Acute toxicity of polyethylene glycol p-isooctylphenol ether in Syrian hamsters exposed by inhalation or bronchopulmonary lavage

Dose-response studies were conducted with Syrian hamsters exposed to polyethylene glycol p-isooctylphenyl ether (Triton X-100) via inhalation or bronchopulmonary lavage. Syrian hamsters were exposed to an aerosol of Triton X-100 with a mass median aerodynamic diameter of 1.5 μm and a concentration of 3.0 mg/liter. Estimated initial lung burdens of Triton X-100 ranged from 800 to 3100 μg. Hamsters were lavaged with concentrations of Triton X-100 ranging from 0.01 to 0.10% in isotonic saline resulting in initial lung burdens of Triton X-100 that ranged from 300 to 3200 μg. The $\text{LD}\text{50}\text{7}$ values were 1700 μg (1300–2100 μg, 95% confidence limits) for the inhalation study and 2100 (1900–2700) μg for the lavage study. The difference between the $\text{LD}\text{50}\text{7}$ values for the two methods of exposure was not significant. However histopathological examination revealed differences in the nature and distribution of pathologic changes observed in animals exposed by the two routes of administration. Animals exposed by inhalation died as a result of ulcerative laryngitis and laryngeal edema with only minimal pulmonary pathologic alterations. Animals exposed by lavage, where the larynx was not exposed to Triton X-100, died from pulmonary edema and acute exudative pneumonia, these results demonstrate the need for careful selection of exposure methods to meet the specific objectives of a toxicology study.     

Some effects of non-ionic detergent triton X-100 on rat liver monoamine oxidase

The non-ionic detergent Triton X-100, an agent used to solubilize mitochondrial membrane monoamine oxidase (EC 1.4.3.4, MAO), has been shown to inhibit markedly MAO activity. The inhibition was non-competitive in nature. Triton X-100 changed the susceptibility of MAO toward clorgyline, a specific type A MAO inhibitor, and deprenyl, a type B inhibitor. Its effect on the temperature dependence of the initial velocity revealed that the transition temperatures for p-tyramine and serotonin (22°) and β-phenylethylamine (16° and 27°) were not changed. The stability of the MAO decreased considerably, however, in the presence of Triton X-100, and its inactivation was particularly pronounced somewhat higher temperatures.     

Characterization of a phosphatidic acid phosphatase from rat brain cell membranes

We have characterized a phosphatidic acid phosphatase (PAP, EC 3.1.3.4) that is associated with cell membranes from rat brain using [³²P] phosphatidic acid as substrate in a simple assay. The enzyme could be activated by Triton X-100, cholic acid and Chaps and inhibited by Lubrol PX and sodium dodecyl sulfate. The optimal pH was between 6.0 and 7.0. Mg²⁺ was not essential for enzyme activity. The enzyme activity was decreased by about 50% by Ca²⁺ at concentrations of 0.1 to 1 mmol/1. Zn²⁺ inhibited the enzyme by 50% at concentrations of about 10 mol/l in the absence of, and 100 nmol/1 in the presence (3 mmol/1) of, Triton X-100. NaF decreased the activity by about 50% at concentrations between 0.3 and 1 mmol/l when Triton X-100 was added, but did not inhibit the enzyme if the detergent was not present. N-Ethylmaleimide (NEM) did not affect the enzyme. In the absence of Triton X-100, propranolol and metoprolol enhanced the PAP activity. In the presence of 3 mmol/1 Triton X-100, the enzyme was inhibited by about 50% by propranolol at a concentration of 10 mmol/l, whereas metoprolol caused only a slight inhibition of PAP. The K _m for phosphatidic acid was 150 mol/1 and was changed to 20 mol/1 by 3 mmol/1 Triton X-100 without the V _max being changed. Enzyme activity could be solubilized by 1–5% (w/v) Triton X-100. Gel filtration chromatography showed a M _r of 320000. This membrane-associated PAP from neuronal tissue probably belongs among the NEM-insensitive forms of PAP enzymes which have been proposed to play a role in transmembrane signal transduction via phospholipase D. Correspondence to: Ariane Hoer at the above address     

On non-specific inhibition of rat liver microsomal UDP glucuronyltransferase by some drugs

The effect of imipramine. desipratnine, harmol, harmalol and some monoamine oxidase inhibitors of the hydrazine type on rat liver UDP glucuronyltransferase (EC 2.4.1.17) activity has been investigated. Substrates of the enzyme were p-nitrophenol and bilirubin; with p-nitrophenol only Triton X-100 activated microsomes were used as enzyme source, with bilirubin both not-activated and Triton X-100 activated microsomes were used. The degree of inhibition obtained with the various inhibitors was dependant on the substrate used and on the pretreatment of the microsomes. It is concluded that most of the effects were caused by the action of the compounds on microsomal membrane structure, which affects UDP glucuronyltransferase activity, and that the physiological relevance of inhibition of the enzyme in vitro is questionable.     

ProZZ-EGFP融合蛋白基因在大肠杆菌中分泌表达条件的优化

目的优化大肠杆菌分泌表达ProZZ-EGFP融合蛋白基因的培养条件。方法采用摇瓶培养,在液体培养基中加入终浓度不同的蔗糖、Triton X-100和甘氨酸,诱导大肠杆菌周质腔内蛋白质“泄漏”到液体培养基中,利用ProZZ-EGFP浓度-荧光强度标准曲线快速检测培养基中目的蛋白质浓度。结果利用大肠杆菌HB101表达ProZZ-EGFP融合蛋白,在培养基中含有终浓度1%Triton X-100及1%甘氨酸,可使ProZZ-EGFP在培养液的分泌表达量提高6倍。结论仅在培养基中加入几种物质即可提高ProZZ-EGFP融合蛋白的分泌表达量,简单易行。     

GABA and benzodiazepine receptors in the offspring of dams receiving diazepam: ontogenetic studies

Ontogenesis of the regulation of ³H-GABA and ³H-diazepam binding to rat brain plasma membranes treated with 0.05% Triton X-100 has been studied. The density of ³H-diazepam and ³H-GABA binding in cortex, cerebellum and corpus striatum at birth was approximately one third of the adult values. They increased at the same rate and reached the adult values between 14–21 days after birth. Study of the binding characteristics showed that the K_D for high and low affinity for ³H-GABA, and for ³H-diazepam did not change during ontogenesis and the increase reflects only an increase of Bmax. The number of Triton X-100 treatments of crude synaptic membrane (CSM) required to maximize ³H-GABA for the high affinity component were different at various postnatal days: only one treatment was required in 1-day old rats, two in 7- and 14-day old rats and three in adult animals. In addition, the capability of muscimol to stimulate ³H-diazepam binding in both frozen-thawed and Triton X-100 treated membrane preparations decreased with increasing age. Binding of ³H-GABA and ³H-diazepam to brain of newborn rats whose dams received diazepam throughout pregnancy (100 mg/kg, × os, bid) was also studied. No significant differences were observed in the ontogenetic development of both bindings. However, in the cortex of these newborn rats the capability of muscimol to stimulate ³H-diazepam binding was greatly reduced in Triton X-100-treated membranes.     

Evaluation of the formazan extraction step of Int-dehydrogenase assay

An essential part of in vivo INT-dehydrogenase assay is quantitation of the intensely colored, water-insoluble iodonitrotetrazolium formazan (INTF) that forms intracellularly as the product of INT reduction. This procedure involves extraction of INTF from microbial cells by an organic solvent, permitting colorimetric measurement of the formazan. In the present study, it was found that dimethylsulfoxide (DMSO) provided the most consistent extraction of INTF from activated sludge and filamentous bacteria. The relative efficiency of 2 + 3 tetrachloroethylene/acetone (TA) was slightly greater than that of DMSO in some cases. However, this solvent only partially extracted INTF from one of the filament types tested (type 1701) and its performance was impaired by adsorption of ferrous ion to sludge. Methanol was generally the least efficient of the solvents tested. It was particularly ineffective in extracting INTF from Fe²⁺-treated sludges. Permeabilization of cells with lysozyme or Triton X-100 improved the INTF yields obtained with methanol. Triton X-100,the more effective of the two permeabilizing agents, increased the INTF yield obtained with methanol to the same level as that obtained with DMSO or TA.     

Inhibition of DNA synthesis and alteration to DNA structure by the phenacetin analog p-aminophenol

p-Aminophenol a structural analog and minor metabolite of phenacetin has previously been shown to be a potent nephrotoxic agent. In this report we have shown that p-aminophenol has a marked effect on DNA function and structure. DNA synthesis was inhibited in a dose-dependent manner in human lymphoblastoid cells after exposure to p-aminophenol. Results suggest that DNA synthesis is inhibited by the action of p-aminophenol on DNA structure. At low concentrations of p-aminophenol a reduction in the degree of supercoiling of cellular DNA is observed, as determined by sedimentation under neutral conditions. However at higher concentrations an increase in sedimentation of nucleoids (supercoiled molecules) is obtained which is indicative of an increased level of supercoiling or a more compact structural form of DNA due to folding or aggregation.The number of single strand breaks in DNA, when determined by sedimentation in alkaline sucrose gradients, increases with increasing dose of p-aminophenol. The increase in strand breakage observed at lower concentrations of p-aminophenol agrees with the reduced sedimentation rate obtained under neutral conditions. At higher concentrations of p-aminophenol the extent of breakage of DNA increases under alkaline conditions but an increase in sedimentation occurs under neutral conditions.     

In Vitro Evaluation of Polymerized Liposomes as an Oral Drug Delivery System

The physical characteristics of polymerized liposomes for potential use as an oral drug delivery system were examined in vitro. The trap efficiency in monomeric liposomes composed of 1,2-di (2,4-octadecadienoyl) phosphatidylcholine was increased from 3% for original multilamellar vesicles to 35% for freeze-thaw treated liposomes. Polymerized liposomes with azobis (isobutyronitrile) and azobis (2-amidinopropane) hydrochloride as radical initiators showed complete stability against solubilization by Triton X-100, a detergent chosen to mimic bile salts. Release rates of ¹⁴C-BSA and ¹⁴C-sucrose in media simulating the gastro-intestinal fluids was 50% less than from regular liposomes composed of hydrogenated egg phosphatidylcholine mixed with cholesterol (molar ratio 1:1), which can be regarded as one of the most stable types of regular liposomes. It was estimated that, when administered orally, polymerized liposomes can reach the intestine while maintaining their vesicle structure and keeping at least 75% of their original content.     

Rapid determination of platinum by flameless atomic absorption spectrophotometry following the administration of cisplatin to cancer patients

An improved flameless atomic absorption spectrophotometric method for the analysis of platinum in plasma is described. Following a simple dilution with Triton X-100, the sample is directly injected into the graphite furnace with a total analysis time of less than 2 min. The assay is precise (CV less than 4.3%) and linear (r greater than 0.9922) in the ranges 0.05-1 and 1-4 mg/L for cisplatin. The applicability of the method is examined by analyzing samples obtained at different intervals from two patients treated with cisplatin.     

The effect of sodium deoxycholate and other surfactants on the mucosal surface pH in proximal jejunum or rat

Summary The mucosal surface pH (acid microclimate) and nucleotide levels of rat proximal jejunum were measured in vivo under various conditions which included exposure to pharmacological agents and to surfactants. Mucosal surface pH was unaffected by sodium nitroprusside, A23187 and amiloride, as was mucosal cGMP content, although amiloride and A23187 reduced cAMP content. In contrast, surfactants elevated the pH of the mucosal surface significantly (P < 0.001): control value 6.23 ± 0.02 (n = 12); Lubrol PX 0.8% (v/v) 6.98 ± 0.02 (n = 5); sodium deoxycholate 2 mmol/l 6.67 ± 0.04 (n = 5); Triton X-100 0.5% (v/v) 7.41 ± 0.03 (n = 5). No significant changes in cGMP levels were noted after surfactant treatment, although DOC and Triton X-100 reduced cAMP levels. The ability of higher concentrations of surfactant to elevate the mucosal surface pH beyond neutrality to values similar to plasma pH contrasts with the action of Escherichia coli heat-stable (STa) enterotoxin which at high concentrations could not elevate the mucosal surface pH beyond neutrality. Consistent with the known effects on tight junction permeability, surfactants may act by allowing plasma-like subepithelial fluid to neutralise the microclimate. Send offprint requests to M. L. Lucas at the above address     

Lactate dehydrogenase isoenzymes in hamster lung lavage fluid after lung injury

Lactate dehydrogenase (LD) levels and isoenzyme patterns were determined in the cell-free supernatant fractions of lung lavage fluid from hamsters exposed to alpha-quartz, iron oxide, Triton X-100, 100% O₂, or 200 ppm SO₂. The isoenzyme patterns were compared to those derived from hamster lung homogenates, serum, polymorphonuclear neutrophils (PMNs), pulmonary macrophages, and red blood cells. The isoenzyme patterns from alpha-quartz- and iron oxide-exposed animals resembled each other and were similar to that of PMNs. In contrast, the pattern seen after Triton X-100 exposure was similar to those of whole lung homogenates and of red blood cells. A 96-hr exposure to 100% O₂ yielded an LD isoenzyme pattern in lung lavage fluid similar to that of serum. Exposure to SO₂ did not alter LD levels, showing that upper airways damage is not reflected by changes in LD in lung lavage fluid. We conclude that LD isoenzyme patterns of lung lavage fluid can be used to differentiate among types of pulmonary injury and may help identify the sites of injury.     

Evaluation of electrophoretic method versus chromatographic, potentiometric and absorptiometric methodologies for determing pKa values of quinolones in hydroorganic mixtures

The advantages of using capillary electrophoresis (CE) over other methodologies for determining pK_a values of drugs in hydroorganic media are discussed. The focus of the discussion based upon the pK_a values of a series of quinolones determined in acetonitrile (MeCN)–water mixtures by CE, liquid chromatography, potentiometric, and spectrophotometric methods.     

A comparative study of the deacetylation of paracetamol by urodele and anuran amphibian organ cultures

1. p-Aminophenol is the major metabolite produced by urodele amphibian tissues in vitro. The deacetylation enzyme system involved is located in the liver microsomal fraction of Amphiuma means.

2. Paracetamol was deacetylated to p-aminophenol by organ cultures of liver, kidney, pancreas and stomach epithelium from adult A. means, by liver, stomach epithelium, bladder and lung cultures from neotenic larval Ambystoma tigrinum, and by adult Triturus cristatus carnifex in vivo.

3. Liver cultures from all seven urodele amphibian species metabolized paracetamol and produced p-aminophenol, but no evidence was found of paracetamol metabolism by liver cultures from any of five anuran amphibian species.

4. The significance of p-aminophenol production from paracetamol and of this difference within the Amphibia are discussed.     

Binding of botulinum neurotoxin to the synaptosome fraction of rat brain

Summary The radioactive ¹²⁵I-labelled neurotoxin of C. botulinum type A, when incubated with rat brain homogenate, is bound selectively to the synaptosome fraction. Intact toxin was liberated from the synaptosome fraction by treatment with Triton X-100, SDS, trypsin or neuraminidase.     

Functions, structures and Triton X-100 effect for the catalytic subunits of heterodimeric phospholipases A2 from Vipera nikolskii venom

Phospholipases A₂ (PLA₂s) from snake venoms have diverse pharmacological functions including neurotoxicity, and more studies are necessary to understand relevant mechanisms. Here we report the different crystal structures for two enzymatically active basic subunits (HDP-1P and HDP-2P) of heterodimeric neurotoxic PLA₂s isolated from Vipera nikolskii venom. Structural comparisons with similar PLA₂s clearly show some flexible regions which might be important for the catalytic function and neurotoxicity. Unexpectedly, Triton X-100 molecule bound in the hydrophobic channel of HDP-1P and HDP-2P was observed, and its binding induced conformational changes in the Ca²⁺ binding loop. Enzymatic activity measurements indicated that Triton X-100 decreased the activity of PLA₂, although with comparatively low inhibitory activity. For the first time exocytosis experiments in pancreatic β cells were used to confirm the presynaptic neurotoxicity of relevant snake PLA₂. These experiments also indicated that Triton X-100 inhibited the influence of HDP-1P on exocytosis, but the inhibition was smaller than that of MJ33, a phospholipid-analogue inhibitor of PLA₂. Our studies performed at a cellular level are in good agreement with earlier findings that enzymatic activity of the snake presynaptic PLA₂ neurotoxins is essential for effective block of nerve terminals.     

Biochemical characterisation of para-aminophenol-induced nephrotoxic lesions in the F344 rat

The acute biochemical effects of the nephrotoxin p-aminophenol (PAP) were studied in detail using a combination of conventional bioanalytical and ¹H-NMR spectroscopic methods. Dosing PAP (25–100 mg/kg) to male F344 rats resulted in a dose-related proximal nephropathy with consequent elevations in urinary enzymes, glucose, and urine total protein as shown by conventional methodology. ¹H-NMR spectroscopy at 400 MHz of urine from PAP-treated rats also revealed a characteristic glycosuria, with concomitant amino aciduria. The increased excretion of these compounds indicates functional defects in the proximal tubule and reduced solute reabsorption efficiency. In addition, ¹H-NMR urinalysis and conventional enzymatic analysis showed a dose-related lactic aciduria. Other changes detected by ¹H-NMR included a dose-related reduction in the excretion of citrate (confirmed by a conventional biochemical method) and an increase in the excretion of acetate. The degree of abnormalities shown by ¹H-NMR urinalysis agreed well with histopathological observations and conventional biochemical indices of nephrotoxicity. ¹H-NMR urinalysis therefore serves to highlight changes in the excretion of low MW urine components not routinely studied by conventional biochemical analysis.Abbreviations ALP alkaline phosphatase - APAP paracetamol - BUN blood urea nitrogen - GFR glomerular filtration rate - GOT glutamate oxaloacetate transaminase - LAP leucine aminopeptidase - LDH lactate dehydrogenase - MW molecular weight - NAG N-acetyl--D-glucosaminidase - PAP p-aminophenol - ppm parts per million - TMAO trimethylamine N-oxide - UFR urine flow rate     

Studies of monoamine oxidases: Effect of Triton X-100 and bile salts on monoamine oxidase in brain mitochondria

Triton X-100 and the bile salts, cholate and deoxycholate, detergents often used in the solubilization of monoamine oxidase (MAO) from mitochondria, have been found to cause an inhibition of the enzyme activity. With beef brain mitochondria, it was found that there was a differential effect of Triton X-100 on the putative MAO types A and B, with MAO-A being more susceptible to inhibition by Triton X-100. This was indicated by the greater loss of serotonin-deaminating than of phenyl ethylamine-deaminating activity in the presence of Triton X-100. Although the bile salts also caused substantial inactivation at concentrations above 0.1%, no differentiation between MAO types could be made. Kinetic studies of the inhibition by Triton X-100 indicated two different mechanisms were occurring with the two MAO types. The inhibition was competitive for MAO-A, but uncompetitive for MAO-B. Removal of Triton X-100 by co-polymer beads restored some, but not all of the activity for both MAO-A and MAO-B types. This suggests that the activity loss may have been due in part to inactivation when the enzyme was separated from the mitochondrial membrane.     

Effect of ginseng saponin on gap junction channel reconstituted with connexin32

Panax-ginseng saponin has been known to exert various pharmacological effects on cellular metabolism. This study was performed to determine the effect of ginseng saponin on gap junction channel-mediated intercellular communication, using an establishedin vitro system of reconstituted gap junction channels. Gap junction channels are a specialized plasma membrane fraction, which are permeable to relatively large water-soluble molecules. The sucrose permeable property of reconstituted gap junction channels was completely inhibited with 0.1% (w/v) of ginseng saponin. We also compared the effect of ginseng saponin with that of Triton X-100, a nonionic detergent, on the same system. Triton X-100 showed significantly different effect on sucrose-permeability of gap junction channel from that was affected by ginseng saponin. The structures of liposomes containing gap junction channels was significantly destroyed by Triton X-100.     

Tetrafluoroethane (RFC 134A) Propellant-Driven Aerosols of Proteins

Purpose. Develop metered-dose propellant-driven aerosols of proteins using tetrafluoroethane (HFC 134A) as propellant. Methods. Proteins were lyophilized with the propellant-soluble surfactants Triton X-100, Triton X-405, Laureth-9, Brij-30, Nonidet-40, and diethylene glycol monoethylether and then charged with propellants. Results. Small particle aerosols of the experimental protein bovine gamma globulin were produced. The fraction of aerosolized respirable-sized protein particles (<4–5 m) increased after dispersion of particles in propellant with agitation by shaking. Scanning electron microscopy of respirable-sized protein aerosols demonstrated bead-like particles in grape-like clusters. Vigorous shaking of propellant-suspended particles for 2 minutes or more reduced the size of clusters and reduced the diameters of the protein-containing subparticles that constituted the clusters. A 50:50 ratio of HFC 134A and dimethylether (DME) propellants improved the respirability of protein aerosols compared to HFC 134A as the sole propellant. Protein/surfactant particles first dispersed in DME and then diluted in HFC 134A propellant most efficiently produced respirable-sized, propellant-driven, protein aerosols. Conclusions. Metered-dose aerosols of respirable-sized proteins can be generated using HFC 134A and HFC 134A:DME blended propellants as an alternative to nebulized aqueous aerosols for delivering peptide-based pharmaceuticals to the respiratory tract.