Characterization of a rat lymphocyte culture system for assessing sister chromatid exchange. II. Effects of 5-bromodeoxyuridine concentration, number of white blood cells in the inoculum, and inoculum volume.

Abstract

We have examined the effects of variation in 5-bromodeoxyuridine (BrdU) concentration, the number of white blood cells (WBCs) in the inoculum, and inoculum volume on sister chromatid exchange (SCE) frequency, mitotic activity, and cell cycle kinetics of phytohemagglutinin-stimulated rat lymphocytes in whole blood culture. BrdU (1 to 5 microM) caused a dose-dependent increase in SCEs/metaphase (7.6 to 12.0) and a dose-dependent decrease in mitotic activity (5.3 to 3.1%). With 2 microM BrdU, as the number of cells in the inoculum increased (0.25 x 10(6) to 2.5 x 10(6)), the mitotic activity decreased (7.7 to 1.8%). At all BrdU concentrations examined, changes in the number of WBCs in the inoculum caused significant changes in the SCE frequency. Thus, high concentrations of BrdU do not stabilize the SCE frequency in the rat lymphocyte culture system. However, high BrdU concentrations (greater than or equal to 25 microM) did affect adversely the cells in culture as evidence by increased chromosome breakage, poor chromosome spreading and morphology, and up to an 8-fold reduction in mitotic activity. At BrdU concentrations greater than or equal to 33 microM, the mitotic indices were depressed (2.4 - 0.7%) at all WBC concentrations, and cell cycle delay was evident. Inoculum volumes greater than or equal to 0.4 ml, necessitated by low WBC counts in the animals, resulted in reduced mitotic activity and in some cases culture failure. Thus, we recommend the use of 2 microM BrdU and the addition of 1 x 10(6) WBCs/2 ml culture from animals with WBC counts ranging from 4-7 10(6)/ml to ensure adequate mitotic activity and stable baseline SCE frequencies.