Cerium oxide nanoparticle uptake kinetics from the gas-phase into lung cells in vitro is transport limited

Nowadays, aerosol processes are widely used for the manufacture of nanoparticles (NPs), creating an increased occupational exposure risk of workers, laboratory personnel and scientists to airborne particles. There is evidence that possible adverse effects are linked with the accumulation of NPs in target cells, pointing out the importance of understanding the kinetics of particle internalization.In this context, the uptake kinetics of representative airborne NPs over 30 min and their internalization after 24 h post-exposure were investigated by the use of a recently established exposure system. This system combines the production of aerosolized cerium oxide (CeO₂) NPs by flame spray synthesis with its simultaneous particle deposition from the gas-phase onto A549 lung cells, cultivated at the air-liquid interface.Particle uptake was quantified by mass spectrometry after several exposure times (0, 5, 10, 20 and 30 min). Over 35% of the deposited mass was found internalized after 10 min exposure, a value that increased to 60% after 30 min exposure. Following an additional 24 h post-incubation, a time span, after which adverse biological effects were observed in previous experiments, over 80% of total CeO₂ could be detected intracellularly.On the ultrastructural level, focal cerium aggregates were present on the apical surface of A549 cells and could also be localized intracellularly in vesicular structures. The uptake behaviour of aerosolized CeO₂ is in line with observations on cerium suspensions, where particle mass transport was identified as the rate-limiting factor for NP internalization.     

高效液相色谱串联质谱法同时定量测定人血清中脱氢表雄酮、睾酮及雄酮

目的:建立一种快速、灵敏地同时测定人血清中脱氢表雄酮、睾酮及雄酮的高效液相色谱串联质谱(电喷雾电离模式)定量方法。方法:血清样品选用甲基叔丁基醚(MTBE)萃取后,取上清液用盐酸羟胺对3种激素进行柱前衍生化后进样测定。色谱柱为Waters YMC ODS-AQ(2.0 mm×150 mm,3.0μm),流动相为70%乙腈和30%醋酸(0.02%)溶液,流速为0.2 mL·min-1,柱温为50℃。本文采用电喷雾离子源进行正离子离子化,使用MRM检测模式选择性监测脱氢表雄酮(m/z 304.2/253.2)、睾酮(m/z 304.2/124.0)、雄酮(m/z 306.2/255.2)及内标炔诺酮(m/z 314.1/124.2)。结果:脱氢表雄酮、睾酮与雄酮的最低定量限(LLOQ)分别为0.10,0.05,0.10 ng·mL-1;标准曲线相关系数(r)均大于0.99;3种激素的日间及日内方法回收率均在±115%之内,日内及日间精密度(RSD)均小于15%。结论:实现在ESI电离模式下,用HPLC-MS/MS同时测定脱氢表雄酮、睾酮与雄酮3种甾体激素;本方法具有良好的特异性、灵敏度、准确度及精密度,能满足人血清样品定量分析需要。     

HPLC-MS/MS法测定人体血浆中丙泊酚的浓度及其药动学研究(英文)

目的:采用HPLC-MS/MS法检测人体血浆中丙泊酚的浓度,并将该方法用于临床药动学研究。方法:采用Zorb-ax Eclipse XDB-C18(50 mm×4.6 mm,5μm)色谱柱分离;流动相为甲醇和0.1%氨水二元梯度洗脱系统;流速为0.3 mL.min-1;通过电喷雾离子化电离源(ESI),采用负离子检测。结果:线性范围为0.010～12μg.mL-1,相关系数≥0.99,定量下限为0.010μg.mL-1。日内及日间精密度不高于15%,准确度的相对误差(RE)在-2.0%～5.0%。药代动力学符合三室模型,清除率为0.029 L.min-1.kg-1,中央室分布容积为0.60 L.kg-1,血药浓度消除半衰期t1/2α为0.84 min,t1/2β为6.7 min,t1/2γ为128.7 min。结论:该方法高效、灵敏,特异性强,准确度、精密度好,适用于丙泊酚药物动力学研究。     

Lignan constituents of Tilia amurensis and their biological evaluation on antitumor and anti-inflammatory activities

In the recent decade, numerous lignan derivatives isolated from plants have been proven to have the potential as an anti-cancer substance. On the search for anti-cancer compounds from Korean medicinal plants, the methanolic extract from the trunk of Tilia amurensis Rupr. (Tiliaceae) was found to have significant cytotoxicity against A549 (lung carcinoma), SK-OV-3 (ovary malignant ascites), SK-MEL-2 (skin melanoma), and HCT-15 (colon adenocarcinoma) in our screening test. Hence, a bioassay-guided fractionation and chemical investigation of the methanolic extract resulted in the isolation and identification of 10 lignan derivatives (1–10) including two new lignan glycosides named tiliamurosides A (1) and B (2). The structures of these new compounds were determined by spectroscopic methods, namely 1D and 2D nuclear magnetic resonance (NMR) techniques, high resolution mass spectrometry (HRMS), circular dichroism (CD) data, and chemical methods. Tiliamuroside B (2) and schizandriside (3) showed significant cytotoxicity against A549, SK-OV-3, SK-MEL-2, and HCT-15 cell lines with inhibitory concentration (IC₅₀) values of 3.26–8.89 μM. Moreover, (−)-syringaresinol (8) and (−)-pinoresinol 4-O-β-d-glucopyranoside (10) significantly inhibited nitric oxide (NO) production in murine microglia BV-2 with IC₅₀ values of 15.05 and 34.35 μM, respectively.     

Multidrug resistance protein (MRP) 4 attenuates benzo[a]pyrene-mediated DNA-adduct formation in human bronchoalveolar H358 cells

Multi-drug resistance protein (MRP) 4, an ATP-binding cassette (ABC) transporter, has broad substrate specificity. It facilitates the transport of bile salt conjugates, conjugated steroids, nucleoside analogs, eicosanoids, and cardiovascular drugs. Recent studies in liver carcinoma cells and hepatocytes showed that MRP4 expression is regulated by the aryl hydrocarbon receptor (AhR) and nuclear factor E2-related factor 2 (Nrf2). The AhR has particular importance in the lung and is most commonly associated with the up-regulation of cytochrome P-450 (CYP)-mediated metabolism of benzo[a]pyrene (B[a]P) to reactive intermediates. Treatment of H358, human bronchoalveolar, cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or (−)-benzo[a]pyrene-7,8-dihydro-7,8-diol (B[a]P-7,8-dihydrodiol), the proximate carcinogen of B[a]P, revealed that MRP4 expression was increased compared to control. This suggested that MRP4 expression might contribute to the paradoxical decrease in (+)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene-2′-deoxyguanosine ((+)-anti-trans-B[a]PDE-dGuo) DNA-adducts observed in TCDD-treated H358 cells. We have now found that decreased MRP4 expression induced by a short hairpin RNA (shRNA), or chemical inhibition with probenecid, increased (+)-anti-trans-B[a]PDE-dGuo formation in cells treated with (−)-B[a]P-7,8-dihydrodiol, but not the ultimate carcinogen (+)-anti-trans-B[a]PDE. Thus, up-regulation of MRP4 increased cellular efflux of (−)-B[a]P-7,8-dihydrodiol, which attenuated DNA-adduct formation. This is the first report identifying a specific MRP efflux transporter that decreases DNA damage arising from an environmental carcinogen.     

以ESI为例论科研评价工具的功能设计

介绍ESI数据库及其指标体系的特点,分析ESI数据库的应用情况,对科学研究评价工具的功能设计思路进行思考,提出设立权重平衡学科差异、建立人名机构规范档、提高可检索性及可视化程度等建议。     

Hippocampal synapsin isoform levels are linked to spatial memory enhancement by SGS742

Synapsins are essential proteins for synaptic plasticity and there is no information available for their role in cognitive enhancement (CE) of spatial memory formation. It was therefore the aim of the study to link individual synapsin proteins and their isoforms to spatial memory formation enhanced by SGS742 in the mouse. Extracted hippocampal proteins from a cognitive study treating OF1 mice with the cognitive enhancer SGS742 and tested in the Morris water maze, were run on two‐dimensional gel electrophoresis. Subsequently, protein spots were unambiguously identified by qQ‐TOF mass spectrometry. Quantification of proteins from four groups (NaCl‐treated mice, SGS742‐treated mice, SGS742‐treated yoked controls, and NaCl‐treated yoked controls) was carried out according to an in‐gel stable isotope labeling method. A total of 17 protein spots representing synapsin isoforms were identified and quantified. Using quantification of individual synapsin isoforms showed that these can be clearly assigned to CE by the GABA_B antagonist SGS742. Quantitative determination of individual synapsin isoform showed an increase in SGS742‐treated mice (mean ± SD) of ratios between light and heavy stable isotope labeled synapsin protein (SGS742 vs. controls: 2.19 ± 0.41 for synapsin Ia, and 1.41 ± 0.81 for synapsin IIa). Synapsins Ib and IIb were not linked to CE. The NaCl‐treated controls and the use of yoked controls that were ruling out swimming‐ and stress‐mediated changes of synapsins, unequivocally allow to propose a role for synapsins Ia and IIa in the mechanism of CE of spatial memory formation. © 2009 Wiley‐Liss, Inc.     

盐酸曲美他嗪健康人中的药动学研究和生物等效性评价

目的:建立LC-MS方法测定盐酸曲美他嗪在健康人血浆中的浓度,并进行药物动力学研究和生物等效性评价。方法:18名男性健康受试者,随机分成2组,交叉口服受试制剂和参比制剂各20 mg,采用LC-MS法测定人血浆中曲美他嗪的浓度。结果:受试制剂与参比制剂的各主要药动学参数:t_(1/2)分别为(7.31±2.36)和(6.74±2.17)h,t_(max)分别为(2.1±0.9)和(1.8±0.6)h,C_(max)分别为(92.05±19.64)和(96.71±22.31)ng·ml~(-1),AUC_(0-24)分别为(691.24±171.82)和(706.31±189.60) ng·h·ml~(-1)。对两种制剂的参数C_(max)、AUC_(0-24)对数转换后先进行方差分析,再进行双单侧t检验。双单侧t检验结果表明上述两个参数生物等效,t_(max)经非参数法检验差异没有显著性意义(P>0.05),这表明两种制剂生物等效,相对生物利用度(101.5±26.7)%。结论:两种制剂具有生物等效。     

Biotransformation and in vitro assessment of metabolism-associated drug–drug interaction for CRx-102, a novel combination drug candidate

CRx-102 is an oral synergistic combination drug which contains the cardiovascular agent, dipyridamole (DP) and a very low dose of the glucocorticoid, prednisolone (PRED). CRx-102 works through a novel mechanism of action in which DP selectively amplifies the anti-inflammatory activity of PRED without replicating its side effects. CRx-102 is in clinical trials for the treatment of osteoarthritis. Here we delineate the in vitro metabolism and explore the potential for a drug–drug interaction between the active agents in CRx-102. Our study using human hepatocyte suspensions showed that both DP and PRED were metabolized by CYP3A4 isozymes, resulting in the formation of diverse arrays of both oxidative and oxidative-reduced metabolites. Within phase 1 biotransformation, CYP3A4 was one of the pathways responsible for the metabolism of PRED, while phase 2 biotransformation played a significant role in the metabolism of DP. Glucuronidation of DP was substantial and was catalyzed by many UGT members, specifically those in the UGT1A subfamily. Based on the tandem mass (MS/MS) product ion spectra (PIS) acquired, the major metabolites of both agents, namely, monooxygenated, mono-N-deethanolaminated, dehydrogenated and O-glucuronidated metabolites of DP and the monooxygenated (e.g., 6-hydroxyl), dehydrogenated (prednisone) and reduced (20-hydroxyl) metabolites of PRED, were identified and elucidated. The affinities for DP biotransformation, including CYP3A4-mediated oxidative pathways and UGT-mediated O-glucuronidation, appeared high (K_m < 10 μM), as compared with the modest affinities of PRED biotransformation catalyzed by CYP3A4 (K_m ∼ 40–170 μM). DP, but not PRED, exerted a minimal inhibitory effect on the drug-metabolizing CYP isoforms, including CYP3A4, which was determined using a panel of CYP isoform-preferred substrate activities in pooled human liver microsomal (HLM) preparations and microsomal preparations containing the recombinant enzymes (K_i ∼ 2–12 μM). Using the DP maximal plasma concentration (C_max) observed in the clinic and a predictive mathematical model for metabolism-associated drug–drug interaction (DDI), we have demonstrated that there is little likelihood of a pharmacokinetic interaction between the two active agents in CRx-102.     

Phenolic acids determination by HPLC–DAD–ESI/MS in sixteen different Portuguese wild mushrooms species

Analysis of phenolic compounds in sixteen Portuguese wild mushrooms species has been carried out by high-performance liquid chromatography coupled to photodiode array detector and mass spectrometer (HPLC–DAD–ESI/MS). No flavonoids were detected in the analysed samples, but diverse phenolic acids namely protocatechuic, p-hydroxybenzoic and p-coumaric acids, and two vanillic acid isomers were found and quantified. A related non-phenolic compound, cinnamic acid, was also detected in some samples, being the only compound found in Cantharellus cibarius (14.97 mg/kg, dry matter), Lycoperdon perlatum (14.36 mg/kg) and Macrolepiota procera (21.53 mg/kg). p-Hydroxybenzoic acid was found in the majority of the samples, being the most abundant compound in Agaricus silvicola (238.7 mg/kg). Ramaria botrytis showed the highest phenolic acids concentration (356.7 mg/kg) due to the significant contribution of protocatechuic acid (342.7 mg/kg).