Aryl Hydrocarbon Receptor Mediates Benzene-Induced Hematotoxicity

Byung-Il Yoon*,1, Yoko Hirabayashi*,1, Yasushi Kawasaki*, Yukio Kodama*, Toyozo Kaneko*, Jun Kanno*, Dae-Yong Kim{dagger}, Yoshiaki Fujii-Kuriyama{ddagger} and Tohru Inoue*,2

* Division of Cellular and Molecular Toxicology, National Institute of Health Sciences, 1-18-1 Kamiyohga, Setagayaku, Tokyo 158-8501, Japan; {dagger} Department of Veterinary Pathology, College of Veterinary Medicine and Agricultural Biotechnology, Seoul National University, Suwon, Republic of Korea; {ddagger} Department of Chemistry, Tohoku University, Graduate School for Science, Sendai, Japan

Received June 20, 2002; accepted August 13, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Benzene can induce hematotoxicity and leukemia in humans and mice. Since a review of the literature shows that the CYP2E1 knockout mouse is not known to possess any benzene toxicity, the metabolism of benzene by CYP2E1 in the liver is regarded to be prerequisite for its cytotoxicity and genotoxicity, although the mechanism is not fully understood yet. Because it was found some years ago that benzene was also a substrate for CYP1A1, we investigated the involvement of the aryl hydrocarbon receptor (AhR) in benzene hematotoxicity using AhR wild-type (AhR+/+), heterozygous (AhR+/–), and homozygous (AhR–/–) male mice. Interestingly, following a 2-week inhalation of 300 ppm benzene (a potent dose for leukemogenicity), no hematotoxicity was induced in AhR–/– mice. Further, there were no changes in cellularity of peripheral blood and bone marrow (BM), nor in levels of granulocyte-macrophage colony-forming units in BM. This lack of hematotoxicity was associated with the lack of p21 overexpression, which was regularly seen in the wild-type mice following benzene inhalation. Combined treatment with two major benzene metabolites, phenol and hydroquinone, induced hemopoietic toxicity, although it was not known whether this happened due to a surprising lack of expression of CYP2E1 by AhR knockout, or due to a lack of other AhR-mediated CYP enzymes, including 1A1 (i.e., a possible alternative pathway of benzene metabolism). The former possibility, evaluated in the present study, failed to show a significant relationship between AhR and the expression of CYP2E1. Furthermore, a subsequent evaluation of AhR expression after benzene inhalation tended to show higher but less significant expression in the liver, and none in the BM, compared with sham control. Although this study failed to identify the more likely of the above-mentioned two possibilities, the study using AhR knockout mice on benzene inhalation presents the unique possibility that the benzene toxicity may be regulated by AhR signaling.

Key Words: aryl hydrocarbon receptor; benzene; CYP2E1; hematotoxicity; mice.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Benzene is a commonly used industrial solvent and an environmental contaminant. It is present in mineral oil, natural gas, coal tar, gasoline, motor vehicle emissions, and tobacco smoke (Koss and Tesseraux, 1999Go). It is well known that benzene and its metabolites induce hemopoietic toxicity, which is characterized by the decrease of bone marrow (BM) cellularity and the induction of leukopenia, aplastic anemia, and leukemia (Aksoy et al., 1974Go, 1976Go; Cronkite et al., 1984Go, 1989Go; Farris et al., 1997Go; Snyder et al., 1980Go). However, to date, the mechanism by which benzene induces hematotoxicity is not fully understood. Recently, we reported that benzene suppresses hemopoietic progenitor cell cycle by the overexpression of p21, a cyclin-dependent kinase inhibitor (Yoon et al., 2001bGo).

The production of reactive benzene metabolites is believed to be essential for mediating the hematotoxicity and leukemogenicity of benzene. Thus, differences in the capacity of the organism to metabolize benzene into reactive metabolites may account for interspecies and intraspecies differences in susceptibility to benzene exposure (Henderson, 1996Go; Longacre et al., 1981Go; Snyder and Hedli, 1996Go). Benzene is metabolized in the liver to phenol (PH), hydroquinone (HQ), catechol, and trans, trans-mucoic acid by hepatic cytochrome P450 enzymes, particularly CYP2E1 (Koss and Tesseraux, 1999Go; Snyder and Hedli, 1996Go). The toxic effects of benzene on BM and peripheral blood cells have been attributed to the interaction of the phenolic metabolites (Eastmond et al., 1987Go; Guy et al., 1990Go). Among the descriptions of mechanisms of benzene toxicity (Ross, 2000Go), the benzene metabolites PH and HQ are considered to play a major role in the induction of myelotoxicity, which is specifically focused (Eastmond et al., 1987Go; Subrahmanyam et al., 1990Go). This may explain the high sensitivity of mice to benzene toxicity compared with the relative resistance of rats (Henderson, 1996Go).

DBA/2 mice have been reported to be more susceptible to benzene than C57BL/6 mice (Longacre et al., 1981Go). On the other hand, DBA/2 mice have been known to be less susceptible to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), possibly because of a mutation in the C-terminal region of the aryl hydrocarbon receptor (AhR) of the strain (reviewed by Okey et al., 1994Go; Safe, 1995Go). It is interesting to note that DBA/2 mice possess high susceptibility to benzene on one hand but low susceptibility to TCDD on the other. The involvement of AhR in the mechanism of benzene hematotoxicity has not yet been studied.

To elucidate the role of AhR on the most simple aryl hydrocarbon, benzene, in collaboration with CYP2E1 and also 1A1 for alternative pathway, in this study we investigated the involvement and mechanistic role of AhR in benzene-induced hematotoxicity using AhR+/+, AhR+/–, and AhR–/–mice.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals.
AhR knockout (AhR–/–) mice were biotechnologically derived from the 129/SvJ strain by the Fujii-Kuriyama group in Japan (Mimura et al., 1997Go) and were crossed back into C57BL/6 strain more than six generations. Knockout mice were maintained as heterozygous mice at the animal facility of the National Institute of Health Sciences (NIHS), Tokyo, Japan. Breeding AhR+/– males with AhR+/– females generated AhR+/+, AhR+/–, and AhR–/–mice. The neonates were genotyped by polymerase chain reaction (PCR) screening of DNA from the tail. Male mice 8 to 9 weeks old were used for the study. The study mice were housed in stainless steel wire cages (Sanki, Ltd., Tokyo, Japan) kept in 1.3 m3 inhalation chambers (Sibata Scientific Technology, Ltd., Tokyo, Japan) and maintained on a 12-h light-dark cycle. Basal pellet diet (Funabashi Farm, Co., Ltd., Chiba, Japan) was provided ad libitum, except during the 6-h daily period of benzene inhalation. Water was supplied ad libitum automatically through the tubing throughout the study. The temperature and humidity in the chambers were maintained automatically at 24 ± 1°C and 55 ± 10%, respectively.

Benzene exposure.
Benzene (Wako Chemical, Co., Ltd., Osaka, Japan) vapor was generated by heating liquid benzene to 16°C, and channeled into the inhalation chambers (Sibata Scientific Technology Ltd., Tokyo, Japan). Groups of 5–7 experimental and control mice were exposed, respectively, to benzene inhalation (300 ppm, 6 h/day, 5 days/week x 2 weeks) or ambient air. Benzene concentration in the chambers was monitored at half-hour intervals during daily exposures, using a gas chromatograph (Shimadzu Co., Kyoto, Japan).

Benzene metabolite exposure.
The combined but not single-agent treatment of mice with PH and HQ was previously found to give rise to hematotoxic effects similar to those following benzene exposure (Eastmond et al., 1987Go). The possibility of alterations in benzene metabolism due to the lack of AhR was evaluated by treating AhR+/+, AhR+/–, and AhR–/–mice with a combination of PH and HQ (Sigma, St. Louis, MO) instead of benzene, according to a modification of the method of Eastmond et al. (1987)Go. Experimental mice (three mice/group) were injected ip with freshly prepared saline solutions of PH (75 mg/kg) + HQ (75 mg/kg), given twice daily at 6-h intervals for 3 days. These doses were calculated to be equivalent to 309 ppm/day for 3 days of benzene exposure at 100% absorption rate and a 100% metabolic ratio. Control mice were injected with saline instead of PH and HQ, using the same protocol. Following the final dose, peripheral blood and BM parameters were measured.

Blood and BM parameters.
Peripheral blood was collected from the orbital sinus. Peripheral blood leukocyte (WBC) and red blood cell (RBC) numbers were measured using a Coulter Counter (Sysmex M-2000, Sysmex Co., Kobe, Japan). Hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) were also measured. BM cellularity was evaluated by harvesting BM cells from the femurs of each mouse (Yoon et al., 2001bGo). The animals were sacrificed; a 27-gauge needle was inserted into the femoral bone cavity through the proximal and distal edges of the bone shafts, and BM cells were flushed out under pressure by injecting 2 ml DMEM without phenol red (Gibco BRL, Rockville, MD). A single-cell suspension was obtained by gently and repeatedly drawing out the BM cells through the 27-gauge needle, and the cells were then counted using the Sysmex M-2000.

Progenitor assay (CFU-GM assay).
Because there is no appropriate assay method for lymphocyte progenitor, we only observed CFU-GM in semisolid methylcellulose culture. Briefly, 8 x 104 BM cells suspended in 100 µl DMEM were added to 3.9 ml of culture medium containing 0.8% methylcellulose (Nakarai Tesque, Co., Ltd., Kyoto, Japan), 30% fetal calf serum (HyClone Laboratories, Inc., Logan, UT), 1% bovine serum albumin (Sigma, St. Louis, MO), 10–4 M mercaptoethanol (Sigma, St. Louis, MO), and 10 ng/ml murine granulocyte-macrophage colony stimulating factor (GM-CSF, R&D Systems, Inc., Minneapolis, MN). One-milliliter aliquots containing 2 x 104 cells were plated in triplicate in a 35-mm tissue-culture plate (Nalgen Nunc International, Rochester, NY), and incubated for 6 days in a fully humidified incubator at 37°C and 5% CO2 in air. Colonies were counted under an inverted microscope (Olympus Optical, Co., Ltd., Tokyo, Japan).

Western blot assays for p21 and CYP2E1.
Protein extracts of the BM were prepared by sonicating femoral BM cells in a cell lysis buffer containing 20% sodium dodecyl sulfate (SDS), 2 mM phenylmethylsulfonyl fluoride, and a protease/phosphatase inhibitor (Yoon et al., 2001bGo). Protein concentration was quantified using a protein assay reagent (Bio-Rad Laboratories, Hercules, CA) and a Beckman spectrophotometer (Beckman Instruments, Inc., Fullerton, CA). Protein extracts of the hepatic tissue were prepared as described elsewhere (Valentine et al., 1996Go). Briefly, 50 mg of hepatic tissues from each mouse was homogenized in 0.7 ml buffer containing 0.25 M sucrose, 10 mM Tris-HCl, 1 mM EDTA, 0.1% ethanol, and 1 tablet of protease inhibitor cocktail (Roche Diagnostics Co., Indianapolis, IN). Homogenates were centrifuged at 13,000 rpm for 1 min (Microcentrifuge MX-150, Tomy Industrial Co., Tokyo, Japan) to remove nuclei and cell debris, and protein concentration of the supernatant was measured using a Beckman sepectrophotometer. Protein extracts from BM and the liver (15 µg for p21 and 10 µg for CYP2E1) were denatured, subjected to 12% (w/v) SDS-polyacrylamide gel electrophoresis, then transferred to HybondTM polyvinylidene fluoride membranes (Amersham Life Science, Buckinghamshire, UK). After blocking nonspecific binding sites by incubating the membranes with 5% nonfat dried milk and 0.1% Tween 20 in Tris-buffered saline (TTBS, pH 7.4) for 1 h at room temperature, the membranes were incubated overnight at 4°C in the presence of diluted primary antibodies, rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), 1:500, for p21 analysis; and goat antirat CYP2E1 polyclonal antibody (Daiichi Pure Chemicals Co., Ltd., Tokyo, Japan), 1:1000, for CYP2E1 quantification. The membranes were then washed with TTBS and incubated with 1:2000 horseradish-peroxidase–conjugated secondary antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 50 min at room temperature. To visualize the bands, the membranes were treated with a detection reagent (Amersham Life Science, Buckinghamshire, UK) for 1 min. The band densities were measured using an image analyzer (Fuji Film, Co., Ltd., Tokyo, Japan).

RT-PCR for AhR mRNA in the liver and bone marrow.
AhR mRNA expression was measured in the hepatic tissue and the BM cells from AhR+/+ mice exposed to 300 ppm benzene for 1 or 2 weeks. Total RNA was extracted using ISOGEN (Wako Chemical Co., Ltd., Osaka, Japan) according to the manufacturer’s instructions. Five micrograms of total RNA was reverse-transcribed for 50 min at 42°C with 200 units of M-MLV (Moloney murine leukemia virus) reverse transcriptase (RT) (Gibco BRL, Rockville, MD). Amplification by PCR was performed using oligonucleotide primers specific for the mouse Ah receptor [5‘ primer (5‘-GAT GCC TTG GTC TTC TAT-3‘) and 3‘ primer (5‘-TCA TGC CAC TTT CTC CAG TCT-3‘)]. One microliter cDNA was added to each tube containing 25 µl (final volume) of the sense and antisense primers (4 µM), 10x PCR buffer (Gibco BRL, Rockville, MD), 2.5 mM dNTP (TAKARA Shuzo Co., Kyoto, Japan) and advantageous cDNA polymerase (BD Biosciences Clontech, Palo Alto, CA). The mixture was preincubated for 5 min at 80°C. Twenty-eight cycles of PCR were carried out for liver sample, or 30 cycles for BM sample, with denaturation at 95°C for 30 sec, annealing at 55°C for 1 min, and extension at 72°C for 1 min. After the final cycle, the products were incubated at 72°C for 7 min. A 10-µl aliquot was electrophoresed on 1.5% agarose gel in tris-acetate/EDTA electrophoresis buffer.

Statistical analysis.
The two-tailed Student t-test was carried out to evaluate the significance of differences between control and benzene-exposed or PH + HQ–exposed groups. Differences greater than p < 0.05 were considered significant.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Blood Parameters after Two-Week Benzene Exposure
After a 2-week exposure of AhR+/+ and AhR+/– mice to 300 ppm benzene, there were significant decreases in the number of peripheral WBC (p < 0.01, p < 0.05, respectively) and RBC (p < 0.01, p < 0.05, respectively). The marked decrease of lymphocyte number attributes to the significant decrease of WBC. The decrease in lymphocytes was 23.4% to the control, whereas that in granulocytes was 67.0% to the control (data not shown; see Yoon et al, 2001bGo). Significant decreases in the values of HGB (p < 0.01, p < 0.05) and HCT (p < 0.01, p < 0.05) were also noted in AhR+/+ mice and AhR+/– mice. In contrast, no significant decreases in peripheral blood parameters were observed in AhR–/– mice (Fig. 1Go).



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FIG. 1. Changes in peripheral blood parameters and bone marrow cellularity in AhR wild-type (AhR+/+), heterozygous (AhR+/–), and homozygous mice (AhR–/–) exposed to 300 ppm benzene for 2 weeks. *Significantly different from corresponding control group, p < 0.05. **Significantly different from corresponding control group, p < 0.01.

 
BM Cellularity and CFU-GM Levels after Two-Week Benzene Exposure
Significant decreases in BM cellularity (to 49.4 ± 8.3%, 53.8 ± 1.0%, and 81.6 ± 1.5%, respectively, of the corresponding control values) were noted in AhR+/+, AhR+/–, and AhR–/– mice after 2-week benzene exposure (p < 0.05) (Fig. 1Go). However, the decrease was significantly less pronounced in AhR–/– mice than in AhR+/+ and AhR+/– mice (p < 0.01) (Fig. 1Go). Furthermore, the cellularity of CFU-GM per femur was decreased significantly (to 28.3 ± 3.9% and 52.0 ± 2.2% of respective control levels) in AhR+/+ and AhR+/–mice (p < 0.05), but remained virtually unchanged in AhR–/– mice (Fig. 2Go).



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FIG. 2. Changes in total number of CFU-GM per two femurs in AhR wild-type (AhR+/+), heterozygous (AhR+/–), and homozygous mice (AhR–/–) exposed to 300 ppm benzene for 2 weeks. The mean values for bone marrow cellularity of AhR wild-type, heterozygous, and homozygous mice were 4.8, 5.6, and 4.8 x 107/two femurs, respectively, and the mean numbers of CFU-GM per 8 x 104 bone marrow cells were 79, 78, and 72, respectively. *Significantly different from corresponding control group at p < 0.05.

 
Peripheral Blood and BM Cellularity after PH + HQ Treatment
In contrast to the above, the concomitant treatment with PH (75 mg/kg bw) and HQ (75 mg/kg bw) was extremely toxic, even in AhR-deficient mice. One AhR+/– mouse died, and one AhR–/– mouse was moribund after only 3 days of PH + HQ exposure. In this study, PH and HQ were injected ip twice per day at 6-h intervals for 3 days. The number of WBC was significantly decreased (to 57.9 ± 19.8%, 30.5 ± 8.9%, and 26.3 ± 7.6%, respectively, of corresponding control values) in AhR+/+, AhR+/–, and AhR–/– mice (p < 0.05; Fig. 3Go). HGB and HCT values were decreased significantly in AhR–/–mice to 91.1 ± 3.5% and 88.8 ± 1.6%, respectively, of control values (p < 0.05), but remained unchanged in AhR+/+ and AhR+/– mice (Fig. 3Go). A significant decrease in BM cellularity was observed in all three AhR mouse genotypes after PH + HQ treatment (to 28.6 ± 4.7%, 25.8 ± 7.2% and 25.8 ± 5.4%, respectively, of the corresponding control values; p < 0.05) (Fig. 3Go).



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FIG. 3. Changes in peripheral blood parameters and bone marrow cellularity in AhR wild-type (AhR+/+), heterozygous (AhR+/–), and homozygous mice (AhR–/–) after treatment with PH + HQ. PH (75 mg/kg bw) and HQ (75 mg/kg bw) were injected ip twice a day at 6-h intervals for 3 days. The dose of metabolites PH and HQ was calculated based on the dose of benzene inhalation equivalent to 300 ppm, 6 h/day, and the calculated exposure dose was 309 ppm, 6 h for 3 days at 100% absorption rate and 100% metabolic ratio (see Materials and Methods). *Significantly different from corresponding control value at p < 0.05.

 
Expression of p21 by BM Cells after Two-Week Benzene Exposure
As shown in Figure 4Go, p21 was upregulated in AhR+/+ and AhR+/– mice, but not in AhR–/– mice, after the 2-week benzene exposure.



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FIG. 4. Western blot analysis showing expression of p21 in bone marrow cells of AhR wild-type (AhR+/+), heterozygous (AhR+/–), and homozygous mice (AhR–/–) exposed to 300 ppm benzene for 2 weeks; p21 was upregulated in AhR wild-type and heterozygous mice but not in AhR homozygous mice. Fifteen µg protein per sample was loaded.

 
Expression of CYP2E1 in Liver after Two-Week Benzene Exposure
The expression of CYP2E1 was enhanced significantly to about twice the control value (p < 0.05) in the livers of benzene-exposed AhR+/+ mice. In contrast, the expression of CYP2E1 in AhR–/– hepatic tissue showed a statistically nonsignificant slight increase (Fig. 5Go).



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FIG. 5. Expression of CYP2E1 in the livers of AhR wild-type (AhR+/+) and homozygous mice (AhR–/–) exposed to 300 ppm benzene for 2 weeks. Note the highly attenuated expression of CYP2E1 enzyme in AhR homozygous mice.

 
AhR mRNA Level in the Hepatic Tissue and the BM
The level of AhR mRNA expression in the liver of benzene-exposed AhR+/+ mice manifested a 1.5-fold increase compared with the control value, although there was no statistical significance (Fig. 6Go). No notable change in the BM levels of AhR mRNA was observed in AhR+/+ mice exposed to 300 ppm benzene for 2 weeks (Fig. 7Go).



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FIG. 6. AhR mRNA level in liver tissues of wild-type mice exposed to 300 ppm benzene for 5 days (D5) and 12 days (D12).

 


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FIG. 7. AhR mRNA level in the bone marrow cells of wild-type mice exposed to 300 ppm benzene for 2 weeks.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The hematotoxicity of benzene is characterized by the suppression of erythromyelopoiesis, resulting in the depression of leukocyte and erythrocyte levels in peripheral blood and BM (Cronkite et al., 1989Go; Farris et al., 1997Go). We recently reported that the suppression of the hemopoietic progenitor cell cycle due to overexpression of p21 is one mechanism of benzene-induced hematotoxicity (Yoon et al., 2001bGo). However, currently the mechanism of benzene toxicity is not fully understood.

In this study, we demonstrated that AhR, a ligand-activated basic helix-loop-helix transcription factor, mediates benzene-induced hematotoxicity, at least in part, by participating in the induction of CYP2E1, a hepatic P450 enzyme critical for the conversion of benzene to its phenolic metabolites.

Our present study confirms the results of previous studies indicating that exposure of chemosensitive (AhR+/+) mice to benzene severely reduces peripheral blood cellularity, BM cellularity, and levels of CFU-GM in the BM. Moreover, these hematotoxic effects were supported by the marked upregulation of p21 in benzene-exposed AhR+/+ mice, as shown in the Western blot analysis (Fig. 4Go). These findings are consistent with those of previous studies. On the other hand, AhR–/– mice appeared to be resistant to benzene-induced hematotoxicity, as reflected by their markedly attenuated hematotoxic effects (Figs. 1 and 2GoGo) and lack of p21 upregulation (Fig. 4Go). Interestingly, the resistance of AhR–/– mice to hematotoxicity disappeared when the mice were concomitantly treated with two principal benzene metabolites, PH and HQ, instead of benzene itself (Fig. 3Go). The comparative dose of benzene inhalation corresponding to the total exposure dose of PH and HQ may be challenging to calculate because of the difficulty in estimation of bioavailability. It may be fair to conclude that the total exposure dose of PH and HQ may be estimated not to exceed the inhalation dose, at least. Not only does this confirm earlier findings by another group (Eastmond et al., 1987Go) that concomitant treatment with PH and HQ severely suppresses murine BM cellularity, but it also strongly suggests a mechanistic role of AhR in the metabolism of benzene.

It has been demonstrated that the breakdown of benzene to phenolic metabolites is responsible for the cytotoxicity and genotoxicity of benzene, and that CYP2E1 plays a critical role in the metabolism of benzene in the liver (Gut et al., 1996Go; Johansson and Ingelman-Sundberg, 1988Go; Koop et al., 1989Go). CYP2E1 knockout mice, which have reduced capacity to metabolize benzene, manifest no myelotoxic and cytotoxic effects following benzene exposure (Valentine et al., 1996Go). The high expression of CYP2E1 following benzene exposure further reflects the important role played by this enzyme in benzene metabolism and toxicity (Gut et al., 1996Go). In this study, the induction of CYP2E1 was upregulated in the livers of AhR+/+ mice exposed to benzene. This is in agreement with earlier findings by others (Snyder and Hedli, 1996Go; Valentine et al., 1996Go). Our results suggest that AhR participates in the induction of CYP2E1 after benzene exposure. However, that the induction of CYP2E1 was not achieved efficiently in AhR–/– mice (Fig. 5Go) may not simply explain the possible regulation of AhR for CYP2E1, because the increase in CYP2E1 in AhR–/– mice after benzene exposure was not significantly attenuated compared with the increase in wild-type mice. In addition, there is a possibility that the AhR–/– mouse may present an altered version of CYP2E1 expression, because the basal level of CYP2E1 expression in AhR–/– mice tends to be higher than that in the wild-type mice. The statistical significance is not clear. Nevertheless, the finding that CYP2E1 induction was not completely inhibited by the lack of AhR could explain, at least in part, why benzene exposure induces mild hematotoxicity in AhR–/– mice. No particular relevant relationship to the present study is yet known; however, CYP1A2 is also reduced to its basal expression level in AhR null mice (Schmidt et al., 1996Go).

The high susceptibility of AhR-deficient (AhR+/– and AhR–/–) mice to the benzene metabolite combination used in this study is probably associated with the decreased activity of the phase II detoxification process. This hypothesis is in agreement with an earlier report that UDP glucuronosyl transferase (UGT1a6), a phase II enzyme that protects organisms against toxins by glucuronidation of PH and HQ, is part of the Ah battery (Nebert and Duffy, 1997Go). It should also be noted that the enzyme NQO1 is a part of the Ah battery and may influence detoxification of increased amounts of quinones after treatment with PH and HQ in AhR-deficient mice. Histological examination of mice in our study revealed that combined PH and HQ treatment produced severe periportal hepatocyte necrosis leading to hepatic failure and death in AhR-deficient mice. This suggests that phenol is a preferred substrate, thereby leaving more free HQ available for activation (data not shown).

In this study, we also investigated the regulation of AhR mRNA expression level after benzene exposure. The expression pattern of AhR mRNA after the administration of dioxin-like chemicals whose toxicities are mediated by AhR has been assumed to be tissue or cell type specific (FitzGerald et al., 1996Go). For instance, AhR mRNA expression levels are increased in liver, but downregulated in craniofacial tissue, after TCDD treatment (Abbott et al., 1994Go; Sommer et al., 1999Go). Additionally, Sommer et al. (1999)Go and Yoon et al. (2001a)Go reported, respectively, no changes in the mRNA expression level of TCDD-exposed reproductive organs and BM. Thus, it is not clear that upregulation of AhR mRNA should be expected to occur in this study. The results of RT-PCR analysis in this study indicate that benzene does not significantly affect the expression level of AhR mRNA in the liver and BM tissue of AhR+/+ mice (Figs. 6 and 7GoGo). It should be noted, however, that the hepatic AhR mRNA expression levels tended to increase in the liver of benzene-exposed AhR+/+ mice in direct proportion to the duration of benzene exposure, in a manner analogous to the dose-dependent increase of the AhR mRNA expression level in the livers of TCDD-exposed rats (Sommer et al., 1999Go). Alternatively, increased message half-life or increased protein stability need to be investigated.

In summary, this study demonstrated for the first time that AhR mediates benzene-induced hematotoxicity, at least in part, by participating in the induction of CYP2E1 enzyme. Studies aimed at developing a binding assay for determining whether benzene binds directly to AhR and for elucidating the molecular mechanism whereby AhR mediates benzene metabolism are promising future endeavors.


    ACKNOWLEDGMENTS
 
We thank Ms. E. Tachihara and Ms. T. Kaneta for their excellent technical assistance. We also thank Lilian Delmonte, DSc, for her constructive peer review. This work was supported by Grants-in-Aid for Scientific Research (11670234, 13670236) from Japan Society for the Promotion of Science.


    NOTES
 
1 Authors contributed equally to this study. Back

2 To whom correspondence should be addressed. Fax: +81-3-3700-1622. E-mail: tohru{at}nihs.go.jp. Back


    REFERENCES
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 ABSTRACT
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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