Bilirubin Glucuronidation Research Articles

Cabozantinib Induces Isolated Hyperbilirubinemia in Renal Cell Carcinoma Patients carrying the UGT1A1*28 Polymorphism

Genetic variants of UGT1A1, involved in glucuronidation and clearance of bilirubin, are associated with reduced bilirubin metabolization and drug-induced isolated hyperbilirubinemia. We studied the impact of the UGT1A1*28 polymorphism on drug-induced isolated hyperbilirubinemia in metastatic renal cell carcinoma patients treated with pazopanib, cabozantinib, and axitinib. We genotyped the UGT1A1*28 TA6/TA6-TA6/TA7-TA7/TA7 polymorphism and correlated with median baseline, on-treatment and peak bilirubin levels during therapy, incidence of grade-1- or -2 (G1/2)-hyperbilirubinemia and time-to-G1-hyperbilirubinemia. Of the 66 patients treated with pazopanib, 29 received axitinib and 28 cabozantinib upon progression. Median baseline bilirubin was higher in TA7/TA7-carriers versus TA6/TA6+TA6/TA7-carriers at start of pazopanib (P < .0001), cabozantinib (P < .0001), and axitinib (P = .007). During pazopanib therapy, median bilirubin increased 1.4-fold in TA7/TA7+TA6/TA7-carriers but not in TA6/TA6-carriers. On cabozantinib, bilirubin increased 1.5-fold in TA7/TA7-carriers but not in TA6/TA6+TA6/TA7-carriers. Axitinib did not increase bilirubin in any genotype. Peak bilirubin in TA7/TA7- versus TA6/TA6+TA6/TA7-carriers was higher on pazopanib (P < .0001) or cabozantinib (P < .0001). With pazopanib, G1-hyperbilirubinemia occurred in 57% of TA7/TA7- and 12% of TA6/TA6+TA6/TA7-carriers (P = .0009) and G2-hyperbilirubinemia in 36% and 6% of the patients, respectively (P = .004). On cabozantinib, G1-hyperbilirubinemia occurred in 100% of TA7/TA7- and 5% of TA6/TA6+TA6/TA7-carriers (P < .0001) and G2-hyperbilirubinemia in 33% and 0% of the patients, respectively (P = .04). On axitinib, no correlation between the genotypes and G1/2-hyperbilirubinemia was observed. We validate the previously described impact of the UGT1A1*28 polymorphism on isolated bilirubin increase on pazopanib. We report for the first time that cabozantinib also interferes with UGT1A1 and causes isolated bilirubin increase.

Bilirubin derivatives in bile, blood, and liver of rats upon correction of experimental fatty hepatosis

The relevance of this study lies in the lack of reliable information on the key aspects of the molecular mechanisms of fatty hepatosis, a common mammalian disease, and its dangerous complications, such as fibrosis, cirrhosis, and hepatocellular carcinoma. These circumstances substantially complicate the prompt diagnosis of this hepatopathology and reduce the effectiveness of treatment. The metabolism of the heme derivative bilirubin is unique and inherent in the liver, which undergoes transformation due to hepatocyte enzyme systems to form neutral compounds, such as glucurono-conjugates and sulphoconjugates, which undergoes changes and requires analysis in fatty hepatosis in animals. The aim of the study was to determine the characteristic changes in the content of unconjugated bilirubin and its sulphoconjugate, glucuronide, monoglucuronide, monoglucoside, and diglucuronide in the blood, bile, and liver of rats under the modelling of fatty hepatosis and the administration of rehabilitation therapy. For this, the thin-layer chromatography method was used. It was found that under tetracycline-induced fatty hepatosis in laboratory rats, the concentration of unconjugated bilirubin in the blood decreased by 39.3%, bilirubin glucuronide by 44.4%, and the total fraction of bilirubin monoglucuronide and monoglycoside by 78.9%. Oral administration of the milk phospholipid complex in the composition of the BAS “FLP-MD” to sick animals ensured the maintenance of these indicators at the control level. The content of bilirubin glucuronide and bilirubin diglucuronide decreased in the liver tissue of the treated rats. When these animals were administered a phospholipid-containing bioadditives, the level of unconjugated bilirubin in the liver tissue increased by 22.2%, but the content of its conjugated forms decreased. The content of all the studied bilirubin fractions decreased in the bile of sick rats during self-rehabilitation. The components of the BAS “FLP-MD” in fatty hepatosis in rats did not affect the reduction of unconjugated bilirubin in bile but eliminated the inhibitory effect of the modelled pathology on the content of all conjugated forms of bilirubin. The findings can be used as biochemical markers to monitor the pigment metabolism in the development of fatty hepatosis in mammals

Dysregulated Glucuronidation of Bilirubin Exacerbates Liver Inflammation and Fibrosis in Schistosomiasis Japonica through the NF-κB Signaling Pathway.

Hepatic fibrosis is an important pathological manifestation of chronic schistosome infection. Patients with advanced schistosomiasis show varying degrees of abnormalities in liver fibrosis indicators and bilirubin metabolism. However, the relationship between hepatic fibrosis in schistosomiasis and dysregulated bilirubin metabolism remains unclear. In this study, we observed a positive correlation between total bilirubin levels and the levels of ALT, AST, LN, and CIV in patients with advanced schistosomiasis. Additionally, we established mouse models at different time points following S. japonicum infection. As the infection time increased, liver fibrosis escalated, while liver UGT1A1 consistently exhibited a low expression, indicating impaired glucuronidation of bilirubin metabolism in mice. In vitro experiments suggested that SEA may be a key inhibitor of hepatic UGT1A1 expression after schistosome infection. Furthermore, a high concentration of bilirubin activated the NF-κB signaling pathway in L-O2 cells in vitro. These findings suggested that the dysregulated glucuronidation of bilirubin caused by S. japonicum infection may play a significant role in schistosomiasis liver fibrosis through the NF-κB signaling pathway.

Structural basis for the modulation of MRP2 activity by phosphorylation and drugs

Multidrug resistance-associated protein 2 (MRP2/ABCC2) is a polyspecific efflux transporter of organic anions expressed in hepatocyte canalicular membranes. MRP2 dysfunction, in Dubin-Johnson syndrome or by off-target inhibition, for example by the uricosuric drug probenecid, elevates circulating bilirubin glucuronide and is a cause of jaundice. Here, we determine the cryo-EM structure of rat Mrp2 (rMrp2) in an autoinhibited state and in complex with probenecid. The autoinhibited state exhibits an unusual conformation for this class of transporter in which the regulatory domain is folded within the transmembrane domain cavity. In vitro phosphorylation, mass spectrometry and transport assays show that phosphorylation of the regulatory domain relieves this autoinhibition and enhances rMrp2 transport activity. The in vitro data is confirmed in human hepatocyte-like cells, in which inhibition of endogenous kinases also reduces human MRP2 transport activity. The drug-bound state reveals two probenecid binding sites that suggest a dynamic interplay with autoinhibition. Mapping of the Dubin-Johnson mutations onto the rodent structure indicates that many may interfere with the transition between conformational states.

A phase I study of ATR inhibitor gartisertib (M4344) as a single agent and in combination with carboplatin in patients with advanced solid tumours

BackgroundGartisertib is an oral inhibitor of ataxia telangiectasia and Rad3-related protein (ATR), a key kinase of the DNA damage response. We aimed to determine the safety and tolerability of gartisertib ± carboplatin in patients with advanced solid tumours.MethodsThis phase I open-label, multicenter, first-in-human study comprised four gartisertib cohorts: A (dose escalation [DE]; Q2W); A2 (DE; QD/BID); B1 (DE+carboplatin); and C (biomarker-selected patients).ResultsOverall, 97 patients were enroled into cohorts A (n = 42), A2 (n = 26), B1 (n = 16) and C (n = 13). The maximum tolerated dose and recommended phase II dose (RP2D) were not declared for cohorts A or B1. In cohort A2, the RP2D for gartisertib was determined as 250 mg QD. Gartisertib was generally well-tolerated; however, unexpected increased blood bilirubin in all study cohorts precluded further DE. Investigations showed that gartisertib and its metabolite M26 inhibit UGT1A1-mediated bilirubin glucuronidation in human but not dog or rat liver microsomes. Prolonged partial response (n = 1 [cohort B1]) and stable disease >6 months (n = 3) did not appear to be associated with biomarker status. Exposure generally increased dose-dependently without accumulation.ConclusionGartisertib was generally well-tolerated at lower doses; however, unexpected liver toxicity prevented further DE, potentially limiting antitumour activity. Gartisertib development was subsequently discontinued.ClinicalTrials.govNCT02278250.

Temporally and spatially resolved molecular profiling in fingerprint analysis using indium vanadate nanosheets-assisted laser desorption ionization mass spectrometry

This study presents the first-ever synthesis of samarium-doped indium vanadate nanosheets (IVONSs:Sm) via microemulsion-mediated solvothermal method. The nanosheets were subsequently utilized as a nano-matrix in laser desorption/ionization mass spectrometry (LDI-MS). It was discovered that the as-synthesized IVONSs:Sm possessed the following advantages: improved mass spectrometry signal, minimal matrix-related background, and exceptional stability in negative-ion mode. These qualities overcame the limitations of conventional matrices and enabled the sensitive detection of small biomolecules such as fatty acids. The negative-ion LDI mechanism of IVONSs:Sm was examined through the implementation of density functional theory simulation. Using IVONSs:Sm-assisted LDI-MS, fingerprint recognitions based on morphology and chemical profiles of endogenous/exogenous compounds were also achieved. Notably, crucial characteristics such as the age of an individual’s fingerprints and their physical state could be assessed through the longitudinal monitoring of particular biomolecules (e.g., ascorbic acid, fatty acid) or the specific biomarker bilirubin glucuronide. Critical information pertinent to the identification of an individual would thus be facilitated by the analysis of the compounds underlying the fingerprint patterns.Graphical

Clinical case of combined genetic pathology in a child with jaundice syndrome

Diagnosis of a combined genetic pathology that occurs with jaundice syndrome is a difficult task, because of common clinical manifestations for several diseases having different genesis and requiring specific diagnosis and treatment tactics. Icteric staining of the skin due to hyperbilirubinemia at any age requires immediate examination to establish its genesis and differential diagnosis between infectious and non-infectious diseases. The spectrum of diseases accompanied by jaundice syndrome includes more than 50 different diseases. The main mechanisms of suprahepatic jaundice are: firstly, a violation of the capture and glucuronidation of bilirubin in the liver, and secondly, an increased destruction of red blood cells due to hemolysis or during the disposal of blood during internal bleeding. The first mechanism is realized in Gilbert’s syndrome, while the second mechanism of suprahepatic jaundice is often due to hemolytic anemia.In presented clinical case, the implementation of two pathogenetic forms of jaundice in one patient is shown — suprahepatic, caused by type 2 hereditary stomatocytosis and Gilbert’s syndrome, as well as obstructive jaundice, caused by cholelithiasis. The special attention should be paid to precise patient examination, if disease manifestations are not consistent with classical clinical picture. The use of a new generation sequencing method, along with traditional diagnostics methods, made it possible to establish an accurate diagnosis and determine the tactics of patient management.

Evaluating variations in bilirubin glucuronidation activity by protease inhibitors in canine and human primary hepatocytes cultured in a 3D culture system

Bilirubin is excreted into the bile from hepatocytes, mainly as monoglucuronosyl and bisglucuronosyl conjugates, reflecting bilirubin glucuronidation activity. However, there is limited information on the in vitro evaluation of liver cell lines or primary hepatocytes. This study aimed to investigate variations in the bilirubin metabolic function of canine and human hepatocyte spheroids formed in a three-dimensional (3D) culture system indicated by the formation of bilirubin glucuronides when protease inhibitors such as atazanavir, indinavir, ritonavir, and nelfinavir were treated with bilirubin. The culture supernatant was collected for bilirubin glucuronidation assessment and the cells were used to evaluate viability. On day 8 of culture, both canine and human hepatocyte spheroids showed high albumin secretion and distinct spheroid formation, and their bilirubin glucuronidation activities were evaluated considering cell viability. Treatment with atazanavir and ritonavir remarkably inhibited bilirubin glucuronide formation, wherein atazanavir showed the highest inhibition, particularly in human hepatocyte spheroids. These results may reflect the effects on cellular uptake of bilirubin and its intracellular metabolic function. Thus, primary hepatocytes cultured in a 3D culture system may be a useful in vitro system for the comprehensive evaluation of bilirubin metabolic function and risk assessment in bilirubin metabolic disorders for drug development.

НАСЛЕДСТВЕННЫЕ НЕКОНЪЮГИРОВАННЫЕ ГИПЕРБИЛИРУБИНЕМИИ

Congenital hereditary non-conjugate hyperbilirubinemias include Gilbert’s syndrome, Crigler-Najjar type 1 and Crigler-Najjar type 2 syndromes (or Arias’ disease). They are caused by a hereditary deficiency of the enzyme - bilirubinuridine-5’-diphosphate glucuronosyltransferase (UGT1A1), involved in the glucuronization of bilirubin. The enzyme deficiency is due to mutations in the UGT1A1 gene, which provides UGT1A1 activity. Complete or almost complete loss of (Crigler-Najjar syndrome type 1) or decreased UGT1A1 activity (Gilbert’s syndrome and Crigler-Najjar syndrome type 2) lead to impaired conversion of bilirubin in the liver with the accumulation of unconjugated bilirubin in the blood. Syndromes are distinguished by the level of bilirubin in blood plasma, the reaction to the introduction of phenobarbital, the presence or absence of bilirubin glucuronides in bile.

Organic anion-transporting polypeptide 2B1 knockout and humanized mice; insights into the handling of bilirubin and drugs

Organic anion transporting polypeptide 2B1 (OATP2B1/SLCO2B1) facilitates uptake transport of structurally diverse endogenous and exogenous compounds. To investigate the roles of OATP2B1 in physiology and pharmacology, we established and characterized Oatp2b1 knockout (single Slco2b1-/- and combination Slco1a/1b/2b1-/-) and humanized hepatic and intestinal OATP2B1 transgenic mouse models. While viable and fertile, these strains exhibited a modestly increased body weight. In males, unconjugated bilirubin levels were markedly reduced in Slco2b1-/- compared to wild-type mice, whereas bilirubin monoglucuronide levels were modestly increased in Slco1a/1b/2b1-/- compared to Slco1a/1b-/- mice. Single Slco2b1-/- mice showed no significant changes in oral pharmacokinetics of several tested drugs. However, markedly higher or lower plasma exposure of pravastatin and the erlotinib metabolite OSI-420, respectively, were found in Slco1a/1b/2b1-/- compared to Slco1a/1b-/- mice, while oral rosuvastatin and fluvastatin behaved similarly between the strains. In males, humanized OATP2B1 strains showed lower conjugated and unconjugated bilirubin levels than control Slco1a/1b/2b1-deficient mice. Moreover, hepatic expression of human OATP2B1 partially or completely rescued the impaired hepatic uptake of OSI-420, rosuvastatin, pravastatin, and fluvastatin in Slco1a/1b/2b1-/- mice, establishing an important role in hepatic uptake. Expression of human OATP2B1 in the intestine was basolateral and markedly reduced the oral availability of rosuvastatin and pravastatin, but not of OSI-420 and fluvastatin. Neither lack of Oatp2b1, nor overexpression of human OATP2B1 had any effect on fexofenadine oral pharmacokinetics. While these mouse models still have limitations for human translation, with additional work we expect they will provide powerful tools to further understand the physiological and pharmacological roles of OATP2B1.

Liver Transplantation in a Patient with Crigler-Najjar Syndrome Type 1: A Case Report of Two Cases

Crigler Najjar syndrome(CNS); is a disease in which the diphosphate glucuronosyltransferase (bilirubin-UGT) enzyme function, which plays a role in the glucuronidation of bilirubin, is deficient as a result of mutation in the uridine 5'-diphosphate-glucuronosyltransferase 1A1 (UGT1A1) gene.1 As a result, non-hemolytic unconjugated hyperbilirubinemia is seen. Orthotopic liver transplantation (OLT) is seen as a curative treatment option in Crigler Najjar syndrome type 1 (CNS1). In this case report, we present our patients who were 11 months old and 8 years old with a diagnosis of CNS1, whose bilirubin levels were controlled by preoperative daily phototherapy and plasmapheresis, and who had OLT from their parents to two siblings. We wanted to show the importance of a close follow-up and multidisciplinary treatment approach in the early period before OLT in CNS1 patients and thus the benefit to the patient's prognosis in the postoperative period.

Modern view on pathogenesis, diagnosis and treatment of Gilbert's syndrome

Gilbert’s syndrome is an autosomal recessive inherited disorder of bilirubin glucuronidation characterized by unconjugated hyperbilirubinaemia in the absence of hepatocellular injury or hemolysis. Gilbert’s syndrome is characterized by mutations to the UGT1A1 gene which encodes the enzyme uridine disphosphate glucuronоsyltransferase which is required for the conjugation and subsequent excretion of bilirubin. Most Gilbert’s syndrome patients experience recurrent and short-term episodes of yellowing of the skin and sclera of eyes caused by bilirubin accumulation in the blood. Approximately 30 % of patients with Gilbert's syndrome do not experience any symptoms, but are detected by blood tests that show elevated levels of unconjugated bilirubin. In addition to traditional methods, genetic analysis of the UGT1A1 gene allows us to confirm the diagnosis of Gilbert's syndrome. The article presents modern concepts of the pathogenesis, diagnosis, differential diagnosis and treatment of Gilbert's syndrome. It is of interest to gastroenterologists, internists and general practitioners who treat such patients.

Gilbert syndrome as a risk factor for the development of cholelithiasis in children

Introduction/Objective. Gilbert syndrome (GS) is the most common hereditary hyperbilirubinemia. As well as mild unconjugated hyperbilirubinemia, it is characterized by the excess of bilirubin monoglucuronide over diglucuronide in the bile and thus increases the risk of biliary calculosis. The aim of the study was to determine the importance of GS as a risk factor in the development of cholelithiasis in children. Methods. The study included a sample of 31 children (14 male and 17 female, mean age 12.16 ? 4.11 years, range 3?16.75 years) with symptomatic cholelithiasis. The diagnosis of cholelithiasis was based on an ultrasonographic finding, and for GS the diagnosis was based on at least a double increase of unconjugated bilirubin fraction after a three-day hypocaloric diet (400 kcal per day). Results. GS was confirmed in five or 16.13% of patients (three male and two female, mean age 14.71 ? 0.55 years, range 14?15.3 years). In addition to GS, in the history of the disease they all had some of the additional risk factors for the development of cholelithiasis. One of them had an identical problem as its mother, one had hereditary elliptocytosis, one had sudden weight loss, one was overweight, and one had premature birth and sepsis. Conclusion. GS registers in one-sixth of children with cholelithiasis, but in none of them as the only risk factor for developing this disease. This finding suggests that GS is a risk factor for the development of cholelithiasis, but not sufficient in itself in that respect.

Cyanidin 3-glucoside targets a hepatic bilirubin transporter in rats

One of the organ-specific functions of the liver is the excretion of bilirubin into the bile. Membrane transport of bilirubin from the blood to the liver is not only an orphan function, because there is no link to the protein/gene units that perform this function, but also a poorly characterised function. The aim of this study was to investigate the pharmacology of bilirubin uptake in the liver of the female Wistar rat to improve basic knowledge in this neglected area of liver physiology. We treated isolated perfused livers of female rats with repeated single-pass, albumin-free bilirubin boli. We monitored both bilirubin and bilirubin glucuronide in perfusion effluent with a bio-fluorometric assay. We tested the ability of nine molecules known as substrates or inhibitors of sinusoidal membrane transporters to inhibit hepatic uptake of bilirubin. We found that cyanidin 3-glucoside and malvidin 3-glucoside were the only molecules that inhibited bilirubin uptake. These dietary anthocyanins resemble bromosulfophthalein (BSP), a substrate of several sinusoidal membrane transporters. The SLCO-specific substrates estradiol-17 beta-glucuronide, pravastatin, and taurocholate inhibited only bilirubin glucuronide uptake. Cyanidin 3-glucoside and taurocholate acted at physiological concentrations. The SLC22-specific substrates indomethacin and ketoprofen were inactive. We demonstrated the existence of a bilirubin-glucuronide transporter inhibited by bilirubin, a fact reported only once in the literature. The data suggest that bilirubin and bilirubin glucuronide are transported to the liver via pharmacologically distinct membrane transport pathways. Some dietary anthocyanins may physiologically modulate the uptake of bilirubin into the liver.

Activation of Alternative Bilirubin Clearance Pathways Partially Reduces Hyperbilirubinemia in a Mouse Model Lacking Functional Ugt1a1 Activity.

Bilirubin is a heme catabolite and Ugt1a1 is the only enzyme involved in the biological elimination of bilirubin. Partially functional or non-functional Ugt1a1 may result in neuronal damage and death due to the accumulation of unconjugated bilirubin in the brain. The understanding of the role of alternative bilirubin detoxification mechanisms that can reduce bilirubin toxicity risk is crucial for developing novel therapeutic strategies. To provide a proof-of-principle showing whether activation of alternative detoxification pathways could lead to life-compatible bilirubin levels in the absence of Ugt1a1 activity, we used Ugt1−/− hyperbilirubinemic mice devoid of bilirubin glucuronidation activity. We treated adult Ugt1−/− mice with TCPOBOP, a strong agonist of the constitutive androstane receptor (CAR). TCPOBOP treatment decreased plasma and liver tissue bilirubin levels by about 38%, and resulted in the transcriptional activation of a vast array of genes involved in bilirubin transport and metabolism. However, brain bilirubin level was unaltered. We observed ~40% degradation of bilirubin in the liver microsomes from TCPOBOP treated Ugt1−/− mice. Our findings suggest that, in the absence of Ugt1a1, the activation of alternative bilirubin clearance pathways can partially improve hyperbilirubinemic conditions. This therapeutic approach may only be considered in a combinatorial manner along with other treatments.

Comparative analysis of bilirubin glucuronidation activity in canine and human primary hepatocytes using a 3D culture system.

Species differences in bilirubin glucuronidation activity are observed between humans and dogs through liver microsomes and recombinant UDP-glucuronosyltransferase 1A1. Humans exhibit higher activity than that of dogs. In this study, bilirubin glucuronidation activity was examined in canine and human primary hepatocyte spheroids formed using a 3D culture system. When spheroid development in canine and human primary hepatocytes was evaluated on days 7 and 14 after the start of culture, canine primary hepatocyte spheroids had a more distinct spherical shape than human hepatocyte spheroids, irrespective of the culture period. Furthermore, mono- and di-glucuronide generation detected in spheroids were significantly higher (P < 0.05) in human primary hepatocytes than in canine primary hepatocytes after 24h of incubation with bilirubin for each culture period. These results suggest that there are species differences in the bilirubin glucuronidation activity of primary hepatocytes with spheroid formation between humans and dogs, with the activity being higher in humans than in dogs.

Effect of the genetic mutant G71R in uridine diphosphate-glucuronosyltransferase 1A1 on the conjugation of bilirubin.

We aimed to investigate the effect of the genetic mutant G71R (c. 211G > A) in uridine diphosphate (UDP)-glucuronosyltransferase 1A1 (UGT1A1) on the glucuronidation of unconjugated bilirubin. The UGT1A1 wild-type and mutant G71R gene sequences were inserted into the lentiviral vector GV358 plasmid and then transfected into COS-7 cells. Real-time polymerase chain reaction and western blot analyses were used to determine mRNA and protein expression levels of UGT1A1, respectively. High-performance liquid chromatography was used to quantitate the levels of conjugated bilirubin. The results showed no significant difference in the mRNA and protein expression levels between the UGT1A1 wild-type and G71R homozygous and heterozygous mutants. The level of conjugated bilirubin reached a maximum in wild-type UGT1A1-transfected COS-7 cells. However, relative to the UGT1A1 wild-type, conjugated bilirubin concentrations were 71 and 22% with G71R heterozygous- and G71R homozygous-transfected COS-7 cells, respectively. In conclusion, we successfully established in vitro cell models of the UGT1A1 wild-type and the G71R homozygous and heterozygous mutants using a lentiviral vector. Furthermore, the catalytic activity for unconjugated bilirubin was lower in the mutant G71R than the UGT1A1 wild-type enzyme, and a weaker effect was observed in the homozygote.

Inhibition of Human UDP-Glucuronosyltransferases1A1-Mediated Bilirubin Glucuronidation by the Popular Flavonoids Baicalein, Baicalin, and Hyperoside Is Responsible for Herb (Shuang-Huang-Lian)-Induced Jaundice.

Bilirubin-related adverse drug reactions (ADRs) or malady (e.g., jaundice) induced by some herbs rich in certain flavonoids have been widely reported. However, the causes and mechanisms of the ADRs are not well understood. The aim of this paper was to explore the mechanism of Shuang-huang-lian (SHL) injections and its major constituents-induced jaundice via inhibiting human UDP-glucuronosyltransferases1A1 (hUGT1A1)-mediated bilirubin glucuronidation. The inhibitory effects of SHL and its major constituents in the herbal medicine, including baicalein (BAI), baicalin (BA), and hyperoside (HYP), on bilirubin glucuroBBREVInidation were investigated. This study indicated that the average formation rates of bilirubin glucuronides [i.e., mono-glucuronide 1 (BMG1), BMG2, and bilirubin diglucuronide] displayed significant differences (P < 0.05). Specifically, the formation of BMGs was favored regardless of whether an inhibitor was absent or present. SHL, BAI, BA, and HYP dose-dependently inhibit bilirubin glucuronidation, showing the IC50 values against total bilirubin glucuronidation were in the range of (7.69 ± 0.94)-(37.09 ± 2.03) μg/ml, (4.51 ± 0.27)-(20.84 ± 1.99) μM, (22.36 ± 5.74)-(41.35 ± 2.40) μM, and (15.16 ± 1.12)-(42.80 ± 2.63) μM for SHL, BAI, BA, and HYP, respectively. Both inhibition kinetics assays and molecular docking simulations suggested that SHL, BAI, BA, and HYP significantly inhibited hUGT1A1-mediated bilirubin glucuronidation via a mixed-type inhibition. Collectively, some naturally occurring flavonoids (BAI, BA, and HYP) in SHL have been identified as the inhibitors against hUGT1A1-mediated bilirubin glucuronidation, which well explains the bilirubin-related ADRs or malady triggered by SHL in clinical settings. SIGNIFICANCE STATEMENT: Herbal products and their components (e.g., flavonoids), which been widely used across the entire world, may cause liver injury. As a commonly used herbal products rich in flavonoids, SHL injections easily lead to symptoms of liver injury (e.g., jaundice) owing to significant inhibition of hUGT1A1-mediated bilirubin glucuronidation by its flavonoid components (i.e., baicalein, baicalin, and hyperoside). Herb-induced bilirubin-related ADRs and the associated clinical significance should be seriously considered.

4-Phenylbutyrate protects against rifampin-induced liver injury via regulating MRP2 ubiquitination through inhibiting endoplasmic reticulum stress

ABSTRACT Rifampin (RFP), a first-line anti-tuberculosis drug, often induces cholestatic liver injury and hyperbilirubinemia which limits its clinical use. Multidrug resistance-associated protein 2 (MRP2) localizes to the hepatocyte apical membrane and plays a pivotal role in the biliary excretion of bilirubin glucuronides. RFP is discovered to reduce MRP2 expression in liver cells. 4-Phenylbutyrate (4-PBA), a drug used to treat ornithine transcarbamylase deficiency (DILI), is reported to alleviate RFP-induced liver cell injury. However, the underlying mechanism still remains unclear. In the current study, we discovered that RFP induced HepG2 cell viability reduction, apoptosis and MRP2 ubiquitination degradation. Administration of 4-PBA alleviated the effect of RFP on HepG2 cell viability reduction, apoptosis and MRP2 ubiquitination degradation. In mechanism, 4-PBA suppressed RPF-caused intracellular Ca2+ disorder and endoplasmic reticulum (ER) stress, as well as the increases of Clathrin and adapter protein 2 (AP2). ER stress marker protein C/EBP homologous protein took part in the modulation of AP2 and clathrin. Besides, 4-PBA reduced the serum bilirubin level in RFP-induced cholestasis mouse model, along with raised the MRP2 expression in liver tissues. These findings indicated that 4-PBA could alleviate RFP-induced cholestatic liver injury and thereby decreased serum total bilirubin concentration via inhibiting ER stress and ubiquitination degradation of MRP2, which provides new insights into the mechanism of 4-PBA in the treatment of RFP-induced cholestasis and liver damage.

Ontogeny of Pediatric Pharmacogenetics: Celebrating the Past and Vision for the Future.

Ontogeny of Pediatric Pharmacogenetics: Celebrating the Past and Vision for the Future.