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Vol. 302, Issue 2, 416-423, August 2002
Wadsworth Center, New York State Department of Health, and School of Public Health, State University of New York at Albany, Albany, New York
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Abstract |
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 Top
 Abstract
 Introduction
Experimental Procedures
Results
Discussion
References
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Human cytochrome P450 2A13 (CYP2A13), which is highly efficient in the metabolic activation of a major tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), may play important roles in xenobiotic toxicity and tobacco-related tumorigenesis in the respiratory tract. The aim of this study was to identify any genetic polymorphisms of the CYP2A13 gene, which may alter the metabolic capacities of the enzyme. Polymerase chain reaction (PCR) single-strand conformational polymorphism analysis was used to identify single-nucleotide polymorphisms (SNPs) in all of the exons and at the exon-intron boundaries, and PCR-restriction fragment length polymorphism analysis and DNA sequencing were used to determine the frequencies of the newly identified variant alleles in the four major ethnic groups. Blood spot DNA from more than 100 individuals was used for these analyses. Seven variant alleles were found, but only one SNP was detected in the coding region, in exon 5, leading to an Arg257Cys amino acid change. The frequencies of the Arg257Cys allele in white, black, Hispanic, and Asian individuals are 1.9%, 14.4%, 5.8%, and 7.7%, respectively. Functional analysis of the variant protein was performed following its heterologous expression. The Arg257Cys variant was 37 to 56% less active than the wild-type Arg-257 protein toward all substrates tested. With NNK, Cys-257 had higher Km and lower Vmax values than did Arg-257, with a >2-fold decrease in catalytic efficiency. The Arg257Cys mutation could provide some protection against xenobiotic toxicity in the respiratory tract to individuals who are homozygous for the Cys-257 allele.
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Introduction |
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Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Two
functional genes have been identified in the human CYP2A
gene subfamily. They are CYP2A6 and CYP2A13
(Fernandez-Salguero and Gonzalez, 1995
; Su et al., 2000), which share a
93.5% identity in deduced amino acid sequences. Both enzymes are
active toward a number of toxicants and carcinogens. CYP2A6 is mainly
expressed in the liver and is the major hepatic coumarin 7-hydroxylase
and nicotine C-oxidase (Fernandez-Salguero and Gonzalez, 1995; Messina et al., 1997). It is also active in the metabolic activation of such
compounds as aflatoxin B1, NNK, N-nitrosodiethylamine,
N-nitrosonornicotine, 2,6-dichlorobenzonitrile, and
hexamethylphosphoramide (Fernandez-Salguero and Gonzalez, 1995; Liu et
al., 1996; Patten et al., 1997). Although the substrate specificity of
CYP2A13 has not been studied as extensively, this enzyme, which is
predominantly expressed in the respiratory tract, is the most efficient
P450 enzyme known in the metabolic activation of the tobacco-specific
nitrosamine NNK (Su et al., 2000). In addition, CYP2A13 is also more
active than CYP2A6 in the metabolic activation of
hexamethylphosphoramide, N,N-dimethylaniline, and
N-nitrosomethylphenylamine (Su et al., 2000).
Both the CYP2A6 and CYP2A13 genes are located in
a P450 gene cluster on chromosome 19 (Fernandez-Salguero et al.,
1995, Hoffman et al., 2001). A number of genetic polymorphisms have
been described for the CYP2A6 gene, including SNPs in the
coding region that lead to inactivation or decreases in enzymatic
function, such as Gly479Val in CYP2A6.5 (Oscarson et al., 1999a) and
Arg128Gln in CYP2A6.6 (Kitagawa et al., 2001), as well as large
deletions of the gene (CYP2A6*4) that lead to a
nonfunctional allele (e.g., Nunoya et al., 1998; Oscarson et al.,
1999b). These genetic polymorphisms in the CYP2A6 gene are
responsible for large interindividual variation observed in hepatic
metabolism of nicotine and other drugs that are CYP2A6 substrates
(e.g., Nunoya et al., 1999a; Inoue et al., 2000). However, conflicting
results have been reported on the proposed role of CYP2A6
genotypes in lung cancer risk in different ethnic populations (Miyamoto
et al., 1999; Loriot et al., 2001; Tan et al., 2001). Notably, these
studies did not examine the potential contributions of polymorphisms in
extrahepatic P450 genes involved in the metabolic activation of
tobacco-associated carcinogens. In that respect, little is known of the
extent of individual differences in the expression or function of the
CYP2A13 gene, which is believed to play important roles in
xenobiotic toxicity and tobacco-related tumorigenesis in the human
respiratory tract (Su et al., 2000) because of its selective expression
in the target tissue and the high efficiency of CYP2A13 in NNK activation.
The aim of this study was to identify potential genetic polymorphisms of the CYP2A13 gene, which may alter the metabolic activities of the enzyme toward xenobiotic compounds. Human genomic DNA samples were obtained from newborn blood spots randomly sampled from the New York State Newborn Screening Program. PCR-SSCP was used to identify SNPs in all the exons and at the exon-intron boundaries. PCR primers were designed according to intron sequences, when possible, to avoid amplification of the other CYP2A sequences. PCR-RFLP was also used in some cases to determine the frequencies of distribution of the newly identified variant alleles in the four major ethnic groups. Finally, the function of a frequent variant allele (Arg257Cys) was determined following heterologous expression of the enzyme in Sf9 cells.
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Experimental Procedures |
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Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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DNA Samples.
Anonymous human genomic DNA samples were
isolated from newborn blood spots using a protocol described recently
(Sheng et al., 2000). Approximately equal numbers of random samples
were obtained from each of the four major ethnic groups (white, black,
Hispanic, and Asian) in the New York State Newborn Screening Program.
PCR-SSCP Analysis.
PCR primers (Table
1) were designed according to the
sequence in clone U22028 (Fernandez-Salguero et al., 1995) to amplify each of the exons and exon-intron boundaries in the CYP2A13
gene. The CYP2A13 sequence from the completed human genome
data base (accession no. AC008962) was used as a reference for
documenting the location of the PCR primers and the identified SNPs.
According to the recommended nomenclature system for human gene
mutations (Antonarakis, 1998), the A of the ATG start codon is
designated as +1. PCR amplification was carried out in a PerkinElmer
thermal cycler 9600 instrument (Applied Biosystems, Foster City,
CA) in a total volume of 50 µl, with Taq polymerase
(Promega, Madison, WI) and 25 µl of an aqueous solution of the
genomic DNA from a blood spot. PCR conditions are summarized in Table
2. A CYP2A13 genomic clone
(number 27292; obtained from Dr. Harvey Mohrenweiser of Lawrence
Livermore National Laboratory, Livermore, CA) was used as a positive
control. An H2O blank (no template) control was
routinely used to detect potential contamination of reagents. All PCR
products were gel-purified using the QIAquick Gel Extraction Kit
(QIAGEN, Chatsworth, CA).
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-32P]ATP (3000 Ci/mM) and 1.25 units of T4 polynucleotide kinase (Promega). After end labeling, PCR
products of 200 bp or smaller were directly used for SSCP, whereas
those bigger than 200 bp were digested, after labeling, with an
appropriate restriction enzyme (Table 2) before SSCP.
SSCP analysis of the purified and end-labeled PCR fragments was
performed after optimization of multiple parameters, including electrophoresis temperature, concentration of acrylamide, ratio of
acrylamide to bis-acrylamide, and addition of glycerol. DNA samples
were mixed with 1 volume of 95% formamide, containing 20 mM EDTA,
0.05% bromphenol blue, and 0.05% xylene cyanol. The mixture was
heated at 97°C for 5 min, followed immediately by chilling on ice.
Then, 2.5 to 3.0 µl of the mixture was loaded on a 6% nondenaturing
polyacrylamide gel (31 cm wide, 38.5 cm long, and 0.35 cm thick)
containing 2.5% glycerol and with a ratio of acrylamide to
bis-acrylamide of 99:1. Electrophoresis was carried out in 1×
Tris/borate/EDTA buffer (89 mM Tris, 89 mM boric acid, and 2 mM
EDTA; pH 8.0), with a sequencing gel electrophoresis apparatus from
Invitrogen (Carlsbad, CA) (model S2001), at 3 W constant power for
about 13 h at room temperature with a cooling fan. After
electrophoresis, the gel was transferred to filter paper, dried, and
subjected to autoradiography at 
80°C for 4 to 24 h. When a
fragment was found to display a shifted band, compared with the
fragments from the CYP2A13 genomic clone, the PCR-SSCP
procedure was repeated to eliminate potential PCR errors. For
identification of the sequence change in a variant allele, the
remaining samples of purified PCR products with a confirmed band shift
were subjected to direct sequencing, in both directions, with use of
the primers shown in Table 1, with an automated DNA sequencer from
Applied Biosystems (model 373A) at the Molecular Genetics Core of the
Wadsworth Center.
PCR-RFLP Analysis. PCR-RFLP assays, which were more convenient than SSCP, were developed to analyze additional DNA samples for a better estimate of the frequencies of the newly identified exon 3 and exon 5 variant alleles in different ethnic groups. No attempts were made to design similar assays for the other variants. For exon 3, the purified 173-bp PCR products were digested with HaeII, which cuts the wild-type allele to give two bands (57 and 116 bp) and the 1662G>C allele to give three bands (42, 57, and 74 bp). The digestion products were analyzed on a 12% polyacrylamide gel and stained with ethidium bromide. For exon 5, the purified PCR products were digested with HhaI, which cuts the wild-type allele to give two bands (99 and 233 bp), but does not cut the 3375C>T allele, thus producing only one band (332 bp). In addition, all samples were also digested with ApalI, which does not cut the wild-type allele but cuts the 3375C>T allele to give two bands (101 and 231 bp). The digestion products were analyzed on a 2% agarose gel.
Site-Directed Mutagenesis.
The 3375C>T point mutation found
in exon 5 of the CYP2A13 gene was introduced into the
wild-type CYP2A13 cDNA clone (Su et al., 2000) in pCR-Script vector
(Stratagene, La Jolla, CA), using the Transformer site-directed
mutagenesis kit (BD Biosciences Clontech, Palo Alto, CA). Two
mismatched oligonucleotide primers were used: one containing the
intended site of mutation in exon 5 of CYP2A13
(5'-gagcacaaccagtgcacgctggatc-3'), and the other containing
mutations in the unique EcoRI site in the vector
(5'-gggctgcaggatatcgatatcaagc-3'). The introduced
single-nucleotide mutation was confirmed by restriction digestion with
ApalI and by sequencing.
Heterologous Expression of the Arg257Cys CYP2A13 Variant in Sf9
Cells.
The mutated CYP2A13 cDNA was released using
NotI and ClaI and reinserted into a pCR-Script
vector at the NotI and SmaI sites to add a unique
EcoRI site at the 3' end of the cDNA. The
NotI-EcoRI fragment was then inserted into the
multiple cloning site of the baculoviral transfer vector pVL1392 (BD
Biosciences PharMingen, San Diego, CA). The integrity of the cloning
sites as well as the entire cDNA insert was confirmed by sequencing.
Recombinant viruses were made by cotransfecting insect Sf9 cells with
the P450-encoding transfer plasmid and linearized BaculoGold viral DNA
(BD Biosciences PharMingen). The preparation and titering of virus
stocks and the detection of P450 expression were performed according to
the procedures described previously for the expression of CYP2A6 and
CYP2A13 (Liu et al., 1996; Su et al., 2000). Cells were harvested at
72 h postinfection and resuspended in 100 mM Tris-acetate buffer
(pH 7.4) containing 1 mM EDTA and 150 mM potassium chloride. Microsomal
fractions were prepared as described previously (Liu et al., 1996) and
stored at 85°C until use. P450 expression was confirmed by
immunoblot analysis of microsomal preparations with a rabbit anti-mouse
CYP2A5 antibody (Gu et al., 1998).
Purification of Arg-257 and Cys-257 CYP2A13.
Procedures for
CYP2A13 purification were modified from those used previously for the
purification of other microsomal P450s (Ding et al., 1991; Gu et al.,
1998). Sf9 cell microsomes (about 90-130 nmol of total P450; at about
3 mg of protein/ml) were solubilized with Tergitol-NP-10
(Sigma-Aldrich, St. Louis, MO) and sodium cholate (0.5% and 1.0%,
respectively; w/v) and fractionated by polyethylene glycol (PEG)
precipitation. The 6% PEG 8000 (Sigma-Aldrich) supernatant fraction,
to which 20% glycerol and 10% NP-10 were added to make the final
concentration of PEG about 2% and that of NP-10, 0.5%, was loaded
onto a 25- to 40-ml HTP column (Bio-Rad, Hercules, CA) previously
equilibrated with 10 mM phosphate buffer, pH 7.4, containing 0.5%
NP-10 and 20% glycerol (buffer A). After washing with 10 volumes of
buffer A, the HTP column was eluted with a linear gradient of potassium
phosphate (10-300 mM, pH 7.4) in buffer A. CYP2A13 was recovered at
about 240 mM phosphate, as determined by immunoblot analysis. The
P450-containing fraction was dialyzed overnight with two changes of 10 mM phosphate buffer, pH 6.4, containing 1 mM EDTA, 0.5% NP-10, and
20% glycerol (buffer B), and loaded onto an 8- to 12-ml S Sepharose
column (Sigma-Aldrich) previously equilibrated with buffer B. The
column was washed with buffer B and eluted with a pH gradient from 6.4 (buffer B) to 7.4 (200 mM phosphate in buffer A). A nearly homogeneous
preparation of CYP2A13 was recovered at about pH 6.9, dialyzed
overnight against 10 mM phosphate buffer, pH 7.4, 0.2% NP-10, and 20%
glycerol (buffer C), and applied to a second HTP column (3-ml) to
remove NP-10, as described previously (Ding and Coon, 1988). The
detergent-free CYP2A13 was eluted and dialyzed as described for the
purification of CYP2A5 (Gu et al., 1998). The final preparations, which
showed CYP2A13 as the predominant band upon SDS-polyacrylamide gel
electrophoretic analysis, had specific P450 contents of 5.0 to 9.5 nmol/mg as determined by CO-difference spectroscopy using an absorption
coefficient of 91 mM
1cm1 (Omura and Sato,
1964).
Determination of Catalytic Activity.
Formaldehyde formed
from hexamethylphosphoramide, 2'-methoxyacetophenone,
N,N-dimethylaniline, and
N-nitrosomethylphenylamine was measured with CYP2A13 in Sf9
microsomes as described recently (Su et al., 2000), according to the
method of Nash (1953). The rates of product formation were
corrected for zero time blanks that were quenched before the addition
of NADPH. Reactions were carried out at 37°C for 10 to 30 min. The
enzyme activities were linear with incubation time under the conditions
used. NNK metabolism was assayed by high-pressure liquid chromatography
with an on-line radioactivity detector as described previously (Su et
al., 2000), except that purified and reconstituted CYP2A13 was used and
that cytochrome b5 was not included.
Reactions were carried out at 37°C and terminated with 50 µl each
of 25% zinc sulfate and saturated barium hydroxide. The contents of
individual reaction mixtures are described in the legends to the tables.
Other Methods and Materials.
[5-3H]NNK (1.9 Ci/mmol; purity > 98%)
and unlabeled NNK were obtained from Chemsyn Science Laboratories
(Lenexa, KS). CO-difference spectra of microsomal P450 were recorded at
room temperature using a Varian model Cary 3E spectrometer, according
to the procedure described by Omura and Sato (1964). Protein
concentrations were determined by the bicinchoninic acid method (Pierce
Chemical, Rockford, IL) using bovine serum albumin as a standard.
Polyclonal rabbit antibodies to CYP2A5 have been described previously
(Gu et al., 1998). Immunoblot analysis was performed with an enhanced chemiluminescence kit from Amersham Biosciences (Piscataway, NJ). Rat and rabbit NADPH-cytochrome P450 reductase was obtained as described elsewhere (Ding and Coon, 1994; Zhang et al., 1998). Chi-square (uncorrected) or Fisher's exact test was used to examine the significance of differences in allele frequency between two different ethnic groups and the significance of linkage disequilibrium between SNP pairs, with use of SigmaStat software (SPSS Science, Chicago, IL). Student's t test was used to examine the
significance of differences in metabolic activity between wild-type and
variant CYP2A13.
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Results |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Identification of SNPs in the CYP2A13 Gene and Determination of Allele Frequency in Major Population Groups. Variant alleles were first detected in the CYP2A13 coding region and exon-intron boundaries by PCR-SSCP or direct DNA sequencing of PCR products. Primers for exons 1, 2, 3, 5, 6, 7, and 8 (Table 1) did not amplify either CYP2A6 or CYP2A7, although those for exons 1, 2, and 7 generated an additional unidentified band (not shown), which was removed during gel purification prior to SSCP analysis. The primers for exons 4 and 9 produced a single PCR band, which, nevertheless, contained both CYP2A6 and CYP2A13 sequences as revealed by SSCP analysis and DNA sequencing.
The SSCP patterns of six of the seven allelic variants detected are shown in Fig. 1. For 1662G>C, 3375C>T, and 3441C>A, the variant and wild-type alleles were well separated. For 523C>T and 6404C>G, the two bands representing two different alleles were distinguishable, although the resolution is not apparent in Fig. 1. The 6424C>T + 6432C>T allele was initially identified by SSCP; the two bands can only be distinguished, with difficulty, by comparison of homozygous individuals. Sequence analysis revealed that the band shift was a result of the double mutation, since individuals with only the 6424C>T mutation did not show a band shift (not shown) and those with only the 6432C>T mutation were not detected.
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Functional Characterization of the Arg257Cys Variant.
A cDNA
for the Arg257Cys variant was generated by site-directed mutagenesis of
the wild-type CYP2A13 plasmid (Su et al., 2000) and cloned
into a baculoviral transfer vector for expression in insect Sf9 cells.
The sequence of the Arg257Cys cDNA insert in the baculoviral expression
vector and the sequences at the cloning sites were confirmed. The yield
of P450 in lysate from recombinant virus-infected Sf9 cells ranged from
10 to 30 nmol/l in different batches of cells cultured with addition of
hemin. The level of the cytochrome in the microsomal fraction ranged
from 0.20 to 0.39 nmol/mg of protein.
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Discussion |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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This is the first report on genetic polymorphisms in the CYP2A13 gene. Of the seven variant alleles found, only four qualify as SNPs based on the allele frequency, including the Arg257Cys variant and the three variants in intron 7. It is interesting that six of the seven changes occur at a cytosine. In contrast, the single-nucleotide changes in the CYP2A6 gene occur at either a guanine or a thymidine (home page of the Human Cytochrome P450 Allele Nomenclature Committee, http://www.imm.ki.se/cypalleles).
The 3375C>T allele was found most frequently in black (14.4%) and
least frequently in white newborns (1.9%). The decreased catalytic
efficiency of the corresponding Arg257Cys CYP2A13 protein in NNK
bioactivation suggests that the Cys-257 genotype may contribute to a
lower risk of respiratory tract xenobiotic toxicity. However, the
activity differences between the Arg-257 and Cys-257 proteins are
relatively small, which will make it difficult to find potential associations between the Cys-257 genotype alone and the incidence of
tobacco-related tumorigenesis in the respiratory tract. On the other
hand, the role of this SNP may be more apparent when analyzed
in conjunction with the genotypes of other biotransformation enzymes
implicated in the metabolism of tobacco-related chemical carcinogens,
particularly CYP2A6. CYP2A6 is believed to play the principal role in
determining systemic clearance of nicotine and, thus, the number of
cigarettes smoked in addicted individuals (Tyndale and Sellers, 2001).
An association of the CYP2A6 deletion genotype with reduced
lung cancer incidence was found in a Japanese population (Miyamoto et
al., 1999), although apparently conflicting results have been reported
by others (Loriot et al., 2001; Tan et al., 2001). It would be
interesting to see whether a stronger association can be found in
subgroups when the data are stratified according to the
CYP2A13 genotypes. A loss of hepatic CYP2A6 in conjunction
with a Cys-257 genotype may further reduce the incidence of
tobacco-related tumors in the respiratory tract. In this regard, due to
the limited quantities of DNA available from the blood spot samples,
the subjects analyzed in the present study were not screened for
CYP2A6 SNPs. However, other efforts to evaluate potential
linkage between CYP2A6 and CYP2A13 SNPs are under way.
Mechanistically, the decrease in activity by the Arg257Cys mutation is
intriguing. The Arg at the 257 position, which is conserved in the
CYP2As, seems to be located near the carboxyl end of the G-helix
according to alignments based on sequence conservation (Gotoh, 1992;
Hasemann et al., 1995). The changed residue corresponds to Lys253 of
CYP2C5, according to an alignment and homology modeling (not shown)
based on the coordinates available from the PDB for CYP2C5 (1DT6). The
residue is expected to be located on the surface of the protein, away
from any of the proposed substrate access channels (Williams et al.,
2000). However, conformational changes that occur with substrate
binding may require the various helices involved in substrate binding
to rotate and move, as found in the structure of P450 BM3 with a bound
substrate analog (Li and Poulos, 1997). Thus, it is possible that a
mutation in the loop region impedes such changes and, consequently,
alters substrate binding or product release in a substrate-independent
fashion. In addition, a recent study (Lehnerer et al., 2000) indicated that an Arg253Ala mutation near the end of the G helix interfered with
the interaction of rabbit CYP2B4 with the P450 reductase, leading to an
approximately 50% decrease in activity. The similar extent of impact
of the Arg257Cys change on the turnover by CYP2A13 of all substrates
tested is consistent with the possible involvement of such general,
substrate-independent mechanisms.
The functional consequence of the Gly144Arg substitution was not
examined in this study. Sequence alignment and modeling suggest that it
corresponds to Ser140 in CYP2C5, which is located just before helix D
and also on the surface of the protein (Williams et al., 2000). The
other exon mutation, 523C>T in exon 2, is silent. However, it remains
to be determined whether this and the Gly144Arg and Arg257Cys mutations
are within an exonic splicing enhancer or exonic splicing silencer
sequence (Blencowe, 2000). Mutations in these regulatory sequences have
been implicated in human genetic diseases (e.g., Liu et al., 2001).
The 3441C>A variant sequence detected in intron 5 is part of the 5'
splice site (AGGTACAT); "A" is
actually the consensus sequence at this position, although "C" is
also found in some genes (Senapathy et al., 1990). Notably, genetic
polymorphisms that affect proper splicing of human P450 transcripts
have been reported previously, such as the mutations found recently in
the CYP3A5 gene that lead to alternative splicing and
protein truncation (Chou et al., 2001; Kuehl et al., 2001). For
CYP2A13, it will be interesting to determine whether the
3441C>A variant allele is expressed at higher levels than the 3441C
allele, possibly due to a more efficient splicing of the pre-mRNA.
The three SNPs in intron 7 are less likely to be involved in splicing.
The changed sequences do not generate consensus splicing donor or
acceptor sites. Analysis of potential binding sites for regulatory
proteins in this region through a search of the TRANSFAC data base
(Heinemeyer et al., 1998), using the TFSEARCH program (Y. Akiyama:
"TFSEARCH: Searching Transcription Factor Binding Sites",
http://www.rwcp.or.jp/papia/), indicated that the 6424C>T change
would destroy a putative stress response element (score 91.5, on the
minus strand) (Schuller et al., 1994) and a putative binding site for
alcohol dehydrogenase gene regulator 1 (score 90.8, on the minus
strand), whereas the 6404C>G and 6432C>T changes would not impact any
known DNA sequences for transcription factor binding (Cheng et al.,
1994).
SSCP analysis may not detect all SNPs in a given gene. Thus, it is
possible that additional variant alleles occur in the
CYP2A13 exons, which remain to be identified using other
techniques, such as direct DNA sequencing. Although not detected in the
present study (data not shown), a potential variant allele was
implicated by a recently reported CYP2A13 exon 1 sequence
(Nunoya et al., 1999a), which had two amino acid differences, Arg30Lys
and Leu33Val, from the known CYP2A13 sequence. Notably,
potential variants in the flanking regions were not examined in the
present study; such variants are also likely according to the findings
on other P450 genes, such as CYP2A6 (Pitarque et al., 2001).
However, large deletions such as those found in CYP2A6,
which appear to arise from homologous unequal crossover of the
neighboring CYP2A6 and CYP2A7 genes (Nunoya et
al., 1999b), may be less likely, since the CYP2A13 gene is
located relatively far away from the other CYP2A genes in
the CYP2 gene cluster on chromosome 19 (Fernandez-Salguero et al., 1995; Hoffman et al., 2001). It should also be noted that, although the present study suggests significant differences in allelic
variant frequencies among broadly defined ethnic groups, these results
are preliminary because the number of subjects examined in each group
is relatively small. In any case, additional studies along these lines
are warranted and may provide useful models for determining the in vivo
function of CYP2A13 in xenobiotic bioactivation and toxicity
in the respiratory tract.
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Acknowledgments |
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We thank Drs. Laurence Kaminsky and Adriana Verschoor for reading the manuscript and Drs. Eric Johnson and James Halpert for helpful discussions. We also gratefully acknowledge the use of the Molecular Genetics Core facility and the Tissue Culture Core facility of the Wadsworth Center.
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Footnotes |
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Accepted for publication March 25, 2002.
Received for publication February 21, 2002.
This work was supported in part by Research Grant ES07462 from the National Institute of Environmental Health Sciences, National Institutes of Health (Bethesda, MD).
Address correspondence to: Dr. Xinxin Ding, Wadsworth Center, New York State Department of Health, Empire State Plaza, Box 509, Albany, NY 12201-0509. E-mail: xding{at}wadsworth.org
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Abbreviations |
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NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; P450, cytochrome P450; SNP, single-nucleotide polymorphism; PCR, polymerase chain reaction; SSCP, single-strand conformational polymorphism; NP-10, Nonidet P-10; RFLP, restriction fragment length polymorphism; bp, base pair(s); HMPA, hexamethylphosphoramide; PEG, polyethylene glycol; HTP, hydroxyapatite.
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References |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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 B. D. Smith, J. L. Sanders, P. R. Porubsky, G. H. Lushington, C. D. Stout, and E. E. Scott Structure of the Human Lung Cytochrome P450 2A13 J. Biol. Chem., June 8, 2007; 282(23): 17306 - 17313. [Abstract] [Full Text] [PDF]
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 S.-L. Wang, X.-Y. He, J. Shen, J.-S. Wang, and J.-Y. Hong The Missense Genetic Polymorphisms of Human CYP2A13: Functional Significance in Carcinogen Activation and Identification of A Null Allelic Variant Toxicol. Sci., November 1, 2006; 94(1): 38 - 45. [Abstract] [Full Text] [PDF]
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L. B. von Weymarn, Q.-Y. Zhang, X. Ding, and P. F. Hollenberg Effects of 8-methoxypsoralen on cytochrome P450 2A13 Carcinogenesis, March 1, 2005; 26(3): 621 - 629. [Abstract] [Full Text] [PDF]
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X.-Y. He, J. Shen, X. Ding, A. Y. H. Lu, and J.-Y. Hong IDENTIFICATION OF CRITICAL AMINO ACID RESIDUES OF HUMAN CYP2A13 FOR THE METABOLIC ACTIVATION OF 4-(METHYLNITROSAMINO)-1-(3-PYRIDYL)-1-BUTANONE, A TOBACCO-SPECIFIC CARCINOGEN Drug Metab. Dispos., December 1, 2004; 32(12): 1516 - 1521. [Abstract] [Full Text] [PDF]
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X. Zhang, M. Caggana, T. L. Cutler, and X. Ding Development of a Real-Time Polymerase Chain Reaction-Based Method for the Measurement of Relative Allelic Expression and Identification of CYP2A13 Alleles with Decreased Expression in Human Lung J. Pharmacol. Exp. Ther., October 1, 2004; 311(1): 373 - 381. [Abstract] [Full Text] [PDF]
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 H. Wang, W. Tan, B. Hao, X. Miao, G. Zhou, F. He, and D. Lin Substantial Reduction in Risk of Lung Adenocarcinoma Associated with Genetic Polymorphism in CYP2A13, the Most Active Cytochrome P450 for the Metabolic Activation of Tobacco-Specific Carcinogen NNK Cancer Res., November 15, 2003; 63(22): 8057 - 8061. [Abstract] [Full Text] [PDF]
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J. R. Jalas, X. Ding, and S. E. Murphy COMPARATIVE METABOLISM OF THE TOBACCO-SPECIFIC NITROSAMINES 4-(METHYLNITROSAMINO)-1-(3-PYRIDYL)-1-BUTANONE AND 4-(METHYLNITROSAMINO)-1-(3-PYRIDYL)-1-BUTANOL BY RAT CYTOCHROME P450 2A3 AND HUMAN CYTOCHROME P450 2A13 Drug Metab. Dispos., October 1, 2003; 31(10): 1199 - 1202. [Abstract] [Full Text] [PDF]
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X. Zhang, Y. Chen, Y. Liu, X. Ren, Q.-Y. Zhang, M. Caggana, and X. Ding SINGLE NUCLEOTIDE POLYMORPHISMS OF THE HUMAN CYP2A13 GENE: EVIDENCE FOR A NULL ALLELE Drug Metab. Dispos., September 1, 2003; 31(9): 1081 - 1085. [Abstract] [Full Text] [PDF]
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G. B. J. Smith, J. R. Bend, L. L. Bedard, K. R. Reid, D. Petsikas, and T. E. Massey BIOTRANSFORMATION OF 4-(METHYLNITROSAMINO)-1-(3-PYRIDYL)-1-BUTANONE (NNK) IN PERIPHERAL HUMAN LUNG MICROSOMES Drug Metab. Dispos., September 1, 2003; 31(9): 1134 - 1141. [Abstract] [Full Text] [PDF]
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) and
those homozygous for the C-C allele (C/C