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.

Tissue‐specific metabolic activation and mutagenicity of 3‐nitrobenzanthrone in Muta™Mouse

“…Colonic and intestinal epithelium were stripped from supporting tissue by inverting the tissue in 0.075 M KCl with 20 mM EDTA and repeatedly forcing it in and out of a needleless 5-mL syringe [Newell and Heddle, 2004]. Genomic DNA was isolated as previously described [Vijg and Douglas, 1996;Douglas et al, 1999;White et al, 2003]. Briefly, minced or homogenized tissues were digested overnight in lysis buffer (10 mM Tris pH 7.6, 150 mM NaCl, 10 mM EDTA) with 1% SDS and 1 mg/mL fresh proteinase K at 378C.…”

“…FE1 is a stable epithelial cell line derived from Muta TM Mouse lung [White et al, 2003]. FE1 cells were cultured in a 1:1 mixture of DMEM:F12 supplemented with 2% (v/v) FBS, 2 mM glutamine, 100 U/ mL penicillin G, 100 mg/mL streptomycin sulphate, and 1 ng/mL murine EGF.…”

“…Moreover, this study employed several substrates to examine tissue-specific nitroreductase and acetyltransferase activities in various Muta TM Mouse tissues. Our previous work demonstrated that the FE1 cell line derived from Muta TM Mouse lung epithelium is a useful in vitro tool for environmental mutagenesis research [White et al, 2003], and exposure to pro-mutagens such as benzo [a]pyrene readily generate high MF values (e.g., >800 3 10 25 ) in the absence of exogenous S9. In vitro mutagenicity of 3-NBA in FE1 cells, as well as nitroreductase and acetyltransferase activities, were also examined and compared with that of lung tissue.…”

Linking environmental carcinogen exposure to TP53 mutations in human tumours using the human TP53 knock-in (Hupki) mouse model

“…For such compounds, it could be advantageous to co-incubate cells with hepatic S9 fractions or isolated microsomes, which are enriched in many xenobiotic metabolism enzymes (e.g. CYPs) [100]. Alternatively, Hupki mice could be created (i.e.…”

IL-1 Receptor Regulates microRNA-135b Expression in a Negative Feedback Mechanism during Cigarette Smoke–Induced Inflammation

“…Mouse NIH3T3 cells were obtained from Dr. Mike Wade (Health Canada, Ottawa, ON, Canada). The FE1 cell line is derived from MutaMouse lung epithelial cells (15). mirVana miRNA isolation kits were purchased from Applied Biosystems (Carlsbad, CA).…”