Study On Structure-Toxicity Relationship Of Fluoroquinolones Revealed By Zebrafish

Fluoroquinolones(FQs)are bicyclic heterocyclic aromatic compounds with a carboxyl group at position 3,a ketone group at position 4 and a fluorine atom at position 6 of the nuclear structure.They are highly effective antibiotics with many desirable pharmacokinetic and pharmacodynamic properties including high bioavailability,large volume of distribution,and a broad spectrum of antimicrobial activity.FQs are widely used in the treatment of a variety of infections,including genitourinary tract,respiratiory tract,gastrointestinal infections.Their activity is based on inhibition of bacterial enzymes:DNA gyrase and DNA topoisomerase IV which are necessary to separate bacterial DNA.This activity leads to inhibition of cell replication.They have been associated with several adverse effects,include gastrointestinal tract effects,central nervous system(CNS)effects,arthropathy,heart rate-corrected QT interval prolongation,dysglycemia,and phototoxicity.AEs of the CNS are the second most frequently reported form of FQ toxicity.They include a variety of neurological and psychiatric symptoms sucha as dizziness,depression,headache,insomnia,delirium,agitation,psychosis,seizures/convulsions,and even suicidal behaviors.It is also reported that FQs treatment can cause chronic persistent peripheral neuritis,mainly characterized by paraesthesia and pain.However,the neurotoxicity mechanism of FQs is not fully understood,but studies have shown that it is closely related to the structure.The possible interaction sites of FQs include antagonizing y-aminobutyric acid(GABA)receptors,activating N-methyl-D-aspartic acid(NMDA)receptors,decreasing serotonin levels,altering microRNA expression and so on.Many models have been used to assess neurotoxicity of FQs,but most of these structure-based investigations are more directed to a known protein target(e.g.,GABA-A),but not an entire living organism.Neurobehavior is a sensitive indicator of the influence of toxicants on the integral CNS in animals.It can be used to detect changes in brain functions in a comprehensive and unbiased way.Zebrafish(Danio rerio)can provide a sound basis for human risk assessment in a cost-effective and ethically acceptable way.The detection of bioconcentration is helpful to improve the accuracy of drug safety evaluation.In this study,zebrafish was used as a model system.The uptake potent,embryotoxicity and effects on zebrafish locomotor activity of 17 typical FQs were studied.The relationships between structure,neurotoxicity,and embryotoxicity were investigated.1.A liquid chromatography-tandem mass spectrometry(LC-MS/MS)method in a multiple reaction monitoring(MRM)mode was developed.The curve for each compound was linear over the concentration range with an r value greater than 0.99(except for GMFX,r = 0.9788).The precision for each FQ was less than 15%.Except for GMFX and R-ATFX,accuracy was within ± 15%.Recovery ranged from 80.86%to 113.95%.Matrix effect ranged from 80.95%to 124.17%.2.The LCso of each compound were defined as 100%.Embryos were exposed from six hour post fertilization(6hpf)in 10%concentration and 1%concentration.The drug contents were measured in the three day post fertilization(3dpf)、4dpf、5dpf、6dpf of 10%treated group,and in 6dpf of 1%treated group.The internal concentration(IC)of 3dpf embryos were used as an indicator for the embryotoxicity.Define the exposed concentration as medium concentration(MC).The IC/MC was used to evaluate absorption capacity.Results demonstrate that modifications at different positions of the FQ nucleus can change zebrafish embryotoxicity and absorption properties,the piperazinyl structure at position C-7 is critical to capacity of absorption.The ethylenediamine moiety at position C-7,which is generated by cleavage of the piperazinyl structure and amino moiety at position C-5,significantly enhanced embryotoxicity.3.We studied effects on zebrafish locomotor activity of 17 typical FQs by recording indicators with automated video-tracking.Average distance(AD)over 20 min under dark conditions was chosen as an indicator to assess the effects of FQs on neurobehavior of zebrafish larvae.Furthermore,a 1%ratio(ratio of the AD difference between the control group and 1%dosage group and the corresponding body burden)was used as an index to compare the different substituents and their effects.The results indicate:(1)Effects of FQs on zebrafish neurobehavior can be divided into four categories.Type I has no significant influence on locomotor activity.Type Ⅱ suppresses locomotor activity.TypeⅢ inhibits at low concentration and stimulates at high concentration.Type IV showed an inverted U-shaped reaction with increasing concentration.4.In addition,we calculated the stable conformation of typical FQs in aqueous conditions.There are two toxic functional groups.The piperazinyl structure at position C-7(toxic functional group I)can cause primary reactions which may be by the inhibition of γ-aminobutyric acid(GABA)receptors,and the nucleus containing a carboxyl group at position 3(toxic functional group Ⅱ)might cause a reaction secondary to the effect of toxic functional group Ⅰ and reverse its effects.5.The relationships between structure,neurotoxicity,and embryotoxicity were analyzed.Structural modifications of FQs can change toxicity properties in zebrafish.Cleavage of the C-7 piperazinyl structure decreases neurotoxicity but enhances embryotoxicity.The C-3 decarboxyl formation and 5-NH2 derivatives might enhance embryotoxicity and neurotoxicity.