Genotoxic Effects of Avenoxan on Allium cepa L. and Allium sativum L.

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1 CARYOLOGIA Vol. 59, no. 3: , 2006 Genotoxic Effects of Avenoxan on Allium cepa L. Allium sativum L. Tartar Gul, Fisun Kaymak 1* Fulya D. Gokalp Muranli 2 1 Trakya University, Faculty of Arts Sciences, Department of Biology, Edirne, TURKIYE. 2 Trakya University, Faculty of Arts Sciences, Department of Biology, Edirne, TURKIYE, Tel: , Fax: , fulyad1@yahoo.com. Abstract Abstract - In the present study, the cytogenetic effects of the herbicide Avenoxan, active substance 2,4- D, were investigated in both Allium cepa L. Allium sativum L. The roots of the plants were treated with 0,1%, 0,2% 0,4% concentrations of Avenoxan for 3, 6 12 hours. Root tips after having grown to a certain length were stained according to aceto orcein squash procedure. All of the concentrations of Avenoxan used in the present study significantly induced abnormalities such as c-mitosis, chromosome stickiness, bridges, laggards, multipolar cells compared to control. Also, Avenoxan significantly decreased mitotic index. Inhibition of the mitotic index was dependent on the concentration time of treatment. Similar results were obtained in both Allium cepa L. Allium sativum L. Key words: Chromosome abnormalities, 2,4-D herbicides, genotoxicity. INTRODUCTION The indiscriminate use of pesticides herbicides in agriculture, as well as the increase of pollution in ecosystems due to industrial development, justifies the evaluation of the toxicity of these chemicals. They can be transformed into mutagenic or carcinogenic agents by vegetables, which are the first living beings in the food chain, absorbing the nutrients of polluted environments acting as the toxic agents vectors to humans (Marcano et al. 2004). Many short-term studies are based on in vitro in vivo tests, which are used for the detection monitoring of a wide variety of environmental chemicals with mutagenic carcinogenic potential (Ashby et al.1985;1988). Chromosomal aberrations can be accepted as indicators of genetic damage induced by pesticides (Reddi Reddi 1985). Root tip systems of various plants have been widely used for determining the harmful effects of mutagens (Khilman 1975; Ma Grant 1982; Rank Neilsen 1994). * Corresponding author: phone: ; fax: ; kaymakf@trakya.edu.tr Herbicides are compounds designed to control the development of undesirable plants that may interfere with the growing of commercial crops (Blair et al. 1990). The systemic selective herbicide 2,4 - dichlorophenoxyacetic acid (2,4-D) is applied mainly to eliminate broad leaf species, where it initiates the action of natural plant hormone indole acetic acid when used in small amounts; besides, in high concentrations it induces chromosome abnormalities (Kallak Javekylg 1971; Bushra et al. 2002). It has been suggested that 2,4-D or other phenoxy herbicides may causes Non-Hodgking s Lymphoma (NHL) other cancers (Holl et al. 2002). It has been known that 2,4-D causes chromosomal damage in root cells of plants. Accordingly, recently certain researches have been suggested that the chemicals that are commonly added to pesticides may change the activity of pesticides (Holl et al. 2002). The herbicide Avenoxan is commercial form of 2,4-D it is widely used in agriculture in Turkish Thrace. The Allium test is a very good plant bioassay for chromosome damage both in mitosis meiosis for somatic mutations induced by chemicals radiations. The present study was aimed to determine the effect of Avenoxan on the process of mitosis on Allium cepa L. Allium sativum L. by using Allium test.

2 242 tartar, kaymak gokalp muranli MATERIALS AND METHODS The plant materials used for the genotoxicity test were Allium cepa L. (2n=16) Allium sativum L. (2n=16). Clean healthy bulbs of Allium cepa L. Allium sativum L. were chosen. Before starting to the experiments, dry scales of bulbs were removed, 0.1%, 0.2% 0.4% concentrations of herbicide Avenoxan were used. The solutions were prepared in distilled water. Allium cepa L. Allium sativum L. roots were treated with different concentrations of Avenoxan for 3, 6, 12 hours. Controls were treated with distilled water for the same time periods. Five bulbs were used in the controls in each of the treatment groups. For the microscopic observations, root tips were fixed in Carnoy hydrolyzed in 1NHClfor 5 min in an oven at 60 0 C. This was followed by the preparation of crushed material with aceto orcein dying method (Darlington La Cour 1976). The mitotic index (MI) was determined by examination of 3000 cells. The X 2 test was used to determine the effects of treatment period concentrations of Avenoxan on MI chromosomal aberrations. RESULTS All of the concentrations of Avenoxan used in the present study induced important abnormalities during mitotic division when compared to control in both Allium cepa L. Allium sativum L. These abnormalities were: c-mitosis, chromosome stickiness, bridges, laggard chromosomes, multipolar, micronuclei fragments. The highest abnormality number was observed in root tips of Allium sativum L.in%0,2 concentration with 6 hr treatment period while the highest abnormality number was seen in root tips of Allium cepa L. in4%concentration with 6 hr treatment period. Chromosome abnormalities were not observed as cell division did not occur in these plants with 12 h treatment periods. The results obtained from control series processed plants are shown in Table 1,2. Distributions of abnormalities according to stages were different. The most frequent abnormalities were seen at metaphase at this stage the predominant type of abnormality was c-mitosis. The examples of chromosomal abnormalities induced by different concentrations treatment periods of Avenoxan are shown in Figure 1. The mitotic index reflects the frequency of cell division. In the present study, the mitotic index was seen to decrease with increasing Avenoxan concentrations in both plants. The complete inhibiton was seen at 12 h treatment period. Mitotic index was found significantly different when compared to control by using the X 2 test (Figure 2). DISCUSSION Several epidemiological studies previously reported an association between occupational 2,4-D exposure NHL other cancers (Holl et al. 2002). The plant tests in some ways are more sensitive than both the microscreen assay the Ames test. It can even detect some carcinogenic substances that are negative to the Ames test (Rank Nielsen 1994). It has been suggested that plant chromosome analysis may be of some help in cancer research (Levan 1951). Many authors reported on the cytogenetic effects of 2,4-D that it induced chromosome abnormalities in the meiosis of Vicia faba (Khilman 1975) barley plants (Khalatkar Bharagava 1985). 2,4-D herbicide induced mitotic chromosome aberrations in the roots of some plants (Kumari Vaidyanath 1989; Bodade 1996). It also induced sister chromatid exchanges in cultured immature embryos of wheat species (Pijnaker Ferwerda 1994). Furthermore, 2,4-D used in commercial products may have different peculiarity from pure 2,4-D (Holl et al. 2002). The herbicide Avenoxan is commercial form of 2,4-D. In the present study, Avenoxan significantly induced mitotic chromosome aberrations at all concentrations in both Allium cepa L. Allium sativum L. except 3 h treatment period in Allium sativum L. The most common abnormality observed in the present study was C-Mitosis. Gomurgen (2000) Bushra et al. (2002) have also demonstrated that 2,4-D herbicides induce c-mitosis. Large number of c-mitosis indicates that Avenoxan acts as potent spindle inhibitor due to which all chromosomes lie on the metaphase plate in stead of moving towards their respective poles (Bushra et al. 2002). Also, lagging chromosomes multipolar s were seen in this study. Laggards may be attributed on the failure of spindle apparatus to organise in a normal way (Patil Bhat 1992). Multipolar s are indicating the inhibition of cytokinesis (Gomurgen 2000). According to Fiskesjo (1988) laggards multipolar s are

3 genotoxic effects of avenoxan on allium sp. 243 Table1 Obtained results after root tips of Allium cepa L. were treated with different concentrations periods. *P 0.05 ** P 0.01 ***P Treatment period of the chemical Concentrations of the chemical Number of dividing cells Normal cell number cell number metaphse c-mitosis Bridges at Chromosome Stickiness Lagging chromosome Multipolar M.I. Control ,8 3h 0, *** ,4*** 0, *** ,7*** 0, *** ,6*** 6h 0, *** ,8*** 0, *** ,7*** 0, *** ,4*** 12 h 0, , , Table2 Obtained results after root tips of Allium sativum L. were treated with different concentrations periods. *P 0.05 ** P 0.01 ***P Treatment period Number of dividing cells Normal cell number cell number metaphse c-mitosis Bridges at Micronucleus Concentration Chromosome Stickiness Micronucleus Lagging chromosome Multipolar M.I. Control ,2 3h 0, ,5*** 0, ,6*** 0, ,7*** 6h 0, ** ,1*** 0, *** ,3*** 0, *** ,1*** 12 h 0, , ,

4 244 tartar, kaymak gokalp muranli Fig. 1 A: c-mitosis that is observed after 6 h treatment of 0.1% concentration of Avenoxan on Allium cepa L.; B: c-mitosis that is observed after 3 htreatment of 0.1% concentration of Avenoxan on Allium sativum L.; C: Bridge stickiness in observed after 3htreatment of 0.4 % concentration of Avenoxan on Allium sativum L.; D: Multipolar cell observed after 6 h treatment of 0.4 % concentration of Avenoxan on Allium cepa L.; E: Multipolar bridge observed after3htreatment of 0.1 % concentration of Avenoxan on Allium sativum L.

5 genotoxic effects of avenoxan on allium sp. 245 Fig. 2 Mitotic index values observed in root tips of both Allium cepa L. Allium sativa L. consequences of weak c-mitosis can lead to aneuploidy. In the present study Avenoxan caused sticky chromosomes. Stickiness may be the result of affected chromosomal proteins due to Avenoxan toxicity. In the study of Marcano et al. (2004) it is indicated that stickiness is regarded as physiological effect exerted by herbicides in plants the phenomenon has been reported as indicative of high toxicity. Chromosome stickiness generally leads to cell death (Fiskesjo 1995). According to Fiskesjo (1988) c-mitosis stickiness more generalised type of toxic effects exerted by herbicides in plants. In the present study, other abnormalities that were observed were formation of bridges micronuclei in both Allium cepa L Allium sativum L. The formation of micronuclei may be the result of the product of acentric fragments or chromosomes with inactivated centromere (Amer Farah 1983). Bridges fragments are clastogenic effects, both resulting from chromosome chromatid breaks (Fiskesjo 1997). The clastogen action on chromosomes is generally regarded to involve an action on DNA (Badr Elkington 1982; Chauhan Sundararaman 1990). High number of abnormal cells were observed particularly at 0,4% concentration for 6 h treatment period. Also, higher percentage of mitotic abnormalities at high concentrations due to 2,4-D has been shown in several studies (Kallak Javekylg 1971; Bushra et al. 2002; Dev 1973; Ronchi et al. 1976). The results of the present study are parallel to the results of these investigators. The mitotic abnormalities were not observed at 12 h treatment period. This may be the blocking effect of cell division due to toxicity of the herbicide. Chromosome abnormalities increase proportionally with an increase in concentration the time of exposure until they reach a point that, because of a decrease at the percentage of cell division chromosome abnormalities begins to diminish. Several authors have also reported similar results for diverse biological systems.(alvarez et al. 2001; Rank et al. 2002). The mitotic index reflects the frequency of cell division. In the present study, the mitotic index was seen to decrease with increasing Avenoxan concentration in both Allium cepa L. Allium sativum L. except 3 h treatment period in Allium sativum L.. The complete inhibition was seen at 12 h treatment period. Cytotoxicity is defined as a decrease in mitotic index as increase in the fraction of cells with c-mitosis, multipolar sticky laggards (Fiskesjo 1995; Bushra et al. 2002; Marcano et al. 2004). Also in our study the most common abnormalities were these. The results may be the evidence of toxic effect of Avenoxan on the treated cells. The reduction of mitotic activity seems to be the common effect of most herbicides tested for their action in mitosis (Amer Farah 1983; Badr Ibrahim 1987). The decrease of the mitotic activity could be due to the inhibition of DNA synthesis (Beau et al. 1976). On the other h the inhibi-

6 246 tartar, kaymak gokalp muranli tion of certain cell cycle specific proteins remains as a possible herbicide target site. Thus, inhibition of enzyme DNA polimerase which is necessary for the synthesis of DNA precursors as well as other enzymes more directly involved with spindle production, assembly or orientation could explain the reported antimitotic effect (Hidalgo et al. 1989). In this study occurrence of c-mitosis, lagging chromosomes multipolar s, clearly shows the accumulated effect of Avenoxan on the spindle formation. The phenomenon of chromosome stickiness indicates the toxicity of Avenoxan occurrence of breaks, bridges show that it is a potent clastogen. As a result, the present study shows that Avenoxan, commercial formula of 2,4-D, is genotoxic because this pesticide induced chromosome abnormalities in both Alllium cepa L. Allium sativum L. Since, mitotic index is decreased, Avenoxan has highly cytotoxic effect. The sensitivity of these two plants to the herbicide Avenoxan is similar, because the parallel results have been obtained in both Allium cepa L. Allium sativum L. Hence, more care should be given to the application of Avenoxan in agriculture, in counties where it is widely used. REFERENCES Alvarez M.C., L.A. Santerre, G.G. Zuniga, B.O. Torres, C.E. Padilla V.A. Feria, 2001 Evaluation of genotoxic activity of Maleic hydrazide ethly methane sulfonate N-Nitroso diethylamine in Tradescantia using the commey assay. Salud Publica de Mexico, 43: Amer S.M. O.R. Farah, 1983 Cytological effect of pesticides. XII. Effects of phosphorothioate insecticide Durspan on the mitosis of Vicia faba. Cytologia, 48: Ashby J., F.J. De Serres, M. Draper, M. Ishidate, G.H. Margolin, B.E. Matter M.D. Shelby, 1985 Evaluation of short-term tests for carcinogens, report of the international programme on chemical safety s Collaborative Study on in vitro assay, progress in mutation research. 5. Elsevier, Amsterdam. Ashby J., F.J. De Serres, M.D. Shelby, G.H. Margolin, M. Ishidate G.C. Becking (Eds) Evaluation of short term tests for carcinogens, report of the international programme on chemical safety s Collaborative Study on in vitro assay. Vols I/II, Cambridge University Press, Cambridge. Badr A. T.T. Elkington, 1982 Antimitotic chromotoxic effects of isoproturon in A. cepa H. vulgare. Enironmental Experimental Botany, 22: Badr A. A.G. Ibrahim, 1987 Effects of the herbicide glean on mitosis, chromosomes nucleic acids in Allium cepa Vicia faba root meristems. Cytologia, 52: Beau L., O.J. Schwarz KW. Huges, 1976 Studies of the herbicide paraquat I. Efffects of cell cycles DNA synthesis in Vicia faba. Canadian Journal of Genetics Cytology, 18: Blair A., O. Axelson, C. Franklin, O.E. Paytner, N. Pearce, D. Stevenson, E. Trosko, H. Vainio, G. Williams, J. Woods S.H. Zahm, 1990 Carcinogenic effect of pesticides. In: Scott B, Wilkinson C (Eds.), The effects of pesticides on Human Health. Princeton Scientific Publication Co, USA, Bodade S.N., 1996 The mitotic effect of herbicide 2,4-D in Crotolaria Juncea Linn, Adv. Plant Sci., 9: Bushra A., F.M. Abdul, A.M. Niamat, W. Ahmad, 2002 Clastogenicity of Pentachlorophenol, 2,4-D butachlor evaluated by Allium root tip test. Mutation Research, 514: Chauhan L.K.S. V. Sundararaman, 1990 Effects of substituted ureas on plant cells. I. Cytological effects of isoproturon on the root meristem cells of A.cepa. Cytologia, 55: Darlington C.D. La Cour, L., 1976 The hling of chromosomes (6 th edn.) Allen Unwin, London, pp.201. Dev K., 1973 Effects of some herbicides on the structure behaviour of plant chromosomes. Proc. Indian. Sci. Congr., 60:308. Fiskesjo G., The Allium test-an alternative in environmental studies the relative toxicity of metal ions. Mutation Research., 197: Fiskesjo G., 1995 Allium test. In vitro toxicity testing protochols. Meth. Mol. Biol., 43: Fiskesjo G., 1997 Allium test for screening chemicals evaluation of cytological parameters in: W.Wang, JW Gorsuch, JS Huges (eds.) Plants for Environmental Studies, CRC, Lewis Publishers, New York, pp Gomurgen A.N., 2000 Cytological effect of the herbicide 2,4-D isooctylester 48% on root mitosis of Allium cepa. Cytologia, 65: Hidalgo A., R.J.A. Gonzales, P. Navas H.G. Garcia, 1989 mitosis growth inhibition in Allium cepa roots induced by propham chlorpropham. Cytobios, 57: Holl N.T., P. Duramad, N. Rothman, L.W. Figgs, A. Blair, A. Hubbard M.T. Smith, 2002 Micronucleus frequency proliferation in human lymphocytes after exposure to herbicide 2,4- Dichlorophenoxy acetic acid in vitro in vivo. Mutation Research, 521: Kallak H. L. Javekylg, 1971 Cytogenetic effects of 2,4-D on pea callus in culture. Acta Biol. (Budapest), 22: Khalatkar A.S. Y.R. Bharagava, 1985 Mutagenic effects of 2,4-dichlorophenoxy acetic acid alone with ethyl methane sulphonate in Hordeum vul-

7 genotoxic effects of avenoxan on allium sp. 247 gare L., Environmental Pollution SeriesA-Ecological Biological., 38: Khilman B.A., 1975 Root tips of Vicia faba for the study of the induction of the chromosomal aberrations. Mutation Research, 31: Kumari T.S. K. Vaidyanath, 1989 Testing of genotoxic effects of 2,4-Dichlorophenoxy acetic acid (2,4-D) using multiple genetic assay systems of plants, Mutat.Res., 226: Levan A., 1951 Chemically induced chromosome reactions in Allium cepa Vicia faba. Cold Spring Harbor Symp. Quant. Biol., 16: Ma T.H. V.F. Grant, 1982 The Tradescantiasadventurous plants. Herbarist, 48: Marcano L., I. Carruyo, A. Del Campo X. Montiel, 2004 Cytotoxicity made of action of Maleic hydrazide in root tips of Allium cepa L.. Environmental Research, 94: Patil B.C. B.I. Bhat, 1992 A comparative study of MH EMS in the induction of chromosomal aberrations on lateral root meristem in Clitoria ternetea L.. Cytologia, 57: Pijnaker L.P. M.A. Ferwerda, 1994 Sister chromatid exchanges in cultured immatrue embriyos of wheat species regenerants. Theoretical Applied Genetics, 89: Rank J. M.H. Nielsen, 1994 Evaluation of Allium Anaphase- test in relation to genotoxicity screening of industrial wastewater. Mutation Research, 312: Rank J., L. Lopez, H. Mette J. Moretton, 2002 Genotoxicity of maleic hydrazide, acridine DEHP in allium root cells performed by two different laboratories. Hereditas, 136: Reddi T.V.V.S. V.R. Reddi, 1985 Cytological effects of chemical mutagens in rice. Cytologia, 50: Ronchi V.N., G. Martini M. Buiatti, 1976 Genotype-hormone interaction in the induction of chromosome aberrations: Effect of 2,4-Dichlorophenoxyacetic acid (2,4-D) kinetin on tissue cultures from Nicotiana spp. Mutation Research, 36: Received October 07, 2005; accepted February 02, 2006