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 vitro malathion-treated human T-lymphocytes were characterized by specific genomic deletions in a 125-bp region of exon 3 (Pluth et al., Cancer Research 56 (1996) 2393-2399. We have now extended study to determine whether additional differences in molecular spectrum at a basepair level exist between control and malathion-treated mutations, and investigated whether there is evidence to support the hypothesis that malathion is an alkylating agent. We analyzed 101 hprt mutants (24 from control and 77 from treated cultures) isolated form six in vitro malathion exposures of T-lymphocytes from four healthy male donors. Analysis consisted of: Southern blotting, genomic multiplex PCR, genomic DNA sequencing and reverse transcription of PCR amplification (RT/PCR) and sequencing of the cDNA product. Mutations at several basepair sites were frequent after malathion exposure and were isolated from treated cells from at least two different individuals. Using a human hprt mutation database for comparison, the frequency of mutations at one of these sites (basepair 134) was found to be significantly elevated in the malathion-treated cell (p < 0.0005). Hprt mutations in malathion-treated cells arose preferentially at G:C basepairs, which is consistent with earlier reports that malathion alkylates guanine nucleotides. Assessing molecular changes at both genomic and cDNA levels in the same mutants revealed that many small, partial exon deletions (< 20 bp) in genomic DNA were often represented in the cDNA at 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 observations affirm the importance of genomic sequence analysis in combination with RT/PCR for a more accurate definition of the mutation spectrum.