Gerd A. Kullak-Ublick

Genome-wide meta-analysis increases to 71 the number of confirmed Crohn's disease susceptibility loci

We undertook a meta-analysis of six Crohns disease genome-wide association studies (GWAS) comprising 6,333 affected individuals (cases) and 15,056 controls and followed up the top association signals in 15,694 cases, 14,026 controls and 414 parent-offspring trios. We identified 30 new susceptibility loci meeting genome-wide significance (P < 5 × 10−8). A series of in silico analyses highlighted particular genes within these loci and, together with manual curation, implicated functionally interesting candidate genes including SMAD3, ERAP2, IL10, IL2RA, TYK2, FUT2, DNMT3A, DENND1B, BACH2 and TAGAP. Combined with previously confirmed loci, these results identify 71 distinct loci with genome-wide significant evidence for association with Crohns disease.

St John's Wort induces intestinal P-glycoprotein/MDR1 and intestinal and hepatic CYP3A4.

St Johns Wort (hypericum perforatum) is an herbal medicine that is frequently used for therapy of mild depression. Recently, St Johns Wort was reported to substantially decrease blood/plasma concentrations and efficacy of cyclosporine (INN, ciclosporin), indinavir, and digoxin. In this study we investigated the mechanisms of these St Johns Wort–induced drug interactions.

Drug- and estrogen-induced cholestasis through inhibition of the hepatocellular bile salt export pump (Bsep) of rat liver.

BACKGROUND & AIMS Drug-induced cholestasis is a frequent form of acquired liver disease. To elucidate the molecular pathogenesis of drug-induced cholestasis, we investigated the effects of prototypic cholestatic drugs on the canalicular bile salt export pump (Bsep) of rat liver. METHODS Vesicles were isolated from Bsep-, Mrp2-, and Bsep/Mrp2-expressing Sf9 cells. Canalicular plasma membrane (cLPM) vesicles from rat liver and Sf9 cell vesicles were used to study adenosine triphosphate (ATP)-dependent solute uptake by a rapid filtration technique. RESULTS Bsep-expressing Sf9 cell vesicles showed ATP-dependent transport of numerous monoanionic bile salts with similar Michaelis constant values as in cLPM vesicles, whereas several known substrates of the multispecific organic anion transporter Mrp2 were not transported by Bsep. Cyclosporin A, rifamycin SV, rifampicin, and glibenclamide cis-inhibited Bsep-mediated bile salt transport to similar extents as ATP-dependent taurocholate transport in cLPM vesicles. In contrast, the cholestatic estrogen metabolite estradiol-17beta-glucuronide inhibited ATP-dependent taurocholate transport only in normal cLPM and in Bsep/Mrp2-coexpressing Sf9 cell vesicles, but not in Mrp2-deficient cLPM or in selectively Bsep-expressing Sf9 cell vesicles, indicating that it trans-inhibits Bsep only after its secretion into bile canaliculi by Mrp2. CONCLUSIONS These results provide a molecular basis for previous in vivo observations and identify Bsep as an important target for induction of drug- and estrogen-induced cholestasis in mammalian liver.

Molecular and functional characterization of an organic anion transporting polypeptide cloned from human liver

BACKGROUND & AIMS Based on a recently cloned rat liver organic anion transporter, we attempted to clone the corresponding human liver organic anion transporting polypeptide. METHODS A human liver complementary DNA library was screened with a specific rat liver complementary DNA probe. The human liver transporter was cloned by homology with the rat protein and functionally characterized in Xenopus laevis oocytes. RESULTS The cloned human liver organic anion transporting polypeptide consists of 670 amino acids and shows a 67% amino acid identity with the corresponding rat liver protein. Injection of in vitro transcribed complementary RNA into frog oocytes resulted in the expression of sodium-independent uptake of [35S]bromosulfophthalein (Michaelis constant [Km], approximately 20 mumol/L), [3H]cholate (Km, approximately 93 mumol/L), [3H]taurocholate (Km, approximately 60 mumol/L), [14C]glycocholate, [3H]taurochenodeoxycholate, and [3H]tauroursodeoxycholate (Km, approximately 19 mumol/L). Northern blot analysis showed cross-reactivity with messenger RNA species from human liver, brain, lung, kidney, and testes. Polymerase chain reaction analysis of genomic DNA from a panel of human-rodent somatic cell hybrids mapped the cloned human organic anion transporter to chromosome 12. CONCLUSIONS These studies show that the cloned human liver organic anion transporter is closely related to, but probably not identical to, the previously cloned rat liver transporter. Furthermore, its additional localization in a variety of extrahepatic tissues suggests that it plays a fundamental role in overall transepithelial organic anion transport of the human body.

Current concepts of mechanisms in drug-induced hepatotoxicity.

Drug-induced liver injury (DILI) has become a leading cause of severe liver disease in Western countries and therefore poses a major clinical and regulatory challenge. Whereas previously drug-specific pathways leading to initial injury of liver cells were the main focus of mechanistic research and classifications, current concepts see these as initial upstream events and appreciate that subsequent common downstream pathways and their attenuation by drugs and other environmental and genetic factors also have a profound impact on the risk of an individual patient to develop overt liver disease. This review summarizes current mechanistic concepts of DILI in a 3-step model that limits its principle mechanisms to three main ways of initial injury, i.e. direct cell stress, direct mitochondrial impairment, and specific immune reactions. Subsequently, initial injury initiates further downstream events, i.e. direct and death receptor-mediated pathways leading to mitochondrial permeability transition, which then results in apoptotic or necrotic cell death. For all mechanisms, mitochondria play a central role in events leading to apoptotic vs. necrotic cell death. New treatment targets consequently focus on interference with downstream pathways that mediate injury and therefore determine the ultimate outcome of DILI. Genome wide and targeted pharmacogenetic as well as metabonomic approaches are now used in order to reach the key goals of a better understanding of mechanisms in hepatotoxicity, and to develop new strategies for its prediction and treatment. However, the complexity of interactions between genetic and environmental risk factors is considerable, and DILI therefore currently remains unpredictable for most hepatotoxins.

Sequence analysis of bile salt export pump (ABCB11) and multidrug resistance p-glycoprotein 3 (ABCB4, MDR3) in patients with intrahepatic cholestasis of pregnancy

Intrahepatic cholestasis of pregnancy (ICP) is a liver disorder associated with increased risk of intrauterine fetal death and prematurity. There is increasing evidence that genetically determined dysfunction in the canalicular ABC transporters bile salt export pump (BSEP, ABCB11) and multidrug resistance protein 3 (MDR3, ABCB4) might be risk factors for ICP development. This study aimed to (i). describe the extent of genetic variability in BSEP and MDR3 in ICP and (ii). identify new disease-causing mutations. Twenty-one women with ICP and 40 women with uneventful pregnancies were recruited between April 2001 and April 2003. Sequencing of BSEP and MDR3 spanned 8-10 kb per gene and comprised the promoter region and 100-350 bp of the flanking intronic region around each exon. DNA sequencing of polymerase chain reaction fragments was performed on an ABI3700 capillary sequencer. MDR3 promoter activity of promoter constructs carrying different ICP-specific mutations was studied using reporter assays. A total of 37 and 51 variant sites were detected in BSEP and MDR3, respectively. Three non-synonymous sites in codons for evolutionarily conserved amino acids were specific for the ICP collective (BSEP, N591S; MDR3, S320F and G762E). Furthermore, four ICP-specific splicing mutations were detected in MDR3 [intron 21, G(+1)A; intron 25, G(+5)C and C(-3)G; and intron 26, T(+2)A]. Activity of the mutated MDR3 promoter was similar to that observed for the wild-type promoter. Our data further support an involvement of MDR3 genetic variation in the pathogenesis of ICP, whereas analysis of BSEP sequence variation indicates that this gene is probably less important for the development of pregnancy-associated cholestasis.

Regional Distribution of Solute Carrier mRNA Expression Along the Human Intestinal Tract

Intestinal absorption of drugs, nutrients, and other compounds is mediated by uptake transporters expressed at the apical enterocyte membrane. These compounds are returned to the intestinal lumen or released into portal circulation by intestinal efflux transporters expressed at apical or basolateral membranes, respectively. One important transporter superfamily, multiple members of which are intestinally expressed, are the solute carriers (SLCs). SLC expression levels may determine the pharmacokinetics of drugs that are substrates of these transporters. In this study we characterize the distribution of 15 human SLC transporter mRNAs in histologically normal biopsies from five regions of the intestine of 10 patients. The mRNA expression levels of CNT1, CNT2, apical sodium-dependent bile acid transporter (ABST), serotonin transporter (SERT), PEPT1, and OCTN2 exhibit marked differences between different regions of the intestine: the first five are predominantly expressed in the small intestine, whereas OCTN2 exhibits strongest expression in the colon. Two transporter mRNAs studied (OCTN1, OATP2B1) are expressed at similar levels in all gut sections. In addition, ENT2 mRNA is present at low levels across the colon, but not in the small intestine. The other six SLC mRNAs studied are not expressed in the intestine. Quantitative knowledge of transporter expression levels in different regions of the human gastrointestinal tract could be useful for designing intestinal delivery strategies for orally administered drugs. Furthermore, changes in transporter expression that occur in pathological states, such as inflammatory bowel disease, can now be defined more precisely by comparison with the expression levels measured in healthy individuals.

BSEP and MDR3 haplotype structure in healthy Caucasians, primary biliary cirrhosis and primary sclerosing cholangitis

Primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) are characterized by a cholestatic pattern of liver damage, also observed in hereditary or acquired dysfunction of the canalicular membrane transporters bile salt export pump (BSEP, ABCB11) and multidrug resistance protein type 3 (MDR3, ABCB4). Controversy exists whether a genetically determined dysfunction of BSEP and MDR3 plays a pathogenic role in PBC and PSC. Therefore, 149 healthy Caucasian control individuals (control group) were compared to 76 PBC and 46 PSC patients with respect to genetic variations in BSEP and MDR3. Sequencing spanned ∼10,000 bp including promoter and coding regions as well as 50–350 bp of flanking intronic regions. In all, 46 and 45 variants were identified in BSEP and MDR3, respectively. No differences between the groups were detected either in the total number of variants (BSEP: control group: 37, PBC: 37, PSC: 31; and MDR3: control group: 35; PBC: 32, PSC: 30), or in the allele frequency of the common variable sites. Furthermore, there were no significant differences in haplotype distribution and linkage disequilibrium. In conclusion, this study provides an analysis of BSEP and MDR3 variant segregation and haplotype structure in a Caucasian population. Although an impact of rare variants on BSEP and MDR3 function cannot be ruled out, our data do not support a strong role of BSEP and MDR3 genetic variations in the pathogenesis of PBC and PSC. (HEPATOLOGY 2004;39:779–791.)

Mechanisms of cholestasis.

From the multiple mechanisms of cholestasis presented in this article, a unifying hypothesis may be deduced by parsimony. The disturbance of the flow of bile must inevitably lead to the intracellular retention of biliary constituents. Alternatively, the lack of specific components of bile unmasks the toxic potential of other components, as in the case of experimental mdr2 deficiency. In the sequence of events that leads to liver injury, the cytotoxic action of bile salts is pivotal to all forms of cholestasis. The inhibition of the bsep by drugs, sex steroids, or monohydroxy bile salts is an example of direct toxicity to the key mediator in canalicular bile salt excretion. In other syndromes, the dysfunction of distinct hepatocellular transport systems is the primary pathogenetic defect leading to cholestasis. Such dysfunctions include the genetic defects in PFIC and the direct inhibition of gene transcription by cytokines. Perturbations in the short-term regulation of transport protein function are exemplified by the cholestasis of endotoxinemia. The effect of bile salts on signal transduction, gene transcription, and transport processes in hepatocytes and cholangiocytes has become the focus of intense research in recent years. The central role of bile salts in the pathogenesis of cholestasis has, ironically, become all the more evident from the improvement of many cholestatic syndromes with oral bile salt therapy.

Transporters involved in the hepatic uptake of 99mTc-mebrofenin and indocyanine green

BACKGROUND & AIMS (99m)Tc-mebrofenin hepatobiliary scintigraphy (HBS) and the indocyanine green (ICG) clearance test are used for the assessment of hepatic function before and after liver surgery. The hepatic uptake of (99m)Tc-mebrofenin and ICG is considered similar to the uptake of organic anions such as bilirubin and bile acids. Little is known about hepatic uptake mechanisms of both compounds and recent evidence suggests that the hepatic transporters for ICG and (99m)Tc-mebrofenin are distinct. The aim of this study was to identify the specific human hepatic transporters of (99m)Tc-mebrofenin and ICG. METHODS The uptake of (99m)Tc-mebrofenin was investigated in cRNA-injected Xenopus laevis oocytes expressing human OATP1B1, OATP1B3, OATP2B1, or NTCP. Chinese hamster ovary (CHO) cells stably expressing OATP1B1, OATP1B3, OATP2B1, or NTCP were used as a mammalian expression system. ICG transport into CHO cells was additionally imaged with confocal microscopy. RESULTS We demonstrated that OATP1B1 and OATP1B3 are involved in the transport of (99m)Tc-mebrofenin. OATP1B1 showed an approximately 1.5-fold higher affinity for (99m)Tc-mebrofenin compared to OATP1B3. ICG is transported by OATP1B3 and NTCP. CONCLUSIONS The transporter specificity of (99m)Tc-mebrofenin and ICG partially overlaps as both compounds are transported by OATP1B3. (99m)Tc-mebrofenin is also taken up by OATP1B1, whereas ICG is additionally transported by NTCP.