p53 Heterozygosity Alters the mRNA Expression of p53 Target Genes in the Bone Marrow in Response to Inhaled Benzene

Scott E. Boley*,1, Victoria A. Wong*, John E. French{dagger} and Leslie Recio*,2

* CIIT Centers for Health Research, Research Triangle Park, North Carolina 27709; and {dagger} National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709

Received October 1, 2001; accepted December 20, 2001


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
C57BL/6 Trp53 heterozygous (N5) mice (p53+/– mice) show an increased sensitivity to tumorigenesis following exposure to genotoxic compounds and are being used as an alternate animal model for carcinogenicity testing. However, there is relatively little data regarding the effect of p53 heterozygosity on the genomic and cellular responses of target tissues in these mice to toxic insult, especially under chronic exposure conditions used in carcinogenicity bioassays. We hypothesized that heterozygosity at the p53 locus in p53+/– mice alters the expression of bone marrow p53-regulated genes involved in cell cycle control and apoptosis during chronic genotoxic stress. We used real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) to examine gene expression alterations in bone marrow cells from C57BL/6 p53+/+ and isogenic p53+/– mice chronically exposed for 15 weeks to genotoxic and carcinogenic levels (100 ppm) of inhaled benzene. Examination of mRNA levels of p53-regulated genes involved in cell cycle control (p21, gadd45, and cyclin G) or apoptosis (bax and bcl-2) showed that during chronic genotoxic stress, bone marrow cells from p53+/+ mice expressed significantly higher levels of a majority of these genes compared to p53+/– bone marrow cells. Our results indicate that p53 heterozygosity results in a haploinsufficient phenotype in p53+/– bone marrow cells as evident by significantly altered mRNA levels of key genes involved in the p53-regulated DNA damage response pathway.

Key Words: p53; haploinsufficiency; DNA damage; altered expression; benzene; tumor; mouse.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The p53 tumor suppressor gene plays a central role in the genomic response to DNA damage by acting as a transcription factor for genes involved in cell cycle control or apoptosis (for review see Agarwal et al., 1998Go; Oren and Rotter, 1999Go; Prives and Hall, 1999Go; see Fig. 1Go). Following genotoxic stress, the p53 protein is activated by specific phosphorylation events that lead to increased levels of p53-responsive target genes, resulting in the cell cycle being interrupted and preventing replication of damaged DNA. If the amount of damage is severe, apoptotic pathways are activated, resulting in the elimination of the damaged cell. These p53-regulated biological defense pathways function to prevent the replication and proliferation of genetically altered cells. Loss of p53 function leads to genomic instability in cultured cells (Harvey et al., 1993Go; Livingstone et al., 1992Go) and is a common event in the development of human cancer (Greenblatt et al., 1994Go; Hainaut et al., 1998Go).



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FIG. 1. Illustration of the p53-mediated genomic response to DNA damage. The p53 protein is activated in response to DNA damage. In its active state, p53 regulates the transcription of a variety of genes involved in cell cycle control and/or apoptosis. In addition, p53 also regulates the expression of mdm-2, which in turn can inhibit the activity of p53 in a feedback loop. This inhibitory effect can be nullified by p19.

 
Much of the data thus far concerning the role of p53 in the genomic response to DNA damage has been obtained following acute exposures to genotoxic compounds or ionizing radiation (Bouvard et al., 2000Go; Venkatachalam et al., 1998Go, 2001Go). However, chemical carcinogenesis in humans and rodents typically requires chronic exposure to a carcinogen and very little data concerning the role of p53 in the genomic response to chronic DNA damage are available. We are using inhaled benzene as a prototype hematotoxic and genotoxic carcinogen to determine genomic and cellular responses to chronic in vivo genotoxic stress in a relevant target tissue, bone marrow.

In the present study we used isogenic C57BL/6 p53+/+ and C57BL/6-Trp53 heterozygous (N5) mice (p53+– mice) to characterize the effect of p53 heterozygosity on the genomic response of bone marrow cells to chronic genotoxic insult. This was accomplished by comparing the expression level of p53-regulated target genes involved in cell cycle control or apoptosis in bone marrow samples from p53+/+ and p53+/– mice exposed to inhaled benzene for 15 weeks. The level of inhaled benzene used in this study (100 ppm) induced the same level of bone marrow genotoxicity in p53+/+ and p53+/–, as assessed by peripheral blood micronuclei (Healy et al., 2001Go). Furthermore, under the benzene exposure regimen used in the present study, inhaled benzene induced an 87% (39/45) incidence of thymic lymphomas in p53+/– mice compared to a 4% (2/45) incidence in p53+/+ at 36 weeks (L. Recio et al., in preparation). Since p53+/– mice are being considered as an alternative model for carcinogenicity assessment (Robinson and MacDonald, 2001Go), it is of importance to understand the basic biological and molecular basis for tumor susceptibility in this model during chronic bioassay conditions using prototype genotoxic carcinogens such as inhaled benzene.

We hypothesized that heterozygosity at the p53 locus in p53+/– mice alters the expression of bone marrow p53-regulated genes involved in cell cycle control and apoptosis during chronic genotoxic stress (Fig. 1Go). To address this question, we used real-time quantitative RT-PCR to determine the mRNA levels of p53-responsive genes involved in p53 regulation (p53, mdm-2, and p19; Haupt et al., 1997Go; Kubbutat et al., 1997Go; Stott et al., 1998Go), cell cycle control (p21, gadd45, and cyclin G; el-Deiry, 1998Go; Kastan et al., 1992Go; Okamoto and Beach, 1994Go; Shimizu et al., 1998Go; Wang et al., 1999Go), or apoptosis (bax and bcl-2; Korsmeyer, 1999Go; Miyashita et al., 1994Go; Oltvai et al., 1993Go) in the bone marrow of p53+/+ and p53+/– mice chronically exposed to inhaled benzene for 15 weeks. Our results indicate that p53 heterozygosity results in a haploinsufficient phenotype in p53+/– bone marrow cells as evident by significantly altered mRNA levels of key genes involved in the p53-regulated DNA damage response pathway.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals and animal care.
C57BL/6 (wild-type p53+/+) and C57BL/6 p53+/–(N5) mice were obtained from Taconic Farms (Germantown, NY) and acclimated to wire caging within the inhalation chambers 1 week prior to start of benzene exposure. At the start of benzene exposure the ages of mice ranged from 8 to 9 weeks. Water (reverse osmosis-treated) and commercially available rodent diet were available ad libidum, and feed exposed to benzene was discarded following each exposure period. Mice were kept in a reverse day-night cycle (lights on at 0100 h, off at 1300 h); exposures took place during the light cycle. The exposure or control group was housed in separate 8-m3 inhalation chambers, and all mice were individually housed in stainless steel wire mesh cages. The institutional Animal Use and Care Committee of the CIIT Centers for Health Research approved all conditions and animal use.

Inhalation exposures to benzene.
The details of the benzene inhalation exposures used in this study are described elsewhere (Healy et al., 2001Go). The control group and benzene-exposed group were placed in whole body 8-m3 inhalation chambers. The benzene-exposed mice (100 ppm) were exposed on an every other day schedule, 3 days/week (Monday, Wednesday, Friday; MWF) 10 h/day. Animals were exposed for 15 weeks in 8-m3 inhalation chambers to target benzene concentrations of 100 ppm. Benzene concentrations were generated and carried in a nitrogen stream to the HEPA-filtered 8-m3 chamber air supply, where it was diluted to the 100 ppm target concentration. The unexposed control chambers (0 ppm) operated under similar environmental conditions. The environmental conditions (temperature, humidity) were monitored continuously; 30-min averages were recorded and printed daily.

Benzene exposure concentrations were measured every 30 min with a calibrated infrared spectrophotometer (MIRAN 1A, The Foxboro Co., Foxboro, MA). The benzene exposure concentration was transmitted to the Andover Infinity Building Automation System (Andover Controls Corp., Andover, MA), logged, and printed in a daily report. The distribution of benzene was checked prior to initiation of exposures at nine locations in each of the exposure chambers and varied by less than 8%. The average exposure concentrations ± SD of benzene was 100 ppm (± 0.7). The average temperatures ranged from 71.1 to 72.8°F and average relative humidity ranged from 49 to 57%.

Isolation of RNA from bone marrow of mice.
Briefly, 5 p53+/+ and p53+/– mice were exposed to 100 ppm inhaled benzene for 10 h per day, 3 times per week, on MWF for 15 weeks (total number of approximately 45 10-h exposures). As an unexposed control, equivalent numbers of mice of both genotypes were exposed to air under identical conditions. Following 15 weeks of exposure, the mice were euthanized on the morning of a benzene exposure (MWF). Femoral bone marrows were flushed with 1 ml of RNA Later (Ambion, Austin, TX) and stored at 4°C until needed. Total RNA was isolated using TriReagent (Sigma, St. Louis, MO) following the manufacturer's recommended protocol, stored in RNA Secure (Ambion), and quantitated using a spectrophotometer.

Quantitative RT-PCR.
Prior to RT-PCR, genomic DNA was removed from all RNA samples by DNase I treatment following the manufacturer's protocol (Ambion). RNA for individual bone marrow samples was reverse transcribed (RT) using SuperScript II reverse transcriptase (Gibco, Rockville, MD) with 2 µg total RNA as template and 500 ng oligo dT (Pharmacia, Piscataway, NJ) as a primer following the manufacturer's protocol. Dependent on the amount of RNA available, up to three RT reactions were performed for each RNA preparation and included the appropriate negative controls. All primers were designed using Primer ExpressTM software (Perkin Elmer, Foster City, CA), and their sequences are listed in Table 1Go. Primer specificity and efficiencies were determined following the manufacturer's recommended protocol.


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TABLE 1 Sequence of Primers Used in Quantitative RT-PCR Analysis of Bone Marrow Samples from Benzene-Exposed p53+/+ and p53+/– Mice
 
Quantitative RT-PCR was performed using SYBR® Green (Perkin Elmer) with the ABI PRISM® 7700 Sequence Detection System (Perkin Elmer). Reaction conditions and data analysis were performed according to the manufacturer's recommended protocol (User Bulletin #2, ABI PRISM® 7700 Sequence Detection System, Perkin Elmer). All samples were run in triplicate, using GAPDH as the calibrator gene since GAPDH levels did not change significantly across genotypes or as a result of benzene exposure (data not shown).

To determine the effect of benzene exposure on the expression of a gene, the threshold cycle (Ct) values for the triplicate reactions were averaged and the average GAPDH Ct value for each sample was subtracted from the average Ct value for the gene of interest to obtain a normalized Ct value. Since there was very little variation among the control animals (data not shown), the normalized Ct values for the control animals were averaged and this average was subtracted from the normalized Ct value for each treated sample to obtain a relative Ct value. To obtain the relative expression of a particular gene as a result of benzene exposure, the following function was used: fold change = 2-(relative Ct). All expression levels reported were expressed relative to the expression levels determined in air-exposed animals. In Figure 2Go, values of less than one indicate that benzene exposure resulted in a decreased expression for that particular gene and values greater than one indicate that benzene exposure induced the expression of that gene.



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FIG. 2. Quantitative analysis of mRNA levels for genes involved in p53 regulation (A), cell cycle control (B), or apoptosis (C) in bone marrow cells of benzene-exposed mice relative to control mice. Bars represent the average with the SEM, of 4–5 mice. The y-axis represents the fold change in the expression of a gene relative to unexposed bone marrow cells. Grey bars represent p53+/+ bone marrow cells and black bars represent p53+/– bone marrow cells; asterisks represent a significance difference (*p <0.05 by Student's t-test) in expression level between p53+/+ and p53+/– mice.

 
Cell cycle analysis.
To examine the cell cycle distribution of bone marrow cells from p53+/+ and p53+/– mice exposed to benzene, bone marrow samples were obtained from the humeri of exposed and control mice of both genotypes. Cells were prepared for flow cytometric analysis by dispersing the cells in 70% ethanol using a 1 ml syringe with a 20 gauge needle. The cells were then centrifuged at 1500 rpm at 4°C for 3 min. Following resuspension in 2 ml of cold PBS (Gibco), the cells were centrifuged as described above. The wash was repeated once more followed by filtering the resuspended cells through a silk screen. The filtered cells were centrifuged, followed by resuspension in 570 µl cold 0.9% saline and 1.42 ml cold 95% ethanol. The cells were centrifuged, resuspended in 2 ml cold PBS, and centrifuged again. The pelleted cells were resuspended in 800 µl cold PBS, 15 µl RNaseA (4 mg/ml), and 50 µl propidium iodide (0.5 mg/ml; Sigma). The resuspended cells were incubated at 37°C for 20 min and kept on ice in the dark until analyzed. Analysis was performed on a FACSVantageTM (Becton Dickinson, San Jose, CA) flow cytometer with an Enterprise laser (Coherent, Palo Alto, CA) that emits at 488 nm, with a 630/22 dichromatic filter. Analysis of DNA content was performed using Modfit LT for Macintosh V 2.0 (Becton Dickinson). G0/G1 and G2/M peaks were identified manually, and doublet discrimination was performed based on peak area versus width.

Statistics.
To determine statistical significance, quantitative RT-PCR data for p53+/+ and p53+/– bone marrow cells were subjected to a one-tailed Student's t-test analysis. Significance in figures was noted for p values less than 0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Expression of Genes Involved in p53 Regulation
Analysis of p53 mRNA levels showed a significantly higher amount of p53 mRNA in benzene-exposed p53+/+ bone marrow cells compared to p53+/– bone marrow cells (Fig. 2AGo). Heterozygosity of p53 also had an effect on benzene-induced mRNA levels of mdm-2 and p19, with p53+/+ bone marrow cells showing significantly higher levels of these transcripts compared to p53+/– bone marrow cells (Fig. 2AGo).

Expression of Cell Cycle Control Genes
Benzene exposure resulted in p21 mRNA levels being induced more than 17-fold in p53+/+ bone marrow cells (Fig. 2BGo) compared to a slightly more than 5-fold induction of p21 mRNA in p53+/– bone marrow cells (Fig. 2BGo). Two other p53-responsive genes involved in cell cycle control are gadd45 and cyclin G. Analysis of gadd45 mRNA levels revealed a 2-fold induction in bone marrow cells from p53+/+ mice compared to no change in gadd45 mRNA levels in p53+/– bone marrow cells (Fig. 2BGo). Heterozygosity of p53 had no significant effect on the level of benzene-induced cyclin G mRNA levels; both genotypes showed a 3- to 5-fold induction (Fig. 2BGo).

Expression of Apoptosis Genes
Quantitative analysis of the mRNA level of bax in bone marrow cells from p53+/+ and p53+/– mice revealed that bone marrow cells from benzene-exposed p53+/+ mice contained nearly twice as much bax mRNA as bone marrow cells from p53+/– mice (Fig. 2CGo). The difference in the level of bax between p53+/+ mice compared to p53+/– mice was significant at p < 0.07. Analysis of bcl-2 levels revealed a slightly lower level of bcl-2 mRNA in bone marrow cells from p53+/+ mice compared to p53+/– mice (Fig. 2CGo).

Cell Cycle Analysis
To examine whether the alterations in gene expression described above had a functional impact on cell cycle, flow cytometry was used to examine samples of total bone marrow cells from benzene-exposed p53+/+ and p53+/– mice. This allowed the effect of benzene exposure on the cell cycle profile of each sample to be determined. Analysis of bone marrow cells from p53+/+ mice showed a significant increase in the G0/G1 fraction in response to benzene exposure as well as a significant decrease in the S fraction of cells (Fig. 3AGo). Identical analysis of p53+/– bone marrow cells indicated a significant increase in the G2/M fraction and a decrease in the S fraction (Fig. 3BGo).



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FIG. 3. Flow cytometry analysis of bone marrow cells from p53+/+ (A) and p53+/– (B) mice. Bars represent the average value with the SEM for five mice. Open bars represent control bone marrow cells; closed bars represent bone marrow cells from benzene-exposed animals. Asterisks designate a significant difference (p < 0.05, Student's t-test) between control and benzene-exposed bone marrow cells.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Benzene is a well-known hematotoxicant, and exposure to inhaled benzene results in DNA damage to the bone marrow (Eastmond et al., 2001Go; Farris et al., 1997Go). In the bone marrow of mice, the primary mode of benzene-induced genotoxicity is due to chromosome breaks (Eastmond et al., 2001Go). The level of benzene used in this study induced an equivalent level of micronuclei in the peripheral blood of exposed p53+/+ and p53+/– mice, indicating the genotype of the mouse had no effect on the initial level of DNA damage induced by benzene (Healy et al., 2001Go). We hypothesized that the equal levels of micronuclei induction in p53+/+ and p53+/– mice were likely due to similar metabolic activation of benzene to genotoxic metabolites in the two strains (Healy et al., 2001Go). Benzene metabolism in these strains of mice is similar (Sanders et al., 2001Go). Since p53 is activated in response to DNA damage (Fig. 1Go), benzene exposures that are genotoxic would be expected to activate the p53-regulated DNA damage response in the bone marrow. We hypothesized that heterozygosity at the p53 locus would compromise the p53-mediated DNA damage response in p53+/– bone marrow, leading to accelerated tumor development.

Genotoxic damage leads to a p53-dependent increase in p21 mRNA and protein that results in cell cycle arrest at the border between the G1 and S phases (for review see El-Deiry, 1998). Therefore, measuring p21 mRNA levels is an accepted measure of the functional status of the p53-dependent response to DNA damage. The significant induction of p21 mRNA expression in p53+/+ bone marrow cells in response to benzene exposure indicates that the p53 pathway is functional in these cells (Fig. 2BGo). However, this response is compromised in p53+/– bone marrow cells since they showed only a 5-fold induction of p21 in response to benzene (Fig. 2BGo) exposure. The lower level of p21 mRNA induction in p53+/– bone marrow cells could result from either lower levels of active p53 or p53-independent mechanisms of p21 induction (Gartel and Tyner, 1999Go; Michieli et al., 1994Go). The results from p21 mRNA analysis suggest that p53 heterozygosity results in p53+/– bone marrow cells exhibiting an altered response to DNA damage. This is supported by the failure of p53+/– bone marrow cells to induce mRNA expression of two genes involved in the p53-dependent DNA damage response, mdm-2 and gadd45, to the level seen in p53+/+ bone marrow cells.

One possible explanation for the difference in the bone marrow genomic response to inhaled benzene might be that the p53 protein forms a tetramer prior to becoming active as a transcription factor. Therefore, fewer active p53 tetramers would be expected to form in p53+/– bone marrow cells compared to p53+/+ bone marrow cells, limiting the cellular availability of p53 protein. Our results showed bone marrow cells from benzene-exposed p53+/+ mice have approximately twice as much p53 mRNA as bone marrow cells from p53+/– mice, consistent with p53+/– cells containing approximately half as much p53 protein compared to p53+/+ cells (Donehower et al., 1992Go). The limited availability of p53 protein can lead to an altered p53-regulated transcriptional response to DNA damage (Venkatachalam et al., 2001Go).

Elevated levels of cyclin G can sensitize mouse cells to the induction of apoptosis (Okamoto and Prives, 1999Go) and increased levels of gadd45 can induce apoptosis in mouse cells (Sheikh et al., 2000Go). Our data showed that bone marrow cells from benzene-exposed p53+/+ mice contained a higher level of gadd45 mRNA than bone marrow cells from p53+/– mice, suggesting that p53+/+ bone marrow cells are more likely to undergo apoptosis in response to benzene exposure than p53+/– bone marrow cells. In addition, the ratio of the p53-dependent apoptosis genes bax and bcl-2 is often used as a predictive factor for apoptosis (Oltvai et al., 1993Go). The elevated level of bax in p53+/+ bone marrow cells, combined with a lower level of bcl-2 in response to benzene exposure, suggests p53+/+ bone marrow cells are more likely to undergo apoptosis than p53+/– bone marrow cells (Korsmeyer, 1999Go; Oltvai et al., 1993Go). This is further supported by reports showing that p53 heterozygosity results in a significant decrease in radiation-induced apoptosis in mouse bone marrow (Cui et al., 1995Go) and thymocytes (Clarke et al., 1993Go; Lowe et al., 1993Go).

This study was designed as a pilot study to identify p53 target genes for planned dose-response and time course studies to define the impact of p53 heterozygosity on chronic DNA damage response in the bone marrow. We are currently developing immunohistochemical methods to examine the protein levels for these target genes in bone marrow and to examine the bone marrow for apoptosis and proliferation in target cell populations. However, since the primary function for p53 protein in response to DNA damage is to activate the transcription of genes analyzed in this study (for review see Agarwal et al., 1998Go; Oren and Rotter, 1999Go; Prives and Hall, 1999Go), examining the mRNA expression levels of p53 target genes supports the presence of an alteration in p53 function (haploinsufficiency) between bone marrow cells from p53+/– and p53+/+ mice in response to chronic DNA damage (Venkatachalam et al., 2001Go). Additional support comes from the flow cytometry data (Fig. 3Go) that showed a significant increase in the fraction of G0/G1 cells in p53+/+ bone marrow cells compared to p53+/– bone marrow cells and is in agreement with published data in bone marrow stem cells (Yoon et al., 2001Go). The lack of G1 arrest and the presence of a G2/M arrest in p53+/– mice is also consistent with literature demonstrating the presence of a G2/M arrest following radiation regardless of p53 status (Greenblatt et al., 1994Go; Hwang and Muschel, 1998Go; Passalaris et al., 1999Go). Although p53 is typically linked with G1 arrest, it is now apparent that multiple overlapping p53-dependent and p53-independent pathways participate in cell cycle checkpoints in response to genotoxic stress (Agarwal et al., 1998Go; Oren and Rotter, 1999Go; Prives and Hall, 1999Go; Shackelford et al., 1999Go; Taylor and Stark, 2001Go). Further support is found in the study of various p53 target genes in p53+/+ and p53–/– mice following acute radiation exposure showing that loss of a single p53 allele results in a defective p53-dependent DNA damage response in a variety of tissues (Bouvard et al., 2000Go).

It is important to note that p53 heterozygosity altered the p53-dependent transcriptional response to benzene-induced DNA damage but had no effect on the initial level of DNA damage induced (Healy et al., 2001Go). Given the importance of p53 in the response to genotoxic insult, alteration of the p53-mediated response pathway would result in altered cell cycle control and inhibited apoptosis, leading to enhanced mutagenesis and contributing to tumorigenesis (Smith et al., 2000Go). In addition, since the bone marrow is a complex population of cells existing in a variety of differentiation states, additional studies need to be performed to determine whether the results described here are reflective of the entire bone marrow or of sensitive subpopulations of cells within the bone marrow.

These results have significance for human cancer since many human cancers progress from a homozygous wild type p53 state through a heterozygous state for p53, eventually resulting in a complete loss of p53 function. The data presented here indicate that p53 heterozygosity severely compromises the genomic response to further genotoxic insult, placing these cells at a greater risk for the accumulation of additional genetic alterations and advancing to a malignant state. Combined, the data supports our hypothesis that p53 heterozygosity results in a haploinsufficient phenotype in p53+/– bone marrow cells as evidenced by the significantly altered expression of key genes involved in the genomic DNA damage response pathway during chromic exposures. This compromised response to DNA damage is likely a central factor in accounting for the increased susceptibility of p53+/– mice to accelerated tumorigenesis following exposure to genotoxic carcinogens.


    ACKNOWLEDGMENTS
 
The authors would like to thank Linda Pluta, Wade Lehman and the Inhalation Research Team, the Animal Care Unit, and Necropsy Histopathlogy Research Support Group at CIIT for their expert technical assistance. In addition, we would like to thank Drs. Byron Butterworth, Chris Corton, and Barbara Kuyper for their critical review of the manuscript. This study was funded in part by the American Chemistry Council through the Long Range Research Initiative.


    NOTES
 
1 Present address: Nonclinical Safety Assessment, Eli Lilly and Company, Greenfield Laboratories GL 43, Greenfield, IN. Back

2 To whom correspondence should be addressed at CIIT Centers for Health Research, 6 Davis Drive, Research Triangle Park, NC 27709. Fax: (919) 558-1300. E-mail: recio{at}ciit.org. Back


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 DISCUSSION
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