Antacid, sucralfate, and prostaglandin E2 effects on the growth and potential for translocation of Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus in an in vitro gastric simulation

Viable bacteria in the gut of thermally injured patients may be translocated through the gut mucosa, causing widespread infection. Increased flora from optimization of bacterial growth by pH elevation, coupled with the decreased intestinal motility common among patients whose mucosal integrity has been compromised, may increase the incidence of translocation. Gastric pH in these patients is monitored and maintained around pH 6 by various agents to reduce susceptibility to stress ulceration. Whole milk, given as a nutrient source, also raises pH. An in vitro trial simulating gastric fluid under conditions found in patients with burns was conducted to evaluate the growth of commonly ingested bacteria. Bicarbonate buffer containing pepsin and adjusted to pH 2, 4, or 7 with HCl was dosed with magnesium and aluminum hydroxide antacid (Maalox) (10 ml), sucralfate (Carafate) (0.4 gm), or prostaglandin E2 (PGE2) (10 ng) before inoculation with Escherichia coli (3 x 10(2) organisms), Pseudomonas aeruginosa (3 x 10(2) organisms), or Staphylococcus aureus (2 x 10(1) organisms). Bacterial growth and pH were determined periodically over the 24-hour trial. Milk was added at intervals in half the samples to simulate patient feeding. Maalox increased pH in all samples containing milk (initially pH 2, 4, or 7) to over 7.0 in 2 hours, and increased pH more slowly without milk. Carafate had a moderating effect, increasing pH 2 and pH 4 and decreasing pH 7, with a narrower pH range found in the milk groups. PGE2 treatments combined with milk also increased pH 2 and pH 4, but slightly elevated pH 7 within 24 hours. Without milk, PGE2 did not alter pH from initial values.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanisms of anti-carcinogenesis by indole-3-carbinol: effect on the distribution and metabolism of aflatoxin B1 in rainbow trout

Indole-3-carbinol (I3C), a component of cruciferous vegetables,was previously shown to inhibit aflatoxin B₁ (AFB₁) carcinogenesisin trout. The purpose of this study was to examine the effectof I3C on AFB₁ metabolism and hepatic DNA adduct formation invivo and in vitro. When fed at 0.2%, I3C produced a 70% reductionin average in vivo hepatic DNA binding of injected AFB₁ overa 21-day period when compared to controls. A 24-h distributionstudy of injected tritiated AFB₁ in I3C fish showed less totalradioactivity in the blood and liver at all times examined,compared to controls. These reductions were due primarily toreduced levels of AFB₁ bound to red blood cell DNA, reducedplasma levels of the primary metabolite aflatoxicol (AFL), anddecreased levels of AFB₁ and polar metabolites present in theliver of I3C fish. In contrast to blood, total radioactivitywas significantly elevated in the bile of I3C fish resultingfrom a 7-fold increase in aflatoxicol-M₁ glucuronide levelsover controls. No difference was observed in concentration ofAFL glucuronide, the primary conjugate present in control fish.There was no difference in total radioactivity remaining inthe carcass of I3C or control fish. AFB₁ metabolism in freshlyisolated hepatocytes from I3C fish showed 20% less DNA bindingin a 1-h assay, with a 2-fold increase in aflatoxin M₁ production.Addition of I3C to control hepatocytes at levels of 1, 10 or100 µM had no effect on AFB₁ DNA binding. These findingsindicate that I3C inhibition of AFB₁ hepatocar-cinogenesis introut involves substantial changes in the phar-macokineticsof carcinogen distribution, metabolism and elmination, leadingto significantly reduced initial hepatic-nuclear DNA damagein vivo.     

Coumarin-induced changes in delta-aminolaevulinic acid synthase and cytochrome P-450 in chick embryo liver

Coumarin occurs naturally in the diet and inhibits several cytochrome P-450 enzymes in laboratory animals. The effect of coumarin was examined on haem biosynthesis and cytochrome P-450 activities in the 18-day-old chick embryo liver in ovo. At 40 and 50 mumol/embryo coumarin increased delta-aminolaevulinic acid synthase, porphyrins, cytochrome P-450, benzphetamine N-demethylase and benzo[a]pyrene hydroxylase. At 10 mumol/embryo coumarin decreased aniline 4-hydroxylase, and at both 10 and 50 mumol/embryo it decreased 7-ethoxyresorufin O-deethylase, coumarin 7-hydroxylase and nitrosodimethylamine N-demethylase. 7-Hydroxycoumarin and 5, 7-methoxycoumarin at 40 mumol/embryo had none of these effects. Coumarin (5-500 microM) added to liver microsomes inhibited aniline hydroxylase by 45%, but not nitrosodimethylamine N-demethylase, and inhibited 7-ethoxyresorufin O-deethylase in microsomes from 3-methylcholanthrene-treated embryos by 15 and 100% at coumarin concentrations of 250 and 500 microM, respectively. Coumarin 7-hydroxylase activity in chick embryo liver was comparable with that reported for human liver and greater than in the rat. The data indicate that coumarin can both increase and decrease cytochrome P-450 activities in chick embryo liver and can induce haem biosynthesis. Because the chick embryo liver hydroxylates coumarin at position 7 in a manner similar to humans, it may be a more suitable model than the rat for studying some of the metabolic effects of coumarin.     

Interaction of cyclopiazonic acid with rat skeletal muscle sarcoplasmic reticulum vesicles. Effect on Ca2+ binding and Ca2+ permeability

The interaction of cyclopiazonic acid with rat skeletal muscle sarcoplasmic reticulum (SR) vesicles was investigated in order to study the mechanism of cyclopiazonic acid inhibition of the Ca2+-ATPase (Goeger et al., Biochem Pharmacol 37: 978-981, 1988). Cyclopiazonic acid at 25 microM prevented the binding of Ca2+ to the high affinity binding site of mixed (light and heavy) SR vesicles and inhibited, in a dose-dependent manner, the Ca2+-dependent phosphorylation of SR vesicles by ATP. Binding of Ca2+ to the high affinity site of the CA2+-ATPase is necessary for both Ca2+ transport and for phosphorylation of the Ca2+-ATPase. We conclude that inhibition of Ca2+ binding to the high affinity site may be responsible, at least in part, for the activity of cyclopiazonic acid. The mechanism of inhibition remains unclear. The inhibition was not reduced after dialysis and was only partially reversed by gel filtration of SR vesicles treated with cyclopiazonic acid. Neither 1 mM glutathione nor dithiothreitol pretreatment had any effect on the inhibition of the Ca2+-ATPase. In addition to its inhibition of Ca2+ uptake and the Ca2+-ATPase, cyclopiazonic acid had significant effects on Ca2+ efflux from both passively and actively loaded SR vesicles. Cyclopiazonic acid impeded the efflux of Ca2+ from passively loaded SR vesicles (in the presence of ruthenium red) when compared to either untreated vesicles or those treated with mersalyl acid, a mercurial which also inhibits the Ca2+-ATPase and is known to induce Ca2+ release by both ruthenium red-sensitive and -insensitive pathways. Treatment of actively loaded vesicles with cyclopiazonic acid resulted in a decreased rate of Ca2+ efflux when compared to SR vesicles in which the Ca2+-ATPase activity was inhibited by ATP depletion with hexokinase and glucose. The results are consistent with the hypothesis that, in mixed SR vesicles, cyclopiazonic acid inhibits both the Ca2+ pump and Ca2+ efflux.     

Aurilides B and C, cancer cell toxins from a Papua New Guinea collection of the marine cyanobacterium Lyngbya majuscula

Cytotoxicity-guided fractionation of a strain of the marine cyanobacterium Lyngbya majuscula collected from Papua New Guinea led to the isolation of aurilides B (1) and C (2). The planar structures of 1 and 2 were established by spectroscopic analysis, including HR-FABMS, 1D (1)H and (13)C NMR, and 2D COSY, HSQC, HSQC-TOCSY, and HMBC spectra. The absolute configuration was determined by spectroscopic analysis and chiral HPLC analysis of acid hydrolysates of 1 and 2. Both aurilides B and C showed in vitro cytotoxicity toward NCI-H460 human lung tumor and the neuro-2a mouse neuroblastoma cell lines, with LC(50) values between 0.01 and 0.13 microM. Aurilide B (1) was evaluated in the NCI 60 cell line panel and found to exhibit a high level of cytotoxicity (the mean panel GI(50) concentration was less than 10 nM) and to be particularly active against leukemia, renal, and prostate cancer cell lines.     

Coumarin chemoprotection against aflatoxin B1-induced gene mutation in a mammalian cell system: A species difference in mutagen activation and protection with chick embryo and rat liver S9

Coumarin (1,2-benzopyrone), a natural food constituent, prevents polycyclic aromatic hydrocarbon-induced neoplasms in rats and mice, but has not been studied with other chemical carcinogens. We examined coumarin chemoprotection against aflatoxin B₁ using the 6-thioguanine resistance mutation assay in two different Chinese hamster ovary cell lines (K₁BH₄ and AS52) with liver S9 from rats and 19-day-old chick embryos for aflatoxin B₁ bioactivation. Laboratory rodents metabolize coumarin through 3-hydroxylation, whereas 7-hydroxylation predominates in chick embryos and humans. Chick embryo liver S9 was approximately 25-fold more effective in activating aflatoxin B₁ to the mutagenic and cytotoxic metabolite(s) than rat liver S9. Coumarin added at 50 and 500 μM with chick embryo liver S9 reduced the mutant frequency of 1 μM aflatoxin B₁ by 40 and 85%, respectively. Coumarin up to 500 μM had no effect on aflatoxin B₁ mutagenicity with rat liver S9. When liver S9 from chick embryos pretreated with coumarin was used for aflatoxin B₁ bioactivation, mutant frequency and cytotoxicity were decreased compared to liver S9 from vehicle-treated controls. Liver S9 from coumarin-treated rats did not significantly affect mutant frequency or cytotoxicity. HPLC analysis of chick embryo liver S9 incubated with 1 μM aflatoxin B₁ showed a dose-dependent decrease by coumarin of aflatoxin B₁ activation to the 8,9-epoxide ranging from 70% of controls at 5 μM coumarin to 4% of controls at 500 μM coumarin. In contrast, coumarin produced a dose-dependent increase in 20 μM aflatoxin B₁ activation by rat liver S9, reaching twice the control levels at 500 μM coumarin. These findings, using a mammalian cell system as a mutagenic endpoint, demonstrate marked species differences in chemoprotection by coumarin. Environ. Mol. Mutagen. 32:64–74, 1998 © 1998 Wiley-Liss, Inc.     

Fibroblast growth factor reserves the bacterial retardation of wound contraction.

Chronic granulating wounds were established in rats by excising burns inoculated with Escherichia coli. Recombinant human basic fibroblast growth factor was applied at dosages of 1, 10, and 100 micrograms/cm2 to the wounds of three groups of 20 animals on days 5, 9, 12, 15, and 18 after injury. The rate of wound closure was compared with that of similarly wounded animals treated with saline vehicle alone. High levels of bacteria caused significant retardation of wound contraction. The addition of basic fibroblast growth factor at the 100 micrograms/cm2 dosage level markedly improved the rate of wound closure whereas inert vehicles applied alone were ineffective. Since bacterial counts did not decrease in the basic fibroblast growth factor treated wounds, basic fibroblast growth factor was not inherently bactericidal. Histologic examination of the wounds treated with basic fibroblast growth factor showed increased cellularity with increased numbers of fibroblasts and round cells. These results suggest basic fibroblast growth factor can overcome the defect in healing created by bacterial infection, and this peptide may have efficacy in the management of the contaminated wound.     

Biochemical characterization of coumarin 7-hydroxylase activity in chick embryo liver microsomes.

Coumarin occurs naturally in the diet and can induce and inhibit cytochrome P450 enzymes. Hepatic coumarin 7-hydroxylase activity is the major pathway for coumarin metabolism in humans but not in rats, most strains of mice, or other laboratory animals. Coumarin 7-hydroxylase activity and the effects of chemical inhibitors and inducers on this activity were studied in 19-day-old chick embryo liver microsomes. Activity was between 35 and 75 nmol/mg protein/hr which is approximately 2-fold higher than reported for human liver microsomes. The pH optimum was 7.8 and the Km determined by both an ether extraction and a high performance liquid chromatography method was 7.3 +/- 0.9 (+/- SD) microM. Substrate inhibition was evident at coumarin concentrations above 250 microM (activities at 1000 and 4000 microM coumarin were 84 and 40% of Vmax, respectively). The Ki values (+/- SD) for inhibitors of microsomal coumarin 7-hydroxylase activity in vitro were: alpha-naphthoflavone, 46.9 +/- 19.8 nM; metyrapone, 0.8 +/- 0.9 microM; aniline, 12.3 +/- 8.2 microM; cimetidine, 70.9 +/- 27.9 microM; N-nitrosodimethylamine, 0.7 +/- 0.9 mM; and dimethyl sulfoxide, 7.9 +/- 1.9 mM. Treatment of chick embryos with pyrazole (40 mumol) increased coumarin 7-hydroxylase by 50% at 24 hr, but this activity was unaffected by treatment of embryos with 3-methylcholanthrene (2 mumol) or glutethimide (8 mumol). Thus, hepatic coumarin 7-hydroxylase activity in 19-day-old chick embryos is higher than in most laboratory animals and has similar biochemical properties as the enzyme in humans and mice. The chick embryo liver may be a useful system for studies on the biochemical effects of coumarin and the regulation of cytochrome P450-dependent coumarin 7-hydroxylase.     

Removal of plasma porphyrins with high-flux hemodialysis in porphyria cutanea tarda associated with end-stage renal disease.

Plasma porphyrin levels are markedly increased in patients with porphyria cutanea tarda (PCT) associated with end-stage renal disease. Conventional hemodialysis (CHD) with lower blood flow rates (less than 250 mL/min) and cuprophan or cellulose acetate membranes is ineffective in removing significant amounts of porphyrins in this condition. Changes in plasma porphyrin levels and porphyrin clearances during hemodialysis with higher blood flow rates and more-permeable, high-efficiency cellulose acetate and high-flux polysulfone dialyzers were evaluated in a chronic hemodialysis patient with PCT and markedly elevated plasma porphyrins. The polysulfone membrane achieved significantly better fractional porphyrin removal (P = 0.02) and porphyrin clearances (P less than 0.01) than did the high-efficiency cellulose acetate membrane. After conversion from maintenance CHD with a standard cellulose acetate dialyzer to a 4-wk period of high-flux hemodialysis (HFHD) with a polysulfone dialyzer, predialysis plasma porphyrins fell by 37%. After returning to CHD, plasma porphyrins returned to the higher prestudy levels. These observations suggest that HFHD with more permeable membranes and higher blood flow rates removes porphyrins more effectively than does CHD. HFHD may be a useful adjunct to other measures used in treating dialysis patients with PCT.