55, 284 –292 (2000)
Copyright © 2000 by the Society of Toxicology
TOXICOLOGICAL SCIENCES
Metabolism of 3-Methylindole by Porcine Liver Microsomes:
Responsible Cytochrome P450 Enzymes
Gonzalo J. Diaz 1,2 and E. James Squires
Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada
Received November 17, 1999; accepted March 1, 2000
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 ⴝ 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.
Skatole (3-methylindole, 3MI) is a naturally occurring microbial metabolite produced from tryptophan in the gastrointestinal tract of ruminants (Yokoyama and Carlson, 1979),
humans (Fordtran et al., 1964), and pigs (Jensen et al., 1995).
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., 1995). 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., 1995). 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., 1994).
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.
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: [email protected].
2
Permanent address: Facultad de Medicina Veterinaria y de Zootecnia,
Universidad Nacional de Colombia, Apartado Aéreo 76948, Santafé de
Bogotá, Colombia. E-mail: [email protected].
MATERIALS AND METHODS
Chemicals. Alpha-naphthoflavone (ANF), 4-methylpyrazole (4-MP),
8-methoxypsoralen (8-MOP), diethyldithiocarbamate (DDTC), menthofuran,
quinidine, sulfaphenazole, troleandomycin (TAO), coumarin, 7-hydroxycou-
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3-METHYLINDOLE METABOLISM IN PORCINE MICROSOMES
marin, 3-methylindole (3MI), indole-3-carbinol (I3C), 2-aminoacetophenone,
␤-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., 1999). Hydroxymethylindolenine (HMI) was isolated from large-scale microsomal incubations and
purified by preparative HPLC as described before (Diaz and Squires, 2000).
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 ⫻ Swedish Yorkshire
boars with Swedish Yorkshire sows (Squires and Lundström, 1997). The
samples were frozen in liquid nitrogen and stored at – 80 0C. 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 MgCl 2, 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-␮l
aliquot of the clear supernatant was analyzed by high-performance liquid
chromatography (HPLC).
Chromatography. HPLC analysis was performed as described previously
(Diaz et al., 1999) using a Spectra-Physics system (Spectra-Physics, San Jose,
CA) consisting of a SP8800 gradient pump, a SP8880 autosampler with a 50
␮l 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., 1999). Inhibitors were also incubated without substrate and
analyzed under the same conditions to ensure that the presence of the inhibitors
285
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 ␮l of microsomal suspension containing 0.4 mg microsomal protein were mixed with 200 ␮l of
coumarin hydroxylase reaction mix (0.05 M Tris buffer pH 7.4, 5 mM MgCl 2
and 0.2 mM coumarin). The reaction was started by adding 15 ␮l of 25 mM
NADPH and the samples were incubated at 37 0C for 15 min in a shaking water
bath. The reaction was terminated by the addition of 50 ␮l of 20% trichloroacetic acid, followed by centrifugation for 2 min at 10,000 g. After centrifugation, 200 ␮l 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 ␮g/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 (ECL™ 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 Analyst™, 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).
RESULTS
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., 1999) is shown in Figure 1. The
CYP2E1 inhibitor 4-methylpyrazole (Clejan and Cederbaum,
1990; Feierman and Cederbaum, 1986) 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 produc-
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DIAZ AND SQUIRES
3-METHYLINDOLE METABOLISM IN PORCINE MICROSOMES
tion of 3MI metabolites: DDTC (Guenguerich et al., 1991)
decreased the production of HMI, HMOI, 3MOI, 5-hydroxy3-methylindole, and 2-aminoacetophenone. Eight-MOP (Koenigs et al., 1997) decreased the production of I3C, 2-aminoacetophenone, and the hydroxyskatoles. Menthofuran
(Khojasteh-Bakht et al., 1998) 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 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.
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
(r 2 ⫽ – 0.68, p ⬍ 0.01) with 3MI fat content (Squires and
Lundström, 1997). 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 3. 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
287
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 4 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.
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 1. 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.
DISCUSSION
The role of specific human cytochrome P450 enzymes in the
metabolism of 3MI has been investigated (Thornton-Manning
et al., 1991, 1996); 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, 1997) and the use of
CYP2E1 inhibitors (Babol et al., 1998). 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
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.
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DIAZ AND SQUIRES
3-METHYLINDOLE METABOLISM IN PORCINE MICROSOMES
289
FIG. 3. Back fat 3-methylindole content vs. (A) hepatic microsomal CYP2A6 ortholog activity, and (B) microsomal CYP2A6 ortholog content in pigs
(n ⫽ 30).
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
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).
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DIAZ AND SQUIRES
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.
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 607fold 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 3MIinduced boar taint. More studies are needed in order to deter-
TABLE 1
Microsomal CYP2A6 Content and Activity in Pigs with Different 3-Methylindole Fat Content
Category
3-Methylindole fat
content
High accumulator
Moderate accumulator
Low accumulator
0.2 mg/kg or more
0.11–0.19 mg/kg
0.1 mg/kg or less
a–c
n
Mean (⫾SD) 3-methyl
indole content
(mg/kg)
Mean (⫾ SD) CYP2A6
porcine ortholog content
(density units)
Mean (⫾SD) CYP2A6
porcine ortholog activity
(nmol 7-hydroxy-coumarin/mg protein/min)
6
15
9
0.25 ⫾ 0.04 a
0.15 ⫾ 0.03 b
0.09 ⫾ 0.01 c
0.24 ⫾ 0.25 c
4.08 ⫾ 4.49 b
8.44 ⫾ 2.96 a
4.5 ⫾ 3.5 c
66.2 ⫾ 93.6 b
119.6 ⫾ 50.2 a
Within a column, means lacking a common superscript differ significantly (p ⬍ 0.05).
3-METHYLINDOLE METABOLISM IN PORCINE MICROSOMES
mine 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.
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