ToxSci Advance Access originally published online on December 13, 2005
Toxicological Sciences 2006 90(1):111-119; doi:10.1093/toxsci/kfj076
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A Crucial Role of Nrf2 in In Vivo Defense against Oxidative Damage by an Environmental Pollutant, Pentachlorophenol
* Division of Pathology, National Institute of Health Sciences, 1–18–1, Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan; Faculty of Pharmaceutical Science, Department of Analytical Chemistry, Hoshi University, 2–4–41 Ebara, Shinakawa-ku, Tokyo 142-8501, Japan; Division of Toxicology, National Institute of Health Sciences, 1–18–1, Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan; and Center for Tsukuba Advanced Research Alliance and Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan
1 To whom correspondence should be addressed. E-mail: umemura{at}nihs.go.jp.
Received October 7, 2005; accepted November 18, 2005
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our goal was to elucidate roles of Nrf2 in in vivo defense against pentachlorophenol (PCP), an environmental pollutant and hepatocarcinogen in mice. We examined oxidative stress and cell proliferation, along with other hepatotoxicological parameters, in the livers of nrf2-deficient (wild:+/+, heterozygous:+/–, homozygous:–/–) animals fed PCP in their diet at doses of 0, 150, 300, 600, or 1200 ppm for 4 weeks. For measurement of methoxyresorufin-O-demethylase (CYP 1A2), NAD(P):quinone oxidoreductase 1 (NQO1), and UDP-glucuronosyltransferase (UDP-GT), an additional study was performed with all but the 150-ppm dose. Significant elevation of 8-hydroxydeoxyguanosine (8-OH-dG) levels in the liver DNA was observed only in –/– mice treated with PCP at 1200 ppm. Levels of thiobarbituric-acid-reactive substances (TBARS) were also raised significantly compared to those of the relevant +/+ mice. Bromodeoxyuridine labeling indices (BrdU-LIs) of hepatocytes in –/– mice were significantly higher at all doses than those in the relevant +/+ mice. Relative liver weights were unchanged in mice lacking Nrf2, whereas liver weight in +/+ and +/– mice was increased. Significant elevations of serum ALP activity, but not ALT and AST activity, occurred at 600 ppm and above in –/– mice compared to the relevant +/+ mice. Histopathologically, centrilobular hepatocyte necrosis was severe in the –/– mice that received 600 ppm. Although CYP 1A2 activity was elevated in all treated mice, increases in NQO1 levels and UDP-GT activities did not occur only in –/– mice. These data suggest that Nrf2 plays a key role in prevention of PCP-induced oxidative stress and cell proliferation.
Key Words: Nrf2; pentachlorophenol; 8-hydroxydeoxyguanosine; cell proliferation; NQO1.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chronic exposure to pentachlorophenol (PCP) induces hepatocellular tumors in mice (McConnell et al., 1991
), which is of concern, because large amounts of this compound are used as a wood preservative, herbicide, and insecticide, resulting in ubiquitous environmental pollution (Besser, et al., 2005; Dwyer et al., 2005; Masunaga et al., 2001). PCP also has the potential to induce proliferation of hepatocytes and intrahepatic biliary epithelial cells, consequently exerting promotion as well as carcinogenic activity in the liver (Umemura et al., 1999, 2003b). While PCP itself lacks genotoxicity, tetrachlorohydroquione (TCHQ), its major metabolite, has been reported to cause micronuclei and point mutations in V79 hamster cells (Jansson and Jansson, 1991, 1992), presumably contributing to PCP clastogenicity and carcinogenicity. Autooxidation and/or enzymatic oxidation of TCHQ to tetrachlorobenzoquinone (TCBQ) followed by redox cycling gives rise to reactive oxygen species (Naito et al., 1994; Tsai et al., 2001). In fact, we have shown increased 8-hydroxydeoxyguanosine (8-OH-dG) levels in liver DNA of mice given a carcinogenic dose of PCP (Umemura et al., 1996). Also, formation of TCBQ and tetrachlorosemiquinone (TCSQ) adducts results from a direct reaction with DNA during PCP metabolism (Lin et al., 1999, 2002). Thus, information regarding the mode of action underlying PCP hepatocarcinogenesis has accumulated. Questions, however, remain to be clarified regarding in vivo preventive mechanisms against phenolic liver carcinogens like PCP, and identification of key factors in in vivo defense is clearly necessary for estimation of accurate hazard risk.
Recently, the transcriptional factor Nrf2, which regulates induction of phase 2 and antioxidant enzymes with antioxidant responsive elements (Itoh et al., 1997), has received much attention as a critical role for cellular defense against oxidative stress (Ishii et al., 2000). Given the involvement of NAD(P):quinone oxidoreductase 1 (NQO1) in catalyzing two-electron reduction to detoxify quinones and their derivatives (Ross et al., 2000) in Nrf2-regulated enzymes (Dhakshinamoorthy and Jaiswal, 2001; Nioi et al., 2003; Nioi and Hayes, 2004), it is highly probable that Nrf2 is an important determinant of the hazards with environmental exposure to PCP. Several studies on Nrf2 effects against exogenous chemical exposure have already been demonstrated using nrf2-deficient mice, which proved highly susceptible to benzo(a)pyrene-induced forestomach (Ramos-Gomez et al., 2003) and N-nitrosobutyl (4-hydroxybutyl) amine-induced bladder carcinogenesis (Iida et al., 2004), acetaminophen-hepatotoxicity (Chan et al., 2001; Enomoto et al., 2001) and diesel exhaust–induced lung toxicity (Aoki et al., 2001).
In the present study, to investigate the potential participation of Nrf2 in the biological preventive reaction following PCP exposure, nrf2-deficient mice were employed to examine PCP-induced liver lesions in terms of serum biochemistry indicating liver injury, oxidative DNA damage, lipid peroxidation (LPO), and hepatocyte cell proliferation. Particular attention was paid to methoxyresorufin-O-demethylase (CYP 1A2) and Nrf2-regulating enzymes such as NAD(P):quinone oxidoreductase 1 (NQO1) and UDP-glucuronosyltransferase (UDP-GT).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chemicals.
PCP (purity, 98.6%) was purchased from Wako Pure Chemical Industries (Osaka, Japan), alkaline phosphatase and bromodeoxyuridine (BrdU) were obtained from Sigma Chemical Co. (St. Louis, MO), and nuclease P1 was from Yamasa Shoyu Co. (Chiba, Japan). Anti-BrdU and anti-NQO1 monoclonal antibodies were from DakoCytomation (Glostrup, Denmark) and Abcam Ltd. (Cambridge, UK), respectively.
Animals, diet and housing conditions.
The protocol for this study was approved by the Animal Care and Utilization Committee of the National Institute of Health Sciences. Nrf2-deficient mice with an ICR/129SVJ background established by Itoh et al. (1997) were crossed with ICR mice (Japan SLC, Schizuoka, Japan); then, homozygous (–/–), heterozygous (+/–), and wild-type littermates (+/+) were obtained from the F1 generation and genotyped by the polymerase chain reaction (PCR) with DNA taken from the tail of each mouse. Mice were housed in polycarbonate cages (five mice per cage) with hard wood chips for bedding in a conventional animal facility maintained under conditions of controlled temperature (23 ± 2°C), humidity (55 ± 5%), air change (12 times per hour), and lighting (12-h light/dark cycle) and given free access to a CRF-1 basal diet (Charles River Japan, Kanagawa, Japan) and tap water.
Animal treatments.
Experiment I: A total of 25 7-week-old male mice of each genotype were divided into five groups. PCP was mixed in the basal powder diet at concentrations of 150, 300, 600, and 1200 ppm, and the diets thus prepared were fed to the animals ad libitum. The second highest dose was the same as the highest dose in the NTP bioassay, with the purities being a little lower (McConnell et al., 1991). Control mice were similarly fed a basal powder diet (CRF-1). All mice were killed at week 4 by exsanguination under ether anesthesia; blood was collected for measuring serum alkaline phosphatase (ALP), alanine transaminase (ALT), and aspartate aminotransferase (AST) activities from the orbital venous plexus. All mice received BrdU (100 mg/kg) by ip injection once a day for the final 2 days of exposure and once on the final day, 2 h before killing, as previously described (Umemura et al., 1992). At necropsy, the livers were immediately removed and weighed; slices were fixed in buffered formalin for hematoxylin and eosin (H & E) stain or immunohistochemistry for BrdU. Remaining pieces of the livers were frozen with liquid nitrogen and stored at –80°C until measurement of 8-OH-dG in nuclear DNA and levels of thiobarbituric acid-reactive substances (TBARS). Experiment II: A total of 20 7-week-old male mice of each genotype were divided into four groups, and the treatments were performed as in Experiment I, except that the lowest dose was 300 ppm. The livers were similarly sampled and stored for the measurement of CYP1A2 activity, NQO1 protein levels, and UDP-GT activities in the homogenates.
Measurement of nuclear 8-OH-dG.
To prevent 8-OH-dG formation as a byproduct during DNA isolation (Kasai, 2002), liver DNA was extracted by a slight modification of the method of Nakae et al. (1995). Briefly, nuclear DNA was extracted with a commercially available DNA Extracter WB Kit (Wako Pure Chemical Industries, Ltd.) containing an antioxidant NaI solution to dissolve cellular components. For further prevention of autooxidation in the cell lysis step, deferoxamine mesylate (Sigma Chemical Co.) was added to the lysis buffer (Helbock et al., 1998). The DNA was digested to deoxynucleotides with nuclease P1 and alkaline phosphatase, and levels of 8-OH-dG (8-OH-dG/105 deoxyguanosine) were assessed by high-performance liquid chromatography (HPLC) with an electrochemical detection system (Coulochem II, ESA, Bedford, MA).
Measurement of TBARS.
Malondialdehyde (MDA, nmol/g) was assessed as an index of lipid peroxidation by the method of Uchiyama and Mihara (1978). In brief, a 0.15-g portion of liver was homogenized with 1.35 ml of 1.15% KCl solution. To 0.05 ml of this homogenate, 0.2 ml 8.1% SDS and 3.0 ml 0.4% 2-thiobarbituric acid in 10% acetic acid solution (pH 3.5) were added, followed by heating in a water bath at 95°C for 60 min. After cooling, 5.0 ml of n-butanol and pyridine (15:1 v/v) and 1.0 ml distilled water were added, and then the mixture was centrifuged at 1,870 x g for 10 min. TBARS were measured with a Hitachi F-2500 fluorescence spectrophotometer (Hitachi High-Technologies Co., Tokyo, Japan) at 515 nm (excitation) and 553 nm (emission) in the butanol/pyridine phase.
Immunohistochemical procedures.
For immunohistochemical staining of BrdU, sections were treated sequentially with normal horse serum, monoclonal mouse anti-BrdU (1:80), biotin-labeled horse anti-mouse IgG (1:400), and avidin-biotin-peroxidase complex (ABC) after denaturation of DNA with 4 N HCl. The sites of peroxidase binding were demonstrated by incubation with 3,3'-diaminobenzidine tetrahydrochloride (Sigma Chemical Co., St. Louis, MO). The immunostained sections were lightly counterstained with hematoxylin for microscopic examination.
Cell proliferation quantification.
For each animal, at least 3000 hepatocytes were counted. The labeling index (LI) was calculated as a percentage value derived from the number of labeled cells divided by the total number of cells counted.
Serum biochemistry.
Serum ALP, ALT, and AST levels were measured at SRL Inc. (Tokyo, Japan).
CYP1A2 activity.
The livers were homogenized with a Teflon homogenizer in 67 mM phosphate buffer containing 1.15% KCl. The homogenate was centrifuged for 10 min at 10,000 x g, 4°C and the resulting supernatant was collected and recentrifuged at 105,000 x g for 1 h to obtain microsomal fractions. Protein concentrations were determined with the Advance Protein Assay Reagent (Cytoskeleton Ltd., Denver, CO). The measurement method used in the present study was based on the protocol of McPherson et al. (2001). Methoxyresorufin (50 µM, 20 ml) was incubated in triplicate with sample microsomes ±1.25 mM NADPH in 67 mM phosphate buffer, pH 7.4, containing 25 µM MgCl2 for 20 min at 37°C. The reaction was stopped by the addition of ice cold methanol, the precipitate removed by centrifugation, and the fluorescence of the supernatant assessed using excitation (ex) at 530 nm and emission (em) at 585 nm. The formation of resorufin was determined from a standard curve.
Western blotting for NQO1.
Liver samples were homogenized with a Teflon homogenizer in ice-cold 50 mM Tris–HCl, pH 7.4 containing 0.25 M sucrose and a 1% protease inhibitor cocktail (Sigma Chemical Co.). The homogenate was centrifuged for 10 min at 10,000 x g, 4°C, and the resulting supernatant was collected and recentrifuged at 105,000 x g for 1 h to obtain cytosolic fractions. Protein concentrations were determined with a BCA Protein Assay kit (Pierce Biotechnology Ltd., Rockford, IL). Cytosolic fractions containing 20 µg protein were resolved by SDS–PAGE, transferred to Immobilon-P membranes (Millipore Corporation, Bedford, MA), and analyzed with anti-NQO1 (1:1000) (Asher et al., 2002) and anti-ß-actin as a loading control (1:8000, Sigma Chemical Co.). Appropriate peroxidase-conjugated secondary antibodies (1:2000, Dako Cytomation) were used to detect the proteins with ECL Plus (Amersham Bioscience Corp., Piscataway, NJ) reagents.
Microplate UDP-GT assays.
Liver samples were homogenized with a Teflon homogenizer in ice-cold 0.1 M Tris–HCl buffer containing 2 mM phenylmethylsulfonyl fluoride, pH 7.8. The homogenate was centrifuged for 20 min at 10,000 x g, 4°C, and the resulting supernatant was collected and assessed for protein content with the Advance Protein Assay Reagent (Cytoskeleton Ltd., Denver, CO) before assaying for UGT activity. The measurement method used was based on the protocol of Collier et al. (2000). A 96-well microtiter plate containing 30 µl of S9 (1 mg/ml stock), 105 µl of 4-methylumbelliferone (4-MU, Wako) (100 µM, final concentration) in 0.1 M Tris–HCl buffer containing 5 mM MgCl2 and 0.05% BSA, pH 7.4, was placed in a Fluoroskan Ascent FL (Thermo Electron Corp., Waltham, MA) set to read fluorescence at 355-nm ex and 460-nm em. The cofactor uridine 5'- diphosphate glucuronic acid (UDPGA, Sigma) (15 µl, 2 mM final concentration) was added to initiate the reaction. The final volume was 150 µl, and fluorescence was measured every 2 min for 10 min. Results were transformed to nmol/min/mg of protein using a standard curve generated with 4 MU (
2 = 0.99).
Statistics.
The significance of differences in the results was evaluated with ANOVA, followed by Dunnett's multiple comparison tests.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experiment I
As shown in Figure 1, 8-OH-dG levels in the liver DNA of –/– mice at the highest dose were significantly higher than those in the relevant +/+ mice and the control mice. However, there were no changes among any groups of +/+ and +/– mice. Likewise, an increment in the TBARS level was found only in the livers of –/– mice given 1200 ppm, the values being statistically significant as compared with the relevant +/+ mice (Fig. 2). BrdU-LIs for hepatocytes of mice exposed to PCP are shown in Figure 3. A dose-dependent increase was apparent with all of the genotypes, but BrdU-LIs from a dose of 150 ppm in –/– mice and from a dose of 600 ppm in +/– mice were statistically significantly higher compared with the relevant +/+ mice. Figure 4 shows the changes of relative liver weights of nrf2-deficient mice given PCP at doses of 0, 150, 300, 600, or 1200 ppm in the diet. In contrast to dose-dependent increases of relative liver weights in both +/+ and +/– mice, there were no changes among –/– mice treated with PCP at doses up to 1200 ppm, each value in the treated –/– mice being significantly lower than that in the relevant +/+ mice. Figure 5 summarizes the changes of serum ALP, ALT, and AST activities of mice treated with PCP at doses of 0, 150, 300, 600, and 1200 ppm in their diet. Significant increments of serum ALP activity occurred at 300 ppm and above in +/– mice as compared with the relevant +/+ mice. ALT and AST activities with all genotypes were increased in a dose-dependent manner, both activities at 1200 ppm in +/– mice being higher with statistical significance as compared with the relevant +/+ mice. Histopathological findings related with PCP treatment are summarized in Table 1. Remarkable cytoplasmic hypertrophy of centrilobular hepatocytes was observed in +/+ and +/– mice given PCP from doses of 150 ppm (Fig. 6A), but this change was much less evident in –/– mice (Fig. 6B). Along with hypertrophy, karyomegaly was found at 300 ppm and higher in +/+ and +/– mice (Fig. 6C). In –/– mice, centrilobular hepatocellular necrosis was observed at 600 ppm (Fig. 6D), the severity of which was clearly greater than that in the other genotypes of mice at the same dose (Fig. 6C). At 1200 ppm, ground glass cytoplasm was prominent only in –/– mice (Fig. 6F), there being no changes in terms of the severity of hepatocyte necrosis among the genotypes (Fig. 6E).
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Experiment II
Figure 7 illustrates changes in CYP1A2 activity in the livers of mice given PCP at doses of 0, 300, 600, and 1200 ppm in the diet for 4 weeks. Significant elevation of CYP1A2 activity was observed in all of the treated mice, the values in –/– mice at each dose being significantly higher than those in +/+ mice. Western blot analysis using the anti-NQO1 monoclonal antibody demonstrated a dose-related increase in hepatic protein levels in +/+ and +/– mice treated with PCP, in contrast to a lack of the change in –/– mice (Fig. 8A). Quantitation by densitometric scanning and normalization to ß-actin levels revealed a statistically significant increase in NQO1 protein levels in the wild-type and heterozygous mice at the highest dose as compared with the relevant control. The levels in –/– mice at any dose were significantly lower as compared with those in the relevant +/+ mice (Fig. 8B). Figure 9 illustrates changes in hepatic UDP-GT activities of mice exposed to PCP at doses of 0, 300, 600, and 1200 ppm in their diet. In the +/+ and +/– cases, activity was elevated from 300 ppm in a dose-dependent manner until 600 ppm. Although the values at 1200 ppm were slightly reduced from the peak, all of the values were statistically significant as compared with the relevant controls. As with the NQO1 protein level, there was no change among the groups of –/– mice, the levels at any doses being statistically lower than those in the relevant +/+ mice.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The present study showed clear differences in the sensitivity of nrf2-deficient and wild-type mice to PCP-induced oxidative stress, consistent with a role for Nrf2 in defense mechanisms. It is in fact well known that a number of proteins that impact oxidative stress, such as heme oxygenase-1 (Alam et al., 1999
), peroxiredoxin MSP23 (Ishii et al., 2000), glutamate cysteine ligase (Sekhar et al., 2003), and NQO1 (Nioi et al., 2003), are regulated by Nrf2. Since generation of reactive oxygen species by PCP is considered to occur via its quinone form, we focused specifically on changes in NQO1, by which benzoquinone is supplied with two electrons, consequently skipping the semiquinone to form hydroquinone (HQ) (Ross et al., 2000). In fact, in the present study we clearly demonstrated induction of NQO1 by PCP exposure in +/+ and +/– mice, which was completely eliminated in the absence of Nrf2. It is highly probable that NQO1 induced by PCP in +/+ and +/– mice efficiently bypassed TCSQ/TCBQ redox cycling by means of two-electron reduction of TCBQ, subsequently leading to escape from oxidizing DNA base. In turn, no induction of NQO1 following PCP exposure caused DNA base to incur extensive oxidation. Together with our present data on 8-OH-dG formation, in consideration of the ability of both TCSQ and TCBQ to modify bases directly (Lin et al., 1997, 2001), the liver DNA of nrf2-deficient mice appears to be in danger of attack by oxidative stress derived from PCP-metabolizing pathways. In an NTP bioassay, PCP at a dose of 600 ppm was hepatocarcinogenic in B6C3F1 mice (McConnell et al., 1991), increases of 8-OH-dG in liver DNA being observed in the same strain with exposure at doses from 300 to 1200 ppm (Umemura et al., 1996). Unexpectedly, there were no changes in 8-OH-dG levels among all of the groups of +/+ and +/– mice in the present study, but their background was 75% ICR. The existence of strain differences among BALB/c, ICR, and C57BL/J mice concerning activities of OGG1 and SOD (Mosquera et al., 2003) might provide an explanation.
Nrf2 is also reported to be a transcriptional factor that regulates enzymes associated with excretion of xenobiotics such as glutathione S-transferase and UDP-GT (Thimmulappa et al., 2002). It is generally considered that HQ is subject to glucuronidation and/or sulfation (Siraki et al., 2004), and PCP- and TCHQ-glucuronide conjugations were detected in urine following PCP exposure in rats (Reigner et al., 1991). Our present data pointing to remarkable induction of UDP-GT in +/+ and +/– mice are thus in agreement with PCP toxicokinetics. The activity at a dose of 1200 ppm in either genotype was lower than that at a dose of 600 ppm, albeit the precise rationale was not able to be provided. In consideration of the highest dose being overtly hepatotoxic in +/+ and +/– mice, the reduction might be secondary to hepatocyte damage, by which the same phenomena observed in the case of the hepatotoxicant, sodium valproate (at a higher dose), was interpreted (Roma-Giannikou et al., 1999). The first step in the metabolism of PCP is to form TCHQ, which is mainly dependent on CYP 1A2 (Ommen et al., 1986), and this was induced with all doses in the present study. There have been no reports indicating a possible role of Nrf2 in regulating P450 isozymes other than CYP 2A5 (Abu-Bakar et al., 2004). Although the further studies on PCP toxicokinetics in B6C3F1 mice demonstrated that sulfation conjugation was predominant in the excretion of PCP (Reigner et al., 1992), our data showing significantly higher levels of CYP1A2 in the treated –/– mice than in their +/+ and +/– counterparts might suggest that the lack of UGT-GT induction results in the higher accumulation of PCP.
PCP has the potential to promote DEN-initiated hepatocarcinogenesis in B6C3F1 mice (Umemura et al., 1999), in line with the increase of BrdU incorporation in hepatocytes (Umemura et al., 2003b). Comparing doses inducing increases of serum ALT activity with those causing elevation of hepatocyte BrdU-LI, we proposed earlier that PCP-induced cell proliferation might result not only from a regenerative response, but also from any other causes, including the alteration of the intranuclear redox status by oxidative stress (Umemura et al., 1996). Likewise, the present results showed that PCP exposure to +/– and –/– mice at all doses, and to +/+ mice at doses of 300 ppm and above was capable of inducing a rise in the BrdU-LI, although a statistically significant elevation of serum biochemical parameters indicating hepatotoxicity first appeared at the dose of 600 ppm. It has been reported that PCP is able to exert inhibitory effects on gap junctional intracellular communication in rat hepatocytes (Sai et al., 1998). In any event, despite the fact that the precise mechanisms underlying PCP-induced cell proliferation are unknown, the present data that BrdU-LIs in –/– mice were significantly higher at any dose than those in the relevant +/+ mice allow us to speculate that a lack of Nrf2 cannot afford to hamper the induction. In view of vulnerability to oxidation of DNA, it can be assumed that nrf2-deficient mice are susceptible to PCP-induced hepatocarcinogenicity. To confirm this hypothesis, a carcinogenicity study on PCP using the transgenic mice is now ongoing.
Morphological alteration in the livers of mice exposed to PCP is known to be reflected in marked hepatomegaly (McConnell et al., 1991). In the present study, dose-related increases of relative liver weight occurred in the treated +/+ and +/– mice, which were consistent with histopathological features showing centrilobular hepatocyte hypertrophy. Interestingly, PCP exposure to –/– mice did not affect liver weight or cause severe hepatocyte hypertrophy. Considering the present data that nrf2-regulated enzymes were induced by PCP exposure in +/+ and +/– mice, it seems likely that the observed hepatocyte hypertrophy was a consequence of adaptation against xenobiotics. The hypothesis that the hepatomegaly due to PCP exposure has no relation to oxidative stress was supported by our previous findings that coadministration of green tea with PCP was not able to prevent hepatomegaly despite effective suppression of 8-OH-dG formation and hepatocyte proliferation (Umemura et al., 2003a).
The data on serum ALP activities and the histopathological features at a dose of 600 ppm indicated high susceptibility of –/– mice to PCP hepatotoxicity. Nevertheless, the other hepatotoxicological parameters failed to support this finding. The fact that DNA base oxidation and lipid peroxidation did not occur, except in the –/– mice that received high doses, might suggest no link between oxidative stress and hepatotoxicity induced by PCP. Our data showing more induction of CYP1A2 in –/– mice enable us to hypothesize that metabolism of PCP to TCHQ was accelerated in response to accumulated PCP due to a lack of the excretion enzyme. Accordingly, although TCHQ has been considered to play a key role in PCP-induced hepatotoxicity (Wang et al., 2000, 2001), the overall data suggest that PCP itself might be a major cause in the toxicity, with neither TCHQ itself nor oxidative stress derived from the metabolizing pathways of TCHQ as the starting point. However, certain differences in histopathological changes between +/+ and –/– mice strongly imply a necessity for performing further studies to explore the etiology of the hepatotoxicity.
In conclusion, our present data made it clear that Nrf2 exerts preventive effects on oxidative damage and induction of hepatocyte proliferation by PCP, especially with NQO1 playing a key role in in vivo defense against these contributing factors to the environmental pollutant-induced carcinogenesis. A single nucleotide polymorphism of NQO1 is known to exist in human populations (Traver et al., 1992, 1997), and data are accumulating that individuals carrying this NQO1 polymorphism have high susceptibility to several cancers (Clairmont et al., 1999; Larson et al., 1999; Schulz et al., 1997; Wiemels et al., 1999). Clearly the carcinogenic risk with environmental exposure to PCP should be considered in this context.
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ACKNOWLEDGMENTS |
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We thank Mss. Machiko Maeda, Ayako Kaneko, and Fukiko Takagi for expert technical assistance in performing the animal experiments and processing histological materials. This work was supported in part by a Grant-in-Aid (12–9) for Cancer Research from the Ministry of Health, Labor and Welfare of Japan.
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