Mutagenesis vol. 1 no. 5 pp. 359-365, 1986
© 1986 UK Environmental Mutagen Society/Oxford University Press
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Establishment of lung fibroblastic cell lines from a non-human primate Tupaia belangeri and their use in a forward gene mutation assay at the hypoxanthing—guanine phosphoribosyl transferase locus
Section of Cell Biology and Cytogenetics, Biological Research Center Japan Tobacco Inc., 23 Nakogi Hatano, Kanagawa 257, Japan
The cells obtained from a lung of a new-born male Tupaia belangeri were maintained in mass culture for >400 days. After 55 population doubling levels (100 days in culture), three cell lines were separately established; these lines showed constant growth properties. One line, designated as T-23, was used for a mutation assay. The T-23 cells showed an absolute plating efficiency of 30–50%, and a population doubling time of 18–19 h in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. The cells had a modal chromosome number of 62 (pseudodiploid) with the loss of a chromosome and the gain of an unidentified one. T-23 cells, like human cells, were much more susceptible to ouabain than mouse cells but relatively less susceptible to 8-azaguanine, while, unlike human cells, they were less sensitive to 6-thioguanine (6TG). N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) was less, but 4-nitroquinoline-l-oxide (4NQO) was more toxic to T-23 cells than to human or mouse cells. Benzo[a]pyrene-induced toxicity was almost comparable among the cell types. For the mutation assay, we chose 6TG-resistance (100 µM) as a marker. The optimal expression time (8–13 days) and cell density at selection to eliminate metabolic cooperation (2 x 104 cells/ 60-mm dish) were determined. Some of the cells selected with 6TG showed <0.4% of the total incorporation of [14C]hypox-anthine into wild-type cells, suggesting the mutants under selection were affected at the hypoxanthine-guanine phos-phoribosyl transferase locus. Following the optimal protocol, we performed a quantitative mutation assay with simple alky-lating agents, MNNG, N-ethyl-N'-nitro-N-nitrosoguanidine, methyl methanesulphonate, ethyl methanesulphonate and 4NQO. All induced mutants in a dose-dependent fashion. The ability to induce mutants in T-23 cells by these mutagens was comparable with that in a conventional V79 assay. This is the first report of a gene mutation assay system using non-human primate cells.