Evaluation Of Cytotoxicity, Genotoxicity And Teratogenicity Of Two Carbamate Insecticides, Propoxur And Carbaryl Using Flounder Gill (FG) Cells And Zebrafish Embryos

Two carbamate insecticides, propoxur and carbaryl, having a wide spectrum ofapplications, have intensified the risk of exposure to non-target organisms due totheir indiscriminate use. Propoxur (2-isopro poxy phenyl methylcarbamate) is usedthroughout the world as insecticides, herbicides, nematocides, acaricides, fungicides,rodenticides, avicides, and bird repellents with their application in a wide variety ofhabitats including agricultural lands, forests, rangelands, wetlands, residential areas,and commercial sites. Carbaryl (1-naphthyl-N-methylcarbamate), the most frequentlyused insecticide in the carbamate chemical family, is widely used for the control of avariety of pests on fruits, vegetables, cereals, forage, cotton, forests, lawns,ornamentals and many other crops as well as poultry, livestock and pets. The toxicmode of action of carbamate insecticides for animals is the inhibition of the enzymeacetylcholinesterase (AChE) at synaptic junctions in the nervous system, resulting inthe accumulation of acetylcholine in the nerve synapses, causing uncontrolledmovement, paralysis, convulsions, tetany, and possible death. In addition, bothinsecticides have also been shown to be toxic to non-target species like honeybees,amphibians, birds, fishes and even mammals including humans, though their toxicityvaries according to the species.Toxicity of pesticides on non-target organisms and ecosystems is of worldwideconcern. The widespread applications of these carbamates have attracted increasingconcerns on the safety of aquatic organisms as this pesticide eventually ends up intothe aquatic environment. Fish are an important population of the aquatic ecosystemsand often used for monitoring toxicity in the aquatic environments. Accumulatingevidences showed that propoxur is moderately to slightly toxic to freshwater fishwhereas carbaryl can range from highly to slightly toxic to freshwater fish on anacute basis and is moderately toxic to ocean and estuary fish.The main purpose of the present study was to examine the toxic effects of bothcarbamate insecticides propoxur and carbaryl in the in vitro cultured FG cells and zebrafish embryos with a view to record their cytotoxicity, genotoxicity andteratogenicity. In specific, in vitro studies were performed using FG cell line todetermine the cytotoxic effects of both insecticides by MTT reduction, neutral reduptake (NRU), lactate dehydrogenase (LDH) release, and Hoechst33342andpropidium iodide (PI) double staining assays; genotoxic effects was evaluated bycomet assay. The embryotoxicity test dealt with the assessment of these carbamatesin developing zebrafish embryos by observing diverse general morphologicalendpoints. This work has been focused on various aspects of toxicity measurementsof both insecticides to FG cells and zebrafish embryos, which may provideinformation relevant to other carbamate insecticides.Both propoxur and carbaryl treatments were seen to inhibit the proliferation ofFG cells in a dose-dependent manner, indicating a decrease in the viability of FG cellswith an increase in insecticides concentrations. The MTT, NRU and LDH releaseassays results demonstrated that both compounds exerted acute cytotoxic effects onFG cells, showing24h-IC50values of89.96土1.04lig/ml,103.4土1.14μg/ml and86.59土1.13lig/ml, respectively for propoxur, whereas53.48土1.21lig/ml,59.13土1.19μg/ml and46.21土1.24lig/ml, respectively for carbaryl. The results showed thatLDH leakage and MTT assay were more sensitive than NRU assay in the cytotoxicitydetection of exposed FG cells to both insecticides. The different mechanisms oftoxicity detection for these assays and the toxic mechanism of action of insecticidesin FG cells may account for the different sensitivity. LDH leakage assay detected thereleased LDH enzymes from the dead cells upon loss of their membrane integrity bytoxicants. However, MTT assay determined the perturbation of the mitochondrialfunction of live cells caused by toxicants, whereas NRU assay detected the loss oflysosomal activity of live cells. But the NR accrual and retention were also dependenton intact plasma membrane and adequate energy metabolism in addition to afunctional lysosome. Thus, the interruption of mitochondrial function and injury ofmembrane integrity by both insecticides may to some degree resulted in a relativelylower sensitivity of NRU assay. Taken together, both propoxur and carbaryl imposed acute cytotoxicity on FGcells, and the order of sensitivity for these three assays, based on their24h-IC50values obtained, was LDH> MTT> NRU. In addition, the obtained cytotoxicity by bothinsecticides in FG cells was closely correlated in all these assays, independent of thecytotoxic endpoints employed.The data by LDH release assay were further confirmed by examining themorphological changes of the exposed FG cells. Obvious morphological changes wereobserved in the FG cells after exposed to100lag/ml and above for propoxur, whereas25μg/ml and above for carbaryl after24h. The data revealed that the release of LDHhad happened before observable morphological changes occurred in exposed FGcells to propoxur but it happened simultaneously with the obvious morphologicalchanges for carbaryl. With further increase of the concentration of these compounds,the treated cells started to shrink and distort into irregular shape, and eventuallydetached from the substrate surface and lysed.Based on the observations of the morphology of FG cells and intensity of blueand red fluorescence of the nuclei of FG cells as determined by Hoechst33342andpropidium iodide (PI) double staining assay, the toxicity of both insecticides was seenmanifested in terms of necrosis and in a dose-dependent way rather than apoptosissince apoptotic cells were noticed sparsely. Significant cell damages were observedafter24h exposure at the concentration of>100μg/ml for propoxur and>10μg/mlfor carbaryl (p<0.05).DNA fragmentation assay revealed that there was no effect of these insecticideson the DNA integrity of FG cells as no obvious DNA laddering was induced by bothinsecticides tested in the FG cells.Further, the results obtained in the genotoxicity evaluation of both insecticidesby comet assay showed that propoxur can induce weak DNA damage in exposed FGcells in a dose-dependent manner at levels from10-75μg/ml of propoxur, however,it was found non-significant up to20μg/ml concentration of carbaryl. The DNA damage scores and the results of propoxur demonstrated that the damage gradeswere not significantly different from the control (p>0.05) up to50μg/ml and a littledose-effect relationship was evident. Though not statistically significant up to50l-ig/ml propoxur, the cells were affected the most at a concentration of75lag/ml. Butthe damage grades were not significantly different from the control (p>0.05) at allthe tested concentrations of carbaryl, though a little dose-effect relationship wasevident.The results of genotoxic response for the time-dependent exposure to50μg/mlpropoxur, based on the concentration that produced similar cytotoxicity, from3-96hin FG cells demonstrated that the DNA damage increased with the increase of theexposure periods to propoxur up to certain level. Significant DNA damage was seenfrom24-96h of exposure as compared with the control (p<0.05). But carbaryldemonstrated no statistically significant genotoxic effects at all the testedconcentrations and time of exposure (p>0.05).In addition, the concentration responsive endpoints analyzed in all the tests inFG cells indicated that the significant toxic effects at all the concentrations testedwere observed from75μg/ml for propoxur and10μg/ml for carbaryl (p<0.05)except the effects of carbaryl on the membrane integrity and genotoxic response ofFG cells.Finally, teratogenicity assay has been carried out to evaluate the teratogenicity ofpropoxur and carbaryl based on the zebrafish embryotoxicity test. The resultsobtained from this assay by determining the developmental effects and endpointsassociated to both insecticides exposure showed that the developing zebrafishembryos were more sensitive to carbaryl than propoxur. The egg/embryo mortalitydata demonstrated elevated mortality rates in both dose-and time-dependentmanner by propoxur with the lowest observed effect concentration (LOEC) of100l-ig/ml (p<0.05) and24h-,48h-and96h-LC50values of166.4土1.06lig/ml,146.3土1.07μg/ml and134.8土1.06lig/ml, respectively. But in case of carbaryl, the early mortality of embryos were first observed at8and12hours post fertilization (hpf)with LC50values of80.01土2.5μg/ml and62.08土1.05lig/ml, respectively. Moreover,the LOEC of carbaryl was10μg/ml (p<0.05) with24h-,48h-and96h-LC50values of41.80土1.10lig/ml,17.80土1.04μg/ml and14.46土1.05lig/ml, respectively.Malformations of the exposed embryos were found after12and8hpf forpropoxur and carbaryl, respectively in comparison to control. The earliest developingprocesses were observed disrupted at100and200μg/ml of propoxur exposureconcentrations causing slowdown of epiboly after12hpf. But slowdown of epibolywas observed significantly only at8and12hpf for40and80μg/ml of carbarylconcentrations (p<0.05). Further, the delayed or unfinished epiboly at8and12hpflater resulted in either malformation or mortality of exposed embryos to bothinsecticides.Yolk sac edema was conspicuous and statistically significant at theconcentrations of100and200μg/ml propoxur at24,48and96hpf but pericardialsac edema was found significant at48and96hpf (p<0.05). The significantpercentage effects on yolk and pericardial sac were observed at24hpf for10,20and40μg/ml carbaryl, however these effects were observed only at10and20μg/mlcarbaryl at48and96hpf. Moreover, total body length was markedly affected bycarbaryl at an exposure level of>10lig/ml, causing significant reduction in bodylength at96hpf (p<0.05). Tail flexure was also observed at96hpf and foundsignificantly affected at10and20μg/ml carbaryl. In addition, carbaryl affected bothtotal body length and tail flexure of all survived embryos at the concentration of20Effects of different concentrations of both insecticides on spontaneousmovement of embryos showed that the frequency of movement decreased withincreasing concentrations. Resulting effects were discrete and the distribution wasnot symmetric over the mean. Kolmogorov-Smirnov (KS) test showed that there wasno uniform distribution between the spontaneous movements among all tested concentration groups (p<0.05).Significant decline in heart rates were observed at propoxur concentrations of100and200μg/ml in comparison to control at48,72and96hpf (p<0.05). However,the heart rates were decreased at48and72hpf at carbaryl concentrations of10and20μg/ml but significantly declined at all the tested concentrations from1lag/ml andabove at96hpf. Moreover, it was noted that the heart rates of exposed embryosshowing yolk sac edema and pericardial edema were feeble and irregular andreduced to the lowest beats for both compounds.The statistically significant effects on hatching at72hpf were observed at200l-ig/ml propoxur but these effects were observed at100and200μg/ml propoxur at96hpf. However, embryos exposed to10and20lag/ml carbaryl at96hpf were foundsignificantly affected on their hatching when compared with the control (p<0.05).But the toxicity effects increased with the increasing concentrations and in adose-dependent manner. The median hatching time (HT50) of zebrafish embryos onexposure to both insecticides were calculated from the cumulative hatching rates.Significant differences between the control and exposed groups were observed onlyat100μg/ml of propoxur concentration (HT50=59.19土1.02) and at10μg/ml ofcarbaryl concentration (HT50=60.93土1.05)(p <0.05).In addition, the time and concentration responsive endpoints analyzed in all thetests in zebrafish embryos demonstrated that the significant toxic effects wereobserved at100and200μg/ml of propoxur except the effect on cumulative hatchingrate at72hpf, in which it was found significant only at the concentration of200l-ig/ml (p<0.05). But in case of embryos exposed to carbaryl, the significant toxiceffects were observed from10μg/ml (p<0.05) except the effect of slowdown ofepiboly at8and12hpf, in which it was found significant only at the concentration of40μg/ml along with the effect on heart rate at96hpf showing significant only at1l-ig/ml carbaryl.Both insecticides demonstrated no abnormality in other developmental endpoints, such as tail detachment, otolith formation, somite formation, eyedevelopment and body pigmentation in the treated embryos.In summary, the current study demonstrated that the exposure of FG cells topropoxur and carbaryl for24h could induce acute cytotoxic effects in adose-dependent manner but weak and non-significant genotoxic effects for propoxurand carbaryl, respectively. FG cells also reacted differently to the differentcytotoxicity assays, confirming the suitability of this cell line in the screening ofcytotoxic and genotoxic effects of this type of pesticide. Using zebrafish embryo as amodel, the present research also provided more information on the toxic effects ofboth carbamates on the early embryo development of fish. The embryos exposed toboth insecticides exhibited a series of toxic effects, including mortality, slowdown ofepiboly, decreased spontaneous movement, yolk and pericardial sac edemas,lowering of heart rates and delayed hatching rates along with additional reducedbody length and tail flexure in case of carbaryl exposure. Thus investigation of thecytotoxicity, genotoxicity, and teratogenicity of propoxur and carbaryl using FG cellsand zebrafish embryos will markedly contribute to the safety assessment of theseinsecticides for aquatic organisms.