Spontaneous and chemically induced point mutations in HPRT cDNA of the metabolically competent human lymphoblastoid cell line, MCL-5

Abstract

Thioguanine-resistant clones of the human lymphoblastoid cell, MCL-5, which carries two recombinant plasmids expressing xenobiotic metabolizing enzymes, were obtained spontaneously and after treatment with 0.1 μg/ml benzo[ a]pyrene (BaP), 1.0 μg/ml 4-( N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK), and 10 μg/ml cigarette smoke condensate (CSC). Treatment with the chemicals reduced the cloning efficiency (CE) of MCL-5 cells from about 30% in untreated cultures to about 10% after treatment with NNK and to about 1% or to less than 1% after treatment with CSC or BaP, respectively. At the same time, the mutant frequencies were increased about sevenfold above those of untreated cultures. Among a total of 138 independent mutant clones that had resulted from 55 separate cultures, 60 point mutations were identified within the hypoxanthine guanine phosphoribosyltransferase ( HPRT) reading frame by sequencing full-size reverse transcription polymerase chain reaction (RT-PCR) products from thioguanine-resistant clones. The identified 53 coding errors were distributed among 33 locations and types. Among the 30 types of single basepair substitutions leading to coding errors, 12 had not been described before. In the present set of point mutations, the distribution of base substitution types as well as of mutated sites appeared to be influenced by the treatment with the chemicals. Thus, the ratio of G to T transversions increased from 3 among 19 spontaneous point mutations in the HPRT coding region to 9 among 21 BaP-induced point mutations. The G119T and G208T transversions were found three times each, exclusively after treatment with BaP, while the accumulation of two to eight incidences of the G97T, CG142/3TA, C508T, T583A and G599A mutations was split among different treatments. All eight identified point mutations identified after NNK treatment were at G or T residues on either strand that were followed by additional G or T residues.