From the Department of Pharmacology, Baylor College
of Medicine, Houston, Texas 77030-3498, the ¶ Laboratory of
Metabolism, NCI, National Institutes of Health, Bethesda, Maryland
20892, and the
Department of Pathology, Fox Chase Cancer
Center, Philadelphia, Pennsylvania 19111
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ABSTRACT |
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NAD(P)H:quinone oxidoreductase 1 (NQO1) is a flavoenzyme that catalyzes two-electron reductive metabolism and detoxification of quinones and their derivatives leading to protection of cells against redox cycling and oxidative stress. To examine the in vivo role of NQO1, a NQO1-null mouse was produced using targeted gene disruption. Mice lacking NQO1 gene expression showed no detectable phenotype and were indistinguishable from wild-type mice. However, NQO1-null mice exhibited increased toxicity when administered menadione compared with wild-type mice. These results establish a role for NQO1 in protection against quinone toxicity. The NQO1-null mice are a model for NQO1 deficiency in humans and can be used to determine the role of this enzyme in sensitivity to toxicity and carcinogenesis.
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INTRODUCTION |
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NQO1,1 previously known as DT diaphorase, is a flavoprotein that catalyzes two-electron reduction of quinones and their derivatives including azo dyes and nitroaromatic compounds (1-3). NQO1 is a cytosolic protein that is found ubiquitously in eukaryotes (1-4). In mammals, it is present in many organs but is most abundant in liver (1). However, in man, NQO1 activity is significantly higher in many extrahepatic tissues (5, 6). The expression of the NQO1 gene is induced in response to a variety of agents including xenobiotics, oxidants, antioxidants, UV light, and ionizing radiation (2, 7). The obligatory two-electron reduction of quinones catalyzed by NQO1 competes with the one-electron reduction of quinones by enzymes such as NADPH-cytochrome P450 oxidoreductase and protects cells against redox cycling and oxidative stress (8, 9). This protection is the result of conversion of quinones to hydroquinones compared with semiquinones and reactive oxygen species. The role of NQO1 in cellular protection is well documented for menadione and benzo(a)pyrene quinones (9, 10). However, some hydroquinones can autoxidize to generate reactive oxygen species or alkylate DNA directly (11). In these instances, NQO1 catalyzes activation of such compounds to their ultimate toxic forms. This property of NQO1 along with the observation that NQO1 is expressed at higher levels in certain tumor types compared with normal tissues has been used to develop bioreductive chemotherapeutic agents (12).
Recent studies have characterized a C T (nucleotide position 609, NQO1P203S) mutation in the NQO1 gene which results in loss of NQO1
activity (13). This mutation in the NQO1 gene has been found in a human
bladder carcinoma cell line (14), colon cancer (15), and fibroblasts
taken from a cancer-prone family (16). In addition, Rosvold et
al. (17) demonstrated that the same C
T mutation was
overrepresented significantly in lung cancer. More recently, Rothman
et al. (18) reported an increased frequency of C
T
mutations in the NQO1 gene associated with benzene poisoning. The
presence of a mutation, together with the fact that the physiological
function of NQO1 is to detoxify potentially mutagenic compounds, poses
the question of whether or not a deficiency in NQO1 activity
predisposes individuals to certain types of cancer.
In the present report, we used homologous recombination in
embryonic cells to disrupt the NQO1 gene and generated knockout (NQO1/
) mice that lack expression of the NQO1 gene. NQO1
/
mice
showed no detectable phenotype and were indistinguishable from
wild-type mice or heterozygous (NQO1+/
) and normal littermates. However, NQO1
/
mice exhibited increased toxicity to menadione compared with wild-type mice.
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EXPERIMENTAL PROCEDURES |
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Isolation and Characterization of Mouse NQO1 Gene--
A
full-length rat NQO1 cDNA was used to screen a 129SVJ mouse genomic library from Stratagene (La Jolla, CA) by procedures described
previously (19). Several
clones were characterized, and one,
designated
EMBL3-mNQO1g-10, was used for further studies and
analysis. To determine the nucleotide sequences encompassing the
exon-intron junctions of mouse NQO1 gene, oligonucleotides 15-17 bp in
length were selected at random from the corresponding mouse NQO1
cDNA (20) and used for sequencing NQO1 gene fragments subcloned
into pUC18 by procedures described previously (21). The process of
sequencing continued until the coding region from mouse NQO1 gene was
completely covered.
Construction of the Targeting Vector-- The targeting vector was constructed using the pPNT vector having positive selection neomycin (G418) resistance and negative selection thymidine kinase markers (22). To disrupt the NQO1 gene, a targeting plasmid containing a deletion of exon 6 of the NQO1 gene was constructed. The 2.0-kb BamHI fragment containing a portion of intron 5, exon 6, and a small portion of the 3'-flanking region of the NQO1 gene was replaced by the 2.0-kb BamHI fragment from the pPNT vector containing the bacterial phosphoribosyltransferase II gene conferring G418 resistance (Fig. 1). The targeting vector contained 3.4 kb of homologous 5'-sequence and 1.4 kb of homologous 3'-sequence of the neo-cassette. A herpes simplex virus thymidine kinase (HSV-TK) gene inserted at the 3'-end of the construct allowed the use of a positive-negative selection scheme. To construct the targeting vector, a 7.0-kb XbaI-EcoRI fragment was isolated and subcloned at the XbaI-EcoRI site of pUC19 to convert an XbaI-SalI site. In the plasmid pUC19, the polylinker region contains an SalI site 5' to the XbaI site. Therefore, the SalI site was simply added to the XbaI site during subcloning of the 7.0-kb XbaI-EcoRI fragment in pUC19. The resultant plasmid pUC19-mNQO1 gene (SalI-EcoRI) was opened at the EcoRI site, made blunt ended, and SalI adapters were added. These manipulations resulted in conversion of the XbaI and EcoRI ends to SalI ends and loss of the BamHI site in the polylinker region of pUC19. The loss of the BamHI site from pUC19 was achieved during digestion of pUC19 with XbaI-EcoRI to generate a vector plasmid to subclone the 7.0-kb mNQO1 gene. The BamHI restriction site is located between the XbaI and EcoRI sites of the polylinker region in pUC19. The digestion of pUC19 with XbaI and EcoRI removed the portion of the polylinker region between XbaI and EcoRI containing the BamHI site, resulting in loss of the BamHI site. The plasmid pUC19-mNQO1 gene (SalI-SalI) was digested with BamHI to remove 2.0 kb of the mouse NQO1 gene containing a portion of intron 5, exon 6, and a small portion of the 3'-flanking region. The 2.0 kb of the mNQO1 gene in the plasmid pUC19-mNQO1 gene was replaced with 2.0 kb of neo-cassette (derived from pPNT vector by digestion with XhoI, addition of BamHI linkers, and redigestion with BamHI to obtain a 2.0-kb neo-cassette). The 7.0 kb of the SalI-SalI fragment from the NQO1 gene containing 2.0 kb of the neo-cassette instead of exon 6 of the mNQO1 gene was isolated and subcloned at the SalI site of the pMC1tk-pA plasmid to generate the targeting vector pPNT-mouse NQO1 gene (Fig. 1). Four plasmids were produced with various orientations of neo- and HSV-TK cassettes. The HSV-TK cassette was under the control of the HSV-TK promoter/enhancer.
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Electroporation, Selection of ES Cells, and Generation of
Chimeric Mice--
Genome Systems (St. Louis, MO) mouse embryonic stem
(ES) cells were used for homologous recombination and deletion of exon 6 from the NQO1 gene. The ES cells were thawed and diluted with ES cell
medium (HEPES-buffered Dulbecco's modified Eagle's medium, 15% fetal
bovine serum (Hyclone), 55 µM -mercaptoethanol, 0.1 mM nonessential amino acids, penicillin-streptomycin, 1,000 units of leukemia inhibitory factor/ml), pelleted by centrifugation, and resuspended in ES cell medium. The ES cells were plated onto a
60-mm Petri dish previously seeded with mitotically inactive,
-irradiated mouse embryonic fibroblasts. After 2 days, fresh medium
was added to the ES cells, incubated for 4 h, treated with 0.25%
trypsin and EDTA buffered with HEPES, resuspended at 2.5 × 105/60-mm-diameter plates with electroporation buffer
(Hanks' balanced salt solution, 20 mM HEPES buffer, 0.11 mM
-mercaptoethanol, pH 7.2) and used for
electroporation.
Southern and Northern Blot Analysis--
DNA was isolated from
ES cells and mouse tails by the procedure described by Laird et
al., (23). DNAs were digested overnight with NcoI,
electrophoresed on 1.0% agarose gel, blotted, and hybridized with a
280-bp EcoRI-NcoI fragment from the 3'-flanking
region of the mouse NQO1 gene by standard procedures (24). Southern blots were washed, exposed to x-ray films, and subjected to
autoradiography. In a related experiment, the DNAs from wild-type
(NQO1+/+), heterozygous (NQO1+/), and NQO1
/
mice were digested
with BamHI, run on agarose gel, blotted, and hybridized with
1.1 kb of human NQO1 cDNA (complete coding region) and the 2.0-kb
neo-cassette probes. The NQO1 cDNAs are highly conserved among
humans, rats, and mice and are known to hybridize each other in
Southern analysis (25, 20). The blots were washed and
autoradiographed.
NQO1 Activity and Western Blot Analysis--
The various tissues
(liver, lung, kidney, colon, and skeletal muscle) from wild-type,
NQO1+/, and NQO1
/
mice were homogenized in 50 mM
Tris, pH 7.4, containing 0.25 M sucrose and centrifuged at
105,000 × g for 1 h to obtain cytosolic
fractions. Dicoumarol-sensitive NQO1 activity was measured in all
cytosolic fractions by a method reported earlier (28). The various
cytosolic fractions were also analyzed for the presence or absence of
NQO1 protein by Western blot analysis as described previously using
antibodies against purified rat liver NQO1 protein (28). The rat NQO1
antibody is known to cross-react with mouse and human proteins (28,
29). Western blots were developed with ECL (Amersham) reagents by the procedure suggested by the manufacturer.
Menadione Toxicity--
6-8-week-old wild-type, NQO1+/, and
NQO1
/
mice were used. Menadione (vitamin K3) was dissolved in
dimethyl sulfoxide and was administered intraperitoneally at doses of
0, 2.5, 5, 10, and 20 mg/kg of body weight. Animals were given a single
dose every day for 3 consecutive days. Animals were observed daily for
symptoms of toxicity and mortality. 24 h after the last dose of
menadione, blood was drawn from the surviving animals. The activities
of alanine aminotransferase and aspartate aminotransferase were
analyzed in the serum of each animal using the diagnostic kit purchased
from Sigma.
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RESULTS |
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Genomic Structure of Mouse NQO1 Gene
The structure of the mouse NQO1 gene was found to be similar to that reported for the human NQO1 gene. Like the human NQO1 gene, the mouse NQO1 gene contained six exons interrupted by five introns. The splice junctions and nucleotide sequences in the various exons were highly conserved between the human and mouse genes (Table I).
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Production of NQO1/
Mice
The structure of the targeting vector pPNT-mouse NQO1 gene is
shown in Fig. 1. This construct was used successfully to generate NQO1/
mice. The 5'- and 3'-homologous genomic sequences were 3.4 kb
and 1.4 kb long, respectively. In the targeting vector, a 2.0-kb
BamHI fragment containing exon 6 of the NQO1 gene was replaced with 2.0 kb of neo-cassette. This replacement was engineered to delete the carboxyl 101 amino acids of the NQO1 enzyme. This design
would effectively disrupt NQO1 gene function. The decision to delete
exon 6 was based on two important observations. First, deletion of 73 amino acids from the COOH terminus of the NQO1 cDNA resulted in a
shorter protein and complete loss of NQO1 activity in transfected COS1
cells.2 Second, the C
T
mutation resulting in the loss of NQO1 activity was reported to be in
exon 6 of the NQO1 gene (13).
DNA from selected ES cells was analyzed by Southern blotting to screen
for homologous recombinants (Fig.
2A). The digestion of DNA from
ES cells (NQO1+/+) and wild-type mouse with NcoI and hybridization with 280 bp of EcoRI-NcoI fragment
from the mouse NQO1 gene revealed the presence of a 9.4-kb band (Fig.
2A). Of the 140 ES clones analyzed, one NQO1+/
heterologous ES cell clone (mNQO1g-46) was identified. The presence of
a 2.4-kb NcoI fragment in a genomic Southern (Fig.
2A) indicated that exon 6 is replaced with the neo-cassette
as depicted in Fig. 1. The homologous recombination-positive ES cells
were used to generate chimeric mice, and germ line transmission was
detected. Heterozygous mice from the F1 generation were normal and were
interbred to generate homozygous NQO1
/
mice. The heterozygous and
homozygous mice DNAs were analyzed for the presence of mutant allele(s)
of NQO1 gene carrying deletion of exon 6 (Fig. 2A). The
absence of a 9.4-kb NcoI band and the presence of a 2.4-kb NcoI band clearly indicated that homozygous NQO1
/
mice
were born (Fig. 2A mice). In addition, the absence of exon 6 and the presence of the neo-cassette were confirmed by digestion of
genomic DNA with BamHI followed by Southern analysis and
hybridization with NQO1 cDNA and the neo-cassette probes (Fig.
2B). Southern analysis of DNA from the wild-type mice
digested with BamHI and hybridization with NQO1 cDNA
showed two bands of 8.0 and 2.0 kb (Fig. 2B). The 8.0-kb
band contains exons 1-5 of the mouse NQO1 gene, and the 2.0-kb band
contains exon 6 of the mouse NQO1 gene as shown in Fig. 1. The
wild-type mice DNA did not hybridize to the neo-cassette probe (Fig.
2B). In a similar Southern analysis experiment, the
NQO1
/
mice DNA showed only an 8.0-kb BamHI band hybridizing to the NQO1 cDNA (Fig. 2B). The 2.0-kb
BamHI band containing exon 6 of the mouse NQO1 gene was
absent in NQO1
/
mice DNA upon hybridization with NQO1 cDNA
(Fig. 2B). However, the NQO1
/
mice DNA hybridized with
the neo-cassette probe and, as expected, showed a band of 2.0 kb upon
digestion with BamHI. This clearly indicated that the 2.0-kb
neo-cassette has replaced the 2.0-kb BamHI fragment
containing exon 6 from the mouse NQO1 gene in NQO1
/
mice. In the
same experiment, as expected, NQO1+/
DNA hybridized with both exon 6 and neo-cassette probes.
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Analysis of NQO1/
Mice
Viability and Fertility--
The NQO1/
mice were found to be
normal in appearance and showed no discernible difference in their
weight, development, or in their behavior compared with their wild-type
NQO1+/+ littermates. This was true for both male and female mice. At 6 weeks of age, wild-type NQO1+/+, heterozygous NQO1+/
, and homozygous
NQO1
/
mice were killed for gross and histological examination. The
organs and tissues examined histologically included liver, lung,
kidney, colon, stomach, duodenum, spleen, thymus, lymph nodes, heart, brain, and skeletal muscle. No obvious anatomical differences in these
organs were seen. In addition, the knockout animals appeared to have
normal reproductive capacity compared with wild-type mice.
Northern Analysis--
Analysis of RNA from five different tissues
(liver, lung, kidney, colon, and skeletal muscle) by hybridization with
the exon 6 probe indicated that NQO1 mRNA was present in wild-type
and heterozygous NQO1+/ animals and absent in NQO1
/
mice (Fig. 3A). The NQO1 mRNA was
lower in heterozygous mice compared with wild-type mice.
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Analysis of NQO1 Protein--
Analysis of cytosolic proteins from
the various tissues (liver, lung, kidney, colon, and skeletal muscle)
by SDS-polyacrylamide gel electrophoresis and Western blotting
indicated the absence of the 32-kDa NQO1 protein in all of the tissues
of NQO1/
mice (Fig. 3B). In similar experiments, NQO1
protein was detected in all of the tissues of wild-type mice. The NQO1
protein was also detected in all of the tissues of heterozygous mice.
However, the NQO1+/
mice demonstrated the presence of
intermediate amounts of NQO1 protein between NQO1
/
and wild-type
mice. Western analysis of the various tissues with NQO1 antibody also
showed the presence of a <30-kDa cross-reacting band in all tissues of
wild-type, heterozygous NQO1+/
, and NQO1
/
mice (Fig.
3B).
NQO1 Activity--
The levels of dicoumarol-sensitive cytosolic
NQO1 activity in the various tissues of wild-type, heterozygous
NQO1+/, and NQO1
/
mice are shown in Fig.
4. Among the five tissues tested, the
highest levels of cytosolic NQO1 activity were observed in kidney
followed by colon of wild-type mice. Livers from wild-type mice showed
only one-fifth of the NQO1 activity observed in kidney. The level of
NQO1 activity in lungs and skeletal muscles of wild-type mice was
reduced further to 10% of NQO1 activity in kidney. The kidney and
colon showed the complete absence of NQO1 activity in NQO1
/
mice.
However, liver, lung, and skeletal muscle showed some NQO1 activity,
which was no more than 15% of NQO1 activity in the respective tissues
of wild-type mice. Heterozygous NQO1+/
mice tissues showed levels of
NQO1 which were intermediate between NQO1
/
and wild-type
animals.
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Menadione Toxicity
Survival of Animals--
Exposure of wild-type, heterozygous
(NQO1+/), and knockout (NQO1
/
) mice to different concentrations
of menadione revealed a dose-dependent response for animal
survival. A significant difference in the survival of wild-type and
NQO1
/
mice was observed with a doses of 5, 10, and 20 mg of
menadione/kg of body weight (Fig. 5A). The survival rate was
determined to be 100% wild-type, 70% heterozygous NQO1+/
, and 30%
knockout NQO1
/
mice with 10 mg of menadione/kg of body weight.
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Levels of Aspartate Aminotransferase and Alanine Aminotransferase
in Serum of Wild-type, Heterozygous, and Knockout Mice as an Indication
of Liver Damage--
The levels of aspartate aminotransferase and
alanine aminotransferase in the serum of wild-type, heterozygous, and
NQO1/
mice are shown in Fig. 5B. Both of these enzymes
were found elevated in NQO1
/
mice with doses of 2.5 and 5 mg of
menadione/kg of body weight. The corresponding doses of menadione had
no effect on these levels in the serum of wild-type mice. Heterozygous
mice showed intermediate sensitivities to menadione. 10 mg of
menadione/kg of body weight produced elevated levels of aspartate
aminotransferase and alanine aminotransferase in the serum of both
wild-type and NQO1
/
mice. However, the magnitude of elevation was
significantly more in the NQO1
/
mice compared with wild-type
mice.
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DISCUSSION |
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Several lines of evidence indicate that modification of the NQO1
gene by replacing exon 6 with the neo-cassette resulted in a null
mutation. NQO1 mRNA and protein were not detected in NQO1/
mice. The NQO1 activity dropped from very high levels in kidney and
colon of wild-type mice to zero in NQO1
/
mice. The NQO1 activity
also dropped significantly (>85%) in other tissues including liver,
lung, and skeletal muscle. We believe that residual amounts of NQO1
activity observed in liver, lung, and kidney tissues of NQO1-null mice
are caused by NQO1-related protein(s) rather than NQO1. This is clearly
evident from the fact that the NQO1 protein was absent in these tissues
of NQO1-null animals as determined by Western analysis. These
NQO1-related protein(s) must be tissue-specific because NQO1 activity
was not detectable in kidney and colon tissues of the NQO1-null
animals. The small amount of NQO1 activity detected in liver, lung, and
skeletal muscle of these mice is probably not caused by a <30-kDa band
detected just below the 32-kDa NQO1 bands in Western blot analysis
because this band was also present in kidney and colon tissues of
NQO1-null mice when no measurable NQO1 activity was found. The <30-kDa
band may be NQO1-related protein with no NQO1 activity or an artifact
of cross-reaction of the NQO1 antibody with an unrelated protein. It
remains a possibility that the <30-kDa band is cytosolic NQO2 (29).
Human NQO2 is a 27-kDa protein that cross-reacts with antibody against
NQO1 (29). It is also known that NQO2 requires reduced
dihydronicotinamide riboside instead of NAD(P)H as cofactor (30). Its
activity with NAD(P)H as cofactor is also very low as measured in the
present studies. The identification of the <30-kDa band as NQO2
awaits additional experimentation.
The loss of NQO1 in knockout mice did not affect the development and
viability of mice. This was expected because a small percentage of the
adult human population is known to be homozygous for mutant alleles of
NQO1 (14-17). The C T mutation in exon 6 of these individuals
results in a proline
serine change and the loss of NQO1 activity.
Humans with the NQO1-null genotype exhibited no developmental or
physiological abnormalities. However, it is expected that NQO1
/
mice and humans with the NQO1
/
-null genotype would be more
susceptible to free radical damage and development of toxicity from
exposure to quinones, their derivatives, and redox cycling
compounds.
Interestingly, the NQO1 was shown to play an important role in the
maintenance of the reduced form of coenzyme Q in membranes which
provides protection to the membranes against free radical damage (31,
32). Thus, NQO1/
mice lacking the expression of NQO1 would be
expected to have a significantly lower capacity to reduce coenzyme Q,
leading to free radical-induced damage to membranes and DNA and
premature aging of the animals. However, the extent of damage would
also depend on the presence of other NQO1-related protein(s) that
catalyze the reduction of coenzyme Q. This is clearly an area of great
future interest.
NQO1 activity is also a part of defensive network within the cells
which protects against redox cycling, oxidative stress, and other toxic
effects caused by exposure to quinones and their derivatives (2, 3, 33,
34). NQO1 activity was shown to prevent the formation of highly
reactive quinone metabolites (35), detoxify benzo(a)pyrene quinones
(10, 36), and reduce Cr(VI) toxicity (37). Recently, benzene poisoning,
a risk factor for hematological malignancy, has been shown to be
associated with the NQO1 C T mutation (18). It is a well known fact
that induction of NQO1 and other detoxifying enzymes is one mechanism of critical importance in chemoprevention (2, 3, 38, 39). Therefore,
many compounds that block toxic, mutagenic, and neoplastic effects of
carcinogens share in common the ability to elevate levels of
detoxifying enzymes including NQO1, glutathione
S-transferase, and UDP-glucuronosyltransferases (2, 3, 40,
41). Studies with NQO1-null mice lacking the expression of NQO1 gene
clearly support a protective role of NQO1 against menadione toxicity. NQO1-null mice were more sensitive to menadione than wild-type mice.
70% of the null mice died when exposed to 10 mg of menadione/kg of
body weight. On the other hand, no deaths were observed with a similar
dose of menadione administered to wild-type mice expressing the NQO1
gene. In addition, serum levels of the liver enzymes aspartate
aminotransferase and alanine aminotransferase as markers for
hepatotoxicity were found to be significantly different in NQO1-null
animals compared with wild-type animals. Elevation of these liver
enzymes, which are considered to be a measure of liver cell death, were
detected at doses of 2.5 and 5 mg/kg of body weight in NQO1-null
animals but were more or less unchanged at these doses in wild-type
mice. These data indicated that liver damage is involved in mediating
the toxicity of menadione in knockout mice and at higher doses in
wild-type mice. The toxicity of menadione in other organs of the
NQO1-null mice will be expected but remains to be determined.
In conclusion, a NQO1-null mutant mouse was produced which develops
normally and is completely viable and fertile. However, NQO1/
mice
exhibit significantly increased sensitivities to menadione toxicity
compared with wild-type mice. The generation and establishment of
NQO1
/
mice provide very important tools in determining the in
vivo role of NQO1 in protection against redox cycling-activated
compounds and whether these mice are sensitive to cancer when exposed
to environmental carcinogens including benzo(a)pyrene (benzo(a)pyrene
quinones) and benzene (benzoquinone). The data obtained with NQO1-null
mice may be used to design further studies in humans to determine if
individuals carrying null alleles of the NQO1 gene are at risk for
developing cancer related to exposure to chemicals via diet and
occupation. In addition, the NQO1
/
mice will be an invaluable tool
to study the role of NQO1 in activation of antitumor drugs such as
mitomycin C and indoloquinone. This is especially important knowing
that NQO1 gene is overexpressed in several kinds of tumors (13).
Finally, it will be of interest to determine if mice lacking NQO1 have
life spans that differ from wild-type mice since accumulation of
oxidative damage has been considered as a factor in aging.
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ACKNOWLEDGEMENTS |
---|
We are thankful to Drs. Harris Busch, Kurt Randerath, and Hagop Youssoufian, all from Baylor College of Medicine, Houston, for reading the manuscript critically. We also thank Dr. Hagop Youssoufian for the use of equipment and reagents and for help in preliminary Western immunoblot experiments.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant RO1 ES07943.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ The first two authors contributed equally to this work.
** To whom correspondence should be addressed: Dept. of Pharmacology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030-3498. Tel.: 713-798-7691; Fax: 713-798-3145; E-mail ajaiswal{at}bcm.tmc.edu.
1 The abbreviations used are: NQO1, NAD(P)H:quinone oxidoreductase 1; bp, base pair(s); kb, kilobase pair(s); HSV, herpes simplex virus; TK, thymidine kinase; ES, embryonic stem.
2 P. Joseph and A. K. Jaiswal, unpublished observations
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REFERENCES |
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