Interactive effects of nrf2 genotype and oltipraz on benzo[a]pyrene–DNA adducts and tumor yield in mice

Minerva Ramos-Gomez1,*, Patrick M. Dolan1, Ken Itoh2, Masayuki Yamamoto2 and Thomas W. Kensler1,3

1 Department of Environmental Health Sciences, Bloomberg School of Public Health, The Johns Hopkins University, Baltimore, MD 21202, USA and
2 Center for TARA, Tsukuba University, Tsukuba 305, Japan


    Abstract
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
The cancer chemopreventive actions of oltipraz (4-methyl-5-[2-pyrazinyl]-1,2-dithiole-3-thione) have been primarily associated with the induction of phase 2 detoxifying enzymes through transcriptional activation of the antioxidant response element (ARE) in the promoter regions of these genes. The transcription factor Nrf2 has been shown to bind to and activate AREs. Previously, we demonstrated that nrf2-deficient mice had low basal expression of phase 2 enzymes and were substantially more susceptible to benzo[a]pyrene (B[a]P)-induced neoplasia of the forestomach than wild-type. Moreover, loss of Nrf2 abrogated the chemopreventive action of oltipraz, when administered 48 h before B[a]P, an interval allowing maximal induction of many phase 2 enzymes. Oltipraz also inhibits some cytochrome P450s involved in the bioactivation of B[a]P. In the present study we observed that oltipraz had no protective effect on tumor burden in the forestomach of nrf2-deficient mice when administered 1 h before B[a]P, a timeline that selectively optimizes for possible inhibitory effects on cytochrome P450s. To evaluate the role of nrf2 genotype on B[a]P disposition, levels of B[a]P–DNA adducts were measured as tetrols released from DNA isolated from target (forestomach) and non-target tissues (liver) of wild-type and nrf2-deficient mice treated with either vehicle or oltipraz 1 or 48 h before B[a]P. Levels of B[a]P–DNA adducts in forestomach were significantly higher in nrf2-deficient compared with wild-type mice. Oltipraz treatment at 1 or 48 h before B[a]P had no protective effect on forestomach tetrol levels in nrf2-deficient mice, whereas a significant reduction was observed in wild-type mice treated with oltipraz 48 h, but not 1 h, before carcinogen. Combining all treatments and genotypes, there was a strong correlation (R2 = 0.91) between levels of B[a]P–DNA adducts in forestomach and subsequent yield of tumors. In contrast to the results in forestomach, nrf2 genotype did not modify hepatic B[a]P–DNA adduct levels while both oltipraz treatments were protective, suggesting that Nrf2-independent mechanisms (e.g. P450 inhibition) for oltipraz can also occur in vivo in some tissues.

Abbreviations: ARE, antioxidant response element; B[a]P, benzo[a]pyrene; (t)-trans-B[a]P-7,8-diol, (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene; CYP, cytochrome P450; GST, glutathione S-transferase; HPLC, high-pressure liquid chromatography; NQO1, NAD(P)H:quinone oxidoreductase; oltipraz, 4-methyl-5-[2-pyrazinyl]-1,2-dithiole-3-thione; TA, trans-anti-B[a]P-tetrol.


    Introduction
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Cancer chemoprevention is a strategy for cancer control that utilizes chemical agents to block, retard or even reverse the carcinogenic process through the administration of one or several compounds. Many classes of cancer chemopreventive agents including phenolic antioxidants, 1,2-dithiole-3-thiones, isothiocynates, flavones and coumarins are effective at blocking DNA damaging steps in the carcinogenic process by modulating the metabolic activation and detoxication of carcinogenic substances. Oltipraz (4-methyl-5-[2-pyrazinyl]-1,2-dithiole-3-thione), a substituted 1,2-dithiole-3-thione, is known to inhibit experimental tumorigenicity elicited by many structurally diverse carcinogens in numerous target tissues (13). The chemopreventive actions of oltipraz have been largely associated with the induction of phase 2 detoxifying enzymes, such as, glutathione S-transferase (GST), NAD(P)H:quinone oxidoreductase (NQO1) and UDP-glucuronosyltransferase (UGT), as well as epoxide hydrolase (46).

Increased expression of phase 2 detoxifying enzymes is primarily the result of transcriptional activation of their corresponding genes. This increased expression is mediated, in part, by an ARE (antioxidant response element) in their promoter regions (79) and the Nrf2 transcription factor has been shown to bind to and activate ARE sequences of these genes (10,11). Nrf2, a member of the basic-leucine zipper family of transcription factors, has been shown to heterodimerize with members of the small Maf family proteins (1214). The involvement of Nrf2–Maf heterodimers in the ARE-mediated regulation of phase 2 detoxifying enzymes, as well as of oxidative stress responsive proteins, has been confirmed by studies using nrf2 knockout mice. Significantly reduced expression and inducibility of NQO1, GST subunits and {gamma}-glutamylcysteine synthetase in mice lacking Nrf2 indicates an in vivo role of this transcription factor in the regulation of these genes (11,1519).

Studies in nrf2-disrupted mice have demonstrated the important role of Nrf2 in protection against different chemical toxicities, such as acute pulmonary injury by the food preservative butylated hydroxytoluene (20) or by hyperoxic conditions (21); to DNA adduct formation in lung after exposure to diesel exhaust (22); the vulnerability of nrf2 knockout to acetaminophen hepatotoxicity (23,24); and in inflammation during the wound repair process in skin (25). In addition to these findings, we reported previously that nrf2-deficient mice were more susceptible to benzo[a]pyrene (B[a]P)-induced neoplasia of the forestomach than wild-type mice. This increased susceptibility was associated with low basal expression of several phase 2 gene transcripts as well as low total GST and NQO1 enzymatic activities in the stomach (26). Moreover, we observed that loss of expression of Nrf2 completely abrogated the chemopreventive actions of oltipraz when administered 48 h before B[a]P, an interval allowing maximal induction of many phase 2 enzymes. Using a similar experimental system, Fahey et al. (27) observed that sulforaphane, an isothiocyanate isolated from cruciferous vegetables, did not reduce the number of gastric tumors in nrf2-disrupted mice, but had a protective effect in wild-type mice when administered in the diet before and during B[a]P exposure. Collectively, these results highlight the prime importance of elevated phase 2 gene expression in the cancer chemopreventive actions of oltipraz and sulforaphane.

Nonetheless, multiple studies have shown that oltipraz is also an inhibitor of some cytochrome P450 enzymes, such as CYP1A1/2, CYP1B1, CYP3A4 and CYP2E1 (2831). A time-course analysis in primary cultures of rat hepatocytes showed that inhibition of ethoxyresorufin-O-deethylase (EROD) activity (associated with CYP1A1/2) by oltipraz was detectable after 40 min and maximal between 2 and 12 h of exposure (28). Inhibition of the bioactivation of B[a]P could be envisioned to contribute to the chemopreventive actions of oltipraz. This possibility was assessed in a tumorigenesis study by administering oltipraz 1 h before dosing with B[a]P to nrf2-deficient mice. To further characterize the role of nrf2 genotype on B[a]P disposition, we measured the levels of B[a]P–DNA adducts in target (forestomach) and non-target tissues (liver) of wild-type and nrf2-deficient mice treated with either vehicle or oltipraz 1 or 48 h prior to the carcinogen. Our results on modulation of B[a]P–DNA binding and tumor burden of the forestomach of wild-type and nrf2-deficient mice give further evidence for the in vivo role of the Nrf2 transcription factor in regulating susceptibility to chemical-induced carcinogenesis.


    Material and methods
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Chemicals
B[a]P and other chemicals were purchased from Sigma Chemical Co. (St Louis, MO). B[a]P metabolite standards were obtained from the National Cancer Institute Chemical Carcinogen Reference Standard Repository (Midwest Research Institute, Kansas City, MO). 5-(2-Pyrazinyl)-4-methyl-1,2-dithiole-3-thione (oltipraz) was provided by the Division of Cancer Prevention, National Cancer Institute. High-pressure liquid chromatography (HPLC)-grade water, methanol and ethyl acetate were purchased from J.T. Baker (Phillipsburg, NJ). HCl (Ultrapurex) and NaOH (1 N Titrisol) were obtained from EM Science (Gibbstown, NJ). The enzymes for DNA purification, proteinase K and RNase A were obtained from Boehringer Mannheim Biochemical (Indianapolis, IN).

Animals and treatments
Nrf2-disrupted mice were generated as described by Itoh et al. (11) and genotypes of homozygous wild-type and nrf2-deficient mice were confirmed as reported previously (26). For the DNA adduct studies, female mice (7–9 weeks old), fed AIN-76A diet and water ad libitum, were treated with a single dose of oltipraz (500 mg/kg in 0.1 ml of 1% cremophor and 25% glycerol) or vehicle only by gavage, 1 or 48 h before dosing with B[a]P (100 mg/kg in 0.2 ml corn oil, p.o.). Mice were killed 24 h after B[a]P treatment and DNA was isolated from forestomach and liver tissues as described below. For the carcinogenesis study, female nrf2-deficient mice (7–9 weeks old) were randomized into groups of 20 mice and fed AIN-76A diet and water ad libitum. Animals were given oltipraz or vehicle by gavage and B[a]P was given 1 h later by oral intubation. This sequence of oltipraz and B[a]P administration was repeated once a week for a total of 4 weeks. Animals were weighed weekly and killed 30 weeks after the initial treatment. Forestomach tissues were removed and fixed in 10%-buffered formalin solution. Tumors of the forestomach were counted grossly as described by Wattenberg (32). Experiments on animals were conducted in compliance with protocols approved by the Animal Care and Use Committee of the Johns Hopkins Medical Institutions.

DNA isolation from tissues and hydrolysis
Tissues were homogenized in 50 mM Tris–HCl, 10 mM MgCl2, 25 mM KCl, 0.25 M sucrose buffer, pH 7, and centrifuged at 300 g for 10 min. Supernatants were discarded and pellets resuspended in Triton X-100-Tris–sucrose buffer and centrifuged at 500 g for 10 min, twice. Pellets were resuspended in Tris–sucrose buffer and incubated with 1% sodium dodecyl sulfate and proteinase K (300 µg/ml) for 6 h at 56°C. To improve the recovery of DNA from a single forestomach, the suspension was extracted twice with chloroform:isoamyl alcohol (24:1) and DNA was precipitated from the aqueous phase with ice-cold ethanol. The DNA was then dissolved in Tris buffer, 40 mM NaCl, 5 mM EDTA, pH 7, and incubated with RNase A (100 µg/ml) for 1 h at 37°C. DNA was extracted two more times with chloroform:isoamyl alcohol (24:1), precipitated with ice-cold ethanol after the addition of 1/10 vol of 3 M sodium acetate, pH 5.3, and washed three times with 70% ethanol. DNA was dissolved in H2O and the concentration was estimated spectrophotometrically by measurement of UV absorbance ratio at 260/280 nm.

B[a]P-tetrols were released by acid hydrolysis of DNA (~150 µg) isolated from mouse forestomach and liver at 80°C for 4 h in a final concentration of 0.01 NHCl and stored at -20°C until analyzed by a reverse-phase HPLC-fluorometric method (33). Before injection, an aliquot of the sample was taken for analysis of guanine. The B[a]P-tetrols were extracted with saturated ethyl acetate, dried down with a stream of N2, resuspended in 20% methanol for analysis.

HPLC fluorometric analysis of B[a]P-tetrols and guanine
Analysis of B[a]P-tetrols by HPLC was performed with a Hewlett Packard Series 1050 System with an autosampler coupled to an automated gradient controller and a solvent delivery system. All solvents were filtered through 0.22 µm Millipore membranes, degassed under vacuum prior to use and continuously sparged with high purity He throughout the analyses (34). Separations performed with a Luna C18 (Phenomenex, Torrance, CA) reverse phase column (5 µm, 250x4.6 mm) were carried out at 25°C with a flow rate of 0.7 ml/min using a methanol/water linear gradient. Initial solvent conditions were 55% methanol in water with a linear gradient to 75% in 35 min, a linear gradient to 100% methanol in 10 min, followed by 10 min at 100% methanol, then a linear gradient to 55% methanol in 5 min and 20 min at 55% methanol before the next injection. The B[a]P-tetrols were detected with a HP 1046A programmable fluorescence detector. The excitation wavelength was set at 246 nm and the emission was measured at 393 nm, after trapping and scanning a B[a]P-tetrol standard in the fluorescence flow cell. Quantification was by comparison with a standard curve using authentic B[a]P-tetrol standards analyzed prior to and immediately following the analysis of each set of samples. The lower limit of detection with the instrumentation used was 2 pg of trans-anti-B[a]P-tetrol (TA) and ~3 pg of the other three tetrols. B[a]P-triol (r-7,t-8,c-9-trihydroxy-7,8,9,10-dihydrobenzo[a]pyrene) was used as an internal standard due to its similar chemical properties to the tetrols. The internal standard was added to each sample before ethyl acetate extraction to detect variations in extraction recovery and fluorometric analysis. Silanized, amber, high recovery autosampler vials were prepared so that 100 µg of hydrolyzed DNA would be injected in a 200 (liver) or 220 µl (forestomach) injection. Levels of B[a]P-tetrols released from mouse forestomach and liver were expressed as picomole per micromole guanine.

Guanine from ~1 µg DNA per injection was eluted from a Whatman Partisil 10 SCX column, isocratically, with 50 mM ammonium phosphate buffer pH 2, at a flow rate of 1 ml/min. Guanine was detected with the excitation wavelength set at 246 nm and the emission was measured at 354 nm. Levels of guanine were determined by comparison with a standard curve using authentic guanine standard in 0.01 N HCl (35).

Statistical analysis
Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparison. Experimental values are given as mean ± SE.


    Results
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Effect of oltipraz treatment on B[a]P-induced neoplasia of the forestomach
In order to evaluate the possible role of P450 inhibition by oltipraz on B[a]P-induced neoplasia of the forestomach, female nrf2-deficient mice were treated weekly with oltipraz or vehicle only 1 h before B[a]P administration. This sequence of dosing was done for 4 consecutive weeks and animals were killed 30 weeks after the initial treatment. Initial mean body weights of both vehicle- and oltipraz-treated nrf2-deficient mice (22.8 ± 1.9 and 22.4 ± 1.6 g, respectively) were similar to those in the 48 h oltipraz pre-treatment experiment (22.1 ± 2.1 and 22.4 ± 2.1 g, respectively). The body weight gains at the end of the 30-week experiment were 8.7 and 7.4 g for nrf2-disrupted vehicle- and oltipraz-pre-treated groups (1 h), respectively, compared with 8.6 and 7.9 g in the 48 h pre-treatment experiment (26). Therefore, we observed no difference on body weight gains by oltipraz treatment, pre-treatment intervals or by genotype. As observed previously, several animals from each group died before the end of the 30-week experiment with symptoms of respiratory distress. Upon autopsy, the thymus was found to be enlarged, occupying most of the thoracic space and compressing the lungs against the posterior wall. Overall survival rate and tumor morphology were also similar to those obtained in a previous study (26). In the nrf2-deficient vehicle-treated group, six mice died between the 17th and 28th weeks, each with between 25 and 35 gastric tumors. An additional mouse had a tumor burden too numerous to count. In the nrf2-disrupted oltipraz-treated group, five mice died between weeks 19 and 27, with 27–35 tumors, whereas an additional mouse had a tumor burden too extensive to count. Two animals in the vehicle-treated group and one mouse in the oltipraz-treated group did survive the 30-week experimental period but had such extensive tumor formation that an accurate tumor count was not possible. All animals in both nrf2-deficient groups bore tumors. There were no tumor-related early deaths. However, comparing the tumors of those animals that died before the end of the 30-week experiment, it was observed that tumors progressed from individual large pinpoint excrescences to cauliflower growths. As shown in Table IGo, oltipraz treatment had no protective effect on tumor multiplicity in the forestomach of nrf2-deficient mice under these experimental conditions (P = 0.83). Both nrf2-disrupted vehicle- and oltipraz-treated mice had similar numbers of neoplasms of the forestomach (29.7 ± 1.7 and 29.2 ± 1.7, respectively). For purpose of comparison, the number of gastric tumors per mouse obtained when animals were treated with oltipraz 48 h before dosing with B[a]P in an earlier experiment were also included (26). In this initial experiment, nrf2-deficient mice had more and larger tumors than wild-type mice. Moreover, oltipraz had no effect on gastric tumor burden in the nrf2-deficient mice. The results were the same when tumor yield from animals before the scheduled termination of the experiment were included in the analysis.


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Table I. Effect of oltipraz treatment on B[a]P-induced neoplasia of the forestomach of wild-type and nrf2-deficient mice
 
Effect of nrf2 genotype and oltipraz treatment on B[a]P–DNA adducts
To examine the effect of nrf2 genotype and oltipraz treatment on formation of B[a]P–DNA adducts, an early step in the process of carcinogenesis, wild-type and nrf2-disrupted mice were treated with vehicle or oltipraz 1 or 48 h before the carcinogen and killed 24 h after dosing with B[a]P. Time-course studies by Sticha et al. (34) indicated that 24 h was the point of highest tetrol burden in the liver of B[a]P-treated mice. A representative profile of the tetrol hydrolysis products from forestomach DNA of a nrf2-deficient mouse killed 24 h after B[a]P administration is shown in Figure 1AGo. TA was the major tetrol detected in the forestomach and liver of wild-type and nrf2-disrupted mice, followed by CS. Peaks other than those corresponding to authentic tetrols standards were also detected, especially a shoulder on the TA. This unknown compound, called X, is likely to be a B[a]P metabolite, as it was only found after B[a]P treatment and was not detected in DNA from cremophor-glycerol-vehicle and corn oil-vehicle treated animals, nor in DNA from untreated mice. Interestingly, the area under the curve of this peak, relative to the amount of DNA hydrolysate on column, was larger (~4-fold) in chromatograms obtained from the hydrolysis of DNA isolated from forestomach than those obtained from liver samples of the same animal and of the entire treated group. An HPLC chromatogram of authentic standards is shown in Figure 1BGo. B[a]P-tetrol standards were also taken through ethyl acetate extraction to determine the efficiency of recovery at different concentrations. The recovery for the four tetrols and B[a]P-triol was ~95–99%, over the range of concentrations from 2 to 254 pg.



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Fig. 1 . Representative reverse-phase HPLC-fluorometric profile of the B[a]P-tetrols after hydrolysis of DNA isolated from mouse forestomach or liver. (A) Chromatogram of forestomach DNA (100 µg) from a nrf2-deficient mouse killed 24 h after B[a]P administration. (B) Separation of authentic standards. Details of the animal treatments, and the procedure for isolation and separation of the B[a]P-tetrols are given in Material and methods. TA, trans-anti-, TS, trans-syn-, CA, cis-anti-, CS, cis-syn-B[a]P-tetrol; X, unknown; IS, internal standard.

 
The results of the analyses in forestomach (target organ) showed that, although not statistically significant, 1 h oltipraz treatment increased the levels of total tetrols in the forestomach of wild-type mice by ~35% (Table IIGo). Similarly, levels of total DNA adducts were significantly increased after oltipraz treatment in the forestomach of nrf2-disrupted mice (36%, P < 0.01). Analyses of the individual tetrols showed that TA was the major B[a]P-tetrol increased by oltipraz, accounting for ~50% of the total increased levels. In both nrf2-deficient vehicle- and oltipraz-treated mice levels of total B[a]P-tetrols were higher compared with those observed in wild-type mice. In contrast to the results with a 1 h oltipraz pre-treatment, DNA adduct levels were inhibited by 26% (P = 0.04) by 48 h oltipraz pre-treatment in the forestomach of wild-type mice. However, this inhibitory effect was not observed in the forestomach of nrf2-deficient mice (P = 1.00). Both nrf2-disrupted vehicle- and oltipraz-treated mice again had higher levels of total DNA adducts than wild-type mice (P < 0.01).


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Table II. Effect of nrf2 genotype and oltipraz treatment on BPDE-DNA adducts
 
In contrast to the results in forestomach tissue, the analyses in liver (non-target organ) of wild-type mice showed that B[a]P–DNA adducts were inhibited by 66% (P < 0.01) by 1 h oltipraz pre-treatment. Although not statistically significant, this inhibitory effect was also observed in the liver of nrf2-deficient mice by 48 h oltipraz pre-treatment (36% inhibition). Interestingly, both nrf2-disrupted vehicle- and oltipraz-treated mice had levels of adducts similar to the corresponding wild-type treated mice. No enhancement of hepatic adduct burden was observed in the knockout mice. As in the case of 1 h oltipraz treatment, DNA adduct levels were significantly (P = 0.01) inhibited by 49% with 48 h oltipraz pre-treatment in the liver of wild-type mice. Moreover, DNA adducts were also decreased with 48 h oltipraz pre-treatment in the liver of nrf2 knockout mice (28%, P = 0.13).


    Discussion
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
The forestomach was the primary target for B[a]P-induced carcinogenesis in the mice (ICR/129Sv background) used in this study and tumor burden could be significantly inhibited when oltipraz was administered to wild-type mice 48 h before the carcinogen (26), in accord with the original findings of Wattenberg and Bueding (1). However, as indicated in Table IGo, pre-treatment with oltipraz either 1 or 48 h before B[a]P was devoid of chemoprotective efficacy in nrf2-deficient mice. In addition, these transcription factor knockout mice were intrinsically more sensitive to B[a]P carcinogenesis. Therefore, to further characterize the susceptibility of nrf2-deficient mice to chemical-induced carcinogenesis, we measured by HPLC-fluorescence detection the interactive effects of nrf2 genotype and oltipraz treatment on B[a]P–DNA adduct levels in the forestomach (target organ) and liver (non-target organ) of wild-type and nrf2-deficient mice.

B[a]P and its penultimate metabolite (±)-trans-B[a]P-7,8-diol require metabolic activation by cytochrome P450s to exert many of their adverse biological effects, including binding to DNA, RNA and proteins, toxicity, mutagenicity and carcinogenicity. Several studies in yeast, baculovirus-infected cells, or in bicistronic systems, have shown that recombinant CYP1A1 was the most active enzyme involved in the oxidation of B[a]P, followed by CYP1B1 and to a lesser extent by CYP1A2 and CYP3A4, whereas CYP2C9, CYP2C19 and CYP2E1 formed relatively low amounts of B[a]P products (36,37). More recently, using cDNA-based recombinant systems expressing different forms of human cytochrome P450s and NADPH-P450 reductase, Shimada et al. (38) showed that CYP1A1 and CYP1B1 had similar catalytic activity toward B[a]P and (±)-trans-B[a]P-7,8-diol. Comparing the metabolism of B[a]P and (±)-trans-B[a]P-7,8-diol by recombinant human P450s in the presence of DNA, Kim et al. (37) observed that CYP1A1, CYP1A2 and CYP1B1 each formed the tetrol metabolites of (±)-trans-B[a]P-7,8-diol with similar stereoselectivity by producing the TA tetrol at the greatest rate. TA is derived from the 7R,8S,9S,10R-enatiomer of anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene which is the most carcinogenic metabolite formed from (±)-trans-B[a]P-7,8-diol. Our analyses of B[a]P–DNA adducts in mouse tissues were in agreement with this metabolite pattern, as TA was the major B[a]P-tetrol detected in DNA isolated from both forestomach and liver of wild-type and nrf2-deficient vehicle- and oltipraz-treated mice.

The in vitro studies on the metabolic activation of B[a]P indicate that CYP1A1 should dominate the oxidation of B[a]P in tissues where it is constitutively and inducible expressed. Although, CYP1A1 is expressed predominantly in extrahepatic organs, as well as tumor tissues, it is also highly induced in liver by polyeychic aromatic hydrocarbons (39,40). Studies on the expression patterns of cytochrome P450s failed to detect either constitutive expression of CYP1A1 and CYP1A2 mRNAs by in situ hybridization or their protein catalytic activities (EROD, methoxyresorufin-O-deethylase) in the stomach of female untreated C57Bl/6N mice and the forestomach of vehicle-treated male Swiss albino mice, respectively (41,42). Treatment of mice with 3-methylcholanthrene led to induction of CYP1A1, but not CYP1A2, transcripts in the mucosa of the stomach (41). However, this induction was modest compared with the effect in other tissues of the gastrointestinal tract. Effects on CYP1B1 mRNA expression and protein were not measured. This pattern of tissue expression of cytochrome P450s suggests that enzymes other than CYP1A1 and CYP1A2 are involved in the biotransformation of B[a]P in the mouse forestomach.

Analysis of B[a]P–DNA adduct burden in mouse forestomach showed that the levels of individual or total tetrols were higher in nrf2-deficient mice, whether vehicle or oltipraz treated, compared with the corresponding wild-type mice. The increased levels of B[a]P–DNA adducts in the forestomach of the knockouts presumably reflect the lower basal expression of phase 2 enzymes contributing to B[a]P detoxication. mRNA levels for GSTA1/2, GSTP1/2 and NQO1 and their enzymatic activities are substantially (50–70%) reduced in forestomach of nrf2-deficient mice (26). Moreover, inducibility of these and other gene transcripts by oltipraz, while marked (3–7-fold) in wild-type mice, is lost in the knockout mice. Oltipraz had no protective effect on tetrol levels when administered either 1 or 48 h before B[a]P in the knockout mice. Moreover, reduction in tetrol levels in forestomach DNA of wild-type mice was only observed when oltipraz was administered 48 h, but not 1 h, in advance of the carcinogen. Collectively, these results indicate that the protective actions of oltipraz against B[a]P carcinogenesis in the forestomach were mediated by altering phase 2 gene expression without any contribution through inhibition of cytochrome P450 enzymes. This expectation is consistent with our observation that pre-treatment of mice with oltipraz 1 h before B[a]P had no protective effect against tumorigenesis. The isoforms of CYP inhibited to the greatest extent by oltipraz are not expressed in this target organ.

CYP1A2 is expressed predominantly in liver. Recent studies reported that CYP 1A1 and 1B1 are also constitutively expressed, albeit at very low levels, in the liver of AhR knockout and C57Bl/6J mice (43,44). Hence, this tissue expression pattern suggests that CYP1A1, CYP1B1, CYP1A2, as well as CYP3A4, and CYP2C enzymes activate B[a]P in the liver of wild-type and nrf2-deficient mice and, for some isoforms (CYP1A1, CYP1A2, CYP3A4) could be affected by oltipraz treatment (29). Indeed, in contrast to the findings in forestomach, oltipraz treatment reduced the levels of hepatic B[a]P–DNA adducts in (i) animals pre-treated at both 1 and 48 h and (ii) both genotypes of mice. In fact, reductions in tetrol content of hepatic DNA were slightly greater at 1 h pre-treatment with oltipraz than at 48 h, suggesting that inhibition of cytochrome P450s may contribute substantively to the reductions in DNA damage. This mechanism might assume importance under circumstances in which first pass metabolism of B[a]P in the liver affects the qualitative and quantitative distribution of B[a]P and its metabolites to distal target tissues, such as the lung and bone marrow.

Overall, inhibition of B[a]P–DNA adduct burden was 26% (P = 0.04) in forestomach when oltipraz was administered 48 h before carcinogen; this level of reduction was also seen for each of the four individual tetrol products. However, inhibition of tumor multiplicity was ~50% (P < 0.01) in two studies using identical dosing protocols as the adduct study (1,26). There are other examples where reduction in target organ DNA adduct burden underestimates chemopreventive efficacy of an intervention with oltipraz. Oltipraz is an effective inhibitor of aflatoxin-induced hepatocarcinogenesis; however, dose–response experiments consistently indicate that near complete abrogation of tumor burden is reflected in 60–80% reductions in hepatic levels of aflatoxin–DNA adducts (45). Similarly, Sticha et al. (34) have observed that changes in B[a]P–DNA adduct levels by the chemopreventive agents benzyl isothiocyanate and phenethyl isothiocyanate do not fully account for their effects on B[a]P-induced lung tumorigenesis. The underestimate in the present study may reflect inhibition by oltipraz of other actions of B[a]P metabolites, such as cytotoxicity, on the carcinogenic process. Nevertheless, combining all treatments and genotypes, a strong positive linear correlation (R2 = 0.91) was observed between the levels of total B[a]P-tetrols and the number of gastric tumors per mouse (Figure 2Go).



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Fig. 2 . Correlation between levels of DNA adducts and tumor burden in the mouse forestomach. Circles, wild-type mice; triangle and square symbols, nrf2-deficient mice.

 
In conclusion, using B[a]P as a model chemical carcinogen, our results expand the evidence that the determination of chemical-specific markers for DNA damage, measured here as individual B[a]P-tetrols, provide a mechanistic linkage between exposure and disease outcome as well as a monitor for the efficacy of a chemopreventive intervention. Different effects of oltipraz on the levels of B[a]P–DNA adducts in target and non-target tissues were observed. Although oltipraz appeared to reduce DNA adducts in a manner consistent with inhibition of cytochrome P450 in liver, such an effect was not observed in forestomach due to lack of expression of susceptible cytochrome P450s. The lack of protective effect of oltipraz when administered 1 h before B[a]P, coupled with the complete abrogation of its protective effects when administered to nrf2-disrupted mice 48 h before B[a]P, strongly indicates that the anticarcinogenic activity of oltipraz in forestomach can be fully accounted for by modulation of Nrf2-regulated genes. Further understanding of the factors controlling the fate of Nrf2 and its downstream effects, should provide insight and opportunities for the development of effective chemopreventive agents.


    Notes
 
* Current address: Division de Estudios de Posgrado, Facultad de Quimica, Universidad Anutonoma de Queretaro, Cerro de las Campanas s/n, Queretaro, Qro. C.P. 7608 Mexico Back

3 To whom correspondence should be addressed Email: tkensler{at}jhsph.edu Back


    Acknowledgments
 
This work was supported by National Institutes of Health R01 Grants CA 39416, CA 94076 and Center Grant ES 03819. M.R.-G. was partially supported by the Consejo de Ciencia y Tecnologia del Estado de Queretaro (CONCYTEQ) and the Universidad Autonoma de Queretaro, Mexico.


    References
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 

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Received October 16, 2002; revised November 25, 2002; accepted November 26, 2002.