Spontaneous gallbladder pathology in baboons.

BACKGROUND: Gallbladder pathology (GBP) is a relatively uncommon, naturally occurring morbidity in both baboons and humans. METHODS: A retrospective analysis was performed on 7776 necropsy reports over a 20 year period to determine the prevalence of baboon GBP. RESULTS: Ninety-seven cases of GBP were identified, yielding a 20 year population prevalence of 1.25%. GBP is more common in adult female baboons, occurring with a female to male ratio of nearly 2:1. Among gallbladder pathologies, cholecystitis (35.1%) and cholelithiasis (29.9%) were the most prevalent abnormalities, followed by hyperplasia (16.5%), edema (15.5%), amyloidosis (5.2%), fibrosis (4.1%), necrosis (4.1%), and hemorrhage (1.0%). CONCLUSION: Many epidemiologic similarities exist between GBP in baboons and humans suggesting that the baboon may serve as a reliable animal model system for investigating GBP in humans.

Toxicidade de fármacos nitrofurânicos / Toxicity of nitrofuran drugs

Durante o planejamento estrutural de novos fármacos, é possível prever a influência de grupamentos específicos na atividade farmacológica. Entre estes, encontra-se o grupo nitro, que possui potencial atividade antimicrobiana, estando presente em diversos fármacos como o metronidazol, nitrofural, furazolidona, oxamniquina, cloranfenicol, entre outros. Também, a introdução do grupo nitro na molécula pode alterar as propriedades físico-químicas e eletrônicas da substância, estando presente em fármacos de outras classes terapêuticas como anti-úlcera, ansiolítico, antiinflamatório. Entretanto, restrições têm sido apontadas para o planejamento de novos fármacos contendo este grupo, devido à toxicidade relacionada. Este estudo trata-se da revisão sobre a toxicidadede compostos nitrofurânicos, bem como os possíveis mecanismos e a utilização do método de latenciação na diminuição desta toxicidade.

Síntese e modelagem molecular do novo derivado indolinônico como candidato a antiinflamatório COX-2 seletivo / Synthesis and molecular modeling of a novel indolinone derivative as a candidate anti-inflammatory selectively inhibiting COX-2

Os fármacos antiinflamatórios são sabidamente os mais comercializados em todo o mundo, e apesar disto apresentam sérios efeitos colaterais, sobretudo no que se refere ao trato gastrintestinal. A descoberta de novos protótipos com atividade e segurança terapêutica melhoradas continua sendo uma busca constante. Com o adventoda química computacional torna-se mais fácil o estudo teórico do comportamento fisiológico de uma nova substância bem como a compreensão do possível mecanismo de ação destas novas moléculas. Assim, através de modelos matemáticos de moléculas e receptores estudou-se neste trabalho o composto I (1-(2,6- diclorofenil)indolin-2-ona) quanto à sua possibilidade deinibir seletivamente a isoforma COX-2 da enzima prostaglandina endoperóxido sintase (PGHS), e também as melhores posições para a introdução de grupamentos químicos e modificações moleculares.

In vitro cytotoxicity of the nitric oxide donor, S-nitroso-N-acetyl-penicillamine, towards cells from human oral tissue.

The cytotoxicity of the nitric oxide donor, S-nitroso-N-acetyl-penicillamine (SNAP), towards cultured human cells from oral tissue was evaluated. The toxicity of SNAP to Smulow-Glickman gingival epithelial cells was correlated with the liberation of nitric oxide, as N-acetyl-D,L-penicillamine, the SNAP metabolites, N-acetyl-D,L-penicillamine disulfide and nitrite, and preincubated (denitrosylated) SNAP did not affect viability. Comparing equimolar concentrations of various nitric oxide donors, cytotoxicity appeared to be inversely related to the relative stability (i.e., half-life) of the test compound; the sequence of cytotoxicity for a 4 hr exposure was S-nitrosoglutathione>>spermine NONOate> SNAP>DPTA NONOate>>DETA NONOate. Intracellular reduced glutathione (GSH) was lowered in S-G cells exposed to SNAP. Pretreatment of the cells with the GSH depleter, 1,3-bis-(chloroethyl)-1-nitrosourea (BCNU), enhanced the toxicity of SNAP Similar findings of enhanced sensitivity to SNAP were noted with gingival fibroblasts and periodontal ligament cells pretreated with BCNU. The toxicity of SNAP towards the gingival epithelial cells was decreased by cotreatment with the antioxidants, N-acetyl-L-cysteine, L-ascorbic acid, and (+)-catechin. Cells exposed to SNAP exhibited nuclear aberrations, including multilobed nuclei and multinucleation. SNAP-induced cell death was apparently by apoptosis, as noted by fluorescence microscopy and DNA agarose gel electrophoresis.

In vitro toxicity of sodium nitroprusside to human endothelial ECV304 cells.

The cytotoxicity of sodium nitroprusside (SNP) to the human endothelial cell line, ECV304, was studied. The cytotoxicity of SNP was primarily related to the liberation of nitric oxide (NO). S-nitroso-N-acetyl-d-penicillamine (SNAP), an NO donor, was highly toxic. Other degradation products of SNP either exerted much less toxicity (i.e. cyanide and nitrite) or were non-toxic (i.e. ferricyanide and ferrocyanide). SNP induced multinucleation, inhibited cell proliferation, lowered the endogenous level of reduced glutathione (GSH), and induced apoptotic cell death. The plasma membrane was not the prime site of toxic action, as leakage of lactic acid dehydrogenase (LDH) occurred only at a relatively high concentration of SNP. Cells treated with non-toxic levels of the glutathione-depleting agents, 1-chloro-2,4-dinitrobenzene (CDNB), dl-buthionine-[S,R]-sulfoximine (BSO), and 1,3-bis-(chloroethyl)-1-nitrosourea (BCNU), were hypersensitive to subsequent exposure to SNP. The GSH status of the cells was, therefore, a key factor in determining the cytotoxicity of SNP.

Benzoyl peroxide cytotoxicity evaluated in vitro with the human keratinocyte cell line, RHEK-1.

The human keratinocyte cell line, RHEK-1, was used to evaluate the cytotoxicity of benzoyl peroxide (BZP). As determined with the neutral red (NR) cytotoxicity assay, the 24-h midpoint (NR50) toxicity values, in mM, were 0.11 for BZP and 29.5 for benzoic acid, the stable metabolite of BZP. Irreversible cytotoxicity occurred after a 1-h exposure to 0.15 mM BZP and greater. When exposed to BZP for 7 days, a lag in growth kinetics was first observed at 0.06 mM BZP. Damage to the integrity of the plasma membrane was evident, as leakage of lactic acid dehydrogenase occurred during a 4-h exposure to BZP at 0.05 mM and greater. Intracellular membranes were also affected, as extensive vacuolization, initially perinuclear but then spreading throughout the cytoplasm, was noted in BZP-stressed cells. The generation of reactive free radicals from BZP was suggested by the following: the intracellular content of glutathione was lowered in cells exposed to BZP; cells pretreated with the glutathione-depleting agent, chlorodinitrobenzene, were hypersensitive to a subsequent challenge with BZP; lipid peroxidation by BZP was inducible in the presence of Fe2+; and cells previously maintained in a medium amended with vitamin E, an antioxidant, were more resistant to BZP, showed less lipid peroxidation in the presence of BZP+Fe2+ and did not develop the extensive intracellular vacuolization as compared to non-vitamin E maintained cells.

An in vitro study on the cytotoxicity of chlorhexidine digluconate to human gingival cells.

Chlorhexidine digluconate is the active ingredient in mouthrinses used to prevent dental plaque and gingivitis. The in vitro cytotoxicity of chlorhexidine was evaluated with the Smulow-Glickman (S-G) gingival epithelial cell line. The potency of chlorhexidine was dependent on the length of exposure and composition of the exposure medium. The midpoint cytotoxicity values for 1-, 24-, and 72-h exposures were 0.106, 0.011, and 0.0045 mmol/L, respectively. S-G cells exposed for 2 h to chlorhexidine and then maintained for 48 h in chlorhexidine-free medium were unable to recover from the initial insult. The adverse effects of chlorhexidine on the plasma membrane were suggested by the leakage of lactic acid dehydrogenase from chlorhexidine-treated S-G cells and by the increased permeability of chlorhexidine-treated liposomes to Ca2+. The toxicity of a 24-h exposure to chlorhexidine to the S-G cells was progressively lessened as the content of fetal bovine serum (FBS) in the exposure medium was increased from 2% to 8%. The potency of a 1-h exposure to chlorhexidine was reduced in medium amended with albumin, lecithin, and heat-killed Escherichia coli. These reductions in toxicity were presumably due to the binding of the cationic chlorhexidine to the negatively charged chemical moieties of the components of FBS and of albumin and lecithin and of sites on the surfaces of bacteria. Combinations of chlorhexidine and carbamide peroxide were additive in their cytotoxicities.

In vitro cytotoxicity of the chlorinated naphthoquinone dichlone to human endothelial ECV304 cells.

The cytotoxicity of the pro-oxidant fungicide dichlone (2,3-dichloro-1,4-naphthoquinone), to the human endothelial cell line, ECV304, was evaluated. The sensitivity of these cells to dichlone was intermediate between that of human hepatoblastoma HepG2 cells (least sensitive) and that of human GMO5757 fibroblasts. The midpoint cytotoxicity values for a 24-hr exposure to dichlone was about 0.02 mm when evaluated with the neutral red, acid phosphatase, and XTT tetrazolium assays. Lactic acid dehydrogenase leakage, after a 4-hr exposure, occurred initially at 0.05 mm dichlone. As with other naphthoquinones, cellular metabolism of dichlone presumably could proceed either by a one- or a two-electron reduction reaction. The enhancement of potency of dichlone towards ECV304 cells pretreated with the glutathione-depleting agents, dl-buthionine-[S,R]-sulfoximine, 1-chloro-2,4-dinitrobenzene, and 1,3-bis(chloroethyl)-1-nitrosourea; the reduction in potency of dichlone to cells pretreated with (-)-2-oxo-4-thiazolidine carboxylic acid; the decrease in intracellular glutathione on exposure to dichlone; the subtle damage to the plasma membrane of dichlone-treated cells (as detected by the leakage of lactate dehydrogenase from these cells); and the lack of potentiation of dichlone toxicity by pretreatment with dicoumarol, are all consistent with the one-electron reduction reaction as the dominant pathway and with the subsequent generation of reactive oxygen molecules. The ECV304 cell line proved to be a useful research tool to study cytotoxic injury to endothelial cells.

Nonsteroidal antiinflammatory drugs exert differential effects on neutrophil function and plasma membrane viscosity. Studies in human neutrophils and liposomes.

Nonsteroidal antiinflammatory drugs (NSAIDs) inhibit neutrophil functions via mechanisms separate from their capacity to inhibit prostaglandin synthesis. We have studied discrete events in the process of signal transduction: NSAIDs but not a related analgesic drug (acetaminophen), inhibited aggregation in response to the chemoattractants f-Met-Leu-Phe (FMLP), leukotriene B4, and C5a. NSAIDs, but not acetaminophen, inhibited binding of radiolabeled FMLP to purified neutrophil membranes. Gpp(NH)p, a GTPase insensitive analog of GTP, also inhibited the binding of FMLP but, paradoxically, enhanced superoxide anion generation and lysozyme release. The inhibition of ligand binding by NSAIDs did not correlate with their capacity to inhibit FMLP-induced increments in diacylglycerol (DG): piroxicam, but not salicylate effectively inhibited appearance of label ([3H]arachidonate, [14C]glycerol) in DG. Finally, NSAIDs exerted differential effects on the viscosity of neutrophil plasma membranes and multilamellar vesicles (liposomes): membrane viscosity was increased by piroxicam and indomethacin, decreased by salicylate, and unaffected by acetaminophen. Thus, the different effects of NSAIDs on discrete pathways are not due to their shared capacity to reduce ligand binding but rather to a capacity to uncouple postreceptor signaling events that depend upon the state of membrane fluidity.

Transmembrane calcium movements mediated by ionomycin and phosphatidate in liposomes with Fura 2 entrapped.

A novel liposomal method permits studies of Ca movements across the bilayers of multilamellar vesicles (MLV) which had entrapped the Ca-dependent, fluorescent indicator dye Fura 2. Ionomycin-mediated Ca translocation across MLV of phosphatidylcholine (PC)/dicetyl phosphate (DCP), 9:1, obeyed simple first-order kinetics since log-log plots of initial rates versus ionomycin or Ca concentration yielded slopes of approximately 1. Since Ca is translocated in a Ca-dependent fashion in the course of stimulus-response coupling of cells which form diacylglycerol (DAG) and phosphatidate (PA) from polyphosphoinositides, we compared effects of PA with those of DAG. PA and DAG were preincorporated in PC/DCP vesicles, in which trace amounts of ionomycin provided transmembrane potential (due to Ca2+/H+ exchange). Significant increases in Ca movements were observed in the presence of egg lecithin PA, dioleoyl-PA, and dipalmitoyl-PA when compared with DCP- or DAG-containing MLV. DAGs such as 1-oleoyl-2-acetoylglycerol or 1,2-dioleoylglycerol in liposomes decreased rates of Ca translocation. Ca influx into PA-containing MLV was dependent on the mole percent of the PA in bilayers; the complex kinetics of Ca influx were compatible with the formation of nonbilayer states. Incorporation of cholesterol into the liposomes inhibited initial rates of Ca uptake by MLV presumably by condensing the bilayers. Ca influx increased with increasing pH of the external medium from 6.9 to 7.9 in liposomes with an internal pH of 7.4.(ABSTRACT TRUNCATED AT 250 WORDS)

Permeability behavior of liposomes prepared from fatty acids and fatty acid methyl esters.

The permeability properties of liposomes prepared at pH 8.7 from a fatty acid and either methyl oleate or methyl elaidate, with or without cholesterol, were investigated. The fatty acids used were oleic acid, elaidic acid, and the selenium-containing fatty acids 9-selenaheptadecanoic acid and 13-selenaheneicosanoic acid. The liposomes trapped sucrose and carboxyfluorescein. Their volume change resulting from osmotic shock was directly proportional to the change in absorbance (light scattering). Liposomes prepared from oleic acid and either methyl oleate or methyl elaidate underwent osmotic swelling much more slowly than liposomes prepared from elaidic acid and either methyl oleate or methyl elaidate. Incorporation of cholesterol decreased the initial rate of erythritol permeation, especially in liposomes containing methyl oleate. The swelling rates of liposomes prepared with the selenium-containing fatty acids indicated that incorporation of methyl elaidate gave more tightly packed bilayers than did incorporation of methyl oleate. The effect of cholesterol on the initial rate of erythritol influx was greater in oleic acid and elaidic acid liposomes than in selenium-containing fatty acid liposomes, indicating that the large bulk of the selenium heteroatom suppresses the ability of cholesterol to interact with the hydrocarbon chain.

The stoichiometry of A23187- and X537A-mediated calcium ion transport across lipid bilayers.

Initial rates of ionophore-mediated Ca2+ transport across egg phosphatidylcholine bilayers of large unilamellar vesicles were measured using the absorbance change of arsenazo III at 650 nm as an indicator of Ca2+ translocation. A23187 induced the movement of Ca2+ in a 2:1 ionophore: Ca2+ complex, whereas its methyl ester (CH3A23187) and X537A mediated Ca2+ movement in a 1:1 ionophore: Ca2+ complex. The relative potencies of these ionophores in transporting Ca2+ across lipid membranes were A23187 much greater than X537A greater than CH3A23187.

Organization of unesterified cholesterol in high density lipoproteins probed by filipin.

The initial rate of filipin association with unesterified cholesterol in high density lipoproteins (HDL) was measured by stopped-flow spectrophotometry to assess the roles played by apolipoproteins and phospholipids in modulating the surface exposure of cholesterol. The initial rate of filipin-unesterified cholesterol association was enhanced upon hydrolysis of the glycerophospholipids of human HDL3 by phospholipase A2. Rate enhancements were also observed following trypsin-catalyzed hydrolysis of apolipoprotein A-I in canine HDL and of apolipoproteins A-I and A-II in human HDL3. However, the initial rate of filipin-unesterified cholesterol association was not altered upon incubation of HDL3 with polymorphonuclear cells, which causes hydrolysis of apolipoprotein A-II but leaves apolipoprotein A-I intact. These results are consistent with the general structural model of HDL in which unesterified cholesterol, apolipoproteins and glycerophospholipids are presumed to be localized at the surface of the HDL particle. From these studies and from results indicating that the initial rate of filipin-unesterified association was enhanced in canine HDL hybrids in which 50% of the apolipoprotein A-I had been replaced by apolipoprotein A-II, we also conclude that apolipoprotein A-I in HDL is in closer proximity to unesterified cholesterol than apolipoprotein A-II. Thus, it appears that rapid kinetic measurements of filipin-cholesterol association may be useful in assessing the organization of unesterified cholesterol in serum lipoproteins.

Determination of cholesterol asymmetry by rapid kinetics of filipin-cholesterol association: effect of modification in lipids and proteins.

The rapid kinetic behavior of filipin association with cholesterol was unaffected by binding of water-soluble proteins to vesicle and mycoplasma membranes and by proteolytic digestion of mycoplasma membrane proteins. The kinetic properties were, however, dependent on the membrane phospholipids, in that the initial rate of filipin association with cholesterol was enhanced by phospholipase A2 treatment by the incorporation of lysophosphatidylcholine, and by increasing the degree of unsaturation in phospholipid vesicles and mycoplasma membranes. The second-order rate constant was also dependent on th mol % of cholesterol in small unilamellar vesicles but not in large unilamellar vesicles. The ratio of rate constants in intact mycoplasma cells relative to isolated membranes provides an estimate of cholesterol distribution in membranes [Bittman, R., & Rottem, S. (1076) Biochem. Biophys. Res. Commun. 71, 318; Clejan, S., Bittman, R., & Rottem, S. (1978) Biochemistry 17, 4579]. This ratio was unaffected by proteolytic digestion of intact cells and by the incorporation of exogenous phospholipids into the Mycoplasma capricolum cell membrane. However, on cross-linking of surface proteins of M. capricolum by dimethylsuberimidate, cholesterol was localized predominantly in the outer half of the bilayer. On aging of mycoplasma cultures, the cholesterol distribution remained constant in membranes of M. capricolum cells but was enriched in the outer leaflet of the Mycoplasma gallisepticum cell membrane. The results of these experiments are discussed in relation to the use of the rapid kinetics of filipin binding as a probe of cholesterol distribution.

Cholesterol distribution between the two halves of the lipid bilayer of human erythrocyte ghost membranes.

The binding of filipin with cholesterol in sealed and unsealed ghosts prepared from human erythrocytes and in right-side-out and inside-out vesicles prepared from ghosts follows second order kinetics (first order in each reactant). The second order rate constant of interaction of filipin with cholesterol, determined by stopped flow measurements of the initial rate, is slower in sealed ghosts than in unsealed ghosts by a factor of 2.0, whereas identical rate constants were obtained with right-side-out and inside-out vesicles. These results suggest that the cholesterol accessible to rapid reaction with filipin is distributed symmetrically between the inner and outer halves of the lipid bilayer of erythrocyte ghost membranes.

Stopped-flow kinetic studies of actinomycin binding to DNAs.

Stopped-flow kinetic studies of the association of actinomycins with narural and synthetic DNA duplexes are presented. The actinomycins examined were D (C1), D lactam (in which the pentapeptide rings are closed by lactam instead of lactone linkages), X2, XObeta, and actinomine. The DNAs used included claf-thymus DNA, PM2, DNA, and two synthetic d(A-T)-lide copolymers containing 2,6-diaminopurine (DAP) in place of adenine residues, poly[d(DAP-T)]-poly[d(DAP-T)] and poly[d(DAP-A-T]-poly[d(DAP-A-T)]. Apparent equilibrium constants indicate that the DAP-containing polynucleotides bind actinomycin strongly. Comples formation of actinomycins D, D lactam, X2 and XObeta with these DNAs can be deconvoluted into five rate processes. These steps do not necessarily proceed to completion. The rates of two of these steps display a firstorder dependence on DNA concentration. The large negative entropies of activation of these steps suggest a high degree of restriction to freedom of motion on the respective transition states. The rates of the remaining three steps are independent of DNA concentration. Kinetic parameters of actinimycin binding to DNAs are presented and suggestions are made about some of the molecular evente believed to be responsible for the appearance of the five rate processes. For example, for DNA, poly[d(DAP-A-T)], and poly[d(DAP-T)], the observed order of apparent second-order rate constants, normalized to the concentration of actinomycin binding sites, suggests that binding of the antibiotic occurs most rapidly at binding sites (G-C of d DAT-T) near d(A-T) base pairs, where weakening of the double-helical conformation requires the least energy. Results obtained from studies of actinomycin D binding to heat-denatured poly[d(DAP-A-T)] and of actinomine and actinomycin D lactam binding to DNA suggest that the slow rate processes are related to an actinomycyl-pentapeptide-induced unwinding of the sugar-phosphate backbone of DNA accompanying insertion of the cyclic peptides into DNA.