Research On The Cytotoxicity And Related Mechanisms Of Herbicide Prometryn In Vitro

Objective:Prometryn is a relatively low toxic herbicide belonging to the triazine group which are widely used in agriculture for the control of various weeds.Although there are many studies and evidences pertaining to the human carcinogenic potential of triazine herbicides,the cytotoxicity of prometryn on human pulmonary cells is not clear yet.Studies have confirmed that prometryn exposure can induce mice cell apoptosis and necrosis in thymus,lymph node and spleen.Foods such as milk,seaweed,fish and other aquatic products have higher concentration of prometryn in residual,and many research have detected the presence of prometryn in human urine,milk and blood.Our study aimed to examine the cytotoxicity and related molecular mechanisms of prometryn on human pulmonary cells in vitro.Methods:A549 and BEAS-2B cells were treated with varying concentration(0,25,50,100,200?M)of prometryn for 24 or 48 hours.The cell viability was assessed using the MTT assay.Cell cycle and the rate of apotosis were measured by flow cytometry(FCM)after staning with PI only or Annexin V-PI both.Comet Assay provides a simple and effective method for evaluating DNA damage in A549 and BEAS-2B cells.The intracellular levels of ROS was fluorometrically monitored using DCFH-DA,and fluorescence intensity was measured by FCM.Results:1.During the concentration between 100?M to 200?M,morphological changes caused by prometryn:A549 cells turned round,cell density decreased significantly,and the gaps between cells widened;shrinking or floating,and chromatin condensation were observed in BEAS-2B cells.2.The concentration-dependent decrease in relative cell viability of prometryn-treated A549 and BEAS-2B cells were both observed.3.Prometryn had no obvious effect on the rate of apoptosis of A549 cells,but decreased Bcl2 expression and increased Bax expression were observed.There were obvious apoptosis induced in BEAS-2B cells after prometryn treatment with increased Cleaved-Caspase9?Cleaved-Caspase3 and Cleaved-PARP.4.At the concentration of 100-200?M,prometryn could induce A549 cells G1 arrest with increasing expression of p53,p21 and p27 proteins,and decreasing expression of E2F1,CyclinD1,CDK4,CyclinE and CDK2 proteins.While BEAS-2B cells were arrested in S cell-cycle,accompanied with increased p53 and decreased CyclinA and CDK2.5.Prometryn treatment had no obvious effect on ROS levels of A549 cells even at the concentration of 200?M.When at 200?M concentration,prometryn could induce BEAS-2B cells to produce obvious overload ROS.6.Prometryn could induce a concentration-dependent DNA damage especially double strand breaks(DSBs)both in A549 and BEAS-2B cells.And the protective effect was only observed in BEAS-2B cells by adding NAC before prometryn treatment.7.Prometryn could induce the phosphorylation of H2AX.The fluorescent focus of ?-H2AX tended to away from main nuclear in A549 cells,distributed evenly like scatter or crumb among BEAS-2B nucleus.Conclusions:1.At the concentration of 50-200?M,prometryn could inhibit A549 cells proliferation,induce DNA damage and G1 arrest,and p53,p21 and p27 protein involved in cycle regulation,?-H2AX involved in DNA damage repair.2.When at 100-200?M concentration,prometryn treatment on BEAS-2B cells for 48 hours resulted in a significant dose-dependent inhibition of cell survival,induced S cell cycle arrest,increased formation of intracellular ROS,then induced the oxidative damage of DNA and obvious apoptosis.3.Although prometryn is a slightly to moderately toxic herbicide,the changes on the molecular level and obvious DNA damage can still be observed in our study.Measures should be taken to reduce the contact with prometryn,and to avoid the potential health hazards caused by prometryn indrawing.