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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])
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ABSTRACT: Extensive usage of fluoroquinolone antibiotics in livestock results in
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their occurrence in manure and subsequently in the environment. Fluoroquinolone
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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
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pose a threat to human and ecological health. In this study, the biotransformation of
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enrofloxacin, marbofloxacin and difloxacin by the fungus X. longipes (Xylaria) was
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investigated. The main metabolites were unequivocally identified as the respective N-
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oxides by mass spectrometry and nuclear magnetic resonance spectroscopy. Fungal-
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mediated N-oxidation of fluoroquinolones led to a 77-90% reduction of the initial
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antibacterial activity. In contrast to their respective parent compounds, N-oxides showed
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low cytotoxic potential and had a reduced impact on cell proliferation. Thus,
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biotransformation by X. longipes may represent an effective way for inactivating
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fluoroquinolones.
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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
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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-
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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
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acetate were acquired from Bernd Kraft (Duisburg, Germany), Fisher Scientific
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(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
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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.
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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-
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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,
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Dreieich, Germany) using gradient elution with (A) methanol and (B) 1 mM ammonium
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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
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quaternary pump (YL 91105), an ultraviolet detector (YL 91205) and a CHF 122SC
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fraction collector (Advantec, Dublin, CA) combined with YL-Clarity software, ver.
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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,
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Darmstadt, Germany) by direct injection using a built-in syringe driver (flow rate 10
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µL/min) while the temperature of ESI source was kept at room temperature. Operating
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conditions were as follows: positive ionization mode (ESI+), capillary voltage 5500 V,
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nebulizer gas 14 psi, curtain gas 10 psi. Data acquisition was performed with Analyst
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1.6.2 software (AB Sciex).
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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
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spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a syringe pump
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(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
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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,
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respectively (Figures 4D and E). Consequently, the residual antibacterial activity
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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
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N-oxide or pefloxacin N-oxide have been reported to exhibit little or no antibacterial
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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
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2 and 3 in murine keratinocytes (Figure 5B) decreased cell viability at a concentration
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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
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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
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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
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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-
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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
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reduced antimicrobial activity, but also significantly lowers the cytotoxic effects.
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Formation of N-oxides is one of the principal metabolic pathways of N-alkylated
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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.,
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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
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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
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among fluoroquinolones and mammals, but is generally low. Interestingly in humans,
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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
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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-
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desmethyl metabolite and presumably to danofloxacin N-oxide (