Polycyclic aromatic hydrocarbons (PAHs) are important environmental pollutants that inhibit humoral and cell mediated immunity in mice and perhaps in humans. The precise mechanisms responsible for immunosuppression are not known. Previous studies by this laboratory have shown that a methylated PAH (7,12-dimethylbenz(a)anthracene, DMBA) interferes with early events associated with lymphocyte activation in murine B and T cells exposed in vivo and in vitro. Exposure of resting B and T cells (human and mouse) to low concentrations (3muM) of DMBA causes an increase in free intracellular levels of Ca+2 and a significant increase in the phosphorylation of several cell proteins associated with cell activation. This application examines the significance of these events in the immunotoxicity of PAHs. Higher concentrations of DMBA (10-30 muM) continue to increase baseline Ca+2 in lymphocytes, but inhibit phosphorylation in T cells induced by antigen receptor stimulation. One of the primary goals of this application is to study the mechanisms of alteration of Ca+2 and phosphorylation in B and T cells following in vivo or in vitro exposure to methylated (DMBA and 3-methylcholanthrene) and nonmethylated PAHs (benzo(a)pyrene and benz(a)anthracene) compared to nonimmunosuppressive PAHs (benzo(e)pyrene and anthracene). Studies are proposed to examine the mechanism of PAH-induced cell death in lymphocytes. Data is presented that high doses of DMBA produce lymphocytotoxicity via an apoptosis-like mechanism, and that cell to cell interactions (cell density) may play an important role in this process. In vivo studies will be conducted using B6C3F1 mice treated orally by gavage with PAHs daily for 5 days at doses of 5-150 mg/kg. Studies will also be conducted with lymphoid cells obtained from mice, human/murine lymphoid cell lines, and normal human peripheral blood T cells exposed to PAHs in vitro at concentrations of 3-30 muM. The effects of PAHs on phosphorylation of antigen receptor- associated molecules (pp 32,34,35,37 and phospholipase C-gamma) important in B cell activation produced by anti-IgM/IgD will be studied by SDS-PAGE analysis of P-32 labeled digitoninized cells and anti-phosphotyrosine Western blots. the type of phosphorylation of proteins (ser/thr/tyr) will be characterized in whole cell lysates and in specific immunoprecipitated proteins. Parallel studies of anti-CD3/CD4 induced Ca+2 mobilization examined by flow cytometry and phosphorylation of molecules important in T cell activation (CD3, and phospholipase C-gamma) will also be performed. the effects of PAHs on several kinases (PKA, PKC, and PTK, as well as src- related proto-oncogenes) and phosphatases (including CD45) important in cell activation will be studied. Several possible mechanisms will be explored for PAH-induced increases in resting cell Ca+2 levels, including inhibition of Ca+2-ATPase, direct release of stored Ca+2, and indirect stimulation of release by increased IP3 production. The results of these studies are likely to give broad insights into mechanisms of immunotoxicity produced by environmental and other agents that alter Ca+2 homeostasis and signal transduction in lymphoid cells.
Showing the most recent 10 out of 20 publications
