Genetic Determinants of Drug-induced Cholestasis and Intrahepatic Cholestasis of Pregnancy

Christiane Pauli-Magnus, M.D.; Peter J. Meier, M.D.; Bruno Stieger, Ph.D.


Semin Liver Dis. 2010;30(2):147-159. 

In This Article

Physiology of Bile Formation

The liver is instrumental for maintaining enterohepatic circulation of bile salts. Bile salts are synthesized in a multistep cascade consisting of 16 enzymes catalyzing 17 reactions in hepatocytes[1] and secreted into the canaliculi, from where they enter the biliary tree.[2,3,4] In the biliary tree, the composition of bile with bile salts is modified and drained to the gallbladder, from where it enters the duodenum. In the duodenum, bile salts promote the digestion of fat and absorption of lipids and fat-soluble vitamins.[5,6] Bile salts are reclaimed to more than 90% in the small intestine and transported back to the liver via the portal circulation. In the liver, bile salts are taken up again from the sinusoidal blood plasma and their journey to the intestine restarts.[7] For efficient transport of bile salts from the sinusoids into the canaliculi as well as for controlling this process, hepatocytes are equipped with an elaborate array of transporters and regulatory mechanisms. Regulation of bile salt flow across hepatocytes is crucial, as bile salts are amphipathic molecules and display detergent properties. Hence, any surplus of bile salts within a hepatocyte can become cytotoxic or even lethal to the cell.

In the basolateral plasma membrane, bile salts are taken up predominantly in a sodium- dependent manner and to a minor degree via sodium-independent processes. The sodium-dependent uptake of bile salts is mediated by the sodium-taurocholate cotransporting polypeptide NTCP (SLC10A1) and shows a preference for conjugated bile salts.[7,8] Sodium-independent uptake of bile salts is fostered by organic anion transporting polypeptides or OATPs (SLCOs), namely OATP1B1 and OATP1B3.[2,9] A third OATP, OATP2B1 is also expressed in hepatocytes, but does not mediate transport of conjugated bile salts.[2,10] OATPs are also mediators of hepatocellular drug and xenobiotic uptake whereby OATP1B1 and OATP1B3 exhibit considerable overlap in their substrate specificity. Both NTCP and OATPs are subject to considerable interindividual differences in their hepatocellular expression levels. Knowledge on intracellular transport of bile salts from the basolateral to the apical plasma membrane is still scarce, but it is assumed that binding proteins are involved. Canalicular export occurs against a steep concentration gradient and is mediated by a member of the ATP-binding cassette (ABC) transporter family: the bile salt export pump BSEP (ABCB11).[7,11,12] The rate-limiting step in the overall transport from the portal blood into bile is located at the canalicular membrane of hepatocytes.[2,13] Hence, proper functioning of BSEP is essential for keeping the potentially cytotoxic bile salts at a low intracellular level in hepatocytes. Consequently, mutations leading to a nonfunctional BSEP protein were associated with familial cholestatic syndromes, the so-called progressive familial intrahepatic cholestasis type 2 (PFIC2).[4] Furthermore, as bile formation is an isoosmotic process, bile salts are a major driving force for the generation of canalicular bile flow. In addition to bile salts, canalicular bile contains lipids. Phosphatidylcholine is the major lipid constituent and its release from the extracellular leaflet of the canalicular membrane into bile is mediated by MDR3 or ABCB4. This ABC transporter acts as a phosphatidylcholine translocator supplying phosphatidylcholine to the outer hemileaflet of the canalicular membrane.[14] From there, phosphatidylcholine is released into bile by the detergent action of bile salts.[15] Mutations in the gene coding for MDR3 lead to progressive familial intrahepatic cholestasis type 3.[16] In primary bile, phosphatidylcholine and bile salts form mixed micelles, which act as acceptors for poorly water-soluble substances, such as cholesterol.[15] The release of cholesterol from the canalicular membrane into bile is facilitated by the heterodimeric transporter ABCG5/ABCG8.[17]

A reduction of bile flow represents a pathophysiologic situation and is called cholestasis. Metabolism of bile salts within hepatocytes leads to sulfated and glucuronidated bile salts, particularly in cholestatic conditions.[18,19] Such bile salt derivatives are excreted into bile via the multidrug resistance protein MRP2 (ABCC2),[20] or back into the sinusoids by MRP3 (ABCC3) and MRP4 (ABCC4),[21] two salvage systems that help to reduce the concentration of potentially cytotoxic intracellular bile salts in hepatocytes. In addition, the heterodimeric organic solute transporter OST-–OSTβ is also expressed in the basolateral membrane and might act as an additional salvage system.[22] The relative contribution of these three adaptive efflux systems is at the moment not fully understood and needs to be worked out in detail.

NTCP has a rather restricted substrate specificity and transports in addition to bile salts sulfated compounds such as bromosulfophthalein and sulfated steroid metabolites.[7,8,23,24] Furthermore, in heterologous expression systems NTCP transports bile salt-drug conjugates and sulfated thyroxin. Taken together, NTCP acts as the key hepatocellular bile salt uptake system, but may also contribute to hepatocellular handling of additional compounds and even drugs (see below). NTCP transports one bile salt molecule together with two sodium ions and is therefore electrogenic.[25] Consequently, it can take up bile salts against a concentration gradient into hepatocytes.

OATPs transport a large variety of endogenous substrates, metabolic end products, as well as xenobiotics, such as bile salts, estrogen metabolites, drugs, and toxins.[10,26,27] In hepatocytes, OATP1B1 and OATP1B3 are the two key uptake transporters for unconjugated and conjugated bile salts and for hydrophobic, anionic xenobiotics, whereas OATP2B1 is so far considered to be mainly a transporter for bromosulfophthalein and steroid sulfates. OATP1B1 and OATP1B3 have a large overlap in their substrate specificity. It is therefore difficult to predict the individual contribution of either of the two transporters for the uptake of a given bile salt or a given drug. For example, in heterologous expression systems all hepatocytes OATPs mediate transport of rosuvastatin.[28] Most interestingly, in a recent genome-wide single nucleotide polymorphism (SNP) association study with patients on a high-dose simvastatin treatment only OATP1B1 variants were identified as a risk factor for myopathy.[29] This can be taken as evidence that OATP1B1 is the functionally relevant simvastatin (and most likely also other statin) uptake system in hepatocytes. The transport mechanism of OATPs is not known in detail, but they are believed to act as organic anion exchangers. Glutathione, glutathione-conjugates, oxidized glutathione as well as bicarbonate have been demonstrated to act as counteranions.[30–33] In a recent study, evidence was presented that many OATPs indeed exchange bicarbonate for anions during the transport step and that most OATPs show higher transport rates at low extracellular pH.[34] Elucidation of the exact transport mechanism of the OATPs is however important, as it will predict whether OATPs have the potential to transport drugs against a concentration gradient into hepatocytes. Such a mechanism would certainly contribute to drug toxicity in hepatocytes. In this context, coadministration of the OATP inhibitor rifampicin with glibenclamide in healthy volunteers leads to an increase of the area under the curve and of maximum serum concentration of glibenclamide.[35] In rat studies, glibenclamide was found to be 50 times higher concentrated in the liver as compared with the serum, suggesting a concentrative uptake mechanism into hepatocytes.[36]

BSEP has a narrow substrate specificity and transports mainly monoanionic, conjugated bile salts.[11,12] There is a variation in its substrate pattern between species; for example, human BSEP, but not rat Bsep transports the bile salt metabolite taurolithocholate-3-sulfate.[37,38] BSEP transports barely any unconjugated bile acids.[39] This in vitro finding is supported in vivo by the observation that patients with a defect in bile acid conjugation have very little unconjugated bile acids in their bile.[40] BSEP is an electrogenic transporter and requires hydrolysis of ATP for transport activity.[11,41] BSEP is the sole transporter for monoanionic bile salts across the canalicular membrane. This becomes evident in patients with mutations in the BSEP gene. Such patients develop progressive familial intrahepatic cholestasis or BSEP-deficiency syndrome type 2 and have less than 1% of primary bile salts in their bile.[4,42,43] Furthermore, a comparison of rat Bsep and Mrp2 revealed no overlap in substrate specificities.[37] These findings suggest that inhibition of BSEP, e.g., by drugs, should lead to reduced bile salt secretion and their retention within hepatocytes and consequently lead to cholestasis. Several drugs have been implicated in drug-induced cholestasis and examples of such drugs like cyclosporine, rifampicin, rifamycin, glibenclamide, or bosentan have been found to be competitive inhibitors of BSEP.[37,44,45] The list of BSEP inhibitors is continuously growing.[46] The estradiol metabolite estradiol-17β-glucuronide induces acute cholestasis in rats. It was therefore also tested for its inhibitory potential of rat Bsep. Interestingly, estradiol-17β-glucuronide does not inhibit Bsep expressed in Sf9 cells. If, however, Mrp2 is coexpressed with Bsep in Sf9 cells, estradiol-17β-glucuronide leads to a concentration and time-dependent inhibition of Bsep.[37] This finding was confirmed and extended to sulfated progesterone metabolites.[47,48] In addition, Mrp2 could also interact directly with Bsep in the canalicular membrane in the presence of estradiol-17β-glucuronide and thereby inhibit Bsep.[49]


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