C Basic & Clinical Pharmacology & Toxicology 2005, 97, 115–121.
Printed in Denmark . All rights reserved
Porcine CYP2A Polymorphisms and Activity
Mette T. Skaanild and Christian Friis
Department of Veterinary Pathobiology, Laboratory of Toxicology, The Royal Veterinary and Agricultural University,
(Received January 31, 2005; Accepted March 1, 2005)
Abstract: CYP2A6 in man catalyzes the oxidation of nicotine-forming cotinine and 7-hydroxylation of coumarin, which
is used as test substrate for CYP2A6 in man. Large interindividual differences are found in man and some are due to
genetic polymorphism. The 7-hydroxylation of coumarin is present in pigs, and an inter-individual variation has been
found that might be due to polymorphisms. To enable the finding of polymorphism in pigs, the minipig cDNA was
sequenced. Two cDNAs were found and translated to a 494 and a 487 amino acid long protein, both cDNAs were found
in all but one pig. The 494 a.a. protein showed high homology to the human and 100% homology to the conventional pig
CYP2A19 protein. In the wild type protein, all 6 substrate recognition sites were found, whereas the short protein only
contained the first 5 substrate recognition sites. SSCP analysis revealed 3 polymorphisms. In order to study the effect of
these polymorphisms on enzyme activity, microsomes were incubated with nicotine and coumarin. The polymorphisms
appeared to have no effect on either enzyme activity as the specific enzyme activity towards nicotine and coumarin were
approximately the same for all pigs. The specificity of pig CYP2A was investigated and it was found that the formation
of cotinine correlated with the immunochemical level of CYP2A as did the coumarin hydroxylation. Anti-human CYP2A
inhibitory antibody inhibited coumarin 7-hydroxylation by about 90% and formation of cotinine by 44–60% and 85–
100% at substrate concentrations of 500 mM and 50 mM respectively, showing that coumarin and nicotine (50 mM) are very
specific substrates for CYP2A in pigs, whereas the CYP2A only is responsible for about 50% of the cotinine formation at
a 500 mM nicotine incubation concentration. These results show that the large interindividual differences in porcine
CYP2A activity are not caused by polymorphisms but transcriptional regulation and the coumarin 7-hydroxylation is as
specific a reaction for porcine CYP2A as for human CYP2A6.
Cytochrome P450 2A (CYP2A) represent 4% of human he-
patic P450 (Shimada et al. 1994; Guengereich et al. 1995).
The CYP2A family includes CYP2A6, CYP2A7 and
CYP2A13 (Fernandez-Salguero et al. 1995; Hoffman et al.
1995) where coumarin has been found to be a high affinity
substrate for human CYP2A6. Human CYP2A6 catalysis
the 7-hydroxylation of coumarin, whereas neither CYP2A7
nor CYP2A13 can catalysis this reaction in any tissue (Pel-
konen et al. 1995). In humans, large interindividual differ-
ences in both coumarin hydroxylase activity and the forma-
polymorphisms in the CYP2A6 gene (Inoue et al. 2000;
Tricker 2003). CYP2A6 catalysed 70–80% of the conversion
of nicotine to cotinine via a C-oxidation (Benowits & Jacobs
been evaluated by molecular modelling and crystallography
6 substrate recognition sites (SRS) in the CYP2A protein.
Quantitative structure-activity relationships (QSAR) of wild
type and mutants showed importance of the amino acids
(a.a.) phenylalanine in SRS 2, arginine and threonine in
SRS 4 for coumarin binding. According to Xu et al. 2002 the
phenylalanine F480 in SRS 6 is the major substrate inter-
are responsible for the folding of the protein around SRS 6.
Author for correspondence: Mette T. Skaanild, Department of Vet-
erinary Pathobiology, Ridebanevej 9, DK-1870 Frederiksberg C,
Denmark (fax π45 35 35 35 14, e-mail mts/kvl.dk).
The coumarin 7-hydroxylase activity has also been detected
in mouse, catalysed by Cyp2a5 (van Iersel et al. 1994) and in
pigs, conventional as well as minipigs. (Skaanild & Friis
1999). It has also been shown that the porcine CYP2A is in-
volved in the metabolism of 3-methylindole or skatole
(Diaz & Squires 2000) one of the major contributors to the
boar taint, that can be observed in 5–10% of intact male pig
(Bæk et al. 1995). The activity of porcine CYP2A show large
interindividual differences both for coumarin as well as 3-
methylindole metabolism, but it is at present not known if
these differences are due to polymorphisms in the porcine
The objective of this work is therefore to further analyse
the CYP2A in pigs with regard to cDNA sequence, poly-
morphisms and enzyme activity.
Materials and Methods
Animals. Eight Göttingen minipig (4 female, 4 male, age 4 month)
and 12 conventional pigs (4 female, 4 male, 4 castrates, age 3.5
month) have previously been described. (Skaanild & Friis 1999).
Chemicals. All antibodies were obtained from Gentest (MA, USA),
sequencing and synthesis of all primers were done by TAG Copen-
hagen A/S (Denmark). RNA isolation kit was supplied by Quiagen
(Hilden, Germany). All other chemicals were of analytical grade
obtained either from Amersham Biosciences or Sigma (St. Louis,
Isolation and sequencing of cDNA. Total RNA was isolated from
female Göttingen minipig hepatocytes using Qiagen RNAeasy mini
METTE T. SKAANILD AND CHRISTIAN FRIIS
kit according to protocol. Amplification of 5ø and 3ø cDNA ends
was done using the GeneRacer kit supplied by Invitrogen life tech-
nologies. The amplification was done according to kit protocol
using 3 mg total RNA. The gene specific primers (GSP) used for the
amplification were designed from the CYP2A PCR product de-
scribed earlier (Skaanild & Friis 1999). This fragment was isolated
and sequenced. The following GSP were used: 3ø enden 5ø TCCAC-
GAGATCCAGAGATTCGGAGACA3ø and 5‘enden 5ø CTCATC-
CAGGAAGTGCTGGGGGTTGT 3ø. Pfu turbo DNA polymerase
from Stratagene was used for high fidelity PCR and the PCR reac-
tions were set up according to kit protocol. Two PCR reactions were
set up for each fragment 1) a reaction with RACE primers and GSP
and 2) a reaction using the first PCR reaction as template with
nested RACE primers and GSP. A touch down cycle reaction with
annealing temperatures from 69æ to 64/æ was used for high speci-
ficity annealing. PCR fragments of about 700 and 1300 base pair
(bp) long for the 3ø end and 5ø end respectively were expected. These
products were isolated from the PCR reactions and both strands
were sequenced by TAGCopenhagen A/S. The isolation of RNA
and RACE amplification and sequencing were done 2 times.
Polymorphism assay using SSCP. Total RNA was isolated as al-
ready described and reverse transcription was performed with first
strand synthesis kit from Amersham Biotech according to protocol
using 2 mg of total RNA in each reaction. The cDNA was divided
into 4 fragment 5ø end, 2 middle parts and 3ø end (1, 2, 3, 4) 383
bp, 557 bp, 470 bp and 537 bp respectively (fig. 1). After PCR again
using touch down cycling the PCR products were analysed using
PAGE gel electrophoresis looking for single strand conformation
polymorphisms. The samples were denatured by addition of an
equal volume 50 mM NaOH, 1 mM EDTA and then incubated at
95 æ for 5 min. The gels were run using an Amersham Biotech mul-
tiphor unit and precast CleanGels and the bands were visualised
using silver staining according to kit protocol (Amersham Biosci-
The frequency of the 3ø deletion was analysed setting up PCR
reactions using a primer on each side of the deletion (primer G and
GSP3ø). This will give rise to two PCR fragments of different sizes,
one about 587 bp and the other about 418 bp.
Isolation of liver microsomes. Isolation of microsomes was per-
formed according to Olsen et al. (1997). Briefly the liver was homo-
genized in 50 mM Tris-HCl buffer containing 0.25 M sucrose and
1 mM EDTA. The homogenate was centrifuged and the super-
natant was transferred to new tubes and centrifuged once more at
105,000 ¿g at 4 æ for 60 min. The pellet containing the microsomes
were homogenized in storage buffer and frozen in liquid nitrogen.
Fig. 1. Sequences and locations for primers used in the polymorph-
Microsomal protein concentration was determined using a modi-
fied Lowry method (Petterson 1977).
Enzyme assay. The microsomal mixture for all assays consisted of
a buffer containing 32 mM K-phosphate pH 7.46, 2.5 mM MgCl2,
15 mM glucose-6-phosphate, 10 U glucose-6-P-dehydrogenase/ml,
1.1 mM NADP and 0.91 mg microsomal protein in a total volume
of 1.075 ml. The mixture was pre incubated for 5 min. at 37 æ before
the test substrate was added.
Nicotine C-oxidation assay. The formation of cotinine was meas-
ured according to Yamazaki et al. (1999) with some modifications.
Briefly the microsomal incubation mixture (final total volume 525
ml) was added cytosol (3.3 mg prot./ml) and the reaction was started
by addition of nicotine (500 mM). The samples were incubated at
37 æ for 60 min. and then the reaction was added 550 ml internal
standard (200 ng caffeine/ml MeOH) and stopped by adding 50 ml
of 1 M NaCl2,1 M Na2CO3pH 10.5 buffer and 3 ml CH2Cl2. The
samples were mixed, centrifuged and 2.5 ml of the organic phase
was dried. The residue was dissolved in 200 ml 0.01 N HCl and 50
ml of this suspension was analysed by HPLC using the following
conditions: Column: Symetri 054215 (250¿4.6 mm, Waters); Col-
umn temperature: 30 æ; eluent: MeOH and 0.05 M CH3COOH
(1:4); flow rate 1 ml/min.; Detection: UV at 260 nm. To determine
the amount of cytosol necessary for maximal cotinine formation,
incubations with different concentrations of cytosol were set up.
The results show that the reaction reached the maximum when 200
ml cytosol (3.3 mg protein/ml) were added.
Coumarin 7-hydroxylase assay. To initiate the reaction 50 ml of a
2.2 mM coumarin solution in water was added and the reaction was
incubated for further 10 min. then stopped by addition of 1.1 ml
methanol. The samples were analyzed by HPLC as described earlier
(Skaanild & Friis 1999).
Immunoblotting. Microsomes isolated from the pig livers were used
for blotting according to Skaanild & Friis (1999). Briefly, micro-
somal protein was separated by PAGE SDS gel electrophoresis and
blotted to Hybond-ECL nitro-cellulose membranes. Membranes
were hybridised (1 hr) with a diluted primary anti-human CYP anti-
body. Antibody binding was detected by chemiluminescence using
a biotinylated secondary antibody followed by a streptavidin-horse-
radish peroxide conjugate. After development, the blots were ex-
posed to HyperfilmECL.
Inhibition assay. Polyclonal anti-human CYP2A6 was added to the
microsome solution (20 ml/mg protein) and pre incubated for 5 min.
at 37 æ, where after the reactions were started by adding either of
the substrates (final concentrations 100 mM coumarin and 500 mM
or 50 mM nicotine). The reactions were then analysed as descried
for coumarin hydroxylase and cotinine formation.
Isolation and sequencing of cDNA.
In order to analyse the porcine CYP2A gene for polymorph-
isms it was necessary to sequence the minipig CYP2A. For
this purpose total RNA was isolated from liver cells from a
female minipig as the females have a much higher expression
of this gene than male minipigs (Skaanild & Friis 1999). The
RNA (mRNA) was reverse transcribed and a 3ø end and 5ø
end RACE (rapid amplification of cDNA end) was per-
formed. The 3ø end PCR reaction gave rise to two different
fragment of about 450 bp and 850 bp respectively, whereas
only one 5ø end PCR fragment of about 1300 bp long was
synthesised. After sequencing of the PCR fragments, two
PORCINE CYP2A POLYMORPHISMS
accession number AY380866). Blast analysis revealed that
the long or wild type cDNA was very homologues to the
pig CYP2A19 sequence (GeneBank accession number
AB052255) and the cDNA encoded a protein that was 99%
(493 of 494 amino acids) homologous to the CYP2A19. The
short cDNA sequences were also very similar to the conven-
tional pig CYP2A19 sequence and from base pair 1 to 1426
to 1446, where after there is a deletion of 169 bp in the mini-
pig cDNA compared to the conventional. Base pairs 1427 to
1578 are again 100% homologous to the conventional base
pairs 1615 to 1766. When compared to the human CYP2A6
cDNA (GeneBank accession number NP 0007359) 88%
homologywas foundfrombase pair20 to1406and againthe
minipig cDNA had a deletion compared to the human
cDNA. This short cDNA sequence encoded a protein of 487
amino acids, 7 amino acids shorter than the human and con-
ventional counterpart (fig. 2). The beginning of this protein
from amino acids 1–466 was nearly identical to both the con-
ventional pig CYP2A19 (464 of 466) and the human
CYP2A6 (409 of 466), whereas the rest of the protein se-
whereas only SRS 1 to 5 was found in the short minipig pro-
teins (fig. 2). The 3 amino acid in SRS 2 and 4 important for
coumarin binding were conserved in both conventional pig
and minipigs and the major nicotine interacting amino acid
phenylalanine F480 is present in both the conventional and
wild-type minipig protein, but not in the short protein. The
frequency of this deletion was analyzed and all but 1 pig con-
tained both the wild type and the deleted PCR fragment.
Polymorphism assay and SSCP.
After sequencing, primers for the polymorphism study were
designed as shown in fig. 1 to divide the cDNA in fragment
sizes ideal for single strand conformation polymorphism
(SSCP). The SSCP analysis of the porcine CYP2A revealed
1 polymorphism (pig 9759) in fragment 1 and 1(pig 9743)
polymorphism in fragment 3. Fragment 4 gave rise to one
polymorphism that was found in pig 54919.
Nicotine C-oxidation assay.
In order to investigate the influence of the 3ø end deletion
and the 3 polymorphisms, the formation of cotinine from
nicotine was measured, as the major nicotine recognition
site is situated in SRS 6. The formation of cotinine from
nicotine involve a nicotine C-oxidation catalysed by CYP2A
and a conversion of the metabolite to cotinine catalysed by
cytosolic aldehyde oxidase. The Km and Vm were 380 mM
and 678 pmol cot./mg prot/min. respectively. The cotinine
formation rate for the pigs varied from 212–921 pmol/mg
prot./min. when incubated with 500 mM nicotine (table 1).
Coumarin 7-hydroxylase assay.
The microsomes were incubated with a final concentration
of 100 mM coumarin and the formation of 7-hydroxy cou-
marin was found to vary between 0.4–500 pmol/mg protein/
min, the male minipigs giving the lowest formation rates
The immunoblotting, using anti-human CYP2A6, gave re-
sponses from 40–172 U/mg protein for the conventional
pigs and 118–160 U/mg protein for female minipigs,
whereas for the male minipigs no bands could be observed
(table 1). The specific activity (enzyme activity/apoprotein
level) could then be calculated and it showed insignificant
pig to pig variation (table 1). No specific activity could be
calculated for male minipigs as no immunoblotting results
were obtained. The CYP2A immunochemical level corre-
lated well with both cotinine formation (r2Ω0.7) and cou-
marin 7-hydroxylation (r2Ω0.9), whereas these activities did
not correlate with CYP2B apoprotein level (fig. 3).
To determine how specific CYP2A is for the coumarin 7-
hydroxylation and formation of cotinine, the enzyme reac-
tions were incubated with inhibitory antibody against hu-
man CYP2A6. The inhibition analysis revealed that the
anti-human CYP2A6 inhibited the coumarin 7-hydroxyl-
ation reaction from 87.5–91.9% (table 2). One of the mini-
pigs, however, gave a much lower inhibition, but that is due
to the nearly 0 enzyme activity. The inhibition of cotinine
formation on the other hand was only 44% to 60% when
using 500 mM nicotine whereas the inhibition varied from
85–100% when using 50 mM nicotine substrate concen-
tration (table 2).
In human CYP2A6 at least 17 different polymorphisms
have been established (www.imm.ki.se/CYPallales/cyp2a6).
They give rise to either no, decreased or unchanged enzyme
activity and are responsible for some of the large interindi-
vidual differences found in humans. The porcine cyto-
chrome CYP2A that catalyses the coumarin 7-hydroxyl-
ation also show large interindividual differences (Skaan-
ild & Friis 1999) as does the metabolism of 3-methylindole.
To further study the reasons for these differences in enzyme
activities, the minipig CYP2A was sequenced as only the
CYP2A sequence of the conventional pig is known. Two
cDNA sequences were obtained, a wild type and a deletion
of 169 bp compared to the conventional pig sequence. The
sequences upstream the deletion was very similar for the 3
porcine sequences and the human sequence. When the 3
porcine protein sequences were compared, it could be seen
that they all contained SRS 1–5, whereas SRS 6 was differ-
ent in the short minipig protein due to the deletion at the
end of the cDNA sequence. Both the wild type and deleted
3ø end was found in all but 1 pig. The SSCP analysis re-
vealed further 3 polymorphisms in different segments of the
sequence. The influence of these polymorphisms and the
deletion was studied further, measuring the formation of
METTE T. SKAANILD AND CHRISTIAN FRIIS
Fig. 2. Protein sequence alignment of CYP2A (short and long) in minipig, CYP2A19 from conventional pig and human CYP2A6. Substrate
recognition sites (SRS) are shaded and substrate interacting amino acids are marked in bold.
PORCINE CYP2A POLYMORPHISMS
This table shows the coumarin 7-hydroxylase activity, the cotinine formation activity, their specific activities, and CYP2A protein level.
ND no detectable band. * SSCP Polymophism. ** No deleted PCR fragment.
cotinine from nicotine. The absence of SRS 6 in the deletion
indicated that this protein can not metabolize nicotine as
SRS 6 contains the nicotine recognition site. It was shown
that the human CYP2A6 test substrate nicotine can also be
metabolized by pig microsomes. The Km was found to be
380 mM, which is higher than the human Km of 64.9∫32.7
mM (Messina et al. 1997). The porcine cotinine formation
rate range between 212–922 pmol/mg protein/min. com-
pared to the human Vmaxvalues of 500∫500 pmol/mg pro-
tein/min. (Messina et al. 1997). This shows that the forma-
tion rate of cotinine in humans and pigs is identical,
whereas the Km in pigs is higher than in humans. The dif-
ference in Km values may be caused either by differences in
the binding strength between enzyme and substrate or be-
cause another enzyme also binds and metabolises nicotine.
Correlation analysis revealed that the formation of cotinine
This table shows the inhibition of coumarin 7-hydroxylase activity and cotinine formation after adding anti-human CYP2A6.
% Inh. % Inhb. pmol/mg/min.% Inhb.
* Substrate concentration 500 mM. ** Substrate concentration 50 mM. Con. pigΩconventional pigs.
correlates with the apoprotein concentration of CYP2A, in-
dicating that this enzyme may be responsible for this reac-
tion. However, it can be seen from the graph that although
there is no apoprotein level, there is still a high cotinine
formation rate of 207 pmol cotinine/mg protein/min., indi-
cating that another isoenzyme is also involved in the cotini-
ne formation. This is in accordance with results published
by Yamazaki et al. (1999) using both human microsomes
and human recombinant CYPs. They found that at low
substrate concentrations (50 mM) the CYP2A6 was the pre-
dominant isoenzyme, but at higher substrate concentration
(500 mM) also CYP2B6 and CYP2D6 played an important
role. The correlation analysis showed that human CYP2A6
correlated well with nicotine C-oxidation as did the porcine
CYP2A. Neither human CYP2B6 nor porcine CYP2B pro-
tein levels correlated with the cotinine formation. The speci-
METTE T. SKAANILD AND CHRISTIAN FRIIS
Fig. 3. Correlation blots between cotinine formation, coumarin 7-hydroxylase activity and the level of CYP2A and CYP2B.
fic activity (activity/U apoprotein) of the cotinine formation
was about the same in all pigs showing that the polymorph-
isms do not have any effect on specific activity. This could
either be because the antibody used in immunoblotting
could not recognize the deleted protein or the short PCR
fragment may be a 3ø end of either a CYP2A7 or CYP2A13
like cDNA. Both these cDNAs are very homologous to
CYP2A6 (96% and 94% respectively) and neither of these
genes shows any CYP2A6 activity. However, the significance
of the deletion is not known. No differences were found in
the specific activity of the nicotine oxidation and coumarin
hydroxylation for the polymorphism found in this study.
This indicates that they do not have any effect on the inter-
individual variation in enzyme activity. The variation must
therefore be due to transcriptional regulation, if the sub-
strates are as specific to porcine CYP2A as the correlation
analysis indicates. This was further investigated using in-
hibitory anti-human CYP2A6. The antibody inhibited the
coumarin 7-hydroxylation with about 91%, which is the
same as seen in human coumarin assay where the same anti-
body can inhibit the assay up to 95% (Le Gal et al. 2003).
The cotinine formation on the other hand could only be
inhibited by 40–60% using a substrate concentration of 500
mM indicating again that another isoenzyme may be in-
volved. At a 50 mM substrate concentration the antibody
inhibited the reaction 86–100%. These results are in accord-
ance with results obtained using human microsomes (Yama-
zaki et al. 1999), where a 33–60% inhibition with anti-hu-
man CYP2A6 was found using a substrate concentration of
500 mM, and cotinine formation can be inhibited up to 80%
with the same antibody (Le Gal et al. 2003).
The conclusion of this study is that the observed differ-
ences in porcine coumarin hydroxylation and cotinine for-
mation are not due to polymorphisms, but to transcrip-
tional regulation and that the coumarin 7-hydroxylation ac-
tivity is as specific in pigs as in humans for CYP2A.
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