Participation of Iron and Nitric Oxide in the Mutagenicity of Asbestos in hgprt^-, gpt⁺ Chinese Hamster V79 Cells¹

Crocidolite asbestos is known to cause cellular damage, leading to asbestosis, bronchogenic carcinoma, and mesothelioma in humans. The mechanism responsible for the carcinogenicity of asbestos is not known. Iron associated with asbestos is thought to play a role by catalyzing the formation of reactive oxygen species, which may cause DNA damage, leading to mutations and cancer. Here, we examined whether asbestos can induce mutations in Chinese hamster hgprt⁺ V79 cells or transgenic hgprt^-, gpt⁺ V79 cells (G12). Treatment with 6 µg/cm² crocidolite for 24 h caused a 2-fold increase in the mutation frequency at the gpt locus of G12 cells, but no increase at the hgprt locus of V79 cells. The mutation frequency at the gpt locus of G12 cells increased with increasing treatment dose of crocidolite. The mutations induced by crocidolite appeared to be due to the generation of reactive oxygen species catalyzed by iron associated with the fibers, because treatment of G12 cells in iron-free medium with fibers from which redox active iron had been removed with desferrioxamine B prevented all of the gpt^- mutations above untreated control levels. In addition, treatment of cells with a soluble form of iron, 1.5 mm ferric ammonium citrate, resulted in an increase in mutation frequency at the gpt locus of ∼1.5 fold above that of untreated G12 cells with no increase in mutations at the hgprt locus of V79 cells with ferric ammonium citrate. We also investigated the effect of nitric oxide on the mutagenicity of crocidolite in G12 cells. When G12 cells were treated with 3 µg/cm² of crocidolite in the presence of nitric oxide-generating compound, 200 µm diethyltriamine/NO, the mutation frequency increased to a level that was more than additive for crocidolite or diethyltriamine/NO treatment alone. These results strongly suggest that the presence of iron and nitric oxide may either lead to the generation of another reactive, mutagenic species, such as peroxynitrite, or that nitric oxide inhibits a DNA repair enzyme(s), leading to an increase in mutations.