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 μM) 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 μM BrdU, as the number of cells in the inoculum increased (0.25 × 10⁶ to 2.5 × 10⁶), 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 (⩾ 25 μM) did affect adversely the cells in culture as evidenced by increased chromosome breakage, poor chromosome spreading and morphology, and up to an 8‐fold reduction in mitotic activity. At BrdU concentrations ⩾ 33μM, the mitotic indices were depressed (2.4‐0.7%) at all WBC concentrations, and cell cycle delay was evident. Inoculum volumes ⩾ 0.4 ml, necessitated by low WBC counts in the animal, resulted in reduced mitotic activity and in some cases culture failure. Thus, we recommend the use of 2 μM BrdU and the addition of 1 × 10⁶ WBCs/2 ml culture from animals with WBC counts ranging from 4–7 × 10⁶/ml to ensure adequate mitotic activity and stable baseline SCE frequencies.