MUTAGENICITY AND SPECIFIC MUTATION SPECTRUM INDUCED BY 8-METHOXYPSORALEN PLUS A LOW-DOSE OF UVA IN THE HPRT GENE IN DIPLOID HUMAN FIBROBLASTS

Exposure of cells to 8-methoxypsoralen plus a low dosage of UVA (365 nm) generates mainly monoadducts (PUVA-I treatment), while further irradiation of PUVA-I treated cells after removal of 8-methoxypsoralen (PUVA-II treatment) converts a high frequency of monoadducts to crosslinks. In this study, a comparison was made of the cytotoxicity and mutagenicity of PUVA-I-treated cells obtained here with those induced by PUVA-II treatment in our previous report. PUVA-I treatment slightly affected the colony-forming ability of cells. However, the 6-thioguanine-resistant cells were markedly increased from 3/10(6) clonable cells in UVA-irradiated populations to 47/10(6) clonable cells in PUVA-I-treated populations. Those results indicated that PUVA-I was more mutagenic than PUVA-II at equal cytotoxic doses, implying that psoralen monoadducts are less cytotoxic and as mutagenic as crosslinks. Mutations in the hypoxanthine (guanine) phosphoribosyltransferase gene of independent PUVA-I mutants were characterized by direct sequencing of cDNA and/or genomic DNA that were amplified by polymerase chain reaction. All the 30 sequenced mutants had single base substitutions. Of those mutations, 21 occurred in the coding region and the others were in the consensus sequences at exon-intron boundaries, thereby resulting in aberrant cDNA. The majority of base substitutions were T to A transversions (23/30); 22 were located at the thymine of 5'TA sites. All of the 24 T . A base pair substitutions (including one T to C) had thymine located on the non-transcribed strand. Five of the six G . C base substitutions were located at the 5' T (G) under bar or 5' C (A) under bar sites on the non-transcribed strand, The frequencies of mutations at 5'TA and 5'TG/5'CA sites were similar in PUVA-I- and PUVA-II-induced mutants. However, the specific kind of T . A base pair substitutions induced by PUVA-I is strikingly different from that induced by PUVA-II. While the transient misalignment-realignment model could account for PUVA-II-induced T . A base substitutions, the low cytotoxic effect and the specific T to A substitutions of PUVA-I treatment might be a result of rapid incorporation of nucleotides after insertion of an adenine or a thymine opposite the psoralen monoadducts on the template by DNA polymerases.