Characterization and inhibition by a wide range of xenobiotics of organic anion excretion by primary human hepatocytes

Organic anion secretion by human hepatocytes was characterized using primar y liver parenchymal cell cultures and the anionic fluorescent dye carboxy-2 ',7'-dichrorofluorescein (CF). Probenecid, a well-known common blocker of t he membrane transport process for anions, was shown to increase CF accumula tion in primary human hepatocytes by inhibiting cellular CF efflux in a dos e-dependent manner, thereby establishing the presence of an efflux system f or organic anions in cultured hepatocytes. Outwardly directed transport of CF from hepatocytes was found to be temperature-dependent; it was not alter ed by changes in the ionic composition of the incubation medium used in eff lux experiments. In addition to probenecid, Various structurally and functi onally unrelated xenobiotics such as glibenclamide, rifampicin, vinblastine , MK-571, indomethacin, and cyclosporin A were shown to inhibit secretion o f CF by primary human hepatocytes, thus suggesting that organic anion excre tion by human liver may be impaired by various drugs. Northern blot and Wes tern blot analyses of the expression of multidrug resistance proteins (MRP) , such as MRP1 and MRP2, which are known to mediate cellular outwardly dire cted transport of organic anions indicated that MRP2 was present at substan tial levels in cultured human hepatocytes as well as in their in vivo count erparts, whereas MRP1 expression was only barely detectable. These results therefore suggest that MRP2, unlike MRP1, may contribute to the organic ani on efflux system displayed by primary human hepatocytes and inhibited by a wide range of xenobiotics. BIOCHEM PHARMACOL 60;12:1967-1975, 2000. (C) 200 0 Elsevier Science Inc.