Excessive usage of pesticides airss increased hazards to non mark species including worlds. In the development states, deficiency of proper consciousness about the toxic potency of pesticides makes the husbandman more vulnerable to pesticide coupled toxicities, which could take to diverse pathological conditions. The toxic potency of a pesticide could be determined by their ability to bring on familial mutants and cytotoxicity. Hence, finding of familial mutant and cytotoxicity of each pesticide is ineluctable to pass wellness and safety assessment about pesticides. The purpose of current probe was to find the genotoxic and cytotoxic potency of Endosulfan ( EN ) and Lambda-cyhalothrin ( LC ) ; separately and in combination. MTT check was utilized to find cytotoxicity, while two mutant histidine dependent Salmonella strains ( TA98, TA100 ) were used to find the mutagenicity of EN and LC. Furthermore, mutagenicity check was conducted with and without S9 to measure the effects of metabolic activation on mutagenicity. Eventhough, dose dependent addition in the figure of revertant settlements was detected with EN against both bacterial strains ; extremely important ( p & lt ; 0.05 ) addition in the mutagenicity was detected in TA98 with S9. In comparing, informations obtained from LC revealed less mutagenic potency 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 single pesticide, reasoning that pesticides exposure even at sub-lethal doses can bring forth cytotoxicity and familial mutants, which could take to carcinogenicity.
Keywords: Endosulfan, Lambda-cyhalothrin, Mutagenicity, Cytotoxicity
Pesticides are extensively used in agribusiness throughout universe to safeguard harvests from plagues and different works diseases. Pesticides are besides used to command vectors of different diseases to minimise their spread i.e. Dengue febrility virus. Immense pesticide exposure has posed toxic hazards to non mark species including worlds. Poor working conditions and deficiency of protective equipments and apparels increases the susceptibleness to unwanted effects of pesticides. Depending upon the mechanism of action, host selectivity and continuance of exposure, symptoms of pesticide poisoning varies from purging, concern, tegument jobs to respiratory depression and paroxysms ( Wan et al. 2005 ) . Before bring forthing clinical symptoms, these pesticides may change the familial stuff ensuing in mutagenicity and carcinogenicity ( LaFiura et al. 2007 ) . Several surveies have reported increased hazard of different types of leukaemia ‘s and malignant neoplastic diseases in pesticide exposed husbandmans than the general population ( Cabello et al. 2001 ) .
The usage of pesticides in Pakistan started in 1954 with preparation of more than 254 metric dozenss, which reached to the degree of 16,226 metric dozenss in 1976-77. In early 1890ss, immense measure of pesticides was exercised in the agricultural countries of Punjab state. It has been reported that figure of pesticide spray has reached beyond 10 per harvest ensuing in increased ( 1,169 % ) pesticide ingestion in Pakistan, which is dismaying state of affairs for human wellness ( Ejaz et al. 2004 ) . Presently, ~ 120 types of insect powders, 39 types of weedicides, 30 types of antifungals, 6 different types of rodenticides and 5 types of acricides are being used in Pakistan.
Endosulfan ( EN ) , an organochlorine ( Figure 1a ) , is classified as reasonably risky pesticide by WHO and as a extremely risky by the United States ( US ) Environmental Protection Agency ( EPA ) ( US EPA: US Environmental Protection Agency ) 2002 ) . It has got possible to bioaccumulate in human organic structure due to its lipotropic features ( Aggarwal et al. 2008 ) . Degree of EN residues have been reported higher than the acceptable “ day-to-day consumption ” in Pakistani husbandmans ( Khan et al. 2010 ) . EN is one of the extensively used pesticides in Pakistan and is the top merchandising insect powder in Sindh state ( Anwar et al. 2011 ) . It is used on different harvests veggies and fruits e.g. , cotton, wheat, soy, rice, baccy and murphies and surprisingly, residues of EN were detected in 22 % samples of cotton seed from all Pakistan. Furthermore, residues of EN were besides detected in the imbibing H2O ( Tariq et al. 2004 ) and husbandmans were the most affected persons.
Lambda-cyhalothrin ( LC ) , a wide spectrum man-made pyrethroid ( Figure 2a ) , which is used to command big assortment of insects in agricultural and human milieus, is classified as a category D carcinogen by US EPA ( US EPA: US Environmental Protection Agency ) 2002 ) . It is reasonably relentless in the dirt environment and several surveies have reported genotoxicity of LC utilizing structural chromosomal aberrances assay ( SCA ) , micronucleus trial ( MN ) and comet check ( Celik et al. 2005 ; Naravaneni and Jamil 2005a ) . Like EN, LC is besides widely used in Pakistan on different nutrient and non nutrient harvests. Unfortunately, it is presently used to command vector of Dengue febrility in human population, doing full population at hazard due to miss of statute law sing usage of pesticides ( Rowland et al. 2000 ) .
In modern agribusiness patterns different combinations of pesticides are used to efficaciously kill the plagues, which besides increases the opportunities of human population particularly husbandmans to be to a great extent exposed to the combined effects of different pesticides. Although toxicity of single pesticides has been extensively evaluated but limited informations is available in the literature sing mutagenicity of combinations of pesticides. Nevertheless, toxicity of pesticide mixtures can non be predicted on the footing toxicity of single pesticides ( Marinovich et al. 1996 ) , pesticides mixtures had shown much greater genotoxic effects than the single pesticides. Increased genotoxic potency of different pesticide combinations have been reported in human lymph cells ( Bolognesi 2003 ) but there is barely any informations available on the mutagenicity and/or cytotoxicity of the EN and LC combination. Since, these pesticides are extensively utilised in agribusiness, veterinary pattern and house clasp applications ( Naravaneni and Jamil 2005a ) either separately or in combination, hence, this survey was designed to find the mutagenicity and cytotoxicity potency of these pesticides, both separately every bit good as in combination.
Material and Methods
EN and LC were kindly provided by Ali Akbar endeavor, Lahore, Pakistan. Mutant Salmonella Typhimurium strains TA98, TA100 and S9 activation enzymes were purchased from EBPI ( Environmental bio-detection merchandises Incorporation ) Canada.
Ames Salmonella check was used to measure the mutagenicity as described by Mortlemans and Zeiger ( Mortelmans and Zeiger 2000 ) . Mutagenicity was tested at eight concentrations of each single pesticide ( 0.125, 0.25, 0.5, 1, 2.5, 5, 10, 50 Aµmol/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 Aµmol/plate ) against two mutant histidine dependent strains of Salmonella typhimurium TA98 and TA100 were used. Two separate checks were performed with and without metabolic activation by S-9. Each trial was carried out in triplicate. Positive ( Sodium azide, 5Aµg/plate ) and negative controls were tested individually against the both strains.
Stock solutions of trial chemicals were prepared in Dimethyl sulfoxide ( DMSO ) . Test chemical dilution, hint sum of histidine and vitamin H ( 50AµL of 0.05 mM solution ) and 50AµL bacterial civilization were transferred to sterile glass tubing. Top agar was added to the trial tubing and the attendant mixture was poured on glucose minimum ( GM ) agar home bases, which were incubated at 37A°C for 48 hour. The revertant settlements were counted and consequences were expressed as figure of revertants/plate to run the consequences between weak positive response ( a‰? two creases than negative control ) and positive response ( a‰? three creases than negative control ) .
Cytotoxicity was evaluated by following MTT assay utilizing babe 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 potency at eight different concentrations runing from 2.5 to 1000 Aµmol/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 civilization flasks, which were so incubated for 72hrs to acquire the feeder monolayer of cells. 100 AµL cell suspension ( 105cells/ml ) was dispensed into each well of 96 well home bases and incubated at 37A°C for 72 hour.
Media on the feeder monolayer of cells was on a regular basis changed and 100AµL of each of the several pesticides concentrations were added in triplicate, which were so incubated at 37A°C for 48 hours. Finally, growing medium was removed ; Wellss were washed with PBS and replenished with fresh media. 100 Aµl of MTT solution ( 0.5 % ) was added to each well and home bases were incubated for ~ 4 hour. MTT solution was so removed ; and home bases were incubated at 37A°C for 2 hour after adding 5 % DMSO to each well. Optical denseness ( OD ) was measured at 570 nanometers by ELISA reader.
The information was analyzed for statistical analysis by utilizing SPSS package ( version 13 for Windowss ) . Independent sample t- trial was applied to statistically measure the consequence of metabolic activation by S9 on the mutagenicity of compound, while consequences of MTT assay were expressed as average OD A± S.D. and cell endurance per centum was calculated ( Shakir et al. 2012 ) .
Bacterial mutant check
Bacterial mutant check was utilized to look into the mutagenic potency of selected pesticides and their combination. Numbers of settlements 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 Aµmol/plate ( Figure 1b ) . It has shown weak positive response at 5 and 10Aµmol/plate dilutions ( Figure 1c ) . Mutagenic consequence increased in dose dependant mode up to 10Aµmol/plate, after which decreased mutagenic consequence was observed. Statistically important ( p & lt ; 0.05 ) difference was observed when check was performed with S9 ( +S9 ) . With the metabolic activation by S9, weak positive response was induced at 2.5Aµmol/plate and positive response at 5Aµmol/plate. TA100 was found more sensitive for EN mutagenicity, with weak positive response at 0.5 and 1Aµmol/plate, positive response at 2.5 and 5Aµmol/plate ( Figure 1d ) . Addition of enzyme showed important ( p & lt ; 0.05 ) difference in mutagenicity with weak positive mutagenic response at 1, 2.5Aµmol/plate, and positive response at 5 and 10Aµmol/plate. Detailed consequences are represented in Table 1 and 3.
LC was found not mutagenic ( Figure 2b ) against TA98 from 0.125 to 0.25 Aµmol/Plate. Weak positive response was observed at 0.5 to 10Aµmol/plate. Positive response ( a‰? threefold the background ) was non observed for any concentration tested. No important difference was found when trial was performed against TA98 with +S9 ( Figure 2c ) . With TA100 weak positive response was found at 10Aµmol/plate ( Figure 2d ) . Mutagenic response was non observed at any tried concentration when the check was performed with the add-on of S9. In decision, 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 consequences are represented in Table 1 and 4.
Mutagenicity testing of the compounds in combined signifier ( Figure 3a ) , against TA98 showed weak positive response at 0.03 to 0.06 Aµmol/plate, positive response at 0.125, 0.25 Aµmol/plate. No important difference was observed when the same process was performed with S9 ( Figure 3b ) . Assay with TA100 exhibited weak positive response at 0.125, 0.25 and 0.5 Aµmol/plate and add-on of enzyme produced statically important difference and weak mutagenic effects began to look at 0.25 Aµmol/plate ( Figure 3c ) . The consequences advocate that mixture of EN and LC showed important ( p & lt ; 0.05 ) addition in the figure of revertant settlements in both the strains. Hence, not mutagenic concentrations of EN and LC ( 0.125, 0.25Aµmol/plate ) showed important dosage related mutagenicity, when tested in combination with LC. Detailed consequences are represented in Table 1 and 5.
Eight different concentrations were evaluated to find the cytotoxic effects of EN and LC. Combinations of EN and LC were besides tested to find the cytotoxic potency in combined signifier. In order to find precise cytotoxicity, ELIZA was used to quantify cell survival per centum ( CSP ) and any concentration with CSP value a‰¤ 50 was considered cytotoxic. The elaborate consequences are represented in Table 2 and 6, while the cell survival tendencies of all tested pesticides are illustrated in Figure 4.
Figure 4a clearly high spots that upper limit cell survived with EN concentration from 2.5 to 10 Aµmol/well. Cell survival per centum bit by bit started to diminish with increasing concentration of EN. EN showed cytotoxic potency at 50 Aµmol with CSP dropped to 38 % . The cytotoxic effects were extremely powerful with the subsequent concentrations, dropping CSP values down to borderline. Relatively indistinguishable form of cytotoxicity was perceived with different concentrations of LC ( Figure 4b ) i.e. , upper limit cell survived at doses between 2.5-10 Aµmol, while cytotoxicity started to look from 50 Aµmol/well concentration with CSP dropped to 35 % . Higher cytotoxic effects were observed at subsequent higher concentrations i.e. , 100, 250, 500, 1000 Aµmol/well. Surprisingly, rating of combined concentrations of EN and LC revealed cytotoxicity effects even at concentrations which were non-toxic when given entirely i.e. , 2.5, 5 Aµmol/well, stand foring interactive cytotoxic effects of both pesticides ( Figure 4c ) .
EN is a wide spectrum organochlorine pesticide ( Figure 1a ) , which is extensively used in Pakistan on different harvests veggies and fruits. Toxicity surveies have proposed kidney, liver, and lien as the chief mark sites of EN toxicity ( Singh et al. 2008 ) . High EN residues ( above acceptable day-to-day consumption ) have late been reported in the baccy husbandmans in Sawabi, Pakistan ( Khan et al. 2010 ) . Present survey, while showing the mutagenic potency of EN, revealed that TA100 proved more sensitive mutant histidine dependent strains of Salmonella typhimurium and statistically important difference ( p & lt ; 0.05 ) was observed when check was performed with and without S9 ( Figure 1b ) . Similar phenomenon has been reported by old surveies with the add-on of S9 ( Bajpayee et al. 2006 ) . Mutagenicity increased in dose dependant mode up to 10Aµmol/plate. After which lessening reversion was observed ( Table 1, 3 ) . This reduced mutagenic consequence is believed to be due to toxic consequence of EN on Salmonella ensuing in reduced figure of settlements. Such cytotoxic responses normally consequences in lessening revertant settlements ( Siddiqui and Ahmad 2003 ) . Data obtained in our survey besides re-affirmed the old consequences 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 bring forth base brace permutations. The information obtained in this survey is in understanding with old scientific findings saying that EN bring on familial mutant, traversing over and cistron transition in Saccharomyces cerevisiae ( Yadav et al. 1982 ) .
LC showed weak mutagenic potency against TA98 at 0.5 to 10Aµmol/plate and against TA100 at 10Aµmol/plate turn outing that TA98 is more sensitive to LC ( Figure 2b ) . Metabolic activation by S9 decreased the mutagenic effects and produced lessening reversion, which could be associated to cytotoxic effects of metabolites ( Table 1, 4 ) . Our consequences are in conformity with the old surveies, which showed that LC has “ no or weak ” mutagenic potency ( US EPA: US Environmental Protection Agency ) 2002 ) . Surveies have besides reported negative mutagenic potency of other pyrethroids ( Ila et al. 2008 ) .
Mutagenicity assay revealed that non-mutagenic concentrations of EN ( 0.125, 0.25 and 0.5Aµmol/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 conformity with the old genotoxic surveies on combination of different other pesticides ( Bolognesi 2003 ) saying that pesticides are more deadly when used in combination. Increase mutagenicity suggests complex molecular reaction of EN with other pesticides to bring forth merchandises holding high mutagenic potency. Studies affecting such interactions are critical in set uping the existent image of toxicological features of pesticides and other chemicals that impact the environment and public wellness ( Ejaz et al. 2004 ) .
Different pesticides have been reported to bring on programmed cell death in human mononucleate cells and thymocytes ( Perez-Maldonado et al. 2004 ) . Hence, MTT check was employed to find the cytotoxicity of EN entirely and in combination with LC. In the present undertaking, EN was observed harmless and non-cytotoxic up to 10 Aµmol ( Table 2 ; Figure 4a ) . Dose dependent cytotoxicity was observed at 50, 100, 250, 500 and 1000 Aµmol ( Table 6 ) . Our findings are in conformity with the old surveies on the EN induced programmed cell death in Jurkat T cell line ( Kannan et al. 2000 ) . EN exposure induces oxidative emphasis at bomber lethal doses, which is thought to be responsible for pesticide toxicity and DNA harm in worlds ( Banerjee et al. 2001 ) . EN has shown the possible to impact the metabolic tracts and produce reactive O species ( ROS ) , which in bend may change cellular maps ( Ledirac et al. 2005 ) . Neuronal cell programmed cell death was found to be responsible for EN induced neurotoxicity ( Jia and Misra 2007 ) .
Present survey revealed cytotoxic consequence of LC at 50, 100, 250, 500, 1000 Aµmol in BHK-21 cell line ( Table 2 ; Figure 4b ) and these observations are comparable to the old surveies on cytotoxicity of LC utilizing “ dye exclusion technique ” , which showed dose dependent cytotoxic effects on lymph cells ( 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 awaited mechanism for LC cytotoxicity is production of ROS, Nitric oxide and individual strand interruptions ( Righi and Palermo-Neto 2005 ) . Many surveies have confirmed that ROS production may be an of import factor in doing DNA harm ( Liu et al. 2008 ) . Evaluation of cytotoxicity of LC in the present survey showed cytotoxic consequence at 50 Aµmol, which is in conformity with the abovementioned a survey.
Mixture of EN and LC showed cytotoxicity at 10Aµmol and found more toxic as compared to single.
EN and LC showed dose dependent cytotoxicity. EN was found to hold greater mutagenic potency than LC. Further in vitro and in vivo surveies should be performed to corroborate the mutagenicity of these pesticides.
This research was financially supported by funding from the section of Pharmacology and Toxicology ( Evening plan ) , University of Veterinary and Animal scientific disciplines, Lahore.