Hyperbilirubinemic Rats Research Articles

Vascular network expansion, integrity of blood–brain interfaces, and cerebrospinal fluid cytokine concentration during postnatal development in the normal and jaundiced rat

BackgroundSevere neonatal jaundice resulting from elevated levels of unconjugated bilirubin in the blood induces dramatic neurological impairment. Central oxidative stress and an inflammatory response have been associated with the pathophysiological mechanism. Cells forming the blood–brain barrier and the choroidal blood–CSF barrier are the first CNS cells exposed to increased plasma levels of unconjugated bilirubin. These barriers are key regulators of brain homeostasis and require active oxidative metabolism to fulfill their protective functions. The choroid plexus-CSF system is involved in neuroinflammatory processes. In this paper, we address the impact of neonatal hyperbilirubinemia on some aspects of brain barriers. We describe physiological changes in the neurovascular network, blood–brain/CSF barriers integrities, and CSF cytokine levels during the postnatal period in normobilirubinemic animals, and analyze these parameters in parallel in Gunn rats that are deficient in bilirubin catabolism and develop postnatal hyperbilirubinemia.MethodsGunn rats bearing a mutation in UGT1a genes were used. The neurovascular network was analyzed by immunofluorescence stereomicroscopy. The integrity of the barriers was evaluated by [14C]-sucrose permeability measurement. CSF cytokine levels were measured by multiplex immunoassay. The choroid plexus-CSF system response to an inflammatory challenge was assessed by enumerating CSF leukocytes.ResultsIn normobilirubinemic animals, the neurovascular network expands postnatally and displays stage-specific regional variations in its complexity. Network expansion is not affected by hyperbilirubinemia. Permeability of the blood–brain and blood–CSF barriers to sucrose decreases between one- and 9-day-old animals, and does not differ between normobilirubinemic and hyperbilirubinemic rats. Cytokine profiles differ between CSF and plasma in all 1-, 9-, and 18-day-old animals. The CSF cytokine profile in 1-day-old animals is markedly different from that established in older animals. Hyperbilirubinemia perturbs these cytokine profiles only to a very limited extent, and reduces CSF immune cell infiltration triggered by systemic exposure to a bacterial lipopeptide.ConclusionThe data highlight developmental specificities of the blood–brain barrier organization and of CSF cytokine content. They also indicate that a direct effect of bilirubin on the vascular system organization, brain barriers morphological integrity, and inflammatory response of the choroid plexus-CSF system is not involved in the alteration of brain functions induced by severe neonatal jaundice.

Sexual dimorphism: increased sterol excretion leads to hypocholesterolaemia in female hyperbilirubinaemic Gunn rats.

Circulating bilirubin is associated with reduced serum cholesterol concentrations in humans and in hyperbilirubinaemic Gunn rats. However, mechanisms contributing to hypocholesterolaemia remain unknown. Therefore, this study aimed to investigate cholesterol synthesis, transport and excretion in mutant Gunn rats. Adult Gunn and control rats were assessed for daily faecal sterol excretion using metabolic cages, and water was supplemented with [1‐13C]‐acetate to determine cholesterol synthesis. Bile was collected to measure biliary lipid secretion. Serum and liver were collected for biochemical analysis and for gene/protein expression using RT‐qPCR and western blot, respectively. Additionally, serum was collected and analysed from juvenile rats. A significant interaction of sex, age and phenotype on circulating lipids was found with adult female Gunn rats reporting significantly lower cholesterol and phospholipids. Female Gunn rats also demonstrated elevated cholesterol synthesis, greater biliary lipid secretion and increased total faecal cholesterol and bile acid excretion. Furthermore, they possessed increased hepatic low‐density lipoprotein (LDL) receptor and SREBP2 expression. In contrast, there were no changes to sterol metabolism in adult male Gunn rats. This is the first study to demonstrate elevated faecal sterol excretion in female hyperbilirubinaemic Gunn rats. Increased sterol excretion creates a negative intestinal sterol balance that is compensated for by increased cholesterol synthesis and LDL receptor expression. Therefore, reduced circulating cholesterol is potentially caused by increased hepatic uptake via the LDL receptor. Future studies are required to further evaluate the sexual dimorphism of this response and whether similar findings occur in females with benign unconjugated hyperbilirubinaemia (Gilbert's syndrome).Key points Female adult hyperbilirubinaemic (Gunn) rats demonstrated lower circulating cholesterol, corroborating human studies that report a negative association between bilirubin and cholesterol concentrations.Furthermore, female Gunn rats had elevated sterol excretion creating a negative intestinal sterol balance that was compensated for by elevated cholesterol synthesis and increased hepatic low‐density lipoprotein (LDL) receptor expression.Therefore, elevated LDL receptor expression potentially leads to reduced circulating cholesterol levels in female Gunn rats providing an explanation for the hypocholesterolaemia observed in humans with elevated bilirubin levels.This study also reports a novel interaction of sex with the hyperbilirubinaemic phenotype on sterol metabolism because changes were only reported in females and not in male Gunn rats.Future studies are required to further evaluate the sexual dimorphism of this response and whether similar findings occur in females with benign unconjugated hyperbilirubinaemia (Gilbert's syndrome).

Mitochondrial Function, Fatty Acid Metabolism, and Body Composition in the Hyperbilirubinemic Gunn Rat.

Background: Circulating bilirubin is associated with reduced adiposity in human and animal studies. A possible explanation is provided by in vitro data that demonstrates that bilirubin inhibits mitochondrial function and decreases efficient energy production. However, it remains unclear whether hyperbilirubinemic animals have similar perturbed mitochondrial function and whether this is important for regulation of energy homeostasis. Aim: To investigate the impact of unconjugated hyperbilirubinemia on body composition, and mitochondrial function in hepatic tissue and skeletal muscle. Materials and Methods: 1) Food intake and bodyweight gain of 14-week old hyperbilirubinemic Gunn (n = 19) and normobilirubinemic littermate (control; n = 19) rats were measured over a 17-day period. 2) Body composition was determined using dual-energy X-ray absorptiometry and by measuring organ and skeletal muscle masses. 3) Mitochondrial function was assessed using high-resolution respirometry of homogenized liver and intact permeabilized extensor digitorum longus and soleus fibers. 4) Liver tissue was flash frozen for later gene (qPCR), protein (Western Blot and citrate synthase activity) and lipid analysis. Results: Female hyperbilirubinemic rats had significantly reduced fat mass (Gunn: 9.94 ± 5.35 vs. Control: 16.6 ± 6.90 g, p < 0.05) and hepatic triglyceride concentration (Gunn: 2.39 ± 0.92 vs. Control: 4.65 ± 1.67 mg g−1, p < 0.01) compared to normobilirubinemic controls. Furthermore, hyperbilirubinemic rats consumed fewer calories daily (p < 0.01) and were less energetically efficient (Gunn: 8.09 ± 5.75 vs. Control: 14.9 ± 5.10 g bodyweight kcal−1, p < 0.05). Hepatic mitochondria of hyperbilirubinemic rats demonstrated increased flux control ratio (FCR) via complex I and II (CI+II) (Gunn: 0.78 ± 0.16 vs. Control: 0.62 ± 0.09, p < 0.05). Similarly, exogenous addition of 31.3 or 62.5 μM unconjugated bilirubin to control liver homogenates significantly increased CI+II FCR (p < 0.05). Hepatic PGC-1α gene expression was significantly increased in hyperbilirubinemic females while FGF21 and ACOX1 was significantly greater in male hyperbilirubinemic rats (p < 0.05). Finally, hepatic mitochondrial complex IV subunit 1 protein expression was significantly increased in female hyperbilirubinemic rats (p < 0.01). Conclusions: This is the first study to comprehensively assess body composition, fat metabolism, and mitochondrial function in hyperbilirubinemic rats. Our findings show that hyperbilirubinemia is associated with reduced fat mass, and increased hepatic mitochondrial biogenesis, specifically in female animals, suggesting a dual role of elevated bilirubin and reduced UGT1A1 function on adiposity and body composition.

Curcumin Prevents Cerebellar Hypoplasia and Restores the Behavior in Hyperbilirubinemic Gunn Rat by a Pleiotropic Effect on the Molecular Effectors of Brain Damage

Bilirubin toxicity to the central nervous system (CNS) is responsible for severe and permanent neurologic damage, resulting in hearing loss, cognitive, and movement impairment. Timely and effective management of severe neonatal hyperbilirubinemia by phototherapy or exchange transfusion is crucial for avoiding permanent neurological consequences, but these therapies are not always possible, particularly in low-income countries. To explore alternative options, we investigated a pharmaceutical approach focused on protecting the CNS from pigment toxicity, independently from serum bilirubin level. To this goal, we tested the ability of curcumin, a nutraceutical already used with relevant results in animal models as well as in clinics in other diseases, in the Gunn rat, the spontaneous model of neonatal hyperbilirubinemia. Curcumin treatment fully abolished the landmark cerebellar hypoplasia of Gunn rat, restoring the histological features, and reverting the behavioral abnormalities present in the hyperbilirubinemic rat. The protection was mediated by a multi-target action on the main bilirubin-induced pathological mechanism ongoing CNS damage (inflammation, redox imbalance, and glutamate neurotoxicity). If confirmed by independent studies, the result suggests the potential of curcumin as an alternative/complementary approach to bilirubin-induced brain damage in the clinical scenario.

LED-phototherapy does not induce oxidative DNA damage in hyperbilirubinemic Gunn rats.

Phototherapy (PT) is the standard treatment of neonatal unconjugated hyperbilirubinemia. Fluorescent tube (FT)-emitted PT light is known to induce oxidative DNA damage in neonates. Nowadays, however, FTs have largely been replaced by light-emitting diodes (LEDs) for delivering PT. Until now, it is unknown whether LED-PT causes oxidative DNA damage. We aim to determine whether LED-PT induces oxidative DNA damage in hyperbilirubinemic rats. Adult Gunn rats, with genetically unconjugated hyperbilirubinemia, received LED-PT in the clinically relevant doses of 10 or 30 µW/cm2/nm. Urine was collected at 0, 24, and 48 h of PT. A group of young Gunn rats received intensive LED-PT of 100 µW/cm2/nm for 24 h. Urine was collected every 8 h and analyzed for the levels of oxidative DNA damage marker 8-hydroxy-2'deoxyguanosine (8-OHdG) and creatinine. DNA damage was evaluated by immunohistochemistry (γH2AX) of skin and spleen samples. LED-PT of 10 and 30 µW/cm2/nm did not affect urinary concentrations of 8-OHdG and creatinine or the 8-OHdG/creatinine ratio. Likewise, intensive LED-PT did not affect the 8-OHdG/creatinine ratio or the number of γH2AX-positive cells in the skin or spleen. Our results show that LED-PT does not induce oxidative DNA damage in hyperbilirubinemic Gunn rats either at clinically relevant or intensive dosages.

Chronically elevated bilirubin protects from cardiac reperfusion injury in the male Gunn rat.

Bilirubin is associated with reduced risk of cardiovascular disease, as evidenced in conditions of mild hyperbilirubinaemia (Gilbert's Syndrome). Little is known regarding myocardial stress resistance in hyperbilirubinaemic conditions or whether life-long exposure modifies cardiac function, which might contribute to protection from cardiovascular disease. Hyperbilirubinaemic rats and littermate controls underwent echocardiography at 3, 6 and 12 months of age, with hearts subsequently assessed for resistance to 30 min of ischaemia. Heart tissue was then collected for assessment of bilirubin content. No difference in baseline cardiac function was evident until 6 months onwards, where Gunn rats demonstrated aortic dilatation and reduced peak ejection velocities. Additionally, duration of ventricular ejection increased progressively, indicating a negative inotropic effect of bilirubin in vivo. Ex vivo analysis of baseline function revealed reduced left ventricular pressure development (LVDP) and contractility in hyperbilirubinaemic rats. Furthermore, stress resistance was improved in Gunn hearts: post-ischaemic recoveries of LVDP (76 ± 22% vs. 29 ± 17% Control, P < 0.01) and coronary flow (96 ± 9% vs. 86 ± 16% Control, P < 0.01) were improved in Gunn hearts, accompanied by reduced infarct area (21 ± 5% vs. 47 ± 15% Control, P < 0.01), and ventricular malondialdehyde and protein carbonyl content. Expression of myocardial nitric oxide-regulating genes including Nos1 and Noa1 were not significantly different. These data reveal life-long hyperbilirubinaemia induces age-dependent hypocontractility in male Gunn rats, and improved stress resistance. In addition, bilirubin exerts sex-independent effects on vascular structure, myocardial function and ischaemic tolerance, the latter likely mediated via bilirubin's antioxidant properties.

Comparison of Serum Zinc Level Between Neonates With Jaundice and Healthy Neonates

Background: Neonatal jaundice is the most common cause of hospitalization in the first month of life. Factors that reportedly affect the severity of neonatal jaundice include: maternal, prenatal and neonatal factors as well as environmental factors (such as zinc). Animal study demonstrated a decrease in serum bilirubin level after zinc treatment in hyperbilirubinemic rats. Objectives: The current study aimed to investigate serum zinc level in the neonates with idiopatic jaundice. Patients and Methods: A case-control study was undertaken from 2008 to 2010 on 263 neonates in a neonatal intensive care unit and obstetrics department in Ghaem hospital, Mashhad, Iran. Of the 263 infants, 114 and 149 neonates were in the control and the case groups, respectively. Infants with a gestational age of > 35 weeks or with idiopathic jaundice were included in the study. Exclusion criteria were as follows: neonates with sepsis or any congenital abnormalities or with glucose-6-phosphate dehydrogenase (G-6PD) deficiency, ABO and RH incompatibility. Serum levels of zinc and bilirubin were compared between the control (114 neonates without jaundice) and case (149 neonates with jaundice) groups using atomic absorption spectrometry. The maternal and neonatal information were recorded. Spearman correlation coefficient, chi-square and Mann-Whitney tests were employed to analyze the data by SPSS software. Results: The mean value of the zinc serum level was 1024.74 ± 245.17 μmol/L in the control group and 841.42 ± 211.99 μmol/L in the case group (P 0.05). Also, no significant correlation was observed between serum levels of Na, blood urea nitrogen (BUN), creatinine (Cr), white blood cell (WBC), platelet, hematocrit (Hct) and zinc (P > 0.05). Conclusions: The level of serum zinc in the neonates with hyperbilirubinemia jaundice was lower than that of the ones without jaundice. It seems that zinc has a protective effect. However, more studies are needed for better decision making.

The neuroprotective and anti-apoptotic effects of melatonin on hemolytic hyperbilirubinemia-induced oxidative brain damage.

Melatonin exerts protection in several inflammatory and neurodegenerative disorders. To investigate the neuroprotective effects of melatonin in an experimental hemolysis-induced hyperbilirubinemia, newborn Sprague-Dawley rats (25-40 g, n = 72) were injected with phenylhydrazine hydrochloride (PHZ; 75 mg/kg) and the injections were repeated at the 24th hour. Rats were treated with saline or melatonin (10 mg/kg) 30 min before the first and second PHZ injections and 24 h after the 2nd PHZ injections. Control rats (n = 24) were injected with saline, but not PHZ. At sixth hours after the last injections of saline or melatonin, all rats were decapitated. Tumor necrosis factor (TNF)-α, IL-1β, IL-10 and brain-derived neurotrophic factor (BDNF) and S100B levels in the plasma were measured. Brain tissue malondialdehyde (MDA), glutathione (GSH) levels and myeloperoxidase (MPO) activities were measured, and brain tissues were evaluated for apoptosis by TUNEL method. In the saline-treated PHZ group, hemoglobin, hematocrit levels were reduced, and total/direct bilirubin levels were elevated when compared to control group. Increased plasma TNF-α, IL-1β levels, along with decreased BDNF, S100B and IL-10 values were observed in the saline-treated PHZ group, while these changes were all reversed in the melatonin-treated group. Increased MDA levels and MPO activities in the brain tissues of saline-treated hyperbilirubinemic rats, concomitant with depleted brain GSH stores, were also reversed in the melatonin-treated hyperbilirubinemic rats. Increased TUNEL(+) cells in the hippocampus of saline-treated PHZ group were reduced by melatonin treatment. Melatonin exerts neuroprotective and anti-apoptotic effects on the oxidative neuronal damage of the newborn rats with hemolysis and hyperbilirubinemia.

Unconjugated Bilirubin exerts Pro-Apoptotic Effect on Platelets via p38-MAPK activation.

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.

Effect of Ascorbic Acid on Co-trimoxazole Induced Hyperbilirubinemia in Rats

Aims:&nbsp;To evaluate the effect of ascorbic acid on co-trimoxazole induced hyperbilirubinemia.Place and Duration of Study:&nbsp;Laboratory of Raghavendra Institute of Pharmaceutical Education &amp; Research (RIPER), between July 8, 2014 to July 23, 2014.Methodology:&nbsp;Rats were divided into three groups of six each, Control group were treated with vehicle, Negative control group were treated with co-trimoxazole (36 mg/kg of body weight) and Test group were treated with ascorbic acid (500 mg/kg) and co-trimoxazole (36 mg/kg of body weight). All the groups were treated once daily for a period of 15 days. After one hour of oral administration, blood were collected by retro-orbital method on 1st, 5th, 10th, and 15th&nbsp;days and bilirubin levels were estimated by using semi-auto analyser.Results:&nbsp;Animals treated with co-trimoxazole have none significantly increased on bilirubin level at day 1stbut bilirubin levels were significantly increased on 5th&nbsp;and 15th&nbsp;days of test (P&lt;.001) when compared to normal group.However, Animals treated with ascorbic acid + co-trimoxazole have none significantly decreased on bilirubin level on day 1st&nbsp;but from day 5th&nbsp;today 15th&nbsp;bilirubin levels were significantly decreased (P&lt;.001), when compared to negative control group.Conclusion:&nbsp;On administration of ascorbic acid to rats, the hyperbilirubinemia which was induced by Co-trimoxazole decreases. So, the combination therapy of co-trimoxazole and ascorbic acid has the beneficial effect over hyperbilirubinemia caused by co-trimoxazole.

Anti-hyperbilirubinemic and wound healing activity of aqueous extract of Calotropis procera leaves in Wistar rats.

Aims:The aim of this study was to evaluate the bilirubin lowering and wound healing property of aqueous extract of Calotropis procera (AECP) leaves in Wistar rats.Materials and Methods:Albino Wistar rats of either sex were used for the study. Bilirubin lowering property of C. procera leaves was evaluated using phenylhydrazine and paracetamol as inducing agents followed by measuring the concentration of serum total bilirubin in hyperbilirubinemic rats. Wound healing property was evaluated using incision and excision models by measuring tensile breaking strength, percentage wound contractions, and epithelization days, respectively.Statistical Analysis:Statistical comparison between groups in each experiment was done with one-way analysis of variance followed by Dunnett's test.Results:AECP showed a significant (P < 0.05) decrease in concentrations of serum total bilirubin in hyperbilirubinemic rats as well as significant (P < 0.05) increase in breaking strength and percentage wound contractions with decreased epithelization period when compared to control groups.Conclusions:AECP showed significant bilirubin lowering and wound healing property in Wistar rats.

Key determinants of the circulatory exposure of organic anions: differences in hepatic uptake between multidrug resistance-associated protein 2 (Mrp2)-deficient rats and wild-type rats

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-17β-glucuronide (E217βG). 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.

Comparison of Zinc Level between Neonates with Jaundice and Healathy Neonates

Introduction: Neonatal jaundice is the most common cause of hospitalization.Animal study demonstrated a decrease in serum bililrubin level after zinc treatment in hyperbilirubinemic rats. The objective of this study was the investigation of serum zinc level in the neonates with idiopatic . Materials and Methods: A case-control study was undertaken on 263 neonates in a neonatal intensive care unit and obstetrics department in Ghaem Hospital, Mashhad, Iran. Health Infants with gestational age more than 35 weeks or with idiopathic jaundice were included to the study. Serum levels of zinc and bilirubin were compared for 114 neonates without jaundice jaundice(control group) and 149 neonates with jaundice (case group) using atomic absorption spectrometry. The maternal and neonatal information were recorded. Data were analyzed using SPSS software, spearman correlation coefficient, and chi-square and Mann-Whitney tests. Results: The mean value of the zinc serum level was 1024.74±245.17 μmol/L in the control group and 841.42±211.99μmol/L in the case group (P 0.05). Also, there was no significant correlation between serum levels of Na, BUN, Cr, WBC, platelet, HTC and zinc (P>0.05). Conclusion: The level of serum zinc in the neonates with hyperbilirubinemia jaundice was lower than those without jaundice. It seems that zinc has a protective effect. However, more studies are needed for better decision. Keywords: Laundice, Zinc, Neonate, Hyperbilirubinemia.

Inhibitory Effects of Palm Tocotrienol-Rich Fraction Supplementation on Bilirubin-Metabolizing Enzymes in Hyperbilirubinemic Adult Rats

BackgroundPhenylhydrazine, a hemolytic agent, is widely used as a model of experimental hyperbilirubinemia. Palm tocotrienol-rich fraction (TRF) was shown to exert beneficial effects in hyperbilirubinemic rat neonates.AimTo investigate the effects of palm TRF supplementation on hepatic bilirubin-metabolizing enzymes and ocidative stress status in rats administered phenylhydrazine.MethodsTwenty-four male Wistar rats were divided into two groups; one group was intraperitoneally injected with palm TRF at the dose of 30 mg/kg/day, while another group was only given vehicle (control) (vitamin E-free palm oil) for 14 days. Twenty-four hours after the last dose, each group was further subdivided into another two groups. One group was administered phenylhydrazine (100 mg/kg, intraperitoneally) and another group was administered normal saline. Twenty-four hours later, blood and liver were collected for biochemical parameter measurements.ResultsPhenylhydrazine increased plasma total bilirubin level and oxidative stress in the erythrocytes as well as in the liver, which were reduced by the pretreatment of palm TRF. Palm TRF also prevented the increases in hepatic heme oxygenase, biliverdin reductase and UDP-glucuronyltransferase activities induced by phenylhydrazine.ConclusionPalm tocotrienol-rich fraction was able to afford protection against phenylhydrazine-induced hyperbilirubinemia, possibly by reducing oxidative stress and inhibiting bilirubin-metabolizing enzymes in the liver.

Endogenous Tetrapyrroles Influence Leukocyte Responses to Lipopolysaccharide in Human Blood: Pre-Clinical Evidence Demonstrating the Anti-Inflammatory Potential of Biliverdin.

Sepsis is associated with abnormal host immune function in response to pathogen exposure, including endotoxin (lipopolysaccharide; LPS). Cytokines play crucial roles in the induction and resolution of inflammation in sepsis. Therefore, the primary aim of this study was to investigate the effects of endogenous tetrapyrroles, including biliverdin (BV) and unconjugated bilirubin (UCB) on LPS-induced cytokines in human blood. Biliverdin and UCB are by products of haem catabolism and have strong cytoprotective, antioxidant and anti-inflammatory effects. In the present study, whole human blood supplemented with BV and without was incubated in the presence or absence of LPS for 4 and 8 hours. Thereafter, whole blood was analysed for gene and protein expression of cytokines, including IL-1β, IL-6, TNF, IFN-γ, IL-1Ra and IL-8. Biliverdin (50 μM) significantly decreased the LPS-mediated gene expression of IL-1β, IL-6, IFN-γ, IL-1Ra and IL-8 (P<0.05). Furthermore, BV significantly decreased LPS-induced secretion of IL-1β and IL-8 (P<0.05). Serum samples from human subjects and, wild type and hyperbilirubinaemic Gunn rats were also used to assess the relationship between circulating bilirubin and cytokine expression/production. Significant positive correlations between baseline UCB concentrations in human blood and LPS-mediated gene expression of IL-1β (R=0.929), IFN-γ (R=0.809), IL-1Ra (R=0.786) and IL-8 (R=0.857) were observed in blood samples (all P<0.05). These data were supported by increased baseline IL-1β concentrations in hyperbilirubinaemic Gunn rats (P<0.05). Blood samples were also investigated for complement receptor-5 (C5aR) expression. Stimulation of blood with LPS decreased gene expression of C5aR (P<0.05). Treatment of blood with BV alone and in the presence of LPS tended to decrease C5aR expression (P=0.08). These data indicate that supplemented BV inhibits the ex vivo response of human blood to LPS. Surprisingly, however, baseline UCB was associated with heighted inflammatory response to LPS. This is the first study to explore the effects of BV in a preclinical human model of inflammation and suggests that BV could represent an anti-inflammatory target for the prevention of LPS mediated inflammation in vivo.

Human liver stem/progenitor cells decrease serum bilirubin in hyperbilirubinemic Gunn rat.

To test the ability of adult-derived human liver stem/progenitor cells (ADHLSC) from large scale cultures to conjugate bilirubin in vitro and in bilirubin conjugation deficient rat. ADHLSC from large scale cultures were tested for their phenotype and for their capacity to conjugate bilirubin in vitro after hepatogenic differentiation. In vivo, Gunn rats [uridine diphosphate-glucuronosyltransferase 1A1 (UGT1A1) deficient animal] were injected with ADHLSC and cryopreserved hepatocytes (positive control). Two, 4, 13 and 27 wk post-transplantation, transplanted Gunn rat bilirubin serum levels were determined by high performance liquid chromatography. Human transplanted cell engraftment was assessed 27 wk post-transplantation using immunohistochemistry and RTqPCR. Large scale culture conditions do not modify ADHLSC phenotype, ADHLSC were able to specifically conjugate bilirubin. ADHLSC were intraportally injected into Gunn rats and blood UCB was measured at different times post-transplantation, infused-Gunn rats exhibited a metabolic effect 3 mo post-transplantation and maintained over a 6 mo period. ADHLSC engraftment into Gunn rat's liver was demonstrated by RTqPCR and immunohistochemistry against albumin and UGT1A1. ADHLSC from large scale cultures are efficient in conjugating bilirubin in vitro and in restoring a deficient metabolic function (reducing bilirubin level) in hyperbilirubinemic rats.

Investigation of the role of transporters on the hepatic elimination of an LAT1 selective inhibitor JPH203

JPH203 has been developed as an anticancer drug that inhibits L-type amino acid transporter 1-mediated essential amino acid uptake into tumor cells. This study sought to elucidate which drug transporters may be involved in JPH203 hepatic elimination, and to estimate human hepatic clearance. In Sprague-Dawley rats, JPH203 total body clearance approached blood flow rate. JPH203 biotransformation via phase II metabolism produces N-acetyl-JPH203 (NAc-JPH203). NAc-JPH203 accumulates in the bile, and NAc-JPH203 canalicular efflux was significantly decreased in Mrp2-deficient mutant rats (Eisai hyperbilirubinemic rats). JPH203 and NAc-JPH203 are organic anion transporters [organic anion transporting polypeptide (OATP)1B1, OATP1B3, OATP2B1, and OAT3] substrates. In human cryopreserved hepatocytes, JPH203 uptake was saturable and inhibited by rifampicin, a prototypical OATP inhibitor. JPH203 metabolic clearance was larger than influx clearance and eventually passive clearance; JPH203 uptake appears to be the rate-determining process in overall hepatic elimination. Furthermore, unlike rats, the human hepatic clearance was predicted to be intrinsic clearance rate limited. These results suggest that the hepatic uptake transporters are determinant factors to determine JPH203 systemic exposure.

Ticlopidine, a Cholestatic Liver Injury-Inducible Drug, Causes Dysfunction of Bile Formation via Diminished Biliary Secretion of Phospholipids: Involvement of Biliary-Excreted Glutathione-Conjugated Ticlopidine Metabolites

The antiplatelet drug, ticlopidine (TIC), reportedly causes cholestatic liver injuries. The present study analyzed the effect of TIC on bile formation, revealing that the biliary secretion of phospholipids was significantly decreased in TIC-administered Sprague Dawley (SD) rats. However, the effect of TIC on biliary phospholipids was not observed in SD rats pretreated with diethylaminoethyl diphenylpropylacetate that inhibits cytochrome P450s (P450), or in Eisai hyperbilirubinemic rats (EHBR) lacking functional multidrug resistance-associated protein 2 (MRP2/ABCC2). These results suggest that glutathione-conjugated TIC metabolites (TIC-SGs), which were formed in the liver after P450s-mediated metabolism and were excreted extensively into bile by MRP2, mediated the observed alterations of the bile composition. Administration of TIC caused significant liver injuries in SD rats, with decreased biliary phospholipids, but not in EHBR, consistent with the in vitro observation that phospholipid-bile acid-mixed micelles moderated the cytotoxic effects of bile acids. Further analyses revealed that TIC-SGs did not directly inhibit multidrug resistance 3 P-glycoprotein (MDR3/ABCB4)-mediated phosphatidylcholine efflux in vitro. Because the diminished biliary secretion of phospholipids with TIC administration was restored by taurocholate infusion in SD rats, the decreased biliary concentration of bile acids, due to the stimulation of bile acid-independent bile flow driven by TIC-SGs, might have indirectly attenuated phospholipid secretion. In conclusion, extensive biliary excretion of TIC-SGs decreased the biliary secretion of phospholipids, which might have increased the risk of TIC-induced cholestatic liver injury.

Association of multidrug resistance-associated protein 2 single nucleotide polymorphism rs12762549 with the basal plasma levels of phase II metabolites of isoflavonoids in healthy Japanese individuals

Multidrug resistance-associated protein 2 (MRP2; ABCC2) is an ATP-binding cassette transporter that mediates the efflux of anionic drugs and phase II metabolites. Our aim was to elucidate the impact of a single nucleotide polymorphism, rs12762549 (G>C), on the in-vivo activity of MRP2. Plasma specimens collected from 18 healthy volunteers were subjected to an untargeted metabolomic analysis using liquid chromatography-mass spectrometry. The role of MRP2 in the disposition of substances of interest was then examined in vivo in Mrp2-deficient mutant rats (Eisai hyperbilirubinemic rats; EHBRs) and in vitro in human MRP2-expressing membrane vesicles. A multivariate analysis model using liquid chromatography-mass spectrometry data sets successfully differentiated the GG and the CC genotypes of rs12762549. The C allele is associated with the basal plasma levels of the five phase II metabolites of genistein and dihydrogenistein. Such phase II metabolites were also accumulated in EHBR, compared with normal rats, partly because of the reduced biliary excretion. Following oral administration of genistein and daidzein, the plasma concentrations of total genistein and total daidzein, which were mainly accounted for by sulfoglucuronide conjugates, were markedly higher in EHBR than in normal rats. ATP-dependent uptake of sulfoglucuronides and glucuronides of the isoflavonoids was observed only in MRP2-expressing membrane vesicles. MRP2 plays a significant role in preventing the accumulation of phase II metabolites of isoflavonoids. The rs12762549 is associated with an interindividual difference in the plasma levels of MRP2 substrates, phase II metabolites of isoflavonoids, suggesting that this single nucleotide polymorphism is associated with variations in in-vivo MRP2 activity.

3-Monoglucuronyl-Glycyrrhretinic Acid Is a Substrate of Organic Anion Transporters Expressed in Tubular Epithelial Cells and Plays Important Roles in Licorice-Induced Pseudoaldosteronism by Inhibiting 11β-Hydroxysteroid Dehydrogenase 2

Licorice (glycyrrhiza root) has been used as a herbal medicine worldwide with its main active constituent being glycyrrhizin (GL). Licorice sometimes causes adverse effects such as inducing pseudoaldosteronism by inhibiting type 2 11β-hydroxysteroid dehydrogenase (11β-HSD2) caused by glycyrrhetinic acid (GA), a major metabolite of GL. In this study we compared the inhibitory effects of GA, GL, and 3-monoglucuronyl-glycyrrhetinic acid (3MGA), another metabolite of GL, on 11β-HSD2 activity by using microsomes and rat kidney tissue slices. GA, 3MGA, and GL inhibited 11β-HSD2 in rat kidney microsomes, with IC(50) values of 0.32, 0.26, and 2.2 μM, respectively. However, the inhibitory activity of these compounds was reduced markedly, in the slices, in a medium containing 5% bovine serum albumin. Assays using human embryonic kidney 293 cells with transient transformation in transporter genes showed that 3MGA is a substrate of human organic anion transporter (OAT) 1, human OAT3, and human organic anion-transporting peptide 4C1, whereas GA is not. When GA (100 mg/kg/day) was administered orally for 16 days to Eisai hyperbilirubinemic rats, plasma concentrations and urinary excretion of 3MGA were significantly higher, whereas the activity of 11β-HSD2 in kidney microsomes was significantly lower compared with Sprague Dawley rats. These results suggest that 3MGA is actively transported into tubules through OATs, resulting in the inhibition of 11β-HSD2. Because the plasma level of 3MGA depends on the function of hepatic transporters, monitoring 3MGA levels in plasma or urine may be useful for preventing pseudoaldosteronism when licorice or GL is prescribed to patients.