Microsomal metabolism of ciprofloxacin generates free radicals

Free Radical Biology & Medicine, Vol. 30, No. 10, pp. 1118 –1121, 2001 Copyright © 2001 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/01/$–see front matter

PII S0891-5849(01)00508-1

Original Contribution MICROSOMAL METABOLISM OF CIPROFLOXACIN GENERATES FREE RADICALS AYLIN G¨URBAY,*† BRIGITTE GONTHIER,† DENIS DAVELOOSE,‡ ALAIN FAVIER,†

and

FILIZ HINCAL*

*Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Toxicology, Ankara, Turkey; †Laboratoire de Biologie du Stress Oxydant, CHU de Grenoble, Grenoble, France; and ‡Service de Biophysique, Centre de Recherche du Service de Sante´ des Arme´es, La Tronche, France (Received 6 February 2001; Accepted 15 February 2001)

Abstract—Ciprofloxacin (CPFX) is a widely used fluoroquinolone antibiotic with a broad spectrum of activity. However, clinical experience has shown a possible incidence of undesirable adverse effects including gastrointestinal, skin, hepatic, and central nervous system (CNS) functions, and phototoxicity. Several examples in the literature data indicate that free radical formation might play a role in the mechanism of some of these adverse effects, including phototoxicity and cartilage defects. The purpose of this study is to investigate free radical formation during the metabolism of CPFX in hepatic microsomes using electron spin resonance (ESR) spectroscopy and spin trapping technique. We then investigate the effects of a cytochrome P450 inhibitor, SKF 525A, Trolox, and ZnCl2 on CPFX-induced free radical production. Our results show that CPFX induces free radical production in a dose- and time-dependent manner. The generation of 4-POBN/radical adduct is dependent on the presence of NADPH, CPFX, and active microsomes. Furthermore, free radical production is completely inhibited by SKF 525A, Trolox, or ZnCl2. © 2001 Elsevier Science Inc. Keywords—Ciprofloxacin, Hepatic microsomes, Electron spin resonance spectroscopy, Spin trapping, Free radicals

INTRODUCTION

reactive oxygen species, including hydrogen peroxide and hydroxyl radical [4]. The aim of this study is to investigate free radical formation during metabolism of CPFX in a hepatic microsomal system by electron spin resonance (ESR) spectroscopy using spin-trapping technique. The effects of a cytochrome P450 inhibitor, SKF 525A, Trolox, or ZnCl2 on CPFX-induced free radical production are also investigated.

Fluoroquinolones (FQs), such as ciprofloxacin (CPFX), represent an important class of antimicrobial agents used in the treatment of a wide range of infectious diseases [1]. Compared with other commonly used antimicrobial agents, the FQs can be considered relatively well tolerated. However, it has been reported that they are also associated with a low incidence of adverse effects related to gastrointestinal, skin, hepatic, and central nervous system (CNS) functions, and phototoxicity [2]. Studies to elucidate the mechanism of toxicity of FQs showed that very little is known about the mechanism of these effects. In the literature, there is some evidence that free radical formation might play a role in the pathogenesis of FQ-induced cartilage defects [3]. It has also been shown that their phototoxic effect is related to the generation of

MATERIALS AND METHODS

Chemicals Ciprofloxacin was obtained from Deva Laboratory (Istanbul, Turkey), the spin-trap ␣-(4-pyridyl 1-oxide)N-tert-butyl-nitrone (4-POBN) was from Aldrich (Milwaukee, WI, USA), and the other chemicals were from Sigma (St. Louis, MO, USA). For all experiments, including preparation of microsomes, the buffer and distilled water were treated with Chelex 100 to remove trace amounts of transition metals by the batch method described by Buettner [5].

Address correspondence to: Brigitte Gonthier, Laboratoire de Biologie du Stress Oxydant, CHU de Grenoble, 38043 Grenoble Cedex, France; Tel: ⫹33 476 63 71 07; Fax: ⫹33 476 63 74 23; E-Mail: [email protected]. 1118

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Preparation of microsomes Rat liver microsomes were prepared as described by Cederbaum and Cohen [6]. Male Wistar albino rats (170 –190 g) were allowed free access to food and water. Animals were sacrificed by decapitation and liver tissue was removed. Tissue homogenate was prepared and microsomes were isolated by differential centrifugation, washed two times, resuspended in 10 mM potassium phosphate-0.25 mM sucrose buffer, pH 7.4, and stored at ⫺80°C until use. The protein concentration of microsomal suspensions was determined by the method of Lowry et al. [7]. Measurements of radical adduct formation Experiments were performed at 37°C for various times of incubation. Complete medium contained 4 mg/ml microsomal protein, 50 mM 4-POBN, 2.5 mM NADPH, and different concentrations of CPFX in 10 mM potassium phosphate buffer, pH 7.4. At the end of the incubation period, the reaction mixture was immediately transferred to calibrated capillary tubes and placed in the ESR cavity. Measurements were performed at room temperature. All CPFX solutions were freshly prepared and protected from light by covering with aluminum foil. During all treatment procedures, incubation flasks were protected from the light in the same way. ESR determination ESR measurements were performed using a Bruker ESP 380 spectrometer operating at 100 KHz modulation frequency with accumulation of 9 scans. The instrument parameters were as follows: modulation amplitude, 1.006 gauss; microwave frequency, 9.56 GHz; incident microwave power, 43 mW; signal conversion, 164 ms; time constant, 82 ms; and gain, 2 ⫻ 104. RESULTS

A six-line 4-POBN/radical adduct was detected when hepatic microsomes were incubated with the various concentrations of CPFX at different time intervals in the presence of 4-POBN and NADPH. The hyperfine coupling constants of this adduct were aN ⫽ 15.44 G, aH␤⫽ 2.86 G (Fig. 1). The generation of the 4-POBN/radical adduct was dependent on the presence of NADPH, CPFX, and active microsomes (Fig. 1). In the absence of CPFX, no signal was detected, confirming that the trapped radical derived directly from the antibiotic added. In the same way, blanks without NADPH or with denatured microsomes

Fig. 1. EPR spectra of the 4-POBN/radical adduct generated from CPFX in hepatic microsomal system. Complete system contains rat liver microsomes (4 mg/ml microsomal protein), CPFX (1 mM), 4-POBN (50 mM), and NADPH (2.5 mM) in potassium phosphate buffer (10 mM, pH 7.4) after 1 h incubation. (A), Complete system. (B), same as A, but without CPFX. (C), same as A, but without NADPH. (D), same as A, but without 4-POBN. (E), same as A, but with microsomes boiled 5 min before adding to incubation. (F), same as A, but without microsomes. The instrumental settings are the same as given in Materials and Methods. Representative spectra are shown from five to seven independent measurements.

or buffer instead of microsomes did not give any signal, showing that the radical was formed via an enzymatic pathway. We also observed that the signal was very stable and nearly disappeared in 24 h. The effects of varying incubation times at 37°C and concentration of CPFX on ESR signal intensity are shown in Fig. 2. Radical production induced by CPFX was directly related to the CPFX concentration and to the incubation time. The free radical production was low and reached a plateau until 0.5 mM for 30 min, and until 0.3 mM for 60 min of incubation. For higher concentrations radical production increased more effectively. After 180 min of incubation, the ESR signal intensity increased regularly and dramatically in a dose-dependent manner. On and after 0.4 mM of CPFX, no difference was noticed in radical production for 60 and 180 min. The role of cytochrome P450 in the mechanism of radical formation during CPFX metabolism was examined by adding 0.1 mM SKF 525 A to the microsomal incubation system. The results showed that SKF 525 A inhibited completely the signal production in hepatic microsomal system after 30 min (Fig. 3), and 60 min (data not shown) of incubation. Pretreatment of microsomes with 0.1 mM ZnCl2 for

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Fig. 2. The effect of varying concentrations of CPFX and incubation times on signal production in hepatic microsomal system. Incubation conditions of the microsomal system as for Fig. 1. The instrumental settings are the same as given in Materials and Methods. The results are expressed as the mean ⫾ SD of three independent experiments.

60 min provided complete protection after 30 min (Fig. 3), and 60 min (data not shown) of incubation. Pretreatment of microsomes for 60 min with a hydrosoluble vitamin E analog, Trolox, also provided complete protection against free radical generation after 30 and 60 min incubation periods. When Trolox was added into the system at the same time as other components, only a marked decrease on free radical production was observed for 30 min, and complete protection was provided for 60 min of incubation (Fig. 4). Addition of any of these components into the medium without CPFX failed to obtain any signal.

Fig. 3. Effects of SKF 525A and ZnCl2 on the signal amplitude of 4-POBN/radical adduct generated from CPFX in hepatic microsomal system. Incubation conditions of the microsomal system are as for Fig. 1 except for the addition of different substances. (A) was complete system as described in Fig. 1. (B), same as A, but pre-incubated with SKF 525A (0.1 mM) for 20 min before addition of 4-POBN, CPFX, and NADPH. (C) same as A, but pre-incubated with ZnCl2 (0.1 mM) for 1 h before addition of 4-POBN, CPFX, and NADPH in hepatic microsomes. Representative spectra are shown from three independent measurements.

DISCUSSION

The data obtained in this study suggest that free radical species are produced during the microsomal metabolism of CPFX. To our knowledge, this is the first study demonstrating such a free radical production in the presence of CPFX using a microsomal system and ESR spectroscopy. In the literature, only superoxide production during UVA irradiation of CPFX solutions using spin trapping technique was reported [8]. CPFX is metabolized in the liver by alteration to the piperazine side-chain of the CPFX molecule [9]. The results of the present study showed that enzymatic activity is indeed required by rat microsomes for the formation of 4-POBN/radical adduct from CPFX. The reaction was also CPFX- and NADPH-dependent. According to the hyperfine coupling constant values of the trapped radical, we can assess that a carbon-centered radical was trapped.

Fig. 4. Effect of Trolox on 4-POBN/radical adduct signal intensity in hepatic microsomes. Incubation conditions of the microsomal system as for Fig. 1. (●), complete system; (Œ), same as ●, but Trolox (0.1 mM) added; (■), same as ●, but pre-incubated with Trolox (0.1 mM) for 60 min before addition of 4-POBN, CPFX, and NADPH.

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The effect exerted by the cytochrome P450 inhibitor, SKF 525A, suggests that radical production is dependent upon a cytochrome catalyzed reaction. Inhibition of 4-POBN/radical adduct formation by ZnCl2 in hepatic microsomes might be another explanation of the importance of microsomal system in signal production. It has been reported that zinc inhibits microsomal NADPH oxidation and NADPH-dependent cytochrome c reductase activity [10,11]. In our system, free radical production induced by CPFX was dose- and time-dependent. Having a longlasting radical stability could suggest a free radical chain process, involving formation and propagation reactions such as those occurring for lipid-derived radicals [12]. Results obtained with a hydrosoluble vitamin E analog, Trolox, and also with ZnCl2, might suggest that metabolism of CPFX by rat microsomes generated free radical intermediate(s) that subsequently cause lipid peroxidation. In addition to Trolox, it has been reported that zinc also has antioxidant properties and it may inhibit lipid peroxidation in different systems [13,14]. As a conclusion, these results suggest that CPFX caused radical production in hepatic microsomes. The mechanism of radical formation by CPFX might be a result of metabolization of this drug by cytochrome P450 and/or redox reactions. The protective effects of Trolox and ZnCl2 suggest that CPFX might conduct to lipid peroxidation in this system. The formation of free radical(s) by CPFX in the microsomal system might provide an explanation to the mechanisms of adverse effects observed after administration of this drug. Further studies investigating the effects of various antioxidants and metal chelators will be needed to shed more light on the mechanism of oxidative stress produced by CPFX.

[2] Hooper, D. C.; Wolfson, J. S. The fluoroquinolones: pharmacology, clinical uses, and toxicities in human. Antimicrob. Agents Chemother. 28:716 –721; 1985. [3] Hayem, G.; Petit, P. X.; Levacher, M.; Gaudin, C.; Kahn, M.-F.; Pocidalo, J. J. Cytofluorometric analysis of chondrotoxicity of fluoroquinolone antimicrobial agents. Antimicrob. Agents Chemother. 38:243–247; 1994. [4] Wagai, N.; Tawara, K. Quinolone antibacterial-agent-induced cutaneous phototoxicity: ear swelling reactions in BALB/c mice. Toxicol. Lett. 58:215–223; 1991. [5] Buettner, G. R. In the absence of catalytic metals ascorbate does not autoxidize at pH 7: ascorbate as a test for catalytic metals. J. Biochem. Biophys. Methods 16:27– 40; 1988. [6] Cederbaum, A. I.; Cohen, G. Microsomal oxidation of hydroxyl radical scavenging agents. In: Greenwald, R. A., ed. CRC handbook of methods for oxygen radical research. Boca Raton, FL: CRC Press, Inc.; 1986:81– 87. [7] Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with Folin phenol reagent. J. Biol. Chem. 193:265–275; 1951. [8] Martinez, L. J.; Sik, R.; Chignell, C. F. Fluoroquinolone antimicrobials: singlet oxygen, superoxide and phototoxicity. Photochem. Photobiol. 67:399 – 403; 1998. [9] So¨rgel, F. Metabolism of gyrase inhibitors. Rev. Infect Dis. 11(Suppl. 5):S1119 –S1129; 1989. [10] Jeffery, E. H. The effect of zinc on NADPH oxidation and monooxygenase activity in rat hepatic microsomes. Cell Pharmacol. 23:467– 473; 1983. [11] Chvapil, M.; Ludwig, J. C.; Sipes, I. G.; Misiorowski, R. L. Inhibition of NADPH oxidation and related drug oxidation in liver microsomes by zinc. Biochem. Pharmacol. 25:1787–1791; 1975. [12] Barclay, L. R. C.; Ingold, K. U. Autooxidation of biological molecules. The autoxidation of a model membrane. A comparison of the autoxidation of egg lecithin phosphatidylcholine in water and in chlorohexane. J. Am. Chem. Soc. 103:6478 – 6485; 1981. [13] Coppen, D. E.; Richardson, D. E.; Cousins, R. J. Zinc suppression of free radicals in cultures of rat hepatocytes by iron, t-buthyl hydroperoxyde, and 3-methylindone. Proc. Soc. Exp. Biol. Med. 189:100 –109; 1988. [14] Chvapil, M.; Ryan, J. N.; Elias, S. L.; Peng, Y. M. Protective effect of zinc on carbon tetrachloride-induced liver injury in rats. Exp. Mol. Pathol. 19:186 –196; 1973.

Acknowledgements — This work was supported by grants from the TUBITAK-NATOA2 Scholarship and French Government. We would like to thank Dr. Tambay Taskin from Deva Laboratory for the generous gift of ciprofloxacin.

ABBREVIATIONS

REFERENCES [1] Lietman, P. S. Fluoroquinolone toxicities: an update. Drugs 49(Suppl. 2):156 –163; 1995.

CPFX—Ciprofloxacin CNS— central nervous system ESR— electron spin resonance FQs—fluoroquinolones 4-POBN—␣-(4-pyridyl 1-oxide)-N-tert-butyl-nitrone