RESUMO
Significance: Evaluation of biological chromophore levels is useful for detection of various skin diseases, including cancer, monitoring of health status and tissue metabolism, and assessment of clinical and physiological vascular functions. Clinically, it is useful to assess multiple different chromophores in vivo with a single technique or instrument. Aim: To investigate the possibility of estimating the concentration of four chromophores, bilirubin, oxygenated hemoglobin, deoxygenated hemoglobin, and melanin from diffuse reflectance spectra in the visible region. Approach: A new diffuse reflectance spectroscopic method based on the multiple regression analysis aided by Monte Carlo simulations for light transport was developed to quantify bilirubin, oxygenated hemoglobin, deoxygenated hemoglobin, and melanin. Three different experimental animal models were used to induce hyperbilirubinemia, hypoxemia, and melanogenesis in rats. Results: The estimated bilirubin concentration increased after ligation of the bile duct and reached around 18 mg/dl at 50 h after the onset of ligation, which corresponds to the reference value of bilirubin measured by a commercially available transcutaneous bilirubin meter. The concentration of oxygenated hemoglobin and that of deoxygenated hemoglobin decreased and increased, respectively, as the fraction of inspired oxygen decreased. Consequently, the tissue oxygen saturation dramatically decreased. The time course of melanin concentration after depilation of skin on the back of rats was indicative of the supply of melanosomes produced by melanocytes of hair follicles to the growing hair shaft. Conclusions: The results of our study showed that the proposed method is capable of the in vivo evaluation of percutaneous bilirubin level, skin hemodynamics, and melanogenesis in rats, and that it has potential as a tool for the diagnosis and management of hyperbilirubinemia, hypoxemia, and pigmented skin lesions.
Assuntos
Bilirrubina , Melaninas , Ratos , Animais , Melaninas/análise , Bilirrubina/análise , Bilirrubina/metabolismo , Análise Espectral/métodos , Pele/química , Hipóxia/diagnóstico por imagem , Hemoglobinas/análise , Oxiemoglobinas/análise , Hiperbilirrubinemia/diagnóstico por imagem , Hiperbilirrubinemia/metabolismoRESUMO
This study aimed to investigate the induction of mild unconjugated hyperbilirubinemia in hepatic UGT1A1 inhibition by Morpholinos antisense in CsA-treated BLC57 mice in comparison with the efficacy of chitosan (CH) as an anti-hypolipidemic natural product. Antisense morpholino oligonucleotides were injected intravenously into CsA-treated mice for 14 days thrice a week. Serum biochemical parameters, antioxidant status, and gene expression analysis of eNOS, PPAR-α, NF-kB, cFn, AT1-R, and ETA-R were determined in cardiac tissues with confirmation by histopathology. Inhibition of UGT1A1 significantly elevated serum unconjugated bilirubin within a physiological range. Furthermore, induced mild hyperbilirubinemia reduces hyperlipidemia, improves antioxidant status, and significantly increases the expression of the cardiac PPAR-α gene while decreasing, ETA-R, iNOS, NF-kB, cFn and AT1-R gene expression in CsA-treated mice. Importantly, mild unconjugated hyperbilirubinemia within physiological ranges may be used as a novel therapeutic strategy to lower hyperlipidemia, atherosclerosis, hypertension, and the CVD outcomes in CsA- treated transplant recipients.
Assuntos
Hiperlipidemias , Hipertensão , Camundongos , Animais , Morfolinos , Ciclosporina , NF-kappa B/genética , NF-kappa B/metabolismo , Oligonucleotídeos Antissenso/uso terapêutico , Bilirrubina , Antioxidantes , Receptores Ativados por Proliferador de Peroxissomo , Hiperbilirrubinemia/induzido quimicamente , Hiperbilirrubinemia/genética , Hiperbilirrubinemia/metabolismo , Hiperlipidemias/tratamento farmacológico , Hiperlipidemias/genética , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismoRESUMO
UGT1A1 (UDP glucuronosyltransferase family 1 member A1) is the primary enzyme required for bilirubin conjugation, which is essential for preventing hyperbilirubinemia. Animal models lack key human organic anion transporting polypeptides with distinct epigenetic control over bilirubin metabolism, necessitating a human model to interrogate the regulatory mechanism behind UGT1A1 function. Here, we use induced pluripotent stem cells to develop human liver organoids that can emulate conjugation failure phenotype. Bilirubin conjugation assays, chromatin immunoprecipitation, and transcriptome analysis elucidated the role of glucocorticoid antagonism in UGT1A1 activation. This antagonism prevents the binding of transcriptional repressor MECP2 at the expense of NRF2 with associated off-target effects. Therefore, we introduced functional GULO (L-gulonolactone oxidase) in human organoids to augment intracellular ascorbate for NRF2 reactivation. This engineered organoid conjugated more bilirubin and protected against hyperbilirubinemia when transplanted in immunosuppressed Crigler-Najjar syndrome rat model. Collectively, we demonstrate that our organoid system serves as a manipulatable model for interrogating hyperbilirubinemia and potential therapeutic development.
Assuntos
Síndrome de Crigler-Najjar , Células-Tronco Pluripotentes , Humanos , Animais , Ratos , Bilirrubina/farmacologia , Bilirrubina/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Fígado/metabolismo , Síndrome de Crigler-Najjar/genética , Síndrome de Crigler-Najjar/terapia , Hiperbilirrubinemia/genética , Hiperbilirrubinemia/metabolismo , Hiperbilirrubinemia/terapia , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Células-Tronco Pluripotentes/metabolismoRESUMO
Gilbert's syndrome, also known as benign hyperbilirubinaemia, was described more than 100 years ago. It has usually been considered a physiological abnormality characterised by a mild elevation of the systemic level of unconjugated bilirubin, in the absence of any underlying liver or overt haemolytic disease. However, since the re-discovery of the potent antioxidant effects of bilirubin in the late 1980s, as well as multiple intracellular signalling pathways affected by bilirubin, an ever-increasing body of evidence suggests that individuals with Gilbert's syndrome may benefit from the mild hyperbilirubinaemia and are actually protected from the development of a wide variety of "diseases of civilisation" such as cardiovascular diseases, certain cancers, and autoimmune or neurodegenerative diseases. This review analyses the current state of medical knowledge given recent discoveries in this rapidly developing field, as well as their possible clinical significance, and provides a new perspective on this condition.
Assuntos
Doença de Gilbert , Humanos , Doença de Gilbert/metabolismo , Hiperbilirrubinemia/metabolismo , Fígado/metabolismo , Bilirrubina/metabolismo , AntioxidantesRESUMO
BACKGROUND AND PURPOSE: Liver failure is associated with psychiatric alterations, partly resulting from the increased brain dopamine levels. We investigated the relationship between increased dopamine levels and mental abnormalities using bile duct ligation (BDL) rats and the mechanism by which liver failure increased dopamine levels in SH-SY5Y cells. Behavioural tests were carried out on day 13 and 27 following BDL, along with measurements of dopamine and metabolites, expressions of enzymes and transporters related to dopamine metabolism, and its transport into the cortex and the hippocampus. SH-SY5Y cells were used to investigate whether NH4 Cl, bile acids and bilirubin affected expression of tyrosine hydroxylase or not. Tyrosine hydroxylase (TH) expression in SH-SY5Y cells co-incubated with bilirubin and signal pathway inhibitors was measured. KEY RESULTS: Open-field test results demonstrated BDL rats showed anxiety-like behaviour, accompanied by increased dopamine levels and expression of TH protein in the cortex. Membrane bound long form (MB)-COMT, slightly but significantly decreased. SH-SY5Y cells indicated that increased bilirubin levels was a factor in inducing TH expression. Both inhibitor of NF-κB pathway BAY 11-7082 and silencing NF-κB p65 reversed bilirubin-induced upregulation of TH protein. NF-κB activator TNF-α increased expression of TH protein. Roles of bilirubin in increases of TH protein expressions and dopamine levels were measured using hyperbilirubinemia rats. Anxiety-like behaviour, was associated with increased dopamine levels and TH protein expressions in hyperbilirubinemia rats. CONCLUSION AND IMPLICATIONS: BDL significantly increased dopamine levels in rat cortex partly due to bilirubin-mediated TH induction. Increased bilirubin induced TH expression via activating NF-κB signalling pathway.
Assuntos
Falência Hepática , Neuroblastoma , Ratos , Humanos , Animais , Tirosina 3-Mono-Oxigenase/metabolismo , Dopamina/metabolismo , NF-kappa B/metabolismo , Neuroblastoma/metabolismo , Ductos Biliares/cirurgia , Falência Hepática/metabolismo , Hiperbilirrubinemia/metabolismo , Bilirrubina/metabolismo , Bilirrubina/farmacologia , Ligadura , Córtex Cerebral/metabolismo , Fígado/metabolismoRESUMO
OBJECTIVE: Multidrug resistance protein 2 (MRP2) is a bottleneck in bilirubin excretion. Its loss is sufficient to induce hyperbilirubinaemia, a prevailing characteristic of acute liver failure (ALF) that is closely associated with clinical outcome. This study scrutinises the transcriptional regulation of MRP2 under different pathophysiological conditions. DESIGN: Hepatic MRP2, farnesoid X receptor (FXR) and Forkhead box A2 (FOXA2) expression and clinicopathologic associations were examined by immunohistochemistry in 14 patients with cirrhosis and 22 patients with ALF. MRP2 regulatory mechanisms were investigated in primary hepatocytes, Fxr -/- mice and lipopolysaccharide (LPS)-treated mice. RESULTS: Physiologically, homeostatic MRP2 transcription is mediated by the nuclear receptor FXR/retinoid X receptor complex. Fxr-/- mice lack apical MRP2 expression and rapidly progress into hyperbilirubinaemia. In patients with ALF, hepatic FXR expression is undetectable, however, patients without infection maintain apical MRP2 expression and do not suffer from hyperbilirubinaemia. These patients express FOXA2 in hepatocytes. FOXA2 upregulates MRP2 transcription through binding to its promoter. Physiologically, nuclear FOXA2 translocation is inhibited by insulin. In ALF, high levels of glucagon and tumour necrosis factor α induce FOXA2 expression and nuclear translocation in hepatocytes. Impressively, ALF patients with sepsis express low levels of FOXA2, lose MRP2 expression and develop severe hyperbilirubinaemia. In this case, LPS inhibits FXR expression, induces FOXA2 nuclear exclusion and thus abrogates the compensatory MRP2 upregulation. In both Fxr -/- and LPS-treated mice, ectopic FOXA2 expression restored apical MRP2 expression and normalised serum bilirubin levels. CONCLUSION: FOXA2 replaces FXR to maintain MRP2 expression in ALF without sepsis. Ectopic FOXA2 expression to maintain MRP2 represents a potential strategy to prevent hyperbilirubinaemia in septic ALF.
Assuntos
Fator 3-beta Nuclear de Hepatócito , Falência Hepática Aguda , Proteína 2 Associada à Farmacorresistência Múltipla , Animais , Camundongos , Bilirrubina , Fator 3-beta Nuclear de Hepatócito/metabolismo , Hepatócitos/metabolismo , Hiperbilirrubinemia/metabolismo , Hiperbilirrubinemia/patologia , Lipopolissacarídeos/metabolismo , Fígado/metabolismo , Falência Hepática Aguda/metabolismo , Proteína 2 Associada à Farmacorresistência Múltipla/metabolismo , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
Conjugated hyperbilirubinemia (CHB) and liver transaminase elevation are known complications of acute lymphoblastic leukemia (ALL) therapy, but host risk factors are poorly understood. Among 373 children diagnosed with ALL between 2011 and 2016, clinically significant CHB and transaminase elevation were observed in 15 (4.0%) and 12 (3.2%) children, respectively, during induction and consolidation. Body mass index ≥95th percentile (odds ratio 9.20, 95% confidence interval 2.56-32.96) was the only host factor independently associated with CHB, and no host factors were associated with transaminase elevation. Obese patients warrant closer monitoring of hepatic function to facilitate early intervention prior to the development of severe, adverse hepatic events.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Biomarcadores Tumorais/análise , Hiperbilirrubinemia/epidemiologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Seguimentos , Humanos , Hiperbilirrubinemia/induzido quimicamente , Hiperbilirrubinemia/metabolismo , Hiperbilirrubinemia/patologia , Incidência , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Prognóstico , Fatores de Risco , Texas/epidemiologia , Adulto JovemRESUMO
Infants receiving long-term parenteral nutrition (PN) develop PN-associated liver disease (PNALD). We previously (Ng K et al. JPEN J Parenter Enteral Nutr 40: 656-671, 2016. doi:10.1177/0148607114567900.) showed that PN containing soy-based lipid supplemented with vitamin E (α-tocopherol) prevents the development of PNALD. We hypothesize that this occurs via vitamin E activation of pregnane X receptor (PXR)-mediated pathways involved in bile acid metabolism. Neonatal piglets received PN for 14 days containing Intralipid (IL; soy-based lipid emulsion), IL supplemented with 12.6 mg·kg-1·day-1 vitamin E (VITE), or IL with 10 mg·kg-1·day-1 Rifadin IV (RIF), a PXR agonist. Pigs treated with IL and VITE, but not RIF, developed cholestasis and hyperbilirubinemia, markers of liver disease. The hepatic PXR target genes CYP3A29 and UGT1A6 increased during RIF treatment. RIF also modestly increased metabolism of chenodeoxycholic acid to the more hydrophilic bile acid hyocholic acid. Serum fibroblast growth factor (FGF)-19, a key regulator in suppressing hepatic bile acid synthesis, significantly increased in the RIF group. We conclude rifampicin modified markers of PNALD development by increased metabolism of bile acids and potentially suppressed bile acid synthesis. Vitamin E was ineffective at high lipid doses in preventing PNALD.NEW & NOTEWORTHY Intravenous vitamin E and rifampicin were administered to neonatal piglets receiving parenteral nutrition to determine their efficacy in reducing the progression of parenteral nutrition-associated liver disease (PNALD). Rifampicin increased serum FGF-19 concentrations and synthesis of the bile acid hyocholic acid which led to a reduction of PNALD parameters at 2 wk of administration. This result has potential clinical implications for the use of rifampicin as a safe and inexpensive treatment for short-term development of PNALD.
Assuntos
Ácidos e Sais Biliares/metabolismo , Emulsões Gordurosas Intravenosas , Hepatopatias/prevenção & controle , Fígado/efeitos dos fármacos , Nutrição Parenteral , Fosfolipídeos , Receptor de Pregnano X/agonistas , Rifampina/farmacologia , Óleo de Soja , Vitamina E/farmacologia , Animais , Animais Recém-Nascidos , Ácidos e Sais Biliares/biossíntese , Colestase/etiologia , Colestase/metabolismo , Colestase/prevenção & controle , Citocromo P-450 CYP3A/metabolismo , Modelos Animais de Doenças , Emulsões , Glucuronosiltransferase/metabolismo , Hiperbilirrubinemia/etiologia , Hiperbilirrubinemia/metabolismo , Hiperbilirrubinemia/prevenção & controle , Fígado/metabolismo , Fígado/patologia , Hepatopatias/etiologia , Hepatopatias/metabolismo , Hepatopatias/patologia , Receptor de Pregnano X/metabolismo , Transdução de Sinais , Sus scrofaRESUMO
BACKGROUND: Bilirubin encephalopathy, the most serious complication of hyperbilirubinemia during the neonatal period, with high mortality and morbidity, often causes irreversible neurological damage. Currently, caspase-1, a member of the cysteinyl aspartate-specific protease caspase family, is regarded as a key mediator of inflammatory processes, attracting widespread attention. The purpose of this study was to investigate whether caspase-1 is involved in bilirubin-induced neuronal injury. METHODS: VX-765, a highly potent and selective inhibitor of caspase-1, was used to investigate the effects of unconjugated bilirubin (UCB) on rat cortical neurons, including cell viability, morphological changes in the cell membrane, and nuclear factor-kappa B (NF-κB) activation. RESULTS: Neurons treated with UCB showed increased caspase-1 activity without the secretion of interleukin (IL)-1ß and IL-18, and caspase-1 was significantly inhibited by pretreatment with VX-765. The cell viability of the VX-765-pretreated neurons was improved, and cell membrane rupture was prevented, as detected by lactate dehydrogenase release and ethidium bromide uptake. Moreover, NF-κB activation by UCB exposure, was attenuated by VX-765 pretreatment. CONCLUSION: Bilirubin-induced neuronal injury involves the activation of caspase-1 and NF-κB, leading to membrane leakage, independently of IL-1ß and IL-18.
Assuntos
Bilirrubina/efeitos adversos , Caspase 1/metabolismo , Córtex Cerebral/embriologia , Neurônios/metabolismo , Animais , Apoptose , Astrócitos/metabolismo , Membrana Celular/metabolismo , Sobrevivência Celular , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Dipeptídeos/farmacologia , Feminino , Hiperbilirrubinemia/metabolismo , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , NF-kappa B/metabolismo , Neurônios/efeitos dos fármacos , Ratos , Transdução de Sinais , para-Aminobenzoatos/farmacologiaRESUMO
Hyperbilirubinemia (jaundice) is caused by raised levels of unconjugated bilirubin in the blood. When severe, susceptible brain regions including the cerebellum and auditory brainstem are damaged causing neurological sequelae such as ataxia, hearing loss and kernicterus. The mechanism(s) by which bilirubin exerts its toxic effect have not been completely understood to date. In this study we investigated the acute mechanisms by which bilirubin causes the neurotoxicity that contributes to hearing loss. We developed a novel mouse model that exhibits the neurological features seen in human Bilirubin-Induced Neurological Dysfunction (BIND) syndrome that we assessed with a behavioural score and auditory brainstem responses (ABR). Guided by initial experiments applying bilirubin to cultured cells in vitro, we performed whole genome gene expression measurements on mouse brain tissue (cerebellum and auditory brainstem) following bilirubin exposure to gain mechanistic insights into biochemical processes affected, and investigated further using immunoblotting. We then compared the gene changes induced by bilirubin to bacterial lipopolysaccharide (LPS), a well characterized inducer of neuroinflammation, to assess the degree of similarity between them. Finally, we examined the extent to which genetic perturbation of inflammation and both known and novel anti-inflammatory drugs could protect hearing from bilirubin-induced toxicity. The in vitro results indicated that bilirubin induces changes in gene expression consistent with endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR). These gene changes were similar to the gene expression signature of thapsigargin-a known ER stress inducer. It also induced gene expression changes associated with inflammation and NF-κB activation. The in vivo model showed behavioural impairment and a raised auditory threshold. Whole genome gene expression analysis confirmed inflammation as a key mechanism of bilirubin neurotoxicity in the auditory pathway and shared gene expression hallmarks induced by exposure to bacterial lipopolysaccharide (LPS) a well-characterized inducer of neuroinflammation. Interestingly, bilirubin caused more severe damage to the auditory system than LPS in this model, but consistent with our hypothesis of neuroinflammation being a primary part of bilirubin toxicity, the hearing loss was protected by perturbing the inflammatory response. This was carried out genetically using lipocalin-2 (LCN2)-null mice, which is an inflammatory cytokine highly upregulated in response to bilirubin. Finally, we tested known and novel anti-inflammatory compounds (interfering with NF-κB and TNFα signalling), and also demonstrated protection of the auditory system from bilirubin toxicity. We have developed a novel, reversible, model for jaundice that shows movement impairment and auditory loss consistent with human symptoms. We used this model to establish ER-stress and inflammation as major contributors to bilirubin toxicity. Because of the rapid and reversible onset of toxicity in this novel model it represents a system to screen therapeutic compounds. We have demonstrated this by targeting inflammation genetically and with anti-inflammatory small molecules that offered protection against bilirubin toxicity. This also suggests that anti-inflammatory drugs could be of therapeutic use in hyperbilirubinemia.
Assuntos
Bilirrubina/toxicidade , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Perda Auditiva/etiologia , Kernicterus/etiologia , Síndromes Neurotóxicas/etiologia , Doença Aguda , Animais , Anti-Inflamatórios/farmacologia , Ataxia/etiologia , Ataxia/metabolismo , Bilirrubina/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Potenciais Evocados Auditivos do Tronco Encefálico/efeitos dos fármacos , Feminino , Perda Auditiva/metabolismo , Perda Auditiva/prevenção & controle , Humanos , Hiperbilirrubinemia/complicações , Hiperbilirrubinemia/metabolismo , Inflamação/etiologia , Inflamação/metabolismo , Kernicterus/metabolismo , Lipocalina-2/deficiência , Lipocalina-2/genética , Lipopolissacarídeos/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos CBA , Camundongos Knockout , NF-kappa B/metabolismo , Síndromes Neurotóxicas/metabolismoRESUMO
BACKGROUND & AIMS: Biliverdin and bilirubin were previously considered end products of heme catabolism; now, however, there is evidence for further degradation to diverse bioactive products. Z-BOX A and Z-BOX B arise upon oxidation with unknown implications for hepatocellular function and integrity. We studied the impact of Z-BOX A and B on hepatic functions and explored their alterations in health and cholestatic conditions. METHODS: Functional implications and mechanisms were investigated in rats, hepatocytic HepG2 and HepaRG cells, human immortalized hepatocytes, and isolated perfused livers. Z-BOX A and B were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in acute and acute-on-chronic liver failure and hereditary unconjugated hyperbilirubinemia. RESULTS: Z-BOX A and B are found in similar amounts in humans and rodents under physiological conditions. Serum concentrations increased â¼20-fold during cholestatic liver failure in humans (p<0.001) and in hereditary deficiency of bilirubin glucuronidation in rats (p<0.001). Pharmacokinetic studies revealed shorter serum half-life of Z-BOX A compared to its regio-isomer Z-BOX B (p=0.035). While both compounds were taken up by hepatocytes, Z-BOX A was enriched â¼100-fold and excreted in bile. Despite their reported vasoconstrictive properties in the brain vasculature, BOXes did not affect portal hemodynamics. Both Z-BOX A and B showed dose-dependent cytotoxicity, affected the glutathione redox state, and differentially modulated activity of Rev-erbα and Rev-erbß. Moreover, BOXes-triggered remodeling of the hepatocellular cytoskeleton. CONCLUSIONS: Our data provide evidence that higher-order heme degradation products, namely Z-BOX A and B, impair hepatocellular integrity and might mediate intra- and extrahepatic cytotoxic effects previously attributed to hyperbilirubinemia. LAY SUMMARY: Degradation of the blood pigment heme yields the bile pigment bilirubin and the oxidation products Z-BOX A and Z-BOX B. Serum concentrations of these bioactive molecules increase in jaundice and can impair liver function and integrity. Amounts of Z-BOX A and Z-BOX B that are observed during liver failure in humans have profound effects on hepatic function when added to cultured liver cells or infused into healthy rats.
Assuntos
Heme/metabolismo , Fígado/metabolismo , Insuficiência Hepática Crônica Agudizada/metabolismo , Animais , Bile/metabolismo , Bilirrubina/metabolismo , Biliverdina/metabolismo , Colestase/metabolismo , Glutationa/metabolismo , Hemodinâmica , Células Hep G2 , Humanos , Hiperbilirrubinemia/metabolismo , Técnicas In Vitro , Circulação Hepática , Masculino , Oxirredução , Pirróis/metabolismo , Ratos , Ratos WistarRESUMO
Hyperbilirubinemia is a pathological condition of excessive accumulation of conjugated or unconjugated bilirubin in blood. It has been associated with neurotoxicity and non-neural organ dysfunctions, while it can also be a warning of liver side effects. Hyperbilirubinemia can either be a result of overproduction of bilirubin due to hemolysis or dyserythropoiesis, or the outcome of impaired bilirubin elimination due to liver transporter malfunction or inhibition. There are several reports in literature that inhibition of organic anion transporting polypeptides 1B1 and 1B3 (OATP1B1 and OATP1B3) might lead to hyperbilirubinemia. In this study we created a set of classification models for hyperbilirubinemia, which, besides physicochemical descriptors, also include the output of classification models of human OATP1B1 and 1B3 inhibition. Models were based on either human data derived from public toxicity reports or animal data extracted from the eTOX database VITIC. The generated models showed satisfactory accuracy (68%) and area under the curve (AUC) for human data and 71% accuracy and 70% AUC for animal data. However, our results did not indicate strong association between OATP inhibition and hyperbilirubinemia, neither for humans nor for animals.
Assuntos
Hiperbilirrubinemia/metabolismo , Transportador 1 de Ânion Orgânico Específico do Fígado/antagonistas & inibidores , Transportadores de Ânions Orgânicos Sódio-Independentes/antagonistas & inibidores , Animais , Interações Medicamentosas , Humanos , Transportador 1 de Ânion Orgânico Específico do Fígado/metabolismo , Modelos Biológicos , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de SolutoRESUMO
Unconjugated bilirubin (UCB) is known to be one of the most potent endogenous antioxidant substances. While hyperbilirubinemia has long been recognized as an ominous sign of liver dysfunction, recent data strongly indicate that mildly elevated bilirubin (BLB) levels can be protective against an array of diseases associated with increased oxidative stress. These clinical observations are supported by new discoveries relating to the role of BLB in immunosuppression and inhibition of protein phosphorylation, resulting in the modulation of intracellular signaling pathways in vascular biology and cancer, among others. Collectively, the evidence suggests that targeting BLB metabolism could be considered a potential therapeutic approach to ameliorate a variety of conditions.
Assuntos
Bilirrubina/metabolismo , Transdução de Sinais , Animais , Antioxidantes/metabolismo , Doenças Cardiovasculares/metabolismo , Diabetes Mellitus/metabolismo , Humanos , Hiperbilirrubinemia/metabolismo , Inflamação/metabolismo , Neoplasias/metabolismo , Doenças do Sistema Nervoso/metabolismo , Oxirredução , Estresse OxidativoRESUMO
DDI could be caused by the inhibition of OATP-mediated hepatic uptakes. The aim of this study is to set the risk criteria for the compounds that would cause DDI via OATP inhibition at the drug discovery stage. The IC50 values of OATP inhibitors for human OATP-mediated atorvastatin uptake were evaluated in the expression system. In order to set the risk criteria for OATP inhibition, the relationship was clarified between OATP inhibitory effect and severe adverse effects of OATP substrates, rhabdomyolysis, hyperbilirubinemia and jaundice. Rhabdomyolysis would be caused in the atorvastatin AUC more than 9-fold of that at a minimum therapeutic dose. The atorvastatin AUC was 6- to 9-fold increased with the OATP inhibitors of which IC50 values were ≤1 µmol/L. Hyperbilirubinemia and jaundice would be caused with the OATP inhibitors of which IC50 values were ≤6 µmol/L. This investigation showed that the compounds with IC50 of ≤1 µmol/L would have high risk for OATP-mediated DDI that would cause severe side effects. Before the detailed analysis based on the dosage, unbound fraction in blood and effective concentration to evaluate the clinical DDI potency, this criteria enable high throughput screening and optimize lead compounds at the drug discovery stage.
Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/etiologia , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/metabolismo , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Atorvastatina/efeitos adversos , Atorvastatina/farmacocinética , Atorvastatina/farmacologia , Linhagem Celular , Descoberta de Drogas/métodos , Células HEK293 , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Hiperbilirrubinemia/tratamento farmacológico , Hiperbilirrubinemia/metabolismo , Icterícia/tratamento farmacológico , Icterícia/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Rabdomiólise/tratamento farmacológico , Rabdomiólise/metabolismo , RiscoRESUMO
Hyperbilirubinemia (HB) is sometimes encountered following bile-duct cannulation in rats. It possibly originates from the reduced functioning of multidrug resistance-associated protein 2 (Mrp2) and subsequent adaptive alterations in the expression of Mrp3 and the organic anion transporting polypeptides (Oatps). Our aim was to clarify the importance of excluding bile-duct-cannulated (BDC) rats with HB for proper conduct of drug excretion studies. We detected HB [serum total bilirubin concentration (TBIL) ≥0.20 mg/dl] in 16% of all BDC rats prepared. The serum activities of aspartate aminotransferase, alanine aminotransferase, leucine aminopeptidase, and alkaline phosphatase were within the respective normal ranges in the BDC rats with mild HB (TBIL, 0.20-0.79 mg/dl), indicating the absence of hepatic failure. In the pharmacokinetics of pravastatin, an Oatps/Mrp2 probe drug in the BDC rats, the apparent volume of distribution and the clearance were smaller in the mild HB group as compared with the normal group, suggesting the reduction of apparent hepatic uptake and hepatobiliary elimination. The biliary excretion (percentage of dose) was significantly reduced by 54%, suggesting that the biliary efflux activity via Mrp2 was reduced to a greater extent relative to metabolic activity in hepatocytes. The serum γ-glutamyltransferase (GGT) activity correlated with TBIL and inversely correlated with biliary excretion of pravastatin, a finding which could serve as a clue to uncover the regulatory system involving cooperation between GGT and Mrp2. In conclusion, BDC rats with HB, however mild, should be excluded from drug excretion studies to avoid the risk of underestimation of the biliary excretion of drugs.
Assuntos
Ductos Biliares/metabolismo , Bile/metabolismo , Cateterismo/efeitos adversos , Eliminação Hepatobiliar , Hiperbilirrubinemia/etiologia , Pravastatina/farmacocinética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Bilirrubina/sangue , Biomarcadores/sangue , Hiperbilirrubinemia/sangue , Hiperbilirrubinemia/metabolismo , Fígado/metabolismo , Masculino , Modelos Biológicos , Transportadores de Ânions Orgânicos/metabolismo , Pravastatina/administração & dosagem , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos TestesRESUMO
Humanized UDP-glucuronosyltransferase (UGT)-1 (hUGT1) mice encode the UGT1 locus including the UGT1A1 gene. During neonatal development, delayed expression of the UGT1A1 gene leads to hyperbilirubinemia as determined by elevated levels of total serum bilirubin (TSB). We show in this report that the redox-sensitive NF-κB pathway is crucial for intestinal expression of the UGT1A1 gene and control of TSB levels. Targeted deletion of IKKß in intestinal epithelial cells (hUGT1/Ikkß(ΔIEC) mice) leads to greater neonatal accumulation of TSB than observed in control hUGT1/Ikkß(F/F) mice. The elevation in TSB levels in hUGT1/Ikkß(ΔIEC) mice correlates with a reduction in intestinal UGT1A1 expression. As TSB levels accumulate in hUGT1/Ikkß(ΔIEC) mice during the neonatal period, the increase over that observed in hUGT1/Ikkß(F/F) mice leads to weight loss, seizures and eventually death. Bilirubin accumulates in brain tissue from hUGT1/Ikkß(ΔIEC) mice inducing an inflammatory state as shown by elevated TNFα, IL-1ß and IL-6, all of which can be prevented by neonatal induction of hepatic or intestinal UGT1A1 and lowering of TSB levels. Altering the redox state of the intestines by oral administration of cadmium or arsenic to neonatal hUGT1/Ikkß(F/F) and hUGT1/Ikkß(ΔIEC) mice leads to induction of UGT1A1 and a dramatic reduction in TSB levels. Microarray analysis following arsenic treatment confirms upregulation of oxidation-reduction processes and lipid metabolism, indicative of membrane repair or synthesis. Our findings indicate that the redox state in intestinal epithelial cells during development is important in maintaining UGT1A1 gene expression and control of TSB levels.
Assuntos
Arsênio/farmacologia , Cádmio/farmacologia , Glucuronosiltransferase/genética , Hiperbilirrubinemia/prevenção & controle , NF-kappa B/genética , Convulsões/prevenção & controle , Animais , Animais Recém-Nascidos , Bilirrubina/antagonistas & inibidores , Bilirrubina/biossíntese , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Glucuronosiltransferase/metabolismo , Humanos , Hiperbilirrubinemia/genética , Hiperbilirrubinemia/metabolismo , Hiperbilirrubinemia/patologia , Quinase I-kappa B/deficiência , Quinase I-kappa B/genética , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/efeitos dos fármacos , Intestinos/patologia , Camundongos , Camundongos Transgênicos , NF-kappa B/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Convulsões/genética , Convulsões/metabolismo , Convulsões/patologia , Transdução de Sinais , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Redução de Peso/efeitos dos fármacosRESUMO
Thrombocytopenia is one of the most frequently observed secondary complications in many pathological conditions including liver diseases, where hyperbilirubinemia is very common. The present study sought to find the cause of thrombocytopenia in unconjugated hyperbilirubinemic conditions. Unconjugated bilirubin (UCB), an end-product of heme catabolism, is known to have pro-oxidative and cytotoxic effects at high serum concentration. We investigated the molecular mechanism underlying the pro-apoptotic effect of UCB on human platelets in vitro, and followed it up with studies in phenylhydrazine-induced hyperbilirubinemic rat model and hyperbilirubinemic human subjects. UCB is indeed found to significantly induce platelet apoptotic events including elevated endogenous reactive oxygen species generation, mitochondrial membrane depolarization, increased intracellular calcium levels, cardiolipin peroxidation and phosphatidylserine externalization (p < 0.001) as evident by FACS analysis. The immunoblots show the elevated levels of cytosolic cytochrome c and caspase activation in UCB-treated platelets. Further, UCB is found to induce mitochondrial ROS generation leading to p38 activation, followed by downstream activation of p53, ultimately resulting in altered expression of Bcl-2 and Bax proteins as evident from immunoblotting. All these parameters conclude that elevated unconjugated bilirubin causes thrombocytopenia by stimulating platelet apoptosis via mitochondrial ROS-induced p38 and p53 activation.
Assuntos
Apoptose , Bilirrubina/metabolismo , Plaquetas/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Apoptose/efeitos dos fármacos , Bilirrubina/farmacologia , Plaquetas/efeitos dos fármacos , Cálcio/metabolismo , Cardiolipinas/metabolismo , Modelos Animais de Doenças , Humanos , Hiperbilirrubinemia/metabolismo , Potencial da Membrana Mitocondrial , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fosforilação , Ratos , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismoRESUMO
Biliary excretion of organic anions, such as bile acids (BAs), is the main osmotic driving force for bile formation, and its impairment induces intrahepatic cholestasis. We investigated the involvement of Atp11c in the hepatic transport of organic anions using Atp11c mutant mice, which exhibit hypercholanemia and hyperbilirubinemia. Pharmacokinetic analysis following a constant intravenous infusion in Atp11c mutant mice showed decreased hepatic sinusoidal uptake and intact biliary secretion of [(3)H]17ß estradiol 17ß-d-glucuronide. Consistent with this result, compared with cells and membranes from control mice, isolated hepatocytes, and liver plasma membranes from Atp11c mutant mice had a much lower uptake of [(3)H]17ß estradiol 17ß-d-glucuronide and expression of organic anion-transporting polypeptides, which are transporters responsible for hepatic uptake of unconjugated BAs and organic anions, including bilirubin glucuronides. Uptake of [(3)H]TC into hepatocytes and expression of Na(+)-taurocholate cotransporting polypeptide in liver plasma membranes, which mediates hepatic uptake of conjugated BAs, was also lower in the Atp11c mutant mice. Bile flow rate, biliary BA concentration, and expression of hepatobiliary transporters did not differ between Atp11c mutant mice and control mice. These results suggest that Atp11c mediates the transport of BAs and organic anions across the sinusoidal membrane, but not the canalicular membrane, by regulating the abundance of transporters. Atp11c is a candidate gene for genetically undiagnosed cases of hypercholanemia and hyperbilirubinemia, but not of intrahepatic cholestasis. This gene may influence the pharmacological and adverse effect of drugs because organic anion-transporting polypeptides regulate their systemic exposure.
Assuntos
Adenosina Trifosfatases/genética , Ácidos Cólicos/sangue , Hiperbilirrubinemia/genética , Hiperbilirrubinemia/metabolismo , Fígado/metabolismo , Proteínas de Membrana Transportadoras/genética , Transportadores de Ânions Orgânicos/metabolismo , Erros Inatos do Metabolismo de Esteroides/genética , Erros Inatos do Metabolismo de Esteroides/metabolismo , Animais , Ácidos e Sais Biliares/metabolismo , Ácidos Cólicos/genética , Ácidos Cólicos/metabolismo , Hepatócitos/metabolismo , Fígado/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação/genéticaRESUMO
AIM: Organic anion-transporting polypeptides OATP1B1 and OATP1B3 are sinusoidal membrane transporters mediating liver uptake of a wide range of substrates including conjugated and unconjugated bilirubin, xenobiotics and drugs. Absence of OATP1Bs in the liver causes Rotor syndrome. Our aim was to correlate OATP1B expression with hyperbilirubinemia in common liver diseases. METHODS: Immunoreactivity of five antibodies against human OATP1Bs was tested on frozen and formalin-fixed paraffin-embedded liver tissue of mouse strains transgenic for SLCO1B1 or SLCO1B3 and on human specimens. The proportion of hepatocytes expressing OATP1Bs was then assessed immunohistologically in formalin-fixed paraffin-embedded liver samples obtained from patients with hepatocellular and primary biliary liver diseases. UGT1A1 promoter TATA-box and SLCO1B1 rs4149056 genotyping was performed to rule out individuals predisposed to hyperbilirubinemia. RESULTS: The most specific detection of OATP1B3 was achieved with the H-52 (sc-98981) antibody. OATP1B1 was specifically recognized with the ESL (ab15441) anti-OATP1B1 antibody, but only in frozen sections. The MDQ (ab15442) anti-OATP1B1 antibody cross-reacted with both OATP1B proteins in liver tissue of the transgenic mouse strains. Expression of the OATP1B proteins was decreased in advanced liver diseases and inversely correlated with serum bilirubin levels. The reduction was more pronounced in advanced primary biliary diseases (1.9±1.1 vs. 2.7±0.6; P=0.009). CONCLUSIONS: Down-regulation of OATP1B proteins may contribute to pathogenesis of jaundice accompanying advanced cholestatic liver diseases.
Assuntos
Colestase/metabolismo , Hepatócitos/metabolismo , Hiperbilirrubinemia/metabolismo , Fígado/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Animais , Bilirrubina/sangue , Biomarcadores/sangue , Colestase/diagnóstico , Colestase/genética , Regulação para Baixo , Fixadores , Formaldeído , Secções Congeladas , Hepatócitos/patologia , Humanos , Hiperbilirrubinemia/diagnóstico , Hiperbilirrubinemia/genética , Imuno-Histoquímica , Fígado/patologia , Transportador 1 de Ânion Orgânico Específico do Fígado , Camundongos Transgênicos , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos Sódio-Independentes/genética , Inclusão em Parafina , Estudos Retrospectivos , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto , Fixação de Tecidos/métodosRESUMO
1. Raloxifene-6-glucuronide (R6G) is a substrate of rat multidrug resistance-associated protein 2 (Mrp2), a transporter responsible for biliary excretion of organic anions. 2. Pharmacokinetic modeling of R6G in Eisai hyperbilirubinemic rats (EHBRs), hereditary Mrp2-deficient rats, and wild-type Sprague-Dawley rats (SDRs) indicated that reduction in not only biliary excretion but also hepatic uptake of R6G influenced low clearance in EHBRs. 3. An integration plot study demonstrated that the hepatic uptake of R6G was 66% lower in EHBRs than that in SDRs. A reduction was observed for the other Mrp2 substrate Valsartan (95% lower) but not for estradiol-17ß-glucuronide (E217ßG). This variation may be associated with the difference in substrate specificity of transporters and/or inhibition of hepatic uptake of organic anions by endogenous substances such as bilirubin glucuronides. 4. In conclusion, incidental alteration of the hepatic uptake of organic anions should be considered as an explanation of their enhanced systemic exposure in EHBRs.