INHIBITION OF ARACHIDONIC ACID ESTERIFICATION IN HUMAN AIRWAY EPITHELIAL CELLS EXPOSED TO OZONE IN VITRO

There is evidence that some lung responses to ozone (O3) exposure are mediated through the altered metabolism of arachidonic acid (AA). Increased concentrations of some AA metabolites such as prostaglandin E2 (PGE2) and prostaglandin F2 (PGF2) are observed in the bronchoalveolar lavage fluid recovered from human subjects exposed to O3. Airway epithelial cells may contribute to this increase in eicosanoid formation. Previous reports have shown increased PGE2 and PGF2 production by O3-exposed air way epithelial cells, believed to be mediated at least in part by increased phospholipase activity. We examined other potential biochemical mechanisms for the increased formation of these two prostaglandins by O3-exposed epithelial cells. Cultured normal human bronchial epithelial (NHBE) cells, prelabeled with 3H-AA, were exposed to air or to 0.1 ppm or1.0 ppm O3 for 60 min in a Transwell cell culture system. NHBE cells produced increased amounts of 3H-PGE2 and 3H-PGF2 in response to 0.1 ppm O3 exposure but not with 1.0 ppm exposure as measured in the conditioned media by high-performance liquidchromatography. Other 3H-AA products, including [3H]-15-hydroxyeicosatetraenoic acid, [3H]-12-heptadecatrienoic, and [3H]aldehydic substances derived from the ozonation of 3H-AA, also were observed in increased amounts. Pulsing of cells with 15 n 3H-AA after 0.1 ppm and 1.0 ppm O3 exposure revealed a decrease in 3H-AA esterification into cellular phospholipids, resulting in an increase in free 3H-AA available for metabolism to prostaglandins. No O3-induced alteration in NHBE cell cyclooxyge nase activity was observed with the 0.1 ppm exposures. Impaired esterification of free AA into cellular phospholipids appears to be a very sensitive target for O3-induced effects on AA metabolism, and may play a role in the increased prostaglandin production observed upon O3 inhalation in vivo.     

p -ARAMID RFP DO NOT INDUCE CHROMOSOMAL ABERRATIONS IN A STANDARDIZED IN VITRO GENOTOXICITY ASSAY USING HUMAN LYMPHOCYTES

Genotoxicity evaluations have been proposed as regulatory requirements for establishing German MAK values for inhaled fibrous dusts. The objective of this in vitro assay was to assess the potential for para -aramid (p -aramid) respirable-sized, fiber-shaped particulates (RFP) to induce chromosomal aberrations in cultured human peripheral blood lymphocytes without metabolic activation. The highest concentration tested in this assay was limited by the physical characteristics of p -aramid RFP. The test substance was suspended in fully supplemented RPMI culture medium with 1% Pluronic F68. All dosing was achieved using a dosing volume of 90% (900 µl/ml), and the vehicle control cultures were treated with 900 µl/ml of fully supplemented RPMI culture medium with 1% Pluronic F68. In the chromosomal aberrations assay, the treatments were either 3 or 19 h without metabolic activation. Cultures were harvested 22 h from the initiation of treatment. Replicated cultures of human whole blood lymphocytes were incubated with p -aramid RFP concentrations of 6.30, 12.6, 25.2, 50.4, 101, 201, and 401 µg/ml. Cultures treated with concentrations to 50.4 µg/ml for 3 h and 6.30, 12.6, 25.2, and 201 µg/ml for 19 h were analyzed for structural and numerical chromosomal aberrations. No significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed in the cultures analyzed. The results demonstrated that p -aramid RFP was negative for inducing chromosomal aberrations in cultured human peripheral blood lymphocytes without metabolic activation. In addition, we conclude that the utility of these tests for evaluating the genotoxicity of fibrous or particulate materials is questionable.     

CYTOKINE PROFILE OF HUMAN BRONCHOALVEOLAR MACROPHAGES AND BRONCHIAL EPITHELIAL CELLS IN RESPONSE TO INHALATION PARTICLES OF THE CYCLOSPORINE DERIVATIVE IMM 125

Administration of antiasthmatic drugs in the form of inhalation particles may alter the cytokine network in the airways, independently of their pharmacological actions. Changes induced by drugs not well tolerated may potentially contribute to the immunopathology of the disease, a strongly undesirable effect. In this study, cell viability assays and characterization of the cellular profile of cytokines and chemokines were performed in order to investigate the response of human bronchoalveolar macrophages and bronchial epithelial cells in culture to inhalation particles of the cyclosporine derivative IMM 125. Interleukin 1beta (IL-1beta), tumor necrosis factor alpha (TNFalpha), and IL-8 were assayed by enzyme-linked immunosorbent assay (ELISA) in the supernatants of bronchoalveolar macrophages, and RANTES, granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-8 in those of bronchial epithelial cells. Cells were studied both under basal and stimulated conditions (lipopolysaccharide and TNFalpha were used for activating macrophages and epithelial cells, respectively). The immunosuppressant FK 506 and the glucocorticoid Budesonide served as comparison. IMM 125 did not affect cell viability (except at high concentrations and long time periods). Moreover, IMM 125 did not induce an increase in the secretion of any of the cytokines and chemokines measured with respect to nontreated cells, except for a slight increase in IL-8, an effect that was also observed for FK 506, Budesonide, and inert latex particles, and was therefore regarded as nonspecific. Furthermore, IMM 125 significantly decreased the secretion of TNFalpha, IL-1beta by macrophages, and GM-CSF by epithelial cells, suggesting an antiinflammatory potential. In conclusion, the present in vitro results point to a good tolerance of human airways to IMM 125 inhalation particles.     

EFFECTS OF SODIUM DIETHYLDITHIOCARBAMATE ON TYPE II PULMONARY EPITHELIAL CELLS IN VITRO

Dithiocarbamates (DDTC) are chemicals widely used in the form of pesticides, therapeutic and chelating agents, and scavengers. Since DDTC interfere with SH, Cu, and Zn enzymes due to chelating properties, it was of interest to clarify, in primary culture of type II alveolar pneumocytes, the effect of this compound upon enzymes of glutathione cycle, Cu, Zn-superoxide dismutase, and the membrane structure of cells. DDTC significantly inhibited the activity of superoxide dismutase and the activity of gamma-glutamyl transpeptidase, glutathione reductase, and alkaline phosphatase, whereas an increase in the activity of glutathione peroxidase was found. The membranes of pneumocytes type II were injured. Data show that DDTC adversely affected type II pneumocyte function and structure.     

AN IN VITRO MODEL OF OXIDATIVE STRESS AND HYPOXIA IN RETINAL PIGMENTED EPITHELIAL CELLS

Retinal pigmented epithelium (RPE) is an ocular tissue that performs a variety of functions including metabolism of photoreceptors. It constitutes an electrical and chemical barrier regulating the movement of solutes and ions between neural retina and the choriocapillary network. Reactive oxygen intermediate (ROI) generation has been implicated in the etiology of certain neurodegenerative diseases of the eye, which may be positively altered by treatment with antioxidant drugs. Cell viability under oxidative stress, hypoxia, and ischemic conditions in RPE were studied in vascular epithelium (VE) cells in culture to explore the possible role of vitamin C and N-acetyl-cysteine to prevent cell injury. The results of this study show that RPE cells may be protected against free radicals and products of peroxidation by endogenous scavengers by treatment with antioxidant drugs.     

VOLUME-ACTIVATED TAURINE TRANSPORT IS DIFFERENTIALLY ACTIVATED IN HUMAN CERVICAL CANCER HT-3 CELLS BUT NOT IN HUMAN PAPILLOMAVIRUS-IMMORTALIZED Z183A AND NORMAL CERVICAL EPITHELIAL CELLS

1. Previous studies demonstrate that volume-sensitive chloride currents are distinctly activated in cervical cancer cells, but not in human papillomavirus (HPV)-immortalized and normal cervical cells. In the present study, the Na⁺-independent volume-activated transport of taurine in three cervical cell types was investigated. 2. Osmotic swelling of cervical cancer HT-3 cells suspended i. Na⁺-free hypotonic medium led to increased membrane uptake of taurine. This taurine uptake was effectively blocked by various C1^- channel blockers with a similar potency in blocking volume-sensitive C1^- channels: l,9-dideoxyforskolin > 5-nitro-2-(3-phenyl-propylamino)-benzoic acid (NPPB) > 4-acetamido-4′-isothiocyanastilbene-2,2′-disulphonic acid (SITS) > 4,4′-diisothio-cyanatostilbene-2,2-disulphonic acid (DIDS)>furosemide. The taurine influx was also abolished by pertussis toxin. In contrast, Na⁺-independent volume-activated taurine transport was not significantly activated in HPV-immortalized Z183A cells and in normal cervical cells. 3. Exposure of HT-3 cells to hypotonic medium also resulted in a marked increase in taurine efflux. The volume-activated taurine efflux was osmolality dependent and the pattern of pharmacological inhibition by C1^- channel blockers was indistinguishable from that for taurine uptake. 4. These results suggest that volume-sensitiv. C1^- channels in HT-3 cells can mediate the transport of amino acids. In addition, the pertussis toxin-sensitive G-protein is linked with the activation of this transport mechanism.     

琼脂扩散盒中人癌细胞集落形成试验及应用

应用改良式软琼脂扩散盒(ADC)进行人癌克隆形成试验,检测26例6种手术切除肿瘤标本,培养成功率为65.4%,铺板率在0.003～0.814%之间,接种细胞与克隆形成之间有明显的线性关系(P<0.05)。采用ADC技术筛选铂兰化合物,初步试验显示该药能抑制集落形成,结果与动物实验一致。     

琼脂扩散盒中人癌细胞集落形成试验及应用

应用改良式软琼脂扩散盒(ADC)进行人癌克隆形成试验,检测26例6种手术切除肿瘤标本,培养成功率为65.4%,铺板率在0.003～0.814%之间,接种细胞与克隆形成之间有明显的线性关系(P<0.05)。采用ADC技术筛选铂兰化合物,初步试验显示该药能抑制集落形成,结果与动物实验一致。     

HPLC法测定人血清索他洛尔浓度

应用ＨＰＬＣ方法检测血清中索他洛尔浓度，以阿替洛尔为内标，采用Ｃ１８分析椎琢保护柱，流动相为甲醇：乙腈：ＰＢ。流速为１．０ｍｌ／ｍｉｎ。提取溶剂为三氯甲烷；异丙醇，采用荧光检测器，激光波长及发射波长分别为２３５ｎ，及３１３ｍｍ实验结果表明：索他洛尔与内标分离良好，保留时间分别为５ｍｉｎ一６．７ｍｉｎ，在血清中萃取的绝对回收率分别为８０．０％及８１．５％，索他洛尔在０－５μｇ／ｍｌ范围内线性关系良好     

OZONE-INDUCED DNA SINGLE STRAND-BREAKS IN GUINEA PIG TRACHEOBRONCHIAL EPITHELIAL CELLS IN VIVO

Two-month-old male guinea pigs (Dunkin-Hartley strain, specific pathogen free), 4 in each group, were exposed to 0.00, 0.45, and 1.00 ppm O 3 for 72 h. The trachea with two main bronchi was removed from each animal after O 3 exposure. The trachea lavage fluid was used to measure the protein content as an index of altered tracheobronchial epithelial (TE) cell membrane permeability after O 3 exposure. The TE cells were isolated and employed for the determination of DNA single-strand breaks (SSBs) by fluorometric analysis of DNA unwinding (FADU). The statistical significance level was set atα = .05. The results show that neither the yield nor the viability of the TE cell from various O 3 treatment groups was different from that of controls. Compared to controls, the protein content was elevated significantly after 0.45 ppm O 3 exposure; however, the amount of DNA SSBs was not. The number of DNA SSBs increased significantly in the 1.00 ppm O 3 exposure group when compared to controls. Regardless of the alkali incubation time at 15°C, the double-stranded DNA left in the alkali TE cell lysate was a linear function of O 3 exposure concentrations.     

TOXAPHENE IS ANTIESTROGENIC IN A HUMAN BREAST-CANCER CELL ASSAY

Toxaphene is a complex mixture of chlorinated bornanes, bornenes, and bornadienes and was a heavily used insecticide in the United States until its use was restricted in 1982. There are conflicting reports regarding the potential for toxaphene to induce estrogenic responses in human and nonhuman animals. Due to the public concern over environmental estrogens, the estrogenicity of toxaphene was examined in a human breast-cancer cell assay, the MCF-7 focus assay, which is based on in vitro postconfluent cell proliferation and tissue restructuring. In this assay, 0.1-1 n M 17 b estradiol (E) produces maximum postconfluent proliferation and formation of multicel2 lular nodules or foci. Toxaphene was also tested for its ability (1) to bind the estrogen receptor (ER) in a competitive binding assay using recombinant human ER a (rhER) and in a whole-cell competitive ER binding assay, and (2) to alter the catabolism of E 2 in MCF-7 cell cultures. Results from the MCF-7 focus assay showed: (1) Toxaphene alone was not estrogenic between the concentrations of 0.5 n M and 10 mu M, (2) toxaphene in binary combinations with chlordane, dieldrin, or endosulfan (a or b) was not estrogenic, and (3) toxaphene was weakly antiestrogenic (it reduced the number of foci induced by 0.1 n M and 0.01 n M E₂). Results from the competitive binding assays showed that (1) toxaphene alone did not bind rhER or ER in MCF-7 cells, and (2) toxaphene in binary combinations with other pesticides did not bind rhER. Results from the growth assay and radiometric analysis of E₂ catabolism showed that (1) toxaphene did not alter the growth rate of MCF-7 cell cultures over 13 d, and (2) toxaphene did not alter the catabolism of E₂. In conclusion, results from the MCF-7 focus assay demonstrate that toxaphene is weakly antiestrogenic rather than estrogenic.     

EVALUATION OF ADVERSE OCULAR EFFECTS OF 5-FLUOROURACIL BY USING HUMAN CORNEAL EPITHELIAL CELL CULTURES

5-Fluorouracil (5-FU) is commonly used in ophthalmology for suppressing fibroblast activity after glaucoma surgery. Adverse effects on corneal epithelial cells have been reported to relate to 5-FU therapy. The effects of 5-FU were evaluated in vitro on SV40-immortalized human corneal epithelial cell (HCE) cultures with two cytotoxicity tests: WST-1 assay as an index of cell proliferation, and lactate dehydrogenase (LDH) assay as an index of plasma membrane integrity. The cells were exposed to 5-FU with various concentrations in serum-free medium and in medium containing 15 % (v/v) fetal bovine serum (FBS) for 1, 24, 48 and 72 hours. One-hour exposure had no effects on HCE cells. Longer exposures caused dose-dependent inhibition of cell proliferation. Exposure to 5 mg/ml 5-FU lowered cell number to 50 % of controls after 24-hour treatment and resulted to complete cell death after 72 hours. Serum protected the cells for 24 hours, but after longer exposure times the protective nature of serum disappeared. 5-FU had only minor effects on LDH release. The LDH leakage was at its peak after 48-hour treatment.     

依诺沙星药代动力学和体外抗菌活性研究

健康志愿者6人口服依诺沙星(ENX)400mg,用反相高效液相法测定给药后12小时内血药浓度以研究其药动学特征,同时测定ENX对797株临床分离致病菌的体外抗菌活性。 色谱条件,流动相为甲醇:0.008mol/L磷酸缓冲液:0.5mol/L四丁基溴化铵(25:75:4,V:V:V,pH2.85)。紫外检测波长275nm,0.04AUFS,流速为0.8ml/min,反应STD。本方法线性范围为0.0625～4μg/ml,r=0.9977。 ENX对所试革兰氏阳性和革兰氏阴性细菌均有良好抗菌活性,抑菌率均>65％。 口服ENX400mg后,药物在体内的转运过程符合一级动力学一室开放模型。Tmax,Cmax分别为2.12h和2.47μg/ml,T 1/2Ka、T 1/2Ke分别为1.22和2.35h,Vd为3.07L/Kg,CL为0.86L/h.kg,说明口服后药物吸收迅速,可达较高浓度,且广泛分布于全身多数组织和体液中。     

IN VITRO CALCIUM DEPENDENCE OF ARTERIAL SMOOTH MUSCLE IN HUMAN HYPERTENSION

Digital arteries, removed at autopsy from 12 hypertensives and 11 normotensives, have been compared in vitro for the calcium dependence of contractures produced by potassium chloride and noradrenaline, and the potency of verapamil to antagonize contractures to noradrenaline. No significant differences were found between the vessels from the hypertensives and normotensives for the pD2 values or the maximum response to either potassium chloride or noradrenaline in bathing solutions containing 2.5, 1.0, 0.5 or 0 mmol/l calcium chloride. There were also no significant differences between the vessels, from the hypertensive or normotensives, in the pD2 values for the addition of calcium chloride to arteries exposed to potassium chloride or noradrenaline in a calcium free bathing medium, in the ability of verapamil to shift the pD2 values for noradrenaline, nor in the ability of verapamil to reduce the maximum responses to noradrenaline (except at the two highest concentrations of verapamil tested). It is concluded that it is unlikely to be a primary abnormality of the mechanisms regulating calcium ion entry and release in vascular smooth muscle in human hypertension.     

甘糖酯对体外培养的晶体上皮细胞增殖的影响

后发性白内障主要是由于术后残留的晶体上皮细胞移行到后囊并增殖所致。用培养的新生大鼠晶体上皮细胞实验,以结晶紫染色和~3H-TdR掺入作指标,表明甘糖酯(0.2～0.8mg/ml)对细胞增殖有明显抑制作用,且比同浓度肝素作用略强。提示甘糖酯对防治后发性白内障可能有效。     

甲壳质对氧自由基致人脐静脉内皮细胞损伤的保护作用

在培养的人脐静脉内皮细胞中观察了不同剂量的甲壳质对氧自由基损伤的内皮细胞培养上清液中丙二醛和乳酸脱氢酶含量的影响。结果表明,与损伤对照组相比,甲壳质保护组培养上清液中丙二醛和乳酸脱氢酶的含量均显著下降。提示甲壳质对氧自由基损伤的内皮细胞有一定的保护作用。     

HPLC法测定人血浆中地尔硫及其代谢产物

本文介绍DZ的HPLC测定法,用WATERS BASELINE 810色谱工作站,WATERS径向加压柱(Nova-Pak C-(18)8mm×10cm,4μm.检测波长UV238nm,流动相为CH_aOH:H_2O;CH_2Cl_2:0.1mol/L NH_4Ac(64:23.5:2.5:10),流速2.0ml/min;血样用混合溶剂一次提取,地尔硫(艹卓)和去乙酰地尔硫(艹卓)的提取回收率分别为:85.25％、82.29％,平均方法回收率分别为:99.59％、101.1％,最低检测浓度均为5ng/ml。     

ACTIONS OF INDORAMIN ON RABBIT AND HUMAN VASCULATURE IN VITRO

1. The antagonist effects of indoramin were investigated in rabbit perfused ear artery, common carotid artery, and human perfused temporal artery preparations. 2. Indoramin was a potent competitive antagonist of the constrictor effects of noradrenaline (NA) in all three preparations, pA₂ values being 7.77 for the ear artery, 8.20 for the common carotid artery and 7.46 for the human temporal artery. 3. The constrictor actions of serotonin (5-hydroxytryptamine, 5-HT) were competitively antagonized by indoramin. The pA₂ values obtained in the rabbit common carotid and human temporal artery (5.92 and 6.25, respectively), were lower than those for NA in the same preparations and lower than that obtained in the rabbit perfused ear artery (7.55). 4. Indoramin was a potent competitive antagonist (pA₂:8.31) of histamine-induced vasoconstriction in the rabbit perfused ear artery preparation. 5. These results can be explained on the basis of previous findings that the action of 5-HT in the rabbit ear artery is mediated via an α-adrenoceptor, and that the rabbit common carotid artery contains true 5-HT receptors. The findings suggest that specific 5-HT receptors may be present in the human temporal artery and that α-adrenoceptor antagonism may be the pharmacological property of most relevance to the efficacy of indoramin in migraine prophylaxis.