Currently, there are no reports on the effects of enrofloxacin (EF), a fluoroquinolone antibiotic, on the cytochrome P450 enzymes in fish, although its use as antimicrobial agent in aquaculture has been put forward. Therefore, the invivo and in vitro effects of EF on hepatic P450 enzymes of sea bass, a widespread food-producing fish, have been evaluated. Sea bass pretreated with a single dose of EF (3 mg/kg i.p.) or with three daily doses of EF (1 mg/kg i.p.) markedly depressed the microsomal N-demethylation of aminopyrine, erythromycin, the O-deethylation of 7- ethoxycoumarin, ethoxyresorufin and the 6β-testosterone hydroxylase. In vitro experiments showed that EF at 10μM inhibited the above-mentioned activities and, in particular, the erythromycin N-demethylase (ERND) and 6β- testosterone-hydroxylase, likely dependant on a P450 3A isoform. When the nature of ERND inhibition by EF was specifically studied with sea bass liver microsomes, it was found that EF is a potent mechanism-based inhibitor, with Ki of 3.7 μM and a Kinact of 0.045 min-1. An immunoblot analysis with anti P450 3A27 of trout showed that the P450 3A isoform, constitutively expressed in sea bass, is particularly susceptible to inactivation by EF. In vitro experiments with sea bass microsomes have also demonstrated that EF is oxidative deethylated by the P450 system to ciprofloxacin (CF) and that this compound maintains the ability to inactivate the P450 enzymes. The mechanism by which EF or CF inactivate the P450 enzymes has not been studied but an attack of P450 on the cyclopropan ring, present, both in EF and CF structure, with the formation of electrophilic intermediates (i.e. radicals) has been postulated. In conclusion, the EF seems to be a powerful inhibitor of P450s in the sea bass. Therefore, the clinical use of this antibiotic in aquaculture has to be considered with caution.

Introduction

Enrofloxacin (EF), a fluorinated quinolone carboxylic acid derivative, is a chemotherapic agent with extensive use in veterinary medicine inview of its broad spectrum of activity (Brown, 1996). Its pharmacokinetic behavior has been determined in several animal species (Mengozzi et al., 1996; Intorre et al., 1997) in which the drug has been indicated as safe and effective for the treatment of various systemic bacterial infections. In these animal species, EF had good degree of absorption and great bioavailability after oral and parenteral administration. EF efficiently penetrates all organs and tissues and the distribution pattern is similar in all species. During the last few years EF has received growing attention for its potential use in fish therapy, as it has been suggested that it reaches tissue levels above the minimum inhibition concentrations (MICs) against the most common fish pathogens (Lewbart et al., 1997; Stoffregen et al., 1997; Intorre et al., 2000); nevertheless EF is not yet approved in aquaculture. In both mammalian and non-mammalian species EF is dealkylated to ciprofloxacin (CF), a metabolite that contributes to EF activity which is, by itself, a potent antimicrobial agent used in human medicine (Bergan et al., 1988). Conversion of EF to CF is low in fish compared with homeothermic species; in fact, liver apart, CF was detected only occasionally in plasma and not in edible tissues of the sea bass (Dicentrarchus labrax), an aquatic species farmed in many countries for its commercial production (Intorre et al., 2000). The in vivo metabolism of EF has been studied in various animal species and in humans (Mayeaux and Winston, 1998; Fuhr et al., 1990) and it has been reported that this drug induces an inhibition of cytochrome P450 (P450) isoenzymes (Schlosberg et al., 1995; Vancutsem and Babish, 1996). However, studies to estimate this inhibitory effect in fish are not available. The aim of the present study was to evaluate the effect of the EF administration on the P450 enzymes in sea bass.