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Biomedical subjects

D L Eaton

Publications and source records attributed to D L Eaton.

At least 109 records · Page 6Linked to original sources
PubMed1987

Effects of phenobarbital on the biliary excretion of aflatoxin P1-glucuronide and aflatoxin B1-S-glutathione in the rat.

Direct h.p.l.c. analysis of bile separated at least five major water-soluble metabolites of AFB; the two most prevalent AFB metabolites were identified as AFB-S-glutathione (AFB-GSH) and AFP1-glucuronide, which accounted for 49-57% and 4-15% of total biliary AFB metabolites, respectively. In the two hours following AFB administration, phenobarbital-treated rats eliminated 50% more AFB-derived radioactivity in bile compared with controls. No qualitative differences in the profile of biliary AFB metabolites were noted between phenobarbital-treated and control rats. However, a 90% increase in the rate of excretion of AFB-GSH was found in phenobarbital-treated animals. Phenobarbital treatment had no significant effect on the amount of AFB remaining in the liver after two hours, but decreased the amount of AFB covalently bound to hepatic DNA by 55%. When individual animals from both control and phenobarbital-treated groups were considered, the correlation between the increase in excretion of AFB-GSH and the decrease in covalent binding was significant with a correlation coefficient of 0.77. This finding is consistent with the hypothesis that induction of GSH S-transferase is responsible for the anticarcinogenic effects of phenobarbital towards AFB-induced hepatocarcinogenicity, although changes in the rate of formation of aflatoxin P1 or other biotransformation pathways may also be important.

Aflatoxin B1↗
PubMed1987

Bile loss in the acute intestinal radiation syndrome in rats.

The effects of bile duct ligation (BDL), choledochostomy, bile acid sequestering within the intestinal lumen by cholestyramine, and fluid and electrolyte replacement on survival time and development of diarrhea after whole-body exposure to doses of ionizing radiation that result in death from acute intestinal injury were studied. BDL significantly prolonged survival and delayed the onset of diarrhea after exposure to 137Cs gamma rays, fission neutrons, or cyclotron-produced neutrons in the range of doses that produce intestinal death or death from a combination of intestinal and hematopoietic injuries. Cannulation of the bile duct with exteriorized bile flow (choledochostomy) to protect the irradiated intestine from the mucolytic action of bile salts did not duplicate the effect of BDL in increasing survival time. Choledochostomy without fluid replacement eliminated the occurrence of diarrhea in 15.4 Gy irradiated rats. Diarrhea did occur in irradiated animals with choledochostomy if they received duodenal injections of fluid and electrolytes to replace the fluid lost as a result of bile drainage. Duodenal injection of fluid and electrolytes, however, had no significant effect on survival time in irradiated rats. In contrast, injection of fluid and electrolytes into the peritoneal cavity of irradiated rats resulted in an increase in survival time that was comparable to that observed after BDL. Addition of antibiotics to the peritoneally injected fluid and electrolytes further increased survival time (up to 9 days). This survival time approached that seen in animals receiving the same radiation dose but which had the intestine exteriorized and shielded to minimize radiation injury to the intestine. Postmortem histological examinations of the irradiated small intestine showed mucosal regeneration in these long-term survivors receiving fluid and antibiotic therapy. In contrast, duodenal injection of cholestyramine post irradiation to bind bile acids had no effect on survival time or diarrhea incidence. The conclusions from these experiments are that BDL prolongs survival and postpones the onset of diarrhea in irradiated rats dying from acute intestinal injury primarily by slowing down the loss of fluid and electrolytes and that bile acids play no significant role.

Animals↗
PubMed1986

Construction and characterization of an active factor VIII variant lacking the central one-third of the molecule.

The primary structure of factor VIII consists of 2332 amino acids that exhibit 3 distinct structural domains, including a triplicated region (A domains), a unique region of 909 amino acids (B domain), and a carboxy-terminal duplicated region (C domains), that are arranged in the order A1-A2-B-A3-C1-C2. The B domain (residues 741-1648) of factor VIII is lost when factor VIII is activated by thrombin, which proteolytically processes factor VIII to active subunits of Mr 50,000 (domain A1), 43,000 (domain A2), and 73,000 (domains A3-C1-C2). To determine if the B domain is required for factor VIII coagulant activity, a variant was constructed by using recombinant DNA techniques in which residues 797-1562 were eliminated. This shortened the B domain from 909 to 142 amino acids. This variant factor VIIIdes-797-1652 was expressed in mammalian cells and was found to be functional. The factor VIIIdes-797-1562 protein was purified and shown to be processed by thrombin in the same manner as full-length factor VIII. The factor VIIIdes-797-1562 variant also bound to von Willebrand factor (vWF) immobilized on Sepharose. These results indicate that most of the highly glycosylated B domain of factor VIII is not required for the expression of factor VIII coagulant activity and its interaction with vWF.

Amino Acid Sequence↗
PubMed1986

Kinetic evaluation of carrier-mediated transport of ouabain and taurocholic acid in isolated rat hepatocytes. Evidence for independent transport systems.

Ouabain and taurocholic acid (Tch) share in common the cyclopentanophenanthrene (steroid) nucleus, and both are concentrated in the liver by hepatic sinusoidal carrier-mediated transport processes. Multiple transport systems for Tch uptake have been implicated, and Tch is an effective inhibitor of ouabain uptake. To determine if the ouabain transport system is related to transport of Tch, kinetic studies were conducted to examine the nature of cross-inhibition of ouabain and Tch. Tch was found to inhibit ouabain uptake in a competitive manner, with a Ki approximately ten times less than the Km for ouabain. However, ouabain failed to inhibit total Tch uptake in a competitive manner when added to the system at the same time as the Tch substrate. Preincubation of cells with ouabain resulted in noncompetitive inhibition of sodium-dependent Tch uptake but had no effect on sodium-independent Tch uptake. Ouabain had a weak competitive inhibitory effect on sodium-independent transport of Tch, but the Ki was approximately ten times greater than the Km for ouabain. These results demonstrate that the ouabain transport system is distinct from both the sodium-dependent and sodium-independent transport systems for Tch. Tch apparently binds competitively to the ouabain transport system but is not effectively transported across the cell membrane by this system.

Animals↗
PubMed1986

Effects of single-dose and repeated-dose pretreatment with 2(3)-tert-butyl-4-hydroxyanisole (BHA) on the hepatobiliary disposition and covalent binding to DNA of aflatoxin B1 in the rat.

The effects of two distinctive BHA pretreatment regimens on the biliary excretion of aflatoxin B1 (AFB) metabolites and the covalent binding of AFB to hepatic DNA were studied in vivo in the rat. To differentiate between enzyme induction effects and direct antioxidant effects, BHA was given to rats for 9 days (500 mg/kg/day, sc) or as a single dose (500 mg/kg, po), and [3H]AFB was administered ip. Repeated treatment with BHA enhanced the biliary excretion of both the glutathione conjugate of AFB and the AFP1-glucuronide to 200% of control values, reduced the amount of AFB remaining in the liver to 53% of control and reduced the covalent binding of AFB to hepatic DNA to 16% of control. A single BHA treatment had no effect on the biliary excretion of AFB or the binding of AFB to hepatic macromolecules, even though high concentrations of BHA were present in the liver during the period of AFB metabolism. These results support the hypothesis that BHA inhibits AFB carcinogenesis via the induction of phase II biotransformation pathways such as glutathione S-transferase, which act to reduce the amount of AFB-epoxide available for binding to DNA. We found no evidence of a direct antioxidant effect of BHA in altering the hepatobiliary disposition of AFB.

Aflatoxin B1↗
PubMed1985

Effects of various trace metals on the binding of cadmium to rat hepatic metallothionein determined by the Cd/hemoglobin affinity assay.

The Cd/hemoglobin affinity assay is finding increasing use as an indicator of metallothionein in a variety of biological tissues. Because the assay relies on the ability of heat-stable proteins to bind Cd, it is important to know the relative affinities of different trace metals to bind to such proteins, relative to Cd. This study examines the ability of 15 trace metals to prevent the binding of Cd to metallothionein using the Cd/hemoglobin affinity assay for metallothionein. Cd, Cu, Hg, and Ag were the only metals tested which significantly inhibited the binding of a fixed concentration (2 microM) of 109Cd to a crude preparation of rat hepatic metallothionein. As, Co, Cr, Fe, Mn, Mo, Ni, Pb, Sn, Th, and Zn had no inhibitory effect at the highest concentrations tested. However, As, Sn, Th, and Zn prevented the precipitation of hemoglobin at relatively low concentrations and, thus, could not be fully tested for inhibitory potency. Cu was the most potent inhibitor, producing more than 90% inhibition at 5 microM, followed by Ag, Hg, and Cd, which produced 76, 72, and 65% inhibition of cadmium binding at 5 microM, respectively. These results suggest that caution should be taken in interpreting metallothionein concentrations obtained by the Cd/hemoglobin affinity assay in tissues which contain relatively a high concentration of Cu, Ag, or Hg, relative to that of Cd.

Animals↗
PubMed1984

Purification of human fibroblast urokinase proenzyme and analysis of its regulation by proteases and protease nexin.

Recently we presented evidence that normal human foreskin fibroblasts (HF cells) limit the activity of secreted urokinase by secreting it as a proenzyme and by secreting protease nexin , an inhibitor of urokinase and certain other serine proteases (Scott, R.W., Eaton, D. L. Duran , N., and Baker, J.B. (1983) J. Biol. Chem. 258, 4397-4403). Using immunoaffinity chromatography we have now purified the HF cell urokinase proenzyme. It is a single 52-kDa polypeptide chain that is inactive toward both plasminogen and low molecular weight substrates. After proteolytic activation, this material (specific activity of 3 X 10(4) Committee on Thrombolytic Agents units/mg) is composed of two disulfide-bridged 33- and 19-kDa chains, and is thus similar to the predominant form of urokinase found in urine. Plasmin at 2 X 10(-10) M causes 50% activation of the proenzyme (1 X 10(-9) M) in 30 min at 37 degrees C. Thrombin and trypsin are one-twentieth as effective as plasmin. Activated HF cell 125I-urokinase forms sodium dodecyl sulfate stable complexes with purified protease nexin or protease nexin present in medium conditioned by HF cells. Purified protease nexin inhibits purified HF cell urokinase action on both plasminogen and low molecular weight substrates. The association rate constant for the reaction between protease nexin and HF cell urokinase is approximately 1.7 X 10(5) M-1 S-1. In contrast, the association rate constants for reactions between protease nexin and the one- and two-chain forms of tissue-type plasminogen activator are approximately 2 X 10(3) and approximately 3 X 10(4) M-1 S-1, respectively. The importance of protease nexin as a regulator of HF cell urokinase is supported by the finding that anti-protease nexin antibody potentiates the fibrinolytic activity of HF cell-conditioned medium incubated with plasminogen.

Amyloid beta-Protein Precursor↗
PubMed1984

Studies on the mechanisms of thallium-mediated inhibition of hepatic mixed function oxidase activity. Correlation with inhibition of NADPH-cytochrome c (P-450) reductase.

Thallium (TlCl3) administration to rats produced a dose-dependent loss of hepatic NADPH-cytochrome c (P-450) reductase and microsomal mixed function oxidase activities within 2-4 hr following treatment. These changes occurred independently of apparent effects on microsomal heme or cytochrome P-450 content, both of which remained unchanged with respect to control levels despite transient inhibition of delta-aminolevulinic acid (ALA) synthetase and induction of heme oxygenase. These results are consistent with the recognized properties of thallium as both a flavoprotein antagonist and sulfhydryl inhibitor and differ uniquely from the action of other metals which impair mixed function oxidase activity through compromise of heme biosynthesis and heme depletion. The potential utility of thallium compounds in further evaluating the functional characteristics of NADPH-cytochrome c (P-450) reductase and its role in microsomal oxidative processes is suggested from these observations.

5-Aminolevulinate Synthetase↗
PubMed1984

Biological availability of lead in a paint aerosol. 1. Physical and chemical characterization of a lead paint aerosol.

This study was conducted to determine the physical and chemical characteristics of an aerosol of lead-based paint, generated in an industrial spray operation, that might influence the biological availability of lead present in inhaled aerosols. Paint aerosols were collected, and mass-size distribution was determined using a portable cascade impactor under actual occupational conditions. Approx. 2% of the particulate mass collected was in the respirable range (less than 10 micron mean aerodynamic diameter), although the maximum airborne concentration of lead was found to be 2-3 mg/m3. The lead concentration in a dried aerosol was very resistant to chemical digestion. Analysis by X-ray diffraction, atomic absorption spectroscopy and inductively coupled plasma emission spectroscopy showed approx. 11% lead by dry weight, although the wet weight concentration of lead reported by the manufacturer was 12.8%.

Aerosols↗
PubMed1984

Biological availability of lead in a paint aerosol. 2. Absorption, distribution and excretion of intratracheally instilled lead paint particles in the rat.

Four groups of rats received by intratracheal instillation (1) a lead chromate paint particulate suspension, (2) lead tetraoxide suspension, (3) lead acetate solution, or (4) saline. Lead-dosed animals received an equivalent dose of 1 mg lead/kg body weight. Distribution of lead was monitored through assays of urine, feces, and tissues (lung, bone, muscle kidney, liver) obtained at post-mortem 5 weeks after exposure. Delta-aminolevulinic acid dehydratase (ALA-D) activity was measured to determine the effect of lead on heme biosynthesis. The vast majority of the dosed lead in the paint matrix remained in the lung. In contrast, in the lead acetate-dosed animals, little remained in the lung, but significant elevations were found in bone and kidney. Blood ADA-D was significantly depressed in the lead acetate-treated animals, but was not significantly different from control animals in the animals dosed with lead paint or lead tetraoxide. These findings suggest that lead chromate in an alkyd resin paint matrix is poorly absorbed from the lung compared with lead acetate and lead tetraoxide.

Absorption↗
PubMed1984

Studies on porphyrin metabolism in the kidney. Effects of trace metals and glutathione on renal uroporphyrinogen decarboxylase.

Uroporphyrinogen (urogen) decarboxylase catalyzes the decarboxylation of 8- to 4-carboxyl porphyrinogen during heme biosynthesis in mammalian tissues. The specific activity of renal urogen decarboxylase was shown to be approximately one-third that of the hepatic enzyme and to be readily inactivated by HgCl2 following acute treatment or at concentrations as low as 50 microM in vitro. HgCl2 differentially inhibited the decarboxylation of 8- to 7- and 7- to lesser-carboxylated porphyrinogens in the kidney, suggesting that at least a two-stage process is involved in the catalytic action of the renal enzyme. In contrast, neither lead nor iron compounds inhibited renal urogen decarboxylase in concentrations as high as 1 mM in the reaction mixture. GSH increased renal but not hepatic urogen decarboxylase activity by over 4-fold in vitro when measured as total porphyrinogen products produced, and preferentially accelerated the decarboxylation of 7- to 4-carboxyl porphyrinogen. GSH also protected the renal enzyme from HgCl2 inhibition. These findings suggest that renal urogen decarboxylase catalyzes porphyrin decarboxylation significantly less rapidly than the hepatic enzyme, is readily inactivated by mercuric chloride, and may be GSH-dependent with respect to achieving optimal catalytic activity. These observations may be useful in characterizing the contribution of the kidney to the clinical manifestations of the inherited porphyrias and environmentally induced disorders of porphyrin metabolism.

Animals↗
PubMed1983

Regulation of extracellular plasminogen activator by human fibroblasts. The role of protease nexin.

When the plasminogen activator urokinase was radioiodinated and incubated at 40 ng/ml in medium conditioned by human foreskin (HF) cells, within 30 min over 80% of the added plasminogen activator was complexed to cell-released protease nexin (PN). The urokinase complexed to PN had little if any activity. Incubation of purified PN with urokinase confirmed that PN is an inhibitor of this plasminogen activator. However, a widely used plasminogen-dependent fibrinolysis assay for plasminogen activator indicated that abundant endogenous plasminogen activator activity co-existed with PN in HF cell-conditioned medium. The source of this activity was electrophoretically and immunologically indistinguishable from urokinase. Furthermore, gel exclusion chromatography showed that about 90% of the urokinase antigen detected in conditioned medium had a molecular weight similar to that of free active urokinase. These paradoxical findings are resolved by evidence that this "PN-resistant urokinase-like" plasminogen activator is actually urokinase proenzyme that is activated by plasmin or conditions in the fibrinolysis assay for plasminogen activator. It is shown that the activated form of HF cell plasminogen activator is sensitive to inhibition by PN. PN may thus be an important component in the cellular regulation of endogenous plasminogen activator activity.

Amyloid beta-Protein Precursor↗
PubMed1983

Evidence that a variety of cultured cells secrete protease nexin and produce a distinct cytoplasmic serine protease-binding factor.

Four criteria were used to examine serum-free conditioned cell culture medium for protease nexin (PN):(1) formation of SDS-stable approximately 77 K Da complexes between a medium component and [125I]thrombin; (2) acceleration by heparin of the rate of formation of these complexes; (3) cellular binding of these complexes; and (4) inhibition by heparin of the cellular binding of complexes. Listed in order of decreasing PN production, PN was detected in media conditioned by the following cell types: human foreskin fibroblasts (0.18 micrograms/10(6) cells), rat embryo heart muscle cells (0.13 micrograms/10(6) cells), mouse myotubes (0.1 micrograms/10(6) cells), monkey kidney epithelial cells, human fibrosarcoma cells, human lung fibroblasts, simian virus 40 (SV-40)-transformed human fibroblasts, human epidermoid carcinoma cells, bovine aortic endothelial cells (only after phorbol ester treatment), and mouse myoblasts. No PN was found in medium conditioned by mouse 3T3 cells, SV40 virus-transformed 3T3 cells, human lymphoblasts, or mouse leukemia cells. Eleven of the cell types examined for secretion of PN were also examined for the presence of cytoplasmic thrombin-binding factors. Lysates from all of these cell types contained a factor that formed approximately 60-65 K Da sodium dodecyl sulfate (SDS)-stable complexes with [125I] thrombin. This MW is significantly lower than that of [125I] thrombin-PN complexes, indicating that the factor is distinct from PN. Nevertheless, PN and the cytoplasmic factor share similarities. Production of both PN (by HF cells and WI-26 cells) and the cytoplasmic factor (by HF cells and 3T3 cells) are stimulated by epidermal growth factor and phorbol myristate acetate. Also, both PN and the cytoplasmic factor complex trypsin, plasmin, urokinase, and thrombin, but not pancreatic elastase. Because a number of the cells that produce PN or the cytoplasmic serine protease-binding factor are known to produce plasminogen activators, both PN and the cytoplasmic factor could regulate plasminogen activator activity.

Amyloid beta-Protein Precursor↗
PubMed1983

A simplified method for quantitating metallothionein in biological tissues.

A new method was modified and evaluated as a simple, sensitive and quantitative procedure for measuring metallothionein (MT) in tissues. Heat denatured cytosolic fractions of tissue homogenates were incubated with 109Cd and bovine hemoglobin was added to scavenge Cd not bound to MT. The hemoglobin and excess Cd were separated from the 109Cd-MT by heating. The Cd remaining in the supernatant was a quantitative measure of the MT present. Analysis of Sephadex G-75 and DEAE-ion exchange fractions of liver supernatants from Cd and Zn-treated rats indicated that the assay is specific for MT. Levels of MT in six rat tissues were quantitated in untreated and Cd-treated rats and were consistent with previous studies on tissue levels of MT. Because of its speed and sensitivity, this assay may be useful for measuring MT in large studies where time and expense are limiting factors.

Animals↗
PubMed1983

Phorbol ester and mitogens stimulate human fibroblast secretions of plasmin-activatable plasminogen activator and protease nexin, an antiactivator/antiplasmin.

Tumor-promoting phorbol esters have been reported to greatly increase plasminogen activator (PA) activity produced in numerous cell types. Many of these studies have employed a widely used fibrinolysis assay for PA activity that involves large-scale dilution of cell lysates or conditioned medium (CM) into buffer containing plasminogen and the plasmin substrate 125I-fibrin. This assay indicates that phorbol ester and the mitogens epidermal growth factor (EGF) and thrombin all stimulate secretion of PA activity in our human foreskin fibroblast cultures. However, these effects are not observed in a modified fibrinolysis assay employing undiluted conditioned culture medium unless the medium is first treated at pH 3, which inactivates the secreted protease inhibitor, protease nexin (PN). Moreover, a direct assay for plasminogen activator activity based on cleavage of 125I-plasminogen indicates that conditioned culture medium contains little if any active plasminogen activator either before or after treatment of the cultures with phorbol ester or EGF. Phorbol ester and mitogens do stimulate secretion of (a) an inactive PA that can be activated by plasmin and (b) PN, which inhibits both the activated form of the PA and plasmin. Secretions of the inactive PA and PN are further correlated in that release of both is stimulated most by phorbol ester, somewhat less by EGF, and least by thrombin. Significantly, these effects are not accompanied by increases in total protein secretion. We propose that fibroblasts secrete PA in an inactive form in the presence of PN to confine PA activity to an as yet undefined location or event.

Amyloid beta-Protein Precursor↗