Cytotoxic Characteristics In Mammalian Vero Cells Exposed To Chemical Pollutants And Feasibility Analysis In Bio-toxicity Test

An increasing number of chemical pollutants are found in drinking water recent years, which gradually result in adverse effect on human health and the environment. Therefore, safety evaluation plays an important role in the process. But up to now, no related evaluation methods are established. Cytotoxicity, especially morphology variation of Vero cells universally respond to all chemicals and have the advantage of simple and sensitive. In this way, we study the effects on morphology and proliferation of Vero cells exposed to organic chemicals. By comparing sensitivity of the two features, we analyze the reliability of the method based on morphology change of Vero cells is analyzed. In virtue of Flow Cytometry, the condition of cells with morphology variation and the related reason are initially explored. We also attempt to apply this method to assess the biological safety of products for use in contact with drinking water.Morphology observation results of Vero cells exposed to two typical organic chemicals, 2,4,6-trichlorophenol (TCP) and 1,2-Benzenedicarboxylic acid, bis (2-ethylhexyl) ester (DOP) show: Small number of cells are rounding off when TCP is in 0.5mg·L^-1. With concentration increasing, the rate of rounding off cells raises. As for DOP, in 0.5ppm cell contour becomes clear and lack of transparency. These changes are more obvious when DOP concentration increases. The results of MTT reveal: TCP and DOP can inhibit the proliferation of Vero cells in a concentration higher than 1mg·L^-1 and 1ppm; A positive correlation exists between concentration and the inhibiting rate when up to 5mg·L^-1 and 5ppm. However, lower concentration of TCP (less than 1mg·L^-1) pose non- inhibit effect on Vero cells. The inhibiting rate irregularly changes with the DOP concentration in 0.25ppm²ppm.The viability variation of Vero cells exposed to TCP is analyzed by ANNEXIN V-FITC/PI-Flow Cytometry. The results show that: 1) the performance characteristics of Vero cytotoxicity caused by TCP are directly connect to TCP concentration. Low dose of TCP(less than 1mg·L^-1) have effects on cell morphology, in the form of "rounding off" or other morphology variation to parts of cells. In higher dose (more than 1mg·L^-1), inhibition of cell viability begins to be tested by MTT Assay. 2) Exposure time to TCP plays an important role in determining the performance characteristics of Vero cytotoxicity. Compared to small portion of cell apoptosis, integrity damage of cell membrane is the main form of Vero cytotoxicity after 24h exposed to TCP. But when it comes to 48h, the ratio of apoptosis cells largely increase. The result of cell rejuvenation further confirms that morphology variation with in 24h do not lead to cell necrosis or apoptosis.Consider the results of cell morphology and Flow Cytometry, it can be postulated that cell morphology change may be related to damage of cell membrane integrity, which can probably be a more sensitive method to indicate cytotoxicity of chemicals, compared to proliferation inhibition, cell apoptosis and necrosis.Finally, Vero cell morphology variation is applied to evaluate the cytotoxicity of water supply PVC and single-use plastic cups, MTT assay as reference. The results reveal: 1) water extract liquid of PVC and Plastic can cause morphology change of Vero cells. At certain concentration, rate of variation cells increases along with temperature rising, which indicate high level of temperature lead to more pollutants extracting from products to water. 2) In fixed temperature (37℃), the number of variation cells obviously grows along with the increasing concentration. A fine correlation exists between cell morphology change and extracts liquid concentration.3) water extract liquid of two products have inhibiting effect on cell proliferation, while lack of pertinence. 4) No significant change is tested by MTT if extract liquid of PVC is condensed or diluted. But there is good correlation between concentration and cell proliferation inhibition of Vero cells cultured in extract liquid of plastic. 5) Taking into account the results of morphology variation and proliferation inhibition, we can see the cytotoxicity of simple-use plastics is higher than PVC. 5) Morphology variation of Vero cells is more sensitive than MTT in application to product cytotoxicity testing. In spite of hard to quantify, it could be an effective screening approach to test toxicity of chemicals.