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Biomedical subjects

D Acosta

Publications and source records attributed to D Acosta.

At least 127 records · Page 7Linked to original sources
PubMed1994

Comparative toxicity of tetracaine, proparacaine and cocaine evaluated with primary cultures of rabbit corneal epithelial cells.

Cocaine was first used as a topical anesthetic for the eye by Dr Carl Koller in 1884. It became evident that this agent produced erosion of the corneal epithelium in high doses or with repeated use. Synthetic local anesthetics such as tetracaine and proparacaine were developed which were more potent and less toxic than cocaine, but still produced corneal epithelium defects if used chronically. This investigation was undertaken to compare and rank the cytotoxicity of the most commonly used ocular local anesthetics, tetracaine, proparacaine and cocaine, with primary cultures of rabbit corneal epithelial cells. Cultures were exposed to either low concentrations of local anesthetics for 4-24 hr or to higher concentrations of local anesthetics for 15-120 min. Plasma membrane integrity was evaluated by measuring leakage of the cytosolic enzyme, lactate dehydrogenase, into the medium. Cell shape changes were evaluated by observing morphological changes. Mitochondrial dehydrogenase activity and cell viability were assessed by measuring 3-(4,5-dimethythiazol-2yl)-2,5-diphenyl tetrazolium bromide reduction. The cytotoxicity of the local anesthetics as evaluated by the lactate dehydrogenase leakage cytotoxicity test was ranked as follows: tetracaine (EC50 = 0.96 mM) >> proparacaine (EC50 = 4.4 mM) > cocaine (EC50 = 9.7 mM). The mitochondrial reduction assay seemed to be more sensitive than the lactate dehydrogenase leakage test in predicting toxicity: tetracaine (EC50 = 0.81 mM) >> proparacaine (EC50 = 3.4 mM) > cocaine (EC50 = 7.1 mM). When corneal epithelial cells were treated with local anesthetics, marked morphological changes occurred at concentrations that did not cause a decrease in viability. This was especially true for cocaine-treated cells. Tetracaine and proparacaine have the same anesthetic potency in vivo, although tetracaine is considered to be more irritating than proparacaine. This in vitro study showed that tetracaine was approximately four times more toxic than proparacaine. Cocaine was less toxic in vitro than proparacaine and tetracaine when compared on an equimolar basis, but in vivo it may be more toxic because of the higher concentrations that must be used to obtain the same degree of anesthesia as well as its marked effects on cell morphology.

Anesthetics, Local↗
PubMed1994

Inhibitory effect of cocaine on calcium mobilization in cultured rat myocardial cells.

Cocaine can produce both positive and negative inotropy. The mechanism of cocaine-induced negative inotropy is poorly understood. In order to evaluate the inhibitory effect of cocaine on myocardial contractility, its action on potassium chloride-induced depolarization, release of calcium from sarcoplasmic reticulum, and sarcolemmal sodium-calcium exchange were studied. At a relatively high concentration (10(-3) M), cocaine significantly blocked an elevation of cytosolic calcium during potassium chloride-induced depolarization and significantly inhibited the release of calcium from sarcoplasmic reticulum by caffeine. In contrast, a much lower concentration of cocaine (10(-7) M) significantly reduced sarcolemmal sodium-calcium exchange. Our results suggest that the negative inotropic action of cocaine may be related to a concentration-dependent effect: higher concentrations may inhibit calcium release from sarcoplasmic reticulum and block calcium influx across the sarcolemma. In contrast, lower concentrations would lead to a positive inotropic effect because of an impaired sarcolemmal sodium-calcium exchanger.

Action Potentials↗
PubMed1994

A digitized fluorescence imaging study on the effects of local anesthetics on cytosolic calcium and mitochondrial membrane potential in cultured rabbit corneal epithelial cells.

It has been documented by several investigators that local anesthetics displace calcium from calcium binding sites and alter the functioning of different calcium regulating systems. Local anesthetics have also been shown to have adverse effects on mitochondrial function and interact with cytoskeletal elements. Few studies have addressed the role that a potential disturbance of calcium homeostasis and mitochondrial function may have on the toxicity caused by local anesthetics in corneal epithelial cells. This investigation was undertaken to evaluate the effects of tetracaine (TTC), proparacaine (PPC), and cocaine (CC) on cytosolic calcium and mitochondrial membrane potential in primary cultures of rabbit corneal epithelial cells. Previous studies by our laboratory documented that the local anesthetics produce toxicity after 30 to 60 min of treatment. In this study, the cells were treated for 15 min, a time when minimal cell damage occurred. The following concentrations of local anesthetics were used to treat the cells: TTC, 0.5-2.5 mM; PPC, 1-5 mM; and CC, 4-10 mM. We utilized the technology of digitized fluorescence imaging to measure changes in intracellular calcium ([Ca2+]i) with fura-2 and mitochondrial membrane potential (delta psi) with rhodamine 123. A dose-dependent increase in [Ca2+]i was evident after treatment with each local anesthetic. Concentrations equal or greater than 2.5 mM TTC dissipated delta psi. A rise in [Ca2+]i preceded any loss of delta psi caused by TTC. PPC at high concentrations (4-5 mM) occasionally dissipated delta psi but this was not a consistent finding. The effects of CC on delta psi could not be evaluated accurately because of the extensive morphological alterations that occurred after treatment. We conclude that TTC, PPC, and CC elevate [Ca2+]i before cytotoxicity occurs and disruptions in calcium homeostasis may contribute to their toxicity.

Anesthetics, Local↗
PubMed1994

Comparison of the hepatotoxicity of toxin T-514 of Karwinskia humboldtiana and its diastereoisomer in primary liver cell cultures.

Toxin T-514 of Karwinskia humboldtiana has been demonstrated to be hepatotoxic in vivo and in vitro. Recently a diastereoisomer of T-514 has been isolated. In the present study we have evaluated and compared the in vitro hepatoxicity of the diastereoisomer of T-514 using primary cultures of rat hepatocytes. Cytotoxicity was evaluated by release of cytoplasmic enzyme lactate dehydrogenase (LDH), and mitochondrial metabolic function (MTT reduction). The diastereoisomer was shown to be almost as hepatoxic in vitro as toxin T-514.

Analysis of Variance↗
PubMed1994

Neutralization of the activity of a Fasciola hepatica cathepsin L proteinase by anti-cathepsin L antibodies.

Fasciola hepatica secretes a cathepsin L proteinase that is suggested to play an in vivo role in immunoprotection since the enzyme can cleave host immunoglobulin. In the present report, rabbit anti-cathepsin L IgG was shown to bind to the cathepsin L enzyme and inhibit its ability to cleave IgG molecules. Cathepsin L can prevent the antibody-mediated attachment of eosinophils to newly excysted juveniles in in vitro assays; however, if anti-cathepsin L IgG are mixed with the cathepsin L prior to the addition of the enzyme to the assay, eosinophils attach to the newly excysted juveniles. Thus it is possible to prepare antibodies that can bind and disrupt the biological activity of the F. hepatica cathepsin L.

Animals↗
PubMed1993

A digitized fluorescence imaging study of intracellular free calcium, mitochondrial integrity and cytotoxicity in rat renal cells exposed to ionomycin, a calcium ionophore.

The objective of this study was to explore the role of extracellular Ca2+ and mitochondrial integrity in ionomycin-induced cytotoxicity in primary cultures of rat kidney cortical epithelial cells using digitized fluorescence imaging (DFI), which is a powerful tool for continuously observing the dynamic intracellular biochemistry of single living cells. Using DFI, intracellular free calcium ion concentration ([Ca2+]i), mitochondrial membrane potential and loss of cell viability in individual rat renal cortical epithelial cells were examined temporally by fura-2, rhodamine 123 (Rh-123) and propidium iodide (PI), respectively. Images were taken within 10 min after exposure to 5 and 10 microM ionomycin. These three parameters, [Ca2+]i, mitochondrial membrane potential and cell viability, were also measured in populations of cells by a multiwell fluorescence scanner with fluo-3, Rh-123 and PI, respectively. Cytotoxicity was also assessed by two colorimetric cytotoxicity tests (LDH leakage and mitochondrial MTT reduction). Using DFI, the fluorescence scanner and the colorimetric cytotoxicity tests, we found that exposure of primary cultures of rat kidney cortical epithelial cells to high concentrations of ionomycin (5 and 10 microM) caused a rapid and sustained rise in [Ca2+]i, which preceded dissipation of the mitochondrial membrane potential and loss of cell viability and that chelation of extracellular Ca2+ with EGTA attenuated these responses. We demonstrated the value of using DFI to continuously observe the dynamic intracellular biochemistry of single living cells by establishing a sequence of elevated [Ca2+]i, dissipation of mitochondrial membrane potential and cytotoxicity. We conclude that a combination of the influx of extracellular Ca2+ and loss of mitochondrial integrity may be responsible for the cytotoxicity observed in individual renal cells and populations of renal cells after treatment with ionomycin.

Aniline Compounds↗
PubMed1993

On the role of macrophages in anthrax.

Bacillus anthracis, the causative agent of anthrax, produces systemic shock and death in susceptible animals, primarily through the action of its lethal toxin. This toxin, at high concentrations, induces lysis of macrophages in vitro but shows little or no effect on other cells. We found that when mice were specifically depleted of macrophages by silica injections, they became resistant to the toxin. Sensitivity could be restored by coinjection of toxin-sensitive cultured macrophages (RAW 264.7 cells) but not by coinjection of other cell lines tested. These results implied that macrophages mediate the action of lethal toxin in vivo and led us to investigate their role in death of the mammalian host. Sublytic concentrations of lethal toxin, orders of magnitude lower than those required to induce lysis of RAW 264.7 cells, were found to induce these cells to express interleukin 1 (IL-1) and tumor necrosis factor in vitro. Passive immunization against IL-1 or injection of an IL-1 receptor antagonist protected mice from toxin challenge, whereas anti-tumor necrosis factor provided little, if any, protection. These results imply that systemic shock and death from anthrax result primarily from the effects of high levels of cytokines, principally IL-1, produced by macrophages that have been stimulated by the anthrax lethal toxin.

Animals↗
PubMed1993

An in vitro model of cyclosporine-induced nephrotoxicity.

The predominant hypothesis for cyclosporine-induced acute renal failure is postulated to be prerenal vasoconstriction with concomitant hemodynamic changes; an alternate hypothesis, however, may be that cyclosporine (CsA) affects intrarenal processes, i.e., direct renal parenchymal cell injury. However, reports on this direct effect of CsA on renal parenchymal cells are contradictory. Therefore, the purpose of this study was to address whether CsA is directly toxic to renal parenchymal cells in a primary culture system of rat renal cortical epithelial cells. The cytotoxicity of Sandimmune, the commercial form of CsA in a polyoxyethylated castor oil vehicle (Cremophor), CsA without vehicle, and the Cremophor vehicle was assessed by plasma membrane integrity (lactate dehydrogenase leakage), mitochondrial metabolic activity [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction], and gross morphology (phase-contrast microscopy). The cytotoxicity of Sandimmune was also assessed by lysosomal activity (neutral red uptake), by proximal tubular enzyme activity (alkaline phosphatase), and by three fluorescent probes using a multiwell scanner. The three fluorescent probes were propidium iodide which stains nuclei of nonviable cells; bis-carboxyethyl-carboxyfluorescein which is retained by viable cells; and rhodamine 123, which assesses mitochondrial membrane potential. The results of this study demonstrated that Sandimmune caused dose- (10, 25, and 50 microM) and time- (12, 24, and 48 hr) dependent cytotoxicity, while Cremophor caused cytotoxicity only at high concentrations and long incubations. We conclude that (1) CsA is directly toxic to renal parenchymal cells in vitro and this system potentially represents a sensitive model for further mechanistic studies; (2) CsA plus vehicle (Sandimmune) was more cytotoxic to renal cells than CsA alone (without the polyoxyethylated castor oil vehicle).

Alkaline Phosphatase↗
PubMed1993

Cell membrane fatty acid composition in type 1 (insulin-dependent) diabetic patients: relationship with sodium transport abnormalities and metabolic control.

We have studied the fatty acid composition of erythrocyte membrane phospholipids in nine Type 1 (insulin-dependent) diabetic patients and nine healthy control subjects. Cell membranes from the diabetic patients showed a marked decrease in the total amount of polyunsaturated fatty acids (19.0% +/- 2.2 vs 24.6% +/- 1.4, p < 0.0001) mainly at the expense of docosahexaenoic acid C22:6(n3) (2.9% +/- 1.1 vs 5.3% +/- 1.3, p < 0.001), and arachidonic acid C20:4n6 (12.0% +/- 1.6 vs 15.1% +/- 0.6, p < 0.0005). Conversely, the total amount of saturated fatty acids was significantly increased (p < 0.05) and the polyunsaturated/saturated ratio was decreased in the Type 1 diabetic patients (p < 0.00 005). Neither the time from diagnosis, nor C-peptide levels, correlated with parameters indicating a poor metabolic control of Type 1 diabetes. However, C22:6(n-3) and total n-3 content significantly correlated with HbA1c (r = -0.79 and r = -0.88, respectively, p < 0.01), fructosamine (r = -0.71 and r = -0.74, respectively, p < 0.05), and Na+-K+ ATPase activity (maximal rate/Km quotient) (r = 0.78 and r = 0.71, respectively, p < 0.05). In conclusion we have found marked alterations of cell membrane lipid composition in Type 1 diabetic patients. These cell membrane abnormalities in lipid content were related to sodium transport systems and to poor metabolic control. Either diet, or the diabetic state, might be responsible for the observed cell membrane abnormalities. A dietary intervention study might differentiate the role of diet and diabetes in the reported cell membrane alterations.

Adult↗
PubMed1993

Microbial models of mammalian metabolism: involvement of cytochrome P450 in the N-demethylation of N-methylcarbazole by Cunninghamella echinulata.

1. As previously reported (Yang and Davis 1992), N-methylcarbazole (NMC) is converted to N-hydroxymethylcarbazole (NHMC), and 3-hydroxy-N-hydroxymethylcarbazole (3-OH-NHMC), two relatively stable carbinolamine metabolites by the fungus Cunninghamella echinulata (ATCC 9244). Decomposition of these two carbinolamines yields the corresponding dealkylated metabolites, carbazole and 3-hydroxycarbazole. In the present study, the possible involvement of cytochrome P450 in the requisite N-alkyl hydroxylation reaction was examined. 2. Carbon monoxide, a classical P450 inhibitor, markedly inhibited the formation of NHMC, as did potassium cyanide. 1-Benzylimidazole, piperonyl butoxide and SKF-525A inhibited the formation of both NHMC and 3-OH-NHMC, while beta-naphthoflavone (5,6-benzoflavone) induced their formation. 3. The source of the oxygen atom in the metabolite NHMC was examined by GC/MS analysis of NHMC formed during incubation of NMC in H218O-enriched medium which resulted in no incorporation of labelled oxygen into the metabolite. 4. An intermolecular isotope effect was not observed for the formation of NHMC suggesting that C-H bond cleavage is not a rate limiting step in the formation of this metabolite under the conditions examined. 5. It was concluded that P450 enzymes may be involved in the N-demethylation of NMC catalyzed by this fungal model of mammalian metabolism, and provides further support for biochemical and mechanistic parallels between mammalian metabolism and microbial systems catalyzing phase-1 biotransformations.

Carbazoles↗