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Excessive use of pesticides poses increased risks to non target species including humans. In the developing countries, lack of proper awareness about the toxic potential of pesticides makes the farmer more vulnerable to pesticide linked toxicities, which could lead to diverse pathological conditions. The toxic potential of a pesticide could be determined by their ability to induce genetic mutations and cytotoxicity. Hence, determination of genetic mutation and cytotoxicity of each pesticide is unavoidable to legislate health and safety appraisal about pesticides. The aim of current investigation was to determine the genotoxic and cytotoxic potential of Endosulfan (EN) and Lambda-cyhalothrin (LC); individually and in combination. MTT assay was utilized to determine cytotoxicity, while two mutant histidine dependent Salmonella strains (TA98, TA100) were used to determine the mutagenicity of EN and LC. Moreover, mutagenicity assay was conducted with and without S9 to evaluate the effects of metabolic activation on mutagenicity. Eventhough, dose dependent increase in the number of revertant colonies was detected with EN against both bacterial strains; highly significant (p<0.05) increase in the mutagenicity was detected in TA98 with S9. In comparison, data obtained from LC revealed less mutagenic potential than EN. Surprising, the non-mutagenic individual-concentrations of EN and LC showed dose dependent mutagenicity when combined. Combination of EN and LC synergistically induced mutagenicity both in TA98 and TA100. MTT assay spotlighted comparable dose dependent cytotoxicity effects of both pesticides. Interestingly, combination of the EN and LC produced increase reversion and cytotoxicity at lower doses as compared to individual pesticide, concluding that pesticides exposure even at sub-lethal doses can produce cytotoxicity and genetic mutations, which could lead to carcinogenicity.
Keywords: Endosulfan, Lambda-cyhalothrin, Mutagenicity, Cytotoxicity
Pesticides are extensively used in agriculture throughout world to safeguard crops from pests and different plant diseases. Pesticides are also used to control vectors of different diseases to minimize their spread i.e. Dengue fever virus. Immense pesticide exposure has posed toxic risks to non target species including humans. Poor working conditions and lack of protective equipments and clothes increases the susceptibility to unwanted effects of pesticides. Depending upon the mechanism of action, host selectivity and duration of exposure, symptoms of pesticide intoxication varies from vomiting, headache, skin problems to respiratory depression and convulsions (Wan et al. 2005). Before producing clinical symptoms, these pesticides may alter the genetic material resulting in mutagenicity and carcinogenicity (LaFiura et al. 2007). Several studies have reported increased risk of different types of leukemia's and cancers in pesticide exposed farmers than the general population (Cabello et al. 2001).
The use of pesticides in Pakistan started in 1954 with formulation of more than 254 metric tons, which reached to the level of 16,226 metric tons in 1976-77. In early nineties, huge quantity of pesticides was exercised in the agricultural areas of Punjab province. It has been reported that number of pesticide spray has reached beyond 10 per crop resulting in increased (1,169%) pesticide consumption in Pakistan, which is alarming situation for human health (Ejaz et al. 2004). Currently, ~ 120 types of insecticides, 39 types of weedicides, 30 types of fungicides, 6 different types of rodenticides and 5 types of acricides are being used in Pakistan.
Endosulfan (EN), an organochlorine (Figure 1a), is classified as moderately hazardous pesticide by WHO and as a highly hazardous by the United States (US) Environmental Protection Agency (EPA) (US EPA: US Environmental Protection Agency) 2002). It has got potential to bioaccumulate in human body due to its lipophilic characteristics (Aggarwal et al. 2008). Level of EN residues have been reported higher than the acceptable "daily intake" in Pakistani farmers (Khan et al. 2010). EN is one of the extensively used pesticides in Pakistan and is the top selling insecticide in Sindh province (Anwar et al. 2011). It is used on different crops vegetables and fruits e.g., cotton, wheat, soy, rice, tobacco and potatoes and surprisingly, residues of EN were detected in 22% samples of cotton seed from all Pakistan. Moreover, residues of EN were also detected in the drinking water (Tariq et al. 2004) and farmers were the most affected individuals.
Lambda-cyhalothrin (LC), a broad spectrum synthetic pyrethroid (Figure 2a), which is used to control large variety of insects in agricultural and human surroundings, is classified as a class D carcinogen by US EPA (US EPA: US Environmental Protection Agency) 2002). It is moderately persistent in the soil environment and several studies have reported genotoxicity of LC using structural chromosomal aberrations assay (SCA), micronucleus test (MN) and comet assay (Celik et al. 2005; Naravaneni and Jamil 2005a). Like EN, LC is also widely used in Pakistan on different food and non food crops. Unfortunately, it is currently used to control vector of Dengue fever in human population, making entire population at risk due to lack of legislation regarding use of pesticides (Rowland et al. 2000).
In modern agriculture practices different combinations of pesticides are used to effectively kill the pests, which also increases the chances of human population especially farmers to be heavily exposed to the combined effects of different pesticides. Although toxicity of individual pesticides has been extensively evaluated but limited data is available in the literature regarding mutagenicity of combinations of pesticides. Nevertheless, toxicity of pesticide mixtures cannot be predicted on the basis toxicity of individual pesticides (Marinovich et al. 1996), pesticides mixtures had shown much greater genotoxic effects than the individual pesticides. Increased genotoxic potential of different pesticide combinations have been reported in human lymphocytes (Bolognesi 2003) but there is hardly any data available on the mutagenicity and/or cytotoxicity of the EN and LC combination. Since, these pesticides are extensively utilized in agriculture, veterinary practice and house hold applications (Naravaneni and Jamil 2005a) either individually or in combination, therefore, this study was designed to determine the mutagenicity and cytotoxicity potential of these pesticides, both individually as well as in combination.
Material and Methods
EN and LC were kindly provided by Ali Akbar enterprise, Lahore, Pakistan. Mutant Salmonella Typhimurium strains TA98, TA100 and S9 activation enzymes were purchased from EBPI (Environmental bio-detection products Incorporation) Canada.
Ames Salmonella assay was used to assess the mutagenicity as described by Mortlemans and Zeiger (Mortelmans and Zeiger 2000). Mutagenicity was tested at eight concentrations of each individual pesticide (0.125, 0.25, 0.5, 1, 2.5, 5, 10, 50 Âµmol/plate) and six concentrations of the mixture of EN and LC (0.03:0.03, 0.06:0.06, 0.125:0.125, 0.25:0.25, 0.5:0.5, 1:1 Âµmol/plate) against two mutant histidine dependent strains of Salmonella typhimurium TA98 and TA100 were used. Two separate assays were performed with and without metabolic activation by S-9. Each test was carried out in triplicate. Positive (Sodium azide, 5Âµg/plate) and negative controls were tested separately against the both strains.
Stock solutions of test chemicals were prepared in Dimethyl sulfoxide (DMSO). Test chemical dilution, trace amount of histidine and biotin (50ÂµL of 0.05 mM solution) and 50ÂµL bacterial culture were transferred to sterile glass tube. Top agar was added to the test tube and the resultant mixture was poured on glucose minimal (GM) agar plates, which were incubated at 37Â°C for 48 hrs. The revertant colonies were counted and results were expressed as number of revertants/plate to range the results between weak positive response (â‰¥ two folds than negative control) and positive response (â‰¥ three folds than negative control).
Cytotoxicity was evaluated by adopting MTT assay using baby hamster kidney cells (BHK-21) as described by Freshney and Frame (Freshney and Frame 1982), while 10% DMSO was used as a negative control. EN and LC were evaluated for cytotoxic potential at eight different concentrations ranging from 2.5 to 1000 Âµmol/ml). Combination of the EN and LC were tested at 6 different concentrations (1:1, 2.5:2.5, 5:5, 10:10, 50:50). Lyophilized BHK-21 cells were revived and transferred into cell culture flasks, which were then incubated for 72hrs to get the confluent monolayer of cells. 100 ÂµL cell suspension (105cells/ml) was dispensed into each well of 96 well plates and incubated at 37Â°C for 72 hrs.
Media on the confluent monolayer of cells was regularly changed and 100ÂµL of each of the respective pesticides concentrations were added in triplicate, which were then incubated at 37Â°C for 48 hours. Finally, growth medium was removed; wells were washed with PBS and replenished with fresh media. 100 Âµl of MTT solution (0.5%) was added to each well and plates were incubated for ~ 4 hrs. MTT solution was then removed; and plates were incubated at 37Â°C for 2 hrs after adding 5% DMSO to each well. Optical density (OD) was measured at 570 nm by ELISA reader.
The data was analyzed for statistical analysis by using SPSS software (version 13 for windows). Independent sample t- test was applied to statistically evaluate the effect of metabolic activation by S9 on the mutagenicity of compound, while results of MTT assay were expressed as mean OD Â± S.D. and cell survival percentage was calculated (Shakir et al. 2012).
Bacterial mutation assay
Bacterial mutation assay was utilized to investigate the mutagenic potential of selected pesticides and their combination. Numbers of colonies were counted and compared with their respective controls for each strain. In TA98, EN was non mutagenic at diverse dilutions from 0.125 to 2.5 Âµmol/plate (Figure 1b). It has shown weak positive response at 5 and 10Âµmol/plate dilutions (Figure 1c). Mutagenic effect increased in dose dependent manner up to 10Âµmol/plate, after which decreased mutagenic effect was observed. Statistically significant (p<0.05) difference was observed when assay was performed with S9 (+S9). With the metabolic activation by S9, weak positive response was induced at 2.5Âµmol/plate and positive response at 5Âµmol/plate. TA100 was found more sensitive for EN mutagenicity, with weak positive response at 0.5 and 1Âµmol/plate, positive response at 2.5 and 5Âµmol/plate (Figure 1d). Addition of enzyme showed significant (p<0.05) difference in mutagenicity with weak positive mutagenic response at 1, 2.5Âµmol/plate, and positive response at 5 and 10Âµmol/plate. Detailed results are represented in Table 1 and 3.
LC was found non mutagenic (Figure 2b) against TA98 from 0.125 to 0.25 Âµmol/Plate. Weak positive response was observed at 0.5 to 10Âµmol/plate. Positive response (â‰¥ threefold the background) was not observed for any concentration tested. No significant difference was found when test was performed against TA98 with +S9 (Figure 2c). With TA100 weak positive response was found at 10Âµmol/plate (Figure 2d). Mutagenic response was not observed at any tested concentration when the assay was performed with the addition of S9. In conclusion, TA98 proved to be more sensitive to the mutagenic effects of LC as compared to TA100 and presence of S9 decreased the mutagenic effects, which may be associated to increased cytotoxic effects of metabolites. Detailed results are represented in Table 1 and 4.
Mutagenicity testing of the compounds in combined form (Figure 3a), against TA98 showed weak positive response at 0.03 to 0.06 Âµmol/plate, positive response at 0.125, 0.25 Âµmol/plate. No significant difference was observed when the same procedure was performed with S9 (Figure 3b). Assay with TA100 exhibited weak positive response at 0.125, 0.25 and 0.5 Âµmol/plate and addition of enzyme produced statically significant difference and weak mutagenic effects began to appear at 0.25 Âµmol/plate (Figure 3c). The results advocate that mixture of EN and LC showed significant (p<0.05) increase in the number of revertant colonies in both the strains. Hence, non mutagenic concentrations of EN and LC (0.125, 0.25Âµmol/plate) showed significant dose related mutagenicity, when tested in combination with LC. Detailed results are represented in Table 1 and 5.
Eight different concentrations were evaluated to determine the cytotoxic effects of EN and LC. Combinations of EN and LC were also tested to determine the cytotoxic potential in combined form. In order to determine precise cytotoxicity, ELIZA was used to quantify cell survival percentage (CSP) and any concentration with CSP value â‰¤ 50 was considered cytotoxic. The detailed results are represented in Table 2 and 6, while the cell survival trends of all tested pesticides are illustrated in Figure 4.
Figure 4a clearly highlights that maximum cell survived with EN concentration from 2.5 to 10 Âµmol/well. Cell survival percentage gradually started to decrease with increasing concentration of EN. EN showed cytotoxic potential at 50 Âµmol with CSP dropped to 38%. The cytotoxic effects were highly potent with the subsequent concentrations, dropping CSP values down to borderline. Comparatively identical pattern of cytotoxicity was perceived with different concentrations of LC (Figure 4b) i.e., maximum cell survived at doses between 2.5-10 Âµmol, while cytotoxicity started to appear from 50 Âµmol/well concentration with CSP dropped to 35%. Higher cytotoxic effects were observed at subsequent higher concentrations i.e., 100, 250, 500, 1000 Âµmol/well. Surprisingly, evaluation of combined concentrations of EN and LC revealed cytotoxicity effects even at concentrations which were non-toxic when given alone i.e., 2.5, 5 Âµmol/well, representing synergistic cytotoxic effects of both pesticides (Figure 4c).
EN is a broad spectrum organochlorine pesticide (Figure 1a), which is extensively used in Pakistan on different crops vegetables and fruits. Toxicity studies have proposed kidney, liver, and spleen as the main target sites of EN toxicity (Singh et al. 2008). High EN residues (above acceptable daily intake) have recently been reported in the tobacco farmers in Sawabi, Pakistan (Khan et al. 2010). Present study, while demonstrating the mutagenic potential of EN, revealed that TA100 proved more sensitive mutant histidine dependent strains of Salmonella typhimurium and statistically significant difference (p<0.05) was observed when assay was performed with and without S9 (Figure 1b). Similar phenomenon has been reported by previous studies with the addition of S9 (Bajpayee et al. 2006). Mutagenicity increased in dose dependent manner up to 10Âµmol/plate. After which decrease reversion was observed (Table 1, 3). This decreased mutagenic effect is believed to be due to toxic effect of EN on Salmonella resulting in decreased number of colonies. Such cytotoxic responses commonly results in decrease revertant colonies (Siddiqui and Ahmad 2003). Data obtained in our study also re-affirmed the previous results of Bajpaye et al. (Bajpayee et al. 2005) that TA100 is more sensitive to the mutagenic effects of EN, which indicate that EN is likely to produce base pair substitutions. The data obtained in this study is in agreement with previous scientific findings stating that EN induce genetic mutation, crossing over and gene conversion in Saccharomyces cerevisiae (Yadav et al. 1982).
LC showed weak mutagenic potential against TA98 at 0.5 to 10Âµmol/plate and against TA100 at 10Âµmol/plate proving that TA98 is more sensitive to LC (Figure 2b). Metabolic activation by S9 decreased the mutagenic effects and produced decrease reversion, which could be associated to cytotoxic effects of metabolites (Table 1, 4). Our results are in accordance with the previous studies, which showed that LC has "no or weak" mutagenic potential (US EPA: US Environmental Protection Agency) 2002). Studies have also reported negative mutagenic potential of other pyrethroids (Ila et al. 2008).
Mutagenicity assay revealed that non-mutagenic concentrations of EN (0.125, 0.25 and 0.5Âµmol/plate) showed weak positive (without S9) and positive response (with S9) when it was tested in combination with non mutagenic concentrations of LC (Table 1, 5; Figure 3). Our findings are in accordance with the previous genotoxic studies on combination of different other pesticides (Bolognesi 2003) stating that pesticides are more lethal when used in combination. Increase mutagenicity suggests complex molecular reaction of EN with other pesticides to produce products having high mutagenic potential. Studies involving such interactions are vital in establishing the real picture of toxicological characteristics of pesticides and other chemicals that impact the environment and public health (Ejaz et al. 2004).
Different pesticides have been reported to induce apoptosis in human mononuclear cells and thymocytes (Perez-Maldonado et al. 2004). Hence, MTT assay was employed to determine the cytotoxicity of EN alone and in combination with LC. In the present project, EN was observed harmless and non-cytotoxic up to 10 Âµmol (Table 2; Figure 4a). Dose dependent cytotoxicity was observed at 50, 100, 250, 500 and 1000 Âµmol (Table 6). Our findings are in accordance with the previous studies on the EN induced apoptosis in Jurkat T cell line (Kannan et al. 2000). EN exposure induces oxidative stress at sub lethal doses, which is thought to be responsible for pesticide toxicity and DNA damage in humans (Banerjee et al. 2001). EN has shown the potential to affect the metabolic pathways and produce reactive oxygen species (ROS), which in turn may alter cellular functions (Ledirac et al. 2005). Neuronal cell apoptosis was found to be responsible for EN induced neurotoxicity (Jia and Misra 2007).
Present study revealed cytotoxic effect of LC at 50, 100, 250, 500, 1000 Âµmol in BHK-21 cell line (Table 2; Figure 4b) and these observations are comparable to the previous studies on cytotoxicity of LC using "dye exclusion technique", which showed dose dependent cytotoxic effects on lymphocytes (Naravaneni and Jamil 2005b), macrophage cell line RAW 264.7 (Zhang et al. 2010), and female albino bone marrow cells (Celik et al. 2003). The anticipated mechanism for LC cytotoxicity is production of ROS, Nitric oxide and single strand breaks (Righi and Palermo-Neto 2005). Many studies have confirmed that ROS production may be an important factor in causing DNA damage (Liu et al. 2008). Evaluation of cytotoxicity of LC in the present study showed cytotoxic effect at 50 Âµmol, which is in accordance with the abovementioned a study.
Mixture of EN and LC showed cytotoxicity at 10Âµmol and found more toxic as compared to individual.
EN and LC showed dose dependent cytotoxicity. EN was found to have greater mutagenic potential than LC. Further in vitro and in vivo studies should be performed to confirm the mutagenicity of these pesticides.
This research was financially supported by funding from the department of Pharmacology and Toxicology (Evening program), University of Veterinary and Animal sciences, Lahore.