Transport Gene Expression and Drug Accumulation in Milk

Neonates are exposed to a variety of drugs and environmental contaminants via milk. While most drugs gain access to milk by diffusion, we have found that the accumulation of nitrofurantoin and cimetidine is many-fold greater than predicted by diffusion models. These data are consistent with an active transport process. However, the proteins responsible for the active transport of these drugs into milk are not known, since little is known regarding the expression drug transport proteins in lactating mammary epithelial cells. Preliminary studies from our laboratory indicate that members of the Solute Carrier Family 22 (SLC22) and other cation/anion transporter genes are present in mammary tissue (MRP, OATP, OCT-1 and OCT-3). The goal of this research is to systematically evaluate the role of members of the ABCB, ABCC, SLC21 and SLC22 transporter gene families in the accumulation of drugs into human milk. A quantitative RT-PCR method based on real-time fluorescence will test the hypothesis that cation/anion transporter genes which are responsible for drug transport in other tissues, are expressed in mammary epithelial cells. Cell culture studies will test the hypothesis that immortalized human mammary epithelial cells also express these transporter genes and that a subset of these genes are up-regulated by lactogenic hormones (i.e., prolactin, insulin and glucocorticoids). Northern and Western blot analysis will confirm the expression and relative abundance of ABCB, ABCC, SLC21 and SLC22 RNA and protein levels. Functional uptake and efflux studies in these immortalized cells will substantiate the role of these expressed transporters in the specific accumulation of radiolabeled cimetidine and nitrofurantoin, as well as model substrates (e.g., PAH, TEA). Transporter genes found to be highly expressed in lactating mammary cells will be cloned by RT-PCR methods and transfected into cell lines with low background expression for these genes. The uptake and efflux of several model substrates, including cimetidine and nitrofurantoin, will be tested in the transfected cell lines. The identification of the mammary epithelial carrier proteins, their genes, and factors that control their regulation are the ultimate, long-term goals of this research program.