The long term objective of this research is to investigate the role of 02 radicals and iron in asbestos-induced cancer. Asbestos catalyzes many of the same reactions that iron does and the most carcinogenic forms can contain as much as 36% iron. This proposal is based upon the hypothesis that iron is responsible for the generation of 02 radicals that damage DNA resulting in the biological effects of asbestos. Iron-catalyzed reactions may generate H202 to produce .OH via the Fenton reaction within the target cell, and extracellular H202, released by activated macrophages, may increase concentrations in the target cell to stimulate the Fenton reaction and formation of .OH.
The specific aims are: 1) determine the effect of H202 addition on the quantity of crocidolite-induced DNA damage in vitro; 2) determine the effect of addition of extracellular H202 on the amount of DNA damage, cytotoxicity and transformation in crocidolite-treated SHE cells; 3) determine the effect of buthionine sulfoximine, a chemical that inhibits cellular destruction of H202, on the amount of DNA damage, cytotoxicity, and transformation in crocidolite-treated SHE cells with or without the addition of extracellular H202; 4) determine if iron-depletion of crocidolite affects the amount of .OH and DNA base damage in SHE cells with or without the addition of extracellular H202; and 5) determine the effect of radical scavengers on the amount of DNA damage, cytotoxicity, and transformation in crocidolite-treated SHE cells with or without the addition of extracellular H202. Two types of DNA damage will be studied, DNA single strand breaks (using phi X174 RFI DNA in vitro or in situ nick translation in cells) and DNA base damage (GC/MS-SIM). The role of .OH will be investigated by using specific radical scavengers (salicylate, DMSO, dimethylthiourea). The formation of .OH in cell cultures will be quantified using salicylate. Buthionine sulfoximine will be used to determine the effects of increased intracellular levels of H202.
