interleukin-8 and resazurin

Air-liquid interface (ALI) exposures enable in vitro testing of mixtures of gases and particles such as diesel exhaust (DE). The main objective of this study was to investigate the feasibility of exposing human lung epithelial cells at the ALI to complete DE generated by a heavy-duty truck in the state-of-the-art TNO powertrain test center. A549 cells were exposed at the air-liquid interface to DE generated by a heavy-duty Euro III truck for 1.5h. The truck was tested at a speed of ∼70kmh(-1) to simulate free-flowing traffic on a motorway. Twenty-four hours after exposure, cells were analyzed for markers of oxidative stress (GSH and HO-1), cytotoxicity (LDH and Alamar Blue assay) and inflammation (IL-8). DE exposure resulted in an increased oxidative stress response (significantly increased HO-1 levels and significantly reduced GSH/GSSH ratio), and a decreased cell viability (significantly decreased Alamar Blue levels and slightly increased LDH levels). However, the pro-inflammatory response seemed to decrease (decrease in IL-8). The results presented here demonstrate that we are able to successfully expose A549 cells at ALI to complete DE generated by a heavy-duty truck in TNO's powertrain test center and show oxidative stress and cytotoxicity responses due to DE exposure.

Topics: Air; Air Pollutants; Cell Culture Techniques; Cell Line, Tumor; Cell Survival; Epithelial Cells; Glutathione; Heme Oxygenase-1; Humans; Interleukin-8; L-Lactate Dehydrogenase; Laboratories; Oxazines; Pulmonary Alveoli; Toxicity Tests; Vehicle Emissions; Xanthenes

To investigate the effect of desiccation on secretion of inflammatory cytokines in corneal epithelial cells and in the rat desiccation model.. A human corneal epithelial cell line (CEPI) was grown in keratinocyte growth medium 2 (KGM2) to approximately 80% confluence. The medium was aspirated and dishes were left for 0 to 30 min with the cover left open to dry the cells (short-term desiccation). After desiccation, KGM2 was added to the dishes and collected from the dishes 15 min later to measure the concentrations of cytokines in the medium by sandwich enzyme immunoassay (ELISA). Viability of the cells was estimated with alamer blue. To study the effect of long-term desiccation, cultivated cells on transwells were used. After dessiccation for up to 8 h, the viability of the cells and levels of cytokines in the culture medium were examined. The expression of cytokines in the cornea of the dry eye model rat was measured by real-time PCR.. Short-term dessication of CEPI cells significantly increased the interleukin (IL)-6 level and slightly increased the tumor necrosis factor (TNF)-α level. Anti-IL-6 antibody partially suppressed cell death caused by desiccation. Upon long-term desiccation, IL-6 and IL-8 levels were increased. 
In the dry eye model rats, the IL-6 mRNA level in the cornea significantly increased, whereas TNF-α mRNA level slightly increased.. Desiccation induced IL-6 expression in corneal epithelial cells, suggesting that IL-6 participates in desiccation-induced cell death.

Topics: Animals; Apoptosis; Cell Line; Cornea; Desiccation; Diffusion Chambers, Culture; Dry Eye Syndromes; Epithelial Cells; Epithelium, Corneal; Gene Expression; Interleukin-6; Interleukin-8; Male; Oxazines; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; RNA, Messenger; Tumor Necrosis Factor-alpha; Xanthenes

This paper describes the in vitro cytotoxicity assessment of single walled carbon nanotubes (SWCNT) on A549 cells, a human lung cell line. Cellular viability was determined using the alamar blue (AB), neutral red (NR) and MTT assays, which evaluated metabolic, lysosomal and mitochondrial activity respectively. In addition, the total protein content of the cells was measured using the coomassie brilliant (CB) blue assay. Supernatants were also assayed for Adenylate Kinase (AK) release and Interleukin 8 (IL-8) which indicated a loss of cell membrane integrity and an inflammation response respectively. To investigate the interactions between serum components in the test medium and the test materials, exposures were conducted both in serum containing (5%) and serum-free medium. Results from the cytotoxicity tests (AB, CB, MTT) revealed the SWCNT to have very low acute toxicity to the A549 cells as all but one of the reported 24h EC(50) values exceeded the top concentration tested (800 microg/ml). The SWCNT were found to interfere with a number of the dyes used in the cytotoxicity assessment and we are currently conducting a comprehensive spectroscopic study to further investigate these interactions. Of the multiple cytotoxicity assays used, the AB assay was found to be the most sensitive and reproducible. Transmission electron microscopy (TEM) studies confirmed that there was no intracellular localization of SWCNT in A549 cells following 24h exposure; however, increased numbers of surfactant storing lamellar bodies were observed in exposed cells.

Topics: Adenylate Kinase; Cell Line; Cell Nucleus; Cell Survival; Culture Media, Conditioned; Cytoplasm; Dose-Response Relationship, Drug; Epithelial Cells; Formazans; Humans; Indicators and Reagents; Interleukin-8; Lung; Microscopy, Electron, Transmission; Nanotechnology; Nanotubes, Carbon; Necrosis; Neutral Red; Oxazines; Quartz; Rosaniline Dyes; Tetrazolium Salts; Xanthenes