Preliminary Investigation Into Biological Toxicity Based On FTIR And The Toxic Mechanism Of Imidazolium Based Ionic Liquids

Recently,environmental pollution has become one of the global crises.As the increase of various pollutants have greatly affected our living environment,it is needed to detect and evaluate the pollutants.With the development of toxicological research,the traditional toxicity tests,which were mostly based on the single endpoint,do not fully reflect the toxic impacts induced by the contaminants.Therefore,it is necessary to develop the multi-level and multi-endpoint method to assess the toxic effects of environmental pollutants.In this thesis,Fourier transform infrared spectroscopy was applied in eco-toxicity test of a variety of chemicals.The aims were to demonstrate the application of FTIR by optimization of sample processing method and instrumental conditions,to compare the endpoint with the traditional ones.FTIR spectroscopy showed its capability of monitoring the toxic effects of pollutants by means of biological molecules such as lipid,protein and DNA.Then FTIR was applied to test the biochemical effects and to help understand the action of mode of ionic liquids(ILs).Later,methods of cyto-toxicology coupled with FTIR was used to verify the mechanism of ILs.The results were summarized as follows:(1)The optimization of sample processing method and instrumental conditions.By comparison with the different instrumental condition,the resolution was 8 cm-1,scan times was 64,and the instrumental mode was transmission,under these conditions,more abundant biochemical information could be collected.While analyzing the biological samples,the fixing solutions were always used to ensure the the invariableness of biomolecules.By comparison with the different fixing methods,10% neutral PBS was selected as the best fixing solution as minor alterations in chemical information.(2)Carrier solvents were frequently used in the toxicity test to dissolve the organic contaminants.Misuse of the carrier solvents might cause misleading of the toxic effects.So,the selection of an appropriate carrier solvents was extremly important.By analyzing the biochemical effects of four solvents,DMSO was found to be an appropriate carrier solvent in toxicity testing when the dose was lower than 0.1% v/v.(3)In the exploratory study of applyinng FTIR in toxicity test,the FTIR method not only had a better sensitivity,but it could also help obtain more abundant biochemical information.While analyzing this information,more accurate data ono toxic effects were obtained.Furthermore,the biochemical information also can be used to underpin the mechanisms of chemical toxicity and provide a future direction for toxicity research.In the joint toxicity study of chemicals,the modes of action of the chemicals could affect the effects of the mixture.As the unique advantages of FTIR in the recognition of toxic mechanism,FTIR spectroscopy couple with multivariate analysis was applied to test the mixtures effects of different pollutants.This study presented the possibility to investigate the biochemical mechanistic effects of pollutants,both in single and mixtures.(4)In the study of the toxicity of ionic liquids,E.coli,Selenastrum capricornutum,and Hela cells were selected as the test organisms,imidazolium ionic liquids with different alkyl chains and doses were used.Results indicated that ILs showed the dose dependent and alkyl chain dependent effects.On the biochemical effects of ILs analyzing on the protein,lipid and DNA,results suggested ILs could induce the production of ROS,and excess ROS could damage the cell membrane,and affect its stability and permeability;In addition,the ROS could also react with proteins and then affect the secondary structure,amino acid residuals and protein post-translational modification.Furthermore,the oxidative damage of DNA induced by ILs could affect the structure of DNA,and induce inhibition of the cell proliferation and cause cell death.(5)For testify the toxic mechanisms inferred by biochemical information,a series of cytotoxicity tests were conducted.Firstly,to understand the effects on the inhibtion of cell proliferation,the cell cycle,cell apoptosis,and cell autophagy were studied.Results suggested that the cell cycle arrest,apoptosis and autophagy were all increased with the increasing doses and alkyl chain of ILs.Secondly,the relationship between the ILs and ROS production was analyzed and cell apoptosis and autophagy mediated by ROS were demonstrated.In the ultrastructure and cytoskeleton structure analysis,mitochondria and membrane was more vulnerable to ILs.Results indicated that ILs might induce a decreasing mitochondrial membrane potential,damage of cell membrane and cytoskeleton.In summary,ILs could increase the membrane permeability and the permeability transition pore,and then induce the rapid drops of the membrane potential followed by the increasing production of ROS,ultimately lead to the cell apoptosis and autophagy.The alterations of membrane permeability could also affect the osmotic pressure of mitochondrial matrix,inducing the swelling and rupture of the mitochondria,and affecting the energy metabolism.ILs might destroy the integrity of the nucleus,induce nuclear fragmentation and lead to the cell cycle arrest.In summary,the biochemical mechanisms of ILs toxicity based on FTIR was in accordance with cytotoxicity tests.This new approach showed a variety of advantages including simpleness,quickness,and nondestructiveness.