Stephen D. Zucker

Mechanism of indinavir-induced hyperbilirubinemia

Indinavir is a viral protease inhibitor used for the treatment of HIV infection. Unconjugated hyperbilirubinemia develops in up to 25% of patients receiving indinavir, prompting drug discontinuation and further clinical evaluation in some instances. We postulated that this side-effect is due to indinavir-mediated impairment of bilirubin UDP-glucuronosyltransferase (UGT) activity and would be most pronounced in individuals with reduced hepatic enzyme levels, as occurs in ≈10% of the population manifesting Gilberts syndrome. This hypothesis was tested in vitro, in the Gunn rat model of UGT deficiency, and in HIV-infected patients with and without the Gilberts polymorphism. Indinavir was found to competitively inhibit UGT enzymatic activity (KI = 183 μM) while concomitantly inducing hepatic bilirubin UGT mRNA and protein expression. Although oral indinavir increased plasma bilirubin levels in wild-type and heterozygous Gunn rats, the mean rise was significantly greater in the latter group of animals. Similarly, serum bilirubin increased by a mean of 0.34 mg/dl in indinavir-treated HIV patients lacking the Gilberts polymorphism versus 1.45 mg/dl in those who were either heterozygous or homozygous for the mutant allele. Whereas saquinavir also competitively inhibits UGT activity, this drug has not been associated with hyperbilirubinemia, most likely because of the higher KI (360 μM) and substantially lower therapeutic levels as compared with indinavir. Taken together, these findings indicate that elevations in serum-unconjugated bilirubin associated with indinavir treatment result from direct inhibition of bilirubin-conjugating activity.

Bilirubin inhibits iNOS expression and NO production in response to endotoxin in rats

The inducible isoform of heme oxygenase (HO), HO‐1, has been shown to play an important role in attenuating tissue injury. Because HO‐1 catalyzes the rate‐limiting step in bilirubin synthesis, we examined the hypothesis that bilirubin is a key mediator of HO‐1 cytoprotection, employing a rat model of endotoxemia. Bilirubin treatment resulted in improved survival and attenuated liver injury in response to lipopolysaccharide infusion. Serum levels of NO and tumor necrosis factor α, key mediators of endotoxemia, and hepatic inducible nitric oxide synthase (iNOS) expression were significantly lower in bilirubin‐treated rodents versus control animals. Both intraperitoneal and local administration of bilirubin also was found to ameliorate hindpaw inflammation induced by the injection of λ‐carrageenan. Consistent with in vivo results, bilirubin significantly inhibited iNOS expression and suppressed NO production in lipopolysaccharide (LPS)‐stimulated RAW 264.7 murine macrophages. In contrast, bilirubin treatment induced a threefold increase in LPS‐mediated prostaglandin synthesis in the absence of significant changes in cyclooxygenase expression or activity, suggesting that bilirubin enhances substrate availability for eicosanoid synthesis. Bilirubin had no effect on LPS‐mediated activation of nuclear factor κB or p38 mitogen‐activated protein kinase, consistent with a nuclear factor κB–independent mechanism of action. Taken together, these data support a cytoprotective role for bilirubin that is mediated, at least in part, through the inhibition of iNOS expression and, potentially, through stimulation of local prostaglandin E2 production. In conclusion, our findings suggest a role for bilirubin in mollifying tissue injury in response to inflammatory stimuli and support the possibility that the phenomenon of “jaundice of sepsis” represents an adaptive physiological response to endotoxemia. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270‐9139/suppmat/index.html). (HEPATOLOGY 2004;40:424–433.)

Unconjugated Bilirubin Exhibits Spontaneous Diffusion through Model Lipid Bilayers and Native Hepatocyte Membranes

The liver is responsible for the clearance and metabolism of unconjugated bilirubin, the hydrophobic end-product of heme catabolism. Although several putative bilirubin transporters have been described, it has been alternatively proposed that bilirubin enters the hepatocyte by passive diffusion through the plasma membrane. In order to elucidate the mechanism of bilirubin uptake, we measured the rate of bilirubin transmembrane diffusion (flip-flop) using stopped-flow fluorescence techniques. Unconjugated bilirubin rapidly diffuses through model phosphatidylcholine vesicles, with a first-order rate constant of 5.3 s−1 (t 1 2 = 130 ms). The flip-flop rate is independent of membrane cholesterol content, phospholipid acyl saturation, and lipid packing, consistent with thermodynamic analyses demonstrating minimal steric constraint to bilirubin transmembrane diffusion. The coincident decrease in pH of the entrapped vesicle volume supports a mechanism whereby the bilirubin molecule crosses the lipid bilayer as the uncharged diacid. Transport of bilirubin by native rat hepatocyte membranes exhibits kinetics comparable with that in model vesicles, suggesting that unconjugated bilirubin crosses cellular membranes by passive diffusion through the hydrophobic lipid core. In contrast, there is no demonstrable flip-flop of bilirubin diglucuronide or bilirubin ditaurate in phospholipid vesicles, yet these compounds rapidly traverse isolated rat hepatocyte membranes, confirming the presence of a facilitated uptake system(s) for hydrophilic bilirubin conjugates.

Unconjugated Bilirubin Inhibits VCAM-1-Mediated Transendothelial Leukocyte Migration

During lymphocyte migration, engagement of VCAM-1 stimulates the generation of endothelial cell-derived reactive oxygen species (ROS) and activation of matrix metalloproteinases, facilitating endothelial retraction. Because bilirubin is a potent antioxidant, we examined the hypothesis that this bile pigment inhibits VCAM-1-dependent cellular events. The migration of isolated murine splenic lymphocytes across monolayers of murine endothelial cell lines (which constitutively express VCAM-1) is significantly inhibited by physiological concentrations of bilirubin, in the absence of an effect on lymphocyte adhesion. Bilirubin administration also suppresses VCAM-1-stimulated ROS generation and reduces endothelial cell matrix metalloproteinase activity. In a murine asthma model characterized by VCAM-1-dependent airway inflammation, treatment of C57BL6/J mice with i.p. bilirubin decreases the total leukocyte count in the lung parenchyma and lavage fluid, through specific inhibition of eosinophil and lymphocyte infiltration. Blood eosinophil counts were increased in bilirubin-treated animals, while VCAM-1 expression in the capillary endothelium and cytokine levels in both lung lavage and supernatants from cultured lymph node lymphocytes were unchanged, suggesting that bilirubin inhibits leukocyte migration. Conclusion: bilirubin blocks VCAM-1-dependent lymphocyte migration in vitro and ameliorates VCAM-1-mediated airway inflammation in vivo, apparently through the suppression of cellular ROS production. These findings support a potential role for bilirubin as an endogenous immunomodulatory agent.

Unconjugated bilirubin induces apoptosis in colon cancer cells by triggering mitochondrial depolarization

Bilirubin is the principal end product of heme degradation. Prompted by epidemiologic analyses demonstrating an inverse correlation between serum bilirubin levels and cancer mortality, we examined the effect(s) of bilirubin on the growth and survival of colon adenocarcinoma cells. Adenocarcinoma cell monolayers were treated with bilirubin over a range of bilirubin:BSA molar ratios (0–0.6), and viability was assessed colorimetrically. Apoptosis was characterized by TUNEL assay, annexin V staining and caspase‐3 activation. The mechanism(s) by which bilirubin induces apoptosis was investigated by Western blotting for cytochrome c release, assaying for caspase‐8 and caspase‐9 activation and for mitochondrial depolarization by JC‐1 staining. The direct effect of bilirubin on the membrane potential of isolated mitochondria was evaluated using light‐scattering and fluorescence techniques. Bilirubin decreased the viability of all colon cancer cell lines tested in a dose‐dependent manner. Cells exhibited substantial apoptosis when exposed to bilirubin concentrations ranging 0–50 μM, as demonstrated by an 8‐ to 10‐fold increase in TUNEL and annexin V staining and in caspase‐3 activity. Bilirubin treatment evokes specific activation of caspase‐9, enhances cytochrome c release into the cytoplasm and triggers the mitochondrial permeability transition in colon cancer monolayers. Additionally, bilirubin directly induces the depolarization of isolated rat liver mitochondria, an effect that is not inhibited by cyclosporin A. Bilirubin stimulates apoptosis of colon adenocarcinoma cells in vitro through activation of the mitochondrial pathway, apparently by directly dissipating mitochondrial membrane potential. As this effect is triggered at concentrations normally present in the intestinal lumen, we postulate a physiologic role for bilirubin in modulating colon tumorigenesis.

Randomized, double-blind, controlled study of glycerol phenylbutyrate in hepatic encephalopathy

Glycerol phenylbutyrate (GPB) lowers ammonia by providing an alternate pathway to urea for waste nitrogen excretion in the form of phenylacetyl glutamine, which is excreted in urine. This randomized, double‐blind, placebo‐controlled phase II trial enrolled 178 patients with cirrhosis, including 59 already taking rifaximin, who had experienced two or more hepatic encephalopathy (HE) events in the previous 6 months. The primary endpoint was the proportion of patients with HE events. Other endpoints included the time to first event, total number of events, HE hospitalizations, symptomatic days, and safety. GPB, at 6 mL orally twice‐daily, significantly reduced the proportion of patients who experienced an HE event (21% versus 36%; P = 0.02), time to first event (hazard ratio [HR] = 0.56; P < 0.05), as well as total events (35 versus 57; P = 0.04), and was associated with fewer HE hospitalizations (13 versus 25; P = 0.06). Among patients not on rifaximin at enrollment, GPB reduced the proportion of patients with an HE event (10% versus 32%; P < 0.01), time to first event (HR = 0.29; P < 0.01), and total events (7 versus 31; P < 0.01). Plasma ammonia was significantly lower in patients on GPB and correlated with HE events when measured either at baseline or during the study. A similar proportion of patients in the GPB (79%) and placebo groups (76%) experienced adverse events. Conclusion: GPB reduced HE events as well as ammonia in patients with cirrhosis and HE and its safety profile was similar to placebo. The findings implicate ammonia in the pathogenesis of HE and suggest that GPB has therapeutic potential in this population. (Clinicaltrials.gov, NCT00999167). (Hepatology 2014;59:1073‐1083)

Mechanism of hepatocellular uptake of albumin-bound bilirubin.

We previously demonstrated that unconjugated bilirubin spontaneously diffuses through phospholipid bilayers at a rate which exceeds albumin dissociation, suggesting that solvation from albumin represents the rate-limiting step in hepatic bilirubin clearance. To further examine this hypothesis, we studied the uptake of bovine serum albumin (BSA)-bound bilirubin by cultured hepatoblastoma (HepG2) cells. Uptake of bilirubin was saturable, with a K(m) and V(max) of 4.2+/-0.5 microM (+/-S.E.M.) and 469+/-41 pmol min(-1) mg(-1) at 25 degrees C. Substantial bilirubin uptake also was observed at 4 degrees C (K(m)=7.0+/-0.8 microM, V(max)=282+/-26 pmol min(-1) mg(-1)), supporting a diffusional transport mechanism. Consistent with reported solvation rates, the cellular uptake of bilirubin bound to human serum albumin was more rapid than for BSA-bound bilirubin, indicative of dissociation-limited uptake. Counterintuitively, an inverse correlation between pH and the rate of bilirubin flip-flop was observed, due to pH effects on the rate of dissociation of bilirubin from albumin and from the membrane bilayer. The identification of an inflection point at pH 8.1 is indicative of a pK(a) value for bilirubin in this range. Taken together, our data suggest that hepatocellular uptake of bilirubin is dissociation-limited and occurs principally by a mechanism involving spontaneous transmembrane diffusion.

Influence of glutathione S-transferase B (ligandin) on the intermembrane transfer of bilirubin. Implications for the intracellular transport of nonsubstrate ligands in hepatocytes.

To examine the hypothesis that glutathione S-transferases (GST) play an important role in the hepatocellular transport of hydrophobic organic anions, the kinetics of the spontaneous transfer of unconjugated bilirubin between membrane vesicles and rat liver glutathione S-transferase B (ligandin) was studied, using stopped-flow fluorometry. Bilirubin transfer from glutathione S-transferase B to phosphatidylcholine vesicles was best described by a single exponential function, with a rate constant of 8.0 +/- 0.7 s-1 (+/- SD) at 25 degrees C. The variations in transfer rate with respect to acceptor phospholipid concentration provide strong evidence for aqueous diffusion of free bilirubin. This finding was verified using rhodamine-labeled microsomal membranes as acceptors. Bilirubin transfer from phospholipid vesicles to GST also exhibited diffusional kinetics. Thermodynamic parameters for bilirubin dissociation from GST were similar to those for human serum albumin. The rate of bilirubin transfer from rat liver basolateral plasma membranes to acceptor vesicles in the presence of glutathione S-transferase B declined asymptotically with increasing GST concentration. These data suggest that glutathione S-transferase B does not function as an intracellular bilirubin transporter, although expression of this protein may serve to regulate the delivery of bilirubin, and other nonsubstrate ligands, to sites of metabolism within the cell.

Sodium taurocholate inhibits intestinal adenoma formation in APCMin/+ mice, potentially through activation of the farnesoid X receptor

In light of clinical and biological evidence that bile constituents exert preventive effects against colorectal cancer, we evaluated the influence of oral bilirubin and sodium taurocholate (NaTC) on intestinal tumor formation in APC(Min/+) mice. Mice received bilirubin and/or bovine serum albumin (BSA) and NaTC in the drinking water for 8 weeks, after which the number, size and location of intestinal adenomas were determined. Tissue specimens were analyzed by light microscopy, TUNEL staining, immunohistochemistry for beta-catenin and Ki-67 and quantitative polymerase chain reaction for farnesoid X receptor (FXR)-dependent gene expression. Colon tumor formation also was assessed in azoxymethane (AOM)-treated hyperbilirubinemic Gunn (j/j) and wild-type (+/+) rats. Compared with untreated APC(Min/+) mice, the mean number of intestinal adenomas was markedly lower in both bilirubin (10.5 +/- 0.9 versus 37.0 +/- 5.2; +/-SEM; P < 0.001) and NaTC plus BSA (14.3 +/- 5.4; P = 0.01)-treated animals. Both treatment groups exhibited reduced levels of cellular proliferation in the ileum (by Ki-67 staining), but no differences in TUNEL staining or the percentage of beta-catenin-positive crypts. Bilirubin feeding reduced intestinal inducible nitric oxide synthase expression, but did not alter adenoma multiplicity in APC(Min/+) mice or in AOM-treated j/j versus +/+ rats. Mice receiving NaTC manifested increased intestinal expression of the FXR-regulated genes, Shp, FGF15 and IBABP, and a concomitant decrease in cyclin D1 message. Administering NaTC to APC(Min/+) mice causes a marked reduction in intestinal adenomas. We postulate that this effect is mediated through activation of FXR, leading to increased Shp expression and consequent downregulation of cyclin D1.

Bilirubin inhibits the up-regulation of inducible nitric oxide synthase by scavenging reactive oxygen species generated by the toll-like receptor 4-dependent activation of NADPH oxidase

It has been previously shown that bilirubin prevents the up-regulation of inducible nitric oxide synthase (iNOS) in response to LPS. The present study examines whether this effect is exerted through modulation of Toll-Like Receptor-4 (TLR4) signaling. LPS-stimulated iNOS and NADPH oxidase (Nox) activity in RAW 264.7 murine macrophages was assessed by measuring cellular nitrate and superoxide (O2−) production, respectively. The generation of both nitrate and O2− in response to LPS was suppressed by TLR4 inhibitors, indicating that activation of iNOS and Nox is TLR4-dependent. While treatment with superoxide dismutase (SOD) and bilirubin effectively abolished LPS-mediated O2− production, hydrogen peroxide and nitrate release were inhibited by bilirubin and PEG-catalase, but not SOD, supporting that iNOS activation is primarily dependent upon intracellular H2O2. LPS treatment increased nuclear translocation of the redox-sensitive transcription factor Hypoxia Inducible Factor-1α (HIF-1α), an effect that was abolished by bilirubin. Cells transfected with murine iNOS reporter constructs in which the HIF-1α-specific hypoxia response element was disrupted exhibited a blunted response to LPS, supporting that HIF-1α mediates Nox-dependent iNOS expression. Bilirubin, but not SOD, blocked the cellular production of interferon-β, while interleukin-6 production remained unaffected. These data support that bilirubin inhibits the TLR4-mediated up-regulation of iNOS by preventing activation of HIF-1α through scavenging of Nox-derived reactive oxygen species. Bilirubin also suppresses interferon-β release via a ROS-independent mechanism. These findings characterize potential mechanisms for the anti-inflammatory effects of bilirubin.