Detection of Genetic Damage Induced by Pesticides Using Cytogenetic and Biomarkers Assays in Allium and Pisum

Abstract

Currently, due to an increase in the population worldwide, there has been an urge for increasing the productivity of crops and agricultural by-products. This yield increment has been obtained mainly by a massive use of pesticides to control the impacts of noxious insects, pyhtopathogens and weeds in agriculture. Pesticides are a multimillion dollar market corresponding to an estimated value of 25.6 million dollars per year (Cardoso et al., 2010) and their application is still the most effective and accepted means for the protection of plants from pest. However, a series of deleterious effects on environment safety and human health have become apparent, where teratogenic, carcinogenic, and mutagenic effects have received special attention (Giacomazzi and Cochet, 2004; Nguyen-Ngoc et al., 2009). It is also known that certain pesticides are promutagens which are metabolized to mutagens (Cardoso et al., 2010). Because of the undesirable side effects of pesticides, there has been an increase in consumer awareness to avoid the use of these compounds (Zucchi et al., 2008) and/or public pressure to enhance their regulation for applications in pre- or post-harvest.
The majority of pesticides have been tested in a wide variety of mutagenicity assays covering gene mutation, chromosomal alteration and DNA damage (Inceer and Beyazoglu, 2000; Soliman and Ghoneam, 2004; Sousa et al., 2009; Cardoso et al., 2010; Lamsal et al., 2010). Although a number of biomarkers are available to assess transient and permanent genotoxic responses, biomonitoring studies on non target populations exposed to pesticides have essentially focused on cytogenetic end-points, namely chromosomal aberrations (CA), micronuclei (MN) frequency and sister-chromatid exchanges (SCE).
Several investigators had studied the side effect of the pesticides on the heredity material of different plant cells (Soliman and Ghoneam, 2004; Lamsal et al., 2010). In particular, Allium cepa possess many advantages in the field of environmental mutagenesis for screening of genotoxic agents according to the standard protocol for the plant assays established by the International Program of Chemical Safety (IPCS) and the World Health Organization (Soliman and Ghoneam, 2004). Pisum sativum bioassay has been also shown to be a very good plant bioassay for assessing chromosome damage both in mitosis and meiosis induced by chemicals, radiations and environmental pollutants (Grant and Owens, 2001). The use of plant root tips as a bioassay test system in the genotoxicity of pesticides has shown extremely good correlation with the bacterial and mammalian systems and could exhibits a good predictive value for human beings (Gopalan, 1999; Sadowaska et al., 2001; Soliman and Ghoneam, 2004).
In general, root development is initiated at the apex of the root tip by mitotic divisions in the meristematic regions (about 1 mm) in length above the root cap and the daughter F1 cells about 1mm are moved upward to lengthen the root structure. A very small portion of the meristematic cells divide transversely to increase the girth of the root tip, whereas the majority divides longitudinally. Based upon this ontogenetic scheme (Ma et al., 1995), the majority of micronuclei should be in F1 except on some rare occasions, when there is a mitotic delay. In most of earlier studies, the Micronuclei (MN) frequencies were probably not scored from F1 cells. Thus MN frequencies obtained in this way would lose their fidelity and the efficiency of the test system would be reduced because the sensitivity of chromosomes to clastogens varies greatly throughout the mitotic cycle (Ma et al., 1995). Among the different cytogenetic assays in plants, the most effective and simplest indicator of cytological damage is micronucleus formation (Ma et al., 1995; Minissi and Lombi, 1997). Furthermore, the micronucleus test can also detect very weak mutagenic effects.
Among the pesticides used, herbicides play a crucial role in agricultural fields to avoid crop competition by weeds (Saladin et al., 2003). On the other hand, chemical insecticides are widely used in Egypt and other countries in the modern agriculture in order to minimize the loss in economic crops due to insect invading (Barakat, 1997).
In view of the mentioned reasons, it was thought of interest, in this work to investigate the cytological and biochemical effects of two different pesticides namely, the herbicide bentazone and the insecticide lannate, using the following approaches: 1) Micronucleus assay in root tips of both Allium cepa and Pisum sativum, 2) Monitoring of meiotic irregularities in Pisum plants and 3) biochemical analysis of the yielded M1 seeds of the treated Pisum plants which include assessment of: storage protein banding patterns using SDS-PAGE , protein and nucleic acids content.