Human lymphocytes resistant to 6-thioguanine: restrictions in the use of a test for somatic mutations arising in vivo studied by flow-cytometric enrichment of resistant cell nuclei.

A method is presented that facilitates the enumeration of 6-thioguanine(TG)-resistant human lymphocytes from peripheral blood. By the use of flow cytometry, nuclei from cells stimulated to divide in vitro and having reached their late S and G2 phases of growth, were sorted out from cultures incubated for 48 h, the last 6 h in medium containing [3H]thymidine. The out-sorted nuclei were subjected to autoradiography, and the labelling index was determined. The frequency of TG-resistant cells was calculated from the quotient (labelling index in the presence of TG/labelling index in the absence of TG). Over a period of several months, two blood donors serving as internal standards showed a variation of 20% in their frequencies of resistant lymphocytes. In a referent group of 48 blood donors, inter-individual differences in the frequency of resistant cells (range 10(-5)-10(-4) ) were established. There was a positive correlation between an increased frequency of resistant cells and the age of the blood donors. Patients treated with cytostatics or psoralen and ultraviolet radiation (PUVA) showed elevated frequencies of resistant cells (about 10(-3)-10(-2) ). We suggest that at least a part of the resistant cells scored are phenocopies and thus have not originated from somatic mutation. The high number of phenocopies after treatments with cytostatics or PUVA may depend on a temporary depletion from the cells of functioning HGPRT enzyme owing to a transcriptional block caused by the agents combined with a rather fast turnover of the enzyme. We conclude that there are restrictions in the use of this system for determining genotoxic effects in vivo.