Research On The Toxic Effects, Mechanisms Of Fluoroquinolone To Antioxidant Enzymes And The Drug Residue Determination

Fluoroquinolones (FQs) are the most important group of synthetic broad-spectrum antibiotics that have been widely used as both human and veterinary medicine to treat and prevent various bacterial infections from 1980s. However, the increasing use of FQs led to large amount of them entering the environment and made them the micro-pollutants of greatest concern.. Their potential risk also became the world wide focal point. Therefore, the establishments of practical detection method and perfect toxicology assessment system of fluoroquinolone are very important for the monitor of the existence of fluoroquinolone in environmemt and the further evaluation of their environmental risk and their hazard to human being further.Enzyme-linked Immunosorbent Assay (ELISA) is a biochemical technique involving the reaction of antigen and antibody for the detection of antigens and antibodies. Because of the advantages of strong specificity, high sensitivity, economy and simplicity, it has become one of the most practical methods for the determination of pollutants in water, soil and food, etc. It also has been applied in cellular and animal toxicology as well as ecotoxicology research. The establishment of ELISA method of fluoroquinolone is necessary for understanding their contamination degree, toxicology study and risk assessment.Catalase (CAT) and superoxide dismutase (SOD) are ubiquitous enzymes among aerobically respiring organisms. As primary antioxidant enzymes, they play key roles in eliminating reactive oxygen species and controlling cellular redox state. Changes in their structures and activities are related to cell biology, aging and a number of diseases. Therefore, the investigations on the effects of contaminants on the structures and activities of them are of great important for assessing their toxicity.In this study, the toxic mechanism of sarafloxacin (SARA) to CAT and Cu/ZnSOD, lomefloxacin (LOME) with the existence of Cu2+ to CAT and Cu/ZnSOD were investigated using multi-spectroscopic methods (UV-vis absorption spectroscopy, circular dichroism (CD) spectroscopy, steady state fluorescence and time-resolved fluorescence spectroscopy) combined with isothermal titration microcalorimetry (ITC) and molecular modeling method. The new methods for the determination of danofloxacin (DANO) and LOME by indirect chemiluminescent competitive ELISA were also developed. The main results are as follows:1. The interaction between SARA and CAT was studied. The formation of sarafloxacin-CAT complex alters the micro-environment of amide moieties and tryptophan (TRP) residues, reduces the a-helix content of the enzyme, changes the peripheral substituents on the porphyrin ring of heme and leads to the inhibition of the enzyme activity. Molecular docking study reveals that sarafloxaein is located between two α-helix of CAT near to TRP 182 and TRP 185 residues, which supports the experimental results and helps to have a more clear understanding about the interaction mechanism. The change in the relative position of HIS 74 to heme induced by the variation of secondary structure is considered to be the major reason for the reduction of CAT activity.2. The effects of SARA on Cu/ZnSOD were evaluated via investigating the structure and the structure basis activity changes of Cu/ZnSOD upon sarafloxacin binding using multi-spectroscopic methods, isothermal titration microcalorimetry (ITC) and molecular docking method. SARA binds to a hydrophobic area located on the surface of β-barrel and tends to form hydrogen bonds with TYR 108, PRO 100, ASP 25 and SER 103 residues around it. The binding of sarafloxacin induces structure change in Cu/ZnSOD but does not affect its activity, which can be attributed to the active site and active site channel of Cu/ZnSOD being far away from the binding site and the microenvironment of them, not to be affected.3. The toxic mechanisms of LOME to CAT, Cu2+ to CAT, and LOME with the existence of Cu2+ to CAT were studied. The reasons for the alteration in CAT activity induced by LOME and Cu2+ were discussed. Both LOME and Cu2+ bind to CAT in adjacent to aromatic amino acid. The binding changed the secondary structure of CAT and the micro-environment of the aromatic amino acid near to HEME. The change induced by LOME and Cu2+ together is almost equal to the sum of the changes induced by LOME and Cu2+ alone. ITC results showed, comparing with CAT, it is easier for Cu2+ to react with LOME to form LOME-Cu2+ complex. As LOME coexists with Cu2+, they tend to bind to CAT in the form of LOME-Cu2+ complex and the binding area is same with Cu2+. Considering the spectroscopic conclusion that the binding site of LOME and Cu2+ is same, we inferred that LOME, Cu2+ and LOME-Cu2+ are bind to the same area of CAT. The results of CAT activity detection indicated that LOME slightly inhibited the activity of CAT while Cu2+ decreased the relative activity of CAT to about 80% at concentration of 1× 10-4 mol L-1 The combined action of LOME and Cu2+ led to a further decrease in enzyme activity which obviously greater than the sum of decreases induced by LOME or Cu2+ alone in the same condition, indicating LOME together with Cu2+ can produce synergistic toxic effect. Molecular docking study revealed that LOME (Cu2+) binds to the area of β3、 β4 and a9 of CAT. The alteration in CAT secondary structure further affected the micro-environment of TYR 357 and PRO 335 in the proximal side of HEME should be the main reason for the enzyme activity inhibition. The reason for LOME, Cu2+ and LOME + Cu2+ produced different inhibition in CAT activity should attribute to their different effects to the structure of CAT.4. The joint toxicity of LOME and Cu2+ to Cu/ZnSOD was studied. As LOME coexists with Cu2+, they tend to bind to Cu/ZnSOD in the form of LOME-Cu2+ complex. LOME, Cu2+ and LOME-Cu2+ can bind into the same area of Cu/ZnSOD in adjacent to the TYR residue. The combined effects of LOME and Cu2+ to the conformation of Cu/ZnSOD are almost equivalent to the sum effects of LOME and Cu2+ alone. The results of Cu/ZnSOD activity detection showed that LOME has no effect on the Cu/ZnSOD activity, while Cu2+ increases Cu/ZnSOD activity remarkably. The effects of the coexist of LOME and Cu2+ on Cu/ZnSOD activity are almost same with Cu2+ exists alone. Molecular docking study revealed that LOME binds to the surface of Cu/ZnSOD between V-Loop and GK2, the reason for LOME does not affect the activity of Cu/ZnSOD should due to its location in Cu/ZnSOD is far away from its activity center and activity tunnel. ITC study reveals, there are multi-binding sites for Cu2+ in Cu/ZnSOD. According to the spectroscopic results, part of Cu2+ should bind to the same area of Cu/ZnSOD with LOME. The coexist of LOME did not affect the Cu/ZnSOD activity, indicating that this part of Cu2+ should not be the reason for inducing activity change in Cu/ZnSOD, and LOME also did not affect the binding of Cu2+ to Cu/ZnSOD which ted to the increase in Cu/ZnSOD activity,5. Chemiluminescent enzyme-linked immumoassay methods (CL-ELISA) for the determination of DANO and LOME were developed. Several influence factors including the concentration of coating antigen, the dilution degree of the primary antibody and IgG-HRP antibody as well as the type of microtiter plate etc. were optimized. Under the selected condition, for DANO determination, the IC50 value and the linear working range was 0.1 ng ml-1 and 0.025 - 0.5 ng ml-1 respectively; for LOME determination, the IC50 value and the linear working range was 0.22 ng ml-1 and 0.05-1.0 ng ml-1, respectively. Compared with the traditional colorimetric ELISA using the same antibody, the sensitivity, specificity and the linear range of the new built CL-ELISA methods were improved dramatically. The developed methods were applied in the detection of the recoveries of DANO and LOME in spiked milk. For DANO, the recoveries rates for intra-assay and inter-assay were 96.2%-105.2% and 92.6%-101.2%, with corresponding coefficient of variation (CV%) of 2.4%-8.7% and 2.6%-9.5%; For LOME, the average recovery rates are in the ranges of 88.7%-103.4% and 97.8%-107.2% for intra- and inter-assay with CV% of 6.3%-8.6% and 2.4% -8.0%. indicating that the new built CL-ELISA methods are reliable with higher practical value.This study provided new data for the risk assessment of fluoroquinolone. It also provided reliable method for the detection of LOME and DANO in environment, cells and animals.