* Department of Pathobiological Sciences,
Department of Pharmacology, and
Molecular and Environmental Toxicology Center, University of Wisconsin, Madison, Wisconsin 53706
Received December 17, 2002; accepted April 10, 2003
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ABSTRACT |
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Key Words: PAH; DMBA; apoptosis; B cell; bone marrow; p53.
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INTRODUCTION |
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7,12-Dimethylbenz[a]anthracene (DMBA) and benzo[a]pyrene (B[a]P) are the prototypical and best characterized PAHs. These compounds can have significant effects on both humoral and cell-mediated immunity (Burchiel et al., 1990; Ward et al., 1984
). The mechanisms responsible for the immunotoxicity of these compounds are not completely understood. It has been demonstrated that metabolic activation by the cytochrome P450 1B1 (CYP1B1) enzyme is required for their immunotoxic effect (Heidel et al., 1999
, 2000
; Mann et al., 1999
; White et al., 1985
). The role of the arylhydrocarbon receptor (AhR) in the immunotoxic effects of PAHs is a source of debate. When the AhR binds PAHs, it translocates to the nucleus and induces transcription of a variety of Ah-responsive genes, including P450 cytochromes such as CYP1B1. However, bone marrow stromal cells express a high basal level of CYP1B1 and are able to metabolize PAHs in the absence of a functional AhR (Heidel et al., 2000
). In addition, 2,3,7,8-tetrachlorodibenzo-p-dioxin, or TCDD (i.e., dioxin), binds the AhR with a greater affinity than PAHs but is not metabolized and has different effects on the immune system than do the PAHs (Laupeze et al., 2002
; Okey et al., 1984a
,b
).
PAHs are both carcinogenic and immunotoxic, although the doses required for these two effects may differ (White et al., 1985). The mechanisms of carcinogenicity and the mutagenic properties of PAHs have been studied in greater detail than their effects on the immune system. As is the case for their immunotoxic effects, PAHs must be metabolically activated by the cytochrome P450 family of monooxygenases to be carcinogenic (Badawi et al., 1996
; Cavalieri and Rogan, 1992
). These activated metabolites then form adducts with DNA and cause mutations. Mutations in key regulatory genes, in addition to changes in gene expression mediated by binding of the AhR, are thought to be the major mechanisms responsible for the carcinogenic effects of PAHs (White et al., 1994
). The fact that these two disparate effects of PAHs are interrelated may provide some clues as to the mechanism of immunotoxicity for these compounds.
The p53 tumor-suppressor protein is responsible for regulating cellular responses to DNA damage and progression through the cell cycle (Hickman et al., 2002; Sharpless and DePinho, 2002
). The importance of p53 in maintaining a normal pattern of cellular proliferation is illustrated by p53 being one of the most frequently mutated genes found in tumor biopsies and transformed cell lines (Gupta et al., 1993
). In addition, p53 plays a crucial role as a "gatekeeper" of genomic integrity. If DNA has been damaged in such a way that it cannot be effectively repaired, p53 will initiate apoptosis to prevent the damaged cell from proliferating. Several DNA-damaging agents (e.g., cisplatin, etoposide) have been demonstrated to induce p53 and activate apoptosis in a p53-dependent manner (Karpinich et al., 2002
; Siemer et al., 1999
).
The B-cell lineage constitutes the main component of the humoral immune system. In the bone marrow, there is a specific differentiation pathway for B cells that begins with the common lymphoid progenitor and ends with immature IgM expressing B cells, which then leave the bone marrow to populate the spleen and lymph nodes (Hardy and Hayakawa, 2001). B-cell development is driven by the interaction of the progenitor B cells with bone marrow stromal cells that secrete cytokines and express adhesion molecules that drive the differentiation of the progenitor B cells. Disruption of this interaction via exogenous toxicants could have profound effects on B-cell lymphopoiesis and can result in the impairment of antibody production directed against pathogens. Previously, our laboratory demonstrated that DMBA causes apoptosis in pre-B cells when the latter are co-cultured with bone marrow stromal cells that metabolize DMBA via CYP1B1 (Heidel et al., 1999
, 2000
; Page et al., 2002
). Our hypothesis is that DMBA disrupts hematopoiesis in the bone marrow and affects normal B-cell development by targeting discreet cell populations along the B-cell lineage continuum and further, that this disruption is dependent on the presence of functional p53.
The goal of this study was to determine whether the effects of DMBA on the murine bone marrow are dependent on activation of p53. To accomplish this goal, we utilized homozygous null p53 mice that were treated with a single bolus injection of DMBA. We also investigated the importance of p53 in the progenitor B cell in their response to stromal cells and DMBA in vitro. We present evidence that the effects of DMBA on bone marrow cells in vivo and progenitor B cells in vitro are largely dependent on the presence of functional p53 protein.
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Materials and Methods |
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At 57 weeks of age, male or female p53-/- and p53+/+ C57Bl/6 mice were injected ip with either DMBA (50 mg/kg) or vehicle (olive oil). After 48 h, the mice were euthanized; their femurs, tibias, and sternums were dissected free of muscle tissue; and the ends were removed with a scalpel. For total cell counts, the femurs were flushed with RPMI 1640 with 5% FBS, using a 25-gauge needle. The cells were then sequentially passed through a 25-gauge needle and a 22-gauge needle to generate single-cell suspensions. The cells were then centrifuged, and the RBCs were lysed using ACK buffer (150 mM NH4Cl, 1.0 mM KHCO3, and 100 mM Na2EDTA, pH 7.3). The white cells were resuspended in 5 ml of RPMI 1640 and centrifuged again. The cell pellets were then resuspended in 5 ml of RPMI 1640, and an aliquot was removed for enumeration of viable cells using a hemacytometer and Trypan blue exclusion.
Histopathology and cytology.
The sternums were removed and fixed in ice-cold 4% phosphate-buffered paraformaldehyde, decalcified for 30 min (Formacal-4, Decal Co., Congers, NY), embedded in paraffin, sectioned at 2-µM thickness, and stained with H&E. Bone marrow smears were prepared by cutting a tibia longitudinally, streaking the exposed bone marrow onto a glass slide with a sable-hair brush, and allowing the cell smears to air dry. These were subsequently stained with Wrights stain and examined microscopically, as described previously (Heidel et al., 2000; Page et al., 2002
). The sternum sections and the bone marrow smears were examined microscopically and scored for cell type by an American College of Veterinary Pathologists board-certified pathologist (P.S.M.). For the bone marrow differential cell counts, a total of 500 cells were counted, and the percentage of cells in each of the following categories was enumerated: erythroid precursors, proliferating granulocytes (myeloblast, progranulocytes, myelocytes), mature granulocytes (metamyelocytes, bands, segmented neutrophils), and lymphoid cells. The total cell number for each category was derived by multiplying the percentage of each cell type present in the Wright-stained bone marrow smears of the tibia by the total number of bone marrow cells generated after flushing the marrow from two femurs per mouse. Abnormalities in cellular development in both the erythrocytic and granulocytic lineages were identified and recorded.
Primary culture of bone marrow progenitor B lymphocytes.
Long-term bone marrow cultures (LTBMC) were established according to the protocol of Witte (Whitlock et al., 1984). Briefly, cells were flushed from the mouse femurs with sterile, ice-cold, RPMI 1640 (5% FBS) (Mediatech, Herndon, VA), then centrifuged and resuspended in 1 ml RPMI 1640 (5% FBS). An aliquot of cells was then diluted 1:10 with crystal violet (0.01% w/v crystal violet, 3% acetic acid) and counted using a hemacytometer. Cell density was adjusted to 1 x 106 cells/ml, and the cells were cultured in RPMI 1640 with 5% FBS at 37°C. The media were changed each week (75% fresh media in total volume). After 2 weeks of culture, the media were supplemented with 4 ng/ml of recombinant murine IL-7 (R&D Systems, Minneapolis, MN). By 3 weeks, progenitor B cells were visible in suspension and loosely adherent to the stromal cells.
The phenotype of the progenitor B cells from the LTBMCs was then verified using fluorochrome-conjugated primary antibodies specific for pre-B cell surface markers and flow cytometric analysis (Thurmond and Gasiewicz, 2000). The antibodies used were IgM (clone AF6-78), B220 (clone RA3-6B2), AA4.1 (clone AA4.1), and CD19 (clone 1D3). All antibodies were purchased from Pharmingen (San Diego, CA). Briefly, the cells were isolated from the LTBMCs by gentle agitation, washed twice with PBS + 2% BSA, and fixed with 4% paraformaldehyde. The cells were then washed again and stained with the appropriate antibodies. The cells were then washed twice and stored at 4°C until FACS analysis was performed. The data were analyzed using Cellquest Pro software (BD Biosciences, San Jose, CA). After phenotyping an aliquot of the cells harvested from the LTBMCs, the remainder of the cells were then used for experiments.
Propidium iodide staining.
The immunophenotyped pre-B cells were removed from the LTBMCs and co-cultured with monolayers of bone marrow stromal cells (BMS2 cell line) that were at approximately 75% confluence. The BMS2 cell line was generously provided by Dr. Paul Kincade (Oklahoma Medical Research Foundation, Oklahoma City, OK). The cultures were incubated with the indicated concentrations of DMBA for 24 h; pre-B cells were then removed from the BMS2 monolayers by gentle agitation, as described previously (Page et al., 2002). The detached cells were centrifuged for 5 min at 1200 rpm and washed once in ice-cold PBS + 2% bovine serum albumin. Cells were fixed in 1 ml of 80% ice-cold ethanol for 30 min at -20°C, then centrifuged for 5 min at 1200 rpm and resuspended in phosphate-citric acid buffer (0.192 M Na2HPO4, 4mM citric acid, pH 7.8) for 5 min at room temperature. After centrifugation, the cells were resuspended in 0.5 ml of propidium iodide staining solution (33 µg/ml propidium iodide; Sigma-Aldrich), 1 mg/ml RNase A, 0.2% Triton X-100, in PBS) and analyzed in a Becton/Dickinson FACScan flow cytometer (BD Biosciences, San Jose, CA). Because cells undergoing DNA fragmentation and apoptosis exhibit weaker propidium iodide fluorescence than do cells in the Go/G1 cell cycle, a decrease in PI fluorescence is indicative of the morphological changes consistent with apoptosis (Yamaguchi et al., 1997
).
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RESULTS |
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DISCUSSION |
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We have further characterized the effects of DMBA on the bone marrow with respect to the cell type. Significantly, we noted a p53-dependent decrease in erythroid precursors after DMBA treatment. Functional p53 was required for the depletion of all cell lineages examined (i.e., granulocytes, erythroid precursors, and lymphocytes). This indicates that the majority of DMBA-induced bone marrow toxicity may be mediated through a p53-dependent pathway, even in disparate cell types. To the best of our knowledge, this is the first observation of the p53 dependence of DMBA-mediated bone marrow toxicity in vivo.
The p53 protein plays a major role in the regulation of the apoptotic cascade, so the observation that ablation of its activity protects against DMBA-mediated toxicity is consistent with its known biological function. It has been very difficult to detect apoptosis in the murine bone marrow in vivo after DMBA treatment. One reason for this might be that bone marrow macrophages phagocytose apoptotic cells very efficiently, so that at any given time there is a very small population of apoptotic cells present. The results of this study, in conjunction with our previous observations using cell-culture model systems, support the notion that DMBA causes apoptosis and/or growth arrest in the murine bone marrow.
It has been previously reported that activation of the nuclear transcription factor NF-B protects pre-B cells against PAH-induced apoptosis. Mann et al. reported that PAHs suppress NF-
B and that suppression of NF-
B by specific inhibitors leads to pre-B cell apoptosis (Mann et al., 2001
). NF-
B activation is, in general, an anti-apoptotic signal, whereas p53 activation is pro-apoptotic (Beg and Baltimore, 1996
; Bertrand et al., 1998
; Levine, 1997
). Other investigators have proposed a link between p53 induction and NF-
B regulation (Holmes-McNary et al., 2001
; Webster and Perkins, 1999
). In other systems, the opposing regulation of p53 and NF-
B has been proposed as a mechanism of apoptosis induction (Holmes-McNary et al., 2001
). These findings and the results of this study suggest that PAH metabolites might cause apoptosis in pre-B cells by both inducing p53 and inhibiting the activity of NF-
B.
DMBA is also carcinogenic, and p53 is often mutated in transformed cells (Gupta et al., 1993). The requisite role of p53 in bone marrow toxicity suggests that the decreased cellularity noted in WT mice may reflect the efforts of the immune system to protect itself from the generation of leukemias and lymphomas following PAH exposure. p53-Induced apoptosis occurs when the levels of DNA adduct formation caused by DNA-damaging agents (e.g., radiation, toxins, etc.) overwhelm the DNA repair machinery of the cell. If PAH metabolites form mutation-causing adducts in the p53 gene, the cells of the immune system may lose the protective mechanism of eliminating aberrant cells, as suggested by the absence of bone marrow hypocellularity in the p53 null mice. This could, in turn, lead to a higher incidence of tumor formation in these mice after DMBA exposure. Indeed, it has been reported that p53 null mice have a higher incidence of mammary tumors after DMBA administration and are more susceptible to teratogenic malformation after ionizing radiation than are WT mice (Jerry et al., 2000
; Kato et al., 2001
).
Although it is possible that the toxicity observed in the bone marrow after DMBA exposure might protect against leukemia or lymphoma, disruption of B-cell development can have profound deleterious effects on acquired immunity. Previous studies have documented a decrease in B-cell reactivity and IgM production among workers occupationally exposed to PAHs (Winker et al., 1997). In addition, it has also been demonstrated that the potent AhR agonist, TCDD, alters B-cell populations (Thurmond and Gasiewicz, 2000
). The precise mechanisms underlying these adverse effects on B cells are unknown. In this study, we demonstrate that the effects of DMBA on the murine bone marrow are dependent on functional p53. Previous work in our laboratory found that the interferon-inducible kinase (PKR) is upregulated in pre-B cells in response to DMBA treatment (Page et al., 2002
). This observation is important because PKR has been shown to activate p53 (Cuddihy et al., 1999a
,b
). Coupled together, these observations point to a possible pathway of DMBA-mediated bone marrow toxicity. Our working model is that metabolites of DMBA stimulate bone marrow cells to release a soluble factor (e.g., cytokine or chemokine) that leads to PKR activation, which in turn activates p53 and causes apoptosis in the affected cell types.
In conclusion, this study has demonstrated that p53 protein is necessary for bone marrow depletion of granulocytic, erythroid, and lymphocytic cells after DMBA treatment. In addition, we have demonstrated that pre-B cells derived from the bone marrow of p53 null mice are also resistant to DMBA-mediated apoptosis in vitro. These studies provide new insights into the molecular mechanisms underlying DMBA-induced immunotoxicity.
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ACKNOWLEDGMENTS |
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NOTES |
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