Bromodeoxyuridine labelling as an alternative method to identify 6-thioguanine-resistant mutant lymphocytes in humans

Abstract

6-Thioguanine-resistant (TG R) mutant lymphocytes in human blood are usually enumerated by the cloning assay which allows the molecular characterisation of the HPRT mutations to be detected. A “short-term” alternative approach is provided by the anti-bromodeoxyuridine (anti-BrdU) technique in which TG R lymphocytes are identified immunocytochemically by their ability to synthesise DNA in the presence of 6-thioguanine (TG). We have evaluated the influence of various experimental factors that could affect the frequency of TG R lymphocytes. A standard protocol is proposed, based on 24-h cold storage of isolated lymphocytes at 4°C and 40-h culture with and without TG, the last 16 h with BrdU. The harvested cells are treated with hypotonic (0.075 M) KCl, fixed with methanol:acetic acid (3:1) and put on microscopic slides. For the TG cultures, all cells are prepared on the slides, while slides from the control cultures are made by a 1/50 dilution. DNA is denatured by formamide, and the BrdU label is identified by anti-BrdU antibody detected by immunoperoxidase staining using a peroxidase-conjugated secondary antibody with diaminobenzidine as substrate. In 10 donors, the frequency of TG R lymphocytes (variant frequency, V f) detected by this protocol ranged from 69.65×10 −6 to 83.45×10 −6, and split measurements showed a relatively small intra-assay variation in V f values of each donor. BrdU in DNA was also detected by immunofluorescence using a fluorescein-conjugated anti-BrdU monoclonal antibody. This method, facilitating easy identification of positive cells and rapid microscopic scoring, may serve as a basis for an automated analysis of TG R lymphocytes. V f values detected by the anti-BrdU assay are higher than mutant frequencies obtained by the cloning assay, which has been assigned to the presence of non-mutant phenocopies considered to represent spontaneously cycling lymphocytes. Although the anti-BrdU assay is rapid and easy and has been shown to respond to genotoxic exposures, its true value could be evaluated only when it can be ascertained that phenocopies do not significantly contribute to the V f values obtained.