BACKGROUND

Drug-induced liver injury (DILI) is a major clinical problem and its pathogenesis is poorly understood. The association of DILI with polymorphisms in hepatobiliary transport systems suggests a role for transport proteins in the pathogenesis of DILI.

OBJECTIVE

To investigate expression and tissue distribution of hepatobiliary transport systems in DILI.

METHODS

Expression of the canalicular bile salt export pump BSEP (ABCC11), phospholipid flippase MDR3 (ABCB4) and bilirubin export pump MRP2 (ABCC2) was assessed immunohistochemically in liver biopsies from 23 patients with DILI.

RESULTS

Of 12 patients with cholestatic DILI (mostly due to antibiotics), 8 displayed a marked reduction of MRP2, MDR3 and BSEP expression. Transporter staining was normal in 4 patients with cholestatic DILI. In 11 patients with necroinflammatory hepatocellular injury (most frequently caused by NSAIDs), transporter staining was normal in areas where hepatocyte morphology was preserved. Due to hepatocyte necrosis and the reduction of the hepatocyte number, overall transporter expression was reduced without a reduction in transporter expression at the single hepatocyte level.

CONCLUSIONS

Canalicular ABC transporter expression is profoundly disturbed in most cases of cholestatic DILI. Drug-induced hepatitis does not lead to repression of transporter expression but to hepatocyte drop-outs with a numerical loss of bile canaliculi.

BACKGROUND

The Wnt/β-catenin signaling pathway plays a crucial role in embryonic development, tissue homeostasis, wound healing and malignant transformation in different organs including the liver. The consequences of continuous β-catenin signaling in hepatocytes remain elusive.

RESULTS

Livers of Ctnnb1CA hep mice were characterized by disturbed liver architecture, proliferating cholangiocytes and biliary type of fibrosis. Serum ALT and bile acid levels were significantly increased in Ctnnb1CA hep mice. The primary bile acid synthesis enzyme Cyp7a1 was increased whereas Cyp27 and Cyp8b1 were reduced in Ctnnb1CA hep mice. Expression of compensatory bile acid transporters including Abcb1, Abcb4, Abcc2 and Abcc4 were significantly increased in Ctnnb1CA hep mice while Ntcp was reduced. Accompanying changes of bile acid transporters favoring excretion of bile acids were observed in intestine and kidneys of Ctnnb1CA hep mice. Additionally, disturbed bile acid regulation through the FXR-FGF15-FGFR4 pathway was observed in mice with activated β-catenin.

METHODS

Mice with a loxP-flanked exon 3 of the Ctnnb1 gene were crossed to Albumin-Cre mice to obtain mice with hepatocyte-specific expression of a dominant stable form of β-catenin (Ctnnb1CA hep mice). Ctnnb1CA hep mice were analyzed by histology, serum biochemistry and mRNA profiling.

CONCLUSIONS

Expression of a dominant stable form of β-catenin in hepatocytes results in severe cholestasis and biliary type fibrosis.

The occurrence of cholestasis during pregnancy may be due to several disorders. These include pregnancyspecific diseases, like intrahepatic cholestasis of pregnancy(ICP), as well as to other causes such as oligosymptomatic choledocolitiasis, viral hepatitis and other underlying liver disorders like primary biliary cirrhosis. In recent years, the discovery of mutations in hepatobiliary transporters genes responsible of some rare forms of genetic cholestasis have led to the study of this mutations in pregnant women with cholestasis. Thus, mutations in the hepatic phospholipid transporter(MDR3, ABCB4), in the aminophospholipid transporter ATP8B1 and in the bile salt export pump (BSEP, ABCB11) have been found in patients diagnosed as ICP. However, patients included in these studies belong to a heterogeneous population, which may not represent true cases of ICP since some reports include patients diagnosed in the first trimester of pregnancy, with elevated serum levels of gamma-glutamyl transpeptidase and clear evidence of chronic liver disease. Thus, consideration must be given to the possibility of other rare underlying hepatic disorders may be unmasked during pregnancy with cholestasis as its first manifestation.

Multidrug resistance protein 3 (MDR3) is a hepatocyte canalicular membrane protein encoded by the ABCB4/MDR3 gene located on chromosome 7. Several liver diseases are known to be associated with MDR3 deficiency. The basic defect is reduced secretion of biliary phospholipid causing disturbance in the primary bile composition, leading to injury to biliary epithelium inducing cell death and inflammation. Severe MDR3 deficiency typically presents during the first year of life or early childhood, often progressing to chronic liver disease with cirrhosis and portal hypertension, requiring liver transplantation. Negative MDR3 immunostaining is suggestive of MDR3 deficiency. Herein, we report the clinical and histopathologic features of 10 cases (6 male/4 female) in infants and children with severe MDR3 deficiency (age range of 8 months to 7 years) diagnosed with negative MDR3 immunostaining in hepatic canaliculi. Three cases underwent liver transplantation. The cases showed periportal bridging fibrosis to micronodular cirrhosis, ductular proliferation with bile plugs, and lobular canalicular bile stasis with rosetting. All 3 explant livers demonstrated cystically dilated large ducts with crystallization of cholesterol. One case showed well-differentiated hepatocellular carcinoma. We conclude that MDR3 immunostaining on formalin-fixed and paraffin-embedded sections is a useful tool to diagnose severe MDR3 deficiency in pediatric liver cholestatic disease cases where genetic testing is not available.

We report the case of a 40-years-old female patient with recurrent cholestatic liver disease who presented twice with severe intrahepatic cholestasis of pregnancy and pronounced choledocholithiasis between pregnancies. Bile duct stones were removed endoscopically and a laparoscopic cholecystectomy was performed after the second pregnancy. Liver histology revealed intrahepatic cholestasis with portal inflammation and fibrosis, resembling progressive familial intrahepatic cholestasis (PFIC). Molecular genetic studies identified the heterozygous mutation c.957C>> T in the ABCB4 gene encoding the hepatobiliary phospholipid transporter. This is the first report of this mutation that introduces a stop codon in an index patient with intrahepatic cholestasis of pregnancy and multiple bile duct stones. In addition, we detected the ABCB11 polymorphism V 444A, which is associated with a decreased expression of the bile salt export pump. Whereas homozygous carriers of the ABCB4 mutation develop PFIC type 3, the heterozygous ABC transporter mutations represent genetic risk factors for cholelithiasis and recurrent cholestatic hepatitis upon challenge with oral contraceptives or during pregnancy. Of note, the patient presented with normal serum gamma-glutamyltranspeptidase activities during pregnancy-associated cholestatic episodes but normal liver enzymes after delivery, whereas choledocholithiasis was associated with high gamma-glutamyl transpeptidase levels. It is unknown whether ursodeoxycholic acid prevents cholestasis or gallstones in patients with ABCB4 deficiency.

OBJECTIVE

To estimate the frequency of mutations involving exons 6, 8 and 9 of Adenosine triphosphate-binding cassette, subfamily B, member 4 (ABCB4) gene among children with progressive intrahepatic cholestasis with high γ-GT activity (PFIC3).

METHODS

Cross sectional study was conducted on 30 children with PFIC3. Genotyping was performed by sequencing analysis of exons 6, 8 and exon 9 of ABCB4 gene.

RESULTS

Heterozygous synonymous polymorphic variant was detected in exon 6 (rs 1202283) and in exon 8 (rs 2109505). No mutations in studied exons were detected.

CONCLUSIONS

Exons 6, 8 and 9 mutations of ABCB4 gene are not common among Egyptian children with PFIC3.

Background: Intrahepatic cholestasis of pregnancy (ICP) is characterized by pruritus and cholestasis in late pregnancy and results in adverse pregnancy outcomes, including preterm delivery and birth weight, which are affected by the genetic and environmental background. However, until now, the genetic architecture of ICP has remained largely unclear.

Methods: Twenty-six clinical data points were recorded for 151 Chinese ICP patients. The data generated from whole-exome sequencing (WES) using the BGISEQ-500 platform were further analyzed by Burrows-Wheeler Aligner (BWA) software, Genome Analysis Toolkit (GATK), ANNOVAR tool, etc. R packages were used to conduct t-test, Fisher's test and receiver operating characteristic (ROC) curve analyses.

Results: We identified eighteen possible pathogenic loci associated with ICP disease in known genes, covering ABCB4, ABCB11, ATP8B1 and TJP2. The loci Lys386Gln, Gly527Gln and Trp708Ter in ABCB4, Leu589Met, Gln605Pro and Gln1194Ter in ABCB11, and Arg189Ser in TJP2 were novel discoveries. In addition, WES analysis indicated that the gene ANO8 involved in the transport of bile salts is newly identified as associated with ICP. The functional network of the ANO8 gene confirmed this finding. ANO8 contained 8 rare missense mutations that were found in eight patients among the 151 cases and were absent from 1029 controls. Out of the eight SNPs, 3 were known, and the remaining five are newly identified. These variants have a low frequency, ranging from 0.000008 to 0.00001 in the ExAC, gnomAD - Genomes and TOPMED databases. Bioinformatics analysis showed that the sites and their corresponding amino acids were both highly conserved among vertebrates. Moreover, the influences of all the mutations on protein function were predicted to be damaging by the SIFT tool. Combining clinical data, it was found that the mutation group (93.36 µmol/L) had significantly (P = 0.038) higher total bile acid (TBA) levels than the wild-type group (40.81 µmol/L).

Conclusions: To the best of our knowledge, this is the first study to employ WES technology to detect genetic loci for ICP. Our results provide new insights into the genetic basis of ICP and will benefit the final identification of the underlying mutations.

Keywords: ANO8; Intrahepatic cholestasis of pregnancy; Mutations; Whole-exome sequencing.

The ABCB gene subfamily of ABC (ATP-binding cassette) transporters is responsible for transporting a wide spectrum of molecules including peptides, iron, bile salts, drugs, and phospholipids. In humans, ABCB4 appears to be exclusively expressed on the apical membrane of hepatocytes where it translocates phosphatidylcholine from the inner to the outer leaflet of the canalicular membrane. Functional alterations in the ABCB4 transporter are associated with a number of cholestatic syndromes in humans. Because of its role in biliary lipid homeostasis in humans, investigation of the ABCB4 gene in dogs is warranted. Thus, the full cDNA sequence of canine ABCB4 was elucidated and its mRNA and protein expression levels in tissues were determined. Canine ABCB4 consists of 3804 nucleotides spanning 26 exons and is 89% identical to human ABCB4. Expression of ABCB4 in canine liver supports a potential role for the protein in normal biliary function similar to that in humans. The function of ABCB4 expressed in brain tissue has yet to be determined.

Clinical studies demonstrated that nonalcoholic steatohepatitis is associated with liver-related outcomes in chronic hepatitis B. Furthermore, primary biliary fibrosis and biliary atresia occurred in patients with HBV infection. Interestingly, hepatitis B virus surface protein (HBs) transgenic mice spontaneously develop hepatic steatosis. Our aim is to investigate the effect of Abcb4 knockout-induced cholestasis on liver steatosis in HBs transgenic mice. Hybrids of HBs transgenic and Abcb4^-/- mice were bred on the BALB/c genetic background. Lipid synthesis, storage, and catabolism as well as proteins and genes that control lipid metabolism were analyzed using HPTLC, qPCR, western blot, electrophoretic mobility shift assay (EMSA), lipid staining, and immunohistochemistry. Hepatic neutral lipid depots were increased in HBs transgenic mice and remarkably reduced in Abcb4^-/- and HBs/Abcb4^-/- mice. Similarly, HPTLC-based quantification analyses of total hepatic lipid extracts revealed a significant reduction in the amount of triacylglycerols (TAG), while the amount of free fatty acids (FFA) was increased in Abcb4^-/- and HBs/Abcb4^-/- in comparison to wild-type and HBs mice. PLIN2, a lipid droplet-associated protein, was less expressed in Abcb4^-/- and HBs/Abcb4^-/-. The expression of genes-encoding proteins involved in TAG synthesis and de novo lipogenesis (Agpat1, Gpat1, Mgat1, Dgat1, Dgat2, Fasn, Hmgcs1, Acc1, Srebp1-c, and Pparγ) was suppressed, and AMPK and CREB were activated in Abcb4^-/- and HBs/Abcb4^-/- compared to wild-type and HBs mice. Simulating cholestatic conditions in cell culture resulted in AMPK and CREB activation while FASN and PLIN2 were reduced. A concurrent inhibition of AMPK signaling revealed normal expression level of FASN and PLIN2, suggesting that activation of AMPK-CREB signaling regulates hepatic lipid metabolism, i.e. synthesis and storage, under cholestatic condition. In conclusions, in vivo and mechanistic in vitro data suggest that cholestasis reduces hepatic lipid storage via AMPK and CREB signaling. The results of the current study could be the basis for novel therapeutic strategies as NASH is a crucial factor that can aggravate chronic liver diseases.

Clinical and epidemiological findings implicate genetic predisposition and the effects of elevated steroids in pregnancy in the pathogenesis of intrahepatic cholestasis of pregnancy. To date, a number of studies have identified polymorphisms encoding biliary canalicular transporters, including those encoded by ABCB4 and ABCB11, which are associated with intrahepatic cholestasis of pregnancy. Questions remain regarding divergent findings between populations and the relative contributions of these polymorphisms. In a large study of Western European women with intrahepatic cholestasis of pregnancy, Dixon et al. (this issue) provide further insights into the genetics of this cholestatic syndrome, which contribute to ongoing evaluation of cholestasis generally.

In the past decade the presence of psychopharmaceuticals, including fluoxetine (FLU), in the aquatic environment has been associated with the increasing trend in human consumption of these substances. Aquatic organisms are usually exposed to chronic low doses and, therefore, risk assessments should evaluate the effects of these compounds in non-target organisms. Teleost fish possess an array of active defence mechanisms to cope with the deleterious effects of xenobiotics. These include ABC transporters, phase I and II of cellular detoxification and oxidative stress enzymes. Hence, the present study aimed at characterising the effect of FLU on embryo development of the model teleost zebrafish (Danio rerio) concomitantly with changes in the detoxification mechanisms during early developmental phases. Embryos were exposed to different concentrations of FLU (0.0015, 0.05, 0.1, 0.5 and 0.8μM) for 80hours post fertilization. Development was screened and the impact in the transcription of key genes, i.e., abcb4, abcc1, abcc2, abcg2, cyp1a, cyp3a65, gst, sod, cat, ahr, pxr, pparα, pparβ, pparγ, rxraa, rxrab, rxrbb, rxrga, rxrgb, raraa, rarab, rarga evaluated. In addition, accumulation assays were performed to measure the activity of ABC proteins and antioxidant enzymes (CAT and Cu/ZnSOD) after exposure to FLU. Embryo development was disrupted at the lowest FLU concentration tested (0.0015μM), which is in the range of concentrations found in WWTP effluents. Embryos exposed to higher concentrations of FLU decreased Cu/Zn SOD, and increased CAT (0.0015 and 0.5μM) enzymatic activity. Exposure to higher concentrations of FLU decreased the expression of most genes belonging to the detoxification system and upregulated cat at 0.0015μM of FLU. Most of the tested concentrations downregulated pparα, pparβ, pparγ, and raraa, rxraa, rxrab, rxrbb rxrgb and ahr gene expression while pxr was significantly up regulated at all tested concentrations. In conclusion, this study shows that FLU can impact zebrafish embryo development, at concentrations found in effluents of WWTPs, concomitantly with changes in antioxidant enzymes, and the transcription of key genes involved in detoxification and development. These finding raises additional concerns supporting the need to monitor the presence of this compound in aquatic reservoirs.

The genes encoding drug-metabolizing enzymes and transporters play an important role in maintaining the normal life processes of human body. Their disorder or defect will lead to the occurrence and development of various diseases. Currently, most of studies have focused on genetic variations in these genes, however, in the present study, we analyzed promoter methylation of 11 drug metabolism and transport genes in a cohort of nodular goiter and normal thyroid tissues using methylation-specific PCR (MSP). Our data first revealed a distinct methylation profiling in drug metabolism and transport genes between nodular goiter and normal thyroid tissues, particularly ABCB4, CYP1B1 and CYP24A1 and SLC1A2. Given these genes contribute to the development and progression of various diseases, such as multidrug resistance and tumorigenesis, these epigenetic events may thus play a critical role in the pathogenesis of nodular goiter.

Bile acid imbalance causes progressive familial intrahepatic cholestasis type 2 (PFIC2) or type 3 (PFIC3) - severe liver diseases associated with genetic defects in the biliary bile acid transporter BSEP (ABCB11), or phosphatidylcholine transporter MDR3 (ABCB4), respectively. Mdr2 -/- mice (a PFIC3 model) develop progressive cholangitis, ductular proliferation, periportal fibrosis, and hepatocellular carcinoma (HCC) because the non-micelle-bound bile acids in the bile of these mice are toxic. We asked whether the highly hydrophilic bile acids generated by Bsep -/- mice could protect Mdr2 -/- mice from progressive liver damage. We generated double knockout (DKO: Bsep -/- and Mdr2 -/-) mice. Their bile acid composition resembles that of Bsep-/- mice, with increased hydrophilic muricholic acids, tetrahydroxylated bile acids (THBA), and reduced hydrophobic cholic acid. These mice lack the liver pathology of their Mdr2 -/- littermates. The livers of DKO mice have gene expression profiles very similar to Bsep -/- mice, with 4410 out of 6134 gene expression changes associated with the Mdr2 -/- mutation being suppressed. Feeding with THBA partially alleviates liver damage in the Mdr2 -/- mice. Conclusion: Hydrophilic changes to biliary bile acid composition, including introduction of THBA, can prevent the progressive liver pathology associated with the Mdr2 -/- (PFIC3) mutation.

OBJECTIVE

Metabolism and transport play major roles in life-long exposure to endogenous and exogenous carcinogens. We therefore explored associations between polymorphisms in absorption, distribution, metabolism and elimination genes and the risk and prognosis of castration-resistant prostate cancer (CRPC).

METHODS

A total of 634 genotypes were tested in 74 patients using the Affymetrix DMETv1.0 platform.

RESULTS

No relation to risk was found. Three SNPs were associated with CRPC prognosis in Caucasians: ABCB11 rs7602171G>A (p = 0.003; n = 30; hazard ratio [HR]: 0.307), GSTP1 rs1799811C>T (p = 0.001; n = 38; HR: 0.254) and SLC5A6 rs1395 (p = 0.004; n = 35; HR: 3.15). Two other polymorphisms among Caucasians were associated with interesting trends: ABCB4 rs2302387C>T (p = 0.039) and ABCC5 rs939339A>G (p = 0.018).

CONCLUSIONS

This exploratory study is the first to show that polymorphisms in several absorption, distribution, metabolism and elimination genes may be associated with CRPC prognosis.

The development of drug resistance is one of the main causes of failure in anti-cancer treatments. Tumor cells adopt many strategies to counteract the action of chemotherapeutic agents, e.g., enhanced DNA damage repair, inactivation of apoptotic pathways, alteration of drug targets, drug inactivation, and overexpression of ABC (Adenosine triphosphate-binding cassette, or ATP-binding cassette) transporters. These are broad substrate-specificity ATP-dependent efflux pumps able to export toxins or drugs out of cells; for instance, ABCB1 (MDR1, or P-glycoprotein 1), overexpressed in most cancer cells, confers them multidrug resistance (MDR). The gene coding for sorcin (SOluble Resistance-related Calcium-binding proteIN) is highly conserved among mammals and is located in the same chromosomal locus and amplicon as the ABC transporters ABCB1 and ABCB4, both in human and rodent genomes (two variants of ABCB1, i.e., ABCB1a and ABCB1b, are in rodent amplicon). Sorcin was initially characterized as a soluble protein overexpressed in multidrug (MD) resistant cells and named "resistance-related" because of its co-amplification with ABCB1. Although for years sorcin overexpression was thought to be only a by-product of the co-amplification with ABC transporter genes, many papers have recently demonstrated that sorcin plays an important part in MDR, indicating a possible role of sorcin as an oncoprotein. The present review illustrates sorcin roles in the generation of MDR via many mechanisms and points to sorcin as a novel potential target of different anticancer molecules.

As a maternal and developmental toxicant, cadmium (Cd) possesses weak penetrability through the placental barrier. However, the underlying mechanism remains unclear. To gain insight into the protein molecules associated with Cd toxicity in placenta and explore their roles in Cd transportation, a reproductive animal experiment was carried out using Sprague-Dawley rats. We performed proteomic analysis of the placenta by Difference Gel Electrophoresis (DIGE) combined with Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Tandem Mass Spectroscopy (MALDI-TOF/TOF MS). The DIGE assay identified 15 protein spots that were differentially expressed with a greater than 1.5-fold change in placenta of Cd-treated rats compared to the control rats. Based on the expression patterns and biological functions of the proteins, we selected the ABCG2 and ABCB4 transporter proteins for further analysis. Western blot analysis showed that Cd exposure could down-regulate the expression of ABCG2 and ABCB4 in the placenta. There was a negative dose-response relationship between Cd exposure and the expression of ABCG2 or ABCB4 protein. These results indicated that down-regulation of ABCG2 and ABCB4 transporters may regulate Cd across through placenta and thus affect the in vivo toxic effect of Cd to fetus.

Biliary lipids consist mainly of bile salts, phospholipids and cholesterol, which form mixed micelles and vesicles. Bile salts play various physiological roles but have damaging effects on cell membranes due to their detergent properties. The cytotoxicity of bile salts on hepatocytes leads to liver injuries and is largely determined by the bile salt species, the concentrations of bile salts, phospholipids and cholesterol, and the lipid composition of cell membranes. In bile, monomers and simple micelles of bile salts coexist with mixed micelles and vesicles in dynamic equilibrium, and contribute to the cytotoxicity on hepatocytes. The ATP-binding cassette (ABC) transporter family members, ABCB11, ABCB4 and ABCG5/ABCG8, mediate the biliary secretion of bile salts, phospholipids and cholesterol, respectively. Mutations in ABCB4 result in severe cholestatic diseases, and the biliary phospholipids are necessary for the attenuation of bile salt cytotoxicity. On the other hand, cholesterol reverses the cytoprotective effects of phospholipids against bile salts. In addition, phosphatidylethanolamine N-methyltransferase increases the cell resistance to bile salts by changing the phospholipid composition and structures of the apical membranes. In this review, we focus on the molecular mechanisms for the protection of hepatocytes against bile salt cytotoxicity. Further understanding of these mechanisms will help to develop new therapeutic strategies for cholestatic liver diseases.

The aim of the current study was to clarify the effect of high sucrose diet (HSD) on bile formation (BF) in rats with hereditary hypertriglyceridemia (HHTg). Potentially positive effects were studied for boldine, a natural choleretic agent. Administration of HSD to HHTg rats led to increased triglyceride deposition in the liver. HSD reduced BF as a consequence of decreased biliary secretion of bile acids (BA) and glutathione. Responsible mechanism was down-regulation of hepatic transporters for BA and glutathione, Bsep and Mrp2, respectively. Moreover, gene expressions of transporters for other constituents of bile, namely Abcg5/8 for cholesterol, Abcb4 for phospholipids, and Oatp1a4 for xenobiotics, were also reduced by HSD. Boldine partially attenuated cholestatic effect of HSD by promotion of biliary secretion of BA through up-regulation of Bsep and Ntcp, and by increase in biliary secretion of glutathione as a consequence of its increased hepatic disposition. This study demonstrates mechanisms of impaired BF during nonalcoholic fatty liver disease induced by HSD. Altered function of responsible transporters suggests also potential for changes in kinetics of drugs, which may complicate pharmacotherapy in subjects with high intake of sucrose, and with fatty liver disease. Sucrose induced alterations in BF may be alleviated by administration of boldine.

ABCB4 is an ATP-binding cassette transporter that extrudes phosphatidylcholine into the bile canaliculi of the liver. Its dysfunction or inhibition by drugs can cause severe, chronic liver disease or drug-induced liver injury. We determined the cryo-EM structure of nanodisc-reconstituted human ABCB4 trapped in an ATP-bound state at a resolution of 3.2 Å. The nucleotide binding domains form a closed conformation containing two bound ATP molecules, but only one of the ATPase sites contains bound Mg²⁺. The transmembrane domains adopt a collapsed conformation at the level of the lipid bilayer, but we observed a large, hydrophilic and fully occluded cavity at the level of the cytoplasmic membrane boundary, with no ligand bound. This indicates a state following substrate release but prior to ATP hydrolysis. Our results rationalize disease-causing mutations in human ABCB4 and suggest an 'alternating access' mechanism of lipid extrusion, distinct from the 'credit card swipe' model of other lipid transporters.

Background&aims: Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a rare lethal autosomal recessive liver disorder caused by loss-of-function variations of the ABCB4 gene, encoding a phosphatidylcholine transporter (ABCB4/MDR3). Currently, no effective treatment exists for PFIC3 outside of liver transplantation.

Methods: We have produced and screened chemically- and genetically-modified mRNA variants encoding human ABCB4 (hABCB4 mRNA) encapsulated in lipid nanoparticles (LNPs). We examined their pharmacological effects in a cell-based and in a new in vivo mouse model resembling human PFIC3 due to homozygous disruption of the Abcb4 gene in fibrosis-susceptible BALB/c.Abcb4^-/- mice.

Results: We show that treatment with liver-targeted hABCB4 mRNA resulted in de novo expression of functional hABCB4 protein and restored phospholipid transport in cultured cells and in PFIC3 mouse livers. Importantly, repeated injections of the hABCB4 mRNA effectively rescued the severe disease phenotype in young Abcb4^-/- mice, with rapid and dramatic normalization of all clinically relevant parameters such as inflammation, ductular reaction and liver fibrosis. Synthetic mRNA therapy also promoted favorable hepatocyte-driven liver regeneration to restore normal homeostasis, including liver weight, body weight, liver enzymes, and portal vein blood pressure.

Conclusion: Our data provide strong preclinical proof-of-concept for hABCB4 mRNA therapy as a potential treatment option for patients with PFIC3.

Keywords: cholangiopathy; cholangitis; congenital biliary cirrhosis; gene therapy; liver fibrosis.

Microcystin-LR (MC-LR) is one of the most common microcystins (MCs), which are hepatotoxic and released into a water body during a period of cyanobacterial blooms. These toxicants can be accumulated in aquatic animals and transferred along the food chain and thus pose adverse effects on aquatic environment and public health. Zebrafish Abcb4 is reported to mediate the cellular efflux of ecotoxicologically relevant compounds including galaxolide, tonalide and phenanthrene; however, it remains unclear whether Abcb4 functions in the detoxification of MC-LR. Here, we demonstrated the role of zebrafish Abcb4 in cellular efflux of MC-LR. Transcripts of zebrafish abcb4 were detected in all of adult tissues examined. MC-LR was able to induce the expression of abcb4 gene and overexpression of Abcb4 significantly decreased the cytotoxicity and accumulation of MC-LR in LLC-PK1 cells and developing embryos. In contrast, overexpression of an Abcb4-G1177D mutant abolished its transporter function but not substrate binding activity, and sensitized LLC-PK1 cells and developing embryos to this cyanobacterial toxin. Moreover, ATPase activity in developing embryos can be induced by MC-LR. Thus, zebrafish Abcb4 plays crucial roles in cellular efflux of MC-LR and is a potential molecular marker for the monitoring of cyanobacteria contamination in the aquatic environment.

Altered bile acid (BA) signaling is associated with hepatotoxicity. The farnesoid X receptor (FXR) is a nuclear receptor that transcriptionally regulates BA homeostasis. Mice with FXR ablation present hepatocarcinoma (HCC) due to high toxic BA levels. Mice with Abcb4 ablation accumulate toxic BA within the bile ducts and present HCC. We have previously shown that intestinal specific activation of FXR by transgenic VP16-FXR chimera is able to reduce BA pool size and prevent HCC. Here we tested chemical FXR activation by administering for 15 months the dual FXR/ membrane G protein-coupled receptor (TGR5) agonist INT-767 (6α-ethyl-3α,7α,23-trihydroxy-24-nor-5β-cholan-23-sulphate) to Fxr-/- and Abcb4-/- mice. HCC number and size were significantly reduced by INT-767 administration. In contrast, no changes in HCC tumor number and size were observed in Fxr-/- mice fed with or without INT-767. Notably, INT-767 preserved the hepatic parenchyma, improved hepatic function and down-regulated pro-inflammatory cytokines. Moreover, in Abcb4-/- mice INT-767 prevented fibrosis by reducing collagen expression and deposition. Thus, long term activation of FXR is able to reduce BA pool, reprogram BA metabolism and prevent HCC. These data provide the impetus to address the bona fide therapeutic potential of FXR activation in disease with BA-associated development of HCC.