Study On The Biological Toxicity Of Three Fluoroquinolones

Fluoroquinolones?FQs?are highly effective broad-spectrum antibacterial agents for the treatment of bacterial infections in humans and animals.Their usages are very large and the applications are widespread.Most FQs cannot be fully absorbed and utilized after taking by humans and animals.These parts of FQs entering into the environment as prodrug or active metabolite have become the new pseudo-persistent pollutants.They pose a serious threat to the environment and human health,and the resulting environmental and ecological risks have attracted widespread attention from the international community.This study selected three kinds of FQs as representatives.Ciprofloxacin?CPFX?is the most used one,gatifloxacin?GTFX?is the latest generation and peflaxacin?PFLX?can be used for both humans and animals.Their interactions with human serum albumin?HSA?and desoxyribonucleic acid?DNA?were analyzed by fluorescence spectroscopy,simultaneous fluorescence and computational simulation,which explored the mode of action and potential mechanism of the above three FQs drugs and biomacromolecules from the molecular level.Embryos and larvae of zebrafish were selected as exposure targets,and the developmental toxicity of the above three FQs pollutants were studied.The molecular mechanism of FQs on the cardiovascular system of zebrafish was elucidated from gene expression.The research content mainly includes the following four aspects.?1?Transmembrane effects of CPFX,GTFX and PFLX were studied by embryo exposure experiments.The specific results were as follows:The kinetic studies reveal that CPFX,GTFX and PFLX migration are divided into two stages:rapid diffusion and slow transit.CPFX and PFLX diffuse rapidly,GTFX diffuses slowly.In the transmembrane process,all three FQs can interact with the embryonic membrane by non-covalent bonds.The effects of CPFX and PFLX on the embryonic membrane are consistent with the Langmuir isotherm adsorption model.The maximum binding ratio are 1.22?mol/embryo and 3.79?mol/embryo,the thermodynamic binding constant are 55.3 L/?mol and 4.00×10² L/?mol,respectively.Due to the hydrophobicity of GTFX,the reaction with embryos is divided into two stages.At the low concentrations?23.12?M-69.4?M?,the classical Pesavento distribution rule is met,and the partition coefficient of GTFX on the embryo is 29.60?L/embryo.At the high concentrations?116?M-694?M?,the results agree with the Langmuir adsorption model,the maximum binding ratio is 0.17?mol/embryo and the thermodynamic binding constant is 4.93×10² L/?mol.CPFX and PFLX are more likely to adsorb and aggregate outside the embryonic membrane,only a small amount will be carried into the cytosol through membrane protein transport.While GTFX is more likely to cross the membrane lipids into the cytosol by hydrophobic interaction,but at the high concentrations it may accumulate outside the embryonic membrane due to self-polymerization and hydrogen bonding.Studies have elucidated the interaction between FQs and cell membranes in transmembrane processes.?2?The interactions of CPFX,GTFX and PFLX with HSA and DNA were studied,and their possible toxicity types were analyzed.The specific results are as follows:The interactions of CPFX,GTFX and PFLX with HSA and DNA mainly are hydrophobic interaction.The interactions of GTFX with HSA and DNA are typical hydrophobic effect;the interactions of CPFX and PFLX in ionic form with HSA and DNA are mainly hydrophobic and electrostatic.Thermodynamic calculations are consistent with molecular simulation results.The forces strong-to-weak sequence of FQs-HSA and FQs-DNA are the same,which is GTFX>CPFX>PFLX.GTFX is more accessible to HSA internal hydrophobic regions and DNA base pairs due to hydrophobic packing and hydrogen bonding,which may cause metabolic toxicity and gene dysfunction.While CPFX and PFLX do not induce metabolic toxicity.The study provides a theoretical basis for the analysis of toxic effects.?3?Exposure experiments of zebrafish embryos and larvae have been taken by using animal model exposure.The developmental toxicity of CPFX,GTFX and PFLX were evaluated from embryonic autonomic movement,hatching rate,embryo and larval survival rate and teratogenic effects.The specific results are as follows:The order of autonomous exercise toxicity?suppression?is GTFX>CPFX>PFLX,which is consistent with the transmembrane difficulty sequences of GTFX,CPFX and PFLX.Therefore,it is preliminarily determined that the easier transmembrane FQs are more toxic to zebrafish autonomic exercise.CPFX?0.68 mM-3.40 mM?exposure delays embryo hatching,GTFX?2.66 mM-5.33 mM?stimulates hatching,but show little effect on the final hatchability?p>0.05?.While PFXC?2.13 mM-15.97 mM?exposure significantly inhibited embryo hatching?p<0.05?.This result may have a certain relationship with the permeability of the embryonic membrane.The number of bounding molecules between PFXC and membrane is three times that of CPFX,which means PFXC is more likely to accumulate in the formation of embryonic membranes,blocking membrane channels,thereby affecting normal hatching.The survival rates of the three FQs exposed embryos and larvae decrease with the increase of time and concentration.Larvae,losing chorionic protection,showed higher sensitivity to FQs exposure.The LC₅₀values of CPFX 3 d exposed embryos and larvae are 2.271 mM and 1.629 mM,respectively.The LC₅₀ values of GTFX 5 d exposed embryos and larvae are 5.224 mM and 3.169 mM,respectively.The LC₅₀ values of PFLX 4 d exposed embryos and larvae were 8.446 mM and 5.728 mM,respectively.GTFX exposure significantly inhibited embryonic heart rate?p<0.05?,teratogenic effects are significant.The embryo has severe symptoms such as pericardial edema,yolk sac enlargement and axial malformation.This is because the adsorped GTFX on the surface of the embryonic membrane easily enters into the embryonic membrane through hydrophobic action.GTFX has strong binding ability to serum protein?SA?in the blood,and it is easy to affect gene expression through binding to DNA,which leads to metabolic toxicity and gene dysfunction.While CPFX and PFLX have no effect on embryonic heart rate?p>0.05?and no teratogenic toxicity for the embryo.This is because most of CPFX and PFLX are adsorbed on the surface of the embryonic membrane,and only tiny amounts of them are carried into the membrane through the transport of membrane proteins.And their bonging ability to SA and DNA was weak,which is not enough to cause deformity.?4?Studies have shown that three FQs exposure have an effect on zebrafish heart rate.Since the binding,migration and toxic effects of CPFX and PFLX are basically the same,so CPFX and GTFX are selected as target pollutants to further deepen the influence of FQs exposure on the cardiovascular system of zebrafish.The specific results are as follows:CPFX exposure?0.41 mM-5.05 mM?does not induce zebrafish pericardial edema,thrombosis and hemorrhage,but it caused impaired heart function,which is characterized by decreased heart rate,cardiac output and blood flow velocity.GTFX exposure?1.12mM-11.18 mM?induces typical cardiovascular morphological abnormalities?pericardial edema?,leading to impaired cardiovascular function?bradycardia and circulatory abnormalities?,but does not cause changes in ventricular structure of zebrafish atrium.The atp2a1l?the calcium ion transport,encoding the calcium ion receptor ATP-enzyme-related gene?,cacna1ab?controling voltage-dependent calcium channel-related gene?and tnnc1a?cardiac troponin C regulatory gene?expression profile have been analyzed.The results indicate that CPFX can significantly inhibit the expression of tnnc1a and atp2a1l,and promote the expression of cacna1ab gene,while GTFX can significantly inhibit the expression of atp2a1l gene,which has a tendency to inhibit the expression of tnnc1a gene,but does not affect the expression of cacna1ab gene.It is speculated that the molecular mechanism of CPFX and GTFX induced cardiovascular toxicity may be mainly through the down-regulation of atp2a1l to inhibit calcium transport and encode sarcoplasmic calcium receptor function,and the down-regulation of tnnc1a to inhibit myocardial contraction.For the first time,the article comprehensively analyzes the correlation between three FQs and embryonic membrane,biomacromolecules and individual toxicity characteristics,and conducts systematic research from microscopic?gene?to individual?molecular,embryo,and population?.The toxic mechanism of FQs environmental pollutants was revealed from four levels:membrane adsorption,molecular action,gene expression and toxic performances.The results provide more information for the ecotoxicology of FQs,and it is expected to establish the experimental foundation for the formulation of environmental quality standards for FQs in water environment,environmental drug regulation and risk assessment.