Full Text Available Portal hyperperfusion after extended hepatectomy or small-for-size liver transplantation may induce organ dysfunction and failure. The underlying mechanisms, however, are still not completely understood. Herein, we analysed whether hepatectomy-associated portal hyperperfusion induces a hepatic arterial buffer response, i.e., an adaptive hepatic arterial constriction, which may cause hepatocellular hypoxia and organ dysfunction.Sprague-Dawley rats underwent 30%, 70% and 90% hepatectomy. Baseline measurements before hepatectomy served as controls. Hepatic arterial and portal venous flows were analysed by ultrasonic flow measurement. Microvascular blood flow and mitochondrial redox state were determined by intravital fluorescence microscopy. Hepatic tissue pO2 was analysed by polarographic techniques. Hepatic function and integrity were studied by bromosulfophthalein bile excretion and liver histology.Portal blood flow was 2- to 4-fold increased after 70% and 90% hepatectomy. This, however, did not provoke a hepatic arterial buffer response. Nonetheless, portal hyperperfusion and constant hepatic arterial blood flow were associated with a reduced mitochondrial redox state and a decreased hepatic tissue pO2 after 70% and 90% hepatectomy. Microvascular blood flow increased significantly after hepatectomy and functional sinusoidal density was found only slightly reduced. Major hepatectomy further induced a 2- to 3-fold increase of bile flow. This was associated with a 2-fold increase of bromosulfophthalein excretion.Portal hyperperfusion after extended hepatectomy does not induce a hepatic arterial buffer response but reduces mitochondrial redox state and hepatocellular oxygenation. This is not due to a deterioration of microvascular perfusion, but rather due to a relative hypermetabolism of the remnant liver after major resection.
Ziegler, K; Grundmann, E; Veil, L B; Frimmer, M
To exclude an involvement of ligandin in the uptake and storage of phalloidin in hepatocytes equilibrium-dialysis studies were made with phalloidin, cholic acid and bromosulfophthalein (BSP). Binding studies with isolated ligandin indicated that the affinity of ligandin for phalloidin is low (KD = 0.8 X 10-3 M). Phalloidin neither displaced BSP (KD = 1.3 X 10-7 M) or cholic acid (KD = 7.6 X 10-5 M) from ligandin, when preloaded with these substrates. Hepatocytes prepared from rats after daily treatment with phenobarbital during 5 days contained 3-4-fold concentrations of ligandin and bound greater amounts of BSP than controls, Nevertheless the velocity of the uptake both of [3H]-demethylphalloin ([3H]-DMP) and of [35S]-BSP was not augmented. Also the sensitivity of liver cells to phalloidin was not drastically modified after induction with phenobarbital and agrees with earlier findings in vivo. We conclude that ligandin plays a negligible role in the uptake and a minor role in a storage of phallotoxins in liver cells.
Full Text Available Animal studies and premarketing clinical trials have revealed hepatotoxicity of statins, primarily minor elevations in serum alanine aminotransferase levels. The combined chronic use of medicines and eventual ethanol abuse are common and may present a synergistic action regarding liver injury. Our objective was to study the effect of the chronic use of atorvastatin associated with acute ethanol administration on the liver in a rat model. One group of rats was treated daily for 5 days a week for 2 months with 0.8 mg/kg atorvastatin by gavage. At the end of the treatment the livers were perfused with 72 mM ethanol for 60 min. Control groups (at least 4 animals in each group consisted of a group of 2-month-old male Wistar EPM-1 rats exposed to 10% ethanol (v/v ad libitum replacing water for 2 months, followed by perfusion of the liver with 61 nM atorvastatin for 60 min, and a group of animals without chronic ethanol treatment whose livers were perfused with atorvastatin and/or ethanol. The combination of atorvastatin with ethanol did not increase the release of injury marker enzymes (alanine aminotransferase, aspartate aminotransferase, and lactic dehydrogenase from the liver and no change in liver function markers (bromosulfophthalein clearance, and oxygen consumption was observed. Our results suggest that the combination of atorvastatin with ethanol is not more hepatotoxic than the separate use of each substance.
Smith, B F; LaMont, J T
Hydrophobic binding properties of purified bovine gallbladder mucin were studied by fluorescence spectroscopy using 1-anilino-8-naphthalene sulfonate (ANS) and N-phenyl-1-naphthylamine. The purified glycoprotein contained 75.5%, dry weight, as carbohydrate, 16.3% as protein, and 3.7% as sulfate; Mr = 2.2 X 10(6) was estimated by chromatography on Sephacryl S-500. Mucin contained a large number of low-affinity binding sites for these hydrophobic ligands. The dissociation constant, KD of mucin-ANS binding was 2.7 X 10(-5); each mucin molecule had approximately 42 binding sites for ANS. These binding sites were deduced to be on the unglycosylated portion of the protein core, as Pronase digestion completely eliminated binding. Reduction of mucin with 2-mercaptoethanol increased the fluorescence yield by formation of subunits with increased binding sites for the ligand. Increasing NaCl concentration (0.125 to 2.0 M) and decreasing pH (9 to 3) progressively increased fluorescence with the charged ligand ANS, suggesting that the binding site may have acidic groups which are shielded at high ionic strength or low pH. The fluorescent yield with N-phenyl-1-naphthylamine, an uncharged ligand, was an order of magnitude higher than with ANS. Bilirubin and bromosulfophthalein inhibited mucin-induced ANS fluorescence, but bile acids did not. Gallbladder mucin contains hydrophobic binding domains in the nonglycosylated peptide core that are involved in polymer formation and binding of biliary lipids and pigment.
Cai, Shi-Ying; Lionarons, Daniël A.; Hagey, Lee; Soroka, Carol J.; Mennone, Albert; Boyer, James L.
The sea lamprey (Petromyzon marinus) is a genetically programmed animal model for biliary atresia as it loses its bile ducts and gallbladder during metamorphosis. However, in contrast to patients with biliary atresia or other forms of cholestasis who develop progressive disease, the post-metamorphosis lampreys grow normally to adult size. To understand how the adult lamprey thrives without the ability to secrete bile, we examined bile salt homeostasis in larval and adult lampreys. Adult livers were severely cholestatic with levels of bile salts >1 mM, but no evidence of necrosis, fibrosis, or inflammation. Interestingly, both larvae and adults had normal plasma levels (~10 μM) of bile salts. In larvae, petromyzonol sulfate (PZS) was the predominant bile salt, whereas the major bile salts in adult liver were sulfated C27 bile alcohols. Cytotoxicity assays revealed that PZS was highly toxic. Pharmacokinetic studies in free-swimming adults revealed that ~35% of intravenously injected bromosulfophthalein (BSP) was eliminated over a 72 hr period. Collection of urine and feces demonstrated that both endogenous and exogenous organic anions, including biliverdin, bile salts and BSP, were predominantly excreted via the kidney with minor amounts also detected in feces. Gene expression analysis detected marked up-regulation of orthologs of known organic anion and bile salt transporters in the kidney with lesser effects in the intestine and gills in adults compared to larvae. These findings indicate that adult lampreys tolerate cholestasis by altering hepatic bile salt composition, while maintaining normal plasma bile salt levels predominantly through renal excretion of bile products. Therefore, we conclude that strategies to accelerate renal excretion of bile salt and other toxins should be beneficial for patients with cholestasis. PMID:23175353
Rojanathammanee, Lalida; Rakoczy, Sharlene
Ames dwarf mice are deficient in growth hormone (GH), prolactin, and thyroid-stimulating hormone and live significantly longer than their wild-type (WT) siblings. The lack of GH is associated with stress resistance and increased longevity. However, the mechanism underlying GH’s actions on cellular stress defense have yet to be elucidated. In this study, WT or Ames dwarf mice were treated with saline or GH (WT saline, Dwarf saline, and Dwarf GH) two times daily for 7 days. The body and liver weights of Ames dwarf mice were significantly increased after 7 days of GH administration. Mitochondrial protein levels of the glutathione S-transferase (GST) isozymes, K1 and M4 (GSTK1 and GSTM4), were significantly higher in dwarf mice (Dwarf saline) when compared with WT mice (WT saline). GH administration downregulated the expression of GSTK1 proteins in dwarf mice. We further investigated GST activity from liver lysates using different substrates. Substrate-specific GST activity (bromosulfophthalein, dichloronitrobenzene, and 4-hydrox-ynonenal) was significantly reduced in GH-treated dwarf mice. In addition, GH treatment attenuated the activity of thioredoxin and glutaredoxin in liver mitochondria of Ames mice. Importantly, GH treatment suppressed Trx2 and TrxR2 mRNA expression. These data indicate that GH has a role in stress resistance by altering the functional capacity of the GST system through the regulation of specific GST family members in long-living Ames dwarf mice. It also affects the regulation of thioredoxin and glutaredoxin, factors that regulate posttranslational modification of proteins and redox balance, thereby further influencing stress resistance. PMID:24285747
Full Text Available Carbonic anhydrases (CAs are ubiquitous metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate and a proton. CAs are involved in numerous physiological and pathological processes, including acid-base homeostasis, electrolyte balance, oxygen delivery to tissues and nitric oxide generation. Given that these processes are found to be dysregulated during ischemia reperfusion injury (IRI, and taking into account the high vulnerability of steatotic livers to preservation injury, we hypothesized a new role for CA as a pharmacological agent able to protect against ischemic damage. Two different aspects of the role of CA II in fatty liver grafts preservation were evaluated: 1 the effect of its addition to Institut Georges Lopez (IGL-1 storage solution after cold ischemia; 2 and after 24h of cold storage followed by two hours of normothermic ex-vivo perfusion. In all cases, liver injury, CA II protein concentration, CA II mRNA levels and CA II activity were determined. In case of the ex-vivo perfusion, we further assessed liver function (bile production, bromosulfophthalein clearance and Western blot analysis of phosphorylated adenosine monophosphate activated protein kinase (AMPK, mitogen activated protein kinases family (MAPKs and endoplasmic reticulum stress (ERS parameters (GRP78, PERK, IRE, eIF2α and ATF6. We found that CA II was downregulated after cold ischemia. The addition of bovine CA II to IGL-1 preservation solution efficiently protected steatotic liver against cold IRI. In the case of reperfusion, CA II protection was associated with better function, AMPK activation and the prevention of ERS and MAPKs activation. Interestingly, CA II supplementation was not associated with enhanced CO2 hydration. The results suggest that CA II modulation may be a promising target for fatty liver graft preservation.
Banerjee, Nilasha; Wu, T Robert; Chio, Jason; Kelly, Ryan; Stephenson, Karin A; Forbes, John; Allen, Christine; Valliant, John F; Bendayan, Reina
Organic Anion Transporting Polypeptides (OATP) are a family of membrane associated transporters that facilitate estrone-3-sulphate (E3S) uptake by hormone dependent, post-menopausal breast cancers. We have established E3S as a potential ligand for targeting hormone dependent breast cancer cells, and in this study sought to prepare and investigate radioiodinated E3S as a tool to study the OATP system. 2- and 4-Iodoestrone-3-sulfates were prepared from estrone via aromatic iodination followed by a rapid and high yielding sulfation procedure. The resulting isomers were separated by preparative HPLC and verified by (1)H NMR and analytical HPLC. Transport studies of 2- and 4-[(125)I]-E3S were conducted in hormone dependent (i.e. MCF-7) and hormone independent (i.e. MDA-MB-231) breast cancer cells in the presence or absence of the specific transport inhibitor, bromosulfophthalein (BSP). Cellular localization of OATP1A2, OATP2B1, OATP3A1 and OATP4A1 were determined by immunofluorescence analysis using anti-Na(+)/K(+) ATPase-α (1:100 dilution) and DAPI as plasma membrane and nuclear markers, respectively. Significantly (pbreast cancer cells. In contrast 4-[(125)I]-E3S did not show cellular accumulation in either case. The efficiency of 2-[(125)I]-E3S transport (expressed as a ratio of Vmax/Km) was 2.4 times greater in the MCF-7 as compared to the MDA-MB-231 breast cancer cells. OATP1A2, OATP3A1 and OATP4A1 expression was localized in plasma membranes of MCF-7 and MDA-MB-231 cells confirming the functional role of these transporters in radioiodinated E3S cellular uptake. An efficient method for the preparation of 2- and 4-[(125)I]-E3S was developed and where the former demonstrated potential as an in vitro probe for the OATP system. The new E3S probe can be used to study the OATP system and as a platform to create radiopharmaceuticals for imaging breast cancer. Copyright © 2014 Elsevier Inc. All rights reserved.