Moderate neonatal jaundice is the most common clinical condition during newborn life. However, a combination of factors may result in acute hyperbilirubinemia, placing infants at risk of developing bilirubin encephalopathy and death by kernicterus. While most risk factors are known, the mechanisms acting to reduce susceptibility to bilirubin neurotoxicity remain unclear. The presence of modifier genes modulating the risk of developing bilirubin-induced brain damage is increasingly being recognised. The Abcb1 and Abcc1 members of the ABC family of transporters have been suggested to have an active role in exporting unconjugated bilirubin from the central nervous system into plasma. However, their role in reducing the risk of developing neurological damage and death during neonatal development is still unknown.To this end, we mated Abcb1a/b-/- and Abcc1-/- strains with Ugt1-/- mice, which develop severe neonatal hyperbilirubinemia. While about 60% of Ugt1-/- mice survived after temporary phototherapy, all Abcb1a/b-/-/Ugt1-/- mice died before postnatal day 21, showing higher cerebellar levels of unconjugated bilirubin. Interestingly, Abcc1 role appeared to be less important.In the cerebellum of Ugt1-/- mice, hyperbilirubinemia induced the expression of Car and Pxr nuclear receptors, known regulators of genes involved in the genotoxic response.We demonstrated a critical role of Abcb1 in protecting the cerebellum from bilirubin toxicity during neonatal development, the most clinically relevant phase for human babies, providing further understanding of the mechanisms regulating bilirubin neurotoxicity in vivo. Pharmacological treatments aimed to increase Abcb1 and Abcc1 expression, could represent a therapeutic option to reduce the risk of bilirubin neurotoxicity.

• MeSH
• bilirubin toxicita   MeSH
• glukuronosyltransferasa fyziologie   MeSH
• lidé MeSH
• modely nemocí na zvířatech * MeSH
• mozeček účinky léků   patologie   MeSH
• myši knockoutované MeSH
• myši MeSH
• neurotoxické syndromy etiologie   metabolismus   patologie   MeSH
• novorozená zvířata MeSH
• novorozenecká hyperbilirubinemie komplikace   metabolismus   patologie   MeSH
• P-glykoprotein genetika   metabolismus   MeSH
• proteiny spojené s mnohočetnou rezistencí k lékům genetika   metabolismus   MeSH
• viabilita buněk MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

In this study, vibrational circular dichroism (VCD) spectroscopy was employed for the first time to study the bilirubin (BR) interaction with model membranes and models for membrane proteins. An enantioselective interaction of BR with zwitterionic 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and sphingomyelin (SPM) liposomes was observed by VCD and electronic circular dichroism (ECD) complemented by absorption and fluorescence spectroscopy. The M-form of BR was preferentially recognized in the BR/DMPC system at concentration above 1×10(-4)M, for lower concentrations the P-form of BR was recognized by the DMPC liposomes. The VCD spectra also showed that the SPM liposomes, which represent the main component of nerve cell membrane, were significantly more disturbed by the presence of BR than the DMPC liposomes-a stable association with a strong VCD signal was observed providing the explanations for the supposed BR neurotoxicity. The effect of time and pH on the BR/DMPC or SPM liposome systems was shown to be essential while the effect of temperature in the range of 15-70°C was negligible demonstrating the surprisingly high temperature stability of BR when interacting with the studied membranes. The influence of a membrane protein was tested on a model consisting of poly-l-arginine (PLAG) bound in the α-helical form to the surface of 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) liposomes and sodium dodecyl sulfate micelles. VCD and also ECD spectra showed that a variety of BR diastereoisomers interacted with PLAG in such systems. In a system of PLAG with micelles composed of sodium dodecyl sulfate, the M-form of bound BR was observed.

• MeSH
• bilirubin chemie   metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• cholesterol metabolismus   MeSH
• cirkulární dichroismus metody   MeSH
• fluorescenční spektrometrie MeSH
• fosfatidylcholiny metabolismus   MeSH
• lipidové dvojvrstvy chemie   metabolismus   MeSH
• liposomy MeSH
• micely MeSH
• molekulární modely MeSH
• spektroskopie infračervená s Fourierovou transformací MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Unconjugated bilirubin (UCB) in newborns may lead to bilirubin neurotoxicity. Few studies investigated the activation of endoplasmic reticulum stress (ER stress) by UCB. We performed an in vitro comparative study using undifferentiated SH-SY5Y, differentiated GI-ME-N neuronal cells and human U87 astrocytoma cells. ER stress and its contribution to inflammation and apoptosis induced by UCB were analyzed. Cytotoxicity, ER stress and inflammation were observed only in neuronal cells, despite intracellular UCB accumulation in all three cell types. UCB toxicity was enhanced in undifferentiated SH-SY5Y cells and correlated with a higher mRNA expression of pro-apoptotic CHOP. Mouse embryonic fibroblast knockout for CHOP and CHOP siRNA-silenced SH-SY5Y increased cells viability upon UCB exposure. In SH-SY5Y, ER stress inhibition by 4-phenylbutyric acid reduced UCB-induced apoptosis and decreased the cleaved forms of caspase-3 and PARP proteins. Reporter gene assay and PERK siRNA showed that IL-8 induction by UCB is transcriptionally regulated by NFкB and PERK signaling. These data suggest that ER stress has an important role in the UCB-induced inflammation and apoptosis, and that targeting ER stress may represent a potential therapeutic approach to decrease UCB-induced neurotoxicity.

• MeSH
• apoptóza MeSH
• astrocytom metabolismus   MeSH
• bilirubin metabolismus   MeSH
• buněčná diferenciace MeSH
• fenylbutyráty farmakologie   MeSH
• interleukin-8 metabolismus   MeSH
• kaspasa 3 metabolismus   MeSH
• lidé MeSH
• myši knockoutované MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• neuroblastom metabolismus   MeSH
• neurony metabolismus   patologie   MeSH
• stres endoplazmatického retikula * MeSH
• transkripční faktor CHOP genetika   MeSH
• umlčování genů MeSH
• viabilita buněk MeSH
• zánět patologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

The bilirubin (BR) photo-conversion in the human body is a protein-dependent process; an effective photo-isomerization of the potentially neurotoxic Z,Z-BR as well as its oxidation to biliverdin in the antioxidant redox cycle is possible only when BR is bound on serum albumin. We present a novel analytical concept in the study of linear tetrapyrroles metabolic processes based on an in-depth mapping of binding sites in the structure of human serum albumin (HSA). A combination of fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and molecular modeling methods was used for recognition of the binding site for BR, its derivatives (mesobilirubin and bilirubin ditaurate), and the products of the photo-isomerization and oxidation (lumirubin, biliverdin, and xanthobilirubic acid) on HSA. The CD spectra and fluorescent quenching of the Trp-HSA were used to calculate the binding constants. The results of the CD displacement experiments performed with hemin were interpreted together with the findings of molecular docking performed on the pigment-HSA complexes. We estimated that Z,Z-BR and its metabolic products bind on two independent binding sites. Our findings support the existence of a reversible antioxidant redox cycle for BR and explain an additional pathway of the photo-isomerization process (increase of HSA binding capacity; the excess free [unbound] BR can be converted and also bound to HSA).

• MeSH
• bilirubin analogy a deriváty   chemie   metabolismus   MeSH
• biliverdin analogy a deriváty   chemie   metabolismus   MeSH
• cirkulární dichroismus MeSH
• fluorescenční spektrometrie MeSH
• fotochemické procesy * MeSH
• kompetitivní vazba MeSH
• lidé MeSH
• ligandy MeSH
• molekulární konformace MeSH
• molekulární modely * MeSH
• oxidace-redukce MeSH
• sérový albumin chemie   metabolismus   MeSH
• simulace molekulového dockingu MeSH
• stereoizomerie MeSH
• taurin analogy a deriváty   chemie   metabolismus   MeSH
• tryptofan chemie   MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Therapies to prevent severe neonatal unconjugated hyperbilirubinemia and kernicterus are phototherapy and, in unresponsive cases, exchange transfusion, which has significant morbidity and mortality risks. Neurotoxicity is caused by the fraction of unconjugated bilirubin not bound to albumin (free bilirubin, Bf). Human serum albumin (HSA) administration was suggested to increase plasma bilirubin-binding capacity. However, its clinical use is infrequent due to difficulties to address its potential preventive and curative benefits, and to the absence of reliable markers to monitor bilirubin neurotoxicity risk. We used a genetic mouse model of unconjugated hyperbilirubinemia showing severe neurological impairment and neonatal lethality. We treated mutant pups with repeated HSA administration since birth, without phototherapy application. Daily intraperitoneal HSA administration completely rescued neurological damage and lethality, depending on dosage and administration frequency. Albumin infusion increased plasma bilirubin-binding capacity, mobilizing bilirubin from tissues to plasma. This resulted in reduced plasma Bf, forebrain and cerebellum bilirubin levels. We showed that, in our experimental model, Bf is the best marker to determine the risk of developing neurological damage. These results support the potential use of albumin administration in severe acute hyperbilirubinemia conditions to prevent or treat bilirubin neurotoxicity in situations in which exchange transfusion may be required.

• MeSH
• bilirubin krev   MeSH
• fototerapie metody   MeSH
• lidé MeSH
• modely nemocí na zvířatech MeSH
• mozeček účinky léků   MeSH
• myši MeSH
• nemoci nervového systému etiologie   prevence a kontrola   MeSH
• novorozenecká hyperbilirubinemie krev   komplikace   MeSH
• novorozenecká žloutenka krev   komplikace   MeSH
• sérový albumin aplikace a dávkování   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Unconjugated hyperbilirubinemia, a feature of neonatal jaundice or Crigler-Najjar syndrome, can lead to neurotoxicity and even death. We previously demonstrated that unconjugated bilirubin (UCB) can be eliminated via transintestinal excretion in Gunn rats, a model of unconjugated hyperbilirubinemia, and that this is stimulated by enhancing fecal fatty acid excretion. Since transintestinal excretion also occurs for cholesterol (TICE), we hypothesized that increasing fecal cholesterol excretion and/or TICE could also enhance fecal UCB disposal and subsequently lower plasma UCB concentrations. METHODS: To determine whether increasing fecal cholesterol excretion could ameliorate unconjugated hyperbilirubinemia, we treated hyperbilirubinemic Gunn rats with ezetimibe (EZE), an intestinal cholesterol absorption inhibitor, and/or a liver X receptor (LXR) and farnesoid X receptor (FXR) agonist (T0901317 (T09) and obeticholic acid (OCA), respectively), known to stimulate TICE. RESULTS: We found that EZE treatment alone or in combination with T09 or OCA increased fecal cholesterol disposal but did not lower plasma UCB levels. CONCLUSIONS: These findings do not support a link between the regulation of transintestinal excretion of cholesterol and bilirubin. Furthermore, induction of fecal cholesterol excretion is not a potential therapy for unconjugated hyperbilirubinemia. IMPACT: Increasing fecal cholesterol excretion is not effective to treat unconjugated hyperbilirubinemia. This is the first time a potential relation between transintestinal excretion of cholesterol and unconjugated bilirubin is investigated. Transintestinal excretion of cholesterol and unconjugated bilirubin do not seem to be quantitatively linked. Unlike intestinal fatty acids, cholesterol cannot "capture" unconjugated bilirubin to increase its excretion. These results add to our understanding of ways to improve and factors regulating unconjugated bilirubin disposal in hyperbilirubinemic conditions.

• MeSH
• bilirubin chemie   MeSH
• cholesterol metabolismus   MeSH
• Criglerův-Najjarův syndrom metabolismus   terapie   MeSH
• dietní tuky farmakokinetika   MeSH
• ezetimib farmakologie   terapeutické užití   MeSH
• feces chemie   MeSH
• fluorované uhlovodíky farmakologie   terapeutické užití   MeSH
• haptoglobiny analýza   MeSH
• hyperbilirubinemie terapie   MeSH
• jaterní receptor X metabolismus   MeSH
• krysa rodu rattus MeSH
• kyselina chenodeoxycholová analogy a deriváty   farmakologie   terapeutické užití   MeSH
• lipidy krev   MeSH
• náhodné rozdělení MeSH
• potkani Gunn MeSH
• PPAR delta metabolismus   MeSH
• receptory cytoplazmatické a nukleární metabolismus   MeSH
• střeva účinky léků   metabolismus   MeSH
• sulfonamidy farmakologie   terapeutické užití   MeSH
• žluč chemie   MeSH
• žlučové kyseliny a soli metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Severe hyperbilirubinemia is toxic during central nervous system development. Prolonged and uncontrolled high levels of unconjugated bilirubin lead to bilirubin-induced neurological damage and eventually death by kernicterus. Bilirubin neurotoxicity is characterized by a wide array of neurological deficits, including irreversible abnormalities in motor, sensitive and cognitive functions, due to bilirubin accumulation in the brain. Despite the abundant literature documenting the in vitro and in vivo toxic effects of bilirubin, it is unclear which molecular and cellular events actually characterize bilirubin-induced neurodegeneration in vivo. METHODS: We used a mouse model of neonatal hyperbilirubinemia to temporally and spatially define the response of the developing cerebellum to the bilirubin insult. RESULTS: We showed that the exposure of developing cerebellum to sustained bilirubin levels induces the activation of oxidative stress, ER stress and inflammatory markers at the early stages of the disease onset. In particular, we identified TNFα and NFKβ as key mediators of bilirubin-induced inflammatory response. Moreover, we reported that M1 type microglia is increasingly activated during disease progression. Failure to counteract this overwhelming stress condition resulted in the induction of the apoptotic pathway and the generation of the glial scar. Finally, bilirubin induced the autophagy pathway in the stages preceding death of the animals. CONCLUSIONS: This study demonstrates that inflammation is a key contributor to bilirubin damage that cooperates with ER stress in the onset of neurotoxicity. Pharmacological modulation of the inflammatory pathway may be a potential intervention target to ameliorate neonatal lethality in Ugt1-/-mice.

• MeSH
• degenerace nervu etiologie   metabolismus   patologie   MeSH
• glukuronosyltransferasa nedostatek   MeSH
• modely nemocí na zvířatech MeSH
• mozeček patologie   MeSH
• myši knockoutované MeSH
• myši MeSH
• novorozená zvířata MeSH
• novorozenecká hyperbilirubinemie komplikace   patologie   MeSH
• zánět etiologie   metabolismus   patologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Bilirubin, the principal bile pigment, is the end product of heme catabolism. For many years, bilirubin was thought to have no physiological function other than that of a waste product of heme catabolism--useless at best and toxic at worst. Although hyperbilirubinemia in neonates has been shown to be neurotoxic, studies performed during the past decade have found that bilirubin has a number of new and interesting biochemical and biological properties. In addition, there is now a strong body of evidence suggesting that bilirubin may have a beneficial role in preventing oxidative changes in a number of diseases including atherosclerosis and cancer, as well as a number of inflammatory, autoimmune, and degenerative diseases. The results also suggest that activation of the heme oxygenase and heme catabolic pathway may have beneficiary effects on disease prevention either through the action of bilirubin or in conjunction with bilirubin. If so, it may be possible to therapeutically induce heme oxygenase, increase bilirubin concentrations, and lower the risk of oxidative stress-related diseases.

• MeSH
• financování organizované MeSH
• hem metabolismus   MeSH
• lidé MeSH
• nádory metabolismus   MeSH
• nemoci cév metabolismus   MeSH
• ochranné látky metabolismus   MeSH
• oxidační stres genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• přehledy MeSH