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Efficient Reduction of Antibacterial Activity and Cytotoxicity of Fluoroquinolones by Fungal-mediated N-Oxidation Marina Rusch, Astrid Spielmeyer, Jessica Meißner, Manfred Kietzmann, Holger Zorn, and Gerd Hamscher J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b01246 • Publication Date (Web): 01 Apr 2017 Downloaded from http://pubs.acs.org on April 2, 2017

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Journal of Agricultural and Food Chemistry

Efficient Reduction of Antibacterial Activity and Cytotoxicity of Fluoroquinolones by Fungal-mediated N-Oxidation Marina Rusch,† Astrid Spielmeyer,† Jessica Meißner,‡ Manfred Kietzmann,‡ Holger Zorn,† § and Gerd Hamscher† *

†

Justus Liebig University Giessen, Institute of Food Chemistry and Food

Biotechnology, Heinrich-Buff-Ring 17, 35392 Giessen, Germany ‡

University of Veterinary Medicine Hannover Foundation, Institute of Pharmacology,

Toxicology and Pharmacy, Buenteweg 17, 30559 Hannover, Germany §

Fraunhofer IME, Project Group Bioresources, 35392 Giessen, Germany

*Corresponding author (Tel: +49 6419934950; Fax: +49 6419934909; E-mail: [email protected])

1 ACS Paragon Plus Environment


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ABSTRACT: Extensive usage of fluoroquinolone antibiotics in livestock results in

2

their occurrence in manure and subsequently in the environment. Fluoroquinolone

3

residues may promote bacterial resistance and are toxic to plants and aquatic organisms.

4

Moreover, fluoroquinolones may enter the food chain through plant uptake, if manure is

5

applied as fertilizer. Thus, the presence of fluoroquinolones in the environment may

6

pose a threat to human and ecological health. In this study, the biotransformation of

7

enrofloxacin, marbofloxacin and difloxacin by the fungus X. longipes (Xylaria) was

8

investigated. The main metabolites were unequivocally identified as the respective N-

9

oxides by mass spectrometry and nuclear magnetic resonance spectroscopy. Fungal-

10

mediated N-oxidation of fluoroquinolones led to a 77-90% reduction of the initial

11

antibacterial activity. In contrast to their respective parent compounds, N-oxides showed

12

low cytotoxic potential and had a reduced impact on cell proliferation. Thus,

13

biotransformation by X. longipes may represent an effective way for inactivating

14

fluoroquinolones.

15 16

KEYWORDS: enrofloxacin, marbofloxacin, difloxacin, N-oxide, Xylaria longipes


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INTRODUCTION

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Fluoroquinolones represent an important group of potent synthetic, broad spectrum,

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highly effective antibacterial agents used in both human and veterinary medicine.1 They

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act by selective inhibition of the bacterial enzymes gyrase and topoisomerase IV which

21

are essentially required for replication of DNA.2 Thus, fluoroquinolones exhibit a high

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bactericidal activity against most Gram-negative bacteria such as Mycoplasma spp. and

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against a wide variety of Gram-positive organisms, including Staphylococcus spp.3

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In human medicine, the amount of antibiotics applied in Germany is estimated to

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account for 700-800 tons per year.4 Beside ß-lactams fluoroquinolones were the most

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frequently used antibiotics in hospitals in 2014.4 Regarding veterinary medicine, data

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about the antibiotics distributed to veterinarians are published by the German Federal

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Office of Consumer Protection and Food Safety since 2012.5 From 2011 to 2015, the

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total sales volume of antibiotics dropped by approximately 53% (1,706 tons in 2011 vs.

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805 tons in 2015). However, during the same period the sales volume of

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fluoroquinolones, which are identified by the World Health Organization6 and by the

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World Organization for Animal Health7 as “Highest Priority Critically Important

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Antimicrobials”, increased up to 29%.5 In veterinary medicine, enrofloxacin, 1, and

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marbofloxacin, 2 (Figure 1) are primarily applied (78% and 18%, respectively).4 In

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general, fluoroquinolones are excreted almost unchanged or only partially metabolized.8

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Within the fluoroquinolones studied in this work difloxacin, 3 (Figure 1) is mainly

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eliminated as parent compound (95%) in pigs. Beside the major metabolite sarafloxacin

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(5%), the N-desmethyl analog of 3 which also exhibits potent antibacterial activity, trace

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metabolites were detected.9 In the case of 1, 74% of the orally applied dose were

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excreted as unchanged compound in broiler chickens.10 Approximately 25% of 1 were



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metabolized via deethylation of the piperazine moiety leading to the highly potent

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fluoroquinolone ciprofloxacin, one of the most frequently prescribed fluoroquinolone in

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human medicine. Aside from ciprofloxacin as the main metabolite,11-12 minor

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metabolites like desethylene-enrofloxacin and desethylene-ciprofloxacin are also

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known.10 For 2, about 30% and 40% of the administered dose was excreted as

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unchanged parent compound in the urine of chickens and dogs, respectively.13-14 N-

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desmethyl-marbofloxacin and N-oxide-marbofloxacin are known metabolites of 2.13, 15-

48

17

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Thus, the extensive usage of these substances leads to their direct release into the

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environment, mostly via application of either liquid manure from livestock or sewage

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sludge from wastewater treatment plants as agricultural fertilizer. The occurrence of

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both unchanged fluoroquinolone residues and their potentially antibiotic active

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metabolites in the environment may result in the development of antibiotic resistance. In

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addition, fluoroquinolones show toxicity to plants and aquatic organisms.18

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Furthermore, after application of manure as fertilizer, fluoroquinolones may be

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transferred to food plants. For example, carry-over of 1 from soil to carrot roots has

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been demonstrated.19 Thus, the presence of fluoroquinolones in the environment may

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pose a serious risk for food safety, human and ecological health. Therefore, it is

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necessary to find an efficient way for degradation or at least inactivation of these

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compounds.

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In order to reduce the entry of fluoroquinolones in the environment, microbial

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degradation of fluoroquinolones has been studied with numerous fungal species.20-23 A

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recent study reported the biotransformation of the fluoroquinolone danofloxacin by the

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ascomycete Xylaria longipes.22 X. longipes quantitatively transformed danofloxacin into


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a single metabolite which was unequivocally identified as danofloxacin N-oxide. The

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residual antibacterial activity of danofloxacin N-oxide was less than 20% of the parent

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compound. Based on these findings the biotransformation of the fluoroquinolones 1, 2

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and 3 by X. longipes was investigated. Beside the elucidation of the respective

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structures, the residual antibacterial activity of produced metabolites was determined.

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Furthermore, cytotoxicity of each fluoroquinolone inclusive danofloxacin and their

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corresponding metabolites was investigated.

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MATERIAL AND METHODS

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Microorganism. Xylaria longipes (Xylaria, Xylariaceae) is a soft rot

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ascomycete and saprophyte growing on dead woods, including fallen branches and

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stumps. The fungus is kept in the fungal culture collection of the Institute of Food

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Chemistry and Food Biotechnology in the Justus Liebig University Giessen, Germany.

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The strain was stored on solid medium at 4 °C as previously described.22

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Chemicals. Difloxacin hydrochloride (CAS 91296-86-5, purity ≥ 98.0%) was

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acquired from Sigma-Aldrich (Steinheim, Germany), while marbofloxacin (CAS

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115550-35-1, purity ≥ 98.0%) and enrofloxacin (CAS 93106-60-6, purity ≥ 98.0%)

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were obtained from TCI (Eschborn, Germany). HPLC grade methanol was obtained

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from VWR (Darmstadt, Germany), formic acid, trifluoroacetic acid and ammonium

83

acetate were acquired from Bernd Kraft (Duisburg, Germany), Fisher Scientific

84

(Schwerte, Germany) and Merck (Darmstadt, Germany), respectively. All chemicals

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were of highest purity available. High-purity water was generated in-house by a

86

laboratory water system (Sartorius, Goettingen, Germany).



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Experimental procedures. Biotransformation experiments. The in vivo

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biotransformation experiments including the preparation of precultures and main

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cultures were carried out as previously described.22 Briefly, 100 mL Erlenmeyer flasks

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containing 4 mL of inoculum and 40 mL of a chemically defined medium were used.

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Fluoroquinolones were dissolved in 0.5% formic acid, sterile filtered (0.22 µm) (Carl

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Roth, Karlsruhe, Germany) and added to the main culture to give a final concentration

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of approximately 10 mg/L. The flasks were shaken at 150 rpm in the dark at 24 °C for 8

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d. Samples were withdrawn at regular intervals (0, 2, 4, 6 and 8 d) and stored at –20 °C

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before analyses. Abiotic and biotic controls were run in parallel and triplicates of each

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condition were carried out.

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Large scale cultivation. For metabolite isolation, the biotransformation

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experiments were repeated on a larger scale. For each substance, ten 250 mL

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Erlenmeyer flasks, each containing 100 mL of the medium and approximately 60 mg/L

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of one of the fluoroquinolones were inoculated and incubated as previously described.22

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When the maximum peak area of the corresponding biotransformation product was

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detected by high-performance liquid chromatography with diode array detection

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(HPLC-DAD), the fungal mycelium was separated by filtration and centrifugation.

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Culture supernatants were frozen (–20 °C) and afterwards thawed in order to precipitate

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high molecular weight polysaccharides produced by the fungus and to remove them by

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filtration. Combined supernatants were concentrated to dryness at 35-37 °C under

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reduced pressure using an evaporator. The residue was resuspended in 25-30 mL 0.5%

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formic acid and used for preparative isolation.

109 110

Analytical procedures. HPLC (analytical). A LaChrom HPLC system equipped with a low-pressure pump (L-7100), an autosampler (L-7200) and a diode


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array detector (DAD) (L-7455) combined with D-7000 HSM software, ver. 4.1 (Merck-

112

Hitachi, Darmstadt, Germany) was utilized for analyses. Measurements were conducted

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on a 150 mm x 4.6 mm i.d., 5 µm, Hypersil GOLD PFP column (Thermo Scientific,

114

Dreieich, Germany) using gradient elution with (A) methanol and (B) 1 mM ammonium

115

acetate in 0.5% formic acid at a flow rate of 1.0 mL/min and detection at 280 nm (for 1

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and 3) and 299 nm (for 2). The binary gradient was as follows: 0-1 min, 5% A; 1-4 min,

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ramp to 60% A; 4-6 min, ramp to 80% A; 6-13 min, ramp to 88% A; 13-14 min, ramp

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to 100% A; 14-16 min, hold at 100% A; 16-16.5 min, ramp to 5% A; 16.5-20 min, hold

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at 5% A. UV spectra were recorded from 200-370 nm, with an interval of 1.6 s and a

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step of 1 nm.

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For quantitation of each fluoroquinolone, stock solutions of each standard (1 mg/mL, in

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methanol) were diluted in medium resulting in concentrations ranging from 0.1 to 30

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mg/L. Linearity was evaluated with eight different concentrations (n = 3) and calculated

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by linear regression. A coefficient of determination (r2) higher than 0.9995 was accepted

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for quantitation. Limit of detection (LOD) and limit of quantitation (LOQ) were

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calculated by the signal/noise ratio (S/N) using the peak-to-peak method. S/N of 3:1 and

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10:1 were used for estimating the LOD and LOQ, respectively.

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HPLC (preparative). A Young Lin preparative HPLC system equipped with

129

quaternary pump (YL 91105), an ultraviolet detector (YL 91205) and a CHF 122SC

130

fraction collector (Advantec, Dublin, CA) combined with YL-Clarity software, ver.

131

3.0.4.444 (Young Lin Instruments, Anyang, Korea) was used for preparative isolation

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of biotransformation products. The column used was a 250 mm x 16 mm i.d., 7 µm,

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Nucleosil C18, with a 10 mm x 16 mm i.d. guard column of the same material

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(Macherey-Nagel, Düren, Germany). The mobile phase consisted of (A) methanol and



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(B) 1 mM ammonium acetate containing 0.05% trifluoroacetic acid. Unlike previously

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described,22 trifluoroacetic acid instead of formic acid was used as eluent additive for

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isolation by HPLC, since formic acid contributed to interfering signals in the NMR

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spectra. The injection volume of the redissolved concentrate was 0.7 mL. The following

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gradient was used: 0-0.3 min, 45% A; 0.3-13 min, ramp to 70% A, 13-13.3 min, ramp to

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45% A; 13.3-19 min, hold at 45% A. The flow rate was set at 8 mL/min and detection

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was carried out at 280 nm and 299 nm. After isolation, fractions containing each

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individual metabolite were pooled, concentrated under reduced pressure and

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lyophilized.

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Mass spectrometry. Electrospray ionization – mass spectrometry (ESI-MS)

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and ESI – tandem mass spectrometry (ESI-MS2) data of fluoroquinolones and their

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biotransformation products were obtained on the 3200 QTRAP System (AB Sciex,

147

Darmstadt, Germany) by direct injection using a built-in syringe driver (flow rate 10

148

µL/min) while the temperature of ESI source was kept at room temperature. Operating

149

conditions were as follows: positive ionization mode (ESI+), capillary voltage 5500 V,

150

nebulizer gas 14 psi, curtain gas 10 psi. Data acquisition was performed with Analyst

151

1.6.2 software (AB Sciex).

152

For accurate mass measurements of the biotransformation products, ESI – time of flight

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– MS (ESI-TOF-MS) measurements were conducted using a micrOTOF mass

154

spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a syringe pump

155

(Cole Parmer, Vernon Hills, IL). Data were processed via Bruker Daltonics Data

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Analysis software, ver. 3.4. Operating conditions were set to the following parameters:

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positive ionization mode, scan range m/z 50-1200, capillary voltage 4500 V, nebulizer

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gas flow 0.4 bar, dry gas flow 4.0 L/min, temperature 180 °C. Mass calibration was


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performed immediately before sample measurements using a 10 mM sodium formate

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solution in isopropanol/water (50:50, v/v).

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The compound 2 was further identified by high resolution mass spectrometry using a

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TripleTOF 5600+ instrument mass spectrometer (AB Sciex) by direct infusion (flow

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rate 10 µL/min). Data were acquired using an ion spray voltage of 5500 V for ESI+

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mode; ion spray temperature was set at room temperature; nebulizer gas 15 psi,

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auxiliary gas 15 psi, curtain gas 10 psi. Mass calibration was performed using an APCI

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Positive Calibration Solution (AB Sciex). Data analysis was performed using Analyst

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TF 1.7.1 and PeakView 2.2 software (AB Sciex). For all MS experiments, compounds

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were dissolved in methanol/0.5% formic acid (50:50, v/v).

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NMR spectroscopy. 1D and 2D NMR experiments including 1H and 13C NMR,

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DEPT-135, 1H, 1H-COSY, 1H, 13C-HSQC and 1H, 13C-HMBC were carried out on an

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Avance III HD 400 MHz spectrometer (Bruker BioSpin, Rheinstetten, Germany). NMR

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analyses of biotransformation products of 2 and 3 were conducted on an Avance III HD

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600 MHz spectrometer (Bruker). All spectra were processed with the Bruker software

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TopSpin 3.5. A mixture of methanol-d4 (VWR, Darmstadt, Germany), water-d2

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(Deutero GmbH, Kastellaun, Germany) and formic acid-d2 (Acros Organics, Geel,

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Belgium) (90:10:1, v/v/v) was used as solvent. Chemical shifts (δ) expressed in ppm

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were referred to the residual solvent peak.

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Photometric analyses. UV absorption spectra were recorded on a Specord 50

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UV/Vis spectrophotometer combined with WinAspect software, ver. 2.2.1.0 (Analytik

180

Jena, Jena, Germany) using Suprasil quartz cuvettes (pathlength 10 mm) (Hellma,

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Müllheim, Germany).



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Residual antibacterial activity. The antibacterial activities of

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fluoroquinolones and their biotransformation products were tested by the brilliant black

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reduction (BRT) maximum residue level (MRL) screening test (AiM, Munich,

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Germany) as previously described.22 The results of the positive and negative control

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were set at 0% and 100%, respectively, whereas all other data were expressed as

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percentage of control. For the determination of the antibacterial activity in fungal

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culture supernatants, the procedure was slightly modified as previously described.22

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Toxicological investigations. The cytotoxicity of both the fluoroquinolones

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and their biotransformation products was determined using the CellTiter 96 AQueous

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One Solution Cell Proliferation Assay (MTS) (Promega, Mannheim, Germany) and the

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Crystal violet staining assay (CVS) (Sigma-Aldrich, Steinheim, Germany). In both

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assays two cell lines, namely the murine fibroblast cell line L929 (CellLine Service,

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Eppelheim, Germany) and the murine keratinocyte cell line MSC-P5 (CellLine Service,

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Eppelheim, Germany) were used.

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The MTS-assay is a colorimetric assay for determining cytotoxicity. Stock solutions of

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fluoroquinolones and their biotransformation products were prepared in RPMI cell

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culture medium (Sigma, Steinheim, Germany) with 0.5% methanol and were added in a

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high concentration of 500 µg/mL onto both cell lines. The MTS-assay was performed in

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a 96-well plate on confluent cell layers, which were seeded with 10,000 cells/well 5 d

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before the test was conducted. Two wells were used for each test substance and the

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RPMI medium with 0.5% methanol served as a viable control. After 24 h of incubation

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with the test substances, the MTS-assay was performed. Viable cells were determined at

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490 nm. Each experiment was performed three times (n = 3).


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The CVS-assay was used in order to determine the effect of the test substances on the

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cell proliferation of L929-cells and MSC-cells. Therefore, the cells were seeded with a

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density of 10,000 cells/well in a 96-well plate. After 4 h, the respective test substance

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(500 µg/mL) or control (RPMI medium with 0.5% methanol) was applied onto two

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wells. The wells were incubated for 24 h and 48 h and the CVS-assay was performed

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according to the manufacturer´s protocol. The amount of cells proliferated was

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determined at 570 nm. Each experiment was performed three times (n = 3). For both

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experiments, the results of the control were set as 100%; all other data were expressed

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as percentage of control.

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Statistical analyses of the results were performed using SigmaPlot, ver. 12.5 (Systat

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Software, Inc., Erkrath, Germany). Results of each fluoroquinolone were compared with

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the corresponding biotransformation product, and differences were analyzed using t-

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test. P values of 60% compared to the positive control (Figures 4C

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and F). Thus, 6 accounted for approximately 14% of the parent compounds activity.

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Compounds 1 and 2, both caused an inhibition of >60% at a concentration of 3.5 mg/L

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(Figures 4A and B). Thus, they represent more potent drugs than 3, confirming

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literature references.3, 35 In contrast, the corresponding N-oxides 4 and 5 produced

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similar inhibition effects at different concentrations of 15 mg/L and 35 mg/L,

333

respectively (Figures 4D and E). Consequently, the residual antibacterial activity

334

compared to the parent drugs was approximately 23% for 4 and 10% for 5. These

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results are in good agreement with the findings obtained for the direct analysis of fungal

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culture supernatants (Figures 3D-F). In a recent study, X. longipes was shown to

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transform danofloxacin quantitatively to its N-oxide with a residual antibacterial activity

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of 20%.22 Similarly, fluoroquinolone N-oxides such as ofloxacin N-oxide, amifloxacin

339

N-oxide or pefloxacin N-oxide have been reported to exhibit little or no antibacterial

340

activity.36-39

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In vitro toxicity. Besides the residual antimicrobial activity, the in vitro

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cytotoxic effects of the N-oxides compared to their parent compounds were evaluated

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(Figure 5). In the MTS assay (Figures 5A-B), all fluoroquinolones with the exception of

344

2 and 3 in murine keratinocytes (Figure 5B) decreased cell viability at a concentration

345

of 500 µg/mL and thus showed cytotoxic effects to different extents in both cell lines.

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Danofloxacin caused the highest reduction in cell viability (approx. 50% of control). In

347

contrast, all N-oxides caused no reduction in cell viability of both cell lines. In the CVS


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assay, all substances tested led to an inhibition of the cell proliferation to different

349

extents in both cell lines after 24 h and 48 h of incubation (Figures 5C-F). However, the

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N-oxides showed a reduced impact on cell proliferation compared to the parent

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compounds. This correlates with the mode of action of fluoroquinolones which involves

352

inhibition of DNA gyrase and topoisomerase IV.2 In the literature, fluoroquinolones

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have been reported to exhibit cytotoxicity and to inhibit cell proliferation in a dose-

354

dependent manner in different cell lines.40-42 The cytotoxic potential of fluoroquinolone

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N-oxides has not been described so far. Thus, fungal N-oxidation not only leads to

356

reduced antimicrobial activity, but also significantly lowers the cytotoxic effects.

357

Formation of N-oxides is one of the principal metabolic pathways of N-alkylated

358

fluoroquinolones in mammals. Only fluoroquinolones with an alkyl group (such as

359

methyl or ethyl) attached to the terminal nitrogen of the substituted piperazine ring (e.g.,

360

1, 2 or 3) can be oxidized to their respective N-oxides. In addition to N-oxidation, N-

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dealkyl- and 3-oxo-metabolites are formed. In contrast, metabolism of non N-alkylated

362

derivatives (such as ciprofloxacin) is characterized by formation of N-formyl-, 3-oxo-,

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N-sulfate- and desethylene-metabolites.43 However, the extent of N-oxidation differs

364

among fluoroquinolones and mammals, but is generally low. Interestingly in humans,

365

pefloxacin is metabolized to its N-oxide to a greater extent (~17-20%) than amifloxacin

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(~6-9%), ofloxacin (~1%) and fleroxacin (3-6%).43-45 Similar results have been reported

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for ofloxacin in animals.46 In metabolism of 3, the metabolite 6 was detected only at

368

trace levels beside the major metabolite sarafloxacin (5%).9 Similarly, 2 is transformed

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to its N-oxide in very small quantities.17

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Fluoroquinolone N-oxides are known as fungal biotransformation products from several

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studies.23 M. ramannianus transformed 1 to its N-oxide as major metabolite (62%)



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beyond N-acetylciprofloxacin (8%) and desethylene-enrofloxacin (3.5%) in 21 d.24

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Within 7 d, Rhizopus arrhizus metabolized danofloxacin to a lesser extent to the N-

374

desmethyl metabolite and presumably to danofloxacin N-oxide (

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