Polycyclic aromatic hydrocarbons (PAHs), such as benzo(a)pyrene (BaP) and 7,12-dimethylbenz(a)anthracene (DMBA), as well as halogenated aromatic hydrocarbons (HAHs), such as 2,3,7,8,-tetrachlorodibenzo-rho-dioxin, (TCDD), are important immunotoxicants that are known to be carcinogenic and immunosuppressive in animals, and also likely in humans. Our work has shown that PAHs, PAH-metabolites (PAH-M, and perhaps HAHs, alter Ca2+-dependent signaling pathways associated with T and B cell antigen and mitogen receptor activation of lymphocytes.
In Aim 1, we will further explore mechanisms by which PAHs and some HAHs (TCDD) alter ca2+ homeostasis in human peripheral blood mononuclear cells (HPBMC), as well as certain human B (Daudi), T (HPB- ALL and Jurkat), and monocyte (Monomac) cell lines. We have shown that PAHs activate Src-related protein tyrosine kinases (PTKs), which may indirect due to inhibition of protein tyrosine phosphatases (PTPases, such as CD45). PAHs also inhibit sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) activity. These studies will focus on the effects of PAHs and PAH-M on PTKs, PTPases, and SERCA examined using highly purified (immunoprecipitated) and/or cloned proteins. We will also examine the effects of AHs on channel-mediated Ca2+ influx. We have shown that alpha-napthoflavone, a PR50 inhibitor, reverses the immunotoxity of BaP and DMBA in T cell mitogen-simulated cultures of HPBMC, suggesting that metabolites may be responsible for the immunotoxicity produced by these PAHs. Therefore, an underlying hypothesis in these studies is that oxidative PAH-M may be responsible for attacking sulphydryl-sensitive proteins associated with Ca2+ signaling.
In AIM 2, we will explore the effects of PAH/PAH- M on downstream signaling pathways in B and T cells to determine the significance of Ca2+ signaling on protein kinase C activation and the expression of certain transcription factors (Fos/Jun, NF-kappaB, NF-AT).
In AIM 3, will continue studies on the molecular mechanisms associated with PAH- induced cell death examining HPBMC for DNA strand breaks- repair by the TUNEL assay, as well as p53 and Bc1-2 expression. Finally, in Aim 4 we will assess the role of monocyte and P450 expression by Western blot and RT-PCR methods in subsets of HPBMC and human cell lines (above).
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