Molecular analysis of styrene oxide-induced hprt mutation in human T-lymphocytes.

Abstract

Styrene-7,8-oxide (SO), the mutagenic in vivo metabolite of the widely used chemical monomer styrene, has been classified as a probable human carcinogen (IARC, 1994). We examined mutations in the hypoxanthine-guanine phosphoribosyl transferase (hprt) gene of primary human T-lymphocytes 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 from two experiments in which the mutation frequencies increased 3.6 and 4.8 times respectively, and 44 control mutations from untreated T-cell cultures. Base substitutions were the dominating type of mutation in both groups, with 35 and 23 independent changes, of which nine and six respectively, have not previously been described in human T-cells. Frameshift mutations (+/-1 bp) and small deletions (2-200 bp) were less frequent and splicing mutations more frequent among the SO-induced than among the control mutations. In SO-treated mutants, base substitutions 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 sites were AT bp. Altogether six independent mutations were found at site 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 splicing regions were identified at 20 sites, eight of which were AT bp. In published data on hprt mutation in untreated T-cells in vivo and in vitro, 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.