International Journal of Biotechnology and Bioengineering Research. ISSN 2231-1238, Volume 4, Number 7 (2013), pp. 703-710 © Research India Publications http://www.ripublication.com/ ijbbr.htm

Determination of Mutagenic Potential of Imidacloprid in Salmonella Typhimurium–TA 98 and TA 100 Following Bacterial Reverse Mutation Assay

Ajay Kumar1, Kavita Sharma2, Monika Tomar3, Vinay Malik4 and Sudhir Kumar Kataria5

1,2,3,4Maharshi Dayanand University, Rohtak. 5Regional Institute of Education, Ajmer.

Abstract

India is an agrarian economy where use of pesticides has increased enormously to cater the food demands of increasing population. Imidacloprid is a relatively new systemic neonicotinoid insecticide with neurotoxic action as an alternative for commonly used organophosphorus insecticides in more than 100 countries. Imidacloprid is a nicotinic acetylcholine receptor agonist is an extensively used insecticide for crop protection worldwide since last decade. The mutagenicity of Imidacloprid was tested using Salmonella typhimurium TA98 and TA100 following the bacterial reverse mutation assay (Salmonella mutagenicity assay-Ames test). The Imidacloprid mutagenicity was determined by the number of revertant colonies in the presence of different concentrations (1µg to 20mg) along with DMSO & Sodium Azide (10 µg) as negative & positive control respectively. There was observed a dose dependent increase in the number of revertant colonies in both strains of Salmonella typhimurium - TA98 and TA100. This leads to the conclusion that Imidacloprid causes potential genotoxic damage to non target organisms including sprayer farmers and/or their school going children exposed to the drug.

Keywords: Salmonella typhimurium; bacterial reverse mutation assay; Imidacloprid; sodium azide; neonicotinoids.

704 Sudhir Kumar Kataria et al

1. Introduction The identification of substances capable of inducing mutations has become an important procedure in safety assessment and such chemicals can potentially damage the germ line leading to fertility problems and establishing defects due to mutations in future generations. Mutagenic chemicals are also capable of inducing cancer, and this concern has driven most of the mutagenicity testing programs. Earlier studies have reported that some pesticides have mutagenic and clastogenic activities in several biological test systems (Shirasu et al 1976; Degraeve and Moutschen, 1984; Ashby et al, 1993; Jayashree et al, 1994; Sierra-Tores et al, 1998; Siroki et al, 2001; Celik, 2003; Stivaktakis et al, 2010, Costa et al, 2009; Malik et al, 2012; Moulas C et al, 2013). The children are exposed to pesticides and are more prone to have their toxic effects due their exposure (National Academy of Sciences, 1993; Vincent, 2004). Imidacloprid (IC) is a relatively new systemic insecticide produced by Bayer Company under the trade name Confidor with neurotoxic action has gained registration in about 120 countries (Drobne et al, 2008). Imidacloprid is a nicotinic acetylcholine receptor agonist is an extensively used due to its low soil persistence and high insecticidal activity at very low application rate (Chao and Casida, 1997). Some studies have reported low imidacloprid toxicity to mammals (Tomizawa and Casida, 2003, 2005; Anatra-Cordone and Durkin, 2005). Human imidacloprid poisoning and two fatal intoxication cases have recently been reported (David et al, 2007; Proenca et al, 2005). In determining mutagenicity of chemicals, the Ames test has shown a variety of chemicals to be either mutagenic or anti-mutagenic, and has been shown to be over 90% accurate in predicting genotoxicity (Weisburger, 2001). In the Ames test, Salmonella typhimurium strains that have a mutation in the his-operon are used to detect the mutagenicity of chemicals (Maron and Ames, 1983). The use of insecticides has become increasingly widespread throughout the world so additional studies are needed to evaluate the potential toxic risk of insecticides (Imidacloprid) on non-target organisms through bacterial mutational/ames test.

2. Materials and Methods To test the mutagenic potential of imidacloprid, strains of Salmonella typhimurium i.e. TA 98 and TA 100 (MTCC 1251 & 1252) strains were purchased from Institute of Microbial Technology, Chandigarh and the Ames/Bacterial reversion assay was performed according to Maron and Ames (1983). The cultures of the strains were generated aseptically from ampoules and maintained in nutrient broth (Vogel Bonner medium). DMSO was used as negative control and sodium azide as a positive control. The testing of the imidacloprid was done on the subculture of the bacteria with a density of 1-2 x 109cells/ml and using a Petroff-Hauser counting chamber bacteria were counted and averaged, and the bacteria/cm3 was calculated using the following equation: (bacteria/square) (25squares) (1/chamber depth) (103). The density was also confirmed spectrophotometrically as the absorbance of 1ml of the S. typhimurium solution was determined at OD 600nm at t=0 as 0.012. The cells needed to be incubated with 12ml of nutrient broth between 90 and 120 min with an absorbance

Determination of Mutagenic Potential of Imidacloprid in Salmonella 705 between 0.174 and 0.350 to grow to a density of 1-2 x 109cells/ml (Maron and Ames, 1983). The mutagenicity test was used to determine the number of spontaneous revertants and positive control revertants. Spontaneous revertants are determined without the addition of test chemical and are used as a baseline to compare with the test chemicals (07 concentrations)/positive control on TA98 and TA100. Sodium azide is used for a positive control to confirm the reversion properties and specificity of the bacteria. The assay was performed on Glucose minimal plates which were prepared prior to the experiment and then top agar was heated in the microwave until liquefied and added 2ml of L-Histidine/D-Biotin (0.5 mM), 0.1ml of the fresh overnight bacteria culture (TA98 or TA100), 0.5 ml of sodium phosphate buffer and 0.1 ml of test chemical (1μg, 10μg, 100μg, 1mg, 5mg, 10mg and 20mg per plate), mixed and added to minimal glucose agar plate. In the positive control assay, 0.1ml of sodium azide (10μg/plate) was used in place of test chemical and 0.1 ml of DMSO was used in case of negative control. All plates were incubated at 37ºC for 48hr, and the numbers of revertant colonies were determined and the entire experiment was replicated in triplicate.

3. Results and Discussion The bacterial reverse mutation test uses amino-acid requiring strains of S. typhimurium to detect point mutations, which involve substitution, addition or deletion of one or a few DNA base pairs. Point mutations are the cause of many human genetic diseases and their occurrence in oncogenes and tumor suppressor genes of somatic cells are involved in tumor formation in experimental animals and humans (Olga Malev, 2012). The Ames test is a basic toxicological test that looks for gene mutations in bacterial strain of Salmonella typhimurium with a mutation in his-operon that causes it to be unable to synthesize histidine was plated in medium along with drug - Imidacloprid to be tested. The his-operon mutation can easily be back mutated when cultured along with any substance with a genotoxic (mutagenic) ability and cause the bacteria to again synthesize histidine resulting in revertant colonies formation as revealed in the mutagenicity assay on TA 98 and TA100 strains of Salmonella typhimurium. In the present studies, Ames plate incorporation assay with the different concentrations of Imidacloprid indicated a mutagenic response with both the TA 98 & TA100 tester strains. Dose dependent increase in the number of revertant colonies in both strains of Salmonella typhimurium - TA98 and TA100 was observed. In addition, the number of spontaneous revertants in negative control (DMSO) was less than all of the different treatment averages of Imidacloprid. The numbers of colonies in sodium azide were much higher than the spontaneous revertants as well as all of the treatment averages (Table 1). The results are in corroboration as different concentrations range of imidacloprid produced a linear dose-response curve with respect to formation of number of revertants per plate indicating its chemical mutagenicity. The Salmonella mutants of the tester strains with the rfa mutation allows larger molecules to pass through the cell wall thereby increasing its ability to detect mutants and the uvrB mutation allows for an increase in detection capability, as it deletes the gene that codes

706 Sudhir Kumar Kataria et al for the DNA excision repair system and required for efficiently or accurately detecting mutagens. This has been reported that insecticides were able to induce G-C base pair mutations causing a frame shift reversion of the histidine-dependent tester strains (TA98 and TA100) to the wild type (his+) (Maron and Ames, 1983). In this study imidacloprid also reverted the histidine negative to wild type which enables them to grow in the absence of histidine and biotin which suggest that imidacloprid induced base pair substitution in TA100 strain, and frame-shift mutations in strain TA98 which confirmed mutagenic potential of imidacloprid. These results are in agreement with Karabay and Oguz (2005) where potential genotoxic effect of imidacloprid on TA98 and TA 100 stains of the Salmonella has been reported. Moulas C et al, (2013) reported that imidacloprid have prominent effect on both the bacterial and fungal colonies. A fundamental maxim of pediatric medicine is that ‘‘children are not little adults’’. This observation is especially relevant to discussion of children and their exposures to pesticides. Children are at risk for pesticide exposures from different sources and at levels different than adults in the same exposure scenario. Children’s respiratory rate, heart rate, and metabolism are significantly different from adults (Bearer, 1995). Food consumption and food consumption patterns place children in a special dietary pesticide risk category (National Academy of Sciences, 1993). The urine samples of the preschool children are even detected positive for pesticides and hence their toxic effects can’t be ruled out (Chensheng Lu et al, 2001). Several researches have shown that many pesticides elicit mutagenic effects on living beings (Alavanja et al, 1994; Coats, 1990; Herrera et al, 1992; Woodruff et al, 1983; Mathew et al, 1990).Several reports confirmed the cytotoxicity and genotoxicity potential of imidacloprid in vivo and in vitro in different animal model organisms like human lymphocytes (Stivaktakis et al, 2010, Costa et al, 2009); rat (Demsia et al, 2007); mice (Malik et al, 2012); human peripheral blood lymphocytes (Feng et al, 2005) using chromosomal aberration assay, micronucleus formation and sister chromatid exchange etc. G. Frantzios et al (2007) reported that imidacloprid is genotoxic for Drosophila larvae both acutely and chronically for larvae. Several reports have shown high clinical toxicity of Imidacloprid in human suicidal attempts (Wu et al, 2001; Proenca et al, 2005; David et al, 2007).

Table 1: The number of revertant colonies formed following Salmonella mutagenicity test.

Treatment Concentration Mean his + revertants/plate (X ± S EM) TA 98 TA100 DMSO 100 μl 42 ± 0.06 141 ± 1.13 Sodium Azide 10 μg 1148 ± 0.16 1182 ± 0.26 Imidacloprid 1 μg 134 ± 0.07 150 ± 0.29 10 μg 140 ± 0.07 154 ± 0.33 100 μg 146 ± 0.06 159 ± 0.29

Determination of Mutagenic Potential of Imidacloprid in Salmonella 707

1 mg 158 ± 0.09 161 ± 0.27 5 mg 160 ± 0.06 165 ± 0.28 10 mg 165 ± 0.01 186 ± 0.27 20 mg 178 ± 0.04 199 ± 0.29

Additional mutagenicity tests should be conducted to validate further mutagenicity detection. It has been suggested that certain compounds may increase the number of revertants in Salmonella tester strains via mechanisms that would correspond to multicellular eukaryotes (Gocke and Albertini, 1996). For example, the umu test may be used in conjunction with the Ames test, as it indicates carcinogens as those chemicals that induce the expression of the umu operon (Reifferschied and Heil, 1996). Furthermore, an in vivo genotoxicity test should be conducted, such as the comet assay that can measure DNA damage of a single cell in any organ (Sasaki et al, 2000). These tests would help confirm the findings of the Ames test and establish predictions for the chemical’s effect in an organism.

4. Conclusion The genotoxic potential of the imidacloprid has been revealed and the toxicity is more prone to the young age population of any species. The rural school going children are more prone to the damage being exposed frequently. The other mutagenicity detection tests will certainly add to the validity of toxic potential of the drug significantly. Accordingly the unavoidable use and prescribed precautions be adhered to by the farmers and/or their children being non target organism so as to minimize the possible health risks associated with the extensive use of pesticides in order to have healthy future generation.

5. Acknowledgement Financial assistance as Major Research Project by UGC, N. Delhi is acknowledged.

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