Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
Gene expression profile in bone marrow and hematopoietic stem cells in mice exposed to inhaled benzene
Introduction
Benzene is a lipid-soluble, volatile organic compound that is rapidly absorbed following short-term inhalation exposures in humans [1]. Benzene is a prototypical human and rodent hematotoxic and genotoxic carcinogen and a ubiquitous environmental pollutant. Chronic benzene exposure results in progressive depression of bone marrow (BM) function, leading to a reduction in the number of circulating red and white blood cells (WBC) [2], [3]. Epidemiological studies show that high-level occupational exposure to benzene results in an increased risk of aplastic anemia, acute myeloid leukemia, and chronic lymphocytic leukemia [4], [5], [6], [7].
Oxidation of benzene in the liver by cytochrome P450 2E1 (CYP2E1) to benzene oxide and other reactive intermediates is a prerequisite for cellular toxicity [5]. Benzene oxide can be oxidized via the microsomal epoxide hydrolase (mEH) enzyme to form catechol [8], can undergo ring opening to produce trans–trans-muconaldehyde, or can spontaneously rearrange to form phenol, which is then hyroxylated in the liver to form hydroquinone (HQ). Once in the bone marrow, it is believed that HQ and catechol are converted by myeloperoxidase to 1,4-benzoquinone (BQ) and 1,2-BQ, respectively, which can be detoxified by reduction via NAD(P)H:quinone oxidoreductase-1 (NQO1). These reactive quinones are capable of binding to macromolecules, including DNA, tubulin, histones, and topoisomerase II. Various benzene metabolites can cause oxidative DNA damage [9], lipid peroxidation in vivo [10], formation of hydroxylated deoxyguanosine residues in BM DNA [9], [11], and DNA strand breaks [12], [13] thus, implicating a role for reactive oxygen species (ROS) in benzene-induced toxicity. Formation of DNA double-strand breaks (DSB) by ROS and other mechanisms can lead to increased mitotic recombination, chromosomal translocations, and aneuploidy [5], [8]. Such genetic consequences may result in protooncogene activation, tumor suppressor gene inactivation, gene fusions, and other deleterious changes in stem cells that can ultimately result in leukemic responses. Thus, DNA damage following benzene exposure must be properly repaired or the affected cells must be eliminated to prevent proliferation of mutated cells and subsequent transformation into malignancies.
Various studies have suggested that hematopoietic stem cells (HSC) are the target cells for benzene-induced alterations. In the BM, HSC are a small population (<0.05% of BM cells) of self-renewing, pluripotent cells that give rise to all blood cells [14]. Enumeration of HSC can be performed by various in vivo and in vitro colony-forming assays. Inhalation exposure to benzene significantly reduced the number of transplantable spleen colony-forming units (CFU-S), granulocyte/monocyte colony-forming units (CFU-GM), and erythroid colony-forming units (CFU-E) in the bone marrow of male and female mice [15], [16], [17], indicating a decrease in the number of HSC following exposure to benzene. More recently, benzene was found to affect cell cycle kinetics as the fraction of CFU-GM in S phase was suppressed in male mice exposed to 300 ppm benzene for 2 weeks compared to unexposed control mice [18]. In addition, persistent benzene-induced DNA damage was observed as an increased frequency of aneuploidy in the long-term self-renewing population of HSC (Lin−, c-kit+, Sca-1+) from male and female mice 8 months after gavage with benzene compared to the corn-oil-exposed control mice [19]. Thus, benzene has short- and long-term deleterious effects on HSC.
Proper repair of benzene-induced DNA lesions in the target cells or initiation of programmed cell death of severely damaged cells is essential for preventing possible malignant transformation; thus, understanding the nature of the DNA repair process in HSC exposed to benzene is critical. Several DNA repair pathways exist in mammals to restore genome integrity following genotoxic stress. Lesions that affect only one of the DNA strands, such as oxidized DNA and adduct formation, can be repaired by base excision repair and/or nucleotide excision repair pathways [20]. DSB are repaired by nonhomologous end joining or, after replication when a second identical DNA copy is present, homologous recombination [20]. Cells with DNA damage that cannot be completely repaired by these pathways may then undergo apoptosis.
Here, we utilized gene expression profiling to examine the DNA damage response and repair pathways in total bone marrow and enriched HSC fractions after in vivo exposure of male 129/SvJ mice to inhaled benzene. Male mice were used because they are more susceptible to benzene-induced toxicity than female mice [21], [22], [23], [24]. Microarray analysis of HSC samples from benzene-exposed mice showed 44 sequences significantly up-regulated and 75 sequences significantly down-regulated by at least 1.5-fold compared to air-exposed HSC samples. From the microarray findings, several genes were further analyzed by quantitative real-time RT-PCR (qRT-PCR) in both HSC and total bone marrow samples. In addition, qRT-PCR analysis of several DNA repair genes was performed despite seeing no alteration of these genes on the microarrays. Our study showed that mRNA levels of several key DNA repair genes are not altered, whereas mRNA levels of some apoptosis, cell cycle, and growth control genes are changed in HSC and total BM in response to benzene-induced DNA damage. Gene expression patterns in benzene-exposed mice were different between HSC and total BM, emphasizing the need to perform microarray experiments on the target cell population rather than on whole tissue samples.
Section snippets
Mice
Male 129/SvJ (129X1/SvJ) mice were purchased from the Jackson Laboratory (Bar Harbor, Maine). Mice were housed in polycarbonate cages in a humidity- and temperature-controlled room and given water and NIH-07 rodent diet (Zeigler Brothers, Gardners, PA.) ad libitum. The Institutional Animal Use and Care Committee of CIIT Centers for Health Research approved all animal use.
Benzene exposures
Forty-two male 129/SvJ mice ranging from 6 to 10 weeks old were acclimated to stainless steel wire mesh cages in a
Analysis of hematotoxicity in mice exposed to benzene
As anticipated, a significant decrease in white blood cell and red blood cell counts occurred in male 129/SvJ mice following exposure to 100 ppm benzene for 2 weeks (Fig. 1). In addition, the mice had decreased hemoglobin concentrations, platelet counts, and percentage of lymphocytes and eosinophils in the peripheral blood following exposure to benzene (data not shown). The percentage of granulocytes and neutrophils was significantly increased in the benzene-exposed mice (data not shown).
Histopathological analysis of the sternal bone marrow and thymus
In the
Discussion
Benzene has been recognized as a human health concern since the mid-1970s when several reports linked benzene exposure with an increased risk of various leukemias [4], [35], [36]. In addition to workplace exposure, environmental exposure to benzene is significant since benzene is a constituent of gasoline and cigarettes and therefore poses a potential health threat to a broad spectrum of individuals, including those exposed to second-hand cigarette smoke [37], [38]. The mechanism that leads to
Acknowledgements
The authors would like to thank Expression Analysis (Durham, NC) for conducting the microarray experiments; Drs. David Dorman and Kevin Gaido for their constructive comments; Diane Abernethy, Linda Pluta, Kay Roberts, and Jason Rose for their excellent technical assistance; and Dr. Barbara Kuyper for her editorial review of the manuscript.
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Cited by (0)
- 1
GlaxoSmithKline Research & Development, Safety Assessment, Research Triangle Park, NC 27709, USA.
- 2
Department of Anesthesiology, Duke University, Durham, NC 27710, USA.