标题: Exposure of Hematopoietic Stem Cells to Benzene or 1,4-Benzoquinone Induces Gend [打印本页] 作者: 江边孤钓 时间: 2009-3-5 10:36 标题: Exposure of Hematopoietic Stem Cells to Benzene or 1,4-Benzoquinone Induces Gend
CIIT Centers for Health Research, Research Triangle Park, North Carolina, USA " B5 P& K3 Y3 t* E* E 3 ^& s- Y% b3 `2 d4 a k$ L' [" lKey Words. Hematopoietic stem cell ? Benzene ? Toxicity ? In vitro culture ? Leukemia Quantitative reverse transcription–polymerase chain reaction ; B2 |* X: C4 c+ K: ^9 d0 t/ z. T$ i) t, ?. r& H
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@gsk.com ! Z( Z! X5 Y) \' k8 Q, s6 P; v- F& _) r; ?0 K" p
ABSTRACT8 Q( s% p! Z2 R9 J" r+ `
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Benzene is a lipid-soluble, volatile organic compound and ubiquitous environmental pollutant that is rapidly absorbed after short-term inhalation exposures in humans . 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 . 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 . 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 . 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 . 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 .$ c- }3 M" i" w7 b& {; u4 V
2 x H% ]8 L3 X& Z0 s" B/ o2 gA 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 . Benzene oxide can be oxidized to form catechol , 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) . 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." w2 N+ n; i! j9 {0 J
+ F. W% ?* F2 t0 R* zVarious 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 ( 1 G& N" I6 V( ~4 A0 |0 Q2 q8 ]2 j7 X1 X
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 . 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) .1 V6 t% V0 W- r8 `" b6 h
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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 . 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 . 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., t: d* L( k- i( g2 y, n3 t
q# n/ Q7 Y' G8 E* o3 o" b' L" ZMATERIALS AND METHODS 7 [* ?/ \( i! m/ N ) a9 q) c6 Z* {& z. @3 {9 [Cytotoxicity of 1,4-BQ in HSCs from Male and Female 129/SvJ Mice K! {9 L1 K6 J6 V( F* `0 A5 K
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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 . 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 . 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, 1B). 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. 1A; 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. 1B). 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).! Z/ g& e2 j8 M( ]6 @5 r
7 X' x* {9 s( _& zFigure 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 ) i, A6 s( k! S; N/ y: H; E
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Gene Expression Analysis of HSCs Exposed to 1,4-BQ In Vitro# Z2 Z D4 i( C
, o& Y9 j& \7 sBased on the microarray analysis of HSCs from benzene-exposed male mice 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, 2B). 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, 2B). 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).5 O: V9 Z& H: [: S% s0 {# L
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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 7 D- g# \/ u; M; H" f# T1 ~& y2 Q
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Gene Expression Analysis of HSCs from Mice Exposed to Benzene In Vivo) f! z& C( x- l3 E/ B! x3 [& J' \
2 t# }0 S& [! L! wThe 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, 3B). 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 Pol?, 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, 3B). 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.# E! Z* k; A' V+ Y1 L
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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 - g% `6 ~ y5 \& a- j) F; S/ ~- A1 M
DISCUSSION* q5 a1 O3 L! L! |4 U, n1 u$ Q
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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. 9 _8 ~7 T. b% U8 M1 M7 s0 x' f% f3 r+ I2 W* o
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