CIIT Centers for Health Research, Research Triangle Park, North Carolina 27709
Received November 13, 2003; accepted January 27, 2004
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ABSTRACT |
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Key Words: benzene; benzoquinone; CD34; hematopoietic progenitor cells; DNA repair; cytotoxicity.
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INTRODUCTION |
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Although the mechanisms responsible for benzene toxicity remain unclear, metabolic activation of benzene is essential for initiating benzene toxicity (Cooper, 1988). Hydroquinone (HQ), one of the active metabolites resulting from CYP2E1 catalyzed metabolism of benzene to phenol, is postulated to be a likely candidate responsible for benzene hematotoxicity (Ross, 1996
). HQ can be further metabolized in bone marrow by myeloperoxidases (MPO) to highly toxic and mutagenic 1,4-benzoquinone (BQ; Golding and Watson, 1999
; Snyder, 2000
). In addition, the benzene metabolite catechol can be metabolized by MPO in bone marrow to 1,2-benzoquinone (Snyder, 2000
). These reactive quinones may form oxidative products in bone marrow that initiate damage in DNA, lipids, or proteins, leading to cell death or mutagenic events that may induce leukemogenesis (Thomas et al., 1990
; Levay et al., 1993
; Winn, 2003
).
Benzene and the metabolites catechol, phenol, and hydroquinone induce cell transformation and gene mutations in mammalian cells in culture (Tsutsui et al., 1997). In addition, rodent bioassays showed benzene to be a multipotential carcinogen that induces numerous neoplasms in male and female rats and mice, including malignant lymphoma in B6C3F1 mice and leukemia in RF/J mice (Maltoni et al., 1989
; National Toxicology Program, 1986
). Several benzene metabolites, not including 1,4-benzoquinone, have been tested for carcinogenic potential in long-term rodent assays. In a 51-week study in F344 male rats, catechol was shown to enhance forestomach and glandular stomach carcinogenesis, possibly having complete carcinogenic potential for the glandular stomach (Hirose et al., 1987
). Phenol was not carcinogenic to male or female rats or mice (National Toxicology Program, 1980
). Hydroquinone was carcinogenic, inducing adenomas of the kidney in male rats, mononuclear cell leukemia in female rats, and hepatocellular adenomas in female mice (Kari et al., 1992
).
All leukemias arise clonally from hematopoietic stem cells (HSC) or hematopoietic progenitor cells (HPC; Irons and Stillman, 1996). HSC are self-renewing bone marrow cells that give rise to all blood cells; early during the development of lymphoid and myeloid lineages, HSC differentiate into lineage-committed HPC that have lost the capacity for self-renewal (Kondo et al., 2003
). The occurrence of DNA damage in these cells as a result of chemical exposure could ultimately lead to the expansion of a fully differentiated pool of myeloid or lymphoid cells carrying mutations that result in leukemia and other blood dyscrasias (Golding and Watson, 1999
; Irons and Stillman, 1996
). Proper repair of benzene-induced DNA lesions and deletion of HSC or HPC with DNA damage are important mechanisms to prevent malignant transformation. Thus, it is important to understand the role that DNA repair plays in modulating the toxic and leukemogenic effects of benzene and its metabolites.
Several studies suggest that HSC or HPC may be important targets for benzene-induced toxicity and carcinogenesis. Human CD34+ cells contain both primitive HSC and more committed HPC (reviewed in Kondo et al., 2003) and have been used to study the effects of many compounds, including benzene and its metabolites. Treatment of CD34+ cells with HQ and HL60 human promyelocytic leukemia cells with catechol and HQ led to an increased percentage of cells undergoing apoptosis (Moran et al., 1996
). Aneusomy of chromosome 5, 7, and 8 in CD34+ cells was also reported following HQ exposure (Smith et al., 2000
; Stillman et al., 2000
). Likewise, eight months after oral exposure to benzene, mouse HSC fractions contained up to 14% aneuploid cells with chromosomal aberrations affecting chromosome 2 or 11 (Giver et al., 2001
). However, the molecular mechanisms associated with benzene-induced DNA damage and repair in these HSC and HPC remain unclear. Following DNA damage, phosphorylation and acetylation of p53 activates its DNA binding and transcriptional activity (Prives and Hall, 1999
). Transcription of p21, a cyclin-dependent kinase that is at least partially regulated by p53, and several other genes is upregulated in response to p53 activation (Prives and Hall, 1999
; Taylor and Stark, 2001
). A recent study has demonstrated that such up-regulation of p21 occurs as a result of DNA damage in the M07 HPC line (Campanini et al., 2001
). In addition, we have shown that p21 mRNA levels in murine total bone marrow increase following benzene inhalation (Bauer et al., 2003a
,b
; Boley et al., 2002
; Faiola et al., 2003
). The p53 transcription factor also regulates DNA repair in addition to cell cycle and cell death. A generalized reduction in DNA repair capacity in CD34+ cells compared to more committed cells has been reported (Buschfort-Papewalis et al., 2002
).
We have begun to examine the use of human CD34+ HPC to assess the cytotoxicity, genotoxicity, and expression of DNA damage response genes resulting from exposure to a metabolite of the human leukemogen benzene. In the present study, we evaluated the toxic effects of the active metabolite 1,4-BQ in cultured human bone marrow CD34+ progenitor cells and we investigated expression of key DNA damage response and repair genes in these cells. The current study was more comprehensive than previous toxicity studies on human CD34+ cells (Moran et al., 1996; Smith et al., 2000
; Stillman et al., 2000
) due to the use of both genders, the defined age and nonsmoking status of the subjects, and use of individual (n = 10 males and 10 females) rather than pooled samples. CD34+ bone marrow cells were found to be sensitive targets of 1,4-BQ cytotoxicity and genotoxicity, and these toxicities were found to be gender-independent. Treatment of human CD34+ cells with 1,4-BQ resulted in induction of the cyclin-dependent kinase inhibitor p21 at the mRNA level, confirming the critical role of this p53-dependent gene in 1,4-BQ-induced toxicity. No changes in mRNA expression were observed for p53 or the DNA repair genes rad51, xpc, xpa, ku80, and ape1. CD34+ cells provide a unique system for examining the toxic effects of a wide variety of hematotoxic substances, including carcinogens.
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MATERIALS AND METHODS |
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Cell culture.
Human bone marrow CD34+ progenitor cells were obtained from Poietics (BioWhittaker, Walkersville, MD). Ten healthy, nonsmoking men and women ages 1830 were selected for the study (Table 1). All donors were negative for HIV as well as hepatitis B and C. CD34+ cells (approximately 11.5 x 106 cells) from one male and one female donor were shipped separately for each experiment. Upon arrival, cells were resuspended in complete medium and seeded at 2.5 x 105 viable cells per well (4 ml/well; 56 wells per donor) in six cell culture plates (Corning Inc. Life Sciences, Acton, MA). Complete culture media consisted of Iscove's Modified Dulbecco's Medium (IMDM; BioWhittaker, Walkersville, MD) supplemented with 15% fetal bovine serum (BioWhittaker, Walkersville, MD), 1% penicillin/streptomycin (Gibco Invitrogen Corp., Carlsbad, CA), 1% L-glutamine (Gibco Invitrogen Corp.), granulocyte-macrophage colony-stimulating factor (GM-CSF; 10 ng/ml) (R & D Systems, Minneapolis, MN), stem cell factor (SCF; 25 ng/ml) (R & D Systems, Minneapolis, MN), interleukin-3 (IL-3; 10 ng/ml) (R & D Systems, Minneapolis, MN), and interleukin-6 (IL-6; 10ng/ml) (R & D Systems, Minneapolis, MN). The cultures were maintained in a humidified incubator at 37°C and 5% CO2.
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Apoptosis assay.
From cultures containing at least 1.2 x 105 viable cells after chemical treatment, 7 x 104 viable cells were transferred to separate 1.5 ml microcentrifuge tubes and washed once with cold PBS. Cells were suspended in 100 µl 1x cold annexin V binding buffer (BD Biosciences, San Diego, CA) and 50 µl of the suspension was transferred to each of two 5 ml polystyrene round-bottom tubes. One tube served as the unstained control sample. To the other tube, 5 µl of Annexin V-FITC (BD Biosciences, San Diego, CA) and 10 µl of 0.1 mg/ml 7-aminoactinomycin D (7-AAD) (Molecular Probes, Inc., Eugene, OR) were added. After the samples were incubated for 15 min at room temperature in the dark, 0.2 ml of 1x binding buffer was added to each tube, and the cells were analyzed on a FACSVantage flow cytometer (Becton Dickinson, San Jose, CA). Quadrants were set based on the analysis of single-stained samples. The percentage of apoptotic cells was equal to the percentage of Annexin V+/7-AAD cells in the dual-stained sample less the percentage of Annexin V+/7-AAD cells in the unstained control sample. The percentage of dead cells was equal to the percentage of Annexin V+/7-AAD+ cells in the dual-stained sample less the percentage of Annexin V+/7-AAD+ cells in the unstained control sample.
Micronuclei analysis.
Aliquots of 50,000100,000 viable cells from each sample were spun onto glass slides at 1000 rpm for 6 min using a Cytocentrifuge (Cytospin ®, Shandon, Pittsburgh, PA). The slides were air dried and stored at 80°C. Prior to scoring micronuclei (MN), cells were fixed and stained using Protocol Hema 3® Stain Set (Biochemical Sciences, Inc., Swedesboro, NJ). Slides were read using an Olympus BX61 Microscope (Olympus America, Melville, NY). MN were scored according to standardized criteria on size, shape, and position with regard to the main nucleus (Fenech, 2000). MN counted were 1/161/3 the diameter of the parent nucleus, round or oval with smooth edges, and stained reddish in color. The total number of MN counted per 25006500 cells was calculated for each sample.
Isolation of RNA.
Aliquots of CD34+ cells in PBS containing 10% FBS were pelleted by centrifugation, resuspended in RNAlaterTM (Ambion, Austin, TX), and stored at 4°C. Total RNA was isolated using the Qiagen RNeasy® Kit (Qiagen, Valencia, CA) following the manufacturer's recommended protocol. DNase digestion using the Qiagen RNase-Free DNase Set was included in the RNA extraction protocol. Total RNA was eluted in H2O and stored at 80°C.
Quantitative RT-PCR.
cDNA was generated from individual total RNA samples using Taqman® Multiscribe Reverse Transcriptase and random hexamers as primers following the manufacturer's recommended protocol (Applied Biosystems, Inc., Foster City, CA). Real-time quantitative RT-PCR (qRT-PCR) was performed using SYBR®Green (Applied Biosystems, Inc., Foster City, CA) on the ABI Prism 7700 Sequence Detection System (Applied Biosystems, Inc., Foster City, CA). qRT-PCR analysis was performed on samples from each donor that exhibited approximately 60% relative toxicity (a 40% reduction in the viable cell count) compared to unexposed controls following 1,4-BQ treatment.
All qRT-PCR primers (Table 2) used in the study were designed using Primer Express software (Applied Biosystems, Inc., Foster City, CA). The efficiency (E) of each primer set was tested on various dilutions of cDNA from untreated CD34+ male and female samples and was calculated as follows: E = 10(1/sl°pe), where slope is determined from the plot of threshold cycle versus cDNA input (ng) (Pfaffl, 2001). Primer specificity was confirmed by performing a dissociation curve analysis, which showed a single product with the appropriate Tm for each primer set.
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Statistical analysis.
Data are presented as means ± standard error of the mean (SEM). For all end points, statistical analyses were done using JMP 5.0.1 statistical software (SAS Institute, Inc., Cary, NC). A two-factor analysis of variance (ANOVA) was performed on each variable with the two factors being gender and 1,4-BQ treatment. The interaction of these factors was also tested. Gender was found to be a nonsignificant factor, and there was no interaction between gender and 1,4-BQ treatment. Therefore, subsequent analysis was performed on combined male and female data using a one-way ANOVA to assess 1,4-BQ concentration for each response. Significant differences by ANOVA were further analyzed by Student's t-test or Dunnett's multiple comparison methods. MN data were analyzed using a paired sample t-test. The level of significance used for all statistical tests was p < 0.05.
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RESULTS |
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DISCUSSION |
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The purpose of the current study was to investigate the toxicity in human bone marrow CD34+ hematopoietic progenitor cells and the induction of various key DNA repair genes following exposure to 1,4-benzoquinone; in dosing so, the utility of this in vitro culture system for examining the toxicity of potential hematotoxic agents was demonstrated. 1,4-BQ is an active metabolite of benzene and has been shown to be genotoxic in a variety of cell types (Glatt et al., 1989; Ludewig et al., 1989
; Winn, 2003
), including murine bone marrow cells (Corti and Snyder, 1998
). The data from the current study demonstrate that 1,4-BQ was cytotoxic and genotoxic to human CD34+ bone marrow cells. The response of the small sample size examined here (n = 10 M and 10 F) was remarkably similar between individuals and genders with no obvious outliers. A significant dose-dependent decrease in cell viability was observed 72 h after CD34+ cells were exposed to 1,4-BQ. Decreased cell counts were likely due to a combination of cell cycle arrest due to increased expression of p21 and increased levels of apoptosis. We also detected significant increases in micronucleated CD34+ cells treated with 1,4-BQ compared to untreated controls. These observations were consistent with reports of 1,4-BQ-induced cytotoxicity and genotoxicity in V79 Chinese hamster lung fibroblast cells (Glatt et al., 1989
; Ludewig et al., 1989
) and murine bone marrow cells (Corti and Snyder, 1998
). In addition, 1,4-BQ and HQ are toxic to murine bone marrow stromal cells (Gaido and Wierda, 1984
). The combined toxic effects on both stromal cells and HPC likely lead to the hematotoxic and leukemogenic effects of benzene exposure.
The toxic response of CD34+ HPC to 1,4-BQ was similar for both genders. This is in contrast to numerous murine studies that show males to be more sensitive than females to benzene and its metabolites (Bauer et al., 2003a,b
; Corti and Snyder, 1998
; Faiola et al., 2003
). However, studies in our laboratory on the toxicity of 1,4-BQ to mouse HSC (Lin, Sca-1+, c-kit+ cells) resulted in similar findings to the human CD34+ HPC, with no observable gender difference in the response (Faiola et al., 2004, submitted manuscript). These in vitro findings indicate that a benzene metabolite or metabolites other than 1,4-BQ may be responsible for the observed gender difference in the mouse response to inhaled benzene. The effect of gender on the response to benzene in humans is less clear. Most of the epidemiological studies on benzene-associated malignancies did not include women (Aksoy et al., 1974
; McCraw et al., 1985
; Rinsky et al., 1981
) or pooled the data for men and women together (Rothman et al., 1996
). Results of a large cohort showed significant excesses of mortality due to leukemia and lymphoma in benzene-exposed workers, with similar relative risk for men and women (Yin et al., 1996
). However, this same study noted significant gender differences for other malignancies such as an increased risk among men for lung cancer (Yin et al., 1996
). One may speculate that gender differences in the metabolism of benzene to HQ and 1,4-BQ exist in humans and mice, thus confounding the interpretation of in vitro, in vivo, and epidemiological studies. Indeed, in vitro and in vivo studies of human CYP2E1, the major enzyme involved in the metabolism of benzene in the liver, have shown significant interindividual variation in the protein level and catalytic activity, possibly due to one or more of the numerous genetic polymorphisms in the CYP2E1 gene (Fairbrother et al., 1998
)
The current study was designed to directly examine the molecular mechanisms associated with DNA damage and repair in a known target cell population for benzene-induced DNA damage and the resulting pathologies. To that end, expression of several genes involved in the DNA damage response, including p21, p53, ku80, rad51, xpa, xpc, and ape1, was analyzed in CD34+ cells treated with 1,4-BQ. Induction of p53 was not seen in CD34+ cells following short-term exposure to 1,4-BQ however, since p53 is regulated at the post-transcriptional level, this finding was not unexpected. Of the genes analyzed, only p21 was induced at the mRNA level following exposure to 1,4-BQ for 72 h. A recent study showed increased homologous recombination (HR) due to exposure to 1,4-BQ and other benzene metabolites (Winn, 2003); however, we did not observe an increase in expression of rad51, which is involved in HR. Perhaps induction of the DNA repair machinery occurred early after exposure and had returned to baseline levels by 72 h. To investigate this possibility, we performed a small follow-up study in which CD34+ cells from one male and one female donor used in the original 72 h experiments were exposed to 5 µM 1,4-BQ for only 24 h and the same genes were analyzed by quantitative real time RT-PCR. Twenty-four hours after exposure to 1,4-BQ, the only gene that showed an alteration at the mRNA level was p21 (increased 2.0 ± 0.13 fold over untreated cells). The gene expression pattern seen following 24 h exposure to 1,4-BQ was similar to that observed after 72 h exposure. Thus, the baseline expression of the DNA repair genes may be adequate to achieve efficient repair of the benzene-induced lesions. Alternatively, regulation of the DNA repair process may occur at the protein level and not be reflected as a change in mRNA levels. In addition, the increased level of apoptosis may be sufficient to remove the damaged cells and prevent malignant transformation.
In conclusion, hematopoietic disorders associated with exposure to benzene may be due in part to the direct toxic effects of 1,4-BQ on human CD34+ HPC. The DNA damage resulting from 1,4-BQ exposure appears to induce a p53 damage response but does not lead to increased mRNA expression of the DNA repair genes tested. Studies with human CD34+ cells 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. In addition, interindividual variability of the metabolic capacity of human CD34+ cells should be assessed with regard to benzene metabolism. Whole genome microarray experiments would be most informative; however, the extremely small amount of RNA isolated from the small number of human CD34+ cells available to us as well as cost were limitations in our study. Development of this in vitro system using CD34+ progenitor cells will prove to be a valuable tool for assessing the genotoxic and carcinogenic potential of a variety of substances. In addition, interindividual variation in the response of these cells to various agents may help elucidate genetic susceptibility loci.
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ACKNOWLEDGMENTS |
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NOTES |
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2 Present address: Merck Research Laboratories, Department of Genetic and Cellular Toxicology WP45-324, West Point, PA 19486.
3 To whom correspondence should be addressed at present address: GlaxoSmithKline, 5 Moore Drive, Research Triangle Park, NC 27709. Fax: (919) 483-6858. E-mail: brenda.x.faiola{at}gsk.com
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