Hepatic Fraction Research Articles

Iron-binding biomolecules in the soluble hepatic fraction of the northern pike (Esox lucius): two-dimensional chromatographic separation with mass spectrometry detection.

Iron plays vital roles in important biological processes in fish, but can be toxic in high concentrations. The information on metalloproteins that participate in maintenance of Fe homeostasis in an esocid fish, the northern pike, as an important freshwater bioindicator species, are rather scarce. The aim of this study was to identify main cytosolic constituents that sequester Fe in the northern pike liver. The method applied consisted of two-dimensional HPLC separation of Fe-binding biomolecules, based on anion-exchange followed by size-exclusion fractionation. Apparent molecular masses of two main Fe-metalloproteins isolated by this procedure were ~360 kDa and ~50 kDa, with the former having more acidic pI, and indicated presence of ferritin and hemoglobin, respectively. MALDI-TOF-MS provided confirmation of ferritin subunit with a m/z peak at 20.65 kDa, and hemoglobin with spectra containing main m/z peak at 16.1 kDa, and smaller peaks at 32.1, 48.2, and 7.95 kDa (single-charged Hb-monomer, dimer, and trimer, and double-charged monomer, respectively). LC-MS/MS with subsequent MASCOT database search confirmed the presence of Hb-β subunits and pointed to close relation between esocid and salmonid fishes. Further efforts should be directed towards optimization of the conditions for metalloprotein analysis by mass spectrometry, to extend the knowledge on intracellular metal-handling mechanisms.

The study of acidic/basic nature of metallothioneins and other metal-binding biomolecules in the soluble hepatic fraction of the northern pike (Esox lucius)

Since fish metalloproteins are still not thoroughly characterized, the aim of this study was to investigate the acidic/basic nature of biomolecules involved in the sequestration of twelve selected metals in the soluble hepatic fraction of an important aquatic bioindicator organism, namely the fish species northern pike (Esox lucius). For this purpose, the hyphenated system HPLC-ICP-MS was applied, with chromatographic separation based on anion/cation-exchange principle at physiological pH (7.4). The results indicated predominant acidic nature of metal-binding peptides/proteins in the studied hepatic fraction. More than 90 % of Ag, Cd, Co, Cu, Fe, Mo, and Pb were eluted with negatively charged biomolecules, and >70 % of Bi, Mn, and Zn. Thallium was revealed to bind equally to negatively and positively charged biomolecules, and Cs predominantly to positively charged ones. The majority of acidic (negatively charged) metalloproteins/peptides were coeluted within the elution time range of applied standard proteins, having pIs clustered around 4–6. Furthermore, binding of several metals (Ag, Cd, Cu, Zn) to two MT-isoforms was assumed, with Cd and Zn preferentially bound to MT1 and Ag to MT2, and Cu evenly distributed between the two. The results presented here are the first of their kind for the important bioindicator species, the northern pike, as well as one of the rare comprehensive studies on the acidic/basic nature of metal-binding biomolecules in fish, which can contribute significantly to a better understanding of the behaviour and fate of metals in the fish organism, specifically in liver as main metabolic and detoxification organ.

Atlantic Salmon Gill Epithelial Cell Line (ASG-10) as a Suitable Model for Xenobiotic Biotransformation.

Fish are exposed to xenobiotics in the water. Uptake occurs mainly through the gills, which function as an exchange point with the environment. The gills' ability to detoxify harmful compounds by biotransformation is an essential protection mechanism. The enormous numbers of waterborne xenobiotics requiring ecotoxicological assessment makes it necessary to replace in vivo fish studies with predictive in vitro models. Here, we have characterized the metabolic capacity of the ASG-10 gill epithelial cell line from Atlantic salmon. Inducible CYP1A expression was confirmed by enzymatic assays and immunoblotting. The activities of important cytochrome P450 (CYP) and uridine 5'-diphospho-glucuronosyltransferase (UGT) enzymes were established using specific substrates and metabolite analysis by liquid chromatography (LC) triple quadrupole mass spectrometry (TQMS). Metabolism of the fish anesthetic benzocaine (BZ) in ASG-10 confirmed esterase and acetyl transferase activities through the production of N-acetylbenzocaine (AcBZ), p-aminobenzoic acid (PABA) and p-acetaminobenzoic acid (AcPABA). Moreover, we were able to determine hydroxylamine benzocaine (BZOH), benzocaine glucuronide (BZGlcA) and hydroxylamine benzocaine glucuronide (BZ(O)GlcA) by LC high-resolution tandem mass spectrometry (HRMS/MS) fragment pattern analysis for the first time. Comparison to metabolite profiles in hepatic fractions, and in plasma of BZ-euthanized salmon, confirmed the suitability of the ASG-10 cell line for investigating biotransformation in gills.

Blunted cardiovascular effects of beta-blockers in patients with cirrhosis: Relation to severity?

AimsPatients with cirrhosis and portal hypertension are at high risk of developing complications such as variceal hemorrhage, ascites, and cardiac dysfunction, the latter of which is known as cirrhotic cardiomyopathy. Since non-selective beta-blockers (NSBB) may aggravate hemodynamic complications we investigated the effect of real-time propranolol infusion on cardiac function in patients with varying degrees of cirrhosis.MethodsThirty-eight patients with Child-Pugh A (n = 17), B (n = 17) and C (n = 4) underwent liver vein catheterization and cardiac magnetic resonance imaging. We assessed the effect of real-time propranolol infusion on the hepatic venous pressure gradient, cardiac index, stroke volume, ejection fraction, heart rate, and contractility.ResultsNineteen patients were classified as responders to beta-blocker therapy. In pooling Child-Pugh B and C patients, the reduction in cardiac index by beta-blockade was weaker than in Child-Pugh A patients (-8.5% vs. -20.5%, p = 0.043). The effect of NSBB on portal pressure was inversely correlated to changes in the left atrium where the left atrial volume changed by 4 mL±18 in responders compared to 15 mL±11 in non-responders (p = 0.03). Finally, the baseline ejection fraction correlated inversely with the reduction in portal pressure (r = -0.39, p = 0.02).ConclusionWe found the effect of beta-blockade on cardiac index in patients with advanced cirrhosis to be less potent than in patients with early cirrhosis, indicating that underlying cirrhotic cardiomyopathy increases, and the cardiac compensatory reserve becomes more compromised, with disease progression. The differential effects of beta-blockade in the left atrium may be used to predict the effect of beta-blockers on portal pressure, but further studies are needed to investigate this possibility.

Qualitative and Quantitative Changes in Total Lipid Concentration and Lipid Fractions in Liver Tissue of Periparturient German Holstein Dairy Cows of Two Age Groups.

Fatty liver syndrome (FLS) is a common disease in high-producing dairy cows. Studies in humans suggest that the different hepatic lipid fractions play a role in this context. In dairy cows, little is known about the composition of fat stored in the liver, its periparturient dynamics, and the effect of cows' age. Therefore, our goal was to generate primary data in healthy cows to serve as reference values for future studies. Eight healthy German Holstein cows (2nd lactation, n = 3; ≥3rd lactation, n = 5) were examined 14 d antepartum and 7, 28, and 42 d postpartum. The examinations included clinical assessment, liver biopsy, blood sampling, and recording of milk yield. Total lipids (TL) in liver tissue were measured gravimetrically. The TL were separated into lipid fractions (triacylglycerol, TAG; phospholipids, PL; non-esterified fatty acids, NEFA; and cholesterol esters) using thin-layer chromatography, followed by gas chromatography for fatty acid determination. Concentrations of NEFA, ß-hydroxybutyrate, and cholesterol were analyzed in blood. Concentrations of TL, TAG, NEFA, and cholesterol esters in liver tissue and NEFA in blood increased in the periparturient period. The older cows had higher hepatic TL, TAG, and PL concentrations, higher relative hepatic concentrations of TAG in TL, higher NEFA concentrations in blood, a greater decrease in body condition, and higher milk yields between d 9 and 40 than the younger cows. We proposed that due to higher milk yield, older cows mobilized and deposited more fat in the liver, and the increase in hepatic TAG concentration was longer-lasting than in younger cows. Higher levels of structural lipids (PL) in older cows could be explained by higher demand for storage of TAG and cholesterol esters in lipid droplets or for the export of TAG via very-low-density lipoproteins. Results show that hepatic fat storage is a reversible process and does not necessarily cause clinical disease. Nevertheless, older cows have a more sustained and greater increase in hepatic TAG concentration, which may explain their increased risk of FLS. The results are limited in their extrapolation due to the small sample size and thereby possible selection bias but present a valuable basis for future studies.

GTL synthetic paraffin oil shows low liver and tissue retention compared to mineral oil

Oral exposure to mineral oil may result in a narrow fraction of mineral oil saturated hydrocarbon (MOSH) being retained in tissues. Excess of MOSH hepatic retention may lead to the formation of lipogranuloma caused by predominantly multiring cycloalkanes (naphthenics) in a critical range of C25–C35. Although hepatic lipogranuloma is of low pathological concern, MOSH tissue deposition could be minimized by using an oil of similar quality but devoid of naphthenic structures to decrease hepatic retention.Synthetic Gas to liquid (GTL) oils offer an alternative to petroleum derived mineral oils, because they do not contain naphthenic structures. To demonstrate this point, SD rats were fed either GTL oil (99% iso-alkanes) or naphthenic mineral oil (84% cycloalkanes) at 200 mg/kg bw/day for 90 or 134 days with a recovery group. Liver, fat and mesenteric lymph nodes were analyzed for alkane sub-type levels using Online-HPLC-GC-FID and GCxGC-TOF-MS.Results indicate that at equal external dose, GTL hydrocarbons result in lower tissue levels and more rapid excretion than MOSH. GTL retained hepatic fractions were also qualitatively different than MOSH constituents. Because chemical composition differences, GTL oil show low absorption and tissue retention potential and thus an advantageous alternative to conventional mineral oil.

Dynamic 18F-FDG PET imaging of liver lesions: evaluation of a two-tissue compartment model with dual blood input function

BackgroundDynamic PET with kinetic modeling was reported to be potentially helpful in the assessment of hepatic malignancy. In this study, a kinetic modeling analysis was performed on hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) from dynamic FDG positron emission tomography/computer tomography (PET/CT) scans.MethodsA reversible two-tissue compartment model with dual blood input function, which takes into consideration the blood supply from both hepatic artery and portal vein, was used for accurate kinetic modeling of liver dynamic 18F-FDG PET imaging. The blood input functions were directly measured as the mean values over the VOIs on descending aorta and portal vein respectively. And the contribution of hepatic artery to the blood input function was optimization-derived in the process of model fitting. The kinetic model was evaluated using dynamic PET data acquired on 24 patients with identified hepatobiliary malignancy. 38 HCC or ICC identified lesions and 24 healthy liver regions were analyzed.ResultsResults showed significant differences in kinetic parameters {K}_{1}-{k}_{4}, blood supplying fraction {f}_{A}, and metabolic rate constant {K}_{i} between malignant lesions and healthy liver tissue. And significant differences were also observed in {K}_{1}, {k}_{3}, {f}_{A} and {K}_{i} between HCC and ICC lesions. Further investigations of the effect of SUV measurements on the derived kinetic parameters were conducted. And results showed comparable effectiveness of the kinetic modeling using either SUVmean or SUVmax measurements.ConclusionsDynamic 18F-FDG PET imaging with optimization-derived hepatic artery blood supply fraction dual-blood input function kinetic modeling can effectively distinguish malignant lesions from healthy liver tissue, as well as HCC and ICC lesions.

Hepatic blood flow in adult Göttingen minipigs and pre-pubertal Danish Landrace x Yorkshire pigs.

The liver receives dual blood supply from the hepatic artery and portal vein. The pig is often used as an animal model in positron emission tomography (PET) and pharmacokinetic studies because of the possibility for extensive and direct blood sampling. In this study, we compared measurements of hepatic blood flow in 10 female adult Göttingen minipigs and 10 female pre-pubertal Danish Landrace x Yorkshire (DLY) pigs. Ultrasound transit time flow meter probes were placed around the hepatic artery and portal vein through open surgery, hepatic blood flow measurements were performed, and the liver was weighed. Total hepatic blood flow was on average 363 ± 131 mL blood/min in Göttingen minipigs and 988 ± 180 mL blood/min in DLY pigs (p < 0.001). The mean hepatic blood perfusion was 623 mL blood/min/mL liver tissue and 950 mL blood/min/mL liver tissue (p = 0.005), and the liver weight was 0.58 kg and 1.04 kg, respectively. The mean arterial flow fraction in Göttingen minipigs was 12 ± 7% and lower than in DLY pigs, where it was 24 ± 7% (p = 0.001). Using the gold standard for blood flow measurements, we found that both total hepatic blood flow and blood perfusion were significantly lower in Göttingen minipigs than in DLY pigs. The hepatic blood perfusion and arterial flow fraction in DLY pigs were comparable to normative values from humans. Differences in hepatic blood flow between adult Göttingen minipigs and humans should be considered when performing physiological liver studies in this model.

The use of an in vitro approach to assess marine invertebrate carboxylesterase responses to chemicals of environmental concern

Carboxylesterases (CEs) are key enzymes which catalyse the hydrolysis reactions of multiple xenobiotics and endogenous ester moieties. Given their growing interest in the context of marine pollution and biomonitoring, this study focused on the in vitro sensitivity of marine invertebrate CEs to some pesticides, pharmaceuticals, personal care products and plastic additives to assess their potential interaction on this enzymatic system and its suitability as biomarkers. Three bivalves, one gastropod and two crustaceans were used and CEs were quantified following current protocols set for mammalian models. Four substrates were screened for CEs determination and to test their adequacy in the hepatic fraction measures of the selected invertebrates. Two commercial recombinant human isoforms (hCE1 and hCE2) were also included for methodological validation. Among the invertebrates, mussels were revealed as the most sensitive to xenobiotic exposures while gastropods were the least as well as with particular substrate-specific preferences. Among chemicals of environmental concern, the plastic additive tetrabromobisphenol A displayed the highest CE-inhibitory capacity in all species. Since plastic additives easily breakdown from the polymer and may accumulate and metabolise in marine biota, their interaction with the CE key metabolic/detoxification processes may have consequences in invertebrate’s physiology, affect bioaccumulation and therefore trophic web transfer and, ultimately, human health as shellfish consumers.

Abstract 15268: Development of a Reliable Means of Assessment of Liver Function After the Fontan Operation: Dual Cholate Clearance Assay

Introduction: Currently there is no biomarker or test to accurately measure liver function in Fontan-associated liver disease. The dual cholate test (HepQuant SHUNT) is a noninvasive, flow-dependent assay measuring hepatic clearance of a bile acid, cholate, and may serve as a useful measure of liver function in the Fontan. We aim to measure cholate clearance in a cohort of Fontan patients and compare to normal controls. Methods: Single center, prospective pilot study of Fontan patients ≥ 18 years. Hepatic clearance of orally administered d4-cholate and intravenously administered 13C-cholate were measured in peripheral venous samples after 5, 20, 45, 60, and 90 minutes. Portal hepatic filtration rate (HFR), systemic HFR, shunt fraction (systemic HFR/portal HFR), and disease severity index (DSI) were calculated. Decline in HFRs and increase in shunt fraction or DSI is indicative of impaired liver function. Two-sided t-tests were used to compare values between Fontans and controls. Results: Twelve Fontan patients were enrolled (33% female, median age 29.9 [range 23.6 - 41.0] years). Median total bilirubin was 0.8 (range 0.3-2.1) mg/dL, AST 30.5 (range 16-55) U/L, ALT 32 (range 11-53) U/L, alkaline phosphatase 87 (range 42-191) U/L and platelets 177 (range 130-428) 10 3 /μL. Mean cardiac index was 3.0 ± 0.5 L/min/m 2 . Cholate clearance was lower in Fontans compared to controls (Figure). Mean portal HFR (mL/min/kg) in Fontans was lower than controls (15.1 ± 10.9 vs 29.1 ± 9.0; p&lt;0.001) as well as systemic HFR (3.7 ± 1.3 vs 6.5 ± 1.5; p&lt;0.001) whereas shunt fraction (%) was not (29.2 ± 11.4 vs 24.1 ± 7.5; p=NS). DSI was higher in Fontans compared to controls (19.4 ± 6.1 vs 9.2 ± 3.4; p&lt;0.001). Conclusions: Fontan patients demonstrate reduced hepatic function compared to normal controls but there is considerable variability. Future studies using the dual cholate test will examine the relationship between liver and cardiac function, as well as risk of adverse clinical outcome, in the Fontan.

Haemodynamic changes in cirrhosis following terlipressin and induction of sepsis\u2014a preclinical study using caval subtraction phase-contrast and cardiac MRI

ObjectivesEffects of liver disease on portal venous (PV), hepatic arterial (HA), total liver blood flow (TLBF), and cardiac function are poorly understood. Terlipressin modulates PV flow but effects on HA, TLBF, and sepsis/acute-on-chronic liver failure (ACLF)-induced haemodynamic changes are poorly characterised. In this study, we investigated the effects of terlipressin and sepsis/ACLF on hepatic haemodynamics and cardiac function in a rodent cirrhosis model using caval subtraction phase-contrast (PC) MRI and cardiac cine MRI.MethodsSprague-Dawley rats (n = 18 bile duct–ligated (BDL), n = 16 sham surgery controls) underwent caval subtraction PCMRI to estimate TLBF and HA flow and short-axis cardiac cine MRI for systolic function at baseline, following terlipressin and lipopolysaccharide (LPS) infusion, to model ACLF.ResultsAll baseline hepatic haemodynamic/cardiac systolic function parameters (except heart rate and LV mass) were significantly different in BDL rats. Following terlipressin, baseline PV flow (sham 181.4 ± 12.1 ml/min/100 g; BDL 68.5 ± 10.1 ml/min/100 g) reduced (sham − 90.3 ± 11.1 ml/min/100 g, p < 0.0001; BDL − 31.0 ± 8.0 ml/min/100 g, p = 0.02), sham baseline HA flow (33.0 ± 11.3 ml/min/100 g) increased (+ 92.8 ± 21.3 ml/min/100 g, p = 0.0003), but BDL baseline HA flow (83.8 ml/min/100 g) decreased (− 34.4 ± 7.5 ml/min/100 g, p = 0.11). Sham baseline TLBF (214.3 ± 16.7 ml/min/100 g) was maintained (+ 2.5 ± 14.0 ml/min/100 g, p > 0.99) but BDL baseline TLBF (152.3 ± 18.7 ml/min/100 g) declined (− 65.5 ± 8.5 ml/min/100 g, p = 0.0004). Following LPS, there were significant differences between cohort and change in HA fraction (p = 0.03) and TLBF (p = 0.01) with BDL baseline HA fraction (46.2 ± 4.6%) reducing (− 20.9 ± 7.5%, p = 0.03) but sham baseline HA fraction (38.2 ± 2.0%) remaining unchanged (+ 2.9 ± 6.1%, p > 0.99). Animal cohort and change in systolic function interactions were significant only for heart rate (p = 0.01) and end-diastolic volume (p = 0.03).ConclusionsCaval subtraction PCMRI and cardiac MRI in a rodent model of cirrhosis demonstrate significant baseline hepatic haemodynamic/cardiac differences, failure of the HA buffer response post-terlipressin and an altered HA fraction response in sepsis, informing potential translation to ACLF patients.Key PointsCaval subtraction phase-contrast and cardiac MRI demonstrate:• Significant differences between cirrhotic/non-cirrhotic rodent hepatic blood flow and cardiac systolic function at baseline.• Failure of the hepatic arterial buffer response in cirrhotic rodents in response to terlipressin.• Reductions in hepatic arterial flow fraction in the setting of acute-on-chronic liver failure.

Biotransformation of phytoestrogens from soy in enzymatically characterized liver microsomes and primary hepatocytes of Atlantic salmon.

Efficient aquaculture is depending on sustainable protein sources. The shortage in marine raw materials has initiated a shift to "green aquafeeds" based on staple ingredients such as soy and wheat. Plant-based diets entail new challenges regarding fish health, product quality and consumer risks due to the possible presence of chemical contaminants, natural toxins and bioactive compounds like phytoestrogens. Daidzein (DAI), genistein (GEN) and glycitein (GLY) are major soy isoflavones with considerable estrogenic activities, potentially interfering with the piscine endocrine system and affecting consumers after carry-over. In this context, information on isoflavone biotransformation in fish is crucial for risk evaluation. We have therefore isolated hepatic fractions of Atlantic salmon (Salmo salar), the most important species in Norwegian aquaculture, and used them to study isoflavone elimination and metabolite formation. The salmon liver microsomes and primary hepatocytes were characterized with respect to phase I cytochrome P450 (CYP) and phase II uridine-diphosphate-glucuronosyltransferase (UGT) enzyme activities using specific probe substrates, which allowed comparison to results in other species. DAI, GEN and GLY were effectively cleared by UGT. Based on the measurement of exact masses, fragmentation patterns, and retention times in liquid chromatography high-resolution mass spectrometry, we preliminarily identified the 7-O-glucuronides as the main metabolites in salmon, possibly produced by UGT1A1 and UGT1A9-like activities. In contrast, the production of oxidative metabolites by CYP was insignificant. Under optimized assay conditions, only small amounts of mono-hydroxylated DAI were detectable. These findings suggested that bioaccumulation of phytoestrogens in farmed salmon and consumer risks from soy-containing aquafeeds are unlikely.

Predicting successful/unsuccessful extrapolation for in vivo total clearance of model compounds with a variety of hepatic intrinsic metabolism and protein bindings in humans from pharmacokinetic data using chimeric mice with humanised liver

1. Immunodeficient chimeric mice with humanised liver have been useful in predicting total clearance values of drugs in humans. However, their usefulness may currently be limited for specific compounds with interspecies differences.2. In vivo total clearance and in vitro hepatic intrinsic clearance values of 16 model compounds were determined in control/humanised-liver mice and in mouse and human hepatocytes, respectively, for extrapolating the total clearance values of compounds in humans.3. The predictability of in vivo total clearance values of 11 model compounds in humans was adequate using pharmacokinetic data from humanised-liver mice. The predictability of total clearance values using humanised-liver mice was better than conventional allometric scaling for compounds with large interspecies differences in in vitro hepatic intrinsic clearance or plasma unbound fractions.4. There were trends that total clearance values in control and humanised-liver mice were similar to or higher than reported hepatic blood flow rates in normal mice among four compounds with poor predictability. Diazepam, with the poorest predictability, showed 38-fold-higher hepatic intrinsic clearance in mice than in humans.5. These results could lead to guidelines describing that compounds may be suited or unsuited to extrapolating total clearance values in humans from pharmacokinetics in humanised-liver mice.

Evaluation of total liver perfusion imaging of CT for efficacy of transcatheter arterial chemoembolization combined with apatinib on rabbit VX2 liver tumors

To investigate the value of the total liver CT perfusion imaging in the evaluation of rabbit VX2 liver tumors treated with TACE and apatinib. Methods: Thirty-six rabbit VX2 liver cancer models were established and randomly divided into 4 groups. Group A: simple TACE group; Group B: simple oral administration of apatinib mesylate; Group C: TACE + oral apatinib mesylate; Group D: control group, administration of saline. CT perfusion imaging (CTPI) was performed before treatment and on the 7 and 14 days after the treatment to acquire perfusion parameters including blood flow (BF), blood volume (BV), MTT (mean transit time), surface permeability (PS), and hepatic artery fraction (HAF). One tumor rabbit was sacrificed in each group after the first perfusion scan, and the remaining tumor rabbits were sacrificed after the last perfusion scan on the 14th day of the treatment. The borders of the tumors were stained immunohistochemically, and microvascular density (MVD) was measured by anti-CD34. The differences of perfusion parameters were compared to evaluate the liver hemodynamic changes, and statistical repeated measurement variance analysist correlation analysis were performed. Results: There were no significant differences in CTPI parameters of BF, BV, MTT, HAF and PS between the 4 groups before treatment (P>0.05). After the treatment, HB, HAF and PS were decreased significantly in Group A, B, and C and slightly increased in the Group D. The value of MVD after 14 d treatment was 80.1±16.4 in Group A, 50.2±11.2 in Group B, 27.4±9.7 in Group C, 68.7±12.7 in Group D, respectively. The value of MVD in the Group C were significantly lower than that in Group A, B, and D. It showed positive correlation between BF, HAF, PS and MVD in Group B, C, and D, and there was no significant correlation between BV, MTT and MVD. It showed no significant correlation between MVD and each CTPI parameter in Group A. Conclusion: Total liver CT perfusion can quantitatively evaluate the blood perfusion information of rabbit liver VX2 tumor after TACE. TACE combined with oral apatinib can effectively inhibit tumor growth and improve the therapeutic effect of VX2 tumor.

Organotropism of total mercury (THg) in Cichla pinima, ecological aspects and human consumption in fish from Amazon region, Brazil

Specimens of the Cichla pinima are of ecological and economic importance in the Amazon region and are a good bioindicator species of Hg bioaccumulation. Adult specimens of Cichla pinima were obtained by fisheries in the Tapajós river region's impacted gold mining area. Tissues such as muscle, liver, skin, and gill were submitted for total mercury (THg) analysis. For hepatic bioavailability, assays were carried out in the whole liver and in the sub-cellular hepatic fraction. The weight-length relationship showed an equation of W = 2E - 06L3.3002 (R2 = 0.856) indicating an allometric growth. The mean THg values found in the muscle tissue of 676 ± 258μgkg-1 were below the maximum limit established for human consumption, but were similar to Cichla fish from other areas impacted by Hg in the Amazon region. The trends of levels in the tissues were as follows: liver>muscle>gonads>skin>gill, with no significant differences between the gills and skin. A significant and positive correlation as rs = 0.65 was obtained between the THg contents in cytosolic fraction and the total hepatic THg (liver homogenate). However, only 7% of the THg were found and were available to the sub-cellular hepatic fraction. This profile can be an indicative of a hepatic cellular transference in fish exposed to high Hg levels in the Amazon region now that low concentrations of Hg have been found in the sub-cellular fraction. However, data of differential THg partition in the subcellular compartments should be considered, since others' hepatic fractions can act in the Hg linkage into the cell.

In Vitro and In Vivo Correlation of Hepatic Fraction of Metabolism by P450 in Dogs

1-Aminobenzotriazole (ABT) has been widely used as a nonspecific mechanism-based inhibitor of cytochrome P450 (P450) enzymes. It is extensively used in preclinical studies to determine the relative contribution of oxidative metabolism mediated by P450 in vitro and in vivo. The aim of present study was to understand the translation of fraction metabolized by P450 in dog hepatocytes to in vivo using ABT, for canagliflozin, known to be cleared by P450-mediated oxidation and UDP-glucuronosyltransferases–mediated glucuronidation, and 3 drug discovery project compounds mainly cleared by hepatic metabolism. In a dog hepatocyte, intrinsic clearance assay with and without preincubation of ABT, 3 Lilly compounds exhibited a wide range of fraction metabolized by P450. Subsequent metabolite profiling in dog hepatocytes demonstrated a combination of metabolism by P450 and UDP-glucuronosyltransferases. In vivo, dogs were pretreated with 50 mg/kg ABT or vehicle at 2 h before intravenous administration of canagliflozin and Lilly compounds. The areas under the concentration-time curve (AUC) were compared for the ABT-pretreated and vehicle-pretreated groups. The measured AUCABT/AUCveh ratios were correlated to fraction of metabolism by P450 in dog hepatocytes, suggesting that in vitro ABT inhibition in hepatocytes is useful to rank order compounds for in vivo fraction of metabolism assessment.

Discovery of 6-(pyrimidin-5-ylmethyl)quinoline-8-carboxamide negative allosteric modulators of metabotropic glutamate receptor subtype 5

Based on previous work that established fused heterocycles as viable alternatives for the picolinamide core of our lead series of mGlu5 negative allosteric modulators (NAMs), we designed a novel series of 6-(pyrimidin-5-ylmethyl)quinoline-8-carboxamide mGlu5 NAMs. These new quinoline derivatives also contained carbon linkers as replacements for the diaryl ether oxygen atom common to our previously published chemotypes. Compounds were evaluated in a cell-based functional mGlu5 assay, and an exemplar analog 27 was >60-fold selective versus the other seven mGlu receptors. Selected compounds were also studied in metabolic stability assays in rat and human S9 hepatic fractions and exhibited a mixture of P450- and non-P450-mediated metabolism.

Biomarkers and bioindicators of the environmental condition using a fish species (Pimelodus maculatus Lacepède, 1803) in a tropical reservoir in Southeastern Brazil.

The Funil Reservoir receives a large amount of xenobiotics from the Paraíba do Sul River (PSR) from large number of industries and municipalities in the watershed. This study aimed to assess environmental quality along the longitudinal profile of the Paraíba do Sul River-Funil Reservoir system, by using biomarkers and bioindicators in a selected fish species. The raised hypothesis is that Funil Reservoir acts as a filter for the xenobiotics of the PSR waters, improving river water quality downstream the dam. Two biomarkers, the ethoxyresorufin-O-deethylase activity (EROD), measured as fluorimetricly in S9 hepatic fraction, and the micronuclei frequency (MN), observed in erythrocytes of the cytoplasm, and three bioindicators, the hepatosomatic index (HSI), gonadosomatic index (GSI) and condition factor (CF) were used in Pimelodus maculatus, a fish species widely distributed in the system. Four zones were searched through a longitudinal gradient: 1, river upstream from the reservoir; 2, upper reservoir; 3, lower reservoir; 4, river downstream of the reservoir. EROD activity and HSI and GSI had significant differences among the zones (P<0.05). The upper reservoir had the lowest EROD activity and HSI, whereas the river downstream of the reservoir had the highest EROD and lowest GSI. The river upstream from the reservoir showed the highest HSI and GSI. It is suggested that the lowest environmental condition occur at the river downstream of the reservoir, where it seems to occur more influence of xenobiotics, which could be associated with hydroelectric plant operation. The hypothesis that Funil reservoir acts as a filter decanting pollution from the Paraíba do Sul River waters was rejected. These results are novel information on this subject for a native fish species and could be useful for future comparisons with other environments.

Evaluation of Antiatherogenic Properties of Ezetimibe Using 3H-Labeled Low-Density-Lipoprotein Cholesterol and 99mTc-cAbVCAM1-5 SPECT in ApoE-/- Mice Fed the Paigen Diet.

The addition of ezetimibe, an intestinal cholesterol absorption inhibitor, to statin therapy has recently shown clinical benefits in the Improved Reduction of Outcomes: Vytorin Efficacy International Trial by reducing low-density-lipoprotein (LDL) cholesterol levels more than statin therapy alone. Here, we investigated the mechanisms by which inhibition of intestinal cholesterol absorption might contribute to the clinically observed reduction in cardiovascular events by evaluating its effect on inflammatory plaque development in apolipoprotein E-/- mice. Methods: Apolipoprotein E-/- mice were fed the Paigen diet (1.25% cholesterol, 0.5% cholic acid, and 15% fat) without or with ezetimibe (7 mg/kg/d) for 6 wk. In a first set of mice (n = 15), we intravenously injected 3H-cholesteryl oleate-labeled human LDL to test whether ezetimibe promotes LDL-derived cholesterol fecal excretion. In a second set (n = 20), we used the imaging agent 99mTc-cAbVCAM1-5 to evaluate expression of an inflammatory marker, vascular cell adhesion molecule 1 (VCAM-1), in atherosclerotic plaques. In a third set (n = 21), we compared VCAM-1 expression with 99mTc-cAbVCAM1-5 uptake in various tissues. Results: Mice treated with ezetimibe showed a 173% higher LDL-cholesteryl ester plasma disappearance rate (P < 0.001 vs. control) after 3H-cholesteryl oleate-labeled LDL injection. At 96 h after injection, the hepatic fraction of 3H-tracer was 61% lower in mice treated with ezetimibe (P < 0.001). Meanwhile, LDL-derived 3H-cholesterol excretion in the feces was 107% higher (P < 0.001). The antiatherogenic effect of ezetimibe monitored by 99mTc-cAbVCAM1-5 SPECT showed a 49% reduction in aortic tracer uptake (percentage injected dose per cubic centimeter, 0.95 ± 0.04 vs. 1.87 ± 0.11; P < 0.01). In addition to hypercholesterolemia, the proinflammatory Paigen diet significantly increased VCAM-1 expression with respect to the control group in various tissues, including the aorta, and this expression correlated strongly with 99mTc-cAbVCAM1-5 uptake (r = 0.75; P < 0.05). Conclusion: Inhibition of intestinal cholesterol absorption with ezetimibe promotes antiatherosclerotic effects through increased LDL cholesterol catabolism and LDL-derived cholesterol fecal excretion and reduces inflamed atherosclerotic plaques. These mechanisms may contribute to the benefits of adding ezetimibe to a statin therapy.

CT perfusion parameters evaluate the liver reserve function of hepatic fibrosis-an experimental research

Objective To discuss the value of the CT perfusion parameters evaluate the liver reserve function in rabbit hepatic fibrosis model. Methods 48 New Zealand white rabbit, male or female, aged 3 months, weighing 2.5-3.0kg, were included in this study. Compound feed including carbon tetrachloride and phenobarbitone was employed to establish the liver fibrosis models. Liver samples of pathology were scored for fibrosis and biochemical indicators of liver function, indocyanine green retention rate at 15 minutes(R15ICG), CT perfusion imaging were measured at the end of 0th, 4th, 8th, 12th, 16th, 20th, weeks, respectively. Hepatic arterial fraction (HAF), blood flow (BF), blood volume (BV), mean transit time (MTT), permeability surface (PS) were measured. Correlation between the biochemical indicators and R15ICG, regression analysis of quantitative perfusion parameters and R15ICG were tested. Results (1) At the end of 20th week, there were 10, 9, 9, 8, 7 cases in stage S0 to S4 of hepatic fibrosis, respectively. (2) From stage S0 to S4, R15ICG were increased (5.31±2.71)%, (12.73±7.87)%, (19.85±12.64)%, (23.13±11.39)%, (31.59±23.88)%, P 0.05). Conclusion Multi-slice CT Perfusion can detect the changes of blood flood of liver fibrosis, R15ICG and hepatic fibrosis liver function is a linear relationship; BF, HAF appears to be the most promising perfusion parameter value affect the liver reserve function R15ICG in hepatic fibrosis. Key words: Hepatic fibrosis; Rabbit; CT perfusion imaging; Liver reserve function; Indole cyanide green 15 minutes retention rate