Characterization of the physiological roles of Multidrug Resistance Protein (MRP)1-6 by in vivo screening for their substrates

Numerous xenobiotics as well as endobiotic waste products are eliminated from cells by the sequence of oxidation, conjugation to an anionic group (e.g. glutathione, glucuronate or sulphate) and transport across the plasma membrane into the extracellular space. The family of multidrug resistance proteins (MRPs) contains nine members (MRP1-9) belonging to the C-branch of the ABC (ATP-binding Cassette) superfamily of membrane transporters. They have been shown to transport several glutathione, glucuronate and sulphate conjugated compounds in vitro and it has been recently recognized that MRPs play a major role in drug distribution, metabolism and elimination and thus influence the pharmacokinetics/dynamics of many drugs. The main purpose of this research project is to investigate the physiological and pharmacological role of MRP1-6. In the last decade, knockout mouse models have become available for MRP1-6, which have contributed to our knowledge of their physiological function. Most Mrp knockout mouse models, however, do not show an overt phenotype in the protected environment of our experimental animal housing facility. Although we have a fair idea of the main substrates transported by MRP1 and 2 in vivo, it remains unclear what the physiological substrates of MRP3-6 are and, hence, their physiological function remains to be elucidated. Moreover, despite the fact that the absence of MRP6 results in a clear phenotype, the key in vivo substrates of this transporter still need to be identified. Most knowledge about the substrate specificity of the MRPs is derived from in vitro studies. For many of the MRP substrates tested in vitro it is debatable whether the MRPs would encounter these substrates in vivo. Determination of the substrates transported by the MRPs in vivo would, therefore, greatly contribute to our understanding of their physiological function(s). The main goals of the present project are to: 1) set up methods employing mass spectrometry coupled to liquid chromatography to make metabolic fingerprints of sulphated, glucuronidated and glutathione-conjugated compounds in biological matrices of mice. 2) use these metabolomic fingerprints of biological matrices of wild type and Mrp knockout mice to identify compounds that are differentially present in wild type and Mrp knockout mice, thereby identifying the substrates transported by the Mrps in vivo. We have set up a preliminary screen for glucuronidated compounds and used this screen on plasma samples of wild type and Mrp3(-/-) mice to determine the relative abundance of several glucuronides in plasma. This resulted in the identification of a new group of Mrp3 substrates: the glucuronate conjugates of phytoestrogens, compounds that have attracted much attention lately because several of these compounds have been suggested to have health beneficial effects. This preliminary glucuronide screen provides a strong proof of principle for the proposed approach.