High-throughput multiplex assays with mouse macrophages on pillar plate platforms.

It is challenging to rapidly identify immune responses that reflect the state and capability of immune cells due to complex heterogeneity of immune cells and their plasticity to pathogens and modulating molecules. Thus, high-throughput and easy-to-use cell culture and analysis platforms are highly desired for characterizing complex immune responses and elucidating their underlying mechanisms as well. In response to this need, we have developed a micropillar chip and a 384-pillar plate, printed mouse macrophage, RAW 264.

High-content imaging of 3D-cultured neural stem cells on a 384-pillar plate for the assessment of cytotoxicity.

The assessment of neurotoxicity has been performed traditionally with animals. However, in vivo studies are highly expensive and time-consuming, and often do not correlate to human outcomes. Thus, there is a need for cost-effective, high-throughput, highly predictive alternative in vitro test methods based on early markers of mechanisms of toxicity.

High-Throughput Assessment of Metabolism-Induced Toxicity of Compounds on a 384-Pillar Plate.

A variety of oxidative and conjugative enzymes are involved in the metabolism of compounds including drugs, which can be converted into toxic metabolites by Phase I drug-metabolizing enzymes (DMEs), such as the cytochromes P450 (CYP450s), and/or detoxified by Phase II DMEs, such as UDP-glucuronosyltransferases (UGTs), sulfotransferases (SULTs), and glutathione S-transferases (GSTs). Traditionally, primary hepatocytes containing a complete set of DMEs have been widely used as a gold standard to assess metabolism-induced compound toxicity. However, primary hepatocytes are expensive, have high donor variability in expression levels of DMEs, and rapidly lose liver-specific functions when the cells are maintained under standard in vitro cell culture conditions over time.

3D-cultured neural stem cell microarrays on a micropillar chip for high-throughput developmental neurotoxicology.

Numerous chemicals including environmental toxicants and drugs have not been fully evaluated for developmental neurotoxicity. A key gap exists in the ability to predict accurately and robustly in vivo outcomes based on in vitro assays. This is particularly the case for predicting the toxicity of chemicals on the developing human brain.

High-throughput metabolism-induced toxicity assays demonstrated on a 384-pillar plate.

The US Environmental Protection Agency (EPA) launched the Transform Tox Testing Challenge in 2016 with the goal of developing practical methods that can be integrated into conventional high-throughput screening (HTS) assays to better predict the toxicity of parent compounds and their metabolites in vivo. In response to this need and to retrofit existing HTS assays for assessing metabolism-induced toxicity of compounds, we have developed a 384-pillar plate that is complementary to traditional 384-well plates and ideally suited for culturing human cells in three dimensions at a microscale. Briefly, human embryonic kidney (HEK) 293 cells in a mixture of alginate and Matrigel were printed on the 384-pillar plates using a microarray spotter, which were coupled with 384-well plates containing nine model compounds provided by the EPA, five representative Phase I and II drug metabolizing enzymes (DMEs), and one no enzyme control.

High-content imaging assays on a miniaturized 3D cell culture platform.

The majority of high-content imaging (HCI) assays have been performed on two-dimensional (2D) cell monolayers for its convenience and throughput. However, 2D-cultured cell models often do not represent the in vivo characteristics accurately and therefore reduce the predictability of drug toxicity/efficacy in vivo. Recently, three-dimensional (3D) cell-based HCI assays have been demonstrated to improve predictability, but its use is limited due to difficulty in maneuverability and low throughput in cell imaging.

Deconvolution of images from 3D printed cells in layers on a chip.

Layer-by-layer cell printing is useful in mimicking layered tissue structures inside the human body and has great potential for being a promising tool in the field of tissue engineering, regenerative medicine, and drug discovery. However, imaging human cells cultured in multiple hydrogel layers in 3D-printed tissue constructs is challenging as the cells are not in a single focal plane. Although confocal microscopy could be a potential solution for this issue, it compromises the throughput which is a key factor in rapidly screening drug efficacy and toxicity in pharmaceutical industries.

Prediction of metabolism-induced hepatotoxicity on three-dimensional hepatic cell culture and enzyme microarrays.

Human liver contains various oxidative and conjugative enzymes that can convert nontoxic parent compounds to toxic metabolites or, conversely, toxic parent compounds to nontoxic metabolites. Unlike primary hepatocytes, which contain myriad drug-metabolizing enzymes (DMEs), but are difficult to culture and maintain physiological levels of DMEs, immortalized hepatic cell lines used in predictive toxicity assays are easy to culture, but lack the ability to metabolize compounds. To address this limitation and predict metabolism-induced hepatotoxicity in high-throughput, we developed an advanced miniaturized three-dimensional (3D) cell culture array (DataChip 2.

Suppression of Tobacco Carcinogen-Induced Lung Tumorigenesis by Aerosol-Delivered Glycerol Propoxylate Triacrylate-Spermine Copolymer/Short Hairpin Rab25 RNA Complexes in Female A/J Mice.

Background: Rab25, a member of Rab family of small guanosine triphosphatase, is associated with progression of various types of human cancers, including lung cancer, the leading cause of cancer-associated deaths around the globe.

Methods: In this study, we report the gene therapeutic effect of short hairpin Rab25 RNA (shRab25) on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis in female A/J mice. Initially, mice (6 weeks old) were injected with single dose of NNK (2 mg/0.

Cigarette Smoking Condensate Disrupts Endoplasmic Reticulum-Golgi Network Homeostasis Through GOLPH3 Expression in Normal Lung Epithelial Cells.

Introduction: Cigarette smoke (CS) is associated with a broad range of diseases including lung cancer. Many researchers have suggested that cigarette smoke condensate (CSC) may be more toxic compared to cigarette smoke extract (CSE) because CSC contains the lipid-soluble faction of smoke while CSE contains the hydrophilic or gas phase. The aim of this research is to investigate the effects of CSC on the disruption of endoplasmic reticulum (ER)-Golgi homeostasis in normal lung epithelial cells.

Endoplasmic reticulum-Golgi intermediate compartment protein 3 knockdown suppresses lung cancer through endoplasmic reticulum stress-induced autophagy.

Trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus is elevated in cancer cells. Therefore, proteins of the ER-Golgi intermediate compartment (ERGIC) attract significant attention as targets for cancer treatment. Enhanced cancer cell growth and epithelial-mesenchymal transition by ERGICs correlates with poor-prognosis of lung cancer.

Effects of feeding a diet containing Gymnema sylvestre extract: Attenuating progression of obesity in C57BL/6J mice.

Objective: To investigate the effect of Gymnema sylvestre extract (GS) on initial anti-obesity, liver injury, and glucose homeostasis induced by a high-fat diet (HFD).

Methods: The dry powder of GS was extracted with methanol, and gymnemic acid was identified by high performance liquid chromatography as deacyl gymnemic acid. Male C57BL/6J mice that fed on either a normal diet, normal diet containing 1 g/kg GS (CON+GS), HFD, or HFD containing 1.

Methodological considerations of electron spin resonance spin trapping techniques for measuring reactive oxygen species generated from metal oxide nanomaterials.

Qualitative and quantitative analyses of reactive oxygen species (ROS) generated on the surfaces of nanomaterials are important for understanding their toxicity and toxic mechanisms, which are in turn beneficial for manufacturing more biocompatible nanomaterials in many industrial fields. Electron spin resonance (ESR) is a useful tool for detecting ROS formation. However, using this technique without first considering the physicochemical properties of nanomaterials and proper conditions of the spin trapping agent (such as incubation time) may lead to misinterpretation of the resulting data.

Dihydroergotamine Tartrate Induces Lung Cancer Cell Death through Apoptosis and Mitophagy.

Background: Mitochondria have emerged as a major target for anticancer therapy because of their critical role in cancer cell survival. Our preliminary works have suggested that dihydroergotamine tartrate (DHE), an antimigraine agent, may have effects on mitochondria.

Methods: We examined the effect of DHE on the survival of several lung cancer cells and confirmed that DHE suppressed diverse lung cancer cell growth effectively.

Inhalation of titanium dioxide induces endoplasmic reticulum stress-mediated autophagy and inflammation in mice.

Titanium dioxide (TiO2) nanoparticles are widely used in cosmetics, sunscreen, electronics, drug delivery systems, and diverse bio-application fields. In the workplace, the primary exposure route for TiO2 nanoparticles is inhalation through the respiratory system. Because TiO2 nanoparticles have different physiological properties, in terms of size and bioactivity, their toxic effects in the respiratory system must be determined.

Suppression of tumor growth in lung cancer xenograft model mice by poly(sorbitol-co-PEI)-mediated delivery of osteopontin siRNA.

Small interfering RNA (siRNA)-mediated gene silencing represents a promising strategy for treating diseases such as cancer; however, specific gene silencing requires an effective delivery system to overcome the instability and low transfection efficiency of siRNAs. To address this issue, a polysorbitol-based transporter (PSOT) was prepared by low molecular weight branched polyethylenimine (bPEI) crosslinked with sorbitol diacrylate (SDA). Osteopontin (OPN) gene, which is highly associated with non-small cell lung cancer (NSCLC) was targeted by siRNA therapy using siRNA targeting OPN (siOPN).

Titanium Dioxide Nanoparticles Induce Endoplasmic Reticulum Stress-Mediated Autophagic Cell Death via Mitochondria-Associated Endoplasmic Reticulum Membrane Disruption in Normal Lung Cells.

Nanomaterials are used in diverse fields including food, cosmetic, and medical industries. Titanium dioxide nanoparticles (TiO2-NP) are widely used, but their effects on biological systems and mechanism of toxicity have not been elucidated fully. Here, we report the toxicological mechanism of TiO2-NP in cell organelles.

Enhancement of cancer specific delivery using ultrasound active bio-originated particles.

A hybrid multifunctional particle comprising of a microbbule (MB), liposome (Lipo), and an Fe ion chelated melanin nanoparticle (MNP(Fe)) was applied for ultrasound mediated cancer targeting as a theranostic agent.

Therapeutic effect of Broussonetia papyrifera and Lonicera japonica in ovalbumin-induced murine asthma model.

Broussonetia papyrifera (L.) Vent. and Lonicera japonica Thunb.

Physicochemical determinants of multiwalled carbon nanotubes on cellular toxicity: influence of a synthetic method and post-treatment.

Since the discovery of carbon nanotubes (CNTs), scientists have performed extensive studies on nanotubes in the fields of materials science, physics, and electronic engineering. Because multiwalled CNTs (MWCNTs) are not homogeneous materials, and because it is not feasible to test every newly synthesized MWCNT, this study was aimed at investigating the physicochemical properties that primarily determine the cellular toxicity of MWCNTs. This study analyzed the relationship between cell viability and physicochemical characteristics following exposure to eight different MWCNTs.

Differential toxic responses between pristine and functionalized multiwall nanotubes involve induction of autophagy accumulation in murine lung.

Carbon nanotubes (CNT) are becoming commonly used in industrial applications. However, the toxicity associated with this material remains to be established. The aim of this study was to investigate the potential toxic mechanisms associated with multiwall carbon nanotubes (MWCNT) in normal mouse lung.

Methylmercury induces caspase-dependent apoptosis and autophagy in human neural stem cells.

Methylmercury (MeHg) is a well-known human neurotoxic agent whose exposure sources are mainly environmental and aquatic-derived food. MeHg is reported to induce central nervous system disability. However, the exact mechanism of MeHg-induced neurotoxicity is still unknown.

Role of p53 in the cellular response following oleic acid accumulation in Chang liver cells.

Abnormal accumulation of fatty acids triggers the harmful cellular response called lipotoxicity. In this study, we investigated the cellular response following accumulation of oleic acid (OA), a monounsaturated fatty acid, in human Chang liver cells. OA droplets were distributed freely in the cytoplasm and/or degraded within lysosomes.

Zinc oxide nanoparticle induced autophagic cell death and mitochondrial damage via reactive oxygen species generation.

Zinc oxide nanoparticles (ZnO-np) are used in an increasing number of industrial products such as paint, coating and cosmetics, and in other biological applications. There have been many suggestions of a ZnO-np toxicity paradigm but the underlying molecular mechanisms about the toxicity of ZnO-np remain unclear. This study was done to determine the potential toxicity of ZnO-np and to assess the toxicity mechanism in normal skin cells.

Aerosol delivery of lentivirus-mediated O-glycosylation mutant osteopontin suppresses lung tumorigenesis in K-ras (LA1) mice.

Background: Osteopontin (OPN) is a secreted glycophosphoprotein that has been implicated in the regulation of cancer development. The function of OPN is primarily regulated through post-translational modification such as glycosylation. As yet, however, the relationship between OPN glycosylation and lung cancer development has not been investigated.