The common commercial use of phthalate esters has resulted in significant human exposure to these bioactive compounds. The facts that phthalate ester metabolites, like endogenous PGs, are peroxisome proliferator-activated receptor (PPAR) agonists, and that PPARγ agonists induce lymphocyte apoptosis suggest that phthalate esters are immunosuppressants that could act together with PGs to modulate early B cell development. In this study we examined the effects of a metabolite of one environmental phthalate, mono(2-ethylhexyl)phthalate (MEHP), and 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2), on developing B cells. MEHP inhibited [3H]thymidine incorporation by primary murine bone marrow B cells and a nontransformed murine pro/pre-B cell line (BU-11). Cotreatment with a retinoid X receptor α ligand, 9-cis-retinoic acid, decreased [3H]thymidine incorporation synergistically, thereby implicating activation of a PPARγ-retinoid X receptor α complex. These results were similar to those obtained with the natural PPARγ ligand 15d-PGJ2. At moderate MEHP concentrations (25 or 100 μM for primary pro-B cells and a pro/pre-B cell line, respectively), inhibition of [3H]thymidine incorporation resulted primarily from apoptosis induction, whereas at lower concentrations, the inhibition probably reflected growth arrest without apoptosis. Cotreatment of bone marrow B cells with 15d-PGJ2 and MEHP significantly enhanced the inhibition of [3H]thymidine incorporation seen with MEHP alone, potentially mimicking exposure in the bone marrow microenvironment where PG concentrations are high. Finally, MEHP- and 15d-PGJ2-induced death does not result from a decrease in NF-κB activation. These data demonstrate that environmental phthalates can cooperate with an endogenous ligand, 15d-PGJ2, to inhibit proliferation of and induce apoptosis in developing bone marrow B cells, potentially via PPARγ activation.

Programmed cell death is a critical process in B lymphocyte development. Premature apoptosis in developing B cells could affect the repertoire and number of mature B cells produced. Of particular concern is the ability of environmentally ubiquitous polycyclic aromatic hydrocarbons (PAH) to induce B cell apoptosis within the bone marrow microenvironment in a clonally nonspecific way. Here, models of bone marrow B cell development were used to assess the role of the "extrinsic" apoptosis pathway in PAH-induced apoptosis and to compare PAH-induced apoptosis with that induced during clonal deletion. As

There is growing interest in using peroxisome proliferator-activated receptor (PPAR) gamma agonists as chemotherapeutic agents in hematologic malignancies. PPARgamma agonists of diverse chemical structure induce apoptosis in several malignant B cell lines. However, PPARgamma agonists also induce apoptosis in normal B cells. One such agonist, GW7845, rapidly induces apoptosis in early B cells. Understanding the mechanisms of PPARgamma agonist-induced death is essential to minimizing loss of normal cells during chemotherapy. PPARgamma agonists influence mitogen-activated protein kinase (MAPK) cascades in other systems, and MAPKs can be associated with apoptosis. Therefore, we investigated the activation of MAPKs in primary pro-B cells and cultured pro/pre-B cells and their role in GW7845-induced apoptosis. Treatment of a nontransformed murine pro/pre-B-cell line with GW7845 transiently induced the phosphorylation of extracellular signal-related protein kinase (ERK) 1/2, but strongly and persistently induced the activation of p38 MAPK and c-Jun NH(2)-terminal kinase (JNK). In primary pro-B-cells, p38 MAPK and JNK were activated following treatment with GW7845. Phosphorylation of activating transcription factor-2 (ATF-2) was induced strongly in both B-cell types. In pro/pre-B cells, pretreatment with the p38 MAPK/JNK inhibitor PD169316 potently suppressed multiple facets of GW7845-induced apoptosis signaling. However, when a series of p38 MAPK and JNK inhibitors were used, only SB202190, also a dual inhibitor, completely suppressed GW7845-induced apoptosis. Inhibitors specific for p38 MAPK and JNK were only partially effective, suggesting that suppression of a single MAPK is not sufficient to inhibit death. The results support the hypothesis that GW7845 initiates an apoptotic pathway in early B cells through the activation of a kinase cascade that includes at least p38 MAPK and JNK.

Apoptosis is a critical event in the deletion of B lymphocytes prior to their migration to the periphery. Synthetic peroxisome proliferator activated receptor gamma (PPARgamma) agonists, including the drug GW7845 and the environmental contaminant mono-(2-ethylhexyl) phthalate, as well as an endogenous ligand, 15-deoxy-Delta(12,14)-prostaglandin J(2), induce clonally unrestricted apoptosis in pro/pre-B cells. Considering that PPARgamma agonists are used clinically for the treatment of diabetes and postulated to be useful as chemotherapeutics, we used GW7845 as a model PPARgamma agonist to examine the mechanism of cell death that may contribute to tumor killing as well as normal bone marrow B lymphocyte toxicity. GW7845 induced rapid mitochondrial membrane depolarization and release of cytochrome c, along with nearly concurrent activation of capases-2, -3, -8, and -9 in primary pro-B cells and BU-11 cells, a nontransformed pro/pre-B cell line. GW7845-induced apoptosis was reduced significantly in Bax-deficient and Apaf-1 mutant primary pro-B cells, supporting the conclusion that GW7845-induced apoptosis is mitochondria- and apoptosome-dependent. Using benzyloxycarbonyl-VAD-fluoromethyl ketone (VAD-FMK) as a pan-caspase inhibitor, we demonstrated that an initial cytochrome c release occurred independently of caspase activation and that only caspase-9 activation was partially caspase independent. The attenuation of GW7845-induced apoptosis by multiple FMK-labeled peptide sequences suggests that multiple caspase pathways are responsible for initiating and executing apoptosis. The strong activation of Bid provides a mechanism by which caspases-2, -3, and -8 may amplify the apoptotic signal. These data support the hypothesis that pharmacologic concentrations of PPARgamma agonists induce an intrinsic apoptotic pathway that is driven in normal bone marrow B cells by multiple amplification loops.