Phenethyl isothiocyanate-induced cytoskeletal changes and cell death in lung cancer cells

Isothiocyanates are known for their anticarcinogenic and antitumor potential, however, the exact mechanism of their action has not been fully elucidated. The present study was designed to investigate and compare the effects of phenethyl isothiocyanate on cell morphology, the cytoskeleton and induction of cell death in human non-small cell lung cancer cell lines A549 and H1299 differing in p53 status. Cell viability tests (MTT assay, xCELLigence system) showed that PEITC exhibits lower cytotoxicity to A549 cells containing wild-type p53. The observed growth-inhibitory effect of PEITC was dose-dependent, but time-dependence was observed only at higher concentrations. The results of flow-cytometric and fluorescence-microscopic analyses indicate that PEITC induced disassembly of actin stress fibers and degradation of tubulin which, most likely, contributed to the induction of cell death. Although, 24-h incubation caused G2/M cell cycle arrest, the fraction of G2/M cells decreased in a dose- and time-dependent manner in favor of cells with sub-G1 DNA content. Further experiments (Annexin V staining, electron microscopic observations) confirmed that the apoptosis-inducing potency of PEITC is probably the main factor responsible for cell growth inhibition. However, PEITC treatment also resulted in the appearance of an increased proportion of H1299 cells exhibiting morphological features of mitotic catastrophe.     

Multiple spectroscopic studies on the interaction between olaquindox,a feed additive,and bovine serum albumin

The interaction between olaquindox (OLA) and bovine serum albumin (BSA) was investigated using fluorescence, UV–vis absorption and circular dichroism (CD) spectroscopy. The results showed that the fluorescence quenching of BSA by OLA was a static quenching process induced by the formation of OLA–BSA complex. The binding constant of OLA–BSA complex was calculated to be 1.299 × 10⁴ L mol⁻¹ (293 K). The negative values of ΔH⁰ and ΔS⁰ indicated that hydrogen bond and van der Waals interactions played major roles in stabilizing the complex. Site probe competition experiments and number of binding sites (n) revealed that OLA could bind to site I in subdomain IIA of BSA, and the binding distance (r) was evaluated to be 3.643 nm according to Förster’s non-radiative energy transfer theory. The results of CD and three-dimensional fluorescence spectra suggested some conformational changes of BSA after OLA binding.     

Cytokine production by co-cultures exposed to monodisperse amorphous silica nanoparticles: the role of size and surface area

The aim of this study was to test the influence of nanoparticle size and surface area (SA) on cytokine secretion by co-cultures of pulmonary epithelial cells (A549), macrophages (differentiated THP-1 cells) and endothelium cells (EA.hy926) in a two-compartment system. We used monodisperse amorphous silica nanoparticles (2, 16, 60 and 104 nm) at concentrations of 5 μg/cm2 cell culture SA or 10 cm2 particle SA/cm2. A549 and THP-1 cells were exposed to nanoparticles for 24h, in the presence of EA.hy926 cells cultured in an insert introduced above the bi-culture after 12h. Supernatants from both compartments were recovered and TNF-α, IL-6, IL-8 and MIP-1α were measured. Significant secretion of all cytokines was observed for the 2 nm particles at both concentrations and in both compartments. Larger particles of 60 nm induced significant cytokine secretion at the dose of 10 cm2 particle SA/cm2. The use of multiple cellular types showed that cytokine secretion in single cell cultures is amplified or mitigated in co-cultures. The release of pro-inflammatory mediators by endothelial cells not directly exposed to nanoparticles indicates a possible endothelium activation after inhalation of silica particles. This work shows the role of size and SA in cellular response to amorphous nanosilica.     

Toxicity of silver nanoparticles - nanoparticle or silver ion?

The toxicity of silver nanoparticles (AgNPs) has been shown in many publications. Here we investigated to which degree the silver ion fraction of AgNP suspensions, contribute to the toxicity of AgNPs in A549 lung cells. Cell viability assays revealed that AgNP suspensions were more toxic when the initial silver ion fraction was higher. At 1.5 μg/ml total silver, A549 cells exposed to an AgNP suspension containing 39% silver ion fraction showed a cell viability of 92%, whereas cells exposed to an AgNP suspension containing 69% silver ion fraction had a cell viability of 54% as measured by the MTT assay. In addition, at initial silver ion fractions of 5.5% and above, AgNP-free supernatant had the same toxicity as AgNP suspensions. Flow-cytometric analyses of cell cycle and apoptosis confirmed that there is no significant difference between the treatment with AgNP suspension and AgNP supernatant. Only AgNP suspensions with silver ion fraction of 2.6% or less were significantly more toxic than their supernatant as measured by MTT assays. From our data we conclude that at high silver ion fractions (≥5.5%) the AgNPs did not add measurable additional toxicity to the AgNP suspension, whereas at low silver ion fractions (≤2.6%) AgNP suspensions are more toxic than their supernatant.     

Modifications of the bacterial reverse mutation test reveals mutagenicity of TiO2 nanoparticles and byproducts from a sunscreen TiO2-based nanocomposite

The bacterial reverse mutation test, recommended by the Organization for Economic Co-operation and Development (OECD) to determine genotoxicity of chemical compounds, has been recently used by several authors to investigate nanoparticles. Surprisingly, test results have been negative, whereas in vitro mammalian cell tests often give positive genotoxic responses. In the present study, we used the fluctuation test procedure with the Salmonella typhimurium strains TA97a, TA98, TA100 and TA102 to determine the mutagenic potential of TiO₂ nanoparticles (NP-TiO₂) and showed that, when it is used conventionally, this test is not suitable for nanoparticle genotoxicity assessment. Indeed, the medium used during exposure prevents electrostatic interactions between bacterial cells and nanoparticles, leading to false-negative responses. We showed that a simple pre-exposure of bacteria to NP-TiO₂ in a low ionic strength solution (NaCl 10 mM) at a pH below the nanoparticle isoelectric points (pH 5.5) can strongly improve the accuracy of the test. Thus, based on these improvements, we have demonstrated the genotoxicity of the engineered NP-TiO₂ tested and a NP-TiO₂ byproduct from a sunscreen nanocomposite. It was also shown that strain TA102 is more sensitive than the other strains, suggesting an oxidative stress-mediated mechanism of genotoxicity.     

Study of serum interaction with a cationic nanoparticle: Implications for in vitro endocytosis, cytotoxicity and genotoxicity

We used well-characterized and positively charged nanoparticles (NP⁺) to investigate the importance of cell culture conditions, specifically the presence of serum and proteins, on NP⁺ physicochemical characteristics, and the consequences for their endocytosis and genotoxicity in bronchial epithelial cells (16HBE14o-). NP⁺ surface charge was significantly reduced, proportionally to NP⁺/serum and NP⁺/BSA ratios, while NP⁺ size was not modified. Microscopy studies showed high endocytosis of NP⁺ in 16HBE14o-, and serum/proteins impaired this internalization in a dose-dependent manner. Toxicity studies showed no cytotoxicity, even for very high doses of NP⁺. No genotoxicity was observed with classic comet assay while primary oxidative DNA damage was observed when using the lesion-specific repair enzyme, formamidopyrimidine DNA-glycosylase (FPG). The micronucleus test showed NP⁺ genotoxicity only for very high doses that cannot be attained in vivo. The low toxicity of these NP⁺ might be explained by their high exocytosis from 16HBE14o- cells. Our results confirm the importance of serum and proteins on nanoparticles endocytosis and genotoxicity.     

Biodegradable nanoparticles mimicking platelet binding as a targeted and controlled drug delivery system

This research aims to develop targeted nanoparticles as drug carriers to the injured arterial wall under fluid shear stress by mimicking the natural binding ability of platelets via interactions of glycoprotein Ib-alpha (GPIbα) of platelets with P-selectin of damaged endothelial cells (ECs) and/or with von Willebrand factor (vWF) of the subendothelium. Drug-loaded poly(d,l-lactic-co-glycolic acid) (PLGA) nanoparticles were formulated using a standard emulsion method and conjugated with glycocalicin, the external fraction of platelet GPIbα, via carbodiimide chemistry. Surface-coated and cellular uptake studies in ECs showed that conjugation of PLGA nanoparticles, with GPIb, significantly increased nanoparticle adhesion to P-selectin- and vWF-coated surfaces as well as nanoparticle uptake by activated ECs under fluid shear stresses. In addition, effects of nanoparticle size and shear stress on adhesion efficiency were characterized through parallel flow chamber studies. The observed decrease in bound nanoparticle density with increased particle sizes and shear stresses is also explained through a computational model. Our results demonstrate that the GPIb-conjugated PLGA nanoparticles can be used as a targeted and controlled drug delivery system under flow conditions at the site of vascular injury.     

氟尿嘧啶炭纳米粒制剂在大鼠血液和淋巴中药物浓度比较

目的对氟尿嘧啶(5-Fu)炭纳米粒新型制剂在大鼠体内血液和淋巴中进行药物动力学研究。方法采用反效液相色谱法(RP-HPLC)测定大鼠腹腔注射5-Fu炭纳米粒新剂型与普通剂型(20mg/kg体质量)后在两者体内的血药浓度和淋巴浓度,并对两者代谢时间进行比较。结果 (1)相同时间内,不论是血液中还是淋巴组织中,炭纳米粒新型制血药峰浓度显著大于普通剂型;(2)15min内,血液和淋巴组织中药物浓度比较接近,而30min后淋巴组织中药物浓度明显高于血液中药物浓度,且淋巴组织内药物释放时间更长。结论腹腔内注射5-Fu炭纳米粒,在淋巴组织中能较血液长时间内维持相对较高的药物浓度。     

糖尿病肾病患者尿微量白蛋白与肌酐比值的影响因素研究

目的探讨糖尿病肾病（DN）患者尿微量白蛋白与肌酐比值（UACR）的相关因素。方法将受试者分为早期DN组123例,DN组113例,糖尿病组100例。比较三组代谢相关指标,进行UACR相关因素分析。结果二三组糖尿病病程、UACR、空腹血糖、HbA1c、TG、FIB、尿素氮、血尿酸、血肌酐之间差异有统计学意义（P〈0．05）。UACR、血尿酸、血肌酐、FIB、空腹血糖、尿素氮、糖尿病病程、HbA1c、TG与T2DM患者肾脏病情程度呈正相关（P〈0．05）。血肌酐、FIB、糖尿病病程、血尿酸、尿素氮、空腹血糖、HbA1c、TG、TC均与UACR呈正相关（P〈0．05）。糖尿病病程、FIB、血尿酸、HbA1c、TG经多因素Logistic回归分析为UACR相关因素,呈正相关（P〈0．05）。结论糖尿病病程、FIB、血尿酸、HbA1c、TG与UACR密切相关,通过干预可延缓DN的发生和进展。     

新生儿高胆红素血症神经毒性作用的监测

目的探讨高胆红素血症神经毒性对新生儿神经行为的影响。方法回顾性分析我院2008年1月。2011年10月收治入院的足月新生儿高胆红素血症患儿160例（观察组）临床资料,另选择我院同期正常足月生理性黄疸新生儿160例作为对照组,两组均采用总胆红素与白蛋白比值（B／A）与脑干听觉诱发电位（ABR）进行检测．并测查新生儿神经行为（NBNA）进行比对。结果观察组TBC、B／A比值测定结果与对照组比较差异有统计学意义（均P〈0．05）。ABR111波、V波潜伏期及I-Ⅲ、I-V波间期与对照组相比,差异均有统计学意义（均P〈0．05）。①高胆患儿NBNA评分均低于对照组,两者差异均有统计学意义。观察组NBNA评分均低于对照组,两者差异均有统计学意义（均P〈0．05）@B／A与NBNA评分的直线相关分析显示两者之间具有负相关关系（r=-0．46,P〈0．05）。结论①NBNA评分可应用早期诊断高胆所致的脑损伤。②B／A可作为评估胆红素神经毒性危险因素的指标之一。@ABR对了解新生儿早期听神经及脑干功能障碍有重要价值。