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Carcinogenesis |
Wadsworth Center, New York State Department of Health, Albany, New York 12201 [T. S., Q-Y. Z., X. D.]; School of Public Health, State University of New York at Albany, Albany, New York [T. S., X. D.]; and Environmental and Occupational Health Sciences Institute, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08854 [Z. B., T. J. S., J-Y. H.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1–10% of the total microsomal CYP in human
liver (2
, 3) and has also been detected in human nasal
mucosa (4, 5, 6, 7)
and lung (8, 9, 10)
. CYP2A6 is the
major coumarin 7-hydroxylase (compare Ref. 11
) and
nicotine C-oxidase in human liver microsomes (12
, 13)
. The
other known substrates for CYP2A6 include many toxicants and
carcinogens, such as aflatoxin B1, NNK,
N-nitrosodiethylamine, N-nitrosonornicotine,
DCBN, and hexamethylphosphoramide (6
, 11 , 14
, 15)
.
Deletion of the CYP2A6 gene has been found to correlate with
reduced lung cancer risk (16)
. CYP2A7 was first cloned from a hepatic cDNA library (17) . However, heterologously expressed CYP2A7 showed no catalytic activity (17 , 18) . CYP2A13 cDNA has not been isolated previously; the reported protein sequence was deduced from the predicted coding region of a CYP2A13 genomic clone (1) . On the basis of its sequence features that resemble the nonfunctional CYP2A7 and CYP2A6v1 (a genetic variant of CYP2A6) proteins, the CYP2A13 protein was predicted to be nonfunctional in coumarin 7-hydroxylation (1) . Because the deduced amino acid sequence of CYP2A13 shares a 95.4% identity with that of CYP2A6 (1) , antibodies and chemical probes for CYP2A6 may interact with CYP2A13. Therefore, CYP2A13 may confound results obtained in studies on CYP2A6.
The tobacco-specific nitrosamine, NNK, which is formed from the
nitrosation of nicotine during tobacco processing and cigarette
smoking, has been suggested to play an important role in human
tobacco-related cancers (19
, 20)
. NNK induces lung tumors
in all laboratory animals tested as well as nasal cavity, pancreatic,
and liver tumors in rats (19
, 20)
. In order for NNK to
exert its carcinogenicity, it must be metabolically activated. The
metabolic activation of NNK involves 
-hydroxylation of the methyl or
methylene carbon, leading to the formation of electrophiles, which can
pyridyloxobutylate and methylate DNA, respectively (20)
.
Hydroxylation at the methyl position leads to the formation of
formaldehyde and 4-(3-pyridyl)-4-oxobutyldiazohydroxide; the latter
decomposes to form 4-hydroxy-1-(3-pyridyl)-1-butanone (keto alcohol).
Hydroxylation at the methylene carbon of NNK leads to the formation of
4-oxo-1-(3-pyridyl)-1-butanone (keto aldehyde) and
methyldiazohydroxide. The detectable products for the two activation
pathways are keto alcohol and keto aldehyde.
It is well-documented that CYP enzymes catalyze the activation of NNK (21, 22, 23, 24, 25) . Of the human CYPs examined, CYP1A1, CYP2A6, CYP2D6, CYP2E1, and CYP3A4 catalyzed the formation of keto aldehyde, and CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2D6, CYP2E1, CYP2F1, and CYP3A4 had activity in the formation of keto alcohol (21, 22, 23, 24, 25) . Although some of the CYPs display activity in the formation of both keto aldehyde and keto alcohol, the CYP enzymes generally show predominance for one activation pathway over the other. Thus, CYP1A1, CYP2A6, and CYP3A4 have higher activity for keto aldehyde formation, whereas CYP2D6 and CYP2E1 are more selective for keto alcohol formation (21, 22, 23, 24, 25) . When one considers the kinetic properties of the various CYPs, CYP2A6 and CYP1A2 have the lowest Km (120 and 300 µM, respectively) and relatively high Vmax values for the formation of keto aldehyde and keto alcohol, respectively (21 , 23 , 25) . Because human individuals are exposed to relatively low levels of NNK, the metabolic activation of this carcinogen appears to be more likely catalyzed by CYP2A6 and CYP1A2. However, the contribution of these and other CYP enzymes to NNK activation in human target tissues will be dependent both on their metabolic efficiency and their abundance in a given tissue.
In the present study, a CYP2A13 cDNA was cloned from human nasal mucosa, and its sequence was compared with the published CYP2A13 genomic sequence (1) . CYP2A13 protein was obtained by heterologous expression in insect cells and characterized. The tissue distribution of CYP2A13 was also determined by isoform-specific RNA-PCR. We report here that CYP2A13 is highly active toward NNK and several other carcinogens and toxicants and that it is predominantly expressed in the human respiratory tract.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cloning of the Coding Region of CYP2A13 cDNA.
PCR primers (forward primer: 5'-tcccatggccaccatgctg-3';
reverse primer: 5'-tcgctcagcggggcaggaag-3') were designed
according to the CYP2A13 gene sequence (1)
; the
underlined nucleotides represent translation initiation and termination
codons, respectively. First-strand cDNA was synthesized by RT from 2
µg of human nasal mucosal total RNA with use of Moloney murine
leukemia virus reverse transcriptase (Promega; Madison, WI) and an
oligo d(T)16 primer (Perkin-Elmer; Foster City,
CA). The cDNA was amplified by high fidelity PCR using PfuTurbo DNA
polymerase (Stratagene, La Jolla, CA) and cloned into pCR-Script vector
(Stratagene) for sequence analysis. Nucleotide sequence was determined
with vector primers and by primer walking with an automated DNA
sequencer from Applied Biosystems (Model 373A) at the Molecular
Genetics Core of the Wadsworth Center. Each position was analyzed at
least three times in each orientation.
Heterologous Expression of CYP2A13 cDNA.
The 5'-primer used for amplifying the CYP2A13 cDNA sequence contained
an in-frame upstream ATG codon, which is undesirable for
heterologous expression because it may introduce three extra amino acid
residues at the amino terminus. This upstream ATG codon was
later found to be ACT in all (>10) individuals genotyped
and may represent additional errors in the original published genomic
sequence (data not shown). One of the three cDNA clones sequenced had
lost the first five bases in the 5'-primer during ligation into the
pCR-Script vector. The resulting insert began with the TG in
the upstream ATG codon, and the 3'-primer was intact.
Therefore, this clone was used for heterologous expression.
The CYP2A13 cDNA insert was excised from the pCR-Script vector and inserted into the multiple cloning site of the baculovirus expression vector pVL1392 (PharMingen, SanDiego, CA). The integrity of the cloning sites was confirmed by sequencing. Recombinant viruses were made by cotransfecting the transfer plasmid and linearized BaculoGold viral DNA (PharMingen) into insect Sf9 cells. Detailed procedures for preparation and titering of virus stocks and detection of CYP expression have been described recently for the expression of CYP2A3 and CYP2A6 (14) . The cells were harvested at 72 h postinfection, washed with PBS, 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 (14) and stored at -85°C until use.
Determination of Catalytic Activities.
The contents of reaction mixtures are described in the table legends.
The enzyme activities were found in preliminary studies to be linear
with CYP concentrations under the conditions used. The rates of product
formation were corrected for zero-time blanks that were quenched before
the addition of NADPH. Formaldehyde formed from
hexamethylphosphoramide, 2'-methoxyacetophenone,
N,N-dimethylaniline, and
N-nitrosomethylphenylamine was measured according to the
method of Nash (26)
, and 7-hydroxylation of coumarin was
assayed according to the method of Greenlee and Poland
(27)
, with use of a Model LS50B Luminescence Spectrometer
(Perkin-Elmer). Formation of the DCBN-protein adduct was assayed as
described recently (28)
with the use of
2,6-[ring-14C]DCBN (16.7 Ci/mol, Sigma; St.
Louis, MO) as a substrate. Formation of acetaldehyde from
N-nitrosodiethylamine was assayed using HPLC after
derivatization with 2,4-dinitrophenylhydrazine (29)
. MTBE
metabolism was assayed by measuring tert-butyl alcohol
formation by gas chromatography (30
, 31)
.
For NNK metabolism, [5-3 H]NNK (2.4 Ci/mmol; purity >97%) and unlabeled NNK were purchased from Chemsyn Science Laboratories (Lenexa, KS). The radiolabeled NNK was further purified by reverse-phase HPLC before use. Authentic NNK metabolite standards were kindly provided by Dr. Stephen Hecht (University of Minnesota Cancer Center, Minneapolis, MN). Unless otherwise specified, incubation mixtures consisted of 100 mM sodium phosphate (pH 7.4), 1 mM EDTA, 3 mM MgCl2, an NADPH-generating system (5 mM glucose 6-phosphate, 1 mM NADP+, and 1.5 units of glucose 6-phosphate dehydrogenase), 10 µM NNK (containing 1 µCi [5-3 H]NNK), 5 mM sodium bisulfite, and 10 pmol of CYP2A13 or 20 pmol of CYP2A6 in a total volume of 0.4 ml. When b5 was added, the molar ratio of CYP:b5 was 1:4. Reactions were carried out at 37°C for 15 min and terminated with 50 µl each of 25% zinc sulfate and saturated barium hydroxide. The samples were centrifuged and filtered, and 50–200-µl aliquots were coinjected with 5 µl of NNK metabolite standards onto a reverse-phase HPLC system equipped with a Radioflow Detector (Radiomatic, Tampa, FL; Ref. 32 ). The HPLC conditions used were the same as previously described (33) . The identity of the keto aldehyde and keto alcohol peaks was confirmed with two different HPLC conditions, in the presence and absence of sodium bisulfite. For kinetic studies, NNK concentrations of 2–160 µM were used in the incubations.
RNA-PCR Analysis of CYP2A13 Tissue Distribution.
RNA-PCR was carried out with a set of CYP2A13-specific primers (forward
primer: 2A13E6F1, 5'-caccctgcgctacggtttcc-3'; reverse primer: 2A13E9R1,
5'-gtcgatatccttaggcgactgagg-3'). The expected product (923–1416 in
the coding region) is 494 bp in length. RT reaction was performed as
described above. PCR mixtures contained 1x reaction buffer
(Perkin-Elmer), 2 mM MgCl2, 0.4
mM dNTPs, 5 µM of each primer, 2.5 µl of a
RT mixture, and 1.25 units of Taq DNA polymerase in a total volume of
25 µl. PCR was performed using a two-step protocol (30 s at 94°C;
45 s at 68°C) for 35 cycles. PCR products were analyzed on
agarose gels and visualized by staining with ethidium bromide.
Quantitative Analysis of CYP2A13 and CYP2A6 mRNA Levels by
Competitive RNA-PCR.
CYP2A13 and CYP2A6 PCR-mimics were prepared using a kit from Clontech.
Competitive RNA-PCR was performed according to a protocol from Ambion
(Austin, TX), using the same two-step protocol as described above for
35 cycles with a constant amount of RT reaction mixtures (2.5 µl) and
serial dilutions of a PCR-mimic. The equality of the amounts of total
RNA from different tissues used for RT was confirmed by determining the
level of cyclophilin mRNA in each RNA sample by competitive RNA-PCR
with a RT-Check kit from Ambion. Isoform-specific PCR primers used for
amplification of CYP2A13 and the CYP2A13-mimic were the same as
described above. The primers for amplification of CYP2A6 and the
CYP2A6-mimic (forward primer: 5'-gggccaagatgccctacatg-3'; reverse
primer: 5'-cgtcaatgtccttaggtgactgg-3') were designed according to the
CYP2A6 cDNA sequence (17)
. Ethidium bromide-stained PCR
products were quantified by a Molecular Dynamics Model 595 Fluorimager
at the Molecular Immunology Core of the Wadsworth Center. The levels of
CYP2A13 or CYP2A6 mRNAs in different human tissues were calculated by
extrapolating from the amounts of the PCR-mimics used in PCR reactions
that generated products with equal intensity of the PCR-mimic band
(CYP2A13-mimic: 622 bp; CYP2A6-mimic: 443 bp) and the CYP2A band
(CYP2A13: 494 bp; CYP2A6: 381 bp).
Other Methods and Materials.
Total RNA was prepared from frozen tissues according to the method of
Chomczynski (34)
, with use of TRIzol Reagent (Life
Technologies, Inc., Grand Island, NY). Preparation of
heterologously expressed CYP2A6 in Sf9 cells was described
previously (14)
. Protein concentrations were determined by
the bicinchoninic acid method (Pierce, Rockford, IL) using BSA as the
standard. Microsomal CYP was determined according to the procedure of
Omura and Sato (35)
. CO-difference spectra of expressed
CYP were recorded at room temperature using a Varian Model Cary 3E
spectrometer. Polyclonal rabbit antibodies to CYP2A5 were prepared in a
recent study (36)
. Immunoblot analysis was performed with
an enhanced chemiluminescence kit from Amersham (Piscataway, NJ).
Rabbit and rat NADPH-CYP reductase and rat b5
were obtained as described previously (30
, 31
, 36)
.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The nucleotide differences between the gene and cDNA sequences are at
(gene
cDNA, numbered from the translation start codon): 141 (CG,
exon 1, silent change), 624 (GC, exon 4), 638 (GC, exon 4), 1193
(AT, exon 8), 1226 (AG, exon 8), 1232 (GT, exon 8), 1235
(GA, exon 8), and 1255 (GA, exon 8). However, in experiments not
presented, DNA sequencing or RFLP analysis of the PCR products
amplified from the exon 8 of the CYP2A13 gene in 23 Chinese
and 29 Caucasians detected only the nucleotide sequences corresponding
to the cDNA, but not the genomic clones (1)
. Subsequently,
an original CYP2A13 genomic clone (no. 27292) was
resequenced, and the results confirmed that the eight nucleotide
differences represent sequencing errors in the published gene sequence,
but not allelic variations. The silent nucleotide difference in exon 1
was also confirmed in a recent report (37)
in which the
CYP2A13 exon 1 sequence was obtained from a different genomic clone.
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. The peak at about 415 nm did not arise from P420 and was routinely
detected in microsomal fractions from control Sf9 cells or cells
infected with the wild-type virus, which did not show any CYP spectrum
(not shown). The specific content of CYP in CYP2A13-expressing Sf9
microsomes ranged from 0.26 to 0.56 nmol of CYP/mg protein. Expression
of the CYP2A13 protein was confirmed by immunoblot analysis with an
antibody to mouse CYP2A5 (36)
. As shown in the inset of
Fig. 2
, a single band of the expected size was detected in the
microsomal fraction of CYP2A13-expressing Sf9 cells, but not in control
cells. The recombinant CYP2A13 migrated to the same position as did a
CYP2A6 standard (not shown), suggesting that it was not degraded or
posttranslationally modified.
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, although CYP2A13 was only about one-tenth as active as CYP2A6 in
catalyzing coumarin 7-hydroxylation, it was as active as CYP2A6 toward
DCBN and N-nitrosodiethylamine and 1.5- to 3.5-times more
active than CYP2A6 toward N,N-dimethylaniline,
N-nitrosomethylphenylamine, hexamethylphosphoramide, and
2'-methoxyacetophenone at 1 mM substrate
concentrations.
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, CYP2A13 was much more active than CYP2A6 in the formation of both keto
aldehyde and keto alcohol at an NNK concentration of 10
µM. The presence of sodium bisulfite in the incubation
mixture to trap keto aldehyde (38)
did not cause any
inhibition of NNK metabolism catalyzed by either CYP2A6 or CYP2A13
(data not shown). The addition of b5
slightly stimulated the activity of both enzymes, but CYP2A13 was still
much more active than CYP2A6. Formation of the keto alcohol by CYP2A6
was not detectable at this substrate concentration. The same CYP2A13
and CYP2A6 preparations were also used to compare their metabolic
activities toward MTBE, a gasoline ether that is a substrate of CYP2A6
(39)
. The activities of both CYP2A13 and CYP2A6 in the
demethylation of MTBE were similar (Table 2)
.
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, the apparent Km value of CYP2A13 was
11.3 µM for the formation of keto aldehyde and
13.1 µM for the formation of keto alcohol.
These Km values are much lower than
those of CYP2A6 or any other human CYP enzymes examined previously
(21
, 25) . The catalytic efficiency, expressed as the ratio
of
Vmax:Km,
of CYP2A13 was 29 and 44 times (for keto alcohol and keto aldehyde
formation, respectively) higher than those found for CYP2A6 under
similar conditions (25)
. These results indicate that
CYP2A13 is a highly efficient enzyme for the metabolic activation of
NNK.
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Consequently, tissue distribution of CYP2A13 mRNA was determined by
RNA-PCR with isoform-specific primers. As shown in Fig. 3
, a band of expected size for CYP2A13 (494 bp) was detected in liver,
lung, nasal mucosa, and trachea, but not in heart or kidney. The lack
of product in several tissues and in additional negative controls (not
shown) in which RNA was omitted provided evidence that the PCR reaction
was specific, i.e., no cross-tissue contamination or
nonspecific amplification occurred in these experiments. The CYP2A13
specificity of the primers was confirmed by direct sequencing of the
PCR products, which did not reveal any sequences corresponding to
CYP2A6 or CYP2A7 (All of the products sequenced were identical to 2A13;
data not shown). In other experiments not shown, the same PCR product
was also detected in brain, mammary gland, prostate, testis, and
uterus, although at very low band intensity, but not in bone marrow,
colon, small intestine, spleen, stomach, thymus, or skeletal muscle.
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, the highest level of CYP2A13 was found in the nasal mucosa, which was
100 times higher than the CYP2A13 level in the liver and nearly 5 times
as high as the level of CYP2A6 in the same tissue. On the other hand,
CYP2A6 was found to have the highest level in the liver, almost
2000-fold higher than the level of CYP2A13 in the same tissue and about
16 times higher than CYP2A13 level in the nasal mucosa. The level of
CYP2A13 mRNA was much lower in lung and trachea than in the nasal
mucosa, but was still >10 times higher than in liver and 3–5 times
higher than in testis (data not shown). The other positive tissues,
including brain, uterus, prostate, and mammary gland, had CYP2A13
levels even lower than in the liver. In the lung, the level of CYP2A13
mRNA was about eight times higher than that of CYP2A6, and in the
trachea, CYP2A13 was also more abundant than CYP2A6. Thus, in contrast
to the previously known liver-predominant expression of CYP2A6, CYP2A13
was preferentially expressed in the respiratory tract.
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DISCUSSION |
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There is an inherent uncertainty associated with studying gene expression in autopsied or biopsied human samples in which the transcript levels are potentially influenced by individual differences in age, medical history, tissue preservation, and genetic polymorphism. In addition, the competitive RNA-PCR method is semiquantitative in nature. Nevertheless, the large differences in CYP2A13 mRNA levels observed between different tissues and the relative levels of CYP2A6 and CYP2A13 determined in the same RNA samples provide strong evidence that CYP2A13 is predominantly expressed in the respiratory tract and that, whereas CYP2A6 is a major CYP2A isoform in the liver, CYP2A13 is more abundant than CYP2A6 in the nasal mucosa and the lung. Previous immunohistochemical studies have detected abundant CYP2A-related proteins in human nasal mucosa; the immunoreactivity was localized to the supporting cells in the olfactory epithelium and acinar cells of Bowman’s glands in the olfactory mucosa and in the ciliated epithelial cells and the serous gland acinar cells in the respiratory mucosa (4) . The present finding that CYP2A13 mRNA is much more abundant than CYP2A6 mRNA in the nasal mucosa suggests that these CYP2A-immunoreactivities mainly represent CYP2A13, but not CYP2A6. However, it remains to be determined whether the two isoforms are located in different cellular populations or different regions in the nasal cavity. Thus, efforts to produce isoform-specific antibodies for additional immunocytochemical studies are warranted.
The detection of CYP2A13 in the human nasal mucosa was reported by Koskela et al. (7) during the preparation of this manuscript. These authors, using qualitative isoform-specific RNA-PCR, detected CYP2A13 in both liver and nasal mucosa. Although they did not quantify the mRNA levels of CYP2A13 in these two tissues and did not compare the relative mRNA levels of CYP2A6 and CYP2A13 in the nasal mucosa, their study did indicate that the level of CYP2A13 in the liver was below the limit of detection under conditions that allowed quantitative comparison of the levels of CYP2A6 and CYP2A7. This is consistent with the results from the present study indicating very low levels of CYP2A13 expression in the liver. Of interest, neither CYP2A6 nor CYP2A13 was detected in other human tissues examined by Koskela et al. (7) , including the lung. The lack of detection of CYP2A6 in lung is in contrast to the present finding and previous reports on the detection of CYP2A6 in lung (8 , 9) .
Cigarette smoking is the single most important etiological factor of lung cancer (41 , 42) . NNK, which induces lung and nasal tumors in the rodents (43) , is believed to play a significant role in the induction of lung cancer in smokers (44) . The incidence of nasal tumors is also higher in smokers (45) . Human liver and lung microsomes were both active in the metabolism of NNK; the activities were significantly inhibited by carbon monoxide, which is consistent with CYP involvement (21) . The major CYP isoform(s) responsible for NNK activation in human target tissues have not been identified. An earlier study indicated that CYP2E1 and CYP2A6 are major catalysts for activation of NNK in human liver microsomes (46) . Antibodies and inhibitors to CYP1A and CYP2E1 inhibited metabolic activation in liver, but not in lung, whereas antibodies to CYP2A1, 2C8, 2D1, or 3A4 had little or no effect on the metabolism of NNK in either tissue (21) . NNK can be activated by CYP2A6 to form DNA adducts (47) . In lung microsomes, CYP2A6 or a related enzyme was implicated in the activation, and lipoxygenases and lipid hydroperoxides possibly also were involved (22) . Thus, the present finding that CYP2A13 is much more active than CYP2A6 and other human CYPs in the activation of NNK is of particular interest.
In conclusion, we demonstrated that among all of the human CYP enzymes examined thus far, CYP2A13 is the most active form, with the highest efficiency, for the metabolic activation of NNK. Together with its predominant expression in the nasal mucosa and lung, we hypothesize that CYP2A13 may play an important role in tobacco-related tumorigenesis in human respiratory tract.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by Public Health Service
Grants ES07462 (to X. D.) and ES10048 (to J-Y. H.) from the NIH. 
2 To whom requests for reprints should be
addressed, at (for J-Y. H.) Environmental and Occupational Health
Sciences Institute, University of Medicine and Dentistry of New Jersey,
170 Frelinghaysen Road, Piscataway, NJ 08854-8020. Phone: (732)
445-7047; Fax: (732) 445-0119; E-mail: jyhong{at}rci.rutgers.edu; or (for
3 The abbreviations used are: CYP, cytochrome
P450; b5, cytochrome
b5; DCBN, 2,6-dichlorobenzonitrile; NNK,
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; MTBE, methyl
tert-butyl ether; HPLC, high performance liquid
chromatography; RT, reverse transcription.
4 J. Sheng and X. Ding, unpublished
observations.
Received 2/ 3/00. Accepted 7/20/00.
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T. Fukami, M. Nakajima, H. Sakai, M. Katoh, and T. Yokoi CYP2A13 Metabolizes the Substrates of Human CYP1A2, Phenacetin, and Theophylline Drug Metab. Dispos., March 1, 2007; 35(3): 335 - 339. [Abstract] [Full Text] [PDF]
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 G. Ling, Y. Wei, and X. Ding Transcriptional Regulation of Human CYP2A13 Expression in the Respiratory Tract by CCAAT/Enhancer Binding Protein and Epigenetic Modulation Mol. Pharmacol., March 1, 2007; 71(3): 807 - 816. [Abstract] [Full Text] [PDF]
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 A. M. Jeffrey, M. J. Iatropoulos, and G. M. Williams Nasal Cytotoxic and Carcinogenic Activities of Systemically Distributed Organic Chemicals Toxicol Pathol, December 1, 2006; 34(7): 827 - 852. [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.-R. Zhu, P. E. Thomas, G. Lu, K. R. Reuhl, G.-Y. Yang, L.-D. Wang, S.-L. Wang, C. S. Yang, X.-Y. He, and J.-Y. Hong CYP2A13 in Human Respiratory Tissues and Lung Cancers: An Immunohistochemical Study with A New Peptide-Specific Antibody Drug Metab. Dispos., October 1, 2006; 34(10): 1672 - 1676. [Abstract] [Full Text] [PDF]
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L. B. von Weymarn, J. A. Chun, and P. F. Hollenberg Effects of benzyl and phenethyl isothiocyanate on P450s 2A6 and 2A13: potential for chemoprevention in smokers Carcinogenesis, April 1, 2006; 27(4): 782 - 790. [Abstract] [Full Text] [PDF]
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L. B. von Weymarn, K. M. Brown, and S. E. Murphy Inactivation of CYP2A6 and CYP2A13 during Nicotine Metabolism J. Pharmacol. Exp. Ther., January 1, 2006; 316(1): 295 - 303. [Abstract] [Full Text] [PDF]
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H. Yamanaka, M. Nakajima, T. Fukami, H. Sakai, A. Nakamura, M. Katoh, M. Takamiya, Y. Aoki, and T. Yokoi CYP2A6 AND CYP2B6 ARE INVOLVED IN NORNICOTINE FORMATION FROM NICOTINE IN HUMANS: INTERINDIVIDUAL DIFFERENCES IN THESE CONTRIBUTIONS Drug Metab. Dispos., December 1, 2005; 33(12): 1811 - 1818. [Abstract] [Full Text] [PDF]
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I. Girault, N. Rougier, C. Chesne, R. Lidereau, P. Beaune, I. Bieche, and I. de Waziers SIMULTANEOUS MEASUREMENT OF 23 ISOFORMS FROM THE HUMAN CYTOCHROME P450 FAMILIES 1 TO 3 BY QUANTITATIVE REVERSE TRANSCRIPTASE-POLYMERASE CHAIN REACTION Drug Metab. Dispos., December 1, 2005; 33(12): 1803 - 1810. [Abstract] [Full Text] [PDF]
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X. Zhang, Q.-Y. Zhang, D. Liu, T. Su, Y. Weng, G. Ling, Y. Chen, J. Gu, B. Schilling, and X. Ding EXPRESSION OF CYTOCHROME P450 AND OTHER BIOTRANSFORMATION GENES IN FETAL AND ADULT HUMAN NASAL MUCOSA Drug Metab. Dispos., October 1, 2005; 33(10): 1423 - 1428. [Abstract] [Full Text] [PDF]
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M. G. Lee, A. Phimister, D. Morin, A. Buckpitt, and C. Plopper In Situ Naphthalene Bioactivation and Nasal Airflow Cause Region-Specific Injury Patterns in the Nasal Mucosa of Rats Exposed to Naphthalene by Inhalation J. Pharmacol. Exp. Ther., July 1, 2005; 314(1): 103 - 110. [Abstract] [Full Text] [PDF]
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 S. T. Saarikoski, H. A.-L. Wikman, G. Smith, C. H. J. Wolff, and K. Husgafvel-Pursiainen Localization of Cytochrome P450 CYP2S1 Expression in Human Tissues by In Situ Hybridization and Immunohistochemistry J. Histochem. Cytochem., May 1, 2005; 53(5): 549 - 556. [Abstract] [Full Text] [PDF]
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S.-L. Wang, X.-Y. He, and J.-Y. Hong HUMAN CYTOCHROME P450 2S1: LACK OF ACTIVITY IN THE METABOLIC ACTIVATION OF SEVERAL CIGARETTE SMOKE CARCINOGENS AND IN THE METABOLISM OF NICOTINE Drug Metab. Dispos., March 1, 2005; 33(3): 336 - 340. [Abstract] [Full Text] [PDF]
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 J. Hukkanen, P. Jacob III, and N. L. Benowitz Metabolism and Disposition Kinetics of Nicotine Pharmacol. Rev., March 1, 2005; 57(1): 79 - 115. [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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Z. Bao, X.-Y. He, X. Ding, S. Prabhu, and J.-Y. Hong METABOLISM OF NICOTINE AND COTININE BY HUMAN CYTOCHROME P450 2A13 Drug Metab. Dispos., February 1, 2005; 33(2): 258 - 261. [Abstract] [Full Text] [PDF]
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L. Wu, J. Gu, H. Cui, Q.-Y. Zhang, M. Behr, C. Fang, Y. Weng, K. Kluetzman, P. J. Swiatek, W. Yang, et al. Transgenic Mice with a Hypomorphic NADPH-Cytochrome P450 Reductase Gene: Effects on Development, Reproduction, and Microsomal Cytochrome P450 J. Pharmacol. Exp. Ther., January 1, 2005; 312(1): 35 - 43. [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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 S. Ali, B. F. El-Rayes, L. K. Heilbrun, F. H. Sarkar, J. F. Ensley, O. Kucuk, and P. A. Philip Cytochrome P450 and Glutathione Transferase Expression in Squamous Cell Cancer Clin. Cancer Res., July 1, 2004; 10(13): 4412 - 4416. [Abstract] [Full Text] [PDF]
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 S. S. Hecht, S. G. Carmella, P. M.J. Kenney, S.-H. Low, K. Arakawa, and M. C. Yu Effects of Cruciferous Vegetable Consumption on Urinary Metabolites of the Tobacco-Specific Lung Carcinogen 4-(Methylnitrosamino)-1-(3-Pyridyl)-1-Butanone in Singapore Chinese Cancer Epidemiol. Biomarkers Prev., June 1, 2004; 13(6): 997 - 1004. [Abstract] [Full Text] [PDF]
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R. M. Baldwin, W. T. Jewell, M. V. Fanucchi, C. G. Plopper, and A. R. Buckpitt Comparison of Pulmonary/Nasal CYP2F Expression Levels in Rodents and Rhesus Macaque J. Pharmacol. Exp. Ther., April 1, 2004; 309(1): 127 - 136. [Abstract] [Full Text]
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S. D. Spivack, G. J. Hurteau, M. J. Fasco, and L. S. Kaminsky Phase I and II Carcinogen Metabolism Gene Expression in Human Lung Tissue and Tumors Clin. Cancer Res., December 1, 2003; 9(16): 6002 - 6011. [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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J. Gu, Y. Weng, Q.-Y. Zhang, H. Cui, M. Behr, L. Wu, W. Yang, L. Zhang, and X. Ding Liver-specific Deletion of the NADPH-Cytochrome P450 Reductase Gene: IMPACT ON PLASMA CHOLESTEROL HOMEOSTASIS AND THE FUNCTION AND REGULATION OF MICROSOMAL CYTOCHROME P450 AND HEME OXYGENASE J. Biol. Chem., July 3, 2003; 278(28): 25895 - 25901. [Abstract] [Full Text] [PDF]
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 F. P. Guengerich Cytochromes P450, Drugs, and Diseases Mol. Interv., June 1, 2003; 3(4): 194 - 204. [Abstract] [Full Text] [PDF]
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N. Ariyoshi, M. Miyamoto, Y. Umetsu, H. Kunitoh, H. Dosaka-Akita, Y.-i. Sawamura, J. Yokota, N. Nemoto, K. Sato, and T. Kamataki Genetic Polymorphism of CYP2A6 Gene and Tobacco-induced Lung Cancer Risk in Male Smokers Cancer Epidemiol. Biomarkers Prev., September 1, 2002; 11(9): 890 - 894. [Abstract] [Full Text] [PDF]
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X. Zhang, T. Su, Q.-Y. Zhang, J. Gu, M. Caggana, H. Li, and X. Ding Genetic Polymorphisms of the Human CYP2A13 Gene: Identification of Single-Nucleotide Polymorphisms and Functional Characterization of an Arg257Cys Variant J. Pharmacol. Exp. Ther., August 1, 2002; 302(2): 416 - 423. [Abstract] [Full Text] [PDF]
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T. Su, Q.-Y. Zhang, J. Zhang, P. Swiatek, and X. Ding Expression of the Rat CYP2A3 Gene in Transgenic Mice Drug Metab. Dispos., May 1, 2002; 30(5): 548 - 552. [Abstract] [Full Text] [PDF]
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W. Godoy, R. M. Albano, E. G. Moraes, P. R. A. Pinho, R. A. Nunes, E. H. Saito, C. Higa, I. M. Filho, C. D. P. Kruel, C. C. Schirmer, et al. CYP2A6/2A7 and CYP2E1 expression in human oesophageal mucosa: regional and inter-individual variation in expression and relevance to nitrosamine metabolism Carcinogenesis, April 1, 2002; 23(4): 611 - 616. [Abstract] [Full Text] [PDF]
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 K. E. Anderson, S. S. Hecht, R. L. Bliss, and C. Le RESPONSE: Re: Metabolites of a Tobacco-Specific Lung Carcinogen in Nonsmoking Women Exposed to Environmental Tobacco Smoke J Natl Cancer Inst, October 17, 2001; 93(20): 1576 - 1577. [Full Text] [PDF]
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S. C. Chen, L. Zhou, X. Ding, and S. S. Mirvish Depentylation of the Rat Esophageal Carcinogen, Methyl-n-pentylnitrosamine, by Microsomes from Various Human and Rat Tissues and by Cytochrome P450 2A3 Drug Metab. Dispos., September 1, 2001; 29(9): 1221 - 1228. [Abstract] [Full Text] [PDF]
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R. N. Hines, Z. Luo, T. Cresteil, X. Ding, R. A. Prough, J. L. Fitzpatrick, S. L. Ripp, K. C. Falkner, N.-L. Ge, A. Levine, et al. Molecular Regulation of Genes Encoding Xenobiotic-Metabolizing Enzymes: Mechanisms Involving Endogenous Factors Drug Metab. Dispos., April 13, 2001; 29(5): 623 - 633. [Abstract] [Full Text]
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M. Oscarson Genetic Polymorphisms in the Cytochrome P450 2A6 (CYP2A6) Gene: Implications for Interindividual Differences in Nicotine Metabolism Drug Metab. Dispos., February 1, 2001; 29(2): 91 - 95. [Abstract] [Full Text]
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J. Sheng, Z. Hua, J. Guo, M. Caggana, and X. Ding Identification of a New Human CYP2A Gene Fragment with Close Linkage to CYP2GP1 Drug Metab. Dispos., January 1, 2001; 29(1): 4 - 7. [Abstract] [Full Text]
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E. M. Leslie, K.-i. Ito, P. Upadhyaya, S. S. Hecht, R. G. Deeley, and S. P. C. Cole Transport of the beta -O-Glucuronide Conjugate of the Tobacco-specific Carcinogen 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) by the Multidrug Resistance Protein 1 (MRP1). REQUIREMENT FOR GLUTATHIONE OR A NON-SULFUR-CONTAINING ANALOG J. Biol. Chem., July 20, 2001; 276(30): 27846 - 27854. [Abstract] [Full Text] [PDF]
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