There are about 70,000 synthetic chemicals in current commercial use, and about 1,000 new products are added to this repertoire each year. Since many of these compounds are potentially mutagenic and/or carcinogenic, the need for more effective mutagen screening is imperative. This need has recently been emphasized in a report from the National Academy of Sciences. In this application, we propose to develop a novel mammalian cell assay for mutagens and potential carcinogens that will precisely identify the predominant type of mutations(s) each such compound induces in the DNA of mammalian cells. Our test utilizes the selectable genetic marker adenine phosphoribosyltransferase (APRT), which we have extensively characterized on the DNA, protein and cellular phenotype levels. We will engineer specific mutations (transitions, transversions or point insertions) at sites in a mouse APRT gene such that the gene cannot be expressed, and will introduce these mutant genes into non-reverting, APRT deficient human cell Recipients. These hybrid constructs will represent our basic test medium for detection of mutagenic activity. As a model system, the tester cells will be treated with mutagens known to preferentially induce specific DNA base-pair transitions in prokaryotes and possibly in mammalian cells. Reversion within the appropriate tester cell culture will then confirm or refute the mode of action of these mutagens. As an additiona approach for the identification of mutagens that produce frameshifts, we have deduced, from our knowledge of the nucleotide sequence of the mouse APRT gene, the amino acid sequences of major frameshift peptides. These frameshifts peptides will be synthesized and individually used to elicit antisera. Mutant colonies, arising as a consequence of frameshift mutation, will be identified in situ by virtue of their binding one of the specific antisera which will be coupled to a color-development assay. Once our tester lines have been constructed and our reagents have been produced, we expect to be able to rapidly establish the exact nature of mutation that any mutagen produces in animal cells.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA036897-02
Application #
3174521
Study Section
Chemical Pathology Study Section (CPA)
Project Start
1984-06-01
Project End
1987-05-31
Budget Start
1985-06-01
Budget End
1986-05-31
Support Year
2
Fiscal Year
1985
Total Cost
Indirect Cost
Name
University of Cincinnati
Department
Type
Schools of Medicine
DUNS #
City
Cincinnati
State
OH
Country
United States
Zip Code
45221
Zhu, Y; Bye, S; Stambrook, P J et al. (1995) Aflatoxin B1, 2-aminoanthracene, and 7,12-dimethylbenz[a]anthracene-induced frameshift mutations in human APRT. Environ Mol Mutagen 26:234-9
Zhu, Y; Bye, S; Stambrook, P J et al. (1994) Single-base deletion induced by benzo[a]pyrene diol epoxide at the adenine phosphoribosyltransferase locus in human fibrosarcoma cell lines. Mutat Res 321:73-9
Zhu, Y; Stambrook, P J; Tischfield, J A (1993) Loss of heterozygosity: the most frequent cause of recessive phenotype expression at the heterozygous human adenine phosphoribosyltransferase locus. Mol Carcinog 8:138-44
Chen, J; Sahota, A; Martin, G F et al. (1993) Analysis of germline and in vivo somatic mutations in the human adenine phosphoribosyltransferase gene: mutational hot spots at the intron 4 splice donor site and at codon 87. Mutat Res 287:217-25
Liu, H S; Scrable, H; Villaret, D B et al. (1992) Control of Ha-ras-mediated mammalian cell transformation by Escherichia coli regulatory elements. Cancer Res 52:983-9
Bertino, A M; Tischfield, J A; Stambrook, P J (1992) Reconstitution of an episomal mouse aprt gene as a consequence of recombination. Mol Gen Genet 232:24-32
Chen, J; Sahota, A; Laxdal, T et al. (1991) Identification of a single missense mutation in the adenine phosphoribosyltransferase (APRT) gene from five Icelandic patients and a British patient. Am J Hum Genet 49:1306-11
Sahota, A; Chen, J; Stambrook, P J et al. (1991) Mutational basis of adenine phosphoribosyltransferase deficiency. Adv Exp Med Biol 309B:73-6
Chen, J; Sahota, A; Stambrook, P J et al. (1991) Polymerase chain reaction amplification and sequence analysis of human mutant adenine phosphoribosyltransferase genes: the nature and frequency of errors caused by Taq DNA polymerase. Mutat Res 249:169-76
Sahota, A; Chen, J; Behzadian, M A et al. (1991) 2,8-Dihydroxyadenine lithiasis in a Japanese patient heterozygous at the adenine phosphoribosyltransferase locus. Am J Hum Genet 48:983-9

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