Pyrolysis of tobacco flavinoids deposits 7-280 micrograms per cigarette of the each of the benzene derivatives phenol, hydroquinone (HQ) and catechol into the smoker's lungs. A fourth derivative, p-benzoquinone (p-BQ), is spontaneously produced by oxidation of hydroquinone. The long-term objective of this research project is to determine the mechanisms by which these benzene derivatives block T cell responses in order to gain a better understanding of how cigarette smoke suppresses T cell function in the lungs and promotes respiratory tract infections and cancer. We have found that three biologically active derivatives of benzene function at different stages in T cell activation; p-BQ blocks IL-2 and IFN-gamma production, HQ blocks transferrin-receptor expression and DNA synthesis, and catechol chelates iron needed to T cell proliferation. The inhibitory effects of these benzene derivatives occur in the absence of cytotoxicity. We hypothesize that p-BQ, HQ and catechol inhibit discrete events in early and late T cell activation that are critical for normal immune function. In order to determine the mechanism of action of these benzene metabolites, the following specific aims will be undertaken: (1) To determine if the inhibitory effect of p-BQ on IL-2 and IFN-gamma production is due to effects on mRNA stability and/or translation; (2) To determine by electron paramagnetic resonance if HQ and catechol quench the tyrosyl radical in the M2 subunit of ribonucleotide reductase; (3) To elucidate, at the subcellular level, the mechanism by which HQ down- regulates transferrin receptors on human T lymphoblasts; and (4) To determine if the anti-proliferative effect of ion-chelation by catechol is limited to inhibition of the iron-dependent enzyme, ribonucleotide reductase. Human peripheral blood T cells (resting) and human T lymphoblasts (proliferating) and a Jurkat T cell line will be used. The effect of p-BQ on IL-2 on IL-2 mRNA stability will be determined by Northern analysis at different times after stimulation of normal T cells with alphaCD3+ PMA, and by measuring its effect on the expression of a fluorescent reporter gene product containing the 3'-untranslated instability region cloned from the IL-2 and IFN-gamma genes. The ability of HQ and catechol to reduce the tyrosyl radical in the M2 subunit of ribonucleotide reductase will be measured by EPR spectroscopy.
