Saudi Journal of Biological Sciences (2010) 17, 215–217 King Saud University Saudi Journal of Biological Sciences www.ksu.edu.sa www.sciencedirect.com ORIGINAL ARTICLE Thiocyclam does not induce structural chromosome aberrations in human lymphocytes in vitro Serap Celikler a, Kamel Saleh b, Mohammed A.A. Sarhan b,* a Department of Biological Sciences, Faculty of Science, Uludag University, 16059 Gorukle, Bursa, Turkey b Department of Biology, College of Science, King Khalid University, 61413 Abha, P.O. Box 9004, Saudi Arabia Received 25 November 2008; accepted 14 July 2009 Available online 13 April 2010 * E- 13 re do KEYWORDS Thiocyclam (Evisect); Chromosomal aberrations; Genotoxicity; Mitotic index; In vitro Corresponding author. mail address: mohammed_sa 19-562X ª 2010 King Saud view under responsibility of i:10.1016/j.sjbs.2010.04.004 Production and h rhan@ya Univers King Sau osting by E Abstract Thiocyclam (trade name Evisect) is a broad-spectrum nereistoxin analogue insecticide used widely for agricultural applications. The aim of this investigation was to determine its geno- toxic effects in the chromosome aberration (CA) test and determining of mitotic index (MI), using lymphocytes from peripheral blood samples of healthy human donors. A negative and a positive control (MMC) were also included. Chromosomal analyses of the metaphase plates of the samples treated with 14 different concentrations (from 0.1 to 120 lg/ml) of thiocyclam, indicating the lack effect on chromosomes. Thus thiocyclam is not genotoxic but highly toxic on cell proliferation in human lymphocytes. ª 2010 King Saud University. All rights reserved. 1. Introduction Pesticides represent a heterogeneous group of chemicals, par- ticularly designed to control undesired pests, such as insects, weeds, fungus and rodents. The term ‘‘pesticide’’ includes insecticides, herbicides, rodenticides, as well as disinfectants, fumigants and wood preservatives. These compounds have a vital role in controlling agricultural, industrial, home/garden and public health pests globally. Pesticides are considered hoo.com (M.A.A. Sarhan). ity. All rights reserved. Peer- d University. lsevier harmful pollutants according to numerous publications con- cerning their genotoxicity (Bolognesi, 2003; Grisolia, 2002). The steady increase in the use of pesticides in agriculture has drawn special attention in environmental pollution research (Kong and Ma, 1999). Biological monitoring provides a useful tool to estimate the genetic risk deriving from an integrated exposure to a complex mixture of chemicals (Bolognesi, 2003). It is important to evaluate structural and numerical chro- mosome aberrations this may be caused by pesticides using the in vitro chromosomal aberration assay because humans are exposed by inhalation to a variety of pesticides in living and working environments. The pesticide nereistoxin, a naturally occurring substance which is active at cholinergic synapses (Narahashi, 1973), was first isolated from marine annelid Lumbriconereis heteroptera by Nitta (1934). Okaichi and Hashimoto (1962a,b) synthesized the toxin and deter- mined its chemical structure as 4-N,N-dimethyl-amino-1,2- dithiolane. Much of the interest in the mechanism of action of nereistoxin stems from its high toxicity to insects caused mailto:mohammed_sarhan@yahoo.com http://dx.doi.org/10.1016/j.sjbs.2010.04.004 http://www.sciencedirect.com/science/journal/1319562X 216 S. Celikler et al. by either natural toxin or the synthesized analogues such as the commercial insecticides cartap and thiocyclam. Thiocyclam (trade name Evisect�) is a broad-spectrum nereistoxin analogue insecticide used widely for agricultural in south east region of Saudi Arabia. This insecticide is an antagonist, blocking cholinergic transmission resulting in paralysis and insect death. This insecticide is metabolically converted to nereistoxin in the insect and interacts with nico- tinic acetylcholine receptors. There are few agricultural studies examining the effects of thiocyclam (Civelek and Weintraub, 2003; Saito et al., 1992), but no studies of its genotoxic effect in literature. Thus, the aim of this study was to evaluate the genotoxic potential of thiocyclam in vitro using lymphocytes isolated from the peripheral blood of clinically healthy humans. 2. Materials and methods 2.1. Chemicals The test substance thiocyclam (Evisect�; CAS No. 31895-22-4) was purchased from Sygenta and Mitomycin-C from Kyowa (Hakko, Japan), respectively. 2.2. Insecticide solution and doses Concentrated thiocyclam stock solution (500 lg/ml) was prepared with sterile distilled water. From this stock solution, 14 different selected concentrations ranging from 0.1 to120 lg/ ml were used in the experiments. Thiocyclam solutions were prepared immediately before use, to avoid degradation. Three sets of experiments at each concentration along with equal numbers of controls were carried out for all the in vitro assays. 2.3. Human lymphocytes cultures and cell harvesting Peripheral blood cultures were prepared according to Carballo et al. (1993). Briefly, blood samples were taken with heparinized syringes from healthy, non-smoking volunteers. Whole blood (0.3 ml) was added to RPMI-1640 medium, supplemented with 20% inactivated fetal calf serum, antibiotics penicillin– streptomycin mixture (100 IU/ml; 100 lg/ml, respectively), L-glutamine (0.5 mg/ml) and phytohaemagglutinin (6 lg/ml) to stimulate cell proliferation. Human lymphocytes were incu- Table 1 The results of chromosome aberrations (CAs) and mitotic Treatments MI ± SD Number of chromoso G CB ICB E D. W. 17.66 ± 0.13 3 1 2 0 Thiocyclam 0.1 lg/ml 0.5 ± 0.15 NI NI NI N 0.5 lg/ml 0.2 ± 0.2 NI NI NI N 1–120 lg/ml (12 different doses) 0.0 ± 0.0 NI NI NI N MMCb (0.25 lg/ml) 10.46 ± 3.27 27 19 11 11 G, gap; CB, chromatid break; ICB, iso chromatid break; EXC, exchan minute; NI, not investigated; MI, mitotic index; TA (G+ P), total chromo chromosome aberration excluding gaps and pulverizations; SD, standard a Total 800 cells in each replicate. b 0.25 lg/ml MMC (mitomycin-C, positive control). bated at 37 �C for 72 h and treated with 14 different concentra- tions (0.1–120 lg/ml) of sterile thiocyclam for 24 h. Distilled water and mitomycin-C (0.25 lg/ml) were used as negative and positive control, respectively. Mitomycin-C was dissolved in distilled water. To arrest the cells in metaphase, colcemid at a final concentration of 0.2 lg/ml was added to all dishes 2 h be- fore the end of the incubation period. Chromosome preparates were prepared to routine harvest procedure (Benn and Perle, 1992). 2.4. Microscopic evaluation of chromosome aberration and mitotic index The analysis of cells with CAs was performed in 50 metaphases for each culture at each concentration. Gaps and pulveriza- tions were both included and excluded from total chromosome aberrations. CAs were classified according to the recommenda- tion of EHC (Environmental Health Criteria) 51 for Short- term Tests for Mutagenic and Carcinogenic Chemicals (IPCS, 1985). The mitotic index (MI, number of cells undergoing mitosis/ 1000 cells) was also examined. The end points analyzed were the mitotic index, the presence of structural chromosomal aberrations, which were recorded as chromatid-type (including breaks or fragments, quadriradial and triradial) or chromo- some-type (including breaks or fragments, translocations, dicentrics and rings), and aberrant metaphases. This was done one of the each of negative and positive controls and treatment groups. 3. Results The frequency of CA was determined in human lymphocytes treated with various doses of thiocyclam (0.1; 0.5; 1.0; 2.5; 5.0;10.0; 15.0; 20.0; 25.0; 40.0; 50.5; 80.0; 100.0 and 120.0 lg/ ml) for 72 h. The average of three sets of experiments is pre- sented in Table 1. MI values of treated with thiocyclam cul- tures are also shown in Table 1. The results indicated that thiocyclam had no significant influence on the frequency of CA in human lymphocyte cells as compared with the corresponding control groups. However, remarkable decrease effect of thiocyclam on MI was exhibited. Positive controls (MMC) significantly enhanced the level of the chromosome aberration (Table 1). index (MI) treated with thiocyclam in human lymphocytes. me aberrationa TA (G+ P) ± SD TA (G � P) ± SD XC SE PLV DM 0 0 0 0.06 ± 0.00 0.03 ± 0.01 I NI NI NI 0.00 ± 0.00 0.00 ± 0.00 I NI NI NI 0.00 ± 0.00 0.00 ± 0.00 I NI NI 0.00 ± 0.00 0.00 ± 0.00 8 1 1 0.39 ± 0.13 0.25 ± 0.10 ge figure; SE, spiralization error; PLV, pulverization; DM, double some aberration including gaps and pulverizations; TA (G � P), total deviation. Thiocyclam does not induce structural chromosome aberrations in human lymphocytes in vitro 217 4. Discussion The cytogenetic characterization and classification of different types of chromosomal aberrations in human peripheral blood lymphocytes have an important role in human genetics. The main chromosomal damage identification has proved the importance of this biomarker in the field of carcinogenesis and mutagenesis (Rossner et al., 2005). As seen from Table 1, the present study showed a highly cytotoxic potential of thiocyclam as demonstrated by decreases on MI. The cytotoxicity of a chemical mutagen can be esti- mated from several endpoints and one of the most used is MI (Galloway, 2000). When using lymphocyte cultures, the use of MI is acceptable as a toxicity measure (Galloway et al., 1994). The results of the study reveal that the selected dosages of thiocyclam were not genotoxic on human lymphocytes in vitro. Chromosome aberrations (CAs) were not observed at the low or high doses after 72 h treatment. This finding is in agreement with neural effect of thiocyclam. Nereistoxins af- fect to spinal reflexivity (Chiba and Nagawa, 1971). So, target organs of thiocyclam are spinal cord and cellular receptors, DNA and the other division material are not. In the present study, this effect of thiocyclam or its metabolite nereistoxin was exhibited by blocking the cellular division in all doses of thiocyclam. In this investigation, we assessed the induction of CAs and its cytotoxic effects using MI parameters in human lympho- cytes in vitro. Since the majority of this pesticide has cellular division blocking properties, persons eat crops treated with pesticides and the employees in drug manufacturing which are exposed to this drugs are at risk of cellular and spinal dam- age. Therefore, it is suggested that humans must avoid uncon- sciously use. References Benn, P.A., Perle, M.A., 1992. Chromosome staining and banding techniques. In: Rooney, B.H.C.D.E. (Ed.), Human Cytogenetics: A Practical Approach I–II, vol. 57–83. IRL Press, Oxford. Bolognesi, C., 2003. Genotoxicity of pesticides: a review of human biomonitoring studies. Mutat. Res. 543, 251–272. Carballo, M.A., Alvarez, S., Boveris, S., 1993. Cellular stress by light and Rose Bengal in human lymphocytes. Mutat. Res. 288, 215–222. Chiba, S., Nagawa, Y., 1971. Effects of nereistoxin and its derivatives on the spinal cord and motor nerve terminals. Jpn. J. Pharmacol. 21, 175–184. Civelek, H.S., Weintraub, P.G., 2003. Effects of bensultap on larval serpentine leafminers, Liriomyza tirfolii (Burgess) (Diptera: Agro- myzidae), in tomatoes. Crop Prot. 22, 479–483. Galloway, S.M., 2000. Cytotoxicity and chromosome aberrations in vitro: experience in industry and the case for an upper limit on toxicity in the aberration assay. Environ. Mol. Mutagen. 35, 191– 200. Galloway, S.M., Aardema, M.J., Ishidate Jr., M., Ivett, J.L., Kirkland, D.J., Morita, T., Mosesso, P., Sofuni, T., 1994. Report from working group on in vitro tests for chromosomal aberrations. Mutat. Res. 312, 241–261. Grisolia, C.K., 2002. A comparison between mouse and fish micro- nucleus test using cyclophosphamide, mitomycin C and various pesticides. Mutat. Res. 518 (2), 145–150. IPCS, 1985 International Programme on Chemical Safety: Environ- mental Health Criteria 46. Guidelines for the Study of Genetic Effects in Human Populations. WHO, Geneva, pp. 25–54. Kong, M.S., Ma, T.H., 1999. Genotoxicity of contaminated soil and shallow well water detected by plant bioassays. Mutat. Res. 426 (2), 221–228. Narahashi, T., 1973. Mode of action of nereistoxin on excitable tissues. In: Marine Pharmacognosy. Actions of Marine Biotoxins at the Cellular Level. Academic Press, New York, pp. 107–126. Nitta, S., 1934. Ober Nereistoxin, einen giftigen Bestandteil von Lumbriconereis heteropoda Marenz (Eunicidae). Yakagaku Zasshi 54, 648–652. Okaichi, T., Hashimoto, Y., 1962a. Physiological activities of nereis- toxin. Bull. Jpn. Soc. Fish. 28, 930–935. Okaichi, T., Hashimoto, Y., 1962b. The structure of nereistoxin. Agric. Biol. Chem. 26, 224–227. Rossner, P., BoVetta, P., Ceppi, M., Bonassi, S., Smerhovsky, Z., Landa, K., Juzova, D., Sram, R.J., 2005. Chromosomal aberra- tions in lymphocytes of healthy subjects and risk of cancer. Environ. Health Perspect. 113, 517–520. Saito, T., Oishi, T., Ikeda, F., Sawaki, T., 1992. Effect of insecticides on the serpentine leafminer, Liriomyza trifolii (Burgess) (Diptera: Agromyzidae). Jpn. J. Appl. Entomol. Zool. 36, 183–191. Thiocyclam does not induce structural chromosome aberrations in human lymphocytes in vitro Introduction Materials and methods Chemicals Insecticide solution and doses Human lymphocytes cultures and cell harvesting Microscopic evaluation of chromosome aberration and mitotic index Results Discussion References