Peroral administration of 5-bromo-2-deoxyuridine in drinking water is not a reliable method for labeling proliferating S-phase cells in rats

Abstract

In rodents, peroral (p.o.) administration of 5-bromo-2′-deoxyuridine (BrdU) dissolved in drinking water is a widely used method for labeling newly formed cells over a prolonged time-period. Despite the broad applicability of this method, the pharmacokinetics of BrdU in rats or mice after p.o. administration remains unknown. Moreover, the p.o. route of administration may be limited by the relatively low amount of BrdU consumed over 24h and the characteristic drinking pattern of rats, with water intake being observed predominantly during the dark phase. Therefore, we investigated the reliability of staining proliferating S-phase cells with BrdU after p.o. administration (1mg/ml) to rats using both in vitro and in vivo conditions. Flow cytometric analysis of tumor cells co-cultivated with sera from experimental animals exposed to BrdU dissolved in drinking water or 25% orange juice revealed that the concentration of BrdU in the blood sera of rats throughout the day was below the detection limits of our assay. Ingested BrdU was only sufficient to label approximately 4.2±0.3% (water) or 4.2±0.3% (25% juice) of all S-phase cells. Analysis of data from in vivo conditions indicates that only 7.6±3.3% or 15.5±2.3% of all S-phase cells in the dentate gyrus of the hippocampus was labeled in animals administered drinking water containing BrdU during the light and dark phases of the day. In addition, the intensity of BrdU-positive nuclei in animals receiving p.o. administration of BrdU was significantly lower than in control animals intraperitoneally injected with BrdU. Our data indicate that the conventional approach of p.o. administration of BrdU in the drinking water to rats provides strongly inaccurate information about the number of proliferating cells in target tissues. Therefore other administration routes, such as osmotic mini pumps, should be considered for labeling of proliferating cells over a prolonged time-period.