MOLECULAR ANALYSIS OF STYRENE OXIDE-INDUCED HPRT MUTATION IN HUMAN T-LYMPHOCYTES

Styrene-7,8-oxide (SO), the mutagenic in vivo metabolite of the widely used chemical monomer styrene, has been classified as a probable huma n carcinogen (IARC, 1994), We examined mutations in the hypoxanthine-g uanine phosphoribosyl transferase (hprt) gene of primary human T-lymph ocytes exposed to 0.2 mM SO for 6 days in vitro. PCR amplification and direct DNA sequencing were used to identify 55 SO-induced mutations f rom two experiments in which the mutation frequencies increased 3.6 an d 4.8 times respectively, and 44 control mutations from untreated T-ce ll cultures, Base substitutions were the dominating type of mutation i n both groups, with 35 and 23 independent changes, of which nine and s ix respectively, have not previously been described in human T-cells. Frameshift mutations (+/- 1 bp) and small deletions (2-200 bp) were le ss frequent and splicing mutations more frequent among the SO-induced than among the control mutations, In SO-treated mutants, base substitu tions in the coding region occurred at 15 sites, nine of which were AT bp, and in the splice donor and acceptor regions six of 10 mutated si tes were AT bp, Altogether six independent mutations were found at sit e 539 in cells from the two SO experiments (four GC>AT and two GC>TA), In the control cultures, base substitutions in the coding and splicin g regions were identified at 20 sites, eight of which were AT bp, In p ublished data on hprt mutation in untreated T-cells in vivo and in vit ro, 31 of 88 base substitutions have been reported to occur at AT bp. These results indicate that SO-induced mutations at the hprt locus in human T-lymphocytes are predominantly base substitutions, and suggest that in addition to DNA adducts at guanine bases, adducts at A and/or T bases also deserve attention with regard to the mutagenesis of SO.