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Revista Mexicana de Fitopatología ISSN: 0185-3309 [email protected] Sociedad Mexicana de Fitopatología, A.C. México Alvarez Moya, Carlos; Santerre Lucas, Anne; Zúñiga González, Guillermo; Padilla Camberos, Eduardo; Feria Velasco, Alfredo A Model on Agave tequilana Weber for Detection of Damaged DNA from Diseased Cells and Cells Exposed to Glyphosate Revista Mexicana de Fitopatología, vol. 19, núm. 1, enero-junio, 2001, pp. 78- 83 Sociedad Mexicana de Fitopatología, A.C. Texcoco, México Available in: http://www.redalyc.org/articulo.oa?id=61219111 How to cite Complete issue More information about this article Journal's homepage in redalyc.org Scientific Information System Network of Scientific Journals from Latin America, the Caribbean, Spain and Portugal Non-profit academic project, developed under the open access initiative 7 8 / Volumen 19, Número 1, 2001 A Model on Agave tequilana Weber for Detection of Damaged DNA from Diseased Cells and Cells Exposed to Glyphosate Carlos Alvarez-Moya, Anne Santerre-Lucas, Universidad de Guadalajara, CUCBA, Laboratorio de Genética, Departamento de Biología Celular y Molecular, km 15.5 Carr. Nogales, Zapopan, Jalisco, México CP 45110; Guillermo Zúñiga-González, Centro de Investigación Biomédica de Occidente, Laboratorio de Mutagénesis, IMSS, Sierra Morena 800, Guadalajara, Jalisco, México; Eduardo Padilla-Camberos, and Alfredo FeriaVelasco, División de Patología y Biotecnología Ambiental, CIATEJ (SEP-CONACYT), Av. Normalistas 800, Guadalajara, Jalisco, México. Correspondence to: [email protected] (Received: February 26, 2001 Accepted: June 1, 2001) Abstract. Alvarez-Moya, C., Santerre-Lucas, A., Zúñiga-González, G., Padilla-Camberos, E. and Feria-Velasco, A. 2001. A model on Agave tequilana Weber for detection of damaged DNA from diseased cells and cells exposed to glyphosate. Revista Mexicana de Fitopatología 19:78-83. The comet assay is a valuable tool to assess for genetic damage induced by chemicals. However, except for one report, this test has been used in animal cells only. In this study, the alkaline comet assay was used in order to detect the presence of lesions in genetic material of Agave tequilana plants. To do this, a simple methodology was designed to obtain the nuclei. Three groups of plants were studied: (a) diseased plants (Agave with wilting) directly taken from crops; (b) healthy plants obtained by somatic embryogenesis; and (c) healthy plants exposed separately to a well known mutagen, namely ethyl methane sulfonate and glyphosate, a herbicide. Glyphosate is extensively applied to agave crops. All plant groups, including the diseased plants, showed a statistically significant difference (p < 0.001) in tail length when compared to healthy plants. The results indicate that the comet assay may be used to diagnose genetic damage in any kind of plant not traditionally used as genotoxicity biomonitors. Additional keywords: Plant genotoxicity test, embryogenesis, maleic hidrazide, ethyl methane sulfonate. Resumen. La prueba del cometa es una excelente herramienta para evaluar daño genético inducido por sustancias químicas, sin embargo, salvo por un reporte, ésta ha sido utilizada solamente en células animales. En este estudio se empleó la prueba del cometa alcalino con el propósito de detectar la existencia de lesiones en el material genético de plantas de Agave tequilana, para ello, se diseño una metodología simple para la obtención de los núcleos. Tres grupos de plantas fueron estudiados: (a) plantas enfermas obtenidas directamente de los cultivos; (b) plantas sanas obtenidas por embriogénesis somática y (c) plantas sanas expuestas, por separado, a un mutágeno conocido: etil metanosulfonato y a un herbicida: glyphosato, el cual, se aplica extensamente en los cultivos de agave. Todos los grupos de plantas, incluyendo las enfermas, presentaron una diferencia, respecto a la longitud de la cola, estadísticamente significativa (p < 0.001) cuando se compararon con las plantas sanas. Los resultados indican que la prueba del cometa puede ser utilizada para diagnosticar daño genético en cualquier tipo de planta no utilizadas tradicionalmente como biomonitores de genotoxicidad. Palabras clave adicionales: Pruebas de genotoxicidad en plantas, embriogénesis, hidrazida málica, etil metanosulfonato. To detect genetic damage induced by chemical or physical agents, a large number of animal testing and bacteria are used (Ames et al., 1973; Underbrink et al., 1973; Graf et al., 1984; Heflich, 1991; Galli and Schiestl, 1995). With bioassays, the genotoxic effect of a specific agent on a test organism is determined and results may be extrapolated to other organisms. The design of new assays allowing to detect genetic damage in any organism where genetic abnormalities are suspected might be significant. Narendra et al. (1988) reported a test known as comet assay, used to detect DNA damage in animals from individual cells. This system uses cells immersed in an agarose gel. The cells, after being lysed and the DNA freed are subjected to electrophoresis. The migration is proportional to both time of electrophoresis and DNA damage. When chemical or physical agents are 8 0 / Volumen 19, Número 1, 2001 Verschaeve (1996). Glass slides were first covered with a normal melting point 1% agarose, which was left to solidify and was then removed. After this, 300 ml of normal melting point 0.6% agarose were placed on the slide. Afterwards, 100 ml of low melting point 0.5% agarose were mixed with 10 ml of nuclear suspension and the mixture was placed over the first layer. A third layer with 100 ml of low melting point 0.5% agarose was added to cover the second layer. Slides with nuclei were immersed in a lysis solution (2.5 mM NaCl, 10 mM Na2EDTA, 10 mM Tris-HCl, 1% sodium lauryl sarcosine, 1% Triton X100, and 10% DMSO, pH 10) for 24 h at 4°C. After lysis, slides were placed on a horizontal electrophoresis system with a high pH alkaline buffer (30 mM NaOH, 1 mM Na2EDTA, pH 13) for 3 h at 4°C. Electrophoresis was carried out for 10 min at 200 mA and 9 volts. Once completed, the slides were washed with distilled water for one min and immediately stained with 0.5 ml of ethidium bromide (20 ml/1ml) for 15 min. Thereafter, slides were washed twice with distilled water and a glass cover was placed over the gel for microscopy observation. Comets were observed under a Reicher fluorescence microscope with a 515-560 nm excitation filter. Micrographs of nuclei were taken with a 40X magnification by using a color ASA 400 film. DNA migration was measured with a graded ruler under the microscope eyepiece. Measurements were made from the center of the nucleus to the end of the tail. Statistical Analysis. Data were analyzed with a computer sofware program Co Stat which contains the ANOVA F-test for variance and the Student-Newman-Keuls t-test. The significance of differences between control groups with three test groups was determined by Dunnett´s t statistic at the 0.05 probability (Ma et al., 1994). RESULTS Nuclei of Agave tequilana were stained with orcein (Fig. 1a) and subjected to the comet assay (Fig. 1b). The tail was readily observable and made measuring the length possible. Table 1 shows the tail length in micrometers of controls, glyphosate treated plants and plants diseased with wilting. Data in each column are significantly higher than expected from randomization (p < 0.0001). Even if only differences between the negative and positive (EMS) controls were expected, results also show strong differences between the negative control and the group of diseased plants (p < 0.001) Table 1. Tail length (mm) observed in Agave tequilana nuclei of the groups studied. Number Healthy plants Plants with Plants treated with Plants treated nuclei (Negative control) wilting disease ethylmethane sulfonate with glyphosate 1 95 113 150 200 2 90 113 138 200 3 75 113 175 170 4 75 113 175 170 5 75 113 138 170 6 73 113 175 170 7 70 113 175 160 8 73 113 163 160 9 73 125 150 150 10 88 125 125 150 11 88 125 175 150 12 88 125 175 150 13 95 125 150 150 14 97 125 175 150 15 88 125 16 95 125 17 80 138 18 95 138 19 88 138 20 100 138 21 95 22 88 23 88 24 95 25 95 26 95 Mean 86.808 ± 9.282 122.80 ± 10.402 159.93 ± 17.709 164.29 ± 18.127 Data in each column are significantly higher than expected from randomization (p < 0.0001). 8 2 / Volumen 19, Número 1, 2001 applied to plants either cultivated for commercial purposes or not, which are in contact with pesticides, chemical contaminants or excessive solar incidence. We do not think the test would be useful to detect indirectly presence of biological pathogens because they don’t produce genetic damage (except virus); the comet assay system will detect only damaged DNA. Hence, finding the genetic material status of Agave tequilana crops diseased with wilting, is particularly significant because of the use of a large amount of herbicides, in many cases without the proper technical advice. It is possible that the etiology of agave wilting could be due to excessive application of pesticides. One of the most widely used herbicides in agave crops is glyphosate. It is used in 80% of comercial A. tequilana. The literature has been contradictory about this herbicide; the IPCS (1994) accepts it is only somewhat toxic and not genotoxic, but other authors report certain genotoxic (Sram et al., 1998) and carcinogenic (Moses, 1992) activity. Thus, it was very important to evaluate the effect of glyphosate directly on Agave tequilana cells and, at the same time, find out if genetic damage in sick plant cells is present at all. To achieve this, it was necessary to develop a simple methodology to separate nuclei. This worked out properly (Fig. 1), even, if we expected to see only DNA lesions on cells exposed to EMS, we were surprised to find genetic damage on cells of diseased plants and plants exposed to glyphosate. While there are differences statistically significant between diseased plant cells and cells of plants exposed to glyphosate (p < 0.001), both groups are also different from the negative control (p < 0.001). Even if there is evidence to indicate this, we cannot claim that in diseased Agave tequilana crops, DNA is damaged due to the glyphosate effect because other chemical or physical agents could also have caused it. Because of new agave crops developed from plantules (asexual reproduction), current crops are virtually the same as those available a century ago. This could lead, maybe, to mutations in the Agave tequilana DNA, causing a greater sensitivity to fungi and bacteria (Erwinia and Fusarium) naturally occurring in the plant. At early stage, the mutated plants can´t be differentiated from the healthy ones, so the growers use them indiscriminately for propagation. Thus an early test of DNA damage would be very useful. Moreover, possibly mutated plants resistant to wilting could be obtained too. Although some authors attribute the disease to Erwinia (Romero, 1998), it cannot be ruled out a possible participation of a genetic damage of A. tequilana in the physiopathology of the disease, which needs to be further investigated. LITERATURE CITED Ames, D.N., Durston, W.R., Yamasaki, E., and Lee, F.D. 1973. 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