In vitro kinetics of hepatic albendazole sulfoxidation in channel catfish (ictalurus punctatus), tilapia (oreochromis sp.), rainbow trout (oncorhynchus mykiss) and induction of erod activity in abz-dosed channel catfish

J. vet. Pharmacol. Therap. doi: 10.1111/j.1365-2885.2009.01056.x In vitro kinetics of hepatic albendazole sulfoxidation in channel catfish(Ictalurus punctatus), tilapia (Oreochromis sp.), rainbow trout (Oncorhynchusmykiss) and induction of EROD activity in ABZ-dosed channel catfish Gonza´lez, J. F., Shaikh, B., Reimschuessel, R., Kane, A. S. In vitro kinetics of hepatic albendazole sulfoxidation in channel catfish (Ictalurus punctatus), tilapia (Oreochromis sp.), rainbow trout (Oncorhynchus mykiss) and induction of EROD activity in ABZ-dosed channel catfish. J. vet. Pharmacol. Therap. doi: 10.1111/ Liver microsomes from market-size (n = 6) rainbow trout, channel catfish and *School of Veterinary Medicine & Animal tilapia were used to investigate in vitro biotransformation kinetics of Science Universidad Nacional de Colombia,Bogota´, Colombia;  Center for Veterinary albendazole (ABZ). ABZ was transformed to a single metabolite, ABZ sulfoxide (ABZ-SO). Catfish displayed the highest maximal velocity (Vmax = 264.0 ± 58.6 pmols ABZ-SO ⁄ min ⁄ mg protein) followed by tilapia (112.3 ± 8.2) and Health & Health Professions – University of rainbow trout (73.3 ± 10.3). Vmax in catfish was significantly different (P < 0.05) from the other two species. Michaelis–Menten constant (Km) values(lM) varied significantly among the species: rainbow trout (3.9 ± 0.5), tilapia(9.2 ± 1.7) and catfish (22.0 ± 3.2). However, Vmax ⁄ Km ratios showed nodifference among the three species, making them equally efficient performingthis phase I biotransformation reaction. In a second series of experiments,channel catfish (n = 6 per treatment) were dosed in vivo with gel-foodcontaining ABZ (10 mg ⁄ kg, p.o.). Fish were killed at 24, 48, 72 and 120 hafter dosage. Control fish were fed ABZ-free feed. Induction of ethoxyresorufin-o-deethylase activity was significant (P < 0.05) in all ABZ-dosed treatments ascompared with controls.
(Paper received 17 September 2008; accepted for publication 16 December 2008) Jaime Fernando Gonza´lez, Calle 23 A No. 59-72 T4 Ap 704 Bogota´, Colombia.
E-mail: jfgonzalezma@unal.edu.co, jaimefgonzalez@gmail.com et al., 1995), rabbits (Li et al., 1995), sheep (Cristo`fol et al.,1998; Chiap et al., 2000), goats (Delatour et al., 1991b), cattle Albendazole (ABZ, [5-(propylthio)-1H-benzimidazol-2-yl]-carba- (Lanusse & Prichard, 1992), chicken (Csiko et al., 1996), mate), is a broad spectrum anthelmintic used for the treatment of humans (Rawden et al., 2000) and helminth parasites (Solana liver flukes, tapeworms and lung and gastrointestinal round et al., 2001). Residue depletion of ABZ and its main metabolites worms in human (Cook, 1990; Ottesen et al., 1999) and after oral administration in rainbow trout, tilapia, Atlantic veterinary medicine (Campbell, 1990). ABZ and other benzim- salmon and channel catfish has been studied by Shaikh et al.
idazoles derivatives have been used successfully in experimental trials to treat fish parasites such as microsporidia (Schmahl & Albendazole and other benzimidazoles have been linked to inducing effects of phase I and II biotransformation enzymes.
In a wide variety of species, oxidative bioactivation of ABZ ABZ is reported to induce CYP1A-mediated activity [e.g., yields a first phase I metabolite, ABZ-sulfoxide (ABZ-SO) and is a ethoxyresorufin-o-deethylation (EROD)] and protein content in critical step for the expression of the anthelmintic activity. A rats (Souhaili-ElAmry et al., 1988; Asteinza et al., 2000; second sulphoxidation produces albendazole sulphone (ABZ- Baliharova´ et al., 2003) and in human microsomes and SO2), a pharmacologically inactive metabolite. A third biologi- human hepatoma cell (HepG2) cultures (Rolin et al., 1989).
cally inactive metabolite, ABZ-2-aminosulfone (ABZ-2-NH2SO2), Induction of S9 fraction activity, specifically EROD, MROD, is produced through the deacetylation of ABZ-SO2 (Gottschall pentoxyresorufin-o-dealkylation (PROD) and benzyloxyresoru- et al., 1990). ABZ biotransformation metabolites have been fin-o-dealkylation (BROD) activities and protein contents of rat studied in mice (Douch & Buchanan, 1979), pigs (Souhaili- livers after intraperitoneal ABZ injection is reported by ElAmry et al., 1987), dogs (Delatour et al., 1991a), rats (Moroni Escobar-Garcı´a et al. (2001). Besides CYP1A induction, ABZ Ó 2009 Blackwell Publishing Ltd. No claim to original US government works induces to a lesser extent CYP2A6, CYP2E1 (Souhaili-ElAmry The microsomes were resuspended with 1 mL SET buffer et al., 1988), CYP2B1 and CYP2B2 (Asteinza et al., 2000) and (pH = 7.4) per gram of wet liver. Both cytosolic and microsomal CYP3A4 (Souhaili-ElAmry et al., 1988; Asteinza et al., 2000) fractions were stored at )80 °C for no longer than 3 months in rats. ABZ has also been reported to induce phase II until performing the assays. Protein was measured using the BCA protein assay kit (Pierce – 23227, Rockford, IL, USA) based activity in humans (Rolin et al., 1989) and glutathione-s- on the colorimetric reaction with bicinchoninic acid (Vodicnik transferase (GST) in mice serum and muscle (Derda et al., In vitro metabolism of drugs in fish can help provide scientific evidence linking similarities in drug metabolism among piscinespecies. This study investigated in vitro kinetics of ABZ hepatic Microsomal fractions from channel catfish, tilapia and rainbow biotransformation in three aquaculturally relevant finfish spe- trout specimens were used for ABZ in vitro metabolism according cies: tilapia, channel catfish and rainbow trout. A second series to a modified method from Rawden et al. (2000). Phosphate of experiments examined the induction of phase I (EROD, PROD, buffer (0.1 M), microsomes (100-lg protein), ABZ (1–30 lM) BROD) and phase II GST activities after ABZ dosage (10 mg ⁄ kg (Sigma A-4673, St Louis, MI, USA) and NADPH (1 mM, p.o.) in channel catfish killed 24, 48, 72 and 120 h post- tetrasodium salt; Calbiochem 481973, San Diego, CA, USA) were pipetted for a total 200-lL reaction volume. After 40 min Channel catfish (Ictalurus punctatus), rainbow trout (Oncorhyn- of incubation at room temperature on a shaker, 200 lL of ice- chus mykiss) and red tilapia (Oreochromis sp.) are finfish of great cold acetonitrile were added to stop the reaction. The tubes were importance for aquaculture in the USA and worldwide. Channel centrifuged at 8000 g for 20 min in a refrigerated high-speed catfish is cultured mainly in the south states in USA and is the centrifuge (Biofuge 22R – Heraeaus Instruments, Hanau, species with the highest revenues and production yields in the Germany). Supernatants were stored at )80 °C until HPLC country. Rainbow trout and tilapia are among the most cultured metabolite analysis. Preliminary experiments were done to and produced species around the world. Imports of tilapia in the determine linearity of the metabolic reaction with regard to USA have surged over 350% in the last 8 years (FAO, 2007).
incubation time and protein content.
These three species were chosen for the present study consid-ering their importance in the aquaculture activities around the Albendazole metabolites obtained after in vitro incubation ofmicrosomal fractions were analyzed according to Shaikh et al.
(2003b). The liquid chromatographic system consisted of a Hewlett-Packard Model 1050 (Palo Alto, CA, USA) with aquaternary pump, autosampler and an Agilent series 1100 fluorescence detector (290 and 330 nm excitation and emis- Healthy, market-size specimens (n = 6) of tilapia, channel catfish sion wavelengths respectively). Analytical and guard columns and rainbow trout were obtained from commercial sources. After were 5-lm Luna C18 and ODS C18 respectively. An isocratic capture, the fish were transported to the Aquatic Pathobiology mobile phase consisted of acetonitrile ⁄ methanol ⁄ 0.05 M ammo- Center at the University of Maryland (College Park, MD, USA) nium acetate buffer (pH = 5) in a ratio of 17:8:75. This and the Center for Veterinary Medicine at the Food and Drug mobile phase was used for the analysis of ABZ metabolites.
Administration (USFDA) (Laurel, MD, USA) where they were Solid reference standards (1–10 mg) of ABZ, ABZ-SO, ABZ-SO2 maintained under controlled conditions for at least 3 months and ABZ-2-NH2SO2, were prepared to obtain a range of prior to kill and liver harvesting. Livers were minced coarsely calibration standards according to the level of quantification.
with scissors and rinsed with KCl. After discarding the last KCl Quantification of ABZ-SO from the in vitro incubation medium rinsing, four volumes of ice-cold 0.25 M sucrose (ICN Biomed- was performed based on calibration curves obtained with icals – 821271, Costa Mesa, CA, USA) were added (e.g. 1 g liver = 4 mL 0.25 M sucrose). The minced livers in sucrose weretransferred to a homogenizer and then to an ice-cold, high-speed Calculation of ABZ sulfoxidation kinetics parameters centrifuge tube and spun at 8000 g for 20 min at 4 °C (Biofuge22 R – Heraeus Instruments, Hanau, Germany). The superna- Maximal velocity (Vmax), Michaelis–Menten constant (Km) and tant was spun at 100 000 g for 60 min at 4 °C (Beckman Vmax ⁄ Km were calculated by linear regression after obtaining Ultracentrifuge XL-80, Fullerton, CA, USA).
double-reciprocal, Lineweaver–Burk plots.
Microsomal and cytosolic fractions preparation After ultracentrifugation, the tubes were removed to ice and the The EROD assay was conducted, with modification, according to supernatant (cytosolic fraction) was aliquoted into cryotubes.
Eggens and Galgani (1992) and Haasch et al. (1994). Reaction Ó 2009 Blackwell Publishing Ltd. No claim to original US government works In vitro kinetics of hepatic albendazole sulfoxidation in three fish species mixtures consisted of 50 lL of Tris–HCl buffer (100 mM, mixture). Other metabolites such as ABZ-SO2 and ABZ-2- pH = 7.4), 25 lL of microsomal fraction accounting for NH2SO2 that have been reported in residue depletion studies 100 lg of protein, 10 lL of 7-ethoyxresorufin (7-ER) (Sigma- with fish were not detected (Shaikh et al., 2003b). Michaelis– E3763) (1 lM final concentration) and 25 lL of NADPH (1 mM tetrasodium salt, Calbiochem 481973, San Diego, CA, USA).
reaction kinetics for each species are shown in Fig. 1.
Blanks consisted of reaction mixtures with boiled microsomes.
Apparent maximum velocity (Vmax), Michaelis–Menten con- Reaction was quantified by reading the fluorescence units of stant (Km) and Vmax ⁄ Km values for this reaction are presented in Table 1. Channel catfish had higher Vmax (264.0 ± 58.6 microplate absorbance–fluorescence reader (GeniosÔ – TECAN, pmols ABZ-SO ⁄ min ⁄ mg protein) as compared with tilapia Austria). A resorufin calibration curve (0–0.5 lM) was used for (112.3 ± 8.2) and rainbow trout (73.3 ± 10.3). Km values the quantification of the reaction rate.
(lM) varied significantly (P < 0.05) among the species: rain- Pentoxyresorufin- (PROD) and benzyloxyresorufin-o-dealkyla- bow trout (3.9 ± 0.5), tilapia (9.2 ± 1.7) and catfish (22.0 ± tion (BROD) activities. PROD and BROD activities were assayed 3.2). These results indicate that rainbow trout had the highest following the same protocol as for EROD. A 5 lM substrate binding affinity for the substrate. Statistical analysis of the concentration was tested. Phenobarbital-induced rat microsomes Vmax ⁄ Km ratios showed no difference among the three species: (R1078 – Xenotech, LLC, St Lenexa, KS, USA) served as positive catfish (12.3 ± 1.9), tilapia (13.6 ± 1.7) and rainbow trout Glutathione-s-transferase activity was determined by the method of Habig et al. (1974). Reaction mixtures consisted of165 lL of 100 mM Tris–HCl buffer (pH = 7.4), 7 lL of 1-chloro-2,4-dinitrobenzene (CDNB; Sigma C 6396) (1 mM final concen- tration), 3.5 lL of 60 mM reduced L-glutathione (Sigma G 6529) and 10 lg of cytosolic protein. Blanks consisted of reactionmixtures with exception of the cytosolic fraction. The rate of CDNB conjugation with GSH was evaluated after 5 min ofreaction determining changes in absorbance (k = 340 nm) at room temperature. Absorbance readings were obtained using a molar absorption coefficient for CDNB (€ = 9.6 ⁄ m used for final calculations after adjusting the path length to thecorresponding 96-well plate volume (Styrene microtiterÒ S25- [ABZ] µM
Results from albendazole in vitro metabolism and phase I–IIbiotransformation (Shapiro–Wilcoxon test) and homogeneity of variances (Bar- lettt’s test). Log transformations were calculated for somevariables to comply with statistical assumptions. Data being both normal and homogeneous were compared using a one-way ANOVA test (comparison among species) followed by Tukey’s mean separation test using SAS (Statistical AnalysisSoftware, Cary, NC, USA). Statistical significance was set at a 1/velocity
1/[ABZ] µM
Albendazole was transformed by hepatic microsomes to a Fig. 1. (a) Michaelis–Menten plot of albendazole (ABZ) sulfoxidation by single metabolite, ABZ sulfoxide (ABZ-SO) in the three species.
rainbow trout (RBT), tilapia (TILA) and channel catfish (CC) hepatic This ABZ sulfoxidation reaction was NADPH-dependent as no microsomes (n = 6) (means ± SEM). Velocity expressed in pmols ABZ- ABZ-SO was detected in controls (no NADPH in incubation SO ⁄ min ⁄ mg protein. (b) Lineweaver–Burk (double-reciprocal) plot.
Ó 2009 Blackwell Publishing Ltd. No claim to original US government works Table 1. Vmax, Km and Vmax ⁄ Km values for in vitro ABZ sulfoxidation of catfish, tilapia and trout hepatic microsomes (means ± SEM) (different letters in the same column denote significant differences among the EROD activity
Time after ABZ dosage
Fig. 2. Ethoxyresorufin-o-deethylation activity (pmols resoru-fin ⁄ min ⁄ mg protein) of hepatic microsomes from albendazole-dosedchannel catfish (n = 6) (means ± SEM). Different letters on bars denote Changes in EROD, PROD, BROD and GST activities after ABZ statistically significant difference (P < 0.05).
A significant induction (2.2–2.6-fold) on EROD activity was found in all the ABZ-dosed time points compared with controls (Rawden et al., 2000) in other studies. Shaikh et al. (2003b) (Fig. 2). Neither control fish nor ABZ-dosed fish showed PROD found in residue depletion studies that rainbow trout and or BROD activities in the microsomal fraction. No induction of tilapia depleted ABZ in muscle by 12 h after ABZ ingestion, GST activity was found in either of the ABZ treatments as whereas channel catfish did it after 8 h (Shaikh et al., 2006).
compared with control values. Interestingly, GST activity was Interestingly, we found in the present study that channel lower in the 120-h treatment when compared with control catfish had the highest ABZ sulfoxidation rate of all three species which correlates with the shortest depletion time forABZ in muscle found by Shaikh et al. (2003b). ABZ-SOdepletion in channel catfish was also the shortest (8 h) as compared with the ones found in tilapia (48 h) and rainbowtrout (48 h). Shaikh et al. (2003b) found the longest retention time in muscle for ABZ (24 h) an ABZ-SO (96 h) in Atlantic The present study compared hepatic in vitro ABZ sulfoxidation in salmon. Although we did not include this species in the rainbow trout, tilapia and channel catfish. In vivo, ABZ present study, a low in vitro ABZ sulfoxidation rate should be undergoes negligible phase II-type of biotransformation reactions expected if the same correlation found in the other three and there is no sequential conjugation after the phase I oxidation. As a result, microsomes are considered a good model Km for ABZ sulfoxidation in channel catfish indicates that this for ABZ in vitro metabolism (Wrighton et al., 1995). In the species had the lowest binding affinity (e.g., highest Km value) of present work, the microsomal fractions of all three species the three species. Tilapia and rainbow trout showed greater transformed ABZ to ABZ-SO. No other metabolites, such as ABZ- binding affinities for ABZ. The three fish species studied in the SO2 or ABZ-2-NH2SO2, that are reported in residue depletion present work had higher binding affinities than those reported studies in fish (Shaikh et al., 2003b), were detectable. The for rat (53.6 lM) (Fargetton et al., 1986) and pig microsomes absence of inactive metabolites could be due to limited oxidation (41.7 lM) (Souhaili-ElAmry et al., 1987). Km in rainbow trout of ABZ-SO to ABZ-SO2 in the liver or notably faster metabolism of was even lower, although quite similar to the value reported by ABZ to ABZ-SO not allowing the detection of the sulfone or the Rawden et al. (2000) in human microsomes (4.6 ± 0.8). Fish aminosulfone during the incubation period tested. Higher rates with high biotransformation capacity may cope with extreme of secondary sulfoxidations may occur in other organs (e.g., exposure concentrations better than those with low maximal kidney) that were not evaluated in the present study. Neverthe- velocities (Gallagher et al., 2000). However, for the purpose of less, these data are consistent with previous studies in other drug metabolism, the concentrations that more likely are present animal species. When microsomal fractions of sheep (Galtier at the site of biotransformation are in a lower range than the Km et al., 1986), rat (Fargetton et al., 1986), pig (Souhaili-ElAmry et al., 1987) and human (Rawden et al., 2000), were incubated Vmax ⁄ Km is a pure measure of enzyme activity and is not with ABZ as the parent compound, ABZ-SO was the only influenced by other physiological factors of liver clearance.
This parameter is used as the basis for the extrapolation of Channel catfish had the highest Vmax for the ABZ sulfox- in vitro data to the in vivo situation (Houston, 1994).
idation of all three species. Vmax values in the fish species Estimation of in vivo drug clearance is based on application studied in this work were lower than those obtained for rat of models that account for nonenzymatic, physiological factors (590 pmols ⁄ min ⁄ mg protein) (Fargetton et al., 1986), pig as well as the Vmax ⁄ Km estimate. When Vmax and Km values (580 pmols ⁄ min ⁄ mg protein) (Souhaili-ElAmry et al., 1987) were combined in the present work to analyze the overall Ó 2009 Blackwell Publishing Ltd. No claim to original US government works In vitro kinetics of hepatic albendazole sulfoxidation in three fish species enzyme efficiency of the sulfoxidation process, the three species The expression at the transcriptional level of the CYP1A gene had very similar results. The high Vmax value found in catfish is mediated through a ligand-dependent transcription factor was accompanied by a high Km (e.g., low binding affinity), located in the cytoplasm and known as the AhR receptor. Some whereas an inverse relationship occurred in rainbow trout of the organic contaminants previously mentioned (PAHs, PCBs, (low capacity along with low Km – high affinity). Tilapia TCDD, etc) are recognized as AhR ligands. TCDD is the most results were in between the ranges found for catfish and trout.
potent known AhR ligand. However, endogenous substrates The enzyme efficiency estimate (Vmax ⁄ Km) showed that the such as bilirubin and biliverdin have also been described as AhR three species had quite similar capabilities to activate ABZ ligands. Authors in this area suggest that AhR acts ‘promiscu- ously’ as a transcription factor given that it modulates theexpression of a battery of genes in addition to the CYP1A gene(Whitlock, 1999). This non-discriminatory feature of AhR could Alkoxyresorufins and GST induction after ABZ treatment in channel be suggested as an explanation for the inducing effects of ABZ in the EROD activity found in the present work.
We were interested in investigating whether or not ABZ could induce CYP1A and GST activities in fish after a therapeutic have been used, among other alkoxyresorufins, to characterize regime (single dose of 10 mg ⁄ K p.o.). Other cytochrome P450- responses of cytochrome P450 isoforms in mammals (Burke mediated reactions (e.g., PROD and BROD), for which no isoforms et al., 1985) and fish (Haasch et al., 1994). Induction of S9 responsible for their biotransformation have been identified in fraction activity, specifically EROD, MROD, PROD and BROD fish, were also investigated. Because of logistics, this part of the activities and protein concentration of rat livers after intraperi- study was done only with channel catfish. All of the time points toneal ABZ injection were reported by Escobar-Garcı´a et al.
after the ABZ dosage (24, 48, 72 and 120 h) showed significant (2001). However, neither PROD nor BROD activities were induction of EROD activity, a good indicator of CYP1A expression detected in either controls nor ABZ-dosed fish in the present (Whitlock, 1999). EROD activity at all of the exposure time points work. PROD and BROD induction has been reported in rainbow was significantly different (P < 0.05) from the one found in the trout after intraperitoneal injection of inducing agents such as control (ABZ-free feed). EROD activity has been used as a good isosafrole and dexamethasone (Haasch et al., 1994), but the indicator of CYP1A induction in fish (Whyte et al., 2000) and P450 isoforms involved in such biotransformation reactions other species (Whitlock, 1999). Some of the CYP1A inducers are have not been identified in fish. In the same study by Haasch organic contaminants that may be present in the water column, et al. (1994), non-induced rainbow trout had negligible baseline in sediments or in the food. Among these compounds are PROD ⁄ BROD activities. Although PROD ⁄ BROD inductions were polyaromatic hydrocarbons (PAHs), coplanar polychlorinated reported in rats after ABZ treatment, it is not uncommon to find biphenils (PCBs), polychlorinated dibenzo-p-dioxins (TCDD), discrepancies between inducers and the target P450 isoforms dibenzofurans (PCDD ⁄ PCDF) and other halogenated compounds when different animal species are compared. This makes the present in some pesticides and herbicides. For this reason, the extrapolation of results between species difficult.
induction of CYP1A1 protein has been widely used as a Glutathione-s-transferase activity was significantly induced in biomarker of pollution in many different species. EROD activity mouse serum and muscle after ABZ treatment (Derda et al., induction due to in vivo ABZ treatment in a fish species had not 2003). In the present work, we found that GST did not change in been previously reported. In the present work, ABZ exposure 24, 48 and 72 h after ABZ dosage treatments, when compared evoked higher EROD activity in dosed-catfish (between 2.2- and with controls. On the contrary, GST activity in the 120-h 2.6-fold) than in controls. Haasch et al. (1994) found 66.3- and treatment was significantly lower than the one found in the 38.8-fold induction in EROD activity after rainbow trout were control group. We have no clear explanation for this particular treated with isosafrole and b-naphtoflavone respectively. TCDD is reduction in GST activity in this group.
reported to induce EROD activity up to 200-fold in some fishspecies (Whyte et al., 2000). The induction found in our studywas seen in all the time point treatments. Reports on EROD induction within the first 48 h after treatment with inducers arefound in the literature. The extent of the EROD induction may The in vitro incubation system used in this study provided a good depend on how easily the inducer is metabolized. PAHs, as an indicator of the rate of ABZ sulfoxidation but not the production example of easily metabolized inducers, elicited increases in EROD of the sulfone or aminosulfone. There were significant differences activity in sea bass (Dicentrarchus labrax) after 24 h, followed by in Vmax and Km in ABZ sulfoxidation kinetics among tilapia, dramatic decline after 1 week (Lemaire et al., 1992). Effects due channel catfish and rainbow trout. Interestingly, Vmax values in to halogenated inducers (e.g., TCDD) are reported to persist for the three species correlate with muscle residue depletion times several weeks in fish (Whyte et al., 2000). Although our exposure found in other reports. Vmax ⁄ Km ratios showed no differences protocol only covered 120 h, and the response was more typical among the three species investigated. Albendazole appears to be of an easily metabolized compound, it is worth considering likely a weak inducer of EROD activity in channel catfish when implications of the EROD inducing effect in ABZ-treated fish in the compared with the effect exerted by organic pollutants as cited in Ó 2009 Blackwell Publishing Ltd. No claim to original US government works Further research in ABZ metabolism in fish should be directed Eggens, M.L. & Galgani, F. (1992) Ethoxyresorufin-o-deethylase (EROD) to discern isoforms involved in the biotransformation pathways activity in flatfish: fast determination with a fluoresecence plate reader.
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