Chemico-Biological Interactions 129 (2000) 77–97
NAD(P)H:quinone oxidoreductase 1 (NQO1):
chemoprotection, bioactivation, gene regulation
and genetic polymorphisms
David Rossa,*, Jadwiga K. Kepaa, Shannon L. Winskia,
Howard D. Beallb, Adil Anwara, David Siegela
aDepartment of Pharmaceutical Sciences, School of Pharmacy and Cancer Center, Box C-238,
Uni?ersity of Colorado Health Sciences Center, 4200 East 9th A?enue, Den?er, CO 80262, USA
bDepartment of Pharmaceutical Sciences, School of Pharmacy, Uni?ersity of Montana, Missoula,
MT 59812, USA
NAD(P)H:quinone oxidoreductase 1 (NQO1) is an obligate two-electron reductase that is
involved in chemoprotection and can also bioactivate certain antitumor quinones. This
review focuses on detoxification reactions catalyzed by NQO1 and its role in antioxidant
defense via the generation of antioxidant forms of ubiquinone and vitamin E. Bioactivation
reactions catalyzed by NQO1 are also summarized and the development of new antitumor
agents for the therapy of solid tumors with marked NQO1 content is reviewed. NQO1 gene
regulation and the role of the antioxidant response element and the xenobiotic response
element in transcriptional regulation is summarized. An overview of genetic polymorphisms
in NQO1 is presented and biological significance for chemoprotection, cancer susceptibility
and antitumor drug action is discussed. © 2000 Elsevier Science Ireland Ltd. All rights
Keywords: NQO1 (NAD(P)H:quinone oxidoreductase 1); Quinone; Vitamin E; Ubiquinone; Antitumor
quinones; Gene regulation; Antioxidant response element (ARE); Polymorphisms
* Corresponding author. Tel.: +1-303-3156077; fax: +1-303-3150274.
E-mail address: firstname.lastname@example.org (D. Ross).
0009-2797/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.
D. Ross et al. / Chemico-Biological Interactions 129 (2000) 77–9778
The catalytic properties of NAD(P)H:quinone oxidoreductase 1 (NQO1) were
first reported by Ernster and Navazio in 1958 . NQO1 is an obligate two-electron
reductase that is characterized by its capacity for utilizing either NADH or
NADPH as a reducing cofactor and by its inhibition by dicoumarol . There is
considerable data indicating that NQO1 can protect against natural and exogenous
quinones. One of the first examples of its protective nature was the discovery that
menadione reductase (later discovered to be NQO1) levels were induced in response
to low doses of certain carcinogens and this afforded some protection against
subsequent treatments . NQO1 reduces quinones to hydroquinones in a single
two-electron step. In addition to yielding substrates for Phase II conjugation
reactions and promoting excretion, this two-electron process bypasses the poten-
tially toxic semiquinone radical intermediates. Not all hydroquinones are redox-sta-
ble, however, and in some cases metabolism by NQO1 yields a more active product.
Redox-labile hydroquinones can react with molecular oxygen to form semiquinones
and generate reactive oxygen species, or semiquinones can be generated via
comproportionation reactions . In addition to potentially causing oxidative stress
through this mechanism, the reduction of the quinone moiety can produce a
compound that is capable of alkylating nucleophilic sites including DNA (Fig. 1).
This process has been termed ‘bioreductive alkylation’ and has formed the basis for
research into the design of NQO1-directed antitumor agents.
2. NQO1 catalytic cycle and substrate specificity
NQO1 is an obligate two-electron reductase that is characterized by its capacity
for utilizing either NADH or NADPH as reducing cofactors and its potent
inhibition by dicoumarol . It is primarily a cytosolic enzyme (?90%) and exists
as a homodimer with one molecule of FAD per monomer. Study of the X-ray
Fig. 1. Activation and deactivation resulting from NQO1-mediated reduction of quinones.
D. Ross et al. / Chemico-Biological Interactions 129 (2000) 77–9779
crystal structure confirmed that NQO1 functions via a ping pong mechanism where
the reduced pyridine nucleotide binds, reduces the flavin co-factor and the oxidized
pyridine nucleotide is released prior to binding of the substrate . The pyridine
nucleotide and quinone binding sites were found to have significant overlap
suggesting a molecular explanation for this mechanism . Reduction to the
hydroquinone occurs in a single step bypassing semiquinone radical intermediates.
This mechanism has been supported by ESR experiments which failed to detect
semiquinone radicals during the metabolism of benzoquinone and naphthoquinone
substrates  and confirmed by stop-flow and steady-state kinetic methods . The
mechanism of catalysis has been proposed to involve a hydride transfer between the
NADH and FAD cofactors and from FADH2to the quinone substrate .
NQO1 is capable of reducing a very broad range of substrates including
quinones, quinone-imines, glutathionyl-substituted naphthoquinones, dichlorophe-
nolindolphenol, methylene blue, azo and nitro compounds [2,8,9]. Both ortho and
para quinones are substrates for NQO1 . Metabolism is not limited to quinones
and the enzyme functions efficiently as a nitro-reductase utilizing substrates such as
dinitropyrenes, nitrophenylaziridines and nitrobenzamides [11–13]. In addition to
two-electron reduction, NQO1 is also capable of performing four-electron reduc-
tion of azo dyes and nitro compounds [14,15]. Using heterodimers of NQO1, it was
determined that the NQO1 subunits function independently in metabolizing two-
electron substrates and in a dependent fashion with four-electron substrates .
3. Detoxification reactions catalyzed by NQO1
3.1. Detoxification of substrates by two-electron reduction
Numerous studies have proposed a role for NQO1 in the detoxification of
redox-cycling quinones such as menadione. Two-electron reduction by NQO1
directly competes with cellular one-electron reductases for menadione [17–22].
Reduction of menadione by NQO1 results in the formation of a stable hy-
droquinone that can be readily conjugated and excreted . Alternatively, reduc-
tion of menadione by one-electron reductases results in the formation of a
semiquinone which in the presence of molecular oxygen redox-cycles to form
reactive oxygen species [17,22,24,25]. This hypothesis is supported by recent studies
in mice where disruption of the NQO1 gene resulted in increased menadione
toxicity . Additional evidence for the role of NQO1 in menadione detoxification
has come from studies using compounds that induce NQO1 such as BHA, BHT
and dimethyl fumarate. Induction of NQO1 was shown to protect against mena-
dione-induced hemolytic anemia, but interestingly, in the same study induction of
NQO1 was shown to potentiate the toxicity of 2-hydroxy-1,4-naphthoquinone .
These data highlight the role of NQO1 as either a detoxification enzyme or an
activation enzyme depending upon the stability of the hydroquinone generated
D. Ross et al. / Chemico-Biological Interactions 129 (2000) 77–97 80
Fig. 2. The role of NQO1 in regeneration of antioxidant forms of ubiquinone and Vitamin E.
Another role for NQO1 in quinone detoxification is the removal of potentially
arylating quinones. Quinones can readily undergo addition and substitution reac-
tions and can react directly with protein thiols. In co-transfection studies with
cytochrome P450 reductase, NQO1 has been shown to decrease benzo[a]pyrene
3,6-quinone-induced DNA adduct formation . In these experiments, the authors
suggested that the active DNA binding species was the semiquinone formed by a
one-electron reduction catalyzed by cytochrome P450 reductase. NQO1-mediated
two-electron reduction of benzo[a]pyrene 3,6-quinone resulted in formation of a
stable hydroquinone. Our studies have shown that NQO1 participates in the
detoxification of the benzene-derived metabolite 1,4-benzoquinone. Hydroquinone
was selectively bioactivated in, and toxic to, marrow macrophages rather than
fibroblasts in bone marrow stroma because of increased levels of peroxidases
(leading to increased bioactivation) and lower levels of NQO1 (decreased deactiva-
tion) in macrophages relative to fibroblasts [29–31]. Transfection of human
promyeloblastic leukemia cells with NQO1 significantly decreased benzenetriol-
DNA adduct formation . In addition, we have shown that in human
promyeloblastic leukemia cells and human CD34+hematopoietic progenitor cells,
NQO1 induction by prior exposure to non-lethal concentrations of hydroquinone
protects cells from apoptosis induced by higher concentrations of hydroquinone
3.2. NQO1 as an antioxidant enzyme
Recent work with NQO1 has suggested that the enzyme may play an antioxidant
role via the reduction of endogenous quinones and these compounds, when
reduced, help protect cellular membranes against oxidative damage (Fig. 2). Exper-
iments have demonstrated that rat liver NQO1 can catalyze the reduction of
ubiquinone analogs (coenzyme Q) to their ubiquinol forms in liposomes and rat
hepatocytes [34,35]. The rate of reduction of coenzyme Q derivatives was dependent
upon the length of the carbon side-chain; short-chain homologs were reduced more
efficiently than long-chains. In these studies it was shown that the ubiquinol formed
D. Ross et al. / Chemico-Biological Interactions 129 (2000) 77–9781
following reduction by NQO1 was an effective antioxidant protecting membrane
phospholipids from oxidative damage. ?-Tocopherolquinone, a product of ?-toco-
pherol (vitamin E) oxidation has been shown to have antioxidant properties
following reduction to ?-tocopherolhydroquinone [36–38]. In experiments with
purified human NQO1 it was shown that this enzyme could effectively catalyze the
reduction of ?-tocopherolquinone to ?-tocopherolhydroquinone . We have also
demonstrated that Chinese hamster ovary cells transfected with human NQO1
generated higher levels of ?-tocopherolhydroquinone and were more resistant to
lipid peroxidation than cells lacking NQO1 .
An additional role for NQO1 in ?-tocopherol metabolism has been postulated
where NQO1 maintains physiological levels of ?-tocopherol from the reduction of
?-tocopherones by NQO1 . Oxidation of ?-tocopherol by peroxyl radicals yields
8?-(alkyldioxy) tocopherones which may either hydrolyze to ?-tocopherolquinone
or may be reduced to regenerate ?-tocopherol . Regeneration of ?-tocopherol
from ?-tocopherone by NQO1, however, remains to be demonstrated.
A role for NQO1 as an antioxidant enzyme is further supported by recent
immunohistochemical studies in humans that have shown NQO1 protein is ex-
pressed in many tissues requiring a high level of antioxidant protection [42–44].
These include the epithelial cells of lung, breast and colon, vascular endothelium,
adipocytes, corneal and lens epithelium, retinal pigmented epithelium, optic nerve
and nerve fibers. The high levels of NQO1 suggest that NQO1 may function
primarily in an antioxidant capacity in these cells. Interestingly, biochemical and
immunological-based assays have failed to detect significant levels of NQO1
expression in human liver [42,45] suggesting that in humans, unlike most species,
NQO1 does not play a major role in hepatic xenobiotic metabolism.
4. Bioactivation by NQO1
4.1. Bioacti?ation reactions
The chemical properties of the hydroquinone formed after NQO1 mediated
reduction of a quinone determines whether NQO1 catalyzes activation or deactiva-
tion. Not all hydroquinones are chemically stable and in some cases metabolism by
NQO1 yields a more active product which can autoxidize to produce reactive
oxygen species (Fig. 1) or undergo rearrangement to generate alkylating species. An
example of this is the reduction and activation of nitro compounds found in cooked
foods such as 4-nitroquinoline-1-oxide (4NQO). Sugimura and colleagues reported
the activation of 4NQO to 4-hydroxyaminoquinoline-1-oxide by a rat liver enzyme
. It was discovered later that the predominant enzyme responsible for this was
As with most substrates that are bioactivated by NQO1, naphthoquinones (NQ)
and dinitropyrenes (DNP) can either be activated or deactivated by NQO1 depend-
ing on substituent groups and their location. Induction of NQO1 with butylated
hydroxyanisole, protected rats from the toxic effects of 2-methyl-1,4-NQ but
D. Ross et al. / Chemico-Biological Interactions 129 (2000) 77–9782
potentiated the toxicity of 2-hydroxy-1,4-NQ . For DNPs which are found in
diesel exhaust, the positioning of the nitro groups greatly influences the clastogenic
potential of the activated compounds. In addition NQO1 increases mutagenicity of
1,6- but not 1,3- or 1,8-DNP in Ames Salmonella typhimurium assay .
4.2. Enzyme directed antitumor agents
Drugs that can produce alkylating metabolites after reduction have been termed
‘bioreactive alkylating agents’ . The result of bioreduction is either the produc-
tion of alkylating species or active oxygen species depending on the chemical
properties of the compounds undergoing enzymatic reduction (see Ref.  for
discussion). Selective toxicity of bioreductive alkylating agents to tumors was based
on the premise that hypoxic cells near the necrotic cores of tumors would have a
greater propensity for reductive metabolism . Enzyme-directed antitumor drug
development, however, exploits bioactivating enzymes that are expressed at high
levels in tumors relative to uninvolved tissue. NQO1 is expressed at high levels
throughout many human solid tumors [9,50] and is one possible candidate for the
enzyme-directed approach. A detailed review of enzyme-directed drug discovery
focused on quinones has recently been published . Since NQO1 is present is
present in uninvolved tissues as well as human tumor tissue , it is possible that
toxicity too normal tissue may be an issue in therapy with NQO1-directed antitu-
mor quinones. Mechanistically, DNA is thought to be the target of such enzyme-di-
rected alkylating agents, so the higher growth fraction of tumors may still offer
opportunity for selective toxicity.
The development of quinones which can be efficiently bioactivated by NQO1 as
potential antitumor agents has focuded on aziridinylbenzoquinones, indole-
quinones, motosenses, pyrrolobenzimidazolequinones and cyclopropamitosenses
(Fig. 3). Understanding NQO1 bioactivation in complex cellular systems is compli-
cated by the presence of other reductases. To combat this problem, newer model
systems are now being employed including gene targeting  and enzyme induc-
tion  approaches together with the use of NQO1-transfected cell lines [54–56]
and specific NQO1 inhibitors [57,58]. This work has provided unequivocal evidence
that NQO1 is capable of bioactivation of antitumor quinones in cellular systems
and currently work is ongoing to determine the role of NQO1 in vivo in both
xenograft models and eventually in clinical settings.
4.2.1. Indolequinones and mitosenes
Mitomycin C (MMC) has been one of the most successful single agents used in
the treatment of NSCLC and is currently used in combination cancer therapies .
MMC is bioactivated through reduction and this can be accomplished by NQO1 in
vitro . MMC is a relatively poor substrate for NQO1 [61,62] and newer
compounds have been designed in the hopes of generating more efficient substrates
and therefore better chemotherapeutic agents. In structure–activity relationship
studies using indolequinones and mitosenes, a number of features have been
identified which impact metabolism and cytotoxicity [56–58,63–69] and these have
D. Ross et al. / Chemico-Biological Interactions 129 (2000) 77–9783
been recently summarized . Depending on the substitution patterns in the indole
or mitosene structure, compound design within the structural series can be opti-
mized for potency and selective toxicity to cells containing high NQO1 levels.
Diaziquone (AZQ) is an aziridinylbenzoquinone that was used for the treatment
of glioma . Similar to MMC, AZQ is a relatively poor substrate for NQO1, but
the structure-based metabolism studies have yielded several aziridinylbenzo-
quinones that are better substrates for NQO1 than AZQ . MeDZQ (2,5-di-
aziridinyl-3,6-methyl-1,4-benzoquinone) was discovered in screening a panel of
synthetic aziridinylbenzoquinones. The rate of MeDZQ metabolism by purified
NQO1 was reported to be over 100-fold higher than MMC, and cytotoxicity and
selectivity to NQO1-containing cells was also increased [61,62,72]. MeDZQ also
required enzymatic activation by NQO1 in order to crosslink DNA . Due to
potential formulation problems from the low solubility of MeDZQ, RH1 (2,5-di-
aziridinyl-3-hydroxymethyl-6-methyl-1,4-benzoquinone) was developed as a water-
soluble analog. RH1 was a better substrate for purified NQO1 and more cytotoxic
to NQO1-expressing cell lines than MeDZQ or MMC. Using NQO1-transfected
cell lines [54,56], we have examined the relative cytotoxicity of MeDZQ, MMC and
RH1. In NQO1-transfected Chinese hamster ovary (CHO) cell lines, we found that
Fig. 3. Quinones and other compounds considered as NQO1-directed antitumor agents.
D. Ross et al. / Chemico-Biological Interactions 129 (2000) 77–9784
MeDZQ exhibited increased toxicity relative to the parent cell line but this effect
was not observed with MMC . A similar human-derived model was obtained by
transfecting human NQO1 into human BE colon adenocarcinoma cell lines which
lack NQO1 activity due to a genetic mutation . Because of the absence of
NQO1 activity in parental cells, BE cells stabley transfected with human NQO1
offer a useful model system in which to examine the selective toxicity of NQO1-di-
rected antitumor agents. In this system, MMC, MeDZQ and RH1 exhibited
increased cell killing in the NQO1-transfected BE cells relative to parental controls
. The degree of selective toxicity was in the order of RH1 (×17)?MeDZQ
(×7)?MMC (×3) . RH1 is currently under consideration under consider-
ation for clinical trials by the National Cancer Institute and The Cancer Research
Campaign. Its potent cytotoxicity is expected to offset its short halflife in mice
[73,74]. Recent pharmacokinetic and metabolic studies in mice suggest that RH1 is
likely to exhibit more favourable characteristics than the bioreductive agent EO9
which was disappointing in clinical trials .
4.2.3. Other quinones
Pyrrolobenzimidazolequinones (PBIs) were discovered in the late 1980s and were
designed to alkylate the phosphate backbone of DNA upon reduction which results
in its clevage [75–77]. PBI substrates for NQO1 can be either activated or
deactivated by the enzyme depending on their structure [77,78]. Quinolinequinones
 and benzoquinone mustards  have also been considered as potential agents
for NQO1-directed approaches to chemotherapy and these have recently been
discussed in more detail .
4.2.4. Nitro prodrugs and other agents
Cytotoxicity of 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954) occurs through
reductive activation and crosslinking of DNA . It was noted in the late 1960s
that Walker rat carcinoma cells were particularly sensitive to CB1954 and this
sensitivity was later linked to expression of NQO1 [13,82,83]. NQO1 reduces
CB1954 to a hydroxylamine derivative that is converted to a bifunctional crosslink-
ing agent by thioesters . In cells with moderate levels of NQO1, crosslinking
may play a lesser role and toxicity may be exerted primarily through redox cycling
. Newer analogues of CB1954 that target NQO1 are being developed . The
bioactivationof tirapazamine (3-amino-1,2,4-benzotriazene-1,4dioxide,
SR4233) is complex and NQO1 may play a limited role in its metabolism. TPZ is
mainly activated through one-electron reduction, but NQO1 can activate TPZ in
hypoxic conditions as well . There is evidence that NQO1 deactivates TPZ in
two- and four-electron reductions (for review see Ref. ). 17-Allylamino, 17-
demethoxygeldanamycin (17AAG) is an hsp90 inhibitor which has currently en-
tered clinical trials. A positive correlation between NQO1 expression and growth
inhibitory of properties of 17AAG in cell culture and xenografts has been reported
D. Ross et al. / Chemico-Biological Interactions 129 (2000) 77–97 85
5. NQO1 gene regulation
Isolation of the human , rat  and mouse  cDNAs for NQO1 confirmed
that NQO1 is a single copy gene and is located on human chromosome 16q22.1
[92–94] and mouse chromosome 8 . The human , mouse [52,96] and rat
[97,98] genes have been cloned and structurally characterized. Restriction mapping
and sequencing have revealed that the NQO1 gene consists of six exons and five
introns for an approximate length of 20 kb. Exon 1 encodes the 5?UT, the first two
amino acids and the first nucleotide of the third amino acid, while exons 2–6
encode the remaining 272 amino acids and the 3?UT. There is extensive homology
(85%) between the rat and human NQO1 coding regions . Transcriptional start
sites differ among the rat, human and mouse genes suggesting potential species-spe-
cific regulation of NQO1 .
Biochemical studies have already demonstrated that NQO1 activity is induced by
a wide range of chemicals including polycyclic aromatic hydrocarbons, azo dyes
and phenolic antioxidants [100–104]. Two distinct regulatory elements in the 5?
flanking region of the NQO1 gene that have been studied extensively are the
antioxidant response element (ARE), also called the EpRE (electrophile response
element), and the xenobiotic response element (XRE), also called the AhRE. The
ARE and the XRE have been shown to mediate NQO1 induction as well as
repression, in many cellular systems. The structure–function relationships within
the NQO1 promoter are now being addressed using functional assays, mutational
analysis and transgenic models.
6. ARE induction
ARE-mediated NQO1 gene expression is increased by a variety of antioxidants,
tumor promoters, and H2O2[105–107]. Various transcription factors have been
described, which bind to the ARE (TMAnnRTGAYnnnGCRwww) in-vitro, sug-
gesting that this is a composite regulatory DNA element. The abbreviations follow
standard IUPAC nomenclature; M=A or C, R=A or G, Y=C or T, W=A or
T, S=G or C. The composition of the specific proteins binding to the ARE is
variable. Because the ARE sequence, GTGACnnnGC, is similar to the AP-1
binding site, TGASTMAG, many reports have suggested that AP-1 and other basic
leucine zipper (bZIP) proteins Nrf1 (NF-E2 related transcription factor), Nrf2 and
Maf  drive the induction of ARE-dependent genes such as NQO1. Others have
recently shown that the ERK (extracellular signal regulated protein kinase) path-
way also participates in the ARE-mediated induction of Phase II detoxifying
enzymes . The ERK pathway mediates the induction of ARE-dependent genes
 whereas p38 MAPK (mitogen activated protein kinase) negatively regulates
induction by tBHQ and sulforaphane . AP-1 proteins are sensitive to activa-
tion by hypoxia [111–113]. Waleh et al.  demonstrated that the hNQO1 ARE
was responsive to low oxygen conditions. A useful model to integrate all these
findings is one proposed by Wasserman and Fahl  which illustrates ARE-medi-
D. Ross et al. / Chemico-Biological Interactions 129 (2000) 77–9786
Fig. 4. Schematic representation of ARE-mediated regulation of hNQO1 (modified from Wasserman
and Fahl ). Members of the bZip protein family are proposed to bind to the RTGAYnnn portion
of the ARE consensus. Binding protein 1 (BP-1) interacts with the bZip proteins and binds to the
flanking sequences within the ARE.
ated expression of genes (Fig. 4). The ARE core sequence, RTGAYnnn, binds the
bZIP family of transcription proteins (Jun, Fos, Fra, Nrf, Maf, Raf, NF-E2), while
the flanking sequences are critical in mediating transactivation in a cell or tissue-
AP-1 proteins may not be necessary for ARE-mediated gene expression. The
hNQO1 ARE was induced by BHQ in mouse F9 cells which are known to contain
no significant AP-1 activity . The ARE in the hNQO1 gene bears a close
resemblance to the NF-E2 (nuclear factor-erythroid 2) and allows binding for other
bZip proteins, Nrf1 and Nrf2 . Venugopal et al.  showed that Nrf1 and
Nrf2 are critical for the induction of specific protein binding to the hNQO1 ARE
in extracts of either human liver or monkey kidney cell lines exposed to B-NF or
tBHQ. However, overexpression of cFos or Fra-1 repressed basal ARE driven
promoter activity. Furthermore, Nrf1 and Nrf2 heterodimerize with several Jun
family members in the presence of unknown cytosolic factors, causing induction of
NQO1 in human hepatoma cells .
Induction through the ARE has primarily been studied in hepatoma cells,
however, Moehlenkamp et al.  have shown differing responses to tBHQ and
TPA in human neuroblastoma cells. Activation of ARE sequences by tBHQ in
neuroblastoma cells was shown to be significantly different from HepG2 cells and
a complete 5? palindrome within the ARE was necessary for maximal induction.
Studies in lung cancer cells  have shown that the ARE was also important for
mediating constitutive NQO1 expression in NSCLC, but surprisingly not in SCLC.
Gel supershift assays with various specific Fos/Jun antibodies identified Fra1, Fra2
and Jun B binding activity in NSCLC cells, whereas SCLC do not appear to
express functional Fra or Jun B. Overexpression of cFos is known to repress the
ARE-mediated expression of hNQO1 in transfected human liver cell lines .
Data from transgenic studies in mice indicate disruption of cFos leads to increased
expression of detoxifying enzymes including NQO1 and suggest a negative role for
cFos in their regulation . The combinatorial interactions of all of these
transcription factors introduce specificity into the transcriptional response to extra-
cellular signals. By dimerizing with different partners under diverse circumstances,
bZip proteins may be mixed and matched to assume a greater role in NQO1 gene
D. Ross et al. / Chemico-Biological Interactions 129 (2000) 77–9787
7. Antiestrogens in regulation of NQO1
Classically, estrogen receptors (ER) enhance transcription by binding to estrogen
response elements (ERE) within target genes. While antiestrogens such as tamox-
ifen, exert little activity at EREs, they can induce significant transcriptional activity
at AP-1 sites . Estrogen receptor beta, (ER?) enhances AP-1 activity via
interactions with a p160/p300 coactivator complex . Recently, Montano et al.
have shown a direct interaction of anti-estrogen liganded ER?with the hNQO1
ARE in the MCF-7 human breast cell line . These observations and the fact
that other reported factors, such as Hela cell binding protein 1 (BP-1) , that
interact with AREs are now beginning to be characterized, raises the possibility that
antiestrogens might play a role in regulating other ARE containing genes offering
chemoprotective benefits in ER-expressing tissues.
8. XRE induction
Induction through the XRE involves the liganded aromatic hydrocarbon recep-
tor, AHR. This intracellular member of the PAS (Per, Arnt, Sim) family of
proteins, translocates to the nucleus upon binding to Arnt . The AHR/Arnt
dimer that interacts with the DNA sequences known as XREs. The hNQO1 XRE
shares significant homology with the human CYP1A1 XRE . Both NQO1 and
CYP1A1 genes can be induced by TCDD and polycyclic aromatic hydrocarbons
, while DeLong et al.  and Rushmore and Pickett  have suggested
that the induction of NQ01 is largely dependent on the ability of bifunctional
inducers such as azo dye, Sudan I and B-NF to first undergo conversion to
oxidative labile metabolites through a functional P450-dependent mechanism. Re-
cently, Rajendriane et al.  reported that TCDD induction of hNQO1 in mouse
hepatoma cells was ARE-mediated and not dependent on XRE.
9. Polymorphisms in NQO1
We have characterized a single nucleotide polymorphism in NQO1 which has
profound phenotypic consequences [132,133]. The polymorphism (NQO1*2 allele)
is a C to T change at position 609 of the cDNA which codes for a proline to serine
change in the structure of the human protein. Genotype-phenotype studies of the
NQO1*2 allele have been performed using both cell systems and tissues. No
detectable or only trace levels of mutant NQO1 protein could be observed in cell
lines and in saliva, bone marrow or lung samples from individuals with the
NQO1*2/*2 genotype [133,134]. Although the mutant NQO1 protein purified from
E. coli expression systems has only between 2 and 4% of the activity of the
wild-type protein  because of a diminished ability to bind FAD , the
mechanism underlying the lack of NQO1 activity in NQO1*2/*2 individuals
appears primarily to be due to a lack of protein . Deficient NQO1 protein
D. Ross et al. / Chemico-Biological Interactions 129 (2000) 77–97 88
levels have also been observed in COS-1 cells transfected with mutant NQO1
cDNA . Recently, we have demonstrated that the lack of protein as a result of
the NQO1*2/*2 genotype appears to be due to accelerated degradation of the
mutant NQO1 protein mediated by the ubiquitin/proteasomal system . The
frequency of the NQO1*2/*2 genotype varies across ethnic group from 4% in
Caucasians, 5% in African–Americans, 16% in Mexican Hispanics to 22% in
Chinese populations . Gaedigk et al.  reported that the NQO1*2 allele
frequency was 0.16 in Caucasians, 0.4 in Native Indians, 0.46 in Inuits and 0.49 in
A second polymorphism in NQO1 (NQO1*3 allele) has also been characterized
[140,141]. This is a C465T change coding for an arginine to tryptophan substitution
in the protein. The implications of this polymorphism for phenotype are variable
depending on the substrate and the frequency of the NQO1*3 polymorphism is low.
In a recent study, the NQO1*3 allele frequency varied from 0 to 0.05 in different
ethnic groups with only one homozygous variant detected in 575 samples tested
Because the homozygous NQO1*2 allele is essentially a null phenotype [132–
134], it provides a convenient molecular tool with which to assess the potential
chemoprotective role of NQO1 in-vivo. Previous work on the implications of the
null polymorphism in NQO1 have almost exclusively been examined from the
perspective of the susceptibility to cancer of individuals carrying the NQO1*2/*2
genotype. The NQO1*2 allele has been associated with an increased risk of
urothelial tumors , therapy-related acute myeloid leukemia , cutaneous
basal cell carcinomas  and pediatric leukemias . We have also demon-
strated that the homozygous NQO1*2 allele is a significant risk factor for the
development of benzene-induced hematotoxicity in exposed workers . The
NQO1*2 polymorphism does not appear to be a risk factor for prostate cancer
 and the alternative hypothesis seems to be true in lung cancers with an
over-representation of the wild-type NQO1 allele in lung cancer cases relative to
The NQO1*2 polymorphism may also have relevance for chemotherapy using
antitumor quinones. Mitomycin C is currently the only quinone used extensively in
chemotherapeutic regimens. Although NQO1 is clearly not the only reductase that
can bioactivate mitomycin C, the use of stable transfection, enzyme induction and
gene targeting approaches by ourselves and others [52,55,56,150] clearly demon-
strate that it is a major determinant of the biological effects of mitomycin C. This
suggests that the effectiveness of mitomycin C in therapy would be diminished in
individuals carrying the NQO1*2 polymorphism. Important supporting evidence
for this hypothesis is beginning to appear in the literature. We recently reported
that the response of primary cultures of gastric tumors to mitomycin C was
dependent on NQO1 genotype  with increased response observed in *1/*1
genotypes. More importantly, Fleming et al.  reported significant differences in
survival in patients treated with mitomycin C depending on NQO1 genotype.
Optimal responses in patients with disseminated peritoneal cancer receiving mito-
mycin C were associated with the NQO1*1/*1 genotype rather than NQO1*1/*2 or
D. Ross et al. / Chemico-Biological Interactions 129 (2000) 77–9789
*2/*2 genotypes. This is the first report relating clinical response to mitomycin C
with NQO1 genotype and builds on considerable in-vitro data demonstrating the
lack of effect of mitomycin C in human cell lines carrying the NQO1*2/*2 genotype
The authors acknowledge support from NIH grants CA51210 and ESO9954.
 L. Ernster, F. Navazio, Soluble diaphorase in animal tissues, Acta Chem. Scand. 12 (1958)
 L. Ernster, DT-diaphorase, Methods Enzymol. 10 (1967) 309–317.
 C. Huggins, Experimental Leukemia and Mammary Cancer, University Chicago Press, Chicago,
 E. Cadenas, Antioxidant and prooxidant functions of DT-diaphorase in quinone metabolism,
Biochem. Pharmacol. 49 (1995) 127–140.
 R. Li, M.A. Bianchet, P. Talalay, L.M. Amzel, The three dimensional structure of
NAD(P)H:quinone reductase, a flavoprotein involved in cancer chemoprotection and chemother-
apy: mechanism of the two electron reduction, Proc. Natl. Acad. Sci. USA 92 (1995) 8846–8850.
 T. Iyanagi, I. Yamazaki, Difference in the mechanism of quinone reduction by the NADH
dehydrogenase and the NAD(P)H dehydrogenase (DT-diaphorase), Biochim. Biophys. Acta 216
 G. Tedeschi, S. Chen, V. Massey, DT-diaphorase: redox potential, steady-state, and rapid reaction
studies, J. Biol. Chem. 270 (1995) 1198–1204.
 C. Lind, E. Cadenas, P. Hochstein, L. Ernster, DT-diaphorase: purification properties and
function, Methods Enzymol. 186 (1990) 287–301.
 D. Ross, Quinone reductases, in: F.P. Guengerich (Ed.), Comprehensive Toxicology, vol. 3.
Biotransformation, Pergamon, New York, 1997, pp. 179–198.
 J. Segura-Aguilar, C. Lind, On the mechanism of the Mn-induced neurotoxicity of dopamine:
prevention of quinone derived oxygen toxicity by DT-diaphorase and superoxide dismutase,
Chem. Biol. Interact. 72 (1989) 309–324.
 A.K.D.Hajos,G.W. Winston, Dinitropyrene
NAD(P)H:quinone oxidoreductase: role in rat liver cytosol and induction by Aroclor-1254
pretreatment, Carcinogenesis 12 (1991) 697–702.
 A.J. Lambert, F. Friedlos, M.P. Boland, R.J. Knox, A CB 1954 analog that is highly cytotoxic in
human tumor cell lines, Br. J. Cancer 65 (Suppl. XVI) (1992) 59.
 R.J. Knox, M.P. Boland, F. Friedlos, B. Coles, C. Southan, J.J. Roberts, The nitroreductase
enzyme in Walker cells that activates 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) to 5-
(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide is a form of NAD(P)H dehydrogenase (quinone)
(EC 1. 6. 99. 2.), Biochem. Pharmacol. 37 (1988) 4671–4677.
 M.P. Boland, R.J. Knox, J.J. Roberts, The differences in kinetics of rat and human DT-di-
aphorase result in a differential sensitivity of derived cell lines to CB 1954 (5-(aziridin-1-yl)-2,4-
dinitrobenzamide), Biochem. Pharmacol. 41 (1991) 867–875.
 M.T. Huang, G.T. Miwa, N. Cronheim, A.Y.H. Lu, Rat liver cytosolic azoreductase, J. Biol.
Chem. 254 (1979) 11223–11227.
 K. Cui, A.Y.H. Lu, C.S. Yang, Subunit functional studies of NAD(P)H:quinone oxidoreductase
with a heterodimer approach, Proc. Natl. Acad. Sci. USA 92 (1995) 1043–1047.
D. Ross et al. / Chemico-Biological Interactions 129 (2000) 77–9790
 H. Thor, M.T. Smith, P. Hartzell, G. Bellomo, S.A. Jewell, S. Orrenius, The metabolism of
menadione (2-methyl-1,4-naphthoquinone) by isolated hepatocytes. A study of the implication of
oxidative stress in intact cells, J. Biol. Chem. 257 (1982) 12419–12425.
 S.J. Duthie, M.H. Grant, The role of reductive and oxidative metabolism in the toxicity of
mitoxantrone, adriamycin and menadione in human liver derived Hep G2 hepatoma cells, Br. J.
Cancer 60 (1989) 566–571.
 S. Shimada, H.K. Mishima, H. Nikaido, S. Kitamura, K. Tatsumi, Metabolism of drugs in the
eye. Menadione-dependent reduction of tertiary amine N-oxide by preparations from bovine
ocular tissues, Curr. Eye Res. 8 (1989) 1309–1313.
 Z. Djuric, T.H. Corbett, F.A. Valeriote, L.K. Heilbrun, L.H. Baker, Detoxification ability and
toxicity of quinones in mouse and human tumor cell lines used for anticancer drug screening,
Cancer Chemother. Pharmacol. 36 (1995) 20–26.
 T.J. Chiou, Y.T. Wang, W.F. Tzeng, DT-diaphorase protects against menadione-induced oxida-
tive stress, Toxicology 139 (1999) 103–110.
 C. Lind, P. Hochstein, L. Ernster, DT-diaphorase as a quinone reductase: a cellular control device
against semiquinone and superoxide radical formation, Arch. Biochem. Biophys. 216 (1982)
 R. Losito, C.A. Owen, E.V. Flock, Metabolism of [14C]menadione, Biochemistry 6 (1967) 62–68.
 H. Wefers, H. Sies, Hepatic low-level chemiluminescence during redox cycling of menadione and
the menadione-glutathione conjugate: relation to glutathione and NAD(P)H:quinone reductase
(DT-diaphorase) activity, Arch. Biochem. Biophys. 224 (1983) 568–578.
 W.S. Utley, H.M. Mehendale, Phenobarbital-induced cytosolic cytoprotective mechanisms that
offset increases in NADPH cytochrome P450 reductase activity in menadione-mediated cytotoxic-
ity, Toxicol. Appl. Pharmacol. 99 (1989) 323–333.
 V. Radjendirane, P. Joseph, Y.H. Lee, S. Kimura, A.J. Klein-Szanto, F.J. Gonzalez, A.K. Jaiswal,
Disruption of the DT diaphorase (NQO1) gene in mice leads to increased menadione toxicity, J.
Biol. Chem. 273 (1998) 7382–7389.
 R. Munday, B.L. Smith, C.M. Munday, Effect of inducers of DT-diaphorase on the toxicity of
2-methyl- and 2-hydroxy-1,4-naphthoquinone to rats, Chem. Biol. Interact. 123 (1999) 219–237.
 P. Joseph, A.K. Jaiswal, NAD(P)H:quinone oxidoreductase1 (DT diaphorase) specifically prevents
the formation of benzo[a]pyrene quinone-DNA adducts generated by cytochrome P4501A1 and
P450 reductase, Proc. Natl. Acad. Sci. USA 91 (1994) 8413–8417.
 D.J. Thomas, A. Sadler, V.V. Subrahmanyam, D. Siegel, M.J. Reasor, D. Wierda, D. Ross, Bone
marrow stromal cell bioactivation and detoxification of the benzene metabolite hydroquinone:
comparison of macrophages and fibroblastoid cells, Mol. Pharmacol. 37 (1990) 255–262.
 D. Ross, D. Siegel, N.W. Gibson, D. Pacheco, D.J. Thomas, M. Reasor, D. Wierda, Activation
and deactivation of quinones catalyzed by DT-diaphorase. Evidence for bioreductive activation of
diaziquone (AZQ) in human tumor cells and detoxification of benzene metabolites in bone marrow
stroma, Free Radic. Res. Commun. 8 (1990) 373–381.
 L.G. Ganousis, D. Goon, T. Zyglewska, K.K. Wu, D. Ross, Cell-specific metabolism in mouse
bone marrow stroma: studies of activation and detoxification of benzene metabolites, Mol.
Pharmacol. 42 (1992) 1118–1125.
 J. Wiemels, J.K. Wiencke, A. Varykoni, M.T. Smith, Modulation of the toxicity and macromolec-
ular binding of benzene metabolites by NAD(P)H:quinone oxidoreductase in transfected HL-60
cells, Chem. Res. Toxicol. 12 (1999) 467–475.
 J.L. Moran, D. Siegel, D. Ross, A potential mechanism underlying the increased susceptibility of
individuals with a polymorphism in NAD(P)H:quinone oxidoreductase 1 (NQO1) to benzene
toxicity, Proc. Natl. Acad. Sci. USA 96 (1999) 8150–8155.
 R.E. Beyer, J. Segura-Aguilar, S. Di Bernardo, M. Cavazzoni, R. Fato, D. Fiorentini, M. Galli,
M. Setti, L. Landi, G. Lenaz, The role of DT-diaphorase in the maintenance of the reduced
antioxidant form of coenzyme Q in membrane systems, Proc. Natl. Acad. Sci. USA 93 (1996)
 L. Landi, D. Fiorentini, M.C. Galli, J. Segura-Aguilar, R.E. Beyer, DT-Diaphorase maintains the
reduced state of ubiquinones in lipid vesicles thereby promoting their antioxidant function, Free
Radic. Biol. Med. 22 (1997) 329–335.
D. Ross et al. / Chemico-Biological Interactions 129 (2000) 77–9791
 A. Bindoli, M. Valente, L. Cavallini, Inhibition of lipid peroxidation by ?-tocopherolquinone and
?-tocopherolhydroquinone, Biochem. Int. 10 (1985) 753–761.
 T. Hayashi, A. Kanetoshi, M. Nakamura, M. Tamura, H. Shirahama, Reduction of alpha-toco-
pherolquinone to alpha-tocopherol hydroquinone in rat hepatocytes, Biochem. Pharmacol. 44
 I. Kohar, M. Baca, C. Suarna, R. Stocker, P.T. Southwell-Keely, Is alpha-tocopherol a reservoir
for alpha-tocopheryl hydroquinone?, Free Radic. Biol. Med. 19 (1995) 197–207.
 D. Siegel, E.M. Bolton, J.A. Burr, D.C. Liebler, D. Ross, The reduction of alpha-toco-
pherolquinone by human NAD(P)H: quinone oxidoreductase: the role of alpha-tocopherolhy-
droquinone as a cellular antioxidant, Mol. Pharmacol. 52 (1997) 300–305.
 E. Cadenas, P. Hochstein, L. Ernster, Pro- and antioxidant functions of quinones and quinone
reductases in mammalian cells, Adv. Enzymol. Relat. Areas Mol. Biol. 65 (1992) 97–146.
 D.C. Liebler, K.L. Kaysen, T.A. Kennedy, Redox cycles of vitamin E: hydrolysis and ascorbic
acid dependent reduction of 8a-alkyldioxytocopherones, Biochemistry 28 (1989) 9772–9779.
 D. Siegel, D. Ross, Immunodetection of NAD(P)H:quinone oxidoreductase 1 (NQO1) in human
tissues, Free Radic. Biol. Med., 2000, in press.
 D. Siegel, W.A. Franklin, D. Ross, Immunohistochemical staining for NAD(P)H:quinone oxidore-
ductase (NQO1) in normal lung and lung tumors, Proc. Am. Assoc. Cancer Res. 39 (1998) 804.
 L.P. Schelonka, D. Siegel, M.W. Wilson, A. Meininger, D. Ross, Immunohistochemical localiza-
tion of NQO1 in epithelial dysplasia and neoplasia and donor eyes. 41 1617–1622.
 J.J. Schlager, G. Powis, Cytosolic NAD(P)H:quinone acceptor oxidoreductase in human normal
and tumor tissue: effects of cigarette smoking and alcohol, Int. J. Cancer 45 (1990) 403–409.
 T. Sugimura, K. Okabe, M. Nagao, The metabolism of 4-nitroquinoline-1-oxide, a carcinogen by
an enzyme catalyzing the conversion of 4-nitroquinoline-1-oxide to 4-hydroxylaminoquinoline-1-
oxide in rat liver and hepatomas, Cancer Res. 26 (1966) 1717–1721.
 A.M. Benson, Conversion of 4-nitroquinoline 1-oxide (4NQO) to 4-hydroxyaminoquinoline
1-oxide by a dicumarol-resistant hepatic 4NQO nitroreductase in rats and mice, Biochem.
Pharmacol. 46 (1993) 1217–1221.
 A.K. Hajos, G.W. Winston, Purified NAD(P)H-quinone oxidoreductase enhances the mutagenic-
ity of dinitropyrenes in vitro, J. Biochem. Toxicol. 6 (1991) 277–282.
 A.C. Sartorelli, Therapeutic attack of hypoxic cells of solid tumors: presidential address, Cancer
Res. 48 (1988) 775–778.
 D. Ross, H.D. Beall, D. Siegel, R.D. Traver, D.L. Gustafson, Enzymology of bioreductive drug
activation, Br. J. Cancer 74 (Suppl. XXVII) (1996) S1–S8.
 H.D. Beall, S.L. Winski, Mechanisms of action of quinone-containing alkylating agents: NAO1-di-
rected drug development, Front. Biosci. 5 (2000) D639–648.
 T. Yoshida, H. Tsuda, Gene targeting of DT-diaphorase in mouse embryonic stem cells:
establishment of null mutant and its mitomycin C-resistance, Biochem. Biophys. Res. Commun.
214 (1995) 701–708.
 G.P. Doherty, M.K. Leith, X. Wang, T.J. Curphey, A. Begleiter, Induction of DT-diaphorase by
1,2-dithiole-3-thiones in human tumour and normal cells and effect on anti-tumour activity of
bioreductive agents, Br. J. Cancer 77 (1998) 1241–1252.
 D.L. Gustafson, H.D. Beall, E.M. Bolton, D. Ross, C.A. Waldren, Expression of human NQO1
(DT-diaphorase) in Chinese hamster ovary cells: effect on the toxicity of antitumor quinones, Mol.
Pharmacol. 50 (1996) 728–735.
 K. Mikami, M. Naito, A. Tomida, M. Yamada, T. Sirakusa, T. Tsuruo, DT-diaphorase as a
critical determinant of sensitivity to mitomycin C in human colon and gastric carcinoma cell lines,
Cancer Res. 56 (1996) 2823–2826.
 S. Winski, R.H.J. Hargreaves, J. Butler, D. Ross, A new screening system for NAD(P)H:quinone
oxidoreductase(NQO1)-directed antitumor quinones: identification of a new aziridinylbenzo-
quinone, RH1, as a NQO1-directed antitumor agent, Clin. Cancer Res. 4 (1998) 3083–3088.
 H.D. Beall, A.R. Hudnott, S. Winski, D. Siegel, E. Swann, D. Ross, C.J. Moody, Indolequinone
antitumor agents: relationship between quinone structure and rate of metabolism by recombinant
human NQO1, Bioorg. Med. Chem. Lett. 8 (1998) 545–548.
D. Ross et al. / Chemico-Biological Interactions 129 (2000) 77–97 92
 R.M. Phillips, M.A. Naylor, M. Jaffar, S.W. Doughty, S.A. Everett, A.G. Breen, G.A. Choudry,
I.J. Stratford, Bioreductive activation of a series of indolequinones by human DT-diaphorase:
structure–activity relationships, J. Med. Chem. 42 (1999) 4071–4080.
 R.C. Spain, The case for mitomycin in non-small cell lung cancer, Oncology 50 (Suppl. 1) (1993)
 D. Siegel, H. Beall, C. Senekowitsch, M. Kasai, H. Arai, N.W. Gibson, D. Ross, Bioreductive
activation of mitomycin C by DT-diaphorase, Biochemistry 31 (1992) 7879–7885.
 H.D. Beall, A.M. Murphy, D. Siegel, R.H.J. Hargreaves, J. Butler, D. Ross, Nicotinamide adenine
dinucleotide (phosphate):quinone oxidoreductase (DT-diaphorase) as a target for bioreductive
antitumor quinones: Quinone cytotoxicity and selectivity in human lung and breast cancer cell
lines, Mol. Pharmacol. 48 (1995) 499–504.
 H.D. Beall, R.T. Mulcahy, D. Siegel, R.D. Traver, N.W. Gibson, D. Ross, Metabolism of
bioreductive antitumor compounds by purified rat and human DT-diaphorases, Cancer Res. 54
 H.D. Beall, S. Winski, E. Swann, A.R. Hudnott, A.S. Cotterill, N. O’Sullivan, S.J. Green, R. Bien,
D. Siegel, D. Ross, C.J. Moody, Indolequinone antitumor agents: correlation between quinone
structure, rate of metabolism by recombinant human NAD(P)H:quinone oxidoreductase, and in
vitro cytotoxicity, J. Med. Chem. 41 (1998) 4755–4766.
 R.M. Phillips, Inhibition of DT-diaphorase (NAD(P)H:quinone oxidoreductase, EC 1. 6. 99. 2) by
5,6-dimethylxanthenone-4-acetic acid (DMXAA) and flavone-8-acetic acid (FAA): implications for
bioreductive drug development, Biochem. Pharmacol. 58 (1999) 303–310.
 M.A. Naylor, E. Swann, S.A. Everett, M. Jaffar, J. Nolan, N. Robertson, S.D. Lockyer, K.B.
Patel, M.F. Dennis, M.R. Stratford, P. Wardman, G.E. Adams, C.J. Moody, I.J. Stratford,
Indolequinone antitumor agents: reductive activation and elimination from (5-methoxy-1-methyl-
4,7-dioxoindol-3-yl)methyl derivatives and hypoxia-selective cytotoxicity in vitro, J. Med. Chem.
41 (1998) 2720–2731.
 R.M. Phillips, Bioreductive activation of a series of analogues of 5-aziridinyl-3-hydroxymethyl-1-
methyl-2-[1H-indole-4,7-dione] prop-?-en-?-ol (EO9) by human DT-diaphorase, Biochem. Phar-
macol. 52 (1996) 1711–1718.
 A.S. Cotterill, C.J. Moody, R.J. Mortimer, C.L. Norton, N. O’Sullivan, M.A. Stephens, N.R.
Stradiotto, E. Swann, I.J. Stratford, Cyclopropamitosenes, novel bioreductive anticancer agents.
Synthesis, electrochemistry, and biological activity of 7-substituted cyclopropamitosenes and
related indolequinones, J. Med. Chem. 37 (1994) 3834–3843.
 M.A. Naylor, M. Jaffar, J. Nolan, M.A. Stephens, S. Butler, K.B. Patel, S.A. Everett, G.E.
Adams, I.J. Stratford, 2-Cyclopropylindoloquinones and their analogues as bioreductively acti-
vated antitumor agents: structure–activity in vitro and efficacy in vivo, J. Med. Chem. 40 (1997)
 M. Jaffar, M.A. Naylor, N. Robertson, S.D. Lockyer, R.M. Phillips, S.A. Everett, G.E. Adams,
I.J. Stratford, 5-substituted analogues of 3-hydroxymethyl-5-aziridinyl-1-methyl-2-[1H-indole-4,7-
dione]prop-2-en-1-ol (EO9, NSC 382459) and their regioisomers as hypoxia-selective agents:
structure–cytotoxicity in vitro, Anticancer Drug Des. 13 (1998) 105–123.
 G.A. Curt, J.A. Kelley, C.V. Kufta, B.H. Smith, P.L. Kornblith, R.C. Young, J.M. Collins, Phase
IIand pharmacokineticstudyof aziridinylbenzoquinone
boethoxyamino)-1,4-benzoquinone, diaziquone, NSC 182986] in high-grade gliomas, Cancer Res.
43 (1983) 6102–6105.
 N.W. Gibson, J.A. Hartley, J. Butler, D. Siegel, D. Ross, Relationship between DT-diaphorase-
mediated metabolism of a series of aziridinylbenzoquinones and DNA damage and cytotoxicity,
Mol. Pharmacol. 42 (1992) 531–536.
 C.S. Lee, J.A. Hartley, M.D. Berardini, J. Butler, D. Siegel, D. Ross, N.W. Gibson, Alteration in
DNA cross-linking and sequence selectivity of a series of aziridinylbenzoquinones after enzymatic
reduction by DT-diaphorase, Biochemistry 31 (1992) 3019–3025.
 P.M. Loadman, R.M. Phillips, L.E. Lim, M.C. Bibby, Pharmacological properties of a new
aziridinylbenzoquinone, RH1 (2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone), in
mice, Biochem. Pharmacol. 59 (2000) 831–837.
D. Ross et al. / Chemico-Biological Interactions 129 (2000) 77–9793
 P. Khan, S. Abbas, R.H. Hargreaves, R. Caffrey, V.F. Megram, A.T. McGown, Development and
validation of a sensitive solid phase extraction and HPLC assay for RH1 in human and mouse
plasma, J. Chromatogr., 2000, in press.
 E.B. Skibo, W.G. Schulz, Pyrrolo[1,2-a]benzimidazole-based aziridinyl quinones. A new class of
DNA cleaving agent exhibiting G and A base specificity, J. Med. Chem. 36 (1993) 3050–3055.
 W.G. Schulz, R.A. Nieman, E.B. Skibo, Evidence for DNA phosphate backbone alkylation and
cleavage by pyrrolo[1,2-a]benzimidazoles: small molecules capable of causing base-pair-specific
phosphodiester bond hydrolysis, Proc. Natl. Acad. Sci. USA 92 (1995) 11854–11858.
 E.B. Skibo, S. Gordon, L. Bess, R. Boruah, M.J. Heileman, Studies of pyrrolo[1,2-
a]benzimidazolequinone DT-diaphorase substrate activity, topoisomerase II inhibition activity,
and DNA reductive alkylation, J. Med. Chem. 40 (1997) 1327–1339.
 W.A. Craigo, B.W. LeSueur, E.B. Skibo, Design of highly active analogues of the pyrrolo[1,2-
a]benzimidazole antitumor agents, J. Med. Chem. 42 (1999) 3324–3333.
 T. Fryatt, D.T. Goroski, Z.D. Nilson, C.J. Moody, H.D. Beall, Novel quinolinequinone antitumor
agents: structure-metabolism studies with NAD(P)H:quinone oxidoreductase (NQO1), Bioorg.
Med. Chem. Lett. 9 (1999) 2195–2198.
 J. Fourie, C. Leschuck, X. Wang, Jr, F. Guziec, L. Guziec, A. Begleiter, Structural modification
of model bioreductive antitumor agents: effects on DT-diaphorase dependent bioreduction and
cytotoxicity, Clin. Cancer Res. 5 (S1) (1999) 3781s.
 F.Y. Bois, M.T. Smith, R.C. Spear, Mechanisms of benzene carcinogenesis: application of a
physiological model of benzene pharmacokinetics and metabolism, Toxicol. Lett. 56 (1991)
 L.M. Cobb, T.A. Connors, L.A. Elson, A.H. Khan, B.C. Mitchley, W.C. Ross, M.E. Whisson,
2,4-Dinitro-5-ethyleneiminobenzamide (CB 1954): a potent and selective inhibitor of the growth of
the Walker carcinoma 256, Biochem. Pharmacol. 18 (1969) 1519–1527.
 R.J. Knox, F. Friedlos, M. Jarman, J.J. Roberts, A new cytotoxic DNA interstrand crosslinking
agent, 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide, is formed from 5-(aziridin-l-yl)-2,4-
dinitrobenzamide (CB 1954) by a nitroreductase enzyme in Walker carcinoma cells, Biochem.
Pharmacol. 37 (1988) 4661–4669.
 V. Miskiniene, E. Sergediene, A. Nemeikaite, J. Segura-Aguilar, N. Cenas, Role of redox cycling
and activation by DT-diaphorase in the cytotoxicity of 5-(aziridin-1-yl)-2,4-dinitrobenzamide
(CB-1954) and its analogs, Cancer Lett. 146 (1999) 217–222.
 L.K. Mehta, S. Hobbs, S. Chen, R.J. Knox, J. Parrick, Phthalimide analogs of CB 1954: synthesis
and bioactivation, Anticancer Drugs 10 (1999) 777–783.
 H.-F. Wang, T.-C. Lee, Glutathione S-transferase ? facilitates the excretion of arsenic from
arsenic-resistant Chinese hamster ovary cells, Biochem. Biophys. Res. Commun. 192 (1993)
 A.V. Patterson, M.P. Saunders, E.C. Chinje, L.H. Patterson, I.J. Stratford, Enzymology of
tirapazamine metabolism: a review, Anticancer Drug Des. 13 (1998) 541–573.
 L.R. Kelland, S.Y. Sharp, P.M. Rogers, T.G. Myers, P. Workman, DT-Diaphorase expression
and tumor cell sensitivity to 17-allylamino, 17-demethoxygeldanamycin, an inhibitor of heat shock
protein 90, J. Natl. Cancer Inst. 91 (1999) 1940–1949.
 A.K. Jaiswal, O.W. McBride, M. Adesnik, D.W. Nebert, Human dioxin inducible cytosolic
NAD(P)H:menadione oxidoreductase, J. Biol. Chem. 263 (1988) 13572–13578.
 J.A. Robertson, H. Chen, D.W. Nebert, NAD(P)H:menadione oxidoreductase. Novel purification
of enzyme, cDNA and complete amino acid sequence and gene regulation, J. Biol. Chem. 261
 V. Vasiliou, M.J. Theurer, A. Puga, S.F. Reuter, D.W. Nebert, Mouse dioxin-inducible NAD(P)H:
menadione oxidoreductase: NMO1 cDNA sequence and genetic differences in mRNA levels,
Pharmacogenetics 4 (1994) 341–348.
 A.K. Jaiswal, Human NAD(P)H:quinone oxidoreductase (NQO1) gene structure and induction by
dioxin, Biochemistry 30 (1991) 10647–10653.
 L.Z. Chen, P.C. Harris, S. Apostolou, E. Baker, K. Holman, S.A. Lane, J.K. Nancarrow, S.A.
Whitmore, R.L. Stallings, C.E. Hildebrand, R.I. Richards, G.R. Sutherland, D.F. Callen, A
refined physical map of the long arm of human chromosome 16, Genomics 10 (1991) 308–312.
D. Ross et al. / Chemico-Biological Interactions 129 (2000) 77–9794
 Los Alamos National Laboratory, Center for Human Genome Studies, Hybrid Breakpoint Map
of Chromosome 16. http://www-ls.lanl.gov/data/map16.txt, 1995.
 D.D. Petersen, F.J. Gonzalez, V. Rapic, C.A. Kozak, J.Y. Lee, J.E. Jones, D.W. Nebert, Marked
increases in hepatic NAD(P)H:oxidoreductase gene transcription and mRNA levels correlated with
a mouse chromosome 7 deletion, Proc. Natl. Acad. Sci. USA 86 (1989) 6699–6703.
 D.W. Nebert, L. Weider, M.J. Theuer, A. Puga, S. Kimura, V. Vasiliou, Organization and
characterization of the murine dioxin-inducible NAD(P)H: menadione oxidoreductase (NMO1)
gene, Toxicologist 15 (1995) A1363.
 J.B. Williams, A.Y.H. Lu, R.G. Cameron, C.B. Pickett, Rat liver NAD(P)H: quinone reductase,
J. Biol. Chem. 261 (1986) 5524–5528.
 R.M. Bayney, J.A. Rodkey, C.D. Bennett, A.Y.H. Lu, C.B. Pickett, Rat liver NAD(P)H: quinone
reductase nucleotide sequence analysis of a quinone reductase cDNA clone and prediction of the
amino acid sequence of the corresponding protein, J. Biol. Chem. 262 (1987) 572–575.
 J.K. Kepa, R.D. Traver, D. Siegel, S.L. Winski, D. Ross, Regulation and function of
NAD(P)H:quinone oxidoreductase (NQO1), Rev. Toxicol. 1 (1997) 53–73.
 P. Joseph, A.K. Jaiswal, C.C. Stobbe, J.D. Chapman, The role of specific reductases in the
intracellular activation and binding of 2-nitroimidazoles, Int. J. Radiat. Oncol. Biol. Phys. 29
 A.M. Benson, M.J. Hunkeler, P. Talalay, Increase of NAD(P)H:quinone reductase by dietary
antioxidants. Possible role in protection against carcinogenesis and toxicity, Proc. Natl. Acad. Sci.
USA 77 (1980) 5216–5220.
 P. Talalay, M.J. De Long, H.J. Prochaska, Identification of a common chemical signal regulating
the induction of enzymes that protect against chemical carcinogenesis, Proc. Natl. Acad. Sci. USA
85 (1988) 8261–8265.
 P. Talalay, H.J. Prochaska, Mechanisms of induction of NAD(P)H:quinone reductase, Chem.
Scripta 27A (1987) 61–66.
 M.J. De Long, H.J. Prochaska, P. Talalay, Induction of NAD(P)H:quinone reductase in murine
hepatoma cells by phenolic antioxidants, azo dyes and other chemoprotectors: A model system for
the study of anticarcinogens, Proc. Natl. Acad. Sci. USA 83 (1986) 787–791.
 Y. Li, A.K. Jaiswal, Human antioxidant response element mediated regulation of type 1
NAD(P)H: quinone oxidoreductase gene expression, Eur. J. Biochem. 226 (1994) 31–39.
 A.K. Jaiswal, Antioxidant response element, Biochem. Pharmacol. 48 (1994) 439–444.
 T. Prestera, W.D. Holtzclaw, Y. Zhang, P. Talalay, Chemical and molecular regulation of enzymes
that detoxify carcinogens, Proc. Natl. Acad. Sci. USA 90 (1993) 2965–2969.
 K. Itoh, T. Chiba, S. Takahashi, T. Ishii, K. Igarashi, Y. Katoh, T. Oyake, N. Hayashi, K. Satoh,
I. Hatayama, M. Yamamoto, Y. Nabeshima, An Nrf2/small Maf heterodimer mediates the
induction of phase II detoxifying enzyme genes through antioxidant response elements, Biochem.
Biophys. Res. Commun. 236 (1997) 313–322.
 R. Yu, W. Lei, S. Mandlekar, M.J. Weber, C.J. Der, J. Wu, A.T. Kong, Role of a mitogen-acti-
vated protein kinase pathway in the induction of phase II detoxifying enzymes by chemicals, J.
Biol. Chem. 274 (1999) 27545–27552.
 R. Yu, S. Mandlekar, W. Lei, W.E. Fahl, T.H. Tan, A.T. Kong, p38 mitogen-activated protein
kinase negatively regulates the induction of phase II drug-metabolizing enzymes that detoxify
carcinogens, J. Biol. Chem. 275 (2000) 2322–2327.
 W.A. Ausserer, B. Bourrat-Floeck, C.J. Green, K.R. Laderoute, R.M. Sutherland, Regulation of
c-jun expression during hypoxic and low-glucose stress, Mol. Cell Biol. 14 (1994) 5032–5042.
 R.A. Rupec, P.A. Baeuerle, The genomic response of tumor cells to hypoxia and reoxygenation.
Differential activation of transcription factors AP-1 and NF-kappa B, Eur. J. Biochem. 234 (1995)
 J.M. Muller, B. Krauss, C. Kaltschmidt, P.A. Baeuerle, R.A. Rupec, Hypoxia induces c-fos
transcription via a mitogen-activated protein kinase-dependent pathway, J. Biol. Chem. 272 (1997)
 N.S. Waleh, J. Calaoagan, B.J. Murphy, A.M. Knapp, R.M. Sutherland, K.R. Laderoute, The
redox-sensitive human antioxidant responsive element induces gene expression under low oxygen
conditions, Carcinogenesis 19 (1998) 1333–1337.
D. Ross et al. / Chemico-Biological Interactions 129 (2000) 77–97 95
 W.W. Wasserman, W.E. Fahl, Functional antioxidant responsive elements, Proc. Natl. Acad. Sci.
USA 94 (1997) 5361–5366.
 K. Yoshioka, T. Deng, M. Cavigelli, M. Karin, Antitumor promotion by phenolic antioxidants:
inhibition of AP-1 activity through induction of Fra expression, Proc. Natl. Acad. Sci. USA 92
 R. Venugopal, A.K. Jaiswal, Nrf1 and Nrf2 positively and c-Fos and Fra1 negatively regulate the
human antioxidant response element-mediated expression of NAD(P)H:quinone oxidoreductase1
gene, Proc. Natl. Acad. Sci. USA 93 (1996) 14960–14965.
 R. Venugopal, A.K. Jaiswal, Nrf2 and Nrf1 in association with Jun proteins regulate antioxidant
response element-mediated expression and coordinated induction of genes encoding detoxifying
enzymes, Oncogene 17 (1998) 3145–3156.
 J.D. Moehlenkamp, J.A. Johnson, Activation of antioxidant/electrophile-responsive elements in
IMR-32 human neuroblastoma cells, Arch. Biochem. Biophys. 363 (1999) 98–106.
 J.K. Kepa, D. Ross, DT-diaphorase activity in NSCLC and SCLC cell lines: a role for fos/jun
regulation, Br. J. Cancer 79 (1999) 1679–1684.
 J. Wilkinson, V. Radjendirane, G.R. Pfeiffer, A.K. Jaiswal, M.L. Clapper, Disruption of c-Fos
leads to increased expression of NAD(P)H:quinone oxidoreductase 1 and glutathione S-trans-
ferase, Biochem. Biophys. Res. Commun. 253 (1998) 855–858.
 K. Paech, P. Webb, G.G. Kuiper, S. Nilsson, J. Gustafsson, P.J. Kushner, T.S. Scanlan,
Differential ligand activation of estrogen receptors ERalpha and ERbeta at AP1 sites, Science 277
 P. Webb, P. Nguyen, C. Valentine, G.N. Lopez, G.R. Kwok, E. McInerney, B.S. Katzenellenbo-
gen, E. Enmark, J.A. Gustafsson, S. Nilsson, P.J. Kushner, The estrogen receptor enhances AP-1
activity by two distinct mechanisms with different requirements for receptor transactivation
functions, Mol. Endocrinol. 13 (1999) 1672–1685.
 M.M. Montano, A.K. Jaiswal, B.S. Katzenellenbogen, Transcriptional regulation of the human
quinone reductase gene by antiestrogen-liganded estrogen receptor-alpha and estrogen receptor-
beta, J. Biol. Chem. 273 (1998) 25443–25449.
 B. Wang, G. Williamson, Detection of a nuclear protein which binds specifically to the antioxidant
responsive element (ARE) of the human NAD(P)H: quinone oxidoreductase gene, Biochim.
Biophys. Acta 1219 (1994) 645–652.
 J.P. Landers, N.J. Bunce, The Ah receptor and the mechanism of dioxin toxicity, Biochem. J. 276
 D.W. Nebert, J.E. Jones, Regulation of the mammalian cytochrome P1-450 (CYP1A1) gene, Int.
J. Biochem. 3 (1989) 243–252.
 K. Kumaki, N.M. Jensen, J.G.M. Shire, D.W. Nebert, Genetic differences in induction of cytosol
reduced-NAD(P): menadione oxidoreductase and microsomal aryl hydrocarbon hydroxylase in the
mouse, J. Biol. Chem. 252 (1977) 157–165.
 M.J. De Long, A.B. Santamaria, P. Talalay, Role of cytochrome P1-450 in the induction of
NAD(P)H: quinone reductase in a murine hepatoma cell line and its mutants, Carcinogenesis 8
 T.H. Rushmore, C.B. Pickett, Transcriptional regulation of the rat glutathione S-transferase Ya
subunit gene, J. Biol. Chem. 265 (1990) 14648–14653.
 V. Radjendirane, A.K.Jaiswal, Antioxidant
chlorodibenzo-p-dioxin (TCDD) induction of human NAD(P)H:quinone oxidoreductase 1 gene
expression, Biochem. Pharmacol. 58 (1999) 1649–1655.
 R.D. Traver, T. Horikoshi, K.D. Danenberg, T.H.W. Stadlbauer, P.V. Danenberg, D. Ross, N.W.
Gibson, NAD(P)H:quinone oxidoreductase gene expression in human colon carcinoma cells:
Characterization of a mutation which modulates DT-diaphorase activity and mitomycin sensitiv-
ity, Cancer Res. 52 (1992) 797–802.
 R.D. Traver, D. Siegel, H.D. Beall, R.M. Phillips, N.W. Gibson, W.A. Franklin, D. Ross,
Characterization of a polymorphism in NAD(P)H:Quinone oxidoreductase (DT-diaphorase), Br.
J. Cancer 75 (1997) 69–75.
D. Ross et al. / Chemico-Biological Interactions 129 (2000) 77–9796
 D. Siegel, S.M. McGuinness, S. Winski, D. Ross, Genotype-phenotype relationships in studies of
a polymorphism in NAD(P)H:quinone oxidoreductase 1, Pharmacogenetics 9 (1999) 113–121.
 K. Wu, P.S.K. Deng, S. Chen, Catalytic properties of a naturally occurring mutant of human
NAD(P)H:quinone oxidoreductase (DT-diaphorase), Pro 187 to Ser, Proc. Am. Assoc. Cancer
Res. 38 (1997) 3758.
 V. Misra, H.J. Klamut, A.M. Rauth, Transfection of COS-1 cells with DT-diaphorase cDNA: role
of a base change at position 609, Br. J. Cancer 77 (1998) 1236–1240.
 D. Siegel, A. Anwar, S. Winski, J.K. Kepa, D. Ross, The NQO1*2 polymorphism in
NAD(P)H:quinone oxidoreductase 1 targets the protein for proteasomal degradation, Proc. Am.
Assoc. Cancer Res. 41 (2000) 227.
 K.T. Kelsey, J.K. Wiencke, D.C. Christiani, Z. Zuo, M.R. Spitz, X. Xu, B.K. Lee, B.S. Schwartz,
R.D. Traver, D. Ross, Ethnic variation in the prevalence of a common NAD(P)H:quinone
oxidoreductase polymorphism and its implications for anticancer chemotherapy, Br. J. Cancer 76
 A. Gaedigk, R.F. Tyndale, M. Jurima-Romet, E.M. Sellers, D.M. Grant, J.S. Leeder,
NAD(P)H:quinone oxidoreductase; polymorphisms and allele frequencies in Caucasian, Chinese
and Canadian Native Indian and Inuit populations, Pharmacogenetics 8 (1998) 305–313.
 S.S. Pan, G.L. Forrest, S.A. Akman, L.-T. Hu, NAD(P)H:quinone oxidoreductase expression and
mitomycin C resistance developed by human colon cancer HCT 116 cells, Cancer Res. 55 (1995)
 L.T. Hu, J. Stamberb, S.S. Pan, The NAD(P)H:quinoneoxidoreductase locus in human colon
carcinoma HCT 116 cells resistant to mitomycin C, Cancer Res. 56 (1996) 5253–5259.
 W.A. Schulz, A. Krummeck, I. Rosinger, P. Eickelmann, C. Neuhas, T. Ebert, B. Schmitz-Dra ¨ger,
H. Sies, Increased frequency of a null allele for NAD(P)H:quinone oxidoreductase in patients with
urological malignancies, Pharmacogenetics 7 (1997) 235–239.
 R.A. Larson, Y. Wang, M. Banerjee, J. Wiemels, C. Hartford, M.M. Beau, M.T. Smith,
Prevalence of the inactivating 609C?T polymorphism in the NAD(P)H:Quinone oxidoreductase
(NQO1) gene in patients with primary and therapy-related myeloid leukemia, Blood 94 (1999)
 A. Clairmont, H. Sies, S. Ramachandran, J.T. Lear, A.G. Smith, B. Bowers, P.W. Jones, A.A.
Fryer, R.C. Strange, Association of NAD(P)H:quinone oxidoreductase (NQO1) null with numbers
of basal cell carcinomas: use of a multivariate model to rank the relative importance of this
polymorphism and those at other relevant loci, Carcinogenesis 20 (1999) 1235–1240.
 J.L. Wiemels, A. Pagnamenta, G.M. Taylor, O.B. Eden, F.E. Alexander, M.F. Greaves, A lack of
a functional NAD(P)H:quinone oxidoreductase allele is selectively associated with pediatric
leukemias that have MLL fusions. United Kingdom Childhood Cancer Study Investigators,
Cancer Res. 59 (1999) 4095–4099.
 N. Rothman, M.T. Smith, R.B. Hayes, R.D. Traver, B.A. Hoener, S. Campleman, G.L. Li, M.
Dosemeci, M. Linet, L.P. Zhang, L.Q. Xi, S. Wacholder, W. Lu, K.B. Meyer, N. Titenko-Hol-
land, J.T. Stewart, S.N. Yin, D. Ross, Benzene poisoning, a risk factor for hematological
malignancy, is associated with the NQ01
chlorzoxazone, Cancer Res. 57 (1997) 2839–2842.
 M. Steiner, M. Hillenbrand, M. Borkowsi, H. Seiter, P. Schuff-Werner, 609C?T polymorphism
in NAD(P)H:quinone oxidoreductase gene in patients with prostatic adenocarcinoma or benign
prostatic hyperplasia, Cancer Lett. 135 (1999) 67–71.
 J.T. Wiencke, M.R. Spitz, A. McMillan, K.T. Kelsey, Lung cancer in Mexican-Americans and
African-Americans is associated with the wild type genotype of the NAD(P)H:quinone oxidore-
ductase polymorphism, Cancer Epidemiol. Biomarkers Prev. 6 (1997) 87–92.
 H. Chen, A. Lum, A. Seifried, L.R. Wilkens, L. Le Marchand, Association of the
NAD(P)H:quinone oxidoreductase 609C?T polymorphism with a decreased lung cancer risk,
Cancer Res. 59 (1999) 3045–3048.
 X. Wang, G.P. Doherty, M.K. Leith, T.J. Curphey, A. Begleiter, Enhanced cytotoxicity of
mitomycin C in human tumour cells with inducers of DT-diaphorase, Br. J. Cancer 80 (1999)
609C?T mutation and rapid fractional excretion of
D. Ross et al. / Chemico-Biological Interactions 129 (2000) 77–9797 Download full-text
 M. Yano, Y. Akiyama, H. Shiozaki, M. Inoue, Y. Doki, Y. Fujiwara, D. Ross, A. Sadler, M.
Monden, Genetic polymorphism of NAD(P)H: quinone oxidoreductase and mitomycin C sensitiv-
ity in gastric cancer, Proc. Am. Assoc. Cancer Res. 41 (2000) 4–5.
 D. Sachs, R.A. Flemming, M.S. Miller, Biochemical modulation of mitomycin C by induction of
NADP(H):quinone oxidoreductase 1, Proc. Am. Assoc. Cancer Res. 41 (2000) 4–5.
 D. Siegel, N.W. Gibson, P.C. Preusch, D. Ross, Metabolism of mitomycin C by DT-diaphorase:
role in mitomycin C-induced DNA damage and cytotoxicity in human colon carcinoma cells,
Cancer Res. 50 (1990) 7483–7489.
 S. Winski, R.H.J. Hargreaves, J. Butler, D. Ross, A new screening system for NAD(P)H:quinone
oxidoreductase(NQO1)-directed antitumor quinones. Identification of a new aziridinylbenzo-
quinone, RH1, as a NQO1-directed antitumor agent, Clin. Cancer Res. 4 (1998) 3083–3088.
 K. Mikami, M. Naito, A. Tomida, M. Yamada, T. Sirakusa, T. Tsuruo, DT-diaphorase as a
critical determinant of sensitivity to mitomycin C in human colon and gastric carcinoma cell lines,
Cancer Res. 56 (1996) 2823–2826.