Investigation On The Micromechanism Of The Toxicity Of Tetracyclines

Protein is the material base for life, being involved in every cell and all important part of body and closely linked to various forms of life activities. The cell is the basic unit of the metabolic activity of biological organism. The harmful factors in the external environment, such as physical (ionizing radiation, noise, ultraviolet, etc.), chemical and biological (bacteria, viruses, etc.) ones, can act on the body through various means (such as respiratory tract, digestive tract, skin and wound), inducing changes in body structure and function, all reflecting those in cell structure and function. So the changes in cell structure and function are the basis for the injuries to the body induced by exogenous harmful substances.Tetracyclines are widely used in animal husbandry and aquaculture. Because of their low bioavailability,50-80%of the applied dose enters the environment without metabolism, becomes a pollutant. The tetracycline residues in the environment such as soil, surface water, groundwater, even drinking water, and animal food (egg, milk, meat, etc.) can enter human bodies by food chain or diet, being potentially harmful. The pollution of tetracycline residues and their toxicity has attracted worldwide attention. There have been many related reports, but the existing research results can not completely explain the inner mechanism of the residues in the environment to ecology and human health. In this thesis, on the basis of the existing related research results, with the targets of the tetracyclines induced toxicity (functional proteins and cell) as the object, we investigated the micromechanism of the toxicity of the common tetracyclines (oxytetracycline (OTC), tetracycline (TC) and chlortetracycline (CTC)) and obtained the following findings:1The interaction mechanism of OTC and TC with serum albuminsSerum albumins are the carrier of a wide variety of endogenous and exogenous compounds in blood. We studied the interaction of OTC and TC with serum albumins based on the elimination of inner filter effect with the correction equation. The site marker competition experiments revealed that OTC and TC bind to site Ⅱ (subdomain ⅢA) of serum albumins, mainly with electrostatic interaction indicated by the calculated negative ΔH°and positive ΔS°. The molecular modeling methods were applied to further define that the binding site for OTC and TC was the positively charged amino acid residues in site II (ARG433or ARG436for BSA, ARG410and LYS414for HSA). The UV-visible absorption, synchronous fluorescence and circular dichroism results showed that the bound OTC and TC can change the secondary structure and the microenvironment of the tryptophan residues of BSA and HSA. The research results were published on Biomacromolecules and Journal of Agricultural and Food Chemistry.2The interaction mechanism of tetracyclines with the functional enzymes related to tetracyclines toxicityIt have been reported that tetracyclines can result in the decrease of the activity of catalase, lysozyme and trypsin. We investigated the micromechanism of the toxicity of tetracyclines by multispectroscopic techniques and molecular modeling method. The experimental results indicated that:(1) OTC can interact with catalase to form a complex mainly by van der Waals'interactions and hydrogen bonds with one binding site. The binding of OTC can result in change of the secondary structure and the microenvironment of the tryptophan residues of catalase. The activity of catalase was also inhibited for the bound OTC.(2) All the three tetracyclines OTC, TC and CTC can bind into lysozyme cleft and interact with the key active-site residues Glu35or Asp52with one binding site, with the affinity order:CTC>TC>OTC, resulting in competitive inhibition of lysozyme activity. Both the spectroscopic technique and the molecular modeling method revealed that tetracyclines interact with lysozyme mainly through electrostatic forces. The binding of tetracyclines can cause the secondary structure and the microenvironment of the tryptophan residues of lysozyme.(3) All the three tetracyclines (OTC, TC and CTC) can interact with trypsin with one binding site to form tetracyclines-trypsin complex, mainly through van der Waals'interactions and hydrogen bonds with the affinity order:TC>OTC>CTC. The bound tetracyclines can result in inhibition of trypsin activity with the inhibition order:CTC>OTC>TC. Both the molecular docking study and the trypsin activity experiment revealed that OTC and TC bound into S1binding pocket, competitively inhibiting the enzyme activity, and CTC was a non-competitive inhibitor which bound to a non-active site of trypsin, different from TC and OTC due to the Cl atom on the benzene ring of CTC which hinders CTC entering into the S1binding pocket. The secondary structure and the microenvironment of the tryptophan residues of trypsin were also changed because of the bound tetracyclines. However, the effect of CTC on the secondary structure content of trypsin was contrary to those of TC and OTC. The related research results were published on Science of the Total Environment, Chemosphere and PLoS ONE.3The toxic interaction mechanism of tetracyclines with human red blood cellsThe common tetracyclines OTC and TC can enter red blood cells (RBCs). We studied the micromechanism of the toxicity of tetracyclines to RBCs. The experimental results revealed that:(1) OTC can reduce the antioxidant capacity of RBCs and induce oxidative stress, causing the following toxic effects:OTC can change the morphology of RBCs and further resulting in hemolysis (OTC concentration higher than8×10-5mol L-1). At low OTC dose, the ATPase activity increased. However, at higher dose, OTC can inhibit the activity of ATPase, affecting cell function.(2) Utilizing the flow cytometry, we established the detection methods for the indicators of antioxidant activity of RBCs (glutathione and reactive oxygen species (ROS)) and investigated the impact of TC on their content in RBCs. The experimental results indicated that TC can result in the reduction of glutathione content in RBCs to reduce the function of the antioxidant defense system of RBCs. The cellular oxidative/antioxidant balance was destroyed, so the ROS concentration in RBCs increased, causing oxidative stress which can lead to the damage of cellular structure and function.(3) OTC and TC can interact with hemoglobin with one binding site to form complex, mainly through van der Waals and hydrogen bond interactions. Both the synchronous fluorescence experiment and the molecular modeling study revealed that OTC and TC bind into hemoglobin central cavity. The bound OTC and TC can change the secondary structure and the microenvironment of the tyrosine residues of hemoglobin. A part of the research results were published on Journal of Hazardous Materials. The thesis makes exploratory research on the micromechanism of the toxicity of tetracyclines and obtains innovative research results, which is conducive to in-depth understanding of the toxicity of tetracyclines. The established new methods for the research on the toxic mechanism of tetracyclines can provide the methodological reference and technical support for the toxicity evaluation of other pollutants.