Published in Cancer Detection and Prevention 2000; 24(Supplement 1).

Interlocus TCR V-gamma and TCR J-beta 1 V-J recombination measures of genomic instablity in human lymphocytes treated with eugenol and carbamide peroxide

FG Velasco DDS 1, P Peitl Jr PhD 2, ET Sakamoto-Hojo PhD 2, GAS Passos PhD 1

1 Molecular Immunogenetics Group,, 2 Laboratory of Cytogenetics and Mutagenesis,, 3 Section of Genetics, DMEF-FORP Faculty of Medicine, University of São Paulo, Ribeirao Preto, Brasil,

The V(D)J recombination of T-cell receptors (TCR alpha/beta and gamma/delta) gene segments is a site specific intralocus mechanism promoting diversity in the immune system. However, illegitimate interlocus recombination generates hybrid genes between different TCR genes. The measurement of TCR hybrid gene is a model to study the mechanism of chromosomal abnormalities and monitoring the genotoxic potential of substances. AIMS: To evaluate the occurrence of illegitimate recombination on human chromosome 7 (TCR V gamma on 7p14-15 and TCR J beta 1 on 7q35) in normal lymphocytes treated in vitro with eugenol or carbamide peroxide. METHODS: DNA from lymphocyte cultures treated with the chemicals or without treatment (negative control) and from the fibroblast cell line AT-5BIVA derived from an ataxia telangiectasia patient (used as a positive control) were prepared and used in PCR with primers Vg - Jb. For a second nested PCR we used a internal primer set Vgb. The products of the second nested-PCR were dot-blotted to nylon N+ membrane and probed with an 33-P-labeled oligonucleotide internal to the TCR Vg-Jb hybrid gene. RESULTS: The presence of hybrid antigen-receptor genes was demonstrated in lymphocytes cultured in the presence of chemical compounds during a period when a high percentage of cells were undergoing the S-phase of the cell cycle. The hybrid genes could be found at higher frequency in DNA obtained from experimental cultures and from the AT-5BIVA cell line compared to negative control. CONCLUSIONS: We demonstrate an increased frequency of hybrid antigen-receptor genes in lymphocytes cultured in the presence of eugenol and carbamide peroxide, two compounds widely used in dental practice. Although the illegitimate recombination generating the TCR V gamma-J beta hybrid gene has no defined transforming activity and thus likely to result in an "innocent" translocation, it is a powerful method to measure induced genomic instability in human cells, since the essential mechanisms for interlocus rearrangements we measured and those generating "malignant" translocations appear to be similar.

KEY WORDS: Genotoxicity, DNA-damage, chromosomal aberrations, mutagens, carcinogens.

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Paper presented at the International Symposium on Impact of Biotechnology on Cancer Diagnostic & Prognostic Indicators; Geneva, Switzerland; October 28 - 31, 2000; in the section on genetic instability.