利用牙鲆鳃细胞FG和斑马鱼胚胎对氨基甲酸酯类杀虫剂残杀威和西维因的细胞毒性、遗传毒性和致畸性的毒性评估研究
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-isopropoxyphenyl 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 Hoechst 33342 andpropidium 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, showing 24h-IC50values of 89.96±1.04μg/ml, 103.4±1.14 μg/ml and86.59±1.13 μg/ml, respectively for propoxur, whereas 53.48±1.21μg/ml, 59.13±1.19μg/ml and 46.21±1.24μg/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 their 24 h-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 to 100 μg/ml and above for propoxur, whereas25 μg/ml and above for carbaryl after 24 h. 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 Hoechst 33342 andpropidium 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 observedafter 24 h exposure at the concentration of ≥ 100 μg/ml for propoxur and ≥ 10 μg/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 from 10–75 μg/ml of propoxur, however,it was found non-significant up to 20 μg/ml concentration of carbaryl. The DNAdamage scores and the results of propoxur demonstrated that the damage gradeswere not significantly different from the control (p> 0.05) up to 50 μg/ml and a littledose-effect relationship was evident. Though not statistically significant up to 50μg/ml propoxur, the cells were affected the most at a concentration of 75 μg/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 to 50μg/mlpropoxur, based on the concentration that produced similar cytotoxicity, from 3–96 hin FG cells demonstrated that the DNA damage increased with the increase of theexposure periods to propoxur up to certain level. Significant DNA damage was seenfrom 24–96 h 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.
牙鲆鳃细胞;斑马鱼胚胎;氨基甲酸酯类杀虫剂;残杀威;西维因;细胞毒性;遗传毒性;致畸性
中国海洋大学
博士
海洋生物学
Shicui Zhang
2013
中文
S917.4
153
2013-09-02（万方平台首次上网日期，不代表论文的发表时间）