Assessment of styrene oligomers eluted from polystyrene-made food containers for estrogenic effects in in vitro assays

Recently, several substances from among the huge numbers of chemicals used by mankind have been implicated as instigators of disrupted endocrine function and related human health problems. Polystyrene (PS) is one of the most frequently used resins in the world, and the styrene oligomer dissolved out from PS has been designated as a potential trigger of estrogen-like activity in the Wingspread Declaration and the Japan Environment Agency's SPEED98 [JEA (Japan Environment Agency) Strategic Problem on Environmental Endocrine Disruptors '98 (SPEED) '98), http://www.env.go.jp/en/pol/speed98/sp98.html]. In order to assess the endocrine disrupting effect of styrene oligomers, we tested one styrene monomer (SM), three styrene dimers (SDs) and seven styrene trimers (STs), newly isolated from optical isomers, known to dissolve in small amounts from cup noodle containers made of polystyrene by the estrogen receptor binding assay, luciferase reporter gene assay, and human breast cancer cell MCF-7 proliferation assay. In all three tests, none of the SM, SDs and STs showed any significant activity. Accordingly, we concluded that these substances have no estrogenic activity.     

In vitro validation of bioluminescent monitoring of disease progression and therapeutic response in leukaemia model animals

Purpose The application of in vivo bioluminescence imaging to non-invasive, quantitative monitoring of tumour models relies on a positive correlation between the intensity of bioluminescence and the tumour burden. We conducted cell culture studies to investigate the relationship between bioluminescent signal intensity and viable cell numbers in murine leukaemia model cells. Methods Interleukin-3 (IL-3)-dependent murine pro-B cell line Ba/F3 was transduced with firefly luciferase to generate cells expressing luciferase stably under the control of a retroviral long terminal repeat. The luciferase-expressing cells were transduced with p190 BCR-ABL to give factor-independent proliferation. The cells were cultured under various conditions, and bioluminescent signal intensity was compared with viable cell numbers and the cell cycle stage. Results The Ba/F3 cells showed autonomous growth as well as stable luciferase expression following transduction with both luciferase and p190 BCR-ABL, and in vivo bioluminescence imaging permitted external detection of these cells implanted into mice. The bioluminescence intensities tended to reflect cell proliferation and responses to imatinib in cell culture studies. However, the luminescence per viable cell was influenced by the IL-3 concentration in factor-dependent cells and by the stage of proliferation and imatinib concentration in factor-independent cells, thereby impairing the proportionality between viable cell number and bioluminescent signal intensity. Luminescence per cell tended to vary in association with the fraction of proliferating cells. Conclusion Although in vivo bioluminescence imaging would allow non-invasive monitoring of leukaemia model animals, environmental factors and therapeutic interventions may cause some discrepancies between tumour burden and bioluminescence intensity.     

Establishment of an In Vitro Test System to Evaluate the Down-Regulatory Activities of Natural Products on IL-4

Interleukin-4 (IL-4), a representative TH2 cytokine, plays a pathologic role in the onset of various allergic diseases including atopic dermatitis, atopic rhinitis, and asthma. Several drug candidates that down-regulate IL-4 expression have been studied for their possible use as antiallergic agents in clinical settings. Therefore, an in vitro test to evaluate IL-4 promoter activities might be useful for selecting candidates of novel natural therapeutics. The promoter region (-741 to +56) of IL-4 was cloned upstream of a luciferase gene in the plasmid pGL4.14 with a hygromycin resistance gene as a selection marker to generate pGL4.14-IL-4. Treatment with PMA and A23187 highly increased luciferase activity by approximately 10-fold compared with the control in both EL-4 thymoma and RBL-2H3 cells transiently transfected with pGL4.14-IL-4, as well as in stable cell lines constantly expressing pGL4.14-IL-4. Cyclosporin A and dexamethasone, well-known anti-allergic agents, significantly down-regulated the activity in a dose-dependent manner. The feasibility of this system was evaluated by measuring the down-regulatory activities of various extracts from the TBRC plant library on PMA- and A23187-induced luciferase activities of IL-4 promoter, and by measuring IL-4 production in cultured cells using ELISA assays. The results of this study suggest that this primary screening system is simple and time-saving, and might be suitable for the selection of natural therapeutic candidates for allergic disease by measuring the down-regulatory effects of natural products on the IL-4 promoter.     

Invesgations [correction investigations] on the influence of halide substituents on the estrogen receptor interaction of 2, 4, 5-tris(4-hydroxyphenyl)imidazoles

Previously, we reported on the synthesis and estrogen receptor (ER) interaction of imidazoles, which had to be 1-alkyl-4, 5-bis(2-halo-4-hydroxyphenyl) substituted for a high relative binding affinity (RBA >1 %). This led to the assumption that a shielding of the polar heterocyclic system is a prerequisite for ER binding. In continuation of this study we synthesized 2, 4, 5-tris(4-hydroxyphenyl)imidazoles with Cl-or F-atoms in the ortho-positions of the aromatic rings and evaluated whether they mediate sufficient hydrophobicity for ER interaction. 2-(2, 6-Dichloro-3/4-hydroxyphenyl)-4, 5-bis(2-halo-4-hydroxyphenyl)imidazoles were synthesized by reaction of the respective methoxy-substituted benzil with either the 2, 6-dichloro-4-methoxy-or the 2, 6-dichloro-3-methoxybenzaldehyde in ammonium acetate solution. The required ether cleavage was performed subsequently with BBr(3). In the competition experiment with [(3)H]estradiol the imidazoles with the a C2-standing (2, 6-dichloro-4-hydroxyphenyl) ring showed an RBA >0.02 %, but did not activate the luciferase gene in estrogen receptor positive MCF-7-2a breast cancer cells stably transfected with the plasmid ERE(wtc)luc. In the test for antagonistic potency only the 2-(2, 6-dichloro-4-hydroxyphenyl)-4, 5-bis(4-hydroxyphenyl)imidazole 3 antagonized the effects of 1 nM estradiol slightly. From these data, it can be concluded that a C2-standing 2, 6-dichloro-4-hydroxyphenyl ring is not appropriate to optimize the ER interaction of 4, 5-(4-hydroxyphenyl)imidazoles.     

A note on poly-L-lysine-mediated gene transfer in HeLa cells

Poly-L-lysines of chain lengths varying from 70 to 300 residues are shown to bring about luciferase pRSVL DNA uptake and expression in HeLa cells. Transfection was approximately 50% that of the cationic liposome DOTAB. Expression was higher in the presence of chloroquine. Of interest was the fact that luciferase activity depended on the polysine/DNA charge ratio (+/ -). Maximum activity occurred at a charge ratio (+/ -) of 3, while at a charge ratio of 1 (conjugate electrically neutral) activity was much lower. At the higher charge ratios (+/ -) of 4 and 5, luciferase activity decreased. The results obtained are discussed.     

稳定表达荧光素酶的产酸克雷伯菌在评估不同灭菌方法中的应用

目的构建稳定表达荧光素酶基因（lux）的产酸克雷伯菌,旨在更直观地观察产酸克雷伯菌（K. oxytoca）在宿主体内的分布以及评估不同杀菌方法的效果。 方法扩增pBAV1k-T5-Lux质粒的荧光素酶基因簇（Lux A/B/C/D/E）,构建含有Lux A/B/C/D/E基因簇的pBBR1质粒（pBBR1-lux）,并获得能够表达荧光素基因基团的大肠埃希菌（E. coli-pBBR1-lux）。将pBBR1-lux质粒电转化至产酸克雷伯菌感受态细胞,经荧光强度鉴定及菌株的3次传代,筛选能够稳定表达lux基因的产酸克雷伯菌（K. oxytoca-pBBR1-lux）。 结果连接成功的环状质粒产物命名为pBBR1-lux。与大肠埃希菌对照株相比,含E. coli-pBBR1-lux的Luminesence荧光信号值显著升高[15 345（14 676,18 654） vs. 63（60,82）],差异具有统计学意义（t = 21.14、P = 0.035）。K. oxytoca-pBBR1-lux的Luminesence荧光信号值[399 303（265 245,617 192）]显著高于产酸克雷伯菌对照菌株[83（63.5,86.75）],差异具有统计学意义（t = 7.07、P = 0.014）。E. coli-pBBR1-lux和K. oxytoca-pBBR1-lux均能够在Veritas微孔板光度计和小动物成像仪上检测到Luminesence荧光信号。将所得菌株按照1︰10稀释6个梯度,荧光值检测结果显示,Luminesence值随菌落浓度降低而下降。多次传代后pBBR1-lux能够在产酸克雷伯菌中稳定表达荧光素酶,不同理化杀菌方法对产酸克雷伯菌杀菌效果不同,紫外线和84消毒液（10%）是最有效的杀灭产酸克雷伯菌方法。 结论本研究获得了可被微孔板光度计和小动物成像仪检测到的稳定表达荧光素酶的K. oxytoca-pBBR1-lux。     

小鼠HMGB1突变型启动子荧光素酶报告基因的构建

目的构建小鼠HMGB1突变型启动子荧光素酶报告基因。方法 PCR扩增小鼠HMGB1启动子DNA,构建小鼠HMGB1野生型启动子荧光素酶报告基因pGL3-HMGB1-Y。重叠延伸PCR突变HSE核心序列,构建HMGB1突变型启动子荧光素酶报告基因pGL3-HMGB1-T,序列比对。结果序列对比结果显示pGL3-HMGB1-Y中HSE核心碱基-TTCGAGAA-已突变为-TACGAGCC-。结论成功构建小鼠HMGB1突变型启动子荧光素酶报告基因,为研究热休克转录因子1对HMGB1的转录调控作用奠定了基础。     

A rapid luciferase transfection assay for transcription activation effects and stability control of estrogenic drugs in cell cultures

A rapid assay system for measuring the potential of estrogenic drugs is introduced. Luciferase induction could be measured in estrogen-receptor-positive human MCF-7 breast cancer cells, which had been transfected with a novel luciferase reporter plasmid ERE luc. The minimal requirement was 1 h exposure to the inducing drug and 3.5 h of incubation after removal of the drug. The assay system was used to measure the stability of the drug diaqua-[1,2-bis (2,6-dichloro-4-hydroxyphenyl) ethylenediamine] platinum(II) sulfate, containing an estrogenic ligand and reactive platinum. Luciferase activity was observed only when the drug was in the culture medium and cells for short times, whereas the estrogenic ligand alone remained active. It is assumed that binding of the platinum moiety to macromolecular constituents of the culture or cells renders the drug inaccessible for binding to the estrogen receptor.     

Silver nanoparticles induced changes in the expression of NF-κB related genes are cell type specific and related to the basal activity of NF-κB

Silver nanoparticles (AgNPs) are widely used in industry and medicine but the recent evidence for their cytotoxicity rise a concern about the safety of their use. We have previously shown that human A549 cells are resistant to AgNPs cytotoxicity, as compared with similarly treated HepG2 cells. In order to check for the role of the NF-κB signaling pathway in response of A549 and HepG2 cell lines to the treatment with 20 nm and 200 nm AgNps, we analyzed the expression of 84 key genes related to the functionality of the NF-κB signaling pathway. We observed considerable alternations in gene expression in HepG2 cells treated with 20 nm AgNPs, and minor changes when exposed to 200 nm AgNPs. Surprisingly, no changes in gene expression were observed in A549 cells treated with both size AgNPs. Using the NF-κB luciferase reporter system, we further tested the basal activity and inducibility of the NF-κB pathway in both cell lines and found that the inducibility of NF-κB signaling in A549 cells is approximately 5 times lower than this of HepG2 cells, but the basal activity is approximately 3.5 times higher. In accordance, the NF-κB activation after AgNPs treatment was observed in HepG2 but not in A549. Altogether indicate that NF-kB mediated cellular response to AgNPs is cell type specific and related to the basal activity of NF-κB.     

活体生物发光成像技术及其在消化道研究领域中的应用

活体生物发光成像技术作为一种非放射性、非损伤性、高灵敏特异性、实时动态的分子生物学检测技术,实现了在活体内对生物学反应实时、原位、动态和无损伤的观察,相比CT、MRI、PET和SPECT等传统影像学成像技术,活体发光成像技术显示出了巨大的优越性.活体生物发光成像技术已成为检测小动物体内分子及细胞事件的强有力工具,越来越广泛地应用于基础生命科学的研究领域.近年来,生物发光成像技术因其高度的特异性和敏感性,在肿瘤学的研究领域也逐渐得以应用,并显示出了良好的应用前景.本文就活体生物发光成像技术的成像特点、技术原理及其在消化道研究领域的应用做一综述.