Kinetics and in vitro origin of the temperature-dependent transition of the estrogen receptor monomer

Partitioning of estrogen receptors in aqueous two-phase polymer systems has provided the basis for a detailed kinetic analysis of the effects of temperature on estrogen receptor (ER) structure in vitro. Exposure to temperatures of 0-30 degrees C increased the rate of change in ER partition coefficients by up to 100-fold but did not affect the final extent of the process. The temperature-dependent change in ER partition coefficients was characterized by a linear Arrhenius plot and an activation energy of 25 kcal/mol. The rate of the temperature-dependent ER transition (28 degrees C) was found to be unaffected by greater than 50-fold changes in receptor concentration, which indicates that the temperature-dependent change in partition coefficients reflects a first-order process. The partition coefficients of heated ER were unaffected by subsequent 18-h incubations at 0 degree C, indicating that the temperature-dependent ER transition is irreversible in vitro. Direct heating of the unoccupied ER resulted in both a change in ER partition coefficients and a loss of ER binding sites. The temperature-dependent change in unoccupied ER partition coefficients was complete within 30 min at 28 degrees C and yielded a first-order rate constant that was the same as that obtained for heating the receptor-estradiol complex at 28 degrees C. In contrast, the loss of unoccupied ER binding sites that occurred during 28 degrees C incubations did not reach completion after 150 min of heating and was found to behave as a second-order process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasma binding of 1-butanol, phenol, nitrobenzene and pentachlorophenol in the rainbow trout and rat: a comparative study

1. The in vitro binding of 1-butanol, phenol, nitrobenzene, and pentachlorophenol in trout plasma and rat plasma was determined. 2. Binding to rainbow trout plasma proteins agreed within 9% of that observed in rat plasma. 3. Percentage bound to rainbow trout (2-99%) or rat (10-99%) plasma proteins increased as the log octanol/water partition coefficient of the chemicals increased within the Log P 1-3 range, and was suggestive of hydrophobic interactions in binding.     

Development of a physiologically based toxicokinetic model for butadiene and four major metabolites in humans: global sensitivity analysis for experimental design issues

1,3-Butadiene (BD) is metabolized in humans and rodents to mutagenic and carcinogenic species. Our previous work has focused on developing a physiologically based toxicokinetic (PBTK) model for BD to estimate its metabolic rate to 1,2-epoxy-3-butene (EB), using exhaled breath BD concentrations in human volunteers exposed by inhalation. In this paper, we extend our BD model to describe the kinetics of its four major metabolites EB, 1,2:3,4-diepoxybutane (DEB), 3-butene-1,2-diol (BDD), and 3,4-epoxy-1,2-butanediol (EBD), and to test whether the extended model and experimental data (to be collected for BD and metabolites in humans) are together adequate to estimate the metabolic rate constants of each of the above chemicals. Global sensitivity analyses (GSA) were conducted to evaluate the relative importance of the model parameters on model outputs during the 20min of exposure and the 40min after exposure ended. All model parameters were studied together with various potentially measurable model outputs: concentrations of BD and EB in exhaled air, concentrations of BD and all metabolites in venous blood, and cumulated amounts of urinary metabolites excreted within 24h. Our results show that pulmonary absorption of BD and subsequent distribution and metabolism in the well-perfused tissues compartment are the critical processes in the toxicokinetics of BD and metabolites. In particular, three parameters influence numerous outputs: the blood:air partition coefficient for BD, the metabolic rate of BD to EB, and the volume of the well-perfused tissues. Other influential parameters include other metabolic rates, some partition coefficients, and parameters driving the gas exchanges (in particular, for BD outputs). GSA shows that the impact of the metabolic rate of BD to EB on the BD concentrations in exhaled air is greatly increased if a few of the model's important parameters (such as the blood:air partition coefficient for BD) are measured experimentally. GSA also shows that all the transformation pathways described in the PBTK model may not be estimable if only data on the studied outputs are collected, and that data on a specific output for a chemical may not inform all the transformations involving that chemical.     

Relationships among carcass weight, acid-base-balance of the blood, meat quality parameters and body composition of cultured rainbow trout (Oncorhynchus mykiss)

The relationshis among carcass weight, acid-base-balance of the blood as an indicator for metabolic load susceptibility, meat quality parameters, and body Composition were investigated in rainbow trout. Examinations were carried out in two replicates using a total of 300 trout from a Danish population. Within replicates, the trout were of aproximately the same age. In Replicate I trout, increasing carcass weight was accompanied by a more arkaline reaction of the blood. Carcass weight was significantly correlated with blood-pH and bicarbonate concentration (.19 and 26). Regression coefficients, however, confirmed only an increase of bicarbonate concentration with increasing carcass weight (b = .013 mmol/L per g, s_b= 0.005). In meat quality parameters, muscular pH₂₄ values decreased linearly in both replicates with increasing carcass weight, as shown by significant correlation estimates (-20 and -.27) and regression coefficients (b = -.0006, s_b=.0002, and b = -.001, s_b= .0003). Relationshis of other meat quality parameters to inmeasing carcass weight were evaluated differently by correlation and regression coefficients. No relationship was found between carcass weight and body composition. In general, correlation estimates describing interrelations between acid-base and meat quality parameters were on a very low level. Independent from carcass weight, there were clearly metabolically loaded and unloaded trout as well as trout with a mixed acid-base-status. Acid-base parameters were significantly correlated with each other. Generally, estimates for correlations between meat quality parameters were on a very low level. Body composition arameters, dry weight and crude fat content were highly significantly correlated (0.47) in Replicate II; in Replicate I there was no interrelation.     

Reappraisal of H2S/sulfide concentration in vertebrate blood and its potential significance in ischemic preconditioning and vascular signaling

Hydrogen sulfide (H(2)S) is rapidly emerging as a biologically significant signaling molecule. Studies published before 2000 report low or undetectable H(2)S (usually as total sulfide) levels in blood or plasma, whereas recent work has reported sulfide concentrations between 10 and 300 microM, suggesting it acts as a circulating signal. In the first series of experiments, we used a recently developed polarographic sensor to measure the baseline level of endogenous H(2)S gas and turnover of exogenous H(2)S gas in real time in blood from numerous animals, including lamprey, trout, mouse, rat, pig, and cow. We found that, contrary to recent reports, H(2)S gas was essentially undetectable (<100 nM total sulfide) in all animals. Furthermore, exogenous sulfide was rapidly removed from blood, plasma, or 5% bovine serum albumin in vitro and from intact trout in vivo. To determine if blood H(2)S could transiently increase, we measured oxygen-dependent H(2)S production by trout hearts in vitro and in vivo. H(2)S has been shown to mediate ischemic preconditioning (IPC) in mammals. IPC is present in trout and, unlike mammals, the trout myocardium obtains its oxygen from relatively hypoxic systemic venous blood. In vitro, myocardial H(2)S production was inversely related to Po(2), whereas we failed to detect H(2)S in ventral aortic blood from either normoxic or hypoxic fish in vivo. These results provide an autocrine or paracrine mechanism for myocardial coupling of hypoxia to H(2)S in IPC, i.e., oxygen sensing, but they fail to provide any evidence that H(2)S signaling is mediated by the circulation.     

Inhibition of hemolytic activity of Aeromonas salmonicida GCAT in rainbow trout red blood cells by a monoclonal antibody

The present study shows that a monoclonal antibody (MAb) directed to Aeromonas salmonicida glycerophospholipid:cholesterol acyltransferase (GCAT) is capable of protecting rainbow trout red blood cells from the cytotoxic effects of the enzyme in vitro.     

Dynamic quenchers in fluorescently labeled membranes. Theory for quenching in a three-phase system.

The theory for quenching of fluorescently labeled membranes by dynamic quenchers is described for a three-phase system: a fluorescently labeled membrane, a nonlabeled membrane, and an aqueous phase. Two different experimental protocols are possible to determine quenching parameters. Using the first protocol, partition coefficients and bimolecular quenching constants were determined for a hydrophobic quencher in carbazole-labeled membranes in the presence of an unlabeled reference membrane. These parameters determined for 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) using this three-phase analysis were in good agreement with values determined by a two-phase analysis without the reference lipid. Hence, the theory was verified. In the second protocol, the quencher partition coefficient was determined for unlabeled membranes in the presence of a carbazole-labeled reference membrane. Partition coefficients for DDE determined by this method were the same as partition coefficients determined for carbazole-labeled membranes using the two-phase analysis. The greater ease in determining partition coefficients and bimolecular quenching constants by the three-phase analysis and, in particular, the ability to determine the partition coefficient in unlabeled membranes make the three-phase analysis especially useful. This method was used to study the effect varying the membrane lipid composition has on the partition coefficient. The data indicate that partition coefficients of DDE in fluid membranes are not dramatically dependent upon polar head group composition, fatty acid composition, or cholesterol content. However, partitioning into gel-phase lipids is at least 100-fold less than fluid-phase lipids.     

The effects of heat shock and acclimation temperature on hsp70 and hsp30 mRNA expression in rainbow trout: in vivo and in vitro comparisons

Heat shock (25° C) of 10° C-acclimated rainbow trout Oncorhynchus mykiss led to increases in heat shock protein 70 (hsp70) mRNA in blood, brain, heart, liver, red and white muscle, with levels in blood being amongst the highest. Hsp30 mRNA also increased with heat shock in all tissues with the exception of blood. When rainbow trout blood was heat shocked in vitro , both hsp70 and hsp30 mRNA increased significantly. In addition, these in vitro experiments demonstrated that blood from fish acclimated to 17° C water had a lower hsp70 mRNA heat shock induction temperature than did 5° C acclimated fish (20 v. 25° C). The hsp30 mRNA induction temperature (25° C), however, was unaffected by thermal acclimation. While increases in hsp70 mRNA levels in blood may serve as an early indicator of temperature stress in fish, tissue type, thermal history and the particular family of hsp must be considered when evaluating stress by these molecular means.     

The distribution of serum albumin in human normal and degenerate articular cartilage.

A method for studying the distribution of a high molecular weight solute (serum albumin) between physiological saline and human articular cartilage is described. Samples of normal and fibrillated articular cartilage from both femoral condyles and femoral heads have been studied. Limited studies have also been performed where the glycosaminoglycan content of normal cartilage has been reduced by chemical or enzymic methods. With naturally occurring cartilage a wide range of partition coefficients (0.3 to less than 0.002) was obtained. The partition coefficients are very dependent upon proteoglycan concentration, with the partition coefficient decreasing with increasing fixed charge density. An attempt is made to interpret the observed partitioning in terms of the steric exclusion by the proteoglycans.     

Subfractionation of cell populations by partitioning in dextran-poly (ethylene glycol) aqueous phases

Partitioning of cells in dextran-poly(ethylene glycol) aqueous two-phase systems depends on the interaction between the surface properties of the cells and the physical properties of the phases. The latter can be manipulated to a considerable extent by selection of polymer concentrations and ionic composition and concentration. If salts (e.g., phopshate) are used that have an unequal affinity for the two phases, an electrostatic potential difference between the phases results and, at appropriately high polymer concentrations, the partition coefficient of cells is determined predominantly by membrane charge-associated properties. By “balancing” the magnitude of the electrostatic potential difference against that of the interfacial tension (primarily a function of polymer, but also phosphate, concentrations) one can obtain phase systems that give usable partition coefficients for most cell populations (1). In work under way in our laboratory on the effects of different chemical and enzymatic modifications on the relative surface properties of rat red blood cells of different ages, we have now found that certain phase compositions did not resolve such treated cell subpopulations while other phase compositions did. Thus not all charged phase systems in which cell populations as a whole have usable partition coefficients are equally capable of detecting or subfractionating cell subpopulations. It is therefore essential, before drawing conclusions on the nonseqarability of cell subpopulations, to test cell separability in charged phase systems of different compositions if the system initially chosen does not afford a subfractionation.     

PKQuest: volatile solutes - application to enflurane,nitrous oxide,halothane, methoxyflurane and toluene pharmacokinetics

BACKGROUND: The application of physiologically based pharmacokinetic models (PBPK) to human studies has been limited by the lack of the detailed organ information that is required for this analysis. PKQuest is a new generic PBPK that is designed to avoid this problem by using a set of "standard human" default parameters that are applicable to most solutes. RESULTS: PKQuest is used to model the human pharmacokinetics of the volatile solutes. A "standard human" value for the lipid content of the blood and each organ (klip) was chosen. This set of klip and the oil/water partition coefficient then specifies the organ/blood partition for each organ. Using this approach, the pharmacokinetics of inert volatile solute is completely specified by just 2 parameters: the water/air and oil/water partition coefficients. The model predictions of PKQuest were in good agreement with the experimental data for the inert solutes enflurane and nitrous oxide and the metabolized solutes halothane and toluene. METHODS: The experimental data that was modeled was taken from previous publications. CONCLUSIONS: This approach greatly increases the predictive power of the PBPK. For inert volatile solutes the pharmacokinetics are determined just from the water/air and oil/water partition coefficient. Methoxyflurane cannot be modeled by this PBPK because the arterial and end tidal partial pressures are not equal (as assumed in the PBPK). This inequality results from the "washin-washout" artifact in the large airways that is established for solutes with large water/air partition coefficients.PKQuest and the worked examples are available on the web www.pkquest.com.     

Effect of lipophilic character on the biological activity of synthetic angiotensin peptides.

[Ile5]angiotensin II (angiotensin) derivatives bearing acetyl, propionyl, butyryl, pentanoyl or hexanoyl moieties at the N-terminal amino group were synthesized. The myotropic effects in vitro (on guinea-pig ileum and rat uterus) of desamino-angiotensin and of the above compounds did not correlate with their partition coefficients in butan-1-ol/acetic acid/water. The pressor effects in vivo in rats showed a negative correlation with the partition coefficients, discouraging further attempts to raise the pressor potency of angiotensin analogues by increasing their hydrophobicity. The half-times for onset and reversal of the biological responses also did not correlate with partition coefficients, but reversal was retarded by the presence of a free amino group. It is concluded that partition between aqueous medium and the lipophilic receptor environment is not a limiting factor for angiotensin activity.     

Rainbow trout (Oncorhynchus mykiss) brain cells biosynthesize novel docosahexaenoic acid-derived resolvins and protectins-Mediator lipidomic analysis

Docosahexaenoic acid (DHA; C22:6 n-3) is an abundant fatty acid in fish phospholipids. In the present study, we employed liquid chromatography-ultraviolet spectrometry-tandem mass spectrometry and dissociated rainbow trout (Oncorhynchus mykiss) brain cells to determine whether fish utilize endogenous DHA to produce the recently uncovered novel lipid mediators termed the resolvins and protectins, generated by mammalian cells [Serhan CN, Hong S, Gronert K, et al. Resolvins: a family of bioactive products of omega-3 fatty acid transformation circuits initiated by aspirin treatment that counter proinflammation signals. J Exp Med 2002; 196:1025-37; Hong S, Gronert K, Devchand P, Moussignac R-L, Serhan, CN. Novel docosatrienes and 17S-resolvins generated from docosahexaenoic acid in murine brain, human blood, and glial cells. J Biol Chem 2003;278:14677-87]. Trout brain cells biosynthesize a range of recently identified di- and tri-hydroxy-containing bioactive products from endogenous sources of DHA when challenged in vitro. We identified neuroprotectin D1, resolvin D5, resolvin D1 and resolvin D2 from trout brain cells. Each compound was identified on the basis of its characteristic physical chemical properties that included MS, MS-MS, UV spectra and chromatographic behavior. The monohydroxy products from DHA, signatures of DHA conversion by lipoxygenases, were also identified. These included both 14S-hydroxy-docosahexaenoic acid and 17S-hydroxy-docosahexaenoic acid. The biosynthesis of these novel bioactive lipid mediators, namely resolvins and protectins, by fish cells provides the first evidence for the conservation of these structures from fish to humans as chemical signals in diverse biological systems.     

The Transport and Affinity of Substituted Benzenes in Soybean Stems

The sorption of non-ionized substituted benzenes in the xylemtissue of excised soybean stems was studied. A positive pressureperfusion technique was used to force solutions containing chemicalsand tritiated water through 50-mm stem segments. The stem effluentwas collected at timed intervals and analysed for each chemicaland tritium activity. A theoretical mass transport model wasdeveloped and the experimental data were analysed to calculatethe flux of water, chemical sorption, and first order rate constants.Sorption of hydrophobic chemicals in the xylem tissue appearedto be the dominant interaction responsible for impeding solutemovement. Linear relationships between sorption and accumulationof the chemicals in the xylem tissue, and the chemical octanol/waterpartition coefficients were demonstrated. The mathematical derivationof the mass transport model is described. Key words: Mass transport, adsorption, partition coefficients     

Evaluation of the lipophilic properties of opioids, amphetamine-like drugs, and metabolites through electrochemical studies at the interface between two immiscible solutions

For the first time, the partition coefficients of the ionized forms of several opioids, amphetamine-like drugs, and their metabolites were determined by studying their ionic transfer process across the bare interface water/organic solvent. The ionic partition coefficients of the monocationic forms of 12 compounds--heroin, 6-monoacetylmorphine (6-MAM), morphine, acetylcodeine, codeine, dihydrocodeine, methamphetamine, amphetamine, 3,4-methylenedioxymethamphetamine (MDMA or "ecstasy"), 3,4-methylenedioxyamphetamine (MDA), 3-methoxy-alpha-methyldopamine (3-OMe-alpha-MeDA), and alpha-methyldopamine (alpha-MeDA)-were attained using electrochemical measurements, by cyclic voltammetry, at the interface between two immiscible electrolyte solutions (ITIES). Then the acquired lipophilicity values were correlated to the chemical structure of the compounds and with the metabolic pathways central to each class of drugs. Although the mechanisms of biotoxicity of this type of drugs are still unclear, the data obtained evidence that the lipophilicity of metabolites may be a contributing factor for the qualitative differences found in their activity. In addition, the partition coefficients of the ionic drugs were calculated using three available software packages: ModesLab, Dragon, and HyperChem. As shown by cross-comparison of the experimental and calculated values, HyperChem was the most reliable software for achieving the main goal. The data obtained so far seem to be correlated to the proposed metabolic pathways of the drugs and could be of great value in understanding their pharmacological and/or toxicological profiles at the molecular level. This study may also contribute to gaining an insight into the mechanisms of biotransportation of this type of compounds given that the ionic partition coefficients reflect their ability to cross the membrane barriers.     

A monoclonal antibody recognising a surface marker on rainbow trout (Oncorhynchus mykiss) monocytes

The characterisation of a monoclonal antibody (mab 45) reacting with phagocytic leucocytes isolated from blood and spleen of rainbow trout (Oncorhynchus mykiss, Walbaum) is described. The surface marker labelled by this mab is expressed at relative low levels on the membrane of large, nearly nongranulated trout leucocytes, and having the typical morphology of monocytes in flow cytometry (Kfoury et al., 1999, Fish Pathology, 34, 1-6). No reaction of mab 45 with granulocytes, lymphocytes or thrombocytes was detected. In spleen and head kidney, large, polymorphonuclear leucocytes were immunostained. The mab most strongly recognised an antigen of 48 kDa prepared from trout leucocytes of different organs, but not in trout plasma. In an in vitro phagocytosis assay trout monocytes were stained with mab 45 after phagocytosis of Aeromonas salmonicida labelled with the lipophilic fluorescent cell surface linker PKH26. However, previous binding of mab 45 on trout leucocytes did not inhibit the phagocytosis of A. salmonicida particles. Using mab 45, the dynamics of monocytes in blood, spleen and peritoneal cavity could be demonstrated after intraperitoneal injection of trout with inactivated A. salmonicida. The described mab serves as a useful tool to investigate the involvement of monocytes/macrophages in immune reactions of trout to a variety of pathogens.     

Conformational and electrostatic properties of unoccupied and liganded estrogen receptors determined by aqueous two-phase partitioning

The technique of aqueous two-phase partitioning (ATPP) has been used to characterize conformational and electrostatic properties of unoccupied and liganded rat uterine estrogen receptors. The adaptation of the hydroxylapatite receptor assay with ATPP systems has permitted estrogen receptor (ER) partition coefficients to be accurately determined, even when the partitioning process results in significant loss of ER binding capacity. The pH and salt dependences of estrogen receptor partition coefficients indicate that the theory governing partitioning behavior can be accurately applied to partitioning data obtained with crude cytosols. This technique has revealed a ligand-induced change in the properties of the unoccupied receptor that precedes the process of heat-induced transformation in vitro. The difference in partitioning behavior between unoccupied and nontransformed estrogen receptor is observed in all combinations of buffers and salts tested and is of equal magnitude as the difference between partition coefficients of nontransformed and transformed ER. The partition coefficients of both unoccupied and nontransformed ER are constant over the ER concentration range in which binding cooperativity has been previously demonstrated. The combined effects of salt and pH on ER partition coefficients indicate a pI of approximately 5.5 for both unoccupied and nontransformed estrogen receptors. However, the partition coefficients at the pI differ. It is concluded that estradiol binding to its unoccupied receptor results in a change in surface properties of the ER monomer that is independent of receptor transformation and makes the receptor less hydrophobic.     

The distribution of serum albumin in human normal and degenerate articular cartilage

A method for studying the distribution of a high molecular weight solute (serum albumin) between physiological saline and human articular cartilage is described. Samples of normal and fibrillated articular cartilage from both femoral condyles and femoral heads have been studied. Limited studies have also been performed where the glycosaminoglycan content of normal cartilage has been reduced by chemical or enzymatic methods. With naturally occuring cartilage large a wide range of partition coefficients (0.3 to less than 0.002) was obtained. The partition coefficients are very dependent upon proteoglycan concentration, with the partitiion coefficient decreasing with increasing fixed charge density. An attempt is made to interpret the observed partitioning in terms of the steric exclusion by the proteoglycans.     

Proteomic analysis of the lake trout (Salvelinus namaycush) heart and blood: The beginning of a comprehensive lake trout protein database

Lake trout (Salvelinus namaycush) are a top-predator species in the Laurentian Great Lakes that are often used as bioindicators of chemical stressors in the ecosystem. Although many studies are done using these fish to determine concentrations of stressors like legacy persistent, bioaccumulative and toxic chemicals, there are currently no proteomic studies on the biological effects these stressors have on the ecosystem. This lack of proteomic studies on Great Lakes lake trout is because there is currently no complete, comprehensive protein database for this species. Here, we employed proteomics approaches to develop a lake trout protein database that could aid in future research on this fish, in particular exposomics and adductomics. The current study utilized heart tissue and blood from two lake trout. Our previous work using lake trout liver revealed 4194 potential protein hits in the NCBI databases and 3811 potential protein hits in the UniProtKB databases. In the current study, using the NCBI databases we identified 838 proteins for the heart and 580 proteins for the blood tissues in the biological replicate 1 (BR1) and 1180 potential protein hits for the heart and 561 potential protein hits for the blood in BR2. Similar results were obtained using the UniProtKB databases. This study builds on our previous work by continuing to build the first comprehensive lake trout protein database and provides insight into protein homology through evolutionary relationships. This data is available via the PRIDE partner repository with the dataset identifier PXD023970.