Liver Of Different Species Research Articles

Metabolic transformation of cyclopiazonic acid in liver microsomes from different species based on UPLC-Q/TOF-MS

To investigate the metabolic transformation of cyclopiazonic acid (CPA) in the liver of different species and to supplement accurate risk assessment information, the metabolism of CPA in liver microsomes from four animals and humans was studied using the ultra–high–performance liquid chromatography–quadrupole/time–of–flight method. The results showed that a total of four metabolites were obtained, and dehydrogenation, hydroxylation, methylation, and glucuronidation were identified as the main metabolic pathways of CPA. Rat liver microsomes exhibited the highest metabolic capacity for CPA, with dehydrogenated (C20H18N2O3) and glucuronic acid-conjugated (C26H28N2O10) metabolites identified in all liver microsomes except chicken, indicating significant species metabolic differences. Moreover, C20H18N2O3 was only detected in the incubation system with cytochromes P450 3A4 (CYP3A4). The hydroxylated (C20H20N2O4) and methylated (C21H22N2O3) metabolites were detected in all incubation systems except for the CYP2C9, with CYP3A4 demonstrating the strongest metabolic capacity. The “cocktail” probe drug method showed that CPA exhibited a moderate inhibitory effect on the CYP3A4 (IC50 value = 8.658 μM), indicating that the substrate had a negative effect on enzyme activity. Our results provide new insights to understand the biotransformation profile of CPA in animals and humans.

A COMPARISON OF THE PROTEIN PROFILE OF TRIAENOPHORUS NODULOSUS PLEROCERCOIDS FROM DIFFERENT INTERMEDIATE HOSTS

The spectrum of proteins was studied in plerocercoids of parasitic cestodes Triaenophorus nodulosus collected from the liver of different species of fish: perch Perca fluviatilis L ., ruffe Gymnocephalus cernuus L. and burbot Lota lota L. A comparison of protein extracts separated by 2D electrophoresis revealed 18 proteins whose concentrations differed among larvae of different fish species. Of these, 4 proteins were identified by using mass-spectrometric methods (MALDI-TOF/TOF, LC–MS): a carbohydrate metabolism enzyme triosephosphate isomerase, the proteins of microtubules tubulin α and β, and the G-protein signaling molecule. The elevated content of components of the G-protein pathway of the cytoskeleton and cell morphology regulation (tubulin and G-protein) in plerocercoids from perch in comparison with plerocercoids from burbot indicate a more dynamic cytoskeleton, increased cell proliferation and differentiation processes, and possibly more mature larvae in the former species. A higher level of expression of the key enzyme of glycolysis and signal transmission in plerocercoids from perch compared to plerocercoids from burbot indicates a greater metabolic activity in larvae from perch. The obtained results indicate that the expression of some proteins in T. nodulosus larvae is influenced by the habitat.

Improved rat liver decellularization by arterial perfusion under oscillating pressure conditions.

One approach of regenerative medicine to generate functional hepatic tissue in vitro is decellularization and recellularization, and several protocols for the decellularization of livers of different species have been published. This appears to be the first report on rat liver decellularization by perfusion under oscillating pressure conditions, intending to optimize microperfusion and minimize damage to the ECM. Four decellularization protocols were compared: perfusion via the portal vein (PV) or the hepatic artery (HA), with (+P) or without (-P) oscillating pressure conditions. All rat livers (n = 24) were perfused with 1% Triton X-100 and 1% sodium dodecyl sulphate, each for 90 min with a perfusion rate of 5 ml/min. Perfusion decellularization was observed macroscopically and the decellularized liver matrices were analysed by histology and biochemical analyses (e.g. levels of DNA, glycosaminoglycans and hepatocyte growth factor). Livers decellularized via the hepatic artery and under oscillating pressure showed a more homogeneous decellularization and less remaining DNA, compared with the livers of the other experimental groups. The novel decellularization method described is effective, quick (3 h) and gentle to the extracellular matrix and thus represents an improvement of existing methodology. Copyright © 2014 John Wiley & Sons, Ltd.

Concentrations and composition of organochlorine contaminants in different species of cephalopod molluscs from the Italian waters (Adriatic Sea)

Persistent organochlorines, such as polychlorinated biphenyls including coplanar congeners and DDT compounds were measured in the liver of different species of mollusc cephalopods: broadtail squid ( Illex coindetii), horned octopus ( Eledone moschata) and spider octopus ( Octopus salutii) collected in two different areas of the Adriatic Sea (central and southern Adriatic Sea). The concentration of PCBs exceeded DDT ones in horned octopus (PCBs: 617.0 ng g −1 lipid weight, DDTs: 188.0 ng g −1 lipid weight) and in spider octopus (PCBs: 590.0 ng g −1 lipid weight, DDTs: 293.0 ng g −1 lipid weight), while in broadtail squid PCB load (748.0 ng g −1 lipid weight) was similar to those of DDTs (514.5 ng g −1 lipid weight). No significant difference in PCB levels was observed among species, while DDT concentrations were higher in broadtail squid than in horned octopus and spider octopus. DDTs composition showed slight differences in relation to species, thought p, p′-DDE was the predominant compound in all cephalopod species (broadtail squid: 88.1%, horned octopus 94.1%, spider octopus: 97.5%). PCB isomer profiles were similar among species being hexachlorinated isomers the most abundant, followed by penta- and heptachlorobiphenyls, while tri- and tetrachlorobiphenyls made up a small percentage of the total PCB residues. Regarding coplanar congeners, non- ortho PCBs were below the detection limit in all samples and the 2,3,7,8-TCDD toxic equivalent (TEQ) concentrations were very low. The influence of biological and ecological factors (size/weight and location) on the bioaccumulation of organochlorine compounds has been discussed.

Polychlorinated biphenyl residues in deep-sea fish from Mediterranean Sea

Polychlorinated biphenyls including coplanar congeners were measured in the liver of different species of skates and ghostsharks . The mean concentrations of PCBs in skates (314 ng/g wet wt) were comparable with those in ghostsharks (285 ng/g wet wt). The specific analysis of PCB congeners in skates hepatic tissue revealed a profile dominated by hexa-, penta-, and heptachlorinated congeners followed by trichlorobiphenyls, tetra- and decachlorobiphenyls. A different profile dominated by hexa-, tri-, and pentachlorinated congeners, followed by heptachlorobiphenyl and decachlorobiphenyls was found in ghostsharks liver. The estimated 2,3,7,8-tetrachlorodibenzo- p-dioxin toxic equivalents (TEQs) of coplanar PCBs in skates and ghostsharks were 0.48 and 0.33 pg/g wet weight, respectively, with mono- ortho congeners PCB105, 118 and 156 contributing more than 90% to the TEQs.

Species differences in the metabolism of olefins: implications for risk assessment

Olefinic compounds are commercially valuable because they form useful polymeric substances. The same chemical property (presence of double bonds) that makes the olefins useful may also cause them to be toxic in the body. The double bonds of olefins can be oxidized by cytochrome P450 enzymes to epoxides, which are electrophiles that can react with DNA and may cause alterations in the genetic information carried by that macromolecule. Epoxides can be rendered inactive toward DNA by binding to proteins, by hydrolysis to diols through epoxide hydrolase enzymes (EHs), or by forming conjugates with glutathione via glutathione S-transferase (GST) activities. The balance between the oxidizing enzymatic activities and the hydrolyzing or conjugating enzymatic activities in the livers of different species can influence the potential toxicity of the olefins. The location of the enzymes and the potential for concerted reactions in which epoxides are inactivated immediately after formation will also influence the potential toxicity of the olefins. Cytochrome P450 enzymes and EHs are in microsomes located in the rough endoplasmic reticulum surrounding the nucleus where the DNA is located. GST is in the cytoplasm of the cell. In the case of 1,3-butadiene (BD), such enzymatic differences may strongly influence the toxicity in different species. The mouse, in which BD is a potent multi-site carcinogen, has the lowest microsomal EH activity of any species. This allows the monoepoxides formed in the microsomes by cytochrome P450 enzymes to be further oxidized to the highly genotoxic diepoxide (DEB), and both epoxides can either be released into the blood for distribution throughout the body or can react with DNA in the nucleus. The rat, in which BD is a weak carcinogen, has much higher levels of microsomal EH, and only trace amounts of DEB enter the bloodstream. Major BD metabolites in primates suggest that the hydrolysis pathway is even more prominent in primates than in rats. Data suggest that BD will be much less toxic in primates than in mice. Considering these quantitative differences in metabolism may help to reduce the uncertainties in extrapolating animal data on olefin toxicity to health risk assessments for humans exposed to the compounds.

Current status of the cytosolic sulfotransferases in the metabolic activation of promutagens and procarcinogens.

Cytosolic sulfotransferases (SULT) catalyze the sulfation of structurally diverse drugs, endogenous compounds and xenobiotics. These reactions involve the transfer of a sulfuryl group from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to the hydroxyl/amino groups of acceptor molecules. Although sulfate conjugation is generally considered as a detoxication pathway producing more water-soluble and often less toxic metabolites, sulfation of certain classes of compounds produce sufficiently electrophilic metabolites that can covalently bind to cellular macromolecules, DNA and RNA. The important roles of electrophilic sulfate ester metabolites in the metabolic activation, mutagenicity and ultimate carcinogenicity of many xenobiotics have been considerably elucidated. Examples include the class of hydroxymethyl polycyclic aromatic hydrocarbons, allylic alcohols, N-hydroxy derivatives of carcinogenic arylamines and heterocyclic amines. Results obtained by many scientists during the last two decade correlate with a hypothesis that electrophilic sulfate esters may be the major ultimate carcinogenic forms of many, if not most, procarcinogens derived from benzylic/allylic alcohols and hydroxy arylamines. Careful analysis of these results suggest that the activities of human hydroxysteroid sulfotransferase (hHST), and a related form in rat liver, rat hydroxysteroid sulfotransferase a (STa), as well as aryl sulfotransferases both from rat and human liver, account for a substantial portion of the activation of benzylic/allylic alcohols in these species. Moreover, aryl sulfotransferases have also been indicated as the responsible SULT family in the bioactivation of hydroxy arylamines in the liver of different species including human. Molecular cloning of the individual sulfotransferases and expression of these individual forms in heterologous expression systems have allowed us to better understand the role of SULTs in the bioactivation of different procarcinogens and the form of sulfotransferase involved in their bioactivation. Additional structure-activity studies with homogeneous forms of rat liver STa and AST IV have also yielded comparative insight into some of the parameters important in recognition of substrates and inhibitors by these enzymes.

Detection and regulation of the messenger for a putative bovine endometrial 9-keto-prostaglandin E(2) reductase: effect of oxytocin and interferon-tau.

During reproductive processes, prostaglandin (PG) E(2) (PGE(2)) and PGF(2alpha) play important roles in which they often exert opposite effects. At the time of recognition of pregnancy in vivo, PGF(2alpha) is recognized as the luteolytic factor in ruminants and in most species including human, whereas PGE(2) may exert a luteoprotective action. We have previously demonstrated that recombinant interferon-tau (rIFN-tau), the embryonic signal responsible for recognition of pregnancy in ruminants, stimulated in vitro the production of PGE(2) and prostaglandin-endoperoxide synthase 2 (Ptgs2; also called cyclooxygenase-2) gene expression in both epithelial and stromal endometrial cells. Since PGE(2) is the major prostaglandin produced by stromal cells, the effect on Ptgs2 could explain the increase in PGE(2) output. At high concentrations, however, recombinant ovine (ro) IFN-tau acts on epithelial cells by changing the primary PG produced from PGF(2alpha) to PGE(2). This change in the primary PG produced could be explained by a decrease in PGF synthase (PGFS) activity or an increase in PGE synthase activity, or by modulation of a putative PGE(2)-9-ketoreductase, which converts PGE(2) into PGF(2alpha). Therefore, we have investigated the regulation of the mRNAs for PGFS and PGE(2)-9-ketoreductase (9K-PGR), two enzymes that lead to the production of PGF(2alpha). Others have described 9K-PGR activity in uterus, ovaries, kidney, and liver of different species and have established that this enzyme could possess both 9K-PGR and 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) activity. Some have concluded that 9K-PGR and 20alpha-HSD are identical enzymes. Using primers sequences chosen from homologous nucleotide sequences of published rabbit 20alpha-HSD/9K-PGR and rat 20alpha-HSD cDNAs, a 317-base pair (bp) fragment was amplified by reverse transcription-polymerase chain reaction (RT-PCR), cloned, and sequenced. Homologies of 83% and 78% were found with rabbit and rat 20alpha-HSD, respectively. The presence of 20alpha-HSD/9K-PGR and prostaglandin F synthase (PGFS) mRNA expression was studied semiquantitatively in cultured epithelial cells using RT-PCR. Stimulation of cells with roIFN-t resulted in a biphasic response, an inhibition of PGF(2alpha) production at low dose (1 ng/ml) and a stimulation of PGE(2) at high dose (10 microg/ml). The increase of PGE(2) was accompanied by reduced 9K-PGR and PGFS mRNA gene expression. The effect of oxytocin (OT) was also studied, and the presence of OT had no effect on either 9K-PGR or PGFS gene expression. The 20alpha-HSD/9K-PGR transcript was also detected in other bovine tissues at different intensity (liver > kidney > testis > ovaries). We believe that the 9K-PGR and PGFS can be key enzymes in the regulation of specific PGs in the endometrium during the periimplantation period.

Multiresidue method for the determination of avermectin and moxidectin residues in the liver using HPLC with fluorescence detection.

A method using high-performance liquid chromatography (HPLC) and fluorescence detection is presented for the simultaneous determination of the antiparasitic agents avermectins (abamectin, ivermectin and doramectin) and moxidectin in liver. Samples are extracted using acetonitrile and cleaned up using solid phase extraction on a C18 column, followed by derivatization with trifluoroacetic anhydride. The samples are injected without further cleanup into the HPLC column and any avermectins or moxidectin present are detected using fluorescence detector set at 361 nm excitation and 465 nm emission wavelengths. The limits of quantification are below the stipulated EU Maximum Residue Limit for each of the avermectins and moxidectin. Mean recoveries in bovine liver ranged from 77.5 to 90.8%, with a RSD from 2.7 to 7.7%. The procedure provides a rapid, reliable and sensitive method for determinating avermectin and moxidectin residues in liver of different species (cattle, sheep, pigs). It is also applicable to muscle without modification.

Species-dependent enantioselective glucuronidation of carprofen

1. The stereoselective glucuronidation of carprofen, a non-steroidal anti-inflammatory drug, was investigated in vitro using microsomes prepared from liver of different species (rat, dog, horse, sheep and man) or UGT2B1 expressed in fibroblasts. 2. The Km towards the drug was very similar among these species and for the two menantiomers, whereas the Vmax varied substantially according to the animal used. The rat exhibited a high stereoselective glucuronidation whereas other species, including man, presented a low stereoselectivity. The R-enantiomer was glucuronidated at a more efficient rate than its enantiomorph, and was a better substrate (in terms of Vmax/Km). 3. To explain the enantioselective disposition of carprofen in man and in the different species,the ratio of the enzymatic efficacies (Vmax/Km) were compared with the ratio of the max m pharmacokinetic parameters AUCs. The basic hypothesis that the intrinsic clearance reflect the enantioselective behaviour of carprofen seemed substantiated when we focused onmanand rat glucuronidation,but the in vivo-in vitro correlationwas not possibleinother species. 4. In conclusion, the chiral pharmacokinetics of carprofen is less dependent on the stereoselective glucuronidationthanotherstereoselective processes such as protein binding of carprofen, enzymatic hydrolysis, or renal elimination of glucuronides.

Studies on the retention of individual polychlorinated dibenzofurans (PCDFs) in the liver of different species

A mixture of nineteen individual PCDF congeners (of which 17 were analyzed) was given as a single oral dose (0.1 μg of each congener/kg body weight) to rats, guinea pigs and hamsters. Postadministration levels of PCDFs in the liver were determined for days 1, 5, 10, 30 and 90. The analytical technique applied for the determination included clean-up of the liver tissues using basic and acidic silica, alox and Carbopak C columns and HRGC-HRMS analyses. This procedure resulted in a method detection level below 1 ppt (0.1 - 1 ppt depending on sample or congener). The fates of the 17 PCDFs were found to be highly species and congener dependent. In rats and hamsters, almost only 2,3,7,8-substituted congeners were detected in liver at this detection level at either timepoint. In contrast, the guinea pigs retained most of the congeners administrated. Elimination of the retained congeners followed first order kinetics with elimination half-lives of 2 – 85 days.

Purification and some properties of aldehyde reductases from pig liver.

Two aldehyde reductases (EC 1.1.1.2), I and II, have been isolated from pig liver. Both are monomeric (Mr approximately equal to 35 000) and NADPH-dependent. Their activity is inhibited by barbiturates. The enzymes reduce essentially aromatic aldehydes, with a preference for those bearing an electron-withdrawing group in the para position. Substrates with a carboxyl group are specially good substrates for reductase I. This may indicate the presence of a positively charged group in the substrate binding site. The binding of NADPH to reductase I causes a red shift of the coenzyme absorption; this shift is characteristic of B-stereospecific dehydrogenases. Nevertheless, this is not confirmed by the stereochemical study with labelled NADPH. The pro-R hydrogen of NADPH is transferred to the re face of the aldehyde. The stereochemical course of reductase I is identical to that of liver alcohol dehydrogenase, but the two enzymes differ by the absence of Zn and of reactive thiol in reductase I, and by the action of pyrazole on the activity. Considerable differences in substrate specificity and immunological properties have been found between reductase I and II but reductases I from liver of different species have some relationship. Reductase I from pig brain and pig kidney seem to be identical to reductase I from pig liver.

Independent lines of evidence suggesting a major gap junctional protein with a molecular weight of 26,000.

Several polypeptides have been described in the past as components of gap junction fractions. Of these, a peptide of Mr 26,000 is found in gap junctions isolated from livers of different species under conditions that minimize proteolysis. Tryptic digestion of purified "intact" junctions causes the rapid disappearance of this peptide with a concomitant appearance of a band of Mr 10,000, which has previously been found to be characteristic of junctional fractions isolated with the aid of proteolytic treatment. Both the peptides of Mr 26,000 and 10,000 are missing from gap junction preparations after partial hepatectomy, when gap junctions are absent from the surface of the hepatocytes. They have reappeared in fractions from the livers of animals killed 3 days postoperatively, when gap junctions are again present in vivo.

Quantification of taurine synthesis in the intact rat and cat liver.

Data from studies with liver homogenates indicate livers of different species differ in their ability to synthesize taurine from various substrates. Rat liver synthesizes taurine rapidly from cystine sulfinic acid (CSA) (1, 2), whereas cat and human livers synthesize taurine slowly (1, 2) or not at all (3, 4) from CSA. Recently a system utilizing inorganic sulfate for taurine synthesis has been described and it has been suggested that this pathway may be important in species which lack an active CSA pathway (5,6). No data exist concerning rates or pathways of taurine synthesis in the intact liver of any species. We studied the taurine synthetic capacity of isolated perfused rat and cat livers to quantify total taurine synthesis and to estimate the relative importance of taurine synthesis from CSA and from inorganic sulfate.Materials and methods. Male Sprague-Dawley rats, 250-350 g, and young cats, 350-500 g, male and female, were used. In perfusions involving [14C]taurine, 2 μCi of isotope was injected in...

Alanine and aspartate aminotransferase activities in serum and various subcellular fractions from the livers of different species

1. 1. Aspartate aminotransferase activity in serum and liver mitochondria and cytosols of rats, guinea-pigs, chickens and cattle was considerably higher than alanine aminotransferase activity. 2. 2. Chicken liver mitochondrial aspartate aminotransferase activity was 6% of the total activity found in the cytosol and remained like the chicken liver cytosol ASPT activity, unchanged after sonication, and could not be attributed to absorption of any cytosol enzyme. 3. 3. K m values of chicken liver mitochondrial and cytosol aspartate aminotransferases and rat liver aspartate aminotransferase for α-ketoglutarate were 0.22, 0.24 and 0.23 mM respectively. 4. 4. Chicken liver mitochondrial and cytosol aminotransferase activities were destroyed to the same extent when heated to the same temperatures. 5. 5. Conversion of aspartate into blood glucose was observed with fasted rats and guineapigs, but not with fasted chickens.

The acylation of glycerol 3-phosphate in different rat organs and in the liver of different species (including man)

1. 1. The organ distribution of glycerol 3-phosphate acylating enzymes has been investigated in rat. The highest activities (per mg of protein) were found in adipose tissue, adrenal gland and liver. 2. 2. For mitochondria isolated from different rat organs and from the liver of different species, a preference for saturated fatty acids in the acylation of glycerol 3-phosphate was found. Monoacylglycerophosphate (lysophosphatidic acid) was a major mitochondrial reaction product. 3. 3. For microsomes, both saturated and unsaturated long-chain fatty acids were efficient substrates, and diacylglycerophosphate (phosphatidic acid) was the main reaction product.

Translation of rabbit hemoglobin meessenger RNA in vitro with purified and partially purified components from brain or liver of different species.

Two mammalian systems have been developed for efficient in vitro translation of exogenous messenger RNA (rabbit globin mRNA). One system is completely derived from guinea pig brain, and the other is from liver of different species. Both systems consist of purified 60S and 40S ribosomal sununits, unseparated initiation factors partially purified by ammonium sulfate precipitation and DEAE-cellulose chromatography, and pH 5 enzyme fractions as sources of elongation and termination factors, aminoacyl-tRNA synthetases, and transfer RNA. Translation depends completely upon exogenous mRNA and initiation factors. Extraction of initiation factors from microsomes or ribosomes has been improved for these tissues by inclusion of Mg(++) ions in the 0.5 M KCl extraction solution. Both systems synthesize complete rabbit alpha- and beta-globin chains, but in variable ratios. The overall efficiencies of the two systems are about 60% (liver) and 40% (brain) of a comparable system with rabbit reticulocyte initiation factors.

Metabolism of high doses of methylprednisolone by the isolated perfused liver of different species

The metabolism of methylprednisolone has been studied in the isolated perfused liver of normal rabbits, pigs, and dogs as well as in dogs that had recovered from hepatic necrosis. A considerable difference in the rate of degradation was noted among the species, the rate being slowest in the rabbit and most rapid in the pig. Previous damage to canine livers that had subsequently recovered resulted in a very moderate decrease in the rate of steroid breakdown. The rate of breakdown of methylprednisolone in the pig approximates most closely that observed in vivo in human volunteers.

Immunochemical studies on malate dehydrogenase (decarboxylating) (NADP)

1. 1. Malate dehydrogenase (decarboxylating) (NADP) ( l-malate:NADP oxidoreductase (decarboxylating), EC 1.1.1.40) was purified from the supernatant fraction of rat liver. 2. 2. Antibody against this purified enzyme was prepared. This antibody inhibited the activity of the enzyme and precipitated it. 3. 3. Malate dehydrogenase (decarboxylating) (NADP) in supernatant fractions from kidney, heart, brain and adipose tissue of rats reacted with the antibody in the same manner as that of the ra tliver enzyme in the inhibition tests. Supernantant malate dehydrogenases from all these tissues except adipose tissue formed single precipitin bands with the antibody on Ouchterlony double-diffusion analysis which fused with each other. 4. 4. The enzyme from the livers of different species differed immunologically from that of rat liver. 5. 5. Mitochondrial enzyme was immunologically quite distinct from supernatant enzyme. 6. 6. Increase and decrease in enzyme activities in the cytoplasm of liver under various dietary and hormonal conditions were accompanied by proportional changes in the quantity of immunologically reactive protein.

Characterization of ribosomal proteins from different tissues and species of animals by electrophoresis on polyacrylamide gel.

Proteins from ribosomes of different tissues and animals were characterized by polyacrylamide disc electrophoresis. The proteins from ribosomes of different tissues from the same animal are qualitatively similar. The results of the experiments with ribosomes from the livers of different species of animals exhibit clear differences, in the electrophoretic patterns of the proteins.