mix. In TA100, Demond EC25 was found to be mutagenic only 800 μg/plate doses without S9 mix. The other doses of this insecticide was not found to be mutagenic in both test strains. Keywords. Allium test, Ames test, cytotoxicity, Demond EC25, mutagenicity, pesti- cide. INDRODUCTION Pyrethroids are among the most commonly used insecticides in agricul- ture; they are also widely used indoors in pet shampoo, lice treatment, and even insect repellent (Saillenfait et al. 2015). They are therefore frequently present in food, air and dust of dwellings and thus can lead to both dietary and non-dietary exposure (Morgan 2012). Pyrethroids are botanical insecti- cides which are synthetic derivatives of pyrethrins and have been used for many years. However, most of pyrethroids are defined as moderately hazard- ous (Class II) by the World Health Organization (WHO 2009) (Jensen et al. 2011). The residues of pyrethroids have been detected in fruits, vegetables, 22 Arzu Özkara tea, pasteurized milk and porcine muscle (Nakamuraet al. 1993). Wider use of pyrethroids posed a serious risk to environment and human. Therefore, it may be an urgent need to evaluate the possible adverse effects of their use (Miao et al. 2017) Pyrethroid pesticides disrupt the nervous system of insects and, to a lesser degree, of mammals, and thus raise human health concerns. (Oulhote and Bouchard 2013; Viel et al. 2015). Pyrethroid residual insecticides exert their toxic effects by targeting the nervous system of insects. Pyrethroids interfere with sodium channels in nerve fiber membrane and organophosphates bind to inhibit the activity of AChE found in the synaptic junction. Both actions result in continued nerve signal- ing and over-stimulation of nerve cells. Poisoned insect exhibits tremors and convulsions, eventually leading to death (ATSDR 2003; Valles and Koehler 2003). It is essential to carefully study and analyze the hazards of pyrethroids on human health including their genotoxic and cytotoxic properties. Hereby, it can be take adequate measures to prevent humans from potential mutagenic and carcinogenic effects. (Nagy et al. 2014). Deltamethrin is a synthetic pyrethroid insecticide, sold by Safa Tarım Limited with trade names Demond EC 25 in local market. To our knowledge, there is no study mutagenicity of Demond EC 25 except in the pre- sent paper. The aim of this experiment was to evalu- ate both the mutagenic and cytotoxic effects of different doses of Demond EC 25 by the bacterial reverse mutation assay in S. typhimurium TA98 and TA100 strains with or without S9 mix and Allium cepa test, respectively. MATERIAL METHOD Chemicals The test substance Demond EC25 was purchased from a local market in Afyonkarahisar/Turkey and dis- solved in sterille distilled water. Allium cepa onion bulbs, 25–30 mm diameter, were obtained from a local market without any treatments. The other chemicals were obtained from Merck and Riedel. Test strains The LT-2 TA98 and TA100 histidine demanding auxotrophs of S. typhimurium were kindly obtained from Prof. B.N. Ames (University of California, Berke- ley). These strains were incubated for 16h in liquid nutri- ent broth and kept at -80°C. Their genetic markers and other properties, such as the numbers of spontaneous revertants and responses to positive controls, were con- trolled as described by Maron and Ames (1983). Allium Test EC50 determination and mitotic index analysis The procedure of the root inhibition test as described by Fiskesjo (1985) was followed with some modifications. The Allium root inhibition test was car- ried out to determine suitable concentrations for the genotoxicity assay. The outer scales of the bulbs and the dry bottom plate were removed without destroying the root primordia. The onions were grown in freshly dis- tilled water for the first 24h and afterwards exposed for 96h to the Demond EC25 solutions (12.5, 25, 50, 100, and 200 ppm, respectively). In order to determine the EC50 values, the roots from each bundle were cut off on the fifth day and the length of each root was measured from both the Demond EC25 exposed bulbs and the control group. The EC50 value was considered as the con- centration which retards the growth of the root 50% less when compared to the control group. The EC50 value for Demond EC25 was approxi- mately 100 ppm. In order to demonstrate possible con- centration-dependent effects of this pesticide, the root tips were treated with 50 ppm (EC50/2), 100 ppm (EC50), 200 ppm (EC50x2) concentrations of Demond EC25, and all application groups were tested 24, 48, and 72h treat- ment periods. Additionally we also used positive control group by using methyl methanesulfonate (MMS). After the treatment, the roots were washed in distilled water and fixed in 3:1 ethanol: glacial acetic acid for 24h and then the roots were transferred into 70% alcohol and stored at +4°C. The root tip cells were stained with Feul- gen and five slides were prepared for each test group. Ames Salmonella/Microsome Assay The mutagenicity of the Demond EC25 was deter- mined using the standard plate incorporation assay. Salmonella typhimurium strains TA98 and TA100 were used with or without S9 mix in this test (Ames et al. 1975; Maron and Ames 1983). The tester strains were tested for the presence of the strain-specific markers as described by Maron and Ames (1983). The cytotoxic doses of the Demond EC25 (800, 400, 200, 100, 50 µg/ plate) were determined by the method of Dean et al. (1985). The stock solutions of the test materials were dis- solved in sterile distilled water and stored at 4°C. The S. typhimurium strains were incubated in nutrient broth at 37°C for 16h with shaking. The positive controls were 23Assessment of cytotoxicity and mutagenicity of insecticide Demond EC25 in Allium cepa and Ames Test 4-nitro-o-phenylenediamine (NPD) for the TA 98 and sodium azide (SA) for the TA100, used without meta- bolic activation, and 2-aminofluorene (AF) for TA 98 and 2-aminoanthracene (2AA) for the TA 100 used with metabolic activation. The test plates for the assays without the S9 mix were prepared by adding 0.1 ml of the test suspension for each concentration, 0.1 ml bacterial suspension from an overnight culture, and 0.5 ml phosphate buffer to 2 ml top agar (kept in 45°C water bath). The mixture was shaken for 3 s using a vortex mixer and then poured into the minimal agar. The test plates with the S9 mix were prepared by adding 0.5 ml of S9 mix instead of the phosphate buffer. All the test plates were incubated for 72h at 37°C, and then the revertant colonies on each plate were counted. The experiments were run in tripli- cate for each concentration and all the results from the two independent parallel experiments were used for the statistical analysis. Statistical analysis The data obtained for the root length, MI, and mitotic phases were expressed as percentages. The levels of difference in the treatment groups were analyzed sta- tistically by using the SPSS 15.0 version for Windows. In the analyses, the Dunnett-t test (2 sided) was performed on both the Allium and Ames tests. RESULTS Allium root growth test results are summarized in Table 1 and Table 2 gives the effect of Demond EC25 on MI and mitotic phase in the root meristematic cells of A. cepa treated for 24, 48 and 72h. The effective con- centration (EC50) was determined as 100 ppm in Allium test. At all concentrations treated in the incubations of root decreased MI compared to negative control at all exposure time. The reduced of MI results (p<0.05) were found statistically significant with all concentra- tions and all treatment time. All doses of Demond EC25 applied in the experiment caused changes in the per- centage of particular phases’ distribution in comparison to the control. Table 1. Allium root growth inhibition test. Test Substance Concentrations (ppm) Mean of root length±SD Negative Control - 3.57±0.24 Positive Control - 1.03±0.15* Demond EC25 12.5 3.12±0.42* 25 2.02±0.15* 50 1.68±0.41* 100 1.45±0.23* 200 1.12±0.22* *Significantly different from negative control (p<0.05 Dunnet-t test, 2-sided), SD: Standart deviation. Table 2. The effects of Demond EC25 on MI and mitotic phases in the root cells of A. cepa. Concentration (ppm ) Treatment Time Counted Cell Number Mitotic Index ± SD Mitotic Phases (%) ± SD Prophase Metaphase Anaphase Telophase Negative control 24 hour 4965 82.45±6.71 79.12±9.42 1.80±0.32 1.12±0.32 0.92±0.57 Positive control 5001 68.78±5.46* 34.24±4.62* 0.48±0.70* 0.49±0.54* 0.52±0.81 50 4889 51.48±4.09* 34.05±2.17* 1.00±0.72* 0.79±0.21* 1.10±0.62 100 4963 46.25±4.74* 32.54±4.20* 0.94±0.42* 0.68±0.34* 1.03±0.42 200 5013 45.21±2.69* 30.21±2.54* 0.82±0.40* 0.52±0.21* 0.59±0.74 Negative control 48 hour 5007 67.28±3.47 70.11±6.74 1.62±0.21 1.27±0.24 1.21±0.26 Positive control 4997 63.11±3.14* 31.75±3.45* 0.45±0.31* 0.52±0.16* 0.65±0.13 50 5101 52.25±3.45* 30.26±3.35* 0.71±0.92* 0.62±0.21* 1.19±0.21 100 5051 42.42±3.65* 28.45±2.70* 0.68±0.40* 0.54±0.02* 1.06±0.32 200 5113 36.20±1.25* 24.45±2.92* 0.52±0.32* 0.45±0.18* 0.89±0.14 Negative control 72 hour 5142 38.21±2.65* 57.52±3.41 1.43±0.41 1.21±0.21 1.09+±0.52 Positive control 5123 26.36±3.02* 29.04±2.28* 0.31±0.43* 0.60±0.42* 0.68±0.32 50 5263 19.23±1.75* 25.45±3.85* 0.58±0.23* 0.52±0.45* 0.49±0.41 100 5047 14.12±2.42* 21.42±2.56* 0.49±0.41* 0.46±0.71* 0.50±0.72 200 4985 13.21±2.21* 16.47±2.31* 0.34±0.42* 0.39±0.43* 0.56±0.61 * Significantly different from negative control (p< 0.05 Dunnet-t test. 2-sided) SD: Standart deviation. 24 Arzu Özkara The results of the Ames test are shown in Table 3. In this experiment, first, the cytotoxic doses of Demond EC25 were determined. As seen in Table 3, spontane- ous revertants were within the normal values in all the strains examined. All of the doses with and without S9 mix in TA98 and TA100 slightly increased when com- pared to the negative control. On the other hand, the plates containing positive control mutagens displayed very significant increases in the spontaneous mutation rate in two strains tested. Most of the results, whether increasing or decreasing relative to the negative control group, were not statistically significant at P<0.05 (Dun- nett-t test, 2 sided) in the examined strains, except for in the 800 and 400 μg/plate doses of the Demond EC25 in the TA98 without S9 mix and 800 μg/plate doses with S9 mix. Additionally it was obtained mutagenic in the TA100 without S9 mix 800 μg/plate doses. DISCUSSION Pyrethroid insecticides are commonly used in agri- culture, veterinary medicine, and to control insect pests in human dwellings because of their high selective toxic- ity for insects and relatively low acute toxicity to mam- mals (Casida and Quistad 1998). These insecticides are favored because of their effective role and have replaced organophosphorus pesticides in many areas of applica- tions (Ministry of the Environment in Japanese 2011). Because of their advantages, pyrethroid insecticides including Demond EC25 are becoming widespread and, therefore, studies on the biological effects of these pes- ticides are of immediate concern. Numerous studies on their toxicity, both in insects and mammals, have been reported in the literature. Although pyrethroid insecti- cides have consistently shown negative results in micro- bial genotoxicity tests, the outcome of other assays has been variable and it has not been possible to draw defi- nite conclusions about the genotoxicity of this group of pesticides (Grossman 2007; Surralles et al. 1995). In determining mutagenicity of chemicals, the Ames test has shown a variety of chemicals to be either mutagenic or anti-mutagenic, and has been shown to be over 90% accurate in predicting genotoxicity (Weis- burger 2001). In the Ames test, S. typhimurium strains that have a mutation in the his-operon are used to detect the mutagenicity of chemicals (Maron and Ames 1983). In the present study, Demond EC25 was studied for its mutagenic activity with the Ames test and results can be concluded that Demond EC25 induced mutations in the 800 and 400 μg/plate doses of the TA98 without S9 mix and 800 μg/plate doses with S9 mix and in the TA100 without S9 mix. Under our experimental conditions, Demond EC25 showed to produce point mutations in the Ames test, both in the absence and presence of the S9 metabolic activation system in high concentrations of both test strains. In order to characterize the possible mechanism of mutagenicity, the important bacterial strains, sensitive to different mutational events due to their specific geno- types, were used. Particularly, S. typhimurium TA98 is characterized by the -1 frameshift deletion hisD3052, which affects the reading frame of a nearby repetitive –C–G– sequence Table 3.The mutagenicity assay results of Demond EC25 for S. tyhimurium TA98 and TA100 strains Test Substance Concentration (µg/ plate) No of His+ revertants/plate, mean±SD TA98 TA100 - S9 + S9 - S9 + S9 Demond EC25 800 95.32±5.41* 116.42±5.52* 206.45±9.44* 215.52±12.85 400 88.04±4.13* 102.21±3.96 178.42±7.45 203.12±10.25 200 68.12±4.63 92.54±4.25 142.45±6.74 184.32±9.54 100 52.09±3.86 78.09±4.52 121.22±6.61 168.35±8.65 50 47.31±3.38 56.24±4.45 102.10±5.08 123.09±6.85 Neg. Control 100 36.07±3.36 49.14±3.70 90.10±13.42 114.23±7.38 SA 10 2965.56±56.35* 2AA 5 2628.42±60.41* 2AF 200 1002.40±16.65* NPD 200 1575.50±24.56* *Mean statistically significant at p<0.05 (Dunnett t-test), SA:Sodium azide, NPD: 4-nitro-o-phenylendiamine, 2AF: 2-aminofluorene, 2AA: 2-aminoanthracene, SD: Standard deviation, Negative control: distilled water. 25Assessment of cytotoxicity and mutagenicity of insecticide Demond EC25 in Allium cepa and Ames Test and can be reverted by frameshift mutagens. TA100 con- tains the marker hisG46, which results from a base-pair substitution of a leucine (GAG/CTC) by a proline (GGG/ CCC): this mutation is reverted by mutagens caus- ing base substitutions at G-C base pairs (Di Sotto et al. 2008). Taking into account these bacterial features, our results highlighted that the Demond EC25 mutagenic- ity, in the absence and presence of S9 in TA98, was likely due to frameshift mutations, and in the absence S9 in TA100 due to base-change mechanisms. The data reported on the genotoxicity of synthetic pyrethroids are rather controversial, depending on the genetic system used (Akintonwa et al. 2008; Saleem et al. 2014). Studies have shown an important relationship between a substance’s chemical structure and its biologi- cal activity (Oztas¸ 2005) chlor. Several factors, includ- ing rings, the functional groups, and the positions of binding locations in the chemical structure may affect a chemical’s binding ability. Mitotic index proved to be a useful parameter that allows one to detect the frequency of the cellular divi- sion (Marcano et al. 2004). The estimation of the poten- tial cytotoxicity of the compounds is generally related to the inhibition of the mitotic activities (Smaka-Kincl et al. 1996). In this study, the used concentrations of Demond EC25 also caused significant inhibition of the mitotic index. The significant decline in the mitotic index could be due to the inhibition of the DNA syn- thesis or the blocking of the G1 suppressing the DNA synthesis or effecting the test compound at the G2 phase of the cell cycle (Sudhakar et al. 2001; Majewska et al. 2003). When a pesticide penetrates the cells and reaches a critical concentration, it could be in an active form, causing lesions during several following cellular cycles (Marcano et al. 2004). The decrease of the mitotic index in our study can be related to this. In this study, all the concentrations of Demond EC25 caused the changes in the percentage of the par- ticular phases’ distribution when compared to the con- trol group. Pesticides accumulate in the cell due to this substance not being able to emerge out of the cell easily after once penetrating the cell and it may be highly toxic in the cell (Antunes-Madeira and Madeira 1979). Deltamethrin, the active ingredient in Demond EC25 has immunosuppressive (Lukowicz and Krech- niak, 1992), reproductive effects on sperm cells (Bhu- nya and Pati 1990; Carrera et al. 1996) and developmen- tal toxicity (Martin 1990). Deltamethrin is reported to cause chromosomal damage in Allium cepa (Chauhan et al. 1986), chromosomal aberrations and micronucleus formation in bone marrow cells of mice exposed in vivo (Chauhan et al. 1997; Gandhi et al. 1995). Saxena et al. (2009) evaluated of cytogenetic effects of deltamethrin in root meristem cells of Allium sativum and Allium cepa and cells analyzed immediately after the exposure showed a significant, concentration-dependent inhibi- tion of mitotic index (MI) and induction of mitotic and chromosomal aberrations in both the test systems. Addi- tionally, in vitro exposure of Deltamethrin is reported to cause DNA damage in Comet assay in human periph- eral blood leukocytes (Villarini et al. 1998). In the pre- sent study with Allium cepa root tip meristem cells how- ever, the three concentrations of Demond EC25 tested induced genotoxicity thus corroborating the findings of these studies. In contrast to our results, no genotoxic response of Deltamethrin was observed in Salmonella typhimu- rium and V79 Chinese hamster ovary cells (Pluijmen et al. 1984). Data on the genotoxicity and carcinogenicity of Deltamethrin are rather controversial, depending on the genetic system or the assay used (Shukla and Taneja, 2000). The safety evaluation of a fragrance material includes a broad range of toxicological information, both for the compound itself and for structurally related chemicals belonging to the same chemical group (Bick- ers et al., 2003). Among toxicological information, geno- toxicity is a systemic consideration, as it can be related to carcinogenicity (Di Sotto et al. 2008). Normally, to evaluate a potential genotoxic risk due to a chemical exposition, in vitro assays for detecting point mutations (Ames test) and extended treatment (e.g., micronucleus assay, Allium test, single cell gel electrophoresis assay or comet assay) are used in the first instance (EMEA 2008; Di Sotto et al. 2013). If the results of these studies are positive, in vivo studies, for example a mammalian cytogenetic study, are performed (EFSA 2014). The tested substances with different test systems can be genotoxic or not genotoxic depending on a num- ber of factors such as chemical structure and biological activity, having rings in the structure and the positions of the binding location (Kutlu et al. 2011). In addition to these, it might be related to differences in test condi- tions, such as exposure time, cell types, concentrations of substances, the dispersal of the materials and physico- chemical characteristics of the compounds (Ema et al. 2012).Therefore, it could be explained why some studies find an increase of genetic damage while in others result as negative. In conclusion, Demond EC25 was found to be cyto- toxic due to decreasing of MI in Allium test and showed mutagenic activity at some doses in the Ames test. Demond EC25 had clear cytotoxic effects and may pose a genotoxic risk for humans. For this reason, further 26 Arzu Özkara investigations are needed to determine the toxicity of this compound using other in vivo and in vitro biological test systems. A single test system is not enough to deter- minate a compound whether it is toxic or non-toxic. In this study we performed two different test methods. Fur- ther investigations are needed to determine the toxicity of this compound using multiple test systems. REFERENCES Agency for Toxic Substances and Disease Registry (ATS- DR). 2003. Toxicological profile for pyrethrins and pyrethroids. Atlanta, GA: Department of Health and Human Services, Public Health Service, Toxicology. Akintonwa A, Awodele O, Olayemi SO, Oreagba IA, Olaniyi OM. 2008. The mutagenic testing of different brands of commonly used insecticides. African J Bio- tech. 7: 2134–2136. Ames BN, McCann J, Yamasaki E. 1975. Methods for detecting carcinogens and mutagens with the Sal- monella/mammalian-microsome mutagenicity test. Mutat Res. 31: 347–364. Antunes-Madeira MC, Madeira VMC. 1979. Interaction of insecticides with lipidmembranes. Biocihim Bio- phys Acta. 550: 384–392. Bhunya SP, Pati PC. 1990. Effect of deltamethrin, a syn- thetic pyrethroid, on the induction of chromosome aberrations, micronuclei and sperm abnormalities in mice. Mutagenesis 5: 229–232. Bickers DR, Calow P, Greim HA, Hanifin JM, Rogers AE, Saurat JH, Sipes IG, Smith RL, Tagamii H. 2003. The safety assessment of fragrance materials. Regul Toxi- col Pharmacol. 37: 218-273. Carrera A, Moos J, Ning XP, Gerton GL, Tesarik J, Kopf GS, Moss SB.1996. Regulation of protein tyrosine phosphorylation in human sperm by a calciumrcalm- odulin dependent mechanism: identification of A kinase anchor proteins as major substrates for tyros- ine phosphorylation. Dev Biol. 180: 284–296. Casida J, Quistad G. 1998. Golden age of insecticide research: past, present, or future? Annu Rev Entomol. 43: 1–16. Chauhan LKS, Agarwal DK, Sundaraman V. 1997. In vivo induction of sister chromatid exchange in mouse bone marrow following oral exposure to commer- cial formulations of alpha-cyano pyrethroids. Toxicol Lett. 93: 153–157. Chauhan LKS, Dikshith TSS, Sundaraman V. 1986. Effect of deltamethrin on plant cells I cytological effects on the root meristem of Allium cepa. Mutat Res. 171: 25–30. Di Sotto A, Evandri MG, Mazzanti G. 2008. Antimuta- genic and mutagenic activities of some terpenes in the bacterial reverse mutation assay. Mutat Res. 653: 130-133 Di Sotto A, Maffei F, Hrelia P, Castelli F, Sarpietro MG, Mazzanti G. 2013. Genotoxicity assessment of b-car- yophyllene oxide. Regul Toxicol Pharmacol. 66: 264- 268. Ema M, Imamura T, Suzuki H. 2012. Evaluation of geno- toxicity of multi-walled carbon nanotubes in a bat- tery of in vitro and in vivo assays. Regul Toxicol Pharmacol. 63: 188–195. EMEA, 2008. Committee on Herbal Medicinal Products (HMPC), Guideline on the Assessment of Geno- toxicity of Herbal Substances/Preparations. EMEA/ HMPC/ 107079/2007. European Food Safety Authority CEF Panel (EFSA Pan- el on Food Contact Materials, Enzymes, Flavour- ings and Processing Aids). 2014. Scientific opin- ion on flavouring group evaluation 213, revision 1 (FGE.213Rev1): consideration of genotoxic potential for a,b-unsaturated alicyclic ketones and precursors from chemical subgroup 2.7 of FGE.19. EFSA J. 12: 3661-3707. Fiskesjo G. 1985. The Allium test as a standart in envi- ronmental monitoring. Hereditas. 102: 99–112. Gandhi G, Chowdhury JB, Sareen PK, Dhillon VP.1995. Genotoxic effects of deltamethrin in the mouse bone marrow micronucleus assay. Mutat Res. 346: 203– 206. Grossman N. 2007. Influence of pyrethroids and pipero- nyl butoxide on histamine release from isolated rat mast cells. Inflamm Res. 56: 473–478. Jensen HK, Konradsen F, Dalsgaard A. 2011. Pesticide use and self-reported symptoms of acute pesticide poisoning among aquatic farmers in Phnom Penh, Cambodia. Int J Toxicol. 1687-8191. Kutlu M, Öztaş E, Aydoğan G. 2011. An investigation of mutagenic activities of some 9-substitued phenan- threne derivatives with Ames/Salmonella/microsome test. Anadolu University J Sci Tech. 1: 83–94. Lukowicz RJ, Krechniak J. 1992. Effects of deltamethrin on the immune system in mice. Environ Res. 59: 467–475. Majewska AE, Wolska E, Sliwinska M, Furmanowa N, Urbanska A, Pietrosiuk A, Zobel A, Kuran M. 2003. Antimitotic effect, G2/M accumulation, chromosom- al and ultrastructure changes in meristematic cells of Allium cepa L. root tips treated with the extract from Rhadiola rosea roots. Caryologia. 56: 337–351. Marcano L, Carruyo I, Del Campo A, Montiel X. 2004. Cytotoxicity and mode of action of maleic hydrazide 27Assessment of cytotoxicity and mutagenicity of insecticide Demond EC25 in Allium cepa and Ames Test in root tips of Allium cepa L. Environ Res. 94: 221- 226. Maron DM, Ames BN. 1983. Revised methods for the Salmonella mutagenicity test. Mutat Res. 113: 173– 215. Martin PA.1990. Effects of carbofuran, chlorpyrifos and deltamethrin on hatchability, deformity, chick size and incu- bation time of Japanese quail Coturnix japonica. eggs, Environ Toxicol Chem. 9: 529–534. Miao J, Wang D, Yan J, Wang Y, Teng M, Zhou Z, Zhu W. 2017. Comparison of subacute effects of two types of pyrethroid insecticides using metabolomics methods. Pest Biochem Physiol. 143: 161–167 Ministry of the Environment. 2011. Compilation of pol- lutant release and transfer register data Ministry of the Environment; in Japanese. Available from: http: //www.env.go.jp/chemi/prtr/result/todokedegaiH22/ suikei/sanko3.pdf. Morgan MK. 2012. Children’s exposures to pyrethroid insecticides at home: a review of data collected in published exposure measurement studies conducted in the United States. Int J Environ Res Public Health. 9: 2964–2985. Nagy K, Racz G, Matsumoto T, Adany R, Adam B. 2014. Evaluation of the genotoxicity of the pyrethroid insecticide phenothrin. Mutat Res Genet Toxicol Environ Mutagen. 770: 1–5. Nakamura Y, Tonogai Y, Tsumura Y, Ito Y. 1993. Deter- mination of pyrethroid residues in vegetables, fruits, grains, beans, and green tea leaves: applications to pyrethroid residue monitoring studies. J AOAC Int. 76: 1348–1361. Oulhote Y, Bouchard MF. 2013. Urinary metabolites of organophosphate and pyrethroid pesticides and behavioral problems in Canadian children. Environ Health Perspect. 121: 1378–1384. Oztas E. 2005. Bazı 9-substitiue fenantren turevlerinin mutajenik aktivitelerinin Ames/Salmonella/mikro- zom testi ile araştırılması. Eskisehir, Yuksek Lisans Tezi, Anadolu Universitesi, Fen Bilimleri Enstitusu. Pluijmen M, Drevon C, Montesano R, Malaveille C, Hautefeuille A, Bartsch H. 1984. Lack of mutagenic- ity of synthetic pyrethroids in Salmonella typhimuri- um strains and in V79, Chinese hamster ovary cells. Mutat Res. 137: 7–15. Saleem U, Ejaz S, Ashraf M, Omer MO, Altaf I, Batool Z, Fatima R, Afzal M. 2014. Mutagenic and cytotoxic potential of Endosulfan and Lambda-cyhalothrin – in vitro study describing individual and combined effects of pesticides. J Environ Sci. 26: 1471–1479. Saxena PN, Chavalı M, Gupta SK. 2009. Evaluation of cytogenetic effects of deltamethrin in root meristem cells of Allium sativum and Allium cepa: A possible mechanism of chromosome damage.Toxicol Environ Chem. 91(3): 577-594. Shukla Y, Taneja P. 2000. Mutagenic evaluation of del- tamethrin using rodent dominant lethal assay. Mutat Res. 467: 119–127. Smaka-Kincl V, Stegnar P, Lovka M, Toman MJ. 1996. The evaluation of waste, surface and ground water quality using the Allium test procedure. Mutat Res. 368: 171-179. Sudhakar R, Gowda N, Venu G. 2001. Mitotic abnormali- ties induced by silk dyeing industry effluents in the cells of Allium cepa. Cytologia. 66: 235–239. Surralles J, Xamena N, Creus A, Catalan J, Norppa H,Marcos R.1995. Induction of micronuclei by five pyrethroid insecticides in whole-blood and isolated human lymphocyte cultures. Mutat Res. 341: 169– 184. Valles SM, Koehler PG. 2003. Insecticides used in the urban environment: mode of action (ENY282). Gainesville, FL: Department of Entomology and Nematology University of Florida. Viel JF, Warembourg C, Le Maner-Idrissi G, Lacroix A, Limon G, Rouget F, Monfort C, Durand G, Cordier S, Chevrier C. 2015. Pyrethroid insecticide exposure and cognitive developmental disabilities in children: the PELAGIE motherchild cohort. Environ Int. 82: 69–75. Villarini M, Moretti M, Pasquini R, Scassellati-Sforzolini G, Fatigoni C, Marcarelli M, Monarca S, Rodroguez AV.1998. In vitro genotoxic effects of the insecticide deltamethrin in human peripheral blood leucocytes: DNA damage ‘comet’ assay in relation to the induc- tion of sister- chromatid exchanges and micronuclei. Toxicol. 130: 129–139. Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Volume 72, Issue 2 - 2019 Firenze University Press Karyotype analysis of a natural Lycoris double-flowered hybrid Jin-Xia Wang1, Yuan-Jin Cao1, Yu-Chun Han1, Shou-Biao Zhou1,2, Kun Liu1,* Insights on cytogenetic of the only strict African representative of genus Prunus (P. africana): first genome size assessment, heterochromatin and rDNA chromosome pattern Justine Germo Nzweundji1, Marie Florence Sandrine Ngo Ngwe2, Sonja Siljak-Yakovlev3,* Assessment of cytotoxicity and mutagenicity of insecticide Demond EC25 in Allium cepa and Ames Test Arzu Özkara Cytogenetic effects of Fulvic acid on Allium cepa L. root tip meristem cells Özlem Sultan Aslantürk Evaluation of the cytotoxic and genotoxic potential of some heavy metals by use of Allium test Ioan Sarac1, Elena Bonciu2,*, Monica Butnariu1, Irina Petrescu1, Emilian Madosa1 Fluorescence In Situ Hybridisation Study of Micronuclei in C3A Cells Following Exposure to ELF-Magnetic Fields Luc Verschaeve1,2,*, Roel Antonissen1, Ans Baeyens3, Anne Vral3, Annemarie Maes1 Phytochemical analysis and in vitro assessment of Polystichum setiferum extracts for their cytotoxic and antimicrobial activities Nicoleta Anca Şuţan1,*, Irina Fierăscu2, Radu Fierăscu2, Deliu Ionica1, Liliana Cristina Soare1 Telomeric heterochromatin and meiotic recombination in three species of Coleoptera (Dorcadion olympicum Ganglebauer, Stephanorrhina princeps Oberthür and Macraspis tristis Laporte) Anne-Marie Dutrillaux, Bernard Dutrillaux* A whole genome analysis of long-terminal-repeat retrotransposon transcription in leaves of Populus trichocarpa L. subjected to different stresses Alberto Vangelisti#, Gabriele Usai#, Flavia Mascagni#, Lucia Natali, Tommaso Giordani*, Andrea Cavallini Differences in C-band patterns between the Japanese house mice (Mus musculus) in Hokkaido and eastern Honshu Hikari Myoshu, Masahiro A. 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