Protective effect of bile acid derivatives in phalloidin-induced rat liver toxicity

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Abstract

Phalloidin causes severe liver damage characterized by marked cholestasis, which is due in part to irreversible polymerization of actin filaments. Liver uptake of this toxin through the transporter OATP1B1 is inhibited by the bile acid derivative BALU-1, which does not inhibit the sodium-dependent bile acid transporter NTCP. The aim of the present study was to investigate whether BALU-1 prevents liver uptake of phalloidin without impairing endogenous bile acid handling and hence may have protective effects against the hepatotoxicity induced by this toxin. In anaesthetized rats, i.v. administration of BALU-1 increased bile flow more than taurocholic acid (TCA). Phalloidin administration decreased basal (− 60%) and TCA-stimulated bile flow (− 55%) without impairing bile acid output. Phalloidin-induced cholestasis was accompanied by liver necrosis, nephrotoxicity and haematuria. In BALU-1-treated animals, phalloidin-induced cholestasis was partially prevented. Moreover haematuria was not observed, which was consistent with histological evidences of BALU-1-prevented injury of liver and kidney tissue. HPLC-MS/MS analysis revealed that BALU-1 was secreted in bile mainly in non-conjugated form, although a small proportion (< 5%) of tauro-BALU-1 was detected. BALU-1 did not inhibit the biliary secretion of endogenous bile acids. When highly choleretic bile acids, – ursodeoxycholic (UDCA) and dehydrocholic acid (DHCA) – were administered, they were found less efficient than BALU-1 in preventing phalloidin-induced cholestasis. Biliary phalloidin elimination was low but it was increased by BALU-1 > TCA > DHCA > UDCA. In conclusion, BALU-1 is able to protect against phalloidin-induced hepatotoxicity, probably due to an inhibition of the liver uptake and an enhanced biliary secretion of this toxin.

Introduction

The accidental ingestion of poisonous mushrooms is the cause of frequent serious life threatening conditions in the emergency rooms of many hospitals every year. Consumption of Amanita phalloides is a common cause of these problems. Toxins contained in this mushroom produce a biphasic illness with early gastrointestinal symptoms and subsequent hepatic injury (Cappell and Hassan, 1992). However, the kidney can be also affected (Beaudreuil et al., 1998).

One of the toxins of this and other species of poisonous mushrooms is the bicyclic heptapeptide phalloidin, which is taken up by hepatocytes via OATP1B1 (previously named OATP-C, gene symbol SLCO1B1) (Fehrenbach et al., 2003, Meier-Abt et al., 2004). The accumulation of phalloidin in hepatocytes is efficient (Petzinger and Frimmer, 1982, Petzinger and Frimmer, 1984) and results in serious damage of this organ characterized by marked cholestasis (Dubin et al., 1978).

This effect has been associated with the ability of phalloidin to induce irreversible polymerization of actin filaments, resulting in a rapid accumulation of polymerized microfilaments, which mainly occurs in the cytoplasmic region close to the canalicular plasma membrane (Gabbiani et al., 1975, Cooper, 1987). The functional consequences of this alteration include reduction in canalicular contractility, loss of integrity in tight junctions, impaired vesicular trafficking and increased paracellular permeability, all leading to a reduction in bile flow (Dumont et al., 1994, Elias et al., 1980, Watanabe et al., 1983), the accumulation of biliary components, and eventually to necrosis of liver cells (Mengs and Trost, 1981). Thus, a common finding after intoxication by phalloidin is an elevation of serum biochemical markers of cholestasis and liver damage, such as alkaline phosphatase, transaminases and bilirubin (Ishizaki et al., 1997).

In spite of the evident interest of preventing the deleterious effect of this toxin, there is no efficient antidote against intoxication by phalloidin. Available treatments include plasmapheresis or a reduction of the phalloidin-induced toxic effects by penicillin or silibinin (Jander and Bischoff, 2000, Jander et al., 2000). However, in many cases liver transplant is the only effective therapy. As an alternative approach, the inhibition of liver uptake followed by stimulated urinary elimination by forced diuresis has been suggested.

In this respect, inhibition of phalloidin uptake by hepatocytes using bile acid derivatives, such as BALU-1 (succinyl amide of the 3β-amino derivative of cholic acid) – able to markedly inhibit OATP1B1 without affecting the main route of endogenous bile acid uptake, i.e., the sodium–taurocholate cotransporting polypeptide (NTCP) –, has been suggested as a potentially useful pharmacological strategy. These OATP1B1 inhibitors might be included as components of antidotes to be used in cases of accidental consumption of poisonous mushrooms, which must also include agents against the toxicity of other mushroom toxins, mainly amanitins. Thus, the present study was undertaken to evaluate the “in vivo” ability of BALU-1 to prevent liver uptake of phalloidin and to evaluate the hepatoprotective effect of this bile acid derivative.

Section snippets

Chemicals

Phalloidin, taurocholic acid (TCA), ursodeoxycholic acid (UDCA), and dehydrocholic acid (DHCA) were purchased from Sigma-Aldrich (Madrid, Spain). As indicated by the supplier, the purity of these compounds was more than 98%, determined by thin-layer chromatography. All other reagents were of analytical grade. Succinyl amide of the 3β-amino derivative of cholic acid (Na2–SuC), named here BALU-1 was obtained by the methods described in previous studies (Soto Tellini et al., 2006, Alvarez Alcalde

Bile flow and bile acid secretion

Phalloidin administration to anaesthetized rats (see experimental design #1 in Fig. 1) resulted in a rapid and progressive decrease in bile flow (Fig. 2). When the animals received a bolus of BALU-1 (1.8 μmol/100 g b.wt.) together with phalloidin (50 μg/100 g b.wt.) (see experimental design #2 in Fig. 1), the impairment in bile flow was not significantly different to that observed in animals receiving phalloidin alone (data not shown). Although initially after BALU-1 administration a rapid and

Discussion

In previous studies we have found that the bile acid derivative BALU-1 was able to markedly inhibit OATP-mediated transport of phalloidin (Herraez et al., unpublished results). This has prompted us to evaluate the ability of this compound to prevent phalloidin-induced liver damage by reducing and/or slowing down the uptake of this toxin by hepatocytes. Indeed, the results obtained here indicate that BALU-1 has a beneficial effect on rats treated with this toxin, although the mechanism may be

Acknowledgments

The authors thank Dr. V.H. Soto and Dr. M. Alvarez for the synthesis and purification of BALU-1. Thanks are also due to L. Muñoz, J.F. Martin, J. Villoria, N. Gonzalez and E. Vallejo for care of the animals. Secretarial help by M. Hernandez, technical help by E. Cruz and revision of the English spelling, grammar and style of the manuscript by N. Skinner are also gratefully acknowledged.

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    Financial Support: This study was supported in part by the Junta de Castilla y Leon (Grants GR75/2008, SA033A08, SA03508, SA03608 and SAN 673/SA07/08), Spain; Ministerio de Ciencia y Tecnologia, Plan Nacional de Investigacion Cientifica, Desarrollo e Innovacion Tecnologica (Grants BFU2006-12577, MAT2001-2911 and MAT2004-04606), Spain. The group is a member of the Network for Cooperative Research on Membrane Transport Proteins (REIT), co-funded by the Ministerio de Educacion y Ciencia, Spain and the European Regional Development Fund (ERDF) (Grants BFU2007-30688-E/BFI) and belongs to the CIBERehd (Centro de Investigacion Biomedica en Red) for Hepatology and Gastroenterology Research (Instituto de Salud Carlos III, Spain).

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