The long-term objective of this research is to investigate the role of reactive oxygen species, now to include nitric oxide (NO), and iron in asbestos-induced cancer. The hypothesis for the proposed research is that asbestos activates cell signalling pathways in target cells which lead to induction of NO synthesis and that NO and iron associated with asbestos participate in chemical reactions leading to DNA damage and mutations.
The Specific Aims for the proposed research are: 1) to determine whether asbestos is activating cell signalling pathways which are responsible for the induction of an inducible form of nitric oxide synthase (iNOS) in human cells; 2) to compare the amount of mRNA for iNOS with respect to time after crocidolite treatment of human cells with cytokine or chrysotile treatments; 3) to determine how iron and NO participate to cause oxidative damage to DNA in crocidolite-treated cells; and 4) to determine the effect of iron and/or NO on the mutagenicity of crocidolite in the gpt+ transgenic hprt V79 cell lines, G10 and G12. The effect of crocidolite-treatment of human lung epithelial cells (A549) and mesothelial cells (JMN and Met-5A) on activation of NF-kB by protein kinases will be determined by cotreatment with various protein kinase inhibitors followed by electrophoretic gel mobility shift assays of nuclear extracts. The time course of NF-kB activation, lipid peroxidation, and DNA oxidation will be compared to elucidate which signalling pathway(s) may be involved. The significant differences between the induction of mRNA for iNOS by crocidolite, chrysotile and cytokines will be determined by Northern analysis of RNA. Immunostaining techniques, using antibodies specific for nitrotyrosine, will be used to determine whether peroxynitrite is being formed in these cells. DNA repair enzymes for 8-hydroxydeoxyguanosine and uracil glycosylase will be assayed in treated cells to determine whether NO has inhibited their repair function. Mutagenesis studies in transgenic V79 cell lines sensitive to reactive oxygen species will be done to determine whether both iron and NO are required for mutations induce by crocidolite.
