Interaction of non-ionic surfactants with hepatic CYP in Prochilodus scrofa

Cytochromes P450 (CYP) constitute a superfamily of hemeproteins that play a vital role in the metabolism of a wide variety of endogenous and xenobiotic compounds. Xenobiotic metabolism and the role of CYP are of particular interest in studies regarding the prevention of the damage caused by chemical pollutants. We investigated, in this study, the interaction of Triton X-100 and Tween 80 with CYP and antioxidant defenses in Curimbatá, a Brazilian fish. Aiming to clarify the effects of non-ionic surfactants in the monooxigenase system of fish through in vitro study, the effects of Triton X-100 and Tween 80 were analyzed using monooxygenases and antioxidant system as experimental model. Total CYP and EROD were strongly inhibited by Triton X-100 and Tween 80 in a concentration-dependent way; the content of CYP was reduced until zero while EROD activity was completely inhibited in the presence of Triton X-100 and more than 40% inhibited in the presence of Tween 80. Each surfactant causes a different effect on each antioxidant enzyme. No effect was detected in SOD activity in the presence of even Triton X-100 or Tween 80. Triton X-100 increase catalase activity, while Tween 80 decreases this enzyme activity. The molecular structure of the surfactants causes the alteration of this system, since they are able to interact with the microsomal protein, especially with monooxigenase's components, altering their conformation and, consequently destroying their function. Our results suggest that surfactants can interact with components of the microsomal system leading to inhibition of CYP. Therefore, CYP activity, which has been used as a biomarker of xenobiotic exposure, should be used as a marker in association with other enzymes.     

Evaluation of surfactant cytotoxicity potential by primary cultures of ocular tissues: I. Characterization of rabbit corneal epithelial cells and initial injury and delayed toxicity studies.

This investigation was undertaken to develop cytotoxicity assay systems using primary cultures of rabbit corneal epithelial cells as an experimental model to evaluate oculotoxic agents and the ability of these in vitro assay systems to predict irritancy potential and delayed toxicity. We have characterized the epithelial nature of the cultures by identifying keratins with antikeratin antibodies (AE1/AE3) and by demonstrating metabolic enzymes important to the integrity of the cells: lactate dehydrogenase, glucose 6-phosphate dehydrogenase and aldolase. Eight surfactants were compared and ranked according to their cytotoxic potential. We evaluated cytotoxicity by measuring leakage of the cytosolic enzyme, lactate dehydrogenase, into the medium, by making morphological observations and by assessing lysosomal neutral red uptake and mitochondrial 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction. The cells were treated for 1 h with the surfactants and the possibility of delayed toxicity was evaluated 24 h after removal of the surfactant. The cytotoxicity of the different types of surfactants as shown by all the tests was cationic > anionic = amphoteric > non-ionic. Triton X-100, a non-ionic surfactant but a severe irritant, had a ranking similar to anionic surfactants. The in vitro rankings corresponded well to reported in vivo Draize rabbit eye test data. The 24-h test for lactate dehydrogenase leakage showed that mild and non-irritating surfactants did not demonstrate any subsequent damage after a 1-h exposure, but the extreme and severe surfactants continued to show further damage after the 1-h exposure. These in vitro findings were similar to reported in vivo results. The neutral red and MTT tests did not adequately predict the prolonged toxicity of the more irritating surfactants, as was demonstrated by the lactate dehydrogenase leakage test. We conclude that in vitro cytotoxicity assays using primary cultures of rabbit corneal epithelial cells may be used to rank the cytotoxic potential of surfactants, but only the lactate dehydrogenase leakage test was able to assess prolonged cell injury.     

Influence of elastase-induced emphysema and the inhalation of an irritant aerosol on deposition and retention of an inhaled insoluble aerosol in Fischer-344 rats

The purpose of this study was to assess the effects of elastase-induced pulmonary emphysema and the inhalation of an irritant aerosol (Triton X-100, a nonionic surfactant similar to those used in a number of pressurized consumer products) on pulmonary deposition and retention of an insoluble test aerosol, 59Fe-labeled Fe2O3. Untreated rats or rats pretreated by intratracheal instillation with elastase were exposed to an aerosol of 59Fe-labeled Fe2O3 either 18 hr or 7 days after exposure to aerosolized Triton X-100 which was administered in doses of 20, 100, or 200 micrograms/g of lung. Rats pretreated with elastase had significantly lower pulmonary deposition of 59Fe than the untreated controls (p less than 0.005). Pulmonary deposition of Fe2O3 was unaffected by pretreatment with Triton X-100. Elastase treatment alone had no effect on retention of Fe2O3. Triton X-100 administered 18 hr prior to exposure of rats to Fe2O3 aerosol resulted in dose-related increases in whole-body retention of 59Fe. When rats were exposed to Triton X-100 7 days before exposure to Fe2O3, increased retention of 59Fe was noted only in those treated at the highest Triton X-100 dose level (200 micrograms/g).     

Effect of age on ocular irritancy as measured with in vitro bovine lenses.

PURPOSE: This project studied the effect of age on optical quality of cultured bovine lenses exposed to a number of common surfactants and alcohol. METHODS: Lenses from calves (8-18 months) and cows (2-3 years) were isolated aseptically and studied optically for 96 h after treatment with various commercial surfactants and an alcohol. Potential eye irritancy was evaluated using a scanning laser in vitro assay system which records the change in focal characteristics (back vertex distance variability or BVDV) of the bovine lenses. Lenses were divided into a total of 14 groups. Both calf and cow lenses (a total of 257 lenses were used) were arranged into control, 0.01% BAK, 1% SDS, 1.0% Triton X-100, 100% ethanol, 10% Tween-20 and 1.0% Tween-20 treatment groups. RESULTS: The cationic surfactant BAK caused the most amount of optical change to the bovine lenses, followed by SDS, Triton X-100, ethanol and then Tween-20. There was also a significant difference in BVDV between the cow and calf groups for all the treated groups, except for Tween-20, with the calf lenses showing greater optical damage. In the case of 10% Tween-20, both cow and calf lenses show equal optical damage while at 1.0% both groups show no effect and are no different from the untreated control lenses. CONCLUSION: Younger bovine lenses are more sensitive to the surfactants and alcohol tested when compared to their older counterparts, indicating that younger eyes may be more sensitive to these chemicals. The results further indicate that age is a factor that should be taken into account in assessing ocular risk.     

表面活性剂作为黄原胶发酵促进剂的应用研究

目的通过加入表面活性剂,提高黄原胶发酵产量。方法初步筛选6种常用的表面活性剂,优选两种优势互补的表面活性剂进行加入量、加入时间、加入比例的优化,采用单因素和正交试验得到最佳工艺参数。结果吐温20、曲拉通X-100协同使用效果最好,发酵0 h加入0.06%曲拉通X-100,48 h加入0.09%吐温20,黄原胶产量提高42.0%。结论表面活性剂能提高黄原胶发酵产量。     

Plasma membrane damage sensing and repairing. Role of heterotrimeric G-proteins and the cytoskeleton

Different toxic agents, derived from bacteria, viruses or cells of the immune system, as well as mechanical forces generated during cell locomotion are able to open pores in the cell plasma membrane. Most of these biological agents operate through specific receptors. We studied the formation and resealing of the “non-specific” plasma membrane pores generated by the mild non-ionic detergent Triton X-100. In HL-60-derived granulocytic cells plasma membrane pore opening after a 1-h treatment with Triton X-100 is documented by entry into the cell of the membrane impermeant dye ethidium bromide. As a consequence of the opening of pores the intracellular K⁺ concentration falls dramatically, the cytosolic pH diminishes and the cell membrane is depolarized. Furthermore the cells acquire a polarized morphology, demonstrating the involvement of the actin cytoskeleton. At the Triton concentration used the membrane lesions are progressively repaired and by 8 h the impermeability to ethidium bromide is restored and the intracellular K⁺ concentration is virtually normal. Following treatments with Triton + Pertussis toxin, Triton + Cytochalasin, or Triton + Pertussis toxin + Cytochalasin the progress of membrane repair is dramatically slowed and is no longer completed by 8 h. It is concluded that the membrane damage activates pertussis-sensitive G-proteins which likely act as sensors of the damage, while both G-proteins and the actin cytoskeleton are involved in the membrane repair mechanism.     

Effects of Triton X-100 nanoaggregates on dimerization and antioxidant activity of morin

Dimerization and antioxidant activity of morin in the Triton X-100 micelles were studied by electronic absorption, ATR-FTIR spectra, cyclic voltammetric, DSC, freeze-fracture TEM, molecular modeling and ab initio quantum calculations. Morin can be solubilized in the Triton X-100 micelles and show selective dimerization in Triton X-100 micelles with different structures. In Triton X-100 spherical micelles, morin always exists in the form of dimer, and in Triton X-100 rodlike micelles, it is always in the form of monomer. The solubilization of morin dimer in Triton X-100 spherical micelles changes the micelle morphology from spherical to cubelike, and the size of the single micelle is also increased, while morin monomer links the Triton X-100 rodlike micelles and forms a kind of network micelle structure with the size of the "rod" unchanged. Solubilized and concentrated in Triton X-100 micelles, morin can protect human serum albumin from the damage induced by hydroxyl radicals effectively and even can form a kind of protein complex with human serum albumin showing more thermal stability.     

高效液相色谱法测定质粒pUDKH中曲拉通X-100的含量

目的检测质粒pUDKH最终产品中去污剂曲拉通X 10 0 (TritonX 10 0 )的残留含量。方法用高效液相色谱法测定TritonX 10 0残留含量 ,色谱柱为C18柱 ,流动相为乙腈 水 (70∶30 ) ,流速为 1.0ml/min ,进样量 10 μl,检测波长 2 2 3nm。结果平均回收率为 10 0 .19% ,日内精密度为 4 .36 % ,日间精密度为 4 .17% ,TritonX 10 0在 1.2 5～2 0 μg/ml浓度范围内呈线性关系。pUDKH产品中的TritonX 10 0含量在 2 .2 7～ 3.0 0 μg/ml之间。TritonX 10 0的最低检测限可达 1.0 μg/ml。 结论此法有良好的准确度与精密度 ,供试品不需预处理 ,不受其它成分的干扰。     

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.     

The Influence of Surfactant on PLGA Microsphere Glass Transition and Water Sorption: Remodeling the Surface Morphology to Attenuate the Burst Release

Purpose The stability of protein unloaded and loaded poly(lactic-co-glycolic acid) (PLGA) microspheres fabricated with surfactant was challenged through exposure to environmental conditions of different relative humidity. Methods Polyvinyl alcohol (PVA) or Triton X-100 was added to the primary emulsion of the double-emulsion solvent evaporation technique. After storage at ambient humidity and 75% relative humidity, the mechanical stability of the polymer was tested to reveal PLGA chain mobility using differential scanning calorimetry. Subsequent surface modifications were examined by atomic force microscopy (AFM), and protein release profiles were collected. Results Residual amounts of PVA and particularly Triton X-100 raised the hydrophilicity of the microspheres. When exposed to ambient humidity or 75% relative humidity, PVA and Triton X-100 had, respectively, an antiplasticizing and a plasticizing effect upon PLGA, and both led to physical aging. The high-resolution AFM imaging of microspheres containing model protein and Triton X-100 showed that the depth of the surface pores was reduced when exposed to 75% relative humidity, and the initial burst release subsequently decreased. Conclusion These studies suggested that the mechanical stability of PLGA was influenced by the addition of surfactants, which, depending on the formulation, led to surface pore remodeling under high humidity, reducing the initial burst release while maintaining the spherical integrity of the microsphere.     

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。     

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     

免疫荧光技术中不同固定剂对细胞p65核移位观察效果的影响

目的观察免疫荧光技术中不同固定剂以及Triton X-100不同渗透时间对小鼠腹腔巨噬细胞p65蛋白核移位的影响,找出最适合观察p65蛋白移位的固定方法及Triton X-100渗透时间。方法采用免疫荧光的方法观察使用不同固定剂(甲醇,1%～4%甲醛)固定,0.1%Triton X-100渗透3～15min的条件下,观察脂多糖刺激2h后,p65蛋白细胞内定位的情况。结果 p65蛋白在对照细胞主要定位于细胞质,脂多糖(LPS)刺激后p65蛋白移入胞核。甲醇固定后细胞荧光染色显色很弱且核区不明显,染色弥散;1%、2%甲醛固定不良可引起细胞收缩;4%甲醛固定后,核区与胞质p65蛋白荧光染色对比明显。0.1%Triton X-100作用3～5min效果最佳,而作用10～15min对照细胞核内也有较强荧光。结论本实验表明,在免疫荧光技术中采用4%甲醛固定20min,0.1%Triton X-100渗透3～5min可更好的观察LPS诱导的小鼠腹腔巨噬细胞p65核移位。     

INHIBITION OF CARP LIVER MITOCHONDRIAL MONOAMINE OXIDASE BY SOME COMMONLY-USED DETERGENTS

The inhibitory effects of some detergents commonly used in biochemical research on carp liver mitochondrial monoamine oxidase were examined. Sodium dodecylsulfate, octyl-β-D-glucopyranoside, sodium cholate and Triton X-100 at relatively low concentrations caused strong dose-dependent inhibition of the activity towards tyramine, but digitonin caused only weak inhibition. Sodium dodecylsulfate, octyl-β-D-glucopyranoside and sodium cholate caused almost complete Inhibition of activity in the concentration ranges tested. The extent of inhibition by Triton X-100 was greater after preincubation at 37°C for 30 min than that without preincubation, but with or without preincubation, the inhibition was not substrate-selective and was not complete at a relatively high concentration (2%) of Triton X-100. Without preincubation, the mode of inhibition by Triton X-100 was competitive and reversible with respect to the oxidations of 5-HT, tyramine and PEA, but after preincubation (37°C for 30 min), it became non-competitive and irreversible, depending on the concentration of detergent used. These findings suggest that it had different actions on the enzyme depending on preincubation. Triton X-100 also slightly changed enzyme sensitivity towards clorgyline and deprenyl, regardless of the preincubation time or the substrate used. Some possible mechanisms of the inhibitory effect of Triton X-100 are discussed.     

聚氧乙烯醚类表面活性剂对大鼠体内细胞色素P450 3A活性的影响

为研究药用辅料对大鼠体内细胞色素P450 3A(CYP3A)药酶活性的影响， 大鼠分别灌胃生理盐水、 酮康唑(75 mg·kg^-1·d^-1)， 曲拉通X-100(30 mg·kg^-1·d^-1)、 聚氧乙烯蓖麻油(EL35, 150 mg·kg^-1·d^-1)、 聚氧乙烯氢化蓖麻油(RH40, 150 mg·kg^-1·d^-1) 5 d后， 12 h禁食再经十二指肠给予上述试药， 20 min后给予咪哒唑仑作为探针。分别测定咪哒唑仑及其代谢物1′-羟基咪哒唑仑在大鼠体内的血药浓度并计算其药代动力学参数， 比较曲拉通X-100、 EL35和RH40处理组与生理盐水组的药代动力学差异。结果显示， 阳性对照药酮康唑对CYP3A有明显的抑制作用； 而曲拉通X-100、 EL35和RH40使1′-羟基咪哒唑仑与咪哒唑仑的AUC_0-∞比值分别从1.14降至0.90、 1.03和0.64，统计学分析表明曲拉通X-100和EL35对CYP3A没有明显的抑制作用， 而RH40对CYP3A有明显的抑制作用。因此， 在药物制剂研究及临床应用中， RH40有可能影响经细胞色素P450 3A转化的药物代谢及处置， 增加药物的生物利用度， 对药物的临床应用产生显著影响。     

Differential effects of triton X-100 on benzodiazepine and GABA binding in a frozen-thawed synaptosomal fraction of rat brain

The effect of Triton X-100 on 3H-GABA and 3H-diazepam binding was measured in a frozen-thawed synaptosomal fraction of rat brain. Specific binding activity (amount bound per mg protein) of both ligands was increased by the treatment. Diazepam binding capacity in the pellet was progressively decreased, while GABA binding was increased, then decreased by increasing Triton X-100. Diazepam binding affinity was unchanged, while GABA binding affinity increased. Triton X-100 appears to preferentially solubilize benzodiazepine binding sites, indicating GABA and benzodiazepine binding sites are on separate macromolecules.     

The effect of a range of Triton non-ionic surfactants on rodent ovaries

Cysts developing on rodent ovaries after the Triton series of non-ionic surfactants have been fed to or applied topically to the animals are subcapsular and associated with obstruction of the ostium. The ability of Triton X-100 to produce subcapsular cysts after being fed to animals has been confirmed but the mechanism by which it does so remains obscure.     

Investigation of the Induction of DNA Double-Strand Breaks by Methylenediphenyl-4,4'-diisocyanate in Cultured Human Lung Epithelial Cells

The question was addressed whether methylenediphenyl-4,4'-di-isocyanate(MDI), a bifunctional electrophile, can induce DNA double-strandbreaks (DSB) by repair of interstrand DNA crosslinks or whetherDSB are the result of cell death. Cultured human lung epithelialcells (A549) were treated with MDI, methylene-4,4'-dianiline(MDA; a potential hydrolysis product of MDI), the nitrogen mustardmelphalan, and the detergent Triton X-100. All chemicals weredissolved in ethylene glycol dimethyl ether which was addedto a cell monolayer covered with phosphate-buffered saline.After 2 h, the treatment solution was exchanged against medium,and 8, 24, and 72 h after treatment initiation, the inductionof DNA double-strand breaks was assessed by pulsed-field gelelectrophoresis. At the same time, the viability was determinedwith the MTT test (intracellular reduction of the tetrazoliumdye MTT). At the 8-h time point, 1 and 10 µM melphalaninduced DSB without concomitant effect on cell viability. Withall other chemicals, the dose-response curves for DNA fragmentationand viability were mirror images. Approximate 50% lethal concentrationswere 200, 3000, and 100 µM for MDI, MDA, and Triton X-100,respectively. For these chemicals, the observed DSB were theconsequence of extragenomic damage in the course of cell deathrather than of an interaction with DNA. The mechanistic differenceof melphalan was supported by analysis of nuclear morphology.Apoptotic bodies were observed only after melphalan treatment,whereas MDI and Triton X-100 produced only irregular clumpingof chromatin (72-h time point). DNA fragment length analysisshowed a time-independent pattern, with sizes between 1 and4 Mbp for melphalan, while MDI, and Triton X-100 induced smallerDNA fragments in a time-dependent manner. It is concluded thatDSB observed in cells treated with MDI are unlikely the resultof DNA crosslink formation.     

Action of detergents on 3H-dihydroergokriptine binding and localization of alpha-adrenoceptors in synaptosomal membranes

3H-dihydroergokriptine (3H-DHE) binding was carried out in synaptosomal membranes from basal ganglia of the cat. A single type of binding site with Kd 3.7 nM, Hill number = 0.95 and Bmax = 1000 pmol/g protein was found. 3H-DHE bound to alpha-adrenoceptors and not to serotonin or dopamine receptors. At very low concentrations, some detergents enhanced binding, but at higher concentrations of those used (Triton X-100, Nonidet P-40, deoxycholate and digitonin), inhibited the binding of 3H-DHE. After binding to the membrane protein, the 3H-DHE-receptor complex was stable to the action of Triton X-100. At concentrations of Triton X-100. At concentrations of Triton X-100 (0.1--0.2%). in which only the presynaptic membrane disintegrated, the 3H-HDE specific radioactivity was reduced. With a more drastic treatment that disintegrated the postsynaptic membrane, 3H-DHE binding was further reduced. These results suggest that alpha-adrenergic receptors may be localized at both the pre- and postsynaptic membranes of central synapses.     

Possible involvement of endothelium in the responses of various vasoactive agents in rabbit isolated perfused kidney.

1. Perfusion of the kidney with methylene blue, a soluble guanylate cyclase inhibitor, significantly enhanced the vasoconstrictor effects of angiotensin II, noradrenaline and phenylephrine but significantly reduced the vasodilator effect of acetylcholine without altering that of iloprost. 2. In the kidneys, which were perfused with Triton X-100 to remove endothelium, acetylcholine-induced vasodilation was completely abolished and angiotensin II-, noradrenaline- and phenylephrine-induced vasoconstriction was greatly reduced. 3. The vasodilator effect of iloprost was unchanged after perfusion of kidney with Triton X-100. 4. Neither methylene blue nor Triton X-100 significantly altered urine volume form normal and angiotensin II induced increase of urine volume. 5. These results were taken as evidence for the involvement of renal vascular endothelium originated EDRF in the responses of various vasoactive agents in the rabbit isolated perfused kidney.