Variations in Prkdc and Susceptibility to Benzene-Induced Toxicity in Mice

Brenda Faiola1, Alison K. Bauer2, Elizabeth S. Fuller3, Victoria A. Wong, Linda J. Pluta, Diane J. Abernethy, James B. Mangum, Jeffrey I. Everitt4 and Leslie Recio5

CIIT Centers for Health Research, Research Triangle Park, North Carolina 27709

Received March 28, 2003; accepted June 25, 2003

ABSTRACT

Benzene, a carcinogen that induces chromosomal breaks, is strongly associated with leukemias in humans. Possible genetic determinants of benzene susceptibility include proteins involved in repair of benzene-induced DNA damage. The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), encoded by Prkdc, is one such protein. DNA-PKcs is involved in the nonhomologous end-joining (NHEJ) pathway of DNA double-strand break (DSB) repair. Here we compared the toxic effects of benzene on mice (C57BL/6 and 129/Sv) homozygous for the wild-type Prkdc allele and mice (129/SvJ) homozygous for a Prkdc functional polymorphism that leads to diminished DNA-PK activity and enhanced apoptosis in response to radiation-induced damage. Male and female mice were exposed to 0, 10, 50, or 100 ppm benzene for 6 h/d, 5 d/week for 2 weeks. Male mice were more susceptible to benzene toxicity compared with females. Hematotoxicity was evident in all male mice but was not seen in female mice. We observed similar, large increases in both micronucleated erythrocyte populations in all male mice. Female mice had smaller but significant increases in micronucleated cells. The p53-dependent response was induced in all strains and genders of mice following benzene exposure, as indicated by an increase in p21 mRNA levels in bone marrow that frequently corresponded with cell cycle arrest in G2/M. Prkdc does not appear to be a significant genetic susceptibility factor for acute benzene toxicity. Moreover, the role of NHEJ, mediated by DNA-PK, in restoring genomic integrity following benzene-induced DSB remains equivocal.

Key Words: DNA-PKcs; Prkdc; benzene; nonhomologous end-joining; susceptibility; toxicity.

Benzene is a prototypical human and rodent hematotoxic and genotoxic carcinogen and a ubiquitous environmental pollutant. As a constituent of gasoline and cigarette smoke, benzene poses a potential health threat to a broad spectrum of individuals (Runion and Scott, 1985Go; Wallace, 1990Go). Benzene is a lipid-soluble, volatile organic compound that is rapidly absorbed following short-term inhalation exposures in humans (Egeghy et al., 2000Go). Chronic benzene exposure results in progressive depression of bone marrow function, leading to a reduction in the number of circulating red and white blood cells (Goldstein and Laskin, 1977Go; Snyder and Kocsis, 1975Go). Epidemiological studies show that high-level occupational exposure of humans to benzene results in an increased risk of aplastic anemia, acute myeloid leukemia, and chronic lymphocytic leukemia (Phibbs, 2001Go; Smith, 1996Go; Snyder, 2000Go; U.S. EPA, 1998Go).

Oxidation of benzene in the liver by cytochrome P450 2E1 (CYP2E1) to benzene oxide and other reactive intermediates is a prerequisite of cellular toxicity (Smith, 1996Go). Benzene oxide can be oxidized by microsomal epoxide hydrolase (mEH) to form catechol (Ross, 2000Go), undergo ring opening to produce trans-trans-muconaldehyde, or spontaneously rearrange to form phenol, which is then hydroxylated in the liver to form hydroquinone (HQ). Once in the bone marrow, HQ and catechol are thought to be converted by myeloperoxidase to 1,4-benzoquinone (BQ) and 1,2-BQ, respectively, which can be detoxified by reduction via NAD(P)H oxidoreductase-1 (NQO1). These reactive quinones are capable of binding to DNA and other macromolecules, thereby generating free radicals and reactive oxygen species (Ross, 2000Go; Smith, 1996Go). The resulting DNA strand breaks can lead to chromosomal aberrations. Thus, DNA damage following benzene exposure must be properly repaired, or the affected cell must undergo apoptosis to prevent proliferation of mutated cells and subsequent transformation into malignancies.

The key genetic determinants associated with benzene-induced toxicity and leukemogenicity are beginning to be characterized. Reports have shown that polymorphisms resulting in high CYP2E1 activity (Valentine et al., 1996Go) or low glutathione transferase activity (Wormhoudt et al., 1999Go) lead to increased benzene sensitivity. Two recent studies have shown that lack of NQO1 or mEH activity leads to gender-specific benzene responses, suggesting that these detoxification enzymes are likely genetic determinants of benzene-induced hematotoxicity (Bauer et al., 2003aGo,bGo).

In addition to enzymes involved in the bioactivation and detoxication of benzene, other possible susceptibility loci include the DNA repair genes involved in restoring genomic integrity following exposure to benzene and its metabolites. One such gene is Prkdc, which encodes the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). DNA-PKcs is recruited to DNA double-strand break (DSB) sites by the Ku70/Ku80 heterodimer to form the active DNA-PK complex (Hartley et al., 1995Go). DNA-PK activity is activated by binding to free DNA ends and catalyzes rejoining of double-strand breaks (DSB) (Gottlieb and Jackson, 1993Go). Thus, DNA-PK activity is essential for nonhomologous end-joining (NHEJ) and V(D)J recombination. Deficiencies in DNA-PK activity are clinically significant. For example, a nonsense mutation in Prkdc is responsible for the severe combined immunodeficiency (SCID) phenotype of some mice, horses, and dogs (Araki et al., 1997Go; Ding et al., 2002Go; Shin et al., 1997Go). Although Prkdc has not been associated with human SCID, a frameshift mutation in Prkdc has been identified in a human glioma-derived cell line (Anderson et al., 2001Go) and approximately 50 single nucleotide polymorphisms (SNP) in human Prkdc have been found according to the National Center for Biotechnology Information database. Analysis of one of these human Prkdc SNPs showed that although it was not associated with breast cancer in a single gene analysis, a joint effect analysis suggested that it acts in association with Ku to increase the risk of breast cancer (Fu et al., 2003Go). A recent study showed that Prkdc is polymorphic in mice, having two variations that result in two amino acid substitutions in DNA-PKcs (Mori et al., 2001Go). These investigators showed that mice homozygous for the altered BALB/c allele at Prkdc, including 129/SvJ mice, exhibited reduced DNA-PK activity as well as DNA-PKcs expression compared to mice homozygous for the wild-type (wt) STS allele, such as C57BL/6 (Mori et al., 2001Go). In addition, enhanced apoptosis occurred in the absence of the wt STS allele, suggesting that Prkdc was a susceptibility gene for radiation-induced apoptosis as well as a candidate susceptibility gene for radiation lymphomagenesis (Mori et al., 2001Go).

Since both radiation and benzene metabolites cause DNA strand breaks, we investigated the role of Prkdc and NEHJ in benzene-induced toxicity. We tested the hypothesis that mice homozygous for the altered BALB/c allele at Prkdc may be more sensitive to benzene-induced toxicity than mice homozygous for the wild-type STS allele due to decreased DNA repair capacity via the NHEJ pathway. Alterations in the level of CYP2E1 protein or its activity could influence the ability of an animal to metabolize benzene therefore, we also assessed whether the mice exhibited any difference in CYP2E1 protein levels and activity.

MATERIALS AND METHODS

Mice.
129/SvJ (129X1/SvJ) mice were purchased from the Jackson Laboratory (Bar Harbor, ME). 129/Sv (Peters et al., 1997Go) mice were initially provided by Frank Gonzalez and maintained as a colony at CIIT through brother-sister mating. C57BL/6 mice were purchased from Taconic (Germantown, NY). Mice were housed in polycarbonate cages in a humidity- and temperature-controlled room and given water and an NIH-07 rodent diet (Zeigler Brothers, Gardners, PA) ad libitum. The Institutional Animal Use and Care Committee of the CIIT Centers for Health Research approved all animal use. Experiments were conducted in accordance with the Guiding Principles in the Use of Animals in Toxicology, as adopted by the Society of Toxicology in 1989.

Genotyping the Prkdc locus in three strains of mice.
To genotype the mice, RFLP regions of exons 48 and 81 of Prkdc were sequenced according to Mori et al.(2001)Go with slight modification. DNA was isolated from bone marrow (BM) of naïve mice using the Puregene DNA isolation kit (Gentra Systems, Minneapolis, MN). The primers used are shown in Table 1Go. PCR amplification of genomic DNA was performed using 50 pmol of each primer for each exon in a 50 µl reaction volume containing 10X PCR buffer with 15 mM MgCl2 (Applied Biosystems, Inc., Foster City, CA), 200 µM dNTPs, and 1.25 units AmpliTaq DNA polymerase (Applied Biosystems, Inc., Foster City, CA). The following cycling conditions were used for exon 48: 1 cycle of 94°C for 3 min, 30 cycles of 94°C for 30 s, 55°C for 1 min, 72°C for 1 min followed by a 4°C soak using a GeneAmp PCR system 9600 or 9700 (Applied Biosystems, Inc., Foster City, CA). The following cycling conditions were used for exon 81: 1 cycle of 94°C for 3 min, 30 cycles of 94°C for 30 s, 62°C for 1 min, 72°C for 1 min followed by a 4°C soak using a GeneAmp PCR system 9600 or 9700 (Applied Biosystems, Inc., Foster City, CA). Following amplification, 10 µl of each reaction was loaded on a 5% NuSieve 3:1 agarose (Biowhittaker Molecular Applications, Rockland, ME) ethidium bromide gel and run for 2 h at 60 volts followed by band detection using the Alpha Innotech Imaging System (San Leandro, CA). The exon 48 primers produced a 212 bp product and the exon 81 primers produced a 104 bp product. The remaining PCR reaction products were purified using QIAquick® columns (Qiagen, Valencia, CA). Each strand was sequenced using the ABI Prism® Big Dye Terminator cycle sequencing ready reaction kit (Applied Biosystems Inc., Foster City, CA) with 3.2 pmol of primer in a 20 µl reaction using the following cycling conditions: 25 cycles of 96°C for 10 s, 50°C for 5 s, 60°C for 4 min followed by a 4°C hold using a GeneAmp PCR system 9600 (Perkin-Elmer, Foster City, CA). Extension products were purified using centri-sep spin columns (Princeton Separations, Adelphi, NJ), dried in a vacuum centrifuge, and rehydrated with 6 µl loading buffer (5:1 deionized formamide: 25 mM EDTA with blue dextran). Samples were denatured at 95°C for 2 min and loaded onto a Long Ranger gel and run on an ABI Prism 377 DNA Sequencer. In addition to sequencing, purified PCR products were digested by BsmBI at 55°C for >7 h (exon 48) or HphI at 37°C for >7 h (exon 81), and the digested DNA fragments were analyzed on 5% NuSieve 3:1 agarose gels.


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TABLE 1 Primers Used for PCR Amplification and Sequencing of Prkdc
 
Benzene exposures.
CIIT has conducted inhalation exposures to benzene previously and methods of test atmosphere generation and characterization are described in detail elsewhere (Healy et al., 2001Go). Male and female mice ranging from 6 to10 weeks old were acclimated to stainless steel wire mesh cages in a Hinners-style whole-body inhalation chamber 2 weeks prior to 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.). Exposures took place during the light cycle (1 A.M. to 7 A.M.) to facilitate sample collection and processing immediately following the end of exposure. Only water was given during the exposures. Each exposure group, including air-exposed controls, was housed in a separate 1 m3 stainless steel and glass inhalation chamber (Hazelton H1000, Lab Products, Seaford, DE). This study was comprised of three benzene exposure groups (10, 50, and 100 ppm) and an air-exposed control group (0 ppm benzene). Mice were exposed for 6 h/day, 5 days/week for 2 weeks. Previous studies at our institution using these exposures had demonstrated significant hematotoxicity and genotoxicity (Bauer et al., 2003aGo,bGo; Farris et al., 1997aGo,bGo; Healy et al., 2001Go). Benzene (Sigma-Aldrich Chemical Co., Milwaukee, WI) purity was assessed by gas chromatography prior to the start of exposure and was determined to be 100%. Exposure atmospheres were generated by metering concentrated benzene vapor through a mass flow controller (MKS Instruments Inc., Andover, MD) and mixing it with HEPA-filtered air upstream of the exposure chamber inlet. The supply airflow through the chambers was controlled by the Infinity Building Automation System (Andover Controls Corp., Andover, MD). Benzene exposure atmospheres in the inhalation chambers were analyzed every 30 min during the exposure period by a gas chromatograph equipped with a 12-port sampling valve. The grand daily mean for each exposure group was maintained within 5% of the target concentrations.

Sample collection.
Mice were euthanized by i.p. injection of 5 mg pentobarbital (Abbott Laboratories, Chicago, IL) and animal weights were recorded. Blood was collected by cardiac puncture and transferred to microtainer® tubes (Becton Dickinson, San Jose, CA) containing EDTA. The sternum and thymus were harvested and fixed while the liver was harvested and frozen in liquid N2. The tibias, femurs, and humeri were removed and placed in complete RPMI 1640 or HBSS containing 10% FBS (Life Technologies, Carlsbad, CA). All tissues and bones were collected within 4 h after termination of exposure.

Hematology.
Complete blood counts were conducted by a contract hematology laboratory (Antech Diagnostics, Cary, NC, Atlanta, GA, or New York, NY) using an Abbott Cell-Dyn 3500 or Bayer Advia 120.

Histopathology of sternum and thymus.
Tissues were removed from the mouse, placed into 10% neutral buffered formalin phosphate (Fisher, Fair Lawn, NJ) for 48 h, and then transferred to 70% ethanol. Sternums underwent decalcification in 7.5% formic acid for 5 days before further processing. Samples were embedded in paraffin, and at least 2 sections per tissue were prepared and stained with hematoxylin and eosin. Bone marrow cellularity was assessed by evaluating five 40x fields (0.069 mm2) per sternum. Images were acquired from standardized regions of sternum using a Spot RT digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI) on a Zeiss Axioscop 2 microscope (Carl Zeiss Inc., Thornwood, NY) and marrow area measurements were obtained using Image-Pro Plus software (Media Cybernetics, Silver Spring, MD).

Micronuclei analysis in blood using flow cytometry.
The Prototype MicroFlow® PLUS Mouse Micronucleus Analysis Kit and protocol (Litron Laboratories, Rochester, NY) was used to evaluate the level of micronucleated polychromatic erythrocytes (MN-PCE) and micronucleated normochromatic erythrocytes (MN-NCE) in mouse blood. Briefly, blood was collected by cardiac puncture, and approximately 50 µl of blood was suspended in 300 µl solution B (anticoagulant). Then 180 µl of the suspension was injected into 2 ml cold methanol (-80°C), struck sharply to discourage aggregate formation, and kept at -80°C until further processing. On the day of analysis, tubes were struck several times to resuspend the cells followed by addition of 12 ml ice-cold solution C. Cells were centrifuged at 600 x g at 4°C for 5 min. The supernatant was decanted and cells were resuspended in residual supernatant. Then 20 µl of each blood sample was transferred to Falcon 2052 tubes containing 80 µl solution D/E (RNase and anti-CD71-FITC conjugated antibody). After 30 min at 4°C and 30 min at room temperature, 1 ml of solution F/C [propidium iodide (PI)] was added to each sample immediately prior to analysis. A FACSVantage flow cytometer (Becton Dickinson, San Jose, CA) was used to carry out the analyses. Malaria-infected cells were used as a reference standard to consistently define the MN analysis windows and to establish proper daily PMT voltages and compensation. Aggregates were excluded based on forward and side scatter. Nucleated leukocytes in the peripheral blood were gated out based on intense PI staining. Polychromatic erythrocytes (PCE) were defined as CD71 positive cells, while mature normochromatic erythrocytes (NCE) were the CD71 negative population. Micronuclei (MN) were defined as PI positive PCE or NCE.

Bone marrow preparation.
Immediately after removing the bones, the marrow was flushed out with complete RPMI 1640 media (Life Technologies, Carlsbad, CA) or HBSS containing 10% fetal bovine serum (Life Technologies, Carlsbad, CA) using a syringe and 23 or 26 gauge needle. At least 2 million bone marrow cells were transferred to 1.5 ml microcentrifuge tubes, pelleted by centriguation, and resuspended in RNAlaterTM (Ambion, Austin, TX) to preserve the RNA. The samples in RNAlaterTM were kept at 4° C and processed within 6 weeks.

Apoptosis assay.
Bone marrow cells (106) were transferred to 1.5 ml microcentrifuge tubes and washed twice with cold PBS. Cells were suspended in 1 ml 1X cold annexin V binding buffer (BD Biosciences, San Diego, CA), and 100 µl of the suspension was transferred to each of two falcon 2052 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, 0.4 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).

Analysis of p21 mRNA expression levels.
Total RNA was isolated from bone marrow cells using the Qiagen RNeasy® kit (Qiagen, Valencia, CA) which includes treatment with DNase I to remove genomic DNA. cDNA was generated from 2 µg of total RNA using the TaqMan® reverse transcription reagents with random hexamers as primers according to the manufacturer’s protocol (Applied Biosystems, Inc., Foster City, CA). Primers for p21 and gapdh (Bauer et al., 2003aGo,bGo; Boley et al., 2002Go) were designed using Primer Express software (Applied Biosystems, Inc., Foster City, CA) and assayed for specificity and efficiency following the manufacturer’s protocol. Quantitative RT-PCR with SYBR® Green (Applied Biosystems, Inc., Foster City, CA) was performed using the ABI PRISM® 7700 Sequence Detection System (Applied Biosystems, Inc., Foster City, CA). All samples were analyzed in triplicate, using gapdh as the reference gene. The primer efficiency (E) of p21 and gapdh were found to be approximately equal (E = 2.052 and 2.053, respectively) and showed no gender or strain difference. Quantitation of mRNA levels for the gene of interest was determined following the manufacturer’s comparative CT method, with the fold change in p21 mRNA level from a benzene-exposed sample compared to an air-control sample given by the following function: fold change = 2(-{Delta}{Delta}CT) (User Bulletin no. 2, ABI Prism 7700 Sequence Detection System, Applied Biosystems, Inc., Foster City, CA).

Cell cycle analysis of BM cells.
One million bone marrow cells were loosely pelleted (1000 x g) and gently washed once in cold PBS (Life Technologies, Carlsbad, CA). The pellet was gently resuspended in 0.5 ml propidium iodide solution, a modified Krishan stain; 3.8 mM sodium citrate, 60 mM propidium iodide, 0.01% NP40, 0.01 mg/ml RNase; (Krishan, 1975Go). Cell cycle stage, based on cellular DNA content, was then analyzed on a FACSVantage flow cytometer (Becton Dickinson, San Jose, CA) at 488 nm with a 630/22 dichromatic filter. The data were then analyzed in the Modfit LT Program (Verity Software House, Topsham, ME).

CYP2E1 protein expression and activity.
Liver microsomes were prepared according to Guengerich (1989)Go. Briefly, livers were thawed slowly, weighed, homogenized in three volumes of ice-cold buffer A (0.05M Tris Base, 0.154M KCl, 0.02mM BHT, pH 7.4), and centrifuged at 10,000 x g for 20 min at 4°C. The supernatant was transferred to an unltracentrifuge tube and centrifuged at 105,000 x g for 60 min at 4°C. Following removal of the supernatant, the pellet was resuspended in buffer B (0.05 M Tris, 0.25 M sucrose, 1 mM EDTA, 0.02 mM BHT, pH 7.4) and homogenized. The microsome solution was centrifuged at 105,000 x g for 60 min at 4°C and the supernatant was discarded. The microsome pellet was resuspended in a volume of buffer C (0.1 M K2HPO4, 0.25 M sucrose, 0.025 M phenylmethanesulfonyl fluoride [PMSF], pH 7.4) equivalent to the liver weight (1 ml buffer for 1 g liver). Aliquots were stored at -80°C until further use. Protein content of the microsome samples was determined by the BCA protein assay (Pierce Biotechnology, Inc., Rockford, IL) according to the manufacturer’s protocol.

CYP2E1 activity was determined by the p-nitrophenol hydroxylase assay, which measures the conversion of p-nitrophenol to p-nitrocatechol (Reinke and Moyer, 1985Go). Briefly, microsomes were incubated with 0.1 M potassium phosphate buffer, pH 7.4, 1 mM p-nitrophenol, and water at a concentration of 0.4 mg/ml in a total volume of 1 ml. Four samples were prepared for each mouse (two used as control samples and two used for enzymatic reaction). Samples were incubated for 3 min at 37°C followed by addition of 200 µl 1.5 N perchloric acid to the control samples or 10 µl of 100 mM NADPH to the other samples. After 4.5 min, 200 µl 1.5 N perchloric acid were added to the samples containing NADPH to stop the reaction, then all samples were removed from the water bath and placed on ice for at least 10 min. After samples were centrifuged for 10 min to remove precipitates, 1 ml of supernatant was transferred to a cuvette and 100 µl of 10 N NaOH was added. Absorbance of the samples was read in a Shimadzu UV-1601 spectrophotometer at 546 nm. Absorbance was converted to nmols of p-nitrocatechol formed by dividing the absorbance by the extinction coefficient of 10.28 mM-1 cm-1 (Reinke and Moyer, 1985Go). Activity is expressed in units of nmol/min/mg protein.

CYP2E1 protein expression was determined by western blot. Laemmli sample buffer (Bio-Rad, Hercules, CA) was added directly to microsomal protein and boiled for 5 min. Eight µg protein were loaded per sample onto a 10% polyacrylamide SDS–PAGE 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 (Life Technologies, Carlsbad, CA) followed by an overnight incubation at 4°C with the primary polyclonal anti-rat cytochrome P450 CYP2E1 antibody (BD GentestTM, Woburn, MA) at a 1:1000 dilution in 0.5% milk in PBS. After three washes in 1% milk plus 0.1% Tween, 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 Dura Chemiluminescence (Pierce Endogen, Rockford, IL).

Statistical analysis.
Statistical analyses were done using SAS 6.12 or JMP 5.0 statistical software (SAS Institute, Inc., Cary, NC). A three factor analysis of variance (ANOVA) was performed on each variable with the three main effect factors being strain, gender, and exposure level and their first-order interactions were tested. Significant differences by ANOVA were further analyzed by pairwise t tests and/or Tukey-Kramer multiple comparison methods. The level of significance used for all statistical tests was p < 0.05.

RESULTS

Variations in Prkdc between Strains of Mice
The Prkdc gene is polymorphic in mice, with the altered Balb/c allele containing two different missense mutations (T6,418C in exon 48 and G11,530A in exon 81) compared to the wild-type STS allele (Mori et al., 2001Go). Both mutations are located in regions that are highly conserved between mice and humans. Mori and colleagues (2001)Go found that 129/SvJ mice are homozygous for the Balb/c allele while C57BL/6 mice are homozygous for the STS allele. To confirm the Prkdc haplotype in the 129/SvJ and C57BL/6 mice used here and to determine which allele our 129/Sv mice carry, the regions containing the mutations were amplified by PCR and then sequenced and subjected to digestion with the appropriate restriction enzyme (BsmBI for exon 48 or HphI for exon 81). PCR-RFLP analysis showed that 129/SvJ mice have the Balb/c haplotype, with the exon 81 fragment cleaved by HphI and the exon 48 product not cleaved by BsmBI (Fig. 1AGo). C57BL/6 and our 129/Sv mice carry the STS haplotype, as the exon 81 PCR product was not digested by HphI while the exon 48 product was cleaved into 166-bp and 46-bp fragments (Fig. 1AGo). Sequence analysis confirmed the presence of homozygous missense mutations in the 129/SvJ strain, while the C57BL/6 and 129/Sv mice were homozygous for the wild-type Prkdc allele (Fig. 1BGo).



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FIG. 1. Prkdc haplotype analysis in three inbred strains of mice. (A) Allele type was determined by PCR-RFLP. PCR products digested with or without the restriction enzyme HphI or BsmBI are indicated by + and - signs. Variation G11,530A in exon 81 (top); Variation T6,418C in exon 48 (bottom). Several mice per strain were tested (129/SvJ n = 5 mice, C57BL/6 n = 6, 129/Sv n = 29). Representative data are shown. (B) A subset of the samples was sequenced to confirm the RFLP results. Variation T6,418C in the sense strand of exon 48 (left) and variation C11,530T in the antisense strand of exon 81 (right) and the resulting amino acid substitutions are indicated by the dotted line. The recognition sequence of the restriction enzyme BsmBI or HphI is indicated by the solid line. Representative data are shown.

 
Analysis of Hematotoxicity in Mice Exposed to Benzene
In our study, a significant decrease in white blood cell (WBC) counts was seen in all three strains of male mice but not in the females of either 129 strain (Fig. 2Go). Differences in WBC counts among the male mice were dependent on exposure level. All three strains showed a significant decrease at the lowest benzene exposure level of 10 ppm, with male C57BL/6 mice more sensitive than the 129 strains. At the 100 ppm level, the C57BL/6 and 129/Sv mice that have the wt Prkdc allele exhibited similar severely reduced WBC counts, while the 129/SvJ mice expressing the altered Prkdc allele were significantly less affected. Female mice showed a slight but insignificant decrease in WBC counts upon exposure to 10, 50 and 100 ppm benzene. All mice had decreased red blood cell counts following exposure to 100 ppm benzene, with male 129 mice also showing a significant decrease in RBC after 50 ppm (data not shown). Overall, male mice were more sensitive to benzene-induced hematotoxicity than females.



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FIG. 2. Hematology in the peripheral blood of mice after exposure to inhaled benzene. (A) Normalized white blood cell counts in the peripheral blood of male 129/SvJ (white bars; n = 7 to 52 mice/exposure), 129/Sv (gray bars; n = 7 to 39 mice/exposure), and C57BL/6 (black bars; n = 12 to 13 mice/exposure) mice. (B) Normalized white blood cell counts in the peripheral blood of female 129/SvJ (white bars; n = 26 to 28 mice/exposure) and 129/Sv (gray bars; n = 30 to 31 mice/exposure) mice. Error bars represent the standard error of the mean (SEM); asterisk indicates significant differences from unexposed mice of the same strain and gender (0 ppm, p < 0.05); ^ indicates a significant difference compared with the other strains of the same gender at the same exposure level (p < 0.05); ND indicates no data.

 
Histopathological Analysis of the Sternal Bone Marrow and Thymus
In the bone marrow, significant reduction in nucleated cell area was seen at 100 ppm in all strains and both genders of mice (Fig. 3Go). However, the two strains of 129 male mice exhibited a >25% reduction in nucleated cell area, whereas the female 129 mice showed only a modest 10–14% reduction. The reduced cellularity appeared to be a pancytopenia. No histological lesions were noted in sections of thymus in any of the strains or either gender exposed.



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FIG. 3. Bone marrow cellularity in mice after exposure to inhaled benzene as a measure of myelotoxicity. Bone marrow cellularity in the sternum of 3 to 10 mice per group. The means ± SEM are shown for unexposed control animals (black bars) and mice exposed to 100 ppm benzene (white bars). Asterisk indicates significant differences from unexposed mice of the same strain and gender (0 ppm, p < 0.05); indicates a significant difference compared with the opposite gender of the same strain at the same exposure level (p < 0.05).

 
Genotoxicity in Mice Exposed to Inhaled Benzene
The enumeration of MN has served as a measure of genotoxicity for many years (MacGregor et al., 1987Go). The frequency of MN-PCE and MN-NCE was determined by a flow cytometric method using blood from mice exposed to 0, 10, 50, or 100 ppm benzene. Since the percentage of MN-PCE and MN-NCE in unexposed mice was different among stains, the data are represented as the fold change in MN cells. An exposure-dependent increase in MN-PCE and MN-NCE occurred in all three strains of male mice (Figs. 4A and 4CGo). Likewise, an exposure-dependent increase in MN-PCE and MN-NCE occurred in both 129 strains of female mice, although the increase was much smaller than that seen in male mice (Figs. 4B and 4DGo). At 100 ppm benzene, the 129/SvJ female mice had a slight increase of MN-PCE and MN-NCE compared to 129/Sv female mice. (MN-PCE = 3.06±0.13 for 129/SvJ and 2.66±0.10 for 129/Sv, p < 0.05; MN-NCE = 1.46±0.02 for 129/SvJ and 1.33±0.02 for 129/Sv, p < 0.05). For male mice however, the 129/Sv showed significantly higher levels of MN-PCE compared to both 129/SvJ and C57BL/6, while 129/SvJ male mice showed significantly less MN-NCE at 100 ppm benzene. At the lowest exposure tested, the 129/SvJ male mice showed a small but significant elevation in MN-PCE compared with the other two strains. The difference in magnitude between the increases in MN-PCE vs. MN-NCE is likely due to the short exposure period (2 weeks), which is not sufficient for maturation of PCE to NCE. Overall, female 129/SvJ mice showed a slightly increased sensitivity to the genotoxic effects of benzene compared to the 129/Sv mice, while male 129/SvJ mice were similarly susceptible to benzene-induced genotoxicity compared to mice with the wt Prkdc allele.



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FIG. 4. A comparison of benzene-induced MN in the PCE and NCE subsets of peripheral blood in male and female mice. (A) Fold change in MN-PCE in male mice. (B) Fold change in MN-PCE in female mice. (C) Fold change in MN-NCE in male mice. (D) Fold change in MN-NCE in female mice. Data represent the means ± SEM (n = 5 to 48 per stain, gender, and exposure level) for 129/SvJ (white bars), C57BL/6 (black bars) and 129/Sv (gray bars) mice. Asterisk indicates significant differences from unexposed mice of the same strain and gender (0 ppm, p < 0.05); indicates a significant difference compared with the other strain(s) of the same gender at the same exposure level (p < 0.05); ND indicates no data.

 
Apoptosis and Cell Death in the Bone Marrow following Benzene Exposure
Since significant levels of hematotoxicity were seen in the male mice, we evaluated the level of apoptosis in the bone marrow as a possible mechanism for the observed decrease in WBC. Interestingly, the percentage of dead cells and the percentage of cells undergoing apoptosis in the bone marrow were not altered following exposure to 100 ppm benzene for 2 weeks (Fig. 5Go). In addition, there were no differences in the percentages of dead cells or cells undergoing apoptosis between the strains or genders.



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FIG. 5. A comparison of dead and apoptotic cells in the bone marrow of mice following exposure to benzene. (A) Percentage of dead cells in the bone marrow in unexposed mice (black bars) and mice exposed to 100 ppm benzene (white bars). (B) Percentage of apoptotic cells in the bone marrow in unexposed mice (black bars) and mice exposed to 100 ppm benzene (white bars). Data represent the means ± SEM (n = 6 per stain, gender, and exposure level).

 
Assessment of a DNA Damage Response in Mice Exposed to Benzene
Previously, our laboratory and others have demonstrated that the cyclin-dependent kinase inhibitor p21 is elevated in response to benzene exposure (Bauer et al., 2003aGo,bGo; Yoon et al., 2001Go). Transcription of 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 (Prives and Hall, 1999Go; Taylor and Stark, 2001Go). We quantified p21 mRNA levels in male and female mice following exposure to various levels of benzene (Fig. 6Go). While both male and female mice showed a dose-dependent increase in p21 mRNA levels, the increase was significantly higher in male than in female mice. In male mice, there was a >10-fold increase in expression of p21 mRNA at the 100 ppm exposure level in all three strains compared to unexposed mice; however, there were no significant differences seen among the three strains of male mice at any given exposure level. Similarly, there were no significant differences seen between the two strains of female mice at all exposure levels, but the increase in p21 mRNA levels was only 3-to 4-fold following exposure to 100 ppm benzene.



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FIG. 6. p21 mRNA expression in bone marrow of mice exposed to benzene. p21 mRNA levels in the bone marrow of male and female mice following exposure to benzene was determined by quantitative real time RT-PCR analysis. Data represents the means ± SEM (n = 3 to 12 mice per group) for the fold change in p21 mRNA expression at various exposure levels (10 ppm = gray bars, 50 ppm = hatched bars, 100 ppm = white bars) relative to unexposed controls (0 ppm, black bars). Asterisk indicates significant differences from unexposed mice of the same strain and gender (0 ppm, p < 0.05); ^ indicates a significant difference compared to the opposite gender of the same strain at the same exposure level (p < 0.05); ND indicates no data.

 
To determine the impact of benzene exposure and the resulting induction of p21 on cell cycle, we analyzed the percentages of bone marrow cells in each phase of the cell cycle. The only significant difference observed in the G1 phase was a slight decrease in 129/Sv male mice exposed to 100 ppm benzene compared to both unexposed 129/Sv male mice and 129/Sv female mice exposed to the same level of benzene (Fig. 7AGo). While there was no significant change in S phase between unexposed and 100 ppm exposed male 129/Sv and C57BL/6 mice, there was a significant reduction in the percentage of cells in S phase in both strains of female mice and the 129/SvJ male mice (Fig. 7BGo). There were significant increases in the fraction of bone marrow cells in G2/M in male 129/Sv and 129/SvJ mice as well as 129/SvJ female mice (Fig. 7CGo). Female 129/Sv mice showed an increasing trend in G2/M, while C57BL/6 male mice showed no change in G2/M (Fig. 7CGo). Thus, the decreases seen in either G1 or S phase were ultimately reflected as an increase in the G2/M phase.



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FIG. 7. Cell cycle analysis of bone marrow cells following exposure to air or benzene. (A) Percentage of bone marrow (BM) cells in G1 phase of the cell cycle in male and female air-exposed control mice (black bars) and mice exposed to 100 ppm benzene (white bars). (B) Percentage of BM cells in S phase of the cell cycle. (C) Percentage of BM cells in G2/M phase of the cell cycle. Data represent the mean ± SEM (n = 6 to 18 mice per group). Asterisk indicates significant differences from unexposed mice of the same strain and gender (0 ppm, p < 0.05); indicates a significant difference compared to the other strains of the same gender at the same exposure level (p < 0.05); ^ indicates a significant difference compared to the opposite gender of the same strain at the same exposure level (p < 0.05).

 
Comparison of CYP2E1 Protein Expression and Activity in Liver Microsomes
CYP2E1 is the major p450 involved in the metabolism of benzene in the liver (Smith, 1996Go). Western blot analysis revealed no significant difference in the level of CYP2E1 protein between strains or genders of 129 mice (data not shown). Microsomes from both male and female mice of all strains exposed to 0 or 100 ppm benzene showed similar activity in converting p-nitrophenol to p-nitrocatechol (Table 2Go). There was a statistically significant increase in the CYP2E1 activity of microsomes from female 129/Sv exposed to 100 ppm benzene compared to unexposed female 129/Sv microsomes. Female 129/SvJ mice also showed a similar trend.


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TABLE 2 p-Nitrophenol Hydroxylase Activity in Liver Microsomes
 
DISCUSSION

The exact mechanism that leads to leukemia in some individuals following benzene exposure is unclear, but it is certain that benzene must undergo biotransformation to exert its toxic effect. In addition, formation of chromosomal breaks is the primary mode of benzene-induced genotoxicity in the bone marrow. Thus, key determinants of inter-individual variability and risk in response to the toxic effects of benzene may likely be the enzyme systems involved in the activation and detoxification reactions of benzene metabolism and the DNA repair enzymes required to restore genomic integrity following 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 toxicity.

It is well established that DSB play an important role in the genotoxicity of benzene and irradiation. Animal models with deficient DNA repair capacity have been used to identify modes of action for these genotoxicants. A polymorphism in murine Prkdc has been used as a model for radiation injury, where animals homozygous for the mutant allele are at increased risk for lymphoma (Mori et al., 2001Go). However, in our study, we failed to demonstrate a role for Prkdc polymorhisms that lead to reduced DNA-PK activity in benzene-induced toxicity.

Male 129/SvJ mice homozygous for the altered Prkdc allele were no more sensitive to inhaled benzene than 129/Sv or C57BL/6 mice homozygous for the wt allele. In fact, the 129/SvJ male mice were less susceptible to benzene-induced hematotoxicity than one or both of the wild-type strains at 10 and 100 ppm exposure levels. In addition, the 129/SvJ and 129/Sv male mice showed equal bone marrow toxicity, cell cycle arrest in G2/M phase, and CYP2E1 protein expression and activity. All three strains of male mice showed similar levels of cells undergoing apoptosis and dead cells in the bone marrow as well as a comparable induction of p21 mRNA. The female 129 mice responded similarly to inhaled benzene as determined by all end points except genotoxicity. The 129/SvJ females, which possess the altered Prkdc allele, were slightly more sensitive to benzene-induced genotoxicity as indicated by higher levels of micronucleated reticulocytes in the blood. Comparison with another wt Prkdc strain would determine whether this result is consistent or simply due to other genetic differences between the strains. Thus, variation in Prkdc does not seem to play a major role in benzene susceptibility in mice.

In addition, our results represent another example of the gender difference seen in response to benzene (Bauer et al., 2003aGo,bGo; Harper et al, 1989Go; Kenyon et al., 1995Go; Siou et al., 1981Go). The explanation for these benzene-specific gender differences is unclear; however, prior studies suggest that male hormones are partly responsible for this phenomenon (Siou et al., 1981Go) and that differences in metabolism of phenol to HQ (Kenyon et al., 1995Go) and other metabolism differences, such as glucuronidases (a Phase II enzyme), could also account for these gender-specific effects (Harper et al., 1989Go). Here we showed a slight induction of CYP2E1 activity following benzene exposure in female but not male mice. However, the modest 1.55-fold increase in 129/Sv and 1.28-fold increase in 129/SvJ CYP2E1 activity in female mice may or may not lead to enhanced production of various benzene metabolites. Liver and lung samples from this study are currently undergoing analysis for various benzene metabolites so that pharmacodynamic differences among the different mouse strains can be determined. If more benzene metabolites are produced in the female mice, one would expect increased toxicity but we observed less toxicity in females. Thus, the increase in CYP2E1 activity is likely not biologically significant.

There was an apparent association between the p53-regulated DNA damage response, measured by p21 mRNA levels, and benzene-induced genotoxicity in our study. p21 mRNA levels in male mice were highly induced (>10-fold), as were MN-PCE (>9-fold) and MN-NCE (2- to 3-fold); in females, p21 mRNA levels increased less (3- to 4-fold), as did MN-PCE (3-fold) and MN-NCE (1.5-fold). With the exception of male C57BL/6 mice, the increase in p21 mRNA correlated with cell cycle arrest in the G2/M phase. Likewise, Irons (1981)Go showed that rats gavaged with benzene for 10 days specifically induced a G2/M arrest in bone marrow cells. A G2/M arrest which causes cells to arrest before they enter mitosis and segregate chromosomes may be more significant in preventing chromosome breaks than a G1 arrest (Taylor and Stark, 2001Go).

An unexpected finding in this study was that the percentage of dead cells and cells undergoing apoptosis in the bone marrow did not change in response to inhaled benzene. Hydroquinone has been shown to inhibit apoptosis (Hazel et al., 1996Go), and it has been postulated that damaged cells that have not been repaired could then proliferate and thereby lead to leukemia (Snyder, 2000Go). Other investigators found that treatment of HL60 human promyelocytic leukemia cells and CD34+ human bone marrow progenitor cells with some benzene metabolites, including hydroquinone, induced time- and concentration-dependent apoptosis (Moran et al., 1996Go). In our study, levels of apoptosis and death may have increased at earlier time points but then stabilized by the end of the 2-week exposure period. A time course study to measure the percentage of dead and apoptotic cells in BM following benzene exposure would allow us to test this hypothesis. Consistent with this hypothesis, increased levels of apoptosis in the thymus and the B lymphocyte compartment of BM have been observed in mice exposed to 200 ppm benzene for only 1 or 5 days (Farris et al., 1997bGo).

In conclusion, this study suggests that variations in Prkdc do not have a major impact on benzene susceptibility in mice overall and thus may have little influence on human susceptibility to benzene as well. However, it is possible that benzene-induced genotoxicity in female mice may be linked to Prkdc. This finding necessitates a more detailed evaluation of this gene as a susceptibility locus for benzene-induced toxicity. Further analyses are ongoing with the two 129 stains of mice to determine strain and gender differences in benzene metabolism as well as the gene expression profiles of hematopoietic stem cells following exposure to benzene or its metabolites. We cannot rule out the possibility that the different responses to benzene are due to other genetic differences between the 129 substrains used here. Therefore, benzene exposure studies with the Prkdc congenic strains of Mori et al.(2001)Go would be most informative. Finally, since Prkdc was found to be a susceptibility gene for radiation-induced apoptosis and possibly lymphomagenesis (Mori et al., 2001Go), the finding that Prkdc is apparently not a susceptibility locus for benzene-induced toxicity suggests that the DNA damage and/or repair of the damage is different from that caused by radiation. A recent report indicated increased homologous recombination due to exposure to several benzene metabolites as a mechanism for benzene-induced toxicity and suggested a role for oxidative stress in this mechanism (Winn, 2003Go). A better understanding of radiation-induced and benzene-induced DNA damage responses may help define other potential benzene susceptibility candidate genes.

ACKNOWLEDGMENTS

The authors would like to thank Dr. David Dorman, Dr. Kevin Gaido, and Dr. Susan Borghoff for their constructive comments; Kay Roberts, Jason Rose, Rosemarie Marchan, Horace Parkinson, Carla Williams, Brian McManus, Frances Trasti, the CIIT inhalation staff, the CIIT animal care staff, and the CIIT necropsy staff for their excellent technical assistance; Dennis House for assistance with statistical analysis; and Dr. Barbara Kuyper for her editorial review of the manuscript. This study was funded in part by the Long-Range Research Initiative of the American Chemistry Council.

NOTES

1 To whom correspondence should be addressed at GlaxoSmithKline Research & Development, Safety Assessment Department, 5 Moore Drive, PO Box 13398, Research Triangle Park, NC 27709-3398. Phone: (919) 483-0000. E-mail: Brenda.Faiola{at}gsk.com. Back

2 Present address: Laboratory of Pulmonary Pathobiology, NIEHS, Research Triangle Park, NC 27709. Back

3 Present address: Department of Anesthesiology, Duke University, Durham, NC 27710. Back

4 Present address: GlaxoSmithKline, Research Triangle Park, NC 27709. Back

5 Present address: Merck Research Laboratories, Department of Genetic and Cellular Toxicology, WP45-324, West Point, PA 19486. Back

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