Development and characterization of a stable epithelial cell line from Muta Mouse lung.

Abstract

We have isolated and characterized a stable epithelial cell line from Muta Mouse lung that is a suitable complement to the in vivo assay system. The cells are contact inhibited, forming a flat monolayer, and retain several epithelial/pulmonary characteristics. The genome is stable across more than 50 generations, with a modal chromosome number of 78. Spontaneous rates of micronuclei (19.2 +/- 1.4 per 1,000), sister chromatid exchanges (0.25 +/- 0.004 per chromosome), and chromosome aberrations ( approximately 4%) are lower than, or comparable to, other transgenic cell lines currently used in mutagenicity research. Fluorescence in situ hybridization analyses showed that 80% of cells contain three lambdagt10lacZ loci. Slot-blot analyses indicated that the average cell contains approximately 17 transgene monomers. Spontaneous mutant frequency at the lacZ transgene is stable (39.8 +/- 1.1 x 10(-5)), and the direct-acting mutagens N-ethyl-N-nitrosourea and ICR-191 yielded increases in mutant frequency of 6.3- and 3.2-fold above control, respectively. Benzo[a]pyrene (BaP) exposure increased mutant frequency more than 25-fold above control and did not require an exogenous metabolic activation mixture. Inhibition of Cyp1A1 by 5 microM alpha-naphthoflavone eliminated BaP mutagenesis. Activation and mutation induction by the heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine required a low concentration (0.05% v/v) of exogenous rat liver S9. High activity of alpha, micro, and pi glutathione-S-transferase isozymes appears to confer resistance to the cytotoxic effects of xenobiotics. The cell line is a suitable complement to the in vivo Muta Mouse assay, and provides an opportunity for routine in vitro mutagenicity testing using an endpoint that is identical to that employed in vivo.