* Division of Cellular and Molecular Toxicology, National Institute of Health Sciences, 1-18-1 Kamiyohga, Setagayaku, Tokyo 158-8501, Japan;
Department of Veterinary Pathology, College of Veterinary Medicine and Agricultural Biotechnology, Seoul National University, Suwon, Republic of Korea;
Department of Chemistry, Tohoku University, Graduate School for Science, Sendai, Japan
Received June 20, 2002; accepted August 13, 2002
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ABSTRACT |
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Key Words: aryl hydrocarbon receptor; benzene; CYP2E1; hematotoxicity; mice.
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INTRODUCTION |
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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, 1996; Longacre et al., 1981
; Snyder and Hedli, 1996
). 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, 1999
; Snyder and Hedli, 1996
). The toxic effects of benzene on BM and peripheral blood cells have been attributed to the interaction of the phenolic metabolites (Eastmond et al., 1987
; Guy et al., 1990
). Among the descriptions of mechanisms of benzene toxicity (Ross, 2000
), 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., 1987
; Subrahmanyam et al., 1990
). This may explain the high sensitivity of mice to benzene toxicity compared with the relative resistance of rats (Henderson, 1996
).
DBA/2 mice have been reported to be more susceptible to benzene than C57BL/6 mice (Longacre et al., 1981). 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., 1994
; Safe, 1995
). 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.
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MATERIALS AND METHODS |
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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 57 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., 1987). 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)
. 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., 2001b). 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), 104 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., 2001b). 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., 1996
). 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-peroxidaseconjugated 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 manufacturers 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 + HQexposed groups. Differences greater than p < 0.05 were considered significant.
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RESULTS |
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DISCUSSION |
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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. 4). 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 2
) and lack of p21 upregulation (Fig. 4
). 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. 3
). 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., 1987
) 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., 1996; Johansson and Ingelman-Sundberg, 1988
; Koop et al., 1989
). CYP2E1 knockout mice, which have reduced capacity to metabolize benzene, manifest no myelotoxic and cytotoxic effects following benzene exposure (Valentine et al., 1996
). 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., 1996
). 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, 1996
; Valentine et al., 1996
). 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. 5
) 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., 1996
).
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, 1997). 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., 1996). For instance, AhR mRNA expression levels are increased in liver, but downregulated in craniofacial tissue, after TCDD treatment (Abbott et al., 1994
; Sommer et al., 1999
). Additionally, Sommer et al. (1999)
and Yoon et al. (2001a)
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 7
). 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., 1999
). 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.
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ACKNOWLEDGMENTS |
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NOTES |
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2 To whom correspondence should be addressed. Fax: +81-3-3700-1622. E-mail: tohru{at}nihs.go.jp.
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REFERENCES |
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Aksoy, M., Erdem, S., and Dincol, G. (1974). Leukemia in shoe-workers exposed chronically to benzene. Blood 44, 837841.[ISI][Medline]
Aksoy, M., Erdem, S., and Dincol, G. (1976). Types of leukemia in chronic benzene poisoning. A study in thirty-four patients. Acta Haematol. 55, 6572.[ISI][Medline]
Cronkite, E. P., Bullis, J., Inoue, T., and Drew, R. T. (1984). Benzene inhalation produces leukemia in mice. Toxicol. Appl. Pharmacol. 75, 358361.[ISI][Medline]
Cronkite, E. P., Drew, R. T., Inoue, T., Hirabayashi, Y., and Bullis, J. E. (1989). Hematotoxicity and carcinogenicity of inhaled benzene. Environ. Health Perspect. 82, 97108.[ISI][Medline]
Eastmond, D. A., Smith, M. T., and Irons, R. D. (1987). An interaction of benzene metabolites reproduces the myelotoxicity observed with benzene exposure. Toxicol. Appl. Pharmacol. 91, 8595.[ISI][Medline]
Farris, G. M., Robinson, S. N., Gaido, K. W., Wong, B. A., Wong, V. A., Hahn, W. P., and Shah, R. S. (1997). Benzene-induced hematotoxicity and bone marrow compensation in B6C3F1 mice. Fundam. Appl. Toxicol. 36, 119129.[ISI][Medline]
FitzGerald, C. T., Fernandez-Salguero, P., Gonzalez, F. J., Nebert, D. W., and Puga, A. (1996). Differential regulation of mouse Ah receptor gene expression in cell lines of different tissue origins. Arch. Biochem. Biophys. 333, 170178.[ISI][Medline]
Gut, I., Nedelcheva, V., Soucek, P., Stopka, P., Vodicka, P., Gelboin, H. V., and Ingelman-Sundberg, M. (1996). The role of CYP2E1 and 2B1 in metabolic activation of benzene derivatives. Arch. Toxicol. 71, 4556.[ISI][Medline]
Guy, R. L., Dimitriadis, E. A., Hu, P. D., Cooper, K. R., and Snyder, R. (1990). Interactive inhibition of erythroid 59Fe utilization by benzene metabolites in female mice. Chem. Biol. Interact. 74, 5562.[ISI][Medline]
Henderson, R. F. (1996). Species differences in the metabolism of benzene. Environ. Health Perspect. 104(Suppl 6), 11731175.[ISI][Medline]
Johansson, I., and Ingelman-Sundberg, M. (1988). Benzene metabolism by ethanol-, acetone-, and benzene-inducible cytochrome P-450 (IIE1) in rat and rabbit liver microsomes. Cancer Res. 48, 53875390.[Abstract]
Koop, D. R., Laethem, C. L., and Schnier, G. G. (1989). Identification of ethanol-inducible P450 isozyme 3a (P450IIE1) as a benzene and phenol hydroxylase. Toxicol. Appl. Pharmacol. 98, 278288.[ISI][Medline]
Koss, G., and Tesseraux, I. (1999). Hydrocarbons. In Toxicology (H. Marquardt, S. G. Schafer, R. McClellan, and F. Welsh, Eds.), pp. 603609. Academic Press, California.
Longacre, S. L., Kocsis, J. J., and Snyder, R. (1981). Influence of strain differences in mice on the metabolism and toxicity of benzene. Toxicol. Appl. Pharmacol. 60, 398409.[ISI][Medline]
Mimura, J., Yamashita, K., Nakamura, K., Morita, M., Takagi, T. N., Nakao, K., Ema, M., Sogawa, K., Yasuda, M., Katsuki, M., et al. (1997). Loss of teratogenic response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice lacking the Ah (dioxin) receptor. Genes Cells 2, 645654.
Nebert, D. W., and Duffy, J. J. (1997). How knockout mouse lines will be used to study the role of drug-metabolizing enzymes and their receptors during reproduction and development, and in environmental toxicity, cancer, and oxidative stress. Biochem. Pharmacol. 53, 249254.[ISI][Medline]
Okey, A. B., Riddick, D. S., and Harper, P. A. (1994). The Ah receptor: Mediator of the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. Toxicol. Lett. 70, 122.[ISI][Medline]
Ross, D. (2000). The role of metabolism and specific metabolites in benzene-induced toxicity: Evidence and issues. J. Toxicol. Environ. Health A 61, 357372.[ISI][Medline]
Safe, S. H. (1995). Modulation of gene expression and endocrine response pathways by 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds. Pharmacol. Ther. 67, 247281.[ISI][Medline]
Schmidt, J. V., Su G. H., Reddy J. K., Simon M. C., and Bradfield C. A. (1996). Characterization of a murine Ahr null allele: Involvement of the Ah receptor in hepatic growth and development. Proc. Natl. Acad. Sci. U.S.A. 93, 67316736.
Snyder, C. A., Goldstein, B. D., Sellakumar, A. R., Bromberg, I., Laskin, S., and Albert, R. E. (1980). The inhalation toxicology of benzene: Incidence of hematopoietic neoplasms and hematotoxicity in ARK/J and C57BL/6J mice. Toxicol. Appl. Pharmacol. 54, 323331.[ISI][Medline]
Snyder, R., and Hedli, C. C. (1996). An overview of benzene metabolism. Environ. Health Perspect. 104 (Suppl 6), 11651171.[ISI][Medline]
Sommer, R. J., Sojka, K. M., Pollenz, R. S., Cooke, P. S., and Peterson, R. E. (1999). Ah receptor and ARNT protein and mRNA concentrations in rat prostate: Effects of stage of development and 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment. Toxicol. Appl. Pharmacol. 155, 177189.[ISI][Medline]
Subrahmanyam, V. V., Doane-Setzer, P., Steinmetz, K. L., Ross, D., and Smith, M. T. (1990). Phenol-induced stimulation of hydroquinone bioactivation in mouse bone marrow in vivo: Possible implications in benzene myelotoxicity. Toxicology 62, 107116.[ISI][Medline]
Valentine, J. L., Lee, S. S., Seaton, M. J., Asgharian, B., Farris, G., Corton, J. C., Gonzalez, F. J., and Medinsky, M. A. (1996). Reduction of benzene metabolism and toxicity in mice that lack CYP2E1 expression. Toxicol. Appl. Pharmacol. 141, 205213.[ISI][Medline]
Yoon, B. I., Hirabaysahi, Y., Kaneko, T., Kodama, Y., Kanno, J., Yodoi, J., Kim, D. Y., and Inoue, T. (2001a). Transgene expression of thioredoxin (TRX/ADF) protects against 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced hematotoxicity. Arch. Environ. Contam. Toxicol. 41, 232236.[ISI][Medline]
Yoon, B. I., Hirabayashi, Y., Kawasaki, Y., Kodama, Y., Kaneko, T., Kim, D.Y., and Inoue, T. (2001b). Mechanism of action of benzene toxicity: Cell cycle suppression in hemopoietic progenitor cells (CFU-GM). Exp. Hematol. 29, 278285.[ISI][Medline]