Study On Cellular Damage Of Human L-02 Hepatocyte Induced By Roundup (41% Glyphosate)

Objective:To study the effects of a glyphosate based herbicide--Roundup on human L-02 hepatocyte strain at different exposure levels,and to explore the possible mechanisms in vitro.Methods:The effects of Roundup on cell survival rate were assessed by the reductions of tetrazolium dye(MTT)in cultured human L-02 hepatocytes. According to cell survival rate,L-02 hep.atocytes were incubated with Roundup at concentrations of 0mg/L,60mg/L,90mg/L,120mg/L,150mg/L and 180mg/L for 24 hours.Giemsa staining and electric microscope were performed to detect the morphological features of administered hepatocytes.The cellular membrane permeability and cytotoxicity were characterized as the activities of laspartate aminotransferase(AST)and alanine aminotransferase(ALT)out of the cytosol;as the indicators of oxidative stress,the activities of superoxide dismutase(SOD),the contents of malonic aldehyde(MDA),reduction of glutathione(GSH)were measured by colorimetry method;mitochondrial membrane potential(△ψm)was measured by fluorescent spectrometry as an indicator of mitochondrial damage.DNA damage was detected by applying the DNA agarose electrophoresiss(DNA-Ladder)in L-02 hepatocytes;AnnexinV-FITC/PI double staining with FCM detection showed the cellular apoptosis and necrosis ratios;applying Western Blotting method to detect Cyt C and AIF expression.Results:1.The concentration-dependent decreases in cell surival rate of Roundup-treated L-02 hepatocytes were observed.The relationship between concentration and survival rate was significant negative correlation(r=-0.947).2.Morphological changes caused by Roundup in L-02 hepatocytes were shrinking or swelling,and chromatin condensation were observed in groups treated by Roundup for 24hours,and the gaps between cells widened.These changes became more obvious of cell density decreased significantly,cell membrane and karyotheca were not intact or disappeared,and basophilic of chromatin declined with the ROUNDUP concentration increased.Loss of membrane microvilli,cytoplasmic hypervacuolization,apoptotic bodies,condensation and margination of nuclear chromatin were observed under Electron Microscope.3.Roundup induced cell membrane penetrability increase and cytotoxicity-were showed as significantly increases of the alanine aminotransferase(ALT)(P＜0.05);typanblue living cell staining showed that blue stained cell increased(P＜0.05)after treated with Roundup;the activities of asparate aminotransferase(AST)increased from group 90mg/L on(P＜0.05);intracellular MDA level increased,while GSH decreased,and SOD activity depressed(P＜0.05);Roundup induced depression of Na~+-K~+ ATPase activity at the dose of 120mg/L(P＜0.05);4.Treatment of Roundup resulted in DNA damages of L-02 hepatocytes,higher dose group represented a more serious DNA fracture; the hepatocyte necrosis rate kept increasing as the dose of Roundup inceased.The apoptosis rate also increased at lower dose,but it started to decrease at 180mg/L group;The fluorescent absorption value was measured to be increase from group 90mg/L on,it demostrated the mitochondrial membrane potential(△ψm)was decreased(P＜0.05);at Roundup 90mg/L group,Cyt C and AIF expression were higher than that of control group(P＜0.05).Conclusion:At 60mg/L-180mg/L,Roundup can obviously result in the decreases of survival rate,the increase of membraneous permeability,the DNA damage,the change of cellular apoptosis and necrosis rate,the decrease of mitochondrial membrane potential,and the leakage of Cyt C and AIF. Its probable mechanism may include the following t pathways,oxidation damage,the inbalance of ionic transport homeostasis and mitochondrial damage.