Bilirubin Glucuronidation Research Articles

Inhibition of Hepatobiliary Transport Activity by the Antibacterial Agent Fusidic Acid: Insights into Factors Contributing to Conjugated Hyperbilirubinemia/Cholestasis.

Conjugated hyperbilirubinemia accompanied by cholestasis is a frequent side effect during chronic treatment with the antimicrobial agent fusidic acid. Previous studies from our laboratory, addressing mechanisms of musculoskeletal toxicity arising from coadministration of fusidic acid with statins, demonstrated the ability of fusidic acid to potently inhibit human organic anion transporting polypeptides OATP1B1 (IC50 = 1.6 μM) and OATP1B3 (IC50 = 2.5 μM), which are responsible for the uptake-limited clearance of statins as well as bilirubin glucuronide conjugates. In the present work, inhibitory effects of fusidic acid were characterized against additional human hepatobiliary transporters [Na+/taurocholate cotransporting polypeptide (NTCP), bile salt export pump (BSEP), and multidrug resistance-associated proteins MRP2 and MRP3] as well as uridine glucuronosyl transferase (UGT1A1), which mediate the disposition of bile acids and bilirubin (and its conjugated metabolites). Fusidic acid demonstrated concentration-dependent inhibition of human NTCP- and BSEP-mediated taurocholic acid transport with IC50 values of 44 and 3.8 μM, respectively. Inhibition of BSEP activity by fusidic acid was also consistent with the potent disruption of cellular biliary flux (AC50 = 11 μM) in the hepatocyte imaging assay technology assay, with minimal impact on other toxicity end points (e.g., cytotoxicity, mitochondrial membrane potential, reactive oxygen species generation, glutathione depletion, etc.). Fusidic acid also inhibited UGT1A1-catalyzed β-estradiol glucuronidation activity in human liver microsomes with an IC50 value of 16 μM. Fusidic acid did not demonstrate any significant inhibition of ATP-dependent LTC4 transport (IC50's > 300 μM) in human MRP2 or MRP3 vesicles. R values, which reflect maximal in vivo inhibition, were estimated from a static mathematical model by taking into consideration the IC50 values generated in the various in vitro assays and clinically efficacious unbound fusidic acid concentrations. The magnitudes of in vivo interaction (R values) resulting from the inhibition of OATP1B1, UGT1A1, NTCP, and BSEP transport were ∼1.9-2.6, 1.1-1.2, 1.0-1.1, and 1.4-1.7, respectively, which are indicative of some degree of inherent toxicity risk, particularly via inhibition of OATP and BSEP. Collectively, these observations indicate that inhibition of human BSEP by fusidic acid could affect bile acid homeostasis, resulting in cholestatic hepatotoxicity in the clinic. Lack of direct inhibitory effects on MRP2 transport by fusidic acid suggests that conjugated hyperbilirubinemia does not arise via interference in MRP2-mediated biliary disposition of bilirubin glucuronides. Instead, it is possible that elevation in the level of bilirubin conjugates in blood is mediated through inhibition of hepatic OATPs, which are responsible for their reuptake and/or downregulation of MRP2 transporter as a consequence of cholestatic injury.

Polymorphisms in UGT1A1 Gene Predispose South Indians to Pigmentous Gallstones

Pigmentous gallstones occur in South Indians despite significant higher levels of circulating cholesterol. This study was conducted to identify the biochemical and/or genetic causes for the formation of pigmentous gallstones in this ethnic group. Plasma lipid profile, bile cholesterol, acids, and phospholipid levels were estimated in patients with gall stone disease and age, sex matched controls using standard protocols. Twenty-seven SNPs related to cholesterol and bilirubin metabolism pathway genes were genotyped in the study population using the Sequenom platform. An equilibrium phase diagram involving bile salt-phospholipid-cholesterol was generated to relate phenotype with the genotype. There were no significant differences in the lipid profiles between the patients (n=305) and controls (n=177). Biliary cholesterol, acids, and phospholipids were significantly different between patients and controls. Single locus analysis revealed association of variants in ABCG6, ABCG8, and UGT1A1 genes with the disease; however when correction was applied as multiple testing was done, only one variant (rs6742078) in UGT1A1 gene was found to be associated with gall stone disease. Equilibrium phase diagram suggested that few samples were in the crystal formation zone. The mutant, but not wild type or heterozygous genotype of SNPs (rs6742078 and rs887829) in UGT1A1 gene, was associated with significantly higher levels of bilirubin. Higher incidence of pigment stones in South Indians could be due to raised serum bilirubin levels that may be ascribed to variant in the UGT1A1 gene involved in glucuronidation of free bilirubin.

Clinical Pharmacogenetics Implementation Consortium (CPIC) Guideline for UGT1A1 and Atazanavir Prescribing.

The antiretroviral protease inhibitor atazanavir inhibits hepatic uridine diphosphate glucuronosyltransferase (UGT) 1A1, thereby preventing the glucuronidation and elimination of bilirubin. Resultant indirect hyperbilirubinemia with jaundice can cause premature discontinuation of atazanavir. Risk for bilirubin-related discontinuation is highest among individuals who carry two UGT1A1 decreased function alleles (UGT1A1*28 or *37). We summarize published literature that supports this association and provide recommendations for atazanavir prescribing when UGT1A1 genotype is known (updates at www.pharmgkb.org).

Impaired Hepatic Uptake by Organic Anion-Transporting Polypeptides Is Associated with Hyperbilirubinemia and Hypercholanemia in Atp11c Mutant Mice.

Biliary excretion of organic anions, such as bile acids (BAs), is the main osmotic driving force for bile formation, and its impairment induces intrahepatic cholestasis. We investigated the involvement of Atp11c in the hepatic transport of organic anions using Atp11c mutant mice, which exhibit hypercholanemia and hyperbilirubinemia. Pharmacokinetic analysis following a constant intravenous infusion in Atp11c mutant mice showed decreased hepatic sinusoidal uptake and intact biliary secretion of [(3)H]17β estradiol 17β-d-glucuronide. Consistent with this result, compared with cells and membranes from control mice, isolated hepatocytes, and liver plasma membranes from Atp11c mutant mice had a much lower uptake of [(3)H]17β estradiol 17β-d-glucuronide and expression of organic anion-transporting polypeptides, which are transporters responsible for hepatic uptake of unconjugated BAs and organic anions, including bilirubin glucuronides. Uptake of [(3)H]TC into hepatocytes and expression of Na(+)-taurocholate cotransporting polypeptide in liver plasma membranes, which mediates hepatic uptake of conjugated BAs, was also lower in the Atp11c mutant mice. Bile flow rate, biliary BA concentration, and expression of hepatobiliary transporters did not differ between Atp11c mutant mice and control mice. These results suggest that Atp11c mediates the transport of BAs and organic anions across the sinusoidal membrane, but not the canalicular membrane, by regulating the abundance of transporters. Atp11c is a candidate gene for genetically undiagnosed cases of hypercholanemia and hyperbilirubinemia, but not of intrahepatic cholestasis. This gene may influence the pharmacological and adverse effect of drugs because organic anion-transporting polypeptides regulate their systemic exposure.

Hepatocellular Shuttling and Recirculation of Sorafenib-Glucuronide Is Dependent on Abcc2, Abcc3, and Oatp1a/1b.

Recently, an efficient liver detoxification process dubbed "hepatocyte hopping" was proposed on the basis of findings with the endogenous compound, bilirubin glucuronide. According to this model, hepatocytic bilirubin glucuronide can follow a liver-to-blood shuttling loop via Abcc3 transporter-mediated efflux and subsequent Oatp1a/1b-mediated liver uptake. We hypothesized that glucuronide conjugates of xenobiotics, such as the anticancer drug sorafenib, can also undergo hepatocyte hopping. Using transporter-deficient mouse models, we show here that sorafenib-glucuronide can be extruded from hepatocytes into the bile by Abcc2 or back into the systemic circulation by Abcc3, and that it can be taken up efficiently again into neighboring hepatocytes by Oatp1a/1b. We further demonstrate that sorafenib-glucuronide excreted into the gut lumen can be cleaved by microbial enzymes to sorafenib, which is then reabsorbed, supporting its persistence in the systemic circulation. Our results suggest broad relevance of a hepatocyte shuttling process known as "hepatocyte hopping"-a novel concept in clinical pharmacology-for detoxification of targeted cancer drugs that undergo hepatic glucuronidation, such as sorafenib.

Amelioration of Hyperbilirubinemia in Gunn Rats after Transplantation of Human Induced Pluripotent Stem Cell-Derived Hepatocytes

SummaryHepatocyte transplantation has the potential to cure inherited liver diseases, but its application is impeded by a scarcity of donor livers. Therefore, we explored whether transplantation of hepatocyte-like cells (iHeps) differentiated from human induced pluripotent stem cells (iPSCs) could ameliorate inherited liver diseases. iPSCs reprogrammed from human skin fibroblasts were differentiated to iHeps, which were transplanted into livers of uridinediphosphoglucuronate glucuronosyltransferase-1 (UGT1A1)-deficient Gunn rats, a model of Crigler-Najjar syndrome 1 (CN1), where elevated unconjugated bilirubin causes brain injury and death. To promote iHep proliferation, 30% of the recipient liver was X-irradiated before transplantation, and hepatocyte growth factor was expressed. After transplantation, UGT1A1+ iHep clusters constituted 2.5%–7.5% of the preconditioned liver lobe. A decline of serum bilirubin by 30%–60% and biliary excretion of bilirubin glucuronides indicated that transplanted iHeps expressed UGT1A1 activity, a postnatal function of hepatocytes. Therefore, iHeps warrant further exploration as a renewable source of hepatocytes for treating inherited liver diseases.

Utility of bilirubins and bile acids as endogenous biomarkers for the inhibition of hepatic transporters.

It is useful to identify endogenous substrates for the evaluation of drug-drug interactions via transporters. In this study, we investigated the utility of bilirubins, substrates of OATPs and MRP2, and bile acids and substrates of NTCP and BSEP, as biomarkers for the inhibition of transporters. In rats administered 20 and 80 mg/kg rifampicin, the plasma levels of bilirubin glucuronides were elevated, gradually decreased, and almost returned to the baseline level at 24 hours after administration without an elevation of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). This result indicates the transient inhibition of rOatps and/or rMrp2. Although the correlation between free plasma concentrations and IC50 values of rOatps depended on the substrates used in the in vitro studies, the inhibition of rOatps by rifampicin was confirmed in the in vivo study using valsartan as a substrate of rOatps. In rats administered 10 and 30 mg/kg cyclosporin A, the plasma levels of bile acids were elevated and persisted for up to 24 hours after administration without an elevation of ALT and AST. This result indicates the continuous inhibition of rNtcp and/or rBsep, although there were differences between the free plasma or liver concentrations and IC50 values of rNtcp or rBsep, respectively. This study suggests that the monitoring of bilirubins and bile acids in plasma is useful in evaluating the inhibitory potential of their corresponding transporters.

Key determinants of the circulatory exposure of organic anions: differences in hepatic uptake between multidrug resistance-associated protein 2 (Mrp2)-deficient rats and wild-type rats

1. Raloxifene-6-glucuronide (R6G) is a substrate of rat multidrug resistance-associated protein 2 (Mrp2), a transporter responsible for biliary excretion of organic anions.2. Pharmacokinetic modeling of R6G in Eisai hyperbilirubinemic rats (EHBRs), hereditary Mrp2-deficient rats, and wild-type Sprague–Dawley rats (SDRs) indicated that reduction in not only biliary excretion but also hepatic uptake of R6G influenced low clearance in EHBRs.3. An integration plot study demonstrated that the hepatic uptake of R6G was 66% lower in EHBRs than that in SDRs. A reduction was observed for the other Mrp2 substrate Valsartan (95% lower) but not for estradiol-17β-glucuronide (E217βG). This variation may be associated with the difference in substrate specificity of transporters and/or inhibition of hepatic uptake of organic anions by endogenous substances such as bilirubin glucuronides.4. In conclusion, incidental alteration of the hepatic uptake of organic anions should be considered as an explanation of their enhanced systemic exposure in EHBRs.

Role of brain cytochrome P450 mono-oxygenases in bilirubin oxidation-specific induction and activity.

In the Crigler-Najjar type I syndrome, the genetic absence of efficient hepatic glucuronidation of unconjugated bilirubin (UCB) by the uridine 5'-diphospho-glucuronosyltransferase1A1 (UGT1A1) enzyme produces the rise of UCB level in blood. Its entry to central nervous system could generate toxicity and neurological damage, and even death. In the past years, a compensatory mechanism to liver glucuronidation has been indicated in the hepatic cytochromes P450 enzymes (Cyps) which are able to oxidize bilirubin. Cyps are expressed also in the central nervous system, the target of bilirubin toxicity, thus making them theoretically important to confer a protective activity toward bilirubin accumulation and neurotoxicity. We therefore investigated the functional induction (mRNA, EROD/MROD) and the ability to oxidize bilirubin of Cyp1A1, 1A2, and 2A3 in primary astrocytes cultures obtained from two rat brain region (cortex: Cx and cerebellum: Cll). We observed that Cyp1A1 was the Cyp isoform more easily induced by beta-naphtoflavone (βNF) in both Cx and Cll astrocytes, but oxidized bilirubin only after uncoupling by 3, 4,3',4'-tetrachlorobiphenyl (TCB). On the contrary, Cyp1A2 was the most active Cyp in bilirubin clearance without uncoupling, but its induction was confined only in Cx cells. Brain Cyp2A3 was not inducible. In conclusion, the exposure of astrocytes to βNF plus TCB significantly enhanced Cyp1A1 mediating bilirubin clearance, improving cell viability in both regions. These results may be a relevant groundwork for the manipulation of brain Cyps as a therapeutic approach in reducing bilirubin-induced neurological damage.

Gene replacement therapy for genetic hepatocellular jaundice.

Jaundice results from the systemic accumulation of bilirubin, the final product of the catabolism of haem. Inherited liver disorders of bilirubin metabolism and transport can result in reduced hepatic uptake, conjugation or biliary secretion of bilirubin. In patients with Rotor syndrome, bilirubin (re)uptake is impaired due to the deficiency of two basolateral/sinusoidal hepatocellular membrane proteins, organic anion-transporting polypeptide 1B1 (OATP1B1) and OATP1B3. Dubin-Johnson syndrome is caused by a defect in the ATP-dependent canalicular transporter, multidrug resistance-associated protein 2 (MRP2), which mediates the export of conjugated bilirubin into bile. Both disorders are benign and not progressive and are characterised by elevated serum levels of mainly conjugated bilirubin. Uridine diphospho-glucuronosyl transferase 1A1 (UGT1A1) is responsible for the glucuronidation of bilirubin; deficiency of this enzyme results in unconjugated hyperbilirubinaemia. Gilbert syndrome is the mild and benign form of inherited unconjugated hyperbilirubinaemia and is mostly caused by reduced promoter activity of the UGT1A1 gene. Crigler-Najjar syndrome is the severe inherited form of unconjugated hyperbilirubinaemia due to mutations in the UGT1A1 gene, which can cause kernicterus early in life and can be even lethal when left untreated. Due to major disadvantages of the current standard treatments for Crigler-Najjar syndrome, phototherapy and liver transplantation, new effective therapeutic strategies are under development. Here, we review the clinical features, pathophysiology and genetic background of these inherited disorders of bilirubin metabolism and transport. We also discuss the upcoming treatment option of viral gene therapy for genetic disorders such as Crigler-Najjar syndrome and the possible immunological consequences of this therapy.

Correction of Hyperbilirubinemia in Gunn Rats by Surgical Delivery of Low Doses of Helper-Dependent Adenoviral Vectors

Helper-dependent adenoviral (HDAd) vectors are attractive for liver-directed gene therapy because they can drive sustained high levels of transgene expression without chronic toxicity. However, high vector doses are required to achieve efficient hepatic transduction by systemic delivery because of a nonlinear dose response. Unfortunately, such high doses result in systemic vector dissemination and dose-dependent acute toxicity with potential lethal consequences. We have previously shown in nonhuman primates that delivery of HDAd in surgically isolated livers resulted in a significantly higher hepatic transduction with reduced systemic vector dissemination compared with intravenous delivery and multiyear transgene expression. Encouraged by these data, we have now employed a surgical vector delivery method in the Gunn rat, an animal model for Crigler-Najjar syndrome. After vector delivery into the surgically isolated liver, we show phenotypic correction at the low and clinically relevant vector dose of 1 × 10(11) vp/kg. Correction of hyperbilirubinemia and increased glucuronidation of bilirubin in bile was achieved for up to 1 year after vector administration. Surgical delivery of the vector was well tolerated without signs of acute or chronic toxicity. This method of delivery could thereby be a safer alternative to liver transplantation for long-term treatment of Crigler-Najjar syndrome type I.

Bilirubin Uridine Diphosphate-Glucuronosyltransferase Variation Is a Genetic Basis of Breast Milk Jaundice

To evaluate the role of bilirubin UDP-glucuronosyltransferase family 1, polypeptide A1 (UGT1A1) gene variations on prolonged unconjugated hyperbilirubinemia associated with breast milk feeding (breast milk jaundice [BMJ]). UGT1A1 gene allelic variation was analyzed in 170 Japanese infants with BMJ with polymerase chain reaction-direct sequencing, and their genotypes compared with serum bilirubin concentrations. In 62 of 170 infants, serum bilirubin concentration was followed after 4 months of life. Genotypes were examined in 55 infants without BMJ. Of 170 infants with BMJ, 88 (51.8%) were homozygous UGT1A1*6. Serum bilirubin concentrations (21.8 ± 3.65 mg/dL) were significantly greater than in infants with other genotypes (P < .0001). The Gilbert UGT1A1*28 allele was not detected in infants with BMJ, except in an infant who was compound heterozygous with UGT1A1*6. At 4 months of age, serum bilirubin concentration improved to >1 mg/dL, except in 2 infants who were homozygous UGT1A1*7. Homozygous UGT1A1*6 was not detected in the control group. One-half of the infants with BMJ were homozygous UGT1A1*6 and exhibited a serum bilirubin concentration significantly greater than other genotypes. This finding indicates that UGT1A1*6 is a major cause of BMJ in infants in East Asia. Previous finding have demonstrated that 5β-pregnane-3α,20β-diol present in breast milk inhibits p.G71R-UGT1A1 bilirubin glucuronidation activity. Thus, prolonged unconjugated hyperbilirubinemia may develop in infants with UGT1A1*6 who are fed breast milk.

Serum Bilirubin Concentration in Healthy Adult North-Europeans Is Strictly Controlled by the UGT1A1 TA-Repeat Variants

The major enzyme responsible for the glucuronidation of bilirubin is the uridine 5′-diphosphoglucose glucuronosyltransferase A1 (UGT1A1) enzyme, and genetic variation in the UGT1A1 gene is reported to influence the bilirubin concentration in the blood. In this study, we have investigated which gene-/haplotype variants may be useful for genetic testing of Gilbert's syndrome. Two groups of samples based on serum bilirubin concentrations were obtained from the Nordic Reference Interval Project Bio-bank and Database (NOBIDA): the 150 individuals with the highest bilirubin (>17.5 µmol/L) and the 150 individuals with normal bilirubin concentrations (<17.5 µmol/L). The individuals were examined for the TA6>TA7 variant in the UGT1A1 promoter and 7 tag-SNPs in an extended promoter region of UGT1A1 (haplotype analysis) and in selected SNPs in candidate genes (SLCO1B3, ABCC2 and NUP153). We found significant odds ratios for high bilirubin level for all the selected UGT1A1 variants. However, in stepwise multivariate logistic regression analysis of all genetic variants together with age, sex, country of origin and fasting time, the repeat variants of UGT1A1 TA6>TA7 and SLCO1B3 rs2117032 T>C were the only variants significantly associated with higher bilirubin concentrations. Most individuals with high bilirubin levels were homozygous for the TA7-repeat (74%) while only 3% were homozygous for the TA7-repeat in individuals with normal bilirubin levels. Among individuals heterozygous for the TA7-repeat, a low frequent UGT1A1-diplotype harboring the rs7564935 G-variant was associated with higher bilirubin levels. In conclusion, our results demonstrate that in testing for Gilbert's syndrome, analyzing for the homozygous TA7/TA7-genotype would be appropriate.

Developmental Onset of Bilirubin-induced Neurotoxicity Involves Toll-like Receptor 2-dependent Signaling in Humanized UDP-glucuronosyltransferase1 Mice

Biological and signaling events that connect developmentally induced hyperbilirubinemia to bilirubin-induced neurological dysfunction (BIND) and CNS toxicity in humans are poorly understood. In mammals, UDP-glucuronosyltransferase 1A1 (UGT1A1) is the sole enzyme responsible for bilirubin glucuronidation, a rate-limiting step necessary for bilirubin metabolism and clearance. Humanized mice that express the entire UGT1 locus (hUGT1) and the UGT1A1 gene, develop neonatal hyperbilirubinemia, with 8-10% of hUGT1 mice succumbing to CNS damage, a phenotype that is presented by uncontrollable seizures. We demonstrate that neuroinflammation and reactive gliosis are prominent features of bilirubin brain toxicity, and a disturbed redox status resulting from activation of NADPH oxidase is an important contributing mechanism found in BIND. Using knock-out mice and primary brain cells, we connect a key pattern recognition receptor, Toll-like receptor 2 (TLR2), to hyperbilirubinemia-induced signaling. We illustrate a requirement for TLR2 signaling in regulating gliosis, proinflammatory mediators, and oxidative stress when neonatal mice encounter severe hyperbilirubinemia. TLR2-mediated gliosis strongly correlates with pronounced neuroinflammation in the CNS with up-regulation of TNFα, IL-1β, and IL-6, creating a pro-inflammatory CNS environment. Gene expression and immunohistochemistry staining show that hUGT1/Tlr2(-/-) mice fail to activate glial cells, proinflammatory cytokines, and stress response genes. In addition, bilirubin-induced apoptosis was significantly enhanced by blocking TLR2 signaling indicating its anti-apoptotic property. Consequently, a higher neonatal death rate (57.1%) in hUGT1/Tlr2(-/-) mice was observed when compared with hUGT1 mice (8.7%). These results suggest that TLR2 signaling and microglia neuroinflammation are linked to a repair and/or protection mode against BIND.

Simultaneous determination of bilirubin and its glucuronides in liver microsomes and recombinant UGT1A1 enzyme incubation systems by HPLC method and its application to bilirubin glucuronidation studies

Bilirubin, an important endogenous substances and liver function index in humans, is primarily eliminated via UGT1A1-catalyzed glucuronidation. Instability of bilirubin and its glucuronides brings substantial technical challenges to conduct in vitro bilirubin glucuronidation assay. In the present study, we developed a simple and robust HPLC method for simultaneous determination of unconjugated bilirubin (UCB) and its multiple glucuronides, i.e. bilirubin monoglucuronides (BMGs, including BMG1 and BMG2 isomers) and diglucuronide (BDG) in rat liver microsomes (RLM), human liver microsomes (HLM) and recombinant human UGT1A1 enzyme (UGT1A1) incubation systems, and applied it to study in vitro bilirubin glucuronidation. UCB, BMG1, BMG2, BDG and their isomers in the incubation mixtures were successfully separated using a C18 column with UV detection at 450nm and mobile phase consisted of 0.1% formic acid in water and acetonitrile by a linear gradient elution program. Assay linearities of bilirubin were confirmed in the range 0.01–2μM. Precision of UCB, BMG1, BMG2 and BDG (n=5) at low, medium and high concentration was within the range of RSD 0.4–3.7%, accuracy expressed in the mean assay recoveries of them (n=5) ranged from 92.8±1.5% to 104.3±2.2% for intra- and inter-day assays and the mean extraction recoveries of them (n=5) were above 91.5±1.0%. Stability of bilirubin and its glucuronides was satisfactory at 37°C in the incubation solutions during the reaction (30min), 25°C for 24h and −70°C for 7d in the processed incubation samples with methanol. Furthermore, we established stable, reliable in vitro incubation systems and optimized the incubation conditions to characterize the kinetics of bilirubin glucuronidation by RLM, HLM and UGT1A1, respectively. The kinetic parameters of formation of total bilirubin glucuronides (TBG, the sum of BMG1, BMG2 and BDG) were as follows: Km of 0.45±0.016, 0.40±0.022, 0.44±0.018μM, Vmax of 2.65±0.057, 1.86±0.029, 2.95±0.036nmol/mg/min, CLint of 5.92±0.22, 4.70±0.079, 6.72±0.27mL/mg/min by RLM, HLM and UGT1A1, respectively. Bilirubin glucuronidation obeyed the Hill equation by RLM and the Michaelis–Menten equation by HLM and UGT1A1 in the range of substrate concentration selected, respectively. In addition, the relative proportions between BDG and BMGs were in connection with enzyme sources (e.g. RLM, HLM and UGT1A1) and bilirubin concentration.

The Roles of MRP2, MRP3, OATP1B1, and OATP1B3 in Conjugated Hyperbilirubinemia

Increased concentrations of bilirubin glucuronides in blood plasma indicate hepatocellular dysfunction. Elucidation of the transport processes of bilirubin conjugates across the basolateral (sinusoidal) and the canalicular plasma membrane domains of hepatocytes has decisively contributed to our current understanding of the molecular basis of conjugated hyperbilirubinemia in human liver diseases. Under normal conditions, unconjugated bilirubin is taken up into hepatocytes by transporters of the organic anion-transporting polypeptide (OATP) family, followed by conjugation with glucuronic acid, and ATP-dependent transport into bile. This efflux across the canalicular membrane is mediated by multidrug resistance protein 2 (MRP2 or ABCC2), which is a 190-kDa glycoprotein transporting with high affinity and efficiency monoglucuronosyl bilirubin and bisglucuronosyl bilirubin into bile. MRP2 is hereditarily deficient in human Dubin-Johnson syndrome. Under pathophysiological conditions such as cholestatic liver injury and MRP2 inhibition, the basolateral efflux pump multidrug resistance protein 3 (MRP3 or ABCC3) is responsible for the occurrence of conjugated hyperbilirubinemia. MRP3 is a glycoprotein with a similar molecular mass as MRP2, with 48% amino acid identity, and with overlapping substrate specificity. Human MRP3 is the only basolateral efflux pump shown to transport bilirubin glucuronides. In human and rat hepatocytes, MRP3/Mrp3 is strongly upregulated under conditions of cholestasis and MRP2 deficiency. This is in line with the concept that basolateral efflux pumps of the hepatocyte compensate for impaired canalicular efflux of compounds into bile and contribute to balance the rate of uptake or synthesis of compounds in hepatocytes with the capacity for efflux into bile.

Indications for Laparoscopic Cholecystectomy or Oral Dissolution Therapy with Ursodeoxycholic Acid in Symptomatic Gallstone Disease

Unconjugated bilirubin plays an important role in cholesterol gallstone formation. Patients with symptomatic gallstone disease who have high bilirubin plasma levels and/or are homozygous for the rs6742078 TT variant of the bilirubin glucuronidating gene UGT1A1 should not undergo oral dissolution therapy with ursodeoxycholic acid. A large Danish study has shown that high bilirubin plasma levels and the genetic variant rs6742078 TT of the enzyme bilirubin glucuronidase UGT1A1 are associated with an increased risk of developing symptomatic gallstone disease. Recent reports regarding the significant association between bilirubin levels and symptomatic gallstone disease open a new chapter about the indication and exclusion criteria for oral dissolution therapy of symptomatic gallstone disease. A highly select subgroup of patients with small, single, radiolucent cholesterol gallstones who received oral dissolution therapy with ursodeoxycholic acid (UDCA) had a reported recurrence of symptomatic gallstone disease of 50% over five years. This is probably related to the persistence of other causal risk factors for gallstones in addition to that of cholesterol suprasaturation. A subgroup of patients with high plasma bilirubin levels and the UGT1A1 genetic variant rs6742078 have a greater risk of recurrence. In conclusion, oral dissolution therapy with UDCA might still be appropriate for patients that refuse laparoscopic cholecystectomy provided they have small (< 0.5 cm), radiolucent cholesterol gallstones and a functioning gallbladder, and have mean plasma bilirubin levels below 1.33 mg/dL and are not homozygous for the UGT1A1 rs6742078 TT genotype.

Therapeutic uses of animal biles in traditional Chinese medicine: an ethnopharmacological, biophysical chemical and medicinal review.

Forty-four different animal biles obtained from both invertebrates and vertebrates (including human bile) have been used for centuries for a host of maladies in traditional Chinese medicine (TCM) beginning with dog, ox and common carp biles approximately in the Zhou dynasty (c. 1046-256 BCE). Overall, different animal biles were prescribed principally for the treatment of liver, biliary, skin (including burns), gynecological and heart diseases, as well as diseases of the eyes, ears, nose, mouth and throat. We present an informed opinion of the clinical efficacy of the medicinal uses of the different animal biles based on their presently known principal chemical components which are mostly steroidal detergent-like molecules and the membrane lipids such as unesterified cholesterol and mixed phosphatidylcholines and sometimes sphingomyelin, as well as containing lipopigments derived from heme principally bilirubin glucuronides. All of the available information on the ethnopharmacological uses of biles in TCM were collated from the rich collection of ancient Chinese books on materia medica held in libraries in China and United States and the composition of various animal biles was based on rigorous separatory and advanced chemical identification techniques published since the mid-20(th) century collected via library (Harvard's Countway Library) and electronic searches (PubMed and Google Scholar). Our analysis of ethnomedical data and information on biliary chemistry shows that specific bile salts, as well as the common bile pigment bilirubin and its glucuronides plus the minor components of bile such as vitamins A, D, E, K, as well as melatonin (N-acetyl-5-methoxytryptamine) are salutary in improving liver function, dissolving gallstones, inhibiting bacterial and viral multiplication, promoting cardiac chronotropsim, as well as exhibiting anti-inflammatory, anti-pyretic, anti-oxidant, sedative, anti-convulsive, anti-allergic, anti-congestive, anti-diabetic and anti-spasmodic effects. Pig, wild boar and human biles diluted with alcohol were shown to form an artificial skin for burns and wounds one thousand years ago in the Tang dynasty (618-907 CE). Although various animal biles exhibit several generic effects in common, a number of biles appear to be advantageous for specific therapeutic indications. We attempt to understand these effects based on the pharmacology of individual components of bile as well as attempting to identify a variety of future research needs.

Polyinosinic Acid Blocks Adeno-Associated Virus Macrophage EndocytosisIn Vitroand Enhances Adeno-Associated Virus Liver-Directed Gene TherapyIn Vivo

Adeno-associated virus serotype 8 (AAV8) has been demonstrated to be effective for liver-directed gene therapy in humans. Although hepatocytes are the main target cell for AAV8, there is a loss of the viral vector because of uptake by macrophages and Kupffer cells. Reducing this loss would increase the efficacy of viral gene therapy and allow a dose reduction. The receptor mediating this uptake has not been identified; a potential candidate seems the macrophage scavenger receptor A (SR-A) that is involved in the endocytosis of, for instance, adenovirus. In this study we show that SR-A can mediate scAAV8 endocytosis and that blocking it with polyinosinic acid (poly[i]) reduces endocytosis significantly in vitro. Subsequently, we demonstrate that blocking this receptor improves scAAV-mediated liver-directed gene therapy in a model for inherited hyperbilirubinemia, the uridine diphospho-glucuronyl transferase 1A1-deficient Gunn rat. In male rats, preadministration of poly[i] increases the efficacy of a low dose (1×10¹¹ gc/kg) but not of a higher dose (3×10¹¹ gc/kg) scAAV8-LP1-UT1A1. Administration of poly[i] just before the vector significantly increases the correction of serum bilirubin in female rats. In these, the effect of poly[i] is seen by both doses but is more pronounced in the females receiving the low vector, where it also results in a significant increase of bilirubin glucuronides in bile. In conclusion, this study shows that SR-A mediates the endocytosis of AAV8 in vitro and in vivo and that blocking this receptor can improve the efficacy of AAV-mediated liver-directed gene therapy.

Evidence for epigenetic regulation of UGT1A1 protein expression and activity in healthy human livers

Interindividual variability in UDP‐glucuronosyltransferase 1A1 (UGT1A1) is only partially explained by genetic polymorphisms and enzyme inducers. Here we determined whether cytosine methylation explains an additional proportion of this variability in human liver. UGT1A1 phenotypes, including UGT1A1 protein and bilirubin glucuronidation, and UGT1A1*28 were determined using a human liver bank (n=46). Methylation levels were quantified at 5 CpG sites associated with known transcription factor response elements in the UGT1A1 promoter and distal enhancer, as well as a CpG‐rich island 1.5 kb further upstream. Individual CpG sites showed considerable methylation variability ranging from 10‐ to 29‐fold difference with average methylation levels from 25 to 41%. Multivariate regression analysis identified *28/*28 genotype, −4 CpG site methylation, and alcohol history as significant predictors of UGT1A1 protein content, accounting for 12%, 11%, and 9% of the variability (R2=0.32, p=0.003). Similarly, −4 CpG methylation and alcohol history accounted for 13% and 12% of the variability in bilirubin glucuronidation (R2=0.25, p=0.005). These results suggest that differential methylation of the −4 CpG site located within a known USF response element may explain a proportion of variability in hepatic glucuronidation by UGT1A1. Funded by NIH‐R01‐GM061834 (to MHC) and NIH‐N01‐DK‐7–0004/ HHSN267200700004C.