Significant Concentration-dependent Increases Research Articles

Evaluation of the effects and mechanisms of action of flutriafol, a triazole fungicide, on striatal dopamine release by using in vivo microdialysis in freely moving rats

The purpose of the present work was to assess the effects of flutriafol, a triazole fungicide, on in vivo dopamine (DA) release from rat striatum, using brain microdialysis coupled to high-performance liquid chromatography with electrochemical detection (HPLC–EC). Intrastriatal administration of flutriafol (1, 6 and 12 mM) produced significant concentration-dependent increases in DA levels to 218.5±51%, 1376±245% and 3093±345% compared with basal values, respectively. Those increases in DA levels could be due to an increased DA exocytotic release and/or a change in the activity of DA transporter (DAT). Thus, we investigated the effects of flutriafol (6 mM) under Ca ++- or Na +-free conditions, and after pretreatment with reserpine and TTX. When flutriafol was perfused in either Ca ++- or Na +-free Ringer, the DA levels reduced 92% and 70%, respectively; perfusion of flutriafol in TTX-treated (10 μM) or reserpine-pretreated animals (10 mg/kg), reduced the levels of DA to 73% and 86%, respectively. Co-infusion of flutriafol and nomifensine (20 μM) shows that the flutriafol-induced DA release did not involve the DAT. Our results suggest that flutriafol induces DA release via vesicular-, Ca ++-, Na +- and TTX-dependent mechanism, being independent of DAT.

Mutagenicity and genotoxicity of coal fly ash water leachate

Fly ash is a by-product of coal-fired electricity generation plants. The prevalent practice of disposal is as slurry of ash and water to storage or ash ponds located near power stations. This has lain to waste thousands of hectares of land all over the world. Since leaching is often the cause of off-site contamination and pathway of introduction into the human environment, a study on the genotoxic effects of fly ash leachate is essential. Leachate prepared from the fly ash sample was analyzed for metal content, and tested for mutagenicity and genotoxicity. Analyses of metals show predominance of the metals—sodium, silicon, potassium, calcium, magnesium, iron, manganese, zinc, and sulphate. The Ames Salmonella mutagenicity assay, a short-term bacterial reverse mutation assay, was conducted on two-tester strains of Salmonella typhimurium strains TA97a and TA102. For genotoxicity, the alkaline version of comet assay on fly ash leachate was carried in vitro on human blood cells and in vivo on Nicotiana plants. The leachate was directly mutagenic and induced significant ( P<0.05) concentration-dependent increases in DNA damage in whole blood cells, lymphocytes, and in Nicotiana plants. The comet parameters show increases in tail DNA percentage (%), tail length (μm), and olive tail moment (arbitrary units). Our results indicate that leachate from fly ash dumpsites has the genotoxic potential and may lead to adverse effects on vegetation and on the health of exposed human populations.

Greater Superficial Petrosal Nerve Transection in Rats does not Change Unconditioned Licking Responses to Putatively Sweet Taste Stimuli

The greater superficial petrosal nerve (GSP), innervating taste buds in the palate, is known to be exceptionally responsive to sucrose, especially compared with the responsiveness of the chorda tympani nerve (CT). However, whereas transection of the CT (CTX) alone has little or no effect on unconditioned licking responses to many "sweet" stimuli, the impact of GSP transection (GSPX) alone is equivocal. To further examine the role of the GSP on licking responses to putatively sweet-tasting substances, brief-access taste tests were conducted in nondeprived rats before and after sham surgery (SHAM) or CTX or GSPX. A range of concentrations of sucrose, L-alanine, glycine, and L-serine, with and without 1.0 mM inosine monophosphate (IMP) added, were used. All groups showed significant concentration-dependent increases in licking to all stimuli presurgically and postsurgically. CTX decreased licking responses relative to SHAM rats in the first sucrose test. There was also a group x concentration interaction for L-alanine, but post hoc tests did not reveal its basis. Other than this, there were no significant differences among the surgical groups. Interestingly, rats with GSPX tended to initiate fewer trials than SHAM rats. Overall, after GSPX, the remaining gustatory nerves are apparently sufficient to maintain concentration-dependent licking responses to all stimuli tested here. The disparity between our results and others in the literature where GSPX reduced licking responses to sucrose is possibly related to differences in surgical technique or test trial duration.

The induction of surface β-amyloid binding proteins and enhanced cytotoxicity in cultured PC-12 and IMR-32 cells by advanced glycation end products

During aging the non-enzymatic glycation of proteins and other molecules increases significantly, leading to the accumulation of advanced glycation end-products (AGEs). These AGEs enhance inflammatory and autoimmune reactions with resultant cytotoxicity. We noted in an earlier study that individuals with Alzheimer’s disease exhibit enhanced expression of the receptor for advanced glycation end-products (RAGE) on the surface of their leukocytes. In order to better understand the relationship between AGEs and the cell surface binding of amyloid-β protein (Aβ) 42 we studied the effect of two AGEs: glycated bovine serum albumin (BSA), and ε-carboxymethyllysine-BSA (CML), a glycoxidation product, on the binding of Aβ42 to rat PC-12 and IMR-32 cells. We measured the expression of three potential cell surface receptors binding Aβ42: RAGE, β-amyloid precursor protein (β-APP), and the α7 subtype of the nicotinic acetylcholine receptor (α7nAChR) by using specific antibody probes. Incubation of PC-12 or IMR-32 cells with bovine serum albumin–advanced glycation end-product (BSA-AGE) or with CML induced small but significant concentration-dependent increases in the expression of β-APP, RAGE, and α7nAChRs as measured by flow cytometry or by ELISA. Incubation of the cells with 48 μM of either of the AGEs combined with varying concentrations (138–1100 nM) of Aβ42 resulted in the enhanced binding of the Aβ42 to the cell surface as compared with cells not exposed to the AGE co-treatment. The combination of AGE and Aβ treatment also resulted in the heightened expression of all three potential Aβ binding sites as well as their gene precursors. Exposure of cells to the same regimen of AGE plus Aβ resulted in the production of reactive oxygen species and mitochondrial toxicity. These results are consistent with the ability of AGEs to enhance the cell surface expression of diverse Aβ42 binding sites, a factor that can lead to the enhanced binding of amyloid and subsequent cell death.

Differentiation-dependent responsiveness of bronchial epithelial cells to IL-4/13 stimulation.

The Th2 cytokines interleukin (IL)-4 and IL-13 are thought to play critical roles in the airway inflammation and hyperresponsiveness that characterize asthma. Recent evidence indicates that IL-13 can mediate these effects by acting directly on airway epithelial cells. Here we evaluated early [signal transducer and activator of transcription (STAT)6 phosphorylation] and delayed [granulocyte/macrophage colony-stimulating factor (GM-CSF) and transforming growth factor-beta(2) (TGF-beta(2)) secretion] responses of airway epithelial cells to IL-4 and IL-13 stimulation and the dependence of these responses on the culture technique employed. As expected, normal human bronchial epithelial cells grown on microporous inserts at an air-liquid interface (ALI) expressed a well-differentiated mucociliary phenotype; in contrast, cells grown on plastic in submerged cultures were poorly differentiated. When stimulated with IL-4 or IL-13, the magnitude and duration of STAT6 phosphorylation under the differing culture conditions were statistically indistinguishable. In contrast, cytokine secretion responses to IL-4 and IL-13 were highly dependent on the culture technique; cells cultured on plastic exhibited significant concentration-dependent increases in GM-CSF and TGF-beta(2) secretion, whereas cells grown at ALI showed no statistically significant response. These results demonstrate that the coupling between early signal transduction responses to IL-4 and IL-13 and downstream functions such as cytokine secretion may be critically dependent on the cell culture technique employed and the resulting differentiation status of bronchial epithelial cells.

Interaction of neuropeptide Y and corticotropin-releasing factor signaling pathways in AR-5 amygdalar cells

Corticotropin-releasing factor (CRF) is a 41 amino acid neuropeptide which is involved in the stress response. CRF and neuropeptide Y (NPY) produce reciprocal effects on anxiety in the central nucleus of the amygdala. The molecular mechanisms of possible CRF-NPY interactions in regulating anxiety behavior is not known. In the central nervous system, the action of NPY leads to inhibition of cAMP production while CRF is known to stimulate levels of cAMP in the brain. Consequently, we hypothesized that NPY may antagonize anxiety-like behavior by counter-regulating CRF-stimulated cAMP accumulation and activation of the protein kinase A pathway. We have engineered an immortalized amygdalar cell line (AR-5 cells) which express via RT-PCR, the CRF 2α, Y 1 and Y 5 NPY receptor. In addition, in these cells CRF treatment results in significant concentration-dependent increases in cAMP production. Furthermore, incubation of 3 μM CRF with increasing concentrations of NPY was able to significantly inhibit the increases in cAMP compared to that observed with 3 μM CRF treatment alone. These findings suggest that CRF and NPY may counter-regulate each other in amygdalar neurons via reciprocal effects on the protein kinase A pathway.

Mg(2+)-induced endothelium-dependent relaxation of blood vessels and blood pressure lowering: role of NO.

In vitro extracellular Mg(2+) concentration ([Mg(2+)](0)) produces endothelium-dependent and endothelium-independent relaxations in rat aorta in a concentration-dependent manner. These relaxant effects of Mg(2+) on intact rat aortic rings, but not denuded rat aortic rings, were suppressed by either N(G)-monomethyl-L-arginine (L-NMMA), N(omega)-nitro-L-arginine methyl ester (L-NAME), or methylene blue. The inhibitory effects of L-NMMA and L-NAME could be reversed partly by L-arginine. [Mg(2+)](0)-induced dilatation in vivo in rat mesenteric arterioles and venules was almost completely inhibited by N(G)-nitro-L-arginine and L-NMMA. Removal of extracellular Ca(2+) concentration ([Ca(2+)](0)) or buffering intracellular Ca(2+) concentration in endothelial cells, with 10 microM 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, markedly attenuated the relaxant effects of Mg(2+). Mg(2+) produced nitric oxide (NO) release from the intact aortic rings in a concentration-dependent manner. Removal of [Ca(2+)](0) diminished the increased NO release induced by elevated levels of [Mg(2+)](0). In vivo infusion of increasing doses (1-30 microM/min) of MgSO(4), directly into the femoral veins of anesthetized rats, elicited significant concentration-dependent sustained increases in serum total Mg and concomitant decreases in arterial blood pressure. Before and after employment of various doses of MgSO(4), intravenous administration of either L-NMMA (10 mg/kg) or L-NAME (10 mg/kg) increased (i.e., reversed) the MgSO(4)-lowered blood pressure markedly, and intravenous injection of L-arginine restored partially the increased blood pressure effects of both L-NMMA and L-NAME. Our results suggest that 1) small blood vessels are very dependent on NO release for Mg(2+) dilatations and 2) the endothelium-dependent relaxation induced by extracellular Mg(2+) is mediated by release of endothelium-derived relaxing factor-NO from the endothelium, and requires Ca(2+) and formation of guanosine 3',5'-cyclic monophosphate.

Detection of DNA Strand Breaks in Isolated Mussel (Mytilus edulisL.) Digestive Gland Cells Using the “Comet” Assay

Isolated mussel (Mytilus edulisL.) digestive gland cells were analyzed using the single-cell gel electrophoresis or “comet” assay to assess the ability of potential aquatic contaminants to induce DNA strand breaks (SBs) and to investigate the potential application of this technique as part of an aquatic biomonitoring regime. Freshly prepared cell suspensions from digestive gland were exposedin vitroto hydrogen peroxide (H2O2, 0–200 μM), 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX, 0–200 μM), benzo[a]pyrene (BaP, 0–200 μM), 1-nitropyrene (1-NP, 0–250 μM) and nitrofurantoin (NF, 0–1000 μM) for 1 h in the dark at 15°C in the presence of the DNA repair inhibitor cytosine-β-D-arabinofuranoside (araC). DNA strand breakage was measured using the comet assay. There were significant concentration-dependent increases in the percentage of DNA in the comet tail (mean values±SD) for all doses compared with controls (P<0.05) with H2O2(up to 61.4±5.1% at 100 μM), MX (up to 34.3±2.2% at 200 μM), BaP (up to 24.7±5.1 at 100 μM), 1-NP (up to 54.7±5.0% at 200 μM), and NF (up to 68.1±4.5% at 500 μM). There was a decrease (P<0.05) in viability (eosin Y exclusion) of exposed compared with control cells at 200 μM H2O2and BaP only. This study has demonstrated the potential of the comet assay to detect DNA strand breakage at subcytotoxic concentrations of a range of agents, some of which require metabolic activation. This may provide a sensitive, but nonspecific, molecular biomarker of genotoxicity.

Regulation of decidual cell chemokine production by group B streptococci and purified bacterial cell wall components

Objective: Our purpose was to determine whether cultured human decidual cells produce chemokines in response to different strains of group B streptococci and purified bacterial cell wall components. Study design: Human decidual cells were cultured from term placentas by standard techniques. Different strains of group B streptococci were isolated from neonates with early-onset group B streptococci sepsis. Confluent cell monolayers were incubated with these different strains of group B streptococci and various concentrations of purified bacterial cell wall components (including lipoteichoic acid, sialic acid, lipopolysaccharide, and lipid A) for 16 hours at 37° C. Culture supernatants were collected and assayed for macrophage inflammatory protein-1α and interleukin-8. Statistical analysis was by analysis of variance. Results: We found that cultured human decidual cells produced significant amounts of the two chemokines macrophage inflammatory protein-1α and interleukin-8 in a strain-specific fashion to the various different strains of group B streptococci tested, from 215% to 421% over baseline production ( p < 0.05 by analysis of variance). Also, we found that incubation of decidual cells with various concentrations of lipoteichoic acid, sialic acid, lipopolysaccharide, and lipid A resulted in significant concentration-dependent increases in decidual cell macrophage inflammatory protein-1α and interleukin-8 production ( p < 0.05.) CONCLUSIONS: Decidual cells produced significant amounts of the chemokines macrophage inflammatory protein-1α and interleukin-8 in response to intact group B streptococci in a strain-specific fashion and in response to various concentrations of different bacterial cell wall components. Because chemokines are important mediators signaling migration of different immune effector cells into areas of inflammation, we suggest that decidual cell chemokine production in response to bacteria and bacterial cell wall components may be a key early event in the pathogenesis of infection-associated preterm labor.

Regulation of human decidual cell macrophage inflammatory protein-1 alpha (MIP-1 alpha) production by inflammatory cytokines.

Inflammation of human gestational tissues is a key pathophysiologic event in the genesis of infection-associated preterm labor. Human gestational tissues produce several inflammatory cytokines after stimulation with bacterial products. These include interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF alpha), and IL-6. Another class of cytokines includes chemokines of the "C-C" subclassification such as macrophage inflammatory protein-1 alpha (MIP-1 alpha). The purpose of this study was to determine whether cultured human decidual cells produce MIP-1 alpha in response to other inflammatory cytokines. Various concentrations of IL-1 beta, TNF alpha, IL-6, and IL-4 were incubated with confluent monolayer cultures of decidual cells isolated from normal term placentae for 16 h at 37 degrees C, and MIP-1 alpha concentrations in culture supernatants were measured by ELISA. We found that incubation of decidual cells with IL-1 beta, TNF alpha, and IL-4 resulted in significant concentration-dependent increases in MIP-1 alpha production. IL-6 had no effect on MIP-1 alpha production. Our data are the first to show that human decidual cells in culture produce MIP-1 alpha in response to other inflammatory cytokines. We suggest that decidual cell production of MIP-1 alpha is an important early event in the pathophysiology of infection-associated preterm labor.

Interleukin-6 production by rat granulosa cells in vitro: effects of cytokines, follicle-stimulating hormone, and cyclic 3',5'-adenosine monophosphate.

In the present study we examined the influence of FSH as well as a number of well-established cytokines on interleukin (IL)-6 by rat granulosa cells in culture. Increasing concentrations of FSH, IL-1 alpha, IL-1 beta, tumor necrosis factor alpha (TNF alpha), and lipopolysaccharide (LPS) were incubated for 48 h with undifferentiated granulosa cells obtained from diethylstilbestrol-primed immature rats. The results demonstrate that FSH, IL-1 alpha, IL-1 beta, and LPS, but not TNF alpha, caused significant concentration-dependent increases in IL-6 release. We also examined the effects of dibutyryl-cAMP, forskolin, and 3-isobutyl-1-methyl-xanthine (IBMX) on IL-6 release by granulosa cells. Each of these agents caused a significant concentration-dependent increase in IL-6 production by granulosa cells in either the absence or presence of FSH. Taken together, these results show that the granulosa cell is not only a likely source of IL-6 but that the release of IL-6 can be regulated. Moreover, evidence suggests that cAMP may serve as a second messenger for the stimulated secretion of IL-6 by undifferentiated granulosa cells.

The comparative effects of big endothelin-1, endothelin-1, and endothelin-3 in the human fetal-placental circulation

OBJECTIVE: This study compared the effects of big endothelin-1, endothelin-1, and endothelin-3 and whether endothelin-converting enzyme was present in the human fetal-placental circulation.STUDY DESIGN: Single cotyledons of term placentas were dually perfused in vitro, and increases in fetal-placental perfusion pressure to bolus injections of big endothelin-1, endothelin-1, and endothelin-3 (8 x 10-10 to 1 x 10-7 mol/L) were recorded. Responses to big endothelin-1 (10-7 mol/L) were measured in the same placenta before and after perfusion of the fetal-placental circulation with the neutral metalloprotease inhibitor phosphoramidon (10-5 mol/L), which acts as an endothelin-converting enzyme inhibitor. All experiments were performed in at least five separate placentas.RESULTS: Significant concentration-dependent increases in fetal-placental perfusion pressure were seen with endothelin-1 (p < 0.0005), endothelin-3 (p < 0.0256), and big endothelin-1 (p < 0.0034, analysis of variance). Big endothelin-1 always elicited transient vasodilatation before constriction. Phosphoramidon significantly inhibited the vasoconstrictor effect of big endothelin-1 (p < 0.039, paired t test).CONCLUSION: The three endothelins tested are vasoconstrictors, and endothelin-converting enzyme is present in the fetal-placental circulation. (AM J OBSTET GYNECOL 1992;167:1651-6.)

Electrophysiologic interactions of procainamide and N-acetylprocainamide in isolated canine cardiac Purkinje fibers.

The study objective was to characterize the electrophysiologic interactions of procainamide (PA) and its metabolite, N-acetylprocainamide (NAPA), in canine Purkinje fibers. Cell (N = 43) action potentials were measured in Tyrode's solution (K+ = 4.0 mM, 36 degrees C) at a basic cycle length of 1,000 ms using standard microelectrode techniques. Six PA concentrations (0.020-0.32 mM) and six NAPA concentrations (0.010-0.24 mM) were studied alone and in combination. PA caused concentration-dependent decreases in Vmax and APD50 but did not alter APD90, ERP, or RMP. NAPA caused a small but not significant concentration-dependent decrease in Vmax, no change in RMP, and significant concentration-dependent increases in APD50, APD90, and ERP. Low NAPA concentrations increased, intermediate concentrations did not affect, and high NAPA concentrations again increased PA's effect on Vmax. PA-NAPA combinations resulted in concentration-dependent changes in APD50 that were intermediate between the effects of PA or NAPA alone. PA did not significantly alter NAPA's effects on APD90 at NAPA concentrations less than or equal to 0.040 mM, while it antagonized NAPA's effect at higher concentrations. The effects of PA-NAPA combinations on ERP were generally similar to their effects on APD90. The electrophysiologic effects of PA-NAPA combinations in normal canine Purkinje fibers are complex functions of the relative and absolute concentrations of the two compounds.

Endothelium-derived kinins account for the immediate response of endothelial cells to bacterial lipopolysaccharide.

We have recently shown that cultured endothelial cells produce kinins that can stimulate endothelial nitric oxide (NO) production in an autocrine manner. Because both the kallikrein-kinin system and the L-arginine/NO pathway have been implicated in the pathogenesis of septic shock, we investigated the possible involvement of endothelium-derived kinins in the response of cultured endothelial cells to bacterial lipopolysaccharide (LPS). In primary cultures of human umbilical vein and porcine aortic endothelial cells, LPS (0.3 to 3 micrograms/ml) induced significant concentration-dependent increases in cyclic GMP and 6-keto-PGF1 alpha, both of which were abolished in the presence of the selective bradykinin B2-receptor antagonist HOE 140 (0.1 microM). These LPS-induced increases in cyclic GMP and 6-keto-PGF1 alpha were short lived, being maximal after 5 min but were not apparent after 60 min. In parallel with these effects, LPS (30 micrograms/ml) induced a distinct, HOE 140-sensitive increase in the intracellular calcium concentration of human endothelial cells loaded with indo-1. In summary, these data suggest that the release of endothelium-derived kinin and subsequent stimulation of endothelial cells, followed by the enhanced production of NO and prostacyclin (PGI2), are implicated in the immediate hypotension induced by LPS in vivo.

Comparison of sister-chromatid exchange frequencies in mouse T- and B-lymphocytes after exposure to 4-hydroxycyclophosphamide or phosphoramide mustard

The present study was designed to investigate the genotoxicity of 4-hydroxycyclophosphamide (4-OHCP) and phosphoramide mustard (PAM), both reactive metabolites of cyclophosphamide (CP), for possible differences in SCE-inducing activity in mouse T- and B-lymphocytes. Mouse peripheral blood lymphocytes were isolated and stimulated to divide with either phytohemagglutinin (T-cell mitogen) or lipopolysaccharide (a polyclonal B-cell activator). Significant concentration-dependent increases in SCE frequencies were observed for both 4-OHCP and PAM with both mitogens, with 4-OHCP being almost twice as potent as PAM. There was no difference in SCE response between T- and B-lymphocytes after exposure to either PAM or 4-OHCP. These data do not support the idea that the difference in SCE response in T- and B-lymphocytes by CP in vivo is due to differential responses to either of the proposed putative metabolites of CP.

Prostaglandin biosynthesis by human decidual cells: Effects of inflammatory mediators

There is substantial evidence that decidual activation, in association with infection, is linked with the onset of both preterm and term labor. We therefore undertook the present study to evaluate prostaglandin production and its potential regulation by inflammatory mediators in human decidual cells in primary monolayer culture. Upon attaining confluence, the cells were incubated with endotoxin, interleukin 1α (IL1α), interleukin 1β (IL1β); or tumor necrosis factor (TNF). Production of prostaglandin (PG) E 2 and PGF 2α was determined using specific radioimmunoassays. Endotoxin and these cytokines all induced significant concentration-dependent increases in PGE 2 and PGF 2α production. Our results suggest that term human decidual cells are responsive to endotoxin and cytokines and that generation of these substances in the decidua or nearby (eg. in response to infection) will lead to increased prostaglandin production and uterine contractions.

Effects of encainide on the inducibility of ventricular arrhythmia in normal anesthetized dogs.

Effects of encainide on electrophysiologic variables and ventricular arrhythmia inducibility were studied in normal anesthetized dogs, using programmed electrical stimulation with unipolar cathodal (8 dogs) or bipolar (10 dogs) stimulation modes. Encainide produced significant concentration-dependent increases in ventricular refractory period, and diastolic current threshold with smaller but significant increases in QRS and Q-Tc. It decreased spontaneous heart rate and mean arterial pressure at high concentration. After administration of encainide, ventricular tachyarrhythmias could be induced by bipolar stimulation in half of the dogs (5/10 dogs,P < 0.05, compared to control). While unipolar cathodal stimulation could not cause reproducible ventricular arrhythmias. We conclude that encainide may facilitate the induction of ventricular arrhythmias in normal dogs and that bipolar stimulation may produce different results compared to unipolar stimulation in the presence of encainide.