Reactivity of human fetal and adult immortalized hepatocytes to potentially toxic bilirubin and bile acid species

Dissertação apresentada para a obtenção do Grau de Mestre em Genética Molecular e Biomedicina, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

The role of circadian timing system on drug metabolism and detoxification.

INTRODUCTION: It has been known for a long time that the efficiency and toxicity of drugs change during a 24-h period. However, the molecular mechanisms involved in these processes have started to emerge only recently. AREAS COVERED: This review aims to highlight recent discoveries showing the direct role of the molecular circadian clock in xenobiotic metabolism at the transcriptional and post-transcriptional levels in the liver and intestine, and the different ways of elimination of these metabolized drugs via biliary and urine excretions. Most of the related literature focuses on transcriptional regulation by the circadian clock of xenobiotic metabolism in the liver; however, the role of this timing system in the excretion of metabolized drugs and the importance of the kidney in this phenomenon are generally neglected. The goal of this review is to describe the molecular mechanisms involved in rhythmic drug metabolism and excretion. EXPERT OPINION: Chronopharmacology is used to analyze the metabolism of drugs in mammals according to the time of day. The circadian timing system plays a key role in the changes of toxicity of drugs by influencing their metabolisms in the liver and intestine in addition to their excretion via bile flow and urine.

Efeitos da administração de vitamina A hidrossolúvel sobre as lesões e a disfunção hepática na colestase obstrutiva: estudo experimental em ratos jovens

Pós-graduação em Fisiopatologia em Clínica Médica - FMB

The role of purinergic signaling in the liver and in transplantation: effects of extracellular nucleotides on hepatic graft vascular injury, rejection and metabolism

Extracellular nucleotides (e.g. ATP, UTP, ADP) are released by activated endothelium, leukocytes and platelets within the injured vasculature and bind specific cell-surface type-2 purinergic (P2) receptors. This process drives vascular inflammation and thrombosis within grafted organs. Importantly, there are also vascular ectonucleotidases i.e. ectoenzymes that hydrolyze extracellular nucleotides in the blood to generate nucleosides (viz. adenosine). Endothelial cell NTPDase1/CD39 has been shown to critically modulate levels of circulating nucleotides. This process tends to limit the activation of platelet and leukocyte expressed P2 receptors and also generates adenosine to reverse inflammatory events. This vascular protective CD39 activity is rapidly inhibited by oxidative reactions, such as is observed with liver ischemia reperfusion injury. In this review, we chiefly address the impact of these signaling cascades following liver transplantation. Interestingly, the hepatic vasculature, hepatocytes and all non-parenchymal cell types express several components co-ordinating the purinergic signaling response. With hepatic and vascular dysfunction, we note heightened P2- expression and alterations in ectonucleotidase expression and function that may predispose to progression of disease. In addition to documented impacts upon the vasculature during engraftment, extracellular nucleotides also have direct influences upon liver function and bile flow (both under physiological and pathological states). We have recently shown that alterations in purinergic signaling mediated by altered CD39 expression have major impacts upon hepatic metabolism, repair mechanisms, regeneration and associated immune responses. Future clinical applications in transplantation might involve new therapeutic modalities using soluble recombinant forms of CD39, altering expression of this ectonucleotidase by drugs and/or using small molecules to inhibit deleterious P2-mediated signaling while augmenting beneficial adenosine-mediated effects within the transplanted liver.

Arginine vasopressin in vasodilatory shock: effects on metabolism and beyond

PURPOSE OF REVIEW: To describe the effects of arginine vasopressin other than its vasoconstrictive and antidiuretic potential in vasodilatory shock. RECENT FINDINGS: Arginine vasopressin influences substrate metabolism by stimulation of hepatic glucose release, gluconeogenesis, ureogenesis and fatty acid esterification. Although arginine vasopressin is a secretagogue of different hormones, only prolactin increases during arginine vasopressin therapy. Plasmatic and cellular coagulation are affected by arginine vasopressin, resulting in thrombocyte aggregation. Therefore, platelet count typically decreases following arginine vasopressin infusion in critically ill patients. In addition, arginine vasopressin reduces bile flow and may increase bilirubin concentrations. Despite its potential to decrease serum sodium, no change in electrolytes was observed in critically ill patients receiving arginine vasopressin. Although arginine vasopressin is an endogenous antipyretic, body temperature is not decreased by central venous arginine vasopressin infusion. In addition, arginine vasopressin modulates immune function through V1 receptors. Compared with norepinephrine, arginine vasopressin may have protective effects on endothelial function. Net arginine vasopressin effects on gastrointestinal motility seem to be inhibitory and are dose dependent. SUMMARY: Except for its antidiuretic and vasoconstrictive actions, the effects of arginine vasopressin in patients with vasodilatory shock have so far only been partially examined. Potential influences of arginine vasopressin on metabolism and immune, liver and mitochondrial function remain to be assessed in future studies.

Portal cavernoma cholangiopathy : case report and systematic review

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Abolition of valproate-derived choleresis in the Mrp2 transporter-deficient rat

Valproic acid (VPA) is a major therapeutic agent in the treatment of epilepsy and other neurological disorders. It is metabolized in humans and rats primarily along two pathways: direct glucuronidation to yield the acyl glucuronide (VPA-G) and beta-oxidation. We have shown much earlier in the Sprague-Dawley rat that i.v. administration of sodium valproate (NaVPA) caused a marked choleresis ( mean of 3.3 times basal bile flow after doses of 150 mg/kg), ascribed to the passive osmotic flow of bile water following excretion of VPA-G across the canalicular membrane. Active biliary pumping of anionic drug conjugates across the canalicular membrane is now believed to be attributable to transporter proteins, in particular Mrp2, which is deficient in the TR- ( a mutant Wistar) rat. In the present study, normal Wistar and Mrp2-deficient TR- rats were dosed i.v. with NaVPA at 150 mg/kg. In the Wistar rats, there was a peak choleretic effect of about 3.2 times basal bile flow, occurring at about 30 to 45 min postdose ( as seen previously with Sprague-Dawley rats). In TR- rats given the same i.v. dose, there was no evidence of postdose choleresis. The choleresis was correlated with the excretion of VPA-G into bile. In Wistar rats, 62.8 +/- 7.7% of the NaVPA dose was excreted in bile as VPA-G, whereas in TR- rats, only 2.0 +/- 0.6% of the same dose was excreted as VPA-G in bile ( with partial compensatory excretion of VPA-G in urine). This study underlines the functional ( bile flow) consequences of biliary transport of xenobiotic conjugated metabolites.

Therapeutic effects and possible mechanisms of a snake venom preparation in the fibrotic rat liver

The effects of a Chinese snake venom preparation from Agkistrodon halys pallas, used for treatment of hepatic fibrosis/cirrhosis in China, was investigated in an {in vivo} rat model and using in situ hepatic perfusion. Four groups were used in the experiments: (i) healthy, (ii) healthy/venom-treated, (iii) carbon tetrachloride (CCl4)-treated, and (iv) CCl4/venom-treated. Treatment effects were assessed by determining hepatic histopathology, biochemistry and fibrosis index parameters, bile production, biliary taurocholate recovery, hepatic mRNA expression of four bile salt transporters (Ntcp, Bsep, Oatp-1, and Oatp-3), comparison of hepatic microcirculation, fibrinolytic activity, and antithrombotic effects. Liver histopathology, biochemistry, and fibrosis index showed a dramatic improvement in venom-treated animals. There were significant differences in bile production between healthy/venom-treated and all other experimental groups and between CCl4/venom-treated and CCl4-treated animals, but no significant differences were found between CCl4/venom-treated and healthy animals. Biliary taurocholate recovery was significantly increased in healthy/venom-treated and CCl4/venom-treated animals. The expression of mRNA levels of the four bile salt transporters showed an increase after venom treatment. The hepatic microcirculation studies showed normalized sinusoidal beds in CCl4/venom-treated animals compared to healthy animals, whereas CCl4-treated animals showed abnormal profiles to the healthy and the CCl4/AHPV-treated animals. The fibrinogen and plasma thromboxane B-2 levels of healthy rats decreased with increasing dose after venom treatment. It was concluded that snake venom treatment may be therapeutic in treatment of hepatic fibrosis/cirrhosis by possibly a combination of increased bile flow and improved hepatic microcirculation, changes in bile salt transporter expression, and fibrinolytic and antithrombotic effects of the snake venom preparation.

Bile acids destabilise HIF-1α and promote anti-tumour phenotypes in cancer cell models

BACKGROUND: The role of the microbiome has become synonymous with human health and disease. Bile acids, as essential components of the microbiome, have gained sustained credibility as potential modulators of cancer progression in several disease models. At physiological concentrations, bile acids appear to influence cancer phenotypes, although conflicting data surrounds their precise physiological mechanism of action. Previously, we demonstrated bile acids destabilised the HIF-1α subunit of the Hypoxic-Inducible Factor-1 (HIF-1) transcription factor. HIF-1 overexpression is an early biomarker of tumour metastasis and is associated with tumour resistance to conventional therapies, and poor prognosis in a range of different cancers. METHODS: Here we investigated the effects of bile acids on the cancer growth and migratory potential of cell lines where HIF-1α is known to be active under hypoxic conditions. HIF-1α status was investigated in A-549 lung, DU-145 prostate and MCF-7 breast cancer cell lines exposed to bile acids (CDCA and DCA). Cell adhesion, invasion, migration was assessed in DU-145 cells while clonogenic growth was assessed in all cell lines. RESULTS: Intracellular HIF-1α was destabilised in the presence of bile acids in all cell lines tested. Bile acids were not cytotoxic but exhibited greatly reduced clonogenic potential in two out of three cell lines. In the migratory prostate cancer cell line DU-145, bile acids impaired cell adhesion, migration and invasion. CDCA and DCA destabilised HIF-1α in all cells and significantly suppressed key cancer progression associated phenotypes; clonogenic growth, invasion and migration in DU-145 cells. CONCLUSIONS: These findings suggest previously unobserved roles for bile acids as physiologically relevant molecules targeting hypoxic tumour progression.

Beam-energy Dependence Of The Directed Flow Of Protons, Antiprotons, And Pions In Au+au Collisions.

Rapidity-odd directed flow (v1) measurements for charged pions, protons, and antiprotons near midrapidity (y=0) are reported in sNN=7.7, 11.5, 19.6, 27, 39, 62.4, and 200 GeV Au+Au collisions as recorded by the STAR detector at the Relativistic Heavy Ion Collider. At intermediate impact parameters, the proton and net-proton slope parameter dv1/dy|y=0 shows a minimum between 11.5 and 19.6 GeV. In addition, the net-proton dv1/dy|y=0 changes sign twice between 7.7 and 39 GeV. The proton and net-proton results qualitatively resemble predictions of a hydrodynamic model with a first-order phase transition from hadronic matter to deconfined matter, and differ from hadronic transport calculations.

Bile And Liver Metallothionein Behavior In Copper-exposed Fish.

The present study analyzed metallothionein (MT) excretion from liver to bile in Nile Tilapia (Oreochromis niloticus) exposed to sub-lethal copper concentrations (2mgL(-1)) in a laboratory setting. MTs in liver and bile were quantified by spectrophotometry after thermal incubation and MT metal-binding profiles were characterized by size exclusion high performance liquid chromatography coupled to ICP-MS (SEC-HPLC-ICP-MS). Results show that liver MT is present in approximately 250-fold higher concentrations than bile MT in non-exposed fish. Differences between the MT profiles from the control and exposed group were observed for both matrices, indicating differential metal-binding behavior when comparing liver and bile MT. This is novel data regarding intra-organ MT comparisons, since differences between organs are usually present only with regard to quantification, not metal-binding behavior. Bile MT showed statistically significant differences between the control and exposed group, while the same did not occur with liver MT. This indicates that MTs synthesized in the liver accumulate more slowly than MTs excreted from liver to bile, since the same fish presented significantly higher MT levels in liver when compared to bile. We postulate that bile, although excreted in the intestine and partially reabsorbed by the same returning to the liver, may also release MT-bound metals more rapidly and efficiently, which may indicate an efficient detoxification route. Thus, we propose that the analysis of bile MTs to observe recent metal exposure may be more adequate than the analysis of liver MTs, since organism responses to metals are more quickly observed in bile, although further studies are necessary.

Electrochemical Oxidation Of Landfill Leachate In A Flow Reactor: Optimization Using Response Surface Methodology.

Response surface methodology based on Box-Behnken (BBD) design was successfully applied to the optimization in the operating conditions of the electrochemical oxidation of sanitary landfill leachate aimed for making this method feasible for scale up. Landfill leachate was treated in continuous batch-recirculation system, where a dimensional stable anode (DSA(©)) coated with Ti/TiO2 and RuO2 film oxide were used. The effects of three variables, current density (milliampere per square centimeter), time of treatment (minutes), and supporting electrolyte dosage (moles per liter) upon the total organic carbon removal were evaluated. Optimized conditions were obtained for the highest desirability at 244.11 mA/cm(2), 41.78 min, and 0.07 mol/L of NaCl and 242.84 mA/cm(2), 37.07 min, and 0.07 mol/L of Na2SO4. Under the optimal conditions, 54.99 % of chemical oxygen demand (COD) and 71.07 ammonia nitrogen (NH3-N) removal was achieved with NaCl and 45.50 of COD and 62.13 NH3-N with Na2SO4. A new kinetic model predicted obtained from the relation between BBD and the kinetic model was suggested.

Triboelectric Effect As A New Strategy For Sealing And Controlling The Flow In Paper-based Devices.

We reported here for the first time that triboelectric charges on PET sheets can be used to seal and control the flow rate in paper-based devices. The proposed method exhibits simplicity and low cost, provides reversible sealing and minimizes the effect of sample evaporation.

Effect Of Concurrent Training With Blood Flow Restriction In The Elderly.

The aim of this present study was to investigate on the effects of concurrent training with blood flow restriction (BFR-CT) and concurrent training (CT) on the aerobic fitness, muscle mass and muscle strength in a cohort of older individuals. 25 healthy older adults (64.7±4.1 years; 69.33±10.8 kg; 1.6±0.1 m) were randomly assigned to experimental groups: CT (n=8, endurance training (ET), 2 days/week for 30-40 min, 50-80% VO2peak and RT, 2 days/week, leg press with 4 sets of 10 reps at 70-80% of 1-RM with 60 s rest), BFR-CT (n=10, ET, similar to CT, but resistance training with blood flow restriction: 2 days/week, leg press with 1 set of 30 and 3 sets of 15 reps at 20-30% 1-RM with 60 s rest) or control group (n=7). Quadriceps cross-sectional area (CSAq), 1-RM and VO2peak were assessed pre- and post-examination (12 wk). The CT and BFR-CT showed similar increases in CSAq post-test (7.3%, P<0.001; 7.6%, P<0.0001, respectively), 1-RM (38.1%, P<0.001; 35.4%, P=0.001, respectively) and VO2peak (9.5%, P=0.04; 10.3%, P=0.02, respectively). The BFR-CT promotes similar neuromuscular and cardiorespiratory adaptations as CT.