Download Role of phase 2 enzyme induction in chemoprotection by

Mutation Research 480–481 (2001) 305–315
Role of phase 2 enzyme induction in chemoprotection
by dithiolethiones
Mi-Kyoung Kwak a , Patricia A. Egner a , Patrick M. Dolan a , Minerva Ramos-Gomez a ,
John D. Groopman a , Ken Itoh b , Masayuki Yamamoto b , Thomas W. Kensler a,∗
a
Department of Environmental Health Sciences, Johns Hopkins University Bloomberg School of Public Health,
615 North Wolfe Street, Baltimore, MD 21205, USA
b Center for TARA, Tsukuba University, Tsukuba 305-8577, Japan
Received 19 October 2000; received in revised form 13 December 2000; accepted 05 January 2001
Abstract
One of the major mechanisms of protection against carcinogenesis, mutagenesis, and other forms of toxicity mediated
by carcinogens is the induction of enzymes involved in their metabolism, particularly phase 2 enzymes such as glutathione
S-transferases (GSTs), UDP-glucuronosyl transferases, and quinone reductases. Animal studies indicate that induction of
phase 2 enzymes is a sufficient condition for obtaining chemoprevention and can be achieved by administering any of a
diverse array of naturally-occurring and synthetic chemopreventive agents. Indeed, monitoring of enzyme induction has led to
the recognition or isolation of novel, potent chemopreventive agents such as 1,2-dithiole-3-thiones, terpenoids and the isothiocyanate sulforaphane. For example, oltipraz, a substituted 1,2-dithiole-3-thione originally developed as an antischistosomal
agent, possesses chemopreventive activity against different classes of carcinogens targeting multiple organs. Mechanistic
studies in rodent models for chemoprevention of aflatoxin B1 (AFB1 )-induced hepatocarcinogenesis by oltipraz indicates that
increased expression of phase 2 genes is of central importance, although inhibition of phase 1 activation of AFB1 can also
contribute to protection. Exposure of rodents to 1,2-dithiole-3-thiones triggers nuclear accumulation of the transcription factor
Nrf2 and its enhanced binding to the “antioxidant response element” (ARE), leading to transcriptional activation of a score of
genes involved in carcinogen detoxication and attenuation of oxidative stress. Nrf2-deficient mice fail to induce many of these
genes in response to dithiolethiones; moreover, basal expression of these genes is typically repressed. To test the hypothesis that
enzyme induction is a useful strategy for chemoprevention in humans, three key elements are necessary: a candidate agent, an
at-risk population and modulatable intermediate endpoints. Towards this end, a placebo-controlled, double blind clinical trial
of oltipraz was conducted in residents of Qidong, PR China who are exposed to dietary aflatoxins and who are at high risk for
the development of liver cancer. Oltipraz significantly enhanced excretion of a phase 2 product, aflatoxin–mercapturic acid, a
derivative of the aflatoxin–glutathione conjugate, in the urine of study participants administered 125 mg oltipraz by mouth daily.
Administration of 500 mg oltipraz once a week led to a significant reduction in the excretion of the primary oxidative metabolite
of AFB1 , AFM1 , when measured shortly after drug administration. While this study highlighted the general feasibility of inducing phase 2 enzymes in humans, a longer term intervention is addressing whether protective alterations in aflatoxin metabolism
can be sustained for extended periods of time in this high-risk population. © 2001 Elsevier Science B.V. All rights reserved.
Keywords: Oltipraz; 1,2-Dithiole-3-thiones; Chemoprotection; Nrf2; Glutathione S-transferases; Aflatoxin
∗ Corresponding
author. Tel.: +1-410-955-4712;
fax: +1-410-955-0116.
E-mail address: [email protected] (T.W. Kensler).
0027-5107/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 0 2 7 - 5 1 0 7 ( 0 1 ) 0 0 1 9 0 - 7
306
M.-K. Kwak et al. / Mutation Research 480–481 (2001) 305–315
1. Role of enzyme induction in cancer
chemoprotection
Chemical protection against toxins and carcinogens can be successfully achieved through several
different mechanisms [1,2]. Indeed, chemical agents
and natural products with cancer chemoprotective
activity encompass diverse classes of molecules with
varied biological effects including anti-mutagenic,
antioxidant, anti-inflammatory, and anti-hormonal activity [3,4]. One strategy of chemoprotection that has
proven to be particularly efficacious in experimental systems is the modulation of the metabolism and
processing of carcinogenic substances. This approach
entails a chemical-induced alteration of key metabolic
processes that results in decreased activation of procarcinogens to reactive intermediates and enhanced
elimination of these carcinogenic metabolites. Protection against a diverse array of carcinogens acting at
different target organ sites has been achieved with this
strategy, highlighting the essential role of metabolism
in chemical carcinogenesis. The prominent contribution of carcinogen metabolism to cancer development
is further evidenced by the fact that the presence
or absence of a particular metabolic pathway can
alone determine target organ or species sensitivity to
carcinogens.
Many chemicals require metabolic activation to
electrophilic intermediates to exert carcinogenic activity [5]. If not detoxified, these reactive species
can react with and thereby functionally modify nucleophilic moieties on critical biomolecules. Nucleophilic groups in DNA are among those targeted by
electrophiles; the interaction of carcinogen metabolites with DNA can cause point mutations and other
genetic lesions, which can result in activation of protooncogenes and inactivation or loss of tumor suppressor genes. The processing of chemicals to metabolites
capable of modifying DNA typically involves an initial two-electron oxidation. This phase 1 reaction can
be catalyzed by a number of enzymes, particularly
those comprising the cytochrome P450 super family. A second metabolic step involves the transfer or
conjugation of an endogenous, water-soluble substrate to the functional group introduced during phase
1 biotransformation, thereby facilitating elimination
of the carcinogen. These phase 2 reactions, which
include sulfation, acetylation, glucuronidation, and
conjugation with glutathione, typically (but not always) lead to carcinogen detoxification. The amount
of ultimate carcinogen available for interaction with
its target represents a balance between competing activating and detoxifying reactions. While this balance
is under genetic control, it is readily modulated by
a variety of factors including nutritional status, age,
hormones, and exposure to drugs or other xenobiotics
[6]. Chemopreventive agents can alter the constitutive
metabolic balance between activation and inactivation
of carcinogens through their actions on both phase 1
and 2 enzymes.
Considerable evidence suggests that modulation
of phase 2 enzymes, particularly the glutathione
S-transferases (GSTs), plays a direct role in the carcinogenic process and in chemoprotection against
carcinogens. The expression of GST pi has been
shown to be altered during the carcinogenic process.
For example, overexpression of GST pi is a hallmark of hepatic preneoplastic tumors in rats. Human
tumors in which GST pi has been reported to be overexpressed include lung, colon, ovary, testis, bladder,
oral cavity and kidney [7]. By contrast, a striking
decrease in GST pi expression was found in human
prostatic carcinoma specimens, but not in normal or
benign hyperplasia tissues. Methylation of cytidine
nucleotides in the GST pi regulatory sequence results in decreased expression of GST pi in prostatic
carcinoma specimens and cells [8]. Loss of GST pi
expression increases the sensitivity of human prostate
cells to the cytotoxic and DNA damaging action of the
prostate carcinogen, PhIP. Conversely, overexpression
of GST pi is protective in these cells [9]. Overexpression of the pi class of GSTs has also been associated
with poor prognosis and drug resistance in human
cancers, and with drug resistance in cell culture systems. Forced overexpression of GSTs can engender
a resistant phenotype [10], while transfection of the
GST pi antisense cDNA has been shown to sensitize
drug-resistant colon cancer cells to cytotoxic agents
[11]. A recent study demonstrated that targeted deletion of the murine GST pi gene cluster confers significantly enhanced sensitivity to polycyclic aromatic
hydrocarbon-induced skin carcinogenesis [12]. In this
study, increased susceptibility of the GST pi knockout
mice relative to wild-type animals was evidenced by
both decreased tumor latency and increased papilloma
development in a two-stage carcinogenesis protocol.
M.-K. Kwak et al. / Mutation Research 480–481 (2001) 305–315
Natural sensitivity and resistance to hepatocarcinogens such as aflatoxin B1 (AFB1 ) has been shown to
be associated with expression of particular isoforms
of GSTs [13]. Collectively, these data suggest that
GSTs have critical functions in tumor development in
animals. In keeping with this view, human deficiencies in GST expression have been associated with
increased cancer risk. The lack of GST T1 is related
to a slightly increased risk of cancer of the bladder,
gastro-intestinal tract, skin, and tobacco-related lung
and oral cavity tumors in human. The GST M1 null
genotype in human has also shown to be a risk factor in smoking-related oral and lung cancer [7,14].
However, the overall importance of polymorphisms
of phase 2 enzymes towards cancer risk in humans is
less certain as the biological impact of allelic variants is generally relatively modest with odds ratios of
about 2.
Arguably, the most compelling evidence supporting
the importance of enzyme induction in protection resides in observations that screening of pharmaceutical
agents and natural products has led to the recognition
or isolation of novel classes of chemoprotective agents
and/or more potent analogs of known protective compounds. For example, this strategy led to the isolation
of two anticarcinogenic terpenoids, kahweol palmitate
and cafestrol palmitate, from green coffee beans [15].
Additionally, the isothiocyanate sulforaphane was
isolated from broccoli on the basis of its potency as a
phase 2 enzyme inducer. Sulforaphane, the principal
phase 2 enzyme inducer in broccoli has been demonstrated to block dimethylbenz[a]anthracene-induced
mammary tumorigenesis in rats [16,17]. Broccoli
sprouts were subsequently identified as an exceptionally rich natural source of the glucosinolate precursor
of sulforaphane, and aqueous extracts of these sprouts
were found to effectively inhibit mammary tumorigenesis [18]. Broccoli sprouts are currently in clinical
trials to assess issues of safety and pharmacodynamic
action in humans.
During the course of studies on the mechanisms
of antischistosomal activity of 1,2-dithiole-3-thiones,
Bueding et al. [19] initially noted that administration
of oltipraz (5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3thione), to mice infected with Schistosoma mansoni
caused a reduction in the glutathione stores of the
parasites, but increased levels of glutathione in many
tissues of the host. Subsequent studies demonstrated
307
that oltipraz and many related dithiolethiones were
potent inducers of enzymes concerned with the
maintenance of reduced glutathione pools as well
as enzymes important to the detoxication of electrophiles and free radicals. Genes now recognized
to be induced by dithiolethiones in vivo include alpha, mu and pi isoforms of GSTs, UDP-glucuronosyl
transferases, NAD(P)H: quinone reductase (NQO1),
epoxide hydrolase, aflatoxin aldehyde reductase,
␥-glutamylcysteine synthase, manganese superoxide
dismutase, catalase, heavy and light chains of ferritin,
and leukotriene B4 dehydrogenase [19–22].
2. Protection against experimental
carcinogenesis by dithiolethiones
The biochemical manifestations of oltipraz in
schistosome-infected mice prompted Bueding to
predict that this drug might have cancer chemoprotective properties. The initial confirmation that
1,2-dithiole-3-thiones such as oltipraz may exert
chemoprotective effects in vivo came from the demonstration that oltipraz protected against the hepatotoxicity of carbon tetrachloride and acetaminophen in mice
[23]. Subsequent studies have demonstrated protection by oltipraz against the acute hepatotoxicities of
allyl alcohol and acetaminophen in the hamster [24]
and AFB1 in the rat [25]. Toxin-induced elevations in
liver function tests were blunted in all cases, clearly
indicating protection. Pretreatment with oltipraz also
substantially reduced the mortality produced by either
acute or chronic exposure to AFB1 [25].
To directly test the cancer chemoprotective efficacy
of oltipraz, Wattenberg and Bueding [26] examined
the capacity of oltipraz to inhibit carcinogen-induced
neoplasia in mice. Oltipraz was administered either 24
or 48 h before treatment with each of three chemically
diverse carcinogens: diethylnitrosamine, uracil mustard, and benzo[a]pyrene. This sequence of oltipraz
and carcinogen administration was repeated once a
week for 4–5 weeks. Oltipraz reduced by nearly 70%
the number of both pulmonary adenomas and tumors
of the forestomach induced by benzo[a]pyrene. Pulmonary adenoma formation induced by uracil mustard
or diethylnitrosamine was also significantly reduced
by oltipraz pretreatment, but to a lesser degree. As
reviewed elsewhere [27,28], oltipraz has now shown
308
M.-K. Kwak et al. / Mutation Research 480–481 (2001) 305–315
chemoprotective activity against different classes of
carcinogens targeting the trachea, lung, stomach,
small intestine, colon, pancreas, liver. Urinary bladder,
mammary gland, hematopoietic cells, and skin. The
most dramatic actions occur in the colon and liver,
where dietary administration results in significant
reductions in both tumor incidence and multiplicity.
Dietary concentrations of 200 and 400 ppm oltipraz
significantly reduced tumor incidence and multiplicity
in azoxymethane-induced intestinal carcinogenesis
[29], while 750 ppm oltipraz afforded complete protection against AFB1 -induced hepatocarcinogenesis
in F344 rats [30]. Moreover, dietary concentrations
as low as 100 ppm engendered >90% reduction in the
hepatic burden of presumptive preneoplastic lesions
(GST pi-positive foci) in the aflatoxin model [31].
3. Mechanisms of phase 2 enzyme
induction by dithiolethiones
Initial molecular studies in rats and subsequent studies in humans indicated that increases in mRNA and
protein levels of several phase 2 genes in response
to dithiolethiones and other chemoprotective agents
were mediated through the transcriptional activation
of these genes [32,33]. Two families of phase 2 enzyme inducers exist. Prochaska and Talalay [34] have
coined the terms bifunctional and monofunctional inducers to describe these families. Bifunctional inducers (e.g. polycyclic hydrocarbons, dioxins, azo dyes,
flavones) can all be characterized as large planar polycyclic aromatics and elevate phase 2 as well as selected
phase 1 enzymatic activities such as aryl hydrocarbon hydroxylase. These compounds are potent ligands
for the aryl hydrocarbon (Ah) receptor, and the direct
participation of the Ah receptor in the activation of
aryl hydrocarbon hydroxylase gene transcription has
been demonstrated [35]. Moreover, since phase 2 enzyme inducibility by bifunctional inducers segregates
in mice that possess functional Ah receptors, it is presumed that these enzymes are under the direct control
of the Ah receptor. Monofunctional inducers (phenols,
lactones, isothiocyanates, dithiocarbamates, dithiins,
and 1,2-dithiole-3-thiones) elevate phase 2 enzymatic
activities without significantly elevating the aforementioned phase 1 activities and do not possess an obvious
defining structural characteristic. Several, but not all,
phase 2 enzyme inducers have also been shown to inhibit the activities of some cytochrome P450 enzymes
[36,37], but the contribution of this component to their
chemoprotective actions in vivo may be limited [38].
There is no evidence at this time to suggest
that monofunctional inducers function through a
receptor-mediated pathway. However, Talalay et al.
[39] have identified a chemical signal present in some
monofunctional inducers: the presence or acquisition
of an electrophilic center. Many monofunctional inducers are Michael reaction acceptors (e.g. olefins
or acetylenes conjugated to an electron withdrawing
group such as a carbonyl function) and potency is
generally paralleled by their efficiency as Michael
reaction acceptors. These generalizations can account
for the inducer activity of many types of chemopreventive agents and have led to the identification
of other novel classes of inducers, including acrylates, fumarates, maleates, vinyl ketones and vinyl
sulfones. Other classes of monofunctional inducers,
notably peroxides, vicinal dimercaptans, heavy metals, arsenicals and the 1,2-dithiole-3-thiones, exhibit
a common capacity for reaction with sulfydryls by
either oxidoreduction or alkylation [40].
Several regulatory elements controlling the expression and inducibility of the Ya subunit of rodent
GSTs by bifunctional and monofunctional inducers
have been characterized [41,42]. A 41 bp element in
the 5 -flanking region of the rat GST Ya gene, termed
the “antioxidant response element” (ARE) has been
identified. To date, AREs have been detected in the
promoters of nearly a score of genes. All share a
common RTGACnnnGC motif [43]. Prestera et al.
[40] observed that members of eight distinct chemical
classes of monofunctional inducers stimulate expression of a reporter gene, growth hormone, through
the ARE when an ARE-growth hormone construct is
transfected into murine hepatoma cells. Comparisons
of potency for induction of reporter gene expression and NQO1 activity in the same cells indicated
a striking concordance over a 4-log range for the
two endpoints. Further, when 25 dithiolethiones and
related analogs were evaluated for their activities as
inducers of NQO1 and as activators of the transfected ARE construct in this model system, a strong
correlation was seen in the potencies of 21 active
1,2-dithiole-3-thiones to elicit the two responses [44].
Moreover, no dithiolethiones were inactive in only
M.-K. Kwak et al. / Mutation Research 480–481 (2001) 305–315
one system. Collectively, these results suggest that the
ARE mediates most, if not all, of the phase 2 enzyme
inducer activity of these compounds.
The transcription factors that bind to the ARE consensus sequence have not been fully identified and
are likely to vary between cell types and species. Nrf1
and Nrf2, members of the basic-leucine zipper NF-E2
family of transcription factors that regulate expression
of globin genes during erythroid development [45,46]
are known to bind and activate the ARE. Overexpression of either Nrf1 or Nrf2 in human hepatoma
cells enhances the basal and inducible transcriptional
activity of an ARE reporter gene [47]. Because other
basic-leucine zipper transcription factors typically
form heterodimers, Nrf1/Nrf2 may also dimerize
with other factors in order to activate the ARE. The
tissue specific expression profiles of a number of
transcription factors suggest that an Nrf2/small Maf
heterodimer best mirrors the pattern for induction of
phase 2 genes in vivo. Using recombinant Nrf2 and
mafK proteins in an electromobility shift assay with
the promoter sequence of the murine GST Ya gene,
Yamamoto et al. [48] demonstrated binding of the heterodimer complex to this promoter. Oligonucleotides
containing the ARE effectively competed for the
binding of this heterodimeric complex to the GST Ya
promoter. This issue has also been directly examined
309
by exploring the effects of disruption of the nrf2 gene
in vivo on induction of phase 2 enzymes [48]. Dithiolethiones elevated transcript levels, protein levels and
activities of multiple phase 2 genes in livers of wild
type mice, but not in the homozygous nrf2-mutant
mice [49]. Table 1 indicates the effects of most potent
and effective dithiolethione, 3H-1,2-dithiole-3-thione
(D3T), on induction of NQO1 and several isoforms of
GSTs in these mice. Inducibility of phase 2 enzyme
activities was blunted in the knockout compared to
wild-type mice. Also of note, basal expression of
overall GST activity as well as mRNA levels for several isoforms were diminished in the nrf2-deficient
mice. The functional consequences of the loss of
expression of the Nrf2 transcription factor are highlighted in Fig. 1, in which the knockout mice show a
diminished propensity to excrete the phase 2 metabolite aflatoxin–mercapturic acid compared to wild-type
mice and exhibit concomitantly higher levels of hepatic DNA adducts following administration of the hepatocarcinogen AFB1 . Diminished basal expression of
some GSTs in the knockout mice appears to enhance
DNA damage by AFB1 . Recently, Ramos-Gomez et
al. showed that the multiplicity of gastric neoplasia
induced by benzo[a]pyrene was increased significantly in nrf2-deficient mice compared to wild-type
mice. Oltipraz significantly reduced the gastric
Table 1
Effect of nrf2 genotype on basal and inducible NQO1 and GST activities and levels of mRNA for NQO1 and GST subunitsa
Genotype
Vehicle-treated
Enzyme activity (nmol/min/mg protein)
Levels of mRNA
D3T-treated
+/+
−/−
GST
NQO1
6.34
6.10
3.32b
+/+
9.84b
−/−
8.36
22.90b
4.08d
9.50d
GST Ya
GST Yb
GST Yc
GST Yp
NQO1
1
1
1
1
1
0.63
1.26
0.75
0.28
1.11
6.38b
2.63b
1.07
4.93b
5.95b
0.79
0.87
0.29c
0.66c
0.67
a GST activity was measured in hepatic cytosols prepared from male wild-type or nrf2-deficient mice pretreated with vehicle or D3T
(0.3 mmol/kg, p.o.) for 3 consecutive days using chlorodinitrobenzene as substrate. Relative mRNA levels of GST subunits were analyzed
24 h after a single treatment with vehicle or D3T (0.5 mmol/kg) using northern blot hybridization. Levels of RNA for each gene were
normalized to albumin mRNA levels and expressed as a ratio over vehicle-treated, wild-type control. Each value is the mean of three
individual animals.
b P < 0.05 compared to vehicle-treated +/+ mice.
c P < 0.05 compared to vehicle-treated −/− mice.
d P < 0.05 compared to D3T-treated +/+ mice.
310
M.-K. Kwak et al. / Mutation Research 480–481 (2001) 305–315
Fig. 1. Effect of Nrf2 genotype on aflatoxin–DNA adduct formation
and mercapturic acid excretion. Male wild-type and nrf2-deficient
mice were treated with 0.5 mmol 1,2-dithiole-3-thione/kg, p.o.,
or vehicle 48 h prior to dosing with 250 mg/kg aflatoxin
B1 . Mice were either killed 2 h later for measurement
of hepatic DNA adduct levels (REF) or housed in glass
metabolism cages for 24 h for urine collection. Urinary levels of
aflatoxin–mercapturic acid (AFB–NAC) were determined by sequential immunoaffinity-LC–MS chromatography (REF).
neoplasia in wild-type mice by 55% but had no effect
in nrf2-deficient mice. In parallel with these results,
constitutive and inducible expression of GST and QR
in forestomach were greatly reduced in nrf2-deficient
mice compared to wild-type mice [50]. Thus, this
result supports that the nrf2 knockout mouse will be
associated with enhanced susceptibility to environmental carcinogenesis by altering the expression of
detoxifying enzymes.
In comparing the human and chicken Nrf2 amino
acid sequences, Itoh et al. [51] found six highly conserved regions and named these homology domains
Neh1 to Neh6 (Nrf2-ECH homology). To identify the
transcription activation domain(s) of Nrf2, various
segments of Nrf2 were fused to the DNA binding domain of the yeast transcription factor Gal4 to generate
a series of Gal4-Nrf2 chimeric proteins. Two independent activation domains (Neh4 and Neh5) were
identified. Follow-up studies with deletion mutants in
this system indicated that the Neh2 deletion mutant
was a much more potent transactivator than wild-type
Nrf2, suggesting that the Neh2 domain interacts with
a cellular protein that acts to repress Nrf2 activity. By
using a Gal4-Neh2 fusion protein as bait in a yeast
two-hybrid system with a mouse embryo prey library,
multiple clones for a single protein were captured. Inspection of the conceptually translated primary amino
acid sequence of the newly cloned cDNA revealed the
presence of two canonical protein interaction motifs:
a BTB domain and a double glycine repeat. Database
searches revealed that this unusual combination of
motifs is characteristic of the Drosophila cytoskeleton
binding protein Kelch leading to the naming of this
protein Keap1 (Kelch-like ECH-associated protein).
Transient cotransfection of a reporter for monitoring
Nrf2 transactivation with either wild-type or mutant
nrf2 in the presence of increasing concentrations of
Keap1 expression plasmid indicated that Keap1 could
completely repress the activity of wild-type Nrf2
[51]. However, when Keap1 was cotransfected with
Nrf2 lacking the Neh2 domain, the transactivation
potential of the mutant Nrf2 protein was not affected.
These data demonstrate unambiguously that the repression of Nrf2 by Keap1 is strictly dependent on
the presence and integrity of the Neh2 domain.
Sulfydryl reactive reagents abrogate Keap1 repression of Nrf2 activity. A model system was developed in which QT6 fibroblasts were transfected with
Keap1 and Nrf2 expression plasmids as well as the
reporter plasmid for monitoring Nrf2 activation [51].
The addition of diethyl maleate (DEM) to the culture medium resulted in restoration of Nrf2 activity,
despite the presence of Keap1, in a dose-dependent
manner. The cellular consequences of DEM treatment
were also examined immunochemically by means
Fig. 2. Effect of 1,2-dithiole-3-thione (D3T) and ␤-napthoflavone
(BNF) on hepatic accumulation of Nrf2. Hepatic nuclear extracts
were prepared from livers of male wild-type mice and electrophoresed on a 6% acrylamide gel. Immunoblot analysis was
carried out using an Nrf2 antibody reacting with the N-terminal
of Nrf2. Each sample was prepared from four pooled livers at
6 h after treatment with D3T (0.1 or 0.5 mmol/kg, p.o.) or BNF
(0.2 mmol/kg, p.o.).
M.-K. Kwak et al. / Mutation Research 480–481 (2001) 305–315
Fig. 3. Proposed pathway for induction of phase 2 and antioxidative
enzymes by dithiolethiones. See text for details.
using an anti-Nrf2 antibody. Nrf2 localization was
primarily cytoplasmic when a Keap1 expression plasmid was transfected into the cells. Consistent with the
cotransfection/transactivation assay results, addition
of DEM to the culture medium led to the localization
of Nrf2 in the nucleus, even in the presence of transfected Keap1. As shown in Fig. 2, treatment of mice
with D3T leads to a rapid accumulation of Nrf2 in
hepatic nuclei. Collectively, these studies provide indirect support for the hypothesis that the Keap1/Nrf2
complex constitutes a cytoplasmic sensor system for
enzyme induction by electrophiles and oxidants.
A general scheme for the actions of Keap1 and Nrf2
in regulating gene expression by phase 2 enzyme inducers is shown in Fig. 3. Cysteine residues in Keap1
may serve as molecular sensors for induction. Oxidation or alkylation of these sulfydryls appears to lead
to dissociation of Nrf2 from Keap1, presumably allowing for its translocation to the nucleus where it
can interact with the ARE to activate transcription.
Post-translational modification of Nrf2 through phosphorylation may be important in signaling as pharmacological or genetic manipulation of MAP kinases and
protein kinase C affects enzyme induction [52–54].
Finally, Nrf2 binds with other transcription factors including several members of the small Maf family to
activate gene transcription through the ARE.
4. Clinical studies with dithiolethiones
Phase I clinical trials are designed to characterize
the pharmacokinetics and tolerableness of the chemo-
311
preventive agent [55]. Dose and schedule of administration are based on achieving plasma drug levels that
are very likely to be safe and likely to show effectiveness based upon preclinical studies in in vivo and in
vitro models. Single dose phase I studies with oltipraz
indicated that administration of 500 mg orally would
produce a peak plasma concentration of about 20 ␮M
while 125 mg produced a peak of only 2 ␮M [56].
Dose escalation studies with repeated administration
suggested that 125 mg oltipraz was close to the maximum tolerated dose following administration daily for
6 months [57]. Although the steady-state concentrations of oltipraz are rather low, reflecting the rapid
clearance of the drug from the body, the peak concentrations following administration of 125–500 mg
oltipraz/day are comparable to those required to induce phase 2 enzyme expression in rodent and human cell culture models. Gupta et al. [56] reported a
doubling in the specific activity of GST in peripheral
lymphocytes obtained from phase I study participants
10 h after administration of 125 mg oltipraz. Elevations in levels of glutathione were also observed. In a
dose-finding study with 125, 250, 500 or 1000 mg/m2
oltipraz as a single oral dose, increases in GST activities were seen in peripheral mononuclear cells and
colon mucosa biopsies at the lower, but not higher,
doses [58]. Four to five-fold increases in mRNA transcripts for ␥-glutamylcysteine synthetase and quinone
reductase were seen in colon mucosa at 250 mg/m2 .
Higher doses were not more effective. mRNA content
increased after dosing to reach a peak on day 2 and
declined to baseline levels over the subsequent week.
Collectively, these results demonstrate that oltipraz
triggers the expression of phase 2 enzymes in humans.
To more directly test the hypothesis that oltipraz
can modulate the metabolism of carcinogens in humans, we conducted a phase IIa intervention trial
with oltipraz. The primary goals of phase IIa studies,
in addition to establishing the general feasibility of
conducting biomarker measurements, are to characterize the dose-response of biomarker modulation, the
tolerance or loss of effect of biomarker modulation
over time, and drug toxicity with chronic administration [55]. Study participants for this phase IIa
trial were recruited from residents of Daxin Township, Qidong, PR China, where dietary exposures to
aflatoxins and risk for hepatocellular carcinoma are
312
M.-K. Kwak et al. / Mutation Research 480–481 (2001) 305–315
high [59]. This trial with oltipraz was a randomized,
placebo-controlled, double-blind study. Two-hundred
forty adults in good general health without any history of major chronic illnesses and with detectable
serum aflatoxin–albumin adduct levels at baseline
were randomized into one of three intervention arms
(A) placebo; (B) 125 mg oltipraz administered daily;
or (C) 500 mg oltipraz administered weekly. The
methods, participant characteristics, compliance and
adverse events as well as initial results on modulation of biomarkers from this trial have been reported
[60–62].
Urine samples were collected at 2-week intervals
throughout the active 8-week intervention period
as well as during the 8-week follow-up period. To
date, aflatoxin metabolites have been assayed in
urine samples from one cross-section in time: after
the first month on the active intervention [62]. Sequential immunoaffinity and liquid chromatography
coupled to mass spectrometry and fluorescence detection were used to identify and quantify the phase
1 metabolite, AFM1 , and the phase 2 metabolite,
aflatoxin–mercapturic acid, in these urine samples.
One month of weekly administration of 500 mg
oltipraz led to a significant decrease (51%) in median
levels of AFM1 excreted in urine compared to placebo,
but had no effects on levels of aflatoxin–mercapturic
acid. By contrast, daily intervention with 125 mg
of oltipraz led to a significant, 2.6-fold increase in
the median levels of aflatoxin–mercapturic acid excretion, but had no pronounced effect on excreted
AFM1 levels. Thus, sustained low dose oltipraz increased phase 2 conjugation of aflatoxin, yielding
higher levels of mercapturic acid, but did not appreciably affect formation of AFM1 . Intermittent,
high dose oltipraz inhibited the phase 1 activation
of aflatoxin, as reflected by lowered excretion of
AFM1 . Potential effects of induction of phase 2 enzymes, i.e. GSTs, in this arm appears to be masked
by the inhibition of aflatoxin–8,9-epoxide formation. Indeed, Langouët et al. [36] have reported two
to four-fold increases in the protein levels of alpha
and mu classes of GSTs in primary human hepatocytes treated with 50 ␮M oltipraz, but found this
inductive effect was not associated with an increased
formation of aflatoxin–glutathione conjugates because it was overridden by the inhibitory effect of
oltipraz on AFB1 activation. As previously noted
in experimental models, it would appear that both
mechanisms are likely to contribute to reduced genotoxicity and other chemopreventive actions of this
drug.
In addition to the cross-sectional measurements
of effects of oltipraz on urinary aflatoxin metabolites, longitudinal analyses of effects of slopes of
aflatoxin–albumin adducts have been conducted [60].
There were no consistent changes in albumin-adduct
levels in the placebo arm, nor in the 125 mg oltipraz
daily arm over the 16 week observation period. However, individuals receiving 500 mg oltipraz once a
week for 8 weeks showed a triphasic response to
oltipraz. No effect was observe during the first month
of the intervention, whereas a significant diminution in adduct levels was observed during the second
month of active intervention and during the first month
of follow-up. A partial rebound in adduct levels toward baseline values was observed during the second
month of follow-up. Linear regression models up to
week 13 confirmed a significant weekly decline in
biomarker levels in this group. Because modulation of
aflatoxin–albumin adducts and diminution of AFM1
levels were both observed in the 500 mg weekly arm,
comparisons of the albumin adduct slopes with levels
of AFM1 were made. Individuals ranked in the lowest
tertile of AFM1 levels showed the greatest decline in
aflatoxin–albumin adduct levels. This moderate correlation suggests that inhibition of cytochrome P450
activity could contribute to the observed decline in
albumin adducts.
The bigger question of whether modulation of carcinogen metabolism, either by enzyme inhibition or
enzyme induction, can substantively reduce the risk of
cancer in individuals at high risk for exposure to environmental carcinogens remains open. A phase IIb intervention trial with oltipraz in Qidong was conducted
in 1999 and 2000 to evaluate the efficacy of 250 or
500 mg oltipraz given weekly to modulate levels of
aflatoxin biomarkers over a 1-year-period in comparison to a placebo group. Biomarkers exploring the multiple mechanisms of action of oltipraz are being used.
The phase IIb study should serve as a foundation for
selecting a safe and effective dose for a phase III trial.
Phase III trials are used to actually establish the efficacy of the drug in chemoprevention, and unless the
biomarker is a strong predictor of cancer prevention,
rely on reduced incidence of disease as the endpoint.
M.-K. Kwak et al. / Mutation Research 480–481 (2001) 305–315
Acknowledgements
We gratefully acknowledge support for our work
in protection against cancer from the National Cancer
Institute (CA39416, CA44530, CA77130 and Center
Grant CA06973) and the National Institute of Environmental Health Sciences (ES06052 and Center Grant
ES03819).
References
[1] L.W. Wattenberg, Chemoprevention of cancer, Cancer Res.
45 (1985) 1–8.
[2] S. DeFlora, C. Ramel, Mechanisms of inhibitors of
mutagenesis and carcinogenesis. Classification and overview,
Mutat. Res. 202 (1988) 285–306.
[3] W.K. Hong, M.B. Sporn, Recent advances in chemoprevention
of cancer, Science 278 (1997) 1073–1077.
[4] G.K. Kelloff, E.T. Hawk, J.A. Crowell, C.W. Boone, V.E.
Steele, R.A. Lubet, C.C. Sigman, Progress in clinical
chemoprevention, Sem. Oncol. 24 (1997) 241–252.
[5] E.C. Miller, J.A. Miller, Some historical perspectives on the
metabolism of xenobiotic chemicals to reactive intermediates,
in: M.W. Anders (Ed.), Bioactivation of Foreign Compounds,
Academic Press, New York, 1985, pp. 1–28.
[6] A.H. Conney, Induction of microsomal enzymes by
foreign chemicals and carcinogenesis by polycyclic aromatic
hydrocarbons. G.H.A. Clowes Memorial Lecture, Cancer Res.
42 (1982) 4875–4917.
[7] J. Wilkinson IV., M.L. Clapper, Detoxication enzymes and
chemoprevention, Proc. Soc. Exp. Biol. Med. 216 (1997)
192–200.
[8] W.H. Lee, R.A. Morton, J.I. Epstein, J.D. Brooks, P.A.
Campbell, G.S. Bova, W.S. Hsich, W.B. Isaacs, W.G. Nelson,
Cytidine methylation of regulatory sequences near the pi-class
glutathione S-transferase gene accompanies human prostatic
carcinogenesis, Proc. Natl. Acad. Sci. U.S.A. 91 (1994)
11733–11737.
[9] C.P. Nelson, L.R. Kidd, J. Saubageot, W.B. Issacs, A.
DeMarzo, J.D. Groopman, W.G. Nelson, T.W. Kensler,
Protection
against
2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyrimidine (N–OH–PhIP) cytotoxicity and
DNA adduct formation in human prostate by glutathione
S-transferase P1, Cancer Res. 61 (2001) 103–109.
[10] A.J. Townsend, W.R. Fields, R.L. Haynes, A.J. Doss, Y.
Li, C.S. Johannes-Doehmer, C.S. Morrow, Chemoprotective
functions of glutathione S-transferases in cell lines induced
to express specific isozymes by stable transfection,
Chemico-Biol. Int. 111/112 (1998) 3889–4070.
[11] N. Ban, Y. Takahaski, T. Takayama, T. Kura, T. Katahira, S.
Sakamaki, Y. Niitsu, Transfection of glutathione S-transferase
(GST)-pi antisense complementary DNA increases the
sensitivity of a colon cancer cell line to adriamycin, cisplatin,
melphalan, and etoposide, Cancer Res. 56 (1996) 3577–3582.
313
[12] C.J. Henderson, A.G. Smith, J. Urf, K. Brown, E.J. Bacon,
C.R. Wolf, Increased skin tumorigenesis in mice lacking pi
class glutathione S-transferases, Proc. Natl. Acad. Sci. U.S.A.
95 (1998) 5275–5280.
[13] D.H. Monroe, D.L. Eaton, Comparative effects of butylated
hydroxyanisole on hepatic in vivo DNA binding and in
vitro biotransformation of aflatoxin B1 in the rat and mouse,
Toxicol. Appl. Pharmacol. 90 (1990) 401–409.
[14] S. Landi, Mammalian class theta GST and differential
susceptibility to carcinogens, Mutat. Res. 463 (2000) 247–
283.
[15] L.K.T. Lam, V.L. Sparnins, L.W. Wattenberg, Isolation and
identification of kahweol palmitate and cafestrol palmitate
as active constituents of green coffee beans that enhance
glutathione S-transferase activity in the mouse, Cancer Res.
42 (1982) 1193–1198.
[16] Y. Zhang, P. Talalay, C.-G. Cho, G.H. Posner, A major
inducer of anticarcinogenic protective enzymes from broccoli:
isolation and elucidation of structure, Proc. Natl. Acad. Sci.
U.S.A. 89 (1992) 2399–2403.
[17] Y. Zhang, T. Kensler, C.-G. Cho, G.H. Posner, P. Talalay,
Anticarcinogenic activities of sulforaphane and structurally
related synthetic norbornyl isothiocyanates, Proc. Natl. Acad.
Sci. U.S.A. 91 (1994) 3147–3150.
[18] J.W. Fahey, Y. Zhang, P. Talalay, Broccoli sprouts: an
exceptionally rich source of inducers of enzymes that protect
against chemical carcinogens, Proc. Natl. Acad. Sci. U.S.A.
94 (1997) 10367–10372.
[19] E. Bueding, P. Dolan, J.-P. Leroy, The antischistosomal
activity of oltipraz, Res. Commun. Chem. Pathol. Pharmacol.
37 (1982) 293–303.
[20] T. Primiano, J.A. Gastel, T.W. Kensler, T.R. Sutter, Isolation
of cDNAs representing dithiolethione-responsive genes,
Carcinogenesis 17 (1996) 2297–2303.
[21] T. Primiano, Y. Li, T.W. Kensler, M.A. Trush, T.R.
Sutter, Identification of dithiolethione-inducible gene-1
as a leukotriene B4 12-dehydrogenase: implications for
chemoprevention, Carcinogenesis 19 (1998) 999–1005.
[22] M. Otieno, T.W. Kensler, K. Guyton, Chemoprotective
3H-1,2-dithiole-3-thione induces antioxidant genes in vivo,
Free Rad. Biol. Med. 28 (2000) 944–952.
[23] S.S. Ansher, P. Dolan, E. Bueding, Chemoprotective effects
of two dithiolthiones and of butylhydroxyanisole against
carbon tetrachloride and acetaminophen toxicity, Hepatology
3 (1983) 932–935.
[24] M.H. Davies, G.J. Schamber, R.C. Schnell, Oltipraz-induced
amelioration of acetaminophen hepatotoxicity in hamsters. I.
Lack of dependence on glutathione, Toxicol. Appl. Pharmacol.
109 (1991) 17–28.
[25] L.-Y. Liu, B.D. Roebuck, J.D. Yager, J.D. Groopman,
T.W. Kensler, Protection by 5-(2-pyrazinyl)-4-methyl-1,2dithiol-3-thione (oltipraz) against the hepatotoxicity of
aflatoxin B1 in the rat, Toxicol. Appl. Pharmacol. 93 (1988)
442–451.
[26] L.W. Wattenberg, E. Bueding, Inhibitory effects of 5(2-pyrazinyl)-4-methyl-1,2-dithiol-3-thione (oltipraz) on carcinogenesis induced by benzo[a]pyrene, diethylnitrosamine
and uracil mustard, Carcinogenesis 7 (1986) 1379–1381.
314
M.-K. Kwak et al. / Mutation Research 480–481 (2001) 305–315
[27] G.J. Kelloff, J.A. Crowell, C.W. Boone, V.E. Steele, R.A.
Lubet, P. Greenwald, C.S. Alberts, J.M. Covey, L.A. Doody,
G.G. Knapp, S. Nayfield, D.R. Parkinson, V.K. Prasad, P.C.
Prorok, E.A. Sausville, C.C. Sigman, Strategy and planning
for chemopreventive drug development: clinical development
plans, J. Cell. Biochem. 20S (1994) 55–299.
[28] T.W. Kensler, K.J. Helzlsouer, Oltipraz: clinical opportunities
for chemoprevention, J. Cell. Biochem. 22S (1995) 101–107.
[29] C.V. Rao, A. Rivenson, M. Katiwalla, G.J. Kelloff, B.S.
Reddy, Chemopreventive effect of oltipraz during different
stages of experimental colon carcinogenesis induced by
azoymethane in male F344 rats, Cancer Res. 53 (1993) 2502–
2506.
[30] B.D. Roebuck, Y.-L. Liu, A.R. Rogers, J.D. Groopman,
T.W. Kensler, Protection against aflatoxin B1 -induced hepatocarcinogenesis in F344 rats by 5-(2-pyrazinyl)-4methyl-1,2-dithiole-3-thione (oltipraz): predictive role for
short-term molecular dosimetry, Cancer Res. 51 (1991) 5501–
5506.
[31] T.W. Kensler, P.A. Egner, P.M. Dolan, J.D. Groopman,
B.D. Roebuck, Mechanism of protection against aflatoxin
tumorigenicity in rats fed 5-(2-pyrazinyl)-4-methyl-1,2dithiol-3-thione (oltipraz) and related 1,2-dithiol-3-thiones
and 1,2-dithiol-3-ones, Cancer Res. 47 (1987) 4271–4277.
[32] T.H. Rushmore, R.G. King, K.E. Paulson, C.B. Pickett,
Regulation of glutathione S-transferase Ya subunit gene
expression: identification of a unique xenobiotic-responsive
element controlling inducible expression by planar aromatic
compounds, Proc. Natl. Acad. Sci. U.S.A. 87 (1990) 3826–
3830.
[33] W.B. Pearson, J.J. Windle, J.F. Morrow, A.B. Benson III,
P. Talalay, Increased synthesis of glutathione S-transferase
in response to anticarcinogenic antioxidants. Cloning and
measurement of messenger RNA, J. Biol. Chem. 258 (1983)
2052–2062.
[34] H.J. Prochaska, P. Talalay, Regulatory mechanisms of
monofunctional and bifunctional anticarcinogenic enzyme
inducers in murine liver, Cancer Res. 48 (1988) 4776–4782.
[35] J.M. Fisher, L. Wu, M.S. Denison, J.P. Whitlock Jr.,
Organization and function of a dioxin-responsive enhancer,
J. Biol. Chem. 265 (1990) 9676–9681.
[36] S. Langouët, B. Coles, F. Morel, L. Becquemont, P. Beaune,
F.P. Guengerich, B. Ketterer, A. Guillouzo, Inhibition of
CYP1A2 and CYP3A4 by oltipraz results in reduction of
aflatoxin B1 metabolism in human hepatocytes in primary
culture, Cancer Res. 55 (1995) 5574–5579.
[37] S. Langouët, L.L. Furge, N. Kerriguy, K. Nakamura, A.
Guillouzo, F.P. Guengerich, Inhibition of human cytochrome
P450 enzymes by 1,2-dithiole-3-thione, oltipraz and its
derivatives, and sulforaphane, Chem. Res. Toxicol. 13 (2000)
245–252.
[38] T. Primiano, P.A. Egner, T.R. Sutter, G.J. Kelloff, B.D.
Roebuck, T.W. Kensler, Intermittent dosing with oltipraz:
relationship between chemoprevention of aflatoxin-induced
tumorigenesis and induction of glutathione S-transferases,
Cancer Res. 55 (1995) 4319–4324.
[39] P. Talalay, M.J. DeLong, H.J. Prochaska, Identification of a
common chemical signal regulating the induction of enzymes
[40]
[41]
[42]
[43]
[44]
[45]
[46]
[47]
[48]
[49]
[50]
[51]
[52]
that protect against chemical carcinogenesis, Proc. Natl. Acad.
Sci. U.S.A. 85 (1988) 8261–8265.
T. Prestera, W.D. Holtzclaw, Y. Zhang, P. Talalay,
Chemical and molecular regulation of enzymes that detoxify
carcinogens, Proc. Natl. Acad. Sci. U.S.A. 90 (1993) 2965–
2969.
R.S. Friling, A. Bensimon, Y. Tichauer, V. Daniel, Xenobioticinducible expression of murine glutathione S-transferase Ya
subunit gene is controlled by an electrophile-responsive
element, Proc. Natl. Acad. Sci. U.S.A. 87 (1990) 6258–6262.
A.K. Jaiswal, Antioxidant response element, Biochem.
Pharmacol. 48 (1994) 439–444.
W.W. Wasserman, W.D. Fahl, Functional antioxidant
responsive elements, Proc. Natl. Acad. Sci. U.S.A. 94 (1997)
5361–5366.
P.A. Egner, T.W. Kensler, T.T. Prestera, P. Talalay, A.H.
Libby, H.H. Joyner, T.J. Curphey, Regulation of phase
2 enzyme induction by oltipraz and other dithiolethiones,
Carcinogenesis 15 (1994) 177–181.
J.Y. Chan, X. Han, Y.W. Kan, Cloning of Nrf1, an
NF-E2-related transcription factor, by genetic selection in
yeast, Proc. Natl. Acad. Sci. U.S.A. 90 (1993) 11366–11370.
P. Moi, K. Chan, I. Asunis, A. Cao, Y.W. Kan, Isolation of
NF-E2-related factor 2 (Nrf2), a NF-E2-like basic leucine
zipper transcriptional activator that binds to the tandem
NF-E2/AP1 repeat of the ␤-globin locus control region, Proc.
Natl. Acad. Sci. U.S.A. 91 (1993) 9926–9930.
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. U.S.A. 93 (1996)
14960–14965.
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.
M.-K. Kwak, K. Itoh, M. Yamamoto, T.R. Sutter, T.W.
Kensler, Role of transcription factor Nrf2 in the induction
of hepatic phase 2 and antioxidative enzymes in vivo by the
chemopreventive agent, 3H-1,2-dithiole-3-thione, Mol. Med.
7 (2001) 135–145.
M. Ramos-Gomez, M.-K. Kwak, P.M. Dolan, K. Itoh,
M. Yamamoto, P. Talalay, T.W. Kensler, Sensitivity to
carcinogenesis in increased and chemoprotective efficacy of
enzymes inducers is lost in nrf2 transcription factor-deficient
mice, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 3410–3415.
K. Itoh, N. Wakabayashi, Y. Katoh, T. Ishii, K. Igarashi, J.D.
Engel, M. Yamamoto, Keap1 represses nuclear activation of
antioxidant responsive elements by Nrf2 through binding to
the amino-terminal Neh2 domain, Genes Dev. 13 (1999) 76–
86.
R. Yu, W. Lei, S. Mandlekar, M.J. Weber, C.J. Der, J. Wu,
A.-N.T. Kong, Role of a mitogen-activated protein kinase
pathway in the induction of phase II detoxifying enzymes by
chemicals, J. Biol. Chem. 274 (1999) 27545–27552.
M.-K. Kwak et al. / Mutation Research 480–481 (2001) 305–315
[53] R. Yu, S. Mandlekar, W. Lei, W.E. Fahl, T.-H. Tan, A.N.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.
[54] H.-C. Huang, T. Nguyen, C.B. Pickett, Regulation of the
antioxidant response element by protein kinase C-mediated
phosphorylation of NF-E2-related factor 2, Proc. Natl. Acad.
Sci. U.S.A. 97 (2000) 12475–12480.
[55] G.J. Kelloff, J.R. Johnson, J.A. Crowell, C.W. Boone, J.J.
DeGeorge, V.E. Steele, M.U. Mehta, J.W. Temeck, W.J.
Schmidt, G. Burke, P. Greenwald, R.J. Temple, Approaches to
the development and marketing approval of drugs that prevent
cancer, Cancer Epidemiol. Biomarkers Prev. 4 (1995) 1–10.
[56] E. Gupta, O.I. Olopade, M.J. Ratain, R. Mick, T.M. Baker,
F.K. Berezin, A.B. Benson III, M.E. Dolan, Pharmacokinetics
and pharmacodynamics of oltipraz as a chemopreventive
agent, Clin. Cancer Res. 1 (1995) 1133–1138.
[57] A.B. Benson III, Oltipraz: a laboratory and clinical review,
J. Cell. Biochem. 17F (1993) 278–291.
[58] P.J. O’Dwyer, C.E. Szarka, K.-S. Yao, T.C. Halbherr, G.R.
Pfeiffer, F. Green, J.M. Gallo, J. Brennan, H. Frucht, E.B.
Goosenberg, T.C. Hamilton, S. Litwin, A.M. Balshem, P.F.
Engstrom, M.L. Clapper, Modulation of gene expression in
subjects at risk for colorectal cancer by the chemopreventive
dithiolethione oltipraz, J. Clin. Invest. 98 (1996) 1210–1217.
[59] J.-S. Wang, G.-S. Qian, A. Zarba, X. He, Y.-R. Zhu, B.-C.
Zhang, L. Jacobson, S.J. Gange, A. Muñoz, T.W. Kensler, J.D.
Groopman, Temporal patterns of aflatoxin–albumin adducts
in hepatitis B surface antigen-positive and antigen-negative
residents of Daxin, Qidong County, People’s Republic
315
of China, Cancer Epidemiol. Biomarkers Prev. 5 (1996)
253–261.
[60] L.P. Jacobson, B.-C. Zhang, Y.-R. Zhu, J.-B. Wang, Y. Wu,
Q.-N. Zhang, L.-Y. Yu, G.-S. Qian, S.-Y. Kuang, Y.-F. Li,
X. Fang, A. Zarba, B. Chen, C. Enger, N.E. Davidson,
M.B. Gorman, G.B. Gordon, H.J. Prochaska, P.A. Egner, J.D.
Groopman, A. Muñoz, K.J. Helzlsouer, T.R. Kensler, Oltipraz
chemoprevention trial in Qidong, People’s Republic of China:
study design and clinical outcomes, Cancer Epidemiol.
Biomarkers Prev. 6 (1997) 257–265.
[61] T.W. Kensler, X. He, M. Otieno, P.A. Egner, L.P. Jacobson,
B. Chen, J.-S. Wang, Y.-R. Zhu, B.-C. Zhang, J.-B. Wang, Y.
Wu, Q.-N. Zhang, G.-S. Qian, S.-Y. Kuang, S. Fang, Y.-F. Li,
L.-Y. Yu, H.J. Prochaska, N.E. Davidson, G.B. Gordon, M.B.
Gorman, A. Zarba, C. Enger, A. Muñoz, K.J. Helzlsouer, J.
Groopman, P.A. Egner, L.P. Jacobson, B. Chen, J.-S. Wang,
Y.-R. Zhu, B.-C. Zhang, J.-B. Wang, Y. Wu, Q.-N. Zhang,
G.-S. Qian, S.-Y. Kuang, S. Fang, Y.-F. Li, L.-Y. Yu, H.J.
Prochaska, N.E. Davidson, G.B. Gordon, M.B. Gorman, A.
Zarba, C. Enger, A. Muñoz, K.J. HelzIsouer, J.C. Groopman,
Oltipraz chemoprevention trial in Qidong, People’s Republic
of China: modulation of serum aflatoxin albumin adduct
biomarkers, Cancer Epidemiol. Biomarkers Prev. 7 (1998)
127–134.
[62] J.-S. Wang, X. Shen, X. He, Y.-R. Zhu, B.-C. Zhang, J.B. Wang, G.-S. Qian, S.-Y. Kuang, A. Zarba, P.A. Egner,
L.J. Jacobson, A. Muñoz, K.J. Helzlsouer, J.D. Groopman,
T.W. Kensler, Protective alterations in phase 1 and 2
metabolism of aflatoxin B1 by oltipraz in residents of Qidong,
People’s Republic of China, J. Natl. Cancer Inst. 91 (1999)
347–354.