Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada
Received November 17, 1999; accepted March 1, 2000
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ABSTRACT |
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Key Words: pig; skatole; metabolism; cytochrome P450; CYP2A6; CYP2E1; inhibitor; boar taint.
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INTRODUCTION |
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Cytochrome P450 enzymes play a major role in the metabolism of 3MI in several species, including goats (Huijzer et al., 1989), humans (Thornton-Manning et al., 1996
) and pigs (Babol et al., 1998
). Specific human, mouse and rabbit P450 enzymes responsible for the bioactivation of 3MI into electrophilic metabolites have been identified, including cytochrome P450s 1A2, 2A6, 2F1, 2C8 and 3A4 (Thornton-Manning et al., 1991
, 1996
); however, the only cytochrome P450 considered to be involved in 3MI metabolism in pigs is CYP2E1 (Friis, 1995
; Squires and Lundström, 1997
). Recently, Diaz et al. (1999) reported that seven major metabolites are produced from 3MI in porcine microsomal incubations; however, the specific cytochrome P450 enzymes involved in the production of these metabolites have not been determined. To the authors' knowledge, 3MI metabolites do not contribute to "boar taint".
Chemical inhibitors have been satisfactorily used to define catalytic specificity of cytochrome P450 enzymes. Most of the earlier generation of P450 inhibitors (e.g., SKF 525A, metyrapone) are not particularly useful in this regard, but others have been developed that have considerable selectivity (Halpert et al., 1994). One major advantage of using selective inhibitors of individual P450s is that the fractional inhibition of a reaction in microsomes (or another crude preparation) indicates the extent to which a particular P450 is responsible for a reaction (Halpert et al., 1994
). It is important to note, however, that human P450 inhibitors do not necessarily exhibit the same selectivity when used with microsomes obtained from other species (Eagling et al., 1998
).
The aim of the present study was to further characterize the role of cytochrome P450 enzymes on 3MI metabolism by porcine microsomes, using selective inhibitors of cytochrome P450s 1A1/2, 2A6, 2E1, 2C9, 2D6, and 3A4 as specific probes.
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MATERIALS AND METHODS |
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Preparation of microsomes.
Liver samples were taken from 30 intact male pigs obtained by back-crossing F3 European Wild Pig x Swedish Yorkshire boars with Swedish Yorkshire sows (Squires and Lundström, 1997). The samples were frozen in liquid nitrogen and stored at 800C. For the preparation of microsomes, partially thawed liver samples were finely minced and homogenized with 4 volumes of 0.05 M Tris-HCl buffer pH 7.4 (containing 0.15 M KCl, 1 mM EDTA, and 0.25 M sucrose) using an Ultra-Turax homogenizer (Janke and Kunkel, Staufen, Germany). The homogenate was centrifuged at 10,000 g for 20 min and the resulting supernatant was centrifuged again at 100,000 g for 60 min in order to obtain the microsomal pellet. The pellets were suspended in a 0.05 M Tris-HCl buffer, pH 7.4, containing 20% glycerol, 1mM EDTA, and 0.25 M sucrose to a final concentration of 20 mg protein/ml and stored at 80°C before analysis. Protein concentrations were determined by the method of Smith et al. (1985) using bicinchoninic acid protein assay reagents purchased from Pierce Chemical Co. (Rockford, IL) and bovine serum albumin as standard.
Microsomal incubations.
In order to determine the specific cytochrome P450(s) involved in the production of the different 3MI metabolites, 8 different P450 inhibitors were tested: ANF (CYP1A1/2), 8-MOP (CYP2A6), menthofuran (CYP2A6), sulphaphenazole (CYP2C9), quinidine (CYP2D6), 4-MP (CYP2E1), DDTC (CYP2A6), and TAO (CYP3A4). Production of 3MI metabolites was detected and quantitated by HPLC as described under Chromatography section below. Each inhibitor was tested in 3 randomly selected porcine microsome samples, and each incubation was run in duplicate. Incubations contained 2 mg microsomal protein, 0.4 mM 3MI, 4 mM NADPH, 5 mM MgCl2, 1 mM EDTA and various concentrations of the different inhibitors (Fig. 1) in 0.05 M sodium phosphate buffer (pH 7.4). The final incubation volume was 0.5 ml. The inhibitors were dissolved in buffer or in an appropriate solvent and the organic solvent content did not exceed 1% (v/v) when added to incubation. Incubations were performed at 37°C for 30 min in a shaking water bath. Production of metabolites in control incubations was determined to be linear over a range of 10 to 40 min and 1 to 4 mg microsomal protein. Incubations with no inhibitor added were regarded as controls. Reactions were started by the addition of NADPH after 3-min preincubation periods at 37°C, and stopped with 0.5 ml of ice-cold acetonitrile. After the addition of acetonitrile, the mixture was vortexed and centrifuged at 2000 g for 10 min. A 50-µl aliquot of the clear supernatant was analyzed by high-performance liquid chromatography (HPLC).
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CYP2A6 activity.
Coumarin 7-hydroxylase activity was determined on the total set of porcine microsomal samples (n = 30), in duplicate, based on the procedure described by Aitio (1978), as follows: 20 µl of microsomal suspension containing 0.4 mg microsomal protein were mixed with 200 µl of coumarin hydroxylase reaction mix (0.05 M Tris buffer pH 7.4, 5 mM MgCl2 and 0.2 mM coumarin). The reaction was started by adding 15 µl of 25 mM NADPH and the samples were incubated at 370C for 15 min in a shaking water bath. The reaction was terminated by the addition of 50 µl of 20% trichloroacetic acid, followed by centrifugation for 2 min at 10,000 g. After centrifugation, 200 µl of clear supernatant were mixed with 2 ml of 0.1 M Tris buffer pH 9.0 and the fluorescence determined in a spectrofluorometer with excitation at 390 nm and emission at 440 nm. The enzymatic activity was quantitated by subtracting the fluorescence of the blank and comparing to a standard curve for 7-hydroxycoumarin. The activity was expressed in nmoles of 7-hydroxycoumarin per mg of microsomal protein per min. The production of 7-hydroxycoumarin was linear with the incubation time and microsomal protein concentrations.
CYP2A6 protein blot.
The presence and amount of the CYP2A6 porcine ortholog protein was determined in the same set of microsomal samples used for CYP2A6 activity (n = 30), in duplicate. Microsomes containing CYP2A6 expressed by cDNA-transfected human lymphoblastoid cells were used as a blotting standard. After 10% resolving sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the separated proteins were transferred to a nitrocellulose membrane as described previously (Fowler et al., 1994). The membrane was blocked with 1 µg/ml polyvinyl alcohol in Tris-buffered saline (TBS) for 1 min with gentle shaking, followed by two 5-min periods of washing with TBS-0.5% Tween 20. The membrane was incubated with the primary antibody (monoclonal mouse anti-human CYP2A6 antibody diluted 1:1000 in 0.5% nonfat powdered milk) for 1 h, followed by 3 5-min periods of washing with TBS-0.5% Tween 20. The membrane was then incubated with the secondary antibody (peroxidase-conjugated goat anti-mouse IgG) diluted 1:2000 in 0.5% nonfat powdered milk for 1 h and then washed for 3 5-min periods with TBS-0.5% Tween 20 followed by two 5-min periods with TBS. The CYP2A6 protein was finally detected using a commercially available kit (ECLTM Western blotting detection reagents, Amersham Pharmacia Biotech, Baie d'Urfé, Québec, Canada). CYP2A6 levels were estimated by measuring the intensity of the protein bands using a commercial software package (Molecular AnalystTM, Bio-Rad Laboratories Ltd., Mississauga, ON, Canada). The intensity of the protein bands was found to be linear with protein concentration.
Measurement of 3MI fat content.
For the quantitation of the 3MI fat content, a sample of back fat was taken from each pig and its 3MI content measured with a colorimetric assay (Mortensen and Sørensen, 1984). All analyses were done in duplicate.
Statistical analysis.
The cytochrome P450-mediated production of 3MI metabolites in the presence of inhibitors is expressed as a percentage of the corresponding control values. Pearson correlation coefficients, linear regression analysis and one-way ANOVA were computed using the Statistical Analysis System (SAS Institute, 1995).
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RESULTS |
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Figure 2 shows the effect of the chemical inhibitors at the highest concentration tested on the production of each individual 3MI metabolite. HMI and HMOI production were significantly decreased by the CYP2A6 inhibitors DDTC and menthofuran, whereas the production of 3MOI was significantly reduced by the CYP2A6 inhibitors DDTC and menthofuran and the CYP2E1 inhibitor 4-MP. The production of 2-aminoacetophenone and 5-hydroxyskatole was significantly decreased by the CYP2A6 inhibitors 8-MOP and DDTC and the CYP2E1 inhibitor 4-MP, while I3C and 6-hydroxyskatole production was reduced significantly by the CYP2A6 inhibitor 8-MOP and the CYP2E1 inhibitor 4-MP.
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DISCUSSION |
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In order to predict and/or rationalize species, strain and individual differences in xenobiotic metabolism, it is important to determine the catalytic specificities and regulation of individual P450 forms. In recent years, the emergence of a battery of isoform-selective chemical inhibitors that can be used both in in vivo and in vitro experiments has facilitated the identification of individual cytochromes P450 responsible for specific reactions (Halpert et al., 1994). In the present study, the inhibitory effect observed with the classic competitive inhibitor of CYP2E1, 4-MP (Clejan and Cederbaum, 1990
; Feierman and Cederbaum, 1986
) suggests a role for this enzyme in the metabolism of 3MI. This finding is in agreement with previous studies in which a role for CYP2E1 in 3MI metabolism was shown (Babol et al., 1998
; Friis, 1995
; Squires and Lundström, 1997
). In the present study, 4-MP significantly decreased the production of five of the seven metabolites reported to be produced by porcine microsomes (Diaz et al., 1999
). However, the production of two major metabolites (HMI and HMOI), which combined account for more than 64% of the net intrinsic metabolism of 3MI by porcine liver microsomes in vitro (Diaz et al., 1999
) was not affected by 4-MP, suggesting the involvement of another cytochrome P450.
Menthofuran (Khojasteh-Bakht et al., 1998) and 8-MOP (Koenigs et al., 1997
) are potent, mechanism-based inactivators of CYP2A6. DDTC was reported to be a selective, mechanism-based inhibitor of CYP2E1 (Guenguerich et al., 1991
) but later was found to inhibit both CYP2E1 and CYP2A6 (Yamazaki et al., 1992
); DDTC is currently used mainly as a probe for CYP2A6 and, to a lesser extent, for CYP2E1 activity (Halpert et al., 1994
). In the present study, menthofuran, 8-MOP, and DDTC decreased the production of several 3MI metabolites, strongly suggesting the involvement of a CYP2A6 porcine ortholog in the metabolism of 3MI. Of particular interest was the effect of menthofuran and DDTC, which were the only inhibitors that simultaneously and significantly decreased the production of HMI, HMOI, and 3MOI. These 3 metabolites account for more than 91% of the net intrinsic metabolism of 3MI by porcine liver microsomes in vitro (Diaz et al., 1999
). This finding suggests that the CYP2A6 porcine ortholog may play a more relevant role in the metabolism of 3MI than CYP2E1, although in vivo the overall contribution of a cytochrome will depend both on the intrinsic metabolic activity of the enzyme and its relative abundance. In humans, the average specific content of CYP2A6 is slightly lower than that of CYP2E1 (14 ± 13 vs. 22 ± 12 pmol/mg protein) and the percentage content is about 6.6% for CYP2E1 and 4.0% for CYP2A6 (Shimada et al., 1994
). In the porcine species, however, the relative abundance of these two P450 enzymes has not been determined.
It is important to note that in previous studies menthofuran was unable to inactivate other cytochrome P450s, including CYP2E1 (Khojasteh-Bakht et al., 1998); therefore, the inhibitory effect caused by menthofuran on 3MI metabolism observed in this study can be attributed solely to the inactivation of CYP2A6. This situation, however, does not seem to be the same for the CYP2E1 inhibitor 4-MP. Feierman and Cederbaum (1986) and Clejan and Cederbaum (1990) reported that 4-MP is a competitive inhibitor of CYP2E1; however, in a more recent study, Draper et al. (1997) found that 4-MP is also a potent inhibitor of CYP2A6. This suggests that the decreased production of 3MI metabolites observed when 4-MP was added to the microsomal incubations could be ascribed to the combined inhibition of both CYP2E1 and CYP2A6.
The results of the correlation analysis between the CYP2A6 porcine ortholog content and activity vs. 3MI fat content (Fig. 3) suggest that CYP2A6 is important in the adequate clearance of 3MI. However, the finding that pigs with either low or high levels of CYP2A6 exhibit low levels of 3MI in the fat suggests that other enzymes besides CYP2A6 participate in the clearance of 3MI. Other enzymes considered to be important in the metabolism of 3MI in pigs are CYP2E1 (Squires and Lundström, 1997
) and aldehyde oxidase (Diaz and Squires, 2000
). Squires and Lundström (1997) found that pigs with high hepatic levels of CYP2E1 had low levels of 3MI in fat, but when CYP2E1 levels were low, 3MI levels could be either high or low. This situation is similar to the one found in the present study for the CYP2A6 porcine ortholog content. A possible explanation for the fact that 3MI fat content can be either high or low when CYP2E1 (or CYP2A6) levels are low is that the low capacity to metabolize 3MI will only result in high 3MI levels in fat when the amount of 3MI absorbed is high, as it was postulated by Squires and Lundström (1997).
Yamano et al. (1990) reported up to a 40-fold difference in coumarin 7-hydroxylase activity among human liver microsome specimens. In the present study, up to a 196-fold difference in coumarin 7-hydroxylase activity and a 607-fold difference in CYP2A6 content were detected among the porcine samples tested. The cause of the extremely high variability in the content and activity of CYP2A6 found in the present study is unknown but it may be the result of a genetic polymorphism. Genetic polymorphisms exist in the CYP2A6 human gene. Three alleles have been identified using restriction fragment length polymorphisms and are known as CYP2A6*1, CYP2A6*2 and CYP2A6*3 (Gullstén et al., 1996); wild-type CYP2A6, CYP2A6*1, is responsible for the 7-hydroxylation of coumarin. A new truncated allele has been identified in the Japanese population. Individuals carrying this allele lack CYP2A6 mRNA and protein and exhibit no activity towards coumarin (Nunoya et al., 1998). In mouse, Lindberg and Negishi (1989) demonstrated that a single mutation is sufficient to convert the specificity of CYP2A3 from coumarin 7-hydroxylation to steroid 15-
-hydroxylation. The genomic structure of the porcine CYP2A6 gene has not been investigated. It may be possible that pigs exhibit a similar CYP2A6 polymorphism to humans and mice and that this may be one of the reasons why only a small proportion of the pigs within a given population accumulate large amounts of 3MI in the fat. This area of research requires further studies.
The results of the present study indicate that at least 2 cytochrome P450 enzymes are important in the hepatic metabolism of 3MI in pigs: the CYP2A6 and CYP2E1 porcine orthologs. The significant negative correlation found between the CYP2A6 porcine ortholog content/activity and 3MI levels in fat suggests that CYP2A6 is critical for an adequate clearance of 3MI. Measurement of the CYP2A6 porcine ortholog content/activity could be used as a potential marker for 3MI-induced boar taint. More studies are needed in order to determine whether the high variability in the CYP2A6 content/activity is due to a genetic polymorphism, which could explain the variability in 3MI fat levels observed in pigs kept under the same management conditions.
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ACKNOWLEDGMENTS |
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NOTES |
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2 Permanent address: Facultad de Medicina Veterinaria y de Zootecnia, Universidad Nacional de Colombia, Apartado Aéreo 76948, Santafé de Bogotá, Colombia. E-mail: dgjdiaz{at}veterinaria.unal.edu.co.
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