Jose Pellegrino - Academia.edu (original) (raw)
Papers by Jose Pellegrino
Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1992
Taurolithocholate (TLC), a natural bile salt, induces selective impairment on canalicular membran... more Taurolithocholate (TLC), a natural bile salt, induces selective impairment on canalicular membrane of the hepatocyte, which seems to be a major determinant of its cholestatic effect in experimental animals. In order to extend existing studies about the effects of TLC on bile secretion, we examined in TLC-treated rats the biliary excretion of compounds that are transported to canalicular membrane via vesicles, such as lipids and proteins. The single intravenous injection of TLC (3 mumol/100 g body wt.) inhibited transiently the biliary bile salt excretion, while the biliary excretion of lipids (i.e., cholesterol and phospholipids) and proteins remained inhibited even though the biliary excretion and composition of bile salts were normalized. Under such a condition, TLC also inhibited the transcellular vesicular pathway to the exogenous protein horseradish peroxidase entry into bile, without altering the paracellular biliary access of the protein. The hepatic uptake of horseradish peroxidase was unaffected by TLC-treatment. The results indicate that TLC can inhibit the biliary excretion of compounds that reach the canaliculus via a vesicular pathway, such as lipids and proteins, by a mechanism not related to a defective bile salt excretion. Possible explanations for these findings are discussed.
BioTechniques, 2011
An inexpensive modular perfused chamber (MPC) designed for low- and normal-temperature live-cell ... more An inexpensive modular perfused chamber (MPC) designed for low- and normal-temperature live-cell imaging is presented. The device consists of four lathed pieces of stainless steel assembled as a cylindrical open chamber that can hold either round or square glass coverslips. The chamber is connected to a thermal-bath operating with recirculation. For image acquisition at 4°C, cooled air is blown toward the coverslip surface to prevent condensation. Principal advantages of this device are thermal stability in the sample environment, rapid response to changes in temperature set point, and easy sample insertion. The device enables the study of dynamic processes in cells governed by large temperature differences such as those imposed by hypothermic preservation of cells (0-4°C) followed by rewarming to normothermia (37°C). The capabilities of the MPC were demonstrated by monitoring the internalization of fluorescent quantum dots (QDs) in rat hepatocytes after hypothermic storage and duri...
Growth Factors, 2010
Wnt/beta-catenin/T cell factor (TCF) pathway is activated in several types of human cancers, prom... more Wnt/beta-catenin/T cell factor (TCF) pathway is activated in several types of human cancers, promoting cell growth and proliferation. Forkhead box containing protein class O (FOXO) transcription factors compete with TCF for beta-catenin binding, particularly under cellular oxidative stress conditions. Contrary to beta-catenin/TCF, beta-catenin/FOXO promotes the transcription of genes involved in cell cycle arrest and apoptosis. We have previously demonstrated that in vivo interferon-alpha2b (IFN-alpha2b) administration induces apoptosis in preneoplastic livers, a mechanism mediated by reactive oxygen species (ROS) and transforming growth factor-beta(1) (TGF-beta(1)). This study was aimed to assess the status of the Wnt/beta-catenin/TCF pathway in a very early stage of rat hepatocarcinogenesis and to further evaluate the effects of in vivo IFN-alpha2b treatment on it. We demonstrated that the Wnt/beta-catenin/TCF pathway is activated in preneoplastic rat livers. More important, in vivo IFN-alpha2b treatment inhibits Wnt/beta-catenin/TCF pathway and promotes programed cell death possibly providing a link with FOXO pathway.
Free Radical Research, 2011
Diabetes mellitus is a risk factor for prognosis after liver resection. In previous work, we foun... more Diabetes mellitus is a risk factor for prognosis after liver resection. In previous work, we found a pro-apoptotic state in the diabetic rat liver. In this work, this was also observed 1 hour post-partial hepatectomy (PH) and resulted in a deficient regenerative response 24 hours post-PH. Treatment with insulin and/or Desferoxamine (DES) (iron chelator) or Tempol (TEM) (free radicals scavenger) was effective in preventing the liver reactive oxygen species (ROS) production induced by diabetic state. High levels of ROS play a role in hepatic lipid peroxidation in diabetes before and after PH, and lead to increased pro-apoptotic events, which contribute to a reduced regenerative response. This becomes of relevance for the potential use of antioxidants/free radical scavengers plus insulin for improvement of post-surgical recovery of diabetic patients subjected to a PH.
Drug Metabolism and Disposition, 2006
The ability of the kidney and small intestine to synthesize and subsequently eliminate dinitrophe... more The ability of the kidney and small intestine to synthesize and subsequently eliminate dinitrophenyl-S-glutathione (DNP-SG), a substrate for the multidrug resistance-associated proteins (Mrps), was assessed in bile duct-ligated (BDL) rats 1, 7, and 14 days after surgery, using an in vivo perfused jejunum model with simultaneous urine collection. A single i.v. dose of 30 mol/kg b.wt. of 1-chloro-2,4-dinitrobenzene (CDNB) was administered, and its glutathione conjugate DNP-SG and dinitrophenyl cysteinyl glycine derivative, which is the result of ␥-glutamyl-transferase action on DNP-SG, were determined in urine and intestinal perfusate by high-performance liquid chromatography. Intestinal excretion of these metabolites was unchanged at day 1, and decreased at days 7 and 14 (؊39% and ؊33%, respectively) after surgery with respect to shams. In contrast, renal excretion was increased by 114%, 150%, and 128% at days 1, 7, and 14. Western blot studies revealed decreased levels of apical Mrp2 in liver and jejunum but increased levels in renal cortex from BDL animals, these changes being maximal between days 7 and 14. Assessment of expression of basolateral Mrp3 at day 14 postsurgery indicated preserved levels in renal cortex, duodenum, jejunum, distal ileum, and colon. Analysis of expression of glutathione-S-transferases ␣, , and , as well as activity toward CDNB, indicates that formation of DNP-SG was impaired in liver, preserved in intestine, and increased in renal cortex. In conclusion, increased renal tubular conversion of CDNB to DNP-SG followed by subsequent Mrp2-mediated secretion into urine partially compensates for altered liver function in experimental obstructive cholestasis.
Drug Metabolism and Disposition, 2005
EE induces cholestasis by affecting bile salt-dependent and-independent fractions of the bile flo... more EE induces cholestasis by affecting bile salt-dependent and-independent fractions of the bile flow. The decrease in bile salt-independent flow is thought to be due, in part, to a reduction in the expression of the canalicular transporter Mrp2. The impact of modulation of Mrp2 function by UDC in EE cholestasis is unknown. We evaluated the protective effect of UDC on EE-induced impairment of Mrp2 activity in vivo and in isolated hepatocytes, by using the substrate dinitrophenyl-S-glutathione (DNP-SG). EE was administered to male Wistar rats at a
Journal of Hepatology, 1998
The mechanism by which many cytochrome P450 (CYP) isozymes decrease during liver regeneration is ... more The mechanism by which many cytochrome P450 (CYP) isozymes decrease during liver regeneration is unclear. Peptides and growth factors are thought to be involved. Putrescine, the first polyamine synthesised by ornithine decarboxylase, peaks early following partial hepatectomy and is known to play an essential role in hepatic regeneration. Gamma amino butyric acid was reported as a physiologic inhibitor of ornithine decarboxylase. In this work we studied the possible involvement of putrescine in the CYP reduction during liver regeneration. Hepatectomised, putrescine-treated sham, and GABA-treated hepatectomised rats were used throughout. Total hepatic cytochrome P450, o-dealkylase activities (CYP1A1 and CYP2B1/2), nifedipine oxidase activity (CYP3A4), and Western blot assays of their respective apoproteins were analysed in liver microsomes. Putrescine levels in hepatic tissue were also measured. Partial hepatectomy and putrescine treatment induced a significant diminution in total CYP (50% and 30% of sham-operated rats, respectively). Gamma amino butyric acid treatment prevented this decrease in partially hepatectomised rats. Nifedipine oxidase activity of partially hepatectomised and putrescine-treated rats significantly decreased to 43% and 60% of that in sham-operated rats, respectively. Again, gamma amino butyric acid prevented the diminution in partially hepatectomised rats. No significant changes were observed in o-dealkylase activities. These results show that inducible CYP1A1 and CYP2B1/2, which are important in carcinogen metabolisation, are preserved after partial hepatectomy. However, constitutive CYP3A4, which represents 50% of total CYP and metabolises drugs like nifedipine, warfarin, acetaminophen, cyclosporin and FK-506, is reduced during liver regeneration. Our experiments suggest that endogenous putrescine is, at least, partly responsible for this decrease.
Colloids and surfaces. B, Biointerfaces, 2018
An enzymatic pool from the Amazonian bacterium Bacillus sp. P7 was used as milk coagulant. Discov... more An enzymatic pool from the Amazonian bacterium Bacillus sp. P7 was used as milk coagulant. Discovery of novel coagulants is of great interest in dairy industry for the development of new textures in cheese. Color, mechanical and microstructural characterization of milk gels induced by the bacterial enzymatic pool was carried out. Effect of mineral fortification on these characteristics was studied. Whiter gels with smaller pore diameters were obtained in the presence of Caor Mg. These characteristics seemed to be influenced by the effect of ionic strength on casein structure which was also evidenced by digital texture features analysis. On the other hand, specific affinity of the assayed cations for milk proteins showed to be important in the development of the mechanical texture of the gels. Firmness and fracture force of milk gels obtained in the presence of Znor Cawere higher than in the presence of Mgand Na.
Toxicology, Aug 15, 1997
Colchicine, a microtubule-disrupting agent, induces hepatotoxicity in experimental animals at the... more Colchicine, a microtubule-disrupting agent, induces hepatotoxicity in experimental animals at the doses commonly employed to explore vesicular transport in the liver. The effect of manipulations of the bile salt pool on colchicine-induced hepatotoxicity was studied in rats to determine the role of bile salts in this phenomenon. Leakage of enzyme markers of liver-cell damage into plasma and bile induced by colchicine pre-treatment displayed a sigmoidal log dose-effect curve, the half-maximal effect being reached at 0.12 vmol per 100 g body wt. Lumicolchicine, instead, showed no harmful effect. Maximal increment of biliary LDH discharge induced by colchicine was reduced from 950 9124% to 216929% by bile diversion leading to a marked reduction in bile salt output, and this parameter was further decreased to 1009 13% and 1579 39% by subsequent repletion of the bile salt pool with the hydrophilic bile salts taurodehydrocholate and tauroursodeoxycholate, respectively. Conversely, infusion of taurocholate into non-bile salt depleted, colchicine-treated rats led to cholestasis and massive discharge of enzymes into both blood and bile. Our data show conclusively that colchicine-induced hepatotoxicity depends on the magnitude and composition of the bile salt flux traversing the liver. They also support the view that functional integrity of vesicular mechanisms presumably involved in membrane repair are indispensable to protect the hepatocytes from the damaging effect of bile salts during normal bile formation.
Biochimica Et Biophysica Acta General Subjects, May 24, 1991
Conjugation has been considered the rate-limiting step for bilirubin hepatic transport, and bypas... more Conjugation has been considered the rate-limiting step for bilirubin hepatic transport, and bypass of this metabolic step could explain why photobilirubins can be rapidly cleared by the liver. In this paper we assessed whether photoirradiation may enhance the bilirubin overall hepatic transport in the isolated perfused Wistar rat liver, a model possessing intact transport and conjugating systems. Bilirubin was administered as a bolus so as to reach a pedusate concentration of-l0/t M (bUirubin/albumin molar ratio !: 17). Perfusate light exposure (0.56-i0 ts quanta s-t cm-z) yielded 7-10% of eonfignrational photoisomers, which were further identified as (4Z,15E/4E,ISZ)-bilirubin IXa. Under such conditions, the pedusate removal rate was increased by 39% over that from dark conditions. Likewise, biliary excretion, estimated as total bilirubin recovery at 60 min, was also increased (+48%). This later improvement was mainly produced at the expense of unconjugated bilirubin, which most likely derived from its configurational pbotoisomers that, once excreted into bile, readily re-isomerized to the parent compound, in addition, this increment was partially due to a delayed improvement of monoglncuronide pig~nent excretion. The calculated hepatic pigment content was significantly higher under light conditions. A direct assessment of hepatic content of different bilirubin moieties at 20 min after bUirubin administration confirmed that such an increment was fully accounted for by unconjugated pigment. Our finding that hepatic pigment content rose (despite a higher biliary excretion) when the bilirubin was irradiated suggests a higher net uptake of photoisonters than native pigment. This observation, and the finding that billrubin photoisomers were usually excreted without undergoing conjugation even if the metabolic system is active, contribute to explain the greater appearance of uneonjugated bilirubin in Wistar rat bile under light exposure.
Consideraciones generales sobre lípidos. Clasificación de lípidos neutros y polares. Lípidos de b... more Consideraciones generales sobre lípidos. Clasificación de lípidos neutros y polares. Lípidos de biomembranas Glicerolípidos, esfingolípidos y esteroles. Estructuras de fosfolípidos. Ácidos grasos, nomenclatura y clasificación. Roles de los lípidos en las membranas biológicas. Referencias bibliográficas.
Canadian Journal of Physiology and Pharmacology, Feb 13, 2011
The lysosomal processing of horseradish peroxidase (HRP) was assessed in this study, i.e., its ly... more The lysosomal processing of horseradish peroxidase (HRP) was assessed in this study, i.e., its lysosomal proteolysis and the biliary output of its possible lysosomal metabolites by rat liver in vivo. HRP was covalently linked to [14C]sucrose to provide a label that remains trapped within lysosomes after proteolysis. The [14C]sucrose-labelled HRP was injected into the portal vein of rat, and after 30 min about 34% of the injected radiolabel was present in the liver. Subcellular fractionation by differential centrifugation and further purification of lysosomes in a Percoll gradient showed that radiolabel was concentrated in lysosomes and indicated that about 91% of the total proteolysis of HRP in liver could be attributed to these organelles. The in vivo lysosomal degradation rate of HRP at 30 min was about 40%/h, decreasing over time. The lysosomal inhibitors chloroquine and leupeptin suppressed proteolysis of HRP by about 30 and 60%, respectively. Analysis of the 14C excreted in bile by trichloroacetic acid precipitation and by SDS-polyacrylamide gel electrophoresis showed a minor fraction, which was intact HRP (40 kDa), and a major fraction, which was associated with material smaller than 3 kDa. The biliary output of these low molecular mass products, in contrast to that of intact HRP, did not gradually decline with time and represented about 3% of the corresponding amounts in liver. Chloroquine and leupeptin specifically decreased their biliary excretion (about 60%), giving additional support to their lysosomal origin. In addition, the overall hepatic processing of [14C]sucrose-labelled HRP did not differ from that of the native HRP measured by enzyme assay, indicating no significant alteration caused by the labelling procedure.
Research communications in chemical pathology and pharmacology
El Texto ha sido pensado como un libro introductorio al análisis de lípidos de biomembranas para ... more El Texto ha sido pensado como un libro introductorio al análisis de lípidos de biomembranas para estudiantes de posgrado de Biología, Bioquímica, Biología Molecular y otros carreras afines. En el mismo se ha priorizado el desarrollo experimental de la metodología analítica de lípidos de biomembranas En cada etapa del análisis se presenta una breve introducción para luego desarrollar un método seleccionado por los autores, en cada capítulo se incluye un experimento con sus resultados esperados obtenidos de experimentos realizados por estudiantes del curso. Se agregaron además notas adicionales con algunas técnicas y datos útiles para el analista. Se han seleccionado técnicas simples y económicas que bastan para obtener información suficiente sobre la membrana del eritrocito y otros tejidos y membranas de origen natural. Se ha seleccionado como técnica separativa la cromatografía en capa fina que permite visualizar resultados con una inversión en equipamiento muy económica.
Gene therapy, 2014
Estrogens can cause liver cholestatic disease. As downregulation of hepatocyte canalicular aquapo... more Estrogens can cause liver cholestatic disease. As downregulation of hepatocyte canalicular aquaporin-8 (AQP8) water channels has been involved in estrogen-induced bile secretory failure, we tested whether the archetypal water channel AQP1 improves 17α-ethinylestradiol (EE)-induced cholestasis. EE administration to rats reduced bile flow by 50%. A recombinant adenoviral (Ad) vector encoding human AQP1 (hAQP1), AdhAQP1, or a control vector was administered by retrograde bile ductal infusion. Hepatocyte canalicular hAQP1 expression was confirmed by liver immunostaining and immunoblotting in purified membrane fractions. Accordingly, canalicular osmotic water permeability was markedly increased. Bile flow, either basal or bile salt-stimulated was significantly augmented by over 50%. The choleretic efficiency of endogenous bile salts (that is, volume of bile per μmol of excreted bile salt) was significantly increased by 45% without changes in the biliary bile salt composition. Our data su...
Medicina, 2001
In order to know whether IFN alpha prevents in vivo oncogenesis in the very-early-stage cancer ce... more In order to know whether IFN alpha prevents in vivo oncogenesis in the very-early-stage cancer cells, we evaluated the action of IFN alpha-2b on preneoplastic foci in rats. Animals were divided into six groups: subjected to an initiation-promotion model of cancer development (G1), treated with IFN alpha-2b during: a) initiation-promotion (G2), b) initiation (G3), promotion (G4); subjected only to an initiation stage (G5) and treated with IFN alpha-2b during this period (G6). The number and area of rGST P-positive foci were reduced and the Apoptotic index was increased in G2, 3 and 6. Bcl-2 and Bcl-XL protein levels were decreased in IFN alpha-2b-treated rats. Increased levels of mitochondrial Bax protein were observed in G2, 3 and 6. In conclusion, preneoplastic hepatocytes in the IFN alpha-2b-treated rats undergo programmed cell death as a result of a significant increase of Bax and its translocation to the mitochondria.
The Journal of pharmacology and experimental therapeutics, 2001
The molecular basis of perinatal changes occurring in major UDP-glucuronosyltransferase (UGT) fam... more The molecular basis of perinatal changes occurring in major UDP-glucuronosyltransferase (UGT) family 1 isoforms and in UGT2B1, a relevant isoform belonging to family 2, was analyzed in rat liver. Nonpregnant, pregnant (19-20 days of pregnancy), and two groups of postpartum animals corresponding to early and middle stages of lactation (2-4 and 10-12 days after delivery, respectively) were studied. UGT activity determined in UDP-N-acetylglucosamine-activated microsomes revealed that bilirubin, p-nitrophenol, and ethynylestradiol (17beta-OH and 3-OH) but not androsterone and estrone glucuronidation rates, were decreased in pregnant rats. Decreased enzyme activities returned to control values after delivery. p-Nitrophenol, androsterone, and estrone conjugation rate increased in postpartum rats. Western blot analysis performed with anti-peptide-specific (anti-1A1, 1A5, 1A6, and 2B1) antibodies revealed decreased levels of all family 1 isoforms and UGT2B1 during pregnancy. In postpartum a...
Acta gastroenterologica Latinoamericana, 1979
Enzyme determinations and hepatic histological studies were carried out in normal rats and in rat... more Enzyme determinations and hepatic histological studies were carried out in normal rats and in rats intoxicated with the organophosphorus pesticide parathion. No significative evidence of hepatocellular damage was seen in parathion -- intoxicated rats as responsible of the impaired bile flow and sulfobromophthalein biliary excretion that were previously reported. On the contrary, the isolated perfused rat liver from normal and intoxicated rats showed a similar behavior when the perfusate flow through the liver was maintained at a similar rate. We conclude that altered hepatic blood flow may be a factor determinant of an impairment of bile flow and of biliary excretion of several organic anions like sulphobromopthalein that were observed in parathion -- intoxicated rats.
Archives of Toxicology, 2014
At present, it has not been systematically evaluated whether the functional alterations induced b... more At present, it has not been systematically evaluated whether the functional alterations induced by cholestatic compounds in canalicular transporters involved in bile formation can be reproduced in sandwich-cultured rat hepatocytes (SCRHs). Here, we focused on two clinically relevant cholestatic agents, such as estradiol 17β-D-glucuronide (E17G) and taurolithocholate (TLC), also testing the ability of dibutyryl cyclic AMP (DBcAMP) to prevent their effects. SCRHs were incubated with E17G (200 µM) or TLC (2.5 µM) for 30 min, with or without pre-incubation with DBcAMP (10 µM) for 15 min. Then, the increase in glutathione methyl fluorescein (GS-MF)-associated fluorescence inside the canaliculi was monitored by quantitative time-lapse imaging, and Mrp2 transport activity was calculated by measuring the slope of the time-course fluorescence curves during the initial linear phase, which was considered to be the Mrp2-mediated initial transport rate (ITR). E17G and TLC impaired canalicular bile formation, as evidenced by a decrease in both the bile canaliculus volume and the bile canaliculus width, estimated from 3D and 2D confocal images, respectively. These compounds decreased ITR and induced retrieval of Mrp2, a main pathomechanism involved in their cholestatic effects. Finally, DBcAMP prevented these effects, and its well-known choleretic effect was evident from the increase in the canalicular volume/width values; this choleretic effect is associated in part with its capability to increase Mrp2 activity, evidenced here by the increase in ITR of GS-MF. Our study supports the use of SCRHs as an in vitro model useful to quantify canalicular transport function under conditions of cholestasis and choleresis.
Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1992
Taurolithocholate (TLC), a natural bile salt, induces selective impairment on canalicular membran... more Taurolithocholate (TLC), a natural bile salt, induces selective impairment on canalicular membrane of the hepatocyte, which seems to be a major determinant of its cholestatic effect in experimental animals. In order to extend existing studies about the effects of TLC on bile secretion, we examined in TLC-treated rats the biliary excretion of compounds that are transported to canalicular membrane via vesicles, such as lipids and proteins. The single intravenous injection of TLC (3 mumol/100 g body wt.) inhibited transiently the biliary bile salt excretion, while the biliary excretion of lipids (i.e., cholesterol and phospholipids) and proteins remained inhibited even though the biliary excretion and composition of bile salts were normalized. Under such a condition, TLC also inhibited the transcellular vesicular pathway to the exogenous protein horseradish peroxidase entry into bile, without altering the paracellular biliary access of the protein. The hepatic uptake of horseradish peroxidase was unaffected by TLC-treatment. The results indicate that TLC can inhibit the biliary excretion of compounds that reach the canaliculus via a vesicular pathway, such as lipids and proteins, by a mechanism not related to a defective bile salt excretion. Possible explanations for these findings are discussed.
BioTechniques, 2011
An inexpensive modular perfused chamber (MPC) designed for low- and normal-temperature live-cell ... more An inexpensive modular perfused chamber (MPC) designed for low- and normal-temperature live-cell imaging is presented. The device consists of four lathed pieces of stainless steel assembled as a cylindrical open chamber that can hold either round or square glass coverslips. The chamber is connected to a thermal-bath operating with recirculation. For image acquisition at 4°C, cooled air is blown toward the coverslip surface to prevent condensation. Principal advantages of this device are thermal stability in the sample environment, rapid response to changes in temperature set point, and easy sample insertion. The device enables the study of dynamic processes in cells governed by large temperature differences such as those imposed by hypothermic preservation of cells (0-4°C) followed by rewarming to normothermia (37°C). The capabilities of the MPC were demonstrated by monitoring the internalization of fluorescent quantum dots (QDs) in rat hepatocytes after hypothermic storage and duri...
Growth Factors, 2010
Wnt/beta-catenin/T cell factor (TCF) pathway is activated in several types of human cancers, prom... more Wnt/beta-catenin/T cell factor (TCF) pathway is activated in several types of human cancers, promoting cell growth and proliferation. Forkhead box containing protein class O (FOXO) transcription factors compete with TCF for beta-catenin binding, particularly under cellular oxidative stress conditions. Contrary to beta-catenin/TCF, beta-catenin/FOXO promotes the transcription of genes involved in cell cycle arrest and apoptosis. We have previously demonstrated that in vivo interferon-alpha2b (IFN-alpha2b) administration induces apoptosis in preneoplastic livers, a mechanism mediated by reactive oxygen species (ROS) and transforming growth factor-beta(1) (TGF-beta(1)). This study was aimed to assess the status of the Wnt/beta-catenin/TCF pathway in a very early stage of rat hepatocarcinogenesis and to further evaluate the effects of in vivo IFN-alpha2b treatment on it. We demonstrated that the Wnt/beta-catenin/TCF pathway is activated in preneoplastic rat livers. More important, in vivo IFN-alpha2b treatment inhibits Wnt/beta-catenin/TCF pathway and promotes programed cell death possibly providing a link with FOXO pathway.
Free Radical Research, 2011
Diabetes mellitus is a risk factor for prognosis after liver resection. In previous work, we foun... more Diabetes mellitus is a risk factor for prognosis after liver resection. In previous work, we found a pro-apoptotic state in the diabetic rat liver. In this work, this was also observed 1 hour post-partial hepatectomy (PH) and resulted in a deficient regenerative response 24 hours post-PH. Treatment with insulin and/or Desferoxamine (DES) (iron chelator) or Tempol (TEM) (free radicals scavenger) was effective in preventing the liver reactive oxygen species (ROS) production induced by diabetic state. High levels of ROS play a role in hepatic lipid peroxidation in diabetes before and after PH, and lead to increased pro-apoptotic events, which contribute to a reduced regenerative response. This becomes of relevance for the potential use of antioxidants/free radical scavengers plus insulin for improvement of post-surgical recovery of diabetic patients subjected to a PH.
Drug Metabolism and Disposition, 2006
The ability of the kidney and small intestine to synthesize and subsequently eliminate dinitrophe... more The ability of the kidney and small intestine to synthesize and subsequently eliminate dinitrophenyl-S-glutathione (DNP-SG), a substrate for the multidrug resistance-associated proteins (Mrps), was assessed in bile duct-ligated (BDL) rats 1, 7, and 14 days after surgery, using an in vivo perfused jejunum model with simultaneous urine collection. A single i.v. dose of 30 mol/kg b.wt. of 1-chloro-2,4-dinitrobenzene (CDNB) was administered, and its glutathione conjugate DNP-SG and dinitrophenyl cysteinyl glycine derivative, which is the result of ␥-glutamyl-transferase action on DNP-SG, were determined in urine and intestinal perfusate by high-performance liquid chromatography. Intestinal excretion of these metabolites was unchanged at day 1, and decreased at days 7 and 14 (؊39% and ؊33%, respectively) after surgery with respect to shams. In contrast, renal excretion was increased by 114%, 150%, and 128% at days 1, 7, and 14. Western blot studies revealed decreased levels of apical Mrp2 in liver and jejunum but increased levels in renal cortex from BDL animals, these changes being maximal between days 7 and 14. Assessment of expression of basolateral Mrp3 at day 14 postsurgery indicated preserved levels in renal cortex, duodenum, jejunum, distal ileum, and colon. Analysis of expression of glutathione-S-transferases ␣, , and , as well as activity toward CDNB, indicates that formation of DNP-SG was impaired in liver, preserved in intestine, and increased in renal cortex. In conclusion, increased renal tubular conversion of CDNB to DNP-SG followed by subsequent Mrp2-mediated secretion into urine partially compensates for altered liver function in experimental obstructive cholestasis.
Drug Metabolism and Disposition, 2005
EE induces cholestasis by affecting bile salt-dependent and-independent fractions of the bile flo... more EE induces cholestasis by affecting bile salt-dependent and-independent fractions of the bile flow. The decrease in bile salt-independent flow is thought to be due, in part, to a reduction in the expression of the canalicular transporter Mrp2. The impact of modulation of Mrp2 function by UDC in EE cholestasis is unknown. We evaluated the protective effect of UDC on EE-induced impairment of Mrp2 activity in vivo and in isolated hepatocytes, by using the substrate dinitrophenyl-S-glutathione (DNP-SG). EE was administered to male Wistar rats at a
Journal of Hepatology, 1998
The mechanism by which many cytochrome P450 (CYP) isozymes decrease during liver regeneration is ... more The mechanism by which many cytochrome P450 (CYP) isozymes decrease during liver regeneration is unclear. Peptides and growth factors are thought to be involved. Putrescine, the first polyamine synthesised by ornithine decarboxylase, peaks early following partial hepatectomy and is known to play an essential role in hepatic regeneration. Gamma amino butyric acid was reported as a physiologic inhibitor of ornithine decarboxylase. In this work we studied the possible involvement of putrescine in the CYP reduction during liver regeneration. Hepatectomised, putrescine-treated sham, and GABA-treated hepatectomised rats were used throughout. Total hepatic cytochrome P450, o-dealkylase activities (CYP1A1 and CYP2B1/2), nifedipine oxidase activity (CYP3A4), and Western blot assays of their respective apoproteins were analysed in liver microsomes. Putrescine levels in hepatic tissue were also measured. Partial hepatectomy and putrescine treatment induced a significant diminution in total CYP (50% and 30% of sham-operated rats, respectively). Gamma amino butyric acid treatment prevented this decrease in partially hepatectomised rats. Nifedipine oxidase activity of partially hepatectomised and putrescine-treated rats significantly decreased to 43% and 60% of that in sham-operated rats, respectively. Again, gamma amino butyric acid prevented the diminution in partially hepatectomised rats. No significant changes were observed in o-dealkylase activities. These results show that inducible CYP1A1 and CYP2B1/2, which are important in carcinogen metabolisation, are preserved after partial hepatectomy. However, constitutive CYP3A4, which represents 50% of total CYP and metabolises drugs like nifedipine, warfarin, acetaminophen, cyclosporin and FK-506, is reduced during liver regeneration. Our experiments suggest that endogenous putrescine is, at least, partly responsible for this decrease.
Colloids and surfaces. B, Biointerfaces, 2018
An enzymatic pool from the Amazonian bacterium Bacillus sp. P7 was used as milk coagulant. Discov... more An enzymatic pool from the Amazonian bacterium Bacillus sp. P7 was used as milk coagulant. Discovery of novel coagulants is of great interest in dairy industry for the development of new textures in cheese. Color, mechanical and microstructural characterization of milk gels induced by the bacterial enzymatic pool was carried out. Effect of mineral fortification on these characteristics was studied. Whiter gels with smaller pore diameters were obtained in the presence of Caor Mg. These characteristics seemed to be influenced by the effect of ionic strength on casein structure which was also evidenced by digital texture features analysis. On the other hand, specific affinity of the assayed cations for milk proteins showed to be important in the development of the mechanical texture of the gels. Firmness and fracture force of milk gels obtained in the presence of Znor Cawere higher than in the presence of Mgand Na.
Toxicology, Aug 15, 1997
Colchicine, a microtubule-disrupting agent, induces hepatotoxicity in experimental animals at the... more Colchicine, a microtubule-disrupting agent, induces hepatotoxicity in experimental animals at the doses commonly employed to explore vesicular transport in the liver. The effect of manipulations of the bile salt pool on colchicine-induced hepatotoxicity was studied in rats to determine the role of bile salts in this phenomenon. Leakage of enzyme markers of liver-cell damage into plasma and bile induced by colchicine pre-treatment displayed a sigmoidal log dose-effect curve, the half-maximal effect being reached at 0.12 vmol per 100 g body wt. Lumicolchicine, instead, showed no harmful effect. Maximal increment of biliary LDH discharge induced by colchicine was reduced from 950 9124% to 216929% by bile diversion leading to a marked reduction in bile salt output, and this parameter was further decreased to 1009 13% and 1579 39% by subsequent repletion of the bile salt pool with the hydrophilic bile salts taurodehydrocholate and tauroursodeoxycholate, respectively. Conversely, infusion of taurocholate into non-bile salt depleted, colchicine-treated rats led to cholestasis and massive discharge of enzymes into both blood and bile. Our data show conclusively that colchicine-induced hepatotoxicity depends on the magnitude and composition of the bile salt flux traversing the liver. They also support the view that functional integrity of vesicular mechanisms presumably involved in membrane repair are indispensable to protect the hepatocytes from the damaging effect of bile salts during normal bile formation.
Biochimica Et Biophysica Acta General Subjects, May 24, 1991
Conjugation has been considered the rate-limiting step for bilirubin hepatic transport, and bypas... more Conjugation has been considered the rate-limiting step for bilirubin hepatic transport, and bypass of this metabolic step could explain why photobilirubins can be rapidly cleared by the liver. In this paper we assessed whether photoirradiation may enhance the bilirubin overall hepatic transport in the isolated perfused Wistar rat liver, a model possessing intact transport and conjugating systems. Bilirubin was administered as a bolus so as to reach a pedusate concentration of-l0/t M (bUirubin/albumin molar ratio !: 17). Perfusate light exposure (0.56-i0 ts quanta s-t cm-z) yielded 7-10% of eonfignrational photoisomers, which were further identified as (4Z,15E/4E,ISZ)-bilirubin IXa. Under such conditions, the pedusate removal rate was increased by 39% over that from dark conditions. Likewise, biliary excretion, estimated as total bilirubin recovery at 60 min, was also increased (+48%). This later improvement was mainly produced at the expense of unconjugated bilirubin, which most likely derived from its configurational pbotoisomers that, once excreted into bile, readily re-isomerized to the parent compound, in addition, this increment was partially due to a delayed improvement of monoglncuronide pig~nent excretion. The calculated hepatic pigment content was significantly higher under light conditions. A direct assessment of hepatic content of different bilirubin moieties at 20 min after bUirubin administration confirmed that such an increment was fully accounted for by unconjugated pigment. Our finding that hepatic pigment content rose (despite a higher biliary excretion) when the bilirubin was irradiated suggests a higher net uptake of photoisonters than native pigment. This observation, and the finding that billrubin photoisomers were usually excreted without undergoing conjugation even if the metabolic system is active, contribute to explain the greater appearance of uneonjugated bilirubin in Wistar rat bile under light exposure.
Consideraciones generales sobre lípidos. Clasificación de lípidos neutros y polares. Lípidos de b... more Consideraciones generales sobre lípidos. Clasificación de lípidos neutros y polares. Lípidos de biomembranas Glicerolípidos, esfingolípidos y esteroles. Estructuras de fosfolípidos. Ácidos grasos, nomenclatura y clasificación. Roles de los lípidos en las membranas biológicas. Referencias bibliográficas.
Canadian Journal of Physiology and Pharmacology, Feb 13, 2011
The lysosomal processing of horseradish peroxidase (HRP) was assessed in this study, i.e., its ly... more The lysosomal processing of horseradish peroxidase (HRP) was assessed in this study, i.e., its lysosomal proteolysis and the biliary output of its possible lysosomal metabolites by rat liver in vivo. HRP was covalently linked to [14C]sucrose to provide a label that remains trapped within lysosomes after proteolysis. The [14C]sucrose-labelled HRP was injected into the portal vein of rat, and after 30 min about 34% of the injected radiolabel was present in the liver. Subcellular fractionation by differential centrifugation and further purification of lysosomes in a Percoll gradient showed that radiolabel was concentrated in lysosomes and indicated that about 91% of the total proteolysis of HRP in liver could be attributed to these organelles. The in vivo lysosomal degradation rate of HRP at 30 min was about 40%/h, decreasing over time. The lysosomal inhibitors chloroquine and leupeptin suppressed proteolysis of HRP by about 30 and 60%, respectively. Analysis of the 14C excreted in bile by trichloroacetic acid precipitation and by SDS-polyacrylamide gel electrophoresis showed a minor fraction, which was intact HRP (40 kDa), and a major fraction, which was associated with material smaller than 3 kDa. The biliary output of these low molecular mass products, in contrast to that of intact HRP, did not gradually decline with time and represented about 3% of the corresponding amounts in liver. Chloroquine and leupeptin specifically decreased their biliary excretion (about 60%), giving additional support to their lysosomal origin. In addition, the overall hepatic processing of [14C]sucrose-labelled HRP did not differ from that of the native HRP measured by enzyme assay, indicating no significant alteration caused by the labelling procedure.
Research communications in chemical pathology and pharmacology
El Texto ha sido pensado como un libro introductorio al análisis de lípidos de biomembranas para ... more El Texto ha sido pensado como un libro introductorio al análisis de lípidos de biomembranas para estudiantes de posgrado de Biología, Bioquímica, Biología Molecular y otros carreras afines. En el mismo se ha priorizado el desarrollo experimental de la metodología analítica de lípidos de biomembranas En cada etapa del análisis se presenta una breve introducción para luego desarrollar un método seleccionado por los autores, en cada capítulo se incluye un experimento con sus resultados esperados obtenidos de experimentos realizados por estudiantes del curso. Se agregaron además notas adicionales con algunas técnicas y datos útiles para el analista. Se han seleccionado técnicas simples y económicas que bastan para obtener información suficiente sobre la membrana del eritrocito y otros tejidos y membranas de origen natural. Se ha seleccionado como técnica separativa la cromatografía en capa fina que permite visualizar resultados con una inversión en equipamiento muy económica.
Gene therapy, 2014
Estrogens can cause liver cholestatic disease. As downregulation of hepatocyte canalicular aquapo... more Estrogens can cause liver cholestatic disease. As downregulation of hepatocyte canalicular aquaporin-8 (AQP8) water channels has been involved in estrogen-induced bile secretory failure, we tested whether the archetypal water channel AQP1 improves 17α-ethinylestradiol (EE)-induced cholestasis. EE administration to rats reduced bile flow by 50%. A recombinant adenoviral (Ad) vector encoding human AQP1 (hAQP1), AdhAQP1, or a control vector was administered by retrograde bile ductal infusion. Hepatocyte canalicular hAQP1 expression was confirmed by liver immunostaining and immunoblotting in purified membrane fractions. Accordingly, canalicular osmotic water permeability was markedly increased. Bile flow, either basal or bile salt-stimulated was significantly augmented by over 50%. The choleretic efficiency of endogenous bile salts (that is, volume of bile per μmol of excreted bile salt) was significantly increased by 45% without changes in the biliary bile salt composition. Our data su...
Medicina, 2001
In order to know whether IFN alpha prevents in vivo oncogenesis in the very-early-stage cancer ce... more In order to know whether IFN alpha prevents in vivo oncogenesis in the very-early-stage cancer cells, we evaluated the action of IFN alpha-2b on preneoplastic foci in rats. Animals were divided into six groups: subjected to an initiation-promotion model of cancer development (G1), treated with IFN alpha-2b during: a) initiation-promotion (G2), b) initiation (G3), promotion (G4); subjected only to an initiation stage (G5) and treated with IFN alpha-2b during this period (G6). The number and area of rGST P-positive foci were reduced and the Apoptotic index was increased in G2, 3 and 6. Bcl-2 and Bcl-XL protein levels were decreased in IFN alpha-2b-treated rats. Increased levels of mitochondrial Bax protein were observed in G2, 3 and 6. In conclusion, preneoplastic hepatocytes in the IFN alpha-2b-treated rats undergo programmed cell death as a result of a significant increase of Bax and its translocation to the mitochondria.
The Journal of pharmacology and experimental therapeutics, 2001
The molecular basis of perinatal changes occurring in major UDP-glucuronosyltransferase (UGT) fam... more The molecular basis of perinatal changes occurring in major UDP-glucuronosyltransferase (UGT) family 1 isoforms and in UGT2B1, a relevant isoform belonging to family 2, was analyzed in rat liver. Nonpregnant, pregnant (19-20 days of pregnancy), and two groups of postpartum animals corresponding to early and middle stages of lactation (2-4 and 10-12 days after delivery, respectively) were studied. UGT activity determined in UDP-N-acetylglucosamine-activated microsomes revealed that bilirubin, p-nitrophenol, and ethynylestradiol (17beta-OH and 3-OH) but not androsterone and estrone glucuronidation rates, were decreased in pregnant rats. Decreased enzyme activities returned to control values after delivery. p-Nitrophenol, androsterone, and estrone conjugation rate increased in postpartum rats. Western blot analysis performed with anti-peptide-specific (anti-1A1, 1A5, 1A6, and 2B1) antibodies revealed decreased levels of all family 1 isoforms and UGT2B1 during pregnancy. In postpartum a...
Acta gastroenterologica Latinoamericana, 1979
Enzyme determinations and hepatic histological studies were carried out in normal rats and in rat... more Enzyme determinations and hepatic histological studies were carried out in normal rats and in rats intoxicated with the organophosphorus pesticide parathion. No significative evidence of hepatocellular damage was seen in parathion -- intoxicated rats as responsible of the impaired bile flow and sulfobromophthalein biliary excretion that were previously reported. On the contrary, the isolated perfused rat liver from normal and intoxicated rats showed a similar behavior when the perfusate flow through the liver was maintained at a similar rate. We conclude that altered hepatic blood flow may be a factor determinant of an impairment of bile flow and of biliary excretion of several organic anions like sulphobromopthalein that were observed in parathion -- intoxicated rats.
Archives of Toxicology, 2014
At present, it has not been systematically evaluated whether the functional alterations induced b... more At present, it has not been systematically evaluated whether the functional alterations induced by cholestatic compounds in canalicular transporters involved in bile formation can be reproduced in sandwich-cultured rat hepatocytes (SCRHs). Here, we focused on two clinically relevant cholestatic agents, such as estradiol 17β-D-glucuronide (E17G) and taurolithocholate (TLC), also testing the ability of dibutyryl cyclic AMP (DBcAMP) to prevent their effects. SCRHs were incubated with E17G (200 µM) or TLC (2.5 µM) for 30 min, with or without pre-incubation with DBcAMP (10 µM) for 15 min. Then, the increase in glutathione methyl fluorescein (GS-MF)-associated fluorescence inside the canaliculi was monitored by quantitative time-lapse imaging, and Mrp2 transport activity was calculated by measuring the slope of the time-course fluorescence curves during the initial linear phase, which was considered to be the Mrp2-mediated initial transport rate (ITR). E17G and TLC impaired canalicular bile formation, as evidenced by a decrease in both the bile canaliculus volume and the bile canaliculus width, estimated from 3D and 2D confocal images, respectively. These compounds decreased ITR and induced retrieval of Mrp2, a main pathomechanism involved in their cholestatic effects. Finally, DBcAMP prevented these effects, and its well-known choleretic effect was evident from the increase in the canalicular volume/width values; this choleretic effect is associated in part with its capability to increase Mrp2 activity, evidenced here by the increase in ITR of GS-MF. Our study supports the use of SCRHs as an in vitro model useful to quantify canalicular transport function under conditions of cholestasis and choleresis.