* CIIT Centers for Health Research, Research Triangle Park, North Carolina 27709; and
National Cancer Institute, Bethesda, Maryland 20892
Received October 23, 2002; accepted December 11, 2002
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ABSTRACT |
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Key Words: benzene; bone marrow; genotoxicity; hematotoxicity; leukemia; mEH; micronuclei.
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INTRODUCTION |
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The metabolism of benzene is complex and consists of many possible pathways (Fig. 1). Cytochrome P4502E1 (CYP2E1) is essential for benzene metabolism; without this enzyme, no hematotoxicity or genotoxicity was observed in mice (Valentine et al., 1996
). Many of the metabolites, such as benzene oxide and 1,4-benzoquinone can lead to toxic effects (Ross, 2000
; Smith, 1996
). The pathway most commonly studied with respect to benzene-induced toxicity is the hydroquinone pathway (Ross, 2000
; Smith, 1996
). Microsomal epoxide hydrolase (mEH; E.C. 3.3.2.3) converts benzene oxide to benzene dihydrodiol, which is then converted to catechol by a dehydrogenase (Ross, 2000
). In an in vitro system, mEH can convert benzene oxide to hydroquinone and phenol metabolites (Snyder et al., 1993
). In addition, there is significant controversy regarding the production of trans,trans-muconaldehyde through the dihydrodiol intermediate in the mEH pathway (Witz et al., 1996
). Thus the importance of mEH in benzene metabolism is unclear.
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Mice deficient in mEH expression and activity have a portion of exon 2 deleted and have no unusual phenotype, suggesting that mEH is not essential for reproduction and physiological homeostasis (Miyata et al., 1999). The mEH-deficient mice did not bioactivate 7,12-dimethylbenz[a]anthracene (DMBA) to the carcinogenic metabolite 3,4-diol-1,2-oxide and are therefore highly resistant to DMBA-induced carcinogenesis (Miyata et al., 1999
). This finding supports a role for mEH in bioactivating certain polycyclic aromatic hydrocarbons. We hypothesized that mice deficient in mEH have a reduced response to benzene-induced toxicity due to a decrease in the amount of toxic metabolites produced. In this study, using 129/sv (background) and mEH/ mice, we demonstrate that mEH is critical in benzene-induced genotoxicity, hematotoxicity, and induction of the p53 DNA damage response in male, but not female, mice.
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MATERIALS AND METHODS |
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Experimental animals.
We used male and female mice that were 1012 weeks old at the start of the study. The mice were housed as described above until they were moved for acclimation to stainless steel wire mesh cages in a Hinners-style, whole-body inhalation chamber 2 weeks prior to benzene exposures. During this 2-week acclimation period, the mice were put on a reversed-light schedule (on at 1 A.M. and off at 1 P.M.), with exposures taking place during the light cycle. Only water was given during the exposures. Tissues were collected within 5 h after termination of exposure. Each exposure group, including air-exposed controls, was housed in a separate inhalation chamber. Mice used in the determination of basal CYP2E1 activity were not housed in suspended wire cages but remained in the polycarbonate shoebox caging.
CYP2E1 activity and protein content.
Liver microsomes were prepared using the method of (Guengerich, 1989). Briefly, after mice were euthanized with 5 mg pentobarbital/mouse, livers were removed from naive male and female mice and then homogenized in 4 volumes of ice-cold buffer A (0.154 M KCl, 0.05 M Tris Base [Sigma, St. Louis, MO], pH 7.4). After centrifugation at 10,000 x g for 20 min at 4°C, the supernatant was transferred to an ultracentrifuge tube and spun at 105,000 x g for 60 min at 4°C. After removal of the supernatant, the pellet was resuspended in 7 ml of buffer B (0.05 M Tris, 0.25 M sucrose, 1 mM EDTA [Sigma], pH 7.4). The microsome solution was then centrifuged at 105,000 x g for 60 min at 4°C and the supernatant discarded. The microsome pellet was resuspended in an amount equivalent to the liver weight in buffer C (0.1 M K2HPO4, 0.25 M sucrose, pH 7.4) and frozen at 80°C until further use.
-Nitrophenol (PNP) hydroxylase activity was measured using a spectrophotometric assay (Reinke and Moyer, 1985
). Ninety percent of PNP hydroxylase activity reflects CYP2E1 activity, thus it was used as a measure of CYP2E1 activity (Tierney et al., 1992
). Incubation mixtures that contained 0.1M potassium phosphate buffer, 0.1 mM PNP, 1.0 mM ascorbate, and 0.4 mg/ml liver microsomal protein were preincubated for 3 min at 37°C prior to the addition of 1.0 mM NADPH. In control incubation mixtures, NADPH was replaced with an equivalent amount of deionized H2O. The total incubation volume was 1.0 ml. The incubation was performed for 7.5 min at 37°C in a shaking water bath and terminated by adding 0.2 ml 1.5 N perchloric acid. The sample was diluted 10:1 with 10 N NaOH, and the absorbance was measured at 546 nm on a Beckman DU 650 spectrophotometer (Fullerton, CA). The concentration of 4-nitrocatechol was determined using an extinction coefficient of 10.28 cm2/mol (Reinke and Moyer, 1985
).
Laemmli sample buffer (Bio-Rad, Hercules, CA) was added directly to microsomal protein and boiled for 5 min. Eight µg protein per sample was loaded onto a 10% polyacrylamide SDSPAGE gel. The gel was electrophoresed and then transferred onto Immobilon-P transfer membrane (Millipore, Bedford, MA). The membrane was blocked for 2 h in 5% milk plus 0.1% Tween-20 (Sigma, St. Louis, MO) in PBS (GibcoBRL-Gaithersburg, MD) followed by an overnight incubation at 4°C with the primary polyclonal rat cytochrome P450 CYP2E1 antibody (Gentest Corporation, Woburn, MA) at a 1:1000 dilution in 0.5% milk in PBS. After 3 washes in 1% milk plus 0.1% Tween-20, the secondary antibody anti-goat-horseradish peroxidase (Santa Cruz Biotechnologies, Santa Cruz, CA) was used at a 1:20,000 dilution in 0.5% milk in PBS for a 1-h incubation at room temperature, followed by more washes. The signal was detected using West Pico Chemiluminescence (Pierce Endogen, Rockford, IL). Densitometry was done using the BIO-RAD Quantity One Quantitation software (Ver. 4).
Experimental design.
This study used three benzene exposure groups (10, 50, and 100 ppm benzene) and one control air-exposed group (0 ppm). Exposure levels were chosen based on a pilot study using 0, 10, and 100 ppm benzene exposure levels in older mice (data not shown). Mice were exposed to benzene 5 days/week, for 6 h/day for a 2-week duration, in all experiments based on previous studies (Farris et al., 1997).
A previous time course in male 129/SvJ (Jackson Laboratories) mice demonstrated that the optimal sampling time point for determining peak levels of micronucleated reticulocytes (MN-RET; immature RBC) and p21 mRNA expression was within 5 h postexposure relative to 12 or 24 h post exposure (A.K. Bauer and L. Recio, unpublished data). Following termination of exposure and chamber off gasing, mice were brought to the necropsy facilities and euthanized with an ip injection of 5 mg pentobarbital/mouse (Abbott Laboratories, Chicago, IL). Blood was collected by cardiac puncture into microcontainers (Becton Dickenson, San Jose, CA) containing EDTA for total white blood cell counts. Additional blood was collected for MN analysis. The bones were then removed, femurs followed by humerus.
Benzene exposure.
Benzene exposure methods are described in detail (Healy et al., 2001). Benzene (Sigma, St. Louis, MO) purity was assessed prior to inhalation by the CIIT inhalation facility, and all benzene exposures were controlled by the Andover Infinity system (Andover Controls, Corp., Andover, MA). Benzene exposure atmospheres were measured at least six times per exposure with infrared spectrophotometers (MIRAN 1A, The Foxboro Co., Foxboro, MA). The chambers were operated with a continuous flow of HEPA-filtered air at approximately 1800 L/min. Control mice (0 ppm) were exposed to a continuous flow of conditioned HEPA-filtered air in inhalation chambers.
MN analysis in blood.
The enumeration of MN is a measure of genotoxicity (Hayashi et al., 2000). The Prototype Microflowplus Mouse Micronucleus Analysis Kit and protocol (Litron Laboratories, Rochester, NY) was used to determine the frequency or percentage of MN-RET and MN-normochromatic erythrocytes (NCE) in mouse blood by flow cytometry. Briefly, approximately 50 µl of blood was collected and suspended in 300 µl anticoagulant (solution B; proprietary solution of Litron Laboratory). One hundred eighty µl of the suspension was then injected into 2 ml cold methanol (-80°C) and kept at 80°C until further processing. On the day of analysis, samples were inverted several times, followed by addition of solution C (proprietary solution of Litron Laboratory). Cells were then centrifuged at 4°C for 5 min and incubated with a CD71 (transferrin receptor)-FITC conjugated antibody and RNase. Propidium iodide (PI) solution was then added to the cells immediately prior to flow cytometry analysis on a FACSVantage (Becton Dickenson, San Jose, CA). Malaria-infected cells were used as a reference standard to consistently define the micronucleus analysis windows and to establish proper PMT voltages and compensation (Dertinger et al., 1996
). Nucleated cells in the peripheral blood other than the RET and NCE were gated out. MN were defined as PI-positive cells, RET were identified as CD71-positive cells, and NCE were identified as CD71-negative cells (Serke and Huhn, 1992
).
Hematology.
White blood cells were counted on an Advia 120 Hematology System (Bayer Diagnostics, Tarrytown, NY) by a contract hematology laboratory (Antech Diagnostics, New York, NY).
Bone marrow preparation.
After the bones were removed, the femur marrow cavity was flushed with RNAlater (Ambion, Austin, TX) to preserve the RNA. The RNA samples were kept at 4°C and processed within 6 weeks. Both humeri were flushed with Hanks balanced salt solution (Sigma) plus 5% albumin (Sigma) to preserve the cellular viability while manipulations were done. Total humoral cell counts were determined on a coulter counter (Model ZM, Coulter Electronics Ltd., Hialeah, FL) and used as a measure of myelotoxicity.
Real-time quantitative RT-PCR for p21 mRNA expression.
Total RNA from bone marrow was isolated using the Qiagen RNAeasy Kit (Qiagen, Valencia, CA), including DNase treatment. RNA was quantified by measuring the absorbance at 260 nm on a Beckman DU 650 Spectrophotometer (Beckman Coulter, Fullerton, CA). Reverse-transcription (RT) reactions were performed with 2 µg of total RNA, using the Reverse Transcription Kit from Applied Biosystems (Foster City, CA). The ABI 7700 Prism Sequence Detection System, using Sybr green (Applied Biosystems) was used to quantify p21 mRNA expression. PCR primers specific for p21 and gapdh (glyceraldehyde-3-phosphate dehydrogenase; for normalization) were determined using Primer Express Software (Applied Biosystems; Boley et al., 2002). The primers 5'
3' are p21 (forward) atgtccaatcctggtgatgt, (reverse) tgcagcagggcagaggaagt; gapdh (forward) aatgtgtccgtcgtggatctga, (reverse) gatgcctgcttcaccaccttct (Boley et al., 2002
). The reaction conditions and data analysis were performed according to the manufacturers recommended protocol. All samples were run in triplicate using GAPDH as the calibrator gene, since GAPDH levels do not change across genotypes or with benzene exposure (Boley et al., 2002
).
Statistical analysis.
All data are presented as the mean ± standard error of the mean (SEM). A three-way analysis of variance (ANOVA) was done on each variable with the three main-effect factors of gender, strain, and exposure level and their first-order interactions. If any of the interactions were significant, additional analyses were done so that the nature of the interaction could be understood. Tukeys multiple comparison procedure was used to determine differences among significant factors with three or more levels; p < 0.05 was used as the level of significance for all statistical tests. A Pearson correlation coefficient was calculated for the two variables MN-RET percentages and p21 mRNA fold-increases. Statistical analyses were done using SAS Statistical Software (SAS Institute, Inc., Cary, NC).
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RESULTS |
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DISCUSSION |
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In the present study, we found that male mice deficient in mEH enzyme activity were unresponsive to benzene in contrast to male 129/Sv mice, which developed significant benzene-induced hematotoxicity, myelotoxicity, and genotoxicity. Female 129/Sv mice exhibited very little response to benzene, as revealed by no significant differences in hematotoxicity, myelotoxicity, and genotoxicity between the female 129/Sv and mEH/ mice. Expression of p21 mRNA was greatly elevated in the male 129/Sv mice and to a lesser degree in the female 129/Sv mice, while no changes were seen in either gender of the mEH/ mice.
The gender differences seen in the 129/Sv strain are not unique. Other rodent studies have demonstrated gender differences in MN, sister chromatin exchanges, and benzene metabolism (Kenyon et al., 1995; Luke et al., 1988
; Meyne and Legator, 1980
). The cause of these differences has not been discerned but may involve metabolic differences between the two genders (Kenyon et al., 1995
). Whether gender differences occur in humans with respect to benzene-induced toxicity is not clear. One study observed that women have a slightly lower risk of cancer mortality due to benzene compared to men (Li et al., 1994
). However, most epidemiological studies to date have not studied both genders (Aksoy and Erdem, 1978
; Rothman et al. (see above), 1997; Yin et al., 1996
). The present work suggests that more gender-specific studies are necessary to address this issue.
Our results in the mEH/ mice demonstrating a male-specific effect are consistent with the recent article by Lebailly et al.(2002), describing chromosomal aberrations similar to those seen with benzene in men with the fast-activity phenotype of mEH. The similar chromosome abnormalities seen were translocations at chromosomes 8 and 21 and chromosome 7 deletions on 7q (Lebailly et al., 2002
). These investigators also found that only men with the high-activity phenotype are at a higher risk of developing AML. The Lebailly et al. association study suggests that polymorphisms in mEH may be risk factors for AML in those individuals with the chromosomal abnormalities discussed above. In addition, Cavalieri et al.(2002)
found that the quinone derived from catechol (1,2-benzoquinone) can form depurinating DNA adducts that could initiate cancer, which further supports the importance of this pathway in benzene metabolism. Our data demonstrates that male mice are affected by deleting part of the mEH gene. However, determining the importance of mEH in females is difficult because the females have such a small response to benzene. In addition to mEH, we recently studied mice deficient in NQO1 activity and found that female NQO1/ mice developed benzene-induced genotoxicity and hematotoxicity compared to the NQO1+/+ mice, which were unresponsive (Bauer et al., 2003
). However, male NQO1/ mice were only more sensitive to benzene hematotoxicity compared to the NQO1+/+ mice. These results further support the need to assess the many different enzymes and gender differences involved in benzene metabolism to identify individuals that are at a higher risk of benzene toxicity and possibly leukemogenesis. Additional studies determining differences in the benzene metabolism pathway will be done using the mEH/ mice to further understand the responses described here.
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ACKNOWLEDGMENTS |
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NOTES |
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