In Vitro Cytotoxicity And Mechanism Of The Organotin Compound Bis-(tri-n-butyltin) Oxide And The Neonicotinoid Insecticide Imidacloprid And Acetamiprid To FG Cells, The Gill Cell Line Of Flounder (Paralichthy Olivaceus)

Cell culture of fish has provided us an important tool to study virology,tumorigenesis genetics and immunology,etc. It has also been widely used in toxicological study. The FG cells, a permanent cell line derived from the gill of the flounder, Paralichthy olivaceus, was used to determine the cytotoxicity effects and mechanism of the organotin compound bis-(tri-n-butyltin)oxide(TBTO) and the neonicotinoid insecticide imidacloprid and acetamiprid in the study.The acute cytotoxicity of organotin compound TBTO on FG cells was measured by three endpoint systems: neutral red (NR) uptake assay, tetrazolium (MTT) assay and cell protein assay. It was found that the cell growth rate was markedly reduced at the concentration of ≥1.7×10~-10 mol/L. The ultrastructures of the cells were also altered, as evidenced by dilation of nuclear membranes and mitochondria cristae, and the ribosomes attached to the RER surfaces in the TBTO-exposed cells were chopped away, existing in the cytoplasm as free ribosomes.The cytotoxicity mechanism of TBTO on FG cells was evaluated by studying the effects of TBTO on the antioxidant enzyme activities (superoxide dismutase: SOD; glutathion peroxidase: GSH-Px) and possible DNA damage. Results showed that SOD and GSH-Px activities were increased at 2 hr after exposure at the concentration of 40ng/mL, but their activities were decreased from 2 to 48 hr after exposure. DNA analyses revealed that a multimeric internucleosomal fragmentation pattern, indicative of supra-physiological levels of apoptosis, was detected at 8 to 12 hr after exposure. It is suggested that TBTO might lead to the inhibition of antioxidant enzyme activities, which results in the increase of oxygen-derived free radicals(OFR) in the cells, whichthen induces apotosis.FG was also used to determine the acute cytotoxic effects of neonicotinoid insecticide imidacloprid by the three endpoint systems(NR, MTT and cell protein assay). It showed cytotoxicity at the concentration of 0.5jig/mL or above , and there was no significant difference in cytotoxic effects for the three test systems. The ultrastructures of the cells were also altered, in which mitochondria, the cell energy-generating sites, are the most prominent sites of parathion cytotoxicity in the cells.The cytotoxicity mechanism of neonicotinoid insecticide imidacloprid(IMI) and acetamiprid(ACE) on FG cells was studied. It found that the activities of SOD and GSH-Px of FG cell which exposured on 60u.g /mL both of the two neonicotinoid insecticides were restrained at the beginning of this tests and remained in lower lever. SHowever, the ATPase activities of FG cell was decreased to more than 60% of the control level at 2hr exposured on IMI and ACE at the concentration of 60p.g/mL and retained at lower lever before 24hr. From 24hr the activities of ATPase began to resume in this tests. These suggest that neonicotinoid insecticides can rapidly inhibit ATPase acitvity. But with the extension of time, the self-repair of the mitochondria and self-adjustment of FG cell lead to increasing number of mitochondria, resulting in the ATPase activity resume.The DNA was extracted from FG cells which exposured on IMI at the concentration of 60jj.g/mL at different time and the agarose gel electrophoresis showed that a multimeric internucleosomal fragmentation pattern, indicative of supra-physiological levels of apoptosis, was detected after exposed for 2hr. The results clearly demonstrated that IMI can trigger off cell apoptosis. The damage of mitochondria is the main factor. The conclusion is consistent with that of mitochondria ultrastructure mentioned above. The cytotoxicity mechanism of the other neonicotinoid insecticide, ACE, on FG cells were also studied. The activities changement of SOD and GSH-Px of FG cell which exposured on ACE was similar to IMI. However, the "DNA ladder " was not found when the DNA from FG cells which exposured on ACE at the concentration of 60ug/mL was electrophoresed.The cytotoxic effects of another neonicotinoid insecticide ACE on FG cells werealso studied using the same methods. The cytotoxicity of ACE on FG cells was found at the concentrations ranging from 1 jig/mL to 80(j.g/mL, and the IC50 values for NR, MTT, and cell protein assays were 38.38|j.g /mL, 36.27ug /mL and 32.03 ug /mL, respectively. There was also no significant difference in cytotoxic effects for the three test systems. The ultrastructures of the cells were also markedly altered by ACE, as mitochondria were severely damaged with the cristae swelled or disrupted, while their nuclei and RER appeared still normal.Researches on the mechanism demonstrated that the decrease of SOD, GSH-Px and ATPase activities resulted in the increase of reactive oxygen species (ROS) in the mitochondria, and damage the mtDNA, which eventualy lead to apopsis of the cell.The mechanism concluded from above data demonstrated that the decrease of SOD, GSH-Px and ATPase activities resulted in the increase of oxygen-derived free radicals(OFR) in mitochondria, which made mitochondria be damaged, and eventually leading to the apoptosis of the cell.This is the first report on application of marine fish cell line to the evaluation of the acute in vitro cytotoxicity of the organotin compound and the neonicotinoid insecticide. It indicated that the FG cell line is not only a suitable bioindicator for the screening of the acute toxicity of the organotin compound and the neonicotinoid insecticide but also suits to be a tool to study the mechanism of the organotin compound and the neonicotinoid insecticide.