In vitro inhibition of OATP-mediated uptake of phalloidin using bile acid derivatives
Introduction
The bicyclic heptapeptide phalloidin is one of the major phallotoxins present in Amanita phalloides (Wieland and Wieland, 1959) that causes death in 20% of intoxicated adults and 50% in the case of children (Jander and Bischoff, 2000, Jander et al., 2000). The toxicity of this mushroom is mainly due to amatoxins, which may lead to death within several days after mushroom ingestion. Other toxins contained in this mushroom, such as virotoxins, phallolysin and phallotoxins, are believed to play a lower role in fatal intoxications after its ingestion (Wieland, 1983).
In the case of phalloidin, this is probably due to the poor intestinal absorption of this compound, which occurs by mechanisms different to those mediating bile acid uptake (Petzinger et al., 1982). However, once phalloidin reaches the liver, it causes a severe damage, which is probably due to the hepatic accumulation brought about by the high efficiency of hepatocytes to carry out phalloidin uptake (Petzinger and Frimmer, 1982, Petzinger and Frimmer, 1984). Indeed, phalloidin-induced liver toxicity is reduced when the excretory ability of the liver is decreased, as happens during liver regeneration following partial hepatectomy (Frimmer and Schischke, 1972). Moreover, for some compounds assayed to prevent liver damage that have been found with a moderate antitoxic effect this was produced by a mechanism unrelated to a stimulation of liver microsomal drug-metabolizing systems (Zanoli, 1979). This has suggested that as part of a detoxification approach, prevention of the liver uptake of these toxins with competitive and non-competitive inhibitors may be a useful pharmacological strategy. These studies have been carried out when the exact identity of the transporters involved in phalloidin uptake was not known (Münter et al., 1986). A similar strategy using rifampicin and other compounds has been suggested as a potential partial antidote in the treatment of intoxication by amatoxins due to the ability of rifampicin to inhibit carrier-mediated uptake of these toxins (Letschert et al., 2006).
Currently, it has been elucidated that the carriers involved in phalloidin uptake by hepatocytes are members of the family of organic anion-transporting polypeptides (OATPs). These, together with sodium-dependent transporters NTCP and ASBT, play a key role in the uptake of bile acids and other organic anions by hepatocyes and ileum epithelial cells. For comparative purposes the values of KM for two typical cholephilic organic anions, i.e., taurocholic acid (TCA) and estradiol 17β-D-glucuronide (E217βG) are shown in Table 1. Human OATP1B3 (also named OATP8 in the old nomenclature) and rat Oatp1a1 (Oatp1) and Oatp1a4 (Oatp2) probably play a minor role in phalloidin uptake, whereas human OATP1B1 (OATP-C) and rat Oatp1b2 (Oatp4) have been reported to be the main route for hepatocyte phalloidin uptake (Fehrenbach et al., 2003, Meier-Abt et al., 2004, Lu et al., 2008). This is consistent with previous observations that the presence in the extracellular medium of typical substrates of these transporters, such as bile acids, reduces phalloidin uptake by hepatocytes (Frimmer et al., 1977, Petzinger, 1981). Regarding the export of phalloidin across the canalicular membrane, in vitro (Fehrenbach et al., 2003, Meier-Abt et al., 2004) and in vivo (Gavrilova et al., 2007) evidence suggest that the isoform 2 of the multidrug resistance associated proteins (MRP2) might be involved in the biliary excretion of this toxin.
The aim of the present study was to investigate the ability of several bile acid derivatives to prevent OATP-mediated toxin uptake by the liver without affecting the uptake of natural bile acids by the major route under physiological circumstances, i.e., the Na+-taurocholate cotransporting polypeptide (NTCP), and hence not interfering with bile acid secretion. From preliminary screening using a large series of bile acid derivatives previously synthesized by our group and Dr. Vazquez-Tato's group (Marin et al., 2005, Soto Tellini et al., 2006, Alvarez Alcalde et al., 2008) four compounds named BALU-1, BALU-2, BALU-3 and BALU-4 (Fig. 1) were selected to enter this study based on their ability to inhibit OATP-mediated transport and on their varied structural characteristics, which include mono- and di-anionic compounds and compounds without or with a bulky group bound to either the C3 position or the side chain. Phalloidin was used here as an important and prototypic toxin taken up by human hepatocytes via OATP1B1, whose transport however occurs through mechanisms different from those mediating sodium-dependent bile acid uptake (Petzinger et al., 1982).
Section snippets
Chemicals
Phalloidin, phalloidin labeled with fluorescein isothiocyanate (phalloidin-FITC), and TCA were purchased from Sigma-Aldrich (Madrid, Spain). As indicated by the supplier, the purity of these compounds was more than 98%, as determined by thin-layer chromatography. All other reagents were of analytical grade. The molecular structures of compounds assayed in the present study are shown in Fig. 1. They were succinyl amide of the 3β-amino derivative of cholic acid (Na2-SuC, named here BALU-1),
Effect of BALUs on transporters expressed in X. laevis oocytes
As a first step to screen the potential usefulness of BALUs we determined the effect of these four compounds on bile acid transport by the major carrier proteins involved in the uptake of organic anions, including bile acids, by the liver from sinusoidal blood across the basolateral plasma membrane. Thus, we measured the uptake of TCA and E217βG (10 μM) by human and rat OATPs in the presence or the absence of 50 μM of one of the potential inhibitors.
All BALUs induced a dramatic inhibition in
Discussion
Human OATP1B1 and OATP1B3, and rat Oatp1b2 have been suggested to transport phalloidin from blood into hepatocytes with KM values below 40 μM (Meier-Abt et al., 2004, Fehrenbach et al., 2003). In the present study, using a concentration slightly higher than this value (50 μM), we found that all compounds assayed (BALU-1, BALU-2, BALU-3 and BALU-4) were able to dramatically inhibit the transport of organic anions mediated by OATP1B1, which suggested that these four compounds might also inhibit
Acknowledgments
The authors thank Dr. P. Meier, Dr. B. Steiger and Dr. B. Hagenbuch (Department of Clinical Pharmacology, Zurich University Hospital, Switzerland) for the generous supply of recombinant plasmids and OATP1B1-transfected cells. We also thank Dr. V.H. Soto and Dr. M. Alvarez for the synthesis and purification of compounds BALU-1, BALU-2, BALU-3 and BALU-4. Thanks are also due to L. Muñoz, J.F. Martin, J. Villoria, N. Gonzalez and E. Vallejo for the care of the animals. Secretarial help by M.
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