Investigations On Resistant And Genotoxicity Of Pesticides: Methomyl And Chlorpyrifos

The target of carbamate and organophosphorus insecticides is acetyloholinesterase.The effects of acetyloholinesterase gene (ace) mutations on acetyloholinesterase protein stucture and functions have led to many insects have developed resistance.The research shown that the resistant strains of drosophila melanogaster exhibited 5.25 and 3.73-fold greater resistance to methomyl and chlorpyrifos respectively,than a susceptible strain.The cDNA encoding acetyloholinesterase rom susceptible and resistant strains of drosophila melanogaster had been cloned using RT-PCR.Two novel mutations (Ala284Val/Cys412 Tyr) in which the C in 851 nt was changed to T and the G in 1235 nt was changed to A were identified in acetyleholinesterase from the resistant strain. The homologous modelling of three dimensional structure of drosophila melanogaster acetyleholinesterase implicated that Ala284 might be involved in a ionic bond with Phe288. Modification of charge on the side chain of Ala284Val mutation would be expected to affect the profile of acylpocket via Phe288 losing anchorage.The intensive use of inscetieide not only has led to many insects have developed resistance,but also led to the pollution of environment.The genotoxic action of insecticide that affects human health and damages the genetic material is considered to play an important role in oncogenesis. Carbamate insecticide methomyl and organophosphorus insecticide chlorpyrifos could induce genotoxic effects, including micronuclei, chromosomal aberrations and sister-chromatid exchanges. However, methomyl and chlorpyrifos induction of cytotoxicity through DNA damage is largely unknown. Here we identify potential genotoxicity of methomyl in vitro. We have employed alkaline comet assay, yH2AX foci formation and DNA ladder assay to detected DNA damage and apoptosis of Drosophila S2, HeLa and HEK293 cells. The alkaline comet assay was used to evaluate total DNA strand breaks in the target cells exposed to in vitro to sublethal concentrations of methomyl and chlorpyrifos. As expected, methomyl and chlorpyrifos induced significant concentration dependent increases in DNA damage of target cells compared with the negative control, as measured by increases in tail length (μm), tail DNA (percentage of the comet tail) and tail moment (arbitrary units). In agreement with the comet assay data, the percentage of yH2AX positive reaction in HeLa cells also revealed methomyl and chlorpyrifos caused DNA double strand breaks (DSBs) in a time-dependent manner. Moreover, methomyl and chlorpyrifos induced a significant increase of apoptosis in Drosophila S2, HeLa and HEK293 cells in a dose-and time-dependent manner, as determined by DNA fragmentation analysis. In conclusion, methomyl and chlorpyrifos are strongly genotoxic agents that induce cell DNA damage and apoptosis in vitro.