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Stem Cells 2004;22:750-758 www.StemCells.com
© 2004 AlphaMed Press

Exposure of Hematopoietic Stem Cells to Benzene or 1,4-Benzoquinone Induces Gender-Specific Gene Expression

Brenda Faiola, Elizabeth S. Fuller, Victoria A. Wong, Linda Pluta, Diane J. Abernethy, Jason Rose, Leslie Recio

CIIT Centers for Health Research, Research Triangle Park, North Carolina, USA

Key Words. Hematopoietic stem cell • Benzene • Toxicity • In vitro culture • Leukemia Quantitative reverse transcription–polymerase chain reaction

Correspondence: Brenda Faiola, Ph.D.,GlaxoSmithKline, Research & Development, 5 Moore Drive, PO Box 13398, Research Triangle Park, NC 27709-3398, USA. Telephone: 919-483-5075; Fax: 919-483-6858; e-mail: Brenda.x.Faiola{at}gsk.com


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Chronic exposure to benzene results in progressive decline of hematopoietic function and may lead to the onset of various disorders, including aplastic anemia, myelodysplastic syndrome, and leukemia. Damage to macromolecules resulting from benzene metabolites and misrepair of DNA lesions may lead to changes in hematopoietic stem cells (HSCs) that give rise to leukemic clones. We have shown previously that male mice exposed to benzene by inhalation were significantly more susceptible to benzene-induced toxicities than females. Because HSCs are targets for benzene-induced cytotoxicity and genotoxicity, we investigated DNA damage responses in HSC from both genders of 129/SvJ mice after exposure to 1,4-benzoquinone (BQ) in vitro or benzene in vivo. 1,4-BQ is a highly reactive metabolite of benzene that can cause cellular damage by forming protein and DNA adducts and producing reactive oxygen species. HSCs cultured in the presence of 1,4-BQ for 24 hours showed a gender-independent, dose-dependent cytotoxic response. RNA isolated from 1,4-BQ–treated HSCs and HSCs from mice exposed to 100 ppm benzene by inhalation showed altered expression of apoptosis, DNA repair, cell cycle, and growth control genes compared with unexposed HSCs. Rad51, xpc, and mdm-2 transcript levels were increased in male but not female HSCs exposed to 1,4-BQ. Males exposed to benzene exhibited higher mRNA levels for xpc, ku80, ccng, and wig1. These gene expression differences may partially explain the gender disparity in benzene susceptibility. HSC culture systems such as the one used here will be useful for testing the hematotoxicity of various substances, including other benzene metabolites.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Benzene is a lipid-soluble, volatile organic compound and ubiquitous environmental pollutant that is rapidly absorbed after short-term inhalation exposures in humans [1]. This prototypical human and rodent carcinogen induces chromosomal breaks as a primary mode of genotoxicity in bone marrow (BM). Chronic benzene exposure results in progressive depression of BM function, leading to a reduction in the number of circulating red and white blood cells [2, 3]. Epidemiological studies show that occupational exposure to benzene results in an increased risk of aplastic anemia, myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), chronic lymphocytic leukemia, and other disorders [4–9]. In a cohort of shoe workers with similar numbers of men and women exposed to benzene, the standardized mortality ratio (SMR) for aplastic anemia and leukemia in men was 1566 and 400, respectively, compared with 416 and 0 for women [9]. Results of a larger cohort study showed similar excesses of mortality due to leukemia and lymphoma in both genders of benzene-exposed workers compared with unexposed workers [8]. In this study, significant excess risks were shown for aplastic anemia (nine cases) and MDS (seven cases) because no cases were seen in the unexposed workers; the relative risk for AML plus the precursor MDS was 4.1 [8].

A prerequisite of benzene-induced cellular toxicity is oxidation of benzene in the liver by cytochrome P450 2E1 (CYP2E1) to benzene oxide and other reactive intermediates [5]. Benzene oxide can be oxidized to form catechol [10], undergo ring opening to produce trans-trans-muconaldehyde, or spontaneously rearrange to form phenol (PH), which is then hydroxylated in the liver to form hydroquinone (HQ). In the BM, HQ and catechol are converted by myeloperoxidase to 1,4-benzoquinone (BQ) and 1,2-BQ, respectively, which can be detoxified by reduction via nicotinamide adenine dinucleotide (phosphate): reduced quinone oxidoreductase-1. These reactive quinones are capable of binding to macromolecules, including DNA, and generating free radicals and reactive oxygen species (ROS) [5, 10]. PH and HQ can act synergistically to potentiate the formation of 1,4-BQ and ROS. The resulting DNA strand breaks can lead to chromosomal aberrations. DNA damage after benzene exposure must be properly repaired or the damaged cell must undergo apoptosis to prevent proliferation of mutated cells and subsequent transformation into malignancies.

Various studies have suggested that hematopoietic stem cells (HSCs) are the target cell population for benzene-induced alterations. In the BM, HSCs are a small population (<0.05% of BM) of self-renewing, pluripotent cells that give rise to all blood cells [11]. Inhalation exposure to benzene significantly reduced the number of transplantable spleen colony-forming units, colony-forming units–granulocyte-monocyte (CFU-GM), and erythroid colony-forming units (CFU-E) in the BM of male and female mice [12–14], indicating a decrease in the number of HSCs after exposure to benzene. CFU-E from adult male Swiss Webster mice cultured in the presence of HQ or 1,4-BQ were more susceptible to the cytotoxic effects of the chemical than CFU-E from females, suggesting that gender differences in benzene-induced hematotoxicity may be attributable in part to intrinsic factors at the target cell level [15]. More recently, benzene was found to affect cell-cycle kinetics, because the fraction of CFU-GM in S phase was 16.3% in male C57BL/6 mice exposed to 300 ppm benzene for 2 weeks compared with 37.1% in unexposed control mice [16]. In addition, persistent benzene-induced DNA damage was observed as an increased frequency of aneuploidy in the long-term self-renewing population of HSCs (Lin–, c-kit+, Sca-1+) from male and female mice 8 months after gavage with benzene compared with the vehicle-only control mice [17]. Thus, benzene has short-and long-term deleterious effects on HSCs.

Because proper repair of benzene-induced lesions is one essential mechanism for preventing possible leukemogenic outgrowth, understanding the nature of the DNA repair process in benzene-exposed HSCs is critical. We have shown previously that male 129/SvJ mice are more sensitive than females to benzene-induced hematotoxicity, myelotoxicity, and genotoxicity as demonstrated by decreased white blood cell counts, decreased nucleated cell area in BM (pancytopenia), and increased micronucleated erythrocytes, respectively [18]. In addition, microarray analysis of isolated BM HSCs from male 129/SvJ mice exposed to 100 ppm benzene for 2 weeks showed altered expression of 119 sequences, including increased mRNA for genes involved in cell-cycle control (cyclin G and cyclin F), growth control (wig1), apoptosis (bax), and DNA repair (nibrin and histone H2AX) [19].

In the present study, we assessed the cytotoxic effects of 1,4-BQ on HSC from 129/SvJ male and female mice. In addition, the DNA damage response and repair pathways in BM HSCs after in vivo exposure to benzene or in vitro exposure to 1,4-BQ were examined at the level of transcription. We focused on 1,4-BQ because formation of this stable metabolite has been proposed to be an important component in the mechanism of benzene-induced myelotoxicity and genotoxicity [20]. In addition, cysteine adducts of 1,4-BQ are more abundant than adducts of 1,2-BQ or benzene oxide in mouse hemoglobin and BM proteins [21]. Quantitative real-time reverse transcription–polymerase chain reaction (qRT-PCR) was used to analyze the expression of several key DNA repair genes in each of the four major DNA repair pathways, as well as various apoptosis, cell-cycle control, and growth-control genes. Differences in gene expression patterns were observed between HSCs from male and female mice exposed to benzene by inhalation or cultured in the presence of 1,4-BQ. The gene expression profiles may partially explain the gender-related differences in hematotoxicity and myelotoxicity seen after exposure to benzene.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice
129/SvJ (129X1/SvJ) mice were purchased from Jackson Laboratory (Bar Harbor, ME). Mice were housed in polycar-bonate cages in a humidity- and temperature-controlled room and given water and an NIH-07 rodent diet (Zeigler Brothers, Gardners, PA) ad libitum. All animal use was conducted in facilities accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care. The Institutional Animal Use and Care Committee of the CIIT Centers for Health Research approved all animal use.

In Vivo Exposure to Benzene
Male and female mice ranging from 6–10 weeks of age were acclimated to stainless steel wire mesh cages in a Hinners-style whole-body inhalation chamber 2 weeks before the start of the 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.). Mice were exposed to 0 (air-exposed control) or 100 ppm benzene for 6 hours/day, 5 days/week for 2 weeks during the light cycle. Only water was given during the exposures. Each exposure group was housed in a separate 1-m3 stainless steel and glass inhalation chamber (Hazelton H1000, Lab Products, Seaford, DE). Benzene (Sigma-Aldrich Chemical Co., Milwaukee) purity was assessed by gas chromatography before the start of exposure and determined to be 100%. Generation and monitoring of exposure atmospheres have been described previously [18, 19].

BM Preparation and HSC Isolation
Immediately after benzene inhalation, mice were euthanized by i.p. injection of 5 mg pentobarbital (Abbott Laboratories, Chicago). For in vitro experiments with 1,4-BQ, naive mice were euthanized by asphyxiation with carbon dioxide (CO2). The tibias, femurs, and humeri from seven mice per group were removed and placed in complete RPMI 1640 containing 10% fetal bovine serum (Life Technologies, Carlsbad, CA). All bones were collected within 2 hours after euthanasia. BM was flushed out with complete RPMI 1640 media (Invitrogen Life Technologies, Carlsbad, CA) containing 10% fetal bovine serum (Invitrogen Life Technologies) using a syringe and 23- or 26-gauge needle. HSCs were isolated from the pooled total BM by a combination of magnetic negative selection and fluorescence-activated cell sorting as described previously [19]. Long-term, self-renewing HSCs were sorted on a FACSVantage flow cytometer (Becton, Dickinson, San Jose, CA) as the lineage–, Sca-1+, c-kit+ fraction [11]. HSCs for in vitro experiments were pelleted by centrifugation and suspended in cell culture media (see next section). HSCs from air- and benzene-exposed mice were immediately pelleted by centrifugation and suspended in 0.5 ml RNAlater® (Ambion, Inc., Austin, TX) to preserve the RNA. HSC samples in RNAlater were kept at 4ºC and processed within 4 weeks.

In Vitro Exposure of HSCs to 1,4-BQ
Sorted HSCs were suspended in complete medium and seeded at 8.125 x 103 cells per well in 96-well round-bottom cell culture plates (Corning Incorporated, Corning, NY). Complete media consisted of Iscove’s modified Dulbecco’s medium (Invitrogen Life Technologies) supplemented with 15% heat-inactivated fetal bovine serum (Invitrogen Life Technologies), 1% penicillin/streptomycin (Invitrogen Life Technologies), 1% L-glutamine (Invitrogen Life Technologies), 50 µM ß-mercaptoethanol, 1 x nonessential amino acids (Invitrogen Life Technologies), 1 x sodium pyruvate (Invitrogen Life Technologies), 25 mM HEPES buffer (Invitrogen Life Technologies), recombinant mouse granulocyte-macrophage colony-stimulating factor (10 ng/ml; R & D Systems, Minneapolis), recombinant mouse stem cell factor (50 ng/ml; R & D Systems), recombinant mouse interleukin (IL)-3 (50 ng/ml; R & D Systems), and recombinant mouse IL-6 (25 ng/ml; R & D Systems). The cultures were maintained in a humidified incubator at 37ºC and 5% CO2.

1,4-BQ (CAS #106-51-4; 99.7% pure) was obtained from Sigma Chemical Co. (St. Louis) and stored desiccated at 4ºC before use. A 100-mM stock solution of 1,4-BQ in HPLC-grade 100% methanol (Fisher Scientific, Pittsburgh) and dilutions of the stock solution in fresh complete medium were prepared immediately before addition to cell cultures. After the overnight incubation period (approximately 20 hours), cells were treated with 0, 1, 5, or 10 µM 1,4-BQ. Cells were harvested 24 hours after chemical exposure by centrifugation at 200 x g and resuspended in fresh media. Cell counts and viability were determined using the Guava® ViaCount® assay and the Guava® PCATM system (Guava Technologies, Inc., Hayward, CA) according to manufacturer’s instructions. The remaining cells were pelleted, resuspended in RNAlater, stored at 4°C, and processed within 4 weeks.

Analysis of mRNA Expression Levels
Total RNA was isolated from HSCs using the Qiagen RNeasy® kit (Qiagen, Valencia, CA), which includes treatment with DNase I to remove genomic DNA. cDNA was generated from 0.2 mg of total RNA using TaqMan® RT reagents with random hexamers as primers according to the manufacturer’s protocol (Applied Biosystems, Inc., Foster City, CA). Primers for p21, bax, wig1, p53, gadd45a, ku80, prkdc, rad51, rad54, rpa, apex1, pcna, DNAPolß, xpc, xpg, bcl-2, cyclin G, mdm-2, and gapdh were designed using Primer Express software (Applied Biosystems, Inc.) and assayed for specificity and efficiency following the manufacturer’s protocol. The primer sequences and efficiencies have been previously published [19]. qRT-PCR with SYBR® Green (Applied Biosystems, Inc.) was performed using the ABI PRISM® 7700 Sequence Detection System (Applied Biosystems, Inc.). All samples were analyzed in duplicate or triplicate using gapdh as the reference gene, and most genes were analyzed twice. Quantitation of mRNA levels for the gene of interest was determined, and the fold change in a target gene mRNA level from a treated sample compared with a control sample was determined by the manufacturer’s comparative CT method, where fold change = 2(–{Delta}{Delta}CT) (User Bulletin #2,ABI Prism 7700 Sequence Detection System).

Statistical Analyses
Statistical analyses of cytotoxicity data were done using JMP 5.0.1 statistical software (SAS Institute, Inc., Cary, NC). An analysis of variance (ANOVA) was performed on each variable, with the two main effect factors being gender and exposure level, and their first-order interaction was tested. Significant differences by ANOVA were analyzed additionally by Dunnett’s multiple-comparison test. Statistical significance of gene expression data was determined by a randomization test using the relative expression software tool (REST©) for groupwise comparison, where fold change = (E target){Delta}C T (MEAN control – MEAN sample, target)/(E gapdh){Delta}CT (MEAN control – MEAN sample, gapdh) [22]. The level of significance used for all statistical tests was p < .05. Because mean and median values were similar, all values reported represent the mean ± standard error of the mean.


   RESULTS
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cytotoxicity of 1,4-BQ in HSCs from Male and Female 129/SvJ Mice
The cytotoxic effect of 1,4-BQ on HSCs isolated from naive male and female 129/SvJ mice was assessed in vitro. The concentrations of 1,4-BQ used were selected based on a previous study with human BM CD34+ hematopoietic progenitor cells that used 0 to 20 µM 1,4-BQ [23]. Cytotoxicity was evaluated soon (24 hours) after exposure to 1,4-BQ, because continued culture would have allowed additional differentiation of HSCs along the myeloid pathway. Under the culture conditions used, the six untreated cultures (0 µM 1,4-BQ) underwent an average of 1.84 ± 0.18 cell doublings, which is similar to the 1.53 ± 0.45 doublings seen in our in vitro studies of human CD34+ cells [23]. Because gender was not a significant factor, the data were combined. Exposure of murine HSCs to 1,4-BQ for 24 hours induced a gender-independent, dose-dependent cytotoxic response (Figs. 1A, 1BGo). Significant reduction in cell viability was observed at exposure to concentrations of 1,4-BQ equal to or greater than 5 µM (n = 6), with exposure to 10 µM (n = 5) 1,4-BQ resulting in 53.02 ± 3.75% mean viability (Fig. 1AGo; 54.90% median viability). In addition to reduced viability, the proliferation of cells in culture was compromised by exposure to 1,4-BQ. From 16,250 initially seeded HSCs, the number of viable cells obtained 24 hours after exposure to 1,4-BQ decreased with increasing concentration of the chemical. Significant reduction in the number of viable cells recovered was seen with exposure of HSCs to concentrations of 1,4-BQ equal to or greater than 5 mM (Fig. 1BGo). Compared with untreated cultures, the number of viable cells in cultures exposed to 10 µM 1,4-BQ was reduced by approximately 77%. The combination of decreased proliferation and increased cell death resulted in recovery of fewer viable cells (approximately 13,000) from cultures exposed to 10 µM 1,4-BQ than the number of viable HSCs initially seeded (16,250).



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  		Figure 1. Cytotoxicity of 1,4-benzoquinone to murine HSCs is dose-dependent. HSCs from six independent isolations (three male and three female) were seeded in 96-well round-bottom tissue culture plates (8,125 HSCs/well). After an overnight incubation period, 1,4-benzoquinone was added at the indicated final concentrations. After 24 hours of culture, cells were harvested for cell enumeration and viability assessment using the Guava®Via-Count® assay. Because there was no statistically significant difference between genders, the data from cultures of male and female HSCs were pooled. (A): Data represent the mean percent viability. (B): Data represent the mean number of viable cells recovered after treatment of 16,250 (two wells) initially seeded HSCs (indicated by dashed line). Bars represent the standard error of the mean (n = 6 for 0, 1, and 5 µM; n = 5 for 10 µM because one of three isolations from female mice yielded <65,000 HSCs). * Indicates significant reduction compared with the untreated (0 µM) control group as determined by Dunnett’s test (p < .05). Abbreviation: HSC, hematopoietic stem cell.

 
Gene Expression Analysis of HSCs Exposed to 1,4-BQ In Vitro
Based on the microarray analysis of HSCs from benzene-exposed male mice [19] and our interest in DNA repair pathways responsible for maintaining genome integrity after benzene exposure, the mRNA levels of 18 genes involved in DNA damage responses such as DNA repair, apoptosis, growth control, and cell-cycle control were determined by qRT-PCR. Despite the similar cytotoxicity to HSCs from both genders of mice, the expression patterns of some genes in HSCs exposed to1,4-BQ in vitro were different between genders. Notably, HSCs from male mice showed significantly increased mRNA levels (1.4- to 3.2-fold) for seven genes after exposure to 5 or 10 µM 1,4-BQ compared with unexposed cells, whereas HSCs from female mice exposed to the same concentration of 1,4-BQ exhibited elevated mRNA levels (1.5- to 2.6-fold) for just four of these seven genes (Figs. 2A, 2BGo). Rad51, xpc, and mdm-2 transcript levels were increased in male but not female HSCs, whereas p21, wig1, bax, and ccng were induced to similar levels in HSCs from both genders (Figs. 2A, 2BGo). Of the 18 genes analyzed, 11 genes showed no significant change in the level of mRNA after exposure to any concentration of 1,4-BQ (data for p53 shown; other 10 genes not shown).



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  		Figure 2. Seven genes in HSCs showed altered mRNA levels in at least one gender after in vitro exposure to 1,4-BQ. mRNA levels in HSCs of male (A) and female (B) 129/SvJ mice after exposure to 0, 1, 5, or 10 µM BQ were determined by quantitative real-time reverse transcription–polymerase chain reaction analysis. RNA was isolated from HSCs from six independent HSC separations (three male and three female) after culture in the presence or absence of 1,4-BQ. One of three HSC separations from female mice did not yield enough cells to treat with 10 µM 1,4-BQ. Data represent the mean ± standard error of the mean fold change in mRNA expression in HSCs after exposure to various levels of 1,4-BQ (white bars, 1 µM; gray bars, 5 µM; hatched bars, 10 µM) relative to unexposed controls (black bars, 0 µM; n = 2–6 samples per exposure level per gender because several genes were analyzed twice). * Indicates significant difference in gene expression between 1,4-BQ–treated and untreated cultures from the same gender (p < .05); indicates a significant difference compared with the opposite gender at the same concentration of 1,4-BQ (p < .05). Abbreviations: BQ, 1,4-benzoquinone; HSC, hematopoietic stem cells.

 
Gene Expression Analysis of HSCs from Mice Exposed to Benzene In Vivo
The mRNA levels of the same 18 genes were determined by qRT-PCR in HSCs from male and female 129/SvJ mice exposed to air or 100 ppm benzene for 2 weeks by inhalation, and gender differences were observed in the expression pattern of some genes. Notably, HSCs from male mice exposed to 100 ppm benzene showed significantly higher mRNA levels for ku80, ccng, and wig1 compared with HSCs from benzene-exposed females (Figs. 3A, 3BGo). A gender difference was also noted in xpc mRNA levels, because HSCs from benzene-exposed female but not male mice exhibited a significantly decreased mRNA level for this gene. Several genes, including p53, rad51, bax, mdm-2, DNA Polß, and gadd45a, showed statistically significant alterations of mRNA levels in HSCs from benzene-exposed mice compared with HSCs from air-exposed mice of the same gender; however, the mRNA levels showed no difference between genders (Figs. 3A, 3BGo). Of the 18 genes analyzed, eight showed no significant change in the level of mRNA after exposure to 100 ppm benzene in either gender (data for p21 shown; data for seven genes not shown). One of these eight unaltered genes was p21, which was significantly induced in male and female HSCs exposed to 5 and 10 µM 1,4-BQ in vitro.



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  		Figure 3. Ten genes in HSCs showed altered mRNA levels in at least one gender after in vivo exposure to benzene. mRNA levels in HSCs of male (A) and female (B) 129/SvJ mice after exposure to 0 or 100 ppm benzene were determined by quantitative real-time reverse transcription–polymerase chain reaction analysis. RNA was isolated from 12 separate HSC separations (3 male, 0 ppm; 3 female, 0 ppm; 3 male, 100 ppm; 3 female, 100 ppm). Data represent the mean ± standard error of the mean fold change in mRNA expression at 100 ppm (white bars) relative to 0 ppm controls (black bars; n = 3 or 6 samples per exposure level per gender because analyses of several genes were repeated). * Indicates significant difference in gene expression between HSCs from benzene-exposed and air-exposed mice of the same gender (p < .05); indicates a significant difference compared with the opposite gender (p < .05). Abbreviation: HSC, hematopoietic stem cells.

 

   DISCUSSION
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Benzene has been recognized as a human health concern since the mid-1970s, when several reports linked exposure to this chemical with an increased risk of developing various forms of leukemia [4, 8, 9, 24, 25]. The mechanisms that lead to leukemia in some individuals after benzene exposure are unclear. To exert a toxic effect, biotransformation of benzene to toxic metabolites, including 1,4-BQ, occurs, and resulting DNA damage can lead to chromosomal aberrations [5] and malignant transformation if not properly repaired. Thus, key determinants of interindividual variability and risk in response to the toxic effects of benzene may likely be the DNA repair enzymes required to restore genomic integrity after DNA damage. A better understanding of the mechanisms of benzene-induced toxicity is essential for developing biomarkers that will identify individuals who constitute genetically susceptible subpopulations at increased risk to benzene-induced toxicity and leukemia.

We have shown previously that exposure of 129/SvJ mice to benzene by inhalation induced significant hematotoxicity, myelotoxicity, and genotoxicity in males, whereas females exhibited no hematotoxicity and only slight myelotoxicity and genotoxicity [18]. ROS and 1,4-BQ are proposed ultimate agents of benzene-induced hematotoxicity and possibly leukemogenicity. Therefore, we examined the cytotoxic effect of 1,4-BQ on HSCs, a likely target cell population for benzene-induced DNA damage, from male and female 129/SvJ mice. Exposure of murine HSCs to 1,4-BQ for 24 hours induced a gender-independent, dose-dependent cytotoxic response. Likewise, exposure of male and female human CD34+ hematopoietic progenitor cells to 1,4-BQ for a similar number of cell doublings resulted in a gender-independent, dose-dependent cytotoxic response [23]. These in vitro findings suggest that benzene metabolites other than 1,4-BQ may be responsible for the gender differences seen in the myelotoxic and hematotoxic response of mice to inhaled benzene. An alternative interpretation is that 1,4-BQ may work in concert with steroid hormones or other endogenous factors not present in the in vitro system to lead to gender differences in benzene-induced toxicities in vivo. Therefore, it is possible that molecular differences in the response to 1,4-BQ may exist despite the similar in vitro cellular response between genders. Other investigators showed that treatment of myeloblasts with HQ resulted in increased numbers of cells because of the 1,4-BQ–mediated inhibition of apoptosis in these cells [26]. Thus, 1,4-BQ elicits different effects on the growth of HSCs compared with the more differentiated myeloblast.

To investigate potential molecular differences between genders, we compared the gene expression pattern in HSCs from male and female 129/SvJ mice that had been exposed to 1,4-BQ in vitro or benzene in vivo. Other studies have demonstrated benzene-induced gender differences in micronuclei induction, sister chromatid exchanges, and metabolism [27–32]. The explanation for these gender differences is unclear; however, prior studies suggest that male hormones are partly responsible for this phenomenon [29] and that differences in metabolism of PH to HQ [27] and other metabolism differences, such as glucuronidases (a phase II enzyme), could also account for these gender-specific effects [28]. Our study provides evidence that gene expression in HSCs may reflect the observed gender differences. We found that HSCs from male 129/SvJ mice exposed to benzene by inhalation had higher mRNA levels for several genes, including xpc, ccng, wig1, and ku80 compared with HSCs from benzene-exposed female mice. Likewise, exposure of HSCs from male 129/SvJ mice to 10 µM 1,4-BQ in vitro resulted in higher mRNA levels for rad51, xpc, and mdm-2 compared with similarly treated HSCs from female mice. In vitro or in vivo exposure of male HSCs to 1,4-BQ or benzene, respectively, resulted in altered expression of 7 of 18 genes tested. Of these seven genes, rad51, bax, ccng, and wig1 were upregulated by both exposure scenarios. Female HSCs had only 4 of 18 genes altered by each exposure condition, with wig1 induced by both 1,4-BQ and benzene.

An increased level of bax mRNA and no change in bcl-2 levels was observed in 1,4-BQ–treated HSCs and HSCs from benzene-exposed male mice, suggesting an increased sensitivity to apoptosis compared with HSCs from benzene-exposed females that showed no significant increase in bax. HSCs from benzene-exposed female mice showed a significant decrease in mRNA levels for p53 and mdm-2, a gene under the control of p53, suggesting suppression of p53 function. Results of a study comparing the repopulation ability and clonogenic activity of HSCs from p53 knockout and wild-type mice suggest that suppression of p53 function facilitates hematopoietic reconstitution after cytotoxic treatment by delaying the exhaustion of the most primitive HSC pool and decreasing the sensitivity to apoptosis [32]. Thus, at the time point assessed, HSCs from benzene-exposed female mice may have suppressed p53 function and decreased sensitivity to apoptosis compared with HSCs from benzene-exposed male mice, which may partially explain the very low level of myelotoxicity and hematotoxicity observed in female mice compared with males [18] and the lower SMR for aplastic anemia in females [9] after exposure to benzene.

Wig1, a direct p53 transcriptional target encoding a zinc finger protein that binds to dsRNA and plays a role in growth regulation [33], was the most highly induced gene for both male and female mice in both the in vitro and in vivo experiments. Although 1,4-BQ exposure induced wig1 transcription equally in male and female HSCs in vitro, benzene exposure in vivo induced wig1 fourfold less in HSCs from females, supporting the lesser p53 function in females compared with males. In addition, transcription of the cyclin-dependent kinase inhibitor p21 and several other genes involved in G1/S and G2/M cell-cycle control is upregulated in response to p53 activation due to DNA damage [34, 35]. HSC quiescence requires p21, with a reduction in p21 resulting in increased cell cycling and stem cell proliferation [36]. In HSCs from benzene-exposed male and female mice, p21 mRNA levels were not altered relative to air-exposed mice; this lack of p21 induction may allow HSCs with benzene-induced DNA damage to escape quiescence and proliferate, leading to leukemia. Because gene expression analysis was conducted at one time point (after 2 weeks of benzene exposure), characterization of the temporal pattern of p53 and p21 gene expression earlier in the exposure period may additionally support or refute this hypothesis.

Male and female murine HSCs exposed to 1,4-BQ in vitro showed no alteration in mRNA levels for p53 or mdm-2 but had similarly increased levels of p21 and bax mRNA, indicating a functional p53-dependent DNA damage response with probable cell-cycle arrest and a similar sensitivity to apoptosis. Similar induction patterns for p21 and p53 were also found in human CD34+ HPCs exposed to 1,4-BQ in vitro [23]. Thus, a key to benzene-induced toxicity differences between genders may be the concentration of 1,4-BQ in the BM, with high levels of 1,4-BQ activating a p53 response. Males may generate more 1,4-BQ than females after exposure to benzene, thereby inducing a greater p53 functional response resulting in cell-cycle arrest and apoptosis, leading to the observed greater myelotoxicity and hematotoxicity in mice [18] and the higher SMR for aplastic anemia in humans [8]. Studies are ongoing to investigate the levels of several benzene metabolites in various tissues from benzene-exposed male and female mice.

The incidence of leukemia is similar in males and females exposed to benzene [8], suggesting that BM cells, HSCs, or more differentiated cells with DNA damage induced by 1,4-BQ or other benzene metabolites are ultimately not eliminated or repaired properly and thus may proliferate as leukemic clones. Indeed, a similar level of DNA damage was observed in 1,4-BQ–treated CD34+ human BM cells [23], which are a mixed population of cells that contains some HSCs and other more differentiated cells. The similar level of DNA damage seen in CD34+ cells of both genders [23] and the similar incidence of leukemia [8] suggests that the observed increase in transcription of rad51 and xpc, genes involved in different pathways of DNA repair, may have little overall impact on DNA repair capacity in HSCs from male mice.

In conclusion, hematopoietic disorders associated with exposure to benzene may be attributable in part to the direct toxic effects of 1,4-BQ on HSCs. Studies on HSCs that examine the toxicity and resulting gene expression profile of other benzene metabolites and mixtures of metabolites should be helpful in elucidating the mechanism of benzene-induced carcinogenesis. Finally, HSC culture systems such as the one used here will be a valuable tool for assessing the hematotoxicity of various substances.


   ACKNOWLEDGMENTS
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The authors would like to thank Drs. Dave Dorman and Kevin Gaido for their constructive comments; the CIIT inhalation, necropsy, and animal care staff for their excellent technical assistance; Jeanne Galbo for her editorial assistance; and Dr. Barbara Kuyper for her editorial review of the manuscript. This study was funded in part by the American Chemistry Council through the Long Range Research Initiative.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received November 26, 2003; accepted for publication March 17, 2004.



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