Triggering endogenous immunosuppressive mechanisms by combined targeting of Dipeptidyl peptidase IV (DPIV/CD26) and Aminopeptidase N (APN/ CD13) — A novel approach for the treatment of inflammatory bowel disease

The ectopeptidases Dipeptidylpeptidase IV and Alanyl-Aminopeptidase N, strongly expressed by both, activated and regulatory T cells were shown to co-operate in T cell regulation. Based on the findings that DPIV and APN inhibitors induce the TGF-β1 and IL-10 production and a suppression of T helper cell proliferation in parallel, and that particularly APN inhibitors amplify the suppressing activity of regulatory T cells, both peptidases represent a promising target complex for treatment of diseases associated with an imbalanced T cell response, such as inflammatory bowel diseases (IBD).The aim of the present study was to analyze the therapeutic potential of DPIV and APN inhibitors in vivo in a mouse model of colitis. Balb/c mice received 3% (w/v) dextran sulphate sodium with the drinking water for 7 days. After onset of colitis symptoms, inhibitor treatment started at day 3. Disease activity index (DAI) was assessed daily, supplemented by histological and immunological analysis. While the DPIV inhibitor Lys-[Z(NO])₂]-pyrrolidide or the APN-inhibitor Actinonin alone had marked but no significant therapeutic effects, the simultaneous administration of both inhibitors reduced colitis activity in comparison to placebo treated mice, significantly (DAI 4.8 vs. 7.7, p < 0.005). A newly developed compound IP12.C6 with inhibitory capacity toward both enzymes significantly attenuated the clinical manifestation of colitis (DAI 3.2 vs. 7.6, p < 0.0001). TGF-β mRNA was found to be up-regulated in colon tissue of inhibitor-treated animals.In summary our results strongly suggest that combined DPIV and APN inhibition by synthetic inhibitors represents a novel and efficient approach for the pharmacological therapy of IBD by triggering endogenous immunosuppressive mechanisms.     

caveolae介导的apelin-13促内皮细胞增殖机制探讨

目的 探讨caveolae及其标志蛋白caveolin-1在apelin-13促内皮细胞增殖中的作用,并对apelin-13促内皮细胞增殖信号转导通路进行拓展性研究.方法 MTT比色法观察caveolae在apelin-13促内皮细胞增殖中的作用;Western Blot检测不同因素处理下,内皮细胞中信号蛋白的表达;免疫共沉淀技术检测不同因素处理后,内皮细胞中信号分子复合物的形成情况.结果 β-环糊精作为caveolae结构破坏剂(5 mmol/L,24 h)对apelin-13诱导的内皮细胞增殖具有明显的增强作用;apelin-13处理内皮细胞后,caveolin-1的表达减少,且它的减少在2μmol/L时表现尤为明显;内皮细胞在使用β-环糊精(5 mmol/L)预孵育后,apelin-13下调caveolin-1表达的作用增强;对照组及apelin-13(2μmol/L)处理组caveolin-1与PI3K、ERK1/2均有复合物形成,apelin-13处理组较对照组caveolin-1-PI3K及caveolin-1-ERK1/2两种复合物的形成均有所减少.结论 caveolae及其标志性蛋白caveolin-1参与apelin-13促内皮细胞增殖过程;apelin-13促内皮细胞增殖作用可能与促进caveolin-1与PI3K、ERK1/2解离有关.     

外周血CD34^＋水平与高血压病患者动脉血管功能的关系

目的探讨能否根据外周血内皮祖细胞（endothelial progenitor cells,EPCs）CD34^＋水平评价高血压病患者动脉血管病变程度。方法高血压病患者组62例,对照组20例。高血压病患者采用Framingham心血管危险因素积分分层心血管危险因素,分为低危组18例,中危组14例,高危组17例,极高危组13例。作外周血循环EPCsCD34^＋水平、内皮依赖性血管舒张功能（flow—mediated dialation,FMD）测定并作EPCsCD34^＋水平与Framingham心血管危险因素积分、FMD的相关性分析。结果高血压病患者外周循环EPCsCD34^＋水平随着其心血管危险程度的增加,逐步下降,各组间比较川差异均有显著性（P〈0．05）.高血压病患者肱动脉FMD随着其心血管危险程度的增加显著下降（P〈0．01）。各研究组高血压病患者循环EPCs CD34^＋水平与Framingham心血管危险因素积分呈负相关（r=-0．875,P〈0．01）,与肱动脉FMD呈正相关r=0．859,P〈0．01）.结论高血压病患者循环EPCs CD34^＋水平下降与心血管危险因素、动脉血管功能损伤有显著的相关性。说明循环EPCs CD34^＋水平可以作为评价高血压病患者动脉血管病变的指标.     

脂肪瘤与皮下脂肪细胞药理学特性差异的研究

目的研究脂肪瘤细胞与皮下脂肪细胞药理学特性的差异，为探讨脂肪瘤的形成机制和非手术治疗的方法提供理论依据。方法用不同浓度的异丙肾上腺素、肾上腺素、去甲肾上腺素和ＢＲＬ３７３４４分别激动离体人脂肪瘤和皮下脂肪细胞上的β受体，根据β受体激动后产生的ｃＡＭＰ的量效曲线和最大效应，分析脂肪瘤细胞和皮下脂肪细胞上β受体药理学特性的差异。结果几种不同的β受体激动剂脂肪瘤细胞上的β受体后产生ｃＡＭＰ的最大效应均小于正常皮下脂肪细胞。几种激动剂激动２种脂肪细胞上的β受体作用强度顺序相同，即异丙肾上腺素＞肾上腺素＞去甲肾上腺素＞ＢＲＬ３７３４４。ＢＲＬ３７３４４对２种脂肪细胞上β受体的激动作用都很微弱，且最大效应间无显著差异。结论脂肪瘤细胞上的β肾上腺素受体对β受体激动剂的敏感性低于正常皮下脂肪细胞。ＢＲＬ３７３４４对人脂肪瘤细胞和皮下脂肪细胞上β受体的激动作用微弱。     

Effects of water soluble PM2.5 extracts exposure on human lung epithelial cells (A549): A proteomic study

Exposure to airborne particulate matter (PM)2.5, a PM with aerodynamic diameter of less than 2.5 µm, is known to be associated with a variety of adverse health effects. However, the molecular mechanisms involved in fine PM toxicity are still not well characterized. The present study aims to provide new insights into the cytotoxicity of PM2.5 on human lung epithelial cells (A549) at the proteomic level. Two‐dimensional difference gel electrophoresis revealed a total of 27 protein spots, whose abundance were significantly altered in A549 cells exposed to water‐soluble PM2.5 extracts (WSPE). Among these, 12 spots were upregulated while 15 were downregulated. Twenty‐two proteins were further identified by matrix‐assisted laser desorption/ionization time‐of‐flight tandem mass/mass spectrometry and database search. The results revealed that oxidative stress, metabolic disturbance, dysregulation of signal transduction, aberrant protein synthesis and degradation, as well as cytoskeleton disorganization are major factors contributing to WSPE‐mediated toxicity in human lung cells. It is further proposed that induction of apoptosis through p53, c‐Myc and p21 pathways may be one of the key toxicological events occurred in A549 cells under WSPE stress. The data obtained here will aid our understanding of the toxic mechanisms related to PM2.5, and develop useful biomarkers indicative of inhalable PM2.5 exposure. Copyright © 2013 John Wiley & Sons, Ltd.     

The adaptor Lnk (SH2B3): an emerging regulator in vascular cells and a link between immune and inflammatory signaling

A better knowledge of the process by which inflammatory extracellular signals are relayed from the plasma membrane to specific intracellular sites is a key step to understand how inflammation develops and how it is regulated. This review focuses on Lnk (SH2B3) a member, with SH2B1 and SH2B2, of the SH2B family of adaptor proteins that influences a variety of signaling pathways mediated by Janus kinase and receptor tyrosine kinases. SH2B adaptor proteins contain conserved dimerization, pleckstrin homology, and SH2 domains. Initially described as a regulator of hematopoiesis and lymphocyte differentiation, Lnk now emerges as a key regulator in hematopoeitic and non hematopoeitic cells such as endothelial cells (EC) moderating growth factor and cytokine receptor-mediated signaling. In EC, Lnk is a negative regulator of TNF signaling that reduce proinflammatory phenotype and prevent EC from apoptosis. Lnk is a modulator in integrin signaling and actin cytoskeleton organization in both platelets and EC with an impact on cell adhesion, migration and thrombosis. In this review, we discuss some recent insights proposing Lnk as a key regulator of bone marrow-endothelial progenitor cell kinetics, including the ability to cell growth, endothelial commitment, mobilization, and recruitment for vascular regeneration. Finally, novel findings also provided evidences that mutations in Lnk gene are strongly linked to myeloproliferative disorders but also autoimmune and inflammatory syndromes where both immune and vascular cells display a role. Overall, these studies emphasize the importance of the Lnk adaptor molecule not only as prognostic marker but also as potential therapeutic target.     

Chlorophyll a, an active anti-proliferative compound of Ludwigia octovalvis, activates the CD95 (APO-1/CD95) system and AMPK pathway in 3T3-L1 cells

Ludwigia octovalvis is an aquatic plant widely distributed in Taiwan. It is traditionally used as a diuretic and is consumed as health drink. In this study, we evaluated the anti-proliferative activity of extracts and active constituent (chlorophyll a; CHL-a) of L. octovalvis in 3T3-L1 adipocytes; its mode of action on apoptosis was also investigated. Results showed that, among the different extracts and fractions, the ethylacetate layer (EAL) possessed the most potent anti-proliferative activity. Activity guided fractionation of the EAL obtained the bioactive constituent CHL-a (IC₅₀: 24.10 ± 0.83 nM). At concentrations 5–30 nM, CHL-a exhibited a dose-dependent accumulation of the Sub-G1 peak and caused cell cycle arrest at the G0/G1 phase. At 30 nM, it significantly reduced the cell viability, induced the appearance of DNA fragments, and enhanced the activation of caspase-3. Western blot data revealed that CHL-a decreased the level of Bcl-2, and increased the expression of CD95 (APO-1/CD95) and Bax. Furthermore, CHL-a up-regulated the AMPK and p-AMPK levels, and down-regulated the expression of PPAR-γ. These results conclude that CHL-a possesses potent anti-proliferative activity, and its apoptotic effects on 3T3-L1 adipocytes are mediated through the activation of CD95 (APO-1/CD95) system and the AMPK signaling pathway.     

Interactions between the chemotherapeutic agent eribulin mesylate (E7389) and P-glycoprotein in CF-1 abcb1a-deficient mice and Caco-2 cells

1. Eribulin is a new anticancer agent currently in Phase III clinical trials for the treatment of metastatic breast cancer. In the current studies, we have investigated the effects of P-glycoprotein (P-gp) on the in vivo disposition of eribulin using CF-1 abcb1a-deficient mice, and the influence of eribulin on P-gp-mediated efflux of digoxin in Caco-2 cells.

2. Eribulin was administered intravenously and orally in both CF-1 wild-type and CF-1 abcb1a-deficient mice. P-gp-mediated efflux of digoxin in Caco-2 cell monolayers was measured in the presence of eribulin.

3. The plasma exposure to eribulin was higher in CF-1 abcb1a-deficient mice than that in CF-1 wild-type mice after intravenous (IV) and oral (PO) administrations. The oral bioavailability of eribulin was 62.3% in CF-1 abcb1a-deficient mice compared with 7.6% in wild-type mice. The brain penetration of eribulin in CF-1 abcb1a-deficient mice was 30-fold greater than that in wild-type mice. Eribulin decreased the efflux ratio of digoxin in a concentration-dependent manner, with the result of IC₅₀ greater than 10 µM. The [I]/IC₅₀ of eribulin was estimated to be <0.05.

4. P-gp is likely to limit the oral absorption and brain penetration of eribulin in CF-1 wild-type mice. Eribulin inhibited the efflux of digoxin with IC₅₀ greater than 10 µM in Caco-2 cells. These results suggest that eribulin, given intravenously at the clinically relevant concentrations, may not alter P-gp-mediated disposition of concurrently administered drugs.

     

A comparative study on the effects of 2,3,7,8,-tetrachlorodibenzo-p-dioxin polychlorinated biphenyl126 and estrogen in human bronchial epithelial cells

Epidemiological studies on 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD) exposure indicated high incidences of pulmonary dysfunctions and lung cancer. Animal studies also demonstrated lung cancer development in female, but not in male, rats exposed to TCDD. Such effects, however, have not been reported in polychlorinated biphenyls (PCB) exposure. In our present study, we have investigated the effects of TCDD and PCB126, with or without cotreatment with 17 beta-estradiol (E2), on a human bronchial epithelial cell line BEAS-2B. We found that treatment with either TCDD or PCB126 alone reduced cell numbers as well as thymidine incorporation. Cell death, however, was only detected in PCB126-, but not TCDD-, treated cultures. The TCDD-induced cell reduction, therefore, could not be contributed to cell death. Meanwhile, because TCDD- and PCB126-enhanced CYP1A1 and CYP1B1 expressions were significantly reduced by the AhR antagonist and CYP1 inhibitor alpha-naphthoflavone (ANF), this indicated that the effects of TCDD and PCB126 were AhR and cytochrome p450 1 dependent. We also found that while E2 itself did not alter CYP1A1 and CYP1B1 expressions, cotreatment of E2 with TCDD or PCB126 would significantly enhance TCDD-, but not PCB126-, induced toxicity. We further demonstrated that in the presence of E2, 1 nM TCDD increased the production of E2 metabolites, 2-methoxyestradiol (2-MeOE2) and 4-methoxyestradiol (4-MeOE2). PCB126, however, only increased 2-MeOE2 formation without significant induction of 4-MeOE2. We believe that these metabolites, especially 4-MeOE2, interacted with TCDD to further suppress cell growth. Our data provided the first demonstration on the enhancement of TCDD-induced toxicity in human lung cells via interaction with estrogen.     

Impact of Bacillus thuringiensis toxin Cry1Ab on rumen epithelial cells (REC) – A new in vitro model for safety assessment of recombinant food compounds

The growing use of genetically modified crops necessitates viable screening methods for safety evaluation of recombinant feed, particularly for ruminants. A new sheep rumen epithelial cell culture is introduced as an in vitro cell system for safety evaluation especially focussing on feed and food compounds. We used lactate dehydrogenase (LDH) release, WST-1 conversion, ATP content and caspase 3/7 activity to evaluate cytotoxicity of Cry1Ab, one of the newly expressed Bt-proteins in transgene maize. The results were compared to the effects of valinomycin, a potassium ionophore known to induce cytotoxic effects on a wide range of cells. Whereas no toxicity of Cry1Ab was observed in short as well as in long term experiments, even at non-physiological high concentrations, exposure to valinomycin induced apoptosis and a significant response of all viability parameters after a number of hours. The ATP content and the WST-1 conversion reflecting the energy metabolism of the cells appear to be more sensitive indicators of valinomycin toxicity than the LDH release, a parameter which reflects the membrane integrity. This study presents an in vitro model system, that may be useful as a supplementary tool in toxicity screening before testing substances on animals in vivo.     

敌枯双及其代谢产物对人胚上腭间质细胞生长的抑制作用

<正> 敌枯双[N,N′-methylene-bis-(2-amino-1,3,4-thiadiazole),MATDA]及其代谢产物敌枯唑(amino-1,3,4 thiadiazole,ATDA)对实验动物有致畸作用,前者作用明显大干后者,但至今仍未发现人群食用喷药后稻米有致畸作用,推测可能由于:人对敌枯双致畸作用不敏感,接触水平低和人体内烟酰胺的拮抗作用(已证实烟酰胺可拮抗敌枯     

Loading dendritic cells with gold nanoparticles (GNPs) bearing HIV-peptides and mannosides enhance HIV-specific T cell responses

Gold nanoparticles (GNPs) decorated with glycans ameliorate dendritic cells (DC) uptake, antigen-presentation and T-cells cross-talk, which are important aspects in vaccine design. GNPs allow for high antigen loading, DC targeting, lack of toxicity and are straightforward prepared and easy to handle. The present study aimed to assess the capacity of DC to process and present HIV-1-peptides loaded onto GNPs bearing high-mannoside-type oligosaccharides (P1@HM) to autologous T-cells from HIV-1 patients. The results showed that P1@HM increased HIV-specific CD4⁺ and CD8⁺ T-cell proliferation and induced highly functional cytokine secretion compared with HIV-peptides alone. P1@HM elicits a highly efficient secretion of pro-T_H1 cytokines and chemokines, a moderate production of pro-T_H2 and significant higher secretion of pro-inflammatory cytokines such as TNF‐α and IL-1β. Thus, co-delivery of HIV-1 antigens and HM by GNPs is an excellent vaccine delivery system inducing HIV-specific cellular immune responses in HIV+ patients, being a promising approach to improve anti-HIV-1 vaccines.     

Bisphenol A induced apoptosis via oxidative stress generation involved Nrf2/HO-1 pathway and mitochondrial dependent pathways in human retinal pigment epithelium (ARPE-19) cells

Bisphenol A (BPA) is an estrogen-like compound, and an environmental hormone, that is commonly used in daily life. Therefore, it may enter the human body through food or direct contact, causing BPA residues in blood and urine. Because most studies focused on the analysis of BPA in reproductive cells or tissues, regarding evidence the effect of BPA on human retinal pigment epithelium (ARPE-19) cells unavailable. Accordingly, the present study explored the cytotoxicity of BPA on ARPE-19 cells. After BPA treatment, the expression of Bcl-XL an antiapoptotic protein, in the mitochondria decreased, and the expression of Bax, a proapoptotic protein increased. Then the mitochondrial membrane potential was affected. BPA changed in mitochondrial membrane potential led to the release of cytochrome C, which activated caspase-9 to promote downstream caspase-3 leading to cytotoxicity. The nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and heme oxygenase 1 (HO-1) pathway play a major role in age-related macular degeneration. Our results showed that expression of HO-1 and Nrf2 suppressed by BPA. Superoxide dismutase and catalase, which Nrf2 downstream antioxidants, were degraded by BPA. AMP-activated kinase (AMPK), which can regulate the phosphorylation of Nrf2, and the phosphorylation of AMPK expression was reduced by BPA. Finally, BPA-induced ROS generation and cytotoxicity were reduced by N-acetyl-l -cysteine. Taken together, these results suggest that BPA induced ARPE-19 cells via oxidative stress, which was associated with down regulated Nrf2/HO-1 pathway, and the mitochondria dependent apoptotic signaling pathway.     

A preliminary study of the pharmacodynamics and pharmacokinetics of a novel enkephalin analogue [Tyr-D.Arg-Gly-Phe(4NO2)·Pro·NH2 (BW443C)] in healthy volunteers

Summary We have studied 16 healthy men to evaluate preliminary pharmacodynamics and kinetics of BW443C given by i.v. infusions. Four volunteers received escalating doses at weekly intervals, starting at 0.1 µg·kg^–1 for 60 min and increasing to a maximum of 2.0 µg·kg^–1·min^–1 for 180 min. Subsequently 12 different subjects received single i.v. infusions of 10 µg·kg^–1·min^–1 for 20 min. Subjective effects were reported and objective measurements made of central nervous and cardiovascular effects. Blood was sampled at intervals on all occasions, plasma concentrations were determined by radioimmunoassay and pharmacokinetic profiles were analysed using NONLIN.Dry mouth and some nasal stuffiness were reported and postural hypotension occurred in 5/16 subjects at plasma concentrations >0.8 µg·ml^–1. Supine blood pressure was well maintained in all subjects and hypotension resolved within 60–90 min of discontinuing the infusion. There was no evidence of sedation, mood change, nausea, vomiting, miosis, change in accomodation or respiratory depression. Rapid infusions produced transient feelings of warmth, heavy eyelids, heavy legs, and increased bowel sounds, which resolved despite increasing plasma concentrations.The disposition of the peptide was adequately described by a 2-compartment model with a mean ± SD plasma clearance of 123±18 ml·min^–1 and a half-life of 2.0±0.4 h.     

Effects of tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) on the activation of ERK1/2 MAP kinases and the proliferation of human mammary epithelial cells

Cigarette smoking is a risk factor in the developing of various cancers including breast tumors. There are more than 60 chemical carcinogens in the cigarette smoke; 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) being one of the strongest tobacco-specific carcinogens. In this study, we demonstrated that NNK rapidly activated ERK1 and ERK2 MAP kinases and stimulated proliferation in human normal mammary epithelial cells. MEK1/2 specific inhibitor UO126 completely blocked NNK-induced ERK1/2 activation and cell proliferation, whereas nicotinic receptor nAchR antagonist mecamylamine partially and the selective ₇-nAchR antagonist -bungarotoxin essentially inhibited the NNK-induced ERK1/2 activation and cell proliferation. Surprisingly, receptor tyrosine kinase inhibitor genistein, the selective β₁-adrenergic antagonist atenolol, and the selective β₂-adrenergic antagonist ICI118.551 had a strong inhibitory effect on ERK1/2 activation and cell proliferation induced by NNK. These results suggest that there are at least two different routes in activating ERK1/2 by NNK. One is through nicotinic receptor ₇-nAchR to MEK1/2; the other is from β₁/β₂-adrenergic transactivation of tyrosine kinase containing receptor(s) to MEK1/2. In human cancer mammary epithelial cell lines, we found that ERK MAPK signaling pathway was deregulated: (1) ERK1/2 was constitutively activated at various levels; (2) ERK1/2 was further significantly activated in response to NNK induction; (3) UO126 partially or totally failed to inhibit ERK1/2 activation induced by NNK; (4) The expression levels of ERK1/2 in the cancer cell lines were much higher than those in the normal mammary epithelial cells. The tobacco-specific carcinogen NNK showed a strong proliferative effect on human normal and cancer mammary epithelial cells; the proliferation multitudes of these cells are well correlated with the activation levels of ERK1/2 MAP kinases.     

The effects of baicalein or baicalin on the colloidal stability of ZnO nanoparticles (NPs) and toxicity of NPs to Caco-2 cells

Recent study suggested that the presence of phytochemicals in food could interact with nanoparticles (NPs) and consequently reduce the toxicity of NPs, which has been attributed to the antioxidant properties of phytochemicals. In this study, we investigated the interactions between ZnO NPs and two flavonoids baicalein (Ba) or baicalin (Bn) as well as the influence of the interactions on the toxicity of ZnO NPs to Caco-2 cells. The antioxidant properties of Ba and Bn were confirmed by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assays, with Ba being stronger. However, the presence of Ba or Bn did not significantly affect cytotoxicity, intracellular superoxide or release of inflammatory cytokines of Caco-2 cells after ZnO NP exposure. When Ba was present, the cellular viability of Caco-2 cells after exposure to ZnO NPs was slightly increased, associated with a modest decrease of intracellular Zn ions, but these effects were not statistically different. Ba was more effective than Bn at changing the hydrodynamic sizes, Zeta potential and UV–Vis spectra of ZnO NPs, which indicated that Ba might increase the colloidal stability of NPs. Taken together, the results of the present study indicated that the anti-oxidative phytochemical Ba might only modestly protected Caco-2 cells from the exposure to ZnO NPs associated with an insignificant reduction of the accumulation of intracellular Zn ions. These results also indicated that when assessing the combined effects of NPs and phytochemicals to cells lining gastrointestinal tract, it might be necessary to evaluate the changes of colloidal stability of NPs altered by phytochemicals.     

Antioxidants inhibit endothelin-1 (1-31)-induced proliferation of vascular smooth muscle cells via the inhibition of mitogen-activated protein (MAP) kinase and activator protein-1 (AP-1)

We previously found that human chymase cleaves big endothelins (ETs) at the Tyr(31)-Gly(32) bond and produces 31-amino acid ETs (1-31), without any further degradation products. In the present study, we investigated the effects of various antioxidants on the ET-1 (1-31)-induced change in intracellular signaling and proliferation of cultured rat aortic smooth muscle cells (RASMC). ET-1 (1-31) stimulated rapid and significant activation of the mitogen-activated protein (MAP) kinase family, i.e. extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun NH(2)-terminal kinase (JNK), and p38 MAPK, in RASMC to an extent similar to that of ET-1. All of the antioxidants examined, i.e. N-acetyl-L-cysteine (NAC), diphenyleneiodonium chloride (DPI), and L-(+)-ascorbic acid (ascorbic acid), inhibited both ET-1 (1-31)- and ET-1-induced JNK and p38 MAPK activation but not ERK1/2 activation. Electron paramagnetic resonance (EPR) spectroscopy measurements revealed that NAC, DPI, and ascorbic acid inhibited xanthine oxidase-induced superoxide (O(2)(.-)) generation in a cell-free system. ET-1 (1-31) in addition to ET-1 increased the generation of cellular reactive oxygen species (ROS) in RASMC. ET-1 (1-31)- and ET-1-induced cellular ROS generation was inhibited similarly by NAC, DPI, and ascorbic acid in RASMC. Gel-mobility shift analysis showed that ET-1 (1-31) and ET-1 caused an increase in activator protein-1 (AP-1)-DNA binding activity in RASMC that was inhibited by the above three antioxidants. ET-1 (1-31) increased [3H]thymidine incorporation into cells to an extent similar to that of ET-1. This ET-1 (1-31)-induced increase in [3H]thymidine incorporation was also inhibited by NAC and DPI, but not by ascorbic acid. These results suggest that antioxidants inhibit ET-1 (1-31)-induced RASMC proliferation by inhibiting ROS generation within the cells. The underlying mechanisms of the inhibition of cellular proliferation by antioxidants may be explained, in part, by the inhibition of JNK activation and the resultant inhibition of AP-1-DNA binding.     

Water-soluble and organic extracts of ambient PM2.5 in Tehran air: assessment of genotoxic effects on human lung epithelial cells (A549) by the Comet assay

The main aim of the present study is to investigate the physicochemical characterization of water-soluble extract (WS) and organic extract (OE) of PM_2.5 ambient in Tehran city air in order to evaluate the genotoxicity effects and the potential attribution to these effects. The lung epithelial cells (A549) were exposed to WS and OE, while Comet assays were conducted to analyze the genotoxicity. The results show that the amount of DNA damage in WS fraction and solvent-extractable organic samples is significantly higher than the control group and the increase in concentration significantly contributes to increase in the amount of DNA damage.     

Vectorial transport of the plant alkaloid berberine by double-transfected cells expressing the human organic cation transporter 1 (OCT1, SLC22A1) and the efflux pump MDR1 P-glycoprotein (ABCB1)

An important function of hepatocytes is the biliary elimination of endogenous and xenobiotic small molecules, many of which are organic cations. To study this vectorial transport of organic cations, we constructed a double-transfected Madin-Darby canine kidney strain II (MDCKII) cell line permanently expressing the human organic cation transporter 1 (OCT1, SLC22A1) in the basolateral membrane and MDR1 P-glycoprotein (MDR1 P-gp, ABCB1), an adenosine triphosphate (ATP)-dependent efflux pump for organic cations, in the apical membrane. Additionally, MDCKII single transfectants stably expressing OCT1, MDR1 P-gp, or human organic cation transporter 2 (OCT2, SLC22A2) were generated. Antisera directed against OCT1 or OCT2 specifically detected OCT1 in the basolateral membrane of human hepatocytes, OCT2 in tubular epithelial cells of human kidney, and the respective recombinant transporter in the basolateral membrane of MDCKII transfectants. We identified the lipophilic organic cation berberine, a fluorescent plant alkaloid exhibiting a broad range of biological activities, as substrate of OCT1 and OCT2 with Michaelis-Menten constants of 14.8 μM and 4.4 μM, respectively. Berberine also inhibited the uptake of the prototypic cations tetraethylammonium and 1-methyl-4-phenylpyridinium by MDCK-OCT1 and MDCK-OCT2 transfectants. When transfected cells were grown polarized on permeable filter supports, berberine was transferred from the basolateral to the apical compartments many times faster by MDCK-OCT1/MDR1 P-gp double transfectants than by MDCK-OCT1 or MDCK-MDR1 P-gp single transfectants. The specific MDR1 P-gp inhibitor, zosuquidar trihydrochloride (LY335979), strongly inhibited berberine efflux into the apical compartment. The MDCK-OCT1/MDR1 P-gp double transfectants may be useful to identify additional cationic substrates and inhibitors of OCT1 and MDR1 P-gp, including drug candidates. This study was supported in part by the Deutsche Krebshilfe (Bonn, Germany).