MOLECULAR-BASES OF HPRT MUTATIONS IN MALATHION-TREATED HUMAN T-LYMPHOCYTES

Recently, we reported that 6 of 84 (7.1%) hprt mutants arising in in v itro malathion-treated human T-lymphocytes were characterized by speci fic genomic deletions in a 125-bp region of exon 3 (Pluth et al., Canc er Research 56 (1996) 2393-2399. We have now extended study to determi ne whether additional differences in molecular spectrum at a basepair level exist between control and malathion-treated mutations, and inves tigated whether there is evidence to support the hypothesis that malat hion is an alkylating agent. We analyzed 101 hprt mutants (24 from con trol and 77 from treated cultures) isolated from six in vitro malathio n exposures of T-lymphocytes from four healthy male donors. Analysis c onsisted of: Southern blotting, genomic multiplex PCR, genomic DNA seq uencing and reverse transcription PCR amplification (RT/PCR) and seque ncing of the cDNA product. Mutations at several basepair sites were fr equent after malathion exposure and were isolated from treated cells f rom at least two different individuals. Using a human hprt mutation da tabase for comparison, the frequency of mutations at one of these site s (basepair 134) was found to be significantly elevated in the malathi on-treated cells(p < 0.0005). Hprt mutations in malathion-treated cell s arose preferentially at G:C basepairs, which is consistent with earl ier reports that malathion alkylates guanine nucleotides. Assessing mo lecular changes at both genomic and cDNA levels in the same mutants re vealed that many small, partial exon deletions (< 20 bp) in genomic DN A were often represented in the cDNA as the loss of one or more exons. In addition, it was noted that identical genomic mutations can result in different cDNA products in different T-cell isolates. These observ ations affirm the importance of genomic sequence analysis in combinati on with RT/PCR for a more accurate definition of the mutation spectrum . (C) 1998 Elsevier Science B.V.