Unconjugated bilirubin modulates nitric oxide production via iNOS regulation.

To induce the in vitro endothelial dysfunction model, H5V cells were treated with tumor necrosis factor α (TNFα) and with unconjugated bilirubin (UCB) at two different physiological concentrations. The TNFα-induced reduction of nitric oxide (NO) concentration was reversed by UCB. Endothelial NO synthase (eNOS) gene expression was not influenced by treatments while inducible NO synthase (iNOS) expression was increased at 24 h. Co-treatment of H5V cells with pyrrolidine dithiocarbamate, TNFα (20 ng/mL) and UCB (Bf 15 or 30 nM) for 2 h caused a significant reduction of iNOS gene expression. We conclude that at physiological concentrations UCB prevents endothelial dysfunction by modulating NO concentration probably through inhibition of NF-κB.

Bilirubin inhibits the TNFalpha-related induction of three endothelial adhesion molecules.

Since an increased serum unconjugated bilirubin (UCB) level has been proposed as an independent protective factor against atherosclerotic disease, we investigated the molecular events at the basis of this effect. HUVEC and H5V cells were treated with TNFalpha and UCB and the effects assessed on E-selectin, VCAM-1 and ICAM-1. In HUVEC cells, UCB blunted the TNFalpha-induced gene upregulation of E-selectin VCAM-1 and ICAM-1. The same pattern was observed in H5V cells except for ICAM-1. UCB also inhibited the PMN endothelial adhesion in HUVEC H5V cells. Western blot and FACS analysis confirmed that UCB prevented TNFalpha-induced over-expression of adhesion molecules proteins in H5V cells. These data contribute to further explain the protective effect of bilirubin against development of atherosclerosis.

Bilirubin effect on endothelial adhesion molecules expression is mediated by the NF-kappaB signaling pathway.

We have recently demonstrated that unconjugated bilirubin (UCB) limits the overexpression of adhesion molecules and inhibits the PMN endothelial adhesion induced by the pro-inflammatory cytokine TNFalpha. To understand the molecular events involved we investigated whether the inhibitory effect is determined by a direct influence of UCB on different nuclear pathways. Co-treatment of cells with UCB, TNFalpha, and pyrridoline dithiocarbamate (PDTC), a NF-kappaB inhibitor, additively enhanced the inhibitory effect of UCB. UCB prevented the nuclear translocation of NF-kappaB induced by TNFalpha. The failure of UCB to alter TNFalpha-induced phosphorylation of cAMP-response element-binding protein (CREB) suggested that the CREB pathway is not involved in the UCB inhibition and that UCB blunting effect on the overexpression of adhesion molecules occurs via inhibition of the NF-kappaB transduction pathway. Collectively these data may contribute to explain the protective effect of bilirubin against development of atherosclerosis.

Drug-induced acute cholestatic liver damage in a patient with mutation of UGT1A1.

BACKGROUND: A 54-year-old woman presented with a 3-week history of fatigue and with jaundice that began 2 days before admission. She had been undergoing treatment with flavoxate for urinary incontinence (for 2 months before admission) and with tibolone for climacteric syndrome (for 6 months before admission). Laboratory tests revealed elevated concentrations of aminotransferases, bilirubin, gamma-glutamyltransferase and alkaline phosphatase. Liver biopsy revealed histological evidence of subacute, drug-induced liver damage. INVESTIGATIONS: Physical examination, liver function tests, serology tests, autoantibody tests, genetic analysis of the TATA box of the UGT1A1 gene, ultrasonography and CT scan; MRI cholangiography; liver biopsy. DIAGNOSIS: Drug-related hepatitis in a patient with Gilbert's syndrome. MANAGEMENT: Flavoxate and tibolone were discontinued. Liver function test results improved progressively and normalized after almost 2 months.

Bilirubin and the risk of common non-hepatic diseases.

Bilirubin is a potent antioxidant but can be toxic at high concentrations. This article critically reviews the reported relationships of plasma bilirubin levels to the severity and/or incidence of various common non-hepatic diseases. Plasma bilirubin levels are reportedly negatively related to the risk of atherosclerotic diseases, cancers, demyelinating neuropathies and seasonal affective disorder. By contrast, the incidence and severity of schizophrenia are increased by elevated bilirubin levels. The data strongly suggest that the level of plasma bilirubin should be considered as a risk factor for several common non-hepatic diseases. Additional studies are needed to clarify the mechanisms of this influence, which are thought to be related to unconjugated bilirubin counteracting the oxidative stress underlying these disorders.

The human multidrug-resistance-associated protein MRP1 mediates ATP-dependent transport of unconjugated bilirubin.

Results of previous studies have suggested that UCB (unconjugated bilirubin) may be transported by MRP1/Mrp1 (multidrug-resistance-associated protein 1). To test this hypothesis directly, [3H]UCB transport was assessed in plasma-membrane vesicles from MDCKII cells (Madin-Darby canine kidney II cells) stably transfected with human MRP1 or MRP2; wild-type MDCKII cells served as controls. As revealed by Western blotting, transfection achieved abundant expression of MRP1 and MRP2. [3H]UCB uptake was measured in the presence of 60 microM human serum albumin at a free (unbound) concentration of UCB (B(F)) ranging from 5 to 72 nM and in the presence of 3 mM ATP or 3 mM AMP-PCP (adenosine 5'-[beta,gamma-methylene]triphosphate). MRP1-transfected vesicles showed transport activity three and five times higher respectively compared with MRP2 or wild-type vesicles, whose transport did not differ significantly. [3H]UCB transport was stimulated 4-fold by 1.5 mM GSH, occurred into an osmotically sensitive space, was inhibited by 3 microM MK571 and followed saturative kinetics with K(m)=10+/-3 nM (B(F)) and V(max)=100+/-13 pmol x min(-1) x (mg of protein)(-1). UCB significantly inhibited the transport of LTC4 (leukotriene C4), a leukotriene substrate known to have high affinity for MRP1. Collectively, these results prove directly that MRP1 mediates ATP-dependent cellular export of UCB and supports its role in protecting cells from bilirubin toxicity.

Upregulation in the expression of multidrug resistance protein Mrp1 mRNA and protein by increased bilirubin production in rat.

Earlier studies suggest that Mrp1 may mediate ATP-dependent cellular extrusion of unconjugated bilirubin (UCB). We studied the serial responses of expression of Mrp1 mRNA and protein in rats with increased bilirubin production due to hemolysis induced by phenylhydrazine (PHZ) treatment. Mrp1 mRNA was analyzed by quantitative PCR and protein by Western blot. Hepatic expression of Mrp1 mRNA and protein peaked at day 3 of PHZ treatment. Splenic expression of Mrp1 mRNA peaked within 24h and returned to baseline at day 5 whereas Mrp1 protein expression peaked at day 3. Pretreatment with heme-oxygenase inhibitor, tin mesoporphyrin, blunted the increase in serum UCB and erased the overexpression of Mrp1 both in liver and spleen. Thus, the upregulation of Mrp1 in hemolysis is mediated by UCB and/or other products of heme oxygenase, further supporting a role of Mrp1 in UCB transport and protection from its cellular toxicity.