ToxSci Advance Access originally published online on November 12, 2007
Toxicological Sciences 2008 101(2):226-238; doi:10.1093/toxsci/kfm268
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Arsenite-Induced Thymus Atrophy is Mediated by Cell Cycle Arrest: A Characteristic Downregulation of E2F-Related Genes Revealed by a Microarray Approach
* Environmental Health Sciences Division, National Institute for Environmental Studies, Tsukuba 305-8506, Japan
Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, University of Tokyo, Tokyo 113-0033, Japan
1 To whom correspondence should be addressed. Fax: +81-29-850-2574. E-mail: keikon{at}nies.go.jp.
Received August 30, 2007; accepted October 22, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Thymus atrophy is induced by a variety of chemicals, including environmental contaminants and is used as a sensitive index to detect their adverse effects on lymphocytes. In the present study we adopted a toxicogenomics approach to identify the pathways that mediate the atrophy induced by arsenite. We also analyzed gene expression changes observed in the course of thymus atrophy by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), dexamethasone (DEX), and estradiol (E2), to determine whether arsenite induces atrophy by activating an arsenite-specific pathway or the same pathways as other chemicals. These compounds were intraperitoneally administered to C57BL/6 mice at doses that reduce thymus weight by approximately 30% within 3 days, and gene expression changes in the thymus 24 h after the administration were analyzed by using microarrays and real-time PCR. The microarray analysis showed that arsenite specifically downregulates a variety of E2F target genes that are involved in cell cycle progression. The same genes were also downregulated when mouse B-cell lymphoma A20 cells were exposed to arsenite. Arsenite exposure of the A20 cells was confirmed to induce cell cycle arrest, mainly in the G1 phase, and reduce cell number. Cell cycle arrest in the G1 phase was also confirmed to occur in the thymocytes of the arsenite-exposed mice. These results indicate that arsenite induces thymus atrophy through E2F-dependent cell cycle arrest. The results of this study also show that analysis of gene expression in thymuses is a useful method of obtaining clues to the pathways that mediate the effects of atrophy-inducing chemicals.
Key Words: thymus atrophy; arsenite; cell cycle; E2F; microarray.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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The thymus has long been known to be an organ that is vulnerable to atrophy when exposed to a variety of stimuli, including hormones, immunosuppressive pharmaceuticals, and environmental chemicals and metals (Ashwell et al., 2000
; Drela, 2006; Shanker, 2004; Zoller and Kersh, 2006). It is an important immune organ where T cells differentiate, are selected, and mature (Ladi et al., 2006). Although its function declines with age, the thymus remains indispensable to T-cell-repertoire reconstitution, which ensures immune reactions in various situations until late adulthood (Shanker, 2004). Thus, insults to the thymus by atrophy-inducing compounds could threaten immune function, and the thymus could be a sensitive indicator to detect the immunotoxicity of various stimuli. However, the pathways through which the stimuli cause the atrophy have largely remained unclear.
Exposure to inorganic arsenic in contaminated drinking water is a serious concern in a variety of countries around the world, including Bangladesh, Taiwan, China, Mexico, and Hungary. Other anthropogenic exposures are attributable to mining and pesticide use in agriculture (Schulz et al., 2002). An excessive intake of arsenic compounds is known to increase the risk of cancer of the skin and several other organs, including the liver, lung, and kidney, in humans (Rossman et al., 2004; Yoshida et al., 2004). By contrast, arsenic compounds, such as arsenic trioxide (As2O3), are therapeutically used to treat acute promyelocytic leukemia (APL) (reviewed in Miller et al., 2002), and relatively low doses of arsenic have been reported to trigger apoptosis not only in APL-derived cells, but also in other leukemia cells and normal lymphocytes (Miller et al., 2002). Arsenic has also been reported to exert immunotoxicity, such as suppression of antibody production and thymus atrophy (Burns et al., 1991; Schulz et al., 2002). However, the pathway that mediates arsenic-induced thymus atrophy has never been identified.
Recent studies on nonlymphoid cells, including hepatoma cells and fibroblasts, have demonstrated that arsenic alters gene expression by affecting transcription factors, including nuclear factor erythroid 2-related factor 2 (Nrf2), activator protein-1 (AP-1), nuclear factor-kappaB (NF-
B), peroxisome proliferator activated receptor 
(PPAR), and CCAAT-enhancer binding protein 
(C/EBP) (He et al., 2006; Hu et al., 2002; Ouyang et al., 2006; Wauson et al., 2002). Given the effects of arsenic are induced via activation of these transcription factors in thymocytes, microarray analyses of gene expression changes are expected to be a very effective means of identifying the pathway that mediates their effects. Actually, previous studies using microarray technology have proven its usefulness in identifying the target events caused by arsenic exposure (Su et al., 2006). The thymus is a very homogenous organ and primarily consists of thymocytes, that is, immature T cells, and because they account for more than 90% of all cells in the thymus, thymus atrophy is attributed to the loss or growth suppression of thymocytes. The establishment of thymus atrophy as the anchoring phenotype is also expected to facilitate identification of the genes responsible, because they should be genes involved in the loss or growth suppression of thymocytes.
In the present study, we used microarray analysis to obtain clues for identifying the pathways that mediate arsenic-induced thymus atrophy. We also analyzed gene expression changes induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the synthetic glucocorticoid dexamethasone (DEX), and estradiol (E2), to determine whether arsenite induces atrophy by activating an arsenite-specific pathway or the same pathways as other chemicals. Each of the three other chemicals tested has been demonstrated to cause thymus atrophy by activating different transcription factors or nuclear receptors. The toxic environmental contaminant TCDD induces thymus atrophy through activation of the transcription factor arylhydrocarbon receptor (AhR) (Hundeiker et al., 1999; Staples et al., 1998). Glucocorticoids (GCs) induce apoptosis in thymocytes via gene expression changes through the activation of glucocorticoid receptor (GR), a member of the nuclear receptor superfamily (Ashwell et al., 2000; Delfino et al., 2004). Estrogen-induced thymus atrophy has been reported to be mainly or partly mediated by nuclear receptor estrogen receptor (ER) (Lindberg et al., 2002; Staples et al., 1999).
We performed microarray analyses of the thymuses of mice exposed to these chemicals at a dose that reduced thymus weight by approximately 30% within 3 days. The gene expression changes were measured 24 h after exposure to identify the atrophy-mediated pathway. The results demonstrated that each of the four chemicals induced a different set of genes. Particularly, arsenite prominently downregulated a variety of E2F target genes involved in cell cycle progression. Additional experiments in mouse thymuses and a cell line supported the finding that arsenite causes thymus atrophy by inducing cell cycle arrest through marked changes in E2F action. Thus, the toxicogenomics approach was very effective in identifying the pathway that mediates arsenic-induced thymus atrophy.
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MATERIALS AND METHODS |
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Animal treatment.
Five-week-old male C57BL/6J mice were purchased from Clea Japan (Tokyo) and were acclimatized to the environment for 1 week prior to use. Sodium arsenite (Sigma, St Louis, MO) was dissolved in saline. TCDD (Cambridge Isotope Laboratory, Andover, MA) was dissolved in corn oil containing 2% nonane and 1% dimethyl sulfoxide (DMSO). E2 (Sigma) and DEX (Sigma) were dissolved in corn oil containing 3% DMSO. Mice were intraperitoneally administered the reagents (sodium arsenite, 10 mg/kg; TCDD, 10 µg/kg; DEX, 2.5 mg/kg; E2, 3 mg/kg). Control mice were administered corn oil containing 3% DMSO. Thymus weight and thymocyte number were determined on days 1, 3, and 7. Total RNA was prepared for microarray analysis 24 h after the administration. The mice were handled in a humane manner in accordance with National Institute for Environmental Studies (NIES) guidelines for animal experiments.
Flow cytometry.
A single-cell suspension was prepared from the thymuses and analyzed by flow cytometry as previously described (Nohara et al., 2005). The antibodies fluorescein isothiocyanate–conjugated anti-CD4 mAb (clone GK-1.5) and phycoerythrin-conjugated anti-CD8 mAb (clone 53-6.7) were purchased from PharMingen (San Diego, CA).
Affimetrix GeneChip analysis.
Affymetrix GeneChip (Affymetrix, Santa Clara, CA) analysis was performed as previously described (Nagai et al., 2005). Briefly, total RNA was prepared from three thymuses per group. Double-stranded complementary DNA (cDNA) was synthesized from 1 µg of thymus total RNA, and was used to prepare biotin-labeled cRNA by in vitro transcription. A 15-µg sample of the biotin-labeled cRNA was fragmented and hybridized to a Mouse Expression Array 430A (Affymetrix). After being hybridized for 15 h, the array was washed, stained, and scanned. Data were analyzed with Affymetrix GCOS 1.2 software. Array normalization with MAS 5.0 algorithm was applied to generate expression values using a scaling factor 500. The generated MAS 5.0-type CHP files were loaded into GeneSpring 7.0 (Silicon Genetics, Redwood City, CA) and comparison analyses between gene expression in the chemical-exposed thymus and the control thymus were performed. Four independent experiments were carried out for arsenite-exposed thymuses. Two independent experiments were conducted for the other three chemicals, respectively. Genes whose expression was consistently increased or decreased by twofold or more in the exposed groups compared with the control groups were selected for each chemical. The differences in gene expression changes between the arsenite-exposed groups and the control group were further assessed by t-test (p < 0.05) using GeneSpring 7.0.
Search for E2F binding sites.
To detect potential E2F binding sites in the downregulated genes by arsenite, the 5'-flanking regions (–2000 bp upstream to transcription start; TSS) and the sequences between TSS and +200 bp were selected using the MartView (http://www.ensembl.org/biomart/martview) (Kasprzyk et al., 2004). The sequences for both sense and antisense strands were screened for the E2F-binding sites (TTTC/GC/GCG) using the JASPAR database (http://mordor.cgb.ki.se/cgi-bin/jaspar2005/jaspar_db.pl) (Sandelin et al., 2004).
Reverse transcription-PCR.
Double-stranded cDNA prepared for GeneChip analysis was used for PCR analysis of thymus RNA. For reverse transcription-PCR (RT-PCR) of various tissues from mice and A20 cells, total RNA was prepared with an RNeasy Mini kit (Qiagen, Chatsworth, CA). After checking the quality of the RNA by electrophoresis, RT-PCR was performed as described previously (Nohara et al., 2005). The primer sequences and annealing temperatures used are shown in Table 1.
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Real-time PCR.
Quantitative real-time PCR analysis was performed on LightCycler instrument (Roche Diagnostics, Basel, Switzerland) as described previously (Nohara et al., 2006
). The control cDNA was prepared from the thymus of C57BL/6 mice. The primer sequences and annealing temperatures used for real-time PCR are shown in Table 1.
Cell culture.
B-cell lymphoma A20 cells were obtained from the Cell Resource Center for Biomedical Research (Tohoku University, Sendai, Japan). They were cultured in RPMI1640-based complete medium (Nohara et al., 2006).
Apoptosis analysis.
For the analysis of apoptotic A20 cells, cells were cultured in the presence or absence of sodium arsenite for 24 h, stained with an In situ Apoptosis Detection Kit (Takara Bio, Inc., Tokyo) according to the manufacturer's instructions, and analyzed with a FACSCalibur (BD Biosciences, Franklin Lakes, NJ). As a positive control, cells were treated with DNase I (RNase-free, Qiagen) for 10 min at room temperature after fixed with 4% paraformaldehyde according to a previous study (Schwarz et al., 1999). Cells for negative control were prepared by omitting TdT enzyme reaction. For the detection of apoptotic cells in the thymocytes freshly prepared or cultured for 24 h, cells were stained with an Annexin V–biotin apoptosis detection kit (BioVision, Palo Alto, CA) and streptavidin allophycocyanin conjugate (SA-APC, BD Biosciences) according to the manufacturer's instructions and analyzed with a FACSCalibur.
Cell cycle analysis.
For cell cycle analyses of A20 cells cultured in the presence or absence of sodium arsenite for 24 h, cells were stained with propidium iodide using a CycleTEST PLUS DNA Reagent Kit (BD Biosciences) according to the manufacturer's instructions, and their DNA content was measured using a FACSCalibur. For cell cycle analyses of double negative (DN) cells, the thymocytes were treated with anti-CD4–biotin and anti-CD8–biotin (both from eBioscience), and then with BD IMag Streptavidin Particles Plus-DM, and the particle-labeled cells were removed by applying a magnet according to the manufacturer's instructions. Cell cycle analyses of thymocytes and DN cells were carried out as described above. The percentages of cells in G1, S, and G2/M phases were analyzed using ModFit software (BD Biosciences).
CFSE-labeling analysis.
Cells were labeled with 2.5µM of CFSE using a CellTrace CFSE Cell Proliferation Kit (Molecular Probes, Inc., Eugene, OR) according to the manufacturer's instructions. After cultured in the presence or absence of sodium arsenite for 72 h, the cells were analyzed using a FACSCalibur.
Chromatin immunoprecipitation assay.
Chromatin immunoprecipitation (ChIP) assay was carried out as previously described (Suzuki and Nohara, 2007). Briefly, A20 cells were cultured in the presence or absence of sodium arsenite for 24 h. Cells were treated with 1% formaldehyde to cross-link protein–DNA complexes and then sonicated to give a final DNA size range from 400 to 900 bp. Immunoprecipitation was carried out by incubating with anti E2F1 (C-20) or anti E2F4 (A-20) antibody, followed by incubation with protein A-agarose. Both the antibodies were obtained from Santa Cruz Biotechnology, Inc. After protein–DNA complexes were recovered from the protein A-agarose beads, the formaldehyde cross-linking was reversed and protein was hydrolyzed with proteinase K. DNA fragments were purified by phenol–chloroform extraction, coprecipitated with glycogen, and suspended in 10 µl of Tris-EDTA (TE) buffer. A 1-µl of sample was subjected to real-time PCR to measure E2F binding site in the promoter region of Melk and Dig7. The E2F binding sequences in Melk and Dig7 were searched using the MartView and the JASPAR database as described above and primers were designed using PRIMER3 (frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). Primer sequences and annealing temperatures are shown in Table 1.
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RESULTS |
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Changes in Thymus Weight and Thymocyte Composition
In preliminary studies we determined the dose at which either of the four chemicals, arsenite, TCDD, DEX, and E2, reduces the thymus weight of mice by approximately 30% within 3 days. When mice were given either of the four chemicals at the determined dose and examined until day 7, their body weight was found to be unaffected (data not shown). The changes in thymus weight, thymocyte number, and thymocyte population are shown in Figure 1 and Table 2. The thymocyte number was found to be greatly reduced by arsenite and E2 on day 3, and by TCDD and DEX on day 3 and day 7. The atrophy pattern by TCDD was peculiar with the progressive reduction of thymocyte number over the 7-day period. To identify the pathways that mediate the atrophy induced by each chemical we analyzed the gene expression changes on day 1 as described below.
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Thymocytes are classified into four subpopulations that differ in stage of maturation or differentiation: CD4–CD8– DN cells, CD4+CD8+ double-positive (DP) cells, CD4 single-positive (SP) cells, and CD8 SP cells. Although changes in the proportions of the subpopulations may cause apparent changes in gene expression, on day 1 there were no marked differences in the proportions of the four subpopulations of thymocytes between the control group and any of the chemical-exposed groups (Table 2).
Distinctive Changes in Gene Expression in the Thymus Induced by Chemical Exposure
We investigated gene expression changes in the thymuses of control mice and chemical-exposed mice on day 1 with Affymetrix GeneChips. Four independent comparisons between the arsenite-exposed group and the control group followed by statistical analyses revealed upregulation of one gene and downregulation of 59 genes in the arsenite group (Table 3). Two independent comparisons of the groups exposed to the other three chemicals and the control groups revealed reproducible upregulation of six genes and downregulation of one gene by TCDD, upregulation of 27 genes and downregulation of 13 genes by DEX, and up- and downregulation of only one gene each by E2 (Table 4). The four chemicals were found to cause changes in expression of different sets of genes. Only Pth was commonly upregulated by arsenite and E2, and AK011345
[GenBank]
was commonly downregulated by arsenite and DEX.
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A most remarkable feature of the altered expression pattern by these chemicals was found for arsenite, which downregulated many genes involved in cell cycle progression (Table 3). The changes in expression of some genes were validated by RT-PCR (Fig. 2). The gene coding cyclin E2, which plays a role in G1/S phase progression, is a well-known E2F-regulated gene. E2F family members also regulate various genes known to function in G2/M-phase progression and in mitosis (Ishida et al., 2001
; Ren et al., 2002; Trimarchi and Lees, 2002), and some of the downregulated genes listed in Table 3, such as Ccnb1, Ccnb2, Kntc2, Mad2l, Ttk, and Melk, have been reported to be regulated by E2F family members (Ishida et al., 2001; Ren et al., 2002). A search for E2F binding sites around the 5'-flanking region of the genes downregulated by arsenite revealed the presence of E2F binding sequences in 20 genes out of the 59 genes (Table 3). In contrast, searches conducted under the same condition identified the presence of E2F binding sites in only four genes in the DEX-upregulated genes, three in the DEX-downregulated genes, and one in the E2-downregulated gene. Although significant suppression of E2f1, which is known to be a target of E2F regulation itself, was detected in the arsenite-exposed thymuses by real-time PCR (Fig. 6), E2f1 was not included in the list of downregulated genes shown in Table 3. This was because the downregulation of E2f1 by arsenite was less than twofold according to the GeneChip analysis. The genes downregulated by arsenite strongly suggested involvement of E2 dependent cell cycle arrest in the thymus atrophy.
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The other three chemicals showed different characteristics of affected genes, respectively. TCDD caused changes in expression of a small number of genes, including Cyp1a1, a hallmark gene of AhR activation, and Scin, which has been reported to be induced by TCDD in the thymus (Svensson et al., 2002
) (Table 4). E2 exposure changed expression in only two genes (Table 4). No clear link between the genes affected by TCDD or E2 and thymus atrophy was suggested. The reason for the detection of only small numbers of affected genes may be that TCDD and E2 cause changes in the small population of DN thymocytes (McMillan et al., 2007; Zoller and Kersh, 2006), and the analyses of the entire thymus were unable to detect the changes that occurred in this small subset, as discussed later. Expression of Gilz (GC-induced leucine zipper) was found to be induced by DEX exposure (Table 4). Gilz is a gene that was discovered in DEX-treated murine thymocytes, and transgenic mice that expressing GILZ specifically in T-lineage cells have been reported to develop thymus atrophy (Delfino et al., 2004). Consistent with these findings, Gilz induction was observed in the thymuses of mice exposed to DEX in the present study, but exposure to the three other chemicals did not result in downregulation of the cell cycle–related genes observed in response to arsenite exposure.
Effect of Arsenite on Cell Cycle Progression and the Growth of Lymphoma Cells
We used cultured cells to investigate whether the arsenite-induced changes in expression of the cell cycle–related genes actually caused suppression of cell cycle progression and cell growth. Exposure of A20 cells to 10µM arsenite for 24 h resulted in gene expression changes similar to those observed in the thymuses exposed to arsenite for 24 h, including downregulation of Ttk, Ccnb2, Ccne2, and E2f1 (Fig. 3A), and we therefore used this cell line to examine the effects of arsenite on lymphoid cell growth. Consistent with the downregulation of cell cycle–related genes, exposure to 10µM arsenite clearly increased the percentage of cells in the G0/G1 phase and decreased cells in the S phase (Fig. 3B). Apoptotic cells were not increased by arsenite exposure for 24 h (Fig. 3C). The absence of sub-G1 fraction in the cell cycle analyses (Fig. 3B) also supported the absence of apoptotic cells. CFSE labeling showed that 10µM arsenite clearly delayed cell cycle progression after 72 h (Fig. 3D). These changes were shown to result in the suppression of cell growth (Fig. 3E).
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The members of the E2F family are divided according to function into activators (E2F1, E2F2, and E2F3) and repressors (E2F4 and E2F5) (Trimarchi and Lees, 2002
). We examined the binding of E2F1 and E2F4 to the E2F binding site in the target genes by ChIP assay and found that the binding of transcription-activating E2F1 to Melk or Dlg7 promoter region was suppressed and the binding of transcription-suppressing E2F4 to Melk or Dlg7 promoter region was increased by 10µM arsenite (Fig. 4).
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These results obtained in A20 cells suggested that arsenite induces thymus atrophy by inhibiting cell cycle progression through changes in E2F function and suppressing cell growth.
Cell Cycle Arrest in the Thymus by Arsenite, and Tissue-Specific Sensitivity
We examined the cell cycle of thymocytes to confirm arrest of the cell cycle by arsenite in the mouse thymus. A previous study reported that approximately 20% of thymocytes are dividing and that approximately 90% of the dividing cells in the thymus are CD4+CD8+ DP cells. The other 10% of dividing cells are mostly contained in the minor CD4–CD8– DN fraction (Egerton et al., 1990). We therefore measured the cell cycle of all thymocytes and of DN cells. The representative results are shown in Table 5. An increase in the percentage of cells in the G0/G1 phase and a decrease in cells in the S phase were detected in the thymocytes as a whole (Table 5), and the changes were attributed to the cell cycle arrest in the predominant DP population. The DN cells were also shown to exhibit a similar pattern of arrest in the cell cycle.
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Because the suppression of cell cycle–related genes by arsenite was very remarked in the thymus, we investigated the tissue specificity of the changes in gene expression in the arsenite-exposed mice. Expression of Ccnb2 and Ccne2 was found to be especially high in the thymus of the control mice and relatively high in their spleens, and expression of these genes in the thymus and spleen was significantly suppressed by arsenite exposure (Fig. 5). On the other hand, the expression of both genes, particularly of Ccnb2, was very low in the liver, kidney, lung, brain, and skin in the control mice (Fig. 5). In contrast to the thymus and spleen, expression of Ccne2 in the kidney was significantly increased by arsenite (Fig. 5). We also examined the expression of E2f1 and E2f4 in several tissues. In the control mice, E2f1 and E2f4 expression was highest in the thymus of the tissues examined, and expression of both genes was relatively high in the spleen (Fig. 6). Significant suppression of E2f1 by arsenite exposure was detected in the thymus by real-time PCR (Fig. 6). By contrast, E2f1 was upregulated by arsenite in the kidney (Fig. 6). E2f4 expression levels were unaffected by arsenite in any of the tissues examined (Fig. 6).
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Apoptosis in the Thymus by Arsenite
Finally, we examined whether arsenite induces apoptosis as well in the thymocytes. When freshly isolated thymocytes from control mice and arsenite-exposed mice were analyzed, no increase in apoptotic cells was observed by arsenite exposure (Fig. 7A).
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Because the major part of thymocytes undergo apoptosis and the apoptotic cells are promptly cleared by phagocytic cells in the normal thymus, a low level of increase in the number of apoptotic cells might not be detected in freshly isolated thymocytes. To seek the possibility that apoptotic cells are increased by arsenite in vivo, we cultured the thymocytes prepared from control mice and arsenite-treated mice for 24 h and examined apoptotic cells. After the culture, the cells from arsenite-treated mice showed higher percentage of apoptotic cells than those from control mice (Fig. 7B). These results suggest that apoptosis is also involved in the thymus atrophy by arsenite, whereas the contribution of apoptosis in the atrophy at the early time point, such as 24 h after exposure, in vivo remains clarified.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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The results of the microarray analyses of the thymuses exposed to four atrophy-causing chemicals in the present study showed that each chemical induced changes in expression of a different set of genes (Tables 3 and 4). Of the effects caused by the four chemicals, the pathway affected by arsenite was successfully characterized. Although recent studies in nonleukocyte cells have demonstrated that arsenic alters gene expression by affecting transcription factors, such as Nrf2, AP-1, NF-
B, PPAR, and C/EBP (He et al., 2006; Hu et al., 2002; Ouyang et al., 2006; Wauson et al., 2002), the microarray approach in the present study showed that arsenite characteristically affects E2F-dependent pathways in thymocytes, which leads to thymus atrophy.
Because the arsenic modulation of transcription factors is cell type-specific, the phenotypic changes induced by arsenic vary with the cell type. Exposure to intermediate or lower doses of arsenic (
5µM) increased cell proliferation of nonleukocyte cells, such as keratinocytes and epithelial cells (Hamadeh et al., 2002; Miller et al., 2002; Ouyang et al., 2006; Simeonova et al., 2000). By contrast, intermediate or lower doses of some arsenic compounds, such as arsenic trioxide and arsenite, have been found to induce apoptosis in lymphomas, myeloid leukemia cells, and normal murine thymocytes in vitro (Akao et al., 1998; Hossain et al., 2000; Wang et al., 1998; Zhu et al., 1999). Arsenite also induces aneuploidy in human lymphocytes by disrupting microtubule assembly and spindle formation (Ramirez et al., 1997) or delays cell cycle progression in mitogen-activated human lymphocytes (Galicia et al., 2003). The microarray approach and the following studies in the thymus (Table 5) and A20 cells (Figs. 3 and 4) in the present study illuminated the involvement of E2F pathway and cell cycle arrest in the thymus atrophy by arsenite at a dose that induces thymus weight loss of approximately 30%. Examination of various tissues in the present study also showed that the characteristic suppression of E2F-dependent cell cycle–related genes by arsenite occurs in lymphoid organs, such as thymus and spleen (Figs. 5 and 6).
McCollum et al. (2005) have reported that an intermediate dose (5µM) of arsenite delay cell cycle progression in myelomonocytic U937 cells by affecting all phases of the cell cycle. In the present study, arsenite consistently suppressed expression of genes involved in G1/S, G2/M, and M-phase progression (Table 3), and thus arsenite may affect all the phases of the cell cycle in thymocytes. The cell cycle analyses of the thymocytes in the present study, on the other hand, showed only an increase in the proportion of G1 phase and no apparent increase in G2/M phase cells (Table 5). This may have been because the rate of increase in the G1 phase was much larger than the increase in the G2/M phase, although all the phases were affected by arsenite, the same as reported in regard to U937 cells (McCollum et al., 2005). Consistently, a clear increase in the doubling time by arsenite was demonstrated by the CFSE-labeling experiment as shown in Figure 3D.
In the present study, E2 exposure induced expression changes in only two genes. E2 has been reported to suppress the proliferation of DN3 cells and DN4 cells, the two subsets in the small DN population (Zoller and Kersh, 2006). Because these DN cells are rapidly growing cells, suppression of their proliferation is thought to lead to a massive reduction in thymocyte number at a later DP cell stage. If this is the case, microarray analysis of the thymocytes as a whole may not effectively detect the changes responsible for the induction of thymus atrophy, as observed in the present study (Table 4). On the other hand, changes in expression of many genes were clearly detected in the arsenite-exposed thymuses. The findings strongly suggest that arsenite affects both the DN cells and the predominant DP cells, which lead to thymus atrophy as well as obvious gene expression changes in many genes. TCDD also changed expression of only a few genes in the present study. TCDD has recently been reported to induce changes in expression in a variety of genes, including Klf2 and its target genes, in the DN2 fraction, a small subpopulation of DN cells, in the mouse thymus (McMillan et al., 2007). The same study also reported later acute cell losses of DN3, DN4, and DP cells, which were derived from DN2 cells (McMillan et al., 2007). Because KLF2 is implicated in T-cell proliferation and trafficking, the results obtained by McMillan et al. (2007) suggest that the aberrant expression of Klf2 in the DN2 fraction leads to thymocyte loss and thymus atrophy. We also isolated total DN cells from control mice and from mice 24 h after exposure to 10 µg/kg of TCDD and investigated gene expression changes with GeneChips. The results showed changes in a large number of genes by TCDD, including upregulation of Klf2 and its target genes, such as the genes coding integrin β7 and IL10R, in the DN cells (data not shown). Because we could not detect the changes in these genes by the analyses of entire thymuses (Table 4), these results indicate that identifying target cells and applying microarray analysis to them are essential to identifying significant changes in gene expression. No upregulation of the representative AhR target gene Cyp1a1 was detected in the DN cells in our study (data not shown), a finding that was consistent with the results obtained by McMillan et al. (2007). Thus, the upregulation of Cyp1a1 detected in the whole thymuses in the present study is attributable to the induction in the predominant DP cells and/or other cell types. These results also indicate that genes in the DP cells and/or other cell types in the thymus are refractory to TCDD stimuli, even though they express the AhR.
Although GCs were known to promote apoptosis of DP cells (Ashwell et al., 2000), the genes responsible for initiating the GC-induced apoptosis had never been identified. Transgenic mice that express Gilz specifically in T-lineage cells have been demonstrated to develop thymus atrophy with reduced DP cells, and the same study reported that GILZ decreased antiapoptotic molecule B-cell leukemia XL (Bcl-xL) and promoted apoptosis in the thymocytes in vitro (Delfino et al., 2004). These findings suggest that GCs and DEX induce thymus atrophy by inducing Gilz expression in DP cells and causing apoptosis. Consistent with these findings, Gilz induction was detected in the total thymuses of mice exposed to DEX in the present study (Table 4), and the obvious upregulation of Gilz in the thymocytes as a whole suggested that Gilz is induced in the DP cells and causes DP cell apoptosis.
Although TCDD, E2, and DEX are known to directly interact with their individual receptors, AhR, ER, and GR, respectively, how arsenite affects transcription factors has yet to be determined. Arsenite has been reported to directly bind to nuclear receptors, such as GR (Simons et al., 1990) and ER (Kitchin and Wallace, 2005). On the other hand, although arsenite has been reported to activate Nrf2 by affecting the Nrf2/Keap/Cul complex, it has never been determined whether arsenite directly interacts with this complex. Several mechanisms have been suggested to be involved in the arsenic-induced cell cycle dysregulation in nonleukocyte cell types. Chen et al. (2002) have reported that arsenic induces G2/M arrest through induction of Cdc25C degradation via the ubiquitin–proteasome pathway, and Tsou et al. (2006) have reported arsenic-induced G2/M arrest through activation of the DNA damage responsive kinases ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3 related (ATR) and following accumulation and/or phosphorylation of checkpoint-related molecules. However, the mechanism of arsenic-induced suppression of E2F function in the thymocytes remains to be clarified.
In summary, the microarray approach very effectively identified E2F-dependent cell cycle arrest as the pathway mediating arsenic-induced thymus atrophy in the present study. The results of this study also implied the importance of focusing on the target cells in the microarray analyses. Determining the effects of arsenic-induced cell cycle arrest on thymocyte differentiation, selection, and maturation will require further study.
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FUNDING |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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National Institute for Environmental Studies grant (0406AG337) to K.N.; Ministry of the Environment of Japan, R&D Project for Environmental Nanotechnology grant to K.N.; and Ministry of Education, Science, Sports and Culture, Grant-in-Aid for Scientific Research (19590611) to K.N.
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ACKNOWLEDGMENTS |
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The authors wish to thank Dr M. Yamamoto (National Institute for Minamata Disease) and Dr S. Hirano (NIES) for their helpful discussions, and M. Matsumoto and H. Murai for their excellent technical and secretarial assistance.
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REFERENCES |
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