Elsevier

Journal of Environmental Sciences

Volume 36, 1 October 2015, Pages 102-111
Journal of Environmental Sciences

Analysis of the genotoxic potential of low concentrations of Malathion on the Allium cepa cells and rat hepatoma tissue culture

https://doi.org/10.1016/j.jes.2015.03.034Get rights and content

Abstract

Based on the concentration of Malathion used in the field, we evaluated the genotoxic potential of low concentrations of this insecticide on meristematic and F1 cells of Allium cepa and on rat hepatoma tissue culture (HTC cells). In the A. cepa, chromosomal aberrations (CAs), micronuclei (MN), and mitotic index (MI) were evaluated by exposing the cells at 1.5, 0.75, 0.37, and 0.18 mg/mL of Malathion for 24 and 48 hr of exposure and 48 hr of recovery time. The results showed that all concentrations were genotoxic to A. cepa cells. However, the analysis of the MI has showed non-relevant effects. Chromosomal bridges were the CA more frequently induced, indicating the clastogenic action of Malathion. After the recovery period, the higher concentrations continued to induce genotoxic effects, unlike the observed for the lowest concentrations tested. In HTC cells, the genotoxicity of Malathion was evaluated by the MN test and the comet assay by exposing the cells at 0.09, 0.009, and 0.0009 mg/5 mL culture medium, for 24 hr of exposure. In the comet assay, all the concentrations induced genotoxicity in the HTC cells. In the MN test, no significant induction of MN was observed. The genotoxicity induced by the low concentrations of Malathion presented in this work highlights the importance of studying the effects of low concentrations of this pesticide and demonstrates the efficiency of these two test systems for the detection of genetic damage promoted by Malathion.

Introduction

Pesticides are used to control the proliferation of unwanted organisms and frequently end up into the environment (Galloway and Handy, 2003). As a result, many chemicals may affect species that are not their intended target.

Malathion, O,O-dimethyl-S-(1,2-dicarboethoxyethyl) phosphorodithioate (C10H19O6PS2), is a non-systemic organophosphate insecticide that is widely used in many countries of the world both in agriculture and in the eradication of disease-carrying insects (Flessel et al., 1993). It has a neurotoxic effect that kills insects by inhibiting the enzyme acetylcholinesterase (Kwong, 2002). Because of its widespread use, Malathion can be found even in drinking water and food (Galloway and Handy, 2003, John et al., 2001). According to Bolognesi and Morasso (2000), residues of pesticides in food and drinking water have a great potential to cause long-term changes in DNA, promoters of carcinogenesis. Some evidence suggests that the human exposure to organophosphate concentrations below those that cause inhibition of acetylcholinesterase can cause several health effects (Salvi et al., 2003, Singh and Sharma, 2000).

According to Cox (2003), Malathion is a potent contaminant of water and air. Of the 36 river basins monitored by the U.S. Geological Survey, 34 were contaminated with residues of Malathion. They also found that urban streams are more contaminated and contain higher concentrations of Malathion than agricultural streams. Furthermore, it was verified that Malathion can also contaminate the groundwater (Cox, 2003, Laine and Cheng, 2007). Studies performed by the U.S. Department of Agriculture (USDA, 2000), indicated that the application of Malathion near the water resources causes toxicity in aquatic organisms as vertebrates, amphibious and fishes. The contamination of soil by Malathion can also occur. However, the rate of its degradation will vary with the moisture/alkaline conditions (Patty, 1963).

Due to runoff and agricultural application, Malathion is frequently found in the aquatic environment in low concentrations. In Egypt, Abdel-Halim et al. (2006) evaluated the presence of several organophosphates in a drainage canal surrounding a pesticide factory and found residues of Malathion ranging from 71.9 to 466 ppb. The presence of Malathion was also detected in the studies of Sankararamakrishnan et al. (2005) in surface water samples of the River Ganges in Kanpur with a concentration of 2.6 μg/L. In the ground water located in agricultural and industrial areas, the authors detected Malathion with a maximum concentration of 29.8 μg/L.

Plant species, because they are direct recipients of biological pesticides, are important organisms for genetic testing and environmental monitoring (Ma et al., 1995). Among the plants used for this purpose, Allium cepa has the advantage of having few (2n = 16) and large chromosomes. In addition, an enzyme oxidase system, which is crucial to detect pro-mutagens, is present in this species (Fiskejö, 1985). Several studies have evaluated the genotoxic action of pesticides by the chromosomal aberration (CA) and micronuclei (MN) tests in meristematic cells of A. cepa and have found positive results with the induction of different types of alterations, including those of clastogenic (chromosome breaks and bridges) and aneugenic origin (chromosome loss) (Ateeq et al., 2002, Chauhan et al., 1998, Fernandes et al., 2009, Rank et al., 1993, Sinha and Kumar, 2014, Srivastava and Mishra, 2009), showing that this test system is an appropriate model for these evaluations.

On the other hand, both in vivo and in vitro tests are often used to evaluate pesticide-induced DNA damage in animals (Das et al., 2007, Kumar et al., 2010, Soloneski and Larramendy, 2010). The in vitro tests are reproducible, rapid, and sensitive (Rogero et al., 2003) and offer a good alternative to tests performed on live animals, which require a large number of individuals (Freshney, 2005). The HTC cells are a good in vitro system to detect DNA damage caused by mutagens that are dependent on metabolic activation without the need for an exogenous metabolizing system (Dean et al., 1980, Marcarini et al., 2011, Tsuboy et al., 2010). Genotoxicity assays performed with these cells presented positive responses in studies that assessed the effects of environmental compounds, such as pesticides (Almeida et al., 2008, Malatesta et al., 2008), industrial effluents (Manzano et al., 2015) and mixture of environmental pollutants derived from petroleum (Mazzeo et al., 2013).

This study aimed at investigating the effectiveness of a plant test system and a cultured mammalian cell to detect genotoxic effects of low concentrations of Malathion.

Section snippets

Test models

A. cepa seeds and HTC cells were used as test organisms in the genotoxicity assays of the insecticide Malathion. Seeds of A. cepa (TopSeed, Agristar of Brazil LTDA) (2n = 16) from the same batch and variety (Baia Periform) were commercially acquired. The HTC cells, derived from Rattus norvegicus hepatoma, were purchased from the Cell Bank of Rio de Janeiro/Brazil (UFRJ).

Chemical substances

The chemical used was the commercially available pesticide formulation containing Malathion (Malathion 500 CE®, w/w 50%, Dipil

A. cepa tests

The results of the A. cepa tests are shown in Table 1. The analysis of the MI of A. cepa cells showed a significant reduction in dividing cells for the concentration 0.75 mg/mL of Malathion after 24 hr of exposure (p = 0.003). No cytotoxic effect was observed for the other tested concentrations of Malathion. In the genotoxicity assays, all concentrations of Malathion presented higher frequencies of CA and MN than those observed for the NC, although not all differences were statistically

Discussion

Most of the chemical residues found in the environment end up in the water resources where they become increasingly diluted. As a consequence, they are often found at very low concentrations (Trapp, 2000, Trapp, 2002). Therefore, when trying to assess the effects of environmental exposure to a given compound, analyses using high concentrations are not very relevant (Cavallo et al., 2010).

Many studies using pesticides employ animals (in vivo or in vitro) as test systems to evaluate the effects

Conclusions

Based on the results found in this study and on the observations mentioned earlier, it can be concluded that the commercial Malathion, in low concentrations, has a genotoxic potential in in vitro assays with the HTC cells and in tests with A. cepa. In addition, both test systems were effective in the detection of the genotoxicity induced by the insecticide and can be indicated as good systems for assessing genotoxic effects of low concentrations of pesticide with indirect action on the cells,

Acknowledgments

The authors would like to thank the Fundação de Amparo à Pesquisa do Estado de São Paulo (Foundation for Research Support of the State of Sao Paulo) — FAPESP for funding the research (Process no. 2005/57857-4).

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