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Albendazole repeated administration induces cytochromes P4501A and accelerates albendazole deactivation in mouflon (Ovis musimon)
Research in Veterinary Science 78 (2005) 255–263 www.elsevier.com/locate/rvsc
Albendazole repeated administration induces cytochromes P4501A and accelerates albendazole deactivation in mouflon (Ovis musimon) J. Velı´k, B. Szota´kova´, V. Baliharova´, J. Lamka, M. Sˇavlı´k, V. Wso´l, E. Sˇnejdrova´, L. Ska´lova´ * Faculty of Pharmacy, Department of Biochemical Sciences, Charles University, Heyrovske´ho 1203, Hradec Kra´love´, Czech Republic Accepted 10 August 2004
Abstract Adult mouflon ewes (Ovis musimon) were treated repeatedly with therapeutic doses of albendazole (ABZ, p.o. 7.5 mg/kg of body weight/day, for five consecutive days). Animals (treated or control) were sacrificed 24 h after the fifth dose of ABZ and liver and small intestine were collected to prepare microsomes. The activities of several biotransformation enzymes were measured in both hepatic and intestinal microsomes. A significant increase in the activity and amount of cytochromes P4501A (CYP1A) was observed in both tissues of ABZ treated mouflons compared to control animals. No other biotransformation enzymes tested were affected by five ABZ doses. The in vitro biotransformation of ABZ was studied in hepatic and intestinal microsomes from ABZ treated and control mouflons. Concentrations of two main ABZ metabolites – pharmacologically active ABZ sulfoxide and pharmacologically inactive ABZ sulfone were analysed using HPLC. A significant increase in rate of formation of ABZ sulfone (which is catalysed by CYP1A) was observed in hepatic as well as in intestinal microsomes from ABZ treated animals. The enhancement of ABZ deactivation by its repeated administration may affect the anthelmintic efficacy of this drug and may contribute to the development of parasite resistance. 2004 Elsevier Ltd. All rights reserved. Keywords: Albendazole; CYP1A; Induction; Anthelmintic; Benzimidazole; Parasite resistance
1. Introduction Mouflon (Ovis musimon) is considered as feral form of domestic sheep (O. ammon f. aries) (Hiendlerer et al., 2002). In middle Europe, mouflon represents a popular hunted and food-producing animal species. Historically, it was classified as a cloven-hoofed game only, but later (in 1990s) it became an important farm animal, too. However, it has always been considered as a species very sensitive to parasitic diseases. Therefore, its suc*
Corresponding author. Tel.: +420 495 067322; fax: +420 495 512665. E-mail address: [email protected] (L. Ska´lova´). 0034-5288/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2004.08.007
cessful breeding in free grounds, game enclosures as well as in farms needs regular administration of anthelmintics (Boch and Supperer, 1992; Kutzer, 1995). With regard to the wide spectrum of helminths infecting mouflon (identical with those in domestic sheep), broad-spectral anthelmintics are appropriate to use. Albendazole (ABZ) is a commonly administered benzimidazole derivative for treatment of the most important helminthoses in mouflon production (Jacobs and Taylor, 2001; Mehta, 2000; Plumb, 1999). After parenteral/oral/intraruminal or in vitro administration in sheep (Delatour et al., 1990, 1991; Galtier et al., 1986; Lanusse et al., 1992, 1993a,b; Marriner and Bogan, 1980) or mouflon (Velı´k et al., 2003), as well
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as in all other animals studied (e.g. Velı´k et al., 2004), ABZ is rapidly metabolised through two-step S-oxidation giving firstly chiral albendazole sulfoxide (ABZSO) followed by albendazole sulfone (ABZSO2). While ABZSO has anthelmintic activity, ABZSO2 is pharmacologically inactive (Lacey, 1990). Flavine containing monooxygenases (FMO) and cytochrome P4503A (CYP3A) predominantly participate in the first metabolic step (El Amri et al., 1987; Fargetton et al., 1986; Moroni et al., 1995), whereas the formation of the sulfone is mainly catalysed by cytochrome P4501A (CYP1A) subfamily (Delatour et al., 1991; El Amri et al., 1988). In the end of 1980s ABZ has been described to induce biotransformation enzymes, mainly CYP1A subfamily. ABZ administration led to an increase of catalytic activity and protein content of CYP1A in rats (Asteinza et al., 2000; Baliharova´ et al., 2003a; El Amri et al., 1988). Induction of CYP1A, measured using 7-ethoxyresorufin as a specific substrate, was also reported in human, rabbit and HepG2 cells (Baliharova´ et al., 2003a,b; Galtier, 1991, 1997; Rolin et al., 1989). However, extrapolation of the data obtained in rodents and human to farm animals is not possible because of inter-species differences in regulation of cytochromes P450 (Kleeberg et al., 1999; Weaver et al., 1994; Ska´lova´ et al., 2001). Hence, induction studies in target animal species must be conducted. Our present study was designed to evaluate the effect of repeated administration of ABZ to mouflon on hepatic and intestinal biotransformation enzymes and ABZ metabolism in vitro.
2. Materials and methods 2.1. Animals and biological material Adult female mouflons (O. musimon, 5–8 years old, 28–33 kg) from game enclosure Vlkov (Czech republic) were divided into two groups. Animals in the first group (n = 5) were individually treated with ABZ in five consecutive days (7.5 mg/kg of body weight in each day) by oral route. ABZ suspension (2%) was prepared in our laboratory. Vermitan (20% granulate, Ceva Phylaxia, Hungary) was powdered and dispersed into aqueous gel based on microcrystalline cellulose (Avicel RC-591, FMC, Belgium). Animals in the second group (n = 3) were used as untreated controls. All animals used have not been subjected to any other pharmacological treatments. Mouflon ewes were culled in agreement with Czech slaughtering rules for farm animals (stunning, exsanguinations) 24 h after the last dose. Whole liver and 1.5 m of small intestine (measured from duodenum) were removed immediately. The gut content was washed out with saline and the blood was eliminated from liver
using Eurocollins solution. Both tissues were cut into smaller pieces and stored in liquid nitrogen during transport to laboratory. 2.2. Preparation of subcellular fractions Frozen pieces of liver or small intestine were thawed at room temperature (up to 15 min). Intestinal pieces were cut open, washed and mucosa was scrapped. Liver pieces and mucosa were homogenised at the w/v ratio of 1:6 in 0.1 M sodium phosphate buffer, pH 7.4, using a Potter–Elvehjem homogeniser and sonication with Sonopuls (Bandeline, Germany). The microsomal fractions were isolated by fractional ultracentrifugation of the tissue homogenate with the same buffer. A re-washing step (followed by a second ultracentrifugation) was included at the end of the microsomes preparation procedure. Microsomes were finally resuspended in a buffer containing 20% glycerol (v/v) and stored at 80 C. Protein concentrations were assayed using the bicinchoninic acid method according to the Sigma protocol. 2.3. Enzyme assays Each enzyme assay was performed in triplicate for each animal. The amount of organic solvents in the final reaction mixtures did not exceed 0.1% (v/v). The 7-ethoxyresorufin (EROD) and 7-methoxyresorufin (MROD) O-dealkylase activities were determined using fluorimetric determination of resorufin (Weaver et al., 1994) at 37 C. Each substrate (dissolved in DMSO) was added at a final concentration of 5 lM. The amount of microsomal protein in the reaction mixture ranged between 0.15–0.20 mg. Assays were conducted using a Perkin–Elmer luminescence spectrophotometer LS 50B with the excitation and emission wavelengths of 530 and 585 nm, respectively. The formation of product was monitored continuously during 3 min. The EROD and MROD activities were calculated using the standard amount – addition technique. The 7-methoxy-4-trifluoromethylcoumarin demethylase (MFCD) activity was measured using fluorimetric determination of 4-trifluoromethylumbelliferone. The final concentration of substrate (dissolved in dimethylsulfoxide) was 20 lM. The amount of microsomal protein in the reaction mixture ranged between 0.2 and 0.3 mg. The excitation and emission wavelengths of 410 and 510 nm were respectively used (Cresspi and Stresser, 2000). Testosterone hydroxylase activity (TOH) was assayed using the method described by Reinerinck et al. (1991) with slight modifications (Ska´lova´ et al., 2001). The final concentration of substrate (dissolved in methanol) was 250 lM and the amount of microsomal protein was 0.6–0.8 mg.
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The activity of flavine monooxygenases (FMO) toward thiobenzamide was assayed using the method of Cashman and Hanzlik (1981). The final concentration of substrate (dissolved in acetonitrile) was 1 mM. The formation of the metabolic product (S-oxide of thiobenzamide TBSO) was followed by spectrophotometry at 370 nm. The microsomal UDP-glucuronosyltransferases (UGT) activity was assayed following the method described by Mizuma et al. (1982). Preincubation of microsomes with detergent Slovasol at 4 C lasted 20 min. The reaction mixture (total volume 0.1 ml) contained 10 ll of microsomes (60–80 lg of protein), 3.3 lM UDP-glucuronic acid, 3.3 lM p-nitrophenol (dissolved in redistilled water) in 0.1 M Tris/HCl buffer, pH 7.4. After 20 min of incubation at 37 C, the reaction was stopped by addition of 50 ll of 3% trichloroacetic acid. After shaking and centrifugation, 50 ll of the supernatant was mixed with 50 ll of 1 M NaOH and absorbance was measured by microplate reader BioRad (detection wavelength 400 nm). 2.4. Western blotting The procedure used for electrophoretic separation and transfer of microsomal protein was described previously (Ska´lova´ et al., 2001; Soucˇek et al., 1995). The amount of proteins per line was 20 lg. Blots were incubated 1 h at 37 C with a primary antibody. Polyclonal goat IgG against rat CYP1A1/2 (Daiichi Chemicals, Tokyo, Japan) were used, diluted 1:1000 with buffer containing 20 mM potassium phosphate (pH 7.4) with 150 mM NaCl, 0.05% Tween 20 (w/v) (PBST) and 0.5% blotting milk. After extensive washing, incubation for 30 min at room temperature with rabbit anti-goat IgG conjugated with alkaline phosphatase (Pierce, IL, USA), dilution 1:10000, followed. Blots were washed in 0.1 M Tris buffer pH 9.5, covered with chemiluminescent substrate DuoLux (Vector Lab., Burlingame, USA) and incubated for 5 min. The membranes were then exposed to X-ray film Medix XBU (Foma, Hradec Kra´love´, Czech Republic). Films were developed (standard developing process) and image was recorded with a scanner (Hewlett-Packard Scanjet 3570c). Densitometry was used for quantification (LabImage 1D gel analysis software, Kapelan GmbH, Halle, Germany). 2.5. Incubation of microsomes with ABZ or ABZSO The hepatic or intestinal microsomal fractions were incubated with either ABZ (Sigma, Prague, Czech Republic) (1.67–25 M) or rac-ABZSO (0.167–2.5 lM). The ABZSO was kind gift of Prof. Lanusse (Argentina). The reaction mixture (total volume of 0.3 ml) contained 25 ll of microsomal suspension containing 0.1–0.15 mg (liver) or 0.2–0.3 mg (small intestine) of proteins,
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NADPH (0.1 mM) and 0.1 M Na-phosphate buffer, pH 7.4. The blank samples contained 25 ll of 0.1 M sodium phosphate buffer, pH 7.4, instead of microsomes or 25 ll of 10-min-boiled microsomal suspension. The incubations were carried out at 37 C for 30 min under aerobic conditions. The product formation was linear up to 60 min. At the end of incubation 180 ll of cooled acetonitrile were added, shaken (3 min, vortex) and centrifuged (10 min, 10,000g). Supernatants were analysed using HPLC. 2.6. HPLC analysis Achiral HPLC was carried out using a Spectra Series P200 gradient pump, a HP 1100 Series autosampler, a HP 1100 Series thermostat column compartment, Philips fluorescence detector (kEX = 290 nm, kEM = 320 nm) fitted with a Discovery C18 (5 lm, 150 mm · 4.6 mm) reverse-phase column (Supelco, Bellefonte, USA) equipped with Discovery C18 precolumn (20 mm · 4 mm). Mobile phase A consisted of acetonitrile/ammonium acetate (0.1 M) pH 4.7 in proportion 28:72 (v/v) with a flow rate of 1.0 ml/min in isocratic mode. All experiments were carried out at 25 C. Data were processed using the Chromatography Station for Windows CSW32 version 1.4.2. The compounds were identified with the retention times of reference standards (Schering Plough, NY, USA; purity 99%). The standards were kind gift of Prof. Lanusse (Argentina). Under these chromatographic conditions, the retention times were 3.1 min (total-ABZSO), 4.6 min (ABZSO2), and 25.0 min (ABZ). The limit of detection for ABZ, ABZSO and ABSO2 was 50, 20 and 2 ng/ml, respectively. The linear calibration curves in range 1–10 lg/ml (ABZ), 0.05–4 lg/ml (ABZSO) and 0.003–3 lg/ml (ABZSO2) served for quantification. During the reverse phase HPLC analysis, the totalABZSO chromatographic peak fractions were collected into vials. The collected fractions were evaporated to dryness using Eppendorf 5310 concentrator and redisolved in 250 ll of mobile phase B (0.5% 2-propanol in 0.01 M phosphate buffer solution, pH 6.9). Fifty microlitres of each sample were injected on to a Shimadzu 10 A HPLC system with the fluorescence detection (kEX = 290 nm, kEM = 320 nm) fitted with a chiral stationary phase column (Chiral-AGP column, 5 lm, 150 mm · 3 mm, ChromTech, Ha¨gersten, Sweden). The flow rate of the mobile phase B was 0.5 ml/min. This chiral chromatographic method was adapted from that described previously by Delatour et al. (1990). The relative proportion of each antipode was determined using the integrator software (Class LC 10, Shimadzu Corporation, Kyoto, Japan). ABZSO enantiomers were identified after chromatographic analysis of the racemic standard of this molecule. The retention time was 6.2 min for (–)-ABZSO and 13.5 min for (+)-ABZSO.
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2.7. Statistical analysis One-way ANOVA was used for statistical evaluation of differences between ABZ treated animals and control animals. A probability of p 6 0.05 was considered significant.
3. Results 3.1. Effect of ABZ treatment on CYP1A in liver Activities of CYP1A enzymes (EROD, MROD) were measured in hepatic microsomes from control and ABZ treated animals. Results are shown in Fig. 1(a). Hepatic microsomes from ABZ treated animals exhibited approximately 4-fold higher EROD activity than control microsomes. ABZ treatment of mouflons led also to significant (p 6 0.001) increase of MROD activity (approximately 3-fold). In hepatic microsomes of ABZ treated animals, the amount of protein corresponding to CYP1A (determined immunochemically by western
blotting – see Fig. 2) was enhanced approximately to 680% compared to control microsomes (100%). 3.2. Effect of ABZ treatment on CYP1A in small intestine In microsomes of small intestine EROD and MROD activities were assayed, but only EROD activity was detected. Results are presented in Fig. 1(b). Comparing hepatic and intestinal microsomes, specific EROD activities were approximately 30 times more extensive in liver. ABZ treatment of mouflons caused significant (p 6 0.001) increase of EROD activity (eight times) in intestinal microsomes. Increase of corresponding protein up to 270% was also observed on western blots (Fig. 2). 3.3. Effect of ABZ treatment on activity of other biotransformation enzymes Activities of MFCD, 6b-TOH, TBSO and pn-UGT were measured in hepatic and intestinal microsomes (Table 1). Intestinal microsomes did not exhibit any
Fig. 1. (a,b) 7-ethoxyresorufin deethylase EROD and 7-methoxyresorufin demethylase MROD activity in hepatic a or intestinal b microsomes from control and albendazole ABZ treated mouflons. Data represent the mean ± SD from five ABZ treated or three control individual animals. Each sample was made in triplicate. * = significantly p 6 0.001 different as compared to control.
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Fig. 2. Immunoblotting analyses of samples of hepatic and intestinal microsomes from control (C) and ABZ treated mouflons (ABZ) with CYP1A1/2 antibodies. MWS = molecular weight standard.
Table 1 Activities of other biotransformation enzymes tested in hepatic and intestinal microsomes from control and ABZ treated animals Activity (pmol/s/mg of proteins)
MFCD 6b-TOH TBSO np-UGT
Liver
Small intestine
Control animals
ABZ treated animals
Control animals
ABZ treated animals
7.2 ± 2.1 25.0 ± 8.3 29.4 ± 7.9 142 ± 15
7.1 ± 0.9 17.0 ± 7.5 30.3 ± 7.0 148 ± 8
ND 0.3 ± 0.2 ND 9.7 ± 1.7
ND 0.4 ± 0.2 ND 8.5 ± 3.3
Data represent the mean ± SD from five ABZ treated or three control individual animals. Each sample was made in triplicate. ND = not detected.
MFCD and TBSO activity. 6b-TOH activity was very low, approximately 80 times less extensive than in hepatic microsomes. Also, UGT activity was detected 15-fold lower in intestinal compared to hepatic microsomes. No significant differences were observed in these enzyme activities between control and ABZ-treated animals.
3.4. In vitro metabolism of ABZ in microsomes of control and ABZ treated mouflons Before incubations, presence of ABZ or its metabolites in microsomal suspension from ABZ treated animals were assayed but no traces of these compounds were detected. ABZ (at several concentrations) was incubated with either hepatic and intestinal microsomes of control and ABZ treated mouflons. Concentration of ABZ and total ABZSO was measured in reaction mixture using HPLC. Results are expressed as plots of ABZSO formation velocity versus substrate concentration (see Fig. 3). In blank samples (without microsomes or with boiled microsomes) none ABZ metabolite was detected. The apparent kinetic parameters V 0max and K 0m were calculated using GraphPad Prism 3.0 software (Table 2). ABZ treatment did not significantly affect the velocity of formation of total ABZSO in hepatic microsomes. Values of K 0m and V 0max were similar in microsomes from ABZ treated and control animals. In intestinal microsomes, ABZ treatment led to significant
increase of velocity and V 0max of ABZSO formation. As ABZSO exists in two enantiomeric forms chiral stationary phase was used for separation of ABZSO enantiomers. Effect of ABZ treatment on stereospecificity of in vitro ABZSO formation is shown in Fig. 4. While ABZ treatment did not affected ratio of ABZSO enantiomers formed in liver, in intestinal microsomes, ABZ treatment caused significant (p 6 0.001) shift of stereospecificity toward preferential formation of ()-ABZSO.
3.5. In vitro metabolism of ABZSO in microsomes of control and ABZ treated mouflons To study the second step of ABZ sulfoxidation, ABZSO was used as a substrate in the incubation mixture with hepatic and intestinal microsomes. In blank samples (without microsomes or with boiled microsomes) none ABZSO metabolite was detected and no changes of ABZSO enantiomeric ratio were observed after incubation. Fig. 5 demonstrates the differences in velocity of ABZSO2 formation in microsomes of control and ABZ treated mouflon. Repeated ABZ administration caused approximately 4- and 2-fold increase in velocity of ABZSO2 formation in microsomes from liver and small intestine, respectively. The kinetic parameters were also affected in both tissues tested (see Table 2). Significant (p 6 0.001) increase of V 0max and significant decrease of K 0m were found in microsomes of ABZ treated animals.
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1.6
A BZ treated animals control animals
ABZSO formation (µM/min/g of protein)
1.4 1.2 1 0.8 0.6 0.4 0.2 0 0
5
10
15
20
25
30
-6
(a)
concentration of substrate ABZ (10 M) 0.08
A BZ treated animals control animals
ABZSO formation (µM/min/g of protein)
0.07 0.06 0.05 0.04 0.03 0.02 0.01 0
0
(b)
5
10
15
20
25
30
-6
concentration of substrate ABZ (10 M)
Fig. 3. (a,b) Velocity of formation of ABZSO in dependence of substrate ABZ concentration in hepatic a or intestinal b microsomes from control and ABZ treated mouflons. Mixtures of microsomes from individual animals ABZ treated or control were used. Each sample was made in triplicate.
Table 2 Apparent kinetic parameters of ABZSO formation from ABZ and ABZSO2 formation from ABZSO in microsomes from liver and small intestine of mouflons Liver
Small intestine
Control animals
ABZ treated animals
Control animals
ABZ treated animals
Formation of ABZSO
K 0m (lM) V 0max (lM/min/g)
38.1 ± 1.9 3.4 ± 0.2
39.2 ± 2.1 3.1 ± 0.2
4.6 ± 0.4 0.05 ± 0.01
4.2 ± 0.2 0.07 ± 0.01*
Formation of ABZSO2
K 0m (lM) V 0max (nM/min/g)
0.78 ± 0.05 3.3 ± 0.2
0.24 ± 0.07* 8.4 ± 0.2*
0.67 ± 0.06 0.43 ± 0.03
0.46 ± 0.03* 0.72 ± 0.04*
Data represent the mean ± SD (n = 4). The values significantly p < 0.01 different from control are marked *.
4. Discussion The administration of drugs or other xenobiotics may cause the induction of biotransformation enzymes i.e. an increase in activity of enzymes in response to the presence of a xenobiotic substance (e.g. Barry and Feely, 1990; Nebbia, 2001; Okey, 1990). This increase of activity is mainly a consequence of an increase in respective gene expression and/or lowering of protein degradation. Enzyme induction may have relevant pharmacological and toxicological consequences. The increase of enzyme activities changes both the intensity and half time of simultaneously or successively administered drugs. This can result in ad-
verse drug reactions as consequence of enhanced levels of toxic metabolites. Alternatively, enzyme induction may cause therapeutic failures due to decrease drug concentration in the body. Induction of some biotransformation enzymes (e.g. CYP1A) can also result in increased sensitivity to environmental contaminants and the risk of pathogenesis. With regard to the abovementioned consequences, the induction effect should represent a necessary monitored parameter in all the drugs used. With respect to the well-known species differences in inducibility of biotransformation enzymes, in present study the induction potency of ABZ was tested in target species – wild sheep mouflon. The ABZ dosage scheme
J. Velı´k et al. / Research in Veterinary Science 78 (2005) 255–263
261
100
relative amount of (+)-ABZSO (%)
90 80 70 60 50 40
control-liver
30
control-intestine 20
ABZ treated-liver
10
ABZ treated-intestine
0 0
5
10
15
20
25
30
-6
concentration of substrate ABZ (10 M)
Fig. 4. Stereospecificity of ABZSO formation in hepatic and intestinal microsomes from control and ABZ treated animals. Stereospecificity is expressed as percentage ratio of ABZSO enantiomers formed. Concentration of total ABZSO sum of (+)-ABZSO and ()-ABZSO = 100%.
ABZSO 2 formation (n M/min/g of protein)
10
A BZ treated animals control animals
9 8 7 6 5 4 3 2 1 0 0
0.5
1
1.5
2
2.5
3
-6
(a)
concentration of substrate ABZ SO (10 M) 0.8
ABZSO 2 formation (n M/min/g of protein)
A BZ treated animals control animals 0.6
0.4
0.2
0 0
(b)
0.5
1
1.5
2
2.5
3
-6
concentration of substrate ABZ SO (10 M)
Fig. 5. (a,b) Velocity of formation of ABZSO2 in dependence of substrate ABZSO concentration in hepatic a or intestinal b microsomes from control and albendazole ABZ treated mouflons. Mixtures of microsomes from individual animals ABZ treated or control were used. Each sample was made in triplicate.
used in this study represents the common therapeutic approach used for successful treatment of mouflon helmintoses, especially those caused by small ling worms or flukes (Protostrongyliadae, Dicrocoeliidae) (Boch and Supperer, 1992; Kutzer, 1995).
The biotransformation enzymes tested were selected according to following reasons: (1) their participation in ABZ metabolism (CYP1A, CYP3A, FMO, UGT) (e.g. Delatour et al., 1991; El Amri et al., 1987, 1988; Fargetton et al., 1986; Moroni et al., 1995), (2). their
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inducibility by ABZ in laboratory species or man (mainly CYP1A, in lesser extent CYP3A, UGT) (e.g. Asteinza et al., 2000; Baliharova´ et al., 2003a; El Amri et al., 1988) and (3) their important role in drug–drug interactions in veterinary medicine (CYP3A, CYP2C, UGT) (e.g. Fink-Gremmels and van Miert, 1996). The activities of these biotransformation enzymes were assayed in microsomes not only from liver but also from small intestine because the significant participation of intestinal enzymes in ABZ metabolism have been reported (Galtier, 1991; Redondo et al., 1999; Villaverde et al., 1995). Repeated ABZ administration to mouflons led to significant increase of EROD activity in hepatic as well as in intestinal microsomes. Significant increase of MROD activity was found in liver only. EROD and MROD activity are ascribed to CYP1A1/2 (Weaver et al., 1994). The increase of amount of CYP1A proteins was confirmed by immunodetection. Thus treatment of mouflons with ABZ (in dosage scheme used) significantly induced CYP1A1/2 in liver and small intestine. No other enzymes tested were affected by ABZ repeated administration. In our previous study the induction effect of ABZ on CYP enzymes was tested in mouflon in vitro (primary cultures of hepatocytes). On contrary to in vivo results presented here, no significant induction of mouflon CYP1A was found in vitro (Baliharova´ et al., 2003b). From this point of view it is clear that in vitro results may not correspond to in vivo situation. Disagreement between the in vivo and in vitro results may be caused by different time of ABZ interaction with cells (120 vs. 48 h). Moreover, inhibition of CYP1A activities by ABZ and ABZSO, which was also observed in mouflon microsomes (unpublished results), probably affected in vitro experiment more than in vivo experiment. In addition to activities of biotransformation enzymes toward their selective substrates, in vitro metabolism of ABZ was also studied with the aim to evaluate the influence of ABZ repeated administration to animals on metabolism of ABZ itself. The formation of pharmacologically active ABZSO (first step of ABZ sulfoxidation) is mainly ascribed to CYP3A and FMO (Delatour et al., 1991; El Amri et al., 1987; Fargetton et al., 1986; Moroni et al., 1995). ABZ repeated administration did not affect the ABZSO formation in hepatic microsomes. The ratio of ABZSO enantiomers formed remained without changes in ABZ treated animals. These results are in agreement with testing of ABZ effect on biotransformation enzymes activities as 6b-TOH, TBSO (reflecting CYP3A and FMO activities) which were not affected, too. A significant increase in ABZSO formation in intestinal microsomes was observed in ABZ treated animals. From all tested intestinal CYPs only CYP1A1 was induced by ABZ. With respect to these facts
CYP1A1 seems to participate on ABZSO formation in mouflon small intestine. The formation of pharmacologically inactive ABZSO2 (second step of ABZ sulfoxidation) is mainly catalysed by CYP1A (El Amri et al., 1988). A significant increase in velocity of ABZSO2 formation was observed in hepatic as well as in intestinal microsomes from ABZ treated animals. V 0max of ABZSO sulfoxidation were significantly higher in microsomes from ABZ treated animals. In addition, ABZ treatment led also to significant decrease of the K 0m value for ABZSO oxidation (i.e. increase of enzyme affinity toward substrate). It was surprising, as classical induction (increase of enzyme amount) enhances Vmax value but not affects Km. Our observation could be explain by participation of at least two enzymes in ABZSO2 formation. But only one (CYP1A), which has lower Km, is inducible by ABZ. Consequently, induction of CYP1A by ABZ is manifested by shift of apparent K 0m . In conclusion, ABZ repeated administration to mouflons significantly induced hepatic and intestinal CYP1A. This induction caused a significant increase of formation of inactive ABZSO2. Thus ABZ repeated administration induces its own metabolic degradation in liver and small intestine, which may result in decrease of therapeutic efficacy, contributing to the development of parasitic resistance. In addition, CYP1A induction could alter metabolism of simultaneously or consecutively administered drugs that are metabolised by this enzyme. Although CYP1A enzymes catalyse predominantly biotransformation of organic environmental contaminants, they take part also in metabolism of several veterinary drugs (e.g. fenbendazole, thiabendazole, mebendazole, caffeine, estradiol) (Testa, 1995; Plumb, 1999). A high CYP1A activity could result in faster biotransformation of these drugs. Moreover, a high activity of CYP1A also leads to an increase in activation of certain procarcinogens e.g. polycyclic aromatic hydrocarbons, polychlorined biphenyls etc. In therapy of mouflon all pharmacological and toxicological consequences of ABZ repeated administration above-mentioned should be taken into account.
Acknowledgement Financial support of this project was provided by Grant Agency of the Czech Republic, Grant No. 524/ 03/1361.
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Lanusse, C.E., Nare, B., Gascon, L.H., Prichard, R.K., 1992. Metabolism of albendazole and albendazole sulphoxide by ruminal and intestinal fluids of sheep and cattle. Xenobiotica 22, 419–426. Lanusse, C.E., Nare, B., Prichard, R.K., 1993a. Comparative sulphoxidation of albendazole by sheep and cattle liver microsomes and the inhibitory effect of methimazole. Xenobiotica 23, 285–295. Lanusse, C.E., Prichard, R.K., 1993b. Clinical pharmacokinetics and metabolism of benzimidazole anthelmintics in ruminants. Drug Metab. Rev. 25, 235–279. Marriner, S.E., Bogan, J.A., 1980. Pharmacokinetics of albendazole in sheep. Am. J. Vet. Res. 41, 1126–1129. Mehta, D.K., 2000. Anthelmintics. In: British National Formulary 2000. British Medical Association, London, pp. 309–311. Mizuma, T., Mashida, M., Hayashi, M., Awazu, S., 1982. Correlation of drug conjugative metabolism rates between in vivo and in vitro: glucuronidation and sulfation of p-nitrophenol as a model compound in rat. J. Pharmacobiodyn. 5, 10811–10817. Moroni, P., Buronfosse, T., Longin-Sauvageon, C., Delatour, P., Benoit, E., 1995. Chiral sulfoxidation of albendazole by the flavin adenine dinucleotide-containing and cytochrome P450-dependent monooxygenases from rat liver microsomes. Drug Metab. Dispos. 23, 160–165. Nebbia, C., 2001. Biotransformation enzymes as determinants of xenobiotic toxicity in domestic animals. Vet. J. 161, 238–252. Okey, A.B., 1990. Enzyme induction in the cytochrome P-450 system. Pharmacol. Therap. 45, 241–298. Plumb, J., 1999. Veterinary Drug Handbook, third ed. Ames Iowa State University press. Redondo, P.A., Alvarez, A.I., Garcia, J.L., Larrode´, O.M., Merino, G., Prieto, J.G., 1999. Presystemic metabolism of albendazole: experimental evidence of an efflux process of albendazole sulfoxide to intestinal lumen. Drug Metab. Dispos. 27, 736–740. Reinerinck, E.M.J., Doorn, L., Jansen, E.H.J.M., Van Iersel, A.A.J., 1991. Measurement of enzyme activities of cytochrome P-450 isoenzymes by high-performance liquid chromatographic analysis of products. J. Chromatogr. 553, 233–241. Rolin, S., El Amri, H.S., Batt, A.M., Levy, M., Bagrel, D., Siest, G., 1989. Study of the in vitro bioactiviation of albendazole in human liver microsomes and hepatome cell lines. Cell Biol. Toxicol. 5, 1–14. Ska´lova´, L., Szota´kova´, B., Machala, M., Nee`a, J., Soue`ek, P., Havlasova´, J., Wso´l, V., Køı´dova´, L., Kvasnie`kova´, E., Lamka, J., 2001. Effect of ivermectin on activities of cytochromes P450 in mouflon (Ovis musimon) and fallow deer (Dama dama). Chem. Biol. Interact. 137, 155–167. Soucˇek, P., Martin, M.V., Ueng, Y.F., Guengerich, F.P., 1995. Identification of a common cytochrome P450 epitope near the conserved heme-binding peptide with antibodies raised against recombinant cytochrome P450 family 2 proteins. Biochemistry 34, 16013–16021. Testa, B., 1995. Biochemistry of redox reactions. In: Testa, B., Caldwell (Eds.), The Metabolism of Drugs and Other Xenobiotics. Academic Press, London. Velı´k, J., Baliharova´, V., Szota´kova´, B., Wso´l, V., Lamka, J., 2003. Stereospecific biotransformation of albendazole in mouflon and rat isolated hepatocytes. J. Vet. Pharmacol. Therap. 26, 297–302. Velı´k, J., Baliharova´, V., Fink-Gremmels, J., Bull, S., Lamka, J., Ska´lova´, L., 2004. Benzimidazole drugs and modulation of biotransformation enzymes. Res. Vet. Sci. 76, 95–108. Villaverde, C., Alvarez, A.I., Redondo, P., Voces, J., Del Estal, J.L., Prieto, J.G., 1995. Small intestinal sulphoxidation of albendazole. Xenobiotica 25, 433–441. Weaver, R.J., Thompson, S., Smith, G., Dickins, M., Elcombe, C.R., Mayers, R.T., Burke, M.D., 1994. A comparative study of constitutive and induced alkoxyresorufin O-dealkylation and individual cytochrome P450 forms in cynomolgus monkey Macaca fascicularis, human, mouse, rat and hamster liver microsomes. Biochem. Pharmacol. 47, 763–773.
Albendazole repeated administration induces cytochromes P4501A and accelerates albendazole deactivation in mouflon (Ovis musimon) J. Velı´k, B. Szota´kova´, V. Baliharova´, J. Lamka, M. Sˇavlı´k, V. Wso´l, E. Sˇnejdrova´, L. Ska´lova´ * Faculty of Pharmacy, Department of Biochemical Sciences, Charles University, Heyrovske´ho 1203, Hradec Kra´love´, Czech Republic Accepted 10 August 2004
Abstract Adult mouflon ewes (Ovis musimon) were treated repeatedly with therapeutic doses of albendazole (ABZ, p.o. 7.5 mg/kg of body weight/day, for five consecutive days). Animals (treated or control) were sacrificed 24 h after the fifth dose of ABZ and liver and small intestine were collected to prepare microsomes. The activities of several biotransformation enzymes were measured in both hepatic and intestinal microsomes. A significant increase in the activity and amount of cytochromes P4501A (CYP1A) was observed in both tissues of ABZ treated mouflons compared to control animals. No other biotransformation enzymes tested were affected by five ABZ doses. The in vitro biotransformation of ABZ was studied in hepatic and intestinal microsomes from ABZ treated and control mouflons. Concentrations of two main ABZ metabolites – pharmacologically active ABZ sulfoxide and pharmacologically inactive ABZ sulfone were analysed using HPLC. A significant increase in rate of formation of ABZ sulfone (which is catalysed by CYP1A) was observed in hepatic as well as in intestinal microsomes from ABZ treated animals. The enhancement of ABZ deactivation by its repeated administration may affect the anthelmintic efficacy of this drug and may contribute to the development of parasite resistance. 2004 Elsevier Ltd. All rights reserved. Keywords: Albendazole; CYP1A; Induction; Anthelmintic; Benzimidazole; Parasite resistance
1. Introduction Mouflon (Ovis musimon) is considered as feral form of domestic sheep (O. ammon f. aries) (Hiendlerer et al., 2002). In middle Europe, mouflon represents a popular hunted and food-producing animal species. Historically, it was classified as a cloven-hoofed game only, but later (in 1990s) it became an important farm animal, too. However, it has always been considered as a species very sensitive to parasitic diseases. Therefore, its suc*
Corresponding author. Tel.: +420 495 067322; fax: +420 495 512665. E-mail address: [email protected] (L. Ska´lova´). 0034-5288/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2004.08.007
cessful breeding in free grounds, game enclosures as well as in farms needs regular administration of anthelmintics (Boch and Supperer, 1992; Kutzer, 1995). With regard to the wide spectrum of helminths infecting mouflon (identical with those in domestic sheep), broad-spectral anthelmintics are appropriate to use. Albendazole (ABZ) is a commonly administered benzimidazole derivative for treatment of the most important helminthoses in mouflon production (Jacobs and Taylor, 2001; Mehta, 2000; Plumb, 1999). After parenteral/oral/intraruminal or in vitro administration in sheep (Delatour et al., 1990, 1991; Galtier et al., 1986; Lanusse et al., 1992, 1993a,b; Marriner and Bogan, 1980) or mouflon (Velı´k et al., 2003), as well
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as in all other animals studied (e.g. Velı´k et al., 2004), ABZ is rapidly metabolised through two-step S-oxidation giving firstly chiral albendazole sulfoxide (ABZSO) followed by albendazole sulfone (ABZSO2). While ABZSO has anthelmintic activity, ABZSO2 is pharmacologically inactive (Lacey, 1990). Flavine containing monooxygenases (FMO) and cytochrome P4503A (CYP3A) predominantly participate in the first metabolic step (El Amri et al., 1987; Fargetton et al., 1986; Moroni et al., 1995), whereas the formation of the sulfone is mainly catalysed by cytochrome P4501A (CYP1A) subfamily (Delatour et al., 1991; El Amri et al., 1988). In the end of 1980s ABZ has been described to induce biotransformation enzymes, mainly CYP1A subfamily. ABZ administration led to an increase of catalytic activity and protein content of CYP1A in rats (Asteinza et al., 2000; Baliharova´ et al., 2003a; El Amri et al., 1988). Induction of CYP1A, measured using 7-ethoxyresorufin as a specific substrate, was also reported in human, rabbit and HepG2 cells (Baliharova´ et al., 2003a,b; Galtier, 1991, 1997; Rolin et al., 1989). However, extrapolation of the data obtained in rodents and human to farm animals is not possible because of inter-species differences in regulation of cytochromes P450 (Kleeberg et al., 1999; Weaver et al., 1994; Ska´lova´ et al., 2001). Hence, induction studies in target animal species must be conducted. Our present study was designed to evaluate the effect of repeated administration of ABZ to mouflon on hepatic and intestinal biotransformation enzymes and ABZ metabolism in vitro.
2. Materials and methods 2.1. Animals and biological material Adult female mouflons (O. musimon, 5–8 years old, 28–33 kg) from game enclosure Vlkov (Czech republic) were divided into two groups. Animals in the first group (n = 5) were individually treated with ABZ in five consecutive days (7.5 mg/kg of body weight in each day) by oral route. ABZ suspension (2%) was prepared in our laboratory. Vermitan (20% granulate, Ceva Phylaxia, Hungary) was powdered and dispersed into aqueous gel based on microcrystalline cellulose (Avicel RC-591, FMC, Belgium). Animals in the second group (n = 3) were used as untreated controls. All animals used have not been subjected to any other pharmacological treatments. Mouflon ewes were culled in agreement with Czech slaughtering rules for farm animals (stunning, exsanguinations) 24 h after the last dose. Whole liver and 1.5 m of small intestine (measured from duodenum) were removed immediately. The gut content was washed out with saline and the blood was eliminated from liver
using Eurocollins solution. Both tissues were cut into smaller pieces and stored in liquid nitrogen during transport to laboratory. 2.2. Preparation of subcellular fractions Frozen pieces of liver or small intestine were thawed at room temperature (up to 15 min). Intestinal pieces were cut open, washed and mucosa was scrapped. Liver pieces and mucosa were homogenised at the w/v ratio of 1:6 in 0.1 M sodium phosphate buffer, pH 7.4, using a Potter–Elvehjem homogeniser and sonication with Sonopuls (Bandeline, Germany). The microsomal fractions were isolated by fractional ultracentrifugation of the tissue homogenate with the same buffer. A re-washing step (followed by a second ultracentrifugation) was included at the end of the microsomes preparation procedure. Microsomes were finally resuspended in a buffer containing 20% glycerol (v/v) and stored at 80 C. Protein concentrations were assayed using the bicinchoninic acid method according to the Sigma protocol. 2.3. Enzyme assays Each enzyme assay was performed in triplicate for each animal. The amount of organic solvents in the final reaction mixtures did not exceed 0.1% (v/v). The 7-ethoxyresorufin (EROD) and 7-methoxyresorufin (MROD) O-dealkylase activities were determined using fluorimetric determination of resorufin (Weaver et al., 1994) at 37 C. Each substrate (dissolved in DMSO) was added at a final concentration of 5 lM. The amount of microsomal protein in the reaction mixture ranged between 0.15–0.20 mg. Assays were conducted using a Perkin–Elmer luminescence spectrophotometer LS 50B with the excitation and emission wavelengths of 530 and 585 nm, respectively. The formation of product was monitored continuously during 3 min. The EROD and MROD activities were calculated using the standard amount – addition technique. The 7-methoxy-4-trifluoromethylcoumarin demethylase (MFCD) activity was measured using fluorimetric determination of 4-trifluoromethylumbelliferone. The final concentration of substrate (dissolved in dimethylsulfoxide) was 20 lM. The amount of microsomal protein in the reaction mixture ranged between 0.2 and 0.3 mg. The excitation and emission wavelengths of 410 and 510 nm were respectively used (Cresspi and Stresser, 2000). Testosterone hydroxylase activity (TOH) was assayed using the method described by Reinerinck et al. (1991) with slight modifications (Ska´lova´ et al., 2001). The final concentration of substrate (dissolved in methanol) was 250 lM and the amount of microsomal protein was 0.6–0.8 mg.
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The activity of flavine monooxygenases (FMO) toward thiobenzamide was assayed using the method of Cashman and Hanzlik (1981). The final concentration of substrate (dissolved in acetonitrile) was 1 mM. The formation of the metabolic product (S-oxide of thiobenzamide TBSO) was followed by spectrophotometry at 370 nm. The microsomal UDP-glucuronosyltransferases (UGT) activity was assayed following the method described by Mizuma et al. (1982). Preincubation of microsomes with detergent Slovasol at 4 C lasted 20 min. The reaction mixture (total volume 0.1 ml) contained 10 ll of microsomes (60–80 lg of protein), 3.3 lM UDP-glucuronic acid, 3.3 lM p-nitrophenol (dissolved in redistilled water) in 0.1 M Tris/HCl buffer, pH 7.4. After 20 min of incubation at 37 C, the reaction was stopped by addition of 50 ll of 3% trichloroacetic acid. After shaking and centrifugation, 50 ll of the supernatant was mixed with 50 ll of 1 M NaOH and absorbance was measured by microplate reader BioRad (detection wavelength 400 nm). 2.4. Western blotting The procedure used for electrophoretic separation and transfer of microsomal protein was described previously (Ska´lova´ et al., 2001; Soucˇek et al., 1995). The amount of proteins per line was 20 lg. Blots were incubated 1 h at 37 C with a primary antibody. Polyclonal goat IgG against rat CYP1A1/2 (Daiichi Chemicals, Tokyo, Japan) were used, diluted 1:1000 with buffer containing 20 mM potassium phosphate (pH 7.4) with 150 mM NaCl, 0.05% Tween 20 (w/v) (PBST) and 0.5% blotting milk. After extensive washing, incubation for 30 min at room temperature with rabbit anti-goat IgG conjugated with alkaline phosphatase (Pierce, IL, USA), dilution 1:10000, followed. Blots were washed in 0.1 M Tris buffer pH 9.5, covered with chemiluminescent substrate DuoLux (Vector Lab., Burlingame, USA) and incubated for 5 min. The membranes were then exposed to X-ray film Medix XBU (Foma, Hradec Kra´love´, Czech Republic). Films were developed (standard developing process) and image was recorded with a scanner (Hewlett-Packard Scanjet 3570c). Densitometry was used for quantification (LabImage 1D gel analysis software, Kapelan GmbH, Halle, Germany). 2.5. Incubation of microsomes with ABZ or ABZSO The hepatic or intestinal microsomal fractions were incubated with either ABZ (Sigma, Prague, Czech Republic) (1.67–25 M) or rac-ABZSO (0.167–2.5 lM). The ABZSO was kind gift of Prof. Lanusse (Argentina). The reaction mixture (total volume of 0.3 ml) contained 25 ll of microsomal suspension containing 0.1–0.15 mg (liver) or 0.2–0.3 mg (small intestine) of proteins,
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NADPH (0.1 mM) and 0.1 M Na-phosphate buffer, pH 7.4. The blank samples contained 25 ll of 0.1 M sodium phosphate buffer, pH 7.4, instead of microsomes or 25 ll of 10-min-boiled microsomal suspension. The incubations were carried out at 37 C for 30 min under aerobic conditions. The product formation was linear up to 60 min. At the end of incubation 180 ll of cooled acetonitrile were added, shaken (3 min, vortex) and centrifuged (10 min, 10,000g). Supernatants were analysed using HPLC. 2.6. HPLC analysis Achiral HPLC was carried out using a Spectra Series P200 gradient pump, a HP 1100 Series autosampler, a HP 1100 Series thermostat column compartment, Philips fluorescence detector (kEX = 290 nm, kEM = 320 nm) fitted with a Discovery C18 (5 lm, 150 mm · 4.6 mm) reverse-phase column (Supelco, Bellefonte, USA) equipped with Discovery C18 precolumn (20 mm · 4 mm). Mobile phase A consisted of acetonitrile/ammonium acetate (0.1 M) pH 4.7 in proportion 28:72 (v/v) with a flow rate of 1.0 ml/min in isocratic mode. All experiments were carried out at 25 C. Data were processed using the Chromatography Station for Windows CSW32 version 1.4.2. The compounds were identified with the retention times of reference standards (Schering Plough, NY, USA; purity 99%). The standards were kind gift of Prof. Lanusse (Argentina). Under these chromatographic conditions, the retention times were 3.1 min (total-ABZSO), 4.6 min (ABZSO2), and 25.0 min (ABZ). The limit of detection for ABZ, ABZSO and ABSO2 was 50, 20 and 2 ng/ml, respectively. The linear calibration curves in range 1–10 lg/ml (ABZ), 0.05–4 lg/ml (ABZSO) and 0.003–3 lg/ml (ABZSO2) served for quantification. During the reverse phase HPLC analysis, the totalABZSO chromatographic peak fractions were collected into vials. The collected fractions were evaporated to dryness using Eppendorf 5310 concentrator and redisolved in 250 ll of mobile phase B (0.5% 2-propanol in 0.01 M phosphate buffer solution, pH 6.9). Fifty microlitres of each sample were injected on to a Shimadzu 10 A HPLC system with the fluorescence detection (kEX = 290 nm, kEM = 320 nm) fitted with a chiral stationary phase column (Chiral-AGP column, 5 lm, 150 mm · 3 mm, ChromTech, Ha¨gersten, Sweden). The flow rate of the mobile phase B was 0.5 ml/min. This chiral chromatographic method was adapted from that described previously by Delatour et al. (1990). The relative proportion of each antipode was determined using the integrator software (Class LC 10, Shimadzu Corporation, Kyoto, Japan). ABZSO enantiomers were identified after chromatographic analysis of the racemic standard of this molecule. The retention time was 6.2 min for (–)-ABZSO and 13.5 min for (+)-ABZSO.
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2.7. Statistical analysis One-way ANOVA was used for statistical evaluation of differences between ABZ treated animals and control animals. A probability of p 6 0.05 was considered significant.
3. Results 3.1. Effect of ABZ treatment on CYP1A in liver Activities of CYP1A enzymes (EROD, MROD) were measured in hepatic microsomes from control and ABZ treated animals. Results are shown in Fig. 1(a). Hepatic microsomes from ABZ treated animals exhibited approximately 4-fold higher EROD activity than control microsomes. ABZ treatment of mouflons led also to significant (p 6 0.001) increase of MROD activity (approximately 3-fold). In hepatic microsomes of ABZ treated animals, the amount of protein corresponding to CYP1A (determined immunochemically by western
blotting – see Fig. 2) was enhanced approximately to 680% compared to control microsomes (100%). 3.2. Effect of ABZ treatment on CYP1A in small intestine In microsomes of small intestine EROD and MROD activities were assayed, but only EROD activity was detected. Results are presented in Fig. 1(b). Comparing hepatic and intestinal microsomes, specific EROD activities were approximately 30 times more extensive in liver. ABZ treatment of mouflons caused significant (p 6 0.001) increase of EROD activity (eight times) in intestinal microsomes. Increase of corresponding protein up to 270% was also observed on western blots (Fig. 2). 3.3. Effect of ABZ treatment on activity of other biotransformation enzymes Activities of MFCD, 6b-TOH, TBSO and pn-UGT were measured in hepatic and intestinal microsomes (Table 1). Intestinal microsomes did not exhibit any
Fig. 1. (a,b) 7-ethoxyresorufin deethylase EROD and 7-methoxyresorufin demethylase MROD activity in hepatic a or intestinal b microsomes from control and albendazole ABZ treated mouflons. Data represent the mean ± SD from five ABZ treated or three control individual animals. Each sample was made in triplicate. * = significantly p 6 0.001 different as compared to control.
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Fig. 2. Immunoblotting analyses of samples of hepatic and intestinal microsomes from control (C) and ABZ treated mouflons (ABZ) with CYP1A1/2 antibodies. MWS = molecular weight standard.
Table 1 Activities of other biotransformation enzymes tested in hepatic and intestinal microsomes from control and ABZ treated animals Activity (pmol/s/mg of proteins)
MFCD 6b-TOH TBSO np-UGT
Liver
Small intestine
Control animals
ABZ treated animals
Control animals
ABZ treated animals
7.2 ± 2.1 25.0 ± 8.3 29.4 ± 7.9 142 ± 15
7.1 ± 0.9 17.0 ± 7.5 30.3 ± 7.0 148 ± 8
ND 0.3 ± 0.2 ND 9.7 ± 1.7
ND 0.4 ± 0.2 ND 8.5 ± 3.3
Data represent the mean ± SD from five ABZ treated or three control individual animals. Each sample was made in triplicate. ND = not detected.
MFCD and TBSO activity. 6b-TOH activity was very low, approximately 80 times less extensive than in hepatic microsomes. Also, UGT activity was detected 15-fold lower in intestinal compared to hepatic microsomes. No significant differences were observed in these enzyme activities between control and ABZ-treated animals.
3.4. In vitro metabolism of ABZ in microsomes of control and ABZ treated mouflons Before incubations, presence of ABZ or its metabolites in microsomal suspension from ABZ treated animals were assayed but no traces of these compounds were detected. ABZ (at several concentrations) was incubated with either hepatic and intestinal microsomes of control and ABZ treated mouflons. Concentration of ABZ and total ABZSO was measured in reaction mixture using HPLC. Results are expressed as plots of ABZSO formation velocity versus substrate concentration (see Fig. 3). In blank samples (without microsomes or with boiled microsomes) none ABZ metabolite was detected. The apparent kinetic parameters V 0max and K 0m were calculated using GraphPad Prism 3.0 software (Table 2). ABZ treatment did not significantly affect the velocity of formation of total ABZSO in hepatic microsomes. Values of K 0m and V 0max were similar in microsomes from ABZ treated and control animals. In intestinal microsomes, ABZ treatment led to significant
increase of velocity and V 0max of ABZSO formation. As ABZSO exists in two enantiomeric forms chiral stationary phase was used for separation of ABZSO enantiomers. Effect of ABZ treatment on stereospecificity of in vitro ABZSO formation is shown in Fig. 4. While ABZ treatment did not affected ratio of ABZSO enantiomers formed in liver, in intestinal microsomes, ABZ treatment caused significant (p 6 0.001) shift of stereospecificity toward preferential formation of ()-ABZSO.
3.5. In vitro metabolism of ABZSO in microsomes of control and ABZ treated mouflons To study the second step of ABZ sulfoxidation, ABZSO was used as a substrate in the incubation mixture with hepatic and intestinal microsomes. In blank samples (without microsomes or with boiled microsomes) none ABZSO metabolite was detected and no changes of ABZSO enantiomeric ratio were observed after incubation. Fig. 5 demonstrates the differences in velocity of ABZSO2 formation in microsomes of control and ABZ treated mouflon. Repeated ABZ administration caused approximately 4- and 2-fold increase in velocity of ABZSO2 formation in microsomes from liver and small intestine, respectively. The kinetic parameters were also affected in both tissues tested (see Table 2). Significant (p 6 0.001) increase of V 0max and significant decrease of K 0m were found in microsomes of ABZ treated animals.
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1.6
A BZ treated animals control animals
ABZSO formation (µM/min/g of protein)
1.4 1.2 1 0.8 0.6 0.4 0.2 0 0
5
10
15
20
25
30
-6
(a)
concentration of substrate ABZ (10 M) 0.08
A BZ treated animals control animals
ABZSO formation (µM/min/g of protein)
0.07 0.06 0.05 0.04 0.03 0.02 0.01 0
0
(b)
5
10
15
20
25
30
-6
concentration of substrate ABZ (10 M)
Fig. 3. (a,b) Velocity of formation of ABZSO in dependence of substrate ABZ concentration in hepatic a or intestinal b microsomes from control and ABZ treated mouflons. Mixtures of microsomes from individual animals ABZ treated or control were used. Each sample was made in triplicate.
Table 2 Apparent kinetic parameters of ABZSO formation from ABZ and ABZSO2 formation from ABZSO in microsomes from liver and small intestine of mouflons Liver
Small intestine
Control animals
ABZ treated animals
Control animals
ABZ treated animals
Formation of ABZSO
K 0m (lM) V 0max (lM/min/g)
38.1 ± 1.9 3.4 ± 0.2
39.2 ± 2.1 3.1 ± 0.2
4.6 ± 0.4 0.05 ± 0.01
4.2 ± 0.2 0.07 ± 0.01*
Formation of ABZSO2
K 0m (lM) V 0max (nM/min/g)
0.78 ± 0.05 3.3 ± 0.2
0.24 ± 0.07* 8.4 ± 0.2*
0.67 ± 0.06 0.43 ± 0.03
0.46 ± 0.03* 0.72 ± 0.04*
Data represent the mean ± SD (n = 4). The values significantly p < 0.01 different from control are marked *.
4. Discussion The administration of drugs or other xenobiotics may cause the induction of biotransformation enzymes i.e. an increase in activity of enzymes in response to the presence of a xenobiotic substance (e.g. Barry and Feely, 1990; Nebbia, 2001; Okey, 1990). This increase of activity is mainly a consequence of an increase in respective gene expression and/or lowering of protein degradation. Enzyme induction may have relevant pharmacological and toxicological consequences. The increase of enzyme activities changes both the intensity and half time of simultaneously or successively administered drugs. This can result in ad-
verse drug reactions as consequence of enhanced levels of toxic metabolites. Alternatively, enzyme induction may cause therapeutic failures due to decrease drug concentration in the body. Induction of some biotransformation enzymes (e.g. CYP1A) can also result in increased sensitivity to environmental contaminants and the risk of pathogenesis. With regard to the abovementioned consequences, the induction effect should represent a necessary monitored parameter in all the drugs used. With respect to the well-known species differences in inducibility of biotransformation enzymes, in present study the induction potency of ABZ was tested in target species – wild sheep mouflon. The ABZ dosage scheme
J. Velı´k et al. / Research in Veterinary Science 78 (2005) 255–263
261
100
relative amount of (+)-ABZSO (%)
90 80 70 60 50 40
control-liver
30
control-intestine 20
ABZ treated-liver
10
ABZ treated-intestine
0 0
5
10
15
20
25
30
-6
concentration of substrate ABZ (10 M)
Fig. 4. Stereospecificity of ABZSO formation in hepatic and intestinal microsomes from control and ABZ treated animals. Stereospecificity is expressed as percentage ratio of ABZSO enantiomers formed. Concentration of total ABZSO sum of (+)-ABZSO and ()-ABZSO = 100%.
ABZSO 2 formation (n M/min/g of protein)
10
A BZ treated animals control animals
9 8 7 6 5 4 3 2 1 0 0
0.5
1
1.5
2
2.5
3
-6
(a)
concentration of substrate ABZ SO (10 M) 0.8
ABZSO 2 formation (n M/min/g of protein)
A BZ treated animals control animals 0.6
0.4
0.2
0 0
(b)
0.5
1
1.5
2
2.5
3
-6
concentration of substrate ABZ SO (10 M)
Fig. 5. (a,b) Velocity of formation of ABZSO2 in dependence of substrate ABZSO concentration in hepatic a or intestinal b microsomes from control and albendazole ABZ treated mouflons. Mixtures of microsomes from individual animals ABZ treated or control were used. Each sample was made in triplicate.
used in this study represents the common therapeutic approach used for successful treatment of mouflon helmintoses, especially those caused by small ling worms or flukes (Protostrongyliadae, Dicrocoeliidae) (Boch and Supperer, 1992; Kutzer, 1995).
The biotransformation enzymes tested were selected according to following reasons: (1) their participation in ABZ metabolism (CYP1A, CYP3A, FMO, UGT) (e.g. Delatour et al., 1991; El Amri et al., 1987, 1988; Fargetton et al., 1986; Moroni et al., 1995), (2). their
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inducibility by ABZ in laboratory species or man (mainly CYP1A, in lesser extent CYP3A, UGT) (e.g. Asteinza et al., 2000; Baliharova´ et al., 2003a; El Amri et al., 1988) and (3) their important role in drug–drug interactions in veterinary medicine (CYP3A, CYP2C, UGT) (e.g. Fink-Gremmels and van Miert, 1996). The activities of these biotransformation enzymes were assayed in microsomes not only from liver but also from small intestine because the significant participation of intestinal enzymes in ABZ metabolism have been reported (Galtier, 1991; Redondo et al., 1999; Villaverde et al., 1995). Repeated ABZ administration to mouflons led to significant increase of EROD activity in hepatic as well as in intestinal microsomes. Significant increase of MROD activity was found in liver only. EROD and MROD activity are ascribed to CYP1A1/2 (Weaver et al., 1994). The increase of amount of CYP1A proteins was confirmed by immunodetection. Thus treatment of mouflons with ABZ (in dosage scheme used) significantly induced CYP1A1/2 in liver and small intestine. No other enzymes tested were affected by ABZ repeated administration. In our previous study the induction effect of ABZ on CYP enzymes was tested in mouflon in vitro (primary cultures of hepatocytes). On contrary to in vivo results presented here, no significant induction of mouflon CYP1A was found in vitro (Baliharova´ et al., 2003b). From this point of view it is clear that in vitro results may not correspond to in vivo situation. Disagreement between the in vivo and in vitro results may be caused by different time of ABZ interaction with cells (120 vs. 48 h). Moreover, inhibition of CYP1A activities by ABZ and ABZSO, which was also observed in mouflon microsomes (unpublished results), probably affected in vitro experiment more than in vivo experiment. In addition to activities of biotransformation enzymes toward their selective substrates, in vitro metabolism of ABZ was also studied with the aim to evaluate the influence of ABZ repeated administration to animals on metabolism of ABZ itself. The formation of pharmacologically active ABZSO (first step of ABZ sulfoxidation) is mainly ascribed to CYP3A and FMO (Delatour et al., 1991; El Amri et al., 1987; Fargetton et al., 1986; Moroni et al., 1995). ABZ repeated administration did not affect the ABZSO formation in hepatic microsomes. The ratio of ABZSO enantiomers formed remained without changes in ABZ treated animals. These results are in agreement with testing of ABZ effect on biotransformation enzymes activities as 6b-TOH, TBSO (reflecting CYP3A and FMO activities) which were not affected, too. A significant increase in ABZSO formation in intestinal microsomes was observed in ABZ treated animals. From all tested intestinal CYPs only CYP1A1 was induced by ABZ. With respect to these facts
CYP1A1 seems to participate on ABZSO formation in mouflon small intestine. The formation of pharmacologically inactive ABZSO2 (second step of ABZ sulfoxidation) is mainly catalysed by CYP1A (El Amri et al., 1988). A significant increase in velocity of ABZSO2 formation was observed in hepatic as well as in intestinal microsomes from ABZ treated animals. V 0max of ABZSO sulfoxidation were significantly higher in microsomes from ABZ treated animals. In addition, ABZ treatment led also to significant decrease of the K 0m value for ABZSO oxidation (i.e. increase of enzyme affinity toward substrate). It was surprising, as classical induction (increase of enzyme amount) enhances Vmax value but not affects Km. Our observation could be explain by participation of at least two enzymes in ABZSO2 formation. But only one (CYP1A), which has lower Km, is inducible by ABZ. Consequently, induction of CYP1A by ABZ is manifested by shift of apparent K 0m . In conclusion, ABZ repeated administration to mouflons significantly induced hepatic and intestinal CYP1A. This induction caused a significant increase of formation of inactive ABZSO2. Thus ABZ repeated administration induces its own metabolic degradation in liver and small intestine, which may result in decrease of therapeutic efficacy, contributing to the development of parasitic resistance. In addition, CYP1A induction could alter metabolism of simultaneously or consecutively administered drugs that are metabolised by this enzyme. Although CYP1A enzymes catalyse predominantly biotransformation of organic environmental contaminants, they take part also in metabolism of several veterinary drugs (e.g. fenbendazole, thiabendazole, mebendazole, caffeine, estradiol) (Testa, 1995; Plumb, 1999). A high CYP1A activity could result in faster biotransformation of these drugs. Moreover, a high activity of CYP1A also leads to an increase in activation of certain procarcinogens e.g. polycyclic aromatic hydrocarbons, polychlorined biphenyls etc. In therapy of mouflon all pharmacological and toxicological consequences of ABZ repeated administration above-mentioned should be taken into account.
Acknowledgement Financial support of this project was provided by Grant Agency of the Czech Republic, Grant No. 524/ 03/1361.
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