Doctoral Dissertations

Date of Award

8-2003

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Comparative and Experimental Medicine

Major Professor

Patricia Tithof

Abstract

Polycyclic aromatic hydrocarbons (P AHs) are common environmental pollutants resulting from incomplete combustion of organic matter, emission sources, automobile exhausts, cigarette smoke (CS), residential wood, or coal combustion. PAHs are readily absorbed from the gastrointestinal tract of mammals and have toxic, mutagenic and/or carcinogenic consequences. More than 100 different P AH compounds are identified. Sixteen of the common P AHs typically analyzed have been listed by the Environmental Protection Agency (EPA) among 129 priority pollutants, and five of which are listed among the 25 hazardous substances that are believed to have the most potential threat to human health at priority superfund sites. CS has been implicated as a major risk factor in atherosclerotic heart disease through a mechanism that involves endothelial cell (EC) injury and apoptosis or programmed cell death. Little is known about the specific components of CS responsible for this effect or the mechanisms by which CS induces EC injury and progression of atherosclerosis. However, the phospholipase A2/arachidonic acid (PLA2/ AA) pathway might be involved in SC-induced EC injury. In this research, the long-range goal was to understand the role of several P AHs found in the environment and CS in the EC and smooth muscle cells (SMC) injury. The hypothesis to be tested is that EC and SMC PAR-induced apoptosis is mediated by PLA2/ AA pathway. The objectives of this research were 1) to evaluate the effects on EC injury of different P AHs present in high concentration in both Chattanooga creek and CS. 2) to test the molecular mechanisms by which P AHs induce such effect. 3) to test the effect of IV P AHs on SMC in vitro and to characterize the role of different PLA2 isoforms in such effect. In this research, we found that Chattanooga creek contains extremely high levels of 11 out of 16 EPA priority P AHs. The levels range from 10,625 mg/kg soil (phenanthrene) to 97 mg/kg soil for fluorene compounds. Six out of the 11 compounds studied, found in the creek or in CS, activate the AA cascade and induce apoptosis of EC. Three of the 11 P AHs cause these effects by activating and increasing the expression of two different isoforms of PLA2; the Group VIB and the Group IVy enzymes. Furthermore, three out of 11 activate and increase expression of only the Group IVy PLA2. PLA2 activation and release of AA is associated with an increase in apoptosis as measured by histone fragmentation and cleavage of P ARP. From these results, we hypothesize that the water solubility as well as the molecular structures of the compounds play important roles in the effects of P AHs on EC PLA2 activation and apoptosis. These data suggest that small molecular weight, highly water soluble compounds present in high concentrations in the Chattanooga creek and CS may be potentially toxic confirming the need for further studies. We also found that 8 P AHs present in high concentrations in CS and in urban pollution induce EC apoptosis by a mechanism that involves PLA2-mediated activation of JNK.s, p-38, and caspases-8, -9, and -3. PAHs induce phosphorylation of JNK-1, JNK-2, p-38, and the transcription factor c-Jun, resulting in apoptosis in EC. This effect is inhibited significantly by MAPP (Group IV and VI PLA2 inhibitor) and JNK-1 L form (JNK-1 and -2 inhibitor) but not with SB203580 (p-38 inhibitor). These data provide compelling evidence that P AHs induce apoptosis by a mechanism that involves fatty V acid-induced phosphorylation of JNK-1 and JNK-2. Furthermore, p-38 phosphorylation did not participate in the mechanism by which PAHs induce apoptosis. This study also establishes a link between the discrete structure of these PAHs and their activity. We investigated the effect of 8 different P AHs on SMC PLA2 activation and induction of apoptosis. Our data show that five of the 8 compounds tested cause 3H-AA release in a concentration-dependent fashion. The 5 active compounds also induce apoptosis at a concentration that induce significant 3H-AA release. In addition, specific PLA2 inhibitors that are selective for Group IV and VI enzymes inhibit 3H-AA release and attenuate the apoptotic effect, suggesting that P AHs induce SMC apoptosis by a mechanism thati nvolves PLA2. Results from cell-free PLA2 activity assays, RT-PCR, and Western blotting show that SMC express 4 isoforms of PLA2, Group IV a, J3, y, and Group VIJ3 PLA2. Transfection of SMC with PLA2 isoforms that were the most strongly expressed (Group IVy and Group VIB) causes a significant increase in 3H-AA when stimulated with active PAHs as compared to wild type cells. Our data suggest that both of these enzymes are responsible for PAR-induced AA release and subsequent apoptosis in SMC with the possibility of Group IVa and B involvement in this effect. This study demonstrates the involvement of P AHs in EC and for the first time in SMC. Furthermore, our data suggest that small molecular weight, highly water-soluble compounds present in Chattanooga creek may be potentially toxic confirming the need for further studies.

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