Metabolism of 3-Methylindole by Porcine Liver Microsomes: Responsible Cytochrome P450 Enzymes

Gonzalo J. Diaz1,2 and E. James Squires

Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada

Received November 17, 1999; accepted March 1, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The role of different cytochrome P450 enzymes on the metabolism of 3-methylindole (3MI) was investigated using selective chemical inhibitors. Eight chemical inhibitors of P450 enzymes were screened for their inhibitory specificity towards 3MI metabolism in porcine microsomes: alpha-naphthoflavone (CYP1A1/2), 8-methoxypsoralen (CYP2A6), menthofuran (CYP2A6), diethyldithiocarbamate (CYP2A6), 4-methylpyrazole (CYP2E1), sulphaphenazole (CYP2C9), quinidine (CYP2D6), and troleandomycin (CYP3A4). The production of 3MI metabolites was only affected by the presence of inhibitors of CYP2A6 and CYP2E1 in the microsomal incubations. In a second experiment, a set of porcine microsomes (n = 30) was analyzed for CYP2A6 content by protein immunoblot analysis and for their coumarin 7-hydroxylation activity (CYP2A6 activity). Both CYP2A6 content and enzymatic activity were found to be highly and negatively correlated with 3MI fat content. The results of the present study indicate that the CYP2A6 porcine ortholog plays an important role in the metabolism of 3MI and that measurement of CYP2A6 levels and/or activity could be a useful marker for 3MI-induced boar taint.

Key Words: pig; skatole; metabolism; cytochrome P450; CYP2A6; CYP2E1; inhibitor; boar taint.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Skatole (3-methylindole, 3MI) is a naturally occurring microbial metabolite produced from tryptophan in the gastrointestinal tract of ruminants (Yokoyama and Carlson, 1979Go), humans (Fordtran et al., 1964Go), and pigs (Jensen et al., 1995Go). 3MI is a well known acute pneumotoxin for cattle and it has important implications for pig meat production. Entire male pigs (uncastrated pigs) are used for meat production in several countries, due to a better feed conversion, improved carcass leanness, and a better composition of fatty acids compared with castrated pigs (Bæk et al., 1995Go). However, 5–10% of intact male pigs carry the so-called "boar taint" (a fecal-like odor liberated when the meat is cooked), and 3MI is one of the major contributors to boar taint (Bæk et al., 1995Go). It is not known why only a small percentage of a given population of pigs accumulates 3MI to a level that can be detected by humans but one possibility is that this is due to individual differences in the metabolism of 3MI (Lundström et al., 1994Go).

Cytochrome P450 enzymes play a major role in the metabolism of 3MI in several species, including goats (Huijzer et al., 1989Go), humans (Thornton-Manning et al., 1996Go) and pigs (Babol et al., 1998Go). 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., 1991Go, 1996Go); however, the only cytochrome P450 considered to be involved in 3MI metabolism in pigs is CYP2E1 (Friis, 1995Go; Squires and Lundström, 1997Go). 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., 1994Go). 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., 1994Go). 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., 1998Go).

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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
Alpha-naphthoflavone (ANF), 4-methylpyrazole (4-MP), 8-methoxypsoralen (8-MOP), diethyldithiocarbamate (DDTC), menthofuran, quinidine, sulfaphenazole, troleandomycin (TAO), coumarin, 7-hydroxycoumarin, 3-methylindole (3MI), indole-3-carbinol (I3C), 2-aminoacetophenone, ß-NADPH, polyvinyl alcohol, sulphatase type H-2 from Helix pomatia and peroxidase-conjugated goat anti-mouse IgG (FAB-specific) were purchased from Sigma-Aldrich (Oakville, ON, Canada). Authentic 3-methyloxindole (3MOI) and 3-hydroxy-3-methyloxindole (HMOI) were graciously provided by Dr. G. S. Yost, Department of Pharmacology and Toxicology, University of Utah (Salt Lake City, UT). Authentic 5-hydroxy-3-methylindole and 6-hydroxy-3-methylindole (in the form of 6-sulphatoxyskatole) were donated by Jens Hansen-Møller (Danish Meat Research Institute, Roskilde, Denmark). In order to obtain 6-hydroxy-3-methylindole from 6-sulphatoxyskatole, the compound was hydrolyzed as described before (Diaz et al., 1999Go). Hydroxymethylindolenine (HMI) was isolated from large-scale microsomal incubations and purified by preparative HPLC as described before (Diaz and Squires, 2000Go). Monoclonal antibodies against human CYP2A6 and microsomes containing cDNA-expressed human CYP2A6 were purchased from Gentest Corp. (Woburn, MA). All other reagents and solvents were of high analytical or HPLC grade supplied by Fisher Scientific (Nepean, ON, Canada).

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, 1997Go). 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. 1Go) 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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FIG. 1. Effects of various chemical inhibitors on cytochrome P450-mediated 3-methylindole metabolism in porcine liver microsomes. Metabolites are: (up triangle) 3-hydroxy-3-methylindolenine, (filled up triangle) 3-hydroxy-3-methyloxindole, (square) 3-methyloxindole, (filled square) 2-aminoacetophenone, (down triangle) indole-3-carbinol, (filled down triangle) 5-hydroxy-3-methylindole, and (filled circle) 6-hydroxy-3-methylindole.

 
Chromatography.
HPLC analysis was performed as described previously (Diaz et al., 1999Go) using a Spectra-Physics system (Spectra-Physics, San Jose, CA) consisting of a SP8800 gradient pump, a SP8880 autosampler with a 50 µl injection loop, a SP Spectra 100 UV detector, and a Spectra System FL-2000 fluorescent detector. HPLC analysis for 3MI metabolites was conducted immediately after the incubations. Metabolites were identified and quantitated by comparison with authentic standards as described previously (Diaz et al., 1999Go). Inhibitors were also incubated without substrate and analyzed under the same conditions to ensure that the presence of the inhibitors in the incubation did not interfere with the quantitation of the respective metabolites in the assays.

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., 1994Go). 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, 1984Go). 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, 1995Go).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The effect of the specific P450 inhibitors on the production of the 7 major metabolites synthesized by porcine liver microsomes from 3MI (Diaz et al., 1999Go) is shown in Figure 1Go. The CYP2E1 inhibitor 4-methylpyrazole (Clejan and Cederbaum, 1990Go; Feierman and Cederbaum, 1986Go) significantly decreased the production of 3MOI, 2-aminoacetophenone, I3C and the hydroxyskatoles (5- and 6-hydroxy-3-methylindole). All the CYP2A6 inhibitors tested significantly decreased the production of 3MI metabolites: DDTC (Guenguerich et al., 1991Go) decreased the production of HMI, HMOI, 3MOI, 5-hydroxy-3-methylindole, and 2-aminoacetophenone. Eight-MOP (Koenigs et al., 1997Go) decreased the production of I3C, 2-aminoacetophenone, and the hydroxyskatoles. Menthofuran (Khojasteh-Bakht et al., 1998Go) affected the production of HMI, HMOI and 3MOI. No significant effect on the production of 3MI metabolites was observed when ANF (CYP1A1/2 inhibitor; Chang et al., 1994); sulphaphenazole (CYP2C9 inhibitor; Baldwin et al., 1995); quinidine (CYP2D6 inhibitor; Otton et al., 1988); or troleandomycin (CYP3A4 inhibitor; Yamazaki and Shimada, 1998) were added to the microsomal incubations.

Figure 2Go 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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FIG. 2. Effect of different chemical inhibitors of cytochrome P450 enzymes on the production of the seven major metabolites of 3-methylindole produced by porcine liver microsomes. TAO = troleandomycin (CYP3A4); quinidine (CYP2D6); sulfaphenazole (CYP2C9); ANF = alpha-naphthoflavone (CYP1A1/2); methofuran (CYP2A6); 8-MOP = 8-methoxypsoralen (CYP2A6); DDTC = diethyldithiocarbamate (CYP2A6); and 4-MP = 4-methylpyrazole (CYP2E1). Asterisk indicates significant difference from control (p < 0.05).

 
Production of all metabolites was significantly affected by chemical inhibitors known to specifically inhibit CYP2A6 activity. Accordingly, it was decided to determine both the levels of CYP2A6 protein and enzymatic activity and to correlate these values with 3MI levels in back fat. Even though the production of some metabolites was affected by the CYP2E1 inhibitor 4-MP, it was decided not to investigate the CYP2E1 content in these samples since a previous study already demonstrated that hepatic levels of CYP2E1 correlate negatively (r2 = –0.68, p < 0.01) with 3MI fat content (Squires and Lundström, 1997Go). The plots of the CYP2A6 porcine ortholog activity and levels vs. the 3MI fat content of the 30 pigs used in this study are shown in Figure 3Go. Pigs with high levels of 3MI in fat consistently showed very low levels of both CYP2A6 activity and protein content but pigs with low levels of 3MI had either high or low levels of CYP2A6. The Pearson correlation coefficient between the CYP2A6 porcine ortholog activity and 3MI fat content was found to be –0.57 (p < 0.001), whereas the Pearson correlation coefficient between CYP2A6 content and the 3MI fat content was –0.67 (p < 0.001). The Pearson correlation coefficient between CYP2A6 activity and CYP2A6 content was 0.83 (p < 0.001). The 3MI fat content in all samples ranged from 0.07 to 0.3 mg/kg and had a mean value of 0.15 mg/kg. The CYP2A6 activity ranged from 1.7 to 330.6 nmol 7-hydroxycoumarin/mg protein/min and had a mean value of 69.9 nmol of 7-hydroxycoumarin/mg protein/min. The CYP2A6 porcine ortholog content ranged from 0 to 13.3 density units, and had an average value of 4.6 density units. Figure 4Go shows a typical protein blot for CYP2A6. The porcine CYP2A6 bands appear below the human CYP2A6, indicating that the CYP2A6 porcine ortholog has a lower molecular weight than the human protein. Lundström and Bonneau (1996) have suggested that levels of 3MI of 0.2–0.25 mg/kg or greater cause unacceptable taint by sensory analysis. All pigs having microsomal CYP2A6 activities greater than 17 nmol of 7-hydroxycoumarin/mg protein/min or CYP2A6 content above 2 density units had 3MI levels below the threshold level of 0.2 mg/kg. The variability observed both in the activity and levels of CYP2A6 was very high. The ratios between the highest and lowest detectable values were 194 and 607 for the CYP2A6 porcine ortholog activity and content, respectively.



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FIG. 3. Back fat 3-methylindole content vs. (A) hepatic microsomal CYP2A6 ortholog activity, and (B) microsomal CYP2A6 ortholog content in pigs (n = 30).

 


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FIG. 4. Protein blot of the porcine CYP2A6 ortholog. Lanes 1–3 correspond to microsomes containing cDNA-expressed human CYP2A6 (0.2, 1.0, and 2.0 pmol of CYP2A6 protein, respectively). Lanes 4–8 show the bands obtained with 5 different samples of porcine microsomes. The corresponding coumarin 7-hydroxylation activity for samples in lanes 4 to 9 was 164.8, 9.7, 146.7, 3.1, and 66.7 nmol/mg protein/min, respectively.

 
The results obtained for both CYP2A6 content and activity were grouped in 3 categories according to the 3MI fat content of each pig, as follows: large 3MI accumulators (0.2 mg/kg 3MI or more), moderate 3MI accumulators (0.11 to 0.19 mg/kg 3MI), and low accumulators (0.1 mg/kg 3MI or less). The mean values for 3MI fat content, CYP2A6 porcine ortholog activity and CYP2A6 porcine ortholog content for these 3 categories of pigs are shown in Table 1Go. CYP2A6 activity in low accumulators of 3MI was about 27 times greater than in high accumulators, whereas the CYP2A6 microsomal content was about 35 times greater in low vs. high accumulators.


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TABLE 1 Microsomal CYP2A6 Content and Activity in Pigs with Different 3-Methylindole Fat Content
 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The role of specific human cytochrome P450 enzymes in the metabolism of 3MI has been investigated (Thornton-Manning et al., 1991Go, 1996Go); however, knowledge on the specific P450 enzymes involved in the metabolism of 3MI in pigs is still incomplete. By correlating the metabolic rate of chlorzoxazone 6-hydroxylation with the formation of unknown 3MI metabolites, Friis (1995) postulated that CYP2E1 was important in the metabolism of 3MI in pigs. This finding was substantiated by subsequent in vitro studies involving the immuno-quantitation of CYP2E1 (Squires and Lundström, 1997Go) and the use of CYP2E1 inhibitors (Babol et al., 1998Go). In these previous studies, however, the possible involvement of other cytochrome P450 enzymes was not investigated.

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., 1994Go). In the present study, the inhibitory effect observed with the classic competitive inhibitor of CYP2E1, 4-MP (Clejan and Cederbaum, 1990Go; Feierman and Cederbaum, 1986Go) 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., 1998Go; Friis, 1995Go; Squires and Lundström, 1997Go). 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., 1999Go). 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., 1999Go) was not affected by 4-MP, suggesting the involvement of another cytochrome P450.

Menthofuran (Khojasteh-Bakht et al., 1998Go) and 8-MOP (Koenigs et al., 1997Go) are potent, mechanism-based inactivators of CYP2A6. DDTC was reported to be a selective, mechanism-based inhibitor of CYP2E1 (Guenguerich et al., 1991Go) but later was found to inhibit both CYP2E1 and CYP2A6 (Yamazaki et al., 1992Go); DDTC is currently used mainly as a probe for CYP2A6 and, to a lesser extent, for CYP2E1 activity (Halpert et al., 1994Go). 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., 1999Go). 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., 1994Go). 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., 1998Go); 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. 3Go) 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, 1997Go) and aldehyde oxidase (Diaz and Squires, 2000Go). 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., 1998Go). 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-{alpha}-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.


    ACKNOWLEDGMENTS
 
Thanks are due to Kerstin Lundström and Leif Andersson of the Swedish University of Agricultural Sciences for access to pig liver samples. Financial support for GJD was provided by the University of Guelph, the Colombian Institute for the Development of Science and Technology (Colciencias), and the National University of Colombia. This work was supported by NSERC research grants and grants from Ontario Pork and the Ontario Ministry of Agriculture, Food, and Rural Affairs.


    NOTES
 
1 To whom correspondence should be addressed at University of Guelph, Department of Animal and Poultry Science, Room 208, Guelph, Ontario N1G-2W1, Canada. Fax: (519) 836-9873. E-mail: gdiaz{at}aps.uoguelph.ca. Back

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. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Aitio, A. (1978). A simple and sensitive assay of 7-ethoxycoumarin deethylation. Anal. Biochem. 85, 488–491.[ISI][Medline]

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Bæk, C. E., Hansen-Møller, J., Friis, C., and Hansen. (1995). Identification and quantification of selected metabolites of skatole: Possibilities for metabolic profiling of pigs. Proceedings of a Meeting of the E.A.A.P. Working Group: Production and Utilization of Meat from Entire Male Pigs, pp. 27–29. September 1995; Milton Keynes, UK. Milton Keynes: INRA and MLC (184 p.).

Baldwin, S. J., Bloomer, J. C., Smith, G. J., Ayrton, A. D., Clarke, S. E., and Chenery, R. J. (1995). Ketoconazole and sulphaphenazole as the respective selective inhibitors of P4503A and 2C9. Xenobiotica 25, 261–170.[ISI][Medline]

Chang, T. K., Gonzalez, F. J., and Waxman, D. J. (1994). Evaluation of triacetyloleandomycin, alpha-naphthoflavone and diethyldithiocarbamate as selective chemical probes for inhibition of human cytochromes P450. Arch. Biochem. Biophys. 311, 437–442.[ISI][Medline]

Clejan, L. A. and Cederbaum, A. I. (1990). Oxidation of pyrazole by reconstituted systems containing cytochrome P-450 IIE1. Biochim. Biophys. Acta 1034, 233–237.[ISI][Medline]

Diaz, G. J., Skordos, K. W. Yost, G. S., and Squires, E. J. (1999). Identification of Phase I metabolites of 3-methylindole produced by pig liver microsomes. Drug Metab. Dispos. 27, 1150–1156.[Abstract/Free Full Text]

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