Effects of methyl tertiary butyl ether and diesel exhaust particles on in vitro and in vivo vascular formation:

Hazardous chemicals are released into the environment by a number of natural and anthropogenic activities and may cause adverse effects on human health and the environment. Air pollutants such as volatile organic compounds and respirable particulate material differ in chemical composition and reaction properties, but both can induce acute and chronic effects on human health. Methyl tertiary butyl ether (MTBE) and diesel exhaust particles (DP) are materials that are representative of these two classes of air pollutants. Although MTBE and DP differ physically and chemically, both have the potential to affect the vasculature. Previous studies in this laboratory reported that exposure to MTBE halted embryonic vascular formation in the model teleost Japanese medaka (Oryzias latipes). The embryonic vasculature failed to develop while other non-vascular tissues developed normally. Based upon these findings, studies were undertaken to determine if MTBE would alter vascular formation in other model systems. Exposure of cultured rat brain endothelial cells to MTBE (0.34 - 34.0 mM) resulted in a dose-dependent reduction of microcapillary formation, with the LOAEL at 0.34 mM. Tertiary butyl alcohol (TBA), a major metabolite of MTBE, was also tested. TBA (0.34 - 34.0 mM) had no effect on microcapillary formation. In a mouse Matrigel plug implantation assay, Matrigel plugs containing MTBE (34.0 mM) showed a complete lack of vessel formation compared to plugs containing Endothelial Cell Growth Supplement without MTBE. Treatment of HUVEC grown on Matrigel with 34 mM MTBE resulted in a decrease in phosphorylation of VEGFR-2 at both the Tyr951 and Try996 phosphorylation sites. In addition, a rat reproductive study was used to test the antiangiogenic effects of MTBE in vivo. Timed-pregnant Fisher 344 rats were dosed orally by gavage with MTBE (500 - 1500 mg/kg) from implantation through gestation. Tissues from one half of the pups of each sex from each litter were examined histologically on post partum Day 1 for developmental vascular changes. The remaining pups from each litter continued to be dosed at the same level as the respective dam on post partum Days 1 to 10, and then were examined as described above. No vascular changes were observed in any of the tissues examined. The in vitro data demonstrated that the vasculature is a primary target of MTBE in several model systems if sufficiently high vascular concentrations are obtained and that the observed changes in vascular development may be in part due to inhibition of activation of VEGFR-2. However, in the rodent reproduction assay no histological vascular lesions were observed, which may reflect the inability to achieve high enough circulating MTBE levels. In addition to the work with MTBE, studies were undertaken to explore the possible effects of DP on vascular formation. Exposure of human umbilical vein endothelial cells (HUVEC) in vitro to DP at concentrations of 25 ug/mL or greater resulted in decreases in capillary-like tube formation. Additional in vitro studies in HUVEC determined that an organic extract derived from DP also inhibits vascular formation with a LOAEL of 20 ug/mL and complete inhibition of vascular formation at 40 ug/mL and that the effects are reversible except at the highest concentration of DP tested (100 ug/mL). Through the use of several assays and immunocytochemical staining techniques, it was determined that neither apoptosis nor chemical-induced cell death are responsible for the observed inhibitory effects. However, studies did indicate that decreases in activation of Rho GTPases may be involved in the inhibition of HUVEC vascular tube formation. Vascular formation would appear to be a sensitive target for both MTBE and organic compounds absorbed onto diesel exhaust particles. One of the mechanisms by which they prevent proper endothelial cell migration and tube formation may be due to disruption of the VEGF pathway in association with the cytoskeletal structure of the cells.Ph.D.Includes bibliographical references (p. 141-155)by John C. Kozlosk