Differential modulation of hepatic cytochrome P-450 enzymes in rat and syrian hamster by 4′-trifluoromethyl-2,3,4,5-tetrachlorobiphenyl

The effects of a single injection (40 mg/kg) of 4′-trifluoromethyl-2,3,4,5-tetrachlorobiphenyl (CF3) on hepatic cytochrome P-450 monooxygenases were assessed in rat and syrian hamster. The CF3 treatment significantly increased the total amount of cytochrome P-450 in both species. In rats, CF3 treatment caused marked increases in ethoxyresorufin O-deethylase (EROD), arylhydrocarbon hydroxylase (AHH), and testosterone 7α-hydroxylase activities but significantly reduced the activities of benzphetamine N-demethylase (BzND), erythromycin N-demethylase (ErND), testosterone 6β, 16α, and 16β-hydroxylases, and formation of androstenedione. Administration of CF3 to hamsters strongly induced the activities of EROD, AHH, BzND, testosterone 15α, and 16α-hydroxylases, and androstenedione production, whereas ErND, testosterone 6β, and 7α-hydroxylases were decreased. Administration of CF3 to rats induced the CYP1A family proteins and CYP2A1, while CF3 reduced the level of CYP2B1, and, to a lesser extent, of CYP6β2. In hamsters, CF3 treatment significantly induced the CYP1A2, CYP2A1, CYP2A8, and CYP2B1 isozymes, whereas the CYP6β2 level was decreased. The ability of hepatic microsomes to activate aflatoxin B1 and benzo(a)pyrene was elevated by CF3 treatment in hamsters, while activation of aflatoxin B1 was decreased in microsomes from CF3-treated rats. These results showed differences in the CF3-induced pattern of rat and hamster cytochrome P-450 monooxygenases.     

Fenfluramine-induced pulmonary vasoconstriction: role of serotonin receptors and potassium channels.

The anorexic agent fenfluramine considerably increases the risk of primary pulmonary hypertension. The mechanism of this effect is unknown. The appetite-reducing action of fenfluramine is mediated by its interaction with the metabolism of serotonin [5-hydroxytryptamine (5-HT)] in the brain. We tested the hypothesis that the pulmonary vasoconstrictive action of fenfluramine is at least in part mediated by 5-HT receptor activation. In addition, we sought to determine whether pharmacological reduction of voltage-gated potassium (K(V)) channel activity would potentiate the pulmonary vascular reactivity to fenfluramine. Using isolated rat lungs perfused with Krebs-albumin solution, we compared the inhibitory effect of ritanserin, an antagonist of 5-HT(2) receptors, on fenfluramine- and 5-HT-induced vasoconstriction. Both 5-HT (10(-5) mol/l) and fenfluramine (5 x 10(-4) mol/l) caused significant increases in perfusion pressure. Ritanserin at a dose (10(-7) mol/l) sufficient to inhibit >80% of the response to 5-HT reduced the response to fenfluramine by approximately 50%. A higher ritanserin dose (10(-5) mol/l) completely abolished the responses to 5-HT but had no more inhibitory effect on the responses to fenfluramine. A pharmacological blockade of K(V) channels by 4-aminopyridine (3 x 10(-3) mol/l) markedly potentiated the pulmonary vasoconstrictor response to fenfluramine but was without effect on the reactivity to 5-HT. These data indicate that the pulmonary vasoconstrictor response to fenfluramine is partly mediated by 5-HT receptors. Furthermore, the pulmonary vasoconstrictor potency of fenfluramine is elevated when the K(V)-channel activity is low. This finding suggests that preexisting K(V)-channel insufficiency may predispose some patients to the development of pulmonary hypertension during fenfluramine treatment.     

Thermodynamic studies of the protein-protein interactions between cytochrome P-450 and cytochrome b5. Evidence for a central role of the cytochrome P-450 spin state in the coupling of substrate and cytochrome b5 binding to the terminal hemoprotein

The interactions between purified rat hepatic microsomal cytochrome P-450 and the type I ligands benzphetamine and cytochrome b5 have been studied in the presence of phospholipid using difference spectrophotometry. Cytochrome b5 was shown to interact with cytochrome P-450 to form a tight 1:1 complex (Kd = 275 nM), in which the proportion of high spin cytochrome P-450 was increased from 7 to 30%. The presence of saturating cytochrome b5 was shown to cause a decrease in the apparent Kd for benzphetamine binding from 111 microM to 40 microM. Likewise, the presence of benzphetamine was shown to cause a decrease in the apparent dissociation constant for cytochrome b5 binding to cytochrome P-450 (Kd = 90 nM). The above interactions were resolved into the basic equilibria inter-relating the various ligation states of the hemoprotein in an energetically closed eight-state free energy coupling model and the relative magnitudes of the microequilibria were analyzed to determine the degree of coupling of the interactions between cytochrome P-450 and both benzphetamine and cytochrome b5. Consequently, the spin state changes in cytochrome P-450 induced by benzphetamine and cytochrome b5 binding were shown to arise because these ligands interact 7 and 4 times more tightly with high spin cytochrome P-450, respectively. Furthermore, the data revealed that these ligands interact at independent sites on cytochrome P-450. Thus the effects of cytochrome b5 upon benzphetamine binding and vice versa were rationalized simply in terms of an increase in the proportion of a high spin (high affinity) conformation of cytochrome P-450 brought about by pre-equilibration with the effector ligand, with the intrinsic binding affinities of the two ligands for the low or high spin states remaining relatively unaltered. The thermodynamic parameters associated with the interactions between cytochrome P-450 and cytochrome b5, determined from the temperature dependence of these interactions, revealed that these protein interactions are entropy driven and probably occur by a hydrophobic mechanism.     

Cometabolism of chlorinated solvents by nitrifying bacteria: kinetics, substrate interactions, toxicity effects, and bacterial response

Pure cultures of ammonia-oxidizing bacteria, Nitrosomonas europaea, were exposed to trichloroethylene (TCE), 1,1-dichloroethylene (1,1-DCE), chloroform (CF), 1,2-dichloroethane (1,2-DCA), or carbon tetrachloride (CT), in the presence of ammonia, in a quasi-steady-state bioreactor. Estimates of enzyme kinetics constants, solvent inactivation constants, and culture recovery constants were obtained by simultaneously fitting three model curves to experimental data using nonlinear optimization techniques and an enzyme kinetics model, referred to as the inhibition, inactivation, and recovery (IIR) model, that accounts for inhibition of ammonia oxidation by the solvent, enzyme inactivation by solvent product toxicity, and respondent synthesis of new enzyme (recovery). Results showed relative enzyme affinities for ammonia monooxygenase (AMO) of 1,1-DCE approximately TCE > CT > NH(3) > CF > 1,2-DCA. Relative maximum specific substrate transformation rates were NH(3) > 1,2-DCA > CF > TCE approximately 1,1-DCE > CT (=0). The TCE, CF, and 1,1-DCE inactivated the cells, with 1,1-DCE being about three times more potent than TCE or CF. Under the conditions of these experiments, inactivating injuries caused by TCE and 1,1-DCE appeared limited primarily to the AMO enzyme, but injuries caused by CF appeared to be more generalized. The CT was not oxidized by N. europaea while 1,2-DCA was oxidized quite readily and showed no inactivation effects. Recovery capabilities were demonstrated with all solvents except CF. A method for estimating protein yield, the relationship between the transformation capacity model and the IIR model, and a condition necessary for sustainable cometabolic treatment of inactivating substrates are presented. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 520-534, 1997.     

Src signaling links mediators of inflammation to Cx43 gap junction channels in primary and transformed CFTR-expressing airway cells

Dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) is associated with recurrent pulmonary infections and inflammation. We previously reported that tumor necrosis factor (TNF)-alpha decreases gap junction connectivity in cell lines derived from the airway epithelium of non-cystic fibrosis (non-CF) subjects, a mechanism that was defective in cells derived from CF patients, and identified the tyrosine kinase c-Src as a possible bridge between TNF-alpha and Cx43. To examine whether this modulation also takes place in primary epithelial cells, the functional expression of Cx43 was studied in non-CF and CF airway cells, obtained from surgical polypectomies and turbinectomies, which were grown either on culture dishes or permeable filters. Expression of Cx43 was detected by immunofluorescence on cells grown under both culture conditions. Non-CF and CF airway cells also showed intercellular diffusion of Lucifer Yellow. Dye coupling was rapidly abolished in non-CF cells in the presence of TNF-alpha, lipopolysaccharide and lysophosphatidic acid, and could be prevented by tyrphostin47, an inhibitor of Src tyrosine kinases. This down-regulation, however, was not detected in CF airway cells. These data indicate that CFTR dysfunction is associated with altered Src signaling, resulting in the persistence of gap junction connectivity in primary and transformed CF airway cells.     

Src Signaling Links Mediators of Inflammation to Cx43 Gap Junction Channels in Primary and Transformed CFTR-Expressing Airway Cells

Dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) is associated with recurrent pulmonary infections and inflammation. We previously reported that tumor necrosis factor (TNF)-α decreases gap junction connectivity in cell lines derived from the airway epithelium of non-cystic fibrosis (non-CF) subjects, a mechanism that was defective in cells derived from CF patients, and identified the tyrosine kinase c-Src as a possible bridge between TNF-α and Cx43. To examine whether this modulation also takes place in primary epithelial cells, the functional expression of Cx43 was studied in non-CF and CF airway cells, obtained from surgical polypectomies and turbinectomies, which were grown either on culture dishes or permeable filters. Expression of Cx43 was detected by immunofluorescence on cells grown under both culture conditions. Non-CF and CF airway cells also showed intercellular diffusion of Lucifer Yellow. Dye coupling was rapidly abolished in non-CF cells in the presence of TNF-α, lipopolysaccharide and lysophosphatidic acid, and could be prevented by tyrphostin47, an inhibitor of Src tyrosine kinases. This down-regulation, however, was not detected in CF airway cells. These data indicate that CFTR dysfunction is associated with altered Src signaling, resulting in the persistence of gap junction connectivity in primary and transformed CF airway cells.     

Isolation and characterization of alkane-utilizing Nocardioides sp. strain CF8

A butane-utilizing bacterial strain CF8 was isolated and identified as a member of the genus Nocardioides from chemotaxonomic and 16S rDNA sequence analysis. Strain CF8 grew on alkanes ranging from C(2) to C(16) in addition to butane and various other substrates including primary alcohols, carboxylic acids, and phenol. Butane degradation by strain CF8 was inactivated by light, a specific inactivator of copper-containing monooxygenases. The unique thermal aggregation phenomenon of acetylene-binding polypeptides was also observed for strain CF8. These results suggest that butane monooxygenase in strain CF8 is a third example of the copper-containing monooxygenases previously described in ammonia oxidizers and methanotrophs.     

Aerobic Cometabolism of Chloroform and 1,1,1-Trichloroethane by Butane-Grown Microorganisms

Aerobic cometabolism of chloroform (CF) and 1,1,1-trichloroethane (1,1,1-TCA) was observed by subsurface microorganisms grown on butane. Studies performed in batch incubated microcosms were screened for CF transformation potential using the following cometabolic substrates: ammonia, methane, propane, butane, propene, octane, isoprene, and phenol. CF transformation was observed in microcosms fed ammonia, methane, propane, and butane. The butane microcosms achieved the most effective transformation. The transformation of CF and 1,1,1-TCA was strongly correlated with butane utilization and oxygen consumption. CF transformation ceased in the absence of butane or when oxygen was depleted to low concentrations in the microcosms. No transformation of carbon tetrachloride was observed. With successive additions of CF and butane to the microcosms, complete transformation of CF was achieved at solution concentrations as high as 1 mg/L. High CF concentrations appeared to inhibit butane utilization. Maximum transformation yield (T_y) of 0.01 mg CF trans-formed/mg of butane consumed, were achieved. The results indicate that a monooxygenase enzyme required for butane utilization is likely responsible for the transformation of CF. Chloride measurements demonstrated that CF was completely dechlorinated. Approximately 70% of the chloride in the transformed 1,1,1 -TCA was released into solution, indicating incomplete dechlorination of 1,1,1-TCA. The results indicate that butane is a promising cometabolic substrate for the transformation of chlorinated methanes, chlorinated ethanes, and potentially chlorinated ethenes.     

Expression and characterization of a rabbit liver cytochrome P450 belonging to P450IIB subfamily with the aid of the baculovirus expression vector system

Using baculovirus a cDNA for cytochrome P450 (P450F1) belonging to rabbit P450IIB subfamily was expressed in Spodoptera frugiperda cells, where P450F1 was located on electron-dense structures (derived from the endoplasmic reticulum) present in both the cytoplasm and nucleus. Partially purified P450F1 exhibited absorption spectra similar to those of P450(1), the major phenobarbital-inducible form in rabbit liver. Like P450(1), P450F1 could oxidize aminopyrine, benzphetamine, 7-ethoxycoumarin, 1-nitropropane, and 2-nitropropane, though its activities toward benzphetamine and 7-ethoxycoumarin were about 50 and 5%, respectively, of those of P450(1). It is concluded that the members of P450IIB subfamily can act on a variety of xenobiotics, although substrate preferences are different among them.     

Molecular Modeling of Cytochrome P450 2B1: Mode of Membrane Insertion and Substrate Specificity

A molecular model of a mammalian membrane-bound cytochrome P450, rat P450 2B1, was constructed in order to elucidate its mode of attachment to the endoplasmic reticulum and the structural basis of substrate specificity. The model was primarily derived from the structure of P450BM-3, which as a class II P450 is the most functionally similar P450 of known structure. However, model development was also guided by the conserved core regions of P450cam and P450terp. To optimally align the P450 2B1 and P450BM-3 sequences, multiple alignment was performed using sequences of five P450s in the II family, followed by minor adjustments on the basis of secondary structure predictions. The resulting P450 2B1 homology model structure was refined by molecular dynamics heating, equilibration, simulation, and energy minimization. The model suggests that the F–G loop serves as both a hydrophobic membrane anchor and entrance channel for hydrophobic substrates from the membrane to the P450 active site. To assess the mode of substrate binding, benzphetamine, testosterone, and benzo[a]pyrene were docked into the active site. The hydrophobic substrate-binding pocket is consistent with the preferences of this P450 toward hydrophobic substrates, while the presence of an acidic Glu-105 in this pocket is consistent with the preference of this P450 for the cationic substrate benzphetamine. This model is thus consistent with several known experimental properties of this P450, such as membrane attachment and substrate selectivity.     

Differential incorporation of 3H-thymidine into DNA in cultured skin fibroblasts derived from patients with cystic fibrosis and controls.

Tritiated thymidine (³H-TdR) incorporation into DNA of the fibroblasts derived from subjects with cystic fibrosis (CF) and their controls was studied with scintillation counting and autoradiography. ³H-TdR incorporation at 24 hours postseeding was significantly less (p<0.005) in CF strains in comparison with cells from controls. The percentage of labeled fibroblasts was not significantly different between the two strains (p>0.1). The cell cycle time and the duration of each phase were studied by a mitotic selection and scintillation counting technique. There was no difference in cell cycle time between CF and control fibroblasts, however, the duration of the synthetic phase was significantly (p<0.005) longer in CF subjects.     

Action of 5-trifluoromethyl-2'-deoxyuridine on DNA synthesis.

The action of 5-trifluoromethyl-2'-deoxyuridine (CF3dUrd) on DNA synthesis was investigated in vitro assay systems with purified DNA polymerases. CF3dUrd was incorporated into the DNA of mammalian cells in culture. We studied the incorporation of CF3dUrd 5'-triphosphate (CF3dUTP) into DNA and effect of CF3dUrd residue on DNA synthesis. Therefore, we synthesized oligonucleotides that allow site specific introduction of a CF3dUrd residue into a synthetic DNA oligonucleotide. After CF3dUTP incorporation, the primer was extended for human DNA polymerase alpha (pol. alpha). When CF3dUrd residue was located at an internucleotide site in the template, however, pol. alpha was exhibited a strong arrest band one nucleotide after the CF3dUrd residue site, and Escherichia coli polymerase I (Klenow fragment) also exhibited a weaker arrest band one nucleotide before the CF3dUrd residue. These results suggested that a mechanism of antitumor activity of CF3dUrd is inhibition of DNA replication.     

Restricted passage of reaction intermediates through the ammonia tunnel of carbamoyl phosphate synthetase

The x-ray crystal structure of the heterodimeric carbamoyl phosphate synthetase from Escherichia coli has identified an intermolecular tunnel that connects the glutamine binding site within the small amidotransferase subunit to the two phosphorylation sites within the large synthetase subunit. The tunneling of the ammonia intermediate through the interior of the protein has been proposed as a mechanism for the delivery of the ammonia from the small subunit to the large subunit. A series of mutants created within the ammonia tunnel were prepared by the placement of a constriction via site-directed mutagenesis. The degree of constriction within the ammonia tunnel of these enzymes was found to correlate to the extent of the uncoupling of the partial reactions, the diminution of carbamoyl phosphate formation, and the percentage of the internally derived ammonia that is channeled through the ammonia tunnel. NMR spectroscopy and a radiolabeled probe were used to detect and identify the enzymatic synthesis of N-amino carbamoyl phosphate and N-hydroxy carbamoyl phosphate from hydroxylamine and hydrazine. The kinetic results indicate that hydroxylamine, derived from the hydrolysis of gamma-glutamyl hydroxamate, is channeled through the ammonia tunnel to the large subunit. Discrimination between the passage of ammonia and hydroxylamine was observed among some of these tunnel-impaired enzymes. The overall results provide biochemical evidence for the tunneling of ammonia within the native carbamoyl phosphate synthetase.     

Stereoselective determination of fenfluramine enantiomers in rat liver microsomal incubates

An enantioselective assay for l- and d-fenfluramine in rat liver microsomal incubates was developed. The method involves extraction of fenfluramine from the microsomal incubates, and formation of fenfluramine diastereomeric derivatives with the chiral reagent S-(−)-N-trifluoroacetyl prolyl chloride. Separation and quantitation of the diastereomeric fenfluramine derivatives are carried out by a capillary gas chromatographic system with flame ionization detection. The assay is linear from 1 to 50 μg/ml for each enantiomer. The analytical method affords average recoveries of 92.28 and 96.44% for l- and d-fenfluramine, respectively. The limits of detection and quantitation for the method are 0.1 and 1.0 μg/ml for the l- and d-fenfluramine isomers, respectively. The reproducibility of the assay was <10% (RSD). The method allowed study of the depletion of l- and d-fenfluramine in rat liver microsomal incubates. The stereoselectivity of fenfluramine phase I metabolism was observed.     

Is the Phytophthora citrophthora culture filtrate a reliable tool for the in vitro selection of resistant Citrus variants?

Summary Nucellar calli from four Citrus cultivars with known resistance to the Phytophthora citrophthora pathogen were chosen as experimental material to test the pathogen's response to culture filtrate (CF). Sensitivity of the four calli to CF of the fungus was in reverse order to what is known on the susceptibility of the cultivars in vivo. Sensitivity of protoplasts derived from the same four calli to 2,4-dichlorophenoxyacetic acid (2,4-D) was in the same order as that of calli to CF. Protoplasts derived from calli selected for tolerance to CF showed a higher plating efficiency with increasing concentration of CF in the medium. TLC and GLC determinations showed the presence of indole acetic acid in the culture filtrate. Results indicate that CF of P. citrophthora cannot be used as a selection tool in vitro.Contribution No. 1655-E, 1986 series, from the Agricultural Research Organization, Bet-Dagan, Israel     

Stimulation and inhibition of human T-lymphocyte colony cell proliferation by hemopoietic cell factors.

Human lymphocytes, isolated from peripheral blood and stimulated with phytohemagglutinin M (PHA) prior to being seeded on a two-layer medium of soft agar which contained the mitogen, developed into colonies 3–4 days after seeding in the culture system. The cloning potential of PHA-treated lymphocytes is significantly enhanced by adding, to the soft agar culture, culture fluid (CF) obtained from mitogen-treated lymphocytes or a feeder layer (FL) prepared either from lymphocytes isolated from peripheral blood or from T-cell enriched populations. PHA seems to stimulate the release of lymphocyte colony enhancing factor (LCEF) from the T-sensitized lymphocytes. The addition of CF or FL to the culture medium appears to increase the amount of LCEF, resulting in enhancement of the number and size of lymphocyte colonies. When CF derived from spleen cells or from the peripheral blood adherent-cell population was added to the lower layer of the soft agar culture, the growth and development of lymphocyte colonies was inhibited. This suggests that monocyte-macrophages release a lymphocyte colony inhibiting factor (LCIF) into the CF. The extent of inhibition or stimulation of colony formation is a function of the number and type of cells used to prepare the CF or FL and the concentration of CF in the culture medium. The presence of FL or CF derived from spleen non-adherent cells, white blood cells, bone marrow cells, or a B-cell enriched population had no effect on colonies growing in the culture. This may possibly be due to the paucity of T lymphocytes and monocyte-macrophages present in these materials. A control system in which LCIF, produced by monocyte-macrophages, and LCEF, produced by T lymphocytes, participate in the regulation of lymphocyte production is postulated.     

Fenfluramine-induced thermogenesis in adult domestic fowl (Gallus domesticus): Elimination by propranolol,a beta-blocker

1. Intra-muscular injection of DL-fenfluramine, a 5-hydroxytryptamine agonist, increased heat production by a mean of 16% over the following 6hr in adult domestic fowl.2. Propranolol, a beta-receptor blocker, completely eliminated the effect of fenfluramine on thermogenesis.3. Respiratory quotient (RQ) was significantly reduced by fenfluramine injection, whether or not this was preceded by injection of propranolol. Injection of propranolol alone produced a decline of RQ to 0.71 within 1 hr, followed by an immediate steady increase to 0.90 over the next 5 hr.4. Extension and lowering of the wings, which augments surface area for heat loss, was observed within 30 min of fenfluramine injection and persisted for several hours. This thermolytic effect of fenfluramine was not eliminated by prior injection of propranolol. Polypnea occurred only when both propranolol and fenfluramine had been injected.5. Food intake over the whole day of measurement was significantly reduced by fenfluramine injection, whether or not this had been preceded by injection of propranolol. Water intake over the same period was unaffected by any of the treatments.6. Fenfluramine reduced spontaneous activity by almost half. The reduction was slightly greater when fenfluramine injection followed propranolol. Propranolol given alone had no effect on activity.     

Pharmacological manipulation of sincalide (CCK-8)-induced suppression of feeding

The current study involves an investigation of the possible neurotransmitter systems involved in the ability of exogenously administered sincalide (cholecystokinin octapeptide, CCK-8) to suppress feeding. Male rats previously trained to obtain food either during a daily 3-hr session, or conditioned to obtain food pellets on a fixed-ratio or fixed-interval schedule of reinforcement, were treated IP with CCK-8, following pretreatment with representative drugs of several pharmacological classes. Pretreatment with phenoxybenzamine, tolazoline, yohimbine, morphine, haloperidol or picrotoxin reduced the efficacy of CCK-8. However, pretreatment with naloxone or clonidine potentiated the suppressant action of CCK-8 on feeding. Propranolol, diphenhydramine, cimetidine, atropine, d-amphetamine, fenfluramine or diazepam pretreatment either had no effect or no consistent action in altering the activity of CCK-8. The ability of CCK-8 to suppress feeding was not altered by subacute treatment with the anorectics, d-amphetamine or fenfluramine, using a regimen known to induce tolerance. These data indicate that CCK-8 exerts a different mechanism of action than that of fenfluramine or d-amphetamine, and furthermore, that noradrenergic, dopaminergic, GABAergic or endogenous opioid systems either mediate or can modify the effect of CCK-8 on feeding.     

Inhibition of gastric emptying by peripheral and central fenfluramine in rats: Correlation with anorexia

Rats treated acutely with i.p. fenfluramine showed a profound inhibition of emptying of a chow meal from the stomach. This inhibition was attenuated by previous chronic treatment with fenfluramine. Tolerance to the gastric slowing occured with as few as 2–4 prior injections, a time course which closely matches tolerance to the anorectic effects of the drug. Cerebroventricular injections of fenfluramine and norfenfluramine in anorectic doses also inhibited gastric emptying.