The Effects of Carbon Black on Cell Viability

Carbon black (CB) is a type of nanoparticle that is found in air pollution and is a known environmental toxin. The purpose of this work is to evaluate whether CB exposure activates cell death via apoptosis in cultured cell lines, supporting future work focused upon assessing the signaling pathways that might be induced by this exposure. Using adenocarcinomic human alveolar basal epithelial (A549) and baby hamster kidney (BHK-21) cells, we hypothesized that carbon black exposure causes cell death and potentially stress signaling via the endoplasmic reticulum (ER). The cells were exposed to CB and data collected for varied doses and time points. In order to measure cell apoptosis, the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method was used to detect apoptosis-associated DNA fragmentation. A 5 day exposure of CB at 100 ug/ml generated a significant reduction in cell survival and elevated numbers of TUNEL positive cells. Future work will focus upon assessing the stress pathways induced in these cells

Evaluating ITPR-dependence of Apoptotic Signaling from the Endoplasmic Reticulum

Stress within the endoplasmic reticulum (ER) can be induced by misfolded proteins accumulating in the lumen of this organelle. Signaling of ER stress to other parts of the cell results in altered gene expression, physiological adaptation, and with sustained stress, apoptosis (cell suicide). ER stress is often studied with highly toxic compounds that create severe ER stress rapidly, and a condition that is likely not physiologically relevant within an organism. In this study, we examine the apoptotic signaling induced by moderate ER stress, and in particular the inositol 1,4,5-trisphosphate receptor (ITPR). The ITPR regulates Ca2+ release from the ER lumen, and can induce apoptosis. We hypothesize that moderate levels of ER stress activate apoptosis via an ITPR-dependent signal. To induce moderate ER stress, we expose cells to 20-30nM concentrations of tunicamycin, an inhibitor of N-linked glycosylation in the ER. In this study, inclusion of an ITPR inhibitor (2-aminoethoxyphenyl borate, 2APB) protected cells from moderate ER stress, but did not protect cells from severe ER stress. A second methodology of assessing ITPR regulation of apoptosis includes overexpression of an ER-localized form of Bcl-2. The B-cell lymphoma 2 protein (Bcl-2has the ability to block the activation of cell suicide (apoptosis) by binding and inhibiting pro-apoptotic proteins (Bax family members). Bcl-2 is a membrane localized protein, found primarily in the mitochondrial outer membrane, and the endoplasmic reticulum (ER) membrane. In recent years, ER localized Bcl-2 has been shown to interact with the ITPR and inhibit pro-apoptotic Ca2+ signaling from the ER. We transfected cells with plasmids bearing forms of a Bcl-2 fusion protein to assess the capability of ER-Bcl-2 to protect cells from moderate apoptosis. The results of initial experiments did not show protection to either moderate or severe ER stress though some replicates of the experiment seemed to indicate protection. As this result is inconsistent with other results in our lab, we propose additional replicates of the experiment and using a drug-based mimic of this interaction to assess moderate ER stress signaling (Akl et al., 2013)

The Survivin and cIAP1 Anti-apoptotic Proteins are Differentially Downregulated in Response to Endoplasmic Reticulum Stress

The endoplasmic reticulum (ER) is an organelle tasked with synthesis and transport of 50% of new cellular proteins. Dysfunction within this organelle creates signals for repair, adaptation, and in severe cases, cellular apoptosis. Multiple human diseases have been associated with ER dysfunction, and the activation of apoptosis in important populations of cells. Inhibitor of Apoptosis (IAP) proteins are cytosolic proteins that play an anti-apoptotic role in the cytosol. The relationship between endoplasmic reticulum (ER) stress and the expression/stability of IAPs is not well characterized. The objective of this study was to characterize the affect of ER stress on the expression/stability of five members of the IAP family; XIAP, cIAP1, cIAP2, Survivin, and Livin. We also assessed how inhibition of the PI3kinase/Akt pathway affects expression of these proteins. In model cell lines (BHK21, A549), Survivin and cIAP1 expression was detected by immunoblot. ER stress was shown to induce a reduction of both Survivin and cIAP1 in a time and dose dependent manner, with Survivin displaying a more dynamic response. The phosphatidylinositol-3 kinase (PI3K) pathway has been associated with regulating expression of some IAP proteins. Inhibition of the PI3K decreased Survivin expression in both cell lines. Further research is required to confirm the affects of ER stress upon regulation of IAP expression (PI3K) and upon stability

Carbon Black and Titanium Dioxide Nanoparticles Differentially Activate Oxidative Stress and Apoptosis in A549 Human Alveolar Epithelial Cells

Recent studies have demonstrated that variation between particulate matter compositions have universally adverse effects on cells and living tissues. Carbon black and titanium dioxide are two such particulates that we are continuously exposed to, yet there is limited research to examine the potential deleterious effects on living tissue. The objective of this study is to characterize the effect of carbon black (CB) and titanium dioxide (TiO2) particulates on A549 human alveolar epithelial lung cells. CB and TiO2 powders were dispersed throughout a solution of water and bovine serum albumin by high-powered sonication. The effects of these particulates on A549 cells were analyzed through fluorescent microscope imaging, DAPI nuclear fluorescent staining, western blotting, H2DCFDA fluorescent staining. Death assays with DAPI revealed that both particulate types exhibit toxicity to cells and induce apoptosis. Live cell imaging showed perinuclear localization of both CB and TiO2 particulates. The human A549 cells were tested for reactive oxygen species (ROS) levels, which revealed a significant increase in ROS production induced by CB, but lowered ROS induction by TiO2. Interplay between ROS and intracellular calcium was examined; 2-APB, a calcium blocker was added to cells treated with CB. Results indicated that 50uM 2-APB provided notable protection to cells by decreasing ROS induction levels. Further research is required to determine through which apoptotic signaling pathway CB causes cell death and to definitively identify the signaling pathways involved in TiO2 associated toxicity. Further investigation of the explicit involvement of the ER in particulate induced apoptosis is a promising direction for future research

Effect of Shear Stress on ecNOS Expression and Dilation in Soleus Feed Arteries

Shear stress causes artery dilation and increased expression of endothelial cell nitric oxide synthase (ecNOS) in coronary and placental arteries. We sought to determine the importance of shear stress in maintaining normal dilation and normal levels of ecNOS in rat soleus feed arteries (SFA). SFA were isolated from male Sprague-Dawley rats and cannulated for in vitro microscopy (Fig. 6). SFA were exposed to no shear stress, low shear stress, or high shear stress conditions for 4 hours. After 4 hours, endothelium-dependent dilation (acetylcholine: ACh) and endothelium-independent dilation (sodium nitroprusside: SNP) were tested. Arteries were then uncannulated, mRNA was isolated, and RT-PCR for ecNOS mRNA was performed to determine whether shear stress altered ecNOS gene expression. Shear stress did not alter dilation to ACh, but dilation to SNP was greater in the high shear stress arteries. ecNOS mRNA content was greater in high shear stress arteries than low shear stress arteries. These data indicate that altered wall shear stress conditions alter ecNOS gene expression and vascular smooth muscle cell function

Effect of Shear Stress Direction on Endothelial Function and eNOS Phosphorylation in Soleus Feed Arteries

Blood flow feeding tissues and organs is closely regulated in order to meet metabolic and functional needs. Control of blood flow is accomplished by regulating the diameter of the arteries and arterioles feeding different organs. Several neural, hormonal, chemical and mechanical mechanisms contribute to the constriction and dilation of arteries. Shear stress, the frictional force created by streaming blood on the endothelial layer of arteries, is one of these mechanical mechanisms (1). Shear stress causes both acute and long term effects on endothelial cells (1,2,5). Blood in arteries typically flows away from the heart towards organs (causing antegrade shear stress) during cardiac contraction and briefly flows back toward the heart (causing retrograde shear stress) during cardiac filling. Retrograde flow occurs more often in some disease situations, and studies have shown that retrograde shear stress decreases endothelial cell function (3,4). The specific mechanisms for endothelial dysfunction are unknown, but altered mechanisms could include impaired cell signaling pathways. The most important endothelial cell dilatory signaling pathway is the production of nitric oxide (NO). Retrograde shear stress causes endothelial cells to secrete NO, and increased rates of shear stress cause increased expression and phosphorylation of nitric oxide synthase (eNOS). Regulatory phosphorylation of eNOS can potentially occur on at least four sites: Ser 1177, Ser 116, Ser 635 and Thr 497 (3). The most well characterized of these is Ser 1177, which is phosphorylated by a by PI3K/AKT shear dependent pathway. Regulating phosphorylation of eNOS is critical to endothelial health and maintaining cardiovascular equilibrium. Using rat soleus muscle feed arteries, we seek to determine the effects of changes in shear stress direction on both endothelial cell function and phosphorylation of eNOS at the Ser 1177 site

Ultrafine carbon nanoparticles activate inflammasome signaling and cell death in murine macrophages

Carbon black (CB) is the primary nanoparticulate component of air pollution from fossil fuel combustion. This work examines the cellular impact of ultrafine carbon (carbon black, CB) nanoparticles, that range in size down to 30 nm, upon murine macrophages. The size analysis of the carbon black nanoparticles was performed using atomic force microscopy (AFM) and transmission electron microscopy (TEM) techniques. RAW246.7 macrophage cells were exposed to CB doses ranging from 50 – 200 ug/ml in complete media. Analysis of cell survival over time revealed elevated rates of significant nuclear degradation and cell lifting after 48 hours of exposure, and in a dose dependent pattern. Live cell imaging of cells exposed to nanoparticles revealed a visible uptake of nanoparticles with accumulation over time. To assess inflammasome signaling, both caspase-1 activation and IL-1b production were observed in whole cell lysates. Caspase-1 activation was measured as the appearance of the active (cleaved) form of the protease appearing in immunoblot analysis. The analysis revealed significant activation of caspase-1 with 48-hour CB exposures at doses of 100-200 ug/ml. Similarly, levels of IL-1b were significantly induced by CB exposure, with maximal induction observed after a 48 hour exposure. Macrophage cells were assessed for accumulation of LC3, a marker for autophagosome vesicle accumulation. Immunoblot analysis revealed a significant accumulation of LC3 in response to CB exposure and in response to chloroquine, which inhibits autophagosome/lysosome fusion. Taken together, these results support a model in which CB exposure activates the inflammasome and disrupts autophagy in macrophages

Examining a Relationship Between Chronic Dietary Folic Acid Deficiency and Activation of p53 Gene in Down Syndrome Ts65Dn Mice

Seaver Undergraduate Researc

Genetically induced production of secondary metabolites in Bacillus megaterium

Historically, the most important source of new antibiotic drug leads has been small organic compounds made by bacteria. Many antibiotics have been developed into pharmaceutical agents from these molecules (often called secondary metabolites) produced by soil bacteria. Bacillus species are soil bacteria known for producing various antimicrobials including gramicidin, bacitracin, surfactin, and others. Bacillus megaterium is a widely used model gram positive bacterium. Although there has been extensive research on this organism, little is known about its secondary metabolites. We hypothesized that the production of secondary metabolites in this organism could be induced by replacing promoters controlling the expression of genes within the identified clusters. In this study, gene clusters that are predicted to control the production of secondary metabolites were identified using the antiSMASH bioinformatics platform. Phenotypic changes in the secondary metabolite profile of B. megaterium were observed when culture conditions were varied indicating that target metabolites are accessible for chemical analysis. To increase production of targeted secondary metabolites, the native promoter of identified secondary metabolite gene clusters will be replaced by an inducible promoter using plasmid mediated chromosomal integration