Article Cite This: Chem. Res. Toxicol. 2018, 31, 238−250
pubs.acs.org/crt
Relationship Between Fluoroquinolone Structure and Neurotoxicity Revealed by Zebrafish Neurobehavior Chaoqiang Xiao,†,‡ Ying Han,‡ Ying Liu,‡ Jingpu Zhang,*,† and Changqin Hu*,†,‡ †
Chinese Academy of Medical Sciences and Peking Union Medical College, 100730 Beijing, China National Institutes for Food and Drug Control, 100050 Beijing, China
‡
S Supporting Information *
ABSTRACT: Central nervous system side effects are one of the most frequently reported adverse reactions of fluoroquinolones (FQs). However, the mechanism is not fully understood. In this study, zebrafish (Danio rerio) were used as a model system. We quantified neurobehavior by recording indicators with automated video-tracking and used liquid chromatography-tandem mass spectrometry to detect drug absorption in vivo. We studied embryotoxicity and effects on zebrafish locomotor activity of 17 typical FQs. In addition, we calculated the stable conformation of typical FQs in aqueous conditions. The relationships between structure, neurotoxicity, and embryotoxicity were analyzed. The results indicate: (1) The 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. Type III inhibits at low concentration and stimulates at high concentration. Type IV stimulates and then suppresses (biphasic response). (2) 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. (3) 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 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.
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INTRODUCTION
caused by a CNS-mediated mechanism in combination with direct irritation of the gastrointestinal tract.1 Studies of the relationship between drug adverse reactions and drug structure contribute to structural modifications and an understanding of adverse reaction mechanisms.2 The neurotoxicity mechanism of FQs is not fully understood,14 but studies have shown that it is closely related to FQ structure.2,15 The possible interaction sites of FQs include antagonizing γaminobutyric acid (GABA) receptors, activating N-methyl-Daspartic acid (NMDA) receptors, decreasing serotonin levels, altering microRNA expression, and so on. 13,14,16 The substituted piperazine or piperidine groups at position C-7 of FQs play an important role in CNS effects by inhibiting binding between GABA and its receptor. This decreases GABAergic inhibition and leads to stimulation of the CNS and a series of nervous system AEs.17 The carboxyl groups at position C-3 and carbonyl moiety at position C-4 can chelate Mg2+ in body fluids, which can decrease Mg2+ concentration. This can lead to opening of Ca2+ channels in the cell membrane, which allows more Ca2+ enter the cell, and activation of NMDA glutamate
Fluoroquinolones (FQs), which are synthesized by addition of a fluorine atom at position C-6 of the bicyclic ring structure of quinolone,1,2 are commonly used in treatment of various infections,3−6 although some of the compounds, for example, grepafloxacin, sparfloxacin, and gatifloxacin, etc., have been withdrawn from the European and U.S. markets.1 They exert bactericidal effects by inhibiting DNA gyrase and topoisomerase IV and interfering replication of bacterial DNA. Despite restricted FDA-approved pediatric indications due to safety concerns, some FQs were used to treat a variety of infections in children due to their excellent pharmacokinetic and pharmacodynamics properties.7−9 The most common FQs-related adverse effects (AEs) include gastrointestinal tract effects, central nervous system (CNS) effects, arthropathy, heart ratecorrected QT interval prolongation, dysglycemia, and phototoxicity.10,11 CNS effects, including neurological and psychiatric symptoms, are the second most frequently reported side effects after gastrointestinal tract AEs. CNS reactions include dizziness, depression, headache, insomnia, delirium, agitation, psychosis, seizures/convulsions,12 and even suicidal behaviors.13 Moreover, gastrointestinal AEs, such as nausea and vomiting, are © 2018 American Chemical Society
Received: November 1, 2017 Published: March 21, 2018 238
DOI: 10.1021/acs.chemrestox.7b00300 Chem. Res. Toxicol. 2018, 31, 238−250
Chemical Research in Toxicology
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receptors, which may contribute to AEs observed with FQs such as depressive symptoms.18 Many animal models have been used to assess neurotoxicity of FQs, and most of these studies focused on the relationship between epileptogenicity and inhibition of GABA receptors by FQs with different structures.17,19,20 The results showed that FQs only induce clonic convulsions in rats at a high dosage or when administered concomitantly with 4-biphenylacetic acid (BPAA). Furthermore, sparfloxacin has no antagonistic effect on GABA receptors but can cause CNS AEs such as anxiety and excitement.21−23 These results indicate that the relationship between structures of FQs and neurotoxicity is not clearly understood. Zebrafish (Danio rerio), which is commonly used as a model system to study toxicology, can reveal pathways of developmental neurotoxicity and provide a sound basis for human risk assessment in a cost-effective and ethically acceptable way.24,25 In recent years, zebrafish have been widely used to assess drug toxicity including embryotoxicity, cardiac toxicity, and neurotoxicity.25−29 Zebrafish embryos (ZFE) develop rapidly, and large numbers can be obtained year-round. The ectoderm differentiates from 6 h post-fertilization (hpf), and the neural tube is formed at 12 hpf. GABAergic and glutamatergic neurons differentiate from 2−3 days post-fertilization (dpf). Neurotransmitters, such as GABA, glutamate, serotonin, dopamine, and acetylcholine, can be detected in neurons of zebrafish at 1−5 dpf. The major organ systems are developed at 5 dpf. The blood−brain barrier (BBB) is structurally and functionally similar to that in mammals, and maturation occurs between 3 and 10 dpf.30 Zebrafish are highly sensitive to various CNS drugs and display behavioral and physiological responses that are very similar to those of rodents and humans. As a whole animal model, zebrafish offer a rapid, high-throughput, and low-cost evaluation system for drugs and impurities.31 Neurobehavior is a sensitive indicator of the influence of toxicants on the integral CNS in animals and can reflect effects on sensation, motor control, attention, and motivation. It can be used to detect changes in brain functions in a comprehensive and unbiased way.32 Hyperactivity of zebrafish is typically related to their psychostimulant or convulsant actions. Conversely, hypolocomotion of zebrafish indicates sedative and neuromotor deficient effects of drugs.33 Akinesia and seizure behaviors can be quantified by recording indicators such as velocity, traveling distance, and time with automated videotracking tools.34−36 Compounds tested on zebrafish are typically administered as aqueous solutions. The absorption capacity of a compound is often related to its structure, and toxic effects of a compound are related to its concentration in the zebrafish. Thus, it is necessary to determine drug concentration in the zebrafish when assessing toxicity of different drugs.37,38 In our previous work, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method in a multiple reaction monitoring (MRM) mode was developed to quantify antibiotic concentration in zebrafish with efficient separation as well as excellent sensitivity and selectivity and to evaluate their toxicity.28,39 In this study, zebrafish were used to evaluate embryotoxicity and behavioral effects of FQs. Based on the results of drug absorption, the relationship between CNS-related AEs caused by FQs and their structures are discussed.
Article
MATERIALS AND METHODS
Chemicals. Ciprofloxacin (CPFX), ciprofloxacin impurity C (CPFX-C), decarboxyl-pazufloxacin (de-PZFX), gemifloxacin (GMFX), moxifloxacin (MXFX), lomefloxacin (LMFX), decarboxylomefloxacin (de-LMFX), antofloxacin (ATFX), (R)-antofloxacin (RATFX), pefloxacin (PEFX), sparfloxacin (SPFX), levofloxacin (LVFX), gatifloxacin (GTFX) and its four impurities were national reference substances obtained from the National Institutes for Food and Drug Control (Beijing, China) (Figure 1). Formic acid was
Figure 1. Chemical structures and abbreviations of the analytes. purchased from Fluka (Sigma-Aldrich, Germany). Enrofloxacin (ENFX, 99%) was purchased from J & K Scientific (Beijing, China) and used as an internal standard. Stock solutions of all compounds were prepared in 90% methanol-water and stored at −20 °C. Calibration standards were diluted from the working solutions, which were approximately 1 μg/mL and obtained from the stock solutions by diluting in methanol. The chemicals for ZFE exposure were dissolved in breeding water, which was prepared from Instant Ocean sea salt (CNSG, Tianjin, China). NaOH/HCl were used to maintain pH at 7.0. Embryotoxicity Study in Zebrafish. Zebrafish of the AB wildtype strain were bred at the Institute of Medicinal Biotechnology, Chinese Academy of Medical Science, Beijing Union Medical College (Beijing, China). The experimental protocols were performed according to the Good Laboratory Practice regulations for nonclinical laboratory studies of drugs issued by the National Scientific and Technologic Committee of the People’s Republic of China. Embryos (n = 30) were immersed in 2−3 mL of test drug solution in a 20 mm dish from 6 hpf to 3 dpf, and the drug solution was replaced by freshly 239
DOI: 10.1021/acs.chemrestox.7b00300 Chem. Res. Toxicol. 2018, 31, 238−250
Article
Chemical Research in Toxicology Table 1. MS Parameters for Each Compound no.
a
drug
MW
precursor ion (Da)
1
GMFX
389.15
390.1
2
CPFX
331.35
332.4
3
CPFX-C
305.12
306.2
4
MXFX
401.18
402.2
5
SPFX
392.17
393.2
6
PEFX
333.36
334.4
7
LMFX
351.35
352.3
8
de-LMFX
307.15
308.3
9
GTFX
375.16
376.3
10
NM-GTFX
389.18
390.2
11
MP-GTFX
375.16
376.2
12
HD-GTFX
361.14
362.2
13
F-GTFX
363.14
364.3
14
LVFX
361.37
362.4
15
ATFX
376.15
377.3
16
R-ATFX
376.15
377.3
17
de-PZFX
274.11
275.3
18
ENFX
359.16
360.2
production ion (Da) d
372.2 (F1) 313.2 288.1 (F2)d 245.3 (F6) 288.3 (F1)d 268.3 (F4) 358.2 (F2)d 384 (F1) 349.2 (F2)d 292.2 (F6) 290.1 (F2)d 232.9 (F6) 308.3 (F2)d 265.3 (F6) 265.1 (F6)d 288.2 (F4) 332 (F2)d 289.2 (F6) 346 (F2)d 289.2 (F6) 332.2 (F2)d 289.3 (F6) 318 (F2)d 275 (F6) 320.1 (F2)d 277.3 (F6) 318.4 (F2)d 261 (F6) 333.4 (F2)d 276.2 (F6) 332.9 (F2)d 276.1 (F6) 255.2 (F4)d 217.2 316 (F2)
Cea (V)
Dpb (V)
Cxpc (V)
25.2 38.9 24.8 31.86 22.5 34 27.5 30 27.86 34.05 24.05 33.89 22.6 32.25 29.96 27.49 23.4 29.6 24.9 31.7 25.8 30.7 20.7 29.4 23.3 30.1 26.15 36.01 27.2 34.7 27.2 34.7 24.56 29 25.2
80.3
7.3 16.5 5.7 6.75 5.58 5.1 8.4 8.02 6.68 7.2 5.26 12.89 3.66 5.24 5.58 5.53 17.9 14.8 19.1 15.1 6.32 7.3 8.7 14.8 6.3 5.2 6.25 13.43 8.41 14.75 8.41 14.75 13.18 10.9 16.1
84.8 54.3 96.4 137 98.56 110.86 126 114.3 129.4 114.5 94.3 118.3 117.25 127 127 70.7 80.5
Declustering potential. bCollision energy. cCell exit potential. dIndicates the ion used for quantification.
prepared solution every day. A concentration series (0.05, 0.5, 5, 50 mM) was used for range finding, followed by a narrower concentration series lying in the range between 0% and 100% mortality for LC50 determination. Zebrafish were observed under a microscope (Olympus). Egg condensation and cardiac arrest were used as indicators of embryo death. Each compound was tested three times. Mortality from each treatment group was documented at 3 dpf. Embryos (n = 30) incubated in breeding water were used as controls. Values for LC50 were calculated using the Bliss algorithm.40 Determination of Drug Absorption in Zebrafish by LC-MS/ MS. For embryotoxicity evaluation, the embryos (n = 15) were exposed to 10% LC50 from 6 hpf to 3dpf. For neurotoxicity study, the embryos (n = 15) were exposed to 1% LC50 from 6 hpf to 6 dpf. Before zebrafish samples were collected, the anesthetic MS-222 (3aminobenzoic acid ethyl ester, methanesulfonate salt, Sigma-Aldrich) was added to the breeding water at a final concentration of 0.016%. Next, the zebrafish were washed three times in deionized water with a cell strainer (100 μm, BD-Falcon) and transferred to a 1.5 mL centrifuge tube in which they were triturated in 100 μL deionized water.39 Then, 20 μL internal standard (40 ng/mL) and 20 μL sample homogenate were added to the 400 μL methanol. The samples were vortexed 2 min and centrifuged 10 min at 10,800 g to remove protein. Finally, 7 μL of the supernatant was injected into LC-MS/MS. LC-MS/MS Method. LC-MS/MS was performed using a LC-20A (Shimadzu, Japan) and Qtrap 6500 mass spectrometer (AB Sciex, Germany) equipped with electrospray ionization (ESI) and controlled
by Analyst (version 1.6.2, AB Sciex). HPLC had a thermostated autosampler (set to 15 °C) and a column oven (set to 35 °C). Separation was carried out using a C18 column (2.1 mm × 100 mm, 2.7 μm, Shiseido Capcell Core) with 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B) as eluents. The flow rate was 300 μL/min. The gradient was as follows: 0.0 min, 5% solvent B; 2 min, 5% solvent B; 4.5 min, 30% solvent B; 5.5 min, 95% solvent B; 7.0 min, 95% solvent B; 7.1 min, 5% solvent B; 10 min, 5% solvent B. The test was operated in the positive ion mode, and quantification was performed by MRM scan mode. The ion source parameters were as follows: curtain gas = 40 psi, ion spray voltage = 5000 V, temperature = 500 °C, ion source gas 1 (gs1) = 50 psi, ion source gas 2 (gs2) = 50 psi, collision gas = medium and entrance potential voltage = 12 V. The compound parameters (declustering potential, collision energy, cell exit potential) were optimized and given in Table 1. A 1/x weighted regression was used for calibration curves. Method Validation. Six-level standard solutions were added to the control zebrafish homogenate to construct the calibration curve for each compound. The limits of detection (LOD) were determined at a signal-to-noise ratio of 3:1. The lower limit of quantification (LOQ) was defined as a signal-to-noise ratio of 10:1, and accuracy was within 80−120%. The method precision and accuracy were evaluated by adding three standard FQ solutions (high, middle, and low concentrations) into blank zebrafish homogenates, and results were expressed as the percentage of relative standard (RSD%) and mean 240
DOI: 10.1021/acs.chemrestox.7b00300 Chem. Res. Toxicol. 2018, 31, 238−250
Article
Chemical Research in Toxicology
Figure 2. Primary fragmentation pathway of FQs based on (+)ESI-MS/MS.
Table 2. Standard Curves, LOD, LOQ, and Ranges of FQs 6 dpf drug
LOD (ng/mL)
LOQ (ng/mL)
range (ng/mL)
GMFX CPFX CPFX-C MXFX SPFX PEFX LMFX de-LMFX GTFX NM-GTFX MP-GTFX HD-GTFX F-GTFX LVFX ATFX R-ATFX de-PZFX
0.032 1.28 3.08 3.64 1.71 2.13 3.32 1.54 0.355 1.34 1.46 0.31 0.992 0.089 0.304 1.82 1.54
0.8 3.2 6.16 18.2 4.28 5.32 16.6 3.84 4.44 3.36 3.64 1.55 2.48 5.56 3.8 9.12 7.68
0.8−100 3.2−800 6.16−770 18.2−4550 4.28−1070 5.32−1330 16.6−830 3.84−960 4.44−1110 3.36−8400 3.64−910 1.55−970 2.48−620 5.56−1390 3.8−950 9.12−1140 7.68−960
standard curve y y y y y y y y y y y y y y y y y
= = = = = = = = = = = = = = = = =
observed concentration/spiked concentration × 100%, respectively. Compound recovery was acquired by comparing the peak area of extraction of the three-point samples to analyte added to blank zebrafish homogenate at the same concentration. Matrix effect was estimated by comparing the peak areas of deproteinated samples of blank zebrafish homogenates from three samples with analytes to those of the standard samples at equivalent concentrations. Triplicate sets of control homogenates with analytes were analyzed by LC-MS/ MS. Neurobehavioral Tests. The exposure period was from 6 hpf to 6 dpf, which covered the major stages of organ and nervous system development. This regimen maximized detecting effects of drugs. The LC50 of 3 dpf zebrafish for each chemical was defined as 100%. Zebrafish were exposed to a series of drug solutions with different concentrations diluted from the LC50. Larvae with no obvious malformations at both 3 dpf and 6 dpf were used for further analysis. Individual larvae were placed in each micropore of a 96-well plate, and 12 larvae per concentration group were subjected to behavioral tests using ZEBRALAB (ViewPoint, Zebralab 3.3, France). Detection was performed in the movement plateau period, 12:30−15:30. Zebrafish locomotor activity was recorded under the following specific conditions: (1) dark for 20 min, recorded every 2 min, and (2) light/dark cycle (dark for 5 min/light for 10 s), recorded every 10 s in 4 cycles. Control larvae were used as a normal group. Measurements were repeated at least three times for each FQ. We then calculated
0.235x + 1.83 0.0221x + 0.0964 0.0435x + 0.217 0.0167x + 1.23 0.0853x + 0.158 0.0332x + 0.138 0.0301x + 0.317 0.0262x + 0.0619 0.043x + 0.164 0.062x + 0.0213 0.0119x + −0.00306 0.0213x + 0.388 0.0299x + 0.062 0.0698x + 0.613 0.0349x + 0.124 0.0319x + 0.315 0.126x + 0.532
3 dpf r 0.9788 0.9958 0.9984 0.9958 0.9963 0.9986 0.9965 0.9987 0.997 0.9993 0.9982 0.9993 0.9993 0.9982 0.9976 0.997 0.998
standard curve y y y y y y y y y y y y y y y y y
= = = = = = = = = = = = = = = = =
0.0978x + 0.196 0.0199x + 0.00512 0.054x + 0.022 0.0142x + −0.0355 0.0726x + 0.0525 0.0351x + 0.0321 0.0287x + 0.0377 0.0341x + 0.0134 0.0357x + 0.0697 0.0593x + −0.0486 0.00998x + 0.0181 0.018x + 0.516 0.0291x + 0.0289 0.0757x + 0.743 0.0379x + 0.118 0.0418x + 0.0151 0.128x + 0.184
r 0.9948 0.9997 0.9977 0.9966 0.9995 0.9999 0.9996 0.9992 0.9994 0.9983 0.9982 0.9984 0.9993 0.9994 0.9994 0.9998 0.9992
average distance (AD) moved and average speed over 20 min under the dark condition for each larvae in the different concentration groups. In addition, we calculated average locomotion distance per 10 s and average speed per larvae in the dark and light under the dark/ light cycle conditions. Statistical analyses were performed using IBM SPSS software 20.0. Comparisons were performed by one-factor ANOVA followed by Dunnett’s post hoc analysis. Significance was assumed at P < 0.05, P < 0.01, and P < 0.001. Theoretical Study of Molecular Conformational Analysis in an Aqueous Environment. Based on a previous study,41 the initial 3D structures of FQs were obtained from the PubMed online compound database. The possible charge forms of the molecules were determined based on pKa and modified manually. Conformer generation and minimization were accomplished by Accerlys Discovery Studio 4.0 (DS4.0, Accelrys Software Inc., San Diego, USA). The best algorithm was used to generate conformation. The relative energy threshold between conformers was set to 50 kcal/mol, and the maximum number of conformers generated was 1023. Conformer minimization was performed using the CHARMM force field with generalized Born with the molecular volume implicit solvent model. The dielectric constant was fixed to 80. Typical conformers with lower energy were selected as global minima candidates for quantum chemical optimization. All density functional theory (DFT) calculations were conducted using the ORCA 2.9.1 program.42 BP86 with Grimme’s latest London241
DOI: 10.1021/acs.chemrestox.7b00300 Chem. Res. Toxicol. 2018, 31, 238−250
Article
Chemical Research in Toxicology Table 3. Accuracy, Precision, Recovery, And Matrix Effect of Analytes in 3/6 dpf Zebrafish (n = 3) accuracy (%) FQ GMFX
CPFX
CPFX-C
MXFX
SPFX
PEFX
LMFX
de-LMFX
GTFX
NM-GTFX
MP-GTFX
HD-GTFX
F-GTFX
LVFX
ATFX
R-ATFX
de-PZFX
precision (%)
recovery (%)
matrix effect (%)
drug dose (ng/mL)
3 dpf
6 dpf
3 dpf
6 dpf
3 dpf
6 dpf
3 dpf
6 dpf
2 10 50 20 100 500 10 50 250 200 1000 5000 40 200 1000 50 250 1250 20 100 500 10 50 250 20 100 500 200 1000 5000 10 50 250 4 20 100 20 100 500 50 250 1250 20 100 500 20 100 500 20 100 500
106.00 102.00 106.00 102.00 93.40 101.00 90.20 106.00 97.30 103.00 81.70 101.00 97.20 104.00 99.10 96.80 101.00 101.00 98.70 96.70 96.90 113.00 88.30 99.40 97.40 93.60 97.40 104.00 89.30 91.60 89.10 105.00 89.10 102.00 104.00 90.90 86.20 101.00 94.10 89.10 114.00 106.00 105.00 105.00 97.00 99.30 101.00 97.40 93.80 109.00 90.60
107.00 118.00 98.30 96.70 107.00 92.50 93.00 110.00 99.80 102.00 109.00 97.20 93.80 112.00 94.30 87.10 106.00 97.20 95.20 114.00 105.00 86.10 94.00 106.00 93.90 102.00 105.00 103.00 103.00 98.70 117.00 86.70 89.30 90.70 93.90 97.80 90.70 104.00 97.40 105.00 114.00 95.80 104.00 119.00 98.80 76.20 123.00 104.00 93.80 98.10 107.00
7.20 9.90 6.50 1.12 2.40 7.21 13.70 4.24 13.23 8.37 9.64 12.36 6.18 2.52 2.42 3.85 3.26 1.28 2.99 0.49 3.63 12.08 13.18 1.13 6.18 5.08 3.95 4.11 5.95 2.95 12.74 5.06 6.75 8.68 10.55 5.76 12.31 5.13 3.54 8.19 5.12 4.32 13.87 2.99 1.73 2.88 7.17 2.64 3.10 8.02 9.74
13.90 5.50 10.30 2.80 2.70 10.30 7.50 5.70 6.40 3.10 1.90 4.70 3.60 2.20 8.60 6.30 3.90 4.50 2.60 2.20 1.20 3.20 2.40 5.00 9.20 0.70 1.80 0.40 1.50 2.10 7.40 3.60 8.20 2.50 3.20 4.10 8.10 2.90 3.70 6.50 1.00 4.40 8.80 0.80 1.70 5.00 3.10 5.50 5.90 2.80 7.60
113.04 111.82 111.39 93.83 100.59 105.32 94.95 100.36 113.95 108.68 94.86 104.46 103.15 108.07 107.60 113.58 112.23 112.33 113.45 110.69 111.20 95.75 92.32 91.04 111.70 111.24 103.88 111.76 109.80 105.98 95.92 103.72 104.05 93.04 113.07 109.05 102.19 110.86 110.96 94.74 109.35 102.85 107.68 108.38 103.88 111.37 107.89 104.48 112.27 112.35 109.85
102.05 112.18 108.90 90.84 112.66 105.34 106.32 97.39 88.91 97.07 107.83 101.93 92.39 97.04 103.51 112.52 106.75 84.48 89.95 105.34 98.96 85.51 93.82 99.44 90.79 103.59 99.94 97.01 86.74 80.86 95.18 91.14 89.57 97.85 95.29 88.97 94.47 87.50 83.22 89.72 100.09 97.09 90.79 92.74 95.16 96.01 83.65 88.51 108.47 95.64 89.45
92.53 92.65 103.77 109.26 106.82 114.60 92.34 80.95 91.54 109.68 92.75 113.36 113.30 110.54 113.53 101.48 105.94 107.40 112.44 111.58 112.64 102.16 99.28 108.37 111.93 106.92 114.71 114.21 109.19 115.87 94.92 85.02 98.06 109.46 101.05 107.35 105.45 100.76 109.34 109.30 114.22 117.79 112.71 113.16 109.46 98.90 91.30 88.92 90.34 94.93 99.61
108.82 110.34 106.34 82.52 110.83 110.20 90.33 89.24 108.07 107.80 112.52 105.29 98.83 110.03 99.92 109.66 98.36 114.50 107.17 114.25 106.46 105.97 89.83 97.21 105.51 113.78 104.12 107.78 116.03 120.08 124.17 100.09 110.40 117.28 105.76 110.99 114.72 108.57 114.44 113.54 115.11 102.42 103.20 120.76 108.30 107.50 111.26 97.14 83.69 99.05 103.37
dispersion correction (BP86-D3)43,44 and def2-TZV plus polarization basis sets45 were used. All the conformers were first optimized at the def2-TZVP (-df) basis set level to locate the most stable conformers. Then the def2-TZVP (-f) basis set was used to reoptimize the conformers. Harmonic vibrational frequencies were used to verify that the calculated structure was minimal on the potential energy surface. Resolution of the identity and the auxiliary def2-TZVP/J Coulomb fitting basis set46 were used to accelerate the computational process.
The conductor-like screening model with a dielectric constant of 80 was used throughout the study to simulate the aqueous environment. Molecules Preparation and Docking. The DS 4.0 software package was used for the docking study of selected target and ligands. For protein preparation, the 3D crystallographic structure of zebrafish GABA receptor was modeled through homology modeling server SWISS-MODEL (https://swissmodel.expasy.org/). The template of GABA receptor was human GABAA receptor (PDB ID: 5OJM). 242
DOI: 10.1021/acs.chemrestox.7b00300 Chem. Res. Toxicol. 2018, 31, 238−250
Article
Chemical Research in Toxicology Table 4. Summary of Absorption Results in Zebrafish 3 dpf no.
drug
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
GMFX CPFX CPFX-C MXFX SPFX PEFX LMFX de-LMFX GTFX NM-GTFX MP-GTFX HD-GTFX F-GTFX LVFX ATFX R-ATFX de-PZFX
LC50 (mM) 0.54 1.87 0.81 1.52 3.6 2.9 4.07 0.57 11.7 0.97 4.6 1.67 1.86 15.0 1.67 7.1 0.48
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.06 0.69 0.14 0.50 0.4 0.2 0.73 0.02 5.0 0.21 1.1 0.49 0.50 2.8 0.03 0.02 0.23
6 dpf
Internal concentration (IC, nM)a
IC/MCb
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
85 98 11 302 767 342 68 182 17 4454 61 69 91 215 125 89 61
4.61 18.37 0.89 45.93 276.02 99.23 27.51 10.40 19.97 432.06 28.02 11.56 16.84 322.44 20.91 62.89 2.91
1.16 2.32 0.33 5.56 63.69 14.52 4.11 3.63 2.39 68.13 3.33 0.89 3.98 28.15 2.79 5.33 1.08
body burden (nmol/larva)c 3.14 8.99 0.99 (except for GMFX, r = 0.9788). The results of precision, accuracy, recovery, and matrix effect for each compound are shown in Table 3. The precision for each FQ was GTFX (16931) ≫ NM-GTFX (2094), which indicates that the strength of methylation influence on movement was 2-methyl > 3-methyl ≫ 3,4-dimethyl. R-8 Substituents. The effects of GTFX and HD-GTFX were classified as Type II, while F-GTFX was Type III. The structural features indicate that substituents at position 8 can change effect type. From the 1% ratio, we found that HDGTFX (359359) ≫ GTFX (16931), which suggests that OH at position C-8 has a greater effect on zebrafish movement than CH3O. Based on these results, correlations between FQ structural features and effects on zebrafish neurobehavior are summarized in Figure 5. The piperazinyl structure at position C-7 is a functional group that affects nerves system function of zebrafish. Other modification sites also affect locomotor activity, but only COOH at position C-3 and F at position C-8 can change the effect pattern. Stable Conformation and Larval Zebrafish Behavior. The structure difference between ATFX and R-ATFX is molecular configuration, and they have different effects on zebrafish locomotor activity. Stable conformations of some FQs were analyzed for illustrating the steric hindrance effects. Based on pH of the environment, FQs may exist in cation, anion, neutral, or zwitterion forms. The 3-carboxyl and 7-piperazinyl groups of FQs can be ionized, and pKa1 is generally pH 5−6, while pKa2 is approximately pH 8. Under physiological conditions, a carboxyl group exists in the form of an oxygen anion, and a nitrogen atom at position 4 of the piperazinyl
smaller effect on embryotoxicity. Absorption capacity of the three substituent groups was such that F > OH > CH3O, and the strength of embryotoxicity was such that OH > F > CH3O. CPFX-C, which is generated by cleavage of the piperazinyl structure of CPFX, has an ethylenediamine moiety at position C-7. The uptake concentration of CPFX was 18.37 nM/larva, which was about 20 times greater than that of CPFX-C (0.89 nM/larva). The IC/MC of CPFX was about nine times higher than that of CPFX-C. The results show that the piperazinyl structure at position C-7 increases drug absorption, and the ethylenediamine moiety significantly enhances embryotoxicity. NM-GTFX and MP-GTFX are impurities of GTFX generated by different methyl substituents at the piperazinyl structure. The internal concentration of NM-GTFX was 432.06 nM/larva, which was about 22 times greater than that of GTFX (19.97 nM/larva) and 15 times greater than that of MP-GTFX (28.02 nM/larva). The IC/MC of these chemicals were 17, 4454, and 61, respectively. The results show that the order of embryotoxicity due to substituted piperazine was 3-methyl > 2methyl ≫ 3,4-dimethyl, and the order of absorption capacity was 3,4-dimethyl ≫ 2-methyl > 3-methyl. Thus, methylation of N at position C-4 of piperazine can significantly enhance absorption capacity and reduce embryotoxicity. Correlations between structural features, uptake, and embryotoxicity are summarized (Figure 5) based on the results presented above. Although 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. Effects of FQs on Larval Zebrafish Neurobehavior. The trends of average distance (AD) and average speed of larval locomotor activity under dark and dark/light cycle conditions were generally consistent as shown for the results of the LMFX group (Figure 3A). Although illumination had an inhibitory effect on larval movement, the dose−response relationship was almost the same as that under the dark condition. Because AD over 20 min under dark conditions per larvae was more reliable (lower SEM), we chose AD as an indicator to assess the influence of FQs on neurobehavior of zebrafish larvae. Based on the results of AD (Table 5), effects of FQs on movement of larvae were divided into four categories as shown in Figure 3B. Type I showed no significant influence on locomotor activity (dose−response) compared with the normal group. These drugs included GMFX, CPFX-C, MXFX, dePZFX, and LVFX. Type II suppressed locomotor activity (monotonic dose−response) compared with the normal group and included GTFX, de-LMFX, R-ATFX, NM-GTFX, HDGTFX, and MP-GTFX. Type III inhibited locomotor activity at low concentration and stimulated locomotor activity at high concentration. The inhibitory effect weakened and even disappeared with an increase of exposed drug concentration, resulting in a monotonic response. These drugs included FGTFX, LMFX, and PEFX. Type IV stimulated and then suppressed (biphasic response) locomotor distance under the exposed dose range. This type included SPFX, CPFX, and ATFX. As shown in Table 5, no significant enhancement of AD compared with the control group indicates that the main effect of FQs was to stimulate and inhibit; however, the inhibitory effect on zebrafish movement was dominant. Furthermore, a 246
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Figure 4. Most stable conformations of ATFX, R-ATFX, LMFX, and GTFX and the docking results for these compounds.
structure may be positively charged. The most stable aqueous conformations of representative chemicals are shown in Figure 4. Both the nucleus and piperazinyl structure of ATFX and RATFX are planar structures, but they are in different planes. For the most stable conformation of ATFX, the methyl at position 1,8-ring and piperazinyl at position 7 occupy space positions on the different side of the nuclear plane observed from 1-N to 4C; however, these groups reside on the same side in R-ATFX. The different effects of ATFX (Type IV) and R-ATFX (Type II) on zebrafish locomotor activity indicate that effects of FQs on zebrafish movement may be related to the different space structure. Decarboxylation can change the effect of LMFX from Type III to Type II, which is caused primarily by the piperazinyl at position 7 and shows that the piperazinyl (toxic functional group I) and the nucleus containing a carboxyl at position 3 (toxic functional group II) can function independently. Because the methyl at the 1,8-ring of ATFX and R-ATFX is adjacent to the carboxyl at position 3 and R-ATFX does not show the Type III effect (the function was lost), we hypothesize that the toxic functional group II is influenced by steric hindrance. Finally, we analyzed effects of LMFX and GTFX on zebrafish behavior. Neurobehavior assessment indicated that LMFX, which has an ethyl at position 1, is Type III, while GTFX with an cyclopropyl at position 1 is Type II. Effects of the adjacent substituent group at position 8, which can be F (LMFX, PEFX,
and F-GTFX) or CH3O (GTFX, NM-GTFX, and MP-GTFX), may contribute to significant differences in conformation. Steric hindrance of toxic functional group II is minor when the R-8 substituent is F. Thus, both toxic functional groups can function independently, showing both stimulation and inhibition of zebrafish locomotor activity. However, when the R-8 substituent is CH3O, due to effects of steric hindrance, only toxic functional group I exerts effects on zebrafish behavior. Analysis of the Optimum Molecular Docking Poses of Different Ligands. The docking results for the compounds that showed the optimum docking pose with GABA receptor were summarized in Figure 4 and Table S1. ATFX presented the formation of three hydrogen bonds with residues of the GABA receptor (Figure 4E). The quaternary amine with a positive charge of piperazinyl group at the C-7 position and the imino group at the C-5 position of ATFX restored hydrogen bonds with GLU 400, GLU 402, and ARG 394. The carboxyl group at the C-3 side chain, the imino group at the C-5 position, and the quaternary amine with a positive charge of piperazinyl group at the C-7 position of R-ATFX (Figure 4F) formed four hydrogen bond interactions with the residues LYS 427, LYS 373, and GLU 402. The hydroxide radical at the C-4 position and the quaternary amine of piperazinyl group at the C-7 position of LMFX interacted via the formation of two hydrogen bonds with LYS 373 and GLU 402 (Figure 4G). In addition, the carboxyl group at the C-3 position and the 247
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Figure 5. Summary of relationships between FQ structure, uptake, and embryotoxicity as well as impacts of different substituent groups on locomotor behavior of zebrafish.
functional group I and reverse its effects. It remains to be investigated whether toxic functional group II works through activation of NMDA receptors. Structural modifications of FQs can change toxicity properties (Figure 5). We compared relationships between embryotoxicity and neurotoxicity. 7-piperazinyl cleavage, such as in CPFX-C, led to loss of neurotoxicity but enhanced embryotoxicity. Modification at position 1 might change neurotoxicity and embryotoxicity by altering absorption. The eight substituents may generate different types of steric hindrance that not only impact absorption properties but also alter neurotoxicity patterns such that neurotoxicity is not relevant to embryotoxicity. Moreover, 5-NH2 derivatives and 3decarboxyl formation might enhance embryotoxicity and neurotoxicity. Both SPFX and ATFX are 5-NH2 derivatives of FQs. As the QTc interval prolongation and phototoxicity, SPFX was withdrawn from the European and U.S. markets.1 CNS effects are the most common adverse effects for SPFX in clinical trials.51 ATFX is a novel broad-spectrum FQ approved for use in China in 2009.52 At present, we have not found reports on its CNS effects, but our results suggest that this drug merits close attention in clinical therapy. As drug impurities are considered to be one of the most important causes of drug safety issues, the relationships between structure and toxicity are useful for more objective control of impurity profiling, which is helpful to reduce the adverse drug effects in humans. According to our results, the imputities with 5-NH2 and 3decarboxyl formation should be paid much attention. The GABA-like structure of the piperazinyl moiety at the 7 position of the parent nucleus acts as antagonist of GABA receptors. FQs that possess an unsubstituted piperazine at position 7 exhibit a stronger inhibitory effect on GABA receptors compared with those that have methyl group(s) substituted for piperazine due to steric hindrance from the methyl group.2,17 Our docking studies showed that the 7piperazinyl of FQs could form hydrogen-bond interaction with the residue GLU 402 of GABA receptor and steric hindrance of the compounds influenced upon the binding. Our results also indicate that the piperazinyl moiety is an essential functional group for neurotoxicity of FQs, and effects of chemicals with a
quaternary amine of piperazinyl group at the C-7 position of GTFX formed two hydrogen bonds with the residues LYS 427 and GLU 402 (Figure 4H). From the combined data of several studies, it appears that the R7 side chain substituent has the strongest influence on the GABA binding inhibition.1,17,49,50 The findings suggest that not only the piperazinyl group at the C-7 side chain may play a vital role in the binding with the residue GLU 402 of GABA receptor, but steric hindrance of the compounds was influenced upon the binding.
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DISCUSSION In this study, the zebrafish was used as a model organism, and neurobehavior was used as an evaluation index. We analyzed relationships between FQ structure, embryotoxicity, and effects on larval locomotor activity and explored correlations between substituents and CNS effects. Results demonstrate that there are two toxic functional groups: piperazinyl (toxic functional group I) and the nucleus containing carboxyl at position 3 (toxic functional group II). These groups have opposite effects on the zebrafish CNS. Previous studies have shown that the 7piperazinyl structure of FQs is essential for inhibition of GABA receptors and stimulation of the CNS,17 and carboxyl groups at positions C-3 and carbonyl moiety at C-4 chelate Mg2+ to stimulate NMDA receptors,18 which may correspond to the toxicity mechanisms of functional groups I and II, respectively. In this study, some of the FQs showed single inhibitory effects of functional group I, but single stimulatory effects of toxic functional group II were not observed. Both LMFX and CPFX have two toxic functional groups. Inhibition and stimulation of zebrafish locomotor activity were observed with LMFX (Type III) and CPFX (Type IV). De-LMFX, which is a decarboxylation product of LMFX, only showed inhibitory effects caused by toxic functional group I. However, CPFX-C, which is formed by piperazinyl cleavage, lost toxic functional group I and did not show effects of toxic functional group II. In addition, neither stimulation followed by inhibition nor significant enhancement of movement (Table 5) was found for any of the chemicals in this study. This suggests that toxic functional group I can function alone to inhibit GABA receptors, but reactions of toxic functional group II might be secondary to effects of toxic 248
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(4) Hooper, D. C., and Wolfson, J. S. (1985) The fluoroquinolones: pharmacology, clinical uses, and toxicities in humans. Antimicrob. Agents Chemother. 28, 716−721. (5) Owens, R. C., and Ambrose, P. G. (2000) Clinical use of the fluoroquinolones. Med. Clin. North Am. 84, 1447−1469. (6) Stahlmann, R., and Lode, H. M. (2013) Risks associated with the therapeutic use of fluoroquinolones. Expert Opin. Drug Saf. 12, 497− 505. (7) Patel, K., and Goldman, J. L. (2016) Safety Concerns Surrounding Quinolone Use in Children. J. Clin. Pharmacol. 56, 1060−1075. (8) Savic, R. M., Ruslami, R., Hibma, J. E., Hesseling, A., Ramachandran, G., Ganiem, A. R., Swaminathan, S., McIlleron, H., Gupta, A., Thakur, K., van Crevel, R., Aarnoutse, R., and Dooley, K. E. (2015) Pediatric tuberculous meningitis: Model-based approach to determining optimal doses of the anti-tuberculosis drugs rifampin and levofloxacin for children. Clin. Pharmacol. Ther. 98, 622−629. (9) Pandolfini, C., Marco, S., Paolo, M., and Maurizio, B. (2013) The use of ciprofloxacin and fluconazole in Italian neonatal intensive care units: a nationwide survey. BMC Pediatr. 13, 1−7. (10) Liu, H. H. (2010) Safety Profile of the Fluoroquinolones. Drug Saf. 33, 353−369. (11) Mandell, L., and Tillotson, G. (2002) Safety of fluoroquinolones: An update. Can. J. Infect Dis 13, 54−61. (12) Tomé, A. M., and Filipe, A. (2011) Quinolones: review of psychiatric and neurological adverse reactions. Drug Saf. 34, 465−488. (13) Samyde, J., Petit, P., Hillaire-Buys, D., and Faillie, J. L. (2016) Quinolone antibiotics and suicidal behavior: analysis of the World Health Organization’s adverse drug reactions database and discussion of potential mechanisms. Psychopharmacology 233, 2503−2511. (14) De Sarro, A., and De Sarro, G. (2001) Adverse reactions to fluoroquinolones. an overview on mechanistic aspects. Curr. Med. Chem. 8, 371−384. (15) Suto, M. J., Domagala, J. M., Roland, G. E., Mailloux, G. B., and Cohen, M. A. (1992) Fluoroquinolones: relationships between structural variations, mammalian cell cytotoxicity, and antimicrobial activity. J. Med. Chem. 35, 4745−4750. (16) Halliwell, R. F., Davey, P. G., and Lambert, J. J. (1993) Antagonism of GABAA receptors by 4-quinolones. J. Antimicrob. Chemother. 31, 457−462. (17) Akahane, K., Sekiguchi, M., Une, T., and Osada, Y. (1989) Structure-epileptogenicity relationship of quinolones with special reference to their interaction with gamma-aminobutyric acid receptor sites. Antimicrob. Agents Chemother. 33, 1704−1708. (18) Serafini, G., Pompili, M., Innamorati, M., Dwivedi, Y., Brahmachari, G., and Girardi, P. (2013) Pharmacological properties of glutamatergic drugs targeting NMDA receptors and their application in major depression. Curr. Pharm. Des. 19, 1898−1922. (19) Ilgin, S., Can, O. D., Atli, O., Ucel, U. I., Sener, E., and Guven, I. (2015) Ciprofloxacin-induced neurotoxicity: evaluation of possible underlying mechanisms. Toxicol. Mech. Methods 25, 374−381. (20) Yakushiji, T., Shirasaki, T., and Akaike, N. (1992) Noncompetitive inhibition of GABAA responses by a new class of quinolones and non-steroidal anti-inflammatories in dissociated frog sensory neurones. Br. J. Pharmacol. 105, 13−18. (21) Davey, P. G., Charter, M., Kelly, S., Varma, T. R., Jacobson, I., Freeman, A., Precious, E., and Lambert, J. (1994) Ciprofloxacin and sparfloxacin penetration into human brain tissue and their activity as antagonists of GABAA receptor of rat vagus nerve. Antimicrob. Agents Chemother. 38, 1356−1362. (22) Goa, K. L., Bryson, H. M., and Markham, A. (1997) Sparfloxacin. A review of its antibacterial activity, pharmacokinetic properties, clinical efficacy and tolerability in lower respiratory tract infections. Drugs 53, 700−725. (23) Bharal, N., Pillai, K. K., and Vohora, D. (2006) Effects of sparfloxacin on CNS functions and urinary hydroxyproline in mice. Pharmacol. Res. 54, 111−117. (24) Nishimura, Y., Murakami, S., Ashikawa, Y., Sasagawa, S., Umemoto, N., Shimada, Y., and Tanaka, T. (2015) Zebrafish as a
dimethyl piperazine moiety are weaker than those with a methyl group substituted for piperazine on zebrafish locomotor activity. MXFX has an azabicyclo at position 7, and in contrast to some other FQs, it appears to have a low propensity for causing CNS excitatory effects.53,54 GMFX is also a 7-azacyclo derivate. In a clinical study of 6775 patients, CNS effects occurred in 2.8% of patients, with 1.2% reporting headache and 0.8% reporting dizziness; however, severe CNS reaction (seizures, toxic psychosis, and delirium) have not been reported.55 LMFX has been reported to be most excitatory of the compounds.56 LVFX has the lowest CNS penetration, which may contribute to its lower incidence rate and weaker neurotoxicity.57,58 These results are similar to the observed correlations between FQ structure and effects on zebrafish locomotor activity in our study, which suggests that zebrafish neurobehavior tests can be used to accurately assess neurotoxicity of FQs and provide information complementary to that of mammal experiments.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.7b00300. Additional experimental details and data (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. Tel: +86-10-53851532. *E-mail:
[email protected]. Tel: +86-1063186645. ORCID
Changqin Hu: 0000-0003-1385-6601 Funding
National Major Scientific and Technological Special Project for “Significant New Drugs Development” (nos. 2011ZX09303, 2015ZX09303001, and 2017ZX09101001-007). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Jie Meng for fish husbandry, and she partly participated in the test of zebrafish embryonic toxicity. We thank LetPub (http://www.letpub.com) for its linguistic assistance during the preparation of this manuscript.
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ABBREVIATIONS CNS, central nervous system; FQs, fluoroquinolones; LC-MS/ MS, liquid chromatography-tandem mass spectrometry; AEs, adverse effects
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REFERENCES
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